Cyclosporine and skin allografts for the treatment of thermal injury. II. Development of an experimental massive third-degree burn model demonstrating extensive graft survival

We have previously reported the successful treatment and apparent development of skin allograft tolerance in a patient sustaining massive burns, utilizing skin allografts and cyclosporine. We now report the experimental correlate via successful achievement of a 75% body surface area (BSA) scald burn cyclosporine-skin allograft model in Lewis (LEW) rats. Cyclosporine (8 mg/kg/day) was given to the experimental animals daily for the first 20 days and then three times a week thereafter. Two experimental groups were studied: one received standard posttrauma care and the second critical posttrauma care. Controls (n = 22) and experimental groups 1 (n = 28) and 2 (n = 4) had average survival times of 13.8 +/- 12.8 days, 44.2 +/- 132.5 days, and 172.0 +/- 19.4 days, respectively. The allografts on the surviving experimental animals appeared normal and healthy and had nearly perfect hair growth. These results indicate that the model follows the clinical burn wound course, and treatment of massive burns with primary excision, skin allografts, and low doses of cyclosporine could provide immediate and complete functional repair of the burn wound.     

Innovative technique using heterogenous collagen and intermingled autologous split-thickness skin graft for coverage of raw areas in deep burns with paucity of donor site as a life-saving measure

Burns injuries are extremely complex and elicit physiological and metabolic interactions involving all major organ systems. These pathological changes occur in a time dependent manner. In the last decades, burn care has improved, so the trend in the current treatment extends beyond the preservation of life. Thus, ultimate goal is complete rehabilitation of the burn victims. To investigate the effectiveness of the use of heterogenous collagen and intermingled autologous split-thickness skin for early coverage of raw areas in deep burns with paucity of donorsite as a life saving measure. A total of 10 subjects between the age group of 5 to 30 years (mean age 22 years) who sustained deep burns with total burn area from 60% to 80% (mean 72 %) and full thickness burn from 40% to 50% (mean 44 %) were treated with collagen and autologous split thickness graft sheets. Complete wound epithelialization took between 30 to 50 days.Rejection of collagen within 5 days was observed in only 1 out of 10 subjects and 2 subjects demonstrated incomplete epithelialization. A total of 9 out of 10 patients survived and 1 patient expereienced death due to an acute rejection reaction. The combination of collagen andsplit thickness skin grafting is an effective method to cover the wounds and achieved satisfactory healing pattern, a minimal rejection phenomenon and good survival rates. Moreover, it can be efficient as a life saving measure in deep burns with few donor areas available for grafting specially for developing and third world nations where expensive facilities like CEA are not feasible.     

Pathological changes in allogenic skin in combined radiation-burn injury in rats]

Skin allografts were transplanted to rats inflicted with third degree burns of 15% of BSA alone or combined with 5 Gy gamma ray radiation. Histopathological, histochemical and ultrastructural changes of the allografts were examined at different postoperative periods. In burned rats, allografts began to be rejected on the 8th postoperative day, showing degeneration and necrosis of its epithelial cells. Only in one rat it survived on the 14th day (10%), and all allografts were rejected before the 19th day. In contrast to this, most allografts (60-80%) on the rats with combined injury survived well for a period of 29 days. Epithelial cells and collagen fibers of the allografts degenerated in 1 to 4 days postoperation, and recovered to near normal during 9 to 14 days. During 24 to 29 days postoperation, the epithelial cells and appendices appeared almost normal in texture except an infiltration of a few lymphocytes and monocytes. The restoration of degenerated collagen, however, was slower than that of degenerated epithelium. The result shows that the survival time of skin allografts in severe combined radiation burn injury is prolonged than that in burns.     

