Hindawi Publishing CorporationAdvances in Materials Science and EngineeringVolume 2013 Article ID 149261 8 pageshttpdxdoiorg1011552013149261
Research ArticlePhysicochemical and Microstructural Characterization ofInjectable Load-Bearing Calcium Phosphate Scaffold
Mazen Alshaaer12 Mohammed H Kailani13 Hanan Jafar14
Nidaa Ababneh1 and Abdalla Awidi14
1 Cell Therapy Center (CTC) The University of Jordan Amman 11942 Jordan2Department of Physics College of Science and Humanitarian Studies Salman Bin Abdul Aziz UniversityPO Box 83 Alkharj 11942 Saudi Arabia
3 Department of Chemistry The University of Jordan Amman 11942 Jordan4 Faculty of Medicine The University of Jordan Amman 11942 Jordan
Correspondence should be addressed to Mazen Alshaaer mazen72yahoocom
Received 20 July 2013 Revised 19 October 2013 Accepted 30 October 2013
Academic Editor Wei Wu
Copyright copy 2013 Mazen Alshaaer et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Injectable load-bearing calcium phosphate scaffolds are synthesized using rod-like mannitol grains as porogen These degradableinjectable strong porous scaffolds prepared by calcium phosphate cement could represent a valid solution to achieve adequateporosity requirements while providing adequate support in load-bearing applications The proposed process for preparing porousinjectable scaffolds is as quick and versatile as conventional technologies Using this method porous CDHA-based calciumphosphate scaffolds with macropores sizes ranging from 70 to 300120583m micropores ranging from 5 to 30 120583m and 30 openmacroporosity were prepared The setting time of the prepared scaffolds was 15 minutes Also their compressive strength and e-modulus 49MPa and 400MPa respectively were comparable with those of the cancellous bone Finally the bioactivity of thescaffolds was confirmed by cell growth with cytoplasmic extensions in the scaffolds in culture demonstrating that the scaffold hasa potential for MSC seeding and growth architectureThis combination of an interconnected macroporous structure with pore sizesuitable for the promotion of cell seeding and proliferation plus adequate mechanical features represents a porous scaffold whichis a promising candidate for bone tissue engineering
1 Introduction
Bone defects arise from skeletal diseases congenital mal-formations trauma and tumor resection [1 2] The needfor bone reconstruction is increasing as the population agesTissue engineering approaches are promising alternativesto autogenous bone grafts Studies have shown excitingresults on the use of scaffolds and stem cells for tissueregeneration [3ndash7] Human mesenchymal stem cells (MSCs)can differentiate into adipocytes osteoblasts chondrocytesneurons endothelial cells and so forth [8ndash12] Scaffoldscan serve as templates for cell attachment differentiationand vascularization in vivo and can then degrade and bereplaced by new bone Calcium phosphate (CaP) scaffoldsmimic the bone mineral and can bond to bone to form afunctional interface [13ndash16] Preformed CaP implants require
machining to fit into a bone cavity In contrast calciumphosphate cements can be injected or sculpted and set in situto form a scaffoldwith intimate adaptation to the neighboringbone [17ndash21] Due to its similarity to the mineral phase ofthe bone good biocompatibility excellent bioactivity self-setting characteristics low setting temperature adequatestiffness and easy shaping in complicated geometrics cal-cium phosphate cement (CPC) is regarded as a promisingmaterial for use in minimally invasive surgery to repair bonedefects [22ndash25] Because CPCs are osteotransductive afterimplantation in bone defects they are rapidly integrated intothe bone structure and transformed into new bone by thecellular activity of osteoclasts and osteoblasts in local boneremodeling [24] Amajor disadvantage of current orthopedicimplants is that they are hard requiring the surgeon to fitthe surgical site around the implant or to carve the graft
2 Advances in Materials Science and Engineering
to the desired shape This can lead to increased bone losstrauma to the surrounding tissue and prolonged surgicaltime [23] However CPCs typically set at low temperaturesfollowing the combination of a solid component containingone or several calcium orthophosphate salts and an aqueoussolution and form a solid calcium phosphate in situ Thismeans that CPCs can adapt immediately to each bone cavityand obtain goodosteointegration In addition injectableCPCformulations that are suitable for new minimally invasivesurgical techniques have been developed [26] In the lasttwo decades numerous biomaterials studies have been con-ducted investigating the development of CPCs Howeverthe physicochemical characterization and in vitro evaluationof the final properties of CPCs produced from calciumpyrophosphate powder have not been studied systematicallyIn this study the main reactive ingredient of CPC tetracal-cium phosphate (TTCP Ca
4(PO4)2O) was synthesized by
solid state reaction between calcium pyrophosphate (CaPyroCa2P2O7) and calcium carbonate (CaCO
3) The aim of this
work consists in designing an injectable and strong poroussystemusing biocompatiblematerials able to promote naturalbody healing which degrade after implantation and containbiologically active phases and able to stimulate the regener-ative tissue growth in order to mimic the morphological andmicrostructural properties of bone tissueWe investigated themicrostructural properties phase evaluation pore structuredensities compressive strength and stiffness of the producedscaffolds We also evaluated the bone formation propertiesand cell differentiation in vitro over a time scale of 21 days
2 Materials and Methods
21 Synthesis of TTCP TheTTCP powder used for this studywas fabricated from the reaction of dicalcium pyrophos-phate (Ca
2P2O7) (Merck Germany) and calcium carbonate
(CaCO3) (Merck Germany) with a weight ratio of 1 127The
powders were mixed uniformly in ethanol for 12 h Then themixed powder was dried and crushed using a mortar and apestle followed by calcination in a crucible at 1500∘C for 5 h inair and quenching in air at 25∘C Finally the calcined powder(TTCP phase) was ground into a fine powder
The chemical reaction for the TTCP powder was asfollows
Ca2P2O7+ 2CaCO
3
997888rarr Ca4(PO4)2O (TTCP) + 2CO
2
(1)
22 Fabrication of the Macroporous CPC Scaffold Water-soluble granular rod-like mannitol porogen was incorpo-rated into CPC to create macropores The TTCP powderwas mixed with granulated mannitol with size that variesfrom 70 120583m to 300 120583m (mannitolTTCP weight ratio is 05)This mixture was then mixed in diammonium hydrogenphosphate ((NH
4)2HPO4 333 wt) with hardening solu-
tionTTCP ratio of 034mLg After mixing the CPC for1min the cement paste was uniformly packed in a polymermold which has an opening of 10 times 10mm and 3mm indepth under a pressure of sim14MPa at 37∘C The hardened
CPC was then removed from the mold and immersed inHanksrsquo physiological solution at 37∘C for 2 days to dissolvethe mannitol
23 CPC Setting Time The CPC setting time was measuredusing a previously reported method [27] The CPC paste wasplaced in a 3 times 4 times 25mm mold and placed in a humidifiedatmosphere at 37∘C At 1min intervals the specimen wasscrubbed gently with fingers until the powder componentdid not come off This indicated that the setting reactionhad occurred to a sufficient extent to hold the specimentogether The time from powder-liquid mixing to this pointwas measured as the setting time
24 Microstructural Characterization of Scaffolds
241 Scanning ElectronMicroscopy An Inspect F50 scanningelectronmicroscopy was used to examine the specimensTheMSCs attached to the scaffolds were rinsed twice with 2mLof 1X PBS and fixed with a 2 glutaraldehyde for 24 h at 4∘CSamples were then subjected to graded alcohol dehydrationsair-dried using filter papers sputter-coated with platinumand viewed by SEM Matrices without cells were used ascontrols
242 X-Ray Diffraction (XRD) XRD was used to identifythe crystallographic phases of the reaction products such asthe TTCP powder the set CPC and the sintered CPC Forthe XRD analysis the samples were ground into fine powdersand each powder was mounted in a specimen holder for thediffractometer (Shimadzu XRD-6000 using CuK120572 radiationat 20mA 40 kV) Scans were performed from 5∘ to 80∘ at arate of 2∘min
25 Physical Characterization of Scaffolds
251 Open Porosity and Pore Structure Analysis Pore struc-ture and open porositypores interconnection (1ndash400 mi-crons) were calculated by MIP (mercury intrusion poro-simetry) that is differential mercury intrusion volumerelated to the applied pressure A PoreMaster (USA) was usedfor mercury intrusion porosimetry test The samples wereplaced in a closed cell called penetrometer and evacuatedApplying high pressure via mercury intrusion porosimetrycould damage the structure of the pores [28] Therefore porestructure of the samples was characterized only by applyinglow vacuum level After reaching this low vacuum level (sim3 kPa) the cell was filled with mercury and pressure wasincreased continuously to 03 MPa The surface tension was480 ergcm2 and the contact angle was 140∘
252 Skeletal Density Helium pycnometry is a techniqueused to determine the true density of solids Since heliumcan enter the smallest voids or pores the density obtained isoften referred to as skeletal density (120588o) It is measured witha helium pycnometer specifically Micromeritics AccuPyc1330 He pycnometer The measurements of skeletal densitywere performed as follows helium was first loaded in
Advances in Materials Science and Engineering 3
a calibrated reference volume and then expanded in achamber filled with the sample The change of pressure ofthe helium in the known cell volume without and with thespecimen means that the volume of the specimenrsquos mineralmatrix can be determined Dividing themass of the specimenby this volume yields the true density Four samples wereprepared for measuring the variations of skeletal density (byusing helium pycnometry) as a function of temperature Thevolume of each of them is around 6 cm3 The samples werecrushed into small aggregates Afterward they were heatedfor 24 hours at 10∘C before testing The specimens were putimmediately in the testing chamber to avoid any moistureuptake
253 Total Porosity The total porosity of the sintered porousCPC sample was determined using the following equations
120588119861 =119898
119881 (2)
where 120588119861119898 and119881 refer to bulk density mass of the sampleand volume of the sample respectively
119877119863 =120588119861
120588otimes 100 (3)
where 119877119863 is the relative density and 120588o is the skeletal densityfor solid fraction (120588o) obtained by helium pycnometry
Total porosity = 100 minus 119877119863 (4)
The dimensions and the weight of each sample weremeasured and recorded through a vernier caliper and anelectronic balance respectively
26 Compressive Test and Youngrsquos Modulus For the measure-ments of the compressive strength of the porous samplesrubber padswere placed on the top and the bottom surfaces ofeach sample [13] The rubber padded sample was then placedin a CBR tester (controls) to conduct a compressive testThe rubber pads were used to ensure a uniform distributionof the applied load onto the sample A crosshead speed of04mmmin was used for the compressive tests
In thematerialrsquos stress-strain curve there is a linear regionwhere the material follows Hookersquos law Hence the followingequation stands for this region
120590 = 119864120576 (5)
where E refers to Youngrsquos modulus for compression and 120576 isthe strain caused by the compressive stress
27 In Vitro Characterisation of Scaffolds
271 MSCs Cell Isolation MSCs were isolated from humanbone marrow aspirates via density gradient centrifugationThe cells were expanded in nondifferentiating MSC growthmedium (CCM) consisting of 120572-minimal essential medium(120572-MEM) with 10 foetal bovine serum (FBS) 1 penicillin-streptomycin (PEN-STREP) and 2mMglutamine Cellswere
incubated at 37∘C with 5 CO2 After they reached 90
confluence cells were harvested by rinsingwith 025 trypsinand 003 EDTA solution and expanded into a secondpassage until they reached 80 confluence then they weretrypsinized and cryopreserved in a liquid nitrogen Thesecells were designated as passage 1 (P1) cells
272 Osteogenic Differentiation Cells were seeded in 12mul-tiwell plates for an estimated 80 confluence Each experi-ment comprised 12 cultures 6 under osteogenic conditionsand 6 controls under normal cell cultures conditions (CCM)To induce the osteogenic differentiation of the MSCs thecultures were maintained in osteogenic media which consistof 60120583M ascorbic acid 10mM 120573-glycerol phosphate and100 nM dexamethasone The medium was changed every 2-3 days
273 Cells Seeding on the Bioceramic Scaffolds Cryopre-served (P1) cells were replated at a seeding density of 4000cellscm2 in 120572-MEM as described above At near confluencyP2 cells were harvested with 025 trypsin in EDTA andresuspended at a density of 2 times 105 cellsmL 120572-MEM plus1 penicillinstreptomycin The calcium phosphate scaffoldswere sterilized in 70 ethanol for 24 h and then incubatedwith cell culture media containing 10 FBS for at least4 h Then the MSCs were seeded into the scaffolds witha cell seeding density of 200000 cells per scaffold TheMSCswere culturedwith osteogenicmedium and cell culturemedium as a standard control to verify their proliferative anddifferentiation potential at 37∘C and 5 CO
2 The medium
was changed every 3 or 4 days
3 Results and Discussion
31 Microstructural Morphological and Physical Character-ization The aim of this work is designing an injectableand strong porous system using biocompatible materialsable to promote natural body healing which degrade afterimplantation and contain biologically active phases and ableto stimulate the regenerative tissue growth in order to mimicthe morphological and microstructural properties of bonetissue The starting material of the prepared scaffolds istetracalcium phosphate (TTCP Ca
4(PO4)2O)The analysis of
the XRD diffraction peaks (Figure 1 lower pattern) revealedthat the TTCP was formed as the main phase of CPC powderby firing the mixture of pyrocalcium phosphate (Ca
2P2O7)
and calcium carbonate (CaCO3) at 1500∘C for 5 h followed
by quenching in air Moreover this CPC powder contained afew peaks corresponding to HAThe peaks in the lower XRDpattern are strong and sharp which indicate relatively highcrystallinity of the powder The setting time of the CPC wasaround 15min
After seting CPC while the hydroxyapatite phase waspresent in the form of intensive layer of nano-HA crystalsas shown by the SEM image Figure 2(c) some residualTTCP were still left behind Figure 1 (XRD upper pattern)In addition the residual TTCP phase was depleted to agreat extent for the formation of hydroxyapatite due to the
4 Advances in Materials Science and Engineering
TTCPHA
CPC powder
CPC
(cps
)
10 20 30 40 50 60
1000
500
0
2000
1500
1000
500
0
CPC
2120579 scale
Figure 1 XRD patterns of CPC powder (tetracalcium phosphateTTCP) and hardened CPC (1 day after mixing with the hardeningsolution to form calcium phosphate cement) HA is hydroxyapatite
setting reactions to form calcium phosphate cement (CPC)The high backgrounds of the upper XRD pattern (Figure 1)between 30∘ and 35∘ and 45∘ and 55∘ indicate the presence ofamorphous or poorly crystalline CaP phase mainly CDHA(Calcium-deficient HA) as shown by the EDSmeasurementswith CaP ratio of 16
The SEM micrographs of the fabricated scaffold taken atdifferent magnifications are reported in Figure 2 To discussthese morphologies it is important to define three types ofpores fine pores micropores and macropores These poresare distinguished by their size fine pores have a diameterbelow 5 120583mandmicropores have a diameter typically close tothe range of 5ndash30120583m whereas macropores have a diameterabove 70120583m The threshold is placed at a size of 50mmThe rod-like mannitol particles were thus used only as atemplate to form the scaffold framework with the desiredpore structure for bone tissue engineering applications Thisopen internal pore network is necessary tomaximize nutrientdiffusion interstitial fluid and blood flow to control cellgrowth and function tomanipulate tissue differentiation andto optimize scaffold mechanical function and regeneratedtissue mechanical properties [29] Another feature of theporous CPCwas the presence of well-connectedmacroporeswith diameters between 70 120583m and 300 120583m inside the strutsas shown in Figure 2(a)The CPC contained mainly calcium-deficient hydroxyapatite phase with macropores (as a replicaof the dissolved rod-like mannitol particles) in the overallstructure and open micropores in the struts as shown inFigure 2(b) This design of injectable scaffolds with well-connected pores or highly effective porosity intended tofavor an increase in the mass transport of nutrients andoxygen and removal of waste products for cell growth
In the presence of hardening phosphate solution TTCPhydrolyses through a dissolution precipitation reaction givingrise to the formation of an entangled network of CDHAcrystals which are close to the mineral component of thebone from a structural point of view In the proposed scaf-folds the hydrolysis reaction occurs during the setting of thebone cement This is confirmed by XRD and SEM (Figures 1
and 2(c)) which show a large distribution of CDHA needle-like crystals approximately 1 120583m long and homogeneouslydistributed along the inner surface pores (Figure 2(c)) Thesedo not significantly affect the scaffold porosity in terms ofpore shape and interconnection degree [14] Several workshave demonstrated the feasibility of forming new bone inclose contact with calcium-deficient apatite (CDHA) gran-ulesThis creates a network of woven bone bonded to residualcalcium phosphate particles as confirmed by no adverseinflammatory reactions and formation of multinucleatedgiant cells close to the CDHA granules [16]
To confirm the qualitative morphological evaluation per-formed by SEM a quantitative estimation of porosity char-acteristics was performed by mercury intrusion porosimetry(MIP)The pore size distribution and final open pore volumeof representative scaffolds were characterized by low pressure(to avoid damage of pore structure) mercury intrusionporosimetry (MIP) [28 30] (Figure 3) Pore interconnectivityfirst arises by the dissolved mannitol grains and the contactpoint between adjacent grains Figure 2(b) also due to thepresence of smaller pores (up to 30120583m) induced by thephase conversionmechanism [14] Fine pores network can beobserved within these channel walls as shown in Figure 2(c)
