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Optimization of differentiation time of mesenchymal-stem-cell to tenocyte under a cyclic stretching with a microgrooved culture membrane and selected measurement cells

Morita Y., Yamashita T., Toku Y., Yu Y.

Acta of Bioengineering and Biomechanics

2018

Vol. 20, no. 1

3--10

There is a need for efficient stem cell-to-tenocyte differentiation techniques for tendon tissue engineering. More than 1 week is required for tenogenic differentiation with chemical stimuli, including co-culturing. Research has begun to examine the utility of mechanical stimuli, which reduces the differentiation time to several days. However, the precise length of time required to differentiate human bone marrow-derived mesenchymal stem cells (hBMSCs) into tenocytes has not been clarified. Understanding the precise time required is important for future tissue engineering projects. Therefore, in this study, a method was developed to more precisely determine the length of time required to differentiate hBMSCs into tenocytes with cyclic stretching stimulus. Methods: First, it had to be determined how stretching stimulation affected the cells. Microgrooved culture membranes were used to suppress cell orientation behavior. Then, only cells oriented parallel to the microgrooves were selected and evaluated for protein synthesis levels for differentiation. Results: The results revealed that growing cells on the microgrooved membrane and selecting optimally-oriented cells for measurement improved the accuracy of the differentiation evaluation, and that hBMSCs differentiated into tenocytes in approximately 10 h. Conclusions: The differentiation time corresponded to the time required for cellular cytoskeleton reorganization and cellular morphology alterations. This suggests that cells, when subjected to mechanical stimulus, secrete mRNAs and proteins for both cytoskeleton reorganization and differentiation.

Determination of optimal cyclic uniaxial stretches for stem cell-to-tenocyte differentiation under a wide range of mechanical stretch conditions by evaluating gene expression and protein synthesis levels

Morita Y., Watanabe S., Ju Y., Xu B.

Acta of Bioengineering and Biomechanics

2013

Vol. 15, no. 3

71--79

We examined optimal cyclic uniaxial stretches for stem cell-to-tenocyte differentiation by applying a wide range of cyclic mechanical stimuli. Human bone marrow mesenchymal stem cells (hBMSCs) were subjected to three types of cyclic elongation of 5%, 10%, or 15% at a cyclic frequency of 1 Hz for 24 h or 48 h, and differentiation into tenocytes was assessed by two methods: real-time polymerase chain reaction determination of gene expression levels and western blotting analysis of protein expression levels. The gene expression levels of the differentiation markers type I collagen (Col I), type III collagen (Col III), tenascin-C (Tnc), and scleraxis (Scx), all of which are constituents of tendon tissue, were increased when cells were exposed to 10% stretching stimulation. The levels of Col I and Tnc protein synthesis levels were also higher in the cells with 10% stretching stimulation than in those subjected to other stimuli. The results indicated that 10% stretching stimulus was efficient to induce the differentiation of hBMSCs into tenocytes. In addition, the changes in gene and protein expression levels were strongly correlated with cell orientation angle. The results presented here suggest that mesenchymal stem cell-to-tenocyte differentiation is strongly associated with cumulative elongation load on the cells. This work provides novel insights into the differentiation of tenogenesis in a strain-induced environment and supports the therapeutic potential of hBMSCs.