INTRAMEMBRANOUS BONE-MATRIX IS OSTEOINDUCTIVE

All known bone-derived osteoinductive factors have been isolated from endochondral (EC) bones and all initiate bone induction via EC ossific ation. However, to date no attempt has been made to isolate comparable factors from bones which form initially and completely via intramembr anous (IM) ossification. The purpose of this work was to isolate osteo inductive proteins from IM bones. To accomplish this, we extracted pro teins from bovine frontal bone matrix (intramembranous origin) using m ethods previously described for endochondral (EC) bone matrix (i.e., f emur). Bone powder (<1 mm) was decalcified and proteins extracted with 4 M guanidine hydrochloride. Ultrafiltration was used to isolate and concentrate a 10-100 kilodalton (kDa) fraction, upon which heparin-Sep harose (HS) affinity chromatography was performed. HS-binding (HS-B) a nd nonbinding proteins (HS-NB) were lyophilized with bovine type I col lagen (Vitrogen) to form pellets which were implanted subcutaneously i n rats. Radiology as well as brightfield, fluorescent, and polarizing microscopy were used to assess the formation of ectopic bone at the si te of pellet implantation. In this report we demonstrate that a hepari n-Sepharose binding, osteoinductive factor can be extracted and partia lly purified from bovine intramembranous bone matrix. This factor has a different sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern than a comparable osteoinductive/chondroind uctive factor isolated from EC bone. (C) 1994 Wiley-Liss, Inc.