Novel bilayered Gellan gum-Gellan gum/hydroxyapatite scaffolds for osteochondral tissue engineering...

J Tissue Eng Regen Med 2012; 6 (Suppl. 2): 8–39.Published online 3 October 2012 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1608

Abstracts List

TS001The influence of aerobic exercise on the numberand differentiation of blood rat endotheliumprogenitor cells (EPCs)S Pinto-Pereira1, D Moretto1, A Lopes1, A Paiva1, L Tonelli1,A Andre2, CA Fontes Ribeiro1 and P Tavares11Faculty of Sport Sciences and Physical Education, Faculty ofMedicine -IBILI, University of Coimbra; 2HistocompatibilityCenter of Coimbra; 3Coimbra College of Health Technology

Objectives: It is known that exercise has the ability to change the skele-tal muscle fibre type. The fibre transformation induced by aerobic exer-cise is from type II to type I. It has been described that in theseconditions there is also an increase in the capillary density. However,the source of the endothelial cells in the formation of these capillariesis unclear. A possible explanation is that exercise increases productionand differentiation of EPCs from bone marrow. Therefore, the aim ofthis work is to study the influence of exercise on the number and differ-entiation of EPCs and its correlation with capillary density in soleusmuscle.Methods: In order to achieve our goal we used male Wistar adult rats,which were trained for 8 weeks in a treadmill aerobic protocol. Theanimals were divided in two groups: a group with exercise and a con-trol one. The control group was submitted to a mild exercise (duringthe same time protocol) just to maintain the motor skills of the trainedrats, and avoid central nervous system interferences. Blood was col-lected from the jugular vein (after anaesthesia) using 6% EDTA as anti-coagulant at the end of the exercise protocol (after 8 weeks). At theend of the experiments the animals were sacrificed by anaesthetic over-dose, and the soleus muscle was removed for histological and molecu-lar analysis. The histological analysis included a hematoxilin-eosinestaining (to verify muscles integrity and/or exercise-induced changes)and an ATPase staining to count and measure the different fibre types.The muscle capillaries were analysed by immunoassay. The EPC’s intotal blood were analysed by flow cytometry using CD45, CD34,CD133 and CD146.Results and Discussion: The results showed that exercise increased thenumber of type I with a reduction in type II skeletal muscle fibres. Wealso verified, as expected, that the C:F ratio increases in the soleus mus-cle of the trained group. As for EPC’s in total blood, we found that themarkers for immature cells (CD34 and CD133) maintained the percent-age after training but the cells CD146+ decreased in the exercise groupwhen compared to control group. This decrease was correlated withthe C:F ratio.Conclusions: Thus, our results suggest that exercise increases the EPC’sturnover and differentiation into endothelial cells to form new vessels.

TS002The influence of aerobic exercise on the numberand differentiation of blood rat endotheliumprogenitor cells (EPCs). Effect of simultaneoustreatment with ibuprofenP Tavares1, S Pinto-Pereira1, D Moretto1, A Lopes2, A Paiva2,L Tonelli1, A Andre3 and CA Fontes Ribeiro11Faculty of Sport Sciences and Physical Education, Faculty ofMedicine -IBILI, University of Coimbra; 2HistocompatibilityCenter of Coimbra; 3Coimbra College of Health Technology

Objectives: This work aims at studying the influence of exercise on thenumber and differentiation of EPC’s and its correlation with capillarydensity in soleus muscle. Since many of the gym frequenting uses anti-inflammatory drugs to avoid pain during training, it is also our goal tostudy the influence of ibuprofen in these mechanisms.Methods: We used male Wistar adult rats that were trained during8 weeks in a treadmill aerobic protocol. The animals were divided intothree groups: a group with exercise, exercise plus ibuprofene (40 mg/kg) and a control one. The control group was submitted to a mild exer-cise (during the same time protocol) just to maintain the motor skillsof the trained rats and avoid central nervous system interferences.Blood was collected using 6% EDTA as anticoagulant at the end of theexercise protocol. At the end of the experiments the animals were sacri-ficed by anaesthetics overdose, and the soleus muscle was removed tohistological and molecular analysis. The histological analysis includeda hematoxilin-eosine staining (to verify muscles integrity and/or exer-cise-induced changes) and an ATPase staining to count and measurethe different fibre types. The muscle capillaries were analysed byimmunoassay. The EPCs in total blood were analysed by flow cytome-try using anti-CD45, anti-CD34, anti-CD133 and anti-CD146.Results and Discussion: The results showed that exercise increased thenumber of type I with a reduction in type II skeletal muscle fibres, butless in the ibuprofen group. As expected, the C:F ratio increased in thesoleus muscle of the trained group and in a lower amount in the ibu-profen treated group. As for EPC’s in total blood, we found that themarkers for immature cells (CD34 and CD133) maintained the percent-age after training but the cells CD146+ decreased in the exercise groupwhen compared to control group. In the ibuprofen group the percent-age of cells with CD34+, CD133+ and CD146+ were lower than in thecontrol but equal to the exercise group. This decrease of CD146+ cellswas correlated with the C:F ratio.Conclusions: Our results suggest that exercise increases the EPCs turn-over and differentiation into endothelial cells to form new vessels.However, the junction of ibuprofen to exercise decreases the exerciseeffects when used alone. This may suggest that the endothelium differ-entiation may be related to the COX pathway.

� 2012 The AuthorsJournal of Tissue Engineering and Regenerative Medicine � 2012 John Wiley & Sons, Ltd.

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE ABSTRACT

TS003Fetal stem cells obtained from amniotic fluid andwharton¢s jelly expanded using platelet lysate fortissue engineering applicationsAR Costa-Pinto1,2, I Aleixo1,2, MT Cerqueira1,2, AM Frias1,2,S Fernandes3, A Barros3, RL Reis1,2 and NM Neves1,213B’s Research Group, University of Minho, Avepark, ZonaIndustrial da Gandra, S. Claudio do Barco, Guimaraes, Portugal;2ICVS/ 3B¢s Associate Laboratory, Braga/Guimaraes, Portugal;3Genetics Department, Faculty of Medicine, University of Porto,Portugal

Extra-embryonic tissues, such as amniotic fluid (AF) and Wharton¢sJelly (WJ) of umbilical cord, offer many advantages over both embry-onic and adult stem cell sources. These tissues are routinely discardedat parturition and the extracorporeal nature of these cell sourcesfacilitates its isolation. Also the comparatively large volume of tissuefacilitates the isolation of larger numbers of stem cells. Autologousapproaches to use MSCs, namely from bone marrow, have difficultiesregarding the limited numbers of cells from the patient but also its lim-ited expansion potential. Furthermore, fetal stem cells appear to haveeven more pronounced immunomodulatory properties than adultMSCs. This allogeneic escape mechanism may be of therapeutic value,because transplantation of allogeneic human MSCs in stock would bereadily available, as opposed to the required expansion stage (involvingboth time and logistic effort) of autologous cells. Cell expansion proto-cols are based on the use of media supplemented with fetal bovineserum (FBS) as a source of nutrientes and growth factors. The animalserum is not completely safe, once there is a possibility of contaminationby animal viroses, prions or others source of contaminants. Additionally,it was described that FBS used systematically in MSCs subcultivationinduces more humoral immune response. Moreover, anti-FBS antibod-ies could be detected in patients after receiving MSCs expanded in FBS.Platelet lysate (PL) has enormous possibilities in cell therapy, namelybecause of the high concentration of growth factors that promoteshigher cell expansion. A recent study showed that proliferation of MSCswas much higher on PL gel compared to tissue culture plastic. Theimmunomodulatory properties ofMSCs are maintainedwhen expandedin culture medium supplemented with PL. Based on these premises weisolated fetal stem cells from AF obtained from amniocentesis and WJfrom umbilical cords. These cells were plated and expanded in low den-sity numbers in basal culture medium either supplemented with FBS orwith PL. In each passage cells were counted for proliferation kineticsand prepared for flow cytometry analysis. Expanded populations wereanalysed both for population size and phenotype and for the character-istic MSCs surface markers (CD34, CD45, CD73, CD44, CD106, CD105,CD29, CD90, CD31), markers related with immune response (HLA-DR,80, 83, 86) and embryonic markers SSEA-4 and TRA-1-60.

TS004Cell sheet engineering for reproducing the bonemarrow hematopoietic stem cell nicheRP Pirraco1,2, AP Marques1,2 and RL Reis1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal;2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal

Hematopoietic stem and progenitor cells (HSPC) are multipotent cellsresponsible for the maintenance and renewal of the hematopoietic line-age in the adult body. The fate of these stem cells is closely regulatedby their surrounding microenvironment, or niche. The importance ofthe microenvironment for HSPC function has been long recognized byresearchers that more than 30 years ago attempted to emulate it in 2Dusing a layer of bone marrow stromal cells to culture hematopoieticcells for long time periods (Dexter-type cultures). However, all themodels based on feeder layers are less than perfect in recreating thehematopoietic microenvironment. The use of growth factor cocktailsprovided some promising results concerning the maintenance and pro-liferation of some cell populations but still struggle to deliver the cor-rect microenvironment for the maintenance of suitable HSPCpopulations. Part of the problem of the current systems lies on the lackof the third dimension. At the same time, the proposed three-dimen-sional methodologies using scaffolds to engineer the bone marrow(BM) microenvironment present very limited results probably due tothe scaffolding matrices’ intrinsic limitations. Therefore, an engineeredBM microenvironment capable of acting as a functional HSPC nichewould provide a tremendous tool for the study of hematopoiesis as wellas for obtaining and maintaining HSPC. Using osteogenic cell sheets,we have previously demonstrated that it was possible to induce theectopic formation of mature bone tissue with a clear bone marrow,avoiding the use of scaffolds. In the present work, we studied thepotential of using osteogenic cell sheets to build in vitro, a 3D microen-vironment capable of providing HSPC a suitable niche for their survivaland proliferation. For this, we used bone marrow stromal cells and adi-pose-derived stem cells to produce the osteogenic cell sheets andhuman umbilical cord blood as a source of hematopoietic stem cells.

� 2012 The Authors J Tissue Eng Regen Med 2012; 6 (Suppl. 2): 8–39.Journal of Tissue Engineering and Regenerative Medicine � 2012 John Wiley & Sons, Ltd. DOI: 10.1002/term.1608

Abstracts List 9

TS005Improved regeneration of mice full-thicknessexcisional wounds by human adipose stem cellscell-sheetsMT Cerqueira1,2, TC Santos1,2, RP Pirraco1,2, AM Frias1,2,AR Martins1,2, RL Reis1,2 and AP Marques1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal; 2ICVS-3B’s – PTGovernment Associate Laboratory, Braga/Guimaraes, Portugal

Stem Cells have emerged as a powerful element for generating skin tis-sue and for promoting enhanced wound healing through the differenti-ation into relevant skin cell lineages and/or by paracrine interactions.Adipose stem cells (hASCs) in particular, appear as an attractive cellsource for skin regeneration, due to their natural abundance, relativelyeasy methodology of isolation and secretion of factors important forthe restoration of healthy skin. Common approaches for stem cell deliv-ery, comprehending the use of direct injection of single stem cell sus-pensions or the use of biomaterials-based strategies, comprise in somecases, poor engraftment of those cells or associated inflammatory pro-cesses that lead to a reduced effect over skin restoration. An innovativealternative comprehends the use of Cell Sheet (CS) engineering that,by taking advantage of temperature-responsive culture surfaces, allowsthe non-invasive harvest of cultured cells, as intact sheets, along withtheir deposited extracellular matrix, facilitating the direct transplanta-tion to host tissues. This study exploited the potential of CS Engineer-ing for fabricating 3-layered hASCs CS to fully regenerate mice full-thickness excisional wounds. The outcome of the transplanted cellmachinery and the success of this technology were evaluated at rele-vant timepoints considering wound healing parameters such as re-epi-thelialization, angiogenesis, neotissue quality formation and hASCscontribution for the skin tissue regeneration. Overall, the created con-structs showed good stability in vitro, were easily attached to thewound bed and showed to play a significant and specific role over epi-dermal regeneration through paracrine effects. Acknowledgements Theauthors would like to acknowledge for the financial support by Skingi-neering (PTDC/SAU-OSM/099422/2008), Portuguese Foundation forScience and Technology (FCT) funded project.

TS006Fish scales patterning guiding hASC growthJ Moreira-Silva1,2, D Afonso1,2, TH Silva1,2, F Volpato3,A Motta3, AP Marques1,2, JF Mano1,2, RL Reis1,2 andC Migliaresi313B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal;2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal, 3Department of Materials Engineering andIndustrial Technologies and BIOtech Research Centre, Universityof Trento, Trento, Italy

Marine organisms and materials exhibit interesting properties for theiruse in biomedical applications, following biomimetic approaches [1,2].For instance, the white seabass (Lates Calcarifer) scales exhibit a veryinteresting pattern, which inspire their use as a guiding platform forcellular growth, in a tissue regeneration approach. Moreover, fish scalesare composed of hydroxyapatite and type I collagen fibrils, equivalentto the materials that one can find in human bone, as thus their use inbone tissue engineering is envisaged. The main goal of the presentwork was the assessment of fish scales, combining intrinsic featuressuch as patterns, hydroxyapatite and collagen in different means, ascell culture supports aiming at guiding cell growth and extracellularmatrix deposition and mineralization. In this sense, human adiposederived stem cells (hASCs) were cultured onto seabass scales, underosteogenic and non-osteogenic conditions. Fish scales supported celladhesion and cytoskeleton organization defined by the surface pattern-ing. Moreover, hASCs were able to proliferate along the time of cultureand to differentiate towards the osteogenic lineage depositing and min-eralizing the characteristic extracellular matrix. This work constitutesthe first step to demonstrate the value of the intrinsic properties of sea-bass scales for exploitation in the biomedical field and in particular forbone tissue engineering.References1. TH Silva et al., Materials of marine origin: a review on polymers andceramics of biomedical interest, International Materials Reviews 2012,in press. DOI: 10.1179/1743280412Y.00000000022. TH Silva et al., Biomaterials from Marine-Origin Biopolymers’’, inJ.F. Mano (Ed.): Biomimetic Approaches for Biomaterials Development,Wiley-VCH, 2012, in press.


10 Abstracts List

TS007Mechanical liposuction vs. laser liposuction vs.surgeries – the best method of adipose tissueharvesting for ADSC isolationN Gurtowska1, J Olkowska1, A Bajek1, S Pierscinski2,S Dabrowiecki2, W Bienkowski3, A Katna-Grzesiak4, R Debski5,M Bodnar6, A Marszałek6 and T Drewa1,71Tissue Engineering Department, Nicolaus Copernicus University,Collegium Medicum Bydgoszcz, Poland; 2Department of Generaland Endocrine Surgery, Nicolaus Copernicus University,Collegium Medicum Bydgoszcz, Poland; 3Bienkowski MedicalCenter, Bydgoszcz, Poland; 4Medical Center Laser-Med,Bydgoszcz, Poland; 5Department of Pediatric Hematology andOncology, Nicolaus Copernicus University, Bydgoszcz, Poland;6Department of Clinical Pathomorphology, Nicolaus CopernicusUniversity, Collegium Medicum Bydgoszcz, Poland; 7NicolausCopernicus Hospital in Torun, Urology Department

Objectives: One of the major goals of tissue engineering and regenera-tive medicine is search and characterize the new sources of mesenchy-mal stem cells, which will be better than bone marrow. Adipose tissueis easily accessible and abundant source of stem cells hence their isola-tion creates new opportunities for regeneration of injured tissues. Theaim of this study was the comparison of the Adipose Derived StemCells (ADSCs) quality harvested during mechanical liposuction (ADS-CsML), laser liposuction (ADSCsLL) and surgeries (ADSCsS) and toselect facilely available mesenchymal stem cell source with the highestproliferative potential and ability to differentiate.Methods: The human adipose tissue was obtained during planned sur-gical procedures in patients with pathological obesity (n = 30),mechanical liposuction (n = 8) and laser liposuction (n = 10). ADSCsafter isolation were cultured in DMEM/Ham’s F-12 medium supple-mented with 20% (up to the first passage) or 10% (after first passage)fetal bovine serum (FBS), 1% antibiotic and basic fibroblast growth fac-tor (bFGF). To characterize their properties the proliferation rate, dif-ferentiation potential and phenotype analysis (CD34, CD44, CD45 andCD90 expression) were performed. Results and Discussion: ADSCs har-vested by three methods underwent rapid adhesion to the surface ofculture dishes and began to proliferate. The proliferation rate was sta-ble during 14 days of culture. The cells proliferated at least to the sixthpassage. The success of culture establishment was 86.7%, 100% and100% for ADSCsS, ADSCsLM and ADSCsLL, respectively. The averageof cell number per 1 g/mL of tissue, at the first passage, was 2.3 · 105,1.1 · 105 and 9.7 · 103 for ADSCsS, ADSCsLL and ADSCsLM, respec-tively. ADSCs cultured in medium supplemented with differentiationfactors toward osteogenic, adipogenic and chondrogenic lineagesshowed ability to multilineage differentiation. All ADSCs types wereCD44+, CD90+, CD34+ and CD45- (at third passage).Conclusions: The results obtained in that study support the findingsthat adipose tissue harvested by three considered methods containsrapidly growing cells with high differentiation potential. However, con-cerning average of cells number at the first passage, adipose tissue col-lected during surgeries seems to be the most efficient. Nevertheless,liposucation is less invasive procedure so that ADSCs isolated from lip-oaspirates obtained from a larger number of patients require moredetailed investigation.

TS008Microparticles loaded Gellan gum hydrogelmatrices: engineering tissues for nucleuspulposus regenerationDR Pereira1,2, J Silva-Correia1,2, SG Caridade1,2, JF Mano1,2,RA Sousa1,2, JM Oliveira1,2 and RL Reis1,213B’s Research Group - Biomaterials, Biodegradables andBiomimetics, Univ. Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, S. Claudio de Barco, Taipas, Guimaraes,Portugal; 2ICVS/3B’s - PT Government Associate Laboratory,Braga/Guimaraes, Portugal

The intervertebral disc central core is made by a gel-like tissue struc-ture composed of more than 80% of water, Nucleus Pulposus (NP).Proteoglycans such as versican and especially aggrecan are the mainconstituents of the NP matrix as well as collagen type II. The purposeof this work is creating novel Gellan gum-based (GG) hydrogel formu-lations. GG microparticles (MPs) dispersed in a GG matrix are the nov-elty for finding application as NP substitute. The ongoing experimentcomprises de GG functionalization through methacrylated groups addi-tion. In order to optimize some properties of GG, the functionalizationwill allow us to improve the water solubility and photopolymerizationin situ of the biomaterial. High acyl (HA) and Low acyl (LA) GellanGum (GG) at different ratio [75%:25% (v/v); 50%:50% (v/v),25%:75% (v/v)], HAGG 0.75% and LAGG 2%, were mixed in order toprepare solutions to be used as formulations of GG MPs/hydrogelsmatrix. The GG MP/hydrogel matrix formulations were characterizedby dynamic mechanical analysis (DMA), swelling behaviour and degra-dation rate. The toxic effect of GG MPs/hydrogel discs leachables ontothe cells was investigated in vitro using a mouse lung fibroblast-like cell(L929 cells) line. Live/Dead cell viability assay was performed to assessthe encapsulation efficacy; meanwhile DAPI/Phalloidin staining wasperformed to evaluate cell morphology. The Methacrylated GellanGum (GG-MA) was prepared following the protocol [1]. Mechanismreactions occurred in presence of glycidyl methacrylate by addition to asolution of LAGG at 2% (w/v). The reaction was running over 24 h atroom temperature controlling the pH at 8.5 with sodium hydroxide1 M. GG MPs size was measured using a stereo microscope by stainingthe MPs with Toluidine Blue-O. This method also allowed evaluatingthe MPs dispersion and matrix cohesion. From DMA analysis it wasobserved that the range of 50–500 mg/mL of incorporated MPs is theoptimal concentration to reinforce GG matrices. It was demonstratedthe non–cytotoxic effect of MPs/hydrogels over L929 cells. In fact,L929 cells were successfully encapsulated in all GG formulations GGMPs and remaining viable over 72 h of culturing. The resulting productfrom methacrylation reaction was evaluated by nuclear magnetic reso-nance to assess the reaction efficiency and the degree of substitution.Methacrylated Gellan gum and GG MP/hydrogel matrix are promisinghydrogels to be used in tissue engineering strategies for treatment ofthe degenerated NP.Reference1. Silva-Correia J, 2011 Journal of Tissue Engineering and Regenera-tive Medicine, 5(6): e97–e107.


