Long-Term Preservation of Rat Skin Tissue by ... - SAGE Journals

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Cell Transplantation, Vol. 18, pp. 513–519, 2009 Printed in the USA. All rights reserved. Copyright  2009 Cognizant Comm. Corp.

Long-Term Preservation of Rat Skin Tissue by Epigallocatechin-3-O-Gallate HakHee Kim,* Takeshi Kawazoe,† Kazuaki Matsumura,* Shigehiko Suzuki,‡ and Suong-Hyu Hyon* *Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan †Department of Plastic and Reconstructive Surgery, Kijunkai, Yoshikawa Hospital, Kyoto 606-8392, Japan ‡Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

Skin grafts can be preserved by cryopreservation and refrigerated storage at 4°C. Epigallocatechin-3-Ogallate (EGCG) enhances the viability of stored skin grafts and also extends the storage time up to 7 weeks at 4°C. EGCG, the major polyphenolic constituent present in green tea, has potent antioxidant, antimicrobial, antiproliferative, and free radical scavenging effects. This study examined the effects of EGCG on skin cryopreservation. Skin sample biopsy specimens from GFP rats were previously treated with/without EGCG then moved to −196°C. Skin samples were transplanted to nude mice after 2, 8, and 24 weeks of preservation. Glucose consumption was measured after thawing to assess the metabolic activity. Two weeks later the transplanted skin grafts were excised and histologically analyzed. Histological examinations revealed the degeneration of the epidermal and dermal layers in all groups. In the EGCG groups, the grafts showed higher integrity in the epidermal layer and dermal matrix. The present findings suggest the future clinical usefulness of EGCG for skin preservation; however, the mechanism by which EGCG promotes skin preservation still remains unclear. Key words: Polyphenol; Epigallocatechin-3-O-gallate (EGCG); Skin; Skin grafts; Cryopreservation

INTRODUCTION

cells, articular cartilage, and myocardium, at room temperature (8,10,12,20). Epigallocatechin-3-O-gallate (EGCG), the predominant catechin from green tea, possesses much stronger antioxidant activities than vitamin C by virtue of its peculiar stereochemical structure and plays an important role in preventing cancer and cardiovascular diseases (12). Moreover, it shows beneficial effects, such as anti-inflammatory, antimicrobial, and immunomodulatory activities (9,11). EGCG enhances the viability of stored skin grafts and also extends the storage time up to 7 week at 4°C. The addition of EGCG to conventional freezing medium, “Cell Banker” (CB), could enhance the viability of skin grafts stored at −196°C and also extend the storage time. Metabolic assay has been used as a surrogate measure of overall viability in the grafts. Skin tissue was transplanted from GFP transgenic rat to immunodeficient mice and cell migration in graft tolerance of GFPpositive cells was investigated after transplantation (6).

Autologous and allogenic skin graft preservation and storage may benefit burned patients with limited donor site availabi1ity. Adequate skin storage may reduce the number of harvesting procedures required to achieve skin cover to a single operation and the stored skin can be applied when required as a bedside dressing. Skin grafts can be preserved by cryopreservation and refrigerated storage at 4°C (1,5). Some physicians prefer refrigerator-stored allografts, because of ostensibly higher viability, while others choose cryopreserved allografts for their relative ease of storage and their potentially greater safety (3). In order to provide the best clinical outcome, skin grafts must be processed and stored in a manner that maintains their viability and structural integrity until needed for transplantation (15,16). Usually, dysfunction of transplants occurs as the results of free radicals due to ischemia, which triggers lipid peroxidation of the cell membrane when blood flow is restarted. A good storage solution should prevent this (2,10). The polyphenols in green tea promote the preservation of tissues, such as blood vessels, cornea, nerve, islet

MATERIALS AND METHODS Animals Transgenic Sprague-Dawley rats (8 weeks old, male) expressing the green fluorescent protein (GFP), green

