Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 530712 13 pageshttpdxdoiorg1011552013530712
Research ArticlePoly(lactic-co-glycolic) AcidNanohydroxyapatite ScaffoldContaining Chitosan Microspheres with AdrenomedullinDelivery for Modulation Activity of Osteoblasts and VascularEndothelial Cells
Lin Wang1 Chunyan Li2 Yingxin Chen1 Shujun Dong1 Xuesi Chen3 and Yanmin Zhou2
1 VIP Integrated Department School of Stomatology Jilin University 1500 Qinghua Road Changchun 130021 China2 Implant Center School of Stomatology Jilin University Changchun 130021 China3 State Key Laboratory of Polymer Physics and Chemistry Changchun Institute of Applied Chemistry Chinese Academy of Sciences5625 Renmin Avenue Changchun 130022 China
Correspondence should be addressed to Yanmin Zhou zhouymjlueducn
Received 24 February 2013 Revised 7 May 2013 Accepted 17 May 2013
Academic Editor Andre Van Wijnen
Copyright copy 2013 Lin Wang et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Adrenomedullin (ADM) is a bioactive regulatory peptide that affects migration and proliferation of diverse cell types includingendothelial cells smooth muscle cells and osteoblast-like cells This study investigated the effects of sustained release of ADMon the modulation activity of osteoblasts and vascular endothelial cells in vitro Chitosan microspheres (CMs) were developedfor ADM delivery Poly(lactic-co-glycolic) acid and nano-hydroxyapatite were used to prepare scaffolds containing microsphereswith ADMThe CMs showed rough surface morphology and high porosity and they were well-distributedThe scaffolds exhibitedrelatively uniform pore sizes with interconnected pores The addition of CMs improved the mechanical properties of the scaffoldswithout affecting their high porosity In vitro degradation tests indicated that the addition of CMs increased the water absorptionof the scaffolds and inhibited pH decline of phosphate-buffered saline medium The expression levels of osteogenic-related andangiogenic-related genes were determined in MG63 cells and in human umbilical vein endothelial cells cultured on the scaffoldsrespectivelyThe expression levels of osteogenic-related and angiogenic-related proteins were also detected by western blot analysisTheir expression levels in cells were improved on the ADMdelivery scaffolds at a certain time pointThe in vitro evaluation suggeststhat the microsphere-scaffold system is suitable as a model for bone tissue engineering
1 Introduction
Bone regeneration comprises a well-orchestrated series ofbiological events including the recruitment and proliferationof osteoprogenitors frommesenchymal stem cells cell differ-entiation osteoid formation and ultimately mineralization[1] Complex clinical conditions in which bone regenerationmaterials are required in large quantity exist Scaffolds forbone engineering are degradable matrices designed to sup-port cell adhesion proliferation and differentiation for boneregeneration
The Food and Drug Administration for human biomedi-cal applications approved the use of poly(lactic-co-glycolic)acid (PLGA) as a commercially available biomaterial [2]
PLGA is relatively hydrophobic and obstructs cell adhesionwhich are the common weaknesses for synthetic polymersNevertheless it can form scaffolds with high mechanicalstrength Similar to natural bone mineral hydroxyapatite(HA) is relatively easier to be identified by cells or biomacro-molecules which can improve the bioactivity bioavailabilityand biocompatibility of scaffoldsMoreover the release of cal-cium and phosphorus ions during the degradation ofHAmaybe involved in bone metabolism to promote the formation ofnew bone The combination of PLGA with natural polymerssuch as HA might overcome the limitations of syntheticand naturally derived polymers alone and produce a materialwith properties (eg high porosity and controllability of pore
2 BioMed Research International
size) beneficial for biomedical applications [3] to mimic bonestructure and substructure
The continued enhancement of biomaterial strategies isknown to be highly dependent on the ability to promoterapid and stable vascularization within scaffolds [4] Themost common approach for vascularization in biomaterialsemerged to address biological growth factors into scaffoldsthus paving the way for vascular endothelial cell seeding andpolymer bioactivity [5] Adrenomedullin (ADM) a 52-aminoacid ringed-structure peptide with C-terminal amidation is anewly discovered member of the calcitonin peptide family itwas originally isolated from human pheochromocytoma [6]ADM is also present in many other tissues such as bone [7]kidney lung heart and adrenals [8] Many studies focusedon the cardiovascular and endocrine effects of ADM [9ndash11]However in recent years other significant effects such asosteogenesis [12] angiogenesis [13] and antibacterial effects[14 15] have also been detectedMany studies [16 17] showedthat ADM can stimulate osteoblast proliferation even at lowconcentrations Given its structural and biological homologywith calcitonin gene-related peptide ADM can also stimulatethe proliferation of osteoblasts by increasing the cAMP levelin osteoblast-like cells [18 19] In addition ADM has a reg-ulatory function in angiogenesis by modulating endothelialcell behavior However similar to the dilemma of peptidesin treating bone defects the application of ADM not onlyrequires appropriate temporary release but also requires acertain concentration to be sustained through controlledrelease during bone regeneration An efficient delivery systemmay be required to provide the controlled release of ADMover an extended period
To date a couple of biodegradable polymers have beenused to encapsulate proteins and peptides Chitosan or poly120573-(14)-2-amino-2-deoxy-D-glucose is an excellent naturalhydrophilic cationic polysaccharide derived from chitin Itis widely used for the controlled delivery of polypeptidesand proteins in the form of microspheres or nanospheres[20] Chitosan exhibits favorable biological properties suchas biodegradability biocompatibility nontoxicity hemo-staticity high surface-charge density bacteriostaticity andstrong adhesion [21] It is also used in the field of surgicalsutures wound dressings drug delivery agents defect fillersand tissue-engineering scaffolds [22] Simple adsorption ofgrowth factors into chitosan allows local delivery but thetemporal control over release kinetics is limited [23] An idealdelivery system can be designed as chitosan microspheres(CM) compound with porous scaffolds of proper mechanicalproperties These two components can coordinately enhancetissue regeneration and extend the release time of growthfactors
Previous studies improved the bioactivity of scaffolds bycoating some proteins or peptides to receive and respond tospecific biological signals In this study CMs loaded withADM were prepared by an emulsion-ionic cross-linkingmethod CMs were embedded in a PLGAnanohydroxyap-atite (nHA) scaffold to enhance the compressive strength anddevelop a microsphere-scaffold system with the capacity ofreleasing bioactive factor in a well-controlled manner The invitro ADM release kinetics of microspheres and composite
scaffolds was demonstrated in our previous study [24] Thepresent study aims to investigate the feasibility of using CMsas a carrier for the controlled release of regulatory peptideADMThe surface morphology size distribution and encap-sulation efficiency (EE) of the microspheres were estimatedAfter introducing CMswith ADM into PLGAnHA scaffoldsthe morphological and mechanical features and degrada-tion behavior of the composite scaffolds were evaluatedThe biological capabilities of the PLGAnHA scaffolds wereevaluated by culturingMG63 cells and human umbilical veinendothelial cells (HUVEC) on these scaffolds by real-timepolymerase chain reaction (PCR) and western blot analysis
2 Materials and Methods
21 Materials Chitosan (119872119908= 500 kDa) was purchased
from Jinqiao Chemical Reagents Company (Taizhou Zhe-jiang China) Human ADM (purity = 95 by HPLC)was obtained from Phoenix Pharmaceuticals (BurlingameCanada) Tripolyphosphate (TPP) and span-80were obtainedfrom Aladdin (Shanghai China) PLGA (nLAnGA = 8020)and nHA were obtained from Changchun Institute ofApplied Chemistry Chinese Academy of Sciences MG63 andHUVEC cells were provided by the Basic Medical Collageof Jilin University Liquid paraffin 14-dioxane phosphate-buffered saline (PBS pH 74) and other chemicals were allanalytical grade and used as received
22 Preparation and Characterization of TPP-CMs UsingTPP as cross-linker CMs loaded with ADM were preparedby an emulsion-ionic cross-linking method Briefly 900mgof chitosan was dissolved in 29mL of 2 (vv) aqueousacetic acid and stirred until the solution was transparentMeanwhile 500120583g of ADM was dissolved in 1mL of 2(vv) acetic acid and added into the chitosan solution Themixture was poured into 300mL of liquid paraffin containing2 (wv) of span-80 and stirred mechanically for 2 h Then70mL of 5 (wv) TPP was dropped into the emulsion andstirred for 4 h at room temperature The end emulsion wasrepeatedly washed with excess amounts of petroleum etherisopropyl alcohol and distilled waterThemicrospheres wereobtained after lyophilization (LGJ-18 Sihuan China)
The morphology of the CMs was examined under ascanning electron microscope (SEM XL30ESEM-FEG FELNetherlands) For the measurement the microspheres wereattached to metal stubs and sputter coated with gold undervacuum In addition the diameter of the microspheres wasdetermined by a laser particle size analyzer (LS 13 320Beckman Coulter USA)
A certain amount of ADM-loaded chitosanmicrospheres(CMs-ADM)was dissolved in 5mL of 2 aqueous acetic acidsolution and filtered to remove any undissolved residue Theamounts of ADM in the collected supernatants were mea-sured by HPLC Encapsulated efficiency (EE) was calculatedas follows using the data above
EE () = actual ADM amounttheoretical ADM amount
times 100 (1)
BioMed Research International 3
All measurements were performed in triplicate for eachof the samples
23 Preparation and Characterization of Scaffolds PorousPLGAnHACMs scaffolds were developed by thermallyinduced phase separation (TIPS) PLGA (720mg) was dis-solved in 12mL of 14-dioxane and nHA (360mg) was addedinto the mixture after stirring for half an hour Ultrasonicswas used to completely disperse the nHA for 10min Then240mg of CMs was added to the aforementioned solutionThe mixture was agitated by magnetic stirring to completelydisperse the microspheres and poured into a polytetrafluo-roethene plate Then the solution was frozen overnight ina refrigerator at minus20∘C Finally PLGAnHACM scaffoldswere obtained after lyophilization Pure PLGAnHA scaffoldsprepared by the same method were set as a control for thesucceeding experiments
The pore architecture of the scaffolds which were locatedon the metal stubs and sputter coated with gold was exam-ined by SEM The porosity of the scaffolds was measuredby a mercury intrusion porosimeter (AutoPore IV 9500USA) Percent porosity was provided in the output from theequipment In addition the density of the polymer scaffoldswas tested by a modified liquid displacement method [25]
24 Test of Mechanical Properties The resistance tomechani-cal compression of the scaffolds was tested on an electromag-netic testingmachine (Enduratec Elf 3200 Bose CorporationEden Prairie MN USA) with a 10 kN load cell at roomtemperature The samples were cylinders with a diameter of8mm and a height of 15 mm Compression tests were carriedout under displacement control at a velocity of 01 mms untilthe sample was 50 of the initial height The compressivemodulus and compressive strength were calculated as theaverage of three scaffold measurements
25 In Vitro Degradation Test The cylinder scaffolds with adiameter of 8mm and a height of 5mm were incubated at37∘C in 10mL PBS pH 74 The samples were centrifugedfor 3min to ensure that the entire scaffold was immersedinto the buffer except in water absorption (WA) test andthen incubated at 37∘C under dynamic conditions for 12weeks The incubation buffer was weekly replaced with freshPBS solution except in pH changing test At scheduledfold (once a week) the samples were washed with distilledwater and lyophilized The pH of the PBS solution duringdegradation was monitored by a pH meter (FE20 MettlerToledo Shanghai China)
All data presented in the figures of this paper are theaverage data from six parallel samples
251 Weight Loss (WL) TheWLof scaffolds was gravimetri-cally examined (AL 104 Mettler Toledo Shanghai China) atscheduled times after the samples were freeze dried WL wascomputed as follows
WL () =1198820minus119882119905
1198820
times 100 (119899 = 6) (2)
where1198820and119882
119905are the weights of the samples before and
after incubation respectively
252 WA Property The initial weight of the dry sample wascharacterized as1198821015840
0 The scaffolds were taken out from PBS
at intervals and gravimetrically weighed (Mettler Toledo AL104) after wiping off the surface water to obtain the wet mass1198821015840119905 Water content was computed as follows
WA () =1198821015840119905minus11988210158400
1198821015840119905
times 100 (119899 = 6) (3)
253 pH of DegradationMedium ThepH of the degradationmedium was measured using a pH meter once a weekfor 12 weeks The medium was not refreshed in the entiredegradation period
26 Gene and Protein Expression of MG63 and HUVECCells Cultured on the Scaffolds We determined the expres-sion levels of osteogenic-related (osteopontin (opn) runt-related transcription factor 2 (runx2) transcription factor7 (sp7) and collagen type 1 (col1)) and angiogenic-related(vascular endothelial growth factor (vegf) andG-protein cou-pled activity-modifying protein 2 (ramp2)) genes in MG63and HUVEC cells respectively Meanwhile the expressionlevels of osteogenic-related (RUNX2 and COLLAGEN-1)and angiogenic-related (VEGF) proteins were determined inMG63 and HUVEC cells respectively The polymer scaffoldswith and without CMs-ADMwere prepared as cylinders witha diameter of 10mm and a height of 2mm and then sterilizedwith a 25 kGy Co60 radiation in preparation for cell seeding
MG63 and HUVEC cell lines were respectively main-tained in Dulbeccorsquos modified Eaglersquos medium (Sigma STLouis MO USA) and Iscoversquos modified Dulbeccorsquos medium(Sigma ST Louis MO USA) containing 10 fetal bovineserum in an incubator with humidified atmosphere contain-ing 95 air and 5 CO
2 For subculture cells at 80 to
90 confluence were passaged at a ratio of 1 3 after treatingwith 025 trypsin The sterilized scaffolds were seeded withapproximately 15 times 105 cells of the third passage The cell-scaffold complexes were cultured in 12-well tissue cultureplates for up to 5 d The cells were retrieved for gene andprotein expression determination at specific time points (days1 3 and 5) The cells cultured in normal condition withoutscaffold were selected as control
261 Quantitative Real-Time PCR Total RNA was iso-lated from retrieved MG63 and HUVEC cells of differenttime points using the Trizol reagent (Invitrogen CarlsbadCanada) according to themanufacturerrsquos protocolThe purityof RNA was determined at 260 and 280 nm absorbance toensure that the ratio was beyond 18 Reverse transcriptionwas performed with a Quantscript RT Kit (Tiangen BeijingChina) using 1 120583g total RNA to obtain cDNA Real-timequantitative PCR was performed for the quantification ofgene expression using a Realtime SYBR Green I PCR MasterMix (TOYOBO Japan) in a StepOnePlus Real-Time PCRSystem (ABI Foster City CA USA) Table 1 lists the primers
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designed by Premier Primer 5 and checked by BLAST Therelative expression levels of genes were analyzed using the2minusΔΔCt method [26] by normalizing with GAPDH expressionand presented as fold increase relative to the control group
262 Western Blot Analysis MG63 and HUVEC cells wereretrieved at predetermined time points washed with ice-cold PBS and centrifuged at 5000timesg for 5min at 4∘CThen the cells were lysed in ice-cold lysis buffer (10mMTris pH 74 100mMNaCl 1mM ethylenediaminetetraaceticacid 1mMphenylmethanesulfonyl fluoride 1 Triton X-10010 glycerol 01 sodium dodecyl sulfate (SDS) and 05deoxycholate) on ice for 1 h After centrifugation proteinconcentration was determined using a NanoDrop ND1000(ThermoScientific Wilmington DE USA) spectrophotome-ter The samples (40 120583g protein) were resolved by 10 SDS-polyacrylamide gel electrophoresis and electrophoreticallytransferred to Immun-Blot polyvinylidene difluoride mem-branes (Millipore Bedford MA USA) After blocking inTris-buffered saline with 005 Tween-20 (TBST) containing5 nonfat dry milk for 1 h the membranes were washedthrice with TBST at room temperature Then primary anti-bodies (SantaCruz Biotechnology Santa Cruz CA USA)were added on the membranes and incubated overnightat 4∘C After incubation with the appropriate horseradishperoxidase protein bands coupled with secondary antibody(1 5000 dilution Proteintech Group Chicago USA) werevisualized with an enhanced chemiluminescent system Pro-tein levels from immunoblot were quantified by densitometryusingQuantityOne software (Bio-RadUSA) Target proteinswere normalized against 120573-actin expression
27 Statistical Analysis All data were expressed as mean plusmnSD Statistical significance of differences was assessed by one-way ANOVA and Studentrsquos t-test Statistical significance wasconsidered at 119875 lt 005
3 Results and Discussion
31 Characterization of TPP-CMs and Polymer Scaffolds Anemulsion-ionic cross-linking method was used in preparingthe CMs loaded with ADM in the presence of TPPThe sharpand relative rough surface of TPP-CMs is shown in Figure 1Microsphere cracks were seldom observed in this study Asshown in Figure 2 the size of the microspheres was welldistributed The average diameter of CMs was 4269 120583m andEE was 794 plusmn 23 Chitosan was selected for developingmicrospheres because of its well-known biocompatibilitybiodegradability low toxicity and low cost [27] Proteinsand peptides released from CMs can be controlled by cross-linking the matrix using chemical cross-linking agents suchas glutaraldehydeNaOH and ethylene glycol diglycidyl ether[28] Ionic cross-linking agents have been developed to avoidthe negative effects of chemical cross-linking agents for pro-teins and peptides [29 30] TPP a nontoxic and multivalentanion is widely used as an ionic cross-linking agent in thepharmaceutical industry [31] Polyelectrolyte complex canbe formed by ionic interaction between positively charged
amino groups of chitosan andmultivalent negatively chargedTPP molecules under mild conditions [32 33] EE is ofsignificant importance for controlled delivery The stableentrapment of ADM into CMs was achieved by cross-linkingthe charge and physical interactions The isoelectric point ofADM is approximately 51 Therefore it carries a negativecharge in PBS during the formation of CMsTheADMcan becompletely reacted with positively charged chitosan leadingto high EE
The porous structure of the scaffolds prepared by TIPS isshown in Figure 3(a) Lactide-based scaffolds made by TIPScan host different types of cells because of their multiscaleporosity that supports cell-matrix interactions [34] The poresize determines cell-seeding efficiency into the scaffold verysmall pores prevent the cells from penetrating into thescaffold whereas very large pores prevent cell attachmentbecause of a reduced area to be colonized by cells [35] Theopened and interconnected pores exhibited a uniform sizeIn addition most of these pores were located between 50 and220120583m which were suitable for cell and tissue penetrationScaffolds with micro- and nano-sized architecture similar tothat of native bone are important An ideal scaffold for clin-ical applications should structurally and functionally mimicnative extracellular matrix (ECM) as closely as possible [36]The substructure of natural bone is composed of nHA andcollagen fibers In this study nHA mimics the nanostructureof natural bone 14-Dioxane as a pore-forming agent wasused in the formation of densely packed vertical arraysof dioxane crystals by TIPS which dominated the finalpore structure of the system resulting in interconnectedpore architecture [34] As shown in Figure 3(b) chitosanmicrospheres were successfully loaded and well distributedin the polymer scaffolds The scaffold morphology slightlychanged after the introduction of CMs inferring that theaddition of microspheres did not damage the structureof the scaffold The microspheres in the scaffold can beregarded as some ldquoislandsrdquo which facilitate the adhesion andproliferation of some cells [37 38] Table 2 shows the densityand porosity of the scaffolds With the addition of CMs thedensity of the scaffolds significantly increased from 0045 plusmn0017 gmL to 0083 plusmn 0020 gmL (119875 lt 005) Howeverthe decrease in porosity was not significant (from 9081 plusmn087 to 8893 plusmn 032) The more dense structure ofthe PLGAnHACM scaffolds was possible because the CMsoccupied the available spaces in the prepared scaffolds Inthis study 30 dosage of CMs did not noticeably changethe porosity of the scaffold The addition of 30 CMs in theporous scaffold did not distinctly change the porosity How-ever increasing the CM content by up to 50 can apparentlydecrease the porosity of the composites [39] Theoreticallythe porosity should be influenced by the amount of the addedmicrospheres Nevertheless Huang et al [40] demonstratedthat no significant difference in porosity exists even after theaddition of 50 microspheres In bone tissue engineeringscaffolds must have sufficient porosity for nutrient and gasexchange [41] Satisfactory porosity of more than 80 isa distinct symbol of a perfect scaffold [39] The porosityof PLGAnHA polymer scaffolds with and without CMswas all beyond 80 The high porosity of scaffolds may be
BioMed Research International 5
(a) (b)
Figure 1 SEM images of chitosan microspheres prepared without (a) and with (b) ADM
Table 1 Primers of genes used in quantitative real-time PCR
PLGAnHACMs 0083 plusmn 0020lowast 8893 plusmn 032lowastP lt 005 indicates statistically significant difference compared with thePLGAnHA group
a result of an interconnected 3D pore structure Furthermorethe retention of approximately 90 porosity in the scaffoldsshould enable a large space for the accommodation of high-density cell cultures
32 Mechanical Properties Mechanical properties of thePLGAnHA scaffolds with and without CMs were evaluatedby a universal material testing machine The mechanicalparameters of the scaffolds are summarized in Figure 4 Thecompressive strength of the PLGAnHACM scaffold (154 plusmn020MPa) was obviously higher than that of the PLGAnHAscaffold (098 plusmn 012MPa) Additionally the compressivemodulus of the PLGAnHACM scaffold (2943 plusmn 242MPa)was significantly higher than that of the PLGAnHA scaf-fold (2145 plusmn 145MPa) A well-designed bone-engineeredscaffold has to meet two mechanical requirements to beeffective The scaffold providing a matrix for cell residencemust retain structural integrity and stability when a doctorimplants it into the defective site Then it must providesufficient mechanical support during tissue regeneration[42] The compression strength of the PLGAnHA scaffoldincreases with the introduction of nHA [43] Moreover themore compressive strength of the PLGAnHACMs scaffold
65
6
55
5
45
4
35
3
25
2
15
1
05
0
Volu
me (
)
01 1 5 10 100 1000Diameter (120583m)
Figure 2 Size distribution of TPP-chitosan microspheres loadedwith ADM
proves that the addition of CMs can improve the mechanicalproperties of the scaffold without affecting the porosity
33 Degradation Properties In Vitro The degradation prop-erties of a scaffold are of crucial importance in the long-term success of a tissue-engineered scaffold Scaffolds forbone regeneration are designed to be gradually replacedwith regenerated ECM during bone formation accompaniedwith degradation WL rate is an important parameter used
6 BioMed Research International
(a) (b)
Figure 3 SEM images of PLGAnHA scaffolds prepared without (a) and with (b) chitosan microspheres The arrows show the chitosanmicrospheres in the scaffold
Compressive strengthCompressive modulus
27525
2252
17515
05
125
025
1075
Com
pres
sive s
treng
th (M
Pa)
10
20
30
40
0
Com
pres
sive m
odul
us (M
Pa)
PLGAnHA PLGAnHACMs
lowast
lowast
Figure 4 Compressive strength and compressive modulus ofPLGAnHA with and without 30 CMs lowast119875 lt 005 indicatesstatistically significant difference compared with the PLGAnHAgroup
to examine the degradation performance of scaffolds TheWL of the PLGAnHA scaffolds with and without CMsis presented in Figure 5(a) The mass of the PLGAnHAscaffolds decreased with increasing degradation time Thepure PLGAnHA polymer showed a slower WL during theentire degradation time The WL of the PLGAnHACMscaffold was slightly faster in the first 3 weeks and thenreached a linear mode At week 12 the WL rates of thePLGAnHA scaffolds with and without CMswere 1223 and827 respectively The four steps for the degradation of thePLGA scaffold are as follows (1) swelling and hydration ofthe polymer (2) breakage of the ester bonds (3) diffusionof the soluble degradation products and (4) disappearanceof the polymer scaffold chips [44] The faster WL detectedfor the PLGAnHACM scaffolds can be associated to thehigher capability of CMs to absorb water when soaked in PBSsolution Moreover mass loss of the scaffolds was reportedto correspond with the changes in pH [45] The results ofthis study are consistent with their report Under in vivo
conditions some enzymes (eg proteinase K and lipasePS) liposomes germs and phagocytes can modulate thedegradation of PLGA polymers to obtain faster degradationrates In addition a previous study [46] reported that thefaster degradation is caused by the autocatalytic effect ofthe acidic degradation products accumulated in the mediumsurrounding the implants This effect was minimized forpolymer degradation in PBS by the frequent change of themedium
Given that medium flow is essential for nutrient andmetabolic exchanges the WA properties of a scaffold areanother important feature for developing a suitable scaffoldfor bone regeneration [47] The WA of PLGAnHA scaffoldswith and without CMs is presented in Figure 5(b) The WAof PLGAnHACMs was 669 at week 1 and gradually rosestably to 8215 at week 6 finally reaching 8834 at week12 The WA of the PLGAnHA polymer was slower duringthe entire time and ultimately reached 524 at week 12 TheWA result was generally consistent with the WL result TheWA of the two scaffolds rapidly increased at week 1 Thisincrease may be attributed to the water that diffused throughthe porous structure The much more rapidly increasingWA of the PLGAnHACM scaffolds was possibly attributedto the excellent absorbent capacity of CMs At the initialstage the WA of the scaffold was critical for integratingthe material-bone construct The superior hydrophilicityof the PLGAnHACM scaffolds might easily facilitate cellmigration into the pores following blood immersion in vivoAs previously observed the hydrophilic characteristics ofthe scaffolds can enhance cell adhesion migration andproliferation in vivo compared with hydrophobic scaffolds[48]
