- Email: [email protected]
Skin Substitutes in Burns
Burns 25 (1999) 97±103
Skin Substitutes in Burns Robert L. Sheridan a, b, c, *, Ronald G. Tompkins a, b, c a
Shriners Burns Hospital, 51 Blossom Street, Boston, MA 02114, USA b Trauma and Burn Services, Massachusetts General Hospital, USA c Department of Surgery, Harvard Medical School, USA Accepted 27 May 1998
1. Introduction The natural history of burn injury has been substantially modi®ed over the past few decades. The ®rst recorded recognition of the exaggerated ¯uid requirements of burn patients was probably that of Underhill in 1930 [1]. After this important clinical observation, investigations began to develop methods to manage burn shock. In the aftermath of the Coconut Grove ®re, Moore and colleagues [2] re®ned the concept of burn resuscitation and proposed a formula for intravenous volume repletion based on body weight [3], and in the 1950's the Evans formula was promulgated by the sta at the United States Army Institute of Surgical Research [4]. Subsequent re®nements in burn shock resuscitation have virtually eliminated this as a cause of death. In the 1970's, the advantage of early excision and closure of small burn wounds was recognized [5]. This excisional approach was subsequently taken to patients with large injuries by Burke and others [6±8] who documented truncated hospital stays and enhanced survival in burn patients who were routinely expected to die; a burn over more than a third of the body surface being almost universally lethal at that time. Re®nement of the surgical approaches to large wounds combined with the ongoing evolution of critical care techniques has extended our ability to support patients with increasingly severe injuries through the physiologic trial of staged wound closure. Burn physical and occupational therapy and burn reconstruction have developed in parallel, facilitating our ability to deliver increasingly satisfying long term outcomes. However, further progress is seriously impaired by our lack of a suitable skin substitute. In
* Corresponding author. Tel.: +1-617-726-5633; Fax: +1-617-3678936; E-mail: [email protected]
both the acutely injured and those requiring extensive post burn reconstruction, the absence of a durable skin substitute regularly hinders recovery. The successful development of a permanent skin substitute will have an enormous impact on the care of patients with serious burns. Skin is a complex organ. Functionally, it has two layers with a highly specialized and eective bonding mechanism. The epidermis, consisting of the strata basale, spinosum, granulosum and corneum, provides a vapor and bacterial barrier. The dermis provides strength and elasticity. The thin epidermal layer is constantly replacing itself from its basal layer, with new keratinocytes undergoing terminal dierentiation over approximately 4 weeks to anuclear keratin ®lled cells that make up the stratum corneum, which provides much of the barrier function of the epidermis. The basal layer of the epidermis is ®rmly attached to the dermis by a complex bonding mechanism containing collagen types IV and VII. When this bond fails, serious morbidity results, as demonstrated by the disease processes of toxic epidermal necrolysis [9] and dystrophic epidermolysis bullosa [10]. At present, most full thickness burn wounds are best closed as quickly as possible with split thickness autograft. However, split thickness autograft is an imperfect replacement for full thickness skin, may be limited in quantity and is associated with donor site morbidity. The ideal skin substitute (Table 1): (1) is inexpensive, (2) has a long shelf life, (3) is used o the shelf, (4) is non-antigenic, (5) is durable, (6) is ¯exible, (7) prevents water loss, (8) is a barrier to bacteria, (9) conforms to irregular wound surfaces, (10) is easy to secure, (11) grows with children, (12) is applied in one operation, (13) does not become hypertrophic and (14) does not exist at the present time [11, 12]. Conceptually, skin substitutes are temporary or permanent; epidermal, dermal or composite; and biologic or synthetic. Biologic components are xenogeneic, allo-
0305-4179/99/$19.00 + 0.00 # 1999 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 9 8 ) 0 0 1 7 6 - 4
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Table 1 Characteristics of the ideal skin substitute (1) Inexpensive (2) Long shelf life (3) Used o the shelf (4) Non-antigenic (5) Durable (6) Flexible (7) Prevents water loss (8) Bacterial barrier (9) Drapes well (10) Easy to secure (11) Grows with child (12) Applied in one operation (13) Does not become hypertrophic (14) Does not yet exist
genic or autogenic. There is a research eort centered on many of the possible permutations of these traits. From a practical perspective, the current reality is that skin substitutes are designed to be temporary or permanent. This manuscript will attempt to review the uses and formulations of temporary and permanent substitutes now available and will speculate on future directions in skin substitute research. Whenever possible, proprietary product names will not be used; any products not mentioned are not purposefully excluded. 2. Current temporary substitutes Temporary skin substitutes provide transient physiologic wound closure. Physiologic wound closure implies a degree of protection from mechanical trauma, vapor transmission characteristics similar to skin and a physical barrier to bacteria. These membranes therefore contribute to the creation of a moist wound environment with a low bacterial density. There are four common uses for temporary skin substitutes: (1) as a dressing on donor sites to facilitate pain control and epithelialization from skin appendages, (2) as a dressing on clean super®cial wounds to a similar end, (3) to provide temporary physiologic closure of deep dermal and full thickness wounds after excision while awaiting autografting or healing of underlying widely meshed autografts and (4) as a `test' graft in questionable wound beds. There are a large number of such membranes in common use, classes of which will be described below. 2.1. Porcine xenograft Xenografts have been used as intact split thickness grafts from a variety of species for many years to pro-
