Osteogenic Ability of Canine Adipose-Derived Mesenchymal Stromal Cell Sheets in Relation to Culture Time.

Cell sheets could be used for bone regeneration without requiring a scaffold and can be easily produced from autologous mesenchymal stromal cells (MSCs). We compared the osteogenic potential of MSC-derived cell sheets in relation to culture time. Undifferentiated cell sheets (U-CS) and osteogenic differentiated cell sheets (O-CS) were generated using canine adipose-derived MSCs. Undifferentiated cells (UCs) were used as the control. Osteogenic differentiation was assessed by assaying alkaline phosphatase (ALP) activity. Expression of osteogenesis-related genes was evaluated by reverse transcription-polymerase chain reaction at 4, 7, 14, and 21 days after initiation of culture. The calcium content in cells was measured, and the cells were stained with Alizarin red S (ARS). The mRNA expression of transforming growth factor-β in U-CS and O-CS at day 4 was higher than that in UCs (p < 0.05). The level of bone morphogenetic protein 7 mRNA in O-CS increased significantly at day 4 and was significantly higher than that of U-CS at day 7. The mRNA level of runt-related transcription factor-2 in both sheet types increased significantly at 7 days of culture. The mRNA level of ALP in O-CS and U-CS increased significantly at day 7, and ALP activity was highest at days 7 and 14, respectively (p < 0.05). The mRNA level of osteocalcin in U-CS and O-CS increased significantly at day 21. O-CS and U-CS showed negative ARS staining but their calcium contents increased marginally at day 21. The O-CS cells started to aggregate at days 10-12, and only a partial sheet remained at day 21. The upregulation of expression of genes related to osteogenic differentiation, peak in ALP activity, and morphological changes in cell sheets suggest that the optimal time for application of O-CS and U-CS is between 7 and 10 days and after 14 days of culture, respectively.