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Preservation of Tissues by Freezing in Skin Grafts


New research is constantly being carried out to improve the leather gret. According to researches, green tea polyphenol affects the protection of rat skin and increases the success rate of skin grafts. Although skin allografts are used in the treatment of severe burn injuries, skin disorders and skin defects, their use is limited due to the limited amount of normal skin that can be removed for such grafts. The best graft for functional closure of wounds is autograft. At the end of an autograft procedure, remaining donor skin is routinely stored in a saline solution. And it is applied to the open wound when graft loss or superficial wound disruption occurs postoperatively. However, prolonged storage in saline leads to poor vaccination.
In addition, researchers were asked to examine other protection environments that will extend the storage period of the vessels. Recently, the polyphenols in green tea have been found to promote the preservation of tissues such as blood vessels, cornea, nerves, islet cells, articular cartilage, and myocardium at room temperature. These findings raise the possibility of a new method for tissue banking without freezing.
Preservation of Tissues by Freezing in Skin GraftsAs mentioned earlier, dysfunction of the transplants occurs as a result of free radicals due to ischemia that trigger lipid peroxidation of the cell membrane when blood flow is restarted. It is reported that EGCG (epigallocatechin gallate) inhibits peroxylipid formation. A study using rat skin was conducted to determine whether the addition of EGCG to conventional cell culture media could increase the viability of stored skin grafts and also prolong the storage time. The storage solution chosen for this study included Dulbecco’s Modified Eagle’s Medium, 10% Fetal Calf Serum, supplement and 1% antibiotic solution. EGCG was dissolved in the storage solution at a final concentration of 1 mg / ml.
For anesthesia, pentobarbital (50 mg / Kg) was administered intraperitoneally to GFP transgenic rats. After the rats’ backs were shaved and shed, their dorsal skin was biopsied and the muscle layers were stripped from the skin biopsies. Skin samples, measuring 1 × 1 cm, were stored at 4 and 37 horses in sterile containers with 50 ml preservation solution for up to 8 weeks. Periodically, part of the preserved skin (30 minutes, 1, 2, 4, 6, 7, 8 weeks) was used for grafts in nude mice or was directly examined histologically. From histological examinations of 4 ‘protective skin, a decrease in GFP value in non-EGCG skin was noted during the second week, such as a slight degeneration of the epidermal layer. Degeneration in epidermal and dermal layers 4 & # 8451; It was recorded in all groups starting from the 5th week.
It occurs 4 weeks after inoculation of protected skins into nude mice and thirty minutes of protected skin with EGCG retains hair. On the other hand, skin that does not contain EGCG (b) does not contain hair and the graft size is smaller than the EGCG groups. After 2 weeks of preservation with EGCG at 4 ° C, the graft color was dark as necrosis, but remained only as EGCG. After 2 weeks of preservation, graft size was smaller or only scarring was observed in all groups except for 4 protection with EGCG. Rat skin inoculated into nude mice after 30 minutes of preservation with EGCG showed a better condition than implanted without EGCG, which is hair loss and shrinkage. In groups protected with EGCG, skin color was dark and superficially necrotic, but the color was found to be due to the presence of EGCG. At 4, there is good graft survival after 2 to 7 weeks of protection in EGCG. In other groups, the grafts were reduced or only scarred due to contracture or rejection.
Histological analysis found that grafts preserved with EGCG at 4 were considered complete with both epidermal and dermal layers remaining. On the other hand, GFP positive keratinocytes or fibroblasts were not observed in grafts preserved at 4 without EGCG, and only phagocytes in the dermal layer were not observed. No GFP positive rat cells with or without EGCG were observed in conserved groups. No graft from 37 preserved groups (with or without EGCG) was successful. However, 4 ‘preserved grafts were accepted and the success rate for EGCG treated grafts was 100% even after 4 weeks of preservation.Preservation of Tissues by Freezing in Skin Grafts
Large skin defects caused by burn trauma should be covered with skin grafts, but sometimes skin grafts fail for various reasons such as hematoma. When skin grafts partially fail, small skin defects can be repaired using skin left over from primary inoculation if preserved. What’s more, it causes complex tears or minor flap-like injuries. In such cases, if the skin can be temporarily protected, it can then be used as a part of the graft after the wound condition has healed. Freeze-dried pigskin, xenografts, and frozen allografts are also used for wound coverage, but are generally not included permanently. Therefore, it is very beneficial to develop better technologies for the protection of autografts.
There are several reports on the preservation of the cornea, blood vessels, nerves, pancreatic islet cells, saphenous veins, and the peripheral nervous system EGCG. In the first reported experiment, histological examination revealed that EGCG improves the length of time of protection of a skin sample. Over time, skin samples begin to degenerate from the epidermal layer to the dermal layer. Skin samples preserved at 4 appear to be less degenerated than those in 37 37. However, it is difficult to judge cell viability based solely on histological examinations because GFP is still present in all skin samples. For this reason, the viability of the skin samples was investigated by examining whether the samples could lead to successful grafts. Approximately two weeks after implantation, rejection of dead tissue or skin has been reported.
Immunodeficient mice were used as recipient animals and the success of skin grafts from GFP-Tg rats was evaluated. The results show that it is not possible to protect the skin with 37 even when EGCG is added. Similarly, AE CRAM et al. They reported that only 1/3 of their grafts were successful in ten days after transplantation of skin samples preserved at 4 for two weeks. In studies, 4 G was successful 4 weeks after transplantation of skins protected with 4 weeks EGCG, and one of 3 EGs was successful even after 7 weeks of storage with 4 wt. Therefore, it was concluded that EGCG is beneficial in protecting skin samples by reducing the degeneration of epidermal layer and suppressing postoperative graft contraction.
There may be several reasons why EGCG improves the protection of the skin, and EGCG has potent antioxidative activities. The strong anti-oxidative activity of EGCG can inhibit lipid peroxidation of the cell membrane of the protected skin. M. Kapoor et al. It has been reported that EGCG has anti-inflammatory and free radical scavenging effects in vitro and also controls cell division. A combination of these effects is believed to improve the condition of protected skin. In addition, such as strengthening the scaffolding structure
EGCG’s actions and antibacterial activity also appear to contribute to its protective effects. EGCG also improved the quality of scars after full thickness incisions in rats. Possibly because EGCG increases the expression of iNOS (inducible nitric oxide synthase), COX-2 (cyclooxygenase-2) and VEGF (vascular endothelial growth factor) new blood vessels are formed. EGCG has also been shown to reduce arginase-I activity and protein levels. Although the underlying mechanism of how EGCG exerts its beneficial effects for skin protection is still unclear from the results, the possibility of future clinical use of EGCG for skin protection from freezing is suggested.

