Analysis of cellular and decellular allogeneic dermal grafts for the treatment of full-thickness wounds in a porcine model.

BACKGROUND AND METHODS: The need for a reliable skin substitute to improve burn treatment has been long apparent. We have investigated the use of cryopreserved cellular and decellularized porcine allogeneic dermal grafts (CADGs or DADGs) in conjunction with an overlying thin split-thickness autograft (STSG) in a one-step technique. Control mirror-image wounds were treated with thin STSG alone. Autograft "take" rates and wound contraction were determined; biopsies were taken at weeks 2, 5, and 8, and blinded scar assessment was performed at week 10. RESULTS: The percent take of autografts overlying CADGs or DADGs was comparable to that of control sites (83.0 +/- 8.3% vs. 81.1 +/- 11.5% for CADG vs. control; 93.2 +/- 7.6% vs. 90.2 +/- 11.1% for DADG vs. control). CADGs did not alter wound contraction or cosmetic outcome. By contrast, DADG treatment inhibited wound contraction (42.9 +/- 17.8% vs. 30.9 +/- 10.4% contraction for control vs. DADG at week 10; p < 0.017) and significantly improved cosmetic outcome in 10 of 12 paired wounds (p < 0.012). CONCLUSION: CADGs and DADGs permitted simultaneous engraftment of an overlying thin STSG. Although CADGs had no effect on wound contraction and cosmetic outcome, DADGs significantly reduced wound contraction and improved cosmetic outcome of full-thickness wounds in a porcine model. The use of DADGs may represent a potential improvement in burn care.

Transplantation of cryopreserved cultured epidermal allografts.

OBJECTIVE: To optimize cryopreservation methods for cultured epidermal allografts (CEAs) for transplantation onto wounds. DESIGN: Conditions were determined to optimize the cryopreservation of CEAs. Cryopreserved CEAs were then grafted onto 16 donor sites in a double blind randomized trial. MATERIALS AND METHODS: CEAs were grown in culture. Viability of cryopreserved CEAs was determined by: 1) trypan blue dye exclusion; 2) histology; 3) flow cytometry; and 4) acid lipase activity. Cryopreserved CEAs were grafted onto 16 donor sites. Wound healing assessments included 1) visual assessment of healing; 2) histologic assessment of re-epithelialization and differentiation; and 3) visual scar assessment. MEASUREMENTS AND MAIN RESULTS: CEAs were cryopreserved using a controlled rate freezer with an experimentally determined setting of -7 degrees C for the CEA freezing point. Such freezing conditions resulted in retention of approximately 92% of the original viability, no loss in basal keratinocytes as determined by flow cytometry, and no change in acid lipase activity. CEAs cryopreserved according to this method were grafted onto 16 donor sites in a double blind randomized trial. All donor sites underwent complete healing. Histologic examination of biopsies taken from the center of the wound beds showed that CEA-treated wounds significantly accelerated the rate of re-epithelialization (7.8 +/- 0.6 days vs. 9.2 +/- 0.9 days for CEA- and control-treated wounds, respectively; p = 0.039) and epithelial differentiation (p = 0.023) compared with control sites. Longterm results showed that CEA-treated wounds were comparable to control sites with regard to: 1) pigmentation; 2) scar height; 3) scar pliability; 4) vascularity; and 5) pain. Wounds treated with cryopreserved CEAs remained durable and not prone to blistering after healing. CONCLUSIONS: CEAs can be successfully cryopreserved for long-term storage. Upon retrieval from storage, CEAs may be used to treat partial thickness wounds.

The effects of burn blister fluid on cultured keratinocytes.

