Osteoblast-like cells complete osteoclastic bone resorption and form new mineralized bone matrix in vitro

Bone remodeling involves old bone resorption by osteoclasts and new bone formation by osteoblasts. However, the precise cellular mechanisms underlying these consecutive events remain obscure. To address this question in vitro, we have established a cell culture model in which the resorption lacunae are first created by osteoclasts and osteoblast-like cells accomplish the subsequent bone formation. We isolated osteoclasts from rat bone marrow and cultured them on bovine bone slices for 48 hours to create resorption lacunae. After removing osteoclasts, confluent differentiated primary osteoblast cultures were trypsinized and the cells were replaced on the resorbed bone slices for up to 14 days. The cultures were then examined by confocal microscopy, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). Our data suggest that after osteoclastic bone resorption, osteoblast-like cells, not macrophages, remove the remaining organic matrix in the lacuna. After cleaning the lacuna, osteoblast-like cells deposit new collagen fibrils at the bottom of the lacuna and calcify the newly formed matrix only, as visualized by labeled tetracycline accumulation merely in the lacuna during the osteoblast culture. Furthermore, an electron-dense layer rich in osteopontin separates the old and new matrices suggesting formation of the cement line. Since the morphology of the newly formed matrix is similar to the natural bone with respect to the cement line and osteoid formation as well as matrix mineralization, the present method provides for the first time a powerful in vitro method to study the cellular mechanisms leading to bone remodeling also in vivo.