The use of human deceased donor skin allograft in burn care

Burns are tissue wounds caused by thermal, electrical, chemical cold or radiation injuries. Deep injuries lead to dermal damage that impairs the ability of the skin to heal and regenerate on its own. Skin autografting following burn excision is considered the current gold standard of care, but lack of patient’s own donor skin or unsuitability of the wound for autografting may require the temporary use of dressings or skin substitutes to promote wound healing, reduce pain, and prevent infection and abnormal scarring. These alternatives include deceased donor skin allograft, xenograft, cultured epithelial cells and biosynthetic skin substitutes. Allotransplantation is the transplantation of cells, tissues, or organs, sourced from a genetically non-identical member of the same species as the recipient. Human deceased donor skin allografts represent a suitable and much used temporizing option for skin cover following burn injury. The main advantages for its use include dermoprotection and promotion of reepithelialisation of the wound and their ability to act as skin cover until autografting is possible or re-harvesting of donor sites becomes available. Disadvantages of its use include the limited abundance and availability of donors, possible transmission of disease, the eventual rejection by the host and its handling storing, transporting and associated costs of provision. This paper will explore the role of allograft skin in burn care, defining the indications for its use in burn management and the future potential for allograft tissue banking.     

Hair follicle destruction and regeneration in guinea pig skin after cutaneous freeze injury

We have investigated the histological changes in hair follicles in guinea pig skin after standardized moderate and severe cryosurgery injuries. Hair follicles were permanently destroyed by cryosurgery, but more than one mechanism may be operative during follicle destruction and shedding. The mechanism depends upon the severity of the freeze. After a light freeze injury, the changes are predominantly within the hair follicle. The hair is shed at the surface and there is selective autolysis of follicular cells, but dermal connective tissue is preserved and there is little surrounding damage. However, after a severe cryoinjury as used in "tumor doses," there is destruction of dermal connective tissue and dermal scarring. The necrotic dermis is shed, taking with it the dead follicles and morphologically normal elastic tissue.     

Effects of recombinant human growth hormone on the development of burn scarring.

Anabolic agents, such as recombinant human growth hormone (rhGH), have been used effectively to ameliorate the catabolic response to burn injury and to improve wound-healing. However, in experimental animal models, growth hormone has also been associated with increased renal scarring. The effect of rhGH on the development of human scarring is unknown. Therefore, the purpose of this study was to assess the effect of rhGH on the scarring of human skin after burn injury. A series of 94 patients was studied in a prospective randomized double-blind clinical trial. Patients receiving 0.2 mg/kg/day subcutaneous rhGH during their acute hospital stays presented with the same quality and intensity of scarring as patients receiving a placebo. Similar reconstructive needs also resulted. The treatment of severely burned children with recombinant human growth hormone during the acute-phase hospital course did not increase scarring of the burn wound.     

Morphologic characterization of acute injury to vascular endothelium of skin after frostbite

Cyclo-oxygenase inhibitors and free-radical scavengers protect the skin against necrosis induced by frostbite. However, the tissue component(s) that determine the evolution of skin necrosis and the mechanism of this pharmacologic protection are not precisely defined. We have studied freezing injury to rabbit ears by serial biopsies examined by light and electron microscopy. The morphologic evidence of skin injury due to freezing was localized exclusively in the endothelial cells, particularly in the arterioles. Within 1 hour, the entire microvasculature demonstrated endothelial damage. Intravascular platelet aggregation occurred just after thawing and closely paralleled the endothelial cell injury. Very few neutrophils were seen initially (at 10 minutes). By 1 hour, leukocyte aggregates were present, and they further increased at 6 hours. Swelling of the interstitium started 10 minutes after thawing, while extravasation of erythrocytes began to appear by 6 hours. Parenchymal elements of skin were relatively free of damage. In the ear cartilage, the chondrocytes showed evidence of damage immediately after freezing. The administration of superoxide dismutase (SOD) during thawing (reperfusion) did not qualitatively alter any of the initial morphologic changes induced by freezing. We conclude that the endothelial cell is the initial target of injury induced by freezing, an initial injury that is mediated by a non-free-radical-mediated mechanism. It is likely that this acute injury ultimately compromises blood flow and leads to skin necrosis.     