MIP showed evidence of a bimodal distribution of poresize with diameter above 5 120583m This pore structure is char-acterized by micropores a few micrometers in diameter andmacropores of the order of hundreds micrometers (see alsoFigure 2(a)) It is apparent that the total open microporeand macropore volume is nearly 30 of the scaffold volumeFigure 3 The calculations and measurements of volumeweight and bulk density show that the total porosity is around64 It was possible to observe Figure 2(b) the characteristicsizes of the smaller pores (with diameters ranging from 5 to30 120583m (microporosity)) peak A and of larger pores peakB (with diameters ranging from sim70 to sim300120583m (macro-porosity)) The pores with diameter above 5120583m representaround 50 of the total open porosity In this structure themacropores are suitable for the accommodation of osteoblastand undifferentiated bone mesenchymal stem cells while themicropores offer interconnection bridges between adjacentmacropores able to promote the nutrient and metaboliteexchange [15ndash18] improving the structural interconnectivity
One of the most important requirements of an ideal bonesubstitute is for it to exhibit mechanical behavior matchingthat of the bone tissue that has to be restored The accuratemimesis of specific mechanical properties such as elasticmodulus may be crucial to the effective reproduction ofthe functional response of natural tissue especially in load-bearing applications In the last decade the use of polymerssuch as PLA PGA and PCL has been a popular strategy forthe provision of biodegradable supports for orthopedic appli-cations [13] However their deficient mechanical propertieslimited their use mainly to a restricted number of non-load-bearing applications [18] Figure 4 shows the representativestress-strain responses of the fabricated CPC scaffold Thisdiagram shows that the scaffold exhibits compressive strengthof 49MPa The e-modulus of the scaffold in compressionsim400MPa was obtained from the slope of the stress-straincurve
Advances in Materials Science and Engineering 5
500120583m
(a)
20120583m
(b)
2120583m
(c)
Figure 2 SEM micrograph of a fracture surface of the porous calcium phosphate scaffold with different magnifications (a) macroporenetwork (b) micropore structure of the internal walls of macropore and (c) matrix of needle-like CDHA (calcium deficient hydroxyapatite)crystals covering the surfaces of the pores
35
3
25
2
15
1
05
01 10 100 1000
Pore diameter (120583m)
Pore
vol
ume (
incr
emen
tal (
)) B
A
Total connected macroporosity 30asymp
Figure 3 Low pressure mercury intrusion porosimetry of CPCscaffold Incremental pore size in above 5120583m
500
450
400
350
300
250
200
150
100
050
0000000 0005 0010 0015 0020
Strain ()
Com
pres
sive s
tress
(MPa
)
Ultimate compressive Slope asymp 400MPa strength
Figure 4 Representative stress-strain responses of the producedscaffold to compression test
The CPC paste can intimately adapt to complex-shapedbone defects and bond to neighboring bone to form afunctional interface The good mechanical properties shownin the present study could enable stem cell delivery to mod-erate load-bearing repairs For example in mandibular andmaxillary ridge augmentation the CPC could be molded to
the desired esthetic shape and then set to form amacroporousscaffold containing stem cells for bone regeneration Theseimplants would be subjected to early loading by provisionaldentures Therefore the macroporous CPC scaffold needs tobe resistant to flexure In addition the CPC paste could beused in major reconstructions of the maxilla or mandibleafter trauma or tumor resection which would require theCPC to be fracture resistant [30] The CPC could also beused to support metal dental implants or augment deficientimplant sites where mechanical properties are importantIt should be noted that to repair large defects mechanicalstrength is only one factor to consider Other factors suchas vascularization are also critically important for success[31 32]Themacroporous CPC in the present study possessedbettermechanical properties than other injectable carriers forcell delivery For example previous studies reported that aninjectable polymeric carrier for cell delivery had a strengthof 07MPa [33] and hydrogels had strengths of about 01MPa[34 35] These systems are promising for non-load-bearingapplications However their strengths are much lower thanthe reported strength of about 35MPa for cancellous bone[36] This macroporous CPC with a strength of 49MPamatched that of cancellous bone and hence may be apromising injectable scaffold for stem cell in orthopedic andcraniofacial applications The bulk density of the scaffold is109 gcm3 and the total porosity is 64 The mechanicaland physical properties of the scaffold were in a range ofthose of cancellous bone Therefore it is suggested that thisscaffold is one of promising scaffold materials for hard tissueregeneration Table 1
In the light of recent approaches in bone regenera-tion based on composite materials the proposed strategycertainly offers the best compromise between structuraland functional properties Porous composite scaffolds madeby incorporating bioactive particles [37] do not assure anadequate improvement in the mechanical performance forbone substitution Meanwhile composites materials formedby integrating high modulus PLA fibers coated with calciumphosphate (CaP) into a PCL matrix [38] show mechanicalproperties that are significantly higher than the valuesreported in the literature for PLA-CaP composites but lack
6 Advances in Materials Science and Engineering
50120583m
50120583m
2120583m
2120583m
5120583m
(a) (b)
(c) (d)
(e) (f)
20120583m
Figure 5 Representative SEM image showing cell attachment to the surface of CPC scaffoldwith cytoplasmic extensions (e) after culturing for4 11 and 21 daysmdashimages (a) (c) and (e) respectively The other 3 images ((b) (d) and (f)) show the development of the calcium phosphatematrix over time
Table 1 Comparison between the CPC scaffold and cancellous bones [19ndash21]
structural porosity In contrast the proposed scaffolds showan intermediate level of mechanical response in porousstructure
32 Preliminary Evaluation of Biocompatibility of ScaffoldsThe injectable CPC scaffolds were prepared for preliminaryin vitro cultivation MSCs were seeded on the 3D scaffoldsand maintained with osteogenic medium for 4 days 11 days
and 21 days After 3 days of culturing cells showed no regularorientation and mineralized nodules (A Figure 5(a)) wereformed in the constructs The size of the cells varies from20120583m to 40120583m Figure 5(a) shows that the cells adhered tothe CPC and developed cytoplasmic extensions (e) After 4days a homogeneous needle-like CDHA matrix covers theinternal surfaces of the scaffold Figure 5(b) After 11 daysof culturing most pores were filled with new tissue mass as
Advances in Materials Science and Engineering 7
observed by SEM (Figure 5(c)) The CDHAmatrices visuallydemonstrated high cell density on the surfaces of the matrixwithin the cylindrical macropores as shown in Figures 5(c)and 5(d)
As reported in Figure 5(c) cells attaching to the nano-sized CDHA crystals that make up the scaffold matrixCell adhesion resulted in elongated and highly stretchedcells within the macropores with focal adhesion points andmulticytoplasmic extensions on the scaffolds after 21 daysof culturing (Figure 5(e)) Moreover cells growth in thescaffolds in culture shows the potential of using this scaffoldarchitecture for MSC seeding and growth The bone matrixconsists of CDHA crystals (Figure 5(f)) containing calciumand phosphorus Therefore these factors provide importantindicators of osteogenic differentiation
4 Conclusions
Engineering tissue with cells and a synthetic extracellularmatrix is an alternative approach to the established practiceof transplantation of harvested tissues Degradable injectableporous scaffolds prepared by calcium phosphate cementrepresent a valid solution to achieving adequate porosityrequirements while providing adequate support in load-bearing applications This proposed process for preparinginjectable scaffolds with two pore networks is as quick andversatile as conventional technologies Using this methodporous CDHA-based calcium phosphate with macroporessizes ranging from 70 to 300120583m micropores ranging from5 to 30 120583m and 30 open macroporosity was preparedThe compressive strength and the e-modulus of the poroushydroxyapatite-based calcium phosphate samples 49MPaand 400MPa respectively were comparable with those ofthe cancellous bone Finally the bioactivity of the scaffoldswas confirmed by cells growth and cytoplasmic extensionsin the scaffolds in culture demonstrating that the scaffoldis suitable for MSC seeding and growth architecture Thiscombination of an interconnected macroporous structurewith pore size has a potential for the promotion of cell seedingand proliferation plus adequate mechanical features andrepresents a porous scaffold which is an excellent candidatefor bone tissue engineering with setting time around 15min
Acknowledgments
The financial support of the project ldquoSynthesis and character-ization of scaffolds for bone tissue engineeringrdquo funded by theDeanship of Academic Research at the University of Jordan isgratefully acknowledged The authors would like to take thisopportunity to thank the Hamdi Mango Center for ScientificResearch (HMCSR) University of Jordan for providing thelaboratory facilities
References
[1] J J Mao G Vunjak-Novakovic A G Mikos and A AtalaRegenerative Medicine Translational Approaches and TissueEngineering Artech House Boston Mass USA 2007
[2] M Bohner ldquoDesign of ceramic-based cements and putties forbone graft substitutionrdquo European Cells and Materials vol 20pp 1ndash12 2010
[3] M-P Ginebra C Canal M Espanol D Pastorino and EB Montufar ldquoCalcium phosphate cements as drug deliverymaterialsrdquo Advanced Drug Delivery Reviews vol 64 pp 1090ndash1010 2012
[4] C S Che Nor Zarida O Fauziah A K Arifah et al ldquoIn vitroelution and dissolution of tobramycin and gentamicin fromcalcium phosphaterdquo African Journal of Pharmacy and Phar-macology vol 5 no 20 pp 2283ndash2291 2011
[5] J JMaoWV Giannobile J AHelms et al ldquoCraniofacial tissueengineering by stem cellsrdquo Journal of Dental Research vol 85no 11 pp 966ndash979 2006
[6] P C Johnson A G Mikos J P Fisher and J A Jansen ldquoStra-tegic directions in tissue engineeringrdquo Tissue Engineering vol13 no 12 pp 2827ndash2837 2007
[7] P Habibovic U Gbureck C J Doillon D C Bassett C A vanBlitterswijk and J E Barralet ldquoOsteoconduction and osteoin-duction of low-temperature 3D printed bioceramic implantsrdquoBiomaterials vol 29 no 7 pp 944ndash953 2008
[8] H SWang S C Hung S T Peng C C Huang HMWei Y JGuo et al ldquoMesenchyma lstem cells in theWhartonrsquos jelly of thehuman umbilical cordrdquo Stem Cells vol 22 pp 1330ndash1337 2004
[9] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007
[10] E Verron I Khairoun J Guicheux and J-M Bouler ldquoCalciumphosphate biomaterials as bone drug delivery systems a reviewrdquoDrug Discovery Today vol 15 no 13-14 pp 547ndash552 2010
[11] A Can and S Karahuseyinoglu ldquoConcise review humanumbilical cord stromawith regard to the source of fetus-derivedstem cellsrdquo Stem Cells vol 25 no 11 pp 2886ndash2895 2007
[12] L Wang M Singh L F Bonewald and M S DetamoreldquoSignalling strategies for osteogenic differentiation of humanumbilical cord mesenchymal stromal cells for 3D bone tissueengineeringrdquo Journal of Tissue Engineering and RegenerativeMedicine vol 3 no 5 pp 398ndash404 2009
[13] X Miao L-P Tan L-S Tan and X Huang ldquoPorous cal-cium phosphate ceramics modified with PLGA-bioactive glassrdquoMaterials Science and Engineering C vol 27 no 2 pp 274ndash2792007
[14] V Guarino and L Ambrosio ldquoThe synergic effect of polylactidefiber and calcium phosphate particle reinforcement in poly120576-caprolactone-based composite scaffoldsrdquo Acta Biomaterialiavol 4 no 6 pp 1778ndash1787 2008
[15] A E Watts A J Nixon M G Papich H D Sparks and WS Schwark ldquoIn vitro elution of amikacin and ticarcillin froma resorbable self-setting fiber reinforced calcium phosphatecementrdquo Veterinary Surgery vol 40 no 5 pp 563ndash570 2011
[16] K J L Burg S Porter and J F Kellam ldquoBiomaterial develop-ments for bone tissue engineeringrdquo Biomaterials vol 21 no 23pp 2347ndash2359 2000
[17] S Yang K-F Leong Z Du and C-K Chua ldquoThe design ofscaffolds for use in tissue engineering Part I traditional factorsrdquoTissue Engineering vol 7 no 6 pp 679ndash689 2001
[18] K F Leong C M Cheah and C K Chua ldquoSolid freeform fab-rication of three-dimensional scaffolds for engineering replace-ment tissues and organsrdquo Biomaterials vol 24 no 13 pp 2363ndash2378 2003
8 Advances in Materials Science and Engineering
[19] J Russias E Saiz R K Nalla K Gryn R O Ritchie and AP Tomsia ldquoFabrication and mechanical properties of PLAHAcomposites a study of in vitro degradationrdquo Materials Scienceand Engineering C vol 26 no 8 pp 1289ndash1295 2006
[20] R Z Le Geros ldquoCalcium phosphate-based osteoinductivematerialsrdquo Chemical Reviews vol 108 no 11 pp 4742ndash47532008
[21] C E Wen Y Yamada K Shimojima Y Chino H HosokawaandMMabuchi ldquoCompressibility of porous magnesium foamdependency on porosity and pore sizerdquo Materials Letters vol58 no 3-4 pp 357ndash360 2004
[22] L L Hench and J M Polak ldquoThird-generation biomedicalmaterialsrdquo Science vol 295 no 5557 pp 1014ndash1017 2002
[23] D Williams ldquoBenefit and risk in tissue engineeringrdquo MaterialsToday vol 7 no 5 pp 24ndash29 2004
[24] V Guarino F Causa and L Ambrosio ldquoBioactive scaffolds forbone and ligament tissuerdquoExpert Review ofMedicalDevices vol4 no 3 pp 405ndash418 2007
[25] D W Hutmacher J T Schantz C X F Lam K C Tan and TC Lim ldquoState of the art and future directions of scaffold-basedbone engineering from a biomaterials perspectiverdquo Journal oftissue engineering and regenerative medicine vol 1 no 4 pp245ndash260 2007
[26] L Gerhardt and A R Boccaccini ldquoBioactive glass and glass-ceramic scaffolds for bone tissue engineeringrdquoMaterials vol 3pp 3867ndash3910 2010
[27] HH K Xu S Takagi J B Quinn and L C Chow ldquoFast-settingcalcium phosphate scaffolds with tailoredmacropore formationrates for bone regenerationrdquo Journal of Biomedical MaterialsResearch A vol 68 no 4 pp 725ndash734 2004
[28] MAlshaaerHCuypersH Rahier and JWastiels ldquoProductionof monetite-based Inorganic Phosphate Cement (M-IPC) usinghydrothermal post curing (HTPC)rdquo Cement and ConcreteResearch vol 41 no 1 pp 30ndash37 2011
[29] C Wenchuan Z Hongzhi D W Michael B Chongyun andH H K Xu ldquoUmbilical cord stem cells released from alginate-fibrin microbeads inside macroporous and biofunctionalizedcalcium phosphate cement for bone regenerationrdquo Acta Bioma-terialia vol 8 no 6 pp 2297ndash2306 2012
[30] M Alshaaer H Cuypers G Mosselmans H Rahier and JWastiels ldquoEvaluation of a low temperature hardening InorganicPhosphate Cement for high-temperature applicationsrdquo Cementand Concrete Research vol 41 no 1 pp 38ndash45 2011
[31] J Rouwkema N C Rivron and C A van Blitterswijk ldquoVas-cularization in tissue engineeringrdquo Trends in Biotechnology vol26 no 8 pp 434ndash441 2008
[32] M Lovett K Lee A Edwards and D L Kaplan ldquoVasculariza-tion strategies for tissue engineeringrdquo Tissue Engineering B vol15 no 3 pp 353ndash370 2009
[33] X Shi B Sitharaman Q P Pham et al ldquoFabrication of porousultra-short single-walled carbonnanotube nanocomposite scaf-folds for bone tissue engineeringrdquo Biomaterials vol 28 no 28pp 4078ndash4090 2007
[34] C K Kuo andP XMa ldquoIonically crosslinked alginate hydrogelsas scaffolds for tissue engineering Part 1 structure gelation rateand mechanical propertiesrdquo Biomaterials vol 22 no 6 pp 511ndash521 2001
[35] J L Drury R G Dennis and D J Mooney ldquoThe tensile prop-erties of alginate hydrogelsrdquo Biomaterials vol 25 no 16 pp3187ndash3199 2004
[36] C J Damien and J R Parsons ldquoBone graft and bone graftsubstitutes a review of current technology and applicationsrdquoJournal of Applied Biomaterials vol 2 no 3 pp 187ndash208 1991
[37] Y Lei B Rai K H Ho and S H Teoh ldquoIn Vitro degradationof novel bioactive polycaprolactone-20 tricalcium phosphatecomposite scaffolds for bone engineeringrdquo Materials Scienceand Engineering C vol 27 no 2 pp 293ndash298 2007
[38] C R Kothapalli M T Shaw J R Olson and M Wei ldquoFab-rication of novel calcium phosphatepoly(lactic acid) fibercompositesrdquo Journal of Biomedical Materials Research B vol 84no 1 pp 89ndash97 2008
to the desired shape This can lead to increased bone losstrauma to the surrounding tissue and prolonged surgicaltime [23] However CPCs typically set at low temperaturesfollowing the combination of a solid component containingone or several calcium orthophosphate salts and an aqueoussolution and form a solid calcium phosphate in situ Thismeans that CPCs can adapt immediately to each bone cavityand obtain goodosteointegration In addition injectableCPCformulations that are suitable for new minimally invasivesurgical techniques have been developed [26] In the lasttwo decades numerous biomaterials studies have been con-ducted investigating the development of CPCs Howeverthe physicochemical characterization and in vitro evaluationof the final properties of CPCs produced from calciumpyrophosphate powder have not been studied systematicallyIn this study the main reactive ingredient of CPC tetracal-cium phosphate (TTCP Ca
4(PO4)2O) was synthesized by
solid state reaction between calcium pyrophosphate (CaPyroCa2P2O7) and calcium carbonate (CaCO
3) The aim of this
work consists in designing an injectable and strong poroussystemusing biocompatiblematerials able to promote naturalbody healing which degrade after implantation and containbiologically active phases and able to stimulate the regener-ative tissue growth in order to mimic the morphological andmicrostructural properties of bone tissueWe investigated themicrostructural properties phase evaluation pore structuredensities compressive strength and stiffness of the producedscaffolds We also evaluated the bone formation propertiesand cell differentiation in vitro over a time scale of 21 days
2 Materials and Methods
21 Synthesis of TTCP TheTTCP powder used for this studywas fabricated from the reaction of dicalcium pyrophos-phate (Ca
2P2O7) (Merck Germany) and calcium carbonate
(CaCO3) (Merck Germany) with a weight ratio of 1 127The
powders were mixed uniformly in ethanol for 12 h Then themixed powder was dried and crushed using a mortar and apestle followed by calcination in a crucible at 1500∘C for 5 h inair and quenching in air at 25∘C Finally the calcined powder(TTCP phase) was ground into a fine powder
The chemical reaction for the TTCP powder was asfollows
Ca2P2O7+ 2CaCO
3
997888rarr Ca4(PO4)2O (TTCP) + 2CO
2
(1)
22 Fabrication of the Macroporous CPC Scaffold Water-soluble granular rod-like mannitol porogen was incorpo-rated into CPC to create macropores The TTCP powderwas mixed with granulated mannitol with size that variesfrom 70 120583m to 300 120583m (mannitolTTCP weight ratio is 05)This mixture was then mixed in diammonium hydrogenphosphate ((NH
