- Email: [email protected]
Initial experience with a composite autologous skin substitute
Burns 27 (2001) 421– 424 www.elsevier.com/locate/burns
Initial experience with a composite autologous skin substitute Robert L. Sheridan a,*, Jeffrey R. Morgan b, Jennifer L. Cusick b, Lisa M. Petras a, Martha M. Lydon a, Ronald G. Tompkins a b
a Shriners Burns Hospital, 51 Blossom street, Boston, MA 02114, USA Department of Surgery, Surgical Ser6ices, Har6ard Medical School, Massachusetts General Hospital, Boston, MA, USA
Accepted 28 November 2000
Abstract Patients with large burns are surviving in increasing numbers, but there remains no durable and reliable permanent skin replacement. After initial favorable small animal experiments, a pilot trial of a composite skin replacement was performed in patients with massive burns. A composite skin replacement (CSR) was developed by culturing autologous keratinocytes on acellular allogenic dermis. This material was engrafted in patients with massive burns and compared to a matched wound covered with split thickness autograft. With human studies committee approval, 12 wounds in 7 patients were grafted with CSR while a matched control wound was covered with split thickness autograft. These 7 children had an average age of 6.4 9 1.4 yr and burn size of 75.9 95.0% of the body surface. Nine wounds were acute burns and three were reconstructive releases. Successful vascularization at 14 days averaged 45.7 9 14.2% (range 0 – 100%) in the study wounds and 98 9 1% (range 90 – 100%) in the control sites (P B0.05). Reduced CSR take seemed to correlate with wound colonization. All children survived. While CSR did not engraft with the reliability of standard autograft, this pilot experience is encouraging in that successful wound closure with this material is possible, if not yet dependable. It is hoped that a more mature epidermal layer may facilitate engraftment, and trials to explore this possibility are in progress. © 2001 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Skin substitutes; Burns; Keratinocytes
1. Introduction Despite the well-described liabilities of donor site morbidity and graft hypertrophy, split thickness autograft remains the standard of care for resurfacing acute full thickness burns and for closing defects associated with burn reconstructive procedures [1]. Donor site morbidity includes pain, healing delays, infection and hypertrophic scarring and relates directly with thickness of harvest. Graft hypertrophy is felt to relate directly to thickness of transplanted dermis [2]. Both of these liabilities must be accepted when performing acute and reconstructive burn surgery in those with limited donor sites. Presented at the Annual Meeting of the American Burn Association, March 2000, Las Vegas supported by the Shriners Hospitals for Children. * Corresponding author. Tel.: + 1-617-726-5633; fax: + 1-617-3678936. E-mail address: [email protected] (R.L. Sheridan).
Human allograft is routinely used as temporary cover of excised burn wounds. These grafts generally vascularize within three days, but are subsequently rejected in two or three weeks [3]. The most immunogenic components of transplanted allograft skin are the cellular elements of the epidermis and dermis [4]. If these components are removed, the remaining non-cellular dermal tissue is relatively immunologically inert [5]. Acellular bone grafts have been routinely used in oral and orthopedic surgery for several years [6], lending credence to the claim that such tissues will maintain their structural integrity over time, serving as a scaffold for ingrowth of host cellular elements, resulting in a normally organized tissue. The use of allogenic dermis in combination with autogenous cultured epithelial cells as permanent coverage for full thickness burn wounds has been explored [7–9]. However, this technique requires excision of the upper dermis with sacrifice of epithelial attachment mechanisms [10], perhaps explaining the fragility of the resulting cover [11].
0305-4179/01/$20.00 © 2001 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 0 0 ) 0 0 1 5 6 - X
422
R.L. Sheridan et al. / Burns 27 (2001) 421–424
The clinical problem of the inadequate donor site is particularly acute in patients with very large burns, in whom there is simply not enough donor site available to address their acute coverage and reconstructive needs. In this group, an innovative approach to wound closure has lifesaving potential, both in the setting of the acute injury and in facilitating a more normal life by making enough skin available for reconstructive needs. Previous work in our laboratory with human keratinocytes cultured onto acellular dermis [12] has shown promise, prompting this pilot trial in humans.
