Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture

SummaryBackgroundThe developmental history of the chondrocyte results in a cell whose biosynthetic activities are optimized to maintain the concentration and organization of a mechanically functional cartilaginous extracellular matrix. While useful for cartilage tissue engineering studies, the limited supply of healthy autologous chondrocytes may preclude their clinical use. Consequently, multipotential mesenchymal stem cells (MSCs) have been proposed as an alternative cell source.ObjectiveWhile MSCs undergo chondrogenesis, few studies have assessed the mechanical integrity of their forming matrix. Furthermore, efficiency of matrix formation must be determined in comparison to healthy chondrocytes from the same donor. Given the scarcity of healthy human tissue, this study determined the feasibility of isolating bovine chondrocytes and MSCs, and examined their long-term maturation in three-dimensional agarose culture.Experimental designBovine MSCs were seeded in agarose and induced to undergo chondrogenesis. Mechanical and biochemical properties of MSC-laden constructs were monitored over a 10-week period and compared to those of chondrocytes derived from the same group of animals maintained similarly.ResultsOur results show that while chondrogenesis does occur in MSC-laden hydrogels, the amount of the forming matrix and measures of its mechanical properties are lower than that produced by chondrocytes under the same conditions. Furthermore, some important properties, particularly glycosaminoglycan content and equilibrium modulus, plateau with time in MSC-laden constructs, suggesting that diminished capacity is not the result of delayed differentiation.ConclusionsThese findings suggest that while MSCs do generate constructs with substantial cartilaginous properties, further optimization must be done to achieve levels similar to those produced by chondrocytes

Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model

The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches

Time evolution of in vivo articular cartilage repair induced by bone marrow stimulation and scaffold implantation in rabbits

Purpose: Tissue engineering techniques were used to study cartilage repair over a 12-month period in a rabbit model. Methods: A full-depth chondral defect along with subchondral bone injury were originated in the knee joint, where a biostable porous scaffold was implanted, synthesized of poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymer. Morphological evolution of cartilage repair was studied 1 and 2 weeks, and 1, 3, and 12 months after implantation by histological techniques. The 3-month group was chosen to compare cartilage repair to an additional group where scaffolds were preseeded with allogeneic chondrocytes before implantation, and also to controls, who underwent the same surgery procedure, with no scaffold implantation. Results: Neotissue growth was first observed in the deepest scaffold pores 1 week after implantation, which spread thereafter; 3 months later scaffold pores were filled mostly with cartilaginous tissue in superficial and middle zones, and with bone tissue adjacent to subchondral bone. Simultaneously, native chondrocytes at the edges of the defect started to proliferate 1 week after implantation; within a month those edges had grown centripetally and seemed to embed the scaffold, and after 3 months, hyaline-like cartilage was observed on the condylar surface. Preseeded scaffolds slightly improved tissue growth, although the quality of repair tissue was similar to non-preseeded scaffolds. Controls showed that fibrous cartilage was mainly filling the repair area 3 months after surgery. In the 12-month group, articular cartilage resembled the untreated surface. Conclusions: Scaffolds guided cartilaginous tissue growth