Int J Stem Cells.  2021 Feb;14(1):33-46. 10.15283/ijsc20023.

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

Affiliations
  • 1Department of Immunology, Transplantology and Internal Diseases, Medical University of Warsaw, Warsaw, Poland
  • 2Department of Pharmacodynamics and Pathophysiology, Centre for Preclinical Research and Technology, Medical University of Warsaw, Warsaw, Poland
  • 3Department of Physical Chemistry, Faculty of Pharmacy with the Laboratory Medicine Division, Medical University of Warsaw, Warsaw, Poland
  • 4Department of Regenerative Medicine, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland
  • 5Department of Orthopedics and Traumatology, Medical University of Warsaw, Warsaw, Poland
  • 6Department of Bioinformatics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

Abstract

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, 5th 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.

Keyword

L-ascorbic acid; Serum reduction; Mesenchymal stromal cells; Tenogenic differentiation
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