Abstracts List 11

TS009Dendrimer-based nanoparticles in tissueengineering and regenerative medicineapproachesJM Oliveira1,2, JF Mano1,2 and RL Reis1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, S. Claudio de Barco, Taipas, Guimaraes,Portugal; 2ICVS/3B’s - PT Government Associate Laboratory,Braga/Guimaraes, Portugal

Dendrimeric macromolecules are synthetic and spherical systems thathave been attracting great deal of attention as nanocarriers for intracel-lular drug delivery (DDS) [1]. These possess several advantages asnanocarriers, as it possibly incorporating smart functionalities such astargetability and stimuli-responsive drug release. Our group has beenproposing the use of dexamethasone (Dex)-loaded carboxymethylchi-tosan/polyamidoamine dendrimer nanoparticles (CMCht/PAMAMNPs) [2,3] for controlling the differentiation of stem cells towards theosteogenic lineage. In this work, the biocompatibility of the Dex-loadedCMCht/PAMAM NPs were investigated, in vitro and in vivo. CMCht/PA-MAM NPs were produced as described by Oliveira et al. [4]. Dexameth-asone (Dex) was loaded into the NPs by means of using a precipitationroute. Dex-loaded NPs were chemically bound to fluorescein isothiocy-anate (FITC) for styding their intracellular fate. The cellular uptake ofNPs was investigated using different cell types namely rat bone marrowstromal cells (RBMSCs) and Human Osteosarcoma cell line (SaOS-2),for times up to 14 days of culturing. The internalization of the FITC-labelled and Dex-loaded CMCht/PAMAM dendrimer NPs waere investi-gated using fluorescence microscopy and flow cytometry (FACS) analy-ses. FITC-labelled NPs (1 lg/g and 10 lg/g) were injectedintraveneously on Wistar rats and its biodistribution in different organswas evaluated up to 72 h. Results have revealed that Dex-loadedCMCht/PAMAM dendrimer NPs are non-cytotoxic, in vitro and in vivo.The in vivo studies also revealed that CMCht/PAMAM dendrimer NPsare stable in circulation. The histological study has shown that NP’s areuptaken by cells from different tissues/organs namely, brain, liver, kid-ney and lung. This study has revealed that the dexamethasone-loadedCMCht/PAMAM dendrimer nanoparticles are non-toxic and demon-strated a great ability for being uptaken by different cell types. More-over, we have demonstrated that the dexamethasone-loaded CMCht/PAMAM dendrimer nanoparticles are able to control stem cells osteo-genic differentiation. As a final conclusion, the developed nanocarriersare advantageous as intracellular nanocarrier tools for preprogrammingthe stem cells fate ex vivo. The developed dexamethasone-loadedCMCht/PAMAM dendrimer nanoparticles showed great promise forapplication in bone tissue engineering strategies as it allowed us to pro-duce de novo bone tissue in vivo.References:1. J.M. Oliveira, et al., 2010, Progress in Polymer Science, 35:1163–

1194.2. J.M. Oliveira, et al., 2010, Bone, 46:1424–1435.3. J.M. Oliveira, et al., 2011, Nanomedicine: Nanotechnology, Biology,and Medicine, 7:914–924.4. J.M. Oliveira, et al., 2008, Advanced Functional Materials, 18:1840–1853.

TS010Controlling mesenchymal stem cell proliferationand differentiation through glycosaminoglycanengineered surfacesY Yang, N Ma, A Kowitsch and T GrothBiomedical Materials Group, Martin Luther UniversityHalle-Wittenberg, Halle, Saale, Germany

Proliferation and differentiation of mesenchymal stem cell (MSCs) areregulated by its ‘‘niches’’ - the specific microenvironment that accountsfor tissue maintenance and repair. Glycosaminoglycans (GAGs), ascomponents of niches, bind non-covalently growth factors and triggermultiple downstream signaling pathways controlling cellular activities.Unraveling the chemical structures of GAGs like hyaluronic acid (HA),chondroitin sulfate (CS) and heparin (Hep) has shown that they allhave similar disaccharide units, but differ in the quantity and locationof sulfate groups, which greatly influences their interactions with pro-teins and cells. Here we developed a 2D model to study GAG effects onmesenchymal stem cells’ (MSCs) proliferation and differentiation,which may pave the way for applications in tissue engineering. GAGswere oxidized using periodate to generate dialdehydes, shown by FT-IR and H1-NMR spectroscopy, which were covalently immobilized onamino-silane modified substrate by reductive amination. Surface wetta-bility increase after immobilization of hydrophilic ox-GAG molecules.Swelling surfaces exhibited relative flatten to aggregated structures, asinvestigated by AFM, with the increase of sulfation of immobilized mol-ecules. Meanwhile the roughness increased from 0.35 nm to 1.42 nm.Zeta-potentials of ox-GAGs immobilized surfaces decreased with ox-HA<ox-CS<ox-Hep, indicating that the amount of sulfate groupsincreased. Proliferation of multipotent C3H/10T1/2 mesenchymalmouse cells was enhanced on ox-Hep while inhibited on ox-HA. Confo-cal laser scanning microscopy images showed that cells on ox-Hep hadthe largest spreading area, by 24% beyond the others, whereas thesmallest form factor of 0.59. Alizarin red-S staining showed that reddye bonded to nodules on each surface, indicating osteogenic differen-tiation in the presence of rhBMP-2, with ox-HA<ox-CS<<ox-Hep. Itcan be assumed that the larger amount of sulfates on ox-Hep contrib-uted to the binding of BMP-2, which protected active protein-glycancomplex from being suppressed by antagonists, thus significantlyenhance osteogenesis of MSCs. In summary, GAG immobilized surfaceswith growth factors can mimic natural ECM-protein interactions andenhance growth factor activity, which seems to be a useful method toengineer MSCs’ proliferation and differentiation for tissue repair andregeneration.Acknowledgement: This work was supported in the frame of 7th Frame-work Program of European Union under grant agreement No. NMP4-SL-2009-229292 (‘‘Find & Bind’’). & Deutsche ForschungsgemeinschaftINST 271/250-1


12 Abstracts List

TS011Novel cellulose and chitosan sulfates promotingthe activity of growth Factors FGF-2 and BMP-2T Groth1, D Peschel1, N Aggarwal1, A-K Loeffler1, AWeltrowski1,K Zhang2 and S Fischer21Biomedical Materials Group, Martin Luther UniversityHalle-Wittenberg, Halle, Saale, Germany; 2Institute of Wood andPlant Chemistry, Dresden University of Technology, Tharandt,Germany

Growth factors regulate proliferation and differentiation of cells andplay important role as tools in tissue engineering. Particular emphasishas been put on the application of heparin as component of surfacecoatings and tissue engineering scaffolds to produce systems for con-trolled release of growth factors with heparin binding domains likeFGF, VEGF and BMP. However, heparin is not an ideal material due tothe variability of its sulfate content, molecular weight and bioactivity.Here we present novel derivatives of cellulose and chitosan that possessbioactivity comparable or greater than that of heparin with first insightsinto the mechanism of their bioactivity. Cellulose and chitosan werechemically modified to obtain derivatives that differ in the degree andsubstitutions site of sulfates and partly also carboxylates. The chemicalstructure and derivatisation degree was studied with Raman and NMRspectroscopy. Studies with the growth factors FGF-2 that possesses notonly mitogenic but also moderate angiogenic effect and 3T3 fibroblastsrevealed that the binding and mitogenic activity of derivatives wasenhanced with increasing sulfate content. The introduction of carbox-ylic groups to the O-6-position of glucose in analogy to heparin wasnot effective. Further studies with BMP-2 demonstrated an osteogenicactivity of derivatives with a maximum at intermediate sulfationdegree, which was evident by the alkaline phosphatase activity ofC2C12 mouse myoblast cell line and expression of osteogenic genes.Studies on the mechanism of action of derivatives vs. FGF-2 revealedthat particularly a protection from proteolytic digestion seem to pro-long activity of growth factors. Since all assays described above weresolution based further studies were carried testing whether cellulosesulfates can be used as bioactive surface coatings. Cellulose sulfateswith high bioactivity were selected and used to form multilayers withchitosan using the layer-by-layer technique. It was found that interme-diate sulfated cellulose sulfates were particularly effective because along-term release was detected. Overall, the novel derivatives seem tobe suitable for construction of bioactive surface coatings or tissue engi-neering scaffolds promoting angiogenesis and bone formation.Acknowledgements: This work was supported by Deutsche Forschungs-gemeinschaft by grants INST 271/250-1, GR1290/7-, GR1290/7-2,FI755/4-1 and FI755/4-2.

TS012Development of a bilayered system forperiodontal regeneration using tissueengineering approachesPM Babo1,2, VE Santo1,2, ME Gomes1,2 and RL Reis1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal;2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal

Periodontitis is a prevalent gram negative infection disease that causesthe destruction of the tooth supportive tissues. Effective treatment ofperiodontal disease is important, since periodontal disease is correlatedwith several systemic diseases. However, adult periodontal tissues havea low potential of self-renewing and regeneration. Concerted effortshave been made to accelerate periodontal tissue regeneration, using aplethora of techniques including grafting materials, signalling mole-cules and cell-based tissue engineering. Nevertheless, a strategy forpredictable reconstruction of normal structure and functionality of peri-odontal damaged tissue is still missing. In this work, we propose thedevelopment of a bilayered system for the regeneration of alveolarbone and periodontal ligament. This system consists of a bilayeredcomposite made of calcium phosphate (CaP) cement incorporatinghyaluronic acid microspheres loaded with Platelet Lysates (PL) and ahydrogel layer based on PL, harbouring mesenchymal stem cells(MSCs). The advantage of this strategy lies in the ability to develop asystem that can be easily injected and which provides adequatemechanical support, both initially and during new tissue ingrowth.After the degradation of the HA microspheres incorporated in the CaPcement, a fully interconnected network can be created, which leads torapid penetration of bone-forming cells into the CaP cement. Addition-ally, the distinct degradation rates of the components of the bilayeredsystem allow a controlled release of the entrapped growth factors andfurther accelerate the periodontal tissues remodelling process, mimick-ing the physiologic wound healing process. The data collected suggeststhat it is possible to fabricate the cement composite layer incorporatingPL from which a number of growth factors are released in a controlledmanner. Moreover, the cement composites incorporating HA micro-spheres loaded with PL show low cytotoxic values and induce theexpression of early markers of osteogenic differentiation in human adi-pose-derived stem cells (hACS).


Abstracts List 13

TS013Development of bioactive constructs through cellcrosslinking for tissue regenerationCA Custodio1,2, VE Santo1,2, ME Gomes1,2 and RL Reis1,2,JF Mano1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, AvePark, Zona Industrial daGandra, S. Claudio do Barco, Caldas das Taipas – Guimaraes,Portugal; 2ICVS/3B’s, PT Government Associated Laboratory,Braga/Guimaraes, Portugal

Injectable systems are particularly attractive for a minimally invasiveapproach in tissue engineering applications. Many different methodsfor in situ crosslinking have been investigated however the ability ofcells to promote hydrogel formation has not been fully explored. Thisstudy addressed the hypothesis that cells can promote crosslinking ofchitosan microparticles forming a tridimensional network. Throughcovalent immobilization we were able to functionalize particles withspecific antibodies, used to promote the attachment of cells and growthfactors of interest. CD90 anti-human antibodies that are highlyexpressed by human adipose stem cells (hASCs) were successfully con-jugated with chitosan microparticles. A tridimensional hydrogel wasobtained by the assembly of the modified chitosan microparticles withhASCs and was stabilized by the crosslinks established by theentrapped cells. The degree of crosslinking of the structure could beregulated by the cell concentration in each construct. Moreover webelieve that a combination of microparticles tailored with specificgrowth factors will increase the stability of the construct and promotecell differentiation. It is well established that platelets are an importantsource of autologous growth factors that can modulate cell growth anddifferentiation. In this study we propose antibody-conjugated particlesas a method to select specific growth factors from the mixtureobtained from platelet lysates. The obtained construct simultaneouslyprovides support for stem cell growth as well localized and sustainedpresentation of factors to modulate cell differentiation. We intend todesign a novel multifunctional injectable system that may be custom-ized by combining particles with different growth factors for a specificapplication.

TS014Liquified multilayered capsules incorporatingmicroparticles for cell adhesion sitesCR Correia1,2, RL Reis1,2 and JF Mano1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal; 2ICVS/3B’s - PTGovernment Associate Laboratory, Braga/Guimaraes, Portugal

Polymeric multilayered capsules (PMCs) have found great applicabilityin various applications due to their versatile wall functions, capabilityto load active substances and unique permeability. PMCs are based onthe sequential adsorption of polyelectrolytes by the layer-by-layer(LbL) technique, followed by the elimination or liquefaction of the tem-plate core. The principle of the strategy is to physically isolate a widerange of materials, including cells, proteins and/or therapeutic mole-cules, from the outside environment. This selective permeability ismediated by the LbL membrane, which allows the diffusion of nutri-ents, oxygen, waste products and metabolites, while avoiding theentrance of high molecular weight immune system components. How-ever, when living cells are encapsulated, the existing methodologiesstill have to address a main issue related to the fact that most cells areanchorage-dependent and, thus, cannot grow in suspension. Therefore,although the liquified environment ensures the diffusion of essentialmolecules for cell survival, on the other hand liquified environmentsare deprived from cell adhesion sites. To overcome this main drawback,we hypothesized that liquified and flexible capsules combined withencapsulated microparticles to provide cell adhesion sites are a promis-ing attempt. To test this hypothesis, hierarchical structures featuring (i)an external shell combining three polyelectrolytes, namely poly(L-lysine) (PLL), alginate (ALG) and chitosan (CHT) prepared by LbL, and(ii) incorporating surface functionalized poly(L-lactic) acid (PLLA)microparticles were developed. The construction of the multilayeredstructure by quartz-crystal microbalance with dissipation monitoringwas monitored. Additionally, the mechanical performance of capsuleswas evaluated. Results show that the combined assembly of PLL, ALGand CHT resulted in a more resistant and thicker film with an exponen-tial build-up growth regime compared to the assembly without PLL.The ability of the optimized capsules to support cell survival wasassessed. L929 cells were encapsulated and cell viability and prolifera-tion assays were performed. Results show that capsules containingPLLA microparticles revealed an enhanced metabolic activity, biocom-patibility and proliferation. We believe that the developed approachwill offer new possibilities to the existing bioencapsulation strategies.Different microparticles loaded with growth factors and other biomole-cules of interest can be encapsulated in order to customize and controldifferent cellular functions, such as differentiation of stem cells into thedesired lineage, according to the target application field.


14 Abstracts List

TS015Gellan gum – hydroxyapatite compositehydrogels for bone tissue engineeringMG Manda-Guiba1,2, MB Oliveira1,2, JF Mano1,2, AP Marques1,2,JM Oliveira1,2, VM Correlo1,2 and RL Reis1,213Bs Research Group – Biomaterials, Biodegradables andBiomimetics, AvePark, Zona Industrial da Gandra, S. Claudio doBarco, Caldas das Taipas – Guimaraes, Portugal;2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal

The modification of polymeric matrices by adding calcium-phosphatederivatives has been proven an effective strategy for tailoring the prop-erties of scaffolds employed in bone tissue engineering. In this regardand, considering the biomechanics of bone as well as the durotacticresponse of osteoblasts, this study builds on the hypothesis that thepreparation of novel Gellan Gum (GG)-Hydroxyapatite (HA) hydrogelcomposites could benefit the mechanical profile of matrices as well asthe cell-substrate interaction in favor of cell recruitment and growth.To this purpose, HA microparticles at different concentrations (10 and20%) were successfully incorporated into GG based hydrogels. Thecomposites were characterized by Scanning Electron Microscopy(SEM) coupled with Energy Dispersive Spectroscopy (EDS), FourierTransformed Infrared Spectroscopy (FTIR), X-Ray Diffraction Analysis(XRD), Micro Computed Tomography (l-CT) Analysis and Thermo-gravimetric Analysis (TGA). In vitro degradation and swelling studieswere conducted in PBS solution, while the GG/HA composites weresubjected to Dynamic Mechanical Analysis (DMA). The efficacy of theGG/HA matrices to precipitate apatite in Simulated Body Fluid (SBF)was evaluated, while the cell-matrix interactions were studied by seed-ing the composites with human osteoblast-like cells. The cell-viabilitywas assayed by staining the cell-loaded composites with calcein-AM,Texas Red-Phalloidin and DAPI-blue and observing by confocal micro-scope. The image (SEM, l-CT)-assisted microstructural characteriza-tion of the GG/HA composites reveals hydrogels with high porosity(>80%) and average pore size of 260 lm. GG/HA composites demon-strate high water retention ability during swelling studies, while theweight loss did not exceed 8% during the degradation studies. FTIRand XRD detected peaks typical of hydroxyapatite. The thermal curvesof the composites disclose an initial weight loss due to moistureremoval and two subsequent degradation phases due to the polymericcomponent decomposition. The matrices exhibit increasing storagemodulus (E’) and decreasing loss factor (tan d) as a function of fre-quency during DMA analysis, while composites containing 20% HApossess always higher E’ and lower tan d values. GG/HA compositesdevelop bioactivity in the form of multiple agglomerates of apatite crys-tals since the 3rd post-immersion day in SBF. Viability assays indicatedthat the human osteoblast-like cells seeded on the GG/HA compositeswere metabolically active. Concluding, the modification of GG-basedhydrogels by HA in different concentration appears to result in compos-ites that meet several scaffold-design criteria and to allow the tailoringof their mechanical performance. However, further optimization isrequired in order to improve cell adhesion and to promote cell growth.

TS016Is bladder wall regeneration nowadays feasible?Comparison of different biomaterials in bladderwall augmentationM Pokrywczynska1, A Jundzill1, J Adamowicz2, SKrzyzanowska1, M Bodnar3, A Marszalek3, T Chmielewski4,P Nakielski4, T Kowalczyk4, R Debski5 and T Drewa1,6,71Tissue Engineering Department, CM NCU Bydgoszcz, Poland;2Department of General, Oncologic and Pediatric Urology, A.Jurasz University Hospital, Bydgoszcz, Poland; 3Chair andDepartment of Clinical Pathomorphology, CM NCU, Bydgoszcz,Poland; 4Department of Mechanics, Physics of Fluids, IFTR PAS,Warsaw, Poland; 5Department of Pediatrics, Hematology andOncology, CM NCU, Bydgoszcz, Poland; 6Department of Generaland Oncological Urology,Nicolaus Copernicus Hospital, Torun,Poland; 7Department of Urology, Oncology Center, Kielce, Poland

Introduction: The gold standard for bladder wall creation after radicalcystectomy gastrointestinal segments augmentation. However, bowelreplacement is associated with many complications. The ideal biomate-rial for bladder reconstruction has not been found so far. The purposeof this study was to compare the three scaffolds: artificial L–lactide–co–caprolactone (PLC 7015), natural SIS, and composite of amniotic mem-brane and L–lactide–co–caprolactone (CAM-PLC 7015) in rat bladderwall regeneration.Methods: Adipose tissue was harvested from 8-week 10 male Wistarrats. Adipose derived stem cells (ADSCs) were seeded in density3 · 106 cells/cm2 onto PLC, SIS and CAM-PLC scaffolds and culturedfor 5 days in stem cell culture medium. Before implantation the cellswere labeled with PKH-26 dye. 28 male Wistar rats were divided intoseven equal groups. Augmentation cystoplasty was performed in theearlier created dome defect. Group I: SIS+ 3 · 106ADSCs; II: PLC+3 · 106ADSCs; III: CAM-PLC+ 3 · 106ADSCs; IV: SIS; V: PLC; VI:CAM-PLC; VII: control. The reconstructed bladder walls were evalu-ated in H&E staining and macroscopic estimation. The presence ofimplanted cells was assessed by fluorescence microscopy. Cystographywas performed.Results: All bladder wall layers has been regenerated in bladders aug-mented with cell seeded scaffolds. Cystography and histological analy-sis indicated that the best results were obtained when the bladderswere reconstructed with SIS or amniotic membrane. In the bladderssubstitute with acellular matrices fibrosis and graft contractionoccurred. Fluorescence analysis confirmed the presence of implantedcells 3 months after reconstruction and stated the cell migration tospleen, bone marrow and liver.Conclusions: Adipose tissue is attractive source of stem cells for recon-structive urology. PLC matrix seems to possess poorer regenerationpotential comparing to bladder wall restored with SIS or amnioticmembrane.


Abstracts List 15

TS017Novel bilayered Gellan gum/Gellan gum-hydroxyapatite scaffolds for osteochondral tissueengineering applicationsR Canadas1,2, DR Pereira1,2, J Silva-Correia1,2, AP Marques1,2,JM Oliveira1,2 and RL Reis1,213Bs Research Group – Biomaterials, Biodegradables andBiomimetics, AvePark, Zona Industrial da Gandra, S. Claudio doBarco, Caldas das Taipas – Guimaraes, Portugal;2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal

Osteoarthritis is a major cause of disability during aging. By the age of60, close to 100% of the population will have histologic changes ofdegeneration in their knee cartilage (Loeser, 2000). Because of its avas-cular nature, cartilage has little capacity to self-regenerate. Despite theprogress already achieved in osteochondral regeneration, some limita-tions have to be overcome. The formation of fibrocartilage has to beavoided and the innervation has to be improved. Further, one main fea-ture to be promoted is the induction of vascularization in the bony partbut not in the cartilage part and to avoid de-differentiation processes. Apromising strategy could pass through the development and optimiza-tion of novel culture systems. The ideal approach could integrate scaf-folds presenting regions with different physical characteristics,combined with different growth factors to support different stem cellsfates, regarding the complex tissue physiology to be regenerate. Thiswork aims to develop novel bilayered gellan gum (GG)/gellan gum-hydroxyapatite (HAp) hydrogels based structures for osteochondral tis-sue engineering applications. Bilayered GG/GG-HAp hydrogels wereproduced by joining both solutions of GG 2% (w/v) with and withoutHAp (20% wt.) for bony and cartilage parts, respectively. The solutionswere introduced into a silicone mould with 20:10 mm (height anddiameter, respectively). Gelation of GG was promoted by immersion inPBS solution for 24 h. The architecture of the bilayered scaffolds wasinvestigated by micro-computed tomography. Results have shown thatthe freeze-dried bilayered scaffolds composed by low acyl GG(2%(w/v)/low acyl GG(2%(w/v)-HAp20%(w/w) possess a porosity of83.4 ± 0.8%, pore size of 279.3 ± 38.6 lm and interconnectivity of62.2 ± 5.4%. Degradability assays are being performed with the intentto use this system to culture human adipose derived stem cells inducingcell co-differentiation into chondrocytes and osteoblasts. Ultimately,the developed bilayered scaffolds will provide new therapeutic possibil-ities for the regeneration of osteochondral defects.