The formation of the degraded acidic molecules andtheir release from PLGA degradation is a negative factor forbone engineering The pH variation of PBS buffer during thedegradation of the PLGAnHA scaffolds with and withoutCMswas detected to verify the acid product released from thescaffoldThe result is shown in Figure 6The pH of the degra-dation medium generally decreased with time maintainingnearly at approximately 71 until week 4 under dynamic
BioMed Research International 7
15
10
5
0
Wei
ght l
oss (
)
PLGAnHACMsPLGAnHA
0 12108642Degradation time (weeks)
(a)
Wat
er u
ptak
e (
)
100
80
60
40
20
0
PLGAnHACMsPLGAnHA
210 108642Degradation time (weeks)
(b)
Figure 5 Weight loss (a) and water uptake (b) of PLGAnHA with and without 30 CMs during degradation in PBS solution
conditions The pH of the PBS buffer for the PLGAnHAscaffolds with and without CMs decreased rapidly at week4 and then slightly decreased from week 4 to week 8 ofdegradation After week 8 the pH plateau was reachedfor the PLGAnHACMs scaffold samples whereas the pHfor the PLGAnHA scaffold samples slightly decreased Aslighter decrease in pH for the PLGAnHACMs scaffoldsafter week 8 may be ascribed to alkaline dissolution of theCMs Arnett [49] reported that osteoblast proliferation andcollagen synthesis are unaffected by pH in the range of 74to 69 The pH of PBS for the PLGAnHACMs scaffolds wasabove 69 during the entire degradation time This findingindicated that the composite PLGAnHA polymer with CMswas suitable for bone engineering
34 Gene Expression Bone formation is an intricate andordered cascade of synthesis of matrix proteins and calciumphosphate in a continuously renewed biological environmentand regulated by a cluster of growth factors [50] An artificialscaffold should be designed as a production of ideal struc-ture that can mimic ECM until host cells including bothosteoblasts and vascular endothelial cells can grow in andresynthesize a new natural matrix The progress in replacingscaffold by natural bone is dependent on the cell adhesionproliferation differentiation and vascularization of the scaf-folds Therefore the successful formation of microvascularcells with long-term patency that are not apt to regression isvery important In bone formation osteogenic-related genes(eg opn col1 runx2 and sp7) and angiogenic-related genes(eg vegf and ramp2) are strictly regulated [51]
Using quantitative real-time PCR the present studydetermined whether the structure of the polymer scaffoldloading with chitosan-ADM microspheres and the sustainedrelease of ADM can promote the early differentiation and
PLGAnHACMsPLGAnHA
8
7
6
5
pH v
alue
0 12108642Degradation time (weeks)
Figure 6 pH of incubated PBS buffer for PLGAnHA with andwithout 30 CMs during in vitro degradation study
activation of osteoblasts and vein endothelial cells at themRNA level of the aforementioned genes
On day 1 the mean expression levels of opn in thePLGAnHACMsADM and PLGAnHA groups were 21and 12 times higher respectively than that in the con-trol group (Figure 7(a)) On day 3 the expression lev-els of opn in the PLGAnHACMsADM and PLGAnHAgroups significantly increased by 28 and 17 times higherthan that in the control group The analogous tendencyappeared on day 5 Meanwhile the opn expression ofthe PLGAnHACMsADM group was higher than thatof the PLGAnHA group at each predetermined time Asshown in Figure 7(b) the col1 mRNA expression of thePLGAnHACMsADM group was almost equal to the other
8 BioMed Research International
two on day 1 It rapidly increased on day 3 and thenmaintained on day 5 which was significantly higher than thatof the control group No significant difference was observedbetween the PLGAnHA group and the control group ateach time point As shown in Figure 7(c) no significantdifference in runx2 expression appeared on day 1 Howeveron day 3 the expression of the PLGAnHACMsADMgroupwas 21 times higher than that of the control group It thenslightly decreased on day 5 which were both significantlyhigher than the control group Nevertheless as shown inFigure 7(d) the sp7 mRNA level was upregulated in thePLGAnHACMsADM group with culture time The valueswere 27 and 47 times significantly higher than those of thecontrol group on days 3 and 5 respectively suggesting theupregulation of osteoblastic activity On day 5 the sp7mRNAexpression in the PLGAnHA scaffold was 17 times higherthan that in the control groupThis result proved the bioactivepotential of the interconnected microstructure of polymerscaffolds
Cell-material interactions can be evaluated by detectingthe cellular receptors responsible for adhesion and migrationand the ligands they bind to specifically bone ECM proteins[52 53] Our previous research revealed the proliferation andactivation functions of ADM The ADM encapsulated in thescaffoldmicrosphere construct can stimulate the prolifera-tion ofMG63 cells for 5 d byMTTassay andfluorescent imageobservation [24] Opn as a mineral-binding protein foundin bone ECM is implicated as an important factor in boneremodeling and crystal growth regulation It is associatedwith cell adhesion proliferation and biomineralization ofECM into bone and its high expression demonstrates theproliferation and activation of MG63 cells Another ECMprotein that is related to further differentiation of osteoblastsis col1 which accounts for 90 of the bone matrix proteins[54] Frick et al [55] reported that the mRNA expressionof col1 is stimulated by alkalosis and inhibited by acidosisThe small pH variation induced by the change in calciumconcentration has a significant effect on col1 expression Theincrease in col1 mRNA of the PLGAnHACMsADM groupon day 3 can be due to the increase in pH induced by chitosandegradation or the effect of ADM Transcription factorsrunx2 and sp7 were essential for osteoblastic differentiationand act as regulatory factors involved in osteogenic-relatedgene expression Early studies reported that runx2 bindsthe osteocalcin promoter and is expressed in osteochondralprogenitors as well as in early stages of osteoblastic differen-tiation [56] In humans runx2 haploinsufficiency results incleidocranial dysplasia a skeletal disorder characterized bybone and dental abnormalities [57] Sp7-deficient mice lackbone formation with a phenotype similar to that of runx2-deficientmiceMeanwhile sp7 is either acting downstreamofrunx2 or expressed later in the osteoblast differentiation path-way [58] Based on the aforementioned result the increasein mRNA expression at an early stage suggested that thedifferentiation and activation of MG63 cells on the surfaceof polymer scaffold were probably due to the sustained ADMrelease PLGAnHACMsADMcan induce differentiation inMG63 cells at the early stage of bone formation
The success of tissue-engineering scaffold is highlydependent on whether the materials can promote rapidand stable neovascularization (new blood vessel formation)within the scaffold typically prior to complete materialdegradation [4] Endothelial cells are currently regarded asthe most interesting target for therapies aimed at enhanc-ing or inhibiting angiogenesis [59] VEGF is an importantregulator of endothelial cell proliferation migration anddifferentiation As one of the ADM receptors ramp2 isessential for angiogenesis and vascular integrity Figures7(e) and 7(f) illustrate the vegf and ramp2 expressionlevels of HUVEC in three groups The vegf expression ofthe PLGAnHACMsADM group completely showed equalexpression pattern to the other two groups at each predeter-mined time However on days 3 and 5 the vegf expressionof the PLGAnHACMsADM and PLGAnHA groups wasslightly higher compared with that of the control groupThe ramp2mRNA expression of the PLGAnHACMsADMgroup steadily increased from day 3 and then slightly down-regulated on day 5The values of the PLGAnHACMsADMgroup on days 3 and 5 were significantly higher than those ofthe control group The expression level of ramp2 was rapidlyupregulated fromday 3 in the PLGAnHACMsADMgroupwhich might be induced by sustained-released ADM How-ever vegf expression was not upregulated The results of thepresent study are generally consistent with those of previousstudies [60] That is the activity of ADM was triggered bythe binding of ADM to its ADM receptor In addition theeffect of ADM on HUVEC was no longer detectable afterthe expression of ramp2 in the cells was almost suppressedby gene silencing The results demonstrated that the releasedADM from the scaffold may be involved in vascularizationfrom the gene level at the early stage
35 Western Blot Analysis To obtain further insights COL-LAGEN 1 RUNX2 protein of MG63 cells and VEGF proteinof HUVEC cells from the cell-scaffold complex were selectedfor detection by western blot analysis after cell culture for 13 and 5 d The results are shown in Figure 8 Significantlyhigher levels of COLLAGEN expression were observed inthe PLGAnHACMsADM and PLGAnHA groups thanin the control group on days 1 and 5 Moreover signifi-cantly higher expression levels of runx2 were observed ateach predetermined time on the PLGAnHACMsADMscaffold which was consistent with real-time PCR analysisFor VEGF which was associated with the proliferation anddifferentiation of endothelial cells the expression level ofthe PLGAnHACMsADM group was significantly higherthan that of the control group on days 1 and 5 Meanwhileon day 1 the PLGAnHA group showed significantly higherVEGF expression than the control group suggesting thatthe structure and property of the PLGAnHA scaffold weresuitable for the migration and proliferation of HUVEC at theinitial stage
Western blot analysis showed higher expression ofCOLLAGEN 1 RUNX2 and VEGF on the PLGAnHACMsADM scaffold during culture time This finding sug-gested that composite scaffold loading with ADM promoted
BioMed Research International 9
4
3
2
1
01 3 5
Culture time (days)
lowast
lowast
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of o
pn
(a)
4
3
2
1
01 3 5
lowastlowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of c
olla
gen
1
Culture time (days)
(b)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
unx2 4
3
2
1
01 3 5
lowastlowast
Culture time (days)
(c)
4
3
2
1
01 3 5
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of s
p7
Culture time (days)
(d)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of v
egf
4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5Culture time (days)
(e)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
amp2 4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5
lowastlowast
Culture time (days)
(f)
Figure 7 RelativemRNA expression level ofMG63 cells (opn (a) collagen 1 (b) runx2 (c) and sp7 (d)) andHUVEC cells (vegf (e) and ramp2(f)) of the PLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times lowast119875 lt 005indicates statistically significant difference compared with the control group (119899 = 6)
10 BioMed Research International
Collagen 1
RUNX2
120573-actin
1 53A B C A B C A B C
120KD
57KD
42KD
MG63
ABC
ControlPLGAnHACMsADMPLGAnHACMs
120573-actin
1 53
A B C A B C A B C
42KD
45KDVEGF
HUVEC
ABC
ControlPLGAnHACMsADMPLGAnHACMs
Relat
ive p
rote
in ex
pres
sion
of co
llage
n 1
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowast lowast
lowast lowastlowast
Relat
ive p
rote
in ex
pres
sion
of R
UN
X2
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
Relat
ive p
rote
in ex
pres
sion
of V
EGF
40
60
80
100
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
(a) (b) (c)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
Figure 8 Western blot assessment of collagen 1 runx2 protein of MG63 cells and VEGF protein of HUVEC cells in thePLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times (a b and c) The bandswere quantitated by densitometry and data are expressed as the ratio of aim protein to 120573-actin lowast119875 lt 005 indicates statistically significantdifference compared with the control group (119899 = 3)
the differentiation and activation of MG63 cells by upreg-ulation of the expression of specific osteogenic proteinsHigher expression levels of COLLAGEN 1 and VEGF werealso observed on the PLGAnHA scaffold ADM has a keyfunction during the development of the vascular systemas demonstrated by Shindo et al [61] Specific conditionssuch as hypoxia are reported to be associated with increasedVEGF expression [62] The ADM-induced upregulation ofVEGF at the protein level agrees well with most reports[63 64] but is not consistent with others [60 65] Thevegf gene was not significantly upregulated The inconsistentlevels between vegf gene and protein were possibly causedby the enhancement of translational efficiency and proteinconstancy
4 Conclusions
As a growth factor ADM was first introduced for tissue-engineering materials The combination of emulsion-ioniccross-linking and TIPS was proven to be suitable for scaf-foldmicrosphere construct developing loading with ADMThe microspheres showed a rough surface morphology andwere well distributed either in the presence or absence of
ADMThe scaffolds showed relatively uniformpore sizeswithinterconnected pores The addition of CMs into the scaffoldsimproved the mechanical properties of the scaffolds withoutremarkably changing their high porosity Moreover in vitrodegradation studies revealed that CM incorporation canaccelerate WL rate increase WA and reduce PLGA acidityin hydrolysisThe expression patterns of opn col1 runx2 andsp7 indicated that inductive osteoblast-like cell differentiationin contact with PLGAnHACMsADM scaffolds appearedearly in bone formation Western blot analysis demonstratedthat the PLGAnHACMsADM scaffold had high levels ofCOLLAGEN 1 and RUNX2 expression In addition RT-PCRanalysis showed that the PLGAnHACMsADM scaffoldhad a high level of ramp2 on days 3 and 5 The expres-sion of vegf was unaffected regardless of ADM loadingMeanwhile VEGF protein levels were relatively high for thePLGAnHACMsADM scaffold at the early osteoblast stageas determined by western blot analysis These findings sug-gest that microspherescaffold composite was more effectivein loading peptides and proteins which can improve theosteogenic and angiogenic differentiation of osteoblasts andvascular endothelial cells on the porous scaffolds Thereforesustained-release ADM from microsphere-scaffold system
BioMed Research International 11
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
size) beneficial for biomedical applications [3] to mimic bonestructure and substructure
The continued enhancement of biomaterial strategies isknown to be highly dependent on the ability to promoterapid and stable vascularization within scaffolds [4] Themost common approach for vascularization in biomaterialsemerged to address biological growth factors into scaffoldsthus paving the way for vascular endothelial cell seeding andpolymer bioactivity [5] Adrenomedullin (ADM) a 52-aminoacid ringed-structure peptide with C-terminal amidation is anewly discovered member of the calcitonin peptide family itwas originally isolated from human pheochromocytoma [6]ADM is also present in many other tissues such as bone [7]kidney lung heart and adrenals [8] Many studies focusedon the cardiovascular and endocrine effects of ADM [9ndash11]However in recent years other significant effects such asosteogenesis [12] angiogenesis [13] and antibacterial effects[14 15] have also been detectedMany studies [16 17] showedthat ADM can stimulate osteoblast proliferation even at lowconcentrations Given its structural and biological homologywith calcitonin gene-related peptide ADM can also stimulatethe proliferation of osteoblasts by increasing the cAMP levelin osteoblast-like cells [18 19] In addition ADM has a reg-ulatory function in angiogenesis by modulating endothelialcell behavior However similar to the dilemma of peptidesin treating bone defects the application of ADM not onlyrequires appropriate temporary release but also requires acertain concentration to be sustained through controlledrelease during bone regeneration An efficient delivery systemmay be required to provide the controlled release of ADMover an extended period
To date a couple of biodegradable polymers have beenused to encapsulate proteins and peptides Chitosan or poly120573-(14)-2-amino-2-deoxy-D-glucose is an excellent naturalhydrophilic cationic polysaccharide derived from chitin Itis widely used for the controlled delivery of polypeptidesand proteins in the form of microspheres or nanospheres[20] Chitosan exhibits favorable biological properties suchas biodegradability biocompatibility nontoxicity hemo-staticity high surface-charge density bacteriostaticity andstrong adhesion [21] It is also used in the field of surgicalsutures wound dressings drug delivery agents defect fillersand tissue-engineering scaffolds [22] Simple adsorption ofgrowth factors into chitosan allows local delivery but thetemporal control over release kinetics is limited [23] An idealdelivery system can be designed as chitosan microspheres(CM) compound with porous scaffolds of proper mechanicalproperties These two components can coordinately enhancetissue regeneration and extend the release time of growthfactors
Previous studies improved the bioactivity of scaffolds bycoating some proteins or peptides to receive and respond tospecific biological signals In this study CMs loaded withADM were prepared by an emulsion-ionic cross-linkingmethod CMs were embedded in a PLGAnanohydroxyap-atite (nHA) scaffold to enhance the compressive strength anddevelop a microsphere-scaffold system with the capacity ofreleasing bioactive factor in a well-controlled manner The invitro ADM release kinetics of microspheres and composite
scaffolds was demonstrated in our previous study [24] Thepresent study aims to investigate the feasibility of using CMsas a carrier for the controlled release of regulatory peptideADMThe surface morphology size distribution and encap-sulation efficiency (EE) of the microspheres were estimatedAfter introducing CMswith ADM into PLGAnHA scaffoldsthe morphological and mechanical features and degrada-tion behavior of the composite scaffolds were evaluatedThe biological capabilities of the PLGAnHA scaffolds wereevaluated by culturingMG63 cells and human umbilical veinendothelial cells (HUVEC) on these scaffolds by real-timepolymerase chain reaction (PCR) and western blot analysis
2 Materials and Methods
21 Materials Chitosan (119872119908= 500 kDa) was purchased
from Jinqiao Chemical Reagents Company (Taizhou Zhe-jiang China) Human ADM (purity = 95 by HPLC)was obtained from Phoenix Pharmaceuticals (BurlingameCanada) Tripolyphosphate (TPP) and span-80were obtainedfrom Aladdin (Shanghai China) PLGA (nLAnGA = 8020)and nHA were obtained from Changchun Institute ofApplied Chemistry Chinese Academy of Sciences MG63 andHUVEC cells were provided by the Basic Medical Collageof Jilin University Liquid paraffin 14-dioxane phosphate-buffered saline (PBS pH 74) and other chemicals were allanalytical grade and used as received
22 Preparation and Characterization of TPP-CMs UsingTPP as cross-linker CMs loaded with ADM were preparedby an emulsion-ionic cross-linking method Briefly 900mgof chitosan was dissolved in 29mL of 2 (vv) aqueousacetic acid and stirred until the solution was transparentMeanwhile 500120583g of ADM was dissolved in 1mL of 2(vv) acetic acid and added into the chitosan solution Themixture was poured into 300mL of liquid paraffin containing2 (wv) of span-80 and stirred mechanically for 2 h Then70mL of 5 (wv) TPP was dropped into the emulsion andstirred for 4 h at room temperature The end emulsion wasrepeatedly washed with excess amounts of petroleum etherisopropyl alcohol and distilled waterThemicrospheres wereobtained after lyophilization (LGJ-18 Sihuan China)
The morphology of the CMs was examined under ascanning electron microscope (SEM XL30ESEM-FEG FELNetherlands) For the measurement the microspheres wereattached to metal stubs and sputter coated with gold undervacuum In addition the diameter of the microspheres wasdetermined by a laser particle size analyzer (LS 13 320Beckman Coulter USA)
A certain amount of ADM-loaded chitosanmicrospheres(CMs-ADM)was dissolved in 5mL of 2 aqueous acetic acidsolution and filtered to remove any undissolved residue Theamounts of ADM in the collected supernatants were mea-sured by HPLC Encapsulated efficiency (EE) was calculatedas follows using the data above
EE () = actual ADM amounttheoretical ADM amount
times 100 (1)
BioMed Research International 3
All measurements were performed in triplicate for eachof the samples
23 Preparation and Characterization of Scaffolds PorousPLGAnHACMs scaffolds were developed by thermallyinduced phase separation (TIPS) PLGA (720mg) was dis-solved in 12mL of 14-dioxane and nHA (360mg) was addedinto the mixture after stirring for half an hour Ultrasonicswas used to completely disperse the nHA for 10min Then240mg of CMs was added to the aforementioned solutionThe mixture was agitated by magnetic stirring to completelydisperse the microspheres and poured into a polytetrafluo-roethene plate Then the solution was frozen overnight ina refrigerator at minus20∘C Finally PLGAnHACM scaffoldswere obtained after lyophilization Pure PLGAnHA scaffoldsprepared by the same method were set as a control for thesucceeding experiments
The pore architecture of the scaffolds which were locatedon the metal stubs and sputter coated with gold was exam-ined by SEM The porosity of the scaffolds was measuredby a mercury intrusion porosimeter (AutoPore IV 9500USA) Percent porosity was provided in the output from theequipment In addition the density of the polymer scaffoldswas tested by a modified liquid displacement method [25]
24 Test of Mechanical Properties The resistance tomechani-cal compression of the scaffolds was tested on an electromag-netic testingmachine (Enduratec Elf 3200 Bose CorporationEden Prairie MN USA) with a 10 kN load cell at roomtemperature The samples were cylinders with a diameter of8mm and a height of 15 mm Compression tests were carriedout under displacement control at a velocity of 01 mms untilthe sample was 50 of the initial height The compressivemodulus and compressive strength were calculated as theaverage of three scaffold measurements
25 In Vitro Degradation Test The cylinder scaffolds with adiameter of 8mm and a height of 5mm were incubated at37∘C in 10mL PBS pH 74 The samples were centrifugedfor 3min to ensure that the entire scaffold was immersedinto the buffer except in water absorption (WA) test andthen incubated at 37∘C under dynamic conditions for 12weeks The incubation buffer was weekly replaced with freshPBS solution except in pH changing test At scheduledfold (once a week) the samples were washed with distilledwater and lyophilized The pH of the PBS solution duringdegradation was monitored by a pH meter (FE20 MettlerToledo Shanghai China)
All data presented in the figures of this paper are theaverage data from six parallel samples
251 Weight Loss (WL) TheWLof scaffolds was gravimetri-cally examined (AL 104 Mettler Toledo Shanghai China) atscheduled times after the samples were freeze dried WL wascomputed as follows
WL () =1198820minus119882119905
1198820
times 100 (119899 = 6) (2)
where1198820and119882
119905are the weights of the samples before and
after incubation respectively
252 WA Property The initial weight of the dry sample wascharacterized as1198821015840
0 The scaffolds were taken out from PBS
at intervals and gravimetrically weighed (Mettler Toledo AL104) after wiping off the surface water to obtain the wet mass1198821015840119905 Water content was computed as follows
WA () =1198821015840119905minus11988210158400
1198821015840119905
times 100 (119899 = 6) (3)
253 pH of DegradationMedium ThepH of the degradationmedium was measured using a pH meter once a weekfor 12 weeks The medium was not refreshed in the entiredegradation period
26 Gene and Protein Expression of MG63 and HUVECCells Cultured on the Scaffolds We determined the expres-sion levels of osteogenic-related (osteopontin (opn) runt-related transcription factor 2 (runx2) transcription factor7 (sp7) and collagen type 1 (col1)) and angiogenic-related(vascular endothelial growth factor (vegf) andG-protein cou-pled activity-modifying protein 2 (ramp2)) genes in MG63and HUVEC cells respectively Meanwhile the expressionlevels of osteogenic-related (RUNX2 and COLLAGEN-1)and angiogenic-related (VEGF) proteins were determined inMG63 and HUVEC cells respectively The polymer scaffoldswith and without CMs-ADMwere prepared as cylinders witha diameter of 10mm and a height of 2mm and then sterilizedwith a 25 kGy Co60 radiation in preparation for cell seeding
MG63 and HUVEC cell lines were respectively main-tained in Dulbeccorsquos modified Eaglersquos medium (Sigma STLouis MO USA) and Iscoversquos modified Dulbeccorsquos medium(Sigma ST Louis MO USA) containing 10 fetal bovineserum in an incubator with humidified atmosphere contain-ing 95 air and 5 CO
2 For subculture cells at 80 to
90 confluence were passaged at a ratio of 1 3 after treatingwith 025 trypsin The sterilized scaffolds were seeded withapproximately 15 times 105 cells of the third passage The cell-scaffold complexes were cultured in 12-well tissue cultureplates for up to 5 d The cells were retrieved for gene andprotein expression determination at specific time points (days1 3 and 5) The cells cultured in normal condition withoutscaffold were selected as control
261 Quantitative Real-Time PCR Total RNA was iso-lated from retrieved MG63 and HUVEC cells of differenttime points using the Trizol reagent (Invitrogen CarlsbadCanada) according to themanufacturerrsquos protocolThe purityof RNA was determined at 260 and 280 nm absorbance toensure that the ratio was beyond 18 Reverse transcriptionwas performed with a Quantscript RT Kit (Tiangen BeijingChina) using 1 120583g total RNA to obtain cDNA Real-timequantitative PCR was performed for the quantification ofgene expression using a Realtime SYBR Green I PCR MasterMix (TOYOBO Japan) in a StepOnePlus Real-Time PCRSystem (ABI Foster City CA USA) Table 1 lists the primers
4 BioMed Research International
designed by Premier Primer 5 and checked by BLAST Therelative expression levels of genes were analyzed using the2minusΔΔCt method [26] by normalizing with GAPDH expressionand presented as fold increase relative to the control group
262 Western Blot Analysis MG63 and HUVEC cells wereretrieved at predetermined time points washed with ice-cold PBS and centrifuged at 5000timesg for 5min at 4∘CThen the cells were lysed in ice-cold lysis buffer (10mMTris pH 74 100mMNaCl 1mM ethylenediaminetetraaceticacid 1mMphenylmethanesulfonyl fluoride 1 Triton X-10010 glycerol 01 sodium dodecyl sulfate (SDS) and 05deoxycholate) on ice for 1 h After centrifugation proteinconcentration was determined using a NanoDrop ND1000(ThermoScientific Wilmington DE USA) spectrophotome-ter The samples (40 120583g protein) were resolved by 10 SDS-polyacrylamide gel electrophoresis and electrophoreticallytransferred to Immun-Blot polyvinylidene difluoride mem-branes (Millipore Bedford MA USA) After blocking inTris-buffered saline with 005 Tween-20 (TBST) containing5 nonfat dry milk for 1 h the membranes were washedthrice with TBST at room temperature Then primary anti-bodies (SantaCruz Biotechnology Santa Cruz CA USA)were added on the membranes and incubated overnightat 4∘C After incubation with the appropriate horseradishperoxidase protein bands coupled with secondary antibody(1 5000 dilution Proteintech Group Chicago USA) werevisualized with an enhanced chemiluminescent system Pro-tein levels from immunoblot were quantified by densitometryusingQuantityOne software (Bio-RadUSA) Target proteinswere normalized against 120573-actin expression
