David G. Greenhalgh

Comparative assessment of cultured skin substitutes and native skin autograft for treatment of full-thickness burns.

ObjectiveComparison of cultured skin substitutes (CSSs) and split-thickness autograft (STAG) was performed to assess whether the requirement for autologous skin grafts may be reduced in the treatment of massive bums. Summary Background DataCultured skiasubstitutes consisting of collagen-glycosaminoglycan substrates populated with autologous fibroblasts and keratinocytes have been demonstrated to close full-thickness skin wounds in athymic mice and to express normal skin antigens after closure of excised wounds in burn patients. MethodsData were collected from 17 patients between days 2 and 14 to determine incidence of exudate, incidence of regrafting, coloration, keratinization, and percentage of site covered by graft (n = 17). Outcome was evaluated on an ordinal scale (0 = worst; 10 = best) beginning at day 14, with primary analyses at 28 days (n = 10) and 1 year (n = 4) for erythema, pigmentation, epithelial blistering, surface roughness, skin suppleness, and raised scar. ResultsSites treated with CSSs had increased incidence of exudate (p = 0.06) and decreased percentage of engraftment (p < 0.05) compared with STAG. Outcome parameters during the first year showed no differences in erythema, blistering, or suppleness. Pigmentation was greater, scar was less raised, but regrafting was more frequent in CSS sites than STAG. No differences in qualtative outcomes were found after 1 year, and antibodes to bovine collagen were not detected in patientsera. ConclusionsThese results suggest that outcome of engrafted CSSs is not different from STAG and that increased incidence is related to decreased percentage of initial engraftment. Increased rates of CSSs may lead to improved outcome for closure of burn wounds, allow greater availability of materials for grafting, and reduce requirements for donor skin autogratt.

Skin anatomy and antigen expression after burn wound closure with composite grafts of cultured skin cells and biopolymers.

Closure of large skin wounds (i.e., burns, congenital giant nevus, reconstruction of traumatic injury) with split-thickness skin grafts requires extensive harvesting of autologous skin. Composite grafts consisting of collagen-glycosaminoglycan (GAG) substrates populated with cultured dermal fibroblasts and epidermal keratinocytes were tested in a pilot study on full-thickness burn wounds of three patients as an alternative to split-thickness skin. Light microscopy and transmission electron microscopy showed regeneration of epidermal and dermal tissue by 2 weeks, with degradation of the collagen-GAG implant associated with low numbers of leukocytes, and deposition of new collagen by fibroblasts. Complete basement membrane, including anchoring fibrils and anchoring plaques, is formed by 2 weeks, is mature by 3 months, and accounts for the absence of blistering of healed epidermis. All skin antigens tested (involucrin, filaggrin, laminin, collagens IV and VII, fibronectin, and chondroitin-sulfate) were expressed by 16 days after grafting. This cultured skin analogue provides an experimental alternative to split-thickness skin graft that develops histiotypic markers of skin anatomy and antigen expression after wound closure.

Apoptosis down-regulates inflammation under the advancing epithelial wound edge: Delayed patterns in diabetes and improvement with topical growth factors

BACKGROUNDnWound healing is involved in many aspects of care, ranging from anastomoses and skin incisions to foot ulcers and decubitus. Clinical healing failures are a major challenge to the physician and cause significant morbidity and mortality in select patient populations. As we are starting to understand more fully the mechanisms of impaired wound healing, the diabetic mouse (C57BL/KsJ-db/db) has been a good model for research. The diabetic wound exhibits significant delays in healing, previously identified as impaired cellular infiltration and granulation tissue formation. Apoptosis, or programmed cell death, is intimately involved in the regulation of inflammation and ultimately should play a role in the inflammatory phase of wound healing.nnnMETHODSnTo examine its role in wound healing, patterns of apoptosis in large, full-thickness cutaneous wounds were compared between groups of diabetic and nondiabetic mice.nnnRESULTSnInitially apoptosis was mainly limited to the wound edge and followed the advancing epithelial edge toward the center of the wound as healing progressed. Significant delays in the appearance of the apoptotic pattern were noted in the diabetic mice. Wounds in diabetic mice were then treated with topical application of growth factors. The delay in apoptotic pattern was reversed after treatment with the combination of insulin-like growth factor-II and platelet-derived growth factor, approaching levels in nondiabetic animals.nnnCONCLUSIONSnApoptosis appears concurrently with reepithelialization of the wound and may signal the end of the inflammatory phase of healing at that site in the wound. One can speculate that a signal for apoptosis and down-regulation of inflammation in the wound is derived from the epithelium.

