gms | German Medical Science

Objectives: Multipotent mesenchymal stem cells (MSCs) can be isolated from different adult tissues (e.g. bone marrow and adipose tissue) and represent an interesting source of cells for therapeutic applications. MSCs consist of a heterogenous population of stem/progenitor cells, which exhibit osteogenic, chondrogenic, and adipogenic potentials. An interesting subpopulation is represented by the perivascular cells (pericytes), which can be prospectively identified by the expression of CD146, NG2, and PDGF-Rbeta and absence of hematopoietic, endothelial, and myogenic cell markers.

Methods: We isolated CD146+ NG2+ and CD45- cells from adipose tissue from C57BL/6 mice by magnetic activated cell sorting (MACS). Upon seeding on scaffolds (e.g. cancellous bone, tricalcium-phosphate) CD146+ NG2+ and CD45- isolated cells were differentiated for two weeks in osteogenic medium. Alizarin red staining and RTQ-PCR confirmed the capacity of these cells to generate osteoblasts and induce mineralization. In a last step, we tested the regenerative capacity of these cells in a mouse model for femoral segmental critical-sized defect. This model is based on a 3.5-mm-long segmental bone defect where the bone fixed by a titanium microlocking plate with four locking screws (RISystem AG). The freshly isolated cells were seeded on a collagenous bone scaffold which upon overnight cultivation in vitro was inserted in the segmental bone gap. A control group with collagenous bone scaffold but without cells was used. Eight weeks upon operation the bones were isolated and analyzed by micro-computer tomography.

Results and Conclusion: CD146+NG2+CD45-cells directly upon MACS, without further expansion in vitro, were differentiated toward the osteogenic lineage and alizarin red staining was performed after two weeks of differentiation. We could confirm that CD146+ NG2+ CD45- cells are able to generate osteoblasts and induce mineralization with a higher efficiency compared to unsorted ASCs. In a second step cells were seeded on the different scaffolds (cancellous bone, tricalcium-phosphate) and cultivated for 5 days. Upon staining with MTT we could observe that the cells were viable and that they mainly colonized the holes present on the scaffolds, as shown by scanning electron microscopy. The freshly isolated cells were seeded on a collagenous bone scaffold which upon overnight cultivation in vitro was inserted in the segmental bone gap. A control group with collagenous bone scaffold but without cells was used. At 8 weeks the bones were isolated and after removal of the plates analyzed by micro-computer tomography. Quantification of the data indicated that the perivascular stem cells seeded scaffold had a significantly stronger mineralization as compared with the scaffold only controls.

This indicate that perivascular stem cells (CD146+NG2+CD45-) isolated from ASCs are able to contribute to bone regeneration and might represent a valuable alternative for improving bone healing in critical size bone injuries.