TS018Free-standing multilayer films made of chitosanand alginate for biomedical applicationsSG Caridade1,2,3, C Monge3, JF Mano1,2 and C Picart313Bs Research Group - Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal; 2ICVS/3B’s - PTGovernment Associate Laboratory, Braga/Guimaraes, Portugal;3LMGP, Institut National Polytechnique de Grenoble et CentreNational de la Recherche Scientifique, 3 parvis Louis Neel,Grenoble, France

Objective: The method for preparing multilayer films by the consecu-tive deposition of oppositely charged polyelectrolytes has gained tre-mendous recognition due the user friendly preparation, capability ofincorporating high loads of different types of biomolecules in the films,fine control over the materials’ structure, and robustness of the prod-ucts under ambient and physiological conditions. However the prepara-tion of such films needs the assembly on a substrate and, sometimes,cannot be detached from it, which has limited the application of suchfilms in areas as tissue engineering and regenerative medicine. Herein,we report the production of chitosan/alginate (CTS/ALG) free-standingfilms that can be detached from an underlying inert substrate withoutany postprocessing step allowing the determination of physical proper-ties of fundamental significance such as ion permeation and mechani-cal properties.Methods: In this work, the buildup of free-standing multilayer filmsmade of CTS and ALG was investigated. Several conditions were testedto follow the film growth in order to get thick films. The CTS/ALG free-standing films were characterized by Fourier transform infrared spec-troscopy (FTIR) and by scanning electron microscopy (SEM). Perme-ability tests were performed using FITC-dextran, with severalmolecular weights, as a drug model molecule. Cell adhesion wasassessed using C2C12 myoblast cells over a period of 48 h. Results andDiscussion. The produced membranes can be detached from an under-lying inert substrate without any postprocessing step. Permeabilityexperiments on these membranes revealed that the permeation ofFITC-dextran depended greatly on its molecular weight. The resultsshowed that these films are a good substrate for cell adhesion as spreadcells were observed all over the surface, by actin and heochst staining.Conclusions: The production of free-standing films permits the directexperimental determination of many physical properties of fundamen-tal significance such as ion permeation and mechanical properties thatcan be tuned for real-world applications. These free-standing films areeasy detachable, easy to handle, stable in the presence of physiologicalsolutions and biocompatible, demonstrating potential for applicationsin tissue engineering and regenerative medicine.


16 Abstracts List

TS019Designing silk fibroin-based matrices with ionicliquids for tissue engineering strategies usinghuman adipose stem cellsSS Silva1,2, EG Popa1,2, ME Gomes1,2, MB Oliveira1,2,JF Mano1,2 and RL Reis1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal;2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal

Silk (SF) is an attractive biomaterial to be used in tissue engineeringapplications because of its excellent mechanical properties and biocom-patibility [1, 2]. In this work, the cellular response of silk hydrogelsproduced through dissolution of this protein in ionic liquids (ILs) wasinvestigated. For that, degummed fibers obtained from the cocoons ofmulberry silkworm (Bombyx mori) were dissolved in an IL and thesolution was gelified in ethanol, followed by IL removal from gels usingsoxhlet extraction. The fabricated hydrogels were characterized bio-chemically and biophysically by detecting amino acid composition,FTIR, SEM and mechanical testing (DMA). For in vitro assessment,human adipose stem cells (hASCs) were seeded in the hydrogels andcultured for different time periods. The resulting hydrogels have a rub-bery consistency, homogeneous surface and viscoelastic behavior. Addi-tionally, no differences on amino acid composition were found,indicating that the silk composition was kept. Confocal images con-firmed cell attachment and alignment of actin filaments within the hy-drogel matrix with well-develop nuclei. The MTS assay demonstratedthe metabolic activity of hASCs in contact with hydrogels up to28 days. Furthermore, the results of DNA quantification showed thathASCs are able to proliferate during studied period. These results indi-cated that (i) the efficiency of IL removal resulted in hydrogels withminimal cytotoxicity; and (ii) positive cellular response of the materialssurface for the adhesion and proliferation of hASCs. SEM observationscorroborated with the results obtained from MTS and DNA suggestedthat cells are able to migrate at different levels within the structure.These findings indicated that silk hydrogels produced using ILs may bepotential candidates for tissue engineering strategies, namely cartilageregeneration.Acknowledgments: The authors acknowledge financial support fromPortuguese Foundation for Science and Technology -FCT (Grant SFRH/BPD/45307/2008; SFRH/BD/64601/2009), ‘‘Fundo Social Europeu’’-FSE, and ‘‘Programa Diferencial de Potencial Humano-POPH’’. Thiswork was partially supported by the FEDER through POCTEP 0330_IBEROMARE_1_PReferences:1. Silva, S.S., A. Motta, M.R.T. Rodrigues, A.F.M. Pinheiro, M.E.

Gomes, J.F. Mano, R.L. Reis, and C. Migliaresi. Biomacromolecules,2008. 9(10): p. 2764-2774.

2. Altman, G.H., F. Diaz, C. Jakuba, T. Calabro, R.L. Horan, J.S. Chen,H. Lu, J. Richmond, and D.L. Kaplan. Biomaterials, 2003. 24: p.401–416.

TS020Multilayered polymeric particle production usingsuperhydrophobic surfaces methodology for drugdelivery and tissue engineering applicationsAC Lima1,2, C Custodio1,2, C Alvarez-Lorenzo3 and JF Mano1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, Headquarters of the European Institute of Excellenceon Tissue Engineering and Regenerative Medicine, University ofMinho, Guimaraes, Portugal; 2ICVS/3B’s – PT GovernmentAssociate Laboratory, Braga/Guimaraes, Portugal;3Departamento de Farmacia y Tecnologıa Farmaceutica, Facultadde Farmacia, Universidad de Santiago de Compostela, Santiagode Compostela, Spain

Encapsulating technologies that render spherical particles containingcells or relevant moleculeshave been developed to be used in fieldssuch as tissue engineering, pharmaceutics, cosmetics, agriculture, asalso in other bio-related applications, namely biosensors and bioreac-tors. The multiple bioactive agents release, with an important role intissue regeneration, constitutes an important strategy in tissue engi-neering. The control of bioactive agents release may be achievedincreasing the complexity of the encapsulating particles by adjustingthe chemistry and the architecture. In this context, multi-compartmen-talized systems able to simultaneously deliver various bioactive agentsat different kinetics have emerged and are envisioned to be the nextarea of development. Multilayered particles exhibiting predefineddiameters and layers thickness may offer additional advantages includ-ing higher bioactive agents loads, improved molecules stability, and tai-lored release schedules such as delayed or pulsatile avoiding initialbursts. The most external layers could even act as rate-limiting barriersto further reduce burst release. Since multilayered particles are com-partmented, each particle can load multiple bioactive agents isolatedfrom each other. Similarly, more than one type of cells may be immobi-lized into different compartments. The layers thickness and composi-tion determine the performance of the system. Compared tomonocompartment delivery systems, the development of multi-com-partmented structures is still immature and intensive efforts are beingdone to efficiently produce this type of systems. The production ofmulti-compartmented particles is quite challenging and the existingmethodologies involve wet and aggressive conditions that compromisethe encapsulation efficiency of bioactive agents and the viability ofcells. Herein we report a simple bottom-up approach suitable for pre-paring multilayered polymeric particles in a very fast way, whichinvolves the use of biomimetic superhydrophobic surfaces. In the pres-ent work, concentric multilayered polymeric particles were prepared byadding layers one-by-one, and then their applications as carriers forsequential multiple drug release and as scaffolds for cells immobiliza-tion intended in cell therapies or tissue engineering were explored. Theresults showed that the engineered particles can be loaded with differ-ent molecules confined in different compartments for later sequentialand time-programmed release. They can also immobilize cells main-taining them viable for long time, being potentially useful for cell-basedtherapies.


Abstracts List 17

TS021Mechanical performance and biocompatibilitystudy of methacrylated Gellan gum hydrogelswith potential for nucleus pulposus regenerationJ Silva-Correia1,2, B Zavan3, V Vindigni4, MB Oliveira1,2,JF Mano1,2, H Pereira1,2,5,6, J M Oliveira1,2,JD Espregueira-Mendes1,2,5, G Abatangelo3 and RL Reis1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Guimaraes, Portugal; 2ICVS/3B’s - PTGovernment Associate Laboratory, Braga/Guimaraes, Portugal;3Department of Histology, Microbiology and BiomedicalTechnology, Univ. Padova, Viale G. Colombo 3, Padova, Italy;4Department of Plastic Surgery, Univ. Padova, Via Giustiniani44, Padova, Italy; 5Saude Atlantica Sports Center – F.C. PortoStadium, Minho University and Porto University Research Center,Portugal; 6Orthopedic Department Centro Hospitalar Povoa deVarzim – Vila do Conde, Portugal

Methacrylated gellan gum hydrogels, obtained either by ionic- (iGG-MA) and photo-crosslinking (phGG-MA), have been investigated aspotential biomaterials for supporting nucleus pulposus (NP) regenera-tion and/or repair [1,2]. In previous work, some advantages wereattributed to GG-MA hydrogels, such as: (i) the possibility to controlendothelial cells infiltration and blood vessel ingrowth’s, (ii) tunableand improved mechanical properties, and (iii) in situ gelation, withinseconds to few minutes. In this study, the mechanical and biologicalperformance of these hydrogels was firstly evaluated in vitro. Humanintervertebral disc (hIVD) cells obtained from herniated patients werecultured within both hydrogels, for 1 up to 21 days. Dynamic mechani-cal analysis and biological characterization (calcein-AM staining, ATPand DNA quantification and PCR) were performed after specific timesof culturing. A biocompatibility study was also performed in vivo, bysubcutaneous implantation of acellular iGG-MA and phGG-MA hydro-gels in Lewis rats for the period of 10 and 18 days. Tissue response tothe hydrogels implantation was determined by histological analysis(haematoxylin-eosin staining). The in vitro study showed that both cellloading and culturing time do not have an effect on the mechanicalproperties of the hydrogels. Regarding their biological performance,the iGG-MA and phGG-MA hydrogels showed to be effective on sup-porting hIVD cells encapsulation and viability up to 21 days of cultur-ing. Human IVD cells were homogeneously distributed within thehydrogels and maintained its round-shape morphology during cultur-ing time. The in vivo biocompatibility study showed that iGG-MA andphGG-MA hydrogels do not elicit any deleterious effect, as denoted bythe absence of necrosis and calcification, or acute inflammatory reac-tion. A thin fibrous capsule was observed around the implanted hydro-gels. The results presented in this study indicate that the iGG-MA andphGG-MA hydrogels are stable in vitro and in vivo, support hIVD cellsencapsulation and viability, and were found to be well-tolerated andnon-cytotoxic in vivo, thus being potential candidates for NP regenera-tion.Acknowledgements: Funding from EU FP7 project Disc Regeneration(NMP3-LA-2008-213904).References:1. J. Silva-Correia, et al., J Tissue Eng Regen Med, 2011, 5(6):e97-

e107.2. J. Silva-Correia, et al., Tissue Eng Part A, 2012, 18(11–12):1203–

1212.

TS022Advanced mimetic materials for meniscus tissueengineering: targeting segmental vascularizationJ Silva-Correia1,2, H Pereira1,2,3,4, L-P Yan1,2, V Miranda-Goncalves2,5, AL Oliveira1,2, JM Oliveira1,2, RM Reis2,5,6,JD Espregueira-Mendes1,2,3 and RL Reis1,213B’s Research Group - Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Guimaraes, Portugal; 2ICVS/3B’s - PTGovernment Associate Laboratory, Braga/Guimaraes, Portugal;3Saude Atlantica Sports Center – F.C. Porto Stadium, MinhoUniversity and Porto University Research Center, Portugal;4Orthopedic Department Centro Hospitalar Povoa de Varzim –Vila do Conde, Portugal; 5Life and Health Science ResearchInstitute (ICVS), School of Health Sciences, University of Minho,Campus de Gualtar, Braga, Portugal; 6Molecular OncologyResearch Center, Barretos Cancer Hospital, Barretos, Sao Paulo,Brazil

Meniscus lesions are among the most common orthopaedic injurieswhich can ultimately lead to degeneration of the knee articular carti-lage. The human meniscus has a limited healing potential, partly dueto a poor vasculature, and thus meniscus regeneration using tissueengineering strategies has recently been investigated as a promisingalternative to total/partial meniscectomy [1]. Advanced scaffolds fortissue engineering of meniscus should be able to mimic and preservethe asymmetric vascular network of this complex tissue, i.e. enable con-trolling the segmental vascularization during the regeneration process.Novel scaffolds were produced combining a silk polymeric matrix (12wt%) [2] and the methacrylated gellan gum hydrogel (iGG-MA),which has been shown to be able to prevent the ingrowth of endothe-lial cells and blood vessels into the hydrogels [3,4]. The angiogenic/anti-angiogenic potential of acellular and cell-laden silk-12 scaffoldscombined with iGG-MA hydrogel was investigated in vivo, using thechick embryo chorioallantoic membrane (CAM) assay. For producingthe cell-laden scaffolds, human meniscus cells (HMC¢s) were isolatedfrom morphologically intact human menisci using an enzymatic-baseddigestion and expanded using standard culture conditions. The HMC’s-laden hydrogel/silk scaffolds were produced by encapsulating theHMC’s into a 2 wt% GG-MA hydrogel, followed by impregnation ontothe 12 wt% silk scaffold and ionic-crosslinking in a saline solution. ACAM assay was used to investigate the control of segmental vasculari-zation of the acellular and HMC¢s-laden hydrogel/silk scaffolds by theeffect of GG-MA hydrogel, until day 14 of embryonic development.The in vivo study allowed investigating the number of macroscopicblood vessels converging to the implants. The evaluation of possibleinflammation and endothelial cells ingrowths was performed by histo-logical (haematoxylin and eosin - H&E - staining) and immunohisto-chemical methods (SNA-lectin staining). When the silk-12 scaffold wascombined with the hydrogel, an inhibitory effect was observed as dem-onstrated by the low number of convergent blood vessels. Results haveshown that iGG-MA hydrogel prevented the endothelial cells adhesionand blood vessels infiltration into the HMC’s hydrogel/silk scaffolds,after 4 days of implantation. This study showed that the hydrogel/silkscaffolds enabled controlling the segmental vascularization, thus it canpossibly mimic the native vasculature architecture during meniscusregeneration.References:1. H. Pereira, et al., Arthroscopy, 2011, 27(12):1706–1719.2. L.-P. Yan, et al., Acta Biomater, 2012, 8(1):289–301.3. J. Silva-Correia, et al., J Tissue Eng Regen Med, 2011, 5(6):e97-

e107.4. J. Silva-Correia, et al., Tissue Eng Part A, 2012, 18(11–12):1203–

1212.


18 Abstracts List

TS023Thermosensitive poly-caprolactone scaffolds for3D differentiation of C2C12 cells and humanadipose-derived stem cellsV Hruschka1, A Saeed2, R Cheikh al Ghanami2, G Feichtinger1,S Nurnberger1, A Meinl1, T Nau1, H Redl1, K Shakesheff2 andS Wolbank11Ludwig Boltzmann Institute for Experimental and ClinicalTraumatology, AUVA Research Centre, Austrian Cluster for TissueRegeneration, Vienna, Austria; 2School of Pharmacy, Centre forBiomolecular Sciences, University of Nottingham, Nottingham,United Kingdom

For tissue engineering (TE) applications, the natural process of regener-ation is imitated by using bioresorbable scaffolds that support cellularattachment, migration and proliferation, along with cells capable of dif-ferentiation upon exposure to inductive factors. Based on the idea ofcombining a fully degradable polymer (Poly(�-caprolactone; PCL) witha thermoresponsive polymer (polyethylene glycol methacrylate, PEG-MA) a scaffold was developed, which liquefies at 4 �C and solidifies at37 �C. Adult stem cells, which can be obtained out of human fat tissue,have the ability to be cultured in vitro and the potential to differentiateinto various lineages, including the osteogenic and adipogenic lineage.There are several advantages using adipose-derived stem cells (ASCs),for instance the possibility to isolate large amounts in an easy way orthe possibility to use them in an autologous setting. The mouse C2C12cell line also features stem cell characteristics and has the potential todifferentiate towards the myocyte as well as osteocyte lineage. In thisstudy, the novel thermoresponsive material PCL-PEGMA was combinedwith C2C12 cells or human ASCs to generate an expandable 3D con-struct for soft or bone TE, depending on the in vitro differentiation con-ditions. As a first step, biomaterial seeding for C2C12 cells as well asASCs was optimized and their attachment, survival, distribution andpersistence within the 3D material was characterized using viabilityassays, fluorescence microscopy and scanning electron microscopy.C2C12 cells as well as ASCs attached to the polymers were viable andevenly distributed in all scaffolds. For C2C12 differentiation, the cellswere seeded 3D within the scaffold and stimulated with either osteo-genic or myogenic differentiation medium for 7 days. The ASC-scaffoldconstructs were cultured in media supporting either adipogenic or oste-ogenic differentiation of ASCs for 21 days. 3D differentiation of thecells was examined using qRT-PCR for differentiation specific markers.Interestingely, C2C12 cells differentiated in the myogenic lineage aswell as ASCs treated with adipogenic differentiation medium showedincreased marker expression in 3D compared to 2D, suggesting that thethermoresponsive PCL-PEGMA scaffold qualifies for 3D differentiation.Conclusion: The thermoresponsive scaffold presented in this study is asuitable material for 3D soft tissue engineering. The properties of thematerial show limited potential to support osteogenic differentiation ofC2C12 or ASCs. In contrast, the matrix allows the attachment ofC2C12 cells as well as ASCs and was able to support 3D myogenic dif-ferentiation of C2C12 cells and 3D adipogenic differentiation of ASCs.

TS024Intracellular delivery of methylprednisolone bydendrimer-based nanoparticles improveslocomotor outcomes after spinal cord injurySR Cerqueira1,2,3, JM Oliveira1,2, BL Silva2,3, N Silva1,2,3,JF Mano1,2, N Sousa2,3, AJ Salgado2,3 and RL Reis1,213B’s Research Group - Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Guimaraes, Portugal; 2Life and HealthSciences Research Institute, School of Health Sciences, Universityof Minho, Braga, Portugal; 3ICVS/3B’s Associate Lab, PTGovernment Associated Lab, Braga, Portugal

Spinal cord injuries (SCI) still remain a major challenge in current bio-medical research. In spite of several advances in the understanding ofits mechanisms there has not been an equal significant translation intothe clinics. As a result, there is no effective treatment that can overcomethe biochemical and cellular adverse reactions that lead to a chronicseverely impaired condition. One of the first opportunities to minimizethese drastic consequences is to control the secondary events that fol-low the trauma. We are proposing the local delivery of an anti-inflam-matory corticosteroid - methylprednisolone (MP) – in an attempt tomodulate the noxious effects of the inflammation in the acute SCI. Asustained delivery as the one provided by these nanoparticles (NP) canbe highly advantageous, maximizing the drug’s potency in the targetsite. Therefore, we synthesized MP-loaded NPs composed of an innerpoly/(amido)amine (PAMAM) dendrimeric core and grafted with car-boxymethylchitosan (CMCht). Chemical and biological characteriza-tion studies were carried out showing that the NPs are stable in acidicand neutral buffer solutions. Also, the viability of primary glial cultureswas not compromised by the presence of 200 lg/mL of NP. In turn, anMP action in microglial cultures was observed in dosages above 1 mg/mL showing that MP is being released from the NPs inside the cells.The uptake profile of these NPs is time dependent and reaches its maxi-mum 24 h after incubation with astrocytes, oligodendrocytes andmicroglia. In a preview of a possible therapeutic effect, the NPs wereadministered in hemisected spinal cord injured rats. To assess the effi-cacy of local injections around the lesion site the animals were sacri-ficed 3 h after surgery and frozen sections were observed. Thefluorescently labeled-NPs were detected in the injury and in the sur-rounding spinal tissue indicating a successful delivery of the NP to thespinal tissue. The local injections were repeated in hemisected rats thatwere kept for 1 month, performing the BBB locomotory test weekly.Significant differences in the BBB test were found between the MP-loaded NPs injected rats and the sham group as well as the onesinjected with MP, demonstrating a favorable action of the MP-NPs inthe acute phase of the injury. This work revealed that sustained deliv-ery of MP via a NP system can be highly beneficial in the managementof the secondary injury that follows SCI improving the overall func-tional outcome of the injured animals.