OBJECTIVE: Previous studies have suggested that burn blister fluid (BBF) may be detrimental to the healing of the underlying wound bed. In this study, the effects of burn blister fluid on cultured keratinocyte proliferation and differentiation were examined and quantitated using various techniques. METHODS: At three different concentrations (2%, 10%, 20% in 20% fetal bovine serum/complete culture medium), 19 BBFs were tested in triplicate using 12 populations of cultured keratinocytes. All BBFs were collected from partial thickness burns within 72 hours of injury,. BBF was added on day 4 of the keratinocyte culture. The effect on proliferation and viability was assessed using trypan blue dye exclusion. Multiparameter flow cytometric analysis was used to quantitate population kinetics and cell size distribution. Keratinocyte differentiation was determined using immunohistochemical staining of differentiation markers and quantitation of cornified envelope formation. RESULTS: Relative to control fluid, the BBF caused a variable effect on proliferation, ranging from 67% inhibition to 103% stimulation with an overall 4% inhibition. The range of keratinocyte viability was narrower, with a similar overall 4% reduction. Using flow cytometry to analyze RNA/DNA content and cell size, the BBF caused a subtle shift in keratinocyte population kinetics and cell size distribution toward larger, less rapidly dividing cells. The BBF had no significant effect on expression of the differentiation markers, filaggrin and involucrin. Finally, the BBF did not alter terminal differentiation as it did not influence formation of cornified envelopes (BBF = 9.1 +/- 4.8%, control = 9.9 +/- 6.6%). CONCLUSION: Previous biochemical analysis has shown that BBF consists primarily of human serum filtrate with locally produced acute reactants. Our study suggests that BBF is biologically similar to serum and does not significantly alter keratinocyte proliferation or differentiation in vitro.

The epidemiology and prevention of burn injuries to firefighters.

From 1981 to 1991, 746 firefighters were treated for burn injuries at New York Hospital-Cornell Burn Center. The predominant anatomic areas of deep injury were the hands and lower extremities. An educational program was initiated that urges proper use of the protective gear as designed. A reduction in the number of burns to the hands subsequently was noted, but lower extremity burns persisted. Analysis of the lower extremity burns revealed that firefighters functioning as nozzle operators were particularly prone to injury because of inadequate lower extremity protection. Improved protection of the lower extremities with the introduction of bunker pants could reduce the incidence of the injuries and result in substantial cost savings for the City of New York.

Human keratinocyte growth factor effects in a porcine model of epidermal wound healing.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family (hence the alternative designation FGF-7). It is produced by stromal cells, but acts as a mitogen for epithelial cells. We examined the effects of topically applied KGF on healing of wounds in a porcine model. In partial-thickness wounds, KGF stimulated the rate of reepithelialization (p < 0.0002), associated with a thickening of the epidermis (p < 0.0001). Epidermis from KGF-treated full-thickness wound sites was significantly thicker (0.31 +/- 0.22 mm) compared with mirror image control sites (0.18 +/- 0.12 mm) (p < 0.0001). Moreover, the majority (77%) of KGF-treated wounds exhibited epidermis with a deep rete ridge pattern as compared with control sites. These effects were observed as early as 14 d and persisted for at least 4 wk. KGF treatment also increased the number of serrated basal cells associated with increased deposition of collagen fibers in the superficial dermis adjacent to the acanthotic epidermis. Electron microscopy revealed better developed hemidesmosomes associated with thicker bundles of tonofilaments in the serrated cells. The pattern of epidermal thickening observed in KGF-treated wounds resembled psoriasis. Psoriasis is a disease associated with epidermal thickening, parakeratosis as well as hyperproliferation that extends beyond the basal layer. In striking contrast to psoriasis, KGF-treated wounds exhibited normal orthokeratotic maturation, and proliferation was localized to the basal cells. Our present findings have significant implications concerning the role of KGF as a paracrine modulator of epidermal proliferation and differentiation.

A nasal trumpet orthosis to maintain nares openings and respiratory function for patients with facial burns: a case report.

Management of facial burns is a challenge to the burn team because it may lead to functional and cosmetic compromise. Severe scarring of the nares may lead to nasal occlusion. This article introduces a method of maintaining nasal patency that allows respiratory exchange through the use of a custom-fabricated, semirigid tubular orthosis. The technique for fabrication is reviewed, and the use of the device is addressed through a case report. This inexpensive, readily available device is useful in preventing nasal occlusion that results from scar formation.

Pediatric burns. An overview.

This article describes the management of burn injuries in children. It begins with an epidemiologic description of pediatric burns. Attention is given to emergency care, burn wound evaluation, operative management, and rehabilitative goals.

Resuscitation of the thermally injured patient.

