Cell migration within confined sandwich-like nanoenvironments

Aim: We introduced sandwich-like cultures to provide cell migration studies with 4 representative nano-bio-environments where both ventral and dorsal cell receptors are activated. Methods: We have investigated different nano-environmental conditions by changing the protein coating (fibronectin, vitronectin) and/or materials (using polymers that adsorb proteins in qualitatively different conformations) of this sandwich system to show their specific role in cell migration. Results: Here we show that cell migration within sandwich cultures greatly differs from 2D cultures and shares some similarities with migration within 3D environments. Beyond differences in cell morphology and migration, dorsal stimulation promotes cell remodeling of the ECM over simple ventral 12 receptor activation in traditional 2D cultures.</p

Effects of hydroxyapatite filler on long-term hydrolytic degradation of PLLA/PCL

[EN] Poly(L-lactic acid)(PLLA)/poly(epsilon-caprolactone)(PCL)/hydroxyapatite(HAp) composites appear as promising materials for healing large bone defects. Highly porous PLLA/PCL scaffolds, 80/20, 20/80 weight ratios, porosity >85%, were prepared by a dual technique of freeze extraction and porogen leaching, with and without HAp. A double pore structure was obtained, with interconnected macroporosity together with interconnected microporosity. Subsequent long-term (78 weeks = 1.5 years) hydrolytic degradation behavior was investigated in terms of the samples' mechanical properties, molecular weight (M-w), mass changes, thermal characteristics, X-ray Diffraction and Thermogravimetric Analysis. Elastic modulus and yield strength of as-synthesized scaffolds were higher for PLLA rich blends and including the inorganic phase does not lead to a mechanical strengthening in these materials. Nevertheless, after 30 weeks of degradation, PLLA rich scaffolds lost more than half of their strength and rigidity. On the contrary, the densification modulus of the PLLA based blends increased with degradation time, whereas PCL-based blends had a relatively constant densification modulus. PCL-based samples showed lower hydrolysis coefficients k than PLLA-based samples, as expected from the higher density of ester bonds in the latter. Interestingly, although including HAp leads to a lower hydrolysis coefficient k in PCL rich samples, it increases k in the PLLA-based sample, which is consistent with the other results obtained.The authors are grateful for the support of the Biomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine, an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, and Consolider Program. J. Rodenas-Rochina acknowledges the funding of his PhD by the Valencian Generality through VALi+d grant.Ródenas Rochina, J.; Vidaurre, A.; Castilla Cortázar, MIC.; Lebourg ., MM. (2015). Effects of hydroxyapatite filler on long-term hydrolytic degradation of PLLA/PCL. Polymer Degradation and Stability. 119:121-131. doi:10.1016/j.polymdegradstab.2015.04.01512113111

Dorsal and ventral stimuli in cell–material interactions: effect on cell morphology

Cells behave differently between bidimensional (2D) and tridimensional (3D) environments. While most of the in vitro cultures are 2D, most of the in vivo extracellular matrices are 3D, which encourages the development of more relevant culture conditions, seeking to provide more physiological models for biomedicine (e.g., cancer, drug discovery and tissue engineering) and further insights into any dimension-dependent biological mechanism. In this study, cells were cultured between two protein coated surfaces (sandwich-like culture). Cells used both dorsal and ventral receptors to adhere and spread, undergoing morphological changes with respect to the 2D control. Combinations of fibronectin and bovine serum albumin on the dorsal and ventral sides led to different cell morphologies, which were quantified from bright field images by calculating the spreading area and circularity. Although the mechanism underlying these differences remains to be clarified, excitation of dorsal receptors by anchorage to extracellular proteins plays a key role on cell behavior. This approach—sandwich-like culture—becomes therefore a versatile method to study cell adhesion in well-defined conditions in a quasi 3D environment

Compositional changes to synthetic biodegradable scaffolds modulate the influence of hydrostatic pressure on chondrogenesis of mesenchymal stem cells