Abstracts List 19

TS025Testing and FEA modelling of a modified suturetechnique to accommodate a tissue engineeredtendon in vivoP Sawadkar1,2, S Alexander1, M Tolk3, L Bozec2 and V Mudera11Tissue Repair and Engineering Centre, UCL Division of Surgery,Stanmore Campus, London, UK; 2Biomaterials and TissueEngineering, UCL Eastman Dental Institute, London, UK;3UCL, Department of Physics and Astronomy, Gower Street,London, UK

Introduction: Tendon repair is surgically challenging as the tendonoften retracts resulting in a gap between the torn end and its bonyinsertion. Tendon auto- or allografts are currently used to fill this defi-cit, but both are associated with potential complications. We havedeveloped a highly reproducible, rapid process technique to manufac-ture compressed cell seeded type I collagen tissue engineered con-structs to replace tendon grafts (1).However, the material properties ofthe engineered constructs are currently unsuitable to complete loadbearing in vivo. A modified suture technique has been developed towithstand physiological loading and off load the artificial constructwhilst integration occurs.Methods: Lapine tendons were used to test the strength of differentsuture techniques with different sizes of prolene sutures and tissueengineered collagen constructs in situ.

Figure 1 (A) Standard repair for tendon with modified Kesslersutures, (B) Modified repair Grasp four strand sutures with tissue engi-neered collagen construct The data was compared to standard modi-fied Kessler suture using a standard tendon graft. Mechanical testingwas carried out and a FEA stress distribution model constructed usingCOMSOL 3.5 software.

Results: The break point for modified suture technique with tissueengineered collagen construct was significantly higher(50.62 ± 1.62 N) compared to standard modified Kessler suture[12.49 ± 8.17N (P < 0.05)].

Figure 2 Stress distribution finite element analysis model for modi-fied suture technique and standard technique, Von Mises stress acrosssuture points and collagen construct. In FEA modelling VonMises stressin the middle of the geometry, i.e. in the middle of the collagen con-struct (for the modified technique) or at the point of the runningsutures (for the standard technique) are as follows (at a load of8 · 106 Pa): standard technique 5.6 · 106 Pa; modified technique2.8 · 105 Pa. Hence, it is evident that, stress is 20 times less if the mod-ified technique is applied.

Discussion & Conclusions: Distributing suture tension further proxi-mally and distally from the tendon ends increased the mechanicalstrength of the repairs. Using this proof of concept data, we will now

test this modified suture technique in vivo to test integration of bonemarrow stem cell seeded collagen construct and it is function in a la-pine model.Acknowledgement: I would like to thank government of India for fund-ing this project.Reference:1. V Mudera, M Morgan, U Cheema, S Nazhat, R Brown, Ultra-rapidengineered collagen constructs tested in an in vivo nursery site, J TissueEng Regen M. 2007;1(3):192–8.

TS026Ccbe1: a secreted protein with cardiomyogenicpotentialJ Facucho-Oliveira, T Justo, PNG Pereira, JM Inacio andJA BeloLaboratory of Embryology and Genetic Manipulation oftheRegenerative Medicine Program, Department of BiomedicalSciences and Medicine (CBME) and Institute for Biotechnologyand Bioengineering (IBB), UAlgarve, Faro, Portugal

Cardiovascular diseases are one of the leading causes of death and dis-ability in the world. The interruption of blood supply to a part of theheart during myocardial infarction leads to injury and eventually deathof the cardiac tissue. Since adult cardiomyocytes have very limitedregenerative capacity, the muscle tissue is replaced by other cells typesin a process called cardiac fibrosis or scarring. The routine usage ofembryonic stem cells (ESCs), and the promise that lies on its pluripo-tency to differentiate into any cellular type has encouraged manyresearchers to derive cardiomyocytes and try to use them to improvethe remodeling of the tissue. Concurrently, others have been trying tofind factors that facilitate the cardiac commitment from ESCs and/orthat may stimulate putative adult heart stem cells to form new muscletissue. There are several studies unraveling the function of such factors,but only very few examples of extracellular proteins with such cardi-omyogenic capacity. The function and molecular mechanism of thesecreted protein Collagen and Calcium Binding EGF domains 1 (Ccbe1)remains largely unclear. Recent reports, however, demonstrate thatCcbe1 has a role in lymphangiogenesis and ovary cancer metastasis,potentially trough a role in regulating migration. Furthermore, we haverecently reported that Ccbe1 is expressed in murine heart progenitorregions suggesting a role during heart development. Interestingly,according to our present data, Ccbe1 potentiates the differentiation ofmouse ESCs towards cardiomyocytes, as seen by an increase in thenumber of beating foci and expression of definitive cardiomyocytegene(s). In addition, Ccbe1 is expressed in undifferentiated ESCs but,during differentiation, the peak of Ccbe1 expression accompanies theincrease in the expression of heart-progenitor markers Nkx2.5 and Isl1.Taken together, our data suggests that Ccbe1 may have a role duringcardiogenesis. To understand this further we are currently performingseveral experiments where we are testing the (inducible) gain- andloss-of-function of Ccbe1. This involves, respectively, the generationand transduction of a polycistronic lentiviral vector containing a tetra-cycline-inducible promoter to co-(over)express Ccbe1 and cyan fluores-cent protein (CFP), and the transduction of lentiviral vectors expressingsiRNAs to downregulate the expression of Ccbe1. Overexpression isinduced with doxycycline during different stages of differentiation. Thiswill allow us to dissect when, where and how Ccbe1 is relevant duringcardiac differentiation from mouse ESCs. Nonetheless, it is becomingsafer to infer that Ccbe1 may potentially be used in non-transgenic car-diac regeneration/treatment.


20 Abstracts List

TS027Sulfation degree of glycosaminoglycans triggersdistinct cytoskeleton organisation inmesenchymal stem cellsDS Costa1,2, Y Yang3, S Moller4, T Groth3, RL Reis1,2 andI Pashkuleva1,213B’s Research Group—Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the theEuropean Institute of Excellence on Tissue Engineering andRegenerative Medicine, AvePark, Guimaraes, Portugal; 2ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimaraes,Portugal; 3Biomedical Materials Group, Department ofPharmaceutical Technology and Biopharmacy, Institute ofPharmacy, Martin Luther University Halle-Wittenberg, Germany;4INNOVENTe.V., Biomaterials Department, Germany

Glycosaminoglycans (GAGs) comprise the closest cellular environment:they are building elements of ECM and can be also found on cells sur-face. Their biological activity depends on several parameters amongwhich the negative charge is of prime importance[1]. This charge isgenerally associated with the presence of sulfate groups (-OSO3H).Sulfation is a dynamic modification: it can occur at various positionswithin the glycan and different sulfation patterns have been identifiedfor the same organs and cells during their development. However, themechanisms of coding and transferring information by these function-alities are not yet complete understood, mainly because of (i) the com-plex physiological microenvironment in which GAGs interactions occurand (ii) the inability to access homogeneous GAGs[2]. In this work, wepropose model surfaces bearing GAGs with different sulfation degreeas platform to investigate the pathways by which mesenchymal stemcells (MSCs) sense and respond to this peculiar functionality: the -OSO3H. We have selected two natural GAGs for this study: hyaluronicacid (HA) because it is the only non-sulfated glycan and heparin (HEP)as the GAG with the highest degree of sulfation. To obtain a largerrange of sulfation degrees, we also prepared a synthetic analogue ofHA with a sulfation degree of 1.4 (sHA). All these GAGs were cova-lently bonded to aminothiols deposited on gold surfaces. MSCs, bothfrom bone marrow and adipose tissue, adhered well to all surfaces. For-mation of focal adhesions was observed after only 1h of culture forbone marrow-derived MSCs regardless the used substrate. The pres-ence of -OSO3H groups induced different morphology and cytoskeletonorganisation: formation of longer filopodia and well pronounced actinfibers were visible for MSCs from both sources. Moreover, cells weremore spread after 24 h in contact with -OSO3H-containing surfaces.Cells behaved similarly on both sulfated surfaces (sHA and HEP) anddifferences in cell morphology were less obvious: higher sulfationdegree induced less lamellipodia formation while filopodia numberand length increased. In summary, the present study provides evidencethat sulfation degree of GAGs triggers distinct cytoskeleton organisa-tion in mesenchymal stem cells that may be related with the differenti-ation of those cells. However, further studies at the molecular levelabout the exact mechanism of these processes need to be carried out.Acknowledgement: This work was carried out under the scope of EUproject Find&Bind-FP7/2007-2013 under grant agreement no.NMP4-SL-2009-229292.References1. Capila I. and Linhardt R.J., A. Chemie-International Edition, 2002.

41(3):391–412.2. Soares da Costa D. et al., J. Materials Chemistry, 2012.

22(15):7172–7178.

TS028Organ printing and stem cells as future ofregenerative medicine: influence of the cellencapsulation system on cell behaviorV Liaudanskaya, L Gasperini, A Motta and C MigliaresiUniversity of Trento Department of Materials Engineering andIndustrial Technologies & BIOtech Research Center, University ofTrento, Trento, Italy

Introduction: Many techniques were developed in the area of regenera-tive medicine, however they are still imperfect and generally based onthe creation of tissue replacement scaffolds, while organ printing tendsto build an organ de novo as closer as possible to the origin organ usingstem cells as a source of regenerative cells. This can potentially elimi-nate needs in organ transplants, and therefore, is very promisingapproach in regenerative medicine. Cell encapsulation by electro-hydrodynamic jetting is a perspective technique for organ printing. However,during the encapsulation process, as well as during the further cellgrowth inside beads, several changes in cell proliferation, activity, andprotein expression can happen due to the complexity of the cell encap-sulation system. Thereby, before starting the process of organ printing,it is important to make sure that encapsulated cells are not harmed.Thus, the main aim of the initial project is to characterize cell behaviorduring and after the cell encapsulation process. Glyceraldehide-3-phos-phate dehydrogenase (GAPDH), the housekeeping protein that inter-acts with telomeric DNA and participates in glycolisis, apoptosis, cellaging, cell proliferation, and other vitally important processes wasselected for checking genes activity of encapsulated cells.Materials and methods: B50 rat neuroblastoma cell line was selectedfor the experiment.

1.2 · 106 cells were seeded in a tissue culture plate(S = 12.56 cm2);

2.4 · 106 cells were seeded on the top of 2% alginate coating(S = 12.56 cm2);

5 · 106 cells were microencapsulated in 1 mL of 2% alginate beads,200 lm in size;

105 cells released from the beads were reseeded (S = 4.5 cm2).Cross-linking of alginate occured with the presence of Ca2+ cations.

GAPDH expression was analysed by SDS-PAGE and Western blottinganalysis, whole cell lyses were collected on days 0, 1 and 3; reseededcells were lysed on the day 5. Cell parameterses were checked with Al-amar Blue test, and with cell viability test (Calcein/PropidiumIodidestaining followed by confocal microscopy visualization).Results and conclusions: Cell viability analysis showed that cells remainalive inside the beads even after encapsulation for a long time (D7),however, the activity of entrapped cells slightly decreased. The WBresults showed a strong increase in GAPDH expression, comparing topreceding days of the experiment. Analyzing the results, the studyingof production of the heat shock protein (Hsp70) that is only expressedin response to cells stress, appeared to be essential, as well as the prolif-eration of entrapped cells.


Abstracts List 21

TS029Novel approach to create hybrid and hierarchicalscaffolds aimed for tissue regenerationSM Oliveira1,2, TH Silva1,2, RL Reis1,2 and JF Mano1,213Bs Research Group – Biomaterials, Biodegradables andBiomimetics, AvePark, Zona Industrial da Gandra, S. Claudio doBarco, Caldas das Taipas – Guimaraes, Portugal;2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal

Tissue Engineering scaffolds with a wide range of properties and usingseveral types of materials have been produced using different process-ing techniques. Among those, hybrid scaffolds, made of synthetic bio-degradable and natural-origin polysaccharides, have been arising asthe most adequate 3D structures to support the mechanical solicitationsonce implanted as well as cell adhesion, proliferation and differentia-tion. A well-known methodology to combine micro/nanofibers with/within scaffolds is the combination of electrospinning and bare scaffold.However, in the common approach, fibers are not homogeneously dis-tributed along the 3D scaffold, being limited to its surface; to achievedeposition on the interior of the scaffold, the electrospinning has to beused during bare scaffolds preparation in a complex process that maylead to structure delamination. Herein, we present a novel approach tohybridize and introduce fibrillar structures and coatings inside 3D scaf-folds, rendering truly hierarchical systems. The structures were createdcombining an unconventional layer-by-layer (LbL) electrostatic self-assembly technology with physical crosslinking by freeze-drying. LbL isbased on a simple alternated deposition of polyanions and polycations,i.e. polyelectrolytes (PEs), and the introduction of such materials insidethe scaffolds creates a new environment which allows to control cellbehavior, by enhancing surface area available for cells attachment andthe similarity to extracellular matrix composition and structure, with-out damaging the mechanical integrity and properties of the bare scaf-fold. Alginate and chitosan were used as polyanion and polycation,respectively, and polycaprolactone bare scaffolds were produced byrapid prototyping. Scaffolds were modified with the PEs using a home-made dipping robot to study the effect of several LbL assemblingparameters on the final structure. Characterization of the structuresrevealed that one can obtain nanocoatings or nanocoatings plus fibril-lar structures inside the scaffolds, homogeneously distributed andlinked to the wall of the bare scaffold in a controlled manner. SaOs-2osteoblastic-like cells were used to assess the cytocompatibility of thehybrid scaffolds quantifying dsDNA, ALP activity and observing cell dis-tribution. After 7 days in culture, cells were able to colonize whole lon-gitudinal section of the scaffolds, being able to adhere to the fibrillarstructures and showing similar or higher ALP activity and dsDNA con-tent comparing with the unmodified PCL scaffolds. In conclusion, thisnew methodology virtually allows the modification of any 3D structurewith the introduction of a new hierarchical level in tissue engineeringscaffolds, as coatings or fibrillar structures, which acts as systems tocontrol cell adhesion, proliferation and differentiation.

TS030Differentiation of rat bone marrow mesenchymalstem cells into insulin producing cells – impact ofextracellular matrix proteinsM Pokrywczynska1, S Krzy _zanowska1, A Jundziłł1,M Lewandowska2, M Hołysz3, W Jozwicki2, M Gagat4,A Grzanka4, K Warda1, M Rasmus1 and T Drewa1,5,61Tissue Engineering Department, Collegium Medicum of NicolausCopernicus University, Bydgoszcz, Poland; 2Department of TumorPathology and Pathomorphology, Franciszek LukaszczykOncology Center, Bydgoszcz, Poland; 3Department ofBiochemistry and Molecular Biology, Medical University, PoznanPoland; 4Department of Histology and Embryology, CollegiumMedicum of Nicolaus Copernicus University, Bydgoszcz, Poland;5Urology Department, Nicolaus Copernicus Hospital, Torun,Poland; 6Department of Urology, Institute of Oncology, Kielce,Poland

Introduction: Pancreatic islet transplantation is a promising approachfor type 1 diabetes treatment, however it is limited by a shortage ofpancreas donors. This study was performed to investigate whetherbone marrow mesenchymal stem cells (MSCs) could be transdifferenti-ated into insulin producing cells in vitro and evaluate the role of colla-gen, laminin and vitronectin in this process.Methods: MSCs were isolated from 8-week Wistar rats and cultivatedunder standard conditions. MSCs phenotype was confirmed by flowcytometry. The cells were induced to differentiate into a pancreaticendocrine phenotype by defined culture conditions within 22 dayswith or without extracellular matrix proteins (collagen, laminin andvitronectin). Pancreatic characteristics were evaluated by real timereverse transcription polymerase chain reaction (qRT-PCR) andimmunofluorescence analysis. Insulin content and release in responseto glucose stimulation were tested with radioimmunoassay.Results: Flow cytometry confirmed the homogeneous MSCs pheno-type. MSCs derived from 3rd passage were positive for the CD44(99.5% of cells) and CD90 (99.7% of cells) markers and negative fortypical endothelial and hematopoietic markers CD34 (99.6% of cells)and CD45 (99.2% of cells). The induced cells expressed pancreaticendocrine cell related genes. Collagen and laminin increased theexpression of Nkx6.1, NeuroD1, Pax4 and PCSK. Laminin induced for-mation of the islet-like structures and increased expression of Ins1 andIns2. However combining collagen, laminin and vitronectin did notheighten the expression of endocrine cell markers. Immunofluores-cence analysis revealed the expression of insulin. Insulin secreted fromdifferentiated cells was much higher than that from pre-differentiatedcells.Conclusions: Rat bone marrow MSCs can differentiate into insulin pro-ducing cells in vitro. Laminin plays an important role in formation ofthree dimensional structure and pancreatic endocrine cell maturation


22 Abstracts List

TS031Urinary conduit construction using tissueengineeringT Kloskowski1, A Jundziłł1,2, N Gurtowska1, J Olkowska1,T Kowalczyk3, M Bodnar4, A Marszałek4 and T Drewa1,51Tissue Engineering Department, Collegium Medicum, NicolausCopernicus University, Bydgoszcz, Poland; 2Department ofGeneral and Vascular Surgery, Collegium Medicum, NicolausCopernicus University, Bydgoszcz, Poland; 3Institute ofFundamental Technological Research, Polish Academy of Sciences,Warsaw, Poland; 4Department of Clinical Pathomorphology,Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz,Poland; 5Department of Urology, Institute of Oncology, Torun,Poland

Introduction: Standard procedure after bladder resection is use of ilealsegment for urine transport outside the body. This procedure requireadditional chirurgical method on ileum and is associated with manyside effects. Aim of our study was to construct artificial conduit thatreplace ileal fragment.Material and Methods: Fourteen Wistar rats were used in experiment.Decelluralized aortic arch or policaprolactone scaffold were used forartificial conduit construction. Scaffold was anastomosed end to endwith ureter on one side, and with skin layer on other side. Second ure-ter was undisturbed. Follow-up was to 3 weeks. After that time ratswere sacrificed and artificial conduits were analyzed using hematoxy-lin-eosin staining. Results Decelluralized aortic arch was used in 12rats, in four cases follw-up was under 1 week because of rats death. Po-licaprolactone was used in two rats with 3 week follow-up. In all casesoutflow of urine was stopped, also presence of pus was observed.Hematoxylin-eosin staining showed lack of cell layers regeneration ondecelluralized aortic arch. One policaprolacton scaffold showed lack ofintegration with ureter, in second scaffold regeneration of cell layerswas observed.Discussion: Our results showed that unseeded aortic scaffolds are notsuitable for ureter conduit construction. Results obtained using policap-rolacton scaffolds are promising, but outflow of urine must be kept,and more experiments have to be performed to confirm promisingproperties of this scaffold.

TS032Electron beam cross linking of hydrogels as anovel and versatile technique of scaffoldfabrication in tissue engineering strategiesMA Shamekhi and S DamavandiPolymer Engineering Department, Sarvestan Branch, IslamicAzad University, Sarvestan, Iran

Radiation induced processes have many advantages over other conven-tional methods [1]. Electron Beam processing of thermoplastic poly-mers results in an array of enhancements, such as an increase in tensilestrength, and resistance to abrasions, stress cracking and solvents [2–4]. Indeed, applications of electron beam processing is not limited tothese areas but modification of biomaterials [5–7]. Hydrogels, on theother hand are important candidates in fabrication of suitable scaffoldsfor tissue engineering purposes. Radiation techniques have proved tobe particularly suitable for producing hydrogels for biomedical use. Inthe present research, novel hydrogels were prepared using Poly vinylPyrolydone (PVP), Poly ethylene glycole (PEG) and Water solublechitosan [8]. Homogeneous mixture of hydrogel constituents were irra-diated by 10Mev electron beam in the dose range of 15–40 kGy.According to the results, the gel fraction of hydrogels increased withthe increasing of the dose and decreased with the increasing of the rela-tive content of PEG in hydrogel. The results show that the maximumswelling increases with increasing of the PEG concentration, which isdue to a decrease of the crosslink density. The hydrogels containingPVP and PEG (without chitosan) were brittle and showed poormechanical properties. With addition of chitosan to hydrogels formula-tion, the mechanical properties increased. The hydrogel containing6%PVP, 6%PEG and 3%chitosan showed the best mechanical proper-ties in 25 kGy.References:1. Shamekhi MA, Jafari SH, Khonakdar HA, Ehsani M,’’ Preparation

and Characterization of UV Irradiation Cross-linked LDPE/EVABlends’’. Plastics, Rubber and Composites, 39, 10,(2010),431–436.

2. Goodarzian N, Shamekhi MA, ‘‘Chemical Cross linking Versus HighEnergy Electron Beam Cross linking of HDPE: Electrical PropertiesStudy’’. J. Iran. Chem. Res. 2 (2009) 189–194.

3. Ziaie F, Borhani M, Shamekhi MA, ‘‘Storing Effects on ElectricalProperties of High Energy Electron Beam Irradiated LDPE andHDPE’’. International Journal of Polymeric Materials, 60:939–946,2011.

4. Shamekhi MA et al., ‘‘Novel Polymer Blends Based on Electron BeamCrosslinked TPU/EVA’’, IAU, Sarvestan Branch, research Grant.

5. Yamato M, Shimizu T, Kikuchi A, Okano T. ‘Radiation-Initiated NanoSurface Modification for Tissue Engineering’, IAEA-CT, Seattle, USA,(2002).

6. Yamato M. et al., Thermally Responsive Polymer-Grafted SurfacesFacilitate Patterned Cell Seeding and Co-culture, Biomaterials 23(2002) 561–567.

7. Gao C, Mao Z, Ma Z, ‘‘Surface Modification and Property Analysis ofBiomedical Polymers Used for Tissue Engineering’’ Colloids and Sur-faces B: Biointerfaces 60 (2007) 137–157.

