Osteogenic potential in vitro of human bone marrow cells cultured on macroporous biphasic calcium phosphate ceramic

Calcium phosphate ceramics are synthetic bone substitutes that promote bone formation by osteoconduction. However, they have shown an osteogenic potential in vivo in animal models when associated with bone marrow cells. In order to develop an osteogenic human "hybrid material," we studied the in vitro osteogenic potential of human bone marrow cells cultured on macroporous biphasic calcium phosphate (BCP) pellets in the presence of dexamethasone with or without 1alpha,25 dihydroxyvitamin D3. We were examining, in particular, their mesenchymal stem cell proliferation and hematopoietic potential. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and immunological characterization of the extracellular matrix formed by these cells. The specimens were observed by scanning and transmission electron microscopy. Human mesenchymal stem cells proliferated on macroporous BCP ceramic, with a doubling time delayed at the beginning of the culture as compared to the cultures realized on plastic. These cells maintained a hematopoietic activity identical to that of cultures performed in plastic wells. The proliferating mesenchymal stem cells expressed an osteoblastic phenotype, as shown by alkaline phosphatase activity and the synthesis in ceramic macropores of an extracellular matrix composed of fibronectin, osteocalcin, and collagen I (but not collagen III). In addition, numerous microcrystals of apatite precipitated on the extracellular matrix, producing a mineralized fibrillar network within the ceramic not observed with cell cultures realized on plastic. These results demonstrate that human mesenchymal stem cells cultured on macroporous BCP ceramic express and conserve their osteoblastic phenotype even after one month of culture and that these osteogenic cells are able to form new bone matrix in a BCP ceramic in vitro.