vide temporary cover of wounds [13]. The most commonly utilized species employed in this fashion has been domestic swine [14]. Porcine xenograft is often used as a reconstituted product consisting of homogenized porcine dermis which is fashioned into sheets and meshed [15]. It is widely used for temporary coverage of clean wounds such as super®cial second degree burns and donor sites [16]. Its use has been favorably reported in patients with toxic epidermal necrolysis [9, 17] and it has been combined with silver to suppress wound colonization [18, 19]. Although it does not vascularize, it will adhere to a clean super®cial wound and can provide excellent pain control while the underlying wound epithelializes. It is applied to a cleansed wound and covered with a dry dressing, no sutures being required. 2.2. Synthetic membranes There exist a number of semipermeable membrane dressings of several proprietary types that are designed to provide a vapor and bacterial barrier and control pain while the underlying super®cial wound or donor site re-epithelializes. A number of single layer semi-permeable synthetic membranes exist that provide a mechanical barrier to bacteria and have physiologic vapor transmission characteristics [20, 21]. These can be used with good eect on clean super®cial wounds and split thickness donor sites. There exists a single bilayer synthetic membrane in general use at present. This product, Biobrane1 (Dow-Hickham, Sugarland, TX) consists of an inner layer of nylon mesh that allows ®brovascular ingrowth and an outer layer of silastic that serves as a vapor and bacterial barrier [22]. It has been used to good eect in clean super®cial burns and donor sites. Hydrocolloid dressings as a group attempt to create a moist wound environment while absorbing wound exudate. A moist wound environment has been found to favor wound healing in experimental and clinical trials [23]. These membranes are generally intended for use on selected clean super®cial wounds and donor sites. All synthetic membranes are more or less occlusive. As such they must be used with caution if wounds are not clearly clean and super®cial. If placed over devitalized tissue, submembrane purulence can occur with potentially disastrous results [24]. 2.3. Allogenic temporary substitutes There are ®ve growth factors or families of growth factors that are known at present to play major roles in wound healing: epidermal growth factor, transforming growth factor-beta, insulin like growth factor, platelet derived growth factors and ®broblast growth factors [25, 26]. Various authors have reported the use of allogenic keratinocytes as temporary dressings on
R.L. Sheridan, R.G. Tompkins / Burns 25 (1999) 97±103
super®cial wounds and donor sites [27], although available data suggest that these cells persist for no more than 14 days. It is presumed that substances secreted by the allogeneic cells or released upon their death and dissolution, will provide signals to the host that enhance wound healing. Scienti®c support for this presumption remains elusive. Perhaps the ®rst group to attempt the manufacture of a biologic composite skin substitute to this end was that led by Bell, who developed a completely allogeneic dermal±epidermal product that used a collagen lattice as scaold for culturing both cell types [28]; in an athymic mouse model, this material was shown to successfully engraft [29, 30]. Although it has not been demonstrated to have a clinical role in burn patients, the device is being explored for utility in chronic ulcers of the lower extremity. Other examples of this concept include a composite cultured skin replacement consisting of cultured keratinocytes and ®broblasts seeded onto opposite sides of a bilaminar bovine collagen matrix. Such a device is being investigated as a dressing for second degree burns [31]; its ecacy in this setting remains speculative. Allogenic ®broblasts grown in a biodegradable mesh [32±34] or in the nylon inner layer of DowHickam's Biobrane1 [35±39] are also undergoing evaluation as potentiators of native wound healing. Genetic modi®cation of keratinocytes such that selected growth factors are secreted in excess is now possible, and may lead to directed topical application of speci®c substances in the future [40]. Split thickness human allograft, procured from organ and tissue donors remains the standard by which other temporary skin covers are judged [41±43]. This tissue is procured, processed, stored, distributed and tracked by skin banks. Although commonly used fresh, refrigerated for 7 days or less, it is quite eective when cryopreserved according to well established protocols designed to maximize cell viability. When viable split thickness allograft skin is applied to a clean excised wound, the tissue vascularizes and provides durable biologic cover until it is recognized a foreign by the host. This process, triggered by host recognition of highly antigenic epithelial elements, generally results in loss of the allogenic epithelium approximately 3 or 4 weeks after application in most burn patients. Prolongation of allograft survival can be achieved by administration of antirejection drugs [44], but this is not widely practiced for fear that such therapy will further compromise impaired host resistance to infection [45]. Fears that split thickness allograft skin will transmit viral diseases appear to have been in¯ated. When modern screening techniques are followed, the disease transmission risk appears to be exceptionally low, particularly in light of its highly eective results.