Potential Mechanisms for the Protection of Mammal Tissues and Organs from Freezing with Polyphenols

All of the work done was initiated on the basis of the observation that polyphenols appear to be useful for the physiological preservation of tissues or organs, particularly rat pancreatic islet cells. Since then, evidence has accumulated showing the beneficial effects of polyphenols on cell protection. Extending this observation to the preservation of tissues and even organs for transplantation regulates the concentration of polyphenols contained in storage solutions, enabling them to be stored for longer periods of time. Recently, it has been reported that polyphenols can reduce ischemia / reperfusion injury in mouse lungs and protect a variety of tissues or organs, including canine lungs, rat aorta, rat peripheral nerves and mammalian pancreatic islet cells.
Preservation of Tissues by Freezing in Skin GraftsThis non-frozen protection of mammalian cells, tissues or organs can be mediated by the reversible regulation of cell proliferation and survival through hibernation of cell proliferation and survival. Regarding this hypothesis, it has been reported that the hibernation phenomenon triggered by polyphenols must be attached to the cell membrane or tissue matrix due to their amphipathic properties. And it has already been reported that it may be related to its intrinsic properties, including its penetration. Absorption of polyphenolic compounds into proteins is produced early, but the desorption rate is very slow. Consequently, mammalian cells, tissues or organs can be physiologically preserved through adsorption of compounds to membranous proteins and extracellular matrices.
Polyphenol was able to control the proliferation of various cell types and has been shown to be very useful for long-term storage of various tissues, including islet cells, blood vessels, cartilage, corneas, nerves and skin. Today, approximately 850,000 tissue allografts are transplanted to patients annually in the USA. Most of this tissue is stored frozen. The method of cryopreservation at -196 ° was adopted in April 1999 at Tokyo University Hospital and Osaka National Cardiovascular Center. Cryopreservation methods are used for long-term preservation of blood vessels, cartilage and skin. However, current research is still aimed at improving the effectiveness of cryopreservation and preservation fluids such as the University of Wisconsin solution for tissue transplantation.
It is reported here that preservation of various tissues from freezing for up to three months is now possible with excellent preservation of the histological and biomechanical properties of the tissue. In addition, the rat sciatic nerve, guinea pig periodontal ligament, and rat myocardium were preserved in an unfrozen state for long-term storage prior to preservation. This was made possible by the development of a new protection fluid containing polyphenols as additive antioxidant. Therefore, cryopreservation of many tissues may eventually be replaced by their storage in the newly developed polyphenol tissue deposition fluid. This cell and tissue preservation method is of great benefit not only in Japan but all over the world.

References:
https://jeb.biologists.org/content/jexbio/29/3/454.full.pdf
https://www.sciencedirect.com/science/article/pii/S0011224018

Author: Ozlem Guvenc Agaoglu


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