Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor beta and serum factors.

Abstract

Rabbit chondrocyte cultures on plastic dishes are capable of depositing a cartilaginous matrix, although the matrix does not calcify unless high levels of phosphate are added to the medium. In the present study, we cultivated a pelleted mass of, rabbit growth-plate chondrocytes in the presence of Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 50 ,sg of ascorbic acid per ml in a plastic centrifuge tube. These cells proliferated for several generations and then reorganized into a cartilage-like tissue that calcified without additional phosphate. The deposition of minerals was observed only after synthesis of a short-chain collagen and alkaline phosphatase. Serum factors were required for the increases in alkaline phosphatase and calcium contents. 5-Bromo-2'-deoxyuridine abolished the increases in uronic acid, alkaline phosphatase, and calcium contents. Transforming growth factor J3, at very low concentrations, suppressed the expression of the mineralization-related phenotype by chondrocytes. These results suggest that cartilugematrix calcification can be controlled by growth factor(s) and that chondrocytes induce the mineralization of extracellular matrix when terminal differentiation is permitted in the absence of an artificial substrate.The calcification of the extracellular matrix in growth-plate cartilage is a key event that leads to longitudinal growth ofthe skeleton. Growth-plate cartilage is organized into three cellular zones containing proliferating, maturing, and hypertrophic cells. Maturing chondrocytes synthesize and deposit large amounts of cartilage-specific proteoglycans and type II collagen. The calcification process is confined to the hypertrophic zone where mineralization is initiated in matrix vesicles (1, 2). Hypertrophic chondrocytes produce a shortchain (type X) collagen (3,4) and alkaline phosphatase (4-6), some of which is associated with matrix vesicles (4, 6).Hypertrophic cells eventually die, and the zone is invaded by capillary sprouts. Vascular invasion is believed to play an important role in the onset of mineralization (see refs. 7 and 8 for review), but it remains unclear whether hypertrophic chondrocytes have the capacity to induce extracellular matrix mineralization in the absence of vascular invasion.Chondrocyte cultures have been used extensively during the past 30 years to study the control of their phenotypic expression. However; there have been few studies demonstrating cartilage-matrix calcification in a cell culture system. It has been reported that embryonic chicken chondrocytes (9-11) and rat growth-plate chondrocytes (12) in mass cultures on plastic dishes produce an abundant extracellular matrix, although the matrix does not calcify unless high levels of phosphate (3-fold over the level in standard medium) are included (11,12). The mineral deposition with high phosphate should be regarded as a physicochemical phenomenon (12). Because of the lack of a good in vitro system, no information about the regulation of cartilage calcificati...

Transforming growth factor‐β1 downregulates dexamethasone‐indunced tetranectin gene expression during the in vitro mineralization of the human osteoblastic cell line SV‐HFO

“…The cell system employed in the present study consists of an SV-40 immortalized human osteoblastic cell line (SV-HFO) [23] that undergo differentiation as evidenced by mineralization, upon treatment with dexamethasone [25]. Our results demonstrating that TGF-fl~ inhibits alkaline phosphatase expression and mineralization are consistent with several previous studies showing that TGF-fll appears to be an inhibitor of bone nodule formation and mineralization in osteoblast cultures [8][9][10]13,37]. A number of in vivo models have been established and it has been repeatedly shown that TGF-fl injections stimulated bone formation [4][5][6][7].…”

Adenoviral-mediated transfer of TGF-β1 but not IGF-1 induces chondrogenic differentiation of human mesenchymal stem cells in pellet cultures

“…The process of bone formation through endochondral ossification means that bone forms by replacing a cartilage anlagen after the chondrocytes undergo a process of hypertrophy, calcification, and apoptosis known as chondrocyte terminal differentiation. It has been shown that TGF-β1 inhibits the terminal differentiation of chondrocytes and that TGF-β1 acts to preserve the chondrocyte phenotype [45,46]. Our results suggest that sustained administration of TGF-β1 may be more effective in suppressing terminal differentiation than intermittent dosing.…”

Adeno-associated viral gene transfer of transforming growth factor-β1 to human mesenchymal stem cells improves cartilage repair

“…[15][16][17] The chondrogenic potential of human mesenchymal stem cells (hMSC) is well described. 18,19 Addition of transforming growth factor-beta-1 (TGF-b1) to hMSC has been shown to consistently induce chondrogenesis while avoiding terminal differentiation [20][21][22][23][24] Adenoviral mediated transfer of TGF-b1 genes to hMSC has been shown to similarly induce chondrogenesis and inhibit terminal differentiation. 21 Efficient and sustained gene delivery to mesenchymal stem cells with adenovirus, lentivirus and retrovirus have been described.…”