TY - JOUR
AU - Wenfeng,, Cheng
AB - Abstract Patients with extensive deep burns often lack enough autologous skin to cover the wounds. This study explores a new method using microskin in combination with autologous keratinocytes in the treatment of extensive deep burn. Wounds in the combination group were treated with automicroskin at an area expansion ratio of 20:1 (wound area to automicroskin area) and autologous keratinocyte suspension, which were compared with the following treatments: no autotransplant, only allografts (control group); autologous keratinocyte suspension only (keratinocyte only group); automicroskin at an area expansion ratio of 20:1 (20:1 group); and automicroskin at an area expansion ratio of 10:1 (10:1 group, positive control). The authors used epithelialization rate (epithelialized area on day 21 divided by original wound area), hematoxylin and eosin staining, laminin, and type IV collagen immunohistochemistry to assess wound healing. The epithelialization rate of combination group (74.2% ± 8.0%) was similar to that of 10: 1 group (84.3% ± 11.9%, P = .085) and significantly (P < .05) higher than that of 20:1 group (59.2% ± 10.8%), keratinocyte only group (53.8% ± 11.5%), and control group (22.7% ± 5.5%). The hematoxylin and eosin staining and immunohistochemistry showed the epithelialization in the combination group was better than that in the keratinocyte only group and control group. Microskin in combination with autologous keratinocyte suspension can promote the reepithelialization of full-thickness wounds and reduce the requirements for automircoskin, and it is a useful option in the treatment of extensive deep burns. The prognosis for burn patients has improved during the past decades because of the early excision of necrotic tissue and immediate coverage of the wounds.1 The accepted standard for the coverage of full-thickness skin defects is autologous skin. However, in patients with extensive and deep burns, the rarely available donor sites limit the application of this method. Therefore, the micrograft concept has been introduced to enhance wound coverage.2,3 Microskin can help create smaller skin islands and increase the area of epithelial surface which is in contact with the wound bed. As a result, wound coverage can be achieved by a smaller amount of donor skin. However, when the burn area is too massive, it is difficult to adopt this method due to the small amount of remaining skin of the patients. Based on our experience and previous study, we hypothesized that the microskin in combination with autologous keratinocyte suspension could achieve wound coverage and promote wound healing. One advantage of the new method is that a small biopsy of the skin can provide enough cultured epithelium to cover the body areas, which reduces the requirements for donor skin. In addition, when the keratinocytes in suspensions are transplanted, cells are transferred to the wound bed before they form a sheet. As a result, the cells express a different integrin profile and proliferate more rapidly, which facilitates the attachment of cells migrating to the wound area. Furthermore, the “take” rate of autologous cultured epidermal cells depends on the conditions of the wound bed before grafting. The “take” rate is generally higher in wounds where dermal elements still exist.4 For example, partial-thickness wounds, donor site wounds, and interstices of widely meshed autograft often show “higher” take rates. Previous study showed fibroblasts in the dermal elements strongly promote wound healing.5 Therefore, adding dermal counterparts to autologous cultured epidermal cells is highly desirable to obtain several valuable mechanical properties. This study aims to evaluate the effects of microskin in combination with autologous keratinocyte suspension in extensive deep burn treatments. METHODS Animals and Experimental Groups In this study, Wistar rats weighing 190 to 230 g served as allogeneic donors, and female Sprague Dawley (SD) rats weighing 190 to 230 g were the recipients. These rats were provided by the Animals Center of the First Affiliated Hospital of People's Liberation Army General Hospital. Female SD rats (N = 50) were randomly divided into the following five groups: control group, no autotransplant, only allografts; keratinocyte only group, treated with autologous keratinocyte suspension only; 20:1 group, treated with automicroskin at an area expansion ratio of 20:1 (wound area to automicroskin area); combination group, treated