Received October 31, 2008; final acceptance March 30, 2009. Address correspondence to Suong-Hyu Hyon, Ph.D., Associate Professor, Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel: +81 75 751 4125; Fax: +81 75 751 4141; E-mail: [email protected]

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rats, produced with the constructs used for green mouse (6), were a kind gift from Dr. Masaru Okabe (Genomic Information Research Center, Osaka, Japan). Genetically immunodeficient mice (BALB/C-nu/nu, 8 weeks old, male) were purchased from Slc Japan Inc. (Osaka, Japan). All animals had free access to water and standard rodent chow. Animal experiments were performed according to the criteria of the Guidelines of the Committee on Animal Care and Use of Kyoto University.

Skin Grafting to Nude Mouse A full-thickness excisional square wound (1 × 1 cm) was created on the dorsum of each nude mouse and the GFP rat skin specimens were sutured to the adjacent normal skin with 6-0 ProleneTM thread. After surgery, the mice were housed in separate cages. Skin grafts were excised with 2–3 mm of the surrounding tissue, bisected, and processed for histology after 14 days (n = 4–6).

Preparation of Storage Solution With EGCG The purified EGCG was purchased from DSM Nutritional Products Ltd. and its purity exceeded 95%. The storage solution chosen for this study was Cell Banker (Juji Field Inc., Osaka, Japan) supplemented with 10% fetal calf serum and DMSO. EGCG solution (10 mg/ml) was prepared in PBS and diluted with the storage solution to a final concentration of 1 mg/ml.

Histological Analysis Half of transplanted GFP rat skin samples were fixed in 10% formalin, routinely embedded in paraffin, and stained with hematoxylin and eosin. The other half was placed in the Tissue-Tek Optimum Cutting Temperature (O.C.T.) compound (Sakura Ltd. Tokyo, Japan) for cryosectioning and further GFP fluorescent confirmation. The prepared sections were all observed under an optical/fluorescence microscope (Biozero-8000, Keyence, Osaka, Japan).

Preservation of Rat Skin For anesthesia, pentobarbital (50 mg/kg) was administered intraperitoneally. After shaving and depilating the back of the rats, the back skin was elevated. After procurement, the muscular layer was immediately stripped from the skin biopsies. Skin samples of GFP rats measuring 1 × 1 cm were held in sterile containers and previously refrigerated with PBS solution with or without EGCG for 1 night. Skin samples moved into CB solution with or without EGCG were stored in liquid nitrogen (−196°C) for up to about 24 weeks. Periodically, preserved skin grafts of GFT rat were transplanted into a nude mouse (2, 8, and 24 weeks). Circular skin biopsies (8 mm in diameter) were also made using a sterile dermal biopsy punch (Kai Industries Co., Ltd. Gifu, Japan) and preserved in the same way for in vitro analysis. In Vitro Analysis Prior to preserving the skin tissue, the optimum preserving concentration was determined by measuring the metabolic activity. Skin biopsies were incubated in conventional cell culture medium (DMEM) with various concentration of EGCG at −196°C for 2 weeks and thawed. The glucose consumption was measured after 6 days of incubation under 37°C tissue culture conditions (n = 5 × 2). After 2 and 8 weeks of cryopreservation, the samples were thawed and the freezing medium (CB) with DMSO with or without EGCG was thoroughly washed out immediately. Skin samples were moved to cell culture dishes with cell culture medium. The amount of glucose consumption from the culture medium was measured using a glucose test kit (Glucose C2, Wako Pure Chemical Industries, Ltd. Osaka, Japan) after 2, 4, and 6 days of incubation under favorable tissue culture conditions.