8. Lu SH et al., Preparation of Water-Soluble Chitosan, J Appl PolymSci 91: 3497–3503, 2004


Abstracts List 23

https://www.researchgate.net/publication/239936989_Storing_Effects_on_Electrical_Properties_of_High_Energy_Electron_Beam_Irradiated_LDPE_and_HDPE?el=1_x_8&enrichId=rgreq-eda9368430952960cfe0e989b832a064-XXX&enrichSource=Y292ZXJQYWdlOzIzNDA4MTIwMDtBUzoxMDIxMTE3MDY2MTU4MTlAMTQwMTM1NjczNzM2NA==




https://www.researchgate.net/publication/6155457_Surface_modification_and_property_analysis_of_biomedical_polymers_used_for_tissue_engineering?el=1_x_8&enrichId=rgreq-eda9368430952960cfe0e989b832a064-XXX&enrichSource=Y292ZXJQYWdlOzIzNDA4MTIwMDtBUzoxMDIxMTE3MDY2MTU4MTlAMTQwMTM1NjczNzM2NA==




https://www.researchgate.net/publication/220008565_Chemical_Cross_linking_vs_High_energy_Electron_Beam_Cross_linking_of_HDPE_Electrical_Properties_Study?el=1_x_8&enrichId=rgreq-eda9368430952960cfe0e989b832a064-XXX&enrichSource=Y292ZXJQYWdlOzIzNDA4MTIwMDtBUzoxMDIxMTE3MDY2MTU4MTlAMTQwMTM1NjczNzM2NA==



TS033Integrative studies on the role of Ccbe1 incardiogenesis: from the embryo to ES cell derivedcardiac tissueJ Facucho-Oliveira1,2, M Bento1,2, J Furtado1,2, E Correia1,2,T Justo1,2, R Drago1,2, PNG Pereira1,2, JM Inacio1,2 andJA Belo1,21Regenerative Medicine Program, Biomedical and MedicineSciences Department, University of Algarve; 2IBB-Institute forBiotechnology and Bioengineering, Center for Molecular eStructural Biomedicine, University of Algarve, Campus deGambelas, Faro, Portugal and Instituto Gulbenkian de Ciencia,Oeiras, Portugal

A central challenge for the future perspective of cardiac regenerativemedicine is the generation of large numbers of patient-specific cardiacmyocytes. Ccbe1 encodes a secreted molecule that was firstly identifiedusing an Affymetrix GeneChip differential screen for chick heart precur-sor cells expressed genes (Bento et al., 2011). In mouse and chick,Ccbe1 is expressed in major cardiac progenitor lineages that contributeto distinct heart structures during heart organogenesis (Facucho-Olive-ira et al., 2011). Moreover, analysis of gain and loss of function per-formed in both mouse and chick embryos showed abnormal cardiacmorphogenesis and aberrant chamber formation further elucidatingthe role of Ccbe1 for cardiac development. Similarly, in mouse andhuman ES cells, increased levels of Ccbe1 expression were detectedafter cardiac lineage commitment demonstrating well-coordinatedexpression of various early and late cardiac specific markers and Ccbe1.Knock-Down in mouse and human ES cells demonstrated the require-ment of Ccbe1 for proper cardiogenesis. Modulation of mCcbe1 activityin differentiating mES cells using media supplemented with mCcbe1recombinant protein has demonstrated a remarkable inductive poten-tial of mCcbe1 to enhance cardiogenesis. Taken together, this datastrongly suggest that Ccbe1 has the ability to direct the expression ofcardiac inducers and to control cardiac progenitor expansion in vitroand in vivo, allowing the generation of non-genetically manipulatedcardiac cells from a renewable cell source for regenerative cardiovascu-lar medicine.

TS034Development of a bilayered scaffold based onsilk fibroin and silk fibroin/nano-calciumphosphate for osteochondral regenerationL-P Yan1,2, JM Oliveira1,2, AL Oliveira1,2,3 and RL Reis1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, S. Claudio de Barco, Taipas, Guimaraes,Portugal; 2ICVS/3B’s - PT Government Associate Laboratory,Braga/Guimaraes, Portugal; 3Department of Health Sciences,Portuguese Catholic University, Viseu, Portugal

Objectives: Osteochondral defect is a common condtion in clinic. Satis-factory outcomes are rarely achieved by traditional methods. Tissueengineering might be a promising strategy for this hinder. The aim ofthis study is to mimick the stratified structure of osteochondral tissue,by developing a bilayered scaffold for osteochondral regeneration. Thedeveloped bilayered scaffold is composed of a porous silk fibroin scaf-fold as the cartilage-like layer and a porous silk fibroin/nano-calciumphosphate (CaP) scaffold as the bone-like layer.Methods: The silk and silk/nano-CaP bilayered scaffolds were preparedby a combination of salt-leaching and freeze-drying approaches, as pre-viously reported [1,2]. Briefly, the concentrated silk fibroion aqueoussolution (16%) was mixed with calcium chloride and ammonium phos-phate dibasic solution to generate the silk/nano-CaP suspension. Thebottom layer was prepared by adding the sodium chloride particles(500–1000 lm) into the suspension. A 16% silk fibroin solution wasthen added onto the top of the silk/nano-CaP layer. Sodium chlorideparticles of the same size were added into the silk solution to producethe top layer. After 48 h, sodium chloride was leached out from the lay-ered scaffold and the final bilayered scaffolds were lyophilized. Thegenerated scaffolds were characterized by SEM, micro-CT, and EDX.Results and Discussion: SEM images showed that a macro/micro por-ous structure was observed in both layers. These two layers integratewell, without the formation of a clear interface. Micro-CT analysisallowed observing that the layered scaffolds were of porous structure,with homogeneous porosity distribution in each layer. The CaP washomogeneously distributed in the bottom layer, while there was noCaP detected in the top layer. By EDX analysis, the amount of CaP fromthe bottom to the top layer was mapped, which presented a gradientdecrease in the interface region, indicating a good integration betweenboth layers.Conclusions: Silk fibroin and silk fibroin/nano-CaP bilayered scaffoldswere successfully generated. The porous structure was maintained ineach layer. The CaP was homogeneously distributed only in the bottomlayer, and presented a gradient decrease at the interface. The mostimportant features of this bilayered construct is the good integrationbetween the two layers at the interface region. This is important sincewhen implanted this region can be particularly sensitive to mechanicalstresses. Therefore, this bilayered scaffold may be useful for an osteo-chondral regeneration approach.References:1. Yan LP, et al. Acta Biomater, 2012, 8(1), 289–301. 2. Yan LP, et al.

Nanomedicine, 2012, accepted.


24 Abstracts List

TS035Superhydrophobic patterned chips for thecombinatorial and rapid study of 3Dbiomaterials-cells interactions and proteindelivery systemsMB Oliveira1,2, CL Salgado1,2, WL Song1,2 and JF Mano1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal;2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal

The development of optimized products in the tissue engineering (TE)field is a time and resource consuming process due to the unpredictableinfluence of the combination of variables such as biomaterials, cellsand soluble factors. As multiple combinations can be considered high-throughput (HT) methods were suggested as a way to master complex-ity in TE. Usually, HT systems test cell-2D biomaterials interactionsand, more recently, cell-3D hydrogels interactions. Using a chip consist-ing of superhydrophobic surfaces patterned with wettable regions wetested cells-hydrogels interactions in three-dimensional environment[1]. The versatility of the chip allowed its use for the first time on-chipcombinatorial study of 3D miniaturized porous scaffolds. Arrays of bio-materials were dispensed and processed in situ as porous scaffolds withdistinct composition, surface characteristics, porosity/pore size andmechanical properties. Those characteristics were assessed by adaptingmicrocomputed tomography equipment and a dynamic mechanicalanalyzer. The interactions between cell types of two distinct origins –osteoblast-like and fibroblasts - and the scaffolds modified with distinctamounts of fibronectin were studied by image-based methods and vali-dated by comparison with conventional destructive methods. Physicaland biological on-chip results were coherent with conventional mea-sures, and conclusions about the most favorable media for the growthof both cell types were taken. Growth factors (GF) proved to play animportant role in TE approaches, mainly for determining cell fate inapplications containing stem cells. We developed a chip based on wet-tability contrast with torus-shaped hydrophilic transparent regions dis-posed in an array matrix. Concentrically to these wettable regions asuperhydrophobic circle was maintained, so the hydrogels could beprocessed as protein-loaded spheres with minimum protein loss [2]and fixed with an indentation. A combinatorial system of BSA-FITC – acommonly used GF model – encapsulated in alginate hydrogels wasdesigned. The protein release from the hydrogels could be studied byimage analysis, avoiding manipulation and protein loss. The resultswere compared with conventional protein release tests and similar ten-dencies were observed. We believe that the proposed innovative usesfor the superhydrophobic chip and their upgrade in future applicationsmay constitute a promising breakthrough in integrated technologies forthe rapid development of TE systems.References:1. Salgado CL, Oliveira MB, Mano JF. Combinatorial cell-3D biomateri-

als cytocompatibility screening for tissue engineering using bioin-spired superhydrophobic substrates. Integrative Biology.2012;4:318–27.

2. Song WL, Lima AC, Mano JF. Bioinspired methodology to fabricatehydrogel spheres for multi-applications using superhydrophobic sub-strates. Soft Matter. 2010;6:5868–71.

TS036Nanostructured Multilayer compartments:towards multifunctionality and ‘‘cell-like’’hierarchical complexityRR Costa1,2, EC Otero1,2, FJ Arias3,4, JC Rodrıguez-Cabello3,4

and JF Mano1,21University of Minho, 3B’s Research Group – Biomaterials,Biodegradables and Biomimetics, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, Avepark, Caldas das Taipas, Guimaraes, Portugal;2ICVS/3B’s, PT Government Associated Laboratory, Braga/Guimaraes, Portugal; 3University of Valladolid, G.I.R. Bioforge,Edificio I + D, Paseo de Belen, Valladolid, Spain; 4NetworkingResearch Center on Bioengineering, Biomaterials andNanomedicine (CIBER-BBN), Valladolid, Spain

In living organisms, there are phenomena that require the presence ofspecific biomolecules with distinct function and in variable concentra-tions at a given time, such as the healing and regeneration of tissueand organ lesions. In this work, we propose the use of a compartment-ed drug delivery device for the multiple release of bioactive agents. Itconsists of nanostructured microcapsules confined within a millimetriccontainer that can be easily handled, mimicking the concept of cellswhich possess organelles with specialized functions. Each hierarchicalstructure was conceived using the layer-by-layer (LbL) method to formmicro and macrocapsules that could individually carry either moleculesand release them with distinct kinetics or magnetic nanoparticles(MNPs) to be used in targeted therapies. Furthermore, the internalmicrocontainers were constructed with a temperature-responsive elas-tin-like recombinamer (ELR) to further add smart properties to the pro-posed system. Sacrificial CaCO3 microparticles empty or entrappingeither rhodamine or Fe3O4 MNPs were incubated in chitosan and ELRsolutions using LbL for the conception of the microcapsules. Then, themicrocapsules were suspended in alginate which was ionically cross-linked in CaCl2 drop-wise. Rhodamine could be encapsulated at thispoint in the alginate. The bead was coated with chitosan and alginateto build the external macrocapsule compartment. All structures werecoated with 3 bilayers. The CaCO3 cores were chelated and the alginatebeads liquefied using EDTA. Fluorescence microscopy using FITC andrhodamine markers showed a uniform distribution of the microcap-sules within the macroreservoir. The release of rhodamine from eitherin the micro or macrocapsule was assessed at 25 and 37 �C in PBS.While the release from the macrocapsule follows a profile similar tothat of traditional drug delivery systems, it is more sustained anddelayed when released from the internal compartments. Such retentionis more pronounced at 37 �C (65% of release in comparison to 90%).This is due to the temperature responsive behavior of ELRs, whichundergo a phase transition and make the LbL shell less permeable. Forthe magnetic response, the incorporation of the MNPs was observed bytransmitted light microscopy. The attraction of the devices wasobserved by applying an external magnetic field along a defined trajec-tory. The results let foresee the use of such multilayer devices as com-partmented structures to encapsulate growth factors, MNPs and stemcells for their controlled differentiation and maintenance or for guidedregeneration of tissues and organs.


Abstracts List 25

TS037Surface modification of tissue engineeringscaffolds by UV laserG Amin1, M Sharaf2, Y Badr1 and N Solouma11NILES, Cairo University, Egypt; 2Department of Chemistry,Helwan University, Egypt

Tissue engineering is a promising area of research in which efforts arebeing made to generate human tissue or organs substitutes in case oftheir failure. The need for substitutes to replace or repair tissues ororgans is overwhelming. Scaffold preparation for tissue substrates is avery important issue in tissue engineering. In this study, we used laserenergy to modify the scaffold’s surface according to tissue engineeringneeds. UV laser energy was applied to the surface of the scaffold in atrial to get a surface texturing that allow the most reliable cell adhesionand proliferation. Modification of polycaprolacton (PCL) scaffold sur-faces using specific excimer laser beam parameter produced distributedregular ripples on their surfaces that influence the surface roughness,surface wettability which in turn enhance cell attachment, proliferationand the degradation rate of the scaffold. The differential scanning calo-rimetry (DSC) results showed a significant shift in the endothermicpeak of PCL (the peak shifted from 60 �C to 63 �C), which is an indica-tion of lack of significant changes in the crystalline state of PCL. TheSEM showed very smooth morphology with lack of any rough or rippleor pores of the surface of unmodified scaffold. On the other hand, theexposed surface of PCL to laser exciter with E = 31 m j, N = 100pulses and p.r.f = 5 Hz showed formation of highly distributed, regu-lar ripple on the scaffold surface with size range from 200–600 nm.The Water Contact angle measurement indicated that the modifiedscaffold yields much higher surface energy than unmodified scaffold.Roughness results showed that the modified PCL scaffold revealedhigher surface roughness than the unmodified PCL scaffold. The degra-dation test revealed that the modified PCL scaffold, have good degra-dability in vitro bioactivity, more than the PCL unmodified scaffoldsand it possess the ability to make a direct bond with living cells whenimplanted in the body. From In vitro biological testing imaging, theSEM revealed good adhesion of cells over the modified scaffold in com-parison to the unmodified scaffold. MTTcell viability indicated that themodified PCL scaffold showed excellent cell viability relative to controlrather than the unmodified scaffold. This is due to lower cell adhesionon the unmodified scaffold due to lack of ripple structure on its surface.These ripples enhanced the cell adhesion and cell growth on the sur-face of the modified PCL scaffold.

TS038Scaffold free bone tissue engineering usingmultilayer cell sheet technologyM Kiamehr1, T Yoshida2, H Takahashi2, T Okano2 and XB Yang1,21Biomaterial and tissue engineering group, Leeds Dental Institute,University of Leeds, UK; 2Institute of Advanced BiomedicalEngineering & Science, Tokyo Women’s Medical University, Japan

The restoration of bone loss due to trauma and congenital problems isa major clinical need. The use of stem/osteoprogenitor cells incombination with scaffolds has been the gold standard in bone tissueengineering. However, the most of the materials used are either non-biocompatible or the degraded materials cause side effect. In addition,the stem cells have to be detached by chemical methods which willdamage the cells and remove the extracellular matrix. Cell sheet tech-nology use thermal sensitive polymer coated dishes (UPCell�) to har-vest cells as an intact monolayer without using routine chemicalmethods. This method reduces the cell loss and retains extracellularmatrix so that it can be transplanted to the host tissues (as autograft)without sutures and any scaffold. The aim of this study was to investi-gate the potential of using multilayer cell sheet technology andHDPSCs for bone tissue engineering. Briefly, human Dental Pulp Stemcells (HDPSCs) were isolated from wisdom teeth and cultured in35mm UPCellTM temperature responsive culture dishes (Passage 3;5 · 105 cells per dish) in basal (1 week) and osteogenic media(2 weeks) at 37 �C and 5% CO2. Cells were harvested from dishes byreducing the temperature form 37 �C to 5% CO2. Then three layers ofHDPSCs were assembled using CellShifterTM. Half of the multilayercells sheet were fixed in 98% ethanol for alkaline phosphatises (ALP)staining. Half were fixed in 10% neutral buffered formalin and paraffinsections were stained by Hematoxylin and Eosin (H&E) as well as Al-cian Blue/Sirius Red (A/S). H&E staining confirmed the formation of athree layer cell sheet. Positive ALP staining showed that the HDPSCsmaintained their osteogenic phenotype within the cell sheets. A/S Redstaining revealed collagen matrix formation within the cell layerswhich was confirmed by birefringence. The mineral nodule formationwas also observed. To date, the most of methods for bone tissue engi-neering required 3D scaffold to support the cells growth. In this study,a scaffold free multilayer cell sheet technique has been evaluated.Unlike the other methods, this technology preserves the extracellularmatrix, cell-cell junctions, and cell surface proteins. It can assemble afunctional 3D tissue without any scaffold. The cells within the multi-layer were remaining osteogenic and produced mineralized collagenmatrix. In conclusion, this study indicated the potential of using multi-layer cell sheet technology in combination with HDPSCs for bone tissueengineering which may lead to translational research for bone augmen-tation.


26 Abstracts List

TS039Cardiogenic differentiation of stem cells onpoly(glycerol sebacate)(Pgs)/gelatin/vegfloaded nanofibrous constructs for treatment ofmyocardial infarctionR Ravichandran1,2, JR Venugopal1, S Sundarrajan1,2,S Mukherjee1 and S Ramakrishna1,21Healthcare and Energy Materials Laboratory, Nanoscience andNanotechnology Initiative, Faculty of Engineering, NationalUniversity of Singapore, Singapore; 2Department of MechanicalEngineering, National University of Singapore, Singapore

Heart failure due to myocardial infarction (MI) remains the leadingcause of death in many industrial nations, mainly due to the lack ofability of the myocardial tissue to regenerate. Current therapeuticapproaches used to treat patients with chronic heart disease includeleft ventricle assist devices, pharmacologic therapies and surgery, butstill heart transplantation remains the only possible solution forend-stage heart failure. The recent advances in the field of tissue engi-neering and stem cell biology have paved way for new strategies formyocardial regeneration. The main objective of this study was todevelop a cardiac patch that is biocompatible, has mechanical proper-ties similar to that of the native tissue and is capable of inducing car-diogenic differentiation of mesenchymal stem cells (MSCs) for MIregeneration. PGS/gelatin fibers were fabricated by core/shell elec-trospinning with gelatin as the shell material, responsible for biologi-cal interactions and core material as PGS for favorable mechanicalproperties. VEGF growth factor was also incorporated into the corematerial to favor its control release, thereby providing angiogenicproperties and anti-apoptotic properties at the infarcted site. The fabri-cated fibers were characterized by scanning electron microscopy(SEM), contact angle, Fourier transform infrared spectroscopy, andtensile testing to analyze the morphology, wettability of the mem-brane, surface functional groups and the mechanical properties of thematerial. The cell–scaffold interactions between MSCs and PGS/gela-tin nanofibers were analyzed by cell proliferation, confocal analysis toconfirm the cardiogenic differentiation of MSCs by employing MSCspecific marker protein CD 90 and cardiac-specific marker protein tro-ponin, and SEM to analyze cell morphology. This novel construct com-prising of PGS/gelatin and VEGF can provide the required tensilestrength to support seeded MSCs differentiation into cardiomyocytesfor tissue regeneration to repair the infarcted myocardium andimprove cardiac functions. Such an elastomeric material can resist theheart wall pressure while providing a structure that enhances thepotential for cell proliferation and differentiation of MSCs into cardio-myocytes. This combinatorial epitome might eventually bring cardiactissue engineering into the clinical forefront.

TS040Differentiation of embryonic stem cells can beinfluenced by different processing of gelatin/silkfibroin hydrogelW Sun1,2, T Incitti2, C Migliaresi1, A Quattrone2, S Casarosa2

and A Motta11Department of Materials Engineering and IndustrialTechnologies, Biotech Research Center, University of Trento, Italy;2The Centre for Integrative Biology, University of Trento, Italy

Introduction: ESCs (Embryonic Stem Cells) are Pluripotent cells capa-ble of differentiating into any cell type of the body, which hold greatpromise in treating diseases by replacement of body parts and organsthrough the generation of new tissues in tissue engineering. Howeverthe greatest challenge of using ESCs in clinical applications is the lackof knowledge in directing their differentiation fate. Directing the con-trolled differentiation of stem cells into a lineage-specific mannerrequires a controlled biophysical and biochemical microenvironmentand signals. Here we present how different processing of Gelatin/SilkFibroin hydrogels can influence ESCs differentiation fates. Gelatin isthe denature form of collagen which is the major constituent of allextracellular matrix in animals. Silk Fibroin (SF) has been explored formany biomedical applications due to its impressive biocompatibilityand biodegradability. We created the Gelatin/SF hydrogel by the chem-ical crosslinker genipin and by physical sonication crosslinking. TheESCs cells cultured on these hydrogel surfaces showed distinct ecto-derm differentiation fates.Materials and Methods: E14tg.2a cells were dissociated in single-cellsuspension and 1000 cells/cm2 were plated on gelatin-coated 12-wellplates and on different hydrogel surfaces. Cells were cultured in Knock-out serum medium that was replaced every 2 days.Results: The protocol is a single-step tool to convert ES cells into neu-rons on gelatin-coated plate, we initially analyzed the expression pro-files of genes of neuroectodermal lineage: Nestin, BIIITublin, NCAM.The expression of the three genes was greatly decreased in the cellscultured on the G-SF and Gel5SF compared to the control and sonica-tion crosslinked SF based hydrogels which indicated that on genipincrosslinked hydrogels the neural differentiation was blocked. We thenanalyzed the expression of K18(non-neural ectoderm), GATA4(meso-derm), Sox17(endoderm) in the cells of Genipin crosslinked samples.mESCs cultured on the the G-SF and Gel5SF showed greatly increasedexpression of K18, while mesodermal and endodermal genes expres-sion levels comparable to the control and undifferentiated ES. This sug-gests that the Genipin crosslinked Gelatin/SF hydrogel can change thecell differentiation fate from neuroectodermal lineage to epithelialectoderm lineage.Conclusions: All these indicated that the different processing of Gela-tin/SF hydrogels can influence the differentiation fate of ESCs. Thismay depend on the fact that the genipin crosslinked Gelatin/SF couldactivate the JNK/BMP4 signal pathways to suppress neural differentia-tion and enhance epithelial differentiation.

