REGENERATIVE EFFECTS OF MOUSE ADIPOSE-DERIVED MULTIPOTENT STROMAL CELLS IN A MICROMASS GRAFT FOR THE TREATMENT OF BONE INJURY MODEL

Abstract

Background. Adipose-derived stem cells (ADSCs) are a promising source for the regeneration of bone tissue injuries. At the same time, three-dimensional cultures provide spatial organization of stem cells for optimal intercellular signaling, contact interaction and increase the efficiency of directed osteogenic differentiation prior to further transplantation. The aim of the study was to establish the regenerative potential of mouse adipose-derived stem cells in micromass grafts differentiated into the osteogenic direction to restore the bone injury in mice. Methods. Three-dimensional micromass cultures of murine ADSCs with further differentiation into osteogenic direction were obtained. The migration potential of cells from micromass in vitro and the effectiveness of differentiation by staining for alkaline phosphatase were evaluated. Mice with the model of femoral bone injury were transplanted with ADSCs micromass grafts and 21 days later the lesion site was examined by histological and morphometric methods. Results. The protocols for the cultivation and directed osteogenic differentiation of ADSCs in the three-dimensional micromass culture have been developed. Alkaline phosphatase production was demonstrated in cells that migrated from micromass, confirming the effectiveness of differentiation. In macroscopic examination 21 days after graft transplantation, the defect sites in femur were filled with dense tissue, while in control bones without the use of transplants, the size of the defect by 80 ± 6 % corresponded to the initial diameter and depth of injury. Histological examination of femoral bone lesions in the area of transplantation of micromass grafts revealed the formation of granulation tissue followed by the replacement of defects with newly formed bone tissue with thickening of periosteum and compact bone substance, similar to callus in fracture regeneration. In animals that underwent transplantation of micromass without prior osteogenic differentiation, the diameter of the zone of active regeneration of the diaphysis at the site of injury was 1.3 ± 0.2 mm while in the group with transplantation of directed differentiated graft it was significantly lower (0.37 ± 0.12 mm, p ≤ 0.05). Conclusions. Three-dimensional grafts of adipose-derived multipotent mesenchymal stromal cells cultured in micromass are able to improve bone tissue regeneration in a model of bone injury in mice. In this case, the grafts differentiated into osteogenic direction, provide better morphological indicators of bone recovery, compared with the micromass without prior differentiation.