This article briefly reviews the pathophysiology of burn wounds as a basis for a more detailed discussion on the resuscitation of burn patients with lactated Ringer's solution or other regimens. The complications resulting from such resuscitation are also reviewed.

Growth of melanocytes in human epidermal cell cultures.

Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

Comparison of an occlusive and a semi-occlusive dressing and the effect of the wound exudate upon keratinocyte proliferation.

Three consecutive studies were performed in 58 patients evaluating the effect of occlusion on the healing of partial-thickness wounds. Mirror-image donor sites were covered with the occlusive hydrocolloid dressing (HCD) (DuoDerm) and compared to fine mesh gauze, and the HCD was subsequently compared to a semi-occlusive dressing of polyurethane film, (Op-site). In addition, partial-thickness burn wounds were covered with the HCD and the remaining burn wound was treated with silver sulfadiazine. The donor sites and burn wounds treated with HCD healed significantly faster than those covered with fine mesh gauze or silver sulfadiazine (p less than 0.001) and with less pain. The HCD and polyurethane film were equivalent. There were no clinical infections with the wounds that were occluded. The exudate collected beneath the DuoDerm and Op-site on donor sites was added to the tissue culture system and resulted in a modest increase in keratinocyte proliferation. However, the exudate from burn wounds under HCD resulted in a marked increase in cell proliferation (p less than 0.001).

Occult diaphragmatic injury from stab wounds to the lower chest and abdomen.

Ninety-five patients with stab wounds to the lower chest and abdomen underwent routine abdominal exploration. Eighteen of these patients had diaphragmatic injury and in five patients it was the only injury found. Isolated diaphragmatic injury in asymptomatic patients cannot be reliably delineated by either serial physical examination or peritoneal lavage. Delayed recognition of incarcerated diaphragmatic hernia after stab wounds to the lower left chest and upper abdomen has an associated mortality rate of 36%. The anatomic area of concern can be defined as stab wounds that penetrate the left side of the chest below the fourth intercostal space anteriorly, the sixth intercostal space laterally, and the tip of the scapula posteriorly. Exploratory laparotomy is necessary in these patients until a reliable nonoperative method is established that can exclude injuries to the diaphragm.

Use of vascular clips to approximate skin grafts.

During excisions of acute burn wounds, attention to aesthetic detail often is secondary to the goal of rapid gross coverage. Expeditious approximation of adjacent skin grafts has long presented a problem to surgeons. Some surgeons simply place the grafts next to each other, relying on the intervening areas to "scar in". Others use staples to hold grafts together. These staples, however, can become buried under healed grafts and can cause "foreign body" reactions in the months and years ahead. In addition, staples cause bleeding beneath the newly placed grafts, contributing to hematoma formation. Still other surgeons suture or tape adjacent pieces of skin graft together, a tedious exercise. The cosmetic result of these techniques is often less than optimal resulting in the unfortunately familiar "patchwork quilt" appearance of grafts interweaved among scars. Vascular clips have proven to be useful for holding adjacent pieces of skin graft together.

Mafenide-induced pseudochondritis.

Wound infections following burns of the ear can result in the devastating complication of chondritis, requiring resection of cartilage. To prevent this, it has become common practice to dress the burned ear with mafenide acetate. We have observed six hypersensitivity reactions to the mafenide that occurred following several weeks of continuous use of the drug. The reaction mimics chondritis, causing edematous, erythematous, pruritic ears with a profuse serous exudate. There is no associated fever, systemic signs, or pain on motion of the cartilage. Treatment consists of stopping the mafenide. Recovery occurs within 72 hours. Differentiating between chondritis, with its required surgical and antibiotic treatment, and a hypersensitivity reaction is necessary to avoid further iatrogenic injury.

Grafting of cultured allogeneic epidermis on second- and third-degree burn wounds on 26 patients.

