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

METHODSBMPs 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.

RESULTSThe 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.

CONCLUSIONSThe 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.

Alkaline Phosphatase ; analysis ; Analysis of Variance ; Animals ; Biopsy, Needle ; Bone Matrix ; pathology ; physiology ; Bone Morphogenetic Proteins ; analysis ; metabolism ; Cattle ; Immunohistochemistry ; Mice ; Osteogenesis ; physiology ; Probability ; Sensitivity and Specificity ; Time Factors ; Tissue Culture Techniques

OBJECTIVETo study the expression of bone matrix protein (BMP) induced by bovine bone morphogenetic proteins (BMPs) in vitro.

METHODSType I collagen, osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and bone sialoprotein (BSP) were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28.

RESULTSThe signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the alkaline phosphatase (ALP) activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblasts in vivo and in differentiating osteoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity.

CONCLUSIONNative bovine BMP induces conversion of myoblasts into osteoblasts, produces type I collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C12 in vitro.

Alkaline Phosphatase ; metabolism ; Animals ; Bone Matrix ; metabolism ; Bone Morphogenetic Proteins ; pharmacology ; Cattle ; Cell Line ; DNA ; metabolism ; Gene Expression Regulation ; physiology ; Mice ; Osteoblasts ; drug effects ; metabolism