27 Statistical Analysis All data were expressed as mean plusmnSD Statistical significance of differences was assessed by one-way ANOVA and Studentrsquos t-test Statistical significance wasconsidered at 119875 lt 005
3 Results and Discussion
31 Characterization of TPP-CMs and Polymer Scaffolds Anemulsion-ionic cross-linking method was used in preparingthe CMs loaded with ADM in the presence of TPPThe sharpand relative rough surface of TPP-CMs is shown in Figure 1Microsphere cracks were seldom observed in this study Asshown in Figure 2 the size of the microspheres was welldistributed The average diameter of CMs was 4269 120583m andEE was 794 plusmn 23 Chitosan was selected for developingmicrospheres because of its well-known biocompatibilitybiodegradability low toxicity and low cost [27] Proteinsand peptides released from CMs can be controlled by cross-linking the matrix using chemical cross-linking agents suchas glutaraldehydeNaOH and ethylene glycol diglycidyl ether[28] Ionic cross-linking agents have been developed to avoidthe negative effects of chemical cross-linking agents for pro-teins and peptides [29 30] TPP a nontoxic and multivalentanion is widely used as an ionic cross-linking agent in thepharmaceutical industry [31] Polyelectrolyte complex canbe formed by ionic interaction between positively charged
amino groups of chitosan andmultivalent negatively chargedTPP molecules under mild conditions [32 33] EE is ofsignificant importance for controlled delivery The stableentrapment of ADM into CMs was achieved by cross-linkingthe charge and physical interactions The isoelectric point ofADM is approximately 51 Therefore it carries a negativecharge in PBS during the formation of CMsTheADMcan becompletely reacted with positively charged chitosan leadingto high EE
The porous structure of the scaffolds prepared by TIPS isshown in Figure 3(a) Lactide-based scaffolds made by TIPScan host different types of cells because of their multiscaleporosity that supports cell-matrix interactions [34] The poresize determines cell-seeding efficiency into the scaffold verysmall pores prevent the cells from penetrating into thescaffold whereas very large pores prevent cell attachmentbecause of a reduced area to be colonized by cells [35] Theopened and interconnected pores exhibited a uniform sizeIn addition most of these pores were located between 50 and220120583m which were suitable for cell and tissue penetrationScaffolds with micro- and nano-sized architecture similar tothat of native bone are important An ideal scaffold for clin-ical applications should structurally and functionally mimicnative extracellular matrix (ECM) as closely as possible [36]The substructure of natural bone is composed of nHA andcollagen fibers In this study nHA mimics the nanostructureof natural bone 14-Dioxane as a pore-forming agent wasused in the formation of densely packed vertical arraysof dioxane crystals by TIPS which dominated the finalpore structure of the system resulting in interconnectedpore architecture [34] As shown in Figure 3(b) chitosanmicrospheres were successfully loaded and well distributedin the polymer scaffolds The scaffold morphology slightlychanged after the introduction of CMs inferring that theaddition of microspheres did not damage the structureof the scaffold The microspheres in the scaffold can beregarded as some ldquoislandsrdquo which facilitate the adhesion andproliferation of some cells [37 38] Table 2 shows the densityand porosity of the scaffolds With the addition of CMs thedensity of the scaffolds significantly increased from 0045 plusmn0017 gmL to 0083 plusmn 0020 gmL (119875 lt 005) Howeverthe decrease in porosity was not significant (from 9081 plusmn087 to 8893 plusmn 032) The more dense structure ofthe PLGAnHACM scaffolds was possible because the CMsoccupied the available spaces in the prepared scaffolds Inthis study 30 dosage of CMs did not noticeably changethe porosity of the scaffold The addition of 30 CMs in theporous scaffold did not distinctly change the porosity How-ever increasing the CM content by up to 50 can apparentlydecrease the porosity of the composites [39] Theoreticallythe porosity should be influenced by the amount of the addedmicrospheres Nevertheless Huang et al [40] demonstratedthat no significant difference in porosity exists even after theaddition of 50 microspheres In bone tissue engineeringscaffolds must have sufficient porosity for nutrient and gasexchange [41] Satisfactory porosity of more than 80 isa distinct symbol of a perfect scaffold [39] The porosityof PLGAnHA polymer scaffolds with and without CMswas all beyond 80 The high porosity of scaffolds may be
BioMed Research International 5
(a) (b)
Figure 1 SEM images of chitosan microspheres prepared without (a) and with (b) ADM
Table 1 Primers of genes used in quantitative real-time PCR
PLGAnHACMs 0083 plusmn 0020lowast 8893 plusmn 032lowastP lt 005 indicates statistically significant difference compared with thePLGAnHA group
a result of an interconnected 3D pore structure Furthermorethe retention of approximately 90 porosity in the scaffoldsshould enable a large space for the accommodation of high-density cell cultures
32 Mechanical Properties Mechanical properties of thePLGAnHA scaffolds with and without CMs were evaluatedby a universal material testing machine The mechanicalparameters of the scaffolds are summarized in Figure 4 Thecompressive strength of the PLGAnHACM scaffold (154 plusmn020MPa) was obviously higher than that of the PLGAnHAscaffold (098 plusmn 012MPa) Additionally the compressivemodulus of the PLGAnHACM scaffold (2943 plusmn 242MPa)was significantly higher than that of the PLGAnHA scaf-fold (2145 plusmn 145MPa) A well-designed bone-engineeredscaffold has to meet two mechanical requirements to beeffective The scaffold providing a matrix for cell residencemust retain structural integrity and stability when a doctorimplants it into the defective site Then it must providesufficient mechanical support during tissue regeneration[42] The compression strength of the PLGAnHA scaffoldincreases with the introduction of nHA [43] Moreover themore compressive strength of the PLGAnHACMs scaffold
65
6
55
5
45
4
35
3
25
2
15
1
05
0
Volu
me (
)
01 1 5 10 100 1000Diameter (120583m)
Figure 2 Size distribution of TPP-chitosan microspheres loadedwith ADM
proves that the addition of CMs can improve the mechanicalproperties of the scaffold without affecting the porosity
33 Degradation Properties In Vitro The degradation prop-erties of a scaffold are of crucial importance in the long-term success of a tissue-engineered scaffold Scaffolds forbone regeneration are designed to be gradually replacedwith regenerated ECM during bone formation accompaniedwith degradation WL rate is an important parameter used
6 BioMed Research International
(a) (b)
Figure 3 SEM images of PLGAnHA scaffolds prepared without (a) and with (b) chitosan microspheres The arrows show the chitosanmicrospheres in the scaffold
Compressive strengthCompressive modulus
27525
2252
17515
05
125
025
1075
Com
pres
sive s
treng
th (M
Pa)
10
20
30
40
0
Com
pres
sive m
odul
us (M
Pa)
PLGAnHA PLGAnHACMs
lowast
lowast
Figure 4 Compressive strength and compressive modulus ofPLGAnHA with and without 30 CMs lowast119875 lt 005 indicatesstatistically significant difference compared with the PLGAnHAgroup
to examine the degradation performance of scaffolds TheWL of the PLGAnHA scaffolds with and without CMsis presented in Figure 5(a) The mass of the PLGAnHAscaffolds decreased with increasing degradation time Thepure PLGAnHA polymer showed a slower WL during theentire degradation time The WL of the PLGAnHACMscaffold was slightly faster in the first 3 weeks and thenreached a linear mode At week 12 the WL rates of thePLGAnHA scaffolds with and without CMswere 1223 and827 respectively The four steps for the degradation of thePLGA scaffold are as follows (1) swelling and hydration ofthe polymer (2) breakage of the ester bonds (3) diffusionof the soluble degradation products and (4) disappearanceof the polymer scaffold chips [44] The faster WL detectedfor the PLGAnHACM scaffolds can be associated to thehigher capability of CMs to absorb water when soaked in PBSsolution Moreover mass loss of the scaffolds was reportedto correspond with the changes in pH [45] The results ofthis study are consistent with their report Under in vivo
conditions some enzymes (eg proteinase K and lipasePS) liposomes germs and phagocytes can modulate thedegradation of PLGA polymers to obtain faster degradationrates In addition a previous study [46] reported that thefaster degradation is caused by the autocatalytic effect ofthe acidic degradation products accumulated in the mediumsurrounding the implants This effect was minimized forpolymer degradation in PBS by the frequent change of themedium
Given that medium flow is essential for nutrient andmetabolic exchanges the WA properties of a scaffold areanother important feature for developing a suitable scaffoldfor bone regeneration [47] The WA of PLGAnHA scaffoldswith and without CMs is presented in Figure 5(b) The WAof PLGAnHACMs was 669 at week 1 and gradually rosestably to 8215 at week 6 finally reaching 8834 at week12 The WA of the PLGAnHA polymer was slower duringthe entire time and ultimately reached 524 at week 12 TheWA result was generally consistent with the WL result TheWA of the two scaffolds rapidly increased at week 1 Thisincrease may be attributed to the water that diffused throughthe porous structure The much more rapidly increasingWA of the PLGAnHACM scaffolds was possibly attributedto the excellent absorbent capacity of CMs At the initialstage the WA of the scaffold was critical for integratingthe material-bone construct The superior hydrophilicityof the PLGAnHACM scaffolds might easily facilitate cellmigration into the pores following blood immersion in vivoAs previously observed the hydrophilic characteristics ofthe scaffolds can enhance cell adhesion migration andproliferation in vivo compared with hydrophobic scaffolds[48]
The formation of the degraded acidic molecules andtheir release from PLGA degradation is a negative factor forbone engineering The pH variation of PBS buffer during thedegradation of the PLGAnHA scaffolds with and withoutCMswas detected to verify the acid product released from thescaffoldThe result is shown in Figure 6The pH of the degra-dation medium generally decreased with time maintainingnearly at approximately 71 until week 4 under dynamic
BioMed Research International 7
15
10
5
0
Wei
ght l
oss (
)
PLGAnHACMsPLGAnHA
0 12108642Degradation time (weeks)
(a)
Wat
er u
ptak
e (
)
100
80
60
40
20
0
PLGAnHACMsPLGAnHA
210 108642Degradation time (weeks)
(b)
Figure 5 Weight loss (a) and water uptake (b) of PLGAnHA with and without 30 CMs during degradation in PBS solution
conditions The pH of the PBS buffer for the PLGAnHAscaffolds with and without CMs decreased rapidly at week4 and then slightly decreased from week 4 to week 8 ofdegradation After week 8 the pH plateau was reachedfor the PLGAnHACMs scaffold samples whereas the pHfor the PLGAnHA scaffold samples slightly decreased Aslighter decrease in pH for the PLGAnHACMs scaffoldsafter week 8 may be ascribed to alkaline dissolution of theCMs Arnett [49] reported that osteoblast proliferation andcollagen synthesis are unaffected by pH in the range of 74to 69 The pH of PBS for the PLGAnHACMs scaffolds wasabove 69 during the entire degradation time This findingindicated that the composite PLGAnHA polymer with CMswas suitable for bone engineering
34 Gene Expression Bone formation is an intricate andordered cascade of synthesis of matrix proteins and calciumphosphate in a continuously renewed biological environmentand regulated by a cluster of growth factors [50] An artificialscaffold should be designed as a production of ideal struc-ture that can mimic ECM until host cells including bothosteoblasts and vascular endothelial cells can grow in andresynthesize a new natural matrix The progress in replacingscaffold by natural bone is dependent on the cell adhesionproliferation differentiation and vascularization of the scaf-folds Therefore the successful formation of microvascularcells with long-term patency that are not apt to regression isvery important In bone formation osteogenic-related genes(eg opn col1 runx2 and sp7) and angiogenic-related genes(eg vegf and ramp2) are strictly regulated [51]
Using quantitative real-time PCR the present studydetermined whether the structure of the polymer scaffoldloading with chitosan-ADM microspheres and the sustainedrelease of ADM can promote the early differentiation and
PLGAnHACMsPLGAnHA
8
7
6
5
pH v
alue
0 12108642Degradation time (weeks)
Figure 6 pH of incubated PBS buffer for PLGAnHA with andwithout 30 CMs during in vitro degradation study
activation of osteoblasts and vein endothelial cells at themRNA level of the aforementioned genes
On day 1 the mean expression levels of opn in thePLGAnHACMsADM and PLGAnHA groups were 21and 12 times higher respectively than that in the con-trol group (Figure 7(a)) On day 3 the expression lev-els of opn in the PLGAnHACMsADM and PLGAnHAgroups significantly increased by 28 and 17 times higherthan that in the control group The analogous tendencyappeared on day 5 Meanwhile the opn expression ofthe PLGAnHACMsADM group was higher than thatof the PLGAnHA group at each predetermined time Asshown in Figure 7(b) the col1 mRNA expression of thePLGAnHACMsADM group was almost equal to the other
8 BioMed Research International
two on day 1 It rapidly increased on day 3 and thenmaintained on day 5 which was significantly higher than thatof the control group No significant difference was observedbetween the PLGAnHA group and the control group ateach time point As shown in Figure 7(c) no significantdifference in runx2 expression appeared on day 1 Howeveron day 3 the expression of the PLGAnHACMsADMgroupwas 21 times higher than that of the control group It thenslightly decreased on day 5 which were both significantlyhigher than the control group Nevertheless as shown inFigure 7(d) the sp7 mRNA level was upregulated in thePLGAnHACMsADM group with culture time The valueswere 27 and 47 times significantly higher than those of thecontrol group on days 3 and 5 respectively suggesting theupregulation of osteoblastic activity On day 5 the sp7mRNAexpression in the PLGAnHA scaffold was 17 times higherthan that in the control groupThis result proved the bioactivepotential of the interconnected microstructure of polymerscaffolds
Cell-material interactions can be evaluated by detectingthe cellular receptors responsible for adhesion and migrationand the ligands they bind to specifically bone ECM proteins[52 53] Our previous research revealed the proliferation andactivation functions of ADM The ADM encapsulated in thescaffoldmicrosphere construct can stimulate the prolifera-tion ofMG63 cells for 5 d byMTTassay andfluorescent imageobservation [24] Opn as a mineral-binding protein foundin bone ECM is implicated as an important factor in boneremodeling and crystal growth regulation It is associatedwith cell adhesion proliferation and biomineralization ofECM into bone and its high expression demonstrates theproliferation and activation of MG63 cells Another ECMprotein that is related to further differentiation of osteoblastsis col1 which accounts for 90 of the bone matrix proteins[54] Frick et al [55] reported that the mRNA expressionof col1 is stimulated by alkalosis and inhibited by acidosisThe small pH variation induced by the change in calciumconcentration has a significant effect on col1 expression Theincrease in col1 mRNA of the PLGAnHACMsADM groupon day 3 can be due to the increase in pH induced by chitosandegradation or the effect of ADM Transcription factorsrunx2 and sp7 were essential for osteoblastic differentiationand act as regulatory factors involved in osteogenic-relatedgene expression Early studies reported that runx2 bindsthe osteocalcin promoter and is expressed in osteochondralprogenitors as well as in early stages of osteoblastic differen-tiation [56] In humans runx2 haploinsufficiency results incleidocranial dysplasia a skeletal disorder characterized bybone and dental abnormalities [57] Sp7-deficient mice lackbone formation with a phenotype similar to that of runx2-deficientmiceMeanwhile sp7 is either acting downstreamofrunx2 or expressed later in the osteoblast differentiation path-way [58] Based on the aforementioned result the increasein mRNA expression at an early stage suggested that thedifferentiation and activation of MG63 cells on the surfaceof polymer scaffold were probably due to the sustained ADMrelease PLGAnHACMsADMcan induce differentiation inMG63 cells at the early stage of bone formation
The success of tissue-engineering scaffold is highlydependent on whether the materials can promote rapidand stable neovascularization (new blood vessel formation)within the scaffold typically prior to complete materialdegradation [4] Endothelial cells are currently regarded asthe most interesting target for therapies aimed at enhanc-ing or inhibiting angiogenesis [59] VEGF is an importantregulator of endothelial cell proliferation migration anddifferentiation As one of the ADM receptors ramp2 isessential for angiogenesis and vascular integrity Figures7(e) and 7(f) illustrate the vegf and ramp2 expressionlevels of HUVEC in three groups The vegf expression ofthe PLGAnHACMsADM group completely showed equalexpression pattern to the other two groups at each predeter-mined time However on days 3 and 5 the vegf expressionof the PLGAnHACMsADM and PLGAnHA groups wasslightly higher compared with that of the control groupThe ramp2mRNA expression of the PLGAnHACMsADMgroup steadily increased from day 3 and then slightly down-regulated on day 5The values of the PLGAnHACMsADMgroup on days 3 and 5 were significantly higher than those ofthe control group The expression level of ramp2 was rapidlyupregulated fromday 3 in the PLGAnHACMsADMgroupwhich might be induced by sustained-released ADM How-ever vegf expression was not upregulated The results of thepresent study are generally consistent with those of previousstudies [60] That is the activity of ADM was triggered bythe binding of ADM to its ADM receptor In addition theeffect of ADM on HUVEC was no longer detectable afterthe expression of ramp2 in the cells was almost suppressedby gene silencing The results demonstrated that the releasedADM from the scaffold may be involved in vascularizationfrom the gene level at the early stage
35 Western Blot Analysis To obtain further insights COL-LAGEN 1 RUNX2 protein of MG63 cells and VEGF proteinof HUVEC cells from the cell-scaffold complex were selectedfor detection by western blot analysis after cell culture for 13 and 5 d The results are shown in Figure 8 Significantlyhigher levels of COLLAGEN expression were observed inthe PLGAnHACMsADM and PLGAnHA groups thanin the control group on days 1 and 5 Moreover signifi-cantly higher expression levels of runx2 were observed ateach predetermined time on the PLGAnHACMsADMscaffold which was consistent with real-time PCR analysisFor VEGF which was associated with the proliferation anddifferentiation of endothelial cells the expression level ofthe PLGAnHACMsADM group was significantly higherthan that of the control group on days 1 and 5 Meanwhileon day 1 the PLGAnHA group showed significantly higherVEGF expression than the control group suggesting thatthe structure and property of the PLGAnHA scaffold weresuitable for the migration and proliferation of HUVEC at theinitial stage
Western blot analysis showed higher expression ofCOLLAGEN 1 RUNX2 and VEGF on the PLGAnHACMsADM scaffold during culture time This finding sug-gested that composite scaffold loading with ADM promoted
BioMed Research International 9
4
3
2
1
01 3 5
Culture time (days)
lowast
lowast
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of o
pn
(a)
4
3
2
1
01 3 5
lowastlowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of c
olla
gen
1
Culture time (days)
(b)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
unx2 4
3
2
1
01 3 5
lowastlowast
Culture time (days)
(c)
4
3
2
1
01 3 5
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of s
p7
Culture time (days)
(d)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of v
egf
4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5Culture time (days)
(e)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
amp2 4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5
lowastlowast
Culture time (days)
(f)
Figure 7 RelativemRNA expression level ofMG63 cells (opn (a) collagen 1 (b) runx2 (c) and sp7 (d)) andHUVEC cells (vegf (e) and ramp2(f)) of the PLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times lowast119875 lt 005indicates statistically significant difference compared with the control group (119899 = 6)
10 BioMed Research International
Collagen 1
RUNX2
120573-actin
1 53A B C A B C A B C
120KD
57KD
42KD
MG63
ABC
ControlPLGAnHACMsADMPLGAnHACMs
120573-actin
1 53
A B C A B C A B C
42KD
45KDVEGF
HUVEC
ABC
ControlPLGAnHACMsADMPLGAnHACMs
Relat
ive p
rote
in ex
pres
sion
of co
llage
n 1
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowast lowast
lowast lowastlowast
Relat
ive p
rote
in ex
pres
sion
of R
UN
X2
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
Relat
ive p
rote
in ex
pres
sion
of V
EGF
40
60
80
100
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
(a) (b) (c)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
Figure 8 Western blot assessment of collagen 1 runx2 protein of MG63 cells and VEGF protein of HUVEC cells in thePLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times (a b and c) The bandswere quantitated by densitometry and data are expressed as the ratio of aim protein to 120573-actin lowast119875 lt 005 indicates statistically significantdifference compared with the control group (119899 = 3)
the differentiation and activation of MG63 cells by upreg-ulation of the expression of specific osteogenic proteinsHigher expression levels of COLLAGEN 1 and VEGF werealso observed on the PLGAnHA scaffold ADM has a keyfunction during the development of the vascular systemas demonstrated by Shindo et al [61] Specific conditionssuch as hypoxia are reported to be associated with increasedVEGF expression [62] The ADM-induced upregulation ofVEGF at the protein level agrees well with most reports[63 64] but is not consistent with others [60 65] Thevegf gene was not significantly upregulated The inconsistentlevels between vegf gene and protein were possibly causedby the enhancement of translational efficiency and proteinconstancy
4 Conclusions
As a growth factor ADM was first introduced for tissue-engineering materials The combination of emulsion-ioniccross-linking and TIPS was proven to be suitable for scaf-foldmicrosphere construct developing loading with ADMThe microspheres showed a rough surface morphology andwere well distributed either in the presence or absence of
ADMThe scaffolds showed relatively uniformpore sizeswithinterconnected pores The addition of CMs into the scaffoldsimproved the mechanical properties of the scaffolds withoutremarkably changing their high porosity Moreover in vitrodegradation studies revealed that CM incorporation canaccelerate WL rate increase WA and reduce PLGA acidityin hydrolysisThe expression patterns of opn col1 runx2 andsp7 indicated that inductive osteoblast-like cell differentiationin contact with PLGAnHACMsADM scaffolds appearedearly in bone formation Western blot analysis demonstratedthat the PLGAnHACMsADM scaffold had high levels ofCOLLAGEN 1 and RUNX2 expression In addition RT-PCRanalysis showed that the PLGAnHACMsADM scaffoldhad a high level of ramp2 on days 3 and 5 The expres-sion of vegf was unaffected regardless of ADM loadingMeanwhile VEGF protein levels were relatively high for thePLGAnHACMsADM scaffold at the early osteoblast stageas determined by western blot analysis These findings sug-gest that microspherescaffold composite was more effectivein loading peptides and proteins which can improve theosteogenic and angiogenic differentiation of osteoblasts andvascular endothelial cells on the porous scaffolds Thereforesustained-release ADM from microsphere-scaffold system
BioMed Research International 11
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
All measurements were performed in triplicate for eachof the samples
23 Preparation and Characterization of Scaffolds PorousPLGAnHACMs scaffolds were developed by thermallyinduced phase separation (TIPS) PLGA (720mg) was dis-solved in 12mL of 14-dioxane and nHA (360mg) was addedinto the mixture after stirring for half an hour Ultrasonicswas used to completely disperse the nHA for 10min Then240mg of CMs was added to the aforementioned solutionThe mixture was agitated by magnetic stirring to completelydisperse the microspheres and poured into a polytetrafluo-roethene plate Then the solution was frozen overnight ina refrigerator at minus20∘C Finally PLGAnHACM scaffoldswere obtained after lyophilization Pure PLGAnHA scaffoldsprepared by the same method were set as a control for thesucceeding experiments
The pore architecture of the scaffolds which were locatedon the metal stubs and sputter coated with gold was exam-ined by SEM The porosity of the scaffolds was measuredby a mercury intrusion porosimeter (AutoPore IV 9500USA) Percent porosity was provided in the output from theequipment In addition the density of the polymer scaffoldswas tested by a modified liquid displacement method [25]
24 Test of Mechanical Properties The resistance tomechani-cal compression of the scaffolds was tested on an electromag-netic testingmachine (Enduratec Elf 3200 Bose CorporationEden Prairie MN USA) with a 10 kN load cell at roomtemperature The samples were cylinders with a diameter of8mm and a height of 15 mm Compression tests were carriedout under displacement control at a velocity of 01 mms untilthe sample was 50 of the initial height The compressivemodulus and compressive strength were calculated as theaverage of three scaffold measurements
25 In Vitro Degradation Test The cylinder scaffolds with adiameter of 8mm and a height of 5mm were incubated at37∘C in 10mL PBS pH 74 The samples were centrifugedfor 3min to ensure that the entire scaffold was immersedinto the buffer except in water absorption (WA) test andthen incubated at 37∘C under dynamic conditions for 12weeks The incubation buffer was weekly replaced with freshPBS solution except in pH changing test At scheduledfold (once a week) the samples were washed with distilledwater and lyophilized The pH of the PBS solution duringdegradation was monitored by a pH meter (FE20 MettlerToledo Shanghai China)
All data presented in the figures of this paper are theaverage data from six parallel samples
251 Weight Loss (WL) TheWLof scaffolds was gravimetri-cally examined (AL 104 Mettler Toledo Shanghai China) atscheduled times after the samples were freeze dried WL wascomputed as follows
