Date Published: April 19, 2019
Publisher: Public Library of Science
Author(s): Eva Hochstrat, Marcus Müller, Andre Frank, Philipp Michel, Uwe Hansen, Michael J. Raschke, Daniel Kronenberg, Richard Stange, Chunfeng Zhao.
Biomechanical research on tendon tissue evaluating new treatment strategies to frequently occurring clinical problems regarding tendon degeneration or trauma is of expanding scientific interest. In this context, storing tendon tissue deep-frozen is common practice to collect tissue and analyze it under equal conditions. The commonly used freezing medium, phosphate buffered saline, is known to damage cells and extracellular matrix in frozen state. Dimethyl sulfoxide, however, which is used for deep-frozen storage of cells in cell culture preserves cell vitality and reduces damage to the extracellular matrix during freezing. In our study, Achilles tendons of 26 male C57/Bl6 mice were randomized in five groups. Tendons were deep frozen in dimethyl sulfoxide or saline undergoing one or four freeze-thaw-cycles and compared to an unfrozen control group analyzing biomechanical properties, cell viability and collagenous structure. In electron microscopy, collagen fibrils of tendons frozen in saline appeared more irregular in shape, while dimethyl sulfoxide preserved the collagenous structure during freezing. In addition, treatment with dimethyl sulfoxide preserved cell viability visualized with an MTT-Assay, while tendons frozen in saline showed no remaining metabolic activity, indicating total destruction of cells during freezing. The biomechanical results revealed no differences between tendons frozen once in saline or dimethyl sulfoxide. However, tendons frozen four times in saline showed a significantly higher Young’s modulus over all strain rates compared to unfrozen tendons. In conclusion, dimethyl sulfoxide preserves the vitality of tendon resident cells and protects the collagenous superstructure during the freezing process resulting in maintained biomechanical properties of the tendon.
Biomechanical and biomolecular research on tendon tissue is of increasing scientific interest and relevance. As part of the musculoskeletal system, tendon tissue is able to transmit energy produced by muscles to the bones due to its typical composition of fibrillar collagens and other proteins, and hence, enables joint and limb movement. It also buffers impact load to protect the muscle from potential damage by storing energy and subsequently releasing it gradually. Tendon tissue can serve these functions due to its typical structure including strict hierarchical collagenous composition as well as low cellularity and vascularization resulting in low metabolic activity. The latter often leads to a protracted healing process and in some cases incomplete recovery following ruptures, cutting damages or tendinopathies and thus a decline of biomechanical properties resulting in re-ruptures, chronic pain and restricted mobility[5,6]. Research focusing on tendon tissue therefore investigates new treatment strategies such as improved suture techniques, tendon engineering and the use of auto-, allo- and xenografts. Improving and evaluating these treatment strategies is commonly executed with animal models, providing the possibility to include gene alterations or pharmacological treatments and test them under standardized conditions. Analysis of animal tendons often includes biomechanical testing, to evaluate functional outcome of the treatment. Therefore, it is common practice to store tendon tissue, mostly deep-frozen, until use to ensure that all harvested tendons are analyzed under equal conditions.
In this study, we compared different protocols for cryoconservation of murine tendons for further evaluation. We demonstrated that freezing of tendon tissue in PBS already causes an impairment of the tendons resident collagen fibrils on a molecular level as well as a nearly complete void of cells. The use of DMSO, on the contrary, led to maintained vitality and collagenous structure within the tendon tissue.
Cryopreservation with DMSO allows the biomechanical analysis of a matrix-cell-compound, since especially in areas rich of cells, such as the enthesis, cell-cell- and cell-matrix interactions could possibly influence the biomechanics, especially when gene alterations or tendon healing models are included in the biomechanical testing. A cryoprotective effect of DMSO on the tendons tissue, which is reflected in the preserved cell viability and the retained intact collagen structure shown by electron microscopy, could be shown. The use of DMSO as a medium during freezing could provide new insight into the impact of stress on cells and ECM in the tendon tissue after regeneration or tendon tissue effected by gene alterations. In summary, our results support the use of DMSO as a cryoproctectant medium during the deep-frozen storage as an alternative for the commonly used PBS, as it provides the possibility of a more physiological biomechanical testing of tendon tissue.