A bone morphogenetic protein (BMP) obtained in solution by digestion of demineralized rabbit cortical bone matrix with bacterial coliagenase retains its biologically active conformation in a neutral salt/ethylene glycol mixture. BMP may be insolubilized by coprecipitation with calcium phosphate and resolubilized by chemical extraction with a neutral salt in the same solvent mixture. Upon concanavalin A-Sepharose chromatography, BMP is boundby hydrophobic interaction and carbohydrate recognition and is recovered by elution with either a-methyl mannoside or ethylene glycol solvent mixture. Implants of both eluates and the extracts of the coprecipitate in double-walled diffusion chambers induce transmembrane bone morphogenesis. BMP is not species specific; rabbit BMP induces new bone formation in the rat. The present observations indicate that BMP is a glycoprotein.One of the most striking and consistently inducible forms of postfetal cell differentiation is the development of cartilage, bone, and bone marrow in an intramuscular implant of dentin or bone matrix (1-4). The initial deposits consist of cartilage and woven bone which are remodeled and replaced by an ossicle of lamellar bone and bone marrow. The quantity of bone is proportional to the mass of preimplanted, demineralized matrix (5). In previous communications (6-9), it was postulated that the new bone develops from somatic migratory mesenchymal type cells under the influence of a bone morphogenetic protein (BMP) that is released from acid-insoluble substance of bone matrix (10, 11) or insoluble bone matrix gelatin (12, 13). This is a preliminary report on a method of solubilization of BMP and of coprecipitation of BMP with calcium phosphate. Fig. 1 summarizes the six-step procedure for separation of BMP from insoluble bone matrix. Rabbit cortical bone (100 g) was demineralized in HC1 at 2°C for 24 hr and lyophilized. In step 1, the lyophilized matrix was sequentially extracted to decrease the content of lipid, proteoglycans, and sialoproteins and to convert the bone collagen to insoluble bone matrix gelatin in 8 M LiCl (12). In step 2, the bone matrix gelatin was incubated for 24 hr at 37°C at pH 7.2 in a 0.00054% purified bacterial collagenase (Worthington, CLSPA) in Hanks' solution (14) containing mM Tris, 300 mM CaCl2, and 3 mM NaN&. The collagenase was purified by the method of Peterkofsky and Diegelmann (15). A low enzyme-to-substrate ratio and a high concentration of Ca2+ were used for suppression of contaminant proteases (16); the NaN3 was used for antimicrobial activity. The pH was readjusted to 7.2 every 2 hr for the first 8 hr. After 24 hr, the total digest was centrifuged at 40,000 X G for 15 min (step 3). MATERIALS AND METHODSStep 4 produced a pellet of insoluble collagenase-resistant substances.In step 5, the supernatant, a clear, slightly opalescent solution, was filtered through a cellulose acetate membrane (pore size, 0.30 Mm). In step 5A, the dialysate was lyophilized. One half of solution SB was transferred, in step 5C, to a me...

Bovine bone morphogenetic protein (bBMP) induces differentiation of mesenchymal-type cells into cartilage and bone. bBMP has an apparent Mr of 18,500 ± 500 and represents <0.001% of the wet weight of bone tissue. A Mr 34,000 protein resembling osteonectin is separated by extraction with Triton X-100. A Mr 24,000 protein and about half of a Mr 22,000 protein are disassociated from bBMP by precipitation in 1.5 M guanidine hydrochloride. Aggregates of bBMP and a Mr 14,000 protein are insoluble in aqueous media; the bBMP becomes soluble when the Mr 14,000 protein is disassociated in 6 M urea and removed from the solution by ultrafiltration. Three separate molecular species with apparent Mrs 18,500, 17,500, and 17,000 are eluted at 0.10, 0.15, and 0.20 M phosphate ion concentrations, respectively, from a hydroxy-apatite column. The Mr 18,500 protein has the amino acid composition of acidic polypeptide and includes four halfcystine residues; the pI is 4.9-5.1. The Mr 22,000 component is a chromoprotein resembling ferritin. The NH12-terminal amino acid sequence of the Mr 17,500 protein simulates histone H2B.The Mr 17,000 protein may possess calmodulin activity. Aggregates of the Mr 18,500 and other proteins induce formation of large deposits of bone; the Mr 18,500 protein alone is rapidly absorbed and induces formation of small deposits. None of the other proteins induces bone formation.Under the influence of bone morphogenetic protein (BMP), perivascular mesenchymal-type cells (pericytes) differentiate into cartilage and woven bone. BMP is an acidic polypeptide (1, 2) embedded in a complex assortment of intra-and extracellular protein aggregates derived from dentin (3), bone (4, 5), and osteosarcoma tissues (6-9). We report here on the purification of BMP by means of a combination of differential precipitation, ultrafiltration, and hydroxyapatite chromatography. MATERIALS AND METHODSTen-kilogram batches of 1-year-old steer long bones were obtained from an abattoir. After the epiphyseal ends were cut away with a band saw, the diaphyses were mechanically scraped clean of soft tissues and extensively washed in cold water solution of 3 mM NaN3. The washed bone was frozen in liquid N2, ground in a Wiley mill to a particle size of 1 mm3, defatted in chloroform/methanol (1:1), and again washed in 10 liters of cold water (step 1). The bone particles were demineralized in 0.6 M HCl at 4°C for 48 hr and again extensively rewashed in NaN3 solution (step 2). The demineralized washed bone particles were chemically extracted to remove soluble noncollagenous protein (i.e., sialoproteins, plasma proteins, y-carboxyglutamyl proteins, and phosphoproteins), simultaneously converting the collagen to insoluble bone matrix gelatin (step 3) by previously described procedures (10). Ten kilograms of whole wet bone produced -'1.4 kg of freeze-dried insoluble bone matrix gelatin. The BMP was extracted from the insoluble bone matrix gelatin in an inorganic/organic solvent mixture of 0.5 M CaCl2 in 6 M urea at 28°C for 24 hr containing 10...

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Human bone morphogenetic protein (hBMP) was chemically extracted from demineralized gelatinized cortical bone matrix by means of a CaClz urea inorganic-organic solvent mixture, differential precipitation in guanidine hydrochloride, and preparative gel electrophoresis. hBMP is isolated in quantities of 1 mg/kg of wet weight of fresh bone, and has the amino-acid composition of an acidic polypeptide. The mol wt is 17 to 18 k-Da (kilodaltons). Implants of the isolated 17-kDa protein are very rapidly adsorbed and produce a smaller volume of bone than protein fractions consisting of 24-, 17-, and 14-kDa proteins. Since the isolated 24-and 14-kDA components lack hBMP activity, the kinetics of the bone morphogenetic processes including the function of other proteins as camer molecules, await investigation. 194