Composite skin graft: frozen dermal allografts support the engraftment and expansion of autologous epidermis

Rapid closure of burn wounds significantly reduces the complications associated with thermal injury. Successful wound coverage, however, is often limited by the lack of suitable autografts. To circumvent this limitation a composite graft was developed which combines the utility and availability of allogeneic skin with the permanence of an autograft. Composite grafts were first employed in a rat wound model and subsequently to treat six patients with thermal injuries. In experiments with rats, full-thickness excised (1") wounds were prepared on thoracic walls, covered with previously frozen allograft skin, dressed, and secured. Five days later, the dead epidermis was removed and trypsin-disaggregated syngeneic epidermal cells applied to the exposed dermal surface. Successful engraftment with complete epidermal coverage could be observed within 7 to 10 days. In eight patients, split-thickness skin bank allografts were placed on full-thickness burn wounds. Four days later the dead epidermis was removed and vacuum blister-prepared sheets of autologous epidermis grafted to the exposed dermal surface. In all eight patients successful engraftment ensued. Increased pigmentation at the site of each original epidermal graft confirmed the stability of underlying allograft dermis. Epidermal expansion ranged from 1:20 to 1:100. All patients were followed from 10 to 12 months with no demonstrated graft loss or significant wound contracture. Composite skin grafts which combine allogeneic dermis and an expanded autologous epidermis can effect rapid wound closure and will remain stable without evidence of rejection or graft breakdown for at least 12 months.     

烧伤创面愈合的理论探索与临床实践

The basic and clinical research in wound healing have made great progress in China in the past 50 years. The method of " intermingle skin transplantation" which was first advocated by surgeons of Ruijin Hospital in 1966 greatly reduced the amount of autologous donor skin, thus making the coverage of an extensive burn wound possible. This method is al so known as " Chinese therapy". In 1986,doctors of Jishuitan Hospital reported successful coverage of an extensive burn wound with mieroautografts and allogeneic skin. The basic research of wound healing has been carried out since 1992,a series of studies showed the characteristics of biological behaviours of cells in concern, extracellular matrix and growth factor, the mechanism underlying progressive injury in deep second burn wound, the effect of " skin island" and the local immune tolerance induced by it (which are the key factors of intermingle transplantation).The induction of local immune tolerance has now become the re search hot subject of skin transplantation immunology. Stem cell research in the field of wound healing has been extensively car ried out. The theory of " dermal template defection" has been proposed as one of the mechanisms of scar formation. On the other hand, great progress has been achieved in the treatment of bums on the basis of clinical researches. Doctors of PLA 304 hospital found that excision of eschar on patients with extensive deep burn injury at early shock stage greatly decreased the occurrence of complications and mortality. Doctors of Ruijin Hospital reported that healing of deep second burn wound could be improved by tangential excision of burn eschar within 24 hours after burn injury. Doctors of Xiang ya Hospital reported patients suffering from deep bums of the hands got satisfied functional restoration when treated with tangential excision of eschar while degraded dermal tissue could be retained with transplantation of autoskin grafts.     

异体脱细胞真皮基质与自体皮片复合移植的临床应用

目的:探讨异体脱细胞真皮基质与自体皮片复合移植修复大面积深度烧伤及瘢痕切除后皮肤缺损创面及其愈合后皮肤的外形和功能。方法:应用异体脱细胞真皮基质与自体刃厚皮片组成复合皮移植,以自体刃厚皮片移植作为对照,采用一步移植法治疗切痂后大面积深度烧伤创面及瘢痕切除后皮肤缺损共56例患者60处创面,观察术后皮片的成活情况、外形及功能恢复情况并随访。结果:60处创面全部愈合,移植皮片生长良好,瘢痕增生不明显,未见明显挛缩,皮肤弹性较好。在6～12个月的观察期内,自体刃厚皮片与异体脱细胞真皮基质复合移植后,功能和形态优于单纯自体刃厚皮片移植;随访2年复合移植未发现明显的排异反应。结论:异体脱细胞真皮基质与自体皮片复合移植修复大面积深度烧伤及瘢痕切除后皮肤缺损创面愈合良好,无瘢痕增生,皮肤外观功能满意,无排异反应。     