Name Sample ratio crosslink

G-SF 100(SF) Genipin

Gel 100(Gelatin) Genipin

Gel/5SF 95/5(Gelatin/SF) Genipin

SF 100(SF) Sonication

SF/5Gel 95/5(SF/Gelatin) Sonication


Abstracts List 27

TS041Decellularized porcine liver scaffolds for stemcell cultivation and differentiationKH Schneider1,2, J Hackethal1,2, A Teuschl1,2,6, C Gabriel2,3,J Grillari2,4,5, R Grillari2,4,5 and H Redl1,21Ludwig Boltzmann Institute for Experimental and ClinicalTraumatology; Vienna Austria; 2Austrian Cluster for TissueRegeneration, Vienna, Austria; 3Red Cross Blood TransfusionService of Upper Austria, Linz, Austria; 4University of NaturalResources and Applied Life Sciences, Vienna, Austria; 5EvercyteGmbH, Vienna, Austria; 6City of Vienna Competence Team TissueEngineering Bioreactors, Department of Biochemical Engineering,University of Applied Sciences Technikum Wien

Decellularized scaffolds are getting more important in TE. Currently,there is no efficient system to expand the functions of human adult he-patocytes in vitro. A three-dimensional structure could present a sup-portive environment for the cells to facilitate the viability andfunctionality of liver cells in culture. The extracellular matrix (ECM) andit compartments can enhance the cell proliferation and differentiation invitro. Especially the use of an ECM derived from the same tissue originshould provide an adequate structure for the cells. One main focus ofthis project is the reduction of the need for animal drug testing modelsby developing new in vitro alternative techniques. Our experiment wasdivided into three parts. Cell free biological scaffolds were prepared dur-ing the pre-cell culture step. 400 lm thick tissues slices (Ø 8 mm) werecut, serving as scaffold. For an effective cell removal the slices were trea-ted with a 3%Triton X-100 solution for 1.5 h. Afterwards the sliceswererinsed with PBS over night to wash out the agent. HE and TMRM stain-ing revealed cell-free conditions after treatment. In the cell culture step,the scaffolds were recellularized with endothelial cells (ECs), humanadipose-derived stem cells (hASCs, p0-p6) and immortalized adipose-derived stem cells (hTERTASCs). Calcein AM staining, HE staining andscanning electron microscopy (SEM) analysis were performed to deter-mine the recellularisation rate. To compare cell qualities hASCswere cul-tivated on fibronectin (FN) coated tissue culture plates (TCPs) in aparallel approach. Following the cell seeding a hepatocytic differentia-tionwas induced using human hepatocytic growth factor (HGF) and on-costatin M (OSM) in serum free media. After up to 28 days ofdifferentiation intracellular and extracellular changes were determinedusing light microscopy and urea quantification assay. Quantitative PCRwas performed to detect the amount of albumin and a-fetoproteinexpression levels. Decellularisation, as well as recellularisation of por-cine liver scaffolds was successfully performed using different cell types.We observed that the cell source seems to be critical for hepatocytic dif-ferentiation. All in all our system showed promising results to developnewmethods for human in vitro drug screening/testing systems.

TS042Platelet lysate – a substitute for fetal calf serumK Ploederl1,2, F Hildner1,2, A Peterbauer-Scherb1,2, H Redl2,3,S Hennerbichler-Lugscheider1,2 and C Gabriel1,21Red Cross Blood Transfusion Service for Upper Austria, Linz,Austria; 2Austrian Cluster for Tissue Regeneration, Austria;3Ludwig Boltzmann Institute for Experimental and ClinicalTraumatology

Fetal calf serum (FCS) is the most common supplement in media usedfor cell expansion. Due to its animal origin, it bears a potential risk totrigger immune reactions and to transmit xenogenic proteins, prionsand viruses. This is a major concern in case of cells intended for humanuse in cell therapy and regenerative approaches. In order to determinealternatives, several studies were already performed comparing FCS tohuman serum or defined media in combination with recombinantgrowth factors (GF) Human platelet lysate (hPL) is known for its highconcentrations in GF representing another potential alternative forFCS. Several cell lines and especially stem cells have already been suc-cessfully cultivated in the presence of hPL. For example, human adi-pose-derived stem cells (ASC) and human articular chondrocytes(HAC) were expanded with 10% FCS compared to 5% hPL. We couldshow that the proliferation potential as well as the chondrogenic differ-entiation potential could be enhanced by using hPL compared to FCS.Still the use of hPL is not very common. The main reason might be theproduction itself. So far, no standardized procedure for large scale pro-duction could be established. An overview on different production pro-cesses will be given. Additionally, we will present results using hPL oncultivation of HAC, ASC and amnion derived cell lines as well as dataof our attempts on product characterization.


28 Abstracts List

TS043Modulation of adipose tissue derived stem cellsencapsulated in injectable multi-functionalhydrogelsDF Coutinho1,2, VE Santo1,2, SG Caridade1,2, JF Mano1,2,NM Neves1,2, ME Gomes1,2 and RL Reis1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal;2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal

Objective: The cellular microenvironment plays an important role incontrolling the cellular behavior. Therefore, in Tissue Engineering strat-egies, systems have been designed so that by combining cells, signalingmolecules and engineered substrates, one can mimic as closely as possi-ble the native microenvironment of the extracellular matrix. In thiscontext, injectable hydrogels have been developed that allow control-ling the cellular spatial distribution and that provide a 3D support.However, many of these systems fail to replicate the mechanical prop-erties of tissues and more significantly to provide a cell-friendly envi-ronment. Therefore, the aim of this study is to evaluate the cellularresponse of human adipose stem cells (hACS) within a growth factor-enriched injectable hydrogel.Methods: Hydrogels were formed by combining platelet lysates (PLs)with a methacrylated gellan gum (MeGG) solution at different ratios.The hydrogels were further stabilized by photopolymerization. Theparameters of photocrosslinkingwere varied and thedynamicalmechan-ical properties evaluated for all the conditions. Total protein contentreleased from the hydrogels was quantified for all the conditions usingmicro-BCA assay. Human ASCs were both seeded on the surface of thehydrogels for 7 days and encapsulatedwithin thematerials for 14 days.Results and Discussion: Hydrogels with tunable mechanical propertieswere fabricated by changing the volume ratio of PLs and MeGG. Thehighest elastic modulus (nearly 500 kPa) was achieved for the condi-tion with the lowest volume of PLs (2MeGG:1PLs), being significantlymore elastic than MeGG alone. The rate of release of proteins presentin the PLs from the PLs-MeGG hydrogels was higher for the conditionwith equal volume of MeGG and PLs (1MeGG:1PLs), as a result of thelower crosslinked polymer network. Human ASCs cultured onto theengineered surfaces showed a good metabolic activity and prolifera-tion. Immunostaining revealed that MeGG:PL combinations fosteredthe attachment and spreading of cells. A strikingly improved cellularbehavior was observed for the formulations with PLs, when comparedto MeGG alone. This behavior was further confirmed at a 3D scale,demonstrated by a significantly higher cellular metabolic activity onthe hydrogels with PLs.Conclusion: Our system combines the mechanical support from MeGGwith the biological cues from PLs to engineer a cell-friendly injectablehydrogel with tunable mechanical properties. Given the simplicity inproducing these rather enriched hydrogels, we envision that it may bebeneficial for various tissue engineering and regenerative medicineapplications.

TS044Cartilage regeneration approach based on squidchitosan scaffolds: in-vitro assessmentLL Reys1,2, SS Silva1,2, RP Pirraco1,2, AP Marques1,2, JF Mano1,2,TH Silva1,2 and RL Reis1,213B’s Research Group - Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal;2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimaraes, Portugal

During the past decades, marine organisms have been the focus of con-siderable attention as potential source of valuable materials. Forinstance, chitosan is a biopolymer with high potential in the biomedicalfield and can be produced from crustacean shells and squid pens [1]. Inthis sense, we propose the use of chitosan to produce scaffolds forregenerative medicine purposes. An alkaline solution was used to de-proteinize squid pens and isolate b-chitin (Chaussard 2004), which wasfurther converted into chitosan through a deacetylation reaction. Chito-san was then processed into porous structures by freeze-drying [3],where chitosan solutions (4%)were submitted to different freezing tem-perature of -80 �C and -196 �C. The produced structures were furthersubmitted to neutralization methods with 4% NaHO, including in somecases a pre-washing step using ethanol/water solutions (100:0; 90:10,80:20; 70:30 and 50:50) [4]. The morphology of scaffolds producedusing either squid or commercial chitosan revealed a lamellar structure,independent of the source and/or freezing temperature. All chitosanscaffolds produced exhibited no-cytotoxic behaviour over L929 cells. Totest the in vitro functionality of the scaffolds, cells from the mouse chon-drogenic cell line ATDC-5 were seeded in the scaffolds and cultured fordifferent time periods. Scaffolds made from squid chitosan were shownto promote better cell adhesion than commercial chitosan scaffolds andcomparable or better cell proliferation. This demonstrates that squidchitosan is a valuable alternative to produce scaffolds for different appli-cations in regenerative medicine, namely the regeneration of cartilage.References:1. Tiago H. Silva, A.A., Bruno M. Ferreira, Joaquim M. Oliveira, Lara L.

Reys, Ricarte J.F. Ferreira, Rui A. Sousa, Simone S. Silva, Joao F.Mano, Rui L. Reis International Materials Reviews DOI: 10.1179/1743280412Y.0000000002, 2011.

2. G. Chaussard, Domard, A., Biomacromolecules, vol. 5, pp. 559–564,(2004).

3. L.L. Reys, S.S. Silva, J.M. Oliveira, S.G. Caridade, J.F. Mano, T.H.Silva, R.L. Reis, ‘‘Unraveling the potential of squid chitosan basedstructures for biomedical applications’’ (submited 2012) [4] V. M.Madihally, Mathee, H.W.T., Biomaterials, vol. 20, pp. 1133–1142,(1999).


Abstracts List 29

https://www.researchgate.net/publication/236957350_Revealing_the_potential_of_squid_chitosan-based_structures_for_biomedical_applications?el=1_x_8&enrichId=rgreq-eda9368430952960cfe0e989b832a064-XXX&enrichSource=Y292ZXJQYWdlOzIzNDA4MTIwMDtBUzoxMDIxMTE3MDY2MTU4MTlAMTQwMTM1NjczNzM2NA==





TS045Tenogenic potential of human stem cells from theamniotic fluid and adipose tissueMT Rodrigues1,2, AI Goncalves1,2, SJ Lee3, A Atala3, JJ Yoo3,RL Reis1,2 and ME Gomes1,213B’s Research Group, University of Minho, Headquarters of theEuropean Institute of Excellence on Tissue Engineering andRegenerative Medicine, Portugal; 2ICVS/3B’s - PT GovernmentAssociate Laboratory, Portugal; 3Wake Forest Institute forRegenerative Medicine, Wake Forest School of Medicine, USA

Introduction: Tendons are highly prone to injury and the intrinsic hypo-cellularity and hypovascularity make their natural healing extremelyslow and inefficient when severed damaged. Surgical repair with graftsis common but unsuccessful in a long term basis. The development oftissue engineering strategies based on stem cells explores a naturalendogenous system of regeneration with potential for tendon applica-tion. We propose to establish biochemical culturing conditions to assessthe tenogenic potential of human adipose stem cells (hASCs) andamniotic fluid-derived stem cells (hAFSCs), known for their prolifera-tive and differentiation capacities. Since several growth factors (GFs)participate in tendon formation and ECM synthesis, these GFs wereadded to the culture medium to stimulate tenogenic differentiation ofthese cells(1, 2). This study also envisions the application of hASCsand/or hAFSCs in cell-based strategies for tendon repair.Materials and Methods: hASCs were enzymatically isolated from lipoas-pirates and expanded in basic medium(a-MEM, 10% FBS, 1% antibi-otic) before being cultured in (i) basic medium, (ii) basic medium withglutamine(2 mM) and ascorbic acid(0.2 mM) plus (iii) EGF(10 ng/mL), (iv) FGF(10 ng/mL), (v) PDGF(10 ng/mL) or (vi) TGF-b(10 ng/mL). hAFSCs were obtained from amniocentesis procedures andexpanded in a-MEM medium plus 15% embryonic screened FBS, 1%glutamine, 1% antibiotic, 18% Chang B and 2% Chang C. Similarly tohASCs, hAFSCs were cultured in media (i), (iii), (iv), (v) and (vi) but(ii) corresponded to hAFSCs expansion medium. Tenogenic differentia-tion was weekly evaluated up to 28 days based on cell morphologyand Tenascin-C and Collagen3 protein expression, as well as on PCRanalysis for tendon related markers (scleraxis, tenascin C, decorin andcollagen 1 and 3. Primary tenoblasts obtained from surgery surpluswere used as cellular control of the experiment.Results: hAFSCs and hASCs showed a tenocyte-like aligned distributionin the different culture media by 14 and 21 days, respectively. But hA-FSCs tend to lose the typical alignment by growing confluent. Also,hASCs expressed higher levels of Tenascin-C protein than hAFSCs.Conclusions: Preliminary data on the aligned morphology and tenas-cin-C expression shows that both cell sources can be biochemicallyinduced towards tenogenic features. Overall, and despite variationsfound, selected GFs do not actively participate in the tenogenic processof these cells. Ongoing studies on molecular biology and other tendonmatrix related markers will clarify which stem cell source has morepotential for tendon regeneration strategies.References:1. Rodrigues, MTet al. (2012) TERM, online published;2. Liu, CF et al.(2011) Tissue Eng Part B Rev. 17: 165

TS046Facs-purified human motor neurons fromamplified human stem cell-derived culturesgenerate robust survival assaysNJ Lamas1,2,3,4,5,6, B Johnson-Kerner3,4,5,6, L Roybon3,4,5,6,7,H Wichterle3,4,5,6 and CE Henderson3,4,5,61Life and Health Science Research Institute, School of HealthSciences, University of Minho, Braga, Portugal; 2ICVS/3B’s-PTGovernment Associate Laboratory, Braga/Guimaraes, Portugal;3Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research,New York, NY, USA; 4Center for Motor Neuron Biology andDisease, Columbia University Center, New York, NY, USA;5Departments of Pathology, Neurology, and Neuroscience, Centerfor Motor Neuron Biology and Disease and Columbia Stem CellInitiative, Columbia University Medical Center, New York, NY,USA; 6Columbia Stem Cell Initiative, Columbia UniversityMedical Center, New York, NY, USA; 7Department ofExperimental Medical Science Wallenberg Neuroscience Center,Lund University, Lund, Sweden

Human Embryonic Stem Cells (hESCs) and Induced Pluripotent StemCells (hiPSC) can virtually give rise to all cell types in the body, consti-tuting an inexhaustible source of relevant cell types. Motor neuronsderived from hESCs and hiPSCs are a potentially important tool tomodel and to unravel mechanisms of pathological motor neuron celldeath. The culturing conditions to specify motor neurons (MNs) fromhuman pluripotent stem cells have been extensively studied and ingeneral work robustly. However, the production of human motor neu-rons on a scale compatible with high-throughput studies remainsimpractical due to the relatively low yield of current in vitro differentia-tion protocols. On the other hand, robust survival assays based on puri-fied human motor neurons have not been developed. Here, wedemonstrate for the first time the presence of ongoing neurogenesis inhuman stem cell-derived cultures of mixed spinal cord identity, whichleads to the continuous generation of new-born motor neurons for sev-eral weeks. We initially exploited this as a means to increase motorneuron yields and screened a collection of bioactive molecules. TheRho-associated kinase (ROCK) inhibitor Y-27632 was identified as acompound that amplifies motor neuron yields up to 4-fold indifferentiated hESC and hiPSC cultures. This likely occurs in a ROCK-independent manner, through the promotion of proliferation ofOlig2-expressing motor neuron progenitors. Since ongoing neurogene-sis constitutes a major potential confound for survival assays, amplifiedmotor neurons expressing the HB9::GFP reporter were therefore FACS-purified and employed to develop a reproducible and robust survivalassay for human motor neurons. Using this assay, we were capable todemonstrate for the first time in human motor neurons a significantsurvival-promoting activity of an array of known neurotrophic factorsand also showed that Y-27632 itself supports motor neuron survival.Together, our results highlight Y-27632 as a useful tool to increaseyields of human motor neurons from pluripotent stem cells for cell-based screening and biochemical applications.


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TS047Bioprocess engineering human pluripotent stemcells for clinical applicationM Serra1,2, C Correia1,2, C Brito1,2, MFQ Sousa1,2, M Silva1,2,J Jensen3, P Bjorquist3, A Fatima4, K Burkert4, MJT Carrondo2,5,J Hescheler4, T Saric4 and PM Alves1,21Instituto de Tecnologia Quımica e Biologica, Universidade Novade Lisboa, Oeiras, Portugal; 2Instituto de Biologia Experimental eTecnologica, Oeiras, Portugal; 3Cellartis AB, Gotemborg, Sweden;4Institute for Neurophysiology University of Cologne, Cologne,Germany; 5Faculdade de Ciencias e Tecnologia, UniversidadeNova de Lisboa, Monte da Caparica, Portugal

Objectives: Human pluripotent stem cells (hPSCs), with their ability forextensive proliferation and pluripotency, are renewable cellularresources with outstanding potential for Cell Therapy and Tissue Engi-neering applications. An imperative pre-requisite for the transition ofhPSCs to these fields is the establishment flexible platforms for thelarge-scale production and storage of hPSC-based products in tightlycontrolled conditions, to deliver high quality cells in relevant quantitiesto satisfy clinical demands. The laborious and time consuming 2D-cul-tures are difficult to control, present poor yields and often lack therequired cell functionality. These characteristics can have severe conse-quences on robustness, reproducibility, scalability and relevance of thecell systems, hampering their possible application in the clinic.Methods: Our work has been focused on the development of integratedbioprocesses capable to guarantee efficient cell expansion, differentia-tion, purification and cryopreservation of clinically relevant hPSCs(embryonic and induced) and/or their derivatives (cardiomyocytes).Different 3D setting have been explored and combined with stirredtank bioreactor technology aiming at controlling hPSC’s fate and cul-ture outcome.Results and Discussion: By combining microcarrier technology withenvironmentally controlled stirred tank bioreactors and further on withcell microencapsulation technology, we were able to improve the finalyield of hPSCs in 15-fold when compared to the standard 2D cultures.This strategy ensured high expansion ratios (20-fold increase in cellconcentration) and high cell recovery yields after cryopreservation(>70%). Alginate was the encapsulation material used due to itsintrinsic properties including biocompatibility, biosafety and permeabil-ity and due to its potential in cell transplantation. For the cardiac differ-entiation of induced PSCs, different bioprocessing parameters wereevaluated and the results showed the importance of controlling pH,pO2 and stirring profile to improve the final yields of functional cardio-myocytes. The incorporation of a perfusion system in the bioreactor isbeing evaluated, providing a promising tool to facilitate and enhancethe purification of induced PSC-derived cardiomyocytes.Conclusion: The knowledge of hPSC bioprocessing gained from ourwork provides important insights for the establishment of more robustproduction platforms, hopefully potentiating the implementation ofnovel hPSC-based therapies.