WL () =1198820minus119882119905
1198820
times 100 (119899 = 6) (2)
where1198820and119882
119905are the weights of the samples before and
after incubation respectively
252 WA Property The initial weight of the dry sample wascharacterized as1198821015840
0 The scaffolds were taken out from PBS
at intervals and gravimetrically weighed (Mettler Toledo AL104) after wiping off the surface water to obtain the wet mass1198821015840119905 Water content was computed as follows
WA () =1198821015840119905minus11988210158400
1198821015840119905
times 100 (119899 = 6) (3)
253 pH of DegradationMedium ThepH of the degradationmedium was measured using a pH meter once a weekfor 12 weeks The medium was not refreshed in the entiredegradation period
26 Gene and Protein Expression of MG63 and HUVECCells Cultured on the Scaffolds We determined the expres-sion levels of osteogenic-related (osteopontin (opn) runt-related transcription factor 2 (runx2) transcription factor7 (sp7) and collagen type 1 (col1)) and angiogenic-related(vascular endothelial growth factor (vegf) andG-protein cou-pled activity-modifying protein 2 (ramp2)) genes in MG63and HUVEC cells respectively Meanwhile the expressionlevels of osteogenic-related (RUNX2 and COLLAGEN-1)and angiogenic-related (VEGF) proteins were determined inMG63 and HUVEC cells respectively The polymer scaffoldswith and without CMs-ADMwere prepared as cylinders witha diameter of 10mm and a height of 2mm and then sterilizedwith a 25 kGy Co60 radiation in preparation for cell seeding
MG63 and HUVEC cell lines were respectively main-tained in Dulbeccorsquos modified Eaglersquos medium (Sigma STLouis MO USA) and Iscoversquos modified Dulbeccorsquos medium(Sigma ST Louis MO USA) containing 10 fetal bovineserum in an incubator with humidified atmosphere contain-ing 95 air and 5 CO
2 For subculture cells at 80 to
90 confluence were passaged at a ratio of 1 3 after treatingwith 025 trypsin The sterilized scaffolds were seeded withapproximately 15 times 105 cells of the third passage The cell-scaffold complexes were cultured in 12-well tissue cultureplates for up to 5 d The cells were retrieved for gene andprotein expression determination at specific time points (days1 3 and 5) The cells cultured in normal condition withoutscaffold were selected as control
261 Quantitative Real-Time PCR Total RNA was iso-lated from retrieved MG63 and HUVEC cells of differenttime points using the Trizol reagent (Invitrogen CarlsbadCanada) according to themanufacturerrsquos protocolThe purityof RNA was determined at 260 and 280 nm absorbance toensure that the ratio was beyond 18 Reverse transcriptionwas performed with a Quantscript RT Kit (Tiangen BeijingChina) using 1 120583g total RNA to obtain cDNA Real-timequantitative PCR was performed for the quantification ofgene expression using a Realtime SYBR Green I PCR MasterMix (TOYOBO Japan) in a StepOnePlus Real-Time PCRSystem (ABI Foster City CA USA) Table 1 lists the primers
4 BioMed Research International
designed by Premier Primer 5 and checked by BLAST Therelative expression levels of genes were analyzed using the2minusΔΔCt method [26] by normalizing with GAPDH expressionand presented as fold increase relative to the control group
262 Western Blot Analysis MG63 and HUVEC cells wereretrieved at predetermined time points washed with ice-cold PBS and centrifuged at 5000timesg for 5min at 4∘CThen the cells were lysed in ice-cold lysis buffer (10mMTris pH 74 100mMNaCl 1mM ethylenediaminetetraaceticacid 1mMphenylmethanesulfonyl fluoride 1 Triton X-10010 glycerol 01 sodium dodecyl sulfate (SDS) and 05deoxycholate) on ice for 1 h After centrifugation proteinconcentration was determined using a NanoDrop ND1000(ThermoScientific Wilmington DE USA) spectrophotome-ter The samples (40 120583g protein) were resolved by 10 SDS-polyacrylamide gel electrophoresis and electrophoreticallytransferred to Immun-Blot polyvinylidene difluoride mem-branes (Millipore Bedford MA USA) After blocking inTris-buffered saline with 005 Tween-20 (TBST) containing5 nonfat dry milk for 1 h the membranes were washedthrice with TBST at room temperature Then primary anti-bodies (SantaCruz Biotechnology Santa Cruz CA USA)were added on the membranes and incubated overnightat 4∘C After incubation with the appropriate horseradishperoxidase protein bands coupled with secondary antibody(1 5000 dilution Proteintech Group Chicago USA) werevisualized with an enhanced chemiluminescent system Pro-tein levels from immunoblot were quantified by densitometryusingQuantityOne software (Bio-RadUSA) Target proteinswere normalized against 120573-actin expression
27 Statistical Analysis All data were expressed as mean plusmnSD Statistical significance of differences was assessed by one-way ANOVA and Studentrsquos t-test Statistical significance wasconsidered at 119875 lt 005
3 Results and Discussion
31 Characterization of TPP-CMs and Polymer Scaffolds Anemulsion-ionic cross-linking method was used in preparingthe CMs loaded with ADM in the presence of TPPThe sharpand relative rough surface of TPP-CMs is shown in Figure 1Microsphere cracks were seldom observed in this study Asshown in Figure 2 the size of the microspheres was welldistributed The average diameter of CMs was 4269 120583m andEE was 794 plusmn 23 Chitosan was selected for developingmicrospheres because of its well-known biocompatibilitybiodegradability low toxicity and low cost [27] Proteinsand peptides released from CMs can be controlled by cross-linking the matrix using chemical cross-linking agents suchas glutaraldehydeNaOH and ethylene glycol diglycidyl ether[28] Ionic cross-linking agents have been developed to avoidthe negative effects of chemical cross-linking agents for pro-teins and peptides [29 30] TPP a nontoxic and multivalentanion is widely used as an ionic cross-linking agent in thepharmaceutical industry [31] Polyelectrolyte complex canbe formed by ionic interaction between positively charged
amino groups of chitosan andmultivalent negatively chargedTPP molecules under mild conditions [32 33] EE is ofsignificant importance for controlled delivery The stableentrapment of ADM into CMs was achieved by cross-linkingthe charge and physical interactions The isoelectric point ofADM is approximately 51 Therefore it carries a negativecharge in PBS during the formation of CMsTheADMcan becompletely reacted with positively charged chitosan leadingto high EE
The porous structure of the scaffolds prepared by TIPS isshown in Figure 3(a) Lactide-based scaffolds made by TIPScan host different types of cells because of their multiscaleporosity that supports cell-matrix interactions [34] The poresize determines cell-seeding efficiency into the scaffold verysmall pores prevent the cells from penetrating into thescaffold whereas very large pores prevent cell attachmentbecause of a reduced area to be colonized by cells [35] Theopened and interconnected pores exhibited a uniform sizeIn addition most of these pores were located between 50 and220120583m which were suitable for cell and tissue penetrationScaffolds with micro- and nano-sized architecture similar tothat of native bone are important An ideal scaffold for clin-ical applications should structurally and functionally mimicnative extracellular matrix (ECM) as closely as possible [36]The substructure of natural bone is composed of nHA andcollagen fibers In this study nHA mimics the nanostructureof natural bone 14-Dioxane as a pore-forming agent wasused in the formation of densely packed vertical arraysof dioxane crystals by TIPS which dominated the finalpore structure of the system resulting in interconnectedpore architecture [34] As shown in Figure 3(b) chitosanmicrospheres were successfully loaded and well distributedin the polymer scaffolds The scaffold morphology slightlychanged after the introduction of CMs inferring that theaddition of microspheres did not damage the structureof the scaffold The microspheres in the scaffold can beregarded as some ldquoislandsrdquo which facilitate the adhesion andproliferation of some cells [37 38] Table 2 shows the densityand porosity of the scaffolds With the addition of CMs thedensity of the scaffolds significantly increased from 0045 plusmn0017 gmL to 0083 plusmn 0020 gmL (119875 lt 005) Howeverthe decrease in porosity was not significant (from 9081 plusmn087 to 8893 plusmn 032) The more dense structure ofthe PLGAnHACM scaffolds was possible because the CMsoccupied the available spaces in the prepared scaffolds Inthis study 30 dosage of CMs did not noticeably changethe porosity of the scaffold The addition of 30 CMs in theporous scaffold did not distinctly change the porosity How-ever increasing the CM content by up to 50 can apparentlydecrease the porosity of the composites [39] Theoreticallythe porosity should be influenced by the amount of the addedmicrospheres Nevertheless Huang et al [40] demonstratedthat no significant difference in porosity exists even after theaddition of 50 microspheres In bone tissue engineeringscaffolds must have sufficient porosity for nutrient and gasexchange [41] Satisfactory porosity of more than 80 isa distinct symbol of a perfect scaffold [39] The porosityof PLGAnHA polymer scaffolds with and without CMswas all beyond 80 The high porosity of scaffolds may be
BioMed Research International 5
(a) (b)
Figure 1 SEM images of chitosan microspheres prepared without (a) and with (b) ADM
Table 1 Primers of genes used in quantitative real-time PCR
PLGAnHACMs 0083 plusmn 0020lowast 8893 plusmn 032lowastP lt 005 indicates statistically significant difference compared with thePLGAnHA group
a result of an interconnected 3D pore structure Furthermorethe retention of approximately 90 porosity in the scaffoldsshould enable a large space for the accommodation of high-density cell cultures
32 Mechanical Properties Mechanical properties of thePLGAnHA scaffolds with and without CMs were evaluatedby a universal material testing machine The mechanicalparameters of the scaffolds are summarized in Figure 4 Thecompressive strength of the PLGAnHACM scaffold (154 plusmn020MPa) was obviously higher than that of the PLGAnHAscaffold (098 plusmn 012MPa) Additionally the compressivemodulus of the PLGAnHACM scaffold (2943 plusmn 242MPa)was significantly higher than that of the PLGAnHA scaf-fold (2145 plusmn 145MPa) A well-designed bone-engineeredscaffold has to meet two mechanical requirements to beeffective The scaffold providing a matrix for cell residencemust retain structural integrity and stability when a doctorimplants it into the defective site Then it must providesufficient mechanical support during tissue regeneration[42] The compression strength of the PLGAnHA scaffoldincreases with the introduction of nHA [43] Moreover themore compressive strength of the PLGAnHACMs scaffold
65
6
55
5
45
4
35
3
25
2
15
1
05
0
Volu
me (
)
01 1 5 10 100 1000Diameter (120583m)
Figure 2 Size distribution of TPP-chitosan microspheres loadedwith ADM
proves that the addition of CMs can improve the mechanicalproperties of the scaffold without affecting the porosity
33 Degradation Properties In Vitro The degradation prop-erties of a scaffold are of crucial importance in the long-term success of a tissue-engineered scaffold Scaffolds forbone regeneration are designed to be gradually replacedwith regenerated ECM during bone formation accompaniedwith degradation WL rate is an important parameter used
6 BioMed Research International
(a) (b)
Figure 3 SEM images of PLGAnHA scaffolds prepared without (a) and with (b) chitosan microspheres The arrows show the chitosanmicrospheres in the scaffold
Compressive strengthCompressive modulus
27525
2252
17515
05
125
025
1075
Com
pres
sive s
treng
th (M
Pa)
10
20
30
40
0
Com
pres
sive m
odul
us (M
Pa)
PLGAnHA PLGAnHACMs
lowast
lowast
Figure 4 Compressive strength and compressive modulus ofPLGAnHA with and without 30 CMs lowast119875 lt 005 indicatesstatistically significant difference compared with the PLGAnHAgroup
to examine the degradation performance of scaffolds TheWL of the PLGAnHA scaffolds with and without CMsis presented in Figure 5(a) The mass of the PLGAnHAscaffolds decreased with increasing degradation time Thepure PLGAnHA polymer showed a slower WL during theentire degradation time The WL of the PLGAnHACMscaffold was slightly faster in the first 3 weeks and thenreached a linear mode At week 12 the WL rates of thePLGAnHA scaffolds with and without CMswere 1223 and827 respectively The four steps for the degradation of thePLGA scaffold are as follows (1) swelling and hydration ofthe polymer (2) breakage of the ester bonds (3) diffusionof the soluble degradation products and (4) disappearanceof the polymer scaffold chips [44] The faster WL detectedfor the PLGAnHACM scaffolds can be associated to thehigher capability of CMs to absorb water when soaked in PBSsolution Moreover mass loss of the scaffolds was reportedto correspond with the changes in pH [45] The results ofthis study are consistent with their report Under in vivo
conditions some enzymes (eg proteinase K and lipasePS) liposomes germs and phagocytes can modulate thedegradation of PLGA polymers to obtain faster degradationrates In addition a previous study [46] reported that thefaster degradation is caused by the autocatalytic effect ofthe acidic degradation products accumulated in the mediumsurrounding the implants This effect was minimized forpolymer degradation in PBS by the frequent change of themedium
Given that medium flow is essential for nutrient andmetabolic exchanges the WA properties of a scaffold areanother important feature for developing a suitable scaffoldfor bone regeneration [47] The WA of PLGAnHA scaffoldswith and without CMs is presented in Figure 5(b) The WAof PLGAnHACMs was 669 at week 1 and gradually rosestably to 8215 at week 6 finally reaching 8834 at week12 The WA of the PLGAnHA polymer was slower duringthe entire time and ultimately reached 524 at week 12 TheWA result was generally consistent with the WL result TheWA of the two scaffolds rapidly increased at week 1 Thisincrease may be attributed to the water that diffused throughthe porous structure The much more rapidly increasingWA of the PLGAnHACM scaffolds was possibly attributedto the excellent absorbent capacity of CMs At the initialstage the WA of the scaffold was critical for integratingthe material-bone construct The superior hydrophilicityof the PLGAnHACM scaffolds might easily facilitate cellmigration into the pores following blood immersion in vivoAs previously observed the hydrophilic characteristics ofthe scaffolds can enhance cell adhesion migration andproliferation in vivo compared with hydrophobic scaffolds[48]
The formation of the degraded acidic molecules andtheir release from PLGA degradation is a negative factor forbone engineering The pH variation of PBS buffer during thedegradation of the PLGAnHA scaffolds with and withoutCMswas detected to verify the acid product released from thescaffoldThe result is shown in Figure 6The pH of the degra-dation medium generally decreased with time maintainingnearly at approximately 71 until week 4 under dynamic
BioMed Research International 7
15
10
5
0
Wei
ght l
oss (
)
PLGAnHACMsPLGAnHA
0 12108642Degradation time (weeks)
(a)
Wat
er u
ptak
e (
)
100
80
60
40
20
0
PLGAnHACMsPLGAnHA
210 108642Degradation time (weeks)
(b)
Figure 5 Weight loss (a) and water uptake (b) of PLGAnHA with and without 30 CMs during degradation in PBS solution
conditions The pH of the PBS buffer for the PLGAnHAscaffolds with and without CMs decreased rapidly at week4 and then slightly decreased from week 4 to week 8 ofdegradation After week 8 the pH plateau was reachedfor the PLGAnHACMs scaffold samples whereas the pHfor the PLGAnHA scaffold samples slightly decreased Aslighter decrease in pH for the PLGAnHACMs scaffoldsafter week 8 may be ascribed to alkaline dissolution of theCMs Arnett [49] reported that osteoblast proliferation andcollagen synthesis are unaffected by pH in the range of 74to 69 The pH of PBS for the PLGAnHACMs scaffolds wasabove 69 during the entire degradation time This findingindicated that the composite PLGAnHA polymer with CMswas suitable for bone engineering
34 Gene Expression Bone formation is an intricate andordered cascade of synthesis of matrix proteins and calciumphosphate in a continuously renewed biological environmentand regulated by a cluster of growth factors [50] An artificialscaffold should be designed as a production of ideal struc-ture that can mimic ECM until host cells including bothosteoblasts and vascular endothelial cells can grow in andresynthesize a new natural matrix The progress in replacingscaffold by natural bone is dependent on the cell adhesionproliferation differentiation and vascularization of the scaf-folds Therefore the successful formation of microvascularcells with long-term patency that are not apt to regression isvery important In bone formation osteogenic-related genes(eg opn col1 runx2 and sp7) and angiogenic-related genes(eg vegf and ramp2) are strictly regulated [51]
Using quantitative real-time PCR the present studydetermined whether the structure of the polymer scaffoldloading with chitosan-ADM microspheres and the sustainedrelease of ADM can promote the early differentiation and
PLGAnHACMsPLGAnHA
8
7
6
5
pH v
alue
0 12108642Degradation time (weeks)
Figure 6 pH of incubated PBS buffer for PLGAnHA with andwithout 30 CMs during in vitro degradation study
activation of osteoblasts and vein endothelial cells at themRNA level of the aforementioned genes
On day 1 the mean expression levels of opn in thePLGAnHACMsADM and PLGAnHA groups were 21and 12 times higher respectively than that in the con-trol group (Figure 7(a)) On day 3 the expression lev-els of opn in the PLGAnHACMsADM and PLGAnHAgroups significantly increased by 28 and 17 times higherthan that in the control group The analogous tendencyappeared on day 5 Meanwhile the opn expression ofthe PLGAnHACMsADM group was higher than thatof the PLGAnHA group at each predetermined time Asshown in Figure 7(b) the col1 mRNA expression of thePLGAnHACMsADM group was almost equal to the other
8 BioMed Research International
two on day 1 It rapidly increased on day 3 and thenmaintained on day 5 which was significantly higher than thatof the control group No significant difference was observedbetween the PLGAnHA group and the control group ateach time point As shown in Figure 7(c) no significantdifference in runx2 expression appeared on day 1 Howeveron day 3 the expression of the PLGAnHACMsADMgroupwas 21 times higher than that of the control group It thenslightly decreased on day 5 which were both significantlyhigher than the control group Nevertheless as shown inFigure 7(d) the sp7 mRNA level was upregulated in thePLGAnHACMsADM group with culture time The valueswere 27 and 47 times significantly higher than those of thecontrol group on days 3 and 5 respectively suggesting theupregulation of osteoblastic activity On day 5 the sp7mRNAexpression in the PLGAnHA scaffold was 17 times higherthan that in the control groupThis result proved the bioactivepotential of the interconnected microstructure of polymerscaffolds
Cell-material interactions can be evaluated by detectingthe cellular receptors responsible for adhesion and migrationand the ligands they bind to specifically bone ECM proteins[52 53] Our previous research revealed the proliferation andactivation functions of ADM The ADM encapsulated in thescaffoldmicrosphere construct can stimulate the prolifera-tion ofMG63 cells for 5 d byMTTassay andfluorescent imageobservation [24] Opn as a mineral-binding protein foundin bone ECM is implicated as an important factor in boneremodeling and crystal growth regulation It is associatedwith cell adhesion proliferation and biomineralization ofECM into bone and its high expression demonstrates theproliferation and activation of MG63 cells Another ECMprotein that is related to further differentiation of osteoblastsis col1 which accounts for 90 of the bone matrix proteins[54] Frick et al [55] reported that the mRNA expressionof col1 is stimulated by alkalosis and inhibited by acidosisThe small pH variation induced by the change in calciumconcentration has a significant effect on col1 expression Theincrease in col1 mRNA of the PLGAnHACMsADM groupon day 3 can be due to the increase in pH induced by chitosandegradation or the effect of ADM Transcription factorsrunx2 and sp7 were essential for osteoblastic differentiationand act as regulatory factors involved in osteogenic-relatedgene expression Early studies reported that runx2 bindsthe osteocalcin promoter and is expressed in osteochondralprogenitors as well as in early stages of osteoblastic differen-tiation [56] In humans runx2 haploinsufficiency results incleidocranial dysplasia a skeletal disorder characterized bybone and dental abnormalities [57] Sp7-deficient mice lackbone formation with a phenotype similar to that of runx2-deficientmiceMeanwhile sp7 is either acting downstreamofrunx2 or expressed later in the osteoblast differentiation path-way [58] Based on the aforementioned result the increasein mRNA expression at an early stage suggested that thedifferentiation and activation of MG63 cells on the surfaceof polymer scaffold were probably due to the sustained ADMrelease PLGAnHACMsADMcan induce differentiation inMG63 cells at the early stage of bone formation
The success of tissue-engineering scaffold is highlydependent on whether the materials can promote rapidand stable neovascularization (new blood vessel formation)within the scaffold typically prior to complete materialdegradation [4] Endothelial cells are currently regarded asthe most interesting target for therapies aimed at enhanc-ing or inhibiting angiogenesis [59] VEGF is an importantregulator of endothelial cell proliferation migration anddifferentiation As one of the ADM receptors ramp2 isessential for angiogenesis and vascular integrity Figures7(e) and 7(f) illustrate the vegf and ramp2 expressionlevels of HUVEC in three groups The vegf expression ofthe PLGAnHACMsADM group completely showed equalexpression pattern to the other two groups at each predeter-mined time However on days 3 and 5 the vegf expressionof the PLGAnHACMsADM and PLGAnHA groups wasslightly higher compared with that of the control groupThe ramp2mRNA expression of the PLGAnHACMsADMgroup steadily increased from day 3 and then slightly down-regulated on day 5The values of the PLGAnHACMsADMgroup on days 3 and 5 were significantly higher than those ofthe control group The expression level of ramp2 was rapidlyupregulated fromday 3 in the PLGAnHACMsADMgroupwhich might be induced by sustained-released ADM How-ever vegf expression was not upregulated The results of thepresent study are generally consistent with those of previousstudies [60] That is the activity of ADM was triggered bythe binding of ADM to its ADM receptor In addition theeffect of ADM on HUVEC was no longer detectable afterthe expression of ramp2 in the cells was almost suppressedby gene silencing The results demonstrated that the releasedADM from the scaffold may be involved in vascularizationfrom the gene level at the early stage
35 Western Blot Analysis To obtain further insights COL-LAGEN 1 RUNX2 protein of MG63 cells and VEGF proteinof HUVEC cells from the cell-scaffold complex were selectedfor detection by western blot analysis after cell culture for 13 and 5 d The results are shown in Figure 8 Significantlyhigher levels of COLLAGEN expression were observed inthe PLGAnHACMsADM and PLGAnHA groups thanin the control group on days 1 and 5 Moreover signifi-cantly higher expression levels of runx2 were observed ateach predetermined time on the PLGAnHACMsADMscaffold which was consistent with real-time PCR analysisFor VEGF which was associated with the proliferation anddifferentiation of endothelial cells the expression level ofthe PLGAnHACMsADM group was significantly higherthan that of the control group on days 1 and 5 Meanwhileon day 1 the PLGAnHA group showed significantly higherVEGF expression than the control group suggesting thatthe structure and property of the PLGAnHA scaffold weresuitable for the migration and proliferation of HUVEC at theinitial stage
Western blot analysis showed higher expression ofCOLLAGEN 1 RUNX2 and VEGF on the PLGAnHACMsADM scaffold during culture time This finding sug-gested that composite scaffold loading with ADM promoted
BioMed Research International 9
4
3
2
1
01 3 5
Culture time (days)
lowast
lowast
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of o
pn
(a)
4
3
2
1
01 3 5
lowastlowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of c
olla
gen
1
Culture time (days)
(b)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
unx2 4
3
2
1
01 3 5
lowastlowast
Culture time (days)
(c)
4
3
2
1
01 3 5
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of s
p7
Culture time (days)
(d)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of v
egf
4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5Culture time (days)
(e)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
amp2 4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5
lowastlowast
Culture time (days)
(f)
Figure 7 RelativemRNA expression level ofMG63 cells (opn (a) collagen 1 (b) runx2 (c) and sp7 (d)) andHUVEC cells (vegf (e) and ramp2(f)) of the PLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times lowast119875 lt 005indicates statistically significant difference compared with the control group (119899 = 6)
10 BioMed Research International
Collagen 1
RUNX2
120573-actin
1 53A B C A B C A B C
120KD
57KD
42KD
MG63
ABC
ControlPLGAnHACMsADMPLGAnHACMs
120573-actin
1 53
A B C A B C A B C
42KD
45KDVEGF
HUVEC
ABC
ControlPLGAnHACMsADMPLGAnHACMs
Relat
ive p
rote
in ex
pres
sion
of co
llage
n 1
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowast lowast
lowast lowastlowast
Relat
ive p
rote
in ex
pres
sion
of R
UN
X2
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
Relat
ive p
rote
in ex
pres
sion
of V
EGF
40
60
80
100
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
(a) (b) (c)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
Figure 8 Western blot assessment of collagen 1 runx2 protein of MG63 cells and VEGF protein of HUVEC cells in thePLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times (a b and c) The bandswere quantitated by densitometry and data are expressed as the ratio of aim protein to 120573-actin lowast119875 lt 005 indicates statistically significantdifference compared with the control group (119899 = 3)
the differentiation and activation of MG63 cells by upreg-ulation of the expression of specific osteogenic proteinsHigher expression levels of COLLAGEN 1 and VEGF werealso observed on the PLGAnHA scaffold ADM has a keyfunction during the development of the vascular systemas demonstrated by Shindo et al [61] Specific conditionssuch as hypoxia are reported to be associated with increasedVEGF expression [62] The ADM-induced upregulation ofVEGF at the protein level agrees well with most reports[63 64] but is not consistent with others [60 65] Thevegf gene was not significantly upregulated The inconsistentlevels between vegf gene and protein were possibly causedby the enhancement of translational efficiency and proteinconstancy
