Phenotypic maintenance of articular chondrocytes in vitro requires BMP activity

Summary

Articular chondrocytes are phenotypically unique cells that are responsible for the maintenance of articular cartilage. The articular chondrocytic phenotype is influenced by a range of soluble factors. In particular, members of the bone morphogenetic protein (BMP) family support the articular chondrocytic phenotype and stimulate synthesis of cartilaginous matrix. This study was carried out to determine the importance of BMPs in supporting the differentiated phenotype of articular chondrocytes in vitro. Exogenous BMP-2 supported expression of collagen type II and aggrecan in monolayer chondrocyte cultures, slowing the dedifferentiation process that occurs under these conditions. In contrast, BMP-2 had little effect on expression of these genes in three-dimensional aggregate cultures. Endogenous BMP-2 expression was lost in monolayer cultures, coincident with the down-regulation of collagen type II and aggrecan mRNAs, whereas BMP-2 mRNA levels were stable in aggregate cultures. Antagonism of endogenous BMP activity in aggregate cultures by Noggin or a soluble form of the BMP receptor resulted in reduced expression of collagen type II and aggrecan mRNAs, reduced collagen type II protein and sulfated glycosaminoglycan (GAG) deposition into the aggregate matrices and reduced secretion of GAGs into the culture media. These results indicate that endogenous BMPs are required for maintenance of the differentiated articular chondrocytic phenotype in vitro. These findings are of importance to cell-based strategies designed to repair articular cartilage. Articular chondrocytes require conditions that will support endogenous expression of BMPs to maintain the specialized phenotype of these cells.