2. Methods
2.1. General methods We enrolled seven children with massive burns and less than 25% of the body surface available for skin harvest who required either acute resurfacing or a reconstructive procedure requiring split thickness autografting in a human studies committee approved protocol. A total of 12 wounds were grafted in these children with the composite skin replacement (CSR), simultaneously grafting a randomly assigned control area with the autograft. Study and control sites were adjacent portions of the same wound, the study site being initially the more cephalic or right, assignments being alternated in sequential patients. Reconstructive wounds were generated by incisional release. Acute excisional wounds were generated by layered excision of full thickness burn wounds, generally to viable subcutaneous fat or by removal of vascularized allograft.
2.2. Study endpoints Study endpoints were (1) engraftment (in %) at seven days, (2) engraftment (in %) at 30 days, (3) the need for regrafting, and (4) Vancouver Scar Scores [13] at 1, 3, 6, and 12 months. This is a scoring system with which we are very practiced and which has shown excellent inter rater reliability in our institution [14]. Study and control site results were compared by t-test.
2.3. Preparation of composite skin replacement Normal human keratinocytes were isolated from full thickness skin biopsies and cultured using the fibroblast feeder layer method of Rheinwald and Green, the original specimen coming from a 1.0× 0.5 cm2 skin biopsy obtained approximately three weeks prior to the study [15]. Keratinocytes were provided in the pre-confluent phase by Genzyme Tissue Repair, Inc. of Cambridge, MA. Keratinocytes were co-cultivated with 3T3-J2 mouse fibroblasts, pretreated with 15 mg/ml mitomycin C to limit the proliferation of the 3T3 cells. Kerati-
nocyte culture medium (KCM) was a 3:1 mixture of Dulbecco’s Modified Eagle’s medium (DMEM) (high glucose) and Ham’s F12 medium; supplemented with 10% fetal bovine serum; adenine, 1.8× 104 M; cholera toxin, 10 − 10 M; hydrocortisone, 0.4 mg/ml; insulin, 5 mg/ml; transferrin, 5 mg/ml; triiodo-L-thyronine, 2× 10 − 9 M, and penicillin– streptomycin, 100 IU/ml-100 mg/ml and was changed every 3–4 days. Beginning with the first medium change, EGF was added at 10 ng/ml. Cultures were incubated in a humidified 10% CO2 atmosphere at 37°C. Cells were subcultured by first removing the feeder layer cells with an EDTA wash (5 mM in PBS), then treating the keratinocytes with trypsin-EDTA and re-seeding them on flasks containing mitomycin C treated 3T3-J2 mouse fibroblasts. To prepare and evaluate composite grafts in vitro, allogenic dermis, from which all cellular elements were removed, was placed into a tissue culture dish, papillary side up prior to adding keratinocytes. This dermal analog was prepared by soaking split thickness human allograft, properly screened, in sterile phosphate buffered saline supplemented with gentamycin (100 mcg/ml), ciprofloxacin (10 mcg/ml), amphotericin B (2.5 mcg/ml), penicillin (100 IU/ml) and streptomycin (100 mcg/ml) for one week at 37°C and then carefully stripping off the alloepithelial layer. This dermal analog was maintained at 4°C for one month in PBS supplemented with antibiotics. The papillary surface of each piece of allogenic dermis was seeded with cultured keratinocytes in KCM (2.5×105 cells/ml, in triplicate). Composite grafts were maintained submerged for one week. Grafts were then washed in DMEM, transported to the operating room in an airtight container containing 10% carbon dioxide and then grafted using standard surgical technique.
