Osteoinductivity assay of the variability of repeated extractions of bone morphogenetic proteins from bovine bone at different times.

Objective: To observe the activity of repeated extracts of bone matrix and the production of purified bone morphogenetic proteins (BMPs).

Methods: BMPs were extracted 1- 4 times from fresh bovine cortical bone by the modified Urist's method, with each collected precipitate separated and lyophilized as partially purified BMPs. Another fresh bovine bone was extracted three times and the precipitates were mixed and lyophilized. Meanwhile,the alkaline phosphatase (ALP) activity was measured by an in vitro assay employing cultured C2C12 mouse myoblast cells through the osteoinductivity of bovine BMPs extracted four times at days 1, 4, 7, and 14, and the correlation between BMPs quantities and costing during extraction processes was analyzed.

Results: The BMPs purified and the cost showed a positive correlation (r=0.969). To separate and lyophilize each collected precipitate as partially purified BMPs raised the cost, and mixed precipitates also cost much. ALP activities of the 1st and mixed extractions of BMPs were shown to be highly osteoinductive and keep a significantly high level (P<0.05-0.01) 4 days after culturing, compared with the 2nd, 3rd and 4th extractions, especially the control group. However, the more times the extraction was done, the less activity of BMPs was shown and more costing was. The x-ray and histological analysis also showed that the 1st extraction of BMPs induced more ossicles and new bone formation.

Conclusions: The results indicated that BMPs enhanced the abilities of osteoinductivity in C2C12 culture in vitro. The first extraction of BMPs from bone is fitfull, the second extraction should be enough, while, the 3rd and 4th extractions are unnecessary for they cost more and waste more time, say nothing of mixed extractions.