4 Conclusions
As a growth factor ADM was first introduced for tissue-engineering materials The combination of emulsion-ioniccross-linking and TIPS was proven to be suitable for scaf-foldmicrosphere construct developing loading with ADMThe microspheres showed a rough surface morphology andwere well distributed either in the presence or absence of
ADMThe scaffolds showed relatively uniformpore sizeswithinterconnected pores The addition of CMs into the scaffoldsimproved the mechanical properties of the scaffolds withoutremarkably changing their high porosity Moreover in vitrodegradation studies revealed that CM incorporation canaccelerate WL rate increase WA and reduce PLGA acidityin hydrolysisThe expression patterns of opn col1 runx2 andsp7 indicated that inductive osteoblast-like cell differentiationin contact with PLGAnHACMsADM scaffolds appearedearly in bone formation Western blot analysis demonstratedthat the PLGAnHACMsADM scaffold had high levels ofCOLLAGEN 1 and RUNX2 expression In addition RT-PCRanalysis showed that the PLGAnHACMsADM scaffoldhad a high level of ramp2 on days 3 and 5 The expres-sion of vegf was unaffected regardless of ADM loadingMeanwhile VEGF protein levels were relatively high for thePLGAnHACMsADM scaffold at the early osteoblast stageas determined by western blot analysis These findings sug-gest that microspherescaffold composite was more effectivein loading peptides and proteins which can improve theosteogenic and angiogenic differentiation of osteoblasts andvascular endothelial cells on the porous scaffolds Thereforesustained-release ADM from microsphere-scaffold system
BioMed Research International 11
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
designed by Premier Primer 5 and checked by BLAST Therelative expression levels of genes were analyzed using the2minusΔΔCt method [26] by normalizing with GAPDH expressionand presented as fold increase relative to the control group
262 Western Blot Analysis MG63 and HUVEC cells wereretrieved at predetermined time points washed with ice-cold PBS and centrifuged at 5000timesg for 5min at 4∘CThen the cells were lysed in ice-cold lysis buffer (10mMTris pH 74 100mMNaCl 1mM ethylenediaminetetraaceticacid 1mMphenylmethanesulfonyl fluoride 1 Triton X-10010 glycerol 01 sodium dodecyl sulfate (SDS) and 05deoxycholate) on ice for 1 h After centrifugation proteinconcentration was determined using a NanoDrop ND1000(ThermoScientific Wilmington DE USA) spectrophotome-ter The samples (40 120583g protein) were resolved by 10 SDS-polyacrylamide gel electrophoresis and electrophoreticallytransferred to Immun-Blot polyvinylidene difluoride mem-branes (Millipore Bedford MA USA) After blocking inTris-buffered saline with 005 Tween-20 (TBST) containing5 nonfat dry milk for 1 h the membranes were washedthrice with TBST at room temperature Then primary anti-bodies (SantaCruz Biotechnology Santa Cruz CA USA)were added on the membranes and incubated overnightat 4∘C After incubation with the appropriate horseradishperoxidase protein bands coupled with secondary antibody(1 5000 dilution Proteintech Group Chicago USA) werevisualized with an enhanced chemiluminescent system Pro-tein levels from immunoblot were quantified by densitometryusingQuantityOne software (Bio-RadUSA) Target proteinswere normalized against 120573-actin expression
27 Statistical Analysis All data were expressed as mean plusmnSD Statistical significance of differences was assessed by one-way ANOVA and Studentrsquos t-test Statistical significance wasconsidered at 119875 lt 005
3 Results and Discussion
31 Characterization of TPP-CMs and Polymer Scaffolds Anemulsion-ionic cross-linking method was used in preparingthe CMs loaded with ADM in the presence of TPPThe sharpand relative rough surface of TPP-CMs is shown in Figure 1Microsphere cracks were seldom observed in this study Asshown in Figure 2 the size of the microspheres was welldistributed The average diameter of CMs was 4269 120583m andEE was 794 plusmn 23 Chitosan was selected for developingmicrospheres because of its well-known biocompatibilitybiodegradability low toxicity and low cost [27] Proteinsand peptides released from CMs can be controlled by cross-linking the matrix using chemical cross-linking agents suchas glutaraldehydeNaOH and ethylene glycol diglycidyl ether[28] Ionic cross-linking agents have been developed to avoidthe negative effects of chemical cross-linking agents for pro-teins and peptides [29 30] TPP a nontoxic and multivalentanion is widely used as an ionic cross-linking agent in thepharmaceutical industry [31] Polyelectrolyte complex canbe formed by ionic interaction between positively charged
amino groups of chitosan andmultivalent negatively chargedTPP molecules under mild conditions [32 33] EE is ofsignificant importance for controlled delivery The stableentrapment of ADM into CMs was achieved by cross-linkingthe charge and physical interactions The isoelectric point ofADM is approximately 51 Therefore it carries a negativecharge in PBS during the formation of CMsTheADMcan becompletely reacted with positively charged chitosan leadingto high EE
The porous structure of the scaffolds prepared by TIPS isshown in Figure 3(a) Lactide-based scaffolds made by TIPScan host different types of cells because of their multiscaleporosity that supports cell-matrix interactions [34] The poresize determines cell-seeding efficiency into the scaffold verysmall pores prevent the cells from penetrating into thescaffold whereas very large pores prevent cell attachmentbecause of a reduced area to be colonized by cells [35] Theopened and interconnected pores exhibited a uniform sizeIn addition most of these pores were located between 50 and220120583m which were suitable for cell and tissue penetrationScaffolds with micro- and nano-sized architecture similar tothat of native bone are important An ideal scaffold for clin-ical applications should structurally and functionally mimicnative extracellular matrix (ECM) as closely as possible [36]The substructure of natural bone is composed of nHA andcollagen fibers In this study nHA mimics the nanostructureof natural bone 14-Dioxane as a pore-forming agent wasused in the formation of densely packed vertical arraysof dioxane crystals by TIPS which dominated the finalpore structure of the system resulting in interconnectedpore architecture [34] As shown in Figure 3(b) chitosanmicrospheres were successfully loaded and well distributedin the polymer scaffolds The scaffold morphology slightlychanged after the introduction of CMs inferring that theaddition of microspheres did not damage the structureof the scaffold The microspheres in the scaffold can beregarded as some ldquoislandsrdquo which facilitate the adhesion andproliferation of some cells [37 38] Table 2 shows the densityand porosity of the scaffolds With the addition of CMs thedensity of the scaffolds significantly increased from 0045 plusmn0017 gmL to 0083 plusmn 0020 gmL (119875 lt 005) Howeverthe decrease in porosity was not significant (from 9081 plusmn087 to 8893 plusmn 032) The more dense structure ofthe PLGAnHACM scaffolds was possible because the CMsoccupied the available spaces in the prepared scaffolds Inthis study 30 dosage of CMs did not noticeably changethe porosity of the scaffold The addition of 30 CMs in theporous scaffold did not distinctly change the porosity How-ever increasing the CM content by up to 50 can apparentlydecrease the porosity of the composites [39] Theoreticallythe porosity should be influenced by the amount of the addedmicrospheres Nevertheless Huang et al [40] demonstratedthat no significant difference in porosity exists even after theaddition of 50 microspheres In bone tissue engineeringscaffolds must have sufficient porosity for nutrient and gasexchange [41] Satisfactory porosity of more than 80 isa distinct symbol of a perfect scaffold [39] The porosityof PLGAnHA polymer scaffolds with and without CMswas all beyond 80 The high porosity of scaffolds may be
BioMed Research International 5
(a) (b)
Figure 1 SEM images of chitosan microspheres prepared without (a) and with (b) ADM
Table 1 Primers of genes used in quantitative real-time PCR
PLGAnHACMs 0083 plusmn 0020lowast 8893 plusmn 032lowastP lt 005 indicates statistically significant difference compared with thePLGAnHA group
a result of an interconnected 3D pore structure Furthermorethe retention of approximately 90 porosity in the scaffoldsshould enable a large space for the accommodation of high-density cell cultures
32 Mechanical Properties Mechanical properties of thePLGAnHA scaffolds with and without CMs were evaluatedby a universal material testing machine The mechanicalparameters of the scaffolds are summarized in Figure 4 Thecompressive strength of the PLGAnHACM scaffold (154 plusmn020MPa) was obviously higher than that of the PLGAnHAscaffold (098 plusmn 012MPa) Additionally the compressivemodulus of the PLGAnHACM scaffold (2943 plusmn 242MPa)was significantly higher than that of the PLGAnHA scaf-fold (2145 plusmn 145MPa) A well-designed bone-engineeredscaffold has to meet two mechanical requirements to beeffective The scaffold providing a matrix for cell residencemust retain structural integrity and stability when a doctorimplants it into the defective site Then it must providesufficient mechanical support during tissue regeneration[42] The compression strength of the PLGAnHA scaffoldincreases with the introduction of nHA [43] Moreover themore compressive strength of the PLGAnHACMs scaffold
65
6
55
5
45
4
35
3
25
2
15
1
05
0
Volu
me (
)
01 1 5 10 100 1000Diameter (120583m)
Figure 2 Size distribution of TPP-chitosan microspheres loadedwith ADM
proves that the addition of CMs can improve the mechanicalproperties of the scaffold without affecting the porosity
33 Degradation Properties In Vitro The degradation prop-erties of a scaffold are of crucial importance in the long-term success of a tissue-engineered scaffold Scaffolds forbone regeneration are designed to be gradually replacedwith regenerated ECM during bone formation accompaniedwith degradation WL rate is an important parameter used
6 BioMed Research International
(a) (b)
Figure 3 SEM images of PLGAnHA scaffolds prepared without (a) and with (b) chitosan microspheres The arrows show the chitosanmicrospheres in the scaffold
Compressive strengthCompressive modulus
27525
2252
17515
05
125
025
1075
Com
pres
sive s
treng
th (M
Pa)
10
20
30
40
0
Com
pres
sive m
odul
us (M
Pa)
PLGAnHA PLGAnHACMs
lowast
lowast
Figure 4 Compressive strength and compressive modulus ofPLGAnHA with and without 30 CMs lowast119875 lt 005 indicatesstatistically significant difference compared with the PLGAnHAgroup
to examine the degradation performance of scaffolds TheWL of the PLGAnHA scaffolds with and without CMsis presented in Figure 5(a) The mass of the PLGAnHAscaffolds decreased with increasing degradation time Thepure PLGAnHA polymer showed a slower WL during theentire degradation time The WL of the PLGAnHACMscaffold was slightly faster in the first 3 weeks and thenreached a linear mode At week 12 the WL rates of thePLGAnHA scaffolds with and without CMswere 1223 and827 respectively The four steps for the degradation of thePLGA scaffold are as follows (1) swelling and hydration ofthe polymer (2) breakage of the ester bonds (3) diffusionof the soluble degradation products and (4) disappearanceof the polymer scaffold chips [44] The faster WL detectedfor the PLGAnHACM scaffolds can be associated to thehigher capability of CMs to absorb water when soaked in PBSsolution Moreover mass loss of the scaffolds was reportedto correspond with the changes in pH [45] The results ofthis study are consistent with their report Under in vivo
conditions some enzymes (eg proteinase K and lipasePS) liposomes germs and phagocytes can modulate thedegradation of PLGA polymers to obtain faster degradationrates In addition a previous study [46] reported that thefaster degradation is caused by the autocatalytic effect ofthe acidic degradation products accumulated in the mediumsurrounding the implants This effect was minimized forpolymer degradation in PBS by the frequent change of themedium
Given that medium flow is essential for nutrient andmetabolic exchanges the WA properties of a scaffold areanother important feature for developing a suitable scaffoldfor bone regeneration [47] The WA of PLGAnHA scaffoldswith and without CMs is presented in Figure 5(b) The WAof PLGAnHACMs was 669 at week 1 and gradually rosestably to 8215 at week 6 finally reaching 8834 at week12 The WA of the PLGAnHA polymer was slower duringthe entire time and ultimately reached 524 at week 12 TheWA result was generally consistent with the WL result TheWA of the two scaffolds rapidly increased at week 1 Thisincrease may be attributed to the water that diffused throughthe porous structure The much more rapidly increasingWA of the PLGAnHACM scaffolds was possibly attributedto the excellent absorbent capacity of CMs At the initialstage the WA of the scaffold was critical for integratingthe material-bone construct The superior hydrophilicityof the PLGAnHACM scaffolds might easily facilitate cellmigration into the pores following blood immersion in vivoAs previously observed the hydrophilic characteristics ofthe scaffolds can enhance cell adhesion migration andproliferation in vivo compared with hydrophobic scaffolds[48]
The formation of the degraded acidic molecules andtheir release from PLGA degradation is a negative factor forbone engineering The pH variation of PBS buffer during thedegradation of the PLGAnHA scaffolds with and withoutCMswas detected to verify the acid product released from thescaffoldThe result is shown in Figure 6The pH of the degra-dation medium generally decreased with time maintainingnearly at approximately 71 until week 4 under dynamic
BioMed Research International 7
15
10
5
0
Wei
ght l
oss (
)
PLGAnHACMsPLGAnHA
0 12108642Degradation time (weeks)
(a)
Wat
er u
ptak
e (
)
100
80
60
40
20
0
PLGAnHACMsPLGAnHA
210 108642Degradation time (weeks)
(b)
Figure 5 Weight loss (a) and water uptake (b) of PLGAnHA with and without 30 CMs during degradation in PBS solution
conditions The pH of the PBS buffer for the PLGAnHAscaffolds with and without CMs decreased rapidly at week4 and then slightly decreased from week 4 to week 8 ofdegradation After week 8 the pH plateau was reachedfor the PLGAnHACMs scaffold samples whereas the pHfor the PLGAnHA scaffold samples slightly decreased Aslighter decrease in pH for the PLGAnHACMs scaffoldsafter week 8 may be ascribed to alkaline dissolution of theCMs Arnett [49] reported that osteoblast proliferation andcollagen synthesis are unaffected by pH in the range of 74to 69 The pH of PBS for the PLGAnHACMs scaffolds wasabove 69 during the entire degradation time This findingindicated that the composite PLGAnHA polymer with CMswas suitable for bone engineering
34 Gene Expression Bone formation is an intricate andordered cascade of synthesis of matrix proteins and calciumphosphate in a continuously renewed biological environmentand regulated by a cluster of growth factors [50] An artificialscaffold should be designed as a production of ideal struc-ture that can mimic ECM until host cells including bothosteoblasts and vascular endothelial cells can grow in andresynthesize a new natural matrix The progress in replacingscaffold by natural bone is dependent on the cell adhesionproliferation differentiation and vascularization of the scaf-folds Therefore the successful formation of microvascularcells with long-term patency that are not apt to regression isvery important In bone formation osteogenic-related genes(eg opn col1 runx2 and sp7) and angiogenic-related genes(eg vegf and ramp2) are strictly regulated [51]
Using quantitative real-time PCR the present studydetermined whether the structure of the polymer scaffoldloading with chitosan-ADM microspheres and the sustainedrelease of ADM can promote the early differentiation and
PLGAnHACMsPLGAnHA
8
7
6
5
pH v
alue
0 12108642Degradation time (weeks)
Figure 6 pH of incubated PBS buffer for PLGAnHA with andwithout 30 CMs during in vitro degradation study
activation of osteoblasts and vein endothelial cells at themRNA level of the aforementioned genes
On day 1 the mean expression levels of opn in thePLGAnHACMsADM and PLGAnHA groups were 21and 12 times higher respectively than that in the con-trol group (Figure 7(a)) On day 3 the expression lev-els of opn in the PLGAnHACMsADM and PLGAnHAgroups significantly increased by 28 and 17 times higherthan that in the control group The analogous tendencyappeared on day 5 Meanwhile the opn expression ofthe PLGAnHACMsADM group was higher than thatof the PLGAnHA group at each predetermined time Asshown in Figure 7(b) the col1 mRNA expression of thePLGAnHACMsADM group was almost equal to the other
8 BioMed Research International
two on day 1 It rapidly increased on day 3 and thenmaintained on day 5 which was significantly higher than thatof the control group No significant difference was observedbetween the PLGAnHA group and the control group ateach time point As shown in Figure 7(c) no significantdifference in runx2 expression appeared on day 1 Howeveron day 3 the expression of the PLGAnHACMsADMgroupwas 21 times higher than that of the control group It thenslightly decreased on day 5 which were both significantlyhigher than the control group Nevertheless as shown inFigure 7(d) the sp7 mRNA level was upregulated in thePLGAnHACMsADM group with culture time The valueswere 27 and 47 times significantly higher than those of thecontrol group on days 3 and 5 respectively suggesting theupregulation of osteoblastic activity On day 5 the sp7mRNAexpression in the PLGAnHA scaffold was 17 times higherthan that in the control groupThis result proved the bioactivepotential of the interconnected microstructure of polymerscaffolds
Cell-material interactions can be evaluated by detectingthe cellular receptors responsible for adhesion and migrationand the ligands they bind to specifically bone ECM proteins[52 53] Our previous research revealed the proliferation andactivation functions of ADM The ADM encapsulated in thescaffoldmicrosphere construct can stimulate the prolifera-tion ofMG63 cells for 5 d byMTTassay andfluorescent imageobservation [24] Opn as a mineral-binding protein foundin bone ECM is implicated as an important factor in boneremodeling and crystal growth regulation It is associatedwith cell adhesion proliferation and biomineralization ofECM into bone and its high expression demonstrates theproliferation and activation of MG63 cells Another ECMprotein that is related to further differentiation of osteoblastsis col1 which accounts for 90 of the bone matrix proteins[54] Frick et al [55] reported that the mRNA expressionof col1 is stimulated by alkalosis and inhibited by acidosisThe small pH variation induced by the change in calciumconcentration has a significant effect on col1 expression Theincrease in col1 mRNA of the PLGAnHACMsADM groupon day 3 can be due to the increase in pH induced by chitosandegradation or the effect of ADM Transcription factorsrunx2 and sp7 were essential for osteoblastic differentiationand act as regulatory factors involved in osteogenic-relatedgene expression Early studies reported that runx2 bindsthe osteocalcin promoter and is expressed in osteochondralprogenitors as well as in early stages of osteoblastic differen-tiation [56] In humans runx2 haploinsufficiency results incleidocranial dysplasia a skeletal disorder characterized bybone and dental abnormalities [57] Sp7-deficient mice lackbone formation with a phenotype similar to that of runx2-deficientmiceMeanwhile sp7 is either acting downstreamofrunx2 or expressed later in the osteoblast differentiation path-way [58] Based on the aforementioned result the increasein mRNA expression at an early stage suggested that thedifferentiation and activation of MG63 cells on the surfaceof polymer scaffold were probably due to the sustained ADMrelease PLGAnHACMsADMcan induce differentiation inMG63 cells at the early stage of bone formation
The success of tissue-engineering scaffold is highlydependent on whether the materials can promote rapidand stable neovascularization (new blood vessel formation)within the scaffold typically prior to complete materialdegradation [4] Endothelial cells are currently regarded asthe most interesting target for therapies aimed at enhanc-ing or inhibiting angiogenesis [59] VEGF is an importantregulator of endothelial cell proliferation migration anddifferentiation As one of the ADM receptors ramp2 isessential for angiogenesis and vascular integrity Figures7(e) and 7(f) illustrate the vegf and ramp2 expressionlevels of HUVEC in three groups The vegf expression ofthe PLGAnHACMsADM group completely showed equalexpression pattern to the other two groups at each predeter-mined time However on days 3 and 5 the vegf expressionof the PLGAnHACMsADM and PLGAnHA groups wasslightly higher compared with that of the control groupThe ramp2mRNA expression of the PLGAnHACMsADMgroup steadily increased from day 3 and then slightly down-regulated on day 5The values of the PLGAnHACMsADMgroup on days 3 and 5 were significantly higher than those ofthe control group The expression level of ramp2 was rapidlyupregulated fromday 3 in the PLGAnHACMsADMgroupwhich might be induced by sustained-released ADM How-ever vegf expression was not upregulated The results of thepresent study are generally consistent with those of previousstudies [60] That is the activity of ADM was triggered bythe binding of ADM to its ADM receptor In addition theeffect of ADM on HUVEC was no longer detectable afterthe expression of ramp2 in the cells was almost suppressedby gene silencing The results demonstrated that the releasedADM from the scaffold may be involved in vascularizationfrom the gene level at the early stage
35 Western Blot Analysis To obtain further insights COL-LAGEN 1 RUNX2 protein of MG63 cells and VEGF proteinof HUVEC cells from the cell-scaffold complex were selectedfor detection by western blot analysis after cell culture for 13 and 5 d The results are shown in Figure 8 Significantlyhigher levels of COLLAGEN expression were observed inthe PLGAnHACMsADM and PLGAnHA groups thanin the control group on days 1 and 5 Moreover signifi-cantly higher expression levels of runx2 were observed ateach predetermined time on the PLGAnHACMsADMscaffold which was consistent with real-time PCR analysisFor VEGF which was associated with the proliferation anddifferentiation of endothelial cells the expression level ofthe PLGAnHACMsADM group was significantly higherthan that of the control group on days 1 and 5 Meanwhileon day 1 the PLGAnHA group showed significantly higherVEGF expression than the control group suggesting thatthe structure and property of the PLGAnHA scaffold weresuitable for the migration and proliferation of HUVEC at theinitial stage
Western blot analysis showed higher expression ofCOLLAGEN 1 RUNX2 and VEGF on the PLGAnHACMsADM scaffold during culture time This finding sug-gested that composite scaffold loading with ADM promoted
BioMed Research International 9
4
3
2
1
01 3 5
Culture time (days)
lowast
lowast
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of o
pn
(a)
4
3
2
1
01 3 5
lowastlowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of c
olla
gen
1
Culture time (days)
(b)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
unx2 4
3
2
1
01 3 5
lowastlowast
Culture time (days)
(c)
4
3
2
1
01 3 5
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of s
p7
Culture time (days)
(d)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of v
egf
4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5Culture time (days)
(e)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
amp2 4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5
lowastlowast
Culture time (days)
(f)
Figure 7 RelativemRNA expression level ofMG63 cells (opn (a) collagen 1 (b) runx2 (c) and sp7 (d)) andHUVEC cells (vegf (e) and ramp2(f)) of the PLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times lowast119875 lt 005indicates statistically significant difference compared with the control group (119899 = 6)
10 BioMed Research International
Collagen 1
RUNX2
120573-actin
1 53A B C A B C A B C
120KD
57KD
42KD
MG63
ABC
ControlPLGAnHACMsADMPLGAnHACMs
120573-actin
1 53
A B C A B C A B C
42KD
45KDVEGF
HUVEC
ABC
ControlPLGAnHACMsADMPLGAnHACMs
Relat
ive p
rote
in ex
pres
sion
of co
llage
n 1
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowast lowast
lowast lowastlowast
Relat
ive p
rote
in ex
pres
sion
of R
UN
X2
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
Relat
ive p
rote
in ex
pres
sion
of V
EGF
40
60
80
100
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
(a) (b) (c)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
Figure 8 Western blot assessment of collagen 1 runx2 protein of MG63 cells and VEGF protein of HUVEC cells in thePLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times (a b and c) The bandswere quantitated by densitometry and data are expressed as the ratio of aim protein to 120573-actin lowast119875 lt 005 indicates statistically significantdifference compared with the control group (119899 = 3)
the differentiation and activation of MG63 cells by upreg-ulation of the expression of specific osteogenic proteinsHigher expression levels of COLLAGEN 1 and VEGF werealso observed on the PLGAnHA scaffold ADM has a keyfunction during the development of the vascular systemas demonstrated by Shindo et al [61] Specific conditionssuch as hypoxia are reported to be associated with increasedVEGF expression [62] The ADM-induced upregulation ofVEGF at the protein level agrees well with most reports[63 64] but is not consistent with others [60 65] Thevegf gene was not significantly upregulated The inconsistentlevels between vegf gene and protein were possibly causedby the enhancement of translational efficiency and proteinconstancy
4 Conclusions
As a growth factor ADM was first introduced for tissue-engineering materials The combination of emulsion-ioniccross-linking and TIPS was proven to be suitable for scaf-foldmicrosphere construct developing loading with ADMThe microspheres showed a rough surface morphology andwere well distributed either in the presence or absence of
ADMThe scaffolds showed relatively uniformpore sizeswithinterconnected pores The addition of CMs into the scaffoldsimproved the mechanical properties of the scaffolds withoutremarkably changing their high porosity Moreover in vitrodegradation studies revealed that CM incorporation canaccelerate WL rate increase WA and reduce PLGA acidityin hydrolysisThe expression patterns of opn col1 runx2 andsp7 indicated that inductive osteoblast-like cell differentiationin contact with PLGAnHACMsADM scaffolds appearedearly in bone formation Western blot analysis demonstratedthat the PLGAnHACMsADM scaffold had high levels ofCOLLAGEN 1 and RUNX2 expression In addition RT-PCRanalysis showed that the PLGAnHACMsADM scaffoldhad a high level of ramp2 on days 3 and 5 The expres-sion of vegf was unaffected regardless of ADM loadingMeanwhile VEGF protein levels were relatively high for thePLGAnHACMsADM scaffold at the early osteoblast stageas determined by western blot analysis These findings sug-gest that microspherescaffold composite was more effectivein loading peptides and proteins which can improve theosteogenic and angiogenic differentiation of osteoblasts andvascular endothelial cells on the porous scaffolds Thereforesustained-release ADM from microsphere-scaffold system