3. Results With human studies committee approval, 12 wounds in 7 patients were grafted with CSR while a matched control wound was covered with split thickness autograft. The 7 children had an average age of 6.491.4 yr and burn size of 75.99 5.0% of the body surface. Nine of the 12 wounds were acute burns and 3 were reconstructive releases. Successful vascularization at 14 days averaged 45.7914.2% (range 0–100%) in the study wounds and 989 1% (range 90–100%) in the control sites (PB0.05). Wounds were followed for an average of 8 months, 6 wounds for 12 months. Evaluation with serial Vancouver Scar Scores between study and control sites revealed no differences at any time point: study and control Vancouver scores being 1.490.4 and 1.39 0.4 at one month, 1.890.5 and 1.69 0.6 at 3 months, 1.690.5 and 1.09 0.8 at 9 months, and 1.29 0.7 and 1.09 0.4 at 12 months. Reduced CSR take
R.L. Sheridan et al. / Burns 27 (2001) 421–424
seemed to correlate with bacterial wound colonization. All enrolled children survived and have been discharged home. The study material did not contribute to their survival given the small areas grafted in this pilot trial.
4. Discussion Refinement of the surgical approaches to large wounds combined with the ongoing evolution of critical care techniques has extended our ability to salvage the lives of those with large deep burns. 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. The development of a permanent skin substitute will have an enormous impact on the care of patients with serious burns. Skin consists of two layers with a highly effective bonding mechanism. The epidermis, consisting of the strata basale, spinosum, granulosum and corneum, provides a vapor and bacterial barrier. The dermis provides elasticity and strength. The thin epidermal layer is constantly replacing itself from its basal layer, with new keratinocytes undergoing terminal differentiation over approximately 4 weeks to anuclear keratin filled cells that make up the stratum corneum, which provides much of the barrier function of the epidermis. The basal layer of the epidermis is firmly attached to the dermis by a complex bonding mechanism involving proteins such as collagen types IV and VII. When this bond fails, serious morbidity results, as demonstrated by the disease processes of toxic epidermal necrolysis and epidermolysis bullosa [16,17]. An ideal skin substitute should have most, if not all, of the following properties: (1) low cost, (2) long shelf life, (3) use off the shelf, (4) non-antigenic, (5) durable, (6) flexible, (7) vapor transmission characteristics mimic epithelium, (8) effective barrier to bacteria, (9) conforms to irregular wound surfaces, (10) easy to secure, (11) grows with children, (12) applied in one operation and (13) does not become hypertrophic [3,18]. It seems likely that the successful permanent skin substitute will be a composite containing some autogenic elements. Not only are both dermal and epidermal elements required for optimal function and appearance, but there are poorly characterized but important interactions between them; one enhancing the maturation of the other [10,19–21]. There are several exciting animal and human investigations going on in this area, yet a successful and durable composite substitute remains an elusive goal. Notable among these is the work by Boyce and Hansbrough, who have produced a completely biologic composite skin substitute, culturing human fibroblasts in a
423
collagen-glycosaminoglycan membrane and then growing keratinocytes upon this [22,23]. Successful in a nude mouse model [24], this device is in clinical trials [25,26]. Another exciting avenue of investigation is described by combination of cultured autologous keratinocytes with one of the currently available dermal analogs. These combinations are produced either on the patient or in the laboratory prior to engraftment. Although limited initial anecdotal experiences with combinations of cultured keratinocytes and IntegraR (Integra Life Sciences, Plainsboro, NJ), AllODermR (LifeCell, The Woodlands, TX) polyglactin mesh, human allogenic dermis and other dermal analogs have not yet demonstrated reliable results, the concept has great appeal [8,27]. Addition of allogenic or autogenic keratinocytes modified by viral transfection to over produce important growth factors may enhance the eventual success of this approach [28]. While the CSR reported here did not engraft with the reliability of split thickness autograft, this pilot experience is encouraging in that successful wound closure with this material is possible, if not yet dependable. It is hoped that a more mature epidermal layer may facilitate engraftment, and trials to evaluate this are in progress.