[EN] Mechanical cues such as hydrostatic pressure (HP) are known to regulate mesenchymal stem cell (MSC) differentiation. The fate of such cells is also strongly influenced by their substrate. The objective of this study was to test how different modifications of polycaprolactone (PCL) scaffolds would influence the response of MSCs to HP. Porcine bone marrow derived MSCs were cultured on PCL, PCL-hyaluronic acid (HA) and PCL-Bioglass (R) (BG) scaffolds for 35 d and stimulated with aHP bioreactor (10 MPa; 1 Hz; 2 h d(-1)). Scaffold composition was found to modulate the response to HP. MSCs seeded onto both PCL and BGscaffolds responded positively to the application of HP, with increases in cartilage extracellular matrix synthesis and a reduction in type I collagen accumulation. This positive effect was not observed onHAscaffolds. The results of this study demonstrate that changes to scaffold composition can have a notable effect on the response of MSCs to bioreactor culture conditions.Joaquin Rodenas-Rochina acknowledges funding of his PhD and his stay at the Trinity Centre for Bioengineering by the Generalitat Valenciana through ACIF grant (ACIF/2010/238) and BEFPI grant (BEFPI/2012/084) respectively. Funding to Daniel Kelly was provided by Science Foundation Ireland (President of Ireland Young Researcher Award: 08/YI5/B1336) and the European Research Council (StemRepair-Project number 258463) Jose L Gomez Ribelles acknowledges the support of the Ministerio de Economia y Competitividad, MINECO, through the MAT2013-46467-C4-1-R project. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Ródenas Rochina, J.; Kelly, DJ.; Gómez Ribelles, JL.; Lebourg, MM. (2016). Compositional changes to synthetic biodegradable scaffolds modulate the influence of hydrostatic pressure on chondrogenesis of mesenchymal stem cells. Biomedical Physics & Engineering Express. 2(3). https://doi.org/10.1088/2057-1976/2/3/035005S23Hunziker, E. B. (2002). Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis and Cartilage, 10(6), 432-463. doi:10.1053/joca.2002.0801Redman, S., Oldfield, S., & Archer, C. (2005). Current strategies for articular cartilage repair. European Cells and Materials, 9, 23-32. doi:10.22203/ecm.v009a04Liao, S., Chan, C. K., & Ramakrishna, S. (2008). Stem cells and biomimetic materials strategies for tissue engineering. Materials Science and Engineering: C, 28(8), 1189-1202. doi:10.1016/j.msec.2008.08.015Huang, A. H., Farrell, M. J., & Mauck, R. L. (2010). Mechanics and mechanobiology of mesenchymal stem cell-based engineered cartilage. Journal of Biomechanics, 43(1), 128-136. doi:10.1016/j.jbiomech.2009.09.018Gelse, K., Ekici, A. B., Cipa, F., Swoboda, B., Carl, H. D., Olk, A., … Klinger, P. (2012). Molecular differentiation between osteophytic and articular cartilage – clues for a transient and permanent chondrocyte phenotype. Osteoarthritis and Cartilage, 20(2), 162-171. doi:10.1016/j.joca.2011.12.004Vinardell, T., Sheehy, E. J., Buckley, C. T., & Kelly, D. J. (2012). A Comparison of the Functionality and In Vivo Phenotypic Stability of Cartilaginous Tissues Engineered from Different Stem Cell Sources. Tissue Engineering Part A, 18(11-12), 1161-1170. doi:10.1089/ten.tea.2011.0544Girotto, D., Urbani, S., Brun, P., Renier, D., Barbucci, R., & Abatangelo, G. (2003). Tissue-specific gene expression in chondrocytes grown on three-dimensional hyaluronic acid scaffolds. Biomaterials, 24(19), 3265-3275. doi:10.1016/s0142-9612(03)00160-1Wu, J., Xue, K., Li, H., Sun, J., & Liu, K. (2013). Improvement of PHBV Scaffolds with Bioglass for Cartilage Tissue Engineering. PLoS ONE, 8(8), e71563. doi:10.1371/journal.pone.0071563Carlisle, E. M. (1988). Silicon as a trace nutrient. Science of The Total Environment, 73(1-2), 95-106. doi:10.1016/0048-9697(88)90190-8Hubka, K. M., Dahlin, R. L., Meretoja, V. V., Kasper, F. K., & Mikos, A. G. (2014). Enhancing Chondrogenic Phenotype for Cartilage Tissue Engineering: Monoculture and Coculture of Articular Chondrocytes and Mesenchymal Stem Cells. Tissue Engineering Part B: Reviews, 20(6), 