4)2HPO4 333 wt) with hardening solu-
tionTTCP ratio of 034mLg After mixing the CPC for1min the cement paste was uniformly packed in a polymermold which has an opening of 10 times 10mm and 3mm indepth under a pressure of sim14MPa at 37∘C The hardened
CPC was then removed from the mold and immersed inHanksrsquo physiological solution at 37∘C for 2 days to dissolvethe mannitol
23 CPC Setting Time The CPC setting time was measuredusing a previously reported method [27] The CPC paste wasplaced in a 3 times 4 times 25mm mold and placed in a humidifiedatmosphere at 37∘C At 1min intervals the specimen wasscrubbed gently with fingers until the powder componentdid not come off This indicated that the setting reactionhad occurred to a sufficient extent to hold the specimentogether The time from powder-liquid mixing to this pointwas measured as the setting time
24 Microstructural Characterization of Scaffolds
241 Scanning ElectronMicroscopy An Inspect F50 scanningelectronmicroscopy was used to examine the specimensTheMSCs attached to the scaffolds were rinsed twice with 2mLof 1X PBS and fixed with a 2 glutaraldehyde for 24 h at 4∘CSamples were then subjected to graded alcohol dehydrationsair-dried using filter papers sputter-coated with platinumand viewed by SEM Matrices without cells were used ascontrols
242 X-Ray Diffraction (XRD) XRD was used to identifythe crystallographic phases of the reaction products such asthe TTCP powder the set CPC and the sintered CPC Forthe XRD analysis the samples were ground into fine powdersand each powder was mounted in a specimen holder for thediffractometer (Shimadzu XRD-6000 using CuK120572 radiationat 20mA 40 kV) Scans were performed from 5∘ to 80∘ at arate of 2∘min
25 Physical Characterization of Scaffolds
251 Open Porosity and Pore Structure Analysis Pore struc-ture and open porositypores interconnection (1ndash400 mi-crons) were calculated by MIP (mercury intrusion poro-simetry) that is differential mercury intrusion volumerelated to the applied pressure A PoreMaster (USA) was usedfor mercury intrusion porosimetry test The samples wereplaced in a closed cell called penetrometer and evacuatedApplying high pressure via mercury intrusion porosimetrycould damage the structure of the pores [28] Therefore porestructure of the samples was characterized only by applyinglow vacuum level After reaching this low vacuum level (sim3 kPa) the cell was filled with mercury and pressure wasincreased continuously to 03 MPa The surface tension was480 ergcm2 and the contact angle was 140∘
252 Skeletal Density Helium pycnometry is a techniqueused to determine the true density of solids Since heliumcan enter the smallest voids or pores the density obtained isoften referred to as skeletal density (120588o) It is measured witha helium pycnometer specifically Micromeritics AccuPyc1330 He pycnometer The measurements of skeletal densitywere performed as follows helium was first loaded in
Advances in Materials Science and Engineering 3
a calibrated reference volume and then expanded in achamber filled with the sample The change of pressure ofthe helium in the known cell volume without and with thespecimen means that the volume of the specimenrsquos mineralmatrix can be determined Dividing themass of the specimenby this volume yields the true density Four samples wereprepared for measuring the variations of skeletal density (byusing helium pycnometry) as a function of temperature Thevolume of each of them is around 6 cm3 The samples werecrushed into small aggregates Afterward they were heatedfor 24 hours at 10∘C before testing The specimens were putimmediately in the testing chamber to avoid any moistureuptake
253 Total Porosity The total porosity of the sintered porousCPC sample was determined using the following equations
120588119861 =119898
119881 (2)
where 120588119861119898 and119881 refer to bulk density mass of the sampleand volume of the sample respectively
119877119863 =120588119861
120588otimes 100 (3)
where 119877119863 is the relative density and 120588o is the skeletal densityfor solid fraction (120588o) obtained by helium pycnometry
Total porosity = 100 minus 119877119863 (4)
The dimensions and the weight of each sample weremeasured and recorded through a vernier caliper and anelectronic balance respectively
26 Compressive Test and Youngrsquos Modulus For the measure-ments of the compressive strength of the porous samplesrubber padswere placed on the top and the bottom surfaces ofeach sample [13] The rubber padded sample was then placedin a CBR tester (controls) to conduct a compressive testThe rubber pads were used to ensure a uniform distributionof the applied load onto the sample A crosshead speed of04mmmin was used for the compressive tests
In thematerialrsquos stress-strain curve there is a linear regionwhere the material follows Hookersquos law Hence the followingequation stands for this region
120590 = 119864120576 (5)
where E refers to Youngrsquos modulus for compression and 120576 isthe strain caused by the compressive stress
27 In Vitro Characterisation of Scaffolds
271 MSCs Cell Isolation MSCs were isolated from humanbone marrow aspirates via density gradient centrifugationThe cells were expanded in nondifferentiating MSC growthmedium (CCM) consisting of 120572-minimal essential medium(120572-MEM) with 10 foetal bovine serum (FBS) 1 penicillin-streptomycin (PEN-STREP) and 2mMglutamine Cellswere
incubated at 37∘C with 5 CO2 After they reached 90
confluence cells were harvested by rinsingwith 025 trypsinand 003 EDTA solution and expanded into a secondpassage until they reached 80 confluence then they weretrypsinized and cryopreserved in a liquid nitrogen Thesecells were designated as passage 1 (P1) cells
272 Osteogenic Differentiation Cells were seeded in 12mul-tiwell plates for an estimated 80 confluence Each experi-ment comprised 12 cultures 6 under osteogenic conditionsand 6 controls under normal cell cultures conditions (CCM)To induce the osteogenic differentiation of the MSCs thecultures were maintained in osteogenic media which consistof 60120583M ascorbic acid 10mM 120573-glycerol phosphate and100 nM dexamethasone The medium was changed every 2-3 days
273 Cells Seeding on the Bioceramic Scaffolds Cryopre-served (P1) cells were replated at a seeding density of 4000cellscm2 in 120572-MEM as described above At near confluencyP2 cells were harvested with 025 trypsin in EDTA andresuspended at a density of 2 times 105 cellsmL 120572-MEM plus1 penicillinstreptomycin The calcium phosphate scaffoldswere sterilized in 70 ethanol for 24 h and then incubatedwith cell culture media containing 10 FBS for at least4 h Then the MSCs were seeded into the scaffolds witha cell seeding density of 200000 cells per scaffold TheMSCswere culturedwith osteogenicmedium and cell culturemedium as a standard control to verify their proliferative anddifferentiation potential at 37∘C and 5 CO
2 The medium
was changed every 3 or 4 days
3 Results and Discussion
31 Microstructural Morphological and Physical Character-ization The aim of this work is designing an injectableand strong porous system using biocompatible materialsable to promote natural body healing which degrade afterimplantation and contain biologically active phases and ableto stimulate the regenerative tissue growth in order to mimicthe morphological and microstructural properties of bonetissue The starting material of the prepared scaffolds istetracalcium phosphate (TTCP Ca
4(PO4)2O)The analysis of
the XRD diffraction peaks (Figure 1 lower pattern) revealedthat the TTCP was formed as the main phase of CPC powderby firing the mixture of pyrocalcium phosphate (Ca
2P2O7)
and calcium carbonate (CaCO3) at 1500∘C for 5 h followed
by quenching in air Moreover this CPC powder contained afew peaks corresponding to HAThe peaks in the lower XRDpattern are strong and sharp which indicate relatively highcrystallinity of the powder The setting time of the CPC wasaround 15min
After seting CPC while the hydroxyapatite phase waspresent in the form of intensive layer of nano-HA crystalsas shown by the SEM image Figure 2(c) some residualTTCP were still left behind Figure 1 (XRD upper pattern)In addition the residual TTCP phase was depleted to agreat extent for the formation of hydroxyapatite due to the
4 Advances in Materials Science and Engineering
TTCPHA
CPC powder
CPC
(cps
)
10 20 30 40 50 60
1000
500
0
2000
1500
1000
500
0
CPC
2120579 scale
Figure 1 XRD patterns of CPC powder (tetracalcium phosphateTTCP) and hardened CPC (1 day after mixing with the hardeningsolution to form calcium phosphate cement) HA is hydroxyapatite
setting reactions to form calcium phosphate cement (CPC)The high backgrounds of the upper XRD pattern (Figure 1)between 30∘ and 35∘ and 45∘ and 55∘ indicate the presence ofamorphous or poorly crystalline CaP phase mainly CDHA(Calcium-deficient HA) as shown by the EDSmeasurementswith CaP ratio of 16
The SEM micrographs of the fabricated scaffold taken atdifferent magnifications are reported in Figure 2 To discussthese morphologies it is important to define three types ofpores fine pores micropores and macropores These poresare distinguished by their size fine pores have a diameterbelow 5 120583mandmicropores have a diameter typically close tothe range of 5ndash30120583m whereas macropores have a diameterabove 70120583m The threshold is placed at a size of 50mmThe rod-like mannitol particles were thus used only as atemplate to form the scaffold framework with the desiredpore structure for bone tissue engineering applications Thisopen internal pore network is necessary tomaximize nutrientdiffusion interstitial fluid and blood flow to control cellgrowth and function tomanipulate tissue differentiation andto optimize scaffold mechanical function and regeneratedtissue mechanical properties [29] Another feature of theporous CPCwas the presence of well-connectedmacroporeswith diameters between 70 120583m and 300 120583m inside the strutsas shown in Figure 2(a)The CPC contained mainly calcium-deficient hydroxyapatite phase with macropores (as a replicaof the dissolved rod-like mannitol particles) in the overallstructure and open micropores in the struts as shown inFigure 2(b) This design of injectable scaffolds with well-connected pores or highly effective porosity intended tofavor an increase in the mass transport of nutrients andoxygen and removal of waste products for cell growth
In the presence of hardening phosphate solution TTCPhydrolyses through a dissolution precipitation reaction givingrise to the formation of an entangled network of CDHAcrystals which are close to the mineral component of thebone from a structural point of view In the proposed scaf-folds the hydrolysis reaction occurs during the setting of thebone cement This is confirmed by XRD and SEM (Figures 1
and 2(c)) which show a large distribution of CDHA needle-like crystals approximately 1 120583m long and homogeneouslydistributed along the inner surface pores (Figure 2(c)) Thesedo not significantly affect the scaffold porosity in terms ofpore shape and interconnection degree [14] Several workshave demonstrated the feasibility of forming new bone inclose contact with calcium-deficient apatite (CDHA) gran-ulesThis creates a network of woven bone bonded to residualcalcium phosphate particles as confirmed by no adverseinflammatory reactions and formation of multinucleatedgiant cells close to the CDHA granules [16]
To confirm the qualitative morphological evaluation per-formed by SEM a quantitative estimation of porosity char-acteristics was performed by mercury intrusion porosimetry(MIP)The pore size distribution and final open pore volumeof representative scaffolds were characterized by low pressure(to avoid damage of pore structure) mercury intrusionporosimetry (MIP) [28 30] (Figure 3) Pore interconnectivityfirst arises by the dissolved mannitol grains and the contactpoint between adjacent grains Figure 2(b) also due to thepresence of smaller pores (up to 30120583m) induced by thephase conversionmechanism [14] Fine pores network can beobserved within these channel walls as shown in Figure 2(c)
MIP showed evidence of a bimodal distribution of poresize with diameter above 5 120583m This pore structure is char-acterized by micropores a few micrometers in diameter andmacropores of the order of hundreds micrometers (see alsoFigure 2(a)) It is apparent that the total open microporeand macropore volume is nearly 30 of the scaffold volumeFigure 3 The calculations and measurements of volumeweight and bulk density show that the total porosity is around64 It was possible to observe Figure 2(b) the characteristicsizes of the smaller pores (with diameters ranging from 5 to30 120583m (microporosity)) peak A and of larger pores peakB (with diameters ranging from sim70 to sim300120583m (macro-porosity)) The pores with diameter above 5120583m representaround 50 of the total open porosity In this structure themacropores are suitable for the accommodation of osteoblastand undifferentiated bone mesenchymal stem cells while themicropores offer interconnection bridges between adjacentmacropores able to promote the nutrient and metaboliteexchange [15ndash18] improving the structural interconnectivity
One of the most important requirements of an ideal bonesubstitute is for it to exhibit mechanical behavior matchingthat of the bone tissue that has to be restored The accuratemimesis of specific mechanical properties such as elasticmodulus may be crucial to the effective reproduction ofthe functional response of natural tissue especially in load-bearing applications In the last decade the use of polymerssuch as PLA PGA and PCL has been a popular strategy forthe provision of biodegradable supports for orthopedic appli-cations [13] However their deficient mechanical propertieslimited their use mainly to a restricted number of non-load-bearing applications [18] Figure 4 shows the representativestress-strain responses of the fabricated CPC scaffold Thisdiagram shows that the scaffold exhibits compressive strengthof 49MPa The e-modulus of the scaffold in compressionsim400MPa was obtained from the slope of the stress-straincurve
Advances in Materials Science and Engineering 5
500120583m
(a)
20120583m
(b)
2120583m
(c)
Figure 2 SEM micrograph of a fracture surface of the porous calcium phosphate scaffold with different magnifications (a) macroporenetwork (b) micropore structure of the internal walls of macropore and (c) matrix of needle-like CDHA (calcium deficient hydroxyapatite)crystals covering the surfaces of the pores
35
3
25
2
15
1
05
01 10 100 1000
Pore diameter (120583m)
Pore
vol
ume (
incr
emen
tal (
)) B
A
Total connected macroporosity 30asymp
Figure 3 Low pressure mercury intrusion porosimetry of CPCscaffold Incremental pore size in above 5120583m
500
450
400
350
300
250
200
150
100
050
0000000 0005 0010 0015 0020
Strain ()
Com
pres
sive s
tress
(MPa
)
Ultimate compressive Slope asymp 400MPa strength
Figure 4 Representative stress-strain responses of the producedscaffold to compression test
The CPC paste can intimately adapt to complex-shapedbone defects and bond to neighboring bone to form afunctional interface The good mechanical properties shownin the present study could enable stem cell delivery to mod-erate load-bearing repairs For example in mandibular andmaxillary ridge augmentation the CPC could be molded to
the desired esthetic shape and then set to form amacroporousscaffold containing stem cells for bone regeneration Theseimplants would be subjected to early loading by provisionaldentures Therefore the macroporous CPC scaffold needs tobe resistant to flexure In addition the CPC paste could beused in major reconstructions of the maxilla or mandibleafter trauma or tumor resection which would require theCPC to be fracture resistant [30] The CPC could also beused to support metal dental implants or augment deficientimplant sites where mechanical properties are importantIt should be noted that to repair large defects mechanicalstrength is only one factor to consider Other factors suchas vascularization are also critically important for success[31 32]Themacroporous CPC in the present study possessedbettermechanical properties than other injectable carriers forcell delivery For example previous studies reported that aninjectable polymeric carrier for cell delivery had a strengthof 07MPa [33] and hydrogels had strengths of about 01MPa[34 35] These systems are promising for non-load-bearingapplications However their strengths are much lower thanthe reported strength of about 35MPa for cancellous bone[36] This macroporous CPC with a strength of 49MPamatched that of cancellous bone and hence may be apromising injectable scaffold for stem cell in orthopedic andcraniofacial applications The bulk density of the scaffold is109 gcm3 and the total porosity is 64 The mechanicaland physical properties of the scaffold were in a range ofthose of cancellous bone Therefore it is suggested that thisscaffold is one of promising scaffold materials for hard tissueregeneration Table 1
In the light of recent approaches in bone regenera-tion based on composite materials the proposed strategycertainly offers the best compromise between structuraland functional properties Porous composite scaffolds madeby incorporating bioactive particles [37] do not assure anadequate improvement in the mechanical performance forbone substitution Meanwhile composites materials formedby integrating high modulus PLA fibers coated with calciumphosphate (CaP) into a PCL matrix [38] show mechanicalproperties that are significantly higher than the valuesreported in the literature for PLA-CaP composites but lack
6 Advances in Materials Science and Engineering
50120583m
50120583m
2120583m
2120583m
5120583m
(a) (b)
(c) (d)
(e) (f)
20120583m
Figure 5 Representative SEM image showing cell attachment to the surface of CPC scaffoldwith cytoplasmic extensions (e) after culturing for4 11 and 21 daysmdashimages (a) (c) and (e) respectively The other 3 images ((b) (d) and (f)) show the development of the calcium phosphatematrix over time
Table 1 Comparison between the CPC scaffold and cancellous bones [19ndash21]
structural porosity In contrast the proposed scaffolds showan intermediate level of mechanical response in porousstructure
32 Preliminary Evaluation of Biocompatibility of ScaffoldsThe injectable CPC scaffolds were prepared for preliminaryin vitro cultivation MSCs were seeded on the 3D scaffoldsand maintained with osteogenic medium for 4 days 11 days
and 21 days After 3 days of culturing cells showed no regularorientation and mineralized nodules (A Figure 5(a)) wereformed in the constructs The size of the cells varies from20120583m to 40120583m Figure 5(a) shows that the cells adhered tothe CPC and developed cytoplasmic extensions (e) After 4days a homogeneous needle-like CDHA matrix covers theinternal surfaces of the scaffold Figure 5(b) After 11 daysof culturing most pores were filled with new tissue mass as
Advances in Materials Science and Engineering 7