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3. Current permanent skin substitutes A useful permanent skin substitute remains the `holy grail' of burn research. Although no ideal substitute exists at present, there are a number of devices currently available that contribute to permanent coverage of burn wounds that will be discussed here. 3.1. Current epidermal substitutes In 1975, Rheinwald and Green demonstrated a very clever and eective method of culturing large numbers of epithelial cells from a small fresh sample of skin [46, 47]. A full thickness biopsy of skin is procured, minced and the epithelial cells separated with trypsin. The epithelial cell suspension is plated in culture dishes containing culture medium with fetal calf serum, insulin, transferrin, hydrocortisone, epidermal growth factor and cholera toxin; this overlying a layer of murine ®broblasts that have been treated with a non lethal dose of radiation that prevents them from multiplying. The murine ®broblasts are essential to inhibit human cells from growing, to provide insoluble matrix proteins that facilitate clonal expansion and perhaps to be a source of other growth factors. Isolated colonies of epithelial cells then expand into broad sheets of undierentiated epithelial cells. These cultures are treated with trypsin and the cells are taken to secondary culture using the same techniques. The resulting sheets are removed from the dishes after treatment with dipase, that digests the proteins attaching the epithelial cells to the dish. The sheets of epithelial cells are attached to petrolatum gauze for ease of handling. The potential application of this technology to burn patients with a paucity of donor sites was immediately apparent [48]; the concept was taken into the clinical setting shortly thereafter [49, 50]. This technology has been widely applied to patients with very large burns; the cells made available by hospital laboratories or commercial vendors. With more frequent use of this method of closing wounds, its liabilities have become more obvious [51, 52]. In the hands of most clinicians, when excised full thickness wounds are closed with epithelial cells alone, engraftment rates are suboptimal and long term durability is less than ideal. However, when faced with a very large wound and minimal donor sites, epithelial cell wound closure is a valuable adjunct to overall patient management. Recognition of the fragility of wounds closed with epithelial cells has lead to several attempts to combine epithelial cells with a functioning dermal layer, investigators feeling that this might improve initial engraftment rates and long term durability. Notable among these eorts are those by Cuono and coworkers who described a technique in which epithelial cells are grafted on wounds previously covered with vascular-
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ized allograft from which the allogenic epithelial cells have been removed by dermabrasion or tangential excision, leaving behind a vascularized but theoretically non-antigenic allogenic dermal layer [53±55]. Although appealing in concept and successful in the hands of some investigators [56, 57], the technique has not proved to be universally eective and has not been widely adopted. Perhaps the epithelial excision either leaves behind nests of antigenic epithelial cells if too super®cial or removes the epidermal±dermal attachment structures if too deep. Limited attempts have been made to combine epithelial cells with other dermal analogs (see below), but experience is as yet far too small to generate meaningful conclusions. 3.2. Current dermal substitutes Developed in the 1980's by Burke and Yannas, Integra1 arti®cial skin was recently released for general use after mass manufacture techniques were developed by Integra LifeSciences Corporation (Plainsboro, NJ) [58]. The research team strove to develop a membrane that would provide a temporary vapor and bacterial barrier to freshly excised wounds while putting in place a material that would serve as a scaold for later dermal regeneration. The inner layer of this material is a 2 mm thick combination of ®bers of collagen isolated from bovine tissue and the glycosaminoglycan chondroitin-6-sulfate which, after exhaustive testing, has a 70 to 200 mm pore size and structure that facilitates ®brovascular ingrowth from the host and then undergoes biodegradation [59, 60]. It's complicated manufacture involves precipitation of the glycosaminoglycan and collagen ®bers which are then freeze dried, cross-linked by gluteraldehyde and carefully washed. The outer layer is 0.009 inch polysiloxane polymer with vapor transmission characteristics that simulate normal epithelium. This material is designed to be placed on freshly excised full thickness burns and the outer silicone membrane replaced with an ultrathin epithelial autograft two to three weeks later [61]. Initial reports of this material were successful in both single [62, 63] and multicenter [64] trials. Post marketing trials of Integra1 arti®cial skin are in progress at this time. Although submembrane purulence must be watched for and promptly treated, the material is likely to play an important role in the management of serious burns. Polygalactin mesh seeded with allogenic ®broblasts has been explored as a dermal analog in burn wounds. The carrier material biodegrades by hydrolysis, inciting a minimal in¯ammatory reaction, while the allogenic ®broblasts are designed to help generate a neo-dermis. Designed to be combined with a thin autograft [65±67], it's utility in full thickness burns remains to be demonstrated.
