Osteogenic potential of cell spheroids composed of varying ratios of gingiva-derived and bone marrow stem cells using concave microwells

The aim of the current study was to evaluate cell viability and osteogenic differentiation potential in cell spheroids composed of varying ratios of gingiva-derived and bone marrow stem cells cultured in concave microwells. Cell spheroids were established from bone marrow and gingiva-derived stem cells in ratios of 6:0 (Group 1), 2:1 (Group 2), 3:3 (Group 3), 1:2 (Group 4), and 0:6 (Group 5). On days 3 and 5, the viability of the cell spheroids was qualitatively analyzed using a calcein acetoxymethyl ester working solution and an ethidium homodimer-1 live/dead assay. On days 1, 3, 5 and 7, a quantitative cell viability analysis was performed using a Cell Counting Kit-8. Alkaline phosphatase activity assays were performed using a commercially available kit on day 7 to assess osteogenic differentiation. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to evaluate runt-related transcription factor 2 (Runx2) and osteocalcin expression. The ratio of gingiva-derived to bone marrow stem cells did not affect the stem cell spheroid morphology. No significant changes in cell viability were noted among the different groups following incubation for 7 days. A consistent alkaline phosphatase activity was measured in co-cultured gingiva-derived and bone marrow stem cell spheroids of varying compositions. Runx2 and osteocalcin expression was increased when co-cultured compared with pure gingiva-derived or bone marrow stem cells. In conclusion, stem cell spheroids established by co-culturing maintained morphology, viability and a high osteogenic differentiation potential during the experimental period of 7 days. These spheroids containing human gingiva-derived and bone marrow stem cells may enhance the osteogenic differentiation potential. The use of multicell spheroids may be a simple and effective strategy for improving stem cell therapy.