Twenty-six individuals with second- and third-degree burn wounds have been grafted with cultured allogeneic epidermal cells. These epidermal cell grafts were grown in culture from cadaver skin according to a technique which we have developed. After being grafted with cultured allogeneic epidermal cells, superficial wounds, e.g., donor sites, healed within 7 days, compared to 14 days for mirror image control sites. Deep second-degree burn wounds which were excised before grafting with cultured cells healed in a mean time of 10 days. Deep second-degree burn wounds which were not excised before grafting healed in a mean time of 14 days. The cultured cells produced rapid healing in 11 of the 12 patients with deep second-degree burn wounds. The deep second-degree wounds grafted with cultured allogeneic epidermal cells healed with results which were comparable to the deep second-degree wounds which were autografted. Grafts of cultured allogeneic epidermal cells placed on full-thickness, or third-degree burn, wounds did not grow well.

Improved burn center survival of patients with toxic epidermal necrolysis managed without corticosteroids.

Fifteen consecutive patients with toxic epidermal necrolysis or the Stevens-Johnson syndrome managed without corticosteroids after transfer to the burn center (group 2) are compared to a previous consecutive group of 15 who received high doses of these drugs (group 1). Group 2 had a 66% survival, which was a significant improvement compared to the 33% survival in group 1 (p = 0.057). In group 1, mortality was associated with loss of more than 50% of the body surface area skin. In group 2, mortality was related to advanced age and associated diseases. Age, extent of skin loss, progression of skin loss after burn center admission, incidence of abnormal liver function tests, and the incidence of septic complications were not significantly different in the two groups (p greater than 0.10). The incidence of detected esophageal slough was similar in both groups. Nonsteroid (group 2) management was associated with a decreased incidence of ulceration of gastrointestinal columnar epithelium, Candida sepsis, and an increased survival after septic complications. The combined experience of these 30 patients suggests that corticosteroids are contraindicated in the burn center management of toxic epidermal necrolysis and the Stevens-Johnson syndrome.

Human keratinocyte culture. Identification and staging of epidermal cell subpopulations.

Stratification of human epidermal cells into multilayered sheets composed of basal and suprabasal layers (resembling the stratum germinativum and stratum spinosum of the epidermis) was studied in a dermal component-free culture system. Although no stratum corneum developed in vitro, this culture system provided a method to study early events in human keratinocyte differentiation. Multiparameter flow cytometric analysis of acridine orange-stained epidermal cells from these cultures revealed three distinct subpopulations differing in cell size, RNA content, and cell cycle kinetics. The first subpopulation was composed of small basal keratinocytes with low RNA content and a long generation time. The second subpopulation consisted of larger keratinocytes, having higher RNA content and a significantly shorter generation time. Finally, the third subpopulation contained the largest cells, which did not divide, and represent the more terminally differentiated keratinocytes. This in vitro approach provides discriminating cytochemical parameters by which the maturity of the epidermal cell sheets can be assessed prior to grafting onto human burn patients.

Respiratory care of the burn patient.

The vast majority of respiratory disorders in thermally injured patients arise from associated inhalation injuries. The major forms of these injuries are carbon monoxide poisoning, injury to the upper airway, and pulmonary parenchymal damage. One hundred per cent oxygen, initiated at the scene of the accident, is the single most effective treatment of carbon monoxide toxicity, which must be assessed by carboxyhemoglobin determinations. Respiratory tract damage is identified by fiberoptic bronchoscopy and xenon ventilation-perfusion scintigrams. The compromised airway is protected by tracheal intubation, and respiratory failure is treated with assisted ventilation and supplemental oxygen. Pulmonary infection requires specific antibiotics based on isolated organisms and their sensitivities to antimicrobials. The upper respiratory tract of patients requiring long-term intubation should be assessed by fiberoptic bronchoscopy and other modalities to prevent fatal late airway occlusion.

Grafting of burn patients with allografts of cultured epidermal cells.

Epidermal cells from cadaver skin grown in culture into confluent sheets of stratified cells were grafted on to partial thickness burn wounds in three patients. The burn areas covered with these allogeneic cultured epidermal allografts were tangentially excised deep second-degree burns which routinely would have been covered with split-thickness autografts. The burn wounds grafted with cultured allografts healed within three days and remained healthy for the 9 months of observation. Since epidermal cell cultures may be grown continuously, cultured allografts may serve as alternative biological dressings, or grafts, for deep second-degree burn wounds. They produce accelerated healing and an excellent cosmetic result, and they reduce the need for split-thickness autografts.