Liposome-encapsulated farnesol accelerated tissue repair in third-degree burns on a rat model

Third-degree or full-thickness burns refer to lesions that extend to the epidermis, dermis, and subcutaneous tissue. The pathophysiology of burn wounds is characterized by tissue inflammation, edema, and hypertrophic scarring. Farnesol is a natural 15-carbon organic compound that possesses many biological effects. We have previously demonstrated that farnesol gel exerts restorative actions on ultraviolet B (UVB)-caused sunburn in vivo. The in vitro results revealed that liposomal farnesol from 0.04 mM to 0.8 mM significantly enhanced collagen production by murine skin fibroblasts, whereas liposomal farnesol at high (0.8 mM) and low concentration (0.04 mM) did not show any suppressions on skin fibroblast proliferation. We treated third-degree burns on a rat model with a formulated gel composed of various ratios of 2% hydroxypropyl methylcellulose (HPMC) and 4 mM liposomal farnesol for 7 and 14 days. On days 7 and 14 post wounding, histopathological observations revealed that the HPMC:farnesol gel ratios of 1:2 and 2:1 exerted the greatest tissue-repairing effects on the skin after third-degree burns compared with skin untreated or treated with a commercial burn gel and HPMC alone. These findings were consistent with the in vivo quantitative collagen-producing assay, wound healing scoring, and IL-6 Western blot results. These findings demonstrated that the fabricated liposomal farnesol gel is potentially able to promote wound healing after third-degree burns.     

Safety and efficacy of basic fibroblast growth factors for deep second–degree burn patients

IntroductionBurn injuries are common afflictions; however, conservative wound care frequently leads to poor treatment compliance and physical disability in deep burn patients. Therefore, regenerative biologic materials, which are more effective for tissue repair, are required, particularly for deep second-degree burns. A novel spray formulation of basic fibroblast growth factors (bFGF) was produced by synthesizing fibroblast growth factor proteins. In this post-marketing surveillance (PMS) study, we assessed the safety and efficacy of bFGF and indirectly compared this formulation with cultured epidermal autografts (CEAs) for treating deep second-degree burns.Materials and methodsA total of 3173 patients treated at 15 hospitals were used for PMS of bFGF in South Korea for six years. In total, 1630 patients with deep second–degree burns were selected for assessing adverse events (AEs) of bFGF treatments. Efficacy was evaluated according to time periods until re-epithelialization, and clinical usefulness of bFGF was indirectly compared with that of CEAs.ResultsAEs occurred in 37 patients (2.3%) and included application site pain (1.7%) and contact dermatitis (0.6%). All AEs were mild and were evaluated as probably unrelated with bFGF. The average time for re-epithelialization was 8 days; this time span was significantly longer after major burns (9.7 days) than after minor (7.8 days) or moderate burns (7.9 days). Most treated burn wounds (99.8%) were assessed as improved. The indirect comparison included 534 patients using the same inclusion criteria for CEA patients (n = 35). The bFGF treatment demonstrated superior efficacy compared to CEAs by significantly reducing the average day to application (5.4 vs. 8.8 days) and re-epithelialization time (7.1 vs. 13.7 days).ConclusionOur study demonstrated that bFGF is a compelling regenerative therapy with competitive clinical efficacy and safety for deep second–degree burns and reduced treatment time, which is expected to reduce medical costs, particularly for deep second–degree burn patients.     

Protective effect of Myrtle (Myrtus communis) on burn induced skin injury

Thermal skin burns cause local injury as well as triggers acute systemic inflammation response where the imbalance between oxidative and antioxidative system occurs.As an alternative treatment, various medicinal herbs are used to treat burn injuries in many countries. In this study, the possible protective role of oral or topical Myrtle (Myrtus communis L.) treatment against burn-induced damage was investigated. The dorsum of the Wistar Albino rats was shaved and exposed to 90 °C water bath in burn group or 25 °C water bath in control group for 10 s under ether anesthesia. Myrtle extract was applied 100 mg/kg/day for 2 days either orally or topically. In skin samples; malondialdehyde and glutathione levels, catalase, superoxide dismutase, nitric oxide and tissue factor activities were determined. Skin tissues were also examined by light microscopy. Severe thermal skin burn injury caused a significant decrease in glutathione level, superoxide dismutase, catalase and tissue factor activities as well as nitric oxide level, which was accompanied with significant increases in skin malondialdehyde level. Myrtle treatment reversed all these biochemical indices except topical Myrtle treated group’s nitric oxide level, as well as histopathological alterations, which were induced by thermal trauma. Both oral and topical Myrtle extract treatment was found to have protective role in the burn induced oxidative injury, which may be attributed to the potential antioxidant effect of Myrtle. As a conclusion, Myrtle significantly diminishes burn-induced damage in skin.     