in vivo, suggesting their importance in stress transmission to the cells for cartilage repair.This study was supported by the Spanish Ministry of Science and Innovation through MAT2010-21611-C03-00 project (including the FEDER financial support), by Conselleria de Educacion (Generalitat Valenciana, Spain) PROMETEO/2011/084 grant, and by CIBER-BBN en Bioingenieria, Biomateriales y Nanomedicina. The work of JLGR was partially supported by funds from the Generalitat Valenciana, ACOMP/2012/075 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.Sancho-Tello Valls, M.; Forriol, F.; Gastaldi, P.; Ruiz Sauri, A.; Martín De Llano, JJ.; Novella-Maestre, E.; Antolinos Turpín, CM.... (2015). Time evolution of in vivo articular cartilage repair induced by bone marrow stimulation and scaffold implantation in rabbits. International Journal of Artificial Organs. 38(4):210-223. https://doi.org/10.5301/ijao.5000404S210223384Becerra, J., Andrades, J. A., Guerado, E., Zamora-Navas, P., López-Puertas, J. M., & Reddi, A. H. (2010). Articular Cartilage: Structure and Regeneration. Tissue Engineering Part B: Reviews, 16(6), 617-627. doi:10.1089/ten.teb.2010.0191Nelson, L., Fairclough, J., & Archer, C. (2009). Use of stem cells in the biological repair of articular cartilage. Expert Opinion on Biological Therapy, 10(1), 43-55. doi:10.1517/14712590903321470MAINIL-VARLET, P., AIGNER, T., BRITTBERG, M., BULLOUGH, P., HOLLANDER, A., HUNZIKER, E., … STAUFFER, E. (2003). HISTOLOGICAL ASSESSMENT OF CARTILAGE REPAIR. The Journal of Bone and Joint Surgery-American Volume, 85, 45-57. doi:10.2106/00004623-200300002-00007Hunziker, E. B., Kapfinger, E., & Geiss, J. (2007). The structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development. Osteoarthritis and Cartilage, 15(4), 403-413. doi:10.1016/j.joca.2006.09.010Onyekwelu, I., Goldring, M. B., & Hidaka, C. (2009). Chondrogenesis, joint formation, and articular cartilage regeneration. Journal of Cellular Biochemistry, 107(3), 383-392. doi:10.1002/jcb.22149Ahmed, T. A. E., & Hincke, M. T. (2010). Strategies for Articular Cartilage Lesion Repair and Functional Restoration. Tissue Engineering Part B: Reviews, 16(3), 305-329. doi:10.1089/ten.teb.2009.0590Hangody, L., Kish, G., Kárpáti, Z., Udvarhelyi, I., Szigeti, I., & Bély, M. (1998). Mosaicplasty for the Treatment of Articular Cartilage Defects: Application in Clinical Practice. Orthopedics, 21(7), 751-756. doi:10.3928/0147-7447-19980701-04Steinwachs, M. R., Guggi, T., & Kreuz, P. C. (2008). Marrow stimulation techniques. Injury, 39(1), 26-31. doi:10.1016/j.injury.2008.01.042Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O., & Peterson, L. (1994). Treatment of Deep Cartilage Defects in the Knee with Autologous Chondrocyte Transplantation. New England Journal of Medicine, 331(14), 889-895. doi:10.1056/nejm199410063311401Richter, W. (2009). Mesenchymal stem cells and cartilagein situregeneration. Journal of Internal Medicine, 266(4), 390-405. doi:10.1111/j.1365-2796.2009.02153.xBartlett, W., Skinner, J. A., Gooding, C. R., Carrington, R. W. J., Flanagan, A. M., Briggs, T. W. R., & Bentley, G. (2005). Autologous chondrocyte implantationversusmatrix-induced autologous chondrocyte implantation for osteochondral defects of the knee. The Journal of Bone and Joint Surgery. British volume, 87-B(5), 640-645. doi:10.1302/0301-620x.87b5.15905Little, C. J., Bawolin, N. K., & Chen, X. (2011). Mechanical Properties of Natural Cartilage and Tissue-Engineered Constructs. Tissue Engineering Part B: Reviews, 17(4), 213-227. doi:10.1089/ten.teb.2010.0572Vikingsson, L., Gallego Ferrer, G., Gómez-Tejedor, J. A., & Gómez Ribelles, J. L. (2014). An «in vitro» experimental model to predict the mechanical behavior of macroporous scaffolds implanted in articular cartilage. Journal of the Mechanical Behavior of Biomedical Materials, 32, 125-131. doi:10.1016/j.jmbbm.2013.12.024Weber, J. F., & Waldman, S. D. (2014). Calcium signaling as a novel method to optimize the biosynthetic response of chondrocytes to dynamic mechanical loading. Biomechanics and Modeling in Mechanobiology, 13(6), 1387-1397. doi:10.1007/s10237-014-0580-xMauck, R. L., Soltz, M. A., Wang, C. C. B., Wong, D. D., Chao, P.