641-654. doi:10.1089/ten.teb.2014.0034Vinatier, C., Mrugala, D., Jorgensen, C., Guicheux, J., & Noël, D. (2009). Cartilage engineering: a crucial combination of cells, biomaterials and biofactors. Trends in Biotechnology, 27(5), 307-314. doi:10.1016/j.tibtech.2009.02.005Kelly, D. J., & Jacobs, C. R. (2010). The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells. Birth Defects Research Part C: Embryo Today: Reviews, 90(1), 75-85. doi:10.1002/bdrc.20173Welter, J. F., Solchaga, L. A., & Baskaran, H. (2012). Chondrogenesis from Human Mesenchymal Stem Cells: Role of Culture Conditions. Stem Cells and Cancer Stem Cells, Volume 5, 269-280. doi:10.1007/978-94-007-2900-1_26Grad, S., Eglin, D., Alini, M., & Stoddart, M. J. (2011). Physical Stimulation of Chondrogenic Cells In Vitro: A Review. Clinical Orthopaedics and Related Research®, 469(10), 2764-2772. doi:10.1007/s11999-011-1819-9Kessler, M. W., & Grande, D. A. (2008). Tissue engineering and cartilage. Organogenesis, 4(1), 28-32. doi:10.4161/org.6116Elder, B. D., & Athanasiou, K. A. (2009). Hydrostatic Pressure in Articular Cartilage Tissue Engineering: From Chondrocytes to Tissue Regeneration. Tissue Engineering Part B: Reviews, 15(1), 43-53. doi:10.1089/ten.teb.2008.0435Wagner, D. R., Lindsey, D. P., Li, K. W., Tummala, P., Chandran, S. E., Smith, R. L., … Beaupre, G. S. (2008). Hydrostatic Pressure Enhances Chondrogenic Differentiation of Human Bone Marrow Stromal Cells in Osteochondrogenic Medium. Annals of Biomedical Engineering, 36(5), 813-820. doi:10.1007/s10439-008-9448-5Meyer, E. G., Buckley, C. T., Steward, A. J., & Kelly, D. J. (2011). The effect of cyclic hydrostatic pressure on the functional development of cartilaginous tissues engineered using bone marrow derived mesenchymal stem cells. Journal of the Mechanical Behavior of Biomedical Materials, 4(7), 1257-1265. doi:10.1016/j.jmbbm.2011.04.012Wong, M., Siegrist, M., & Goodwin, K. (2003). Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes. Bone, 33(4), 685-693. doi:10.1016/s8756-3282(03)00242-4Carroll, S. F., Buckley, C. T., & Kelly, D. J. (2014). Cyclic hydrostatic pressure promotes a stable cartilage phenotype and enhances the functional development of cartilaginous grafts engineered using multipotent stromal cells isolated from bone marrow and infrapatellar fat pad. Journal of Biomechanics, 47(9), 2115-2121. doi:10.1016/j.jbiomech.2013.12.006Steward, A. J., Thorpe, S. D., Vinardell, T., Buckley, C. T., Wagner, D. R., & Kelly, D. J. (2012). Cell–matrix interactions regulate mesenchymal stem cell response to hydrostatic pressure. Acta Biomaterialia, 8(6), 2153-2159. doi:10.1016/j.actbio.2012.03.016Villanueva, I., Weigel, C. A., & Bryant, S. J. (2009). Cell–matrix interactions and dynamic mechanical loading influence chondrocyte gene expression and bioactivity in PEG-RGD hydrogels. Acta Biomaterialia, 5(8), 2832-2846. doi:10.1016/j.actbio.2009.05.039Peter, M., Binulal, N. S., Nair, S. V., Selvamurugan, N., Tamura, H., & Jayakumar, R. (2010). Novel biodegradable chitosan–gelatin/nano-bioactive glass ceramic composite scaffolds for alveolar bone tissue engineering. Chemical Engineering Journal, 158(2), 353-361. doi:10.1016/j.cej.2010.02.003Ródenas-Rochina, J., Ribelles, J. L. G., & Lebourg, M. (2013). Comparative study of PCL-HAp and PCL-bioglass composite scaffolds for bone tissue engineering. Journal of Materials Science: Materials in Medicine, 24(5), 1293-1308. doi:10.1007/s10856-013-4878-5Lennon, D. P., & Caplan, A. I. (2006). Isolation of human marrow-derived mesenchymal stem cells. Experimental Hematology, 34(11), 1604-1605. doi:10.1016/j.exphem.2006.07.014Thorpe, S. D., Buckley, C. T., Vinardell, T., O’Brien, F. J., Campbell, V. A., & Kelly, D. J. (2008). Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells. Biochemical and Biophysical Research Communications, 377(2), 458-462. doi:10.1016/j.bbrc.2008.09.154Connelly, J. T., García, A. J., & Levenston, M. E. (2007). Inhibition of in vitro chondrogenesis in RGD-modified three-dimensional alginate gels. Biomaterials, 28(6), 1071-1083. doi:10.1016/j.biomaterials.2006.10.00