An actively investigated dermal analog designed to contribute to permanent coverage of burn wounds is a cryopreserved allogenic dermis which is designed to be combined with a thin epithelial autograft, marketed as AlloDerm1 (LifeCell Corporation, The Woodlands, TX) [68, 69]. Split thickness skin allografts are procured from appropriately screened cadaver donors. Using hypertonic saline, the epithelial elements of the grafts are removed. The tissue is then treated in a detergent to inactivate any viruses and is freeze-dried. This process results in a theoretically non-antigenic complete dermal scaold with basement membrane proteins, including laminin and type IV and VII collagen, intact. The material is rehydrated in saline prior to application. It is designed to be combined with an ultrathin epithelial autograft at the time of initial wound closure. Clinical experience with this material in acute and reconstructive burn wounds is still early and limited, but appears favorable [70, 71]. 3.3. Composite substitutes Most investigators have come to the conclusion that the optimal skin substitute will provide for immediate replacement of both the lost dermis and epidermis. Not only are both elements required for optimal function and appearance, but there are poorly characterized but important interactions between dermal and epidermal elements; one enhancing the maturation of the other [72±75]. There have been a number of animal and human investigations conducted in this direction; yet a successful de®nitive composite substitute has not yet been characterized. Boyce and Hansbrough have produced a completely biologic composite skin substitute, culturing human ®broblasts in a collagen±glycosaminoglycan membrane and then growing keratinocytes upon this [76, 77]. Although this composite membrane would successfully engraft in a nude mouse model [78], engraftment rates were found to be suboptimal in a small clinical series [79]. Further investigations of this potentially exciting technology continue [80].
4. Future directions There are two particularly important areas in which signi®cant change may occur in this ®eld over the next few years: the development of temporary dressings containing growth factor secreting allogenic tissues that stimulate native wound healing and the realization of a permanent composite skin replacement. Other areas in which progress is likely to be seen include improvements in temporary skin substitutes and re®nements in skin banking techniques.
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Viral transfection techniques have evolved to the degree that genetic modi®cation of keratinocytes is possible. Keratinocytes are being engineered to over express platelet derived growth factor, human growth hormone, insulin like growth factor-1 and other growth factors [40]. It is likely that they will be trialed in animal models and human wounds over the next few years [81]. If they prove ecacious, their application to healing burns and donor sites may prove valuable in accelerating natural healing processes. Further, composite skin substitutes populated by chimeric epidermal combinations of autogeneic epithelial cells with genetically modi®ed allogeneic cells might be possible; such a combination possibly resulting in enhanced engraftment secondary to transient overexpression of critical growth factors during the early stages of graft vascularization. It is generally hoped that a reliable and durable permanent composite skin substitute is within the realm of possibility sometime in the next decade. Just what form that substitute will take is not clear. A promising approach is to combine cultured autologous keratinocytes with one of the currently available dermal analogs; either on the patient or in the laboratory prior to engraftment. Although limited initial anecdotal experiences with combinations of cultured keratinocytes and Integra1, AlloDerm1, polygalactin mesh, human allogenic dermis and other dermal analogs are not encouraging, the concept has great potential promise. This important avenue remains to be systematically studied. Active investigations are underway in laboratories around the world. It is only a matter of time before a successful approach to this important clinical problem is arrived at by one of the many enthusiastic investigators who have chosen to direct their eorts to the well being of our patients.
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epidermal sheet graft containing human keratinocytes on athymic mice. J. Invest. Dermatol. 1993;101:811±9. Woodley DT, Peterson HD, Herzog SR et al. Burn wounds resurfaced by cultured epidermal autografts show abnormal reconstitution of anchoring ®brils. JAMA 1988;259:2566±71. Coulomb B, Lebreton C, Dubertret L. In¯uence of human dermal ®broblasts on epidermalization. J. Invest. Dermatol. 1989;92:122±5. Boyce ST, Christianson DJ, Hansbrough JF. Structure of a collagen±GAG dermal skin substitute optimized for cultured human epidermal keratinocytes. J. Biomed. Mater. Res. 1988;22:939±57. Boyce ST, Hansbrough JF. Biologic attachment, growth, and dierentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate. Surgery 1988;103:421±31.
