Abstract
Tendon calcification is common in the Achilles tendon, and injuries affect not only athletes, but also the general population. However, the underlying cellular mechanisms are not yet fully understood. In this study, we isolated healthy human tenocytes and subjected them to uniaxial mechanical stretching (at 1.0 Hz) for various stretch times (4 h, 8 h, 12 h) or magnitudes (0 %, 4 %, 8 %, 12 %). The extracellular calcium chelator EGTA, calcium channel inhibitor MnCl2, nifedipine, or various doses of exogenous calcium were administered to these cells with or without mechanical overloading. The intracellular calcium concentration was determined by using a Fluo-3/AM fluorescence probe, and the cytoskeleton was revealed by F-actin Phalloidin staining. The intracellular calcium concentration increased in a magnitude- and time-dependent manner following stretching. These increases were suppressed by EGTA, MnCl2, or nifedipine. Additionally, cytoskeleton F-actin was disrupted significantly by stretching in a time-dependent manner. When extracellular calcium was applied, the intracellular calcium concentration increased, and F-actin was disrupted dramatically under mechanical stretching compared with non-stretched cells. Thus, repetitive mechanical overloading induces the accumulation of abnormally high concentrations of intracellular calcium resulting from extracellular calcium influx mediated, at least in part, by membrane calcium channels and finally causes cytoskeleton disorganization and tenocyte dysfunction. These findings provide novel experimental evidence for the pathology of tendon calcification and indicate that the blockade of calcium influx is a potential target for the prevention and treatment of calcific tendinopathy.
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Acknowledgments
The microgroove silicon membrane was kindly provided by Dr. James H.-C. Wang (University of Pittsburgh, USA) who also strictly supervised the custom-made controlling apparatus. We are grateful to Dr. Kaifa Wang (Third Military Medical University, Chongqing, China) for kind assistance with the statistical analysis and to Yang Xiang (Central Laboratory of Southwest Hospital, Chongqing, China) for the collection and measurement of the morphological data from confocal microscopy.
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Wan Chen and Yinshuan Deng contributed equally to this study.
This work was supported by the Natural Science Foundation of China (81230040, 30872620, and 81071464) and Chongqing Science and Technology Committee (CSTC2011BA5010).
The authors declare no conflicts of interest.
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Electronic Supplementary Material Figure S1
The uniaxial cyclic mechanical stretching device with the microgroove silicon membrane system. a The cyclic mechanical stretch device. b This device is set at 4%, 8%, and 12% magnitude (two dishes each) at 1.0 Hz or 0.5 Hz. c The tension unit. d The device in an incubator. (GIF 178 kb)
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Chen, W., Deng, Y., Zhang, J. et al. Uniaxial repetitive mechanical overloading induces influx of extracellular calcium and cytoskeleton disruption in human tenocytes. Cell Tissue Res 359, 577–587 (2015). https://doi.org/10.1007/s00441-014-2018-2
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DOI: https://doi.org/10.1007/s00441-014-2018-2