PARTIAL-PURIFICATION AND CHARACTERIZATION OF BONE MORPHOGENETIC PROTEIN FROM BONE-MATRIX OF THE PREMATURE MOOSE (ALCES-ALCES) - DEGRADATIONOF BONE-INDUCING ACTIVITY DURING STORAGE

In spite of the advances in recombinant techniques in the production o f bone morphogenetic proteins (BMPs), the best clinical results so far have been obtained with human and animal source-extracted BMPs. Also, the poor availability of recombinant products gives rise to continued research with different extracted and purified proteins. In a search for a new source of bone-matrix-derived BMP with high osteoinductive a ctivity, BMP was extracted from fresh bone matrix of the premature moo se (Alces alces). Bone-inducing activity was investigated by implantin g 0.5-20 mg of BMP into thigh muscle pouches of BALB mice. Radiologica lly detectable formation of new bone required 2.0 mg of partially puri fied BMP. Immediately after the extraction, an analytic chromatogram w ith known molecular weight (MW) markers showed three fractions with di fferent MWs. After 15 months of storage at +1 degrees C lyophilized an d desiccated, BMP was fractionated by HPLC gel filtration and bioassay ed. New bone formation was evaluated qualitatively by histology and qu antitatively by radiomorphometry, the quantity of calcified tissue per milligram of implanted agent being determined. Fractions I and III, w ith high (100-700 kD) and low MW (15-25 kD), respectively, were appare ntly more effective inducers of new bone than the second-time-tested p artially purified BMP complex, the activity of which had significantly (p < 0.05) decreased during 15 months of storage compared to initial results after extraction. However, the bone-inducing activity of fract ions I and III corresponded closely to the initial activity of the BMP complex. Fraction II, with medium MW (25-55 kD), caused an apparent i nflammatory reaction and no bone formation, and was thought to be immu nogeneic. Fraction In was considered to include the dominant BMP compo nent with MW 18.5 and fraction I an association of BMP with other non- collagenous bone matrix proteins after onestep gel filtration. The res ults suggest that BMP from the premature moose has high bone-forming a ctivity. With identification and removal of apparently immunogenic pro tein fractions, the inflammatory reaction and inhibitory effect on bon e induction could be eliminated, and still higher bone-forming activit y was attained. Acid protease enzymes were assumed to be responsible f or the observed decline in the inductive activity of semipurified BMP after 15 months of storage, as both osteoinductive fractions proved to be acidic in isoelectric focusing.