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[78] Cooper ML, Hansbrough JF. Use of a composite skin graft composed of cultured human keratinocytes and ®broblasts and a collagen±GAG matrix to cover full-thickness wounds on athymic mice. Surgery 1991;109:198±207. [79] Hansbrough JF, Boyce ST, Cooper ML, Foreman TJ. Burn wound closure with cultured autologous keratinocytes and ®broblasts attached to a collagen±glycosaminoglycan substrate. JAMA 1989;262:2125±30. [80] Hansbrough JF, Morgan JL, Greenleaf GE, Bartel R. Composite grafts of human keratinocytes grown on a polyglactin mesh-cultured ®broblast dermal substitute function as a bilayer skin replacement in full-thickness wounds on athymic mice. J. Burn Care Rehab. 1993;14:485±94. [81] Morgan JR, Yarmush ML. Bioengineered skin substitutes. Sci. Med. 1997;July/August:6±15.
Skin Substitutes in Burns Robert L. Sheridan a, b, c, *, Ronald G. Tompkins a, b, c a
Shriners Burns Hospital, 51 Blossom Street, Boston, MA 02114, USA b Trauma and Burn Services, Massachusetts General Hospital, USA c Department of Surgery, Harvard Medical School, USA Accepted 27 May 1998
1. Introduction The natural history of burn injury has been substantially modi®ed over the past few decades. The ®rst recorded recognition of the exaggerated ¯uid requirements of burn patients was probably that of Underhill in 1930 [1]. After this important clinical observation, investigations began to develop methods to manage burn shock. In the aftermath of the Coconut Grove ®re, Moore and colleagues [2] re®ned the concept of burn resuscitation and proposed a formula for intravenous volume repletion based on body weight [3], and in the 1950's the Evans formula was promulgated by the sta at the United States Army Institute of Surgical Research [4]. Subsequent re®nements in burn shock resuscitation have virtually eliminated this as a cause of death. In the 1970's, the advantage of early excision and closure of small burn wounds was recognized [5]. This excisional approach was subsequently taken to patients with large injuries by Burke and others [6±8] who documented truncated hospital stays and enhanced survival in burn patients who were routinely expected to die; a burn over more than a third of the body surface being almost universally lethal at that time. Re®nement of the surgical approaches to large wounds combined with the ongoing evolution of critical care techniques has extended our ability to support patients with increasingly severe injuries through the physiologic trial of staged wound closure. Burn physical and occupational therapy and burn reconstruction have developed in parallel, facilitating our ability to deliver increasingly satisfying long term outcomes. However, further progress is seriously impaired by our lack of a suitable skin substitute. In
* Corresponding author. Tel.: +1-617-726-5633; Fax: +1-617-3678936; E-mail: [email protected]
both the acutely injured and those requiring extensive post burn reconstruction, the absence of a durable skin substitute regularly hinders recovery. The successful development of a permanent skin substitute will have an enormous impact on the care of patients with serious burns. Skin is a complex organ. Functionally, it has two layers with a highly specialized and eective bonding mechanism. The epidermis, consisting of the strata basale, spinosum, granulosum and corneum, provides a vapor and bacterial barrier. The dermis provides strength and elasticity. The thin epidermal layer is constantly replacing itself from its basal layer, with new keratinocytes undergoing terminal dierentiation over approximately 4 weeks to anuclear keratin ®lled cells that make up the stratum corneum, which provides much of the barrier function of the epidermis. The basal layer of the epidermis is ®rmly attached to the dermis by a complex bonding mechanism containing collagen types IV and VII. When this bond fails, serious morbidity results, as demonstrated by the disease processes of toxic epidermal necrolysis [9] and dystrophic epidermolysis bullosa [10]. At present, most full thickness burn wounds are best closed as quickly as possible with split thickness autograft. However, split thickness autograft is an imperfect replacement for full thickness skin, may be limited in quantity and is associated with donor site morbidity. The ideal skin substitute (Table 1): (1) is inexpensive, (2) has a long shelf life, (3) is used o the shelf, (4) is non-antigenic, (5) is durable, (6) is ¯exible, (7) prevents water loss, (8) is a barrier to bacteria, (9) conforms to irregular wound surfaces, (10) is easy to secure, (11) grows with children, (12) is applied in one operation, (13) does not become hypertrophic and (14) does not exist at the present time [11, 12]. Conceptually, skin substitutes are temporary or permanent; epidermal, dermal or composite; and biologic or synthetic. Biologic components are xenogeneic, allo-
0305-4179/99/$19.00 + 0.00 # 1999 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 9 8 ) 0 0 1 7 6 - 4
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Table 1 Characteristics of the ideal skin substitute (1) Inexpensive (2) Long shelf life (3) Used o the shelf (4) Non-antigenic (5) Durable (6) Flexible (7) Prevents water loss (8) Bacterial barrier (9) Drapes well (10) Easy to secure (11) Grows with child (12) Applied in one operation (13) Does not become hypertrophic (14) Does not yet exist
genic or autogenic. There is a research eort centered on many of the possible permutations of these traits. From a practical perspective, the current reality is that skin substitutes are designed to be temporary or permanent. This manuscript will attempt to review the uses and formulations of temporary and permanent substitutes now available and will speculate on future directions in skin substitute research. Whenever possible, proprietary product names will not be used; any products not mentioned are not purposefully excluded. 2. Current temporary substitutes Temporary skin substitutes provide transient physiologic wound closure. Physiologic wound closure implies a degree of protection from mechanical trauma, vapor transmission characteristics similar to skin and a physical barrier to bacteria. These membranes therefore contribute to the creation of a moist wound environment with a low bacterial density. There are four common uses for temporary skin substitutes: (1) as a dressing on donor sites to facilitate pain control and epithelialization from skin appendages, (2) as a dressing on clean super®cial wounds to a similar end, (3) to provide temporary physiologic closure of deep dermal and full thickness wounds after excision while awaiting autografting or healing of underlying widely meshed autografts and (4) as a `test' graft in questionable wound beds. There are a large number of such membranes in common use, classes of which will be described below. 2.1. Porcine xenograft Xenografts have been used as intact split thickness grafts from a variety of species for many years to pro-