Protein and miRNA profiling of radiation-induced skin injury in rats: the protective role of peroxiredoxin-6 against ionizing radiation

Radiation-induced skin injury is a serious concern during radiotherapy. However, the molecular mechanism underlying the pathogenesis of radiation-induced skin injury has not been extensively reported. Most biological functions are performed and regulated by proteins and noncoding RNAs, including microRNAs (miRNAs). The interplay between mRNA and miRNA has been implicated in disease initiation and progression. Technical advances in genomics and proteomics have enabled the exploration of the etiology of diseases and have the potential to broaden our understanding of the molecular pathogenesis of radiation-induced skin injury. In this study, we compared the protein and miRNA expression in rat skin irradiated with a 45-Gy electron beam with expression from adjacent normal tissues. We found 24 preferentially expressed proteins and 12 dysregulated miRNAs in irradiated skin. By analyzing the protein and miRNA profiles using bioinformatics tools, we identified a possible interaction between miR-214 and peroxiredoxin-6 (PRDX-6). Next, we investigated the expression of PRDX-6 and the consequences of its dysregulation. PRDX-6 is suppressed by radiation-inducible miR-214 and is involved in the pathogenesis of radiation-induced skin injury. Overexpression of PRDX-6 conferred radioresistance on cells, decreased cell apoptosis, and preserved mitochondrial integrity after radiation exposure. In addition, in vivo transfection with PRDX-6 reduced radiation-induced reactive oxygen species and the malondialdehyde concentration and ameliorated radiation-induced skin damage in rats. Our present findings illustrate the molecular changes during radiation-induced skin injury and the important role of PRDX-6 in ameliorating this damage in rats.     

Role of the p38 mitogen‐activated protein kinase signaling pathway in estrogen‐mediated protection following flap ischemia‐reperfusion injury

Ischemia‐reperfusion (I/R) injury often occurs during skin flap transplantation and results in tissue damage. Although estrogen treatment significantly alleviates this I/R injury‐induced damage, the detailed molecular mechanism is not clear. In this study, a superficial epigastric artery flap I/R injury model was created in adult Wistar rats. Severe necrosis was observed in skin tissue after I/R injury. Histological examination of skin tissue revealed that I/R injury damages skin structure and results in neutrophil infiltration. Inflammation‐related parameters, including neutrophil count, tumor necrosis factor‐α, and interleukin‐10 levels, were increased due to I/R injury. These pathological phenomena were reduced by estradiol treatment. Further investigation found that I/R injury triggers the p38 mitogen‐activated protein kinase (p38‐MAPK) pathway. The expression levels of p38‐MAPK and phosphorylated p38‐MAPK were increased after I/R injury. Estradiol increased the expression level of MAPK phosphatase‐2, a putative phosphatase of p38, and reduced the levels of p38‐MAPK and phosphorylated p38‐MAPK. These results suggest that estradiol can improve skin flap survival, possibly by inhibiting neutrophil infiltration and the expression of p38‐MAPK. This study provides an explanation for how estrogen alleviates I/R injury‐induced damage that occurs during skin flap transplantation. In a rat pathological model, I/R injury leads to skin necrosis, skin structure damage, neutrophil infiltration, and inflammatory cytokine secretion, which are probably downstream effects of activation of the p38‐MAPK pathway. On the other hand, estradiol treatment triggers the expression of MAPK phosphatase‐2, a putative phosphatase of p38‐MAPK, and reduced all examined pathological phenomena. Therefore, estrogen may reduce the deleterious effect of I/R injury on skin flap transplantation through modulating the p38‐MAPK pathway.     

Promoting Optic Nerve Regeneration in Adult Mice with Pharmaceutical Approach

Our previous research has suggested that lack of Bcl-2-supported axonal growth mechanisms and the presence of glial scarring following injury are major impediments of optic nerve regeneration in postnatal mice. Mice overexpressing Bcl-2 and simultaneously carrying impairment in glial scar formation supported robust optic nerve regeneration in the postnatal stage. To develop a therapeutic strategy for optic nerve damage, the combined effects of chemicals that induce Bcl-2 expression and selectively eliminate mature astrocytes—scar forming cells—were examined in mice. Mood-stabilizer, lithium, has been shown to induce Bcl-2 expression and stimulate axonal outgrowth in retinal ganglion cells in culture and in vivo. Moreover, astrotoxin (alpha-aminoadipate), a glutamate analogue, selectively kills astrocytes while has minimal effects on surrounding neurons. In the present study, we sought to determine whether concurrent applications of lithium and astrotoxin were sufficient to induce optic nerve regeneration in mice. Induction of Bcl-2 expression was detected in the ganglion cell layer (GCL) of mice that received a lithium diet in compared with control-treated group. Moreover, efficient elimination of astrocytes and glial scarring was observed in the optic nerve of mice treated with astrotoxin. Simultaneous application of lithium and astrotoxin, but not any of the drugs alone, induced robust optic nerve regeneration in adult mice. These findings further support that a combinatorial approach of concurrent activation of Bcl-2-supported growth mechanism and suppression of glial scarring is required for successful regeneration of the severed optic nerve in adult mice. They suggest a potential therapeutic strategy for treating optic nerve and CNS damage. Special issue article in honor of Dr. Ji-Sheng Han.     

