Effects of hypoxia on differentiation of menstrual blood stromal stem cells towards tenogenic cells in a co-culture system with Achilles tendon cells.

Achilles tendons have a very poor capacity for intrinsic regeneration. The cell-based treatment strategy for Achilles tendinitis includes the application of mesenchymal stem cells (MSCs), which have high proliferative and multipotent differentiation ability, and is a promising approach. The aim of the present study was to explore the tenogenic potential of human menstrual blood stromal stem cells (MenSCs) in a co-culture system and to compare the tenogenic capability under normoxic and hypoxic conditions. MenSCs were co-cultured indirectly with Achilles tendon cells in a Transwell co-culture system for 1, 2, or 3 weeks in two different concentrations of oxygen (20 and 2% O2), whereas the control contained only MenSCs. The extracellular matrix of MenSCs in each system was evaluated by Alcian blue staining assay, histological staining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blot analysis. Alcian blue staining assay revealed a significant increase (P<0.05) in proteoglycan secretion by the differentiated MenSCs. Identical results were obtained by RT-qPCR for collagen I, which was validated by western blot analysis. Considerably increased collagen I and collagen III gene expression levels were exhibited by cells in the co-culture treatment group when compared with the control (P<0.05); however, no significant difference was detected between the normoxic (20% O2) and hypoxic treatment (2% O2) groups. RT-qPCR was utilized to determine the expression levels of thrombospondin 4, scleraxis and tenascin C in the differentiated MenSCs; a significant increase in the expression of these specific genes was indicated in the co-culture treatment group compared with the control (P<0.05). Although the expression levels were markedly higher in hypoxia than in normoxia conditions, this difference was not significant. To conclude, the present study indicated that MenSCs manifested a strong proliferative and multipotent capacity for differentiation and differentiated into Achilles tenogenic cells. Therefore, the use of MenSCs may be considered in Achilles tendinitis therapy.