vide temporary cover of wounds [13]. The most commonly utilized species employed in this fashion has been domestic swine [14]. Porcine xenograft is often used as a reconstituted product consisting of homogenized porcine dermis which is fashioned into sheets and meshed [15]. It is widely used for temporary coverage of clean wounds such as super®cial second degree burns and donor sites [16]. Its use has been favorably reported in patients with toxic epidermal necrolysis [9, 17] and it has been combined with silver to suppress wound colonization [18, 19]. Although it does not vascularize, it will adhere to a clean super®cial wound and can provide excellent pain control while the underlying wound epithelializes. It is applied to a cleansed wound and covered with a dry dressing, no sutures being required. 2.2. Synthetic membranes There exist a number of semipermeable membrane dressings of several proprietary types that are designed to provide a vapor and bacterial barrier and control pain while the underlying super®cial wound or donor site re-epithelializes. A number of single layer semi-permeable synthetic membranes exist that provide a mechanical barrier to bacteria and have physiologic vapor transmission characteristics [20, 21]. These can be used with good eect on clean super®cial wounds and split thickness donor sites. There exists a single bilayer synthetic membrane in general use at present. This product, Biobrane1 (Dow-Hickham, Sugarland, TX) consists of an inner layer of nylon mesh that allows ®brovascular ingrowth and an outer layer of silastic that serves as a vapor and bacterial barrier [22]. It has been used to good eect in clean super®cial burns and donor sites. Hydrocolloid dressings as a group attempt to create a moist wound environment while absorbing wound exudate. A moist wound environment has been found to favor wound healing in experimental and clinical trials [23]. These membranes are generally intended for use on selected clean super®cial wounds and donor sites. All synthetic membranes are more or less occlusive. As such they must be used with caution if wounds are not clearly clean and super®cial. If placed over devitalized tissue, submembrane purulence can occur with potentially disastrous results [24]. 2.3. Allogenic temporary substitutes There are ®ve growth factors or families of growth factors that are known at present to play major roles in wound healing: epidermal growth factor, transforming growth factor-beta, insulin like growth factor, platelet derived growth factors and ®broblast growth factors [25, 26]. Various authors have reported the use of allogenic keratinocytes as temporary dressings on
R.L. Sheridan, R.G. Tompkins / Burns 25 (1999) 97±103
super®cial wounds and donor sites [27], although available data suggest that these cells persist for no more than 14 days. It is presumed that substances secreted by the allogeneic cells or released upon their death and dissolution, will provide signals to the host that enhance wound healing. Scienti®c support for this presumption remains elusive. Perhaps the ®rst group to attempt the manufacture of a biologic composite skin substitute to this end was that led by Bell, who developed a completely allogeneic dermal±epidermal product that used a collagen lattice as scaold for culturing both cell types [28]; in an athymic mouse model, this material was shown to successfully engraft [29, 30]. Although it has not been demonstrated to have a clinical role in burn patients, the device is being explored for utility in chronic ulcers of the lower extremity. Other examples of this concept include a composite cultured skin replacement consisting of cultured keratinocytes and ®broblasts seeded onto opposite sides of a bilaminar bovine collagen matrix. Such a device is being investigated as a dressing for second degree burns [31]; its ecacy in this setting remains speculative. Allogenic ®broblasts grown in a biodegradable mesh [32±34] or in the nylon inner layer of DowHickam's Biobrane1 [35±39] are also undergoing evaluation as potentiators of native wound healing. Genetic modi®cation of keratinocytes such that selected growth factors are secreted in excess is now possible, and may lead to directed topical application of speci®c substances in the future [40]. Split thickness human allograft, procured from organ and tissue donors remains the standard by which other temporary skin covers are judged [41±43]. This tissue is procured, processed, stored, distributed and tracked by skin banks. Although commonly used fresh, refrigerated for 7 days or less, it is quite eective when cryopreserved according to well established protocols designed to maximize cell viability. When viable split thickness allograft skin is applied to a clean excised wound, the tissue vascularizes and provides durable biologic cover until it is recognized a foreign by the host. This process, triggered by host recognition of highly antigenic epithelial elements, generally results in loss of the allogenic epithelium approximately 3 or 4 weeks after application in most burn patients. Prolongation of allograft survival can be achieved by administration of antirejection drugs [44], but this is not widely practiced for fear that such therapy will further compromise impaired host resistance to infection [45]. Fears that split thickness allograft skin will transmit viral diseases appear to have been in¯ated. When modern screening techniques are followed, the disease transmission risk appears to be exceptionally low, particularly in light of its highly eective results.
