Chondrogenic differentiation of human mesenchymal stem cells in collagen type I hydrogels.

The chondrogenic differentiation of bone marrow-derived human mesenchymal stem cells (MSCs) in a collagen type I hydrogel, which is in clinical use for matrix-based autologous chondrocyte transplantation (ACT), was investigated. Collagen hydrogels with 2.5 x 10(5) MSCs/mL were fabricated and cultured for 3 weeks in a serum-free, defined, chondrogenic differentiation medium containing 10 ng/mL TGF-beta1 or 100 ng/mL BMP-2. Histochemistry revealed morphologically distinct, chondrocyte-like cells, surrounded by a sulfated proteoglycan-rich extracellular matrix in the TGF-beta1 and BMP-2 treated group, with more elongated cells seen in the BMP-2 treated group. Immunohistochemistry detected collagen type II (Col II) in the TGF-beta1 and BMP-2 treated group. Collagen type X (Col X) staining was positive in the TGF-beta1 but only very weak in the BMP-2 treated group. RT-PCR analyses revealed a specific chondrogenic differentiation with the expression of the cartilage specific marker genes Col II, Col X, and aggrecan (AGN) in the TGF-beta1 and the BMP-2 treated group, with earlier expression of these marker genes in the TGF-beta1 treated group. Interestingly, MSC-gels cultured in DMEM with 10% FBS (control) indicated few isolated chondrocyte-like cells but no expression of Col II or Col X could be detected. The results show, that MSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway, similar to that described for MSCs cultured in high-density pellet cultures. These findings are valuable in terms of ex vivo predifferentiation or in situ differentiation of MSCs in collagen hydrogels for articular cartilage repair.

[1]  J. Farr,et al.  Concomitant Meniscal Allograft Transplantation and Autologous Chondrocyte Implantation , 2007, The American journal of sports medicine.

[2]  R. Rallapalli,et al.  Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene transfer of BMP-2. , 2001, Differentiation; research in biological diversity.

[3]  I. Sekiya,et al.  Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma , 2005, Cell and Tissue Research.

[4]  C. Kaps,et al.  BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture. , 2003, Differentiation; research in biological diversity.

[5]  U. Schneider,et al.  Klinische Drei-Jahres-Ergebnisse der matrixgekoppelten ACT zur Behandlung osteochondraler Defekte am Kniegelenk , 2008 .

[6]  R Cancedda,et al.  Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. , 2000, Journal of cell science.

[7]  Christoph Gaissmaier,et al.  Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads. , 2004, Osteoarthritis and cartilage.

[8]  S. Giannini,et al.  Autologous chondrocyte implantation in the knee joint: open compared with arthroscopic technique. Comparison at a minimum follow-up of five years. , 2008, The Journal of bone and joint surgery. American volume.

[9]  A M Mackay,et al.  Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. , 1998, Tissue engineering.

[10]  W. Woodward,et al.  N-Cadherin expression and signaling in limb mesenchymal chondrogenesis: stimulation by poly-L-lysine. , 1999, Developmental genetics.

[11]  E. Tanaka,et al.  Multidifferentiation potential of mesenchymal stem cells in three-dimensional collagen gel cultures. , 2005, Journal of biomedical materials research. Part A.

[12]  R. Tuan,et al.  Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: II. Stimulation by bone morphogenetic protein-2 requires modulation of N-cadherin expression and function. , 1999, Differentiation; research in biological diversity.

[13]  Freddie H. Fu,et al.  Clinical Outcome of Autologous Chondrocyte Implantation at 5 Years in US Subjects , 2005, Clinical orthopaedics and related research.

[14]  Shigeyuki Wakitani,et al.  Autologous Bone Marrow Stromal Cell Transplantation for Repair of Full-Thickness Articular Cartilage Defects in Human Patellae: Two Case Reports , 2004, Cell transplantation.

[15]  R. Cancedda,et al.  Response of young, aged and osteoarthritic human articular chondrocytes to inflammatory cytokines: molecular and cellular aspects. , 2002, Matrix biology : journal of the International Society for Matrix Biology.

[16]  Richard Tuli,et al.  Adult mesenchymal stem cells and cell-based tissue engineering , 2002, Arthritis research & therapy.

[17]  M. Schünke,et al.  Die Redifferenzierung von dedifferenzierten Gelenkknorpelzellen in Alginatkultur , 2000, Der Orthopäde.

[18]  E B Hunziker,et al.  Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. , 2002, Osteoarthritis and cartilage.

[19]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[20]  S. Ichinose,et al.  In vitro cartilage formation of composites of synovium-derived mesenchymal stem cells with collagen gel , 2005, Cell and Tissue Research.

[21]  M. Schünke,et al.  [Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure]. , 2000, Der Orthopade.

[22]  Joseph M. Mansour,et al.  Mesenchymal Cell-Based Repair of Large Full Thickness Defects of Articular Cartilage , 1994 .

[23]  S. Rennard,et al.  Smad3 mediates TGF-beta1-induced collagen gel contraction by human lung fibroblasts. , 2006, Biochemical and biophysical research communications.

[24]  M. Brittberg,et al.  Articular cartilage engineering with autologous chondrocyte transplantation. A review of recent developments. , 2003, The Journal of bone and joint surgery. American volume.

