Cryopreservation of tendon tissue using dimethyl sulfoxide combines conserved cell vitality with maintained biomechanical features

Abstract

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.

Fig 1. Freezing tendons in DMSO retains metabolic active cells in the tissue.

Dissected tendons were frozen either in PBS or in 20% DMSO containing PBS at -20°C and thawed at room temperature. The cells were incubated in αMEM for 3h containing 10% MTT reagent. Violet coloration shows metabolic active cells.

Fig 2. Freezing tendons in DMSO retain scleraxis positive cells, which can be put in cell culture.

After 7 days of cultivation, tendon-derived cells can be detected either in freshly cultivated as well as in in DMSO frozen tendons (A). Quantification of MTT -assay of tendon derived cells after 8 days of cultivation (B). Quantitative real-time PCR of the resident cells from freshly prepared, in PBS and DMSO-frozen tendons, testing the scleraxis expression(C).

Fig 3. Freezing tendons in DMSO retains cells in-situ, which were destroyed when freezing in PBS.

Transmission electron microscopy of Achilles tendon after freezing once using PBS or 20% DMSO in PBS as medium. Arrows mark matrix cavities housing tenocytes with either intact integrity or remaining debris.

Fig 4. Collagen morphology of Achilles tendon is disturbed after freezing in PBS but not in DMSO.

Transmission electron microscopy of Achilles tendon after freezing once using PBS or 20% DMSO in PBS as medium. Lines mark the orientation of bands caused by the D-period in relation to the fiber orientation in the detail images. Scale bar: 500 nm.

Fig 5. Dynamic biomechanical testing showed significant differences between tendons frozen in PBS four times and freshly tested tendons for the dynamic Young's modulus.

Biomechanical testing was performed using a custom-made axial testing set-up (LM1, ElectroForce/TA Instruments). Dynamic Young's modulus calculated from the amplitudes of the dynamic testing at a frequency of 1Hz for all strain rates (A) is shown for freshly tested tendons, tendons frozen once in PBS or 20% DMSO in PBS as well as tendons frozen four times in PBS or 20% DMSO in PBS. A linear increase at increasing strain levels (B) was observed. The static Young's modulus was calculated from the linear region of the ramp-to-failure-curve (C).