BioMed Research International 11
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
PLGAnHACMs 0083 plusmn 0020lowast 8893 plusmn 032lowastP lt 005 indicates statistically significant difference compared with thePLGAnHA group
a result of an interconnected 3D pore structure Furthermorethe retention of approximately 90 porosity in the scaffoldsshould enable a large space for the accommodation of high-density cell cultures
32 Mechanical Properties Mechanical properties of thePLGAnHA scaffolds with and without CMs were evaluatedby a universal material testing machine The mechanicalparameters of the scaffolds are summarized in Figure 4 Thecompressive strength of the PLGAnHACM scaffold (154 plusmn020MPa) was obviously higher than that of the PLGAnHAscaffold (098 plusmn 012MPa) Additionally the compressivemodulus of the PLGAnHACM scaffold (2943 plusmn 242MPa)was significantly higher than that of the PLGAnHA scaf-fold (2145 plusmn 145MPa) A well-designed bone-engineeredscaffold has to meet two mechanical requirements to beeffective The scaffold providing a matrix for cell residencemust retain structural integrity and stability when a doctorimplants it into the defective site Then it must providesufficient mechanical support during tissue regeneration[42] The compression strength of the PLGAnHA scaffoldincreases with the introduction of nHA [43] Moreover themore compressive strength of the PLGAnHACMs scaffold
65
6
55
5
45
4
35
3
25
2
15
1
05
0
Volu
me (
)
01 1 5 10 100 1000Diameter (120583m)
Figure 2 Size distribution of TPP-chitosan microspheres loadedwith ADM
proves that the addition of CMs can improve the mechanicalproperties of the scaffold without affecting the porosity
33 Degradation Properties In Vitro The degradation prop-erties of a scaffold are of crucial importance in the long-term success of a tissue-engineered scaffold Scaffolds forbone regeneration are designed to be gradually replacedwith regenerated ECM during bone formation accompaniedwith degradation WL rate is an important parameter used
6 BioMed Research International
(a) (b)
Figure 3 SEM images of PLGAnHA scaffolds prepared without (a) and with (b) chitosan microspheres The arrows show the chitosanmicrospheres in the scaffold
Compressive strengthCompressive modulus
27525
2252
17515
05
125
025
1075
Com
pres
sive s
treng
th (M
Pa)
10
20
30
40
0
Com
pres
sive m
odul
us (M
Pa)
PLGAnHA PLGAnHACMs
lowast
lowast
Figure 4 Compressive strength and compressive modulus ofPLGAnHA with and without 30 CMs lowast119875 lt 005 indicatesstatistically significant difference compared with the PLGAnHAgroup
to examine the degradation performance of scaffolds TheWL of the PLGAnHA scaffolds with and without CMsis presented in Figure 5(a) The mass of the PLGAnHAscaffolds decreased with increasing degradation time Thepure PLGAnHA polymer showed a slower WL during theentire degradation time The WL of the PLGAnHACMscaffold was slightly faster in the first 3 weeks and thenreached a linear mode At week 12 the WL rates of thePLGAnHA scaffolds with and without CMswere 1223 and827 respectively The four steps for the degradation of thePLGA scaffold are as follows (1) swelling and hydration ofthe polymer (2) breakage of the ester bonds (3) diffusionof the soluble degradation products and (4) disappearanceof the polymer scaffold chips [44] The faster WL detectedfor the PLGAnHACM scaffolds can be associated to thehigher capability of CMs to absorb water when soaked in PBSsolution Moreover mass loss of the scaffolds was reportedto correspond with the changes in pH [45] The results ofthis study are consistent with their report Under in vivo
conditions some enzymes (eg proteinase K and lipasePS) liposomes germs and phagocytes can modulate thedegradation of PLGA polymers to obtain faster degradationrates In addition a previous study [46] reported that thefaster degradation is caused by the autocatalytic effect ofthe acidic degradation products accumulated in the mediumsurrounding the implants This effect was minimized forpolymer degradation in PBS by the frequent change of themedium
Given that medium flow is essential for nutrient andmetabolic exchanges the WA properties of a scaffold areanother important feature for developing a suitable scaffoldfor bone regeneration [47] The WA of PLGAnHA scaffoldswith and without CMs is presented in Figure 5(b) The WAof PLGAnHACMs was 669 at week 1 and gradually rosestably to 8215 at week 6 finally reaching 8834 at week12 The WA of the PLGAnHA polymer was slower duringthe entire time and ultimately reached 524 at week 12 TheWA result was generally consistent with the WL result TheWA of the two scaffolds rapidly increased at week 1 Thisincrease may be attributed to the water that diffused throughthe porous structure The much more rapidly increasingWA of the PLGAnHACM scaffolds was possibly attributedto the excellent absorbent capacity of CMs At the initialstage the WA of the scaffold was critical for integratingthe material-bone construct The superior hydrophilicityof the PLGAnHACM scaffolds might easily facilitate cellmigration into the pores following blood immersion in vivoAs previously observed the hydrophilic characteristics ofthe scaffolds can enhance cell adhesion migration andproliferation in vivo compared with hydrophobic scaffolds[48]
The formation of the degraded acidic molecules andtheir release from PLGA degradation is a negative factor forbone engineering The pH variation of PBS buffer during thedegradation of the PLGAnHA scaffolds with and withoutCMswas detected to verify the acid product released from thescaffoldThe result is shown in Figure 6The pH of the degra-dation medium generally decreased with time maintainingnearly at approximately 71 until week 4 under dynamic
BioMed Research International 7
15
10
5
0
Wei
ght l
oss (
)
PLGAnHACMsPLGAnHA
0 12108642Degradation time (weeks)
(a)
Wat
er u
ptak
e (
)
100
80
60
40
20
0
PLGAnHACMsPLGAnHA
210 108642Degradation time (weeks)
(b)
Figure 5 Weight loss (a) and water uptake (b) of PLGAnHA with and without 30 CMs during degradation in PBS solution
conditions The pH of the PBS buffer for the PLGAnHAscaffolds with and without CMs decreased rapidly at week4 and then slightly decreased from week 4 to week 8 ofdegradation After week 8 the pH plateau was reachedfor the PLGAnHACMs scaffold samples whereas the pHfor the PLGAnHA scaffold samples slightly decreased Aslighter decrease in pH for the PLGAnHACMs scaffoldsafter week 8 may be ascribed to alkaline dissolution of theCMs Arnett [49] reported that osteoblast proliferation andcollagen synthesis are unaffected by pH in the range of 74to 69 The pH of PBS for the PLGAnHACMs scaffolds wasabove 69 during the entire degradation time This findingindicated that the composite PLGAnHA polymer with CMswas suitable for bone engineering
34 Gene Expression Bone formation is an intricate andordered cascade of synthesis of matrix proteins and calciumphosphate in a continuously renewed biological environmentand regulated by a cluster of growth factors [50] An artificialscaffold should be designed as a production of ideal struc-ture that can mimic ECM until host cells including bothosteoblasts and vascular endothelial cells can grow in andresynthesize a new natural matrix The progress in replacingscaffold by natural bone is dependent on the cell adhesionproliferation differentiation and vascularization of the scaf-folds Therefore the successful formation of microvascularcells with long-term patency that are not apt to regression isvery important In bone formation osteogenic-related genes(eg opn col1 runx2 and sp7) and angiogenic-related genes(eg vegf and ramp2) are strictly regulated [51]
Using quantitative real-time PCR the present studydetermined whether the structure of the polymer scaffoldloading with chitosan-ADM microspheres and the sustainedrelease of ADM can promote the early differentiation and
PLGAnHACMsPLGAnHA
8
7
6
5
pH v
alue
0 12108642Degradation time (weeks)
Figure 6 pH of incubated PBS buffer for PLGAnHA with andwithout 30 CMs during in vitro degradation study
activation of osteoblasts and vein endothelial cells at themRNA level of the aforementioned genes
On day 1 the mean expression levels of opn in thePLGAnHACMsADM and PLGAnHA groups were 21and 12 times higher respectively than that in the con-trol group (Figure 7(a)) On day 3 the expression lev-els of opn in the PLGAnHACMsADM and PLGAnHAgroups significantly increased by 28 and 17 times higherthan that in the control group The analogous tendencyappeared on day 5 Meanwhile the opn expression ofthe PLGAnHACMsADM group was higher than thatof the PLGAnHA group at each predetermined time Asshown in Figure 7(b) the col1 mRNA expression of thePLGAnHACMsADM group was almost equal to the other
8 BioMed Research International
two on day 1 It rapidly increased on day 3 and thenmaintained on day 5 which was significantly higher than thatof the control group No significant difference was observedbetween the PLGAnHA group and the control group ateach time point As shown in Figure 7(c) no significantdifference in runx2 expression appeared on day 1 Howeveron day 3 the expression of the PLGAnHACMsADMgroupwas 21 times higher than that of the control group It thenslightly decreased on day 5 which were both significantlyhigher than the control group Nevertheless as shown inFigure 7(d) the sp7 mRNA level was upregulated in thePLGAnHACMsADM group with culture time The valueswere 27 and 47 times significantly higher than those of thecontrol group on days 3 and 5 respectively suggesting theupregulation of osteoblastic activity On day 5 the sp7mRNAexpression in the PLGAnHA scaffold was 17 times higherthan that in the control groupThis result proved the bioactivepotential of the interconnected microstructure of polymerscaffolds
Cell-material interactions can be evaluated by detectingthe cellular receptors responsible for adhesion and migrationand the ligands they bind to specifically bone ECM proteins[52 53] Our previous research revealed the proliferation andactivation functions of ADM The ADM encapsulated in thescaffoldmicrosphere construct can stimulate the prolifera-tion ofMG63 cells for 5 d byMTTassay andfluorescent imageobservation [24] Opn as a mineral-binding protein foundin bone ECM is implicated as an important factor in boneremodeling and crystal growth regulation It is associatedwith cell adhesion proliferation and biomineralization ofECM into bone and its high expression demonstrates theproliferation and activation of MG63 cells Another ECMprotein that is related to further differentiation of osteoblastsis col1 which accounts for 90 of the bone matrix proteins[54] Frick et al [55] reported that the mRNA expressionof col1 is stimulated by alkalosis and inhibited by acidosisThe small pH variation induced by the change in calciumconcentration has a significant effect on col1 expression Theincrease in col1 mRNA of the PLGAnHACMsADM groupon day 3 can be due to the increase in pH induced by chitosandegradation or the effect of ADM Transcription factorsrunx2 and sp7 were essential for osteoblastic differentiationand act as regulatory factors involved in osteogenic-relatedgene expression Early studies reported that runx2 bindsthe osteocalcin promoter and is expressed in osteochondralprogenitors as well as in early stages of osteoblastic differen-tiation [56] In humans runx2 haploinsufficiency results incleidocranial dysplasia a skeletal disorder characterized bybone and dental abnormalities [57] Sp7-deficient mice lackbone formation with a phenotype similar to that of runx2-deficientmiceMeanwhile sp7 is either acting downstreamofrunx2 or expressed later in the osteoblast differentiation path-way [58] Based on the aforementioned result the increasein mRNA expression at an early stage suggested that thedifferentiation and activation of MG63 cells on the surfaceof polymer scaffold were probably due to the sustained ADMrelease PLGAnHACMsADMcan induce differentiation inMG63 cells at the early stage of bone formation
The success of tissue-engineering scaffold is highlydependent on whether the materials can promote rapidand stable neovascularization (new blood vessel formation)within the scaffold typically prior to complete materialdegradation [4] Endothelial cells are currently regarded asthe most interesting target for therapies aimed at enhanc-ing or inhibiting angiogenesis [59] VEGF is an importantregulator of endothelial cell proliferation migration anddifferentiation As one of the ADM receptors ramp2 isessential for angiogenesis and vascular integrity Figures7(e) and 7(f) illustrate the vegf and ramp2 expressionlevels of HUVEC in three groups The vegf expression ofthe PLGAnHACMsADM group completely showed equalexpression pattern to the other two groups at each predeter-mined time However on days 3 and 5 the vegf expressionof the PLGAnHACMsADM and PLGAnHA groups wasslightly higher compared with that of the control groupThe ramp2mRNA expression of the PLGAnHACMsADMgroup steadily increased from day 3 and then slightly down-regulated on day 5The values of the PLGAnHACMsADMgroup on days 3 and 5 were significantly higher than those ofthe control group The expression level of ramp2 was rapidlyupregulated fromday 3 in the PLGAnHACMsADMgroupwhich might be induced by sustained-released ADM How-ever vegf expression was not upregulated The results of thepresent study are generally consistent with those of previousstudies [60] That is the activity of ADM was triggered bythe binding of ADM to its ADM receptor In addition theeffect of ADM on HUVEC was no longer detectable afterthe expression of ramp2 in the cells was almost suppressedby gene silencing The results demonstrated that the releasedADM from the scaffold may be involved in vascularizationfrom the gene level at the early stage
35 Western Blot Analysis To obtain further insights COL-LAGEN 1 RUNX2 protein of MG63 cells and VEGF proteinof HUVEC cells from the cell-scaffold complex were selectedfor detection by western blot analysis after cell culture for 13 and 5 d The results are shown in Figure 8 Significantlyhigher levels of COLLAGEN expression were observed inthe PLGAnHACMsADM and PLGAnHA groups thanin the control group on days 1 and 5 Moreover signifi-cantly higher expression levels of runx2 were observed ateach predetermined time on the PLGAnHACMsADMscaffold which was consistent with real-time PCR analysisFor VEGF which was associated with the proliferation anddifferentiation of endothelial cells the expression level ofthe PLGAnHACMsADM group was significantly higherthan that of the control group on days 1 and 5 Meanwhileon day 1 the PLGAnHA group showed significantly higherVEGF expression than the control group suggesting thatthe structure and property of the PLGAnHA scaffold weresuitable for the migration and proliferation of HUVEC at theinitial stage
Western blot analysis showed higher expression ofCOLLAGEN 1 RUNX2 and VEGF on the PLGAnHACMsADM scaffold during culture time This finding sug-gested that composite scaffold loading with ADM promoted
BioMed Research International 9
4
3
2
1
01 3 5
Culture time (days)
lowast
lowast
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of o
pn
(a)
4
3
2
1
01 3 5
lowastlowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of c
olla
gen
1
Culture time (days)
(b)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
unx2 4
3
2
1
01 3 5
lowastlowast
Culture time (days)
(c)
4
3
2
1
01 3 5
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of s
p7
Culture time (days)
(d)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of v
egf
4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5Culture time (days)
(e)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
amp2 4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5
lowastlowast
Culture time (days)
(f)
Figure 7 RelativemRNA expression level ofMG63 cells (opn (a) collagen 1 (b) runx2 (c) and sp7 (d)) andHUVEC cells (vegf (e) and ramp2(f)) of the PLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times lowast119875 lt 005indicates statistically significant difference compared with the control group (119899 = 6)
10 BioMed Research International
Collagen 1
RUNX2
120573-actin
1 53A B C A B C A B C
120KD
57KD
42KD
MG63
ABC
ControlPLGAnHACMsADMPLGAnHACMs
120573-actin
1 53
A B C A B C A B C
42KD
45KDVEGF
HUVEC
ABC
ControlPLGAnHACMsADMPLGAnHACMs
Relat
ive p
rote
in ex
pres
sion
of co
llage
n 1
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowast lowast
lowast lowastlowast
Relat
ive p
rote
in ex
pres
sion
of R
UN
X2
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
Relat
ive p
rote
in ex
pres
sion
of V
EGF
40
60
80
100
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
(a) (b) (c)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
Figure 8 Western blot assessment of collagen 1 runx2 protein of MG63 cells and VEGF protein of HUVEC cells in thePLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times (a b and c) The bandswere quantitated by densitometry and data are expressed as the ratio of aim protein to 120573-actin lowast119875 lt 005 indicates statistically significantdifference compared with the control group (119899 = 3)
the differentiation and activation of MG63 cells by upreg-ulation of the expression of specific osteogenic proteinsHigher expression levels of COLLAGEN 1 and VEGF werealso observed on the PLGAnHA scaffold ADM has a keyfunction during the development of the vascular systemas demonstrated by Shindo et al [61] Specific conditionssuch as hypoxia are reported to be associated with increasedVEGF expression [62] The ADM-induced upregulation ofVEGF at the protein level agrees well with most reports[63 64] but is not consistent with others [60 65] Thevegf gene was not significantly upregulated The inconsistentlevels between vegf gene and protein were possibly causedby the enhancement of translational efficiency and proteinconstancy
4 Conclusions
As a growth factor ADM was first introduced for tissue-engineering materials The combination of emulsion-ioniccross-linking and TIPS was proven to be suitable for scaf-foldmicrosphere construct developing loading with ADMThe microspheres showed a rough surface morphology andwere well distributed either in the presence or absence of
ADMThe scaffolds showed relatively uniformpore sizeswithinterconnected pores The addition of CMs into the scaffoldsimproved the mechanical properties of the scaffolds withoutremarkably changing their high porosity Moreover in vitrodegradation studies revealed that CM incorporation canaccelerate WL rate increase WA and reduce PLGA acidityin hydrolysisThe expression patterns of opn col1 runx2 andsp7 indicated that inductive osteoblast-like cell differentiationin contact with PLGAnHACMsADM scaffolds appearedearly in bone formation Western blot analysis demonstratedthat the PLGAnHACMsADM scaffold had high levels ofCOLLAGEN 1 and RUNX2 expression In addition RT-PCRanalysis showed that the PLGAnHACMsADM scaffoldhad a high level of ramp2 on days 3 and 5 The expres-sion of vegf was unaffected regardless of ADM loadingMeanwhile VEGF protein levels were relatively high for thePLGAnHACMsADM scaffold at the early osteoblast stageas determined by western blot analysis These findings sug-gest that microspherescaffold composite was more effectivein loading peptides and proteins which can improve theosteogenic and angiogenic differentiation of osteoblasts andvascular endothelial cells on the porous scaffolds Thereforesustained-release ADM from microsphere-scaffold system
BioMed Research International 11
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
Figure 3 SEM images of PLGAnHA scaffolds prepared without (a) and with (b) chitosan microspheres The arrows show the chitosanmicrospheres in the scaffold
Compressive strengthCompressive modulus
27525
2252
17515
05
125
025
1075
Com
pres
sive s
treng
th (M
Pa)
10
20
30
40
0
Com
pres
sive m
odul
us (M
Pa)
PLGAnHA PLGAnHACMs
lowast
lowast
Figure 4 Compressive strength and compressive modulus ofPLGAnHA with and without 30 CMs lowast119875 lt 005 indicatesstatistically significant difference compared with the PLGAnHAgroup
to examine the degradation performance of scaffolds TheWL of the PLGAnHA scaffolds with and without CMsis presented in Figure 5(a) The mass of the PLGAnHAscaffolds decreased with increasing degradation time Thepure PLGAnHA polymer showed a slower WL during theentire degradation time The WL of the PLGAnHACMscaffold was slightly faster in the first 3 weeks and thenreached a linear mode At week 12 the WL rates of thePLGAnHA scaffolds with and without CMswere 1223 and827 respectively The four steps for the degradation of thePLGA scaffold are as follows (1) swelling and hydration ofthe polymer (2) breakage of the ester bonds (3) diffusionof the soluble degradation products and (4) disappearanceof the polymer scaffold chips [44] The faster WL detectedfor the PLGAnHACM scaffolds can be associated to thehigher capability of CMs to absorb water when soaked in PBSsolution Moreover mass loss of the scaffolds was reportedto correspond with the changes in pH [45] The results ofthis study are consistent with their report Under in vivo
conditions some enzymes (eg proteinase K and lipasePS) liposomes germs and phagocytes can modulate thedegradation of PLGA polymers to obtain faster degradationrates In addition a previous study [46] reported that thefaster degradation is caused by the autocatalytic effect ofthe acidic degradation products accumulated in the mediumsurrounding the implants This effect was minimized forpolymer degradation in PBS by the frequent change of themedium
Given that medium flow is essential for nutrient andmetabolic exchanges the WA properties of a scaffold areanother important feature for developing a suitable scaffoldfor bone regeneration [47] The WA of PLGAnHA scaffoldswith and without CMs is presented in Figure 5(b) The WAof PLGAnHACMs was 669 at week 1 and gradually rosestably to 8215 at week 6 finally reaching 8834 at week12 The WA of the PLGAnHA polymer was slower duringthe entire time and ultimately reached 524 at week 12 TheWA result was generally consistent with the WL result TheWA of the two scaffolds rapidly increased at week 1 Thisincrease may be attributed to the water that diffused throughthe porous structure The much more rapidly increasingWA of the PLGAnHACM scaffolds was possibly attributedto the excellent absorbent capacity of CMs At the initialstage the WA of the scaffold was critical for integratingthe material-bone construct The superior hydrophilicityof the PLGAnHACM scaffolds might easily facilitate cellmigration into the pores following blood immersion in vivoAs previously observed the hydrophilic characteristics ofthe scaffolds can enhance cell adhesion migration andproliferation in vivo compared with hydrophobic scaffolds[48]
The formation of the degraded acidic molecules andtheir release from PLGA degradation is a negative factor forbone engineering The pH variation of PBS buffer during thedegradation of the PLGAnHA scaffolds with and withoutCMswas detected to verify the acid product released from thescaffoldThe result is shown in Figure 6The pH of the degra-dation medium generally decreased with time maintainingnearly at approximately 71 until week 4 under dynamic
BioMed Research International 7
15
10
5
0
Wei
ght l
oss (
)
PLGAnHACMsPLGAnHA
0 12108642Degradation time (weeks)
(a)
Wat
er u
ptak
e (
)
100
80
60
40
20
0
PLGAnHACMsPLGAnHA
210 108642Degradation time (weeks)
(b)
Figure 5 Weight loss (a) and water uptake (b) of PLGAnHA with and without 30 CMs during degradation in PBS solution
conditions The pH of the PBS buffer for the PLGAnHAscaffolds with and without CMs decreased rapidly at week4 and then slightly decreased from week 4 to week 8 ofdegradation After week 8 the pH plateau was reachedfor the PLGAnHACMs scaffold samples whereas the pHfor the PLGAnHA scaffold samples slightly decreased Aslighter decrease in pH for the PLGAnHACMs scaffoldsafter week 8 may be ascribed to alkaline dissolution of theCMs Arnett [49] reported that osteoblast proliferation andcollagen synthesis are unaffected by pH in the range of 74to 69 The pH of PBS for the PLGAnHACMs scaffolds wasabove 69 during the entire degradation time This findingindicated that the composite PLGAnHA polymer with CMswas suitable for bone engineering
34 Gene Expression Bone formation is an intricate andordered cascade of synthesis of matrix proteins and calciumphosphate in a continuously renewed biological environmentand regulated by a cluster of growth factors [50] An artificialscaffold should be designed as a production of ideal struc-ture that can mimic ECM until host cells including bothosteoblasts and vascular endothelial cells can grow in andresynthesize a new natural matrix The progress in replacingscaffold by natural bone is dependent on the cell adhesionproliferation differentiation and vascularization of the scaf-folds Therefore the successful formation of microvascularcells with long-term patency that are not apt to regression isvery important In bone formation osteogenic-related genes(eg opn col1 runx2 and sp7) and angiogenic-related genes(eg vegf and ramp2) are strictly regulated [51]
Using quantitative real-time PCR the present studydetermined whether the structure of the polymer scaffoldloading with chitosan-ADM microspheres and the sustainedrelease of ADM can promote the early differentiation and
PLGAnHACMsPLGAnHA
8
7
6
5
pH v
alue
0 12108642Degradation time (weeks)
Figure 6 pH of incubated PBS buffer for PLGAnHA with andwithout 30 CMs during in vitro degradation study
activation of osteoblasts and vein endothelial cells at themRNA level of the aforementioned genes
On day 1 the mean expression levels of opn in thePLGAnHACMsADM and PLGAnHA groups were 21and 12 times higher respectively than that in the con-trol group (Figure 7(a)) On day 3 the expression lev-els of opn in the PLGAnHACMsADM and PLGAnHAgroups significantly increased by 28 and 17 times higherthan that in the control group The analogous tendencyappeared on day 5 Meanwhile the opn expression ofthe PLGAnHACMsADM group was higher than thatof the PLGAnHA group at each predetermined time Asshown in Figure 7(b) the col1 mRNA expression of thePLGAnHACMsADM group was almost equal to the other
8 BioMed Research International
two on day 1 It rapidly increased on day 3 and thenmaintained on day 5 which was significantly higher than thatof the control group No significant difference was observedbetween the PLGAnHA group and the control group ateach time point As shown in Figure 7(c) no significantdifference in runx2 expression appeared on day 1 Howeveron day 3 the expression of the PLGAnHACMsADMgroupwas 21 times higher than that of the control group It thenslightly decreased on day 5 which were both significantlyhigher than the control group Nevertheless as shown inFigure 7(d) the sp7 mRNA level was upregulated in thePLGAnHACMsADM group with culture time The valueswere 27 and 47 times significantly higher than those of thecontrol group on days 3 and 5 respectively suggesting theupregulation of osteoblastic activity On day 5 the sp7mRNAexpression in the PLGAnHA scaffold was 17 times higherthan that in the control groupThis result proved the bioactivepotential of the interconnected microstructure of polymerscaffolds