References [1] Sheridan RL, Tompkins RG, Burke JF. Management of burn wounds with prompt excision and immediate closure. J Intensive Care Med 1994;9:6 –19. [2] Klein L, Rudolph R. 3 H-collagen tumover in skin grafts. Surgery Gynecol Obstet 1972;135:49 – 57. [3] Pruitt BA Jr., Levine NS. Characteristics and uses of biologic dressings and skin substitutes. Arch Surgery 1984;119:312 – 22. [4] Sedmak DD, Orosz CG. The role of vascular endothelial cells in transplantation. Arch Pathol Lab Med 1991;115:260 – 5. [5] Yukna RA, Turner DW, Robinson LJ. Variable antigenicity of lyophilized allogeneic and lyophilized xenogeneic skin in guinea pigs. J Periodont Res 1977;12:197 – 203. [6] Mellonig JT. Freeze-dried bone allografts in periodontal reconstructive surgery. Dental Clinics N Am 1991;35:505 – 20. [7] Compton CC, Hickerson W, Nadire K, Press W. Acceleration of skin regeneration from cultured epithelial autografts by transplantation to homograft dermis. J Burn Care Rehabil 1993;14:653 – 62. [8] Cuono CB, Langdon R, Birchall N, Barttelbort S, McGuire J. Composite autologous-allogeneic skin replacement: development and clinical application. Plastic Reconstruct Surgery 1987;80:626 – 37. [9] Hickerson WL, Compton C, Fletchall S, Smith LR. Cultured epidermal autografts and allodermis combination for permanent burn wound coverage. Burns 1994;20(Suppl 1):S52 – 5. [10] Woodley DT, Peterson HD, Herzog SR, et al. Burn wounds resurfaced by cultured epidermal autografts show abnormal reconstitution of anchoring fibrils. JAMA 1988;259:2566 –71. [11] Sheridan RL, Tompkins RG. Cultured autologous epithelium in patients with burns of ninety percent or more of the body surface. J Trauma 1995;38:48 – 50.
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[12] Medalie DA, Eming SA, Tompkins RG, Yarmush ML, Krueger GG, Morgan JR. Evaluation of human skin reconstituted from composite grafts of cultured keratinocytes and human acellular dermis transplanted to athymic mice. J Invest Dermatol 1996;107:121 – 7. [13] Sullivan T, Smith J, Kermode J, McIver E, Courtemanche DJ. Rating the burn scar. J Burn Care Rehabil 1990;11:256 – 60. [14] Baryza MJ, Baryza GA. The Vancouver Scar Scale: an administration tool and its interrater reliability. J Burn Care Rehabil 1995;16:535 – 8. [15] Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331 –43. [16] Heimbach DM, Engrav LH, Marvin JA, Harnar TJ, Grube BJ. Toxic epidermal necrolysis. A step forward in treatment. JAMA 1987;257:2171 – 5. [17] Fine JD, Johnson LB, Cronce D, et al. Intracytoplasmic retention of type VII collagen and dominant dystrophic epidermolysis bullosa: reversal of defect following cessation of or marked improvement in disease activity. J Invest Dermatol 1993;101:232 – 6. [18] Tompkins RG, Burke JF. Burn wound closure using permanent skin replacement materials. World J Surgery 1992;16:47 – 52. [19] Briggaman RA, Wheeler CE Jr. The epidermal –dermal junction. J Invest Dermatol 1975;65:71 –84. [20] Cooper ML, Andree C, Hansbrough JF, Zapata-Sirvent RL, Spielvogel RL. Direct comparison of a cultured composite skin substitute containing human keratinocytes and fibroblasts to an epidermal sheet graft containing human keratinocytes on athymic mice. J Invest Dermatol 1993;101:811 – 9.
.