复合皮混合移植治疗深Ⅱ度烧伤患者创面疗效观察

目的观察深Ⅱ度烧伤患者创面削痂术后应用复合皮混合移植治疗的效果。方法对23例烧伤患者的30个深Ⅱ度烧伤肢体在伤后3d内分次行削痂术,削至浅筋膜后移植大张异体脱细胞真皮基质,然后切取大张自体刃厚皮(0.10～0.25mm)覆盖于其上。术后10—12d计算移植皮片的存活率,记录创面愈合时间。观察随访3—6个月时患者的肢体外观及功能恢复情况。取1例患者随访3个月时的愈合创面皮肤标本,行病理学观察。结果本组患者复合皮片成活率为93%,7%的皮片因术中固定较差,移植后自体刃厚皮与异体脱细胞真皮基质分离致皮片坏死,或因感染致皮片溶解。随访3—6个月,移植部位皮肤外观、弹性及功能恢复良好。病理学观察显示,成活皮片表皮、真皮结构正常。结论烧伤后早期削痂立即移植复合皮是治疗深Ⅱ度创面的有效方法。     

Methods of burn treatment. Part II: Technical aspects]

The best treatment for burns and scalds depends on the depth of the skin necrosis. Epidermal and superficial dermal burn injuries (IIa) can heal spontaneously with conservative treatment without scar development, but deep dermal or full-thickness burns constitute an absolute indication for surgery. Full-thickness or split-thickness skin grafts are used for wound closure. In the case of extensive burn injuries allografts are used for temporary wound closure. In certain licensed laboratories autologous keratinocytes can be cultured for transplantation. In circumferential burn injuries affecting the extremities or the trunk the rigid eschar has to be incised to relieve the pressure behind it. Following a debridement conservative treatment of superficial dermal burns involving wound coverage with biosynthetic dressings or nanocristalline silver gauze dressings or use of special disinfecting ointments can be implemented.     

Behandlungskonzepte bei Verbrennungen

The best treatment for burns and scalds depends on the depth of the skin necrosis. Epidermal and superficial dermal burn injuries (IIa) can heal spontaneously with conservative treatment without scar development, but deep dermal or full-thickness burns constitute an absolute indication for surgery. Full-thickness or split-thickness skin grafts are used for wound closure. In the case of extensive burn injuries allografts are used for temporary wound closure. In certain licensed laboratories autologous keratinocytes can be cultured for transplantation. In circumferential burn injuries affecting the extremities or the trunk the rigid eschar has to be incised to relieve the pressure behind it. Following a debridement conservative treatment of superficial dermal burns involving wound coverage with biosynthetic dressings or nanocristalline silver gauze dressings or use of special disinfecting ointments can be implemented.     

Cultured epithelium as a skin substitute

Twenty-five burn patients with full or partial thickness skin loss received cultured epithelium (CE), allografts or autografts as part of their treatment. Overall, a 30 per cent graft 'take' was achieved irrespective of whether the CE was autograft or allograft, fresh or frozen. In the case of deep dermal burns this figure improved to 50 per cent. The surviving grafts merged with split thickness skin grafts (SSG) and advancing wound edges. When full thickness skin loss was grafted, only a patchy take could be achieved and the surviving islands of CE tended not to spread across the wound. No rejection of CE allograft was recorded either clinically or histologically up to 6 months. The technical problems and clinical implications are discussed.     

New grafts for old? A review of alternatives to autologous skin.

Immediate resurfacing of skin defects is a challenging prospect, especially in patients with extensive full-thickness burns. Currently, split-thickness autografts offer the best form of wound coverage, but limited donor sites and their associated morbidity have prompted the search for alternatives. The application of allogeneic skin is restricted by availability and the risk of transmission of infection, whilst synthetic skin substitutes are simply expensive dressings. The problems of limited expansion may be overcome by culturing keratinocytes in vitro. Unlike autologous cells, allogeneic keratinocytes are available immediately, although they survive for less than a week when applied to full-thickness skin defects. Moreover, the absence of a dermal component in these grafts predisposes to instability and contracture. A cross-linked collagen and glycosaminoglycan dermal substitute, covered with thin split-skin grafts or cultured autologous keratinocytes, shows promise in burns patients. An alternative is a collagen matrix populated by allogeneic fibroblasts and overlaid with cultured autologous or allogeneic keratinocytes. The clinical application of cultured grafts remains imperfect but offers the prospect of immediate coverage and massive expansion.     