Statistical Analysis All variables were tested in two or three independent experiments, and each experiment was repeated twice (n = 4–6). Quantitative data were expressed as mean ± SE. Statistical comparisons were carried out with an analysis of variance (ANOVA, SAS Institute Inc., Cary, NC), which was followed by the Fisher’s PLSD test using StatView-J 4.5 software package (Abacus Concepts, Inc., Berkeley, CA). A value of p < 0.05 was considered statistically significant. RESULTS The in vitro results showed that 1 mg/ml EGCG had the highest protective effect on skin samples when it preserved at −196°C for 2 weeks. Over 1.5 mg/ml EGCG delayed the recovery of the metabolic activity of skin samples after thawing (Fig. 1). Figure 2 shows that glucose consumption was gradually increased with time and the skin samples preserved with EGCG showed higher glucose consumption in all groups. The in vitro data showed decreased glucose consumption in the 8week-preserved group in comparison to 2-week-preserved group. Comparing Figures 1 and 2 indicates that DMSO and EGCG in the freezing medium were more effective for maintaining glucose consumption. Glucose consumption by the 2-week-preserved skin samples with only EGCG after 6 days was about 220 mg/dl while the 4-weekpreserved skin sample with EGCG and DMSO was almost 300 mg/dl. After day 7 the possibility of bacterial contamination increased and false-positive data were obtained. All skin samples were washed with PBS containing antibiotics every 2 days. GFP fluorescence appeared to be similar between the

EGCG ENHANCES VIABILITY OF CRYOPRESERVED RAT SKIN TISSUE

Figure 1. The optimum concentration of EGCG for rat skin preservation was determined by measuring the metabolic activity. Skin biopsy samples were preserved in cell culture medium with various concentrations of EGCG and preserved at −196°C for 2 weeks. Glucose consumption was measured after 6 days of 37°C incubation after thawing.

2- and 8-week-preserved groups (Fig. 3). Degradation of the epidermal layer to the dermal layer was observed in all groups. Skin grafts of GFP rats preserved in CB showed only green florescence around the dermis of the transplanted area in the 24-week-preserved group. On the other hand, the fluorescence of the epidermal layer and some viable cells were confirmed in the EGCGpreserved skin grafts. The degradation was improved by EGCG in comparison to the CB group. An evaluation of the viability of the transplanted skin grafts was difficult with GFP confirmation only. The histology of the transplanted skin grafts of GFP

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rats and nude mice was also observed in H&E-stained sections (Fig. 4). The grafts preserved with EGCG showed a dense dermal matrix and an intact epidermal layer as shown in the GFP pictures. Micrographs of CBpreserved skin grafts were not fully rejected; however, separation between transplanted skin of the GFP rat and immunodeficient mouse was observed near the location of the original epidermal layer. The histological score was marked with micrographs of H&E-stained specimens based on the attached area of the transplanted skin graft and the condition of surviving skin grafts (Fig. 5). Dead and completely rejected skin grafts scored 0. Surviving skin grafts with a wide attached area, intact epidermis, and dermis with hair follicles and a dense matrix scored 5. Neither the survival rate of the skin grafts nor the histological scores indicated a clear difference and tendency among the 2-, 8-, and 24-week-preserved groups. Both the survival rate of the skin grafts and the histological scores were increased by EGCG in all groups. EGCG enhances the viability of stored skin grafts and extends storage time up to 7 weeks at 4°C. In this study, the storage time of the skin grafts was extended to 24 weeks by cryopreservation with both DMSO and EGCG. The survival rate of the transplanted skin grafts reached almost 100% in the 24-week-preserved group. DISCUSSION Cryopreserved cells and tissues are increasingly used for stem cell transplantation and tissue engineering. However, they are highly sensitive to freezing, storage, and thawing, which suggests the need for improved cryopreservation methods. When storing any living tissue, the destructive effects

Figure 2. Glucose consumption of the preserved skin biopsies after thawing. Skin biopsy samples were preserved in freezing medium (CB) with or without EGCG at −196°C for 4 and 8 weeks. There was no significant difference; however, the amount of glucose consumption in the EGCG groups showed faster metabolic recovery in comparison to the CB groups.

