Extracellular matrix synthesized by clonal osteogenic cells is osteoinductive in vivo and in vitro: Role of transforming growth factor-beta1 in osteoblast cell-matrix interaction

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Abstract
ROB-C26 (C26) is a multipotential, clonal cell line known to express several members of the TGF-β superfamily and to become more osteoblastic (e.g., express higher levels of alkaline phosphatase) upon treatment with 10−6 M retinoic acid (RA). We hypothesize that the expression of this more osteoblastic phenotype subsequent to RA exposure is the result of the treated cell's extracellular matrix (ECM) becoming a repository and active source of putative osteoinductive growth factors including, specifically, select members of the TGF-β superfamily. To test this hypothesis, we isolated the ECM from RA-treated and untreated C26 cells and assessed them for their ability to promote osteogenic differentiation in vivo and in vitro. We then explored whether the latter activities could be attributed specifically to TGF-β1. We found that the ECM of treated cells isolated by cell lysis and extensive washing induced endochondral bone formation in vivo when implanted into the thigh muscles of athymic nude mice and stimulated alkaline phosphatase (ALP) activity in vitro in freshly plated C26 cells. This latter stimulation was comparable to levels observed with direct RA treatment. This latter in vitro activity was only very partially mimicked by the ECM prepared from untreated cells and not duplicated at all by RA-treated collagen or the ECM from another RA-treated multipotential cell line. Moreover, the in vivo osteoinductive effect of the treated C26 cell ECM was not duplicated by comparable ECM prepared from untreated cells. Finally, we also found that preincubation of the ECM with specific, neutralizing antibodies to either TGF-β's 1, 2, and 3 or TGF-β1 alone substantially reduced the ability of the ECM to stimulate ALP activity. This inhibitory effect was not seen using nonspecific IgG. These data identify the C26 cell line as a valuable model system for exploring cell-matrix interaction in osteogenic differentiation, provide direct support for an autocrine role of the ECM in such differentiation, and suggest that TGF-β1 is an important ECM-based mediator in the regulation of osteoblastic development.
Funding Information
  • NIH/NIDR (T32 DE07204)
  • NIH (AR 37716)