Hydroxyapatite ceramics as a carrier of gene-transduced bone marrow cells.

Abstract

The aim of this study was to develop an efficient exogenous gene delivery system using cultured marrow cells and porous hydroxyapatite ceramics. Bone marrow cells were obtained from the femoral shaft of a Fischer 344 rat and cultured in a medium containing 15% fetal bovine serum until confluent. After trypsinization, cells were subcultured at a cell density of 1 x 10(4) cells/cm2 in the presence of fetal bovine serum. The subcultured bone marrow cells were infected with recombinant retroviruses carrying the lacZ gene. The retrovirus infection was performed seven times from day 1 to day 7 during the culturing procedure. Cells expressing the lacZ gene were stained blue with the X-gal staining and represented approximately 80%. Composites of virus-infected bone marrow cells and hydroxyapatite ceramics were implanted at the subcutaneous site of recipient Fischer 344 rats. Four weeks after the implantation the ceramics were harvested. The histological sections of the ceramics showed abundant bone formation in the pores of the ceramics and obviously blue-stained osteoblasts and osteocytes. Other cell types that were stained blue were some fibroblastic cells and endothelial cells in the newly formed capillaries. These findings indicate that osteoblasts and osteocytes in the newly formed bone were derived from the cultured bone marrow cells, and therefore gene transfection by retroviruses did not disturb the bone formation process. Because of the durability of the newly formed bone tissue, creating composites of cultured bone marrow cells and hydroxyapatite ceramics might be an ideal method for exogenous gene transfection.