[21] Coulomb B, Lebreton C, Dubertret L. Influence of human dermal fibroblasts on epidermalization. J Invest Dermatol 1989;92:122 – 5. [22] 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. [23] Boyce ST, Hansbrough JF. Biologic attachment, growth, and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate. Surgery 1988;103:421 – 31. [24] 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. [25] Hansbrough JF, Boyce ST, Cooper ML, Foreman TJ. Burn wound closure with cultured autologous keratinocytes and fibroblasts attached to a collagen-glycosaminoglycan substrate. JAMA 1989;262:2125 – 30. [26] Hansbrough JF, Morgan JL, Greenleaf GE, Bartel R. Composite grafts of human keratinocytes grown on a polyglactin mesh-cultured fibroblast dermal substitute function as a bilayer skin replacement in full-thickness wounds on athymic mice. J Burn Care Rehabil 1993;14:485 – 94. [27] Sheridan RL, Choucair RJ. Acellular allogenic dermis does not hinder initial engraftment in burn wound resurfacing and reconstruction. J Burn Care Rehabil 1997;18:496 – 9. [28] Sheridan RL, Tompkins RG. Skin substitutes in burns. Burns 1999;25:97 – 103.
Initial experience with a composite autologous skin substitute Robert L. Sheridan a,*, Jeffrey R. Morgan b, Jennifer L. Cusick b, Lisa M. Petras a, Martha M. Lydon a, Ronald G. Tompkins a b
a Shriners Burns Hospital, 51 Blossom street, Boston, MA 02114, USA Department of Surgery, Surgical Ser6ices, Har6ard Medical School, Massachusetts General Hospital, Boston, MA, USA
Accepted 28 November 2000
Abstract Patients with large burns are surviving in increasing numbers, but there remains no durable and reliable permanent skin replacement. After initial favorable small animal experiments, a pilot trial of a composite skin replacement was performed in patients with massive burns. A composite skin replacement (CSR) was developed by culturing autologous keratinocytes on acellular allogenic dermis. This material was engrafted in patients with massive burns and compared to a matched wound covered with split thickness autograft. With human studies committee approval, 12 wounds in 7 patients were grafted with CSR while a matched control wound was covered with split thickness autograft. These 7 children had an average age of 6.4 9 1.4 yr and burn size of 75.9 95.0% of the body surface. Nine wounds were acute burns and three were reconstructive releases. Successful vascularization at 14 days averaged 45.7 9 14.2% (range 0 – 100%) in the study wounds and 98 9 1% (range 90 – 100%) in the control sites (P B0.05). Reduced CSR take seemed to correlate with wound colonization. All children survived. While CSR did not engraft with the reliability of standard autograft, this pilot experience is encouraging in that successful wound closure with this material is possible, if not yet dependable. It is hoped that a more mature epidermal layer may facilitate engraftment, and trials to explore this possibility are in progress. © 2001 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Skin substitutes; Burns; Keratinocytes
1. Introduction Despite the well-described liabilities of donor site morbidity and graft hypertrophy, split thickness autograft remains the standard of care for resurfacing acute full thickness burns and for closing defects associated with burn reconstructive procedures [1]. Donor site morbidity includes pain, healing delays, infection and hypertrophic scarring and relates directly with thickness of harvest. Graft hypertrophy is felt to relate directly to thickness of transplanted dermis [2]. Both of these liabilities must be accepted when performing acute and reconstructive burn surgery in those with limited donor sites. Presented at the Annual Meeting of the American Burn Association, March 2000, Las Vegas supported by the Shriners Hospitals for Children. * Corresponding author. Tel.: + 1-617-726-5633; fax: + 1-617-3678936. E-mail address: [email protected] (R.L. Sheridan).
Human allograft is routinely used as temporary cover of excised burn wounds. These grafts generally vascularize within three days, but are subsequently rejected in two or three weeks [3]. The most immunogenic components of transplanted allograft skin are the cellular elements of the epidermis and dermis [4]. If these components are removed, the remaining non-cellular dermal tissue is relatively immunologically inert [5]. Acellular bone grafts have been routinely used in oral and orthopedic surgery for several years [6], lending credence to the claim that such tissues will maintain their structural integrity over time, serving as a scaffold for ingrowth of host cellular elements, resulting in a normally organized tissue. The use of allogenic dermis in combination with autogenous cultured epithelial cells as permanent coverage for full thickness burn wounds has been explored [7–9]. However, this technique requires excision of the upper dermis with sacrifice of epithelial attachment mechanisms [10], perhaps explaining the fragility of the resulting cover [11].