Keywords

Articular chondrocyte - BMPs - phenotype - cartilage

• References

• 1 Poole AR, Kojima T, Yasuda T. et al. Composition and structure of articular cartilage. Clin Orthop 2001; 391: S26-S33.
• 2 Fortier LA, Mohammed HO, Lust G. et al. Insulinlike growth factor-I enhances cell-based repair of articular cartilage. J Bone Joint Surg Br 2002; 84 B 276-288.
• 3 Hidaka C, Goodrich LR, Chen CT. et al. Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogen- etic protein-7. J Orthop Res 2003; 21: 573-583.
• 4 Kaps C, Bramlage C, Smolian H. et al. Bone mor- phogenetic proteins promote cartilage differentiation and protect engineered artificial cartilage from fibroblast invasion and destruction. Arthritis Rheum 2002; 46: 149-162.
• 5 Sellers RS, Peluso D, Morris EA. The effect of recombinant human bone morphogenetic pro- tein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage. J Bone Joint Surg Am 1997; 79 A 1452-1463.
• 6 Hauselmann HJ, Fernandes RJ, Mok SS. et al. Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. J Cell Sci 1994; 107: 17-27.
• 7 Reginato AM, Iozzo RV, Jimenez SA. Formation of nodular structures resembling mature articular cartilage in long-term primary cultures of human fetal epiphyseal chondrocytes on a hydrogel substrate. Arthritis Rheum 1994; 37: 1338-1349.
• 8 Stewart MC, Saunders KM, Burton-Wurster N. et al. Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation. J Bone Miner Res 2000; 15: 166-174.
• 9 Hering TM, Kollar J, Huynh TD. et al. Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension. Arch Biochem Biophys 1994; 314: 90-98.
• 10 Lefebvre V, Garofalo S, Zhou G. et al. Characterization of primary cultures of chondrocytes from type II collagen/(S-galactosidase transgenic mice. Matrix Biol 1994; 14: 329-335.
• 11 Bobacz K, Gruber R, Soleiman A. et al. Expression of bone morphogenetic protein 6 in healthy and osteoarthritic human articular chondrocytes and stimulation of matrix synthesis in vitro. Arthritis Rheum 2003; 48: 2501-2508.
• 12 Erlacher L, Ng C-K, Ullrich R. et al. Presence of cartilage-derived morphogenetic proteins in articular cartilage and enhancement of matrix replacement in vitro. Arthritis Rheum 1998; 41: 263-273.
• 13 Flechtenmacher J, Huch K, Thonar EJ-MA. et al. Recombinant human osteogenic protein 1 is apo- tent stimulator of the synthesis of cartilage pro- teoglycans and collagens by human articular chondrocytes. Arthritis Rheum 1996; 39: 1896-1904.
• 14 Hiraki Y, Inoue H, Shigeno C. et al. Bone morpho- genetic proteins (BMP-2 and BMP-3) promote growth and expression of the differentiated phe- notype of rabbit chondrocytes and osteoblastic MC3T3-E1 cells in vitro. J Bone Miner Res 1991; 6: 1373-1385.
• 15 Luyten FP, Chen P, Paralkar V. et al. Recombinant bone morphogenetic protein-4, transforming growth factor-(S1, and activin A enhance the cartilage phenotype of articular chondrocytes in vitro. Exp Cell Res 1994; 210: 224-229.
• 16 Luyten FP, Yu YM, Yanagishita M. et al. Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures. J Biol Chem 1992; 267: 3691-3695.
• 17 Nishida Y, Knudson CB, Kuettner KE. et al. Osteogenic protein-1 promotes the synthesis and retention of extracellularmatrix within bovine articular cartilage and chondrocyte cultures. Osteoarthritis Cartilage 2000; 8: 127-136.
• 18 Sailor LZ, Hewick RM, Morris EA. Recombinant human bone morphogenetic protein-2 maintains the articular chondrocyte phenotype in long-term culture. J Orthop Res 1996; 14: 937-945.
• 19 Glansbeek HL, van Beuningen HM, Vitters EL. et al. Bone morphogenetic protein 2 stimulates articular cartilage proteoglycan synthesis in vivo but does not counteract interleukin-1a effects on proteoglycan synthesis and content. Arthritis Rheum 1997; 40: 1020-1028.
• 20 Van Beuningen HM, Glansbeek HL, van der Kraan PM. Differential effects of local application of BMP-2 or TGF-(S1 on both articular cartilage composition and osteophyte formation. Osteoarthritis Cartilage 1998; 6: 306-317.
• 21 Stewart MC, Kadlcek RM, Robbins P. et al. Expression and activity of the CDK inhibitor p57Kip2 in chondrocytes undergoing hypertrophic differentiation. J Bone Miner Res 2004; 19: 123-132.
• 22 Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulfated glyco- saminoglycans by the use of dimethylmethylene blue. BiochimBiophys Acta 1986; 883: 173-177.
• 23 Reddi AH. Interplay between bone morphogen- etic proteins and cognate binding proteins in bone and cartilage development: noggin, chordin and DAN. Arthritis Res 2001; 3: 1-5.
• 24 Stewart MC, Fosang AJ, Bai Y. et al. ADAMTS5-mediated aggrecanolysis in murine epiphyseal chondrocyte cultures. Osteoarthritis Cartilage 2006; 14: 392-402.
• 25 Tsuji K, Harfe BD, Cox K. et al. Absence of BMP2 results in low bone mineral density, osteoarthritis, spontaneous fractures and impaired fracture healing. J Bone Miner Res. 2005 20. S8 (abstract).
• 26 Rountree RB, Schoor M, Chen H. et al. BMP receptor signaling is required for postnatal maintenance of articular cartilage. PLoS Biol 2004; 2: 1815-1827.
• 27 Dell'Accio F, De Bari C, Luyten FP. Molecular markers predictive of the capacity of expanded human articular chondrocytes to form stable cartilage in vivo. Arthritis Rheum 2001; 44: 1608-1619.
• 28 Anderson HC, Hodges PT, Aguilera XM. et al. Bone morphogenetic protein (BMP) localization in developing human and rat growth plate, meta- physis, epiphysis, and articular cartilage. J Histo- chem Cytochem 2000; 48: 1493-1502.
• 29 Chubinskaya S, Merrihew C, Cs-Szabo G. et al. Human articular chondrocytes express osteogenic protein-1. J Histochem Cytochem 2000; 48: 239-250.
• 30 Serra R, Johnson M, Filvaroff EH. et al. Expression of a truncated, kinase-defective TGF-fi type II receptor in mouse skeletal tissue promotes terminal chondrocyte differentiation and osteoar- thritis. J Cell Biol 1997; 139: 541-552.
• 31 Yang X, Chen L, Xu X. et al. TGF-13/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage. J Cell Biol 2001; 153: 35-46.