Mesenchymal Stem Cells (MSCs) are important in regenerative medicine and tissue engineering and will be a very sensible choice for repair and regeneration of tendon. New biological practices, such as cellular therapy using stem cells, are promising for facilitating or expediting tendon therapy. Before using these cells clinically, it is best to check and confirm the optimal conditions for differentiation of these cells in the laboratory. Hence, in the present study, the impacts of PDGF-BB and GDF-6 supplementation on adipose-derived MSCs (ASCs) culture were studied. The frozen ASC were recovered and expanded in basic culture medium (DMEM with 10%FBS). The cells after passage five (P5) were treated with basic medium containing L-Prolin, Ascorbic Acid (AA) and only PDGF-BB or GDF-6 (20 ng/ml) or both of them (mix) as 3 groups for 14 days to investigate efficiency of ASCs differentiation towards tenocytes. The cells culturing in basic medium were used as control group. To validate tenogenic differentiation, H&E and Sirius Red staining were used to assess cell morphology and collagen production, respectively. In addition, mRNA levels of collagen I and III, Scleraxis and Tenomodulin as tenogenic markers were analyzed using qPCR. In all test groups, cells appeared slenderer, elongated cytoplasmic attributes compared to the control cells. The intensity of Sirius Red staining was significantly higher in GDF-6, PDGF-BB alone, than in group without supplements. The optical density (OD) was higher in the GDF-6 than PDGF-BB and mix-group. QPCR results showed that Col I and III gene expression was increased in all groups compared to the control. SCX expression was significantly increased only in the PDGF-BB group. TNMD mRNA expression was not significant among groups. In this study, we have corroborated that human ASCs are reactionary to tenogenic induction by GDF-6 and PDGF-BB alone or in combination. These outcomes will help greater insight into GDF-6 and PDGF-BB driven tenogenesis of ASCs and new directions of discovery in the design of ASC-based treatments for tendon healing.

Keywords