[25]  Maurilio Marcacci,et al.  Articular Cartilage Engineering with Hyalograft® C: 3-Year Clinical Results , 2005, Clinical orthopaedics and related research.

[26]  C. Deng,et al.  TGF-β/Smad3 Signals Repress Chondrocyte Hypertrophic Differentiation and Are Required for Maintaining Articular Cartilage , 2001, The Journal of cell biology.

[27]  C. Toma,et al.  Repair of articular cartilage defects treated by microfracture and a three-dimensional collagen matrix. , 2005, Biomaterials.

[28]  W. Horton,et al.  Nucleotide sequence of the full length cDNA encoding for human type II procollagen. , 1989, Nucleic acids research.

[29]  Wai-Hee Lo,et al.  Chondrogenesis of human mesenchymal stem cells encapsulated in alginate beads. , 2003, Journal of biomedical materials research. Part A.

[30]  Glenn D Prestwich,et al.  Molecular stenting with a crosslinked hyaluronan derivative inhibits collagen gel contraction. , 2006, The Journal of investigative dermatology.

[31]  F Dubrana,et al.  Autologous chondrocyte implantation in a novel alginate-agarose hydrogel: outcome at two years. , 2008, The Journal of bone and joint surgery. British volume.

[32]  B. Steinberg,et al.  Establishment and transformation diminish the ability of fibroblasts to contract a native collagen gel , 1980, The Journal of cell biology.

[33]  U. Schneider,et al.  Die Behandlung femoropatellarer Knorpelschäden mit einem dreidimensionalen Kollagengel: Klinische Ergebnisse im Zwei-Jahres-Verlauf , 2007 .

[34]  J. Puzas,et al.  Transforming growth factor beta: an autocrine regulator of chondrocytes. , 1989, Connective tissue research.

[35]  A. Dimmler,et al.  Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh‐viscosity alginate , 2003, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[36]  B. Hogan,et al.  Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). , 1990, Development.

[37]  A I Caplan,et al.  Characterization of cells with osteogenic potential from human marrow. , 1992, Bone.

[38]  D. Hutmacher,et al.  Scaffolds in tissue engineering bone and cartilage. , 2000, Biomaterials.

[39]  K. Jepsen,et al.  Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro , 2003, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[40]  D. Green,et al.  Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl , 1995, The Journal of experimental medicine.

[41]  U. Schneider,et al.  Erste klinische Erfahrungen mit einem neuartigen dreidimensionalen Kollagengel (CaReS®) zur Behandlung fokaler Knorpeldefekte am Kniegelenk , 2006 .

[42]  H. Lorenz,et al.  Multilineage cells from human adipose tissue: implications for cell-based therapies. , 2001, Tissue engineering.

[43]  E. Schwarz,et al.  Smad2 and 3 Mediate Transforming Growth Factor-β1-Induced Inhibition of Chondrocyte Maturation* *The work was supported by National Health Services Grant AR-38945 (to R.J.O.) and an Orthopaedic Research Education Foundation Award (to C.M.F.). , 2000, Endocrinology.

[44]  V. Goldberg,et al.  The Chondrogenic Potential of Human Bone-Marrow-Derived Mesenchymal Progenitor Cells* , 1998, The Journal of bone and joint surgery. American volume.

[45]  U. Schneider,et al.  Chondrocytes and Collagen Gels , 2003 .

[46]  A I Caplan,et al.  In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. , 1998, Experimental cell research.

[47]  T. Yamamoto,et al.  Temporal and spatial expressions of transforming growth factor-betas and their receptors in epiphyseal growth plate. , 1999, International journal of oncology.

[48]  J. Baron,et al.  Regulation of growth plate chondrogenesis by bone morphogenetic protein-2. , 2001, Endocrinology.

[49]  C. Tickle,et al.  Bone morphogenetic protein-2 (BMP-2) inhibits muscle development and promotes cartilage formation in chick limb bud cultures. , 1996, Developmental biology.

[50]  J A Skinner,et al.  Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study. , 2005, The Journal of bone and joint surgery. British volume.

[51]  R. Tuan,et al.  Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures. , 1999, Differentiation; research in biological diversity.

[52]  J. Glowacki,et al.  In vitro engineering of cartilage: effects of serum substitutes, TGF-beta, and IL-1alpha. , 2005, Orthodontics & craniofacial research.

[53]  P. Schiller,et al.  Inhibition of Gap-Junctional Communication Induces the Trans-differentiation of Osteoblasts to an Adipocytic Phenotype in Vitro * , 2001, The Journal of Biological Chemistry.

[54]  M. Brittberg,et al.  Articular Cartilage Engineering with Autologous Chondrocyte Transplantation , 2003 .

[55]  F. Barry,et al.  Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. , 2001, Experimental cell research.

[56]  C. Ohlsson,et al.  Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. , 1994, The New England journal of medicine.

[57]  A I Caplan,et al.  Tissue-engineered fabrication of an osteochondral composite graft using rat bone marrow-derived mesenchymal stem cells. , 2001, Tissue engineering.

[58]  V. Goldberg,et al.  Culture‐expanded human periosteal‐derived cells exhibit osteochondral potential in vivo , 1991, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[59]  E B Hunziker,et al.  BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel. , 2005, Osteoarthritis and cartilage.