-H. G., Valhmu, W. B., … Ateshian, G. A. (2000). Functional Tissue Engineering of Articular Cartilage Through Dynamic Loading of Chondrocyte-Seeded Agarose Gels. Journal of Biomechanical Engineering, 122(3), 252-260. doi:10.1115/1.429656Palmoski, M. J., & Brandt, K. D. (1984). Effects of static and cyclic compressive loading on articular cartilage plugs in vitro. Arthritis & Rheumatism, 27(6), 675-681. doi:10.1002/art.1780270611Khoshgoftar, M., Ito, K., & van Donkelaar, C. C. (2014). The Influence of Cell-Matrix Attachment and Matrix Development on the Micromechanical Environment of the Chondrocyte in Tissue-Engineered Cartilage. Tissue Engineering Part A, 20(23-24), 3112-3121. doi:10.1089/ten.tea.2013.0676Agrawal, C. M., & Ray, R. B. (2001). Biodegradable polymeric scaffolds for musculoskeletal tissue engineering. Journal of Biomedical Materials Research, 55(2), 141-150. doi:10.1002/1097-4636(200105)55:23.0.co;2-jPérez Olmedilla, M., Garcia-Giralt, N., Pradas, M. M., Ruiz, P. B., Gómez Ribelles, J. L., Palou, E. C., & García, J. C. M. (2006). Response of human chondrocytes to a non-uniform distribution of hydrophilic domains on poly (ethyl acrylate-co-hydroxyethyl methacrylate) copolymers. Biomaterials, 27(7), 1003-1012. doi:10.1016/j.biomaterials.2005.07.030Horbett, T. A., & Schway, M. B. (1988). Correlations between mouse 3T3 cell spreading and serum fibronectin adsorption on glass and hydroxyethylmethacrylate-ethylmethacrylate copolymers. Journal of Biomedical Materials Research, 22(9), 763-793. doi:10.1002/jbm.820220903Kiremitçi, M., Peşmen, A., Pulat, M., & Gürhan, I. (1993). Relationship of Surface Characteristics to Cellular Attachment in PU and PHEMA. Journal of Biomaterials Applications, 7(3), 250-264. doi:10.1177/088532829300700304Lydon, M. ., Minett, T. ., & Tighe, B. . (1985). Cellular interactions with synthetic polymer surfaces in culture. Biomaterials, 6(6), 396-402. doi:10.1016/0142-9612(85)90100-0Campillo-Fernandez, A. J., Pastor, S., Abad-Collado, M., Bataille, L., Gomez-Ribelles, J. L., Meseguer-Dueñas, J. M., … Ruiz-Moreno, J. M. (2007). Future Design of a New Keratoprosthesis. Physical and Biological Analysis of Polymeric Substrates for Epithelial Cell Growth. Biomacromolecules, 8(8), 2429-2436. doi:10.1021/bm0703012Funayama, A., Niki, Y., Matsumoto, H., Maeno, S., Yatabe, T., Morioka, H., … Toyama, Y. (2008). Repair of full-thickness articular cartilage defects using injectable type II collagen gel embedded with cultured chondrocytes in a rabbit model. Journal of Orthopaedic Science, 13(3), 225-232. doi:10.1007/s00776-008-1220-zKitahara, S., Nakagawa, K., Sah, R. L., Wada, Y., Ogawa, T., Moriya, H., & Masuda, K. (2008). In Vivo Maturation of Scaffold-free Engineered Articular Cartilage on Hydroxyapatite. Tissue Engineering Part A, 14(11), 1905-1913. doi:10.1089/ten.tea.2006.0419Martinez-Diaz, S., Garcia-Giralt, N., Lebourg, M., Gómez-Tejedor, J.-A., Vila, G., Caceres, E., … Monllau, J. C. (2010). In Vivo Evaluation of 3-Dimensional Polycaprolactone Scaffolds for Cartilage Repair in Rabbits. The American Journal of Sports Medicine, 38(3), 509-519. doi:10.1177/0363546509352448Wang, Y., Bian, Y.-Z., Wu, Q., & Chen, G.