Denatured acellular dermal matrix seeded with bone marrow mesenchymal stem cells for wound healing in mice

It is the basic task of burn therapy to cover the wound with self-healthy skin timely and effectively. However, for patients with extensive burns, autologous skin is usually insufficient, and allogenic or heterogeneous skin leads to strong immune response. It is vital to choose an appropriate treatment for deep extensive burns. Nowadays, the dermal substitute combined with bone marrow mesenchymal stem cells (BM-MSCs) is a prospective strategy for burn wound healing. Denatured acellular dermal matrix (DADM), as one of dermal substitutes, which prepared by burn skin discarded in escharotomy, not only maintains a certain degree of 3D structure of collagen, but also has good biocompatibility. In this study, the preparation method of DADM was improved and DADM was seeded with BM-MSCs. Then BM-MSCs-seeded DADM (DADM/MSCs) was implanted into mice cutaneous wound, and the effect of DADM/MSCs dermal substitute was assessed on skin regeneration. As a result, BM-MSCs survived well and DADM/MSCs scaffolds significantly promoted wound healing in terms of angiogenesis, re-epithelialization and skin appendage regeneration. DADM/MSCs scaffold may represent an alternative promising therapy for wound healing in deep extensive burns.     

大鼠深Ⅱ度烧伤创面保留变性真皮并覆盖自体皮疗效观察

目的为探讨自体皮覆盖变性真皮修复深Ⅱ度烧伤创面的可行性提供实验依据。方法在大鼠背部造成直径3.5cm深Ⅱ度烧伤创面。伤后2—5d行创面浅层削痂保留变性真皮,同时在局部移植大张自体断层皮片。移植前和移植后不同时相点分别切取植皮区全层皮肤,光镜下观察其形态学及胶原纤维变化,并检测其生物力学改变。取移植大鼠同体背部正常皮肤作为对照指标。结果(1)保留的变性真皮为玻璃样变性。(2)大鼠自体皮移植术后7d,皮片与创面融合无法分离,光镜下可见真皮乳头及网状层。术后21d移植部位皮肤厚度、结构、形态与正常组织相似,有萎缩毛囊,胶原纤维条索密度逐渐增大接近融合。(3)鼠皮抗拉强度、最大应变值在术后逐渐增大,至60d时接近正常。结论将自体皮覆盖于变性真皮上用以修复深Ⅱ度烧伤创面,变性真皮能够逐渐复苏,使其结构、形态接近正常。     

Polyunsaturated phosphatidylcholine protects against wound contraction in experimental skin burn injury.

Polyunsaturated phosphatidylcholine (PP) is a soybean lecithin, whose oral supplementation prevents fibrosis and strictures in animal models. The purpose of the present study was to investigate the effect of PP on wound contraction using a model of skin burn injury. Thirty male Sprague-Dawley rats were randomly divided into three groups: control (C), just burn (JB), and burn treated with PP (BPP). Burns were induced on the dorsum of the rats by a metal plate with a 4-cm(2) contact surface. Ten percent emulsion of PP was given orally for 21 days in BPP group. The effects of PP on burns were evaluated histologically on day 21, and wound contraction was measured by planimetry weekly. Microscopic evaluation included the amount and density of myofibroblasts and collgen type III, the existence of mature collagen fibers in the regenerated dermis, and thickness measurements of the regenerated tissue. There was a significant decrease in mean wound size index in group JB when compared to BPP on days 14 and 21 (p <.05 and p <.01, respectively). After 3 weeks, the granulation tissue was more cell dense, containing high numbers of myofibroblasts in group JB when compared to BPP (p <.05). The wounds had more mature collagen bundles, but less collagen type III in group BPP when compared to JB. Thus, polyunsaturated phospatidylcholine protected against wound contraction and modulated wound healing in the rat model of skin burn injury.     