The role of skin bank in the treatment of severely burnt patients

The accessibility of suitable temporary covers plays the key role in the treatment of severe skin losses. Biological covers have got the longest tradition in the wound healing. Skin banks are engaged in their production and distribution. Already in 1973 J. Moserová developed the methodology of harvesting pig xenografts. Later on, the short-term and the long-term method of storage were verified (B?hm, Konícková, Vogtová). In 1986, the Skin Bank in the Prague Burn Centre was established. In Prague Burn Centre the allografts are used very rarely, usually from the living donors, family members of the patients. Therefore, in our bank, we specialized in harvesting porcine xenografts. They are produced in three different forms--fresh, deep frozen in vapours of liquid nitrogen, and glycerolized. Porcine xenografts serve as a biological cover; they make barrier against infection and evaporation and protect the wound against desiccation. They are used namely for the treatment of superficial burn wounds, as a temporary coverage of excised wounds and as a dressing on release incision. Every year more than 10,000 strips have been used in our Burn Centre, it represents the area 200 m2. Since 1991 cultivation laboratory has been a part of our Skin Bank. We are interested in cultivation of human epidermal cells--keratinocytes. Cultured epidermal grafts became the first human in vitro prepared tissue, which was successfully transplanted to the patient. For the treatment of deep dermal skin losses we use either autologous keratinocytes, which can create permanent cover, or allogeneic cells, which stimulate spontaneous healing. Cultured keratinocytes are used in the treatment of burnt patients as well as in the trophic defects.     

Allogeneic skin substitutes applied to burns patients

Early re-surfacing of burn wounds remains the ideal but is limited by the availability of skin graft donor sites. Cultured grafts overcome these problems and autologous keratinocytes can be grown in culture and placed on a dermal substitute, but this results in delay and requires two operations. We developed an organotypic skin substitute, which achieves cover in one procedure, and have previously found allogeneic cell survival up to 2.5 years after grafting onto clean elective wounds (tattoo removal). Here, we report a short series using the same model applied to burns patients with less than 20% total body surface area affected. The skin substitutes consisted of allogeneic dermal fibroblasts embedded in a collagen gel overlain with allogeneic epidermal keratinocytes, and were grafted to patients with tangentially excised burns. A side-by-side comparison with meshed split-thickness autografts was performed. No grafts became infected. The allogeneic skin substitute showed little effective take at 1 week, and by 2 weeks only small islands of keratinocytes survived. These sites were subsequently covered with meshed split-thickness autograft, which took well. It is concluded that further development of this model is needed to overcome the hostile wound bed seen in burns patients.     

Early burn wound excision and skin grafting postburn trauma restores in vivo neutrophil delivery to inflammatory lesions

This study assessed the effect of early vs delayed postburn wound excision and skin grafting on the in vivo neutrophil delivery to a delayed-type hypersensitivity (DTH) reaction and a bacterial skin lesion (BSL). Male Lewis rats were presensitized to keyhole-limpet hemocyanin. Group 1 comprised sham controls. Groups 2 through 4 were given a 30% 3 degrees scald burn, but the burn wounds were excised, and skin was grafted on days 1, 3, and 7, respectively, after the burn. Group 5 comprised burn controls. Twelve days after burn trauma, all rats were injected at different intervals (during a 24-hour period) with a trio of intradermal injections of keyhole-limpet hemocyanin, Staphylococcus aureus 502A, and saline at different sites. In vivo neutrophil delivery to these dermal lesions was determined by injecting indium in 111 oxyquinoline-labeled neutrophils isolated from similarly treated groups of rats. Neutrophil delivery to DTH and BSL lesions was restored to normal by excision and skin grafting of the burn wound one day after burn trauma. Waiting three days after burn trauma to excise and skin graft the wound partially, but not completely, restored the in vivo neutrophil delivery to DTH and BSL lesions. Waiting one week to excise and skin graft a burn wound resulted in no improvement in neutrophil delivery to DTH and BSL dermal lesions. It was concluded that burn wound excision and skin grafting immediately after burn trauma restored in vivo neutrophil delivery to a BSL and DTH dermal lesion. This may, in part, explain the beneficial effect of early aggressive burn wound debridement in patients with burn injuries.     

Treatment of burns by grafting of cultured epithelium

The most serious problem in the treatment of extensive burns is a lack of sufficient healthy skin for coverage of the affected area. Several methods have been used for the coverage of extensive burn wounds. The grafting of cultured epithelium is a potentially effective method for a permanent covering of the wound, particularly in patients with extensive burns. The condition of the recipient site is the most important factor in the success of cultured epithelium grafting and the preservation of the dermis or dermal components in the burned area will enhance the grafting process. We recommend that prior to the grafting of cultured epithelial cells, the burn wounds should be excised and covered with an allograft or artificial dermis during the first 2 weeks after admission. An allograft of cultured epithelium is also useful. This method accelerates the epithelialization of both the burn and donor sites. It is expected that cultured epithelial cell grafts will prove to be an effective treatment not only for extensive burns but also to epithelialize small area burn wounds and resurfaced burn scars.     

