The Effect of L-Ascorbic Acid and Serum Reduction on Tenogenic Differentiation of Human Mesenchymal Stromal Cells

BACKGROUND AND OBJECTIVES: Despite significant improvement in the treatment of tendon injuries, the full tissue recovery is often not possible because of its limited ability to auto-repair. The transplantation of mesenchymal stromal cells (MSCs) is considered as a novel approach in the treatment of tendinopathies. The question about the optimal culture conditions remains open. In this study we aimed to investigate if serum reduction, L-ascorbic acid supplementation or a combination of both factors can induce tenogenic differentiation of human adipose-derived MSCs (ASCs). METHODS AND RESULTS: Human ASCs from 3 healthy donors were used in the study. The tested conditions were: 0.5 mM of ascorbic acid 2-phosphate (AA-2P), reduced serum content (2% FBS) or combination of these two factors. The combination of AA-2P and 2% FBS was the only experimental condition that caused a significant increase of the expression of all analyzed genes related to tenogenesis (SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3, DECORIN) in comparison to the untreated control (evaluated by RT-PCR, 5(th) day of experiment). Moreover, this treatment significantly increased the synthesis of SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3 proteins at the same time point (evaluated by Western blot method). Double immunocytochemical staining revealed that AA-2P significantly increased the extracellular deposition of both types of collagens. Semi-quantitative Electron Spin Resonance analysis of ascorbyl free radical revealed that AA-2P do not induce harmful transition metals-driven redox reactions in cell culture media. CONCLUSIONS: Obtained results justify the use of reduced content of serum with the addition of 0.5 mM of AA-2P in tenogenic inducing media.