Observations Implicating an Extracellular Enzymic Mechanism of Control of Bone Morphogenesis

Abstract

Data on physicochemical conditions leading to loss of the bone morphogenetic property of bone matrix in neutral buffer solutions support the concept of an enzymic control mechanism better than a chemical blocking reaction or denaturation. The loss is associated with release of 35S-labeled constituents and not prevented by ε-amino caproic acid, an inhibitor of cathepsins. The loss is also associated with release of 35S-cysteine-labeled protein; about 60% of the yield is sustained by the addition of only 3 mmoles/liter of iodoacetic acid. A latent period of about 12 hr, decreased by extraction of bone matrix with CaCl2, is characterized by release of protein polysaccharide and other noncollagenous proteins. Release of sialic acid from the bone matrix by neuraminidase at pH 7.4 has no effect upon bone yield. At 2°C, Tris-HCl buffer or ethylenediaminetetraacetic acid extracts noncollagenous proteins without loss of bone yield; at 37°C, pH 7.4, these solutions also activate endogenous enzymes and reduce bone yield. The component of bone matrix responsible for reduction in bone yield is separable from bone matrix by extraction with phosphate buffer, by catheptic digestion of bone matrix in acidic buffer solutions, by sequential chemical extraction of noncollagenous proteins with cold slightly acidic salt solutions or by extraction-denaturation with chloroform-methanol. Detergents neither extinguish nor denature the morphogenetic property but some solubilize or extract degradative enzymes; hexodecyl trimethyl ammonium bromide, at pH 5.0, is positively charged and extracts hydrophobic proteins, including part of the bone morphogenetic property. A special selection of sulfhydryl chemical inhibitors remarkably different from the selection inhibiting known enzymes preserves the bone morphogenetic property of bone matrix; p-chloromercuribenzoate preservation is reversible by chemical reactions with cysteine. Reduction in bone yield in phosphate buffer is not attributable to a chemical block because chloroform-methanol extraction of the agent does not restore bone yield and is not attributable to denaturation because bone yield sustained by p-chloromercuribenzoate is lost by chemical reactions with cysteine. An hypothetical insoluble bone morphogenetic protein (BMP) firmly bound to collagen is degraded by a soluble neutral proteinase (BMPase). Digestion of the hypothetical BMP occurs without loss of the 640-A electron micrographic image of bone collagen, resembles tryptic digestion and is more selective as well as physiologic in action.

BMP depletion occurs during prolonged acid demineralization of bone: characterization and implications for graft preparation

“…The inductor belongs to the family of bone morphogenetic proteins (BMPs) of which BMPs 2, 4, and 7 are the most active in stimulating endochondral ossification (Bauer and Muschler 2000;Reddi 2001;Sampath and Reddi 1984;Urist et al 1967Urist et al , 1974Urist and Strates 1971;Winn et al 1999). BMPs are principally associated with bone matrix with a minor component associated with bone mineral (Sampath and Reddi 1984).…”

Donor age and gender effects on osteoinductivity of demineralized bone matrix

“…There are many possible factors that may ultimately contribute to the osteoinductivity of human demineralized bone as measured in an ectopic animal model. Particularly important is the fact that in its native form DBM contains both growth factors and endogenous proteases 20–22. Thus, without proper handling and a consistent processing method, DBM osteoinductivity may appear to be unstable and variable.…”

Solubilized and insolubilized bone morphogenetic protein.

“…The quantity of bone is proportional to the mass of preimplanted, demineralized matrix (5). In previous communications (6)(7)(8)(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.…”