Development of Osteoinductive Biomaterials (Purification of Osteoinductive Factor and Its Basic Research for Clinical Application)

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I. PurificationDecalcified bovine bone matrix powder was extracted dessociatively in 4 M guanidine hydrochloride and the water soluble proteins of the extract was collected (Fraction I). Then, the partial purified fraction (Fraction II) was obtained from fraction I by using heparin-sepharose chromatography and gel-exclusion chromatography. Fraction I or II adsorbed to aterocollagen were bioassayed by intramuscular implantation in rats. Fraction II had approximately 680 times activity as much as fraction I, as measured by alkaline phosphatase activity of the mineralized implant. Final fraction was purified by using reverse-phase HPLC. Electrophoresis of this fraction revealed a band with an apparent molecular mass of 18 kDa.II. BioassayThe following methods have been developed to decide osteoinduclive ability in just a few days.1) in vitroA hard tissue is formed in our co-culture system which contains the osteoinductive factor and mesenchymal cells derived from rat skeletal muscle tissue or rat calvaria subperiosteum, which have a high responsibility to that factor.2) in vivoOsteoinductive activity is demonstrated by some histological or/and biochemical parameters of bone tissue, which is induced by implantation of the complex of osteoinductive factor and aterocollagen.III. ImplantationSome new findings about the mechanism of osteoinduction has been revealed by the investigations on the implantation of partial purified osteoinductive factor.1) Acellular mineral deposition, which appeared in bone matrix gelatin preceding bone induction may be significant for differentiation of osteoblast.2) Muscle satellite cells will differentiate into osteoblasts.3) Porous hydroxyapatite block and aterocollagen are valid as a carrier for osteoinductive factor.