Allogenic bone and cartilage morphogenesis. Rat BMP in vivo and in vitro

An allogenic aggregate of bone morphogenetic protein (BMP) and insoluble non-collagenous proteins (NCP) as well as a crude GuHCl extract were isolated from rats diaphyseal bones. Intramuscular implantation of 5 mg and 10 mg rat BMP/NCP in rats formed new ossicles, whereas 20 mg GuHCl extract failed to induce heterotopic bone formation. When 6 samples of inactivated rat bone matrix gelatin (BMG) were reconstituted with 0.75 mg of either BMP/NCP or GuHCl extract all 3 matrices reconstituted with BMP/NCP but only 1 out of 3 samples reconstituted with GuHCl extract induced heterotopic bone formation. Inactivated BMG alone did not show any osteoinductive activity. The small amount of BMP/NCP necessary for osteoinduction when recombined with inactivated BMG suggests that growth factors in bone matrix without inherent bone-forming activity enhance BMP activity. In vitro, connective tissue outgrowths of neonatal rat muscle on a substratum of inactivated rat BMG differentiated into cartilage in response to 0.05 microgram/ml, 0.5 microgram/ml and 5.0 micrograms/ml allogenic BMP/NCP added to the medium during the incubation period of 2 weeks. On day 14 of cultivation S35-sulphate incorporation into glycosaminoglycans (GAG) and H3-thymidine incorporation into DNA were measured, and the results related to the DNA content and the weight of the incubated muscle tissue, respectively. All doses of BMP/NCP increased GAG synthesis statistically significantly (p less than 0.05 to p less than 0.001). In contrast to that, DNA synthesis rate was not influenced by BMP/NCP. This suggests that GAG synthesis was not caused by cell proliferation but by cell differentiation.