with automicroskin at an area expansion ratio of 20:1 and the autologous keratinocyte suspension; 10:1 group, treated with automicroskin at an area expansion ratio of 10:1, which was positive control. Rat Keratinocyte Isolation and Culture The dorsal aspects of the rats in the combination group and keratinocyte only group were used for keratinocyte culture. All rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg). The dorsum was shaved, and the skin was cleaned with povidone-iodine solution. A 2.0 cm × 1.0 cm area was outlined by a surgical skin marker. Skin was removed with a scalpel and washed with phosphate-buffered saline three times. After that, the subcutaneous connective tissue was shaved off carefully and the defect in the dorsum was sutured. All rats were housed in individual cages. The skin was cut into 0.5 cm × 1 cm and incubated in 1.25 g/L dispase solution at 4°C for 12 to 14 hours, and the epidermis was then peeled off from the dermis. Keratinocytes were dissociated from the epidermis using a 0.25% trypsin solution containing 0.01% EDTA for 3 minutes at 37°C. The trypsin was neutralized by fetal bovine serum, and the solution was then passed through a 200 mesh sterile sieve. Next, the solution was centrifuged at 750 rpm for 6 minutes at 37°C, and the keratinocytes were pelleted. Cells were resuspended in keratinocyte serum-free medium and seeded onto type I collagen (354249, BD Biosciences, US)–coated dishes at a density of 6 × 106 cells per 100-mm dish. The medium was changed every 2 days. Usually, after 10 to 12 days, the cells reached 90% confluence. Then, the wound model and treatment were initiated. Wound Model All rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg). The dorsum was shaved, and the skin was cleaned with povidone-iodine solution. A 3 cm × 3.3 cm area was outlined by a surgical skin marker. A full-thickness skin defect was then created, leaving the underlying panniculus carnosus intact. The steel wire frames were sutured with the skin wound margin to reduce wound contraction. Preparing the Automicroskin and Allografts Full-thickness skin grafts from both donors and recipients were trimmed to partial-thickness skin grafts with scissors. The recipient partial-thickness graft was harvested and cut into microskin (pieces smaller than 1 mm3) according to the proportion of the area as described by Lin et al.6 When the wounds (3 cm × 3.3 cm) were treated with automicroskin at an area expansion ratio of 10:1 or 20:1, the automicroskin (1 cm × 0.99 cm or 0.5 cm × 0.99 cm) was harvested from the excised skin. The microskin fragments were kept moist with sterile saline until transplantation onto the recipient wound. Transplantation For the control group, allografts were overlaid onto the wound directly. For the keratinocyte only group, approximately 5 × 105 cells were collected and suspended in 300 μl of keratinocyte serum-free medium. The cells were placed onto the recipient wound using a pipetting gun. Then, allografts were overlaid onto the wound. For the 20:1 or 10:1 group, the automicroskin fragments were spread as evenly as possible on the dermis side of the allografts using surgical forceps. Then, the allografts were overlaid onto the wound. For the combination group, the autologous keratinocyte suspension was placed onto the recipient wound, and allografts with automicroskin were then overlaid onto the wound. A sufficient number of holes were made by scalpel in the allograft for effective drainage and were sutured to the wound. The wound was then dressed with several layers of sterile gauze on the outer surface. Bacterial prophylaxis was implemented by applying chlortetracycline ointment to the area around the wound. After recovering from anesthesia, each rat was housed in an individual cage and received a normal diet ad libitum. The wounds were disinfected with povidone-iodine solution daily to prevent infection. Gross Wound Observation and Epithelialization Rate All rats were anesthetized (intraperitoneal injection of sodium pentobarbital 40 mg/kg) once weekly until day 21 for digital photography and gross wound observations (dehiscence of allograft, separation of allograft with wound, and presence of signs of infection). Photographs of each wound with a scaled ruler placed at each side were taken with a digital camera. The images of the wounds were imported into Image Pro Plus software (Media Cybernetics, Silver Spring, MD) for processing, and planimetry was used to calculate the wound surface