Buried chip skin grafting for treatment of perianal burns

A technique of buried chip skin grafting for perianal burn injury is described. Small chip skin grafts are buried in the granulating wound around the anus and this procedure achieves epithelialization of the perianal and perineal wound within about 5 or 6 cm laterally from the middle with extremely small amounts of skin graft. Graft survival is not disturbed by stools or gently wiping of the wound to remove stools, as grafts are buried in the holes. If infection occurs soon after the operation, graft survival does not appear to be affected because the grafted holes show good drainage. This procedure is considered to be very useful for treating perianal or perineal granulating wounds in extensively and deeply burned patients who have limited autograft donor sites.     

Utilizing biologic assimilation of bovine fetal collagen in staged skin grafting

Seven patients underwent 2-stage skin grafting with bovine fetal collagen (BFC) as an initial wound cover. Split-thickness skin grafts were successfully placed on the wounds after completion of interval management. BFC proved to be a resilient acellular dermal matrix that could proceed to assimilation and skin grafting under a variety of wound conditions. BFC may prove to be a valuable material, as the role of acellular dermal matrices in skin grafting becomes better defined.     

Prospective study of the effect of the recipient bed on skin graft survival after thermal injury

There is little objective information available on the influence of the graft bed on skin graft survival after early excision of the burn wound. Yet surgeons are faced daily with the question of whether to graft onto fat after sequential excision of the burn eschar, or whether to excise deeper and graft onto muscle or fascia. To answer this question 71 consecutive patients were enrolled in a prospective study of the effect of the recipient graft bed on skin graft survival after thermal injury. These patients included 25 pediatric patients (mean TBSA, 19.1%) and 46 adult patients (mean TBSA, 25.1%). Overall mean +/- SD graft take was 94 +/- 11%. No difference between the per cent of graft take of grafts placed on fat (94 +/- 11%) versus fascia (85 +/- 19%) was found (p = 0.21). The results of this prospective study indicate that when the burn is too deep to graft onto dermis, there does not appear to be any advantage to routinely performing a fascial excision to avoid grafting onto a fat or mixed fat/dermal recipient graft bed, since the percentage of graft take and the incidence of regrafting are similar between these groups.     

Aesthetic closure of the donor site of a radial forearm free flap with two local curved skin grafts

The authors have designed an aesthetic and effective coverage technique using local curved skin grafts along with vascular pedicles without additional skin incisions to solve the disadvantages of skin coverage problem of donor site defect after radial forearm free flap (RFFF) harvesting. This has, to the authors’ knowledge, not been previously described.     

Acellular allograft dermal matrix: immediate or delayed epidermal coverage?

In a prospective, randomized study seventeen patients received skin grafts to a freshly excised burn wound. One group was grafted with a deantigenized dermal matrix and immediately overgrafted with thin autograft. The second group was grafted with dermal matrix, which was then covered with bank allograft for protection, and autografted 1 week later. Each group also received a standard split thickness control graft. Assessment was carried out for up to 1 year. There were no statistically significant differences of graft take between any of the groups, or in the Vancouver scar score at follow-up. Thin donor sites used for dermal matrix coverage healed faster than standard control graft sites, P<0.001. Immediate grafting of acellular dermal matrix with thin autograft works well and leads to an acceptable late result, with faster donor site healing than standard split thickness grafts.     

Use of tissue-engineered skin in the dermal atrophy patient with traumatic avulsion injuries

Tissue-engineered skin has been approved by the Food and Drug Administration for use in certain chronic ulcers (venous stasis ulcers and diabetic foot ulcers). Its use has also been reported in acute (surgical) excisional wounds. The authors report the use of tissue-engineered skin in traumatic avulsion wounds in a series of 10 patients with dermal atrophy. All patients were older than 60 years of age and clinically had thin, atrophic skin. Most were older than 70 years of age and were steroid users. Tissue-engineered skin (Apligraf or Living Skin Equivalent; Organogenesis, Inc. [corrected], Canton, MA) was used for subacute and acute wound coverage. All wounds healed 100%. Average healing time overall was 9.2 weeks. No wound has recurred with an average follow-up of 14 months. There were no wound infections. The authors conclude that tissue-engineered skin provides a safe, efficacious, and convenient solution for acute avulsion wounds in the patient with age- and/or steroid-related dermal atrophy.     