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3. Current permanent skin substitutes A useful permanent skin substitute remains the `holy grail' of burn research. Although no ideal substitute exists at present, there are a number of devices currently available that contribute to permanent coverage of burn wounds that will be discussed here. 3.1. Current epidermal substitutes In 1975, Rheinwald and Green demonstrated a very clever and eective method of culturing large numbers of epithelial cells from a small fresh sample of skin [46, 47]. A full thickness biopsy of skin is procured, minced and the epithelial cells separated with trypsin. The epithelial cell suspension is plated in culture dishes containing culture medium with fetal calf serum, insulin, transferrin, hydrocortisone, epidermal growth factor and cholera toxin; this overlying a layer of murine ®broblasts that have been treated with a non lethal dose of radiation that prevents them from multiplying. The murine ®broblasts are essential to inhibit human cells from growing, to provide insoluble matrix proteins that facilitate clonal expansion and perhaps to be a source of other growth factors. Isolated colonies of epithelial cells then expand into broad sheets of undierentiated epithelial cells. These cultures are treated with trypsin and the cells are taken to secondary culture using the same techniques. The resulting sheets are removed from the dishes after treatment with dipase, that digests the proteins attaching the epithelial cells to the dish. The sheets of epithelial cells are attached to petrolatum gauze for ease of handling. The potential application of this technology to burn patients with a paucity of donor sites was immediately apparent [48]; the concept was taken into the clinical setting shortly thereafter [49, 50]. This technology has been widely applied to patients with very large burns; the cells made available by hospital laboratories or commercial vendors. With more frequent use of this method of closing wounds, its liabilities have become more obvious [51, 52]. In the hands of most clinicians, when excised full thickness wounds are closed with epithelial cells alone, engraftment rates are suboptimal and long term durability is less than ideal. However, when faced with a very large wound and minimal donor sites, epithelial cell wound closure is a valuable adjunct to overall patient management. Recognition of the fragility of wounds closed with epithelial cells has lead to several attempts to combine epithelial cells with a functioning dermal layer, investigators feeling that this might improve initial engraftment rates and long term durability. Notable among these eorts are those by Cuono and coworkers who described a technique in which epithelial cells are grafted on wounds previously covered with vascular-
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ized allograft from which the allogenic epithelial cells have been removed by dermabrasion or tangential excision, leaving behind a vascularized but theoretically non-antigenic allogenic dermal layer [53±55]. Although appealing in concept and successful in the hands of some investigators [56, 57], the technique has not proved to be universally eective and has not been widely adopted. Perhaps the epithelial excision either leaves behind nests of antigenic epithelial cells if too super®cial or removes the epidermal±dermal attachment structures if too deep. Limited attempts have been made to combine epithelial cells with other dermal analogs (see below), but experience is as yet far too small to generate meaningful conclusions. 3.2. Current dermal substitutes Developed in the 1980's by Burke and Yannas, Integra1 arti®cial skin was recently released for general use after mass manufacture techniques were developed by Integra LifeSciences Corporation (Plainsboro, NJ) [58]. The research team strove to develop a membrane that would provide a temporary vapor and bacterial barrier to freshly excised wounds while putting in place a material that would serve as a scaold for later dermal regeneration. The inner layer of this material is a 2 mm thick combination of ®bers of collagen isolated from bovine tissue and the glycosaminoglycan chondroitin-6-sulfate which, after exhaustive testing, has a 70 to 200 mm pore size and structure that facilitates ®brovascular ingrowth from the host and then undergoes biodegradation [59, 60]. It's complicated manufacture involves precipitation of the glycosaminoglycan and collagen ®bers which are then freeze dried, cross-linked by gluteraldehyde and carefully washed. The outer layer is 0.009 inch polysiloxane polymer with vapor transmission characteristics that simulate normal epithelium. This material is designed to be placed on freshly excised full thickness burns and the outer silicone membrane replaced with an ultrathin epithelial autograft two to three weeks later [61]. Initial reports of this material were successful in both single [62, 63] and multicenter [64] trials. Post marketing trials of Integra1 arti®cial skin are in progress at this time. Although submembrane purulence must be watched for and promptly treated, the material is likely to play an important role in the management of serious burns. Polygalactin mesh seeded with allogenic ®broblasts has been explored as a dermal analog in burn wounds. The carrier material biodegrades by hydrolysis, inciting a minimal in¯ammatory reaction, while the allogenic ®broblasts are designed to help generate a neo-dermis. Designed to be combined with a thin autograft [65±67], it's utility in full thickness burns remains to be demonstrated.