Stromal vascular fraction improves deep partial thickness burn wound healing

ObjectiveThe practice of early burn wound excision and wound closure by immediate autologous skin or skin substitutes is the preferred treatment in extensive deep partial and full-thickness burns. To date there is no proven definite medical treatment to decrease burn wound size and accelerate burn wound healing in modern clinical practice. Stromal vascular fraction is an autologous mixture that has multiple proven beneficial effects on different kinds of wounds. In our study, we investigated the effects of stromal vascular fraction on deep partial-thickness burn wound healing.MethodsIn this study, 20 Wistar albino rats were used. Inguinal adipose tissue of the rats was surgically removed and stromal vascular fraction was isolated. Thereafter, deep second-degree burns were performed on the back of the rats by hot water. The rats were divided into two groups in a randomized fashion. The therapy group received stromal vascular fraction, whereas the control group received only physiologic serum by intradermal injection. Assessment of the burn wound healing between the groups was carried out by histopathologic and immuno-histochemical data.ResultsStromal vascular fraction increased vascular endothelial growth factor, proliferating cell nuclear antigen index, and reduced inflammation of the burn wound. Furthermore, vascularization and fibroblastic activity were achieved earlier and observed to be at higher levels in the stromal vascular fraction group.ConclusionsStromal vascular fraction improves burn wound healing by increasing cell proliferation and vascularization, reducing inflammation, and increasing fibroblastic activity.     

Porcine dermal collagen as a wound dressing for skin donor sites and deep partial skin thickness burns

Collagen was extracted by pepsin digestion from porcine skin, and collagen membrane was prepared by salt precipitation. The porcine collagen membrane was evaluated as a burn wound dressing in deep partial skin thickness burn wounds in rats. Burn wounds, 4 × 4 cm, were inflicted by exposure of skin to 75°C for 15 s followed by de-epithelialization. Wound healing was assessed by planimetry of epithelialization on day 10 after injury. Open wounds exhibited 24 per cent of wound area re-epithelialized. Collagen membrane dressing significantly improved the healing to 69 per cent of wound area (P < 0.0001). In a completely separate experiment, the porcine collagen membrane was applied as a wound dressing to the donor sites of burn patients, and its effect on wound healing was compared with that of a petroleum jelly gauze dressing. The donor sites covered with petroleum jelly gauze had re-epithelialized by an average of 14.5 days (ranging from 13 to 16 days) after wounding. The wounds dressed with collagen membrane demonstrated a significant increase in the healing rate. Complete re-epithelialization was observed by 10.3 days (ranging from 10 to 12 days) after wounding (P < 0.0001).     

Interferon‐gamma inhibits healing post scald burn injury

Impaired healing after severe burns remains a reason for prolonged hospitalization, opportunistic infections, and debilitating scarring. Interferon‐gamma (IFN‐γ) is an important immune regulator that has been shown to inhibit collagen synthesis by fibroblasts, resulting in delayed healing in incision wounds. To determine whether IFN‐γ plays similar roles in the healing process after severe burn, we induced scald injury in mice deficient or sufficient in IFN‐γ and examined local responses. In the absence of IFN‐γ, scalded areas healed faster. This was associated with attenuated local inflammatory responses, enhanced reepithelialization, increased proliferation of keratinocytes in reepithelialized leading edges, and up‐regulation of growth factors in burned skin areas. Furthermore, angiogenesis and myofibroblast formation commenced and terminated earlier in IFN‐γ^–/– mice compared with wild type (WT) controls. Our observations demonstrate that inhibition of IFN‐γ results in accelerated healing after burn injury by dampening excessive inflammation and facilitating reepithelialization, collagen deposition, and wound contraction.     

Interstitial‐matrix edema in burns: Mechanistic insights from subatmospheric pressure treatment in vivo

Thermal injury disrupts fluid homeostasis and hydration, affecting hemodynamics and local interstitial fluid‐driving forces, leading rapidly to edema. This study explores local mechanisms in vivo, after deep partial‐thickness burns in the dermal matrix. Heat‐damaged skin was obtained from pig corpses, byproducts of unrelated burn treatment protocols approved by the Institutional Animal‐Care‐and‐Use Committee. Hydration potential and flow rates were measured by osmotic stress techniques at 4 and 37 °C, and collagen folding/unfolding was examined by differential scanning calorimetry and diffusion tensor magnetic resonance imaging. Kinetic and equilibrium hydration parameters differed in heat‐damaged and undamaged skin; the mean hydration potential and initial flow rates of damaged skin were negative at 37 but positive at 4 °C, in contrast to the positive mean at either temperature of explants taken from undamaged skin sites on the same animals. After subatmospheric pressure treatment (125 mmHg), parameters in damaged reversed to values similar to those of undamaged, whereas the proportion of folded collagen and unidirectional resistance to water diffusion increased. Together, results support interfacial rather than colloidosmotic fluid transfer mechanisms in burns and confirm in vivo the relevance of collagen folding/unfolding, further suggesting collagen structural transitions as potential therapeutic targets and models for engineered biomimetic materials.     

