Optimization of Tenocyte Lineage-related Factors from Tonsil-derived Mesenchymal Stem Cells using Response Surface Methodology

Researchers should consider various potential factors that affect tenogenic differentiation of mesenchymal stem cells (MSCs); however, this requires numerous experimental settings, which are associated with high cost and time. We aimed to assess the differential effects of transforming growth factor beta 3 (TGF-{beta}3) on the tenogenesis of tonsil-derived MSCs (T-MSCs) and bone marrow-derived MSCs (BM-MSCs) using design of experiments (DoE). Bone marrow and tonsillar tissue was collected from four patients; mononuclear cells were separated and treated with 5 and 10 ng/mL of TGF-{beta}3 with vehicle control. A full-factorial experimental design with a categorical factor of 0 was employed to study the effect of tension based on T-MSCs. Eighty-four trials were utilized, fitted with RSM, and then used to obtain mathematical prediction models. Exposure of T-MSCs and BM-MSCs to TGF-{beta}3 increased the expression of scleraxis (SCX), tenomodulin (TNMD), decorin, collagen I, and tenascin C. Expression of most of these factors reached a maxima after 2-3 days of treatment. Considering all of the tenocyte lineage-related factors that were assessed, the predicted value of the factors from T-MSCs was significantly induced at 2.7 ng/mL of TGF-{beta}3 during 2.5-day culture, whereas the predicted value of the factors from BM-MSCs was significantly induced during 2.3-day culture, regardless of TGF-{beta}3 concentration. This study demonstrated that tenogenic differentiation of T-MSCs and BM-MSCs under TGF-{beta}3 stimulation showed a similar culture time for peak expression of tenocyte-related mRNAs using RSM. This study suggests the potential of using the DoE approach for optimization of the culture protocol for tenogenesis of MSCs.