area. The original wound area 3.0 cm × 3.3 cm and the epithelialized wound area were measured for calculating the epithelialization rate on day 21. We used the epithelialized area on day 21 divided by original wound area (3.0 cm × 3.3 cm) as the epithelialization rate to assess wound healing. Staining With Hematoxylin–Eosin The skin grafts were excised on day 14 (four rats of each group) and on day 21 (six rats of each group) postwounding. Samples were fixed in 10% formalin and embedded in paraffin. Four-micrometer-thick tissue sections were processed for standard staining with hematoxylin and eosin (HE). Images were photographed with a microscope (IX71; Olympus, Tokyo, Japan). Immunohistochemical Analysis The structural proteins of the epidermal basement membrane zone, such as type collagen IV and laminin, were detected using immunohistochemical methods, resulting in some brown products. Sections of normal SD rat skin were used as positive controls for proteins expression. Statistical Analysis The epithelialization rate is presented as the mean ± SD and analyzed by analysis of variance using the least significant difference correction, with P < .05 considered significant. All statistical analyses were performed using SPSS version 19.0 (IBM Corporation, Armonk, NY). Ethics Statement This study was approved by Animal Care and Use Committee and the Ethics Committee of the First Affiliated Hospital of People's Liberation Army General Hospital, Beijing, China. RESULTS Gross Wound Observation and Epithelialization Rate On posttransplantation day 7, all the grafted wounds showed slight contraction. The transplanted skin turned to be ruddy without any obvious bleeding or infection. On posttransplantation day 14, the allogeneic skin grafts became necrotic and dissected away during epithelialization. On posttransplantation day 21, when the allogeneic skin grafts peeled off, the transplanted skin in 20:1 group, combination group, and 10:1 group formed stratified epidermis. However, in the keratinocyte only group, the wound was covered with fragile epithelialized tissue, and in the control group, only few reepithelializations were found (Figure 1A). Figure 1. Open in new tabDownload slide A. Changes in appearances of the wounds treated with different transplantations. Control group: wounds received no autotransplant; keratinocyte only group: wounds treated with autologous keratinocyte suspension only; 20:1 group: wounds treated with automicroskin at an area expansion ratio of 20:1; combination group: wounds treated with automicroskin at an area expansion ratio of 20:1 and autologous keratinocyte suspension; and 10:1 group: wounds treated with automicroskin at an area expansion ratio of 10:1. B. Statistical results in the reepithelialization rate of the wounds treated with different transplantation. *P < .05. †P > .05. Figure 1. Open in new tabDownload slide A. Changes in appearances of the wounds treated with different transplantations. Control group: wounds received no autotransplant; keratinocyte only group: wounds treated with autologous keratinocyte suspension only; 20:1 group: wounds treated with automicroskin at an area expansion ratio of 20:1; combination group: wounds treated with automicroskin at an area expansion ratio of 20:1 and autologous keratinocyte suspension; and 10:1 group: wounds treated with automicroskin at an area expansion ratio of 10:1. B. Statistical results in the reepithelialization rate of the wounds treated with different transplantation. *P < .05. †P > .05. The epithelialization rate of combination group (74.2% ± 8.0%) was significantly (P < .05) higher than that of 20:1 group (59.2% ± 10.8%), keratinocyte only group (53.8% ± 11.5%), and control group (22.7% ± 5.5%). However, no significant differences (P = .085) were found in the combination group and in the 10:1 group (84.3% ± 11.9%; Figure 1B). Histological Observation On posttransplantation day 14, the HE staining demonstrated a neoepithelium originated from the grafts in the combination group, the 20:1 group, the 10:1 group, and the keratinocyte only group but not in the control group. On posttransplantation day 21, the wounds in the combination group, the 20:1 group, and the 10:1 group were covered with stratified epidermis. The neoepidermis was thicker than the normal epidermis. In the keratinocyte only group, the HE staining also proved neoepithelium originated; however, the neoepithelium contained multiple vacuoles. In the control group, we only found granulation