TS048Canine adipose stem cells: the influence of theanatomy and passaging on the stemness andosteogenic differentiation potentialJF Requicha1,2,3, CA Viegas1,2,3, CM Albuquerque2,JM Azevedo4, RL Reis1,3 and ME Gomes1,313B’s Research Group – Biomaterials, Biodegradables andBiomimetics, Department of Polymer Engineering, University ofMinho, Guimaraes, Portugal; 2Department of VeterinarySciences, School of Agrarian and Veterinary Sciences, Universityof Tras-os-Montes e Alto Douro, Vila Real, Portugal; 3ICVS/3B’s –PT Government Associated Laboratory, Braga/Guimaraes,Portugal; 4CECAV – Centre for Studies in Animal and VeterinarySciences, Department of Animal Sciences, School of Agrarian andVeterinary Sciences, University of Tras-os-Montes e Alto Douro,Vila Real, Portugal

Periodontal disease is an inflammatory pathology highly prevalent bothin humans and in dogs, which is characterized by destruction of theperiodontal ligament (PDL), cementum and alveolar bone. The devel-opment of new therapies, such as Tissue Engineering strategies, ismandatory due to the inefficacy of conventional therapies currentlyused. Dog is a very relevant animal model to study several human dis-eases, such as PD and simultaneously an important subject in veteri-nary medicine. Adipose derived stem cells have a great potential forapplication in cell based therapies, such as tissue engineering. The aimof this study was to assess the potential of the canine adipose tissuederived stem cells (cASCs) to differentiate into osteoblasts and chon-droblasts and study the anatomical origin and cell passaging effect onthe cASCs stemness and osteogenic potential. In addition, we aimed atassessing the behavior of cASCs when cultured onto a newly developeddouble-layer scaffold, specifically designed for the regeneration of peri-odontal defects. The adipose tissue was harvested from the abdominalsubcutaneous layer and from the greater omentum from adult healthydogs, according to the animal welfare Portuguese legislation. cASCswere isolated by an enzymatic method and expanded along 4 passagesin basal medium. cASCs were cultured using either osteogenic mediumor chondrogenic medium. The stemness and osteogenic differentiationwas followed by real time RT-PCR analysis of typical markers of MSCs,namely CD73, CD90 and CD105, and osteoblasts, like COLIA1, RUNX2and Osteocalcin. The behavior of the cASCs was then evaluated on apreviously developed double layer scaffold based on a starch and polyc-aprolactone (SPCL) blend that comprises a functionalized 3D fibermesh to promote osteogenesis (an thus support alveolar bone regener-ation) combined with a membrane aiming at acts as a physical barrierand promote PDL regeneration. The cellular proliferation was assessedby dsDNA quantification and SEM observation. The gene expression ofMSCs/osteogenic typical markers was assessed by real time RT-PCR.The obtained data revealed that cASCs exhibit a progressivelydecreased expression of the MSCs markers along passages and also adecreased osteogenic differentiation potential. Moreover, the resultsshowed that the anatomical origin of the adipose tissue has an evidenteffect in the differentiation potential of the cASCs. The resemblanceswith the human ASCs make the cASCs a suitable cell model for studynew cell therapies for humans. Additionally, we reported the highpotential of a newly developed scaffold combined with ASCs for peri-odontal tissue engineering.


Abstracts List 31

TS049Platelet lysates scaffolds with tunable gradientsof mineral microparticles guide osteogenicdifferentiation of human adipose derived stemcellsVE Santo1,2, ARC Duarte1,2, JF Mano1,2, ME Gomes1,2 andRL Reis1,213Bs Research Group - Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal; 2ICVS/3B’s - PTGovernment Associate Laboratory, Braga/Guimaraes, Portugal

Objectives: Platelet lysates (PLs) are an enriched pool of growth factors(GFs) obtained from activation of platelets, promoting the recruitment,growth and morphogenesis of stem cells. PLs can be used as either aGFs source or as a 3D hydrogel for simultaneous GFs and cell delivery.However, most of current PLs-based hydrogels lack stability, exhibitingconstant shrinking in culture and need to be activated with animal-derived thrombin. In this work we report the development of stableand bioactive 3D PLs-based scaffolds, by crosslinking with genipin andincorporating a gradient of Bioglass� microparticles under supercriticalfluid conditions. It is hypothesized that the structure itself is capable ofproviding the adequate support and biological cues to promote osteo-genic differentiation of seeded human adipose derived stem cells (hAS-Cs) envisioning applications in bone tissue engineering.Methods: Briefly, 1.5 mL of lysates solution mixed with genipin (cross-linking agent, 0.18% w/v) and Bioglass� microparticles (2.5% w/v)were placed inside the high-pressure vessel and underwent phaseinversion. Morphological characterization of the scaffolds was per-formed by scanning electron microscopy and micro-computed tomo-graphy. In vitro release quantification of specific GFs (Platelet DerivedGF and Transforming GF – PDGF and TGF, respectively) was also per-formed. Human ASCs were seeded onto the PLs-based hydrogels andcultured in vitro using either basal or osteogenic media. Deposition ofmineralized extracellular matrix (ECM) and expression of osteogenicmarkers were characterized by histological, immunohistochemical andreal time RT-PCR analysis.Results and Discussion: Morphological analysis of the stable PLs-basedscaffolds crosslinked with genipin indicated adequate levels of porosityand interconnectivity. In vitro protein release assays showed a con-trolled profile up to 28 days. When the scaffolds were seeded withhASCs, deposition of osteogenic ECM could be detected in constructscultured in both basal and osteogenic supplemented media, as shownby the alizarin red staining and through the detection of collagen type Iand osteocalcin through immunohistochemical and gene expressionanalysis.Conclusions: The developed PLs-based scaffolds can simultaneously actas a template, with a well-defined gradient of a mineral component,and as a multiple GFs release system. The combination of PLs scaffoldswith hASCs represents a feasible strategy for an autologous-orientedbone engineering approach. The possibility to fine tune the propertiesof the PLs scaffolds has demonstrated, in a previous work, its potentialto be used for cartilage regeneration. Coupling these two systems mayoffer significant advantages in osteochondral regeneration strategies.

TS050Translating human adipose-derived stem cells(hASCs) to clinical practice requiresstandardization of isolation and culture protocolsPP Carvalho1,2, JM Gimble3, IR Dias1,2,4, ME Gomes1,2 andRL Reis1,213B’s Research Group – Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, Avepark, Guimaraes, Portugal; 2ICVS/3B’s PTGovernment Associated Lab, Braga/Guimaraes, Portugal; 3StemCell Biology Laboratory, Pennington Biomedical Research Center,Louisiana State University System, Baton Rouge, LA, USA;4Department of Veterinary Sciences, School of Agrarian andVeterinary Sciences, University of Tras-os-Montes e Alto Douro,Vila Real, Portugal

Adipose-derived stromal/stem cells (ASCs) have been explored inrecent pre-clinical trials to treat diseases in a broad range of tissues.The ultimate goal is to translate these findings to clinical trials to testsafety and efficacy in human subjects. However, multiple steps areneeded to make ASC clinically available: adipose tissue must be stablefor up to 24 h during transit to cGMP laboratories1 for further process-ing. Second, isolation and expansion protocols must eliminate exposureto animal proteins2. This will require the development of animal-freeproducts suitable for the culturing of the cells. Current protocols3,4

employ several animal-derived reagents, such as collagenase for theenzymatic digestion of adipose tissue, FBS for cell culture and expan-sion, trypsin derived from porcine tissue (stomach) for cell detachingand passaging, etc. Likewise, the length of time between adipose tissueharvest and processing will need to be systematically evaluated withrespect to cell yield, viability, and function. The current study exploresthese technical challenges exploring alternative, non-animal sources ofcollagenase and trypsin-like enzymes for the isolation and passage ofASCs and assessing the effect of time delays on the yield and functionof ASCs after collagenase digestion. The results obtained demonstratedthat it’s possible to use purified or animal-free enzymes for digestion ofadipose tissue without decreasing the yield of stromal/stem cells noraffecting their surface markers5 and differentiation potential. Cell yieldand viability prove to be similar amongst any of the products (for bothSVFand ASCs). The differentiation potential was not affected and ASCswere easily induced to adipogenic, osteogenic and chondrogenic lin-eages. Cell surface markers analysis showed no significant differencesamongst any of the different grades of collagenase and trypsin-likeproducts. These outcomes have practical implications with respect tothe development of Standard Operating Procedures for cGMP manufac-ture of clinical grade human ASCs, which are essential for allowingtheir future use in the clinical practice.References:1. Carvalho, P.P., et al., Cells Tissues Organs, 2011. 194(6): p. 494–

500.2. Carvalho, P.P., et al., Cytotherapy, 2011. 13(5): p. 594–7.3. Dubois, S.G., et al., Methods Mol Biol, 2008. 449: p. 69–79.4. Bunnell, B.A., et al., Methods, 2008. 45(2): p. 115–20.5. Dominici, M., et al., Cytotherapy, 2006. 8(4): p. 315–7.


32 Abstracts List

TS051Exploring the osteoconductive effect of silanolgroups by using human adipose stem cellsAI Rodrigues1,2, ME Gomes1,2, IB Leonor1,2 and RL Reis1,213B’s Research Group - Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Taipas, Guimaraes, Portugal; 2ICVS/3B’s - PTGovernment Associate Laboratory, Braga/Guimaraes, Portugal

Current methods for bone tissue engineering are concentrated in the invitro proliferation and osteogenic differentiation of stem cells seededonto 3D scaffolds, as these are key elements to achieve tissue regenera-tion. One critical strategy in bone tissue engineering is to develop 3Denvironments that can provide the necessary cues for guiding cellrecruitment and driving tissue regeneration by mimicking the bonemicroenvironment and eventually eliminate the need to seed cells pre-viously to implantation. Silicon is known to have an influence on cal-cium phosphate deposition and on the differentiation of bone precursorcells1,2. In previous work3 we demonstrated that a wet spun fiber meshbased on a blend of corn-starch with polycaprolactone (30/70 wt.%,SPCL) with silanol (Si-OH) groups sustained human adipose stem cells(hASC) proliferation and osteogenic differentiation when culturedunder dynamic conditions. The present study attempts to analyze inmore detail the role of Si-OH groups on the osteogenic gene expressionof hASC. Thus the in vitro evaluation of SPCL with and without Si-OHgroups was done with human ASCs cultured in either a-MEM or osteo-genic medium for up to 21 days. Results showed that hASC readily pro-liferated and migrated into the interior of the wet-spun fibre meshscaffolds, and calcium and phosphate on the cell surfaces weredetected, within only 14 days of culture. Moreover, the studies in vitroindicated an enhancement of osteogenic gene expression of hASCwhen cultured in the presence of Si-OH groups. The present workshows that combining wet-spinning technology with a calcium silicatesolution as a non-solvent allows designing scaffolds with key cues torender an osteoconductive behavior as the classic ceramic materials forbone regeneration. Additionally, these results suggests that silicon andcalcium either individually or in combination may relevantly contributeto properly drive hASC towards the osteoblastic phenotype and high-light the potential of those ions in the control of cellular response suchas cell differentiation and/or in stem cells recruitment upon implanta-tion of a cell-free scaffold.References:1. Hoppe A., et al., Biomaterials 32: 2757–2774, 2011.2. Beck Jr G.R., et al., Nanomedicine: Nanotechnology, Biology, and

Medicine, 1–11, 2011.3. Rodrigues A.I., et al., Acta Biomaterialia, 2012, DOI: 10.1016/j.act-

bio.2012.05.025.

TS052Induction of human mesenchymal stem cellsosteogenesis by bioactive agent-releasingliposomesN Monteiro1,2, A Martins1,2, D Ribeiro1,2, S Faria3, NA Fonseca4,JN Moreira4, RL Reis1,2 and NM Neves1,213B¢s Research Group – Biomaterials, Biodegradables andBiomimetics; Department of Polymer Engineering, University ofMinho, Headquarters of the European Institute of Excellence onTissue Engineering and Regenerative Medicine, AvePark,Guimaraes, Portugal; 2ICVS/3B’s, PT Government AssociateLaboratory, Braga/Guimaraes, Portugal; 3Research CenterOfficinal Mathematical, Department of Mathematics for Scienceand Technology, University of Minho, Campus de Azurem,Guimaraes, Portugal; 4CNC - Center for Neurosciences and CellBiology, FFUC – Faculty of Pharmacy of the University ofCoimbra, Coimbra, Portugal

Stem cell therapy is a rapidly evolving area of research in regenerativemedicine. Mesenchymal stem cells (MSCs) have received considerableattention by the scientific community because of their potential ofexpansion and the ability to differentiate into various mesodermal tis-sues. Liposomes are well-established non-viral carrier systems, present-ing significant advantages over other nanoparticle-based drug deliverysystems, namely a high load carrying capacity, a relative safety and anease of large-scale production, as well as a versatile nature in terms ofpossible formulation and functionalization. The objectives of the pres-ent study were to evaluate the efficacy of growth differentiation factor-releasing liposomes on the induction of MSCs osteogenesis. For that,dexamethasone (Dex) was encapsulated within the liposome bilayer atdifferent lipid formulations. The obtained liposomes showed a mono-disperse distribution of particles size, and an increased f-potential forthe PEGylated liposomes. Dex encapsulation studies demonstrate thatthe presence of cholesterol (Chol) decreases the Dex loading capacityof the liposome bilayer. The different stabilizing effect of Chol and Dexon the liposomes is due to differences in their interaction with phos-pholipid molecules. Highly lipophilic Chol gets incorporated betweenthe acyl chains and reduces chain movement increasing rigidity andstabilization the membrane. Dex, being more hydrophilic, interacts dif-ferently with phospholipid acyl chains and head groups and destabiliz-es the membrane. In vitro release study demonstrated an initial burstrelease within an initial timeframe of 24 h. Following the initial release,a slower release was observed until 6 days. Afterwards, Dex continuesto be released at a slower but steady rate until day 21. The effect ofDex-loaded liposomes on viability, proliferation and osteogenic differ-entiation of human bone marrow-derived mesenchymal stem cells(hBMSCs) was assessed. The results of the biological activity showedthat the Dex-loaded liposomes do not have any cytotoxic effect and,more importantly, were able to promote an earlier induction ofhBMSCs differentiation into the osteogenic lineage, as demonstratedby the expression of osteoblastic markers at the phenotypic and thegenotypic levels. Concluding, Dex-loaded liposomes represent a novelbiological or nature-inspired nanoparticle strategy for tissue engineer-ing and regenerative medicine applications.


Abstracts List 33

TS053Suitability of silk-based 3D biotextiles seededwith human adipose-derived stem cells for abone tissue engineering approachVP Ribeiro1,2, AR Martins1,2, AP Marques1,2, G Bonifacio3,AL Oliveira1,2,4, RA Sousa1,2 and RL Reis1,213B’s Research Group–Biomaterials, Biodegradables andBiomimetics, University of Minho, Headquarters of the EuropeanInstitute of Excellence on Tissue Engineering and RegenerativeMedicine, AvePark, Caldas das Taipas, Portugal; 2ICVS/3B’s-PTGovernment Associated Laboratory, Braga/Guimaraes, Portugal;3CITEVE, Technological Centre for Textile and Clothing Industry,V. N. Famalicao, Portugal; 4Department of Health Sciences,Portuguese Catholic University, Viseu, Portugal

Human Adipose-derived Stem Cells (hASCs) became an emerging pos-sibility for tissue replacement therapies, such as bone tissue regenera-tion. Due to their osteogenic differentiation potential, easy isolation,expansion and in vitro proliferation, they have become a highly poten-tial source of seed cells to be seeded in bone tissue engineering (TE)constructs and have demonstrated promising prospects in bone regen-eration [1, 2]. To date several strategies have been proposed with moreor less success to prepare porous three-dimensional biodegradable scaf-folds for bone TE. Among them, textile technologies are particularlyinteresting since they can allow for producing finely tuned, fibre-basedcomplex structures, offering superior control over the design (ex: size,shape, porosity, fibre alignment), manufacturing and reproducibility.The aim of this work is to evaluate the potential of recently developedsilk-based biotextile structures [3] to promote hASCs adhesion, prolif-eration and osteoblastic differentiation. Natural silk yarns were pro-cessed into different 3D structures using standard knitting or warp-knitting technologies to increase the scaffold’s tridimensionality. In thelatter case two knitted silk layers are assembled and spaced by a mono-filament of polyethylene terephthalate (PET). These constructs werecharacterized in terms of their morphology by Microcomputed Tomog-raphy (l-CT) and scanning electron microscopy (SEM). The mechani-cal properties were investigated through compressive tests anddynamic mechanical analysis (DMA). All constructs disclose a biocom-patible behavior, assessed using a mouse fibroblastic cell line (L929;ECACC, UK). hASCs were seeded onto the scaffolds and cultured for14, 21 and 28 days in osteogenic medium. All textile constructs wereanalysed in terms of cell adhesion, proliferation and differentiationpotential influence through the biological assays, alkaline phosphatase(ALP), DNA and Ca2+ quantification and histological, confocal, SEMand Real-Time PCR analysis. The obtained constructs present veryreproducible intra-architectural scaffold geometry with high surfacearea and exhibiting a wide range of porosities. By the above mentionedassays it was possible validate the developed constructs as suitable forhASCs adhesion, proliferation and differentiation into an osteoblasticlineage. The positive influence of the developed 2D/3D textile struc-tures on the osteoblastic differentiation potential of hACSs is an impor-tant outcome that validates future bone tissue enginnering approachesusing these fibre-based architectures.References:1. de Girolamo, L., et al., 2009. 11(6): p. 793–803.2. Correia, C., et al., Acta Biomater, 2012. 8(7): p. 2483–92.3. Almeida, L.R., et al., 2011. 34(8): p. 663–663.

TS054Time-regulated drug delivery system on coaxiallyincorporated platelet alphagranules forbiomedical useM Buzgo1,2, R Jakubova1,2, M Plencner1,2, A Mickova1,2,M Rampichova1,2, E Prosecka1,2, P Kochova3, D Lukas4 andE Amler1,21Institute of Experimental Medicine, Academy of Sciences of theCzech Republic, v.v.i, Vıdenska, Prague, Czech Republic;2Department of Biophysics, 2nd Faculty of Medicine, CharlesUniversity in Prague, V Uvalu, Prague, Czech Republic;3Department of Mechanics, Faculty of Applied Sciences, Universityof West Bohemia, Univerzitni 8, Pilsen, Czech Republic;4Department of Nonwovens, Technical University of Liberec,Studentska 2, Liberec, Czech Republic

Aims: Platelet derivatives serve as an efficient source of natural growthfactors. In the current study, we incorporated alpha granules in coaxialnanofibers.Materials and Methods: A nanofiber scaffold containing alpha granuleswas prepared by coaxial electrospinning. The biological potential of thenanofiber scaffold was evaluated in chondrocyte and mesenchymalstem cell cultivation studies; additionally, the concentration of trans-forming growth factor (TGF)-b1 was determined.Results: Microscopy studies showed that intact alpha granules wereincorporated into the coaxial nanofibers. The cultivation tests showedthat the novel scaffold stimulated viability and extracellular matrix pro-duction of chondrocytes and mesenchymal stem cells (MSC). The con-centration of growth factors necessary for induction of cell proliferationwas significantly decreased as well.Conclusions: The system preserved alpha granule bioactivity and stim-ulated cell viability and chondrogenic differentiation of mesenchymalstem cells. Core/shell nanofibers incorporating alpha granules are apromising system for tissue engineering, namely, cartilage engineering.Acknowledgement: Grant Agency of Czech Republic (grant No. P304/10/1307). The Grant Agency of the Charles University (grant. No330611, 384311, 626012, 648112). The Ministry of Education of theCzech Republic – project ERA-NET CARSILA No. ME 10145, GrantAgency of the Czech Ministry of Health (project no. NT12156).


34 Abstracts List

TS055Injectable hydrogel functionalized by nanofibresas a drug delivery system for skeletal tissuesregenerationM Rampichova1,2, M Buzgo1,2, E Prosecka1,2, A Mıckova1,2,E Filova1,2, M Pouzar3 and E Amler1,21Laboratory of Tissue Engineering, Institute of ExperimentalMedicine, Academy of Sciences of the Czech Republic, v. v. i.,Prague, Czech Republic; 2Institute of Biophysics, 2nd Faculty ofMedicine, Charles University in Prague, Czech Republic; 3Instituteof Environmental and Chemical Engineering, University ofPardubice, Czech Republic

Hydrogels are favorite materials in tissue engineering for its big poten-tial to bond water. It enables good transport of nutrients and wasteproduct and also homogenous distribution of cells. In addition, hydro-gels can be injected directly to the defect and fill in defects of differentshapes and sizes and subsequently can be polymerized chemically andphysically. This material can also be enriched by bioactive molecules orparticles and used as a drug delivery system. In our study, injectable hy-drogels based on fibrin, hyaluronic acid (HA) and type I collagen wereseeded with mesenchymal stem cells (MSCs) and tested. Moreover, thescaffolds were enriched with thrombocytes, as a source of nativegrowth factors, and/or capsules from polycaprolactone (PCL) loadedwith dexamethasone and ascorbic acid. The hydrogels were two-com-ponent and solidified after mixing in 37 �C. The first component con-tained fibrin and type I collagen, the second one comprised ofthrombin, MSCs, HA and thrombocytes and/or PCL capsules. The cellswere viable for the whole 14 days of experiment. It was shown, thatcell proliferation and viability was improved by thrombocytes, but notby PCL capsules. Neither combination of both thrombocytes and cap-sules was better than the control. On the other hand, the presence ofcapsules prolonged the disintegration of samples. This way the biome-chanical properties of hydrogels can be improved. Hydrogels based onfibrin, hyaluronic acid and type I collagen enriched by thrombocytesshowed improved cell proliferation and viability in comparison to con-trol. Presence of capsules in hydrogel enhanced biomechanical proper-ties and prolonged scaffold degradation. Moreover, these particles canserve as a drug delivery system for stimulation of cell proliferation anddifferentiation. Further experiments are essential for adjustment ofdrug dose and release kinetics. This work was supported by the GrantAgency of the Charles University (grant No. 330611, 384311, 626012),The Grant Agency of the Czech Republic (grant No. P304/10/1307) andInternal Grant Agency of the Ministry of Health of the Czech Republic(grant no. NT12156).

