Purification and characterization of two cartilage-inducing factors from bovine demineralized bone.

Abstract

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...

Proteolysis of Latent Transforming Growth Factor-β (TGF-β)-binding Protein-1 by Osteoclasts

“…Mature TGF-␤ can bind to heparin (44) and our own unpublished studies indicate that LTBP1 itself binds to heparin. 3 Studies by Tiedemann et al (45) have demonstrated the importance of heparan sulfatecontaining proteoglycans in assembly of fibrillin-1 into the ECM. Thus heparan sulfate-containing proteoglycans may play a similar role in LTBP1 (and by implication TGF-␤) incorporation.…”

“…Matrix metalloproteinases (active MMP2 and MMP9) and the tissue inhibitor of the matrix metalloproteinases (TIMP1) were purchased from Oncogene Research Products (Cambridge, MA). 1,25-Dihydroxyvitamin D 3 (1,25-D 3 ) was purchased from Biomol (Plymouth Meeting, PA). Parathyroid hormone-related peptide (PTHrP) was purchased from Bachem (Torrance, CA).…”

Transforming growth factor‐β and bone morphogenetic protein‐2 act by distinct mechanisms to promote chick limb cartilage differentiation in vitro

“…Both TGF-p1 and TGF-p2 stimulate fibroblastic, mesenchymal-like cells isolated from fetal rat muscle explants to synthesize matrix proteins characteristic of cartilage (Seyedin et al, 1985(Seyedin et al, , 1986(Seyedin et al, , 1987 and both these factors can elicit localized chondrogenesis when injected subperiosteally (Joyce et al, 1990). TGF-p1 and TGF-P2 are potent promoters of the chondrogenic differentiation of embryonic limb mesenchymal cells in vitro (Kulyk et al, 1989a;Carrington and Reddi, 1990;Schofield and Wolpert, 1990;Leonard et al, 1991) as are BMP-3 , BMP-4 (Chen et al, 1991), activin (Jiang et a]., 1993), and inhibin (Chen et al, 1993).…”

Crystallization and preliminary X‐ray analysis of recombinant human transforming growth factor β2

“…isoform, has been purified from various natural sources including porcine platelets [3], human adenocarcinoma [4,5], human glioblastoma [6], simian epithelial kidney cells [7], bovine bone [8,9] and bovine milk [IO, has a molecular weight of approximately 25 kDa and consists of two identical polypeptide chains covalently linked by one or more disulfide bonds [4]. TGF-,82 has been shown to have important roles in mouse embryogenesis [12], wound healing (Cox, D.A.…”