gms | German Medical Science

Questionnaire: For instant stem-cell therapy aiming at site-specific bone regeneration the utilization of bone marrow concentrates (BMC) containing both osteogenic progenitor cells and differentiation factors is appealing. The aim of this study was to evaluate the proliferative and osteogenic differentiation potential of point-of-care concentrated BMC-derived total nuclear cells (TNCs) versus mononucleated cells (MNC) isolated by gradient separation method and to assess the effects of 3D cell-immobilization by combined use of fibrin-based gels and tricalcium phosphate (TCP)-granules.

Methods: The osteogenic differentiation capacities of human gradient-isolated MNCs and BMC purified by a closed point-of-care system (SmartPReP® System, Harvest Technologies) were compared. Bone marrow was harvested from N=3 donors prior to undergoing total hip replacement therapy (local institutional review board protocol no. EK203082008). Moreover, TNCs were isolated from BMC whereas MNCs, served as control, were isolated by manual Ficoll separation. Cells were seeded on TCP-granules (cyclOS®, Mathys AG Bettlach,) and were immobilized by use of fibrin (Tissucol®, Duo S Immuno, Baxter,)- and platelet-rich plasma(PRP)-based gels. Cell cultures were maintained both with (OS+) and without (OS-) osteogenic supplements for up to 7 days. Proliferation was assessed by total DNA quantification. For evaluation of osteogenic differentiation cell-specific alkaline phosphatase (ALP) activity and gene expression levels of the osteogenic markers Runx-2, ALP, and OSP were determined using quantitative real-time PCR.

Results and Conclusions: After 7 days of cultivation embedded within fibrin- or PRP-based gels OS- TNCs and -MNCs showed an increased proliferation rate as compared with OS+ TNCs and -MNCs. In fibrin- based gels the proliferation of OS- TNCs was significantly increased (p<0.05) compared to OS+ TNCs. The proliferation of OS- TNCs was significantly higher (p<0.01) compared to the proliferation of MNCs both in fibrin-based gels. All marker gene expression levels by OS+ TNCs and -MNCs were higher compared to OS- TNCs and -MNCs. There were no significant intergroup differences for Runx2, ALP and OSP expression between TNCs and MNCs.

Point-of-care concentrated TNCs demonstrated a superior proliferative capacity compared to MNCs. Furthermore, we demonstrated the feasibility to immobilize TNCs and MNCs in fibrin-based for a maximum period of 7 days. These results will be used for further optimization of cell-based strategies for bone regeneration.