This proposal on the biochemistry and physiology of Bone Morphogenetic Protein (BMP) encompasses experimental and clinical objectives. One experimental objective is to isolate and characterize BMP. Previous and current research has identified a partially purified bovine BMP (Mr 18.5k) and human BMP (Mr 17.5k). Further efforts to purify BMP preparations by removing small impurities will continue applying (in addition to previously described techniques), conventional hydroxyapatite as well as HPLC chromatography, reverse phase HPLC, FPLC, and antibody affinity chromatography. Osteoinductive activities of molecular aggregates and isolated proteins are confirmed by implantation into muscle pouches and by tissue culture techniques utilizing histological, immunofluorescence, and radioisotopic techniques. The characterization of BMP by amino acid analysis and sequence has been initiated and represents a major effort for the proposal period. Present in vitro investigation employs a cellulose acetate cone which eliminates the previous use of matrix residues for a substratum. Another experimental objective is to isolate polypeptides with Mr in the range of 4 to 7k generated by limited proteolysis with pepsin or trypsin; these polypeptides have osteoinductive activity both in vivo and in vitro. The isolation of bone morphogenetic polypeptide (BMP-p) may facilitate purification , sequenation and synthesis. The first clinical objective is to evaluate delivery systems combining BMP with non-immunogenic polymers for controlled BMP release. In this work, standardized dog skull trephine and dog ulna diaphysectomy defects are used in addition to the mouse muscle pouch assay. The object is to secure FDA institutional approval for a project on implantation of BMP for repair of tumors, pseudarthrosis, traumatic defects and congenital malformations. Twelve patients, who had multiple failed operations for ununited fractures including unsuccessful treatment by electrical stimulation, and were candidates for amputation, were successfully treated with BMP implants. The second clinical objective is to determine BMP and anti-BMP serum levels by radioimmunoassay in normal and BMP-implanted animals utilizing polyclonal and monoclonal antibodies. The formulation of a RIA for serum BMP and anti-BMP antibodies has opened up a theoretical basis for new investigations of physiology of bone in health and disease. Preliminary observations suggest high levels of serum BMP in Paget's Disease and of serum anti-BMP in osteoporosis.