Fibronectin-matrix sandwich-like microenvironments to manipulate cell fate

[EN] Conventional 2D substrates fail to represent the natural environment of cells surrounded by the 3D extracellular matrix (ECM). We have proposed sandwich-like microenvironments as a versatile tool to study cell behaviour under quasi-3D conditions. This is a system that provides a broad range of dorsal and ventral independent spatio-temporal stimuli. Here, we use this sandwich technology to address the role of dorsal stimuli in cell adhesion, cell proliferation and ECM reorganisation. Under certain conditions, dorsal stimuli within sandwich microenvironments prevent the formation of focal plaques as well as the development of the actin cytoskeleton, whereas alpha(5) versus alpha(v) integrin expression is increased compared to the corresponding 2D controls. Cell signaling is similarly enhanced after dorsal stimuli (measured by the pFAK/FAK level) for cells sandwiched after 3 h of 2D ventral adhesion, but not when sandwiched immediately after cell seeding (similar levels to the 2D control). Cell proliferation, studied by the 5-bromo-2-deoxyuridine (BrdU) incorporation assay, was significantly reduced within sandwich conditions as compared to 2D substrates. In addition, these results were found to depend on the ability of cells to reorganise the dorsal layer of proteins at the material interface, which could be tuned by adsorbing FN on material surfaces that results in a qualitatively different conformation and distribution of FN. Overall, sandwich-like microenvironments switch cell behaviour (cell adhesion, morphology and proliferation) towards 3D-like patterns, demonstrating the importance of this versatile, simple and robust approach to mimic cell microenvironments in vivo.The support from ERC through HealInSynergy (306990) and the FPU program AP2009-3626 are acknowledged.Ballester Beltrán, J.; Moratal Pérez, D.; Lebourg, MM.; Salmerón Sánchez, M. (2014). Fibronectin-matrix sandwich-like microenvironments to manipulate cell fate. Biomaterials Science. 2(3):381-389. https://doi.org/10.1039/C3BM60248FS3813892