The treatment of Lewisite burns with laser debridement---'lasablation'

Lewisite (dichloro (2-chlorovinyl) arsine) was first synthesised in 1918 and its potential for use in military confrontations as a vesicant agent has been widely recognised. These agents cause blistering skin reactions with resultant full thickness burns. Effective treatments to date have been delayed by the lack of suitable animal models. Porcine skin has recently been used successfully to model the development and natural history of these burn injuries. A large white pig model (n=6) was employed to investigate the effectiveness of CO(2) and Erbium-YAG lasers (EYL) in laser dermabrasion of established Lewisite burns. Burns underwent treatment at 4 days post-exposure and were assessed at 1, 2 and 3 weeks, thereafter, for the rate of epithelial healing. The re-epithelialisation rates in the laser dermabraded groups were accelerated by a factor of four compared to untreated controls by the first week (analysis of vartiance, ANOVA, P=0.006 for pulsed CO(2) and P=0.011 for Erbium-YAG). Ablation of the burn eschar was thought to accelerate the rate of healing by causing partial debridement. This method has been termed 'lasablation' and represents a significant advance in the clinical management of this type of injury.     

Skin flap survival after superficial and deep partial-thickness burn injury

Whether a flap can be raised successfully in a body region that has been subjected to burn injury remains an issue. The aim of this study was to investigate the survival of skin flaps that were elevated after superficial and deep partial-thickness burn injury in a rat model. Sixty-five rats were divided into five groups: Group 1 (N = 15) was the control group, group 2 (N = 10) included rats with superficial partial-thickness burns that had flaps elevated on day 0, group 3 (N = 15) was comprised up of rats with superficial partial-thickness burns that had flaps elevated on day 4, group 4 (N = 10) included rats with deep partial-thickness burns that had flaps elevated on day 0, and group 5 (N = 15) was comprised of rats with deep partial-thickness burns that had flaps elevated on day 4. Caudally based dorsal flaps consisting of skin and panniculus carnosus were elevated in all groups, and the amount of surviving tissue on each flap was quantified. The surviving areas of flaps elevated on postburn days 0 and 4 in superficial partial-thickness burn zones (groups 2 and 3) were larger than those of flaps that were elevated on postburn days 0 and 4 in deep partial-thickness burn zones (groups 4 and 5). The surviving portions of flaps that were elevated on day 4 in superficial partial-thickness burn zones (group 3) were similar to the surviving areas of flaps in the control group (group 1), and were larger than those of all other groups (groups 2, 4, and 5). In this rat model, flaps were elevated in superficial dermal burn zones with successful outcomes. However, raising flaps in deep dermal burn zones was not a reliable method.     

Effective enzymatic debridement of burn wounds depends on the denaturation status of collagen

The treatment of burn wounds by enzymatic debridement using bromelain has shown promising results in our burn center. However, inadequate debridement occurred in a few cases in which the etiology of the burn was attributed to relatively low temperature burns. We hypothesized that bromelain is ineffective in burns in which collagen denaturation, which occurs approximately at 65°C, has not taken place. Our objective was to assess whether there is a relationship between the denaturation of collagen and the ability of bromelain to debride acute scald burn wounds of different temperatures. Ex vivo human skin from four different donors was cut into 1x1 cm samples, and scald burns were produced by immersion in water at temperatures of 40°C, 50°C, 60°C, 70°C, and 100°C for 20 minutes. Denaturation of collagen was assessed with histology, using hematoxylin and eosin (H&E) staining and a fluorescently labeled collagen hybridizing peptide (CHP), and with second harmonic generation (SHG) microscopy. Burned samples and one control sample (room temperature) were weighed before and after application of enzymatic debridement to assess the efficacy of enzymatic debridement. After enzymatic debridement, a weight reduction of 80% was seen in the samples heated to 70°C and 100°C, whereas the other samples showed a reduction of 20%. Unfolding of collagen, loss of basket‐weave arrangement, and necrosis was seen in samples heated to 60°C or higher. Evident CHP fluorescence, indicative of collagen denaturation, was seen in samples of 60°C, 70°C and 100°C. SHG intensity, signifying intact collagen, was significantly lower in the 70°C and 100°C group (P <.05) compared to the lower temperatures. In conclusion, denaturation of collagen in skin samples occurred between 60°C and 70°C and strongly correlated with the efficacy of enzymatic debridement. Therefore, enzymatic debridement with the use of bromelain is ineffective in scald burns lower than 60°C.     