observed by SEM (Figure 5(c)) The CDHAmatrices visuallydemonstrated high cell density on the surfaces of the matrixwithin the cylindrical macropores as shown in Figures 5(c)and 5(d)
As reported in Figure 5(c) cells attaching to the nano-sized CDHA crystals that make up the scaffold matrixCell adhesion resulted in elongated and highly stretchedcells within the macropores with focal adhesion points andmulticytoplasmic extensions on the scaffolds after 21 daysof culturing (Figure 5(e)) Moreover cells growth in thescaffolds in culture shows the potential of using this scaffoldarchitecture for MSC seeding and growth The bone matrixconsists of CDHA crystals (Figure 5(f)) containing calciumand phosphorus Therefore these factors provide importantindicators of osteogenic differentiation
4 Conclusions
Engineering tissue with cells and a synthetic extracellularmatrix is an alternative approach to the established practiceof transplantation of harvested tissues Degradable injectableporous scaffolds prepared by calcium phosphate cementrepresent a valid solution to achieving adequate porosityrequirements while providing adequate support in load-bearing applications This proposed process for preparinginjectable scaffolds with two pore networks is as quick andversatile as conventional technologies Using this methodporous CDHA-based calcium phosphate with macroporessizes ranging from 70 to 300120583m micropores ranging from5 to 30 120583m and 30 open macroporosity was preparedThe compressive strength and the e-modulus of the poroushydroxyapatite-based calcium phosphate samples 49MPaand 400MPa respectively were comparable with those ofthe cancellous bone Finally the bioactivity of the scaffoldswas confirmed by cells growth and cytoplasmic extensionsin the scaffolds in culture demonstrating that the scaffoldis suitable for MSC seeding and growth architecture Thiscombination of an interconnected macroporous structurewith pore size has a potential for the promotion of cell seedingand proliferation plus adequate mechanical features andrepresents a porous scaffold which is an excellent candidatefor bone tissue engineering with setting time around 15min
Acknowledgments
The financial support of the project ldquoSynthesis and character-ization of scaffolds for bone tissue engineeringrdquo funded by theDeanship of Academic Research at the University of Jordan isgratefully acknowledged The authors would like to take thisopportunity to thank the Hamdi Mango Center for ScientificResearch (HMCSR) University of Jordan for providing thelaboratory facilities
References
[1] J J Mao G Vunjak-Novakovic A G Mikos and A AtalaRegenerative Medicine Translational Approaches and TissueEngineering Artech House Boston Mass USA 2007
[2] M Bohner ldquoDesign of ceramic-based cements and putties forbone graft substitutionrdquo European Cells and Materials vol 20pp 1ndash12 2010
[3] M-P Ginebra C Canal M Espanol D Pastorino and EB Montufar ldquoCalcium phosphate cements as drug deliverymaterialsrdquo Advanced Drug Delivery Reviews vol 64 pp 1090ndash1010 2012
[4] C S Che Nor Zarida O Fauziah A K Arifah et al ldquoIn vitroelution and dissolution of tobramycin and gentamicin fromcalcium phosphaterdquo African Journal of Pharmacy and Phar-macology vol 5 no 20 pp 2283ndash2291 2011
[5] J JMaoWV Giannobile J AHelms et al ldquoCraniofacial tissueengineering by stem cellsrdquo Journal of Dental Research vol 85no 11 pp 966ndash979 2006
[6] P C Johnson A G Mikos J P Fisher and J A Jansen ldquoStra-tegic directions in tissue engineeringrdquo Tissue Engineering vol13 no 12 pp 2827ndash2837 2007
[7] P Habibovic U Gbureck C J Doillon D C Bassett C A vanBlitterswijk and J E Barralet ldquoOsteoconduction and osteoin-duction of low-temperature 3D printed bioceramic implantsrdquoBiomaterials vol 29 no 7 pp 944ndash953 2008
[8] H SWang S C Hung S T Peng C C Huang HMWei Y JGuo et al ldquoMesenchyma lstem cells in theWhartonrsquos jelly of thehuman umbilical cordrdquo Stem Cells vol 22 pp 1330ndash1337 2004
[9] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007
[10] E Verron I Khairoun J Guicheux and J-M Bouler ldquoCalciumphosphate biomaterials as bone drug delivery systems a reviewrdquoDrug Discovery Today vol 15 no 13-14 pp 547ndash552 2010
[11] A Can and S Karahuseyinoglu ldquoConcise review humanumbilical cord stromawith regard to the source of fetus-derivedstem cellsrdquo Stem Cells vol 25 no 11 pp 2886ndash2895 2007
[12] L Wang M Singh L F Bonewald and M S DetamoreldquoSignalling strategies for osteogenic differentiation of humanumbilical cord mesenchymal stromal cells for 3D bone tissueengineeringrdquo Journal of Tissue Engineering and RegenerativeMedicine vol 3 no 5 pp 398ndash404 2009
[13] X Miao L-P Tan L-S Tan and X Huang ldquoPorous cal-cium phosphate ceramics modified with PLGA-bioactive glassrdquoMaterials Science and Engineering C vol 27 no 2 pp 274ndash2792007
[14] V Guarino and L Ambrosio ldquoThe synergic effect of polylactidefiber and calcium phosphate particle reinforcement in poly120576-caprolactone-based composite scaffoldsrdquo Acta Biomaterialiavol 4 no 6 pp 1778ndash1787 2008
[15] A E Watts A J Nixon M G Papich H D Sparks and WS Schwark ldquoIn vitro elution of amikacin and ticarcillin froma resorbable self-setting fiber reinforced calcium phosphatecementrdquo Veterinary Surgery vol 40 no 5 pp 563ndash570 2011
[16] K J L Burg S Porter and J F Kellam ldquoBiomaterial develop-ments for bone tissue engineeringrdquo Biomaterials vol 21 no 23pp 2347ndash2359 2000
[17] S Yang K-F Leong Z Du and C-K Chua ldquoThe design ofscaffolds for use in tissue engineering Part I traditional factorsrdquoTissue Engineering vol 7 no 6 pp 679ndash689 2001
[18] K F Leong C M Cheah and C K Chua ldquoSolid freeform fab-rication of three-dimensional scaffolds for engineering replace-ment tissues and organsrdquo Biomaterials vol 24 no 13 pp 2363ndash2378 2003
8 Advances in Materials Science and Engineering
[19] J Russias E Saiz R K Nalla K Gryn R O Ritchie and AP Tomsia ldquoFabrication and mechanical properties of PLAHAcomposites a study of in vitro degradationrdquo Materials Scienceand Engineering C vol 26 no 8 pp 1289ndash1295 2006
[20] R Z Le Geros ldquoCalcium phosphate-based osteoinductivematerialsrdquo Chemical Reviews vol 108 no 11 pp 4742ndash47532008
[21] C E Wen Y Yamada K Shimojima Y Chino H HosokawaandMMabuchi ldquoCompressibility of porous magnesium foamdependency on porosity and pore sizerdquo Materials Letters vol58 no 3-4 pp 357ndash360 2004
[22] L L Hench and J M Polak ldquoThird-generation biomedicalmaterialsrdquo Science vol 295 no 5557 pp 1014ndash1017 2002
[23] D Williams ldquoBenefit and risk in tissue engineeringrdquo MaterialsToday vol 7 no 5 pp 24ndash29 2004
[24] V Guarino F Causa and L Ambrosio ldquoBioactive scaffolds forbone and ligament tissuerdquoExpert Review ofMedicalDevices vol4 no 3 pp 405ndash418 2007
[25] D W Hutmacher J T Schantz C X F Lam K C Tan and TC Lim ldquoState of the art and future directions of scaffold-basedbone engineering from a biomaterials perspectiverdquo Journal oftissue engineering and regenerative medicine vol 1 no 4 pp245ndash260 2007
[26] L Gerhardt and A R Boccaccini ldquoBioactive glass and glass-ceramic scaffolds for bone tissue engineeringrdquoMaterials vol 3pp 3867ndash3910 2010
[27] HH K Xu S Takagi J B Quinn and L C Chow ldquoFast-settingcalcium phosphate scaffolds with tailoredmacropore formationrates for bone regenerationrdquo Journal of Biomedical MaterialsResearch A vol 68 no 4 pp 725ndash734 2004
[28] MAlshaaerHCuypersH Rahier and JWastiels ldquoProductionof monetite-based Inorganic Phosphate Cement (M-IPC) usinghydrothermal post curing (HTPC)rdquo Cement and ConcreteResearch vol 41 no 1 pp 30ndash37 2011
[29] C Wenchuan Z Hongzhi D W Michael B Chongyun andH H K Xu ldquoUmbilical cord stem cells released from alginate-fibrin microbeads inside macroporous and biofunctionalizedcalcium phosphate cement for bone regenerationrdquo Acta Bioma-terialia vol 8 no 6 pp 2297ndash2306 2012
[30] M Alshaaer H Cuypers G Mosselmans H Rahier and JWastiels ldquoEvaluation of a low temperature hardening InorganicPhosphate Cement for high-temperature applicationsrdquo Cementand Concrete Research vol 41 no 1 pp 38ndash45 2011
[31] J Rouwkema N C Rivron and C A van Blitterswijk ldquoVas-cularization in tissue engineeringrdquo Trends in Biotechnology vol26 no 8 pp 434ndash441 2008
[32] M Lovett K Lee A Edwards and D L Kaplan ldquoVasculariza-tion strategies for tissue engineeringrdquo Tissue Engineering B vol15 no 3 pp 353ndash370 2009
[33] X Shi B Sitharaman Q P Pham et al ldquoFabrication of porousultra-short single-walled carbonnanotube nanocomposite scaf-folds for bone tissue engineeringrdquo Biomaterials vol 28 no 28pp 4078ndash4090 2007
[34] C K Kuo andP XMa ldquoIonically crosslinked alginate hydrogelsas scaffolds for tissue engineering Part 1 structure gelation rateand mechanical propertiesrdquo Biomaterials vol 22 no 6 pp 511ndash521 2001
[35] J L Drury R G Dennis and D J Mooney ldquoThe tensile prop-erties of alginate hydrogelsrdquo Biomaterials vol 25 no 16 pp3187ndash3199 2004
[36] C J Damien and J R Parsons ldquoBone graft and bone graftsubstitutes a review of current technology and applicationsrdquoJournal of Applied Biomaterials vol 2 no 3 pp 187ndash208 1991
[37] Y Lei B Rai K H Ho and S H Teoh ldquoIn Vitro degradationof novel bioactive polycaprolactone-20 tricalcium phosphatecomposite scaffolds for bone engineeringrdquo Materials Scienceand Engineering C vol 27 no 2 pp 293ndash298 2007
[38] C R Kothapalli M T Shaw J R Olson and M Wei ldquoFab-rication of novel calcium phosphatepoly(lactic acid) fibercompositesrdquo Journal of Biomedical Materials Research B vol 84no 1 pp 89ndash97 2008
a calibrated reference volume and then expanded in achamber filled with the sample The change of pressure ofthe helium in the known cell volume without and with thespecimen means that the volume of the specimenrsquos mineralmatrix can be determined Dividing themass of the specimenby this volume yields the true density Four samples wereprepared for measuring the variations of skeletal density (byusing helium pycnometry) as a function of temperature Thevolume of each of them is around 6 cm3 The samples werecrushed into small aggregates Afterward they were heatedfor 24 hours at 10∘C before testing The specimens were putimmediately in the testing chamber to avoid any moistureuptake
253 Total Porosity The total porosity of the sintered porousCPC sample was determined using the following equations
120588119861 =119898
119881 (2)
where 120588119861119898 and119881 refer to bulk density mass of the sampleand volume of the sample respectively
119877119863 =120588119861
120588otimes 100 (3)
where 119877119863 is the relative density and 120588o is the skeletal densityfor solid fraction (120588o) obtained by helium pycnometry
Total porosity = 100 minus 119877119863 (4)
The dimensions and the weight of each sample weremeasured and recorded through a vernier caliper and anelectronic balance respectively
26 Compressive Test and Youngrsquos Modulus For the measure-ments of the compressive strength of the porous samplesrubber padswere placed on the top and the bottom surfaces ofeach sample [13] The rubber padded sample was then placedin a CBR tester (controls) to conduct a compressive testThe rubber pads were used to ensure a uniform distributionof the applied load onto the sample A crosshead speed of04mmmin was used for the compressive tests
In thematerialrsquos stress-strain curve there is a linear regionwhere the material follows Hookersquos law Hence the followingequation stands for this region
120590 = 119864120576 (5)
where E refers to Youngrsquos modulus for compression and 120576 isthe strain caused by the compressive stress
27 In Vitro Characterisation of Scaffolds
271 MSCs Cell Isolation MSCs were isolated from humanbone marrow aspirates via density gradient centrifugationThe cells were expanded in nondifferentiating MSC growthmedium (CCM) consisting of 120572-minimal essential medium(120572-MEM) with 10 foetal bovine serum (FBS) 1 penicillin-streptomycin (PEN-STREP) and 2mMglutamine Cellswere
incubated at 37∘C with 5 CO2 After they reached 90
confluence cells were harvested by rinsingwith 025 trypsinand 003 EDTA solution and expanded into a secondpassage until they reached 80 confluence then they weretrypsinized and cryopreserved in a liquid nitrogen Thesecells were designated as passage 1 (P1) cells
272 Osteogenic Differentiation Cells were seeded in 12mul-tiwell plates for an estimated 80 confluence Each experi-ment comprised 12 cultures 6 under osteogenic conditionsand 6 controls under normal cell cultures conditions (CCM)To induce the osteogenic differentiation of the MSCs thecultures were maintained in osteogenic media which consistof 60120583M ascorbic acid 10mM 120573-glycerol phosphate and100 nM dexamethasone The medium was changed every 2-3 days
273 Cells Seeding on the Bioceramic Scaffolds Cryopre-served (P1) cells were replated at a seeding density of 4000cellscm2 in 120572-MEM as described above At near confluencyP2 cells were harvested with 025 trypsin in EDTA andresuspended at a density of 2 times 105 cellsmL 120572-MEM plus1 penicillinstreptomycin The calcium phosphate scaffoldswere sterilized in 70 ethanol for 24 h and then incubatedwith cell culture media containing 10 FBS for at least4 h Then the MSCs were seeded into the scaffolds witha cell seeding density of 200000 cells per scaffold TheMSCswere culturedwith osteogenicmedium and cell culturemedium as a standard control to verify their proliferative anddifferentiation potential at 37∘C and 5 CO
2 The medium
was changed every 3 or 4 days
3 Results and Discussion
31 Microstructural Morphological and Physical Character-ization The aim of this work is designing an injectableand strong porous system using biocompatible materialsable to promote natural body healing which degrade afterimplantation and contain biologically active phases and ableto stimulate the regenerative tissue growth in order to mimicthe morphological and microstructural properties of bonetissue The starting material of the prepared scaffolds istetracalcium phosphate (TTCP Ca
4(PO4)2O)The analysis of
the XRD diffraction peaks (Figure 1 lower pattern) revealedthat the TTCP was formed as the main phase of CPC powderby firing the mixture of pyrocalcium phosphate (Ca
2P2O7)
and calcium carbonate (CaCO3) at 1500∘C for 5 h followed
by quenching in air Moreover this CPC powder contained afew peaks corresponding to HAThe peaks in the lower XRDpattern are strong and sharp which indicate relatively highcrystallinity of the powder The setting time of the CPC wasaround 15min
After seting CPC while the hydroxyapatite phase waspresent in the form of intensive layer of nano-HA crystalsas shown by the SEM image Figure 2(c) some residualTTCP were still left behind Figure 1 (XRD upper pattern)In addition the residual TTCP phase was depleted to agreat extent for the formation of hydroxyapatite due to the
4 Advances in Materials Science and Engineering
TTCPHA
CPC powder
CPC
(cps
)
10 20 30 40 50 60
1000
500
0
2000
1500
1000
500
0
CPC
2120579 scale
Figure 1 XRD patterns of CPC powder (tetracalcium phosphateTTCP) and hardened CPC (1 day after mixing with the hardeningsolution to form calcium phosphate cement) HA is hydroxyapatite
setting reactions to form calcium phosphate cement (CPC)The high backgrounds of the upper XRD pattern (Figure 1)between 30∘ and 35∘ and 45∘ and 55∘ indicate the presence ofamorphous or poorly crystalline CaP phase mainly CDHA(Calcium-deficient HA) as shown by the EDSmeasurementswith CaP ratio of 16
The SEM micrographs of the fabricated scaffold taken atdifferent magnifications are reported in Figure 2 To discussthese morphologies it is important to define three types ofpores fine pores micropores and macropores These poresare distinguished by their size fine pores have a diameterbelow 5 120583mandmicropores have a diameter typically close tothe range of 5ndash30120583m whereas macropores have a diameterabove 70120583m The threshold is placed at a size of 50mmThe rod-like mannitol particles were thus used only as atemplate to form the scaffold framework with the desiredpore structure for bone tissue engineering applications Thisopen internal pore network is necessary tomaximize nutrientdiffusion interstitial fluid and blood flow to control cellgrowth and function tomanipulate tissue differentiation andto optimize scaffold mechanical function and regeneratedtissue mechanical properties [29] Another feature of theporous CPCwas the presence of well-connectedmacroporeswith diameters between 70 120583m and 300 120583m inside the strutsas shown in Figure 2(a)The CPC contained mainly calcium-deficient hydroxyapatite phase with macropores (as a replicaof the dissolved rod-like mannitol particles) in the overallstructure and open micropores in the struts as shown inFigure 2(b) This design of injectable scaffolds with well-connected pores or highly effective porosity intended tofavor an increase in the mass transport of nutrients andoxygen and removal of waste products for cell growth
In the presence of hardening phosphate solution TTCPhydrolyses through a dissolution precipitation reaction givingrise to the formation of an entangled network of CDHAcrystals which are close to the mineral component of thebone from a structural point of view In the proposed scaf-folds the hydrolysis reaction occurs during the setting of thebone cement This is confirmed by XRD and SEM (Figures 1
and 2(c)) which show a large distribution of CDHA needle-like crystals approximately 1 120583m long and homogeneouslydistributed along the inner surface pores (Figure 2(c)) Thesedo not significantly affect the scaffold porosity in terms ofpore shape and interconnection degree [14] Several workshave demonstrated the feasibility of forming new bone inclose contact with calcium-deficient apatite (CDHA) gran-ulesThis creates a network of woven bone bonded to residualcalcium phosphate particles as confirmed by no adverseinflammatory reactions and formation of multinucleatedgiant cells close to the CDHA granules [16]
To confirm the qualitative morphological evaluation per-formed by SEM a quantitative estimation of porosity char-acteristics was performed by mercury intrusion porosimetry(MIP)The pore size distribution and final open pore volumeof representative scaffolds were characterized by low pressure(to avoid damage of pore structure) mercury intrusionporosimetry (MIP) [28 30] (Figure 3) Pore interconnectivityfirst arises by the dissolved mannitol grains and the contactpoint between adjacent grains Figure 2(b) also due to thepresence of smaller pores (up to 30120583m) induced by thephase conversionmechanism [14] Fine pores network can beobserved within these channel walls as shown in Figure 2(c)
