Group | Protocol | Decellularization procedures |
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| | 5 repetitions of freeze-thawing | Further treatment |
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| | Cooling | Freeze hold | Heating | Thaw hold | |
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1 | Auto-Protocol 1 | -50 °C per min | 3 min at -80 °C | +50 °C per min | 10 min at +20 °C | 48 h distilled water 48 h 1% Triton X-100 Washing steps |
2 | Auto-Protocol 2 | -20 °C per min | 3 min at -80 °C | +20 °C per min | 10 min at +20 °C |
3 | Manual-Protocol | Manual transfer | 2 min in liquid nitrogen | Manual transfer | 10 min in 37 °C PBS |
| Control | No treatment |
- Equine superficial digital flexor tendon samples of group 1 and group 2 were processed by automated freeze-thaw cycles, differing in the performed cooling and heating rates (Auto-Protocol 1 and Auto-Protocol 2). Both of the applied cooling and heating rates describe a temperature change per unit time. For Auto-Protocol 1 as well as for Auto-Protocol 2 the maximum reached temperature was + 20 °C (thaw hold for 10 min) and the minimum reached temperature was -80 °C (freeze hold for 3 min). All temperature regulations of the automated freeze-thaw cycles were carried out by a controlled rate freezer (PLANER® Kryo 360–1.7) that utilizes liquid nitrogen to adjust temperature. Group 3 included manual freeze-thaw cycles. Further steps of decellularization were the same for all sample groups. Tendon samples classified as internal control underwent no decellularization