-Q. (2008). Evaluation of three-dimensional scaffolds prepared from poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for growth of allogeneic chondrocytes for cartilage repair in rabbits. Biomaterials, 29(19), 2858-2868. doi:10.1016/j.biomaterials.2008.03.021Alió del Barrio, J. L., Chiesa, M., Gallego Ferrer, G., Garagorri, N., Briz, N., Fernandez-Delgado, J., … De Miguel, M. P. (2014). Biointegration of corneal macroporous membranes based on poly(ethyl acrylate) copolymers in an experimental animal model. Journal of Biomedical Materials Research Part A, 103(3), 1106-1118. doi:10.1002/jbm.a.35249Diego, R. B., Olmedilla, M. P., Aroca, A. S., Ribelles, J. L. G., Pradas, M. M., Ferrer, G. G., & Sánchez, M. S. (2005). Acrylic scaffolds with interconnected spherical pores and controlled hydrophilicity for tissue engineering. Journal of Materials Science: Materials in Medicine, 16(8), 693-698. doi:10.1007/s10856-005-2604-7Serrano Aroca, A., Campillo Fernández, A. J., Gómez Ribelles, J. L., Monleón Pradas, M., Gallego Ferrer, G., & Pissis, P. (2004). Porous poly(2-hydroxyethyl acrylate) hydrogels prepared by radical polymerisation with methanol as diluent. Polymer, 45(26), 8949-8955. doi:10.1016/j.polymer.2004.10.033Diani, J., Fayolle, B., & Gilormini, P. (2009). A review on the Mullins effect. European Polymer Journal, 45(3), 601-612. doi:10.1016/j.eurpolymj.2008.11.017Mullins, L. (1969). Softening of Rubber by Deformation. Rubber Chemistry and Technology, 42(1), 339-362. doi:10.5254/1.3539210Jurvelin, J. S., Buschmann, M. D., & Hunziker, E. B. (2003). Mechanical anisotropy of the human knee articular cartilage in compression. Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine, 217(3), 215-219. doi:10.1243/095441103765212712Shapiro, F., Koide, S., & Glimcher, M. J. (1993). Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. The Journal of Bone & Joint Surgery, 75(4), 532-553. doi:10.2106/00004623-199304000-00009SELLERS, R. S., ZHANG, R., GLASSON, S. S., KIM, H. D., PELUSO, D., D’AUGUSTA, D. A., … MORRIS, E. A. (2000). Repair of Articular Cartilage Defects One Year After Treatment with Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2)*. The Journal of Bone and Joint Surgery-American Volume, 82(2), 151-160. doi:10.2106/00004623-200002000-00001Hunziker, E. B., Michel, M., & Studer, D. (1997). Ultrastructure of adult human articular cartilage matrix after cryotechnical processing. Microscopy Research and Technique, 37(4), 271-284. doi:10.1002/(sici)1097-0029(19970515)37:43.0.co;2-oAppelman, T. P., Mizrahi, J., Elisseeff, J. H., & Seliktar, D. (2009). The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation. Biomaterials, 30(4), 518-525. doi:10.1016/j.biomaterials.2008.09.063Chung, C., & Burdick, J. A. (2008). Engineering cartilage tissue. Advanced Drug Delivery Reviews, 60(2), 243-262. doi:10.1016/j.addr.2007.08.027HUNZIKER, E. B., & ROSENBERG, L. C. (1996). Repair of Partial-Thickness Defects in Articular Cartilage. The Journal of Bone & Joint Surgery, 78(5), 721-33. doi:10.2106/00004623-199605000-00012Schulze-Tanzil, G. (2009). Activation and dedifferentiation of chondrocytes: Implications in cartilage injury and repair. Annals of Anatomy - Anatomischer Anzeiger, 191(4), 325-338. doi:10.1016/j.aanat.2009.05.003Umlauf, D., Frank, S., Pap, T., & Bertrand, J. (2010). Cartilage biology, pathology, and repair. Cellular and Molecular Life Sciences, 67(24), 4197-4211. doi:10.1007/s00018-010-0498-0Karystinou, A., Dell’Accio, F., Kurth, T. B. A., Wackerhage, H., Khan, I. M., Archer, C. W., … De Bari, C. (2009). Distinct mesenchymal progenitor cell subsets in the adult human synovium. Rheumatology, 48(9), 1057-1064. doi:10.1093/rheumatology/kep192Sakaguchi, Y., Sekiya, I., Yagishita, K., & Muneta, T. (2005). Comparison of human stem cells derived from various mesenchymal tissues: Superiority of synovium as a cell source. Arthritis & Rheumatism, 52(8), 2521-2529. doi:10.1002/art.21212Schaefer, D., Martin, I., Jundt, G., Seidel, J., Heberer, M., Grodzinsky, A., … Freed, L. E. (2002). Tissue-engineered composites for the repair of large osteochondral defects. Arthritis & Rheumatism, 46(9), 2524-2534. doi:10.1002/art.1049