MIP showed evidence of a bimodal distribution of poresize with diameter above 5 120583m This pore structure is char-acterized by micropores a few micrometers in diameter andmacropores of the order of hundreds micrometers (see alsoFigure 2(a)) It is apparent that the total open microporeand macropore volume is nearly 30 of the scaffold volumeFigure 3 The calculations and measurements of volumeweight and bulk density show that the total porosity is around64 It was possible to observe Figure 2(b) the characteristicsizes of the smaller pores (with diameters ranging from 5 to30 120583m (microporosity)) peak A and of larger pores peakB (with diameters ranging from sim70 to sim300120583m (macro-porosity)) The pores with diameter above 5120583m representaround 50 of the total open porosity In this structure themacropores are suitable for the accommodation of osteoblastand undifferentiated bone mesenchymal stem cells while themicropores offer interconnection bridges between adjacentmacropores able to promote the nutrient and metaboliteexchange [15ndash18] improving the structural interconnectivity
One of the most important requirements of an ideal bonesubstitute is for it to exhibit mechanical behavior matchingthat of the bone tissue that has to be restored The accuratemimesis of specific mechanical properties such as elasticmodulus may be crucial to the effective reproduction ofthe functional response of natural tissue especially in load-bearing applications In the last decade the use of polymerssuch as PLA PGA and PCL has been a popular strategy forthe provision of biodegradable supports for orthopedic appli-cations [13] However their deficient mechanical propertieslimited their use mainly to a restricted number of non-load-bearing applications [18] Figure 4 shows the representativestress-strain responses of the fabricated CPC scaffold Thisdiagram shows that the scaffold exhibits compressive strengthof 49MPa The e-modulus of the scaffold in compressionsim400MPa was obtained from the slope of the stress-straincurve
Advances in Materials Science and Engineering 5
500120583m
(a)
20120583m
(b)
2120583m
(c)
Figure 2 SEM micrograph of a fracture surface of the porous calcium phosphate scaffold with different magnifications (a) macroporenetwork (b) micropore structure of the internal walls of macropore and (c) matrix of needle-like CDHA (calcium deficient hydroxyapatite)crystals covering the surfaces of the pores
35
3
25
2
15
1
05
01 10 100 1000
Pore diameter (120583m)
Pore
vol
ume (
incr
emen
tal (
)) B
A
Total connected macroporosity 30asymp
Figure 3 Low pressure mercury intrusion porosimetry of CPCscaffold Incremental pore size in above 5120583m
500
450
400
350
300
250
200
150
100
050
0000000 0005 0010 0015 0020
Strain ()
Com
pres
sive s
tress
(MPa
)
Ultimate compressive Slope asymp 400MPa strength
Figure 4 Representative stress-strain responses of the producedscaffold to compression test
The CPC paste can intimately adapt to complex-shapedbone defects and bond to neighboring bone to form afunctional interface The good mechanical properties shownin the present study could enable stem cell delivery to mod-erate load-bearing repairs For example in mandibular andmaxillary ridge augmentation the CPC could be molded to
the desired esthetic shape and then set to form amacroporousscaffold containing stem cells for bone regeneration Theseimplants would be subjected to early loading by provisionaldentures Therefore the macroporous CPC scaffold needs tobe resistant to flexure In addition the CPC paste could beused in major reconstructions of the maxilla or mandibleafter trauma or tumor resection which would require theCPC to be fracture resistant [30] The CPC could also beused to support metal dental implants or augment deficientimplant sites where mechanical properties are importantIt should be noted that to repair large defects mechanicalstrength is only one factor to consider Other factors suchas vascularization are also critically important for success[31 32]Themacroporous CPC in the present study possessedbettermechanical properties than other injectable carriers forcell delivery For example previous studies reported that aninjectable polymeric carrier for cell delivery had a strengthof 07MPa [33] and hydrogels had strengths of about 01MPa[34 35] These systems are promising for non-load-bearingapplications However their strengths are much lower thanthe reported strength of about 35MPa for cancellous bone[36] This macroporous CPC with a strength of 49MPamatched that of cancellous bone and hence may be apromising injectable scaffold for stem cell in orthopedic andcraniofacial applications The bulk density of the scaffold is109 gcm3 and the total porosity is 64 The mechanicaland physical properties of the scaffold were in a range ofthose of cancellous bone Therefore it is suggested that thisscaffold is one of promising scaffold materials for hard tissueregeneration Table 1
In the light of recent approaches in bone regenera-tion based on composite materials the proposed strategycertainly offers the best compromise between structuraland functional properties Porous composite scaffolds madeby incorporating bioactive particles [37] do not assure anadequate improvement in the mechanical performance forbone substitution Meanwhile composites materials formedby integrating high modulus PLA fibers coated with calciumphosphate (CaP) into a PCL matrix [38] show mechanicalproperties that are significantly higher than the valuesreported in the literature for PLA-CaP composites but lack
6 Advances in Materials Science and Engineering
50120583m
50120583m
2120583m
2120583m
5120583m
(a) (b)
(c) (d)
(e) (f)
20120583m
Figure 5 Representative SEM image showing cell attachment to the surface of CPC scaffoldwith cytoplasmic extensions (e) after culturing for4 11 and 21 daysmdashimages (a) (c) and (e) respectively The other 3 images ((b) (d) and (f)) show the development of the calcium phosphatematrix over time
Table 1 Comparison between the CPC scaffold and cancellous bones [19ndash21]
structural porosity In contrast the proposed scaffolds showan intermediate level of mechanical response in porousstructure
32 Preliminary Evaluation of Biocompatibility of ScaffoldsThe injectable CPC scaffolds were prepared for preliminaryin vitro cultivation MSCs were seeded on the 3D scaffoldsand maintained with osteogenic medium for 4 days 11 days
and 21 days After 3 days of culturing cells showed no regularorientation and mineralized nodules (A Figure 5(a)) wereformed in the constructs The size of the cells varies from20120583m to 40120583m Figure 5(a) shows that the cells adhered tothe CPC and developed cytoplasmic extensions (e) After 4days a homogeneous needle-like CDHA matrix covers theinternal surfaces of the scaffold Figure 5(b) After 11 daysof culturing most pores were filled with new tissue mass as
Advances in Materials Science and Engineering 7
observed by SEM (Figure 5(c)) The CDHAmatrices visuallydemonstrated high cell density on the surfaces of the matrixwithin the cylindrical macropores as shown in Figures 5(c)and 5(d)
As reported in Figure 5(c) cells attaching to the nano-sized CDHA crystals that make up the scaffold matrixCell adhesion resulted in elongated and highly stretchedcells within the macropores with focal adhesion points andmulticytoplasmic extensions on the scaffolds after 21 daysof culturing (Figure 5(e)) Moreover cells growth in thescaffolds in culture shows the potential of using this scaffoldarchitecture for MSC seeding and growth The bone matrixconsists of CDHA crystals (Figure 5(f)) containing calciumand phosphorus Therefore these factors provide importantindicators of osteogenic differentiation
4 Conclusions
Engineering tissue with cells and a synthetic extracellularmatrix is an alternative approach to the established practiceof transplantation of harvested tissues Degradable injectableporous scaffolds prepared by calcium phosphate cementrepresent a valid solution to achieving adequate porosityrequirements while providing adequate support in load-bearing applications This proposed process for preparinginjectable scaffolds with two pore networks is as quick andversatile as conventional technologies Using this methodporous CDHA-based calcium phosphate with macroporessizes ranging from 70 to 300120583m micropores ranging from5 to 30 120583m and 30 open macroporosity was preparedThe compressive strength and the e-modulus of the poroushydroxyapatite-based calcium phosphate samples 49MPaand 400MPa respectively were comparable with those ofthe cancellous bone Finally the bioactivity of the scaffoldswas confirmed by cells growth and cytoplasmic extensionsin the scaffolds in culture demonstrating that the scaffoldis suitable for MSC seeding and growth architecture Thiscombination of an interconnected macroporous structurewith pore size has a potential for the promotion of cell seedingand proliferation plus adequate mechanical features andrepresents a porous scaffold which is an excellent candidatefor bone tissue engineering with setting time around 15min
Acknowledgments
The financial support of the project ldquoSynthesis and character-ization of scaffolds for bone tissue engineeringrdquo funded by theDeanship of Academic Research at the University of Jordan isgratefully acknowledged The authors would like to take thisopportunity to thank the Hamdi Mango Center for ScientificResearch (HMCSR) University of Jordan for providing thelaboratory facilities
References
[1] J J Mao G Vunjak-Novakovic A G Mikos and A AtalaRegenerative Medicine Translational Approaches and TissueEngineering Artech House Boston Mass USA 2007
[2] M Bohner ldquoDesign of ceramic-based cements and putties forbone graft substitutionrdquo European Cells and Materials vol 20pp 1ndash12 2010
[3] M-P Ginebra C Canal M Espanol D Pastorino and EB Montufar ldquoCalcium phosphate cements as drug deliverymaterialsrdquo Advanced Drug Delivery Reviews vol 64 pp 1090ndash1010 2012
[4] C S Che Nor Zarida O Fauziah A K Arifah et al ldquoIn vitroelution and dissolution of tobramycin and gentamicin fromcalcium phosphaterdquo African Journal of Pharmacy and Phar-macology vol 5 no 20 pp 2283ndash2291 2011
[5] J JMaoWV Giannobile J AHelms et al ldquoCraniofacial tissueengineering by stem cellsrdquo Journal of Dental Research vol 85no 11 pp 966ndash979 2006
[6] P C Johnson A G Mikos J P Fisher and J A Jansen ldquoStra-tegic directions in tissue engineeringrdquo Tissue Engineering vol13 no 12 pp 2827ndash2837 2007
[7] P Habibovic U Gbureck C J Doillon D C Bassett C A vanBlitterswijk and J E Barralet ldquoOsteoconduction and osteoin-duction of low-temperature 3D printed bioceramic implantsrdquoBiomaterials vol 29 no 7 pp 944ndash953 2008
[8] H SWang S C Hung S T Peng C C Huang HMWei Y JGuo et al ldquoMesenchyma lstem cells in theWhartonrsquos jelly of thehuman umbilical cordrdquo Stem Cells vol 22 pp 1330ndash1337 2004
[9] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007
[10] E Verron I Khairoun J Guicheux and J-M Bouler ldquoCalciumphosphate biomaterials as bone drug delivery systems a reviewrdquoDrug Discovery Today vol 15 no 13-14 pp 547ndash552 2010
[11] A Can and S Karahuseyinoglu ldquoConcise review humanumbilical cord stromawith regard to the source of fetus-derivedstem cellsrdquo Stem Cells vol 25 no 11 pp 2886ndash2895 2007
[12] L Wang M Singh L F Bonewald and M S DetamoreldquoSignalling strategies for osteogenic differentiation of humanumbilical cord mesenchymal stromal cells for 3D bone tissueengineeringrdquo Journal of Tissue Engineering and RegenerativeMedicine vol 3 no 5 pp 398ndash404 2009
[13] X Miao L-P Tan L-S Tan and X Huang ldquoPorous cal-cium phosphate ceramics modified with PLGA-bioactive glassrdquoMaterials Science and Engineering C vol 27 no 2 pp 274ndash2792007
[14] V Guarino and L Ambrosio ldquoThe synergic effect of polylactidefiber and calcium phosphate particle reinforcement in poly120576-caprolactone-based composite scaffoldsrdquo Acta Biomaterialiavol 4 no 6 pp 1778ndash1787 2008
[15] A E Watts A J Nixon M G Papich H D Sparks and WS Schwark ldquoIn vitro elution of amikacin and ticarcillin froma resorbable self-setting fiber reinforced calcium phosphatecementrdquo Veterinary Surgery vol 40 no 5 pp 563ndash570 2011
[16] K J L Burg S Porter and J F Kellam ldquoBiomaterial develop-ments for bone tissue engineeringrdquo Biomaterials vol 21 no 23pp 2347ndash2359 2000
[17] S Yang K-F Leong Z Du and C-K Chua ldquoThe design ofscaffolds for use in tissue engineering Part I traditional factorsrdquoTissue Engineering vol 7 no 6 pp 679ndash689 2001
[18] K F Leong C M Cheah and C K Chua ldquoSolid freeform fab-rication of three-dimensional scaffolds for engineering replace-ment tissues and organsrdquo Biomaterials vol 24 no 13 pp 2363ndash2378 2003
8 Advances in Materials Science and Engineering
[19] J Russias E Saiz R K Nalla K Gryn R O Ritchie and AP Tomsia ldquoFabrication and mechanical properties of PLAHAcomposites a study of in vitro degradationrdquo Materials Scienceand Engineering C vol 26 no 8 pp 1289ndash1295 2006
[20] R Z Le Geros ldquoCalcium phosphate-based osteoinductivematerialsrdquo Chemical Reviews vol 108 no 11 pp 4742ndash47532008
[21] C E Wen Y Yamada K Shimojima Y Chino H HosokawaandMMabuchi ldquoCompressibility of porous magnesium foamdependency on porosity and pore sizerdquo Materials Letters vol58 no 3-4 pp 357ndash360 2004
[22] L L Hench and J M Polak ldquoThird-generation biomedicalmaterialsrdquo Science vol 295 no 5557 pp 1014ndash1017 2002
[23] D Williams ldquoBenefit and risk in tissue engineeringrdquo MaterialsToday vol 7 no 5 pp 24ndash29 2004
[24] V Guarino F Causa and L Ambrosio ldquoBioactive scaffolds forbone and ligament tissuerdquoExpert Review ofMedicalDevices vol4 no 3 pp 405ndash418 2007
[25] D W Hutmacher J T Schantz C X F Lam K C Tan and TC Lim ldquoState of the art and future directions of scaffold-basedbone engineering from a biomaterials perspectiverdquo Journal oftissue engineering and regenerative medicine vol 1 no 4 pp245ndash260 2007
[26] L Gerhardt and A R Boccaccini ldquoBioactive glass and glass-ceramic scaffolds for bone tissue engineeringrdquoMaterials vol 3pp 3867ndash3910 2010
[27] HH K Xu S Takagi J B Quinn and L C Chow ldquoFast-settingcalcium phosphate scaffolds with tailoredmacropore formationrates for bone regenerationrdquo Journal of Biomedical MaterialsResearch A vol 68 no 4 pp 725ndash734 2004
[28] MAlshaaerHCuypersH Rahier and JWastiels ldquoProductionof monetite-based Inorganic Phosphate Cement (M-IPC) usinghydrothermal post curing (HTPC)rdquo Cement and ConcreteResearch vol 41 no 1 pp 30ndash37 2011
[29] C Wenchuan Z Hongzhi D W Michael B Chongyun andH H K Xu ldquoUmbilical cord stem cells released from alginate-fibrin microbeads inside macroporous and biofunctionalizedcalcium phosphate cement for bone regenerationrdquo Acta Bioma-terialia vol 8 no 6 pp 2297ndash2306 2012
[30] M Alshaaer H Cuypers G Mosselmans H Rahier and JWastiels ldquoEvaluation of a low temperature hardening InorganicPhosphate Cement for high-temperature applicationsrdquo Cementand Concrete Research vol 41 no 1 pp 38ndash45 2011
[31] J Rouwkema N C Rivron and C A van Blitterswijk ldquoVas-cularization in tissue engineeringrdquo Trends in Biotechnology vol26 no 8 pp 434ndash441 2008
[32] M Lovett K Lee A Edwards and D L Kaplan ldquoVasculariza-tion strategies for tissue engineeringrdquo Tissue Engineering B vol15 no 3 pp 353ndash370 2009
[33] X Shi B Sitharaman Q P Pham et al ldquoFabrication of porousultra-short single-walled carbonnanotube nanocomposite scaf-folds for bone tissue engineeringrdquo Biomaterials vol 28 no 28pp 4078ndash4090 2007
[34] C K Kuo andP XMa ldquoIonically crosslinked alginate hydrogelsas scaffolds for tissue engineering Part 1 structure gelation rateand mechanical propertiesrdquo Biomaterials vol 22 no 6 pp 511ndash521 2001
[35] J L Drury R G Dennis and D J Mooney ldquoThe tensile prop-erties of alginate hydrogelsrdquo Biomaterials vol 25 no 16 pp3187ndash3199 2004
[36] C J Damien and J R Parsons ldquoBone graft and bone graftsubstitutes a review of current technology and applicationsrdquoJournal of Applied Biomaterials vol 2 no 3 pp 187ndash208 1991
[37] Y Lei B Rai K H Ho and S H Teoh ldquoIn Vitro degradationof novel bioactive polycaprolactone-20 tricalcium phosphatecomposite scaffolds for bone engineeringrdquo Materials Scienceand Engineering C vol 27 no 2 pp 293ndash298 2007
[38] C R Kothapalli M T Shaw J R Olson and M Wei ldquoFab-rication of novel calcium phosphatepoly(lactic acid) fibercompositesrdquo Journal of Biomedical Materials Research B vol 84no 1 pp 89ndash97 2008
Figure 1 XRD patterns of CPC powder (tetracalcium phosphateTTCP) and hardened CPC (1 day after mixing with the hardeningsolution to form calcium phosphate cement) HA is hydroxyapatite
setting reactions to form calcium phosphate cement (CPC)The high backgrounds of the upper XRD pattern (Figure 1)between 30∘ and 35∘ and 45∘ and 55∘ indicate the presence ofamorphous or poorly crystalline CaP phase mainly CDHA(Calcium-deficient HA) as shown by the EDSmeasurementswith CaP ratio of 16
The SEM micrographs of the fabricated scaffold taken atdifferent magnifications are reported in Figure 2 To discussthese morphologies it is important to define three types ofpores fine pores micropores and macropores These poresare distinguished by their size fine pores have a diameterbelow 5 120583mandmicropores have a diameter typically close tothe range of 5ndash30120583m whereas macropores have a diameterabove 70120583m The threshold is placed at a size of 50mmThe rod-like mannitol particles were thus used only as atemplate to form the scaffold framework with the desiredpore structure for bone tissue engineering applications Thisopen internal pore network is necessary tomaximize nutrientdiffusion interstitial fluid and blood flow to control cellgrowth and function tomanipulate tissue differentiation andto optimize scaffold mechanical function and regeneratedtissue mechanical properties [29] Another feature of theporous CPCwas the presence of well-connectedmacroporeswith diameters between 70 120583m and 300 120583m inside the strutsas shown in Figure 2(a)The CPC contained mainly calcium-deficient hydroxyapatite phase with macropores (as a replicaof the dissolved rod-like mannitol particles) in the overallstructure and open micropores in the struts as shown inFigure 2(b) This design of injectable scaffolds with well-connected pores or highly effective porosity intended tofavor an increase in the mass transport of nutrients andoxygen and removal of waste products for cell growth
In the presence of hardening phosphate solution TTCPhydrolyses through a dissolution precipitation reaction givingrise to the formation of an entangled network of CDHAcrystals which are close to the mineral component of thebone from a structural point of view In the proposed scaf-folds the hydrolysis reaction occurs during the setting of thebone cement This is confirmed by XRD and SEM (Figures 1