An actively investigated dermal analog designed to contribute to permanent coverage of burn wounds is a cryopreserved allogenic dermis which is designed to be combined with a thin epithelial autograft, marketed as AlloDerm1 (LifeCell Corporation, The Woodlands, TX) [68, 69]. Split thickness skin allografts are procured from appropriately screened cadaver donors. Using hypertonic saline, the epithelial elements of the grafts are removed. The tissue is then treated in a detergent to inactivate any viruses and is freeze-dried. This process results in a theoretically non-antigenic complete dermal scaold with basement membrane proteins, including laminin and type IV and VII collagen, intact. The material is rehydrated in saline prior to application. It is designed to be combined with an ultrathin epithelial autograft at the time of initial wound closure. Clinical experience with this material in acute and reconstructive burn wounds is still early and limited, but appears favorable [70, 71]. 3.3. Composite substitutes Most investigators have come to the conclusion that the optimal skin substitute will provide for immediate replacement of both the lost dermis and epidermis. Not only are both elements required for optimal function and appearance, but there are poorly characterized but important interactions between dermal and epidermal elements; one enhancing the maturation of the other [72±75]. There have been a number of animal and human investigations conducted in this direction; yet a successful de®nitive composite substitute has not yet been characterized. Boyce and Hansbrough have produced a completely biologic composite skin substitute, culturing human ®broblasts in a collagen±glycosaminoglycan membrane and then growing keratinocytes upon this [76, 77]. Although this composite membrane would successfully engraft in a nude mouse model [78], engraftment rates were found to be suboptimal in a small clinical series [79]. Further investigations of this potentially exciting technology continue [80].
4. Future directions There are two particularly important areas in which signi®cant change may occur in this ®eld over the next few years: the development of temporary dressings containing growth factor secreting allogenic tissues that stimulate native wound healing and the realization of a permanent composite skin replacement. Other areas in which progress is likely to be seen include improvements in temporary skin substitutes and re®nements in skin banking techniques.
R.L. Sheridan, R.G. Tompkins / Burns 25 (1999) 97±103
Viral transfection techniques have evolved to the degree that genetic modi®cation of keratinocytes is possible. Keratinocytes are being engineered to over express platelet derived growth factor, human growth hormone, insulin like growth factor-1 and other growth factors [40]. It is likely that they will be trialed in animal models and human wounds over the next few years [81]. If they prove ecacious, their application to healing burns and donor sites may prove valuable in accelerating natural healing processes. Further, composite skin substitutes populated by chimeric epidermal combinations of autogeneic epithelial cells with genetically modi®ed allogeneic cells might be possible; such a combination possibly resulting in enhanced engraftment secondary to transient overexpression of critical growth factors during the early stages of graft vascularization. It is generally hoped that a reliable and durable permanent composite skin substitute is within the realm of possibility sometime in the next decade. Just what form that substitute will take is not clear. A promising approach is to combine cultured autologous keratinocytes with one of the currently available dermal analogs; either on the patient or in the laboratory prior to engraftment. Although limited initial anecdotal experiences with combinations of cultured keratinocytes and Integra1, AlloDerm1, polygalactin mesh, human allogenic dermis and other dermal analogs are not encouraging, the concept has great potential promise. This important avenue remains to be systematically studied. Active investigations are underway in laboratories around the world. It is only a matter of time before a successful approach to this important clinical problem is arrived at by one of the many enthusiastic investigators who have chosen to direct their eorts to the well being of our patients.
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