Bridges of biomaterials promote nigrostriatal pathway regeneration

[EN] Repair of central nervous system (CNS) lesions is difficulted by the lack of ability of central axons to regrow, and the blocking by the brain astrocytes to axonal entry. We hypothesized that by using bridges made of porous biomaterial and permissive olfactory ensheathing glia (OEG), we could provide a scaffold to permit restoration of white matter tracts. We implanted porous polycaprolactone (PCL) bridges between the substantia nigra and the striatum in rats, both with and without OEG. We compared the number of tyrosine-hydroxylase positive (TH+) fibers crossing the striatal-graft interface, and the astrocytic and microglial reaction around the grafts, between animals grafted with and without OEG. Although TH+ fibers were found inside the grafts made of PCL alone, there was a greater fiber density inside the graft and at the striatal-graft interface when OEG was cografted. Also, there was less astrocytic and microglial reaction in those animals. These results show that these PCL grafts are able to promote axonal growth along the nigrostriatal pathway, and that cografting of OEG markedly enhances axonal entry inside the grafts, growth within them, and re-entry of axons into the CNS. These results may have implications in the treatment of diseases such as Parkinson's and others associated with lesions of central white matter tracts.Contract grant sponsor: Regional Government Health Department (Conselleria de Sanitat, Generalitat Valenciana) and Carlos III Health Institute of the Ministry of Health and Consumer Affairs (Spain) (Regenerative Medicine Programme) Contract grant sponsor: Spanish ministry of Education and Science; contract grant number: MAT 2006-13554-C02-02 Contract grant sponsor: Red de Terapia Celular TERCEL (RETICS), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovacion (ISCIII); contract grant number: RD12/0019/0010 (to J.A.) Contract grant sponsor: Spanish Science & Innovation Ministery; contract grant number: MAT2008-06434 (to M.M.P.) Contract grant sponsor: "Convenio de Colaboracion para la Investigacion Basica y Traslacional en Medicina Regenerativa," Instituto Nacional de Salud Carlos III, the Conselleria de Sanidad of the Generalitat Valenciana, and the Foundation Centro de Investigacion Principe FelipeGómez Pinedo, U.; Sanchez-Rojas, L.; Vidueira, S.; Sancho, FJ.; Martínez-Ramos, C.; Lebourg, M.; Monleón Pradas, M.... (2019). Bridges of biomaterials promote nigrostriatal pathway regeneration. Journal of Biomedical Materials Research Part B Applied Biomaterials. 107(1):190-196. https://doi.org/10.1002/jbm.b.34110S1901961071Pekny, M., Wilhelmsson, U., & Pekna, M. (2014). The dual role of astrocyte activation and reactive gliosis. Neuroscience Letters, 565, 30-38. doi:10.1016/j.neulet.2013.12.071Bliss, T. M., Andres, R. H., & Steinberg, G. K. (2010). Optimizing the success of cell transplantation therapy for stroke. Neurobiology of Disease, 37(2), 275-283. doi:10.1016/j.nbd.2009.10.003Tam, R. Y., Fuehrmann, T., Mitrousis, N., & Shoichet, M. S. (2013). Regenerative Therapies for Central Nervous System Diseases: a Biomaterials Approach. Neuropsychopharmacology, 39(1), 169-188. doi:10.1038/npp.2013.237Skop, N. B., Calderon, F., Cho, C. H., Gandhi, C. D., & Levison, S. W. (2014). Improvements in biomaterial matrices for neural precursor cell transplantation. Molecular and Cellular Therapies, 2(1), 19. doi:10.1186/2052-8426-2-19Yasuhara, T., Kameda, M., Sasaki, T., Tajiri, N., & Date, I. (2017). Cell Therapy for Parkinson’s Disease. Cell Transplantation, 26(9), 1551-1559. doi:10.1177/0963689717735411Orive, G., Anitua, E., Pedraz, J. L., & Emerich, D. F. (2009). Biomaterials for promoting brain protection, repair and regeneration. Nature Reviews Neuroscience, 10(9), 