0305-4179/01/$20.00 © 2001 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 0 0 ) 0 0 1 5 6 - X
422
R.L. Sheridan et al. / Burns 27 (2001) 421–424
The clinical problem of the inadequate donor site is particularly acute in patients with very large burns, in whom there is simply not enough donor site available to address their acute coverage and reconstructive needs. In this group, an innovative approach to wound closure has lifesaving potential, both in the setting of the acute injury and in facilitating a more normal life by making enough skin available for reconstructive needs. Previous work in our laboratory with human keratinocytes cultured onto acellular dermis [12] has shown promise, prompting this pilot trial in humans.
2. Methods
2.1. General methods We enrolled seven children with massive burns and less than 25% of the body surface available for skin harvest who required either acute resurfacing or a reconstructive procedure requiring split thickness autografting in a human studies committee approved protocol. A total of 12 wounds were grafted in these children with the composite skin replacement (CSR), simultaneously grafting a randomly assigned control area with the autograft. Study and control sites were adjacent portions of the same wound, the study site being initially the more cephalic or right, assignments being alternated in sequential patients. Reconstructive wounds were generated by incisional release. Acute excisional wounds were generated by layered excision of full thickness burn wounds, generally to viable subcutaneous fat or by removal of vascularized allograft.
2.2. Study endpoints Study endpoints were (1) engraftment (in %) at seven days, (2) engraftment (in %) at 30 days, (3) the need for regrafting, and (4) Vancouver Scar Scores [13] at 1, 3, 6, and 12 months. This is a scoring system with which we are very practiced and which has shown excellent inter rater reliability in our institution [14]. Study and control site results were compared by t-test.
2.3. Preparation of composite skin replacement Normal human keratinocytes were isolated from full thickness skin biopsies and cultured using the fibroblast feeder layer method of Rheinwald and Green, the original specimen coming from a 1.0× 0.5 cm2 skin biopsy obtained approximately three weeks prior to the study [15]. Keratinocytes were provided in the pre-confluent phase by Genzyme Tissue Repair, Inc. of Cambridge, MA. Keratinocytes were co-cultivated with 3T3-J2 mouse fibroblasts, pretreated with 15 mg/ml mitomycin C to limit the proliferation of the 3T3 cells. Kerati-
nocyte culture medium (KCM) was a 3:1 mixture of Dulbecco’s Modified Eagle’s medium (DMEM) (high glucose) and Ham’s F12 medium; supplemented with 10% fetal bovine serum; adenine, 1.8× 104 M; cholera toxin, 10 − 10 M; hydrocortisone, 0.4 mg/ml; insulin, 5 mg/ml; transferrin, 5 mg/ml; triiodo-L-thyronine, 2× 10 − 9 M, and penicillin– streptomycin, 100 IU/ml-100 mg/ml and was changed every 3–4 days. Beginning with the first medium change, EGF was added at 10 ng/ml. Cultures were incubated in a humidified 10% CO2 atmosphere at 37°C. Cells were subcultured by first removing the feeder layer cells with an EDTA wash (5 mM in PBS), then treating the keratinocytes with trypsin-EDTA and re-seeding them on flasks containing mitomycin C treated 3T3-J2 mouse fibroblasts. To prepare and evaluate composite grafts in vitro, allogenic dermis, from which all cellular elements were removed, was placed into a tissue culture dish, papillary side up prior to adding keratinocytes. This dermal analog was prepared by soaking split thickness human allograft, properly screened, in sterile phosphate buffered saline supplemented with gentamycin (100 mcg/ml), ciprofloxacin (10 mcg/ml), amphotericin B (2.5 mcg/ml), penicillin (100 IU/ml) and streptomycin (100 mcg/ml) for one week at 37°C and then carefully stripping off the alloepithelial layer. This dermal analog was maintained at 4°C for one month in PBS supplemented with antibiotics. The papillary surface of each piece of allogenic dermis was seeded with cultured keratinocytes in KCM (2.5×105 cells/ml, in triplicate). Composite grafts were maintained submerged for one week. Grafts were then washed in DMEM, transported to the operating room in an airtight container containing 10% carbon dioxide and then grafted using standard surgical technique.