and 2(c)) which show a large distribution of CDHA needle-like crystals approximately 1 120583m long and homogeneouslydistributed along the inner surface pores (Figure 2(c)) Thesedo not significantly affect the scaffold porosity in terms ofpore shape and interconnection degree [14] Several workshave demonstrated the feasibility of forming new bone inclose contact with calcium-deficient apatite (CDHA) gran-ulesThis creates a network of woven bone bonded to residualcalcium phosphate particles as confirmed by no adverseinflammatory reactions and formation of multinucleatedgiant cells close to the CDHA granules [16]
To confirm the qualitative morphological evaluation per-formed by SEM a quantitative estimation of porosity char-acteristics was performed by mercury intrusion porosimetry(MIP)The pore size distribution and final open pore volumeof representative scaffolds were characterized by low pressure(to avoid damage of pore structure) mercury intrusionporosimetry (MIP) [28 30] (Figure 3) Pore interconnectivityfirst arises by the dissolved mannitol grains and the contactpoint between adjacent grains Figure 2(b) also due to thepresence of smaller pores (up to 30120583m) induced by thephase conversionmechanism [14] Fine pores network can beobserved within these channel walls as shown in Figure 2(c)
MIP showed evidence of a bimodal distribution of poresize with diameter above 5 120583m This pore structure is char-acterized by micropores a few micrometers in diameter andmacropores of the order of hundreds micrometers (see alsoFigure 2(a)) It is apparent that the total open microporeand macropore volume is nearly 30 of the scaffold volumeFigure 3 The calculations and measurements of volumeweight and bulk density show that the total porosity is around64 It was possible to observe Figure 2(b) the characteristicsizes of the smaller pores (with diameters ranging from 5 to30 120583m (microporosity)) peak A and of larger pores peakB (with diameters ranging from sim70 to sim300120583m (macro-porosity)) The pores with diameter above 5120583m representaround 50 of the total open porosity In this structure themacropores are suitable for the accommodation of osteoblastand undifferentiated bone mesenchymal stem cells while themicropores offer interconnection bridges between adjacentmacropores able to promote the nutrient and metaboliteexchange [15ndash18] improving the structural interconnectivity
One of the most important requirements of an ideal bonesubstitute is for it to exhibit mechanical behavior matchingthat of the bone tissue that has to be restored The accuratemimesis of specific mechanical properties such as elasticmodulus may be crucial to the effective reproduction ofthe functional response of natural tissue especially in load-bearing applications In the last decade the use of polymerssuch as PLA PGA and PCL has been a popular strategy forthe provision of biodegradable supports for orthopedic appli-cations [13] However their deficient mechanical propertieslimited their use mainly to a restricted number of non-load-bearing applications [18] Figure 4 shows the representativestress-strain responses of the fabricated CPC scaffold Thisdiagram shows that the scaffold exhibits compressive strengthof 49MPa The e-modulus of the scaffold in compressionsim400MPa was obtained from the slope of the stress-straincurve
Advances in Materials Science and Engineering 5
500120583m
(a)
20120583m
(b)
2120583m
(c)
Figure 2 SEM micrograph of a fracture surface of the porous calcium phosphate scaffold with different magnifications (a) macroporenetwork (b) micropore structure of the internal walls of macropore and (c) matrix of needle-like CDHA (calcium deficient hydroxyapatite)crystals covering the surfaces of the pores
35
3
25
2
15
1
05
01 10 100 1000
Pore diameter (120583m)
Pore
vol
ume (
incr
emen
tal (
)) B
A
Total connected macroporosity 30asymp
Figure 3 Low pressure mercury intrusion porosimetry of CPCscaffold Incremental pore size in above 5120583m
500
450
400
350
300
250
200
150
100
050
0000000 0005 0010 0015 0020
Strain ()
Com
pres
sive s
tress
(MPa
)
Ultimate compressive Slope asymp 400MPa strength
Figure 4 Representative stress-strain responses of the producedscaffold to compression test
The CPC paste can intimately adapt to complex-shapedbone defects and bond to neighboring bone to form afunctional interface The good mechanical properties shownin the present study could enable stem cell delivery to mod-erate load-bearing repairs For example in mandibular andmaxillary ridge augmentation the CPC could be molded to
the desired esthetic shape and then set to form amacroporousscaffold containing stem cells for bone regeneration Theseimplants would be subjected to early loading by provisionaldentures Therefore the macroporous CPC scaffold needs tobe resistant to flexure In addition the CPC paste could beused in major reconstructions of the maxilla or mandibleafter trauma or tumor resection which would require theCPC to be fracture resistant [30] The CPC could also beused to support metal dental implants or augment deficientimplant sites where mechanical properties are importantIt should be noted that to repair large defects mechanicalstrength is only one factor to consider Other factors suchas vascularization are also critically important for success[31 32]Themacroporous CPC in the present study possessedbettermechanical properties than other injectable carriers forcell delivery For example previous studies reported that aninjectable polymeric carrier for cell delivery had a strengthof 07MPa [33] and hydrogels had strengths of about 01MPa[34 35] These systems are promising for non-load-bearingapplications However their strengths are much lower thanthe reported strength of about 35MPa for cancellous bone[36] This macroporous CPC with a strength of 49MPamatched that of cancellous bone and hence may be apromising injectable scaffold for stem cell in orthopedic andcraniofacial applications The bulk density of the scaffold is109 gcm3 and the total porosity is 64 The mechanicaland physical properties of the scaffold were in a range ofthose of cancellous bone Therefore it is suggested that thisscaffold is one of promising scaffold materials for hard tissueregeneration Table 1
In the light of recent approaches in bone regenera-tion based on composite materials the proposed strategycertainly offers the best compromise between structuraland functional properties Porous composite scaffolds madeby incorporating bioactive particles [37] do not assure anadequate improvement in the mechanical performance forbone substitution Meanwhile composites materials formedby integrating high modulus PLA fibers coated with calciumphosphate (CaP) into a PCL matrix [38] show mechanicalproperties that are significantly higher than the valuesreported in the literature for PLA-CaP composites but lack
6 Advances in Materials Science and Engineering
50120583m
50120583m
2120583m
2120583m
5120583m
(a) (b)
(c) (d)
(e) (f)
20120583m
Figure 5 Representative SEM image showing cell attachment to the surface of CPC scaffoldwith cytoplasmic extensions (e) after culturing for4 11 and 21 daysmdashimages (a) (c) and (e) respectively The other 3 images ((b) (d) and (f)) show the development of the calcium phosphatematrix over time
Table 1 Comparison between the CPC scaffold and cancellous bones [19ndash21]
structural porosity In contrast the proposed scaffolds showan intermediate level of mechanical response in porousstructure
32 Preliminary Evaluation of Biocompatibility of ScaffoldsThe injectable CPC scaffolds were prepared for preliminaryin vitro cultivation MSCs were seeded on the 3D scaffoldsand maintained with osteogenic medium for 4 days 11 days
and 21 days After 3 days of culturing cells showed no regularorientation and mineralized nodules (A Figure 5(a)) wereformed in the constructs The size of the cells varies from20120583m to 40120583m Figure 5(a) shows that the cells adhered tothe CPC and developed cytoplasmic extensions (e) After 4days a homogeneous needle-like CDHA matrix covers theinternal surfaces of the scaffold Figure 5(b) After 11 daysof culturing most pores were filled with new tissue mass as
Advances in Materials Science and Engineering 7
observed by SEM (Figure 5(c)) The CDHAmatrices visuallydemonstrated high cell density on the surfaces of the matrixwithin the cylindrical macropores as shown in Figures 5(c)and 5(d)
As reported in Figure 5(c) cells attaching to the nano-sized CDHA crystals that make up the scaffold matrixCell adhesion resulted in elongated and highly stretchedcells within the macropores with focal adhesion points andmulticytoplasmic extensions on the scaffolds after 21 daysof culturing (Figure 5(e)) Moreover cells growth in thescaffolds in culture shows the potential of using this scaffoldarchitecture for MSC seeding and growth The bone matrixconsists of CDHA crystals (Figure 5(f)) containing calciumand phosphorus Therefore these factors provide importantindicators of osteogenic differentiation
4 Conclusions
Engineering tissue with cells and a synthetic extracellularmatrix is an alternative approach to the established practiceof transplantation of harvested tissues Degradable injectableporous scaffolds prepared by calcium phosphate cementrepresent a valid solution to achieving adequate porosityrequirements while providing adequate support in load-bearing applications This proposed process for preparinginjectable scaffolds with two pore networks is as quick andversatile as conventional technologies Using this methodporous CDHA-based calcium phosphate with macroporessizes ranging from 70 to 300120583m micropores ranging from5 to 30 120583m and 30 open macroporosity was preparedThe compressive strength and the e-modulus of the poroushydroxyapatite-based calcium phosphate samples 49MPaand 400MPa respectively were comparable with those ofthe cancellous bone Finally the bioactivity of the scaffoldswas confirmed by cells growth and cytoplasmic extensionsin the scaffolds in culture demonstrating that the scaffoldis suitable for MSC seeding and growth architecture Thiscombination of an interconnected macroporous structurewith pore size has a potential for the promotion of cell seedingand proliferation plus adequate mechanical features andrepresents a porous scaffold which is an excellent candidatefor bone tissue engineering with setting time around 15min
Acknowledgments
The financial support of the project ldquoSynthesis and character-ization of scaffolds for bone tissue engineeringrdquo funded by theDeanship of Academic Research at the University of Jordan isgratefully acknowledged The authors would like to take thisopportunity to thank the Hamdi Mango Center for ScientificResearch (HMCSR) University of Jordan for providing thelaboratory facilities
References
[1] J J Mao G Vunjak-Novakovic A G Mikos and A AtalaRegenerative Medicine Translational Approaches and TissueEngineering Artech House Boston Mass USA 2007
[2] M Bohner ldquoDesign of ceramic-based cements and putties forbone graft substitutionrdquo European Cells and Materials vol 20pp 1ndash12 2010
[3] M-P Ginebra C Canal M Espanol D Pastorino and EB Montufar ldquoCalcium phosphate cements as drug deliverymaterialsrdquo Advanced Drug Delivery Reviews vol 64 pp 1090ndash1010 2012
[4] C S Che Nor Zarida O Fauziah A K Arifah et al ldquoIn vitroelution and dissolution of tobramycin and gentamicin fromcalcium phosphaterdquo African Journal of Pharmacy and Phar-macology vol 5 no 20 pp 2283ndash2291 2011
[5] J JMaoWV Giannobile J AHelms et al ldquoCraniofacial tissueengineering by stem cellsrdquo Journal of Dental Research vol 85no 11 pp 966ndash979 2006
[6] P C Johnson A G Mikos J P Fisher and J A Jansen ldquoStra-tegic directions in tissue engineeringrdquo Tissue Engineering vol13 no 12 pp 2827ndash2837 2007
[7] P Habibovic U Gbureck C J Doillon D C Bassett C A vanBlitterswijk and J E Barralet ldquoOsteoconduction and osteoin-duction of low-temperature 3D printed bioceramic implantsrdquoBiomaterials vol 29 no 7 pp 944ndash953 2008
[8] H SWang S C Hung S T Peng C C Huang HMWei Y JGuo et al ldquoMesenchyma lstem cells in theWhartonrsquos jelly of thehuman umbilical cordrdquo Stem Cells vol 22 pp 1330ndash1337 2004
[9] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007
[10] E Verron I Khairoun J Guicheux and J-M Bouler ldquoCalciumphosphate biomaterials as bone drug delivery systems a reviewrdquoDrug Discovery Today vol 15 no 13-14 pp 547ndash552 2010
[11] A Can and S Karahuseyinoglu ldquoConcise review humanumbilical cord stromawith regard to the source of fetus-derivedstem cellsrdquo Stem Cells vol 25 no 11 pp 2886ndash2895 2007
[12] L Wang M Singh L F Bonewald and M S DetamoreldquoSignalling strategies for osteogenic differentiation of humanumbilical cord mesenchymal stromal cells for 3D bone tissueengineeringrdquo Journal of Tissue Engineering and RegenerativeMedicine vol 3 no 5 pp 398ndash404 2009
[13] X Miao L-P Tan L-S Tan and X Huang ldquoPorous cal-cium phosphate ceramics modified with PLGA-bioactive glassrdquoMaterials Science and Engineering C vol 27 no 2 pp 274ndash2792007
[14] V Guarino and L Ambrosio ldquoThe synergic effect of polylactidefiber and calcium phosphate particle reinforcement in poly120576-caprolactone-based composite scaffoldsrdquo Acta Biomaterialiavol 4 no 6 pp 1778ndash1787 2008
[15] A E Watts A J Nixon M G Papich H D Sparks and WS Schwark ldquoIn vitro elution of amikacin and ticarcillin froma resorbable self-setting fiber reinforced calcium phosphatecementrdquo Veterinary Surgery vol 40 no 5 pp 563ndash570 2011
[16] K J L Burg S Porter and J F Kellam ldquoBiomaterial develop-ments for bone tissue engineeringrdquo Biomaterials vol 21 no 23pp 2347ndash2359 2000
[17] S Yang K-F Leong Z Du and C-K Chua ldquoThe design ofscaffolds for use in tissue engineering Part I traditional factorsrdquoTissue Engineering vol 7 no 6 pp 679ndash689 2001
[18] K F Leong C M Cheah and C K Chua ldquoSolid freeform fab-rication of three-dimensional scaffolds for engineering replace-ment tissues and organsrdquo Biomaterials vol 24 no 13 pp 2363ndash2378 2003
8 Advances in Materials Science and Engineering
[19] J Russias E Saiz R K Nalla K Gryn R O Ritchie and AP Tomsia ldquoFabrication and mechanical properties of PLAHAcomposites a study of in vitro degradationrdquo Materials Scienceand Engineering C vol 26 no 8 pp 1289ndash1295 2006
[20] R Z Le Geros ldquoCalcium phosphate-based osteoinductivematerialsrdquo Chemical Reviews vol 108 no 11 pp 4742ndash47532008
[21] C E Wen Y Yamada K Shimojima Y Chino H HosokawaandMMabuchi ldquoCompressibility of porous magnesium foamdependency on porosity and pore sizerdquo Materials Letters vol58 no 3-4 pp 357ndash360 2004
[22] L L Hench and J M Polak ldquoThird-generation biomedicalmaterialsrdquo Science vol 295 no 5557 pp 1014ndash1017 2002
[23] D Williams ldquoBenefit and risk in tissue engineeringrdquo MaterialsToday vol 7 no 5 pp 24ndash29 2004
[24] V Guarino F Causa and L Ambrosio ldquoBioactive scaffolds forbone and ligament tissuerdquoExpert Review ofMedicalDevices vol4 no 3 pp 405ndash418 2007
[25] D W Hutmacher J T Schantz C X F Lam K C Tan and TC Lim ldquoState of the art and future directions of scaffold-basedbone engineering from a biomaterials perspectiverdquo Journal oftissue engineering and regenerative medicine vol 1 no 4 pp245ndash260 2007
[26] L Gerhardt and A R Boccaccini ldquoBioactive glass and glass-ceramic scaffolds for bone tissue engineeringrdquoMaterials vol 3pp 3867ndash3910 2010
[27] HH K Xu S Takagi J B Quinn and L C Chow ldquoFast-settingcalcium phosphate scaffolds with tailoredmacropore formationrates for bone regenerationrdquo Journal of Biomedical MaterialsResearch A vol 68 no 4 pp 725ndash734 2004
[28] MAlshaaerHCuypersH Rahier and JWastiels ldquoProductionof monetite-based Inorganic Phosphate Cement (M-IPC) usinghydrothermal post curing (HTPC)rdquo Cement and ConcreteResearch vol 41 no 1 pp 30ndash37 2011
[29] C Wenchuan Z Hongzhi D W Michael B Chongyun andH H K Xu ldquoUmbilical cord stem cells released from alginate-fibrin microbeads inside macroporous and biofunctionalizedcalcium phosphate cement for bone regenerationrdquo Acta Bioma-terialia vol 8 no 6 pp 2297ndash2306 2012
[30] M Alshaaer H Cuypers G Mosselmans H Rahier and JWastiels ldquoEvaluation of a low temperature hardening InorganicPhosphate Cement for high-temperature applicationsrdquo Cementand Concrete Research vol 41 no 1 pp 38ndash45 2011
[31] J Rouwkema N C Rivron and C A van Blitterswijk ldquoVas-cularization in tissue engineeringrdquo Trends in Biotechnology vol26 no 8 pp 434ndash441 2008
[32] M Lovett K Lee A Edwards and D L Kaplan ldquoVasculariza-tion strategies for tissue engineeringrdquo Tissue Engineering B vol15 no 3 pp 353ndash370 2009
[33] X Shi B Sitharaman Q P Pham et al ldquoFabrication of porousultra-short single-walled carbonnanotube nanocomposite scaf-folds for bone tissue engineeringrdquo Biomaterials vol 28 no 28pp 4078ndash4090 2007
[34] C K Kuo andP XMa ldquoIonically crosslinked alginate hydrogelsas scaffolds for tissue engineering Part 1 structure gelation rateand mechanical propertiesrdquo Biomaterials vol 22 no 6 pp 511ndash521 2001
[35] J L Drury R G Dennis and D J Mooney ldquoThe tensile prop-erties of alginate hydrogelsrdquo Biomaterials vol 25 no 16 pp3187ndash3199 2004
[36] C J Damien and J R Parsons ldquoBone graft and bone graftsubstitutes a review of current technology and applicationsrdquoJournal of Applied Biomaterials vol 2 no 3 pp 187ndash208 1991
[37] Y Lei B Rai K H Ho and S H Teoh ldquoIn Vitro degradationof novel bioactive polycaprolactone-20 tricalcium phosphatecomposite scaffolds for bone engineeringrdquo Materials Scienceand Engineering C vol 27 no 2 pp 293ndash298 2007
[38] C R Kothapalli M T Shaw J R Olson and M Wei ldquoFab-rication of novel calcium phosphatepoly(lactic acid) fibercompositesrdquo Journal of Biomedical Materials Research B vol 84no 1 pp 89ndash97 2008
Figure 2 SEM micrograph of a fracture surface of the porous calcium phosphate scaffold with different magnifications (a) macroporenetwork (b) micropore structure of the internal walls of macropore and (c) matrix of needle-like CDHA (calcium deficient hydroxyapatite)crystals covering the surfaces of the pores
35
3
25
2
15
1
05
01 10 100 1000
Pore diameter (120583m)
Pore
vol
ume (
incr
emen
tal (
)) B
A
Total connected macroporosity 30asymp
Figure 3 Low pressure mercury intrusion porosimetry of CPCscaffold Incremental pore size in above 5120583m
500
450
400
350
300
250
200
150
100
050
0000000 0005 0010 0015 0020
Strain ()
Com
pres
sive s
tress
(MPa
)
Ultimate compressive Slope asymp 400MPa strength
Figure 4 Representative stress-strain responses of the producedscaffold to compression test
The CPC paste can intimately adapt to complex-shapedbone defects and bond to neighboring bone to form afunctional interface The good mechanical properties shownin the present study could enable stem cell delivery to mod-erate load-bearing repairs For example in mandibular andmaxillary ridge augmentation the CPC could be molded to