tissues (Figure 2). Figure 2. Open in new tabDownload slide Hematoxylin–eosin staining in reepithelialization of the wounds treated with different transplantations at 21 days following wounding. Immunohistochemistry observation: collagen IV and laminin were presented at the basal epidermal–dermal junction at 21 days following wounding in each group. The blank arrows show that collagen IV was positive. The blue arrows show that laminin was positive (×100). Figure 2. Open in new tabDownload slide Hematoxylin–eosin staining in reepithelialization of the wounds treated with different transplantations at 21 days following wounding. Immunohistochemistry observation: collagen IV and laminin were presented at the basal epidermal–dermal junction at 21 days following wounding in each group. The blank arrows show that collagen IV was positive. The blue arrows show that laminin was positive (×100). Immunohistochemistry Observation On posttransplantation day 21, in the combination group, the 20:1 group, and the 10:1 group, the immunohistochemistry showed collagen IV and laminin were present at the basal epidermal–dermal junction. No collagen IV or laminin was found in the keratinocyte only group and the control group (Figure 2). DISCUSSION The limited availability of skin donor site greatly restricts the application of autologous skin grafts. This situation becomes more serious when burn areas exceed 60% of the TBSA. Therefore, in the 1980s, the automicroskin transplantation with overlaid allografts was introduced to treat deep and large burns. The skin is minced into pieces smaller than 1 mm3 to cover an area 10 times as large as the donor area. This method has been widely applied in China.2,3 Despite these trials showing promising results, severe burn patients with large burned areas still have limited donor sites.7 Autologous cultured epidermal cells are another approach to manage extensive burns without sufficient donor skin.8 In the present study, a mixture of automicroskin and autologous cultured epidermal cells was used to repair a full-thickness skin defect in rats. Our study showed the epithelialization rate of the combination group was significantly higher than that of 20:1 group. Moreover, compared with the 10:1 group, the combination management had similar remarkable epithelialization rate. Therefore, treating the full-thickness wound with a mixture of automicroskin and autologous cultured keratinocyte can promote the epithelialization process and reduce the demand for automircoskin. The capacity of microskin in initiating cell cultures demonstrates the microskin can serve as a cell source in the process of skin wound repair.9 Compared with allogeneic epidermal cells, autogeneic epidermal cells not only secret growth factors that promote mircoskin expand to heal wound but also serve as seed cells to repair the wound directly.10 Yim et al11 state that application of suspension-type cultured epithelial autografts to treat extensive burns could enhance the take rate of a wide-meshed autograft. The benefit of suspension-type cultured epithelial autografts has also been proved by our study and others.12 The basement membrane is an amorphous laminar structure with intimate contact with various cell types. Type IV collagen is a major constituent of the basement membranes.13 Laminin is a ubiquitous noncollagenous connective tissue glycoprotein that is also a major constituent of the basement membranes.14 The presence of IV collagen and laminin at the basal epidermal–dermal junction provides evidence for the presence of basement membranes in automicroskin treating groups, the combination group, the 20:1 group, and the 10:1 group. According to previous studies, fibroblasts are responsible for laminin and IV collagen production.15 Therefore, implantation of automicroskin that consists of fibroblasts is favorable to the early formation of basement membrane.16,17 The reconstitution of the basement membrane is of utmost importance for the firm adhesion of the epidermis and dermis. It is also conducive to form long-term mechanical stability of the reepithelialization wound. In the keratinocyte only group, wounds treated with autokeratinocyte alone showed a delayed maturation of the basement membrane. This may be an explanation for the reepithelialization of wounds but with a fragile surface. The activity of the allograft is of utmost importance in the successful “take” of the microskin autograft. The allograft served to protect the autograft during the healing