THE OSTEOGENIC POTENTIAL OF A RAT PERIPHERAL BLOOD MONONUCLEAR CELL SUBSET

Abstract

Introduction: The existence of circulating skeletal stem cells in the peripheral blood from different species including adult mouse and human has been found and documented. The circulating skeletal stem cells may provide a new source of stem cells that may be used for bone regeneration and tissue engineering applications. The aim of this study was to investigate the existence of circulating osteogenic stem cells in the rat peripheral blood, and to compare their osteogenic potentials with bone marrow mesenchymal stem cells (BMMSCs).

Methods: Whole blood from twelve female 3-month old SD rats was harvested by cardiac puncture and bone marrows were also collected. Mononuclear cells from both bone marrow and peripheral blood (PBMNCs) were isolated by Lymphoprep density gradient centrifugation method, and plated at a density of 300000 to 400000/cm2 in flasks with á-MEM medium and 15% FCS. The colony forming efficiency (CFE) was calculated after 10–14 days culture. The osteogenic, adipogenic, and chondrogenic differentiation potential of both BMMSCs and peripheral blood mononuclear cell subset were examined and compared under different specific culture conditions. In addition, both BMMSCs and peripheral blood mononuclear cell subset were seeded into absorbable porous calcium phosphate substitute and implanted subcutaneously into SCID mice for 12 weeks, and the implants were examined histologically.

Results: After 10–14 days in culture, the adherent fibroblast-like colonies were formed in the PBMNCs, with CFE ranging from 1.3 to 3.5 per 10000000 cells. Under osteogenic conditions, both BMMSCs and PBMNCs subset were positive for bone markers such as ALP, type I collagen and osteocalcin; bone nodules were formed in BMMSCs and PBMNCs subset long-term culture with positive Von Kossa and Alizarin Red S staining. Under adipogenic conditions, PBMNCs subset and BMMSCs were positive for Oil Red O and C/EBP á immunostaining. For chondrogenic differentiation studies, PBMNCs subset and BMMSCs were positive for type II collagen and they had Alcian blue positive nodules formation. After implantation with calcium phosphate substitutes in SCID mice for 12 weeks, osteoid and bony tissues were evident in the implants both loaded with PBMNCs subset and BMSCSs.

Conclusions: A subset of mononuclear cells that have multi-differentiation potential similar to BMMSCs exists in the rat peripheral blood. Our present study has shown that these circulating stem cells possess osteogenic potential in vitro and in vivo. Further work is ongoing to investigate the roles of PBMNCs subset in fracture healing and their recruiting and homing mechanisms.