Wound healing in the transforming growth factor‐β1—deficient mouse

To investigate the role of transforming growth factor‐β1 in tissue repair, we performed wound healing studies in the transforming growth factor‐β1—deficient mouse with targeted disruption of the transforming growth factor‐β1 gene. Transforming growth factor‐β1—deficient mice exhibit no obvious developmental defects and are phenotypically normal until approximately 3 weeks of age when a severe wasting syndrome develops, accompanied by an overwhelming inflammatory response resulting in multisystem organ failure and death. Full‐thickness 0.5 × 0.5 cm skin wounds were created on the backs of 10‐day‐old mice (wild type or heterozygous controls versus homozygous transforming growth factor‐β1—deficient mutants) and covered with a nonabsorbent dressing (OpSite). Serial wound measurements were made, and percentage of wound closure over time was determined. On day 10, wounds and liver were harvested for histologic and molecular analysis. Histologic scores were assigned (1 [no healing] to 12 [complete healing]) on the basis of granulation tissue formation, vascularity, collagen deposition, and epithelialization. Reverse transcription—polymerase chain reaction was performed to detect messenger RNA transcripts for transforming growth factor‐β1, transforming growth factor‐β2, platelet‐derived growth factor A‐chain and B‐chain, interleukin‐1β and ‐6, and tumor necrosis factor‐α in unwounded skin, day 10 wounds, and liver. No significant differences in wound closure were observed until day 10. Weight gain, however, was significantly decreased in the mutant animals as early as day 6. Histologic scores were significantly lower in the transforming growth factor‐β1—deficient mutants (5.4 ± 0.6 versus 11.1 ± 0.3, p < 0.01, Wilcoxon rank‐sum test) and showed decreased granulation tissue formation, vascularity, collagen deposition, and epithelialization and a marked inflammatory infiltrate. As expected, transforming growth factor‐β1 was expressed in controls but not mutants. Transforming growth factor‐β2, platelet‐derived growth factor A‐chain and B‐chain, and tumor necrosis factor‐α were constitutively expressed in unwounded skin, day 10 wounds, and liver of both controls and mutants. Interleukin‐1β and ‐6, however, were induced after wounding. Early wound healing in the transforming growth factor‐β1—deficient mouse proceeds relatively normally because of upregulation or functional redundancy of other growth factors or possibly because of maternal rescue by means of transforming growth factor‐β1 transmitted in milk. Loss of transforming growth factor‐β1 regulation ultimately results in a marked inflammatory response, as evidenced by the histologic appearance of the wound and increased expression of the inflammatory cytokines (tumor necrosis factor‐α, interleukin‐1β and 6). The severe wasting syndrome (marked by weight loss) undoubtedly has an adverse effect on wound healing.

Synergistic actions of platelet‐derived growth factor and the insulin‐like growth factors in vivo

Topical application of growth factors has been shown to benefit both normal and impaired wound healing. In normal tissue repair, resident cells produce a “cocktail” of various types of growth factors that overlap in function. In vitro studies have proved that growth factor combinations can act synergistically to enhance cellular function beyond that achieved with individual growth factors. To determine whether similar combinations have a synergistic effect in vivo, we applied growth factor combinations topically to full‐thickness skin wounds created in genetically diabetic mice. The C57BL/KsJ‐db/db mouse is obese and has insulin‐resistant diabetes, and it has been proved that this mouse has markedly impaired wound healing. Topical application of platelet‐derived growth factor, insulin‐like growth factor‐I, or insulin‐like growth factor‐II enhances healing in this model. Marked synergism was found when platelet‐derived growth factor and insulin‐like growth factor‐II were combined to produce augmentation in wound closure beyond that achieved by application of the individual growth factors. The synergistic effect allowed for improved tissue repair at doses of platelet‐derived growth factor and insulin‐like growth factor‐II that were ineffective when applied individually. The addition of insulin‐like growth factor‐I or insulin to platelet‐derived growth factor produced no significant synergism. Because multiple growth factors are released in the wound during the healing process, it is not surprising that their combination further enhances healing. Growth factor combinations should become an important addition to the armamentarium for the treatment of chronic, nonhealing wounds.

Surface electrical capacitance as an index of epidermal barrier properties of composite skin substitutes and skin autografts

Restoration of the epidermal barrier is a requirement for burn wound closure. A rapid, reliable, and noninvasive measure of the rate of restoration of the epidermal barrier is not readily available. To monitor the reformation of the epidermal barrier, we measured surface electrical capacitance on cultured skin substitutes (human keratinocytes and fibroblasts attached to collagen‐glycosaminoglycan substrates) and split‐thickness skin autografts grafted to patients. Data were collected from four patients with burns and one pediatric patient with a congenital hairy nevus comprising > 60% total body surface area. Capacitance measurements were performed at days 7, 10, 12, 14, and 28 by direct contact of the capacitance probe for 10 seconds to the cultured skin substitutes or split‐thickness autograft. On postoperative days 7, 10, 12, 14, 21, and 28, the surface electrical capacitance of cultured skin substitutes after 10 seconds of sampling was 2468 ± 268, 1443 ± 439, 129 ± 43, 200 ± 44, 88 ± 20, and 74 ± 19 picofarads (mean ± standard error of the mean), respectively. Surface electrical capacitance for split‐thickness autograft on the same days was 1699 ± 371, 1914 ± 433, 125 ± 16, 175 ± 63, 110 ± 26, 271 ± 77 picofarads, respectively. Surface electrical capacitance in all of the grafts decreased with time. Cultured skin substitutes had approximately the same 10‐second capacitance values as split‐thickness autograft during 3 weeks of healing and approached values for uninjured skin (32 ± 5 picofarads) by 12 days. Measurement of surface electrical capacitance is a direct, inexpensive, and convenient index for noninvasive monitoring of epidermal barrier formation.