Cell-material interactions can be evaluated by detectingthe cellular receptors responsible for adhesion and migrationand the ligands they bind to specifically bone ECM proteins[52 53] Our previous research revealed the proliferation andactivation functions of ADM The ADM encapsulated in thescaffoldmicrosphere construct can stimulate the prolifera-tion ofMG63 cells for 5 d byMTTassay andfluorescent imageobservation [24] Opn as a mineral-binding protein foundin bone ECM is implicated as an important factor in boneremodeling and crystal growth regulation It is associatedwith cell adhesion proliferation and biomineralization ofECM into bone and its high expression demonstrates theproliferation and activation of MG63 cells Another ECMprotein that is related to further differentiation of osteoblastsis col1 which accounts for 90 of the bone matrix proteins[54] Frick et al [55] reported that the mRNA expressionof col1 is stimulated by alkalosis and inhibited by acidosisThe small pH variation induced by the change in calciumconcentration has a significant effect on col1 expression Theincrease in col1 mRNA of the PLGAnHACMsADM groupon day 3 can be due to the increase in pH induced by chitosandegradation or the effect of ADM Transcription factorsrunx2 and sp7 were essential for osteoblastic differentiationand act as regulatory factors involved in osteogenic-relatedgene expression Early studies reported that runx2 bindsthe osteocalcin promoter and is expressed in osteochondralprogenitors as well as in early stages of osteoblastic differen-tiation [56] In humans runx2 haploinsufficiency results incleidocranial dysplasia a skeletal disorder characterized bybone and dental abnormalities [57] Sp7-deficient mice lackbone formation with a phenotype similar to that of runx2-deficientmiceMeanwhile sp7 is either acting downstreamofrunx2 or expressed later in the osteoblast differentiation path-way [58] Based on the aforementioned result the increasein mRNA expression at an early stage suggested that thedifferentiation and activation of MG63 cells on the surfaceof polymer scaffold were probably due to the sustained ADMrelease PLGAnHACMsADMcan induce differentiation inMG63 cells at the early stage of bone formation
The success of tissue-engineering scaffold is highlydependent on whether the materials can promote rapidand stable neovascularization (new blood vessel formation)within the scaffold typically prior to complete materialdegradation [4] Endothelial cells are currently regarded asthe most interesting target for therapies aimed at enhanc-ing or inhibiting angiogenesis [59] VEGF is an importantregulator of endothelial cell proliferation migration anddifferentiation As one of the ADM receptors ramp2 isessential for angiogenesis and vascular integrity Figures7(e) and 7(f) illustrate the vegf and ramp2 expressionlevels of HUVEC in three groups The vegf expression ofthe PLGAnHACMsADM group completely showed equalexpression pattern to the other two groups at each predeter-mined time However on days 3 and 5 the vegf expressionof the PLGAnHACMsADM and PLGAnHA groups wasslightly higher compared with that of the control groupThe ramp2mRNA expression of the PLGAnHACMsADMgroup steadily increased from day 3 and then slightly down-regulated on day 5The values of the PLGAnHACMsADMgroup on days 3 and 5 were significantly higher than those ofthe control group The expression level of ramp2 was rapidlyupregulated fromday 3 in the PLGAnHACMsADMgroupwhich might be induced by sustained-released ADM How-ever vegf expression was not upregulated The results of thepresent study are generally consistent with those of previousstudies [60] That is the activity of ADM was triggered bythe binding of ADM to its ADM receptor In addition theeffect of ADM on HUVEC was no longer detectable afterthe expression of ramp2 in the cells was almost suppressedby gene silencing The results demonstrated that the releasedADM from the scaffold may be involved in vascularizationfrom the gene level at the early stage
35 Western Blot Analysis To obtain further insights COL-LAGEN 1 RUNX2 protein of MG63 cells and VEGF proteinof HUVEC cells from the cell-scaffold complex were selectedfor detection by western blot analysis after cell culture for 13 and 5 d The results are shown in Figure 8 Significantlyhigher levels of COLLAGEN expression were observed inthe PLGAnHACMsADM and PLGAnHA groups thanin the control group on days 1 and 5 Moreover signifi-cantly higher expression levels of runx2 were observed ateach predetermined time on the PLGAnHACMsADMscaffold which was consistent with real-time PCR analysisFor VEGF which was associated with the proliferation anddifferentiation of endothelial cells the expression level ofthe PLGAnHACMsADM group was significantly higherthan that of the control group on days 1 and 5 Meanwhileon day 1 the PLGAnHA group showed significantly higherVEGF expression than the control group suggesting thatthe structure and property of the PLGAnHA scaffold weresuitable for the migration and proliferation of HUVEC at theinitial stage
Western blot analysis showed higher expression ofCOLLAGEN 1 RUNX2 and VEGF on the PLGAnHACMsADM scaffold during culture time This finding sug-gested that composite scaffold loading with ADM promoted
BioMed Research International 9
4
3
2
1
01 3 5
Culture time (days)
lowast
lowast
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of o
pn
(a)
4
3
2
1
01 3 5
lowastlowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of c
olla
gen
1
Culture time (days)
(b)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
unx2 4
3
2
1
01 3 5
lowastlowast
Culture time (days)
(c)
4
3
2
1
01 3 5
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of s
p7
Culture time (days)
(d)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of v
egf
4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5Culture time (days)
(e)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
amp2 4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5
lowastlowast
Culture time (days)
(f)
Figure 7 RelativemRNA expression level ofMG63 cells (opn (a) collagen 1 (b) runx2 (c) and sp7 (d)) andHUVEC cells (vegf (e) and ramp2(f)) of the PLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times lowast119875 lt 005indicates statistically significant difference compared with the control group (119899 = 6)
10 BioMed Research International
Collagen 1
RUNX2
120573-actin
1 53A B C A B C A B C
120KD
57KD
42KD
MG63
ABC
ControlPLGAnHACMsADMPLGAnHACMs
120573-actin
1 53
A B C A B C A B C
42KD
45KDVEGF
HUVEC
ABC
ControlPLGAnHACMsADMPLGAnHACMs
Relat
ive p
rote
in ex
pres
sion
of co
llage
n 1
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowast lowast
lowast lowastlowast
Relat
ive p
rote
in ex
pres
sion
of R
UN
X2
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
Relat
ive p
rote
in ex
pres
sion
of V
EGF
40
60
80
100
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
(a) (b) (c)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
Figure 8 Western blot assessment of collagen 1 runx2 protein of MG63 cells and VEGF protein of HUVEC cells in thePLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times (a b and c) The bandswere quantitated by densitometry and data are expressed as the ratio of aim protein to 120573-actin lowast119875 lt 005 indicates statistically significantdifference compared with the control group (119899 = 3)
the differentiation and activation of MG63 cells by upreg-ulation of the expression of specific osteogenic proteinsHigher expression levels of COLLAGEN 1 and VEGF werealso observed on the PLGAnHA scaffold ADM has a keyfunction during the development of the vascular systemas demonstrated by Shindo et al [61] Specific conditionssuch as hypoxia are reported to be associated with increasedVEGF expression [62] The ADM-induced upregulation ofVEGF at the protein level agrees well with most reports[63 64] but is not consistent with others [60 65] Thevegf gene was not significantly upregulated The inconsistentlevels between vegf gene and protein were possibly causedby the enhancement of translational efficiency and proteinconstancy
4 Conclusions
As a growth factor ADM was first introduced for tissue-engineering materials The combination of emulsion-ioniccross-linking and TIPS was proven to be suitable for scaf-foldmicrosphere construct developing loading with ADMThe microspheres showed a rough surface morphology andwere well distributed either in the presence or absence of
ADMThe scaffolds showed relatively uniformpore sizeswithinterconnected pores The addition of CMs into the scaffoldsimproved the mechanical properties of the scaffolds withoutremarkably changing their high porosity Moreover in vitrodegradation studies revealed that CM incorporation canaccelerate WL rate increase WA and reduce PLGA acidityin hydrolysisThe expression patterns of opn col1 runx2 andsp7 indicated that inductive osteoblast-like cell differentiationin contact with PLGAnHACMsADM scaffolds appearedearly in bone formation Western blot analysis demonstratedthat the PLGAnHACMsADM scaffold had high levels ofCOLLAGEN 1 and RUNX2 expression In addition RT-PCRanalysis showed that the PLGAnHACMsADM scaffoldhad a high level of ramp2 on days 3 and 5 The expres-sion of vegf was unaffected regardless of ADM loadingMeanwhile VEGF protein levels were relatively high for thePLGAnHACMsADM scaffold at the early osteoblast stageas determined by western blot analysis These findings sug-gest that microspherescaffold composite was more effectivein loading peptides and proteins which can improve theosteogenic and angiogenic differentiation of osteoblasts andvascular endothelial cells on the porous scaffolds Thereforesustained-release ADM from microsphere-scaffold system
BioMed Research International 11
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
Figure 5 Weight loss (a) and water uptake (b) of PLGAnHA with and without 30 CMs during degradation in PBS solution
conditions The pH of the PBS buffer for the PLGAnHAscaffolds with and without CMs decreased rapidly at week4 and then slightly decreased from week 4 to week 8 ofdegradation After week 8 the pH plateau was reachedfor the PLGAnHACMs scaffold samples whereas the pHfor the PLGAnHA scaffold samples slightly decreased Aslighter decrease in pH for the PLGAnHACMs scaffoldsafter week 8 may be ascribed to alkaline dissolution of theCMs Arnett [49] reported that osteoblast proliferation andcollagen synthesis are unaffected by pH in the range of 74to 69 The pH of PBS for the PLGAnHACMs scaffolds wasabove 69 during the entire degradation time This findingindicated that the composite PLGAnHA polymer with CMswas suitable for bone engineering
34 Gene Expression Bone formation is an intricate andordered cascade of synthesis of matrix proteins and calciumphosphate in a continuously renewed biological environmentand regulated by a cluster of growth factors [50] An artificialscaffold should be designed as a production of ideal struc-ture that can mimic ECM until host cells including bothosteoblasts and vascular endothelial cells can grow in andresynthesize a new natural matrix The progress in replacingscaffold by natural bone is dependent on the cell adhesionproliferation differentiation and vascularization of the scaf-folds Therefore the successful formation of microvascularcells with long-term patency that are not apt to regression isvery important In bone formation osteogenic-related genes(eg opn col1 runx2 and sp7) and angiogenic-related genes(eg vegf and ramp2) are strictly regulated [51]
Using quantitative real-time PCR the present studydetermined whether the structure of the polymer scaffoldloading with chitosan-ADM microspheres and the sustainedrelease of ADM can promote the early differentiation and
PLGAnHACMsPLGAnHA
8
7
6
5
pH v
alue
0 12108642Degradation time (weeks)
Figure 6 pH of incubated PBS buffer for PLGAnHA with andwithout 30 CMs during in vitro degradation study
activation of osteoblasts and vein endothelial cells at themRNA level of the aforementioned genes
On day 1 the mean expression levels of opn in thePLGAnHACMsADM and PLGAnHA groups were 21and 12 times higher respectively than that in the con-trol group (Figure 7(a)) On day 3 the expression lev-els of opn in the PLGAnHACMsADM and PLGAnHAgroups significantly increased by 28 and 17 times higherthan that in the control group The analogous tendencyappeared on day 5 Meanwhile the opn expression ofthe PLGAnHACMsADM group was higher than thatof the PLGAnHA group at each predetermined time Asshown in Figure 7(b) the col1 mRNA expression of thePLGAnHACMsADM group was almost equal to the other
8 BioMed Research International
two on day 1 It rapidly increased on day 3 and thenmaintained on day 5 which was significantly higher than thatof the control group No significant difference was observedbetween the PLGAnHA group and the control group ateach time point As shown in Figure 7(c) no significantdifference in runx2 expression appeared on day 1 Howeveron day 3 the expression of the PLGAnHACMsADMgroupwas 21 times higher than that of the control group It thenslightly decreased on day 5 which were both significantlyhigher than the control group Nevertheless as shown inFigure 7(d) the sp7 mRNA level was upregulated in thePLGAnHACMsADM group with culture time The valueswere 27 and 47 times significantly higher than those of thecontrol group on days 3 and 5 respectively suggesting theupregulation of osteoblastic activity On day 5 the sp7mRNAexpression in the PLGAnHA scaffold was 17 times higherthan that in the control groupThis result proved the bioactivepotential of the interconnected microstructure of polymerscaffolds
Cell-material interactions can be evaluated by detectingthe cellular receptors responsible for adhesion and migrationand the ligands they bind to specifically bone ECM proteins[52 53] Our previous research revealed the proliferation andactivation functions of ADM The ADM encapsulated in thescaffoldmicrosphere construct can stimulate the prolifera-tion ofMG63 cells for 5 d byMTTassay andfluorescent imageobservation [24] Opn as a mineral-binding protein foundin bone ECM is implicated as an important factor in boneremodeling and crystal growth regulation It is associatedwith cell adhesion proliferation and biomineralization ofECM into bone and its high expression demonstrates theproliferation and activation of MG63 cells Another ECMprotein that is related to further differentiation of osteoblastsis col1 which accounts for 90 of the bone matrix proteins[54] Frick et al [55] reported that the mRNA expressionof col1 is stimulated by alkalosis and inhibited by acidosisThe small pH variation induced by the change in calciumconcentration has a significant effect on col1 expression Theincrease in col1 mRNA of the PLGAnHACMsADM groupon day 3 can be due to the increase in pH induced by chitosandegradation or the effect of ADM Transcription factorsrunx2 and sp7 were essential for osteoblastic differentiationand act as regulatory factors involved in osteogenic-relatedgene expression Early studies reported that runx2 bindsthe osteocalcin promoter and is expressed in osteochondralprogenitors as well as in early stages of osteoblastic differen-tiation [56] In humans runx2 haploinsufficiency results incleidocranial dysplasia a skeletal disorder characterized bybone and dental abnormalities [57] Sp7-deficient mice lackbone formation with a phenotype similar to that of runx2-deficientmiceMeanwhile sp7 is either acting downstreamofrunx2 or expressed later in the osteoblast differentiation path-way [58] Based on the aforementioned result the increasein mRNA expression at an early stage suggested that thedifferentiation and activation of MG63 cells on the surfaceof polymer scaffold were probably due to the sustained ADMrelease PLGAnHACMsADMcan induce differentiation inMG63 cells at the early stage of bone formation
The success of tissue-engineering scaffold is highlydependent on whether the materials can promote rapidand stable neovascularization (new blood vessel formation)within the scaffold typically prior to complete materialdegradation [4] Endothelial cells are currently regarded asthe most interesting target for therapies aimed at enhanc-ing or inhibiting angiogenesis [59] VEGF is an importantregulator of endothelial cell proliferation migration anddifferentiation As one of the ADM receptors ramp2 isessential for angiogenesis and vascular integrity Figures7(e) and 7(f) illustrate the vegf and ramp2 expressionlevels of HUVEC in three groups The vegf expression ofthe PLGAnHACMsADM group completely showed equalexpression pattern to the other two groups at each predeter-mined time However on days 3 and 5 the vegf expressionof the PLGAnHACMsADM and PLGAnHA groups wasslightly higher compared with that of the control groupThe ramp2mRNA expression of the PLGAnHACMsADMgroup steadily increased from day 3 and then slightly down-regulated on day 5The values of the PLGAnHACMsADMgroup on days 3 and 5 were significantly higher than those ofthe control group The expression level of ramp2 was rapidlyupregulated fromday 3 in the PLGAnHACMsADMgroupwhich might be induced by sustained-released ADM How-ever vegf expression was not upregulated The results of thepresent study are generally consistent with those of previousstudies [60] That is the activity of ADM was triggered bythe binding of ADM to its ADM receptor In addition theeffect of ADM on HUVEC was no longer detectable afterthe expression of ramp2 in the cells was almost suppressedby gene silencing The results demonstrated that the releasedADM from the scaffold may be involved in vascularizationfrom the gene level at the early stage
35 Western Blot Analysis To obtain further insights COL-LAGEN 1 RUNX2 protein of MG63 cells and VEGF proteinof HUVEC cells from the cell-scaffold complex were selectedfor detection by western blot analysis after cell culture for 13 and 5 d The results are shown in Figure 8 Significantlyhigher levels of COLLAGEN expression were observed inthe PLGAnHACMsADM and PLGAnHA groups thanin the control group on days 1 and 5 Moreover signifi-cantly higher expression levels of runx2 were observed ateach predetermined time on the PLGAnHACMsADMscaffold which was consistent with real-time PCR analysisFor VEGF which was associated with the proliferation anddifferentiation of endothelial cells the expression level ofthe PLGAnHACMsADM group was significantly higherthan that of the control group on days 1 and 5 Meanwhileon day 1 the PLGAnHA group showed significantly higherVEGF expression than the control group suggesting thatthe structure and property of the PLGAnHA scaffold weresuitable for the migration and proliferation of HUVEC at theinitial stage
Western blot analysis showed higher expression ofCOLLAGEN 1 RUNX2 and VEGF on the PLGAnHACMsADM scaffold during culture time This finding sug-gested that composite scaffold loading with ADM promoted
BioMed Research International 9
4
3
2
1
01 3 5
Culture time (days)
lowast
lowast
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of o
pn
(a)
4
3
2
1
01 3 5
lowastlowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of c
olla
gen
1
Culture time (days)
(b)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
unx2 4
3
2
1
01 3 5
lowastlowast
Culture time (days)
(c)
4
3
2
1
01 3 5
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of s
p7
Culture time (days)
(d)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of v
egf
4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5Culture time (days)
(e)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
amp2 4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5
lowastlowast
Culture time (days)
(f)
Figure 7 RelativemRNA expression level ofMG63 cells (opn (a) collagen 1 (b) runx2 (c) and sp7 (d)) andHUVEC cells (vegf (e) and ramp2(f)) of the PLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times lowast119875 lt 005indicates statistically significant difference compared with the control group (119899 = 6)
10 BioMed Research International
Collagen 1
RUNX2
120573-actin
1 53A B C A B C A B C
120KD
57KD
42KD
MG63
ABC
ControlPLGAnHACMsADMPLGAnHACMs
120573-actin
1 53
A B C A B C A B C
42KD
45KDVEGF
HUVEC
ABC
ControlPLGAnHACMsADMPLGAnHACMs
Relat
ive p
rote
in ex
pres
sion
of co
llage
n 1
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowast lowast
lowast lowastlowast
Relat
ive p
rote
in ex
pres
sion
of R
UN
X2
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
Relat
ive p
rote
in ex
pres
sion
of V
EGF
40
60
80
100
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
(a) (b) (c)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
Figure 8 Western blot assessment of collagen 1 runx2 protein of MG63 cells and VEGF protein of HUVEC cells in thePLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times (a b and c) The bandswere quantitated by densitometry and data are expressed as the ratio of aim protein to 120573-actin lowast119875 lt 005 indicates statistically significantdifference compared with the control group (119899 = 3)
the differentiation and activation of MG63 cells by upreg-ulation of the expression of specific osteogenic proteinsHigher expression levels of COLLAGEN 1 and VEGF werealso observed on the PLGAnHA scaffold ADM has a keyfunction during the development of the vascular systemas demonstrated by Shindo et al [61] Specific conditionssuch as hypoxia are reported to be associated with increasedVEGF expression [62] The ADM-induced upregulation ofVEGF at the protein level agrees well with most reports[63 64] but is not consistent with others [60 65] Thevegf gene was not significantly upregulated The inconsistentlevels between vegf gene and protein were possibly causedby the enhancement of translational efficiency and proteinconstancy
4 Conclusions
As a growth factor ADM was first introduced for tissue-engineering materials The combination of emulsion-ioniccross-linking and TIPS was proven to be suitable for scaf-foldmicrosphere construct developing loading with ADMThe microspheres showed a rough surface morphology andwere well distributed either in the presence or absence of
ADMThe scaffolds showed relatively uniformpore sizeswithinterconnected pores The addition of CMs into the scaffoldsimproved the mechanical properties of the scaffolds withoutremarkably changing their high porosity Moreover in vitrodegradation studies revealed that CM incorporation canaccelerate WL rate increase WA and reduce PLGA acidityin hydrolysisThe expression patterns of opn col1 runx2 andsp7 indicated that inductive osteoblast-like cell differentiationin contact with PLGAnHACMsADM scaffolds appearedearly in bone formation Western blot analysis demonstratedthat the PLGAnHACMsADM scaffold had high levels ofCOLLAGEN 1 and RUNX2 expression In addition RT-PCRanalysis showed that the PLGAnHACMsADM scaffoldhad a high level of ramp2 on days 3 and 5 The expres-sion of vegf was unaffected regardless of ADM loadingMeanwhile VEGF protein levels were relatively high for thePLGAnHACMsADM scaffold at the early osteoblast stageas determined by western blot analysis These findings sug-gest that microspherescaffold composite was more effectivein loading peptides and proteins which can improve theosteogenic and angiogenic differentiation of osteoblasts andvascular endothelial cells on the porous scaffolds Thereforesustained-release ADM from microsphere-scaffold system
BioMed Research International 11
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
two on day 1 It rapidly increased on day 3 and thenmaintained on day 5 which was significantly higher than thatof the control group No significant difference was observedbetween the PLGAnHA group and the control group ateach time point As shown in Figure 7(c) no significantdifference in runx2 expression appeared on day 1 Howeveron day 3 the expression of the PLGAnHACMsADMgroupwas 21 times higher than that of the control group It thenslightly decreased on day 5 which were both significantlyhigher than the control group Nevertheless as shown inFigure 7(d) the sp7 mRNA level was upregulated in thePLGAnHACMsADM group with culture time The valueswere 27 and 47 times significantly higher than those of thecontrol group on days 3 and 5 respectively suggesting theupregulation of osteoblastic activity On day 5 the sp7mRNAexpression in the PLGAnHA scaffold was 17 times higherthan that in the control groupThis result proved the bioactivepotential of the interconnected microstructure of polymerscaffolds
Cell-material interactions can be evaluated by detectingthe cellular receptors responsible for adhesion and migrationand the ligands they bind to specifically bone ECM proteins[52 53] Our previous research revealed the proliferation andactivation functions of ADM The ADM encapsulated in thescaffoldmicrosphere construct can stimulate the prolifera-tion ofMG63 cells for 5 d byMTTassay andfluorescent imageobservation [24] Opn as a mineral-binding protein foundin bone ECM is implicated as an important factor in boneremodeling and crystal growth regulation It is associatedwith cell adhesion proliferation and biomineralization ofECM into bone and its high expression demonstrates theproliferation and activation of MG63 cells Another ECMprotein that is related to further differentiation of osteoblastsis col1 which accounts for 90 of the bone matrix proteins[54] Frick et al [55] reported that the mRNA expressionof col1 is stimulated by alkalosis and inhibited by acidosisThe small pH variation induced by the change in calciumconcentration has a significant effect on col1 expression Theincrease in col1 mRNA of the PLGAnHACMsADM groupon day 3 can be due to the increase in pH induced by chitosandegradation or the effect of ADM Transcription factorsrunx2 and sp7 were essential for osteoblastic differentiationand act as regulatory factors involved in osteogenic-relatedgene expression Early studies reported that runx2 bindsthe osteocalcin promoter and is expressed in osteochondralprogenitors as well as in early stages of osteoblastic differen-tiation [56] In humans runx2 haploinsufficiency results incleidocranial dysplasia a skeletal disorder characterized bybone and dental abnormalities [57] Sp7-deficient mice lackbone formation with a phenotype similar to that of runx2-deficientmiceMeanwhile sp7 is either acting downstreamofrunx2 or expressed later in the osteoblast differentiation path-way [58] Based on the aforementioned result the increasein mRNA expression at an early stage suggested that thedifferentiation and activation of MG63 cells on the surfaceof polymer scaffold were probably due to the sustained ADMrelease PLGAnHACMsADMcan induce differentiation inMG63 cells at the early stage of bone formation