682-692. doi:10.1038/nrn2685Walker, P. A., Aroom, K. R., Jimenez, F., Shah, S. K., Harting, M. T., Gill, B. S., & Cox, C. S. (2009). Advances in Progenitor Cell Therapy Using Scaffolding Constructs for Central Nervous System Injury. Stem Cell Reviews and Reports, 5(3), 283-300. doi:10.1007/s12015-009-9081-1Zhong, Y., & Bellamkonda, R. V. (2008). Biomaterials for the central nervous system. Journal of The Royal Society Interface, 5(26), 957-975. doi:10.1098/rsif.2008.0071Pérez‐GarnezM BarciaJA Gómez‐PinedoU Monleón‐PradasM Vallés‐LluchA.Materials for Central Nervous System Tissue Engineering Cells and Biomaterials in Regenerative Medicine. InTech;2014. Chap 7.Sinha, V. R., Bansal, K., Kaushik, R., Kumria, R., & Trehan, A. (2004). Poly-ϵ-caprolactone microspheres and nanospheres: an overview. International Journal of Pharmaceutics, 278(1), 1-23. doi:10.1016/j.ijpharm.2004.01.044Raisman, G. (2001). Olfactory ensheathing cells — another miracle cure for spinal cord injury? Nature Reviews Neuroscience, 2(5), 369-375. doi:10.1038/35072576Raisman, G., & Li, Y. (2007). Repair of neural pathways by olfactory ensheathing cells. Nature Reviews Neuroscience, 8(4), 312-319. doi:10.1038/nrn2099Fairless, R., & Barnett, S. C. (2005). Olfactory ensheathing cells: their role in central nervous system repair. The International Journal of Biochemistry & Cell Biology, 37(4), 693-699. doi:10.1016/j.biocel.2004.10.010Collins, A., Li, D., Mcmahon, S. B., Raisman, G., & Li, Y. (2017). Transplantation of Cultured Olfactory Bulb Cells Prevents Abnormal Sensory Responses during Recovery from Dorsal Root Avulsion in the Rat. Cell Transplantation, 26(5), 913-924. doi:10.3727/096368917x695353Navarro, X., Valero, A., Gudi�o, G., For�s, J., Rodr�guez, F. J., Verd�, E., … Nieto-Sampedro, M. (1999). Ensheathing glia transplants promote dorsal root regeneration and spinal reflex restitution after multiple lumbar rhizotomy. Annals of Neurology, 45(2), 207-215. doi:10.1002/1531-8249(199902)45:23.0.co;2-kGómez-Pinedo, U., Félez, M. C., Sancho-Bielsa, F. J., Vidueira, S., Cabanes, C., Soriano, M., … Barcia, J. A. (2008). Improved technique for stereotactic placement of nerve grafts between two locations inside the rat brain. Journal of Neuroscience Methods, 174(2), 194-201. doi:10.1016/j.jneumeth.2008.07.008HowardCV ReedMG.Unbiased Stereology: Three‐Dimensional Measurement in Microscopy. Oxford: Bioimaging Group;1998.Collier, T. J., & Springer, J. E. (1991). Co-grafts of embryonic dopamine neurons and adult sciatic nerve into the denervated striatum enhance behavioral and morphological recovery in rats. Experimental Neurology, 114(3), 343-350. doi:10.1016/0014-4886(91)90160-eBourke, J. L., Coleman, H. A., Pham, V., Forsythe, J. S., & Parkington, H. C. (2014). Neuronal Electrophysiological Function and Control of Neurite Outgrowth on Electrospun Polymer Nanofibers Are Cell Type Dependent. Tissue Engineering Part A, 20(5-6), 1089-1095. doi:10.1089/ten.tea.2013.0295Nga, V. D. W., Lim, J., Choy, D. K. S., Nyein, M. A., Lu, J., Chou, N., … Teoh, S.-H. (2015). Effects of Polycaprolactone-Based Scaffolds on the Blood–Brain Barrier and Cerebral Inflammation. Tissue Engineering Part A, 21(3-4), 647-653. doi:10.1089/ten.tea.2013.0779Pérez-Garnés, M., Martínez-Ramos, C., Barcia, J. A., Escobar Ivirico, J. L., Gómez-Pinedo, U., Vallés-Lluch, A., & Monleón Pradas, M. (2012). One-Dimensional Migration of Olfactory Ensheathing Cells on Synthetic Materials: Experimental and Numerical Characterization. Cell Biochemistry and Biophysics, 65(1), 21-36. doi:10.1007/s12013-012-9399-1Diban, N., Ramos-Vivas, J., Remuzgo-Martinez, S., Ortiz, I., & Urtiaga, A. (2015). Poly(&#949;-caprolactone) Films with Favourable Properties for Neural Cell Growth. Current Topics in Medicinal Chemistry, 14(23), 2743-2749. doi:10.2174/156802661466614121515393