Evaluation of the burn wound with perfusion fluorometry

Determination of depth of burn injury using vital dyes has been unsatisfactory. The present study evaluated the ability of the fiberoptic perfusion fluorometer to assess the depth of burn in the early postburn period. Sixty-three burns were examined with the fluorometer after intravenous administration of sodium fluorescein. The fluorescein kinetics were monitored for 1 hour within the first 48 hours and again between the third and sixth days postburn. The rate of fluorescein uptake and burn wound fluorescence was determined and compared to that of normal unburned skin. Depth of burn was confirmed by biopsy and healing characteristics. Fluorometric analysis during both study periods consistently distinguished between partial-thickness and full-thickness burns. Partial-thickness burns uniformly exhibited fluorescence within 10 minutes; full-thickness burns showed nil fluorescence. None of the patients experienced a change in skin color or complications from the small dose of fluorescein given.     

A recombinant human collagen hydrogel for the treatment of partial-thickness burns: A prospective,self-controlled clinical study

IntroductionAllogeneic and xenogeneic skin are recognized as the best coverings for skin burn wounds, but currently face a supply shortage. To solve this problem, our research group developed a standardized manufactured hydrogel dressing based on a new type of highly bioactive recombinant human collagen.Study designProspective self-controlled trial.ObjectiveTo evaluate the efficacy and safety of recombinant human collagen hydrogel in the treatment of partial burn wounds to the skin compared to those of xenogeneic skin.MethodsThis study included twenty-one patients admitted to Shanghai Changhai Hospital within 48 h after receiving partial-thickness skin burns. The wounds were symmetrically separated along the axis and treated with recombinant human collagen hydrogel (RHCH) or a human-CTLA4-Ig gene-transferred pig skin xenotransplant. The condition of the wound surfaces was recorded on days 0 (of enrollment), 5, 10, 15, and 20, and bacterial drug sensitivity testing, hematuria examination, and electrocardiographic tests were conducted on days 0, 10, 20, or on the day of wound healing.ResultsThere were no statistically significant differences in wound healing time between the two groups. The median number of days to healing was 11.00 ± 0.56 for xenogeneic skin vs. 11.00 ± 1.72 for RHCH.ConclusionDuring the observation period, the therapeutic effect of the RHCH developed by our group on partial-thickness burn wounds was not significantly different from that of gene-transferred xenogeneic skin. Thus, our designed RHCH shows potential for clinical use to treat burn wounds on the skin.     

Cold-water treatment of scald injury and inhibition of histamine-mediated burn edema

Using the hairless mouse ear burn model we have studied the effects of immediate short-term cold-water treatment (CWT) of a moderate scald injury (54°C water for 20 sec) to intact skin using 8–10°C water for 5 min. CWT following scald injury caused brief periods of arteriolar vasomotion and shortened the duration of postburn venular dilation by about 50% as compared to untreated burns. CWT significantly (P < 0.05) reduced edema formation of the burned ear for only the first 2 hr after injury as compared to untreated burns. Significant delayed remote edema formation normally observed in abdominal and unburned (contralateral) ear skin after untreated ear scald injury was abolished after CWT to the burned ear. Chemical inhibition of remote burn edema, comparable to that achieved with CWT, was produced by cimetidine pretreatment or histamine depletion prior to injury. CWT also significantly decreased tissue histamine loss of the burned ear after scald injury Ultrastructural demonstration of increased vacuole formations in capillary and venular endothelial cells in edematous, unburned ear skin 2 hr after untreated scald injury suggested the presence of histamine-mediated remote burn edema formation. Predominant pathophysiologic mechanisms of CWT in local and remote burn edema inhibition and perhaps burn shock protection were suggested as: (1) decreased vascular response with a shortened duration of postburn venular dilation, and, most importantly, (2) the inhibition of local and systemic histamine release from thermally injured tissues.