Allogeneic cultured keratinocytes vs. cadaveric skin to cover wide-mesh autogenous split-thickness skin grafts.

Improved shock therapy has extended the limits of survival in patients with massive burns, and nowadays skin coverage has become the major problem in burn management. The use of mesh skin grafts is still the simplest technique to expand the amount of available donor skin. However, very wide-mesh skin grafts take a very long time to heal, often resulting in unaesthetic scar formation. On the other hand, allogeneic cultured keratinocytes have been reported as a natural source of growth factors and thus could be useful to improve wound healing of these wide-mesh grafts. A clinical study was performed to compare the use of cryopreserved allogeneic cultured keratinocytes vs. the traditional cadaveric skin as a double layer over widely expanded autogenous skin grafts. This procedure was performed in 18 pairs of full-thickness burn wounds (with similar depth and location) in 11 severely burned patients. Early clinical evaluation was made at 2, 3, and 4 to 5 weeks. Parameters such as epithelialization, granulation tissue formation, infection, and scar formation were evaluated. Biopsies were taken to compare the histological characteristics of the epidermis, the epidermal-dermal junction, and the dermis. Late evaluations were performed at 6 and 12 months regarding color, softness, thickness, and subjective feeling of the scar tissue. Aside from a faster (p < 0.05) epithelialization in the keratinocyte group at 2 weeks, there were no statistically different results in any of the early evaluated parameters, neither clinically nor histologically. At long-term follow-up, clinical results and scar characteristics were not significantly different in the two compared groups. It is concluded from the results of this study that, during the early phase, epithelialization was faster with allogeneic cultured keratinocytes compared with cadaveric skin. However, taking into account the substantial difference in costs, the described use of cryopreserved allogeneic cultured keratinocytes as a double layer on meshed autogenous split-thickness skin grafts can hardly be advocated.     

The application of new biosynthetic artificial skin for long-term temporary wound coverage

Temporary dressings protect wounds from desiccation and infection. In our previous study, we used meshed acellular porcine dermis (APD) to enhance wound healing and decrease wound contraction; however, the wounds showed meshed scar [Wang HJ, Chen TM, Cheng TY. Use of a porcine dermis template to enhance widely expanded mesh autologous split-thickness skin graft growth: preliminary report. J Trauma 1997;42(2):177–82]. In this study, we produced an artificial skin composed of a cross-linked silicon sheet on the surface of APD which we have called silicone acellular porcine dermis (SAPD). This new artificial skin can protect the wound long enough to promote wound healing either by second intention or covered long enough until cultured epithelium autograft (CEA) or autologous skin graft can be harvested for permanent coverage.

We delivered 4 cm × 5 cm full-thickness wound on the back of 350 g Sprague–Dawley rats. Thirty-six rats were divided into two groups. Eighteen rats had SAPD and the other 18 were covered with Biobrane. The wounds were first examined 2 weeks after grafting and followed weekly for an additional 4 weeks to evaluate the wound and study pathological changes by using H.E. and Masson's stains. Wound size was calculated by ruler and analyzed by Student's t-test.

At the 2-week inspection, both SAPD and Biobrane showed tight adherence to the wound with no change of wound size. Both the SAPD and Biobrane dermal templates were pink. In the Biobrane-covered group, the wounds contracted soon after the tie-over dressing was removed. Its dermal layer is a layer of thin porcine dermal substance, which was promptly digested by tissue hyaluronidase and provides no real dermal template. In the SAPD-covered group however, the wound size was maintained significantly from third to sixth week after grafting (p < 0.001). SAPD was designed with thick epidermal silicone and a well-organized porcine dermis so that it incorporates into the recipient wound. Clinically the silicone layer of SAPD dislodged from APD about 6–7 weeks after grafting and was followed by dermal matrix exposure and infection. In pathological examination, much like a human skin graft, new vessels were found in APD about 1 week after grafting with minimal inflammatory cells infiltrated in the graft and wound. Six weeks after grafting, the collagen of APD incorporated into the wound, showing palisade arrangement and no sign of rejection. In the Biobrane group however, the wounds showed severe inflammation, the porcine dermal matrix was digested and disappeared 3 weeks after coverage.

In conclusion, SAPD is a thick biosynthetic artificial skin, which protects the rat wound significantly longer than Biobrane and prevents contraction. We expect that using of SAPD for temporary wound coverage will provide enough time to grow autologous-cultured epithelium or to reharvest skin grafts.