3. Results With human studies committee approval, 12 wounds in 7 patients were grafted with CSR while a matched control wound was covered with split thickness autograft. The 7 children had an average age of 6.491.4 yr and burn size of 75.99 5.0% of the body surface. Nine of the 12 wounds were acute burns and 3 were reconstructive releases. Successful vascularization at 14 days averaged 45.7914.2% (range 0–100%) in the study wounds and 989 1% (range 90–100%) in the control sites (PB0.05). Wounds were followed for an average of 8 months, 6 wounds for 12 months. Evaluation with serial Vancouver Scar Scores between study and control sites revealed no differences at any time point: study and control Vancouver scores being 1.490.4 and 1.39 0.4 at one month, 1.890.5 and 1.69 0.6 at 3 months, 1.690.5 and 1.09 0.8 at 9 months, and 1.29 0.7 and 1.09 0.4 at 12 months. Reduced CSR take
R.L. Sheridan et al. / Burns 27 (2001) 421–424
seemed to correlate with bacterial wound colonization. All enrolled children survived and have been discharged home. The study material did not contribute to their survival given the small areas grafted in this pilot trial.
4. Discussion Refinement of the surgical approaches to large wounds combined with the ongoing evolution of critical care techniques has extended our ability to salvage the lives of those with large deep burns. 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. The development of a permanent skin substitute will have an enormous impact on the care of patients with serious burns. Skin consists of two layers with a highly effective bonding mechanism. The epidermis, consisting of the strata basale, spinosum, granulosum and corneum, provides a vapor and bacterial barrier. The dermis provides elasticity and strength. The thin epidermal layer is constantly replacing itself from its basal layer, with new keratinocytes undergoing terminal differentiation over approximately 4 weeks to anuclear keratin filled cells that make up the stratum corneum, which provides much of the barrier function of the epidermis. The basal layer of the epidermis is firmly attached to the dermis by a complex bonding mechanism involving proteins such as collagen types IV and VII. When this bond fails, serious morbidity results, as demonstrated by the disease processes of toxic epidermal necrolysis and epidermolysis bullosa [16,17]. An ideal skin substitute should have most, if not all, of the following properties: (1) low cost, (2) long shelf life, (3) use off the shelf, (4) non-antigenic, (5) durable, (6) flexible, (7) vapor transmission characteristics mimic epithelium, (8) effective barrier to bacteria, (9) conforms to irregular wound surfaces, (10) easy to secure, (11) grows with children, (12) applied in one operation and (13) does not become hypertrophic [3,18]. It seems likely that the successful permanent skin substitute will be a composite containing some autogenic elements. Not only are both dermal and epidermal elements required for optimal function and appearance, but there are poorly characterized but important interactions between them; one enhancing the maturation of the other [10,19–21]. There are several exciting animal and human investigations going on in this area, yet a successful and durable composite substitute remains an elusive goal. Notable among these is the work by Boyce and Hansbrough, who have produced a completely biologic composite skin substitute, culturing human fibroblasts in a
423
collagen-glycosaminoglycan membrane and then growing keratinocytes upon this [22,23]. Successful in a nude mouse model [24], this device is in clinical trials [25,26]. Another exciting avenue of investigation is described by combination of cultured autologous keratinocytes with one of the currently available dermal analogs. These combinations are produced either on the patient or in the laboratory prior to engraftment. Although limited initial anecdotal experiences with combinations of cultured keratinocytes and IntegraR (Integra Life Sciences, Plainsboro, NJ), AllODermR (LifeCell, The Woodlands, TX) polyglactin mesh, human allogenic dermis and other dermal analogs have not yet demonstrated reliable results, the concept has great appeal [8,27]. Addition of allogenic or autogenic keratinocytes modified by viral transfection to over produce important growth factors may enhance the eventual success of this approach [28]. While the CSR reported here did not engraft with the reliability of split thickness autograft, this pilot experience is encouraging in that successful wound closure with this material is possible, if not yet dependable. It is hoped that a more mature epidermal layer may facilitate engraftment, and trials to evaluate this are in progress.