process. In our study, several holes were made in the allograft for external drainage to ensure the survival of allogeneic skin. Besides, the wounds were disinfected daily with povidone-iodine solution to prevent infection of the allogeneic skin. Reepithelialization and granulation tissue formation are the major forces involved in human wound healing, whereas in rodent models, the major mechanism of wound closure is contraction; this cannot simulate human wound healing process.18 In our study, a steel wire frame was sutured with the skin wound margin to minimize wound contraction in rats, allowing wound healing by reepithelialization and granulation tissue formation. This varies from polytetrafluoroethylene chambers, which are used in a porcine model.19 The steel wire frame does not prevent epidermal migration from the skin edge to the wound area. That is why wounds also healed lightly in the control group. In conclusion, the automicroskin combined with autologous keratinocyte suspension can promote the reepithelialization of the full-thickness wounds and reduce the requirements for automircoskin. Therefore, automicroskin in combination with autologous keratinocyte suspension is a promising treatment in extensive deep burns and further preclinical experiments are needed to prove our findings. ACKNOWLEDGMENT We are grateful to Miss Lulu Jiao for her kind help in language of this article. REFERENCES 1. Williamson JS Snelling CF Clugston P Macdonald IB Germann E Cultured epithelial autograft: five years of clinical experience with twenty-eight patients . J Trauma 1995 ; 39 : 309 19 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Ming-liang Z Chang-yeh W Zhi-de C et al. Microskin grafting. II. Clinical report . Burns 1986 ; 12 : 544 8 . Google Scholar Crossref Search ADS WorldCat 3. Ming-liang Z Zhi-de C Xun H et al. Microskin grafting. I. Animal experiments . Burns 1986 ; 12 : 540 3 . Google Scholar Crossref Search ADS WorldCat 4. Song G Jia J Ma Y et al. Experience and efficacy of surgery for retaining viable subcutaneous tissue in extensive full-thickness burns . 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Foldager CB Toh WS Christensen BB Lind M Gomoll AH Spector M Collagen type IV and laminin expressions during cartilage repair and in late clinically failed repair tissues from human subjects . Cartilage 2016 ; 7 : 52 61 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Timpl R Rohde H Robey PG Rennard SI Foidart JM Martin GR Laminin – a glycoprotein from basement membranes . J Biol Chem 1979 ; 254 : 9933 7 . Google Scholar PubMed WorldCat 15. Cooper ML Hansbrough JF Use of a composite skin graft composed of cultured human keratinocytes and fibroblasts and a collagen-GAG matrix to cover full-thickness wounds on athymic mice . Surgery. 1991 ; 109 : 198 207 . Google Scholar PubMed WorldCat 16. Varkey M Ding J Tredget EE Superficial dermal fibroblasts enhance basement membrane and epidermal barrier formation in tissue-engineered skin: implications for treatment of skin basement membrane disorders . Tissue Eng Part A 2014 ; 20 : 540 52 . Google Scholar PubMed WorldCat 17. El Ghalbzouri A Ponec M Diffusible factors released by fibroblasts support epidermal morphogenesis and deposition of basement membrane components . Wound Repair Regen 2004 ; 12 : 359 67 . Google Scholar Crossref Search ADS PubMed WorldCat 18. Galiano RD Michaels J 5th Dobryansky M Levine JP Gurtner GC Quantitative and reproducible murine model of excisional wound healing . Wound Repair Regen. 2004 ; 12 : 485 92 . Google Scholar Crossref Search ADS Google Preview WorldCat COPAC 19. Kangesu T Navsaria HA Manek S et al. A porcine model using skin graft chambers for studies on cultured keratinocytes . Br J Plast Surg 1993 ; 46 : 393 400 . Google Scholar Crossref Search ADS PubMed WorldCat Footnotes Y.S. and D.L. contributed equally to this article. This work were supported by the National Natural Science Foundation of China (grant numbers 81373140 and 81641090) and Key Issues for the Scientific Research Project of Logistics of PLA (grant number BWS14J048). Copyright © 2017 by the American Burn Association
TI - The Benefit of Microskin in Combination With Autologous Keratinocyte Suspension to Treat Full Skin Loss In Vivo
JO - Journal of Burn Care & Research
DO - 10.1097/BCR.0000000000000552
DA - 2017-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-benefit-of-microskin-in-combination-with-autologous-keratinocyte-04xdsoe3az
SP - 348
VL - 38
IS - 6
DP - DeepDyve
ER -