Rapid chondrocyte maturation by serum-free culture with BMP-2 and ascorbic acid

Abstract

In serum-containing medium, ascorbic acid induces maturation of prehypertrophic chick embryo sternal chondrocytes. Recently, cultured chondrocytes have also been reported to undergo maturation in the presence of bone morphogenetic proteins or in serum-free medium supplemented with thyroxine. In the present study, we have examined the combined effect of ascorbic acid, BMP-2, and serum-free conditions on the induction of alkaline phosphatase and type X collagen in chick sternal chondrocytes. Addition of either ascorbate or rhBMP-2 to nonconfluent cephalic sternal chondrocytes produced elevated alkaline phosphatase levels within 24-72 h, and simultaneous exposure to both ascorbate and BMP yielded enzyme levels at least threefold those of either inducer alone. The effects of ascorbate and BMP were markedly potentiated by culture in serum-free medium, and alkaline phosphatase levels of preconfluent serum-free cultures treated for 48 h with BMP+ascorbate were equivalent to those reached in serum-containing medium only after confluence. While ascorbate addition was required for maximal alkaline phosphatase activity, it did not induce a rapid increase in type X collagen mRNA. In contrast, BMP added to serum-free medium induced a three- to fourfold increase in type X collagen mRNA within 24 h even in the presence of cyclohexamide, indicating that new protein synthesis was not required. Addition of thyroid hormone to serum-free medium was required for maximal ascorbate effects but not for BMP stimulation. Neither ascorbate nor BMP induced alkaline phosphatase activity in caudal sternal chondrocytes, which do not undergo hypertrophy during embryonic development. These results indicate that ascorbate+BMP in serum-free culture induces rapid chondrocyte maturation of prehypertrophic chondrocytes. The mechanisms for ascorbate and BMP action appear to be distinct, while BMP and thyroid hormone may share a similar mechanism for induction.

Functions of Transforming Growth Factor-β Family Type I Receptors and Smad Proteins in the Hypertrophic Maturation and Osteoblastic Differentiation of Chondrocytes

“…Taken together, these results indicate that SV40 large T expression does not interfere with the biological activities of BMP-2 on specific genes and, thus, that the MC615 cell line provides a suitable system to examine the molecular mechanisms underlying BMP-2 signaling in chondrocytes. Our results are also in line with previous reports showing that BMP-2 induces chondrogenesis and/or osteogenesis in pluripotent mesenchymal precursor cell lines (4, 58 -60) and in teratocarcinoma cells (61), and induces hypertrophic maturation of chondrocytes (62). Here, we present evidence for the first time that BMP-2 induces chondrocytes to transdifferentiate into osteoblastic cells.…”

Effect of parathyroid hormone-related protein in an in vitro hypertrophy model for mesenchymal stem cell chondrogenesis

“…PTHrP may directly suppress ALP expression in the peripheral non-chondrogenic cells (5) application of dexamethasone and TGF-β1 may be necessary to facilitate PTHrP action or act synergistically in standard chondrogenic medium. Dexamethasone has been shown to suppress the increase of ALP activity in chick sternal chondrocytes [25] and TGF-β prevents hypertrophy in epiphyseal chondrocytes [26]. Possible effects of PTHrP in our model that are discussed here are summarised in Fig.…”

Serum‐free media for periosteal chondrogenesis in vitro

“…Such an approach will be of value when investigating the roles of individual growth factors in chondrogenesis. Indeed, our laboratory has already been able to use this information to study the role of IGF-1 and TGF-PI during periosteal chondrogenesis Leboy et al reported that the effects of BMP-2 and ascorbic acid on cultured chondrocytes were potentiated in serum-free conditions [23]. Fortier et al found that the stimulation of proteoglycan synthesis and total proteoglycan accumulation by TGF-P1 equine chondro-~4 1 .…”