the desired esthetic shape and then set to form amacroporousscaffold containing stem cells for bone regeneration Theseimplants would be subjected to early loading by provisionaldentures Therefore the macroporous CPC scaffold needs tobe resistant to flexure In addition the CPC paste could beused in major reconstructions of the maxilla or mandibleafter trauma or tumor resection which would require theCPC to be fracture resistant [30] The CPC could also beused to support metal dental implants or augment deficientimplant sites where mechanical properties are importantIt should be noted that to repair large defects mechanicalstrength is only one factor to consider Other factors suchas vascularization are also critically important for success[31 32]Themacroporous CPC in the present study possessedbettermechanical properties than other injectable carriers forcell delivery For example previous studies reported that aninjectable polymeric carrier for cell delivery had a strengthof 07MPa [33] and hydrogels had strengths of about 01MPa[34 35] These systems are promising for non-load-bearingapplications However their strengths are much lower thanthe reported strength of about 35MPa for cancellous bone[36] This macroporous CPC with a strength of 49MPamatched that of cancellous bone and hence may be apromising injectable scaffold for stem cell in orthopedic andcraniofacial applications The bulk density of the scaffold is109 gcm3 and the total porosity is 64 The mechanicaland physical properties of the scaffold were in a range ofthose of cancellous bone Therefore it is suggested that thisscaffold is one of promising scaffold materials for hard tissueregeneration Table 1
In the light of recent approaches in bone regenera-tion based on composite materials the proposed strategycertainly offers the best compromise between structuraland functional properties Porous composite scaffolds madeby incorporating bioactive particles [37] do not assure anadequate improvement in the mechanical performance forbone substitution Meanwhile composites materials formedby integrating high modulus PLA fibers coated with calciumphosphate (CaP) into a PCL matrix [38] show mechanicalproperties that are significantly higher than the valuesreported in the literature for PLA-CaP composites but lack
6 Advances in Materials Science and Engineering
50120583m
50120583m
2120583m
2120583m
5120583m
(a) (b)
(c) (d)
(e) (f)
20120583m
Figure 5 Representative SEM image showing cell attachment to the surface of CPC scaffoldwith cytoplasmic extensions (e) after culturing for4 11 and 21 daysmdashimages (a) (c) and (e) respectively The other 3 images ((b) (d) and (f)) show the development of the calcium phosphatematrix over time
Table 1 Comparison between the CPC scaffold and cancellous bones [19ndash21]
structural porosity In contrast the proposed scaffolds showan intermediate level of mechanical response in porousstructure
32 Preliminary Evaluation of Biocompatibility of ScaffoldsThe injectable CPC scaffolds were prepared for preliminaryin vitro cultivation MSCs were seeded on the 3D scaffoldsand maintained with osteogenic medium for 4 days 11 days
and 21 days After 3 days of culturing cells showed no regularorientation and mineralized nodules (A Figure 5(a)) wereformed in the constructs The size of the cells varies from20120583m to 40120583m Figure 5(a) shows that the cells adhered tothe CPC and developed cytoplasmic extensions (e) After 4days a homogeneous needle-like CDHA matrix covers theinternal surfaces of the scaffold Figure 5(b) After 11 daysof culturing most pores were filled with new tissue mass as
Advances in Materials Science and Engineering 7
observed by SEM (Figure 5(c)) The CDHAmatrices visuallydemonstrated high cell density on the surfaces of the matrixwithin the cylindrical macropores as shown in Figures 5(c)and 5(d)
As reported in Figure 5(c) cells attaching to the nano-sized CDHA crystals that make up the scaffold matrixCell adhesion resulted in elongated and highly stretchedcells within the macropores with focal adhesion points andmulticytoplasmic extensions on the scaffolds after 21 daysof culturing (Figure 5(e)) Moreover cells growth in thescaffolds in culture shows the potential of using this scaffoldarchitecture for MSC seeding and growth The bone matrixconsists of CDHA crystals (Figure 5(f)) containing calciumand phosphorus Therefore these factors provide importantindicators of osteogenic differentiation
4 Conclusions
Engineering tissue with cells and a synthetic extracellularmatrix is an alternative approach to the established practiceof transplantation of harvested tissues Degradable injectableporous scaffolds prepared by calcium phosphate cementrepresent a valid solution to achieving adequate porosityrequirements while providing adequate support in load-bearing applications This proposed process for preparinginjectable scaffolds with two pore networks is as quick andversatile as conventional technologies Using this methodporous CDHA-based calcium phosphate with macroporessizes ranging from 70 to 300120583m micropores ranging from5 to 30 120583m and 30 open macroporosity was preparedThe compressive strength and the e-modulus of the poroushydroxyapatite-based calcium phosphate samples 49MPaand 400MPa respectively were comparable with those ofthe cancellous bone Finally the bioactivity of the scaffoldswas confirmed by cells growth and cytoplasmic extensionsin the scaffolds in culture demonstrating that the scaffoldis suitable for MSC seeding and growth architecture Thiscombination of an interconnected macroporous structurewith pore size has a potential for the promotion of cell seedingand proliferation plus adequate mechanical features andrepresents a porous scaffold which is an excellent candidatefor bone tissue engineering with setting time around 15min
Acknowledgments
The financial support of the project ldquoSynthesis and character-ization of scaffolds for bone tissue engineeringrdquo funded by theDeanship of Academic Research at the University of Jordan isgratefully acknowledged The authors would like to take thisopportunity to thank the Hamdi Mango Center for ScientificResearch (HMCSR) University of Jordan for providing thelaboratory facilities
References
[1] J J Mao G Vunjak-Novakovic A G Mikos and A AtalaRegenerative Medicine Translational Approaches and TissueEngineering Artech House Boston Mass USA 2007
[2] M Bohner ldquoDesign of ceramic-based cements and putties forbone graft substitutionrdquo European Cells and Materials vol 20pp 1ndash12 2010
[3] M-P Ginebra C Canal M Espanol D Pastorino and EB Montufar ldquoCalcium phosphate cements as drug deliverymaterialsrdquo Advanced Drug Delivery Reviews vol 64 pp 1090ndash1010 2012
[4] C S Che Nor Zarida O Fauziah A K Arifah et al ldquoIn vitroelution and dissolution of tobramycin and gentamicin fromcalcium phosphaterdquo African Journal of Pharmacy and Phar-macology vol 5 no 20 pp 2283ndash2291 2011
[5] J JMaoWV Giannobile J AHelms et al ldquoCraniofacial tissueengineering by stem cellsrdquo Journal of Dental Research vol 85no 11 pp 966ndash979 2006
[6] P C Johnson A G Mikos J P Fisher and J A Jansen ldquoStra-tegic directions in tissue engineeringrdquo Tissue Engineering vol13 no 12 pp 2827ndash2837 2007
[7] P Habibovic U Gbureck C J Doillon D C Bassett C A vanBlitterswijk and J E Barralet ldquoOsteoconduction and osteoin-duction of low-temperature 3D printed bioceramic implantsrdquoBiomaterials vol 29 no 7 pp 944ndash953 2008
[8] H SWang S C Hung S T Peng C C Huang HMWei Y JGuo et al ldquoMesenchyma lstem cells in theWhartonrsquos jelly of thehuman umbilical cordrdquo Stem Cells vol 22 pp 1330ndash1337 2004
[9] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007
[10] E Verron I Khairoun J Guicheux and J-M Bouler ldquoCalciumphosphate biomaterials as bone drug delivery systems a reviewrdquoDrug Discovery Today vol 15 no 13-14 pp 547ndash552 2010
[11] A Can and S Karahuseyinoglu ldquoConcise review humanumbilical cord stromawith regard to the source of fetus-derivedstem cellsrdquo Stem Cells vol 25 no 11 pp 2886ndash2895 2007
[12] L Wang M Singh L F Bonewald and M S DetamoreldquoSignalling strategies for osteogenic differentiation of humanumbilical cord mesenchymal stromal cells for 3D bone tissueengineeringrdquo Journal of Tissue Engineering and RegenerativeMedicine vol 3 no 5 pp 398ndash404 2009
[13] X Miao L-P Tan L-S Tan and X Huang ldquoPorous cal-cium phosphate ceramics modified with PLGA-bioactive glassrdquoMaterials Science and Engineering C vol 27 no 2 pp 274ndash2792007
[14] V Guarino and L Ambrosio ldquoThe synergic effect of polylactidefiber and calcium phosphate particle reinforcement in poly120576-caprolactone-based composite scaffoldsrdquo Acta Biomaterialiavol 4 no 6 pp 1778ndash1787 2008
[15] A E Watts A J Nixon M G Papich H D Sparks and WS Schwark ldquoIn vitro elution of amikacin and ticarcillin froma resorbable self-setting fiber reinforced calcium phosphatecementrdquo Veterinary Surgery vol 40 no 5 pp 563ndash570 2011
[16] K J L Burg S Porter and J F Kellam ldquoBiomaterial develop-ments for bone tissue engineeringrdquo Biomaterials vol 21 no 23pp 2347ndash2359 2000
[17] S Yang K-F Leong Z Du and C-K Chua ldquoThe design ofscaffolds for use in tissue engineering Part I traditional factorsrdquoTissue Engineering vol 7 no 6 pp 679ndash689 2001
[18] K F Leong C M Cheah and C K Chua ldquoSolid freeform fab-rication of three-dimensional scaffolds for engineering replace-ment tissues and organsrdquo Biomaterials vol 24 no 13 pp 2363ndash2378 2003
8 Advances in Materials Science and Engineering
[19] J Russias E Saiz R K Nalla K Gryn R O Ritchie and AP Tomsia ldquoFabrication and mechanical properties of PLAHAcomposites a study of in vitro degradationrdquo Materials Scienceand Engineering C vol 26 no 8 pp 1289ndash1295 2006
[20] R Z Le Geros ldquoCalcium phosphate-based osteoinductivematerialsrdquo Chemical Reviews vol 108 no 11 pp 4742ndash47532008
[21] C E Wen Y Yamada K Shimojima Y Chino H HosokawaandMMabuchi ldquoCompressibility of porous magnesium foamdependency on porosity and pore sizerdquo Materials Letters vol58 no 3-4 pp 357ndash360 2004
[22] L L Hench and J M Polak ldquoThird-generation biomedicalmaterialsrdquo Science vol 295 no 5557 pp 1014ndash1017 2002
[23] D Williams ldquoBenefit and risk in tissue engineeringrdquo MaterialsToday vol 7 no 5 pp 24ndash29 2004
[24] V Guarino F Causa and L Ambrosio ldquoBioactive scaffolds forbone and ligament tissuerdquoExpert Review ofMedicalDevices vol4 no 3 pp 405ndash418 2007
[25] D W Hutmacher J T Schantz C X F Lam K C Tan and TC Lim ldquoState of the art and future directions of scaffold-basedbone engineering from a biomaterials perspectiverdquo Journal oftissue engineering and regenerative medicine vol 1 no 4 pp245ndash260 2007
[26] L Gerhardt and A R Boccaccini ldquoBioactive glass and glass-ceramic scaffolds for bone tissue engineeringrdquoMaterials vol 3pp 3867ndash3910 2010
[27] HH K Xu S Takagi J B Quinn and L C Chow ldquoFast-settingcalcium phosphate scaffolds with tailoredmacropore formationrates for bone regenerationrdquo Journal of Biomedical MaterialsResearch A vol 68 no 4 pp 725ndash734 2004
[28] MAlshaaerHCuypersH Rahier and JWastiels ldquoProductionof monetite-based Inorganic Phosphate Cement (M-IPC) usinghydrothermal post curing (HTPC)rdquo Cement and ConcreteResearch vol 41 no 1 pp 30ndash37 2011
[29] C Wenchuan Z Hongzhi D W Michael B Chongyun andH H K Xu ldquoUmbilical cord stem cells released from alginate-fibrin microbeads inside macroporous and biofunctionalizedcalcium phosphate cement for bone regenerationrdquo Acta Bioma-terialia vol 8 no 6 pp 2297ndash2306 2012
[30] M Alshaaer H Cuypers G Mosselmans H Rahier and JWastiels ldquoEvaluation of a low temperature hardening InorganicPhosphate Cement for high-temperature applicationsrdquo Cementand Concrete Research vol 41 no 1 pp 38ndash45 2011
[31] J Rouwkema N C Rivron and C A van Blitterswijk ldquoVas-cularization in tissue engineeringrdquo Trends in Biotechnology vol26 no 8 pp 434ndash441 2008
[32] M Lovett K Lee A Edwards and D L Kaplan ldquoVasculariza-tion strategies for tissue engineeringrdquo Tissue Engineering B vol15 no 3 pp 353ndash370 2009
[33] X Shi B Sitharaman Q P Pham et al ldquoFabrication of porousultra-short single-walled carbonnanotube nanocomposite scaf-folds for bone tissue engineeringrdquo Biomaterials vol 28 no 28pp 4078ndash4090 2007
[34] C K Kuo andP XMa ldquoIonically crosslinked alginate hydrogelsas scaffolds for tissue engineering Part 1 structure gelation rateand mechanical propertiesrdquo Biomaterials vol 22 no 6 pp 511ndash521 2001
[35] J L Drury R G Dennis and D J Mooney ldquoThe tensile prop-erties of alginate hydrogelsrdquo Biomaterials vol 25 no 16 pp3187ndash3199 2004
[36] C J Damien and J R Parsons ldquoBone graft and bone graftsubstitutes a review of current technology and applicationsrdquoJournal of Applied Biomaterials vol 2 no 3 pp 187ndash208 1991
[37] Y Lei B Rai K H Ho and S H Teoh ldquoIn Vitro degradationof novel bioactive polycaprolactone-20 tricalcium phosphatecomposite scaffolds for bone engineeringrdquo Materials Scienceand Engineering C vol 27 no 2 pp 293ndash298 2007
[38] C R Kothapalli M T Shaw J R Olson and M Wei ldquoFab-rication of novel calcium phosphatepoly(lactic acid) fibercompositesrdquo Journal of Biomedical Materials Research B vol 84no 1 pp 89ndash97 2008
Figure 5 Representative SEM image showing cell attachment to the surface of CPC scaffoldwith cytoplasmic extensions (e) after culturing for4 11 and 21 daysmdashimages (a) (c) and (e) respectively The other 3 images ((b) (d) and (f)) show the development of the calcium phosphatematrix over time
Table 1 Comparison between the CPC scaffold and cancellous bones [19ndash21]
structural porosity In contrast the proposed scaffolds showan intermediate level of mechanical response in porousstructure
32 Preliminary Evaluation of Biocompatibility of ScaffoldsThe injectable CPC scaffolds were prepared for preliminaryin vitro cultivation MSCs were seeded on the 3D scaffoldsand maintained with osteogenic medium for 4 days 11 days
and 21 days After 3 days of culturing cells showed no regularorientation and mineralized nodules (A Figure 5(a)) wereformed in the constructs The size of the cells varies from20120583m to 40120583m Figure 5(a) shows that the cells adhered tothe CPC and developed cytoplasmic extensions (e) After 4days a homogeneous needle-like CDHA matrix covers theinternal surfaces of the scaffold Figure 5(b) After 11 daysof culturing most pores were filled with new tissue mass as
Advances in Materials Science and Engineering 7
observed by SEM (Figure 5(c)) The CDHAmatrices visuallydemonstrated high cell density on the surfaces of the matrixwithin the cylindrical macropores as shown in Figures 5(c)and 5(d)
As reported in Figure 5(c) cells attaching to the nano-sized CDHA crystals that make up the scaffold matrixCell adhesion resulted in elongated and highly stretchedcells within the macropores with focal adhesion points andmulticytoplasmic extensions on the scaffolds after 21 daysof culturing (Figure 5(e)) Moreover cells growth in thescaffolds in culture shows the potential of using this scaffoldarchitecture for MSC seeding and growth The bone matrixconsists of CDHA crystals (Figure 5(f)) containing calciumand phosphorus Therefore these factors provide importantindicators of osteogenic differentiation
4 Conclusions
Engineering tissue with cells and a synthetic extracellularmatrix is an alternative approach to the established practiceof transplantation of harvested tissues Degradable injectableporous scaffolds prepared by calcium phosphate cementrepresent a valid solution to achieving adequate porosityrequirements while providing adequate support in load-bearing applications This proposed process for preparinginjectable scaffolds with two pore networks is as quick andversatile as conventional technologies Using this methodporous CDHA-based calcium phosphate with macroporessizes ranging from 70 to 300120583m micropores ranging from5 to 30 120583m and 30 open macroporosity was preparedThe compressive strength and the e-modulus of the poroushydroxyapatite-based calcium phosphate samples 49MPaand 400MPa respectively were comparable with those ofthe cancellous bone Finally the bioactivity of the scaffoldswas confirmed by cells growth and cytoplasmic extensionsin the scaffolds in culture demonstrating that the scaffoldis suitable for MSC seeding and growth architecture Thiscombination of an interconnected macroporous structurewith pore size has a potential for the promotion of cell seedingand proliferation plus adequate mechanical features andrepresents a porous scaffold which is an excellent candidatefor bone tissue engineering with setting time around 15min
Acknowledgments
The financial support of the project ldquoSynthesis and character-ization of scaffolds for bone tissue engineeringrdquo funded by theDeanship of Academic Research at the University of Jordan isgratefully acknowledged The authors would like to take thisopportunity to thank the Hamdi Mango Center for ScientificResearch (HMCSR) University of Jordan for providing thelaboratory facilities
References
[1] J J Mao G Vunjak-Novakovic A G Mikos and A AtalaRegenerative Medicine Translational Approaches and TissueEngineering Artech House Boston Mass USA 2007
[2] M Bohner ldquoDesign of ceramic-based cements and putties forbone graft substitutionrdquo European Cells and Materials vol 20pp 1ndash12 2010
[3] M-P Ginebra C Canal M Espanol D Pastorino and EB Montufar ldquoCalcium phosphate cements as drug deliverymaterialsrdquo Advanced Drug Delivery Reviews vol 64 pp 1090ndash1010 2012
[4] C S Che Nor Zarida O Fauziah A K Arifah et al ldquoIn vitroelution and dissolution of tobramycin and gentamicin fromcalcium phosphaterdquo African Journal of Pharmacy and Phar-macology vol 5 no 20 pp 2283ndash2291 2011
[5] J JMaoWV Giannobile J AHelms et al ldquoCraniofacial tissueengineering by stem cellsrdquo Journal of Dental Research vol 85no 11 pp 966ndash979 2006
[6] P C Johnson A G Mikos J P Fisher and J A Jansen ldquoStra-tegic directions in tissue engineeringrdquo Tissue Engineering vol13 no 12 pp 2827ndash2837 2007
[7] P Habibovic U Gbureck C J Doillon D C Bassett C A vanBlitterswijk and J E Barralet ldquoOsteoconduction and osteoin-duction of low-temperature 3D printed bioceramic implantsrdquoBiomaterials vol 29 no 7 pp 944ndash953 2008
[8] H SWang S C Hung S T Peng C C Huang HMWei Y JGuo et al ldquoMesenchyma lstem cells in theWhartonrsquos jelly of thehuman umbilical cordrdquo Stem Cells vol 22 pp 1330ndash1337 2004
[9] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007
[10] E Verron I Khairoun J Guicheux and J-M Bouler ldquoCalciumphosphate biomaterials as bone drug delivery systems a reviewrdquoDrug Discovery Today vol 15 no 13-14 pp 547ndash552 2010
[11] A Can and S Karahuseyinoglu ldquoConcise review humanumbilical cord stromawith regard to the source of fetus-derivedstem cellsrdquo Stem Cells vol 25 no 11 pp 2886ndash2895 2007