In Vivo Evaluation of 3-Dimensional Polycaprolactone Scaffolds for Cartilage Repair in Rabbits

Background: Cartilage tissue engineering using synthetic scaffolds allows maintaining mechanical integrity and withstanding stress loads in the body, as well as providing a temporary substrate to which transplanted cells can adhere. Purpose: This study evaluates the use of polycaprolactone (PCL) scaffolds for the regeneration of articular cartilage in a rabbit model. Study Design: Controlled laboratory study. Methods: Five conditions were tested to attempt cartilage repair. To compare spontaneous healing (from subchondral plate bleeding) and healing due to tissue engineering, the experiment considered the use of osteochondral defects (to allow blood flow into the defect site) alone or filled with bare PCL scaffold and the use of PCL-chondrocytes constructs in chondral defects. For the latter condition, 1 series of PCL scaffolds was seeded in vitro with rabbit chondrocytes for 7 days and the cell/scaffold constructs were transplanted into rabbits’ articular defects, avoiding compromising the subchondral bone. Cell pellets and bare scaffolds were implanted as controls in a chondral defect. Results: After 3 months with PCL scaffolds or cells/PCL constructs, defects were filled with white cartilaginous tissue; integration into the surrounding native cartilage was much better than control (cell pellet). The engineered constructs showed histologically good integration to the subchondral bone and surrounding cartilage with accumulation of extracellular matrix including type II collagen and glycosaminoglycan. The elastic modulus measured in the zone of the defect with the PCL/cells constructs was very similar to that of native cartilage, while that of the pellet-repaired cartilage was much smaller than native cartilage. Conclusion: The results are quite promising with respect to the use of PCL scaffolds as aids for the regeneration of articular cartilage using tissue engineering techniques.The support of the Spanish Ministry of Science through projects No. MAT2007-66759-C03-01 and MAT2007-66759C03-02 (including FEDER financial support) is acknowledged. Dr Gomez Tejedor acknowledges the support given by the government of Valencia, the Generalitat Valenciana, through the GVPRE/2008/160 project. The support of Grant 2005SGR 00762 and 2005SGR 00848 (Catalan Department of Universities, Research and the Information Society) is also acknowledged. The Aging and Fragile Elderly cooperative research network (Red Tematica de Investigacion Cooperativa en Envejecimiento y Fragilidad [RETICEF]) and the Bioengineering, Biomaterials and Nanomedicine research network (Centro de Investigacion Biomedica en Red en Bioingenieria, Biomateriales y Nanomedicina [CIBER BBN]) are initiatives of the Instituto de Salud Carlos III (ISCIII). The group of the Centro de Investigacion Principe Felipe (CIPF) acknowledges funding in the framework of the collaboration agreement among the ISCIII, the Conselleria de Sanidad de la Comunidad Valenciana, and the CIPF for the "Investigacion Basica y Traslacional en Medicina Regenerativa."Martinez-Diaz, S.; Garcia-Giralt, N.; Lebourg, MM.; Gómez-Tejedor, JA.; Vila, G.; Caceres, E.; Benito, P.... (2010). In Vivo Evaluation of 3-Dimensional Polycaprolactone Scaffolds for Cartilage Repair in Rabbits. American Journal of Sports Medicine. 38(3):509-519. https://doi.org/10.1177/0363546509352448S50951938

Robust fabrication of electrospun-like polymer mats to direct cell behaviour

Currently, cell culture systems that include nanoscale topography are widely used in order to provide cells additional cues closer to the in vivo environment, seeking to mimic the natural extracellular matrix. Electrospinning is one of the most common techniques to produce nanofiber mats. However, since many sensitive parameters play an important role in the process, a lack of reproducibility is a major drawback. Here we present a simple and robust methodology to prepare reproducible electrospun-like samples. It consists of a polydimethylsiloxane mold reproducing the fiber pattern to solvent-cast a polymer solution and obtain the final sample. To validate this methodology, poly( L-lactic) acid ( PLLA) samples were obtained and, after characterisation, bioactivity and ability to direct cell response were assessed. C2C12 myoblasts developed focal adhesions on the electrospun-like fibers and, when cultured under myogenic differentiation conditions, similar differentiation levels to electrospun PLLA fibers were obtained.The support of ERC through HealInSynergy (306990) and FPU program AP2009-3626 is acknowledged.Ballester Beltrán, J.; Lebourg., MM.; Capella Monsonís, H.; Díaz Lantada, A.; Salmerón Sánchez, M. (2014). Robust fabrication of electrospun-like polymer mats to direct cell behaviour. Biofabrication. 6(3). https://doi.org/10.1088/1758-5082/6/3/035009S6

