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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

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2018

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Vol. 20, no. 1

3--10

EN

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.

2

A new device for dynamic ventilation-analogue mechanostimulation of pliant tissue layers

Gamerdinger K., Schneider M., Smudde E., Guttmann J., Schuman S.

Acta of Bioengineering and Biomechanics

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2012

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Vol. 14, no. 4

53-61

EN

The experimental mechanostimulation of biological cell and tissue test samples has become a standard method in biomechanics research. In order to apply a static or a dynamic mechanical load on biological tissue a variety of different devices for the mechanostimulation have been developed. While cyclic load applications are typically restricted to sinusoidal or rectangular stimulation patterns, a device for more complex dynamic stimulation patterns which would simulate, for instance, the dynamics during mechanical ventilation does not exist. The dynamic alveolar recruitment/derecruitment has been identified as one of the main causes of ventilator-induced lung injury. Therefore, there is a demand for an experimental ventilation-analogue mechanostimulation of the pulmonary cells and tissue. Here, we present our mechanostimulator combined with a new driving system which is able to produce the ventilation-analogue patterns of a dynamic mechanostimulation. In an experimental setting where the test samples were simulated by silicone-membranes in single-, double- and fourfold membrane configuration, we varied the stimulation amplitude from 5% to 60% surface increase and stimulation frequencies ranging from 15/min to 2000/min. Furthermore, the frequency components of mechanical load applied to the sample at sinusoidal, rectangular and ventilation- analogue mechanostimulations were analyzed by means of a Fast Fourier Transform (FFT). The system allows for a homogeneous mechanostimulation with various temporal profiles which may include frequency components of up to 20 Hz. The relative amount of mechanical load applied to the sample at the main stimulation frequency was 76% during sinusoidal stimulation, 35% during the rectangular stimulation, and 29% to 42% during ventilation analogue stimulation.

3

Distribution of radiological density in bone regenerate in relation to cyclic displacements of bone fragments

Filipiak J., Krawczyk A., Morasiewicz L.

Acta of Bioengineering and Biomechanics

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2009

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Vol. 11, nr 3

3-9

EN

We asked how bone fragment displacement could influence the distribution of radiological density in bone regenerate formed during the process of bone lengthening. The metatarsi of 21 sheep were lengthened by 20 mm by the Ilizarov method. The bone fragments were externally fixed with a specially designed ring external fixator equipped with linear actuator driver system. The test sheep were divided into three experimental groups: the G1 and G2 groups (N = 8) and the GR group (N = 5) - the reference group. In the case of sheep from the G1 and G2 groups, the lengthening was supplemented with mechanical stimulation of the regenerate in the form of cyclic bone fragment displacements (CBFDs) with the amplitudes of 1 mm (G1) and 2 mm (G1). Mechanical stimulation was applied over 30 days for 1 h per day with a frequency of 1 Hz. Eight weeks after the procedure the sheep were sacrificed in accordance with the required procedures. The analysis of the degree of bone regenerate mineralization involved the studies based on the CT scanning. The analysis of the results obtained is based on the paramenter called the degree of regenerate mineralization (RMD). The analysis of radiological density was carried out in the selected measurement areas. Such an area was located in three horizontal zones, taking into account the regenerate height, i.e. in its middle part (half regenerate length); the top part, 2 mm from the edge of the proximal fragment; and the bottom part, 2 mm from the edge of the distal fragment. The value of the RMD parameter varies significantly, depending on the bone regenerate area. The results obtained show that the CBFD = 2 mm accelerates the rate of mineralization of an eight-week-old regenerate. In the case of CBFD = 1 mm, the mineralization rate is lower by more than a dozen per cent.