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Figure 3. Cryosections of GFP rat skin graft to a nude mouse (left: 2-week-preserved group, middle: 8-week-preserved group, fight: 24-week-preserved group). These micrographs are representative of four to six independent experiments, and showed similar results.

of hypoxic metabolism must be controlled. A tissue removed from its blood supply will die unless cellular metabolic activity is decreased or nutrients are provided. By reducing metabolism and providing nutrients, viability can be further improved (5). EGCG was reported to control cell division, which causes the energy metabolism to decrease by stopping of the cell cycle. Therefore, the combination of such effects improves the condition of the preserved skin. In addition, EGCG reduces ischemia/reperfusion injury by attenuating nitric oxide synthase expression and activity and improved hypoxic metabolism is predictable in preserved skin (17,18). Furthermore, other actions of EGCG such as strengthening the scaffold structure, an immunomodulatory effect, and antibacterial activity, may also contribute to its preservative effects (11,14). In the present study, the protection afforded by EGCG was similar for all three groups. EGCG and DMSO in the freezing medium were more effective for maintaining glucose consumption. These results suggest that oxygen radicals scavenged by EGCG may be re-

sponsible for damage at the membrane level and thus that exogenous EGCG can protect cell membranes. These phenomena may be related to the intrinsic characteristics of polyphenol compounds, which readily penetrate cell membranes due to their amphipathic properties. These compounds are easily absorbed by lipid bilayers, extracellular matrices (e.g., collagen, fibronectin), and various cell membrane receptors. The absorption of polyphenolic compounds to such proteins is rapid and adsorption rates are slow. Consequently, the skin graft could be protected from freeze–thaw injuries due to absorption of EGCG to various membrane proteins and lipids (7). The glucose consumption of thawed tissues was assessed after 2, 4, and 6 days of incubation under tissue culture conditions. This postthaw incubation period was selected to allow recovery of metabolic activity in lethally damaged cells. These delayed measurements may be more characteristic of the actual viability of the skin allografts than measurements taken immediately after thawing and dilution. The cells may sustain lethal dam-


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Figure 4. Histological observation in skin grafts with H&E-stained micrographs (top: original magnification 5×, bottom: original magnification 20×). Degradation of the epidermal layer to the dermal layer was observed in the CB groups. The degradation was improved by EGCG in comparison to the CB groups. These photographs are representative of four to six independent experiments, and showed similar results.

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Figure 5. (A) The survival rate of grafted GFP rat skin after 2 weeks. (B) Considering the epidermal morphology and dermal integrity of the transplanted grafts, a score was given from 0 to 5. A higher score indicates better condition of the skin grafts. The results are reported as the mean ± SE (n = 4–6) and analyzed by Fisher’s PLSD test. The value marked with an asterisk is significantly (p < 0.05) different from the nontreatment groups.

age during the thawing and dilution process but have not yet completely disintegrated; thus, there may be residual metabolic activity in the lethally damaged cells. The viability of the preserved skin biopsy samples was investigated by examining whether skin specimens could be successfully grafted after preservation. It is reported that dead tissue or skin is rejected in around 2 weeks (4,19). Therefore, immunodeficient mice were used as recipient animals to judge the success of skin grafts from GFP rats (6). GFP fluorescent protein does not need any chemical substrate for visualization. By using a GFP transgenic animal, transplanted cells or tissues having GFP can be detected by direct visualization. Therefore, the GFP transgenic rat is thought to be a suitable skin donor for skin storage and transplantation studies. A histological analysis demonstrated that the preserved skin began to degenerate from the epidermis to the dermis. Epidermis can be easily degenerated during the preservation processes. Considering the histological data in previous studies, the results of 2 weeks of preservation seem to be better at 4°C rather than with cryopreservation. However, in 4°C preserved skin grafts 8 weeks was the time point of skin graft survival becoming 0. EGCG enhances the viability of stored skin grafts and extends the storage time up to 7 weeks at 4°C (13). In this study, the storage time of skin graft was extended to 24 weeks by cryopreservation using EGCG and the survival rate was almost 100% in the 24-week-preserved group. In conclusion, the present findings suggest the future clinical use of EGCG for skin preservation without

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