Two naturally occurring peptides that induce chondrogenesis in culture have been purified to apparent homogeneity. These cartilage-inducing factors (CIF-A and CIF-B) were isolated from bovine demineralized bone by dissociative extraction, gel filtration, cation-exchange chromatography, and reversed-phase HPLC. CIF-A and CIF-B at concentrations of 1-10 ng/ml each induce embryonic rat mesenchymal cells in culture to assume a cartilage morphology and synthesize cartilage-specific proteoglycan and type II collagen. The amino acid compositions of CIF-A and CIF-B are similar but not identical. Both factors have an apparent Mr of 26,000, as determined by NaDodSO4/PAGE. In the presence of 2-mercaptoethanol, both are converted to species of about one-half that Mr, indicating that they are dimers of identical or very similar chains.When fragments of demineralized bone are implanted subcutaneously or intramuscularly, they elicit a biological response that has all the elements of endochondral bone formation (1). These include the attraction, proliferation, and subsequent induction of mesenchymal cells into cartilage cells and matrix which are then replaced by bone (2). Similar findings have been obtained with a powder prepared from a 4 M guanidine HCl (Gdn HCl) extract of demineralized bone particles, suggesting that the response involves a hormonelike factor or group of factors (3, 4).The early chondrogenic induction and cartilage formation of this response can be elicited in vitro (5). By using an ELISA for cartilage markers, the chondrogenic activity of soluble factors can be assayed in a rapid, highly sensitive, and quantitative manner (4). Treatment of embryonic rat mesenchymal cells, isolated from muscle and embedded in agarose gel, with an extract from demineralized bone powder induces the synthesis of cartilage-specific proteoglycan and type II collagen. Using this assay, we have isolated, purified, and characterized two bovine demineralized-bone proteins that are potent inducers of chondrogenesis in vitro and that may be involved in the osteogenic response to demineralized bone in vivo. MATERIALS AND METHODSPreparation of Demineralized Bone Powder. Bovine metatarsal bone was obtained fresh from the slaughterhouse and transported on dry ice. Bones were cleaned of all marrow and periosteum, broken into fragments, and pulverized in a liquid-nitrogen-cooled mill. The pulverized bone was washed twice for 15 min with deionized water (10 ml/g). The bone was then washed overnight with the same volume of 0.01 M HCl at 4°C and defatted as described elsewhere (4). The bone powder was demineralized for 16 hr in 0.5 M HCl (25 ml/g) at 40C. The acid was decanted, and the demineralized bone powder was washed several times with cold deionized water until the wash reached a pH >4. The excess water was removed on a suction filter.Extraction of Chondrogenic Factors. Demineralized bone powder was extracted with 4 M Gdn HCl/1 mM N-ethylmaleimide/10 mM EDTA, pH 6.8, (3.3 ml/g) for 16 hr at 40C.The suspension was suction-filtered...

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Transforming growth factor beta (TGF-beta) has been shown to induce chondrogenesis by embryonic rat mesenchymal cells (Seyedin et al., J. Biol. Chem., 261: 5693, 1986). Here we report the effects of bovine TGF-beta on the phenotypic expression of differentiated primary rat osteoblastic and chondroblastic cells. Culture of rat calvarial osteoblasts with TGF-beta resulted in a dose and time-dependent decrease in alkaline phosphatase activity. Levels of alkaline phosphatase were reduced to less than 10% of control values by 0.4 nM TGF-beta. The decrease became apparent after 24 hours and reached a maximum by 72 hours. Similarly, treatment of chondroblasts with 0.4 nM TGF-beta resulted in decreased production of cartilage-specific macromolecules: type II collagen and cartilage proteoglycan. Both cell types exhibited dramatic changes in cell shape after treatment with TGF-beta. Modulation of these differentiated markers by TGF-beta could be mimicked, in part, by addition of fibronectin. Addition of dihydrocytochalasin B blocked the inhibition of phenotypic expression by TGF-beta. These results indicate that TGF-beta inhibits phenotypic expression by osteoblasts and chondroblasts in vitro and suggest that this activity of TGF-beta may be mediated through interactions between the extracellular matrix and cytoskeletal elements.

We have obtained a cDNA clone coding for human transforming growth factor (TGF)-beta 2. The clone was isolated from a tamoxifen-treated human prostatic adenocarcinoma cell line (PC-3) using oligonucleotide probes based on the partial amino acid sequence of purified TGF-beta 2. The cDNA sequence predicts that TGF-beta 2 is synthesized as a 442-amino-acid polypeptide precursor from which the mature 112-amino-acid TGF-beta 2 subunit is derived by proteolytic cleavage. The proteins coded for by the human TGF-beta 1 and TGF-beta 2 cDNAs show an overall homology of 41%. The mature and amino-terminal precursor regions show 71% and 31% homology, respectively. Northern blot analysis identified TGF-beta 2 transcripts of 4.1, 5.1, and 6.5 kb using mRNA from several different sources. Analysis of polyadenylated RNA from tamoxifen-treated PC-3 cells showed that these cells contain higher numbers of transcripts for TGF-beta 1 than for TGF-beta 2, although they produce more TGF-beta 2 protein than TGF-beta 1. This suggests that there is a post-transcriptional level of regulation for the production of these proteins.