Solubilized and insolubilized bone morphogenetic protein.

Abstract

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...

Growth factors in bone

“…Urist believed that osteoinduction by demineralised bone [72], was caused by a contained factor which he named bone morphogenetic protein (BMP), and in 1979 the group isolated an extract of osteoinductive glycoproteins [73]. In 1988, Wozney et al extracted 40 µg inductive proteins from 40 kg of bone.…”

Demineralized bone matrix, bone morphogenetic proteins, and animal models of spine fusion: an overview

“…Several osteoinductive growth factors, referred to as the bone morphogenetic proteins (BMP), have been shown to induce the transformation of undifferentiated mesenchymal cells into chondroblasts and osteoblasts, resulting in the formation of de novo bone [2, 18, 19, 22, 36, 42, 43, 47, 55,72,73]. Numerous preclinical animal studies have demonstrated the superiority of BMP implants over traditional autogenous bone grafts for a variety of orthopedic applications [17, 25, 29, 32, 37, 49,66].…”

Ability of Hydroxyapatite-Bone Morphologenetic Protein (BMP) Complex to Induce Dentin Formation in Dogs

“…In recent studies on the development of new implantable biomaterials as bone substitutes in the reparation of bone defect, BMP bound to 3-TCP, HAP and human fibrin have been investigated (Urist et al 1979, Kawamura et al 1988, Mieki 1990). Mieki (1990) reported that the osteoinductive activity of HAP/BMP complex was higher than that of BMP alone.…”