A bone morphogenetic protein (BMP) that induces differentiation of mesenchymaltype cell into cartilage and bone was partially purified from bovine tooth. In this study, the activity of BMP was assayed by its ability to induce enchondral ossification after injections of samples into rat thigh muscles.
Frozen teeth were ground to fine particles and demineralized in 0.6 N HCl. The demineralized tooth matrix was treated with 4 M guanidine hydrochloride to extract BMP. Three volumes of cold ethanol were added to the supernatant obtained by filtration and the insoluble materials formed were collected by centrifugation. These materials were redissolved in 4 M guanidine hydrochloride and subjected to gel filtration using a Superose 12 column. The precipitate formed by adding three volumes of cold ethanol to each fraction was collected by centrifugation and BMP activity was assayed.
The molecular weight of BMP of bovine tooth analyzed by electrophoresis on a SDS polyacrylamide gel was 27, 000. The combination of partially purified BMP with 0.5% atelocollagen (in HCl, pH 3) more effectively enlarged the volumes of cartilage and bone than did BMPTalone, indicating usefulness of atelocollagen as a carrier. BMP induced enchondral ossification in the periosteum of the femur as well as in the muscle, suggesting that the periosteum includes cells that differentiate into osteoblasts in response to BMP.