The success of tissue-engineering scaffold is highlydependent on whether the materials can promote rapidand stable neovascularization (new blood vessel formation)within the scaffold typically prior to complete materialdegradation [4] Endothelial cells are currently regarded asthe most interesting target for therapies aimed at enhanc-ing or inhibiting angiogenesis [59] VEGF is an importantregulator of endothelial cell proliferation migration anddifferentiation As one of the ADM receptors ramp2 isessential for angiogenesis and vascular integrity Figures7(e) and 7(f) illustrate the vegf and ramp2 expressionlevels of HUVEC in three groups The vegf expression ofthe PLGAnHACMsADM group completely showed equalexpression pattern to the other two groups at each predeter-mined time However on days 3 and 5 the vegf expressionof the PLGAnHACMsADM and PLGAnHA groups wasslightly higher compared with that of the control groupThe ramp2mRNA expression of the PLGAnHACMsADMgroup steadily increased from day 3 and then slightly down-regulated on day 5The values of the PLGAnHACMsADMgroup on days 3 and 5 were significantly higher than those ofthe control group The expression level of ramp2 was rapidlyupregulated fromday 3 in the PLGAnHACMsADMgroupwhich might be induced by sustained-released ADM How-ever vegf expression was not upregulated The results of thepresent study are generally consistent with those of previousstudies [60] That is the activity of ADM was triggered bythe binding of ADM to its ADM receptor In addition theeffect of ADM on HUVEC was no longer detectable afterthe expression of ramp2 in the cells was almost suppressedby gene silencing The results demonstrated that the releasedADM from the scaffold may be involved in vascularizationfrom the gene level at the early stage
35 Western Blot Analysis To obtain further insights COL-LAGEN 1 RUNX2 protein of MG63 cells and VEGF proteinof HUVEC cells from the cell-scaffold complex were selectedfor detection by western blot analysis after cell culture for 13 and 5 d The results are shown in Figure 8 Significantlyhigher levels of COLLAGEN expression were observed inthe PLGAnHACMsADM and PLGAnHA groups thanin the control group on days 1 and 5 Moreover signifi-cantly higher expression levels of runx2 were observed ateach predetermined time on the PLGAnHACMsADMscaffold which was consistent with real-time PCR analysisFor VEGF which was associated with the proliferation anddifferentiation of endothelial cells the expression level ofthe PLGAnHACMsADM group was significantly higherthan that of the control group on days 1 and 5 Meanwhileon day 1 the PLGAnHA group showed significantly higherVEGF expression than the control group suggesting thatthe structure and property of the PLGAnHA scaffold weresuitable for the migration and proliferation of HUVEC at theinitial stage
Western blot analysis showed higher expression ofCOLLAGEN 1 RUNX2 and VEGF on the PLGAnHACMsADM scaffold during culture time This finding sug-gested that composite scaffold loading with ADM promoted
BioMed Research International 9
4
3
2
1
01 3 5
Culture time (days)
lowast
lowast
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of o
pn
(a)
4
3
2
1
01 3 5
lowastlowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of c
olla
gen
1
Culture time (days)
(b)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
unx2 4
3
2
1
01 3 5
lowastlowast
Culture time (days)
(c)
4
3
2
1
01 3 5
lowast
lowast
lowast
n-fo
ld m
RNA
incr
ease
to co
ntro
l of s
p7
Culture time (days)
(d)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of v
egf
4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5Culture time (days)
(e)
n-fo
ld m
RNA
incr
ease
to co
ntro
l of r
amp2 4
3
2
1
0
PLGAnHACMsADMPLGAnHACMsControl
1 3 5
lowastlowast
Culture time (days)
(f)
Figure 7 RelativemRNA expression level ofMG63 cells (opn (a) collagen 1 (b) runx2 (c) and sp7 (d)) andHUVEC cells (vegf (e) and ramp2(f)) of the PLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times lowast119875 lt 005indicates statistically significant difference compared with the control group (119899 = 6)
10 BioMed Research International
Collagen 1
RUNX2
120573-actin
1 53A B C A B C A B C
120KD
57KD
42KD
MG63
ABC
ControlPLGAnHACMsADMPLGAnHACMs
120573-actin
1 53
A B C A B C A B C
42KD
45KDVEGF
HUVEC
ABC
ControlPLGAnHACMsADMPLGAnHACMs
Relat
ive p
rote
in ex
pres
sion
of co
llage
n 1
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowast lowast
lowast lowastlowast
Relat
ive p
rote
in ex
pres
sion
of R
UN
X2
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
Relat
ive p
rote
in ex
pres
sion
of V
EGF
40
60
80
100
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
(a) (b) (c)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
Figure 8 Western blot assessment of collagen 1 runx2 protein of MG63 cells and VEGF protein of HUVEC cells in thePLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times (a b and c) The bandswere quantitated by densitometry and data are expressed as the ratio of aim protein to 120573-actin lowast119875 lt 005 indicates statistically significantdifference compared with the control group (119899 = 3)
the differentiation and activation of MG63 cells by upreg-ulation of the expression of specific osteogenic proteinsHigher expression levels of COLLAGEN 1 and VEGF werealso observed on the PLGAnHA scaffold ADM has a keyfunction during the development of the vascular systemas demonstrated by Shindo et al [61] Specific conditionssuch as hypoxia are reported to be associated with increasedVEGF expression [62] The ADM-induced upregulation ofVEGF at the protein level agrees well with most reports[63 64] but is not consistent with others [60 65] Thevegf gene was not significantly upregulated The inconsistentlevels between vegf gene and protein were possibly causedby the enhancement of translational efficiency and proteinconstancy
4 Conclusions
As a growth factor ADM was first introduced for tissue-engineering materials The combination of emulsion-ioniccross-linking and TIPS was proven to be suitable for scaf-foldmicrosphere construct developing loading with ADMThe microspheres showed a rough surface morphology andwere well distributed either in the presence or absence of
ADMThe scaffolds showed relatively uniformpore sizeswithinterconnected pores The addition of CMs into the scaffoldsimproved the mechanical properties of the scaffolds withoutremarkably changing their high porosity Moreover in vitrodegradation studies revealed that CM incorporation canaccelerate WL rate increase WA and reduce PLGA acidityin hydrolysisThe expression patterns of opn col1 runx2 andsp7 indicated that inductive osteoblast-like cell differentiationin contact with PLGAnHACMsADM scaffolds appearedearly in bone formation Western blot analysis demonstratedthat the PLGAnHACMsADM scaffold had high levels ofCOLLAGEN 1 and RUNX2 expression In addition RT-PCRanalysis showed that the PLGAnHACMsADM scaffoldhad a high level of ramp2 on days 3 and 5 The expres-sion of vegf was unaffected regardless of ADM loadingMeanwhile VEGF protein levels were relatively high for thePLGAnHACMsADM scaffold at the early osteoblast stageas determined by western blot analysis These findings sug-gest that microspherescaffold composite was more effectivein loading peptides and proteins which can improve theosteogenic and angiogenic differentiation of osteoblasts andvascular endothelial cells on the porous scaffolds Thereforesustained-release ADM from microsphere-scaffold system
BioMed Research International 11
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
Figure 7 RelativemRNA expression level ofMG63 cells (opn (a) collagen 1 (b) runx2 (c) and sp7 (d)) andHUVEC cells (vegf (e) and ramp2(f)) of the PLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times lowast119875 lt 005indicates statistically significant difference compared with the control group (119899 = 6)
10 BioMed Research International
Collagen 1
RUNX2
120573-actin
1 53A B C A B C A B C
120KD
57KD
42KD
MG63
ABC
ControlPLGAnHACMsADMPLGAnHACMs
120573-actin
1 53
A B C A B C A B C
42KD
45KDVEGF
HUVEC
ABC
ControlPLGAnHACMsADMPLGAnHACMs
Relat
ive p
rote
in ex
pres
sion
of co
llage
n 1
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowast lowast
lowast lowastlowast
Relat
ive p
rote
in ex
pres
sion
of R
UN
X2
40
60
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
Relat
ive p
rote
in ex
pres
sion
of V
EGF
40
60
80
100
20
0
Culture time (days)
PLGAnHACMsADMPLGAnHACMs
Control
1 3 5
lowastlowast
lowast
(a) (b) (c)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
( o
f120573-A
ctin
)
Figure 8 Western blot assessment of collagen 1 runx2 protein of MG63 cells and VEGF protein of HUVEC cells in thePLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times (a b and c) The bandswere quantitated by densitometry and data are expressed as the ratio of aim protein to 120573-actin lowast119875 lt 005 indicates statistically significantdifference compared with the control group (119899 = 3)
the differentiation and activation of MG63 cells by upreg-ulation of the expression of specific osteogenic proteinsHigher expression levels of COLLAGEN 1 and VEGF werealso observed on the PLGAnHA scaffold ADM has a keyfunction during the development of the vascular systemas demonstrated by Shindo et al [61] Specific conditionssuch as hypoxia are reported to be associated with increasedVEGF expression [62] The ADM-induced upregulation ofVEGF at the protein level agrees well with most reports[63 64] but is not consistent with others [60 65] Thevegf gene was not significantly upregulated The inconsistentlevels between vegf gene and protein were possibly causedby the enhancement of translational efficiency and proteinconstancy
4 Conclusions
As a growth factor ADM was first introduced for tissue-engineering materials The combination of emulsion-ioniccross-linking and TIPS was proven to be suitable for scaf-foldmicrosphere construct developing loading with ADMThe microspheres showed a rough surface morphology andwere well distributed either in the presence or absence of
ADMThe scaffolds showed relatively uniformpore sizeswithinterconnected pores The addition of CMs into the scaffoldsimproved the mechanical properties of the scaffolds withoutremarkably changing their high porosity Moreover in vitrodegradation studies revealed that CM incorporation canaccelerate WL rate increase WA and reduce PLGA acidityin hydrolysisThe expression patterns of opn col1 runx2 andsp7 indicated that inductive osteoblast-like cell differentiationin contact with PLGAnHACMsADM scaffolds appearedearly in bone formation Western blot analysis demonstratedthat the PLGAnHACMsADM scaffold had high levels ofCOLLAGEN 1 and RUNX2 expression In addition RT-PCRanalysis showed that the PLGAnHACMsADM scaffoldhad a high level of ramp2 on days 3 and 5 The expres-sion of vegf was unaffected regardless of ADM loadingMeanwhile VEGF protein levels were relatively high for thePLGAnHACMsADM scaffold at the early osteoblast stageas determined by western blot analysis These findings sug-gest that microspherescaffold composite was more effectivein loading peptides and proteins which can improve theosteogenic and angiogenic differentiation of osteoblasts andvascular endothelial cells on the porous scaffolds Thereforesustained-release ADM from microsphere-scaffold system
BioMed Research International 11
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
Figure 8 Western blot assessment of collagen 1 runx2 protein of MG63 cells and VEGF protein of HUVEC cells in thePLGAnHACMsADM PLGAnHA and control groups (normal cells without treatment) at predetermined times (a b and c) The bandswere quantitated by densitometry and data are expressed as the ratio of aim protein to 120573-actin lowast119875 lt 005 indicates statistically significantdifference compared with the control group (119899 = 3)
the differentiation and activation of MG63 cells by upreg-ulation of the expression of specific osteogenic proteinsHigher expression levels of COLLAGEN 1 and VEGF werealso observed on the PLGAnHA scaffold ADM has a keyfunction during the development of the vascular systemas demonstrated by Shindo et al [61] Specific conditionssuch as hypoxia are reported to be associated with increasedVEGF expression [62] The ADM-induced upregulation ofVEGF at the protein level agrees well with most reports[63 64] but is not consistent with others [60 65] Thevegf gene was not significantly upregulated The inconsistentlevels between vegf gene and protein were possibly causedby the enhancement of translational efficiency and proteinconstancy
4 Conclusions
As a growth factor ADM was first introduced for tissue-engineering materials The combination of emulsion-ioniccross-linking and TIPS was proven to be suitable for scaf-foldmicrosphere construct developing loading with ADMThe microspheres showed a rough surface morphology andwere well distributed either in the presence or absence of
ADMThe scaffolds showed relatively uniformpore sizeswithinterconnected pores The addition of CMs into the scaffoldsimproved the mechanical properties of the scaffolds withoutremarkably changing their high porosity Moreover in vitrodegradation studies revealed that CM incorporation canaccelerate WL rate increase WA and reduce PLGA acidityin hydrolysisThe expression patterns of opn col1 runx2 andsp7 indicated that inductive osteoblast-like cell differentiationin contact with PLGAnHACMsADM scaffolds appearedearly in bone formation Western blot analysis demonstratedthat the PLGAnHACMsADM scaffold had high levels ofCOLLAGEN 1 and RUNX2 expression In addition RT-PCRanalysis showed that the PLGAnHACMsADM scaffoldhad a high level of ramp2 on days 3 and 5 The expres-sion of vegf was unaffected regardless of ADM loadingMeanwhile VEGF protein levels were relatively high for thePLGAnHACMsADM scaffold at the early osteoblast stageas determined by western blot analysis These findings sug-gest that microspherescaffold composite was more effectivein loading peptides and proteins which can improve theosteogenic and angiogenic differentiation of osteoblasts andvascular endothelial cells on the porous scaffolds Thereforesustained-release ADM from microsphere-scaffold system
BioMed Research International 11
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
may be a promising therapeutic agent for local application inbone tissue engineering
Conflict of Interests
No conflict of interests is present The authors have nofinancial involvement or interest with any organization orcompany on subjects or materials discussed in the paper
Acknowledgments
The authors sincerely appreciate the financial support fromthe State Key Laboratory of Oral Diseases of China (Grant nod2009001) and the Development and ReformCommission ofJilin Province (Grant no 20101935)They also thank P He andX M Li for technical assistance and paper revising
References
[1] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001
[2] R A Jain ldquoThemanufacturing techniques of various drug load-ed biodegradable poly(lactide-co-glycolide) (PLGA) devicesrdquoBiomaterials vol 21 no 23 pp 2475ndash2490 2000
[3] RDorati C Colonna I Genta TModena andBConti ldquoEffectof porogen on the physico-chemical properties and degradationperformance of PLGA scaffoldsrdquo Polymer Degradation andStability vol 95 no 4 pp 694ndash701 2010
[4] G Papavasiliou C Ming-Huei and E M Brey ldquoStrategies forvascularization of polymer scaffoldsrdquo Journal of InvestigativeMedicine vol 58 no 7 pp 838ndash844 2010
[5] J G Nemeno-Guanzon S Lee and J R Berg ldquoTrends in tissueengineering for blood vesselsrdquo Journal of Biomedcine and Bio-technology vol 2012 Article ID 956345 14 pages 2012
[6] K Kitamura K Kangawa M Kawamoto et al ldquoAdrenomedull-in a novel hypotensive peptide isolated from human pheochro-mocytomardquo Biochemical and Biophysical Research Communica-tions vol 192 no 2 pp 553ndash560 1993
[7] J P Hinson S Kapas and D M Smith ldquoAdrenomedullin amultifunctional regulatory peptiderdquo Endocrine Reviews vol 21no 2 pp 138ndash167 2000
[8] Y Ichiki ldquoDistribution and characterization of immunoreactiveadrenomedullin in human tissue and plasmardquo FEBS Letters vol338 no 1 pp 6ndash10 1994
[9] J G Lainchbury G J S Cooper D H Coy et al ldquoAdrenomed-ullin a hypotensive hormone in manrdquo Clinical Science vol 92no 5 pp 467ndash472 1997
[10] W K Samson T Murphy and D A Schell ldquoA novel vasoac-tive peptide adrenomedullin inhibits pituitary adrenocorticot-ropin releaserdquo Endocrinology vol 136 no 5 pp 2349ndash23521995
[11] F Yoshihara S-I Suga N Yasui et al ldquoChronic administrationof adrenomedullin attenuates the hypertension and increasesrenal nitric oxide synthase in Dahl salt-sensitive ratsrdquo Regula-tory Peptides vol 128 no 1 pp 7ndash13 2005
[12] J Cornish D Naot and I R Reid ldquoAdrenomedullinmdasha regula-tor of bone formationrdquo Regulatory Peptides vol 112 no 1ndash3 pp79ndash86 2003
[13] D Ribatti B Nico R Spinazzi A Vacca and G G NussdorferldquoThe role of adrenomedullin in angiogenesisrdquo Peptides vol 26no 9 pp 1670ndash1675 2005
[14] R P Allaker and S Kapas ldquoAdrenomedullin and mucosaldefence interaction between host and microorganismrdquo Regu-latory Peptides vol 112 no 1ndash3 pp 147ndash152 2003
[15] M Groschl O Wendler H-G Topf J Bohlender and HKohler ldquoSignificance of salivary adrenomedullin in the mainte-nance of oral health stimulation of oral cell proliferation andantibacterial propertiesrdquo Regulatory Peptides vol 154 no 1ndash3pp 16ndash22 2009
[16] J Cornish K E Callon D H Coy et al ldquoAdrenomedullin isa potent stimulator of osteoblastic activity in vitro and in vivordquoAmerican Journal of Physiology vol 273 no 6 pp E1113ndashE11201997
[17] J Cornish K E Callon U Bava et al ldquoSystemic administrationof adrenomedullin(27ndash52) increases bone volume and strengthin male micerdquo Journal of Endocrinology vol 170 no 1 pp 251ndash257 2001
[18] V P Michelangeli A E Fletcher E H Allen G C Nicholsonand T J Martin ldquoEffects of calcitonin gene-related peptide oncyclic AMP formation in chicken rat and mouse bone cellsrdquoJournal of Bone andMineral Research vol 4 no 2 pp 269ndash2721989
[19] H Hamada K Kitamura E Chosa T Eto and N Tajima ldquoAd-renomedullin stimulates the growth of cultured normal humanosteoblasts as an autocrineparacine regulatorrdquo Peptides vol 23no 12 pp 2163ndash2168 2002
[20] H-Q Mao K Roy V L Troung-Le et al ldquoChitosan-DNAnanoparticles as gene carriers synthesis characterization andtransfection efficiencyrdquo Journal of Controlled Release vol 70 no3 pp 399ndash421 2001
[21] J Varshosaz ldquoThe promise of chitosanmicrospheres in drug de-livery systemsrdquo Expert Opinion on Drug Delivery vol 4 no 3pp 263ndash273 2007
[22] K G H Desai and H J Park ldquoEncapsulation of vitamin C intripolyphosphate cross-linked chitosan microspheres by spraydryingrdquo Journal of Microencapsulation vol 22 no 2 pp 179ndash192 2005
[23] S TamuraH Kataoka YMatsui et al ldquoThe effects of transplan-tation of osteoblastic cells with bone morphogenetic protein(BMP)carrier complex on bone repairrdquo Bone vol 29 no 2 pp169ndash175 2001
[24] L Wang C-Y Li P He L Fu Y-M Zhou and X-S ChenldquoPreparation and bioactivities of plganano-hydroxyapatitescaffold containing chitosan microspheres for controlled deliv-ery of mutifuncational peptide-adrenomedullinrdquo ChemicalJournal of Chinese Universities vol 32 no 7 pp 1622ndash1628 2011
[25] Y XWan X Cao QWu S Zhang andW Sheng ldquoPreparationand mechanical properties of poly(chitosan-g-DL-lactic acid)fibrousmesh scaffoldsrdquoPolymers for Advanced Technologies vol19 no 2 pp 114ndash123 2008
[26] K J Livak and T D Schmittgen ldquoAnalysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[27] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001
[28] J A Ko H J Park S J Hwang J B Park and J S LeeldquoPreparation and characterization of chitosan microparticlesintended for controlled drug deliveryrdquo International Journal ofPharmaceutics vol 249 no 1-2 pp 165ndash174 2002
12 BioMed Research International
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
[29] S Mobini J JavadpourM Hosseinalipour M Ghazi-KhansariA Khavandi and H R Rezaie ldquoSynthesis and characterisationof gelatin-nano hydroxyapatite composite scaffolds for bonetissue engineeringrdquo Advances in Applied Ceramics vol 107 no1 pp 4ndash8 2008
[30] A H Touny C Laurencin L Nair H Allcock and PW BrownldquoFormation of composites comprised of calcium deficient HApand cross-linked gelatinrdquo Journal of Materials Science vol 19no 10 pp 3193ndash3201 2008
[31] W Zeng J Huang X Hu et al ldquoIonically cross-linked chitosanmicrospheres for controlled release of bioactive nerve growthfactorrdquo International Journal of Pharmaceutics vol 421 no 2pp 283ndash290 2011
[32] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002
[33] K G H Desai and H J Park ldquoPreparation of cross-linkedchitosan microspheres by spray drying effect of cross-linkingagent on the properties of spray dried microspheresrdquo Journal ofMicroencapsulation vol 22 no 4 pp 377ndash395 2005
[34] C Mandoli B Mecheri G Forte et al ldquoThick soft tissuereconstruction on highly perfusive biodegradable scaffoldsrdquoMacromolecular Bioscience vol 10 no 2 pp 127ndash138 2010
[35] F J OrsquoBrien B A Harley I V Yannas and L J Gibson ldquoTheeffect of pore size on cell adhesion in collagen-GAG scaffoldsrdquoBiomaterials vol 26 no 4 pp 433ndash441 2005
[36] J A Jansen J W M Vehof P Q Ruhe et al ldquoGrowth factor-loaded scaffolds for bone engineeringrdquo Journal of ControlledRelease vol 101 no 1ndash3 pp 127ndash136 2005
[37] M J Dalby S Childs M O Riehle H J H Johnstone SAffrossman and A S G Curtis ldquoFibroblast reaction to islandtopography changes in cytoskeleton and morphology withtimerdquo Biomaterials vol 24 no 6 pp 927ndash935 2003
[38] Y Wan Y Wang Z Liu et al ldquoAdhesion and proliferation ofOCT-1 osteoblast-like cells on micro- and nano-scale topogra-phy structured poly(L-lactide)rdquo Biomaterials vol 26 no 21 pp4453ndash4459 2005
[39] XNiuQ FengMWang XGuo andQ Zheng ldquoPorous nano-HAcollagenPLLA scaffold containing chitosan microspheresfor controlled delivery of synthetic peptide derived from BMP-2rdquo Journal of Controlled Release vol 134 no 2 pp 111ndash117 2009
[40] W Huang X Shi L Ren C Du and Y Wang ldquoPHBVmicrospheresmdashPLGAmatrix composite scaffold for bone tissueengineeringrdquo Biomaterials vol 31 no 15 pp 4278ndash4285 2010
[41] K M Kulig and J P Vacanti ldquoHepatic tissue engineeringrdquoTransplant Immunology vol 12 no 3-4 pp 303ndash310 2004
[42] D W Hutmacher ldquoScaffolds in tissue engineering bone andcartilagerdquo Biomaterials vol 21 no 24 pp 2529ndash2543 2000
[43] Y X Huang J Ren C Chen T B Ren and X Y Zhou ldquoPrepa-ration and properties of poly(lactide-co-glycolide) (PLGA)Nano-Hydroxyapatite (NHA) scaffolds by thermally inducedphase separation and rabbit MSCs culture on scaffoldsrdquo Journalof Biomaterials Applications vol 22 no 5 pp 409ndash432 2008
[44] Y Gong Q Zhou C Gao and J Shen ldquoin vitro and invivo degradability and cytocompatibility of poly(l-lactic acid)scaffold fabricated by a gelatin particle leaching methodrdquo ActaBiomaterialia vol 3 no 4 pp 531ndash540 2007
[45] L Wu and J Ding ldquoin vitro degradation of three-dimensionalporous poly(DL-lactide-co- glycolide) scaffolds for tissue engi-neeringrdquo Biomaterials vol 25 no 27 pp 5821ndash5830 2004
[46] L Lu S J Peter M D Lyman et al ldquoin vitro and in vivodegradation of porous poly(DL-lactic-co-glycolic acid) foamsrdquoBiomaterials vol 21 no 18 pp 1837ndash1845 2000
[47] J M Oliveira M T Rodrigues S S Silva et al ldquoNovelhydroxyapatitechitosan bilayered scaffold for osteochondraltissue-engineering applications scaffold design and its perfor-mance when seeded with goat bone marrow stromal cellsrdquoBiomaterials vol 27 no 36 pp 6123ndash6137 2006
[48] M J Kim J-H Kim G Yi S-H Lim Y S Hong and D JChung ldquoin vitro and in vivo application of PLGA nanofiber forartificial blood vesselrdquo Macromolecular Research vol 16 no 4pp 345ndash352 2008
[49] T R Arnett ldquoExtracellular pH regulates bone cell functionrdquoJournal of Nutrition vol 128 no 2 pp S415ndashS418 2008
[50] Z S Al-Aql A S Alagl D T Graves L C Gerstenfeld andT AEinhorn ldquoMolecular mechanisms controlling bone formationduring fracture healing and distraction osteogenesisrdquo Journal ofDental Research vol 87 no 2 pp 107ndash118 2008
[51] H Bahar D Benayahu A Yaffe and I Binderman ldquoMolecularsignaling in bone regenerationrdquo Critical Reviews in EukaryoticGene Expression vol 17 no 2 pp 87ndash101 2007
[52] C H Damsky ldquoExtracellular matrix-integrin interactions inosteoblast function and tissue remodelingrdquo Bone vol 25 no1 pp 95ndash96 1999
[53] S F El-Amin H H Lu Y Khan et al ldquoExtracellular matrixproduction by human osteoblasts cultured on biodegradablepolymers applicable for tissue engineeringrdquo Biomaterials vol24 no 7 pp 1213ndash1221 2003
[54] M Sila-Asna A Bunyaratvej S Maeda H Kitaguchi and NBunyaratavej ldquoOsteoblast differentiation and bone formationgene expression in strontium-inducing bone marrow mes-enchymal stem cellrdquo Kobe Journal of Medical Sciences vol 53no 1 pp 25ndash35 2007
[55] K K Frick J Li and D A Bushinsky ldquoAcutemetabolic acidosisinhibits the induction of osteoblastic egr-1 and type 1 collagenrdquoAmerican Journal of Physiology vol 272 no 5 pp C1450ndashC1456 1997
[56] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997
[57] J H Jonason G Xiao M Zhang L Xing and D Chen ldquoPost-translational regulation of Runx2 in bone and cartilagerdquo Journalof Dental Research vol 88 no 8 pp 693ndash703 2009
[58] S H H Hong X Lu M S Nanes and J Mitchell ldquoRegulationof osterix (Osx Sp7) and the Osx promoter by parathyroidhormone in osteoblastsrdquo Journal of Molecular Endocrinologyvol 43 no 5 pp 197ndash207 2009
[59] R Binetruy-Tournaire CDemangel BMalavaud et al ldquoIdenti-fication of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesisrdquo EMBO Journal vol 19 no 7pp 1525ndash1533 2000
[60] D Guidolin G Albertin R Spinazzi et al ldquoAdrenomedullinstimulates angiogenic response in cultured human vascu-lar endothelial cells involvement of the vascular endothelialgrowth factor receptor 2rdquo Peptides vol 29 no 11 pp 2013ndash20232008
[61] T Shindo Y Kurihara H Nishimatsu et al ldquoVascular ab-normalities and elevated blood pressure in mice lackingadrenomedullin generdquo Circulation vol 104 no 16 pp 1964ndash1971 2001
BioMed Research International 13
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
[62] M Garayoa A Martınez S Lee et al ldquoHypoxia-induciblefactor-1 (HIF-1) up-regulates adrenomedullin expression inhuman tumor cell lines during oxygen deprivation a pos-sible promotion mechanism of carcinogenesisrdquo MolecularEndocrinology vol 14 no 6 pp 848ndash862 2000
[63] N Schwarz D Renshaw S Kapas and J P Hinson ldquoAdren-omedullin increases the expression of calcitonin-like receptorand receptor activity modifying protein 2 mRNA in humanmicrovascular endothelial cellsrdquo Journal of Endocrinology vol190 no 2 pp 505ndash514 2006
[64] T Maki M Ihara Y Fujita et al ldquoAngiogenic roles ofadrenomedullin through vascular endothelial growth factorinductionrdquo NeuroReport vol 22 no 9 pp 442ndash447 2011
[65] S Fernandez-Sauze C Delfino K Mabrouk et al ldquoEffectsof adrenomedullin on endothelial cells in the multistepprocess of angiogenesis involvement of CRLRRAMP2 andCRLRRAMP3 receptorsrdquo International Journal of Cancer vol108 no 6 pp 797ndash804 2004