Expression of interleukin-1alpha, interleukin-6, and basic fibroblast growth factor by cultured skin substitutes before and after grafting to full-thickness wounds in athymic mice.

OBJECTIVESnCultured skin substitutes (CSSs), consisting of human keratinocytes and human fibroblasts attached to collagen-glycosaminoglycan substrates, have been demonstrated to cover wounds, and may release detectable quantities of growth factors that promote wound healing.nnnMATERIALS AND METHODSnBasic fibroblast growth factor (bFGF), interleukin-1alpha (IL-1alpha), and interleukin-6 (IL-6) were assayed by enzyme linked immunosorbent assay and immunohistochemistry in CSSs in vitro and at days 1, 3, 7, 14, and 21 after grafting to full-thickness wounds in athymic mice.nnnMEASUREMENTS AND MAIN RESULTSnWhen isolated cells were tested, IL-1alpha was found to come primarily from the keratinocytes, whereas bFGF was from the fibroblasts. Combinations of both cell types in the CSSs resulted in a synergistic enhancement of IL-6 expression. Quantities of all three cytokines from CSSs were greater in vitro compared with in vivo levels at all time points after grafting. bFGF increased from day 1 to day 7, and then remained relatively constant until day 21. At day 3 maximal levels of IL-1alpha were observed. By day 7, IL-1alpha decreased to approximately 40% of maximal levels, and subsequently increased until day 21. IL-6 levels were highest at day 7 after grafting. All cytokines had reached elevated levels during the time of wound revascularization (days 3-7).nnnCONCLUSIONSnThe sequence of cytokine synthesis in the wounds (i.e., rapid IL-1alpha increase followed by IL-6 expression) parallels serum levels reported after a septic challenge. These findings support the hypothesis that the wound is a source of systemic cytokines.

Pediatric burn patients with respiratory failure: Predictors of outcome with the use of extracorporeal life support

Extracorporeal life support (ECLS) for pediatric burn patients is a viable option for respiratory failure that is unresponsive to maximal conventional therapy. No criteria have been identified that are predictive of the success of the use of ECLS for these patients. This article presents a retrospective review of the pediatric burn patients placed on ECLS at a single pediatric medical center. It was found that 12 patients (mean age, 30.3 months; range 6 to 69 months) were placed on ECLS because of profound pulmonary failure that was unresponsive to aggressive ventilatory support. The mean size of the burns of these patients was 50.2% of the total body surface area (average size of full-thickness burns, 41.8% total body surface area), with 6 patients having scald burns and 6 having flame burns. The overall survival was 67% (8 of 12). Nonsurvivors had greater positive end-expiratory pressure, mean airway pressure, peak inspiratory pressure, and oxygenation index before ECLS. It is felt that ECLS is a life-saving therapy for pediatric patients with thermal injury. Greater ventilator requirements before ECLS are associated with nonsurvival. Early institution of ECLS in pediatric burn patients with severe respiratory failure may prevent excessive barotrauma and thus discourage the onset of irreversible lung injury.

Capillary morphogenesis during healing of full‐thickness skin grafts: An ultrastructural study

Biologic mechanisms by which skin grafts become revascularized after transplantation are poorly understood. To investigate graft revascularization, we examined the pattern of capillary growth in full‐thickness skin grafts at serial time points. Full‐thickness skin (2 × 2 cm) was excised to muscle fascia from the bilateral hind limbs of adult male Lewis rats. The graft/wound base boundary was identified by placement of a polypropylene mesh on the wound beneath the graft. Excised skin was replaced in its original orientation and secured with silk sutures tied over a gauze bolster dressing. After 3, 5, 7, and 10 days, animals were killed, and their aortas were cannulated and infused with an acrylic polymer to generate vascular casts. Grafts were excised, tissues were digested, and casts were examined with the use of scanning electron microscopy. Transmission electron microscopy was performed on tissues infused with the acrylic polymer that were not digested. At day 3, an immature lobular pattern was observed extending from the neovascular plexi on the graft side of the polypropylene mesh. At day 5, defined vessels with lobular ends occurred with high frequency. At day 7, the number of observed lobular structures was greatly reduced, and high frequencies of depressions in acrylic casts suggested protrusion of endothelial cell nuclei. By day 10, lobular structures were rare, well‐defined microvascular plexi were contiguous with larger vessels, and depressions from endothelial cell nuclei appeared more shallow and less frequent. These findings suggest that (1) an immature lobular pattern representing either capillary outgrowth or extracapillary leakage occurs at day 3; (2) these immature lobules decrease, and more discrete capillaries increase by day 5; (3) vascular integrity is reestablished by day 7; (4) vascular plexi has regained full continuity, and there are suggestions that endothelial cell proliferation has subsided by day 10.