| BackgroundTendinopathy is one of common desease of orthopedics surgery,sports medicine and professional injury. It was broadly believed that overuse be directly related with tendinopathy. But there was unclear and some controversial issue in its exact mechanism. There are two opposite theory of the tendinopathy mechanism including the inflammation mediators and degeneration theory.Some experts proposed that the inflammation mediators, which tenocytes produce after overload stretching, plays a important role in the formation of tendinopathy. But another experts objected this proposal.They believed there was not any evidence of inflammation and only some evidence of degeneration.we plan to prove whether tenocytes produce inflammation mediators after overload stretching.It is very difficulty to get the direct information of tendon after overuse in vivo because of the complex environment and the ethical problems.So we have to depend on the cell stretching machine in vitro to prove whether the tenocytes produce the imflammatin mediators after overload stretching. There are many pitfalls in the current cell stretching machine, such as :①the limitation of the stretching magnitude and frequency,the poor automation;②the stretching modes were too simple to copy this situation in vivo including the cell orientation and stressing types.ObjectiveThe objective of our study was to design a new cyclic stretching system of tenocytes in vitro according to the orientation and stressing type of tenocytes in vivo and evaluate its effects. Furthermore, we measure the concentration of prostaglandin E2 (PGE2), cyclooxygenase 2 (COX-2) and interleukin 1β(IL-1β),which human tendocytes produce after overloading stretching in order to prove whether the imflammation mediators plays a important role in the formation of tendinopathy. Finally, we hope to provide an ideal stretching model of tenocytes in vitro and important data for the tendinopathy mechanism.Method1. The microgrooved silicone dishes were made of two silicone components: polydimethylsiloxane and firming agent at a ratio of 10:1. The tencytes were cultured on the microgrooved dishes with coating ProNectin-F on its surface,and the smooth petri dishs as the contrast, to compare their growth and alignment, and to account their adherence rate on the both of dishes.2. The microgrooved silicone dishes were been uniaxial stretched through the cyclic stretching system.The system can be controlled on different stretching frequency, duration through Programmable Logic Controller (PLC) and stretching magnitude was been controlled by the eccentricity of the circular cams,which were rotated by a motor. The stability of machine and the biology of tenocytes were observed under different frequency, duration time and magnitude of stretching.3. The culture human tenocytes were stretched by the new cyclic stretching system of tenocytes in vitro under different stretching magnitude and frequency.At 0.5Hz of stretching frequency and 12 hours of stretching duration,we have measured the concentration of PGE2 and IL-1βat 0,4%,8% and 12% of stretching magnitude through enzyme linked immunosorbent assay(ELISA) method. At 8% of magnitude and 12 hours of stretching duration, we have detected the concentration of PGE2 and IL-1βthrough ELISA method at 0, 0.5Hz, 1.0Hz and 1.5Hz of stretching frequency. The COX-2 expression was detected under different stretching magnitudes.Results1. The parameter of the microgrooved silicone dishes: 10μm ridge and groove width, 3μm groove depth, 3cm×6cm surface area.The dishes have the good elasticity and didn't be torn under the overloading stretching.The culture tenocytes growed very well on the microgrooved silicone dishes and have been arranged as parallel, which were similar in vivo.However, the tenocytes orientation can't be controled on the smooth surface dishes. There is no evidence difference between on both different dishes in the adherence rate of tenocytes;2. The parameter of the new cyclic stretching system of tenocytes in vitro: variable stretching magnitudes:2%, 4%, 8% and 12%; variable stretching frequency: 0.1-2Hz; the degree of adjustment:0.1Hz;control time:0-24 hour,The degree of adjustment:1 minute. The cyclic stretching system worked well and stable under the different of stretching frequency, duration and magnitude;3. At 0.5Hz of stretching frequecy, 12h of duration,there were 499.97±73.71 pg,559.11±55.89 pg,1132.84±42.24 pg,1507.72±40.86 pg per 10,000 cell of PGE2 concentration, 6.35±0.64 pg/ml,7.52±0.66 pg/ml,8.88±1.21 pg/m,16.64±3.25 pg/ml of IL-1βconcentration at 0%,4%,8%,12% of stretching magnitudes. It proved that the PGE2 obvously increased at 8%,12% of stretching magnitudes (P<0.05), whereas IL-1βobvously increased at 12% of stretching magnitudes (P<0.05). At 8% of stretching magnitude, 12h of duration, there were 645.31±24.76pg,1038.162±42.41 pg,1649.06±265.30 pg,1756.70±250.68 pg per 10,000 cell of PGE2 concentration, 6.88±1.91 pg/ml,9.54±0.15pg/ml,13.80±3.63 pg/ml,15.88±3.5 pg/ml of IL-1βconcentration at 0,0.5Hz,1.0Hz,1.5Hz of stretching frequency. It proved that the PGE2 obvously increased at 0.5Hz,1.0Hz and 1.5Hz of stretching frequency (P<0.05), whereas IL-1βobvously increased at 1.5Hz of stretching frequency (P<0.05).The COX-2 expression was increased at 4%,8% and 12% of stretching magnitude(P<0.05).Conclusion1. The microgrooved silicone dishes with good flexibility are satifacted with the demand of different stretching magnitudes,frequency and duration, and can be sterilized repetitively. The culture tenocytes grow very well on the microgrooved silicone dishes and parallel, which are similar in vivo.2. The new system is a good cyclic stretching model of tenocytes in vitro with stable, good-controlled characteristics.3. The inflammation mediators, which the tenocytes can produce under overload stretching, obvously increase. It will be helpful to support the theory of inflammation mediators on tendinopathy.
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