[12] L Wang M Singh L F Bonewald and M S DetamoreldquoSignalling strategies for osteogenic differentiation of humanumbilical cord mesenchymal stromal cells for 3D bone tissueengineeringrdquo Journal of Tissue Engineering and RegenerativeMedicine vol 3 no 5 pp 398ndash404 2009
[13] X Miao L-P Tan L-S Tan and X Huang ldquoPorous cal-cium phosphate ceramics modified with PLGA-bioactive glassrdquoMaterials Science and Engineering C vol 27 no 2 pp 274ndash2792007
[14] V Guarino and L Ambrosio ldquoThe synergic effect of polylactidefiber and calcium phosphate particle reinforcement in poly120576-caprolactone-based composite scaffoldsrdquo Acta Biomaterialiavol 4 no 6 pp 1778ndash1787 2008
[15] A E Watts A J Nixon M G Papich H D Sparks and WS Schwark ldquoIn vitro elution of amikacin and ticarcillin froma resorbable self-setting fiber reinforced calcium phosphatecementrdquo Veterinary Surgery vol 40 no 5 pp 563ndash570 2011
[16] K J L Burg S Porter and J F Kellam ldquoBiomaterial develop-ments for bone tissue engineeringrdquo Biomaterials vol 21 no 23pp 2347ndash2359 2000
[17] S Yang K-F Leong Z Du and C-K Chua ldquoThe design ofscaffolds for use in tissue engineering Part I traditional factorsrdquoTissue Engineering vol 7 no 6 pp 679ndash689 2001
[18] K F Leong C M Cheah and C K Chua ldquoSolid freeform fab-rication of three-dimensional scaffolds for engineering replace-ment tissues and organsrdquo Biomaterials vol 24 no 13 pp 2363ndash2378 2003
8 Advances in Materials Science and Engineering
[19] J Russias E Saiz R K Nalla K Gryn R O Ritchie and AP Tomsia ldquoFabrication and mechanical properties of PLAHAcomposites a study of in vitro degradationrdquo Materials Scienceand Engineering C vol 26 no 8 pp 1289ndash1295 2006
[20] R Z Le Geros ldquoCalcium phosphate-based osteoinductivematerialsrdquo Chemical Reviews vol 108 no 11 pp 4742ndash47532008
[21] C E Wen Y Yamada K Shimojima Y Chino H HosokawaandMMabuchi ldquoCompressibility of porous magnesium foamdependency on porosity and pore sizerdquo Materials Letters vol58 no 3-4 pp 357ndash360 2004
[22] L L Hench and J M Polak ldquoThird-generation biomedicalmaterialsrdquo Science vol 295 no 5557 pp 1014ndash1017 2002
[23] D Williams ldquoBenefit and risk in tissue engineeringrdquo MaterialsToday vol 7 no 5 pp 24ndash29 2004
[24] V Guarino F Causa and L Ambrosio ldquoBioactive scaffolds forbone and ligament tissuerdquoExpert Review ofMedicalDevices vol4 no 3 pp 405ndash418 2007
[25] D W Hutmacher J T Schantz C X F Lam K C Tan and TC Lim ldquoState of the art and future directions of scaffold-basedbone engineering from a biomaterials perspectiverdquo Journal oftissue engineering and regenerative medicine vol 1 no 4 pp245ndash260 2007
[26] L Gerhardt and A R Boccaccini ldquoBioactive glass and glass-ceramic scaffolds for bone tissue engineeringrdquoMaterials vol 3pp 3867ndash3910 2010
[27] HH K Xu S Takagi J B Quinn and L C Chow ldquoFast-settingcalcium phosphate scaffolds with tailoredmacropore formationrates for bone regenerationrdquo Journal of Biomedical MaterialsResearch A vol 68 no 4 pp 725ndash734 2004
[28] MAlshaaerHCuypersH Rahier and JWastiels ldquoProductionof monetite-based Inorganic Phosphate Cement (M-IPC) usinghydrothermal post curing (HTPC)rdquo Cement and ConcreteResearch vol 41 no 1 pp 30ndash37 2011
[29] C Wenchuan Z Hongzhi D W Michael B Chongyun andH H K Xu ldquoUmbilical cord stem cells released from alginate-fibrin microbeads inside macroporous and biofunctionalizedcalcium phosphate cement for bone regenerationrdquo Acta Bioma-terialia vol 8 no 6 pp 2297ndash2306 2012
[30] M Alshaaer H Cuypers G Mosselmans H Rahier and JWastiels ldquoEvaluation of a low temperature hardening InorganicPhosphate Cement for high-temperature applicationsrdquo Cementand Concrete Research vol 41 no 1 pp 38ndash45 2011
[31] J Rouwkema N C Rivron and C A van Blitterswijk ldquoVas-cularization in tissue engineeringrdquo Trends in Biotechnology vol26 no 8 pp 434ndash441 2008
[32] M Lovett K Lee A Edwards and D L Kaplan ldquoVasculariza-tion strategies for tissue engineeringrdquo Tissue Engineering B vol15 no 3 pp 353ndash370 2009
[33] X Shi B Sitharaman Q P Pham et al ldquoFabrication of porousultra-short single-walled carbonnanotube nanocomposite scaf-folds for bone tissue engineeringrdquo Biomaterials vol 28 no 28pp 4078ndash4090 2007
[34] C K Kuo andP XMa ldquoIonically crosslinked alginate hydrogelsas scaffolds for tissue engineering Part 1 structure gelation rateand mechanical propertiesrdquo Biomaterials vol 22 no 6 pp 511ndash521 2001
[35] J L Drury R G Dennis and D J Mooney ldquoThe tensile prop-erties of alginate hydrogelsrdquo Biomaterials vol 25 no 16 pp3187ndash3199 2004
[36] C J Damien and J R Parsons ldquoBone graft and bone graftsubstitutes a review of current technology and applicationsrdquoJournal of Applied Biomaterials vol 2 no 3 pp 187ndash208 1991
[37] Y Lei B Rai K H Ho and S H Teoh ldquoIn Vitro degradationof novel bioactive polycaprolactone-20 tricalcium phosphatecomposite scaffolds for bone engineeringrdquo Materials Scienceand Engineering C vol 27 no 2 pp 293ndash298 2007
[38] C R Kothapalli M T Shaw J R Olson and M Wei ldquoFab-rication of novel calcium phosphatepoly(lactic acid) fibercompositesrdquo Journal of Biomedical Materials Research B vol 84no 1 pp 89ndash97 2008
observed by SEM (Figure 5(c)) The CDHAmatrices visuallydemonstrated high cell density on the surfaces of the matrixwithin the cylindrical macropores as shown in Figures 5(c)and 5(d)
As reported in Figure 5(c) cells attaching to the nano-sized CDHA crystals that make up the scaffold matrixCell adhesion resulted in elongated and highly stretchedcells within the macropores with focal adhesion points andmulticytoplasmic extensions on the scaffolds after 21 daysof culturing (Figure 5(e)) Moreover cells growth in thescaffolds in culture shows the potential of using this scaffoldarchitecture for MSC seeding and growth The bone matrixconsists of CDHA crystals (Figure 5(f)) containing calciumand phosphorus Therefore these factors provide importantindicators of osteogenic differentiation
4 Conclusions
Engineering tissue with cells and a synthetic extracellularmatrix is an alternative approach to the established practiceof transplantation of harvested tissues Degradable injectableporous scaffolds prepared by calcium phosphate cementrepresent a valid solution to achieving adequate porosityrequirements while providing adequate support in load-bearing applications This proposed process for preparinginjectable scaffolds with two pore networks is as quick andversatile as conventional technologies Using this methodporous CDHA-based calcium phosphate with macroporessizes ranging from 70 to 300120583m micropores ranging from5 to 30 120583m and 30 open macroporosity was preparedThe compressive strength and the e-modulus of the poroushydroxyapatite-based calcium phosphate samples 49MPaand 400MPa respectively were comparable with those ofthe cancellous bone Finally the bioactivity of the scaffoldswas confirmed by cells growth and cytoplasmic extensionsin the scaffolds in culture demonstrating that the scaffoldis suitable for MSC seeding and growth architecture Thiscombination of an interconnected macroporous structurewith pore size has a potential for the promotion of cell seedingand proliferation plus adequate mechanical features andrepresents a porous scaffold which is an excellent candidatefor bone tissue engineering with setting time around 15min
Acknowledgments
The financial support of the project ldquoSynthesis and character-ization of scaffolds for bone tissue engineeringrdquo funded by theDeanship of Academic Research at the University of Jordan isgratefully acknowledged The authors would like to take thisopportunity to thank the Hamdi Mango Center for ScientificResearch (HMCSR) University of Jordan for providing thelaboratory facilities
References
[1] J J Mao G Vunjak-Novakovic A G Mikos and A AtalaRegenerative Medicine Translational Approaches and TissueEngineering Artech House Boston Mass USA 2007
[2] M Bohner ldquoDesign of ceramic-based cements and putties forbone graft substitutionrdquo European Cells and Materials vol 20pp 1ndash12 2010
[3] M-P Ginebra C Canal M Espanol D Pastorino and EB Montufar ldquoCalcium phosphate cements as drug deliverymaterialsrdquo Advanced Drug Delivery Reviews vol 64 pp 1090ndash1010 2012
[4] C S Che Nor Zarida O Fauziah A K Arifah et al ldquoIn vitroelution and dissolution of tobramycin and gentamicin fromcalcium phosphaterdquo African Journal of Pharmacy and Phar-macology vol 5 no 20 pp 2283ndash2291 2011
[5] J JMaoWV Giannobile J AHelms et al ldquoCraniofacial tissueengineering by stem cellsrdquo Journal of Dental Research vol 85no 11 pp 966ndash979 2006
[6] P C Johnson A G Mikos J P Fisher and J A Jansen ldquoStra-tegic directions in tissue engineeringrdquo Tissue Engineering vol13 no 12 pp 2827ndash2837 2007
[7] P Habibovic U Gbureck C J Doillon D C Bassett C A vanBlitterswijk and J E Barralet ldquoOsteoconduction and osteoin-duction of low-temperature 3D printed bioceramic implantsrdquoBiomaterials vol 29 no 7 pp 944ndash953 2008
[8] H SWang S C Hung S T Peng C C Huang HMWei Y JGuo et al ldquoMesenchyma lstem cells in theWhartonrsquos jelly of thehuman umbilical cordrdquo Stem Cells vol 22 pp 1330ndash1337 2004
[9] D Baksh R Yao and R S Tuan ldquoComparison of proliferativeand multilineage differentiation potential of human mesenchy-mal stem cells derived from umbilical cord and bone marrowrdquoStem Cells vol 25 no 6 pp 1384ndash1392 2007
[10] E Verron I Khairoun J Guicheux and J-M Bouler ldquoCalciumphosphate biomaterials as bone drug delivery systems a reviewrdquoDrug Discovery Today vol 15 no 13-14 pp 547ndash552 2010
[11] A Can and S Karahuseyinoglu ldquoConcise review humanumbilical cord stromawith regard to the source of fetus-derivedstem cellsrdquo Stem Cells vol 25 no 11 pp 2886ndash2895 2007
[12] L Wang M Singh L F Bonewald and M S DetamoreldquoSignalling strategies for osteogenic differentiation of humanumbilical cord mesenchymal stromal cells for 3D bone tissueengineeringrdquo Journal of Tissue Engineering and RegenerativeMedicine vol 3 no 5 pp 398ndash404 2009
[13] X Miao L-P Tan L-S Tan and X Huang ldquoPorous cal-cium phosphate ceramics modified with PLGA-bioactive glassrdquoMaterials Science and Engineering C vol 27 no 2 pp 274ndash2792007
[14] V Guarino and L Ambrosio ldquoThe synergic effect of polylactidefiber and calcium phosphate particle reinforcement in poly120576-caprolactone-based composite scaffoldsrdquo Acta Biomaterialiavol 4 no 6 pp 1778ndash1787 2008
[15] A E Watts A J Nixon M G Papich H D Sparks and WS Schwark ldquoIn vitro elution of amikacin and ticarcillin froma resorbable self-setting fiber reinforced calcium phosphatecementrdquo Veterinary Surgery vol 40 no 5 pp 563ndash570 2011
[16] K J L Burg S Porter and J F Kellam ldquoBiomaterial develop-ments for bone tissue engineeringrdquo Biomaterials vol 21 no 23pp 2347ndash2359 2000
[17] S Yang K-F Leong Z Du and C-K Chua ldquoThe design ofscaffolds for use in tissue engineering Part I traditional factorsrdquoTissue Engineering vol 7 no 6 pp 679ndash689 2001
[18] K F Leong C M Cheah and C K Chua ldquoSolid freeform fab-rication of three-dimensional scaffolds for engineering replace-ment tissues and organsrdquo Biomaterials vol 24 no 13 pp 2363ndash2378 2003
8 Advances in Materials Science and Engineering
[19] J Russias E Saiz R K Nalla K Gryn R O Ritchie and AP Tomsia ldquoFabrication and mechanical properties of PLAHAcomposites a study of in vitro degradationrdquo Materials Scienceand Engineering C vol 26 no 8 pp 1289ndash1295 2006
[20] R Z Le Geros ldquoCalcium phosphate-based osteoinductivematerialsrdquo Chemical Reviews vol 108 no 11 pp 4742ndash47532008
[21] C E Wen Y Yamada K Shimojima Y Chino H HosokawaandMMabuchi ldquoCompressibility of porous magnesium foamdependency on porosity and pore sizerdquo Materials Letters vol58 no 3-4 pp 357ndash360 2004
[22] L L Hench and J M Polak ldquoThird-generation biomedicalmaterialsrdquo Science vol 295 no 5557 pp 1014ndash1017 2002
[23] D Williams ldquoBenefit and risk in tissue engineeringrdquo MaterialsToday vol 7 no 5 pp 24ndash29 2004
[24] V Guarino F Causa and L Ambrosio ldquoBioactive scaffolds forbone and ligament tissuerdquoExpert Review ofMedicalDevices vol4 no 3 pp 405ndash418 2007
[25] D W Hutmacher J T Schantz C X F Lam K C Tan and TC Lim ldquoState of the art and future directions of scaffold-basedbone engineering from a biomaterials perspectiverdquo Journal oftissue engineering and regenerative medicine vol 1 no 4 pp245ndash260 2007
[26] L Gerhardt and A R Boccaccini ldquoBioactive glass and glass-ceramic scaffolds for bone tissue engineeringrdquoMaterials vol 3pp 3867ndash3910 2010
[27] HH K Xu S Takagi J B Quinn and L C Chow ldquoFast-settingcalcium phosphate scaffolds with tailoredmacropore formationrates for bone regenerationrdquo Journal of Biomedical MaterialsResearch A vol 68 no 4 pp 725ndash734 2004
[28] MAlshaaerHCuypersH Rahier and JWastiels ldquoProductionof monetite-based Inorganic Phosphate Cement (M-IPC) usinghydrothermal post curing (HTPC)rdquo Cement and ConcreteResearch vol 41 no 1 pp 30ndash37 2011
[29] C Wenchuan Z Hongzhi D W Michael B Chongyun andH H K Xu ldquoUmbilical cord stem cells released from alginate-fibrin microbeads inside macroporous and biofunctionalizedcalcium phosphate cement for bone regenerationrdquo Acta Bioma-terialia vol 8 no 6 pp 2297ndash2306 2012
[30] M Alshaaer H Cuypers G Mosselmans H Rahier and JWastiels ldquoEvaluation of a low temperature hardening InorganicPhosphate Cement for high-temperature applicationsrdquo Cementand Concrete Research vol 41 no 1 pp 38ndash45 2011
[31] J Rouwkema N C Rivron and C A van Blitterswijk ldquoVas-cularization in tissue engineeringrdquo Trends in Biotechnology vol26 no 8 pp 434ndash441 2008
[32] M Lovett K Lee A Edwards and D L Kaplan ldquoVasculariza-tion strategies for tissue engineeringrdquo Tissue Engineering B vol15 no 3 pp 353ndash370 2009
[33] X Shi B Sitharaman Q P Pham et al ldquoFabrication of porousultra-short single-walled carbonnanotube nanocomposite scaf-folds for bone tissue engineeringrdquo Biomaterials vol 28 no 28pp 4078ndash4090 2007
[34] C K Kuo andP XMa ldquoIonically crosslinked alginate hydrogelsas scaffolds for tissue engineering Part 1 structure gelation rateand mechanical propertiesrdquo Biomaterials vol 22 no 6 pp 511ndash521 2001
[35] J L Drury R G Dennis and D J Mooney ldquoThe tensile prop-erties of alginate hydrogelsrdquo Biomaterials vol 25 no 16 pp3187ndash3199 2004
[36] C J Damien and J R Parsons ldquoBone graft and bone graftsubstitutes a review of current technology and applicationsrdquoJournal of Applied Biomaterials vol 2 no 3 pp 187ndash208 1991
[37] Y Lei B Rai K H Ho and S H Teoh ldquoIn Vitro degradationof novel bioactive polycaprolactone-20 tricalcium phosphatecomposite scaffolds for bone engineeringrdquo Materials Scienceand Engineering C vol 27 no 2 pp 293ndash298 2007
[38] C R Kothapalli M T Shaw J R Olson and M Wei ldquoFab-rication of novel calcium phosphatepoly(lactic acid) fibercompositesrdquo Journal of Biomedical Materials Research B vol 84no 1 pp 89ndash97 2008
[19] J Russias E Saiz R K Nalla K Gryn R O Ritchie and AP Tomsia ldquoFabrication and mechanical properties of PLAHAcomposites a study of in vitro degradationrdquo Materials Scienceand Engineering C vol 26 no 8 pp 1289ndash1295 2006
[20] R Z Le Geros ldquoCalcium phosphate-based osteoinductivematerialsrdquo Chemical Reviews vol 108 no 11 pp 4742ndash47532008
[21] C E Wen Y Yamada K Shimojima Y Chino H HosokawaandMMabuchi ldquoCompressibility of porous magnesium foamdependency on porosity and pore sizerdquo Materials Letters vol58 no 3-4 pp 357ndash360 2004
[22] L L Hench and J M Polak ldquoThird-generation biomedicalmaterialsrdquo Science vol 295 no 5557 pp 1014ndash1017 2002
[23] D Williams ldquoBenefit and risk in tissue engineeringrdquo MaterialsToday vol 7 no 5 pp 24ndash29 2004
[24] V Guarino F Causa and L Ambrosio ldquoBioactive scaffolds forbone and ligament tissuerdquoExpert Review ofMedicalDevices vol4 no 3 pp 405ndash418 2007
[25] D W Hutmacher J T Schantz C X F Lam K C Tan and TC Lim ldquoState of the art and future directions of scaffold-basedbone engineering from a biomaterials perspectiverdquo Journal oftissue engineering and regenerative medicine vol 1 no 4 pp245ndash260 2007
[26] L Gerhardt and A R Boccaccini ldquoBioactive glass and glass-ceramic scaffolds for bone tissue engineeringrdquoMaterials vol 3pp 3867ndash3910 2010
[27] HH K Xu S Takagi J B Quinn and L C Chow ldquoFast-settingcalcium phosphate scaffolds with tailoredmacropore formationrates for bone regenerationrdquo Journal of Biomedical MaterialsResearch A vol 68 no 4 pp 725ndash734 2004
[28] MAlshaaerHCuypersH Rahier and JWastiels ldquoProductionof monetite-based Inorganic Phosphate Cement (M-IPC) usinghydrothermal post curing (HTPC)rdquo Cement and ConcreteResearch vol 41 no 1 pp 30ndash37 2011
[29] C Wenchuan Z Hongzhi D W Michael B Chongyun andH H K Xu ldquoUmbilical cord stem cells released from alginate-fibrin microbeads inside macroporous and biofunctionalizedcalcium phosphate cement for bone regenerationrdquo Acta Bioma-terialia vol 8 no 6 pp 2297ndash2306 2012
[30] M Alshaaer H Cuypers G Mosselmans H Rahier and JWastiels ldquoEvaluation of a low temperature hardening InorganicPhosphate Cement for high-temperature applicationsrdquo Cementand Concrete Research vol 41 no 1 pp 38ndash45 2011
[31] J Rouwkema N C Rivron and C A van Blitterswijk ldquoVas-cularization in tissue engineeringrdquo Trends in Biotechnology vol26 no 8 pp 434ndash441 2008
[32] M Lovett K Lee A Edwards and D L Kaplan ldquoVasculariza-tion strategies for tissue engineeringrdquo Tissue Engineering B vol15 no 3 pp 353ndash370 2009
[33] X Shi B Sitharaman Q P Pham et al ldquoFabrication of porousultra-short single-walled carbonnanotube nanocomposite scaf-folds for bone tissue engineeringrdquo Biomaterials vol 28 no 28pp 4078ndash4090 2007
[34] C K Kuo andP XMa ldquoIonically crosslinked alginate hydrogelsas scaffolds for tissue engineering Part 1 structure gelation rateand mechanical propertiesrdquo Biomaterials vol 22 no 6 pp 511ndash521 2001
[35] J L Drury R G Dennis and D J Mooney ldquoThe tensile prop-erties of alginate hydrogelsrdquo Biomaterials vol 25 no 16 pp3187ndash3199 2004
[36] C J Damien and J R Parsons ldquoBone graft and bone graftsubstitutes a review of current technology and applicationsrdquoJournal of Applied Biomaterials vol 2 no 3 pp 187ndash208 1991
[37] Y Lei B Rai K H Ho and S H Teoh ldquoIn Vitro degradationof novel bioactive polycaprolactone-20 tricalcium phosphatecomposite scaffolds for bone engineeringrdquo Materials Scienceand Engineering C vol 27 no 2 pp 293ndash298 2007
[38] C R Kothapalli M T Shaw J R Olson and M Wei ldquoFab-rication of novel calcium phosphatepoly(lactic acid) fibercompositesrdquo Journal of Biomedical Materials Research B vol 84no 1 pp 89ndash97 2008
