The Effect of Bone Marrow Transplantation on the Osteoblastic Differentiation of Human Bone Marrow Stromal Cells

Abstract

Osteoporosis is a serious and relatively common complication of transplantation procedures. However, little is known about the exact mechanism or severity of osteoporosis complicated by bone marrow transplantation (BMT). We conducted both ex vivo and clinical studies to identify the mechanism and extent of bone loss after BMT. In a prospective clinical study, we intended to identify the changes in bone turnover markers and bone mineral density (BMD) after BMT. During a 1-yr follow-up, BMD was measured before BMT and 1 yr after BMT in 67 patients undergoing BMT. Biochemical markers of bone formation and resorption were measured in all patients at short-term intervals during the yearlong follow-up. In ex vivo study, we cultured human bone marrow cells of normal controls and BMT recipients in osteogenic medium and compared their osteogenic potential. Using a DNA fingerprinting method, we also investigated the origin of bone marrow stromal cells that were harvested 3–4 wk after BMT. In a clinical study of 67 patients undergoing BMT, the mean serum carboxy-terminal cross-linked telopeptide of type I collagen increased progressively until 4 wk after BMT. Thereafter, it began to decrease and reached basal values after 1 yr. Serum osteocalcin decreased progressively until 3 wk after BMT and reached basal values after 3 months. One year after BMT, lumbar spine BMD had decreased by 3.3% (P < 0.05), and total proximal femoral BMD had decreased by 8.9% (P < 0.001). For the ex vivo study, bone marrow was obtained from healthy donors (n = 7) and transplant recipients (n = 7). Then, mononuclear cells including marrow stromal cells were isolated and cultured to osteoblastic lineage. Alkaline phosphatase activities of each group were measured by the time course of secondary culture, and the mineralizing potentials were compared between the two groups. Cells cultured in our system showed characteristics of osteoblast-like cells differentiated from marrow stromal cells. They were initially in a fibroblastic-like spindle shape and became cuboidal with the formation of nodules that were later confluent. The cells were stained to both alkaline phosphatase histochemistry and Von Kossa histochemistry, demonstrating that these cells were of osteoblastic lineage differentiating from marrow stromal cells. The mean time required for the near-confluence in the primary culture was 15 and 22.9 d in healthy donors and transplant recipients, respectively (P = 0.003). Alkaline phosphatase activity was significantly lower in the bone marrow recipients than in the healthy donors at d 7 and 10 of the secondary cultures. The period at which peak activity of alkaline phosphatase was reached was also delayed in the osteoblasts derived from BMT recipient bone marrow compared with those of healthy donors. Using Von Kossa histochemistry, much more mineralization was observed in osteoblasts of healthy donors than those of BMT recipients. After BMT, although the peripheral mononuclear cells in the recipients were of donor origin, the bone marrow stromal cells were of recipient origin according to the PCR analysis using YNZ 22 mini-satellite probe. In conclusion, the differentiation of bone marrow stromal cells into osteoblasts was impaired after BMT, and this might contribute to post-BMT bone loss.