An immuno-light- and electron-microscopic study of the expression of bone morphogenetic protein-2 during the process of ectopic bone formation in the rat

Immunolocalization of endogenous bone morphogenetic protein-2 (BMP-2) was investigated during the process of ectopic bone formation induced by recombinant human BMP-2 (rhBMP-2). Pellets consisting of 5 μg of rhBMP-2 and 6 mg of atelopeptide type I collagen (AC) were implanted into the calf muscles of 6-week-old rats. On days 7, 10, 14, 21 and 28 after implantation, tissue specimens were removed and examined immunohistochemically by light and electron microscopy after incubation with anti-BMP-2 monoclonal antibodies. Immunolocalization by light microscopy showed BMP-2 in chondrocytes at the pellet rim on days 7 and 10, in osteocyte-like cells in the chondroid matrix on day 14, and in osteocytes in the newly formed bone on days 21 and 28 after implantation. Ultrastructurally, on days 7 and 10 after implantation, immunolabelling for BMP-2 was aggregated in vesicle-like matrices released from mature chondrocytes in the chondroid matrix. On day 14, immunolabelling against BMP-2 had accumulated in vesicle-like matrices embedded in the calcified cartilage, in the cytoplasmic vacuoles of chondroclasts absorbing the matrix, and at the resorption surface of the calcified cartilage. On days 21 and 28, BMP-2 immunolabelling was seen in the osteoid layer and osteocyte lacunae. These results suggest that the chondrocytes and osteocytes induced by rhBMP-2 produce endogenous BMP-2. It seems that part of the endogenous BMP-2 that accumulated in the chondroid matrix was absorbed by chondroclasts and then participated in the osteoblastic differentiation of immature mesenchymal cells. This study indicates that, in addition to the implanted exogenous rhBMP-2, endogenous BMP-2 plays an important part in the maintenance of the bone-formation cascade during ectopic osteoinduction.