Osteogenic differentiation potential of canine bone marrow-derived mesenchymal stem cells under different -glycerophosphate concentrations in vitro

Abstract

Stem cell-based therapy employing bone marrow-derived mesenchymal stem cells has been proposed as a promising approach for bone regenerative treatment. Despite numerous studies supporting the application in human medicine, such information in veterinary practice is still lacking. Thus, this study aimed to investigate in vitro osteogenic differentiation potential of canine bone marrow-derived mesenchymal stem cells under different doses of -glycerophosphate supplement. The isolated cells showed self-renewing ability and expressed pluripotent marker genes, zinc finger protein 42 and octamer-binding transcription factor 4, suggesting their potentiality in vitro. In vitro osteogenic differentiation was performed by using a regular osteogenic induction medium containing 10 mM β-glycerophosphate while 20 mM and 40 mM doses of β-glycerophosphate were used as treatment intervention. At day 14 post-induction, results showed that the levels of increased alkaline phosphatase activity and matrix mineralization upon induction were comparable among the three osteogenic groups. For osteogenic gene marker expression, the -glycerophosphate supplement showed an upregulating trend of gene set related to osteoblastic differentiation in a dose-dependent manner including osterix, collagen type I alpha 1, and osteocalcin. The 20 mM and 40 mM β-glycerophosphate supplements showed a suppressing trend of an osteochondrogenic marker, runt-related transcription factor 2, while an upregulating trend of gene encoding mineralization-inhibiting protein, osteopontin, was found in all doses of β-glycerophosphate supplement. In conclusion, β-glycerophosphate benefits in vitro osteogenic induction of cBM-MSCs. However, a controversial effect in mineralization was found. Further studies aiming at enhancing alkaline phosphatase activity will benefit osteogenic induction in terms of matrix mineralization.