TS056Core/shell nanofibers with embedded liposomesas a drug delivery systemA Mickova1,2, M Buzgo1,2, O Benada3, M Rampichova1,2,Z Fisar4, E Filova2, M Tesarova5, D Bezdekova1,2, D Lukas6

and E Amler1,21Department of Biophysics, 2nd Faculty of Medicine, CU inPrague, Czech Republic; 2Institute of Experimental Medicine,ASCR, v.v.i, Prague, Czech Republic; 3Laboratory of MolecularStructure Characterization, Institute of Microbiology, ASCR,v.v.i., Czech Republic; 4Department of Psychiatry, First Faculty ofMedicine, CU in Prague, Czech Republic; 5Laboratory of ElectronMicroscopy, Institute of Parasitology, Biology Centre of the ASCR,Ceske Budejovice, Czech Republic; 6Department of Nonwovens,Technical University of Liberec, Czech Republic

The broader application of liposomes in regenerative medicine is ham-pered by their short half-life and inefficient retention at the site ofapplication. These disadvantages could be significantly reduced bytheir combination with nanofibers. We produced two different nanofi-ber-liposome systems in the present study, i.e., liposomes blendedwithin nanofibers and core/shell nanofibers with embedded liposomes.Herein, we demonstrate that blend electrospinning does not conserveintact liposomes. In contrast, coaxial electrospinning enables the incor-poration of liposomes into nanofibers. We report polyvinyl alcohol-core/poly-�-caprolactone-shell nanofibers with embedded liposomesand show that they preserve the enzymatic activity of encapsulatedhorseradish peroxidase. To demonstrate the potential of the coaxial sys-tem with embedded liposomes for a drug delivery of growth factors,we examined the viability and proliferation of MSCs on preparednanofibers. MSCs were seeded on polyvinyl alcohol-core/poly-�-capro-lactone-shell nanofibers with the addition of a mixture of growth fac-tors (bFGF, TGF-b and IGF-I) either dispersed in the core solution (GF)or encapsulated in liposomes (LIP-GF). Concentrations of releasedgrowth factors were detected by ELISA. Finally, core/shell nanofibrousscaffolds containing embedded liposomes with encapsulated recombi-nant growth factors were more potent at stimulating MSC proliferationthan coaxial nanofibers without liposomes. In conclusion, intact lipo-somes incorporated into nanofibers by coaxial electrospinning are verypromising drug delivery systems in tissue engineering and regenerativemedicine applications.


Abstracts List 35

TS057Hypoxic preconditioned mesenchymal stem cellsinduces elevated expression of cardioprotectivecytokines and growth factorsK Haneef1, N Naeem1, N Kabir1, S Jamall2 and A Salim1

1Dr. Panjwani Center for Molecular Medicine and Drug Research,International Center for Chemical and Biological Sciences,University of Karachi, Karachi, Pakistan; 2Department ofBiochemistry, University of Karachi, Karachi, Pakistan

Objective: Mesenchymal Stem Cells (MSCs) have ability to differentiateinto various types of lineages which make them attractive candidatesin the field of regenerative medicine. Transdifferentiation process ofMSCs is mainly influenced by hypoxic microenvironment. Hypoxic pre-conditioning play a very important role in myocardial regeneration as itenhances the differentiation potential of stem cells and also upregulatethe expression of different cardioprotective cytokines and growth fac-tors. The present study main objectives were:

Isolation of Mesenchymal cells from the bone marrow of rat.Isolation of cardiomyocytes from 1 day old neonatal rat’s pups.Characterization of these cells based on the presence of cell surface

markers by immunostaining, PCR, flowcytometry and Western blot-ting.

Hypoxia treatment of mesenchymal stem cells with 2, 4 dinitrophe-nol.

Determination of the expression of various cytokines and growthfactors by RT-PCR.

Determination of myogenic and angiogenic potential of these stemcells after co-culturing them with hypoxic cardiomyocytes at differenttime periods.

Determination of myogenic and angiogenic potential of these stemcells after co-culturing them with cytokines transfected cardiomyocytesat different time periods.Methods: Rat bone marrow derived MSCs were cultured and assessedfor their responsiveness to anoxia by an optimized dose of 0.25 mM 2,4, dinitrophenol (DNP) and then allowed to propagate under normalcondition for 2 and 24 h. Analysis of various cytokines and growth fac-tors were done by RT-PCR and Determination of myogenic and angio-genic potential of stem cells co-cultured with hypoxic cardiomyocytesand cytokines transfected MSCs co cultured with normal cardiomyo-cytes were analyzed by flow cytometry.Results: The morphological examination of MSCs has indicated thatthe cells were slightly shrunken immediately after the anoxic insults.Analysis of various cytokines through RT-PCR has shown higher expres-sion of IL-7, SCF, TGFß, VEGF, IGF, HIF and HGF. The expressionincreased exponentially with the duration of anoxic insult. The cocul-ture studies of hypoxic cardiomyocytes with MSCs showed higherfusion in conditioned medium as compared to normal medium. The co-culture studies of cytokines transfected MSCs with cardiomyocytesshowed higher fusion.Conclusions: The results indicate that during hypoxia and ischemia,MSCs secrete cell survival factors that may operate via various signal-ing pathways enhancing their regenerating potential.

TS058The effect of ionizing radiation on differentiationof human embryonic stem cellsEA Remeeva, IV Panyutin, RD Neumann and IG PanyutinRadiology and Imaging Sciences, Clinical Center, NIH, Bethesda,MD, USA

Pluripotent human embryonic stem cells (hESC) are able to differenti-ate to various cell types found in human body, and, thus, carry greatpotential to regenerative cell replacement-based medicine. The fullrealization of the therapeutic potential of hESC in practice may requirelabeling these cells with radionuclides for in vivo analysis of their en-grafting and subsequent tracking of their fate with imaging modalitiesusing low levels of ionizing radiation. In addition, study the effect ofionizing radiation (IR) on hESCs may provide valuable information onresponses to IR exposure of human cells in their most naive state, andon consequences of IR exposure on the development of human organ-ism. We hypothesize that irradiation of hESC may affect the pathwaysof their spontaneous and induced differentiation, and change the pat-terns of gene expression within the resulting lineage-specific cell types.To test this hypothesis we irradiated cultured pluripotent hESC withlow doses of 60Co gamma-radiation. Next we induced differentiation ofirradiated hESC with two different methods; Activin A treatment todefinitive endoderm, and spontaneous embryoid bodies formation. Wethen analyzed time course of the expression of pluripotency markers(Oct4 and Nanog), and early differentiation markers for definitiveendoderm (FOXA2, Sox17), mesoderm (Brachyury), and neuroecto-derm (PAX6) by quantitative RT PCR. Our results will provide impor-tant insights into sensitivity of hESC to IR, and could potentially resultsin development of an assay for detection of the effects of very lowdoses of ionizing radiation on hESC.


36 Abstracts List

TS059The use of embryos in stem cell research:scientific practices, political views and citizens’expectations in portugalS Silva1,2, H Machado3, BR Alves2, C Samorinha1,2, S Sousa2

and T Rodrigues1,21Department of Clinical Epidemiology, Predictive Medicine andPublic Health, University of Porto Medical School, Porto,Portugal; 2Institute of Public Health – University of Porto(ISPUP), Porto, Portugal; 3Department of Sociology, University ofMinho, Braga, Portugal

The use of embryos in stem cell research depends on permissive poli-cies, availability of stem cell research centers and citizens willing todonate embryos. This paper compares the views, actions and expecta-tions mobilized by three different agents in Portugal: (i) in vitro fertil-ization (IVF) heterosexual couples in their decisions to donate embryosfor scientific research; (ii) legislation and political debate about stemcell research; and (iii) scientific projects on embryonic stem cellresearch. The analysis of the interrelationships between science, politicsand citizenship concerning the use of embryos in stem cell researchwas grounded on the content analysis of the following sources: (i)interviews conducted with 451 IVF patients (267 women and 184men) at Hospital de Sao Joao (Porto, Portugal), between August 2011and June 2012, about reasons underlying the donation of embryos forresearch; (ii) arguments used by the Portuguese Government to justifythe approval of a new permissive regime for embryonic stem cellresearch on February 24, 2011 and the parliamentary discussions aboutassisted reproductive technologies conducted in January 2012; (iii) thescientific fields, topics and aims of research, as well as composition ofthe teams, in all embryo research projects funded by Foundation forScience and Technology (major research funding state agency in Portu-gal) between 2000 and 2009. Scientists, stakeholders and IVF patientswere very receptive to medical and technological progress, revealingexpectations concerning the regenerative and therapeutic power ofembryonic stem cells that are founded on high levels of trust and hopeon science and technology and on health professionals. While IVFpatients justified the donation of embryos for research grounded onfeelings of reciprocity towards science, responsibility on contributing toimprove fertility treatments and altruism, stakeholders emphasized thehuman embryonic stem cell research contributions for economic activ-ity and progress of the country, and scientists highlighted the humanhealth as the justification for funding embryonic stem cell research pro-jects. We conclude that it is needed to maximize the available informa-tion on the scientific projects using embryonic stem cells fordissemination among IVF patients and health professionals and tomonitor stakeholders and citizens’ knowledge and expectations aboutthe upcoming therapies within regenerative medicine. The debatesabout research ethics and regulation of embryonic stem cell researchshould include the views and expectations of scientists, stakeholdersand IVF couples, as well as other citizens, aiming to ensure wide partic-ipation and to achieve a consensus decision making process.

TS0603D functionalized collagen/hydroxyapatitescaffold seeded with MSC for bone defectregeneration in vivoE Prosecka1,2, M Rampichova1,2, A Lytvinets2, L Vojtova3,Z Tonar4, M Kralıckova4, P Kochova5, M Plencner1,2, M Buzgo1,2,A Mıckova1,2, J Benesova1,2 and E Amler1,21Institute of Biophysics, 2nd Faculty of Medicine, CharlesUniversity in Prague, V Uvalu, Prague, Czech Republic; 2Instituteof Experimental Medicine, AS CR, Videnska, Prague, CzechRepublic; 3Faculty of Chemistry, Technical University, Purkynova,Brno, Czech Republic; 4Biomedical Centre, Faculty of Medicine inPilsen, Charles University in Prague, Husova 3, Pilsen, CzechRepublic; 5Research Centre New Technologies, University of WestBohemia, Univerzitni 8, Pilsen, Czech Republic

Objectives: The aim of this study was to develop the ideal scaffold forbone defect regeneration.Materials and Methods: In our experiment was prepared a scaffold fromCollagen type I (Col)/ Hydroxyapatite (HA) and combined with Poly-e-caprolactone (PCL) nanofibres. Three variants of this scaffold were pre-pared. The scaffold seeded with autologous Mesenchymal Stem Cells(MSCs) in osteogenic differentiation media (Group1) or scaffoldenriched with platelet rich plasma (PRP) (Group 2) or scaffoldenriched with both MSCs and PRP (Group 3). All groups of scaffoldswere implanted to the rabbit femur condyles where critical size defect6 mm in diameter and 10 ± 0.5 mm in depth was made. Emptydefects were used as a control (Group 4). 12 weeks later rabbits weresacrificed and the femoral condyles were examined by histologicalanalysis. The aim was to analyze the volume fraction and distributionof bone within an experimental defect.Results and Discussion: The highest bone volume fraction within thehealing defect was found in samples with scaffolds enriched with bothMSC and PRP. Bone volume fraction was comparable between the sam-ples with PRP-enriched scaffolds, and MSC-enriched scaffolds, whilesamples without any scaffold contained the lowest bone volume frac-tion. Only samples containing both MSC and PRP showed uniformbone deposition in whole volume of defect. The moduli of elasticityunder compressive test significant increased at the Col/ HA/ PCL scaf-fold compared to Col/ HA scaffold without PCL nanofibres.Conclusion: This smart composite scaffold enriched with PCL nanofi-bres, MSCs and PRP present new possibilities for bone defect regenera-tion.Acknowledgement: Grant Agency of Czech Republic (grant No.P304/10/1307), The Grant Agency of the Charles University (grant No.330611, 164010).


Abstracts List 37

TS0613D cultures of human neural progenitor cells:dopaminergic differentiation and geneticmodificationC Brito1,2, D Simao1,2, C Pinto1,2, P Fernandes1,2, M Serra1,2,G Schiavo3, EJ Kremer4 and PM Alves1,21IBET, Oeiras, Portugal; 2ITQB-UNL, Oeiras, Portugal;3University of Leipzig, Leipzig, Germany; 4Cancer Research UK,London, UK, 5IGMM, Montpellier, France

Central nervous system (CNS) disorders remain a formidable challengefor the development of efficient therapies. Cell and gene therapyapproaches are promising alternatives that can have a tremendousimpact by treating the causes of the disease rather than the symptoms,providing specific targeting and prolonged duration of action. Hamper-ing translation of these novel therapies from experimental to clinicalsettings is the lack of relevant in vitro and in vivo models that can beused in pre-clinical stages.

This work describes the development of 3D in vitromodels of hCNS,using human midbrain-derived neural stem cells (hNSC) as a scalablesupply of neural-subtype cells, in particular dopaminergic neurons. Fol-lowing a systematic technological approach based on stirred-tank biore-actors, with control of chemical and physical environmentalparameters, resulted in reproducible ratios of differentiated cells, asshown by detailed characterization using confocal microscopy, fieldemission scanning electron microscopy, transmission electron micros-copy, qRT-PCR, Western Blot and metabolic profiling.

In order to establish methodologies for gene transfer and manipula-tion of gene expression in the 3D hCNS cell model, canine adenovirustype 2 (CAV-2) vectors were chosen Due to their characteristics: highcloning capacity, long-term transgene expression, low immunogenicityand neuronal tropism. CAV-2-GFP vector was used for optimization oftransduction of differentiated neurospheres and assessment of vectorentry and impact on neurosphere composition. The model systemdeveloped herein constitutes a practical and versatile new in vitroapproach to study hCNS. Furthermore, this culture strategy may beextended to other sources of hNSC, such as human pluripotent stemcells, including patient-derived induced pluripotent stem cells.

The authors acknowledge Dr J Schwarz (U. Leipzig), for the cellsupply; this work was supported by PTDC/EBB-BIO/112786/2009 andPTDC/EBB-BIO/119243/2010, FCT, Portugal; BrainCAV (FP7-222992), EU.

TS062The potential of microtissues in osteogenic tissueengineeringF Langenbach1, C Naujoks1, K Berr1, R Depprich1, G Kogler2,NR Kubler1 and J Handschel11Department for Oral and Maxillofacial Surgery, Heinrich-HeineUniversity, Dusseldorf, Germany; 2Transplantation Diagnosticsand Cell Therapeutics, Heinrich-Heine University, Dusseldorf,Germany

Large bone defects that do not heal spontaneously necessitate thera-peutic measures for bone regeneration. As an alternative to classic ther-apies, osteogenic tissue engineering (TE) is regarded as a promisingtechnology for bone regeneration. As a standard procedure multipotentstem cells are incubated in two-dimensional (2D) cell cultures beforethey are combined with scaffolds, to form a TE bone graft (TEBG) thatis implanted. However, cells in a 3D-environment have more similarproperties to cells in vivo. Multipotent human stem cells from umbilicalcord blood (USSC) have been differentiated to spherical osteogenic Mi-crotissues (MTs), which mineralized faster than 2D-cultures. Moreover,also control microspheres that have been treated without osteoinduc-tive agents mineralized spontaneously (Langenbach et al., Nat. Protoc.2011). Prior to the use of MTs for TEBGs, it was ensured that cells areable to migrate out of osteogenic differentiated MTs to colonize spacesin scaffolds. It was found that the optimum duration for an osteogenicpredifferentiation, while retaining the ability of cells to migrate out ofthe MT into extracellular matrix (ECM)-gel, is 3 days. For the detectionof mineralization, a commercially available hydroxyapatit-specific fluo-rescent dye was adapted for histology, which provided higher sensitiv-ity in mineral detection compared to standard histological stains. Byfurther analysis of MT-minerals, it was found that minerals consisted ofcalcium-deficient hydroxyapatite (CDHA) amorphous-calciumphos-phate (ACP), octa-calciumphosphate, (Mg)Whitlockit and hydroxyapa-tite. Moreover, dystrophic mineralization after apoptosis of cells couldbe excluded by TUNEL-assay. Best biocompatibility with USSC of sev-eral potential biomaterials for TEBGs was obtained for demineralizedspongiosa (ICBM). In contrast to inoculation with cell suspensions a40-fold increased cell number could be integrated in one scaffold byinoculating ICBM with MTs and a more precise administration of largecell numbers is possible. For the investigation of the osteogenic poten-tial of USSC-ICBM- and MT-ICBM-scaffolds the constructs wereimplanted into dorsal muscle bags of immuno-compromised rats. Itwas found that ectopic bone formation was induced in USSC-ICBM-and MT-ICBM-scaffolds, whereas cell-free ICBM did not lead to boneformation. In conclusion, MTs offer improved in vivo-like conditions forstem cells compared to 2D-cultures, resulting in enhanced osteogenicdifferentiation. MT-technology can ameliorate seeding efficiency ofbiomaterials and can increase the cell load of a scaffold. Furthermore,microtissues reduce the risk of unwanted cellular distribution and washout in tissue engineering and scaffold free cell therapy. With their capa-bility of ectopic bone formation MT-ICBM-constructs hold promise forfacilitated, accelerated and improved bone regeneration.


38 Abstracts List

TS063Are statins able to improve EPCs response ofdiabetic patients with myocardial infarction?A Soares1, N Antonio1,2, R Fernandes1, A Lopes3, A Paiva3,L Goncalves2 and CF Ribeiro11Pharmacology and Therapeutics Departament, Faculty ofMedicine, Coimbra University, Coimbra, Portugal; 2CardiologyDepartment, Coimbra’s Hospital Centre and University, Coimbra,Portugal; 3Histocompatibility Centre of Coimbra, Coimbra,Portuga

Aim: Our study aims to compare the response to statins of EPCs of dia-betic vs. nondiabetic patients with myocardial infarction.Methods: Ten patients admitted for acute myocardial infarction in aCoronary Care Unit, between January and March 2012, were includedin this study. Levels of circulating EPCs were quantified from peripheralcirculation by flow cytometry and cell culture was performed in orderto evaluate their functional characteristics. To compare functional dif-ferences of EPCs, 5 diabetic and 5 nondiabetic patients’ cells were iso-lated and cultivated under two different conditions: high glucose(25 mM), normal glucose (5.5 mM). Colony forming units (CFUs)were evaluated after 7 days of incubation as well as proliferation rateof EPCs, which were maintained in culture until the 15th day. To testthe effects of statins on EPCs function of diabetics and non diabetics,cell culture was held under three conditions: control; atorvastatin(0.1 mM) and pravastatin (10 mM).Results: We found that the rate of cell death was significantly higher indiabetic patients, achieving a significantly lower number of EPCs byfield after 15 days in culture in control conditions (glucose concentra-tion of 5.5 mM) (111.5 ± 4.2 vs. 160.4 ± 18.1 EPCs in non diabeticpatients, P < 0.05). Furthermore, the ability to form CFUs was signifi-cantly reduced in diabetic patients by comparison with non diabetics(0.83 ± 0.44 vs 1.82 ± 0.082, P < 0.05). In nondiabetic patients wefound no significant differences in proliferation rate of EPCs when cul-tured with atorvastatin or pravastatin compared with the control condi-tion. However, in this group of patients the ability to form CFUs wassignificantly improved by atorvastatin. For diabetic patients we foundthat EPCs treated with atorvastatin slowed the progression of celldeath, between the 6th and the 10th day of culture, when compared tocontrol conditions. In addition, CFUs from diabetic patients onlyincreased to numbers similar to non-diabetic patients when treatedwith atorvastatin.Conclusions: These findings indicate a beneficial effect of atorvastatinin the function of EPCs from patients with myocardial infarction. More-over, statins showed to improve the function of EPCs, even in diabeticpatients with an endogenous impairment of EPCs.

TS064Critical Assessment of rhBMP-2 Mediated BoneInduction: an In Vitro and In Vivo EvaluationKisiel M1, Ventura M2, George A1, Hilborn J1 and Varghese OP11Division of Polymer Chemistry, Department of Chemistry,Ångstrom Laboratory, Uppsala University, Uppsala, Sweden;2Biomaterials, Radboud University, Nijmegen Medical Centre,Nijmegen, Netherlands

Understanding the influence of formulation and storage conditions onrhBMP-2 bioactivity is extremely important for its clinical application.Reports in the literature show that different research groups employdifferent parameters such as formulation conditions, storage, doses forin vivo applications etc. that makes it difficult to correlate results fromdifferent experiments. We therefore decided to rationalize theseanomalies by performing a basic study on such parameters using twocommercially available BMPs. Our in vitro experiments suggest thatBMPs from different sources have significant differences in their bio-activity. The clinically approved rhBMP-2 (InductOs�; BMP-P) showedsuperior stability, compared to rhBMP-2 from R&D Systems (BMP-R) atphysiological pH (determined by ALP assay). This BMP-P also showedlower binding to polypropylene Eppendorf tubes. The BMP-R almostlost its bioactivity within 30 min at physiological pH and also showsmore adhesion to plastic surfaces. The in vitro results were alsoreflected in the in vivo experiments, in a rat ectopic model with inject-able hyaluronic acid (HA) hydrogel as BMP carrier. After 7 weeks post-implantation we observed larger bone volume with oriented collagenin the BMP-P group but a smaller bone with disoriented collagen in theBMP-R case. Our results highlight the large difference in activitybetween seemingly identical substances and also the importance ofproper handling of such sensitive proteins.


Abstracts List 39

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