References [1] Sheridan RL, Tompkins RG, Burke JF. Management of burn wounds with prompt excision and immediate closure. J Intensive Care Med 1994;9:6 –19. [2] Klein L, Rudolph R. 3 H-collagen tumover in skin grafts. Surgery Gynecol Obstet 1972;135:49 – 57. [3] Pruitt BA Jr., Levine NS. Characteristics and uses of biologic dressings and skin substitutes. Arch Surgery 1984;119:312 – 22. [4] Sedmak DD, Orosz CG. The role of vascular endothelial cells in transplantation. Arch Pathol Lab Med 1991;115:260 – 5. [5] Yukna RA, Turner DW, Robinson LJ. Variable antigenicity of lyophilized allogeneic and lyophilized xenogeneic skin in guinea pigs. J Periodont Res 1977;12:197 – 203. [6] Mellonig JT. Freeze-dried bone allografts in periodontal reconstructive surgery. Dental Clinics N Am 1991;35:505 – 20. [7] Compton CC, Hickerson W, Nadire K, Press W. Acceleration of skin regeneration from cultured epithelial autografts by transplantation to homograft dermis. J Burn Care Rehabil 1993;14:653 – 62. [8] Cuono CB, Langdon R, Birchall N, Barttelbort S, McGuire J. Composite autologous-allogeneic skin replacement: development and clinical application. Plastic Reconstruct Surgery 1987;80:626 – 37. [9] Hickerson WL, Compton C, Fletchall S, Smith LR. Cultured epidermal autografts and allodermis combination for permanent burn wound coverage. Burns 1994;20(Suppl 1):S52 – 5. [10] Woodley DT, Peterson HD, Herzog SR, et al. Burn wounds resurfaced by cultured epidermal autografts show abnormal reconstitution of anchoring fibrils. JAMA 1988;259:2566 –71. [11] Sheridan RL, Tompkins RG. Cultured autologous epithelium in patients with burns of ninety percent or more of the body surface. J Trauma 1995;38:48 – 50.
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R.L. Sheridan et al. / Burns 27 (2001) 421–424
[12] Medalie DA, Eming SA, Tompkins RG, Yarmush ML, Krueger GG, Morgan JR. Evaluation of human skin reconstituted from composite grafts of cultured keratinocytes and human acellular dermis transplanted to athymic mice. J Invest Dermatol 1996;107:121 – 7. [13] Sullivan T, Smith J, Kermode J, McIver E, Courtemanche DJ. Rating the burn scar. J Burn Care Rehabil 1990;11:256 – 60. [14] Baryza MJ, Baryza GA. The Vancouver Scar Scale: an administration tool and its interrater reliability. J Burn Care Rehabil 1995;16:535 – 8. [15] Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331 –43. [16] Heimbach DM, Engrav LH, Marvin JA, Harnar TJ, Grube BJ. Toxic epidermal necrolysis. A step forward in treatment. JAMA 1987;257:2171 – 5. [17] Fine JD, Johnson LB, Cronce D, et al. Intracytoplasmic retention of type VII collagen and dominant dystrophic epidermolysis bullosa: reversal of defect following cessation of or marked improvement in disease activity. J Invest Dermatol 1993;101:232 – 6. [18] Tompkins RG, Burke JF. Burn wound closure using permanent skin replacement materials. World J Surgery 1992;16:47 – 52. [19] Briggaman RA, Wheeler CE Jr. The epidermal –dermal junction. J Invest Dermatol 1975;65:71 –84. [20] Cooper ML, Andree C, Hansbrough JF, Zapata-Sirvent RL, Spielvogel RL. Direct comparison of a cultured composite skin substitute containing human keratinocytes and fibroblasts to an epidermal sheet graft containing human keratinocytes on athymic mice. J Invest Dermatol 1993;101:811 – 9.
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