Comparative study of PCL-HAp and PCL-bioglass composite scaffolds for bone tissue engineering

The aim of this work is to compare the effect of hydroxyapatite (HAp) or bioglass (BG) nanoparticles in a polycaprolactone composite scaffold aimed to bone regeneration. To allow a comparison of the influence of both types of fillers, scaffolds made of PCL or composites containing up to 20 % by weight HAp or BG were obtained. Scaffolds showed acceptable mechanical properties for its use and high interconnected porosity apt for cellular colonization. To study the effect of the different materials on pre-osteoblast cells differentiation, samples with 5 % mineral reinforcement, were cultured for up to 28 days in osteogenic medium. Cells proliferated in all scaffolds. Nevertheless, differentiation levels for the selected markers were higher in pure PCL scaffolds than in the composites; inclusion of bioactive particles showed no positive effects on cell differentiation. In osteogenic culture conditions, the presence of bioactive particles is thus not necessary in order to observe good differentiation.JLGR acknowledges the support of the Spanish Ministry of Science and Education through project No. MAT2010-21611-C03-01 (including the FEDER financial support), and from Generalitat Valenciana, ACOMP/2012/075 Project.. Lebourg acknowledges the support of UPV through Project PAID-O6-10 and thanks CIBER-BBN for funding her post-doc research. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. J. Rodenas acknowledges the funding of his PhD by the Generalitat Valenciana through VALi+d Grant. 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Hydrolytic degradation of PLLA/PCL microporous membranes prepared by freeze extraction

Poly(L-lactic acid) Poly(e-caprolactone) blends (PLLA/PCL) porous membranes were prepared by freeze extraction (a modification of freeze drying) with ratios 100/0, 80/20, 60/40, 40/60, 20/80, 0/100 in weight. Degradation of the membranes in phosphate buffer solution (PBS) up to 65 weeks was studied using weight loss measurements, high performance liquid chromatography (HPLC), differential scanning calorimetry (DSC), mechanical indentation, gel permeation chromatography (GPC), and scanning electron microscopy (SEM). Degradation rate as observed by weight loss and reduction of molecular weight and mechanical properties depended on the composition of the blends. In most blends the degradation was more prominent in the PLLA phase and was accompanied by consequent recrystallization that formed a crystalline phase with increased resistance to hydrolysis. Occurrence of such crystalline phases and degradation of intercrystalline domain led to formation of nearly monodisperse molecular weight populations. Membranes with only 20% PCL presented favorable behavior compared to pure PLLA membranes as reflected in a lower degradation rate and a limited loss of the mechanical properties. At the same time, degradation rate of 80/20 membranes was enhanced with respect to pure PCL, and membranes were stiffer than PCL membranes at all degradation times. This composition could thus be useful for use in tissue engineering for bone or cartilage applications.Luis Andres Gaona wishes to thank "Programa de Doctorados Nacionales 2009" of COLCIENCIAS (Departamento Administrativo de Ciencia, Tecnologia e Innovacion Colombia) and COOPEN Project (Colombia, Costa Rica, Panama and European Network) for supporting his PhD studies. Myriam Lebourg acknowledges UPV for funding through project PAID 06-10, and CIBER-BBN for funding her postdoc research. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Jose Luis Gomez Ribelles acknowledges the support of the Spanish Ministry of Science and Innovation through MAT2010-21611-C03-01 (including the FEDER financial support) and funding in the Centro de Investigacion Principe Felipe in the field of Regenerative Medicine through the collaboration agreement from the Conselleria de Sanidad (Generalitat Valenciana), and the Instituto de Salud Carlos III (Ministry of Science and Innovation). The authors wish also to thank the Microscopy Service and Instituto de Tecnologia Quimica of Universidad Politecnica de Valencia for useful help and advices.Gaona, LA.; Gómez Ribelles, JL.; Perilla, JE.; Lebourg, MM. (2012). Hydrolytic degradation of PLLA/PCL microporous membranes prepared by freeze extraction. Polymer Degradation and Stability. 97(9):1621-1632. https://doi.org/10.1016/j.polymdegradstab.2012.06.031S1621163297