The scarless heart

Over the past several years many mechanisms by which myocardial replacement could be achieved have been described. These include resident cardiac stem cells or circulating stem cells that can either differentiate into, or fuse to cardiomyocytes, or mature cells that can transdifferentiate into cardiomyocytes. However, the fact remains that after injury to the heart, the overriding response is scar formation with little myocardial replacement. One exception to this response is the MRL mouse, which heals with little scarring and shows nearly full myocardial replacement after injury. Results obtained with this model will be discussed.     

Free radical involvement in hypertrophic scar formation

Hypertrophic scarring following thermal injury has become a major problem in Hong Kong. There is evidence that immunological and biochemical changes are associated with thermal injury, including pyridinoline crosslinks which are present in large quantities in hypertrophic scar, but the primary cause of hypertrophic scar formation still remains to be established. It has been reported that free radicals are assosciated with the formation of pyridinoline. In this study, attempts have been made to elucidate the involvement of free radicals in hypertrophic scar formation after thermal injury by determining the concentrations of Complement, free iron and pyridinoline crosslinks in collagen fibres. The results showed that the Complement activation product, C3d, was increased in the first week (i.e., day 7) postburn, indicating an acute inflammatory response. Free radicals, reported to be associated with the formation of pyridinoline crosslinks, and free iron content, were also found to have higher concentration in hypertrophic scar than in normal skin. The data suggest the involvement of free radical in hypertrophic scar formation. The observed increase in serum C3d concentration in about the first week indicates an acute inflammatory response to thermal injury. Both C3d and free iron concentrations (in vitro) are found higher in hypertrophic scar than in normal skin may suggest their roles in the generation of free radicals.     

The inflammatory response after an epidermal burn depends on the activities of mouse mast cell proteases 4 and 5

A second-degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to nonmuscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-Kit(W)/Kit(Wv) mice, which lack MCs in a context of other defects due to a mutation of the Kit receptor. To address further an MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury after a second-degree scald burn, whereas mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficient mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first hour postburn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing nonredundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.     

Side effects and complications of variable-pulsed erbium:yttrium-aluminum-garnet laser skin resurfacing: extended experience with 50 patients

Recent advances in technology have provided laser surgeons with new options for cutaneous laser resurfacing. Despite its popularity, there is limited information on the short-term and long-term side effects and complications of variable-pulsed erbium:yttrium-aluminum-garnet (erbium:YAG) laser skin resurfacing. The purpose of this study was to prospectively evaluate postoperative wound healing, side effects, and complications of multiple-pass, variable-pulsed erbium:YAG laser skin resurfacing for facial photodamage, rhytides, and atrophic scarring. Fifty consecutive patients with facial photodamage, rhytides, or atrophic scarring were treated with a variable-pulsed erbium:YAG laser. Side effects and complications relating to postoperative healing, erythema, and pigmentary changes were tabulated. Patients were evaluated at postoperative days 3 through 7 and at 1, 3, 6, and 12 months after laser skin resurfacing. The average time for reepithelialization was 5.1 days. Prolonged erythema (>1 month) was observed in three patients (6 percent). Transient hyperpigmentation occurred in 20 patients (40 percent), with an average duration of 10.4 weeks. No cases of hypopigmentation or scarring were seen. In summary, a variable-pulsed erbium:YAG laser can safely be used for the treatment of facial photodamage, rhytides, and atrophic scarring. Although more postoperative erythema is seen after variable-pulsed erbium:YAG laser treatment than is usually produced with a short-pulsed erbium:YAG system, the side-effect profile and recovery period after variable-pulsed erbium:YAG laser skin resurfacing still are more favorable than after multiple-pass carbon dioxide laser skin resurfacing.     

Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors

Severe traumatic injury to the adult mammalian CNS leads to life-long loss of function. By contrast, several non-mammalian vertebrate species, including adult zebrafish, have a remarkable ability to regenerate injured organs, including the CNS. However, the cellular and molecular mechanisms that enable or prevent CNS regeneration are largely unknown. To study brain regeneration mechanisms in adult zebrafish, we developed a traumatic lesion assay, analyzed cellular reactions to injury and show that adult zebrafish can efficiently regenerate brain lesions and lack permanent glial scarring. Using Cre-loxP-based genetic lineage-tracing, we demonstrate that her4.1-positive ventricular radial glia progenitor cells react to injury, proliferate and generate neuroblasts that migrate to the lesion site. The newly generated neurons survive for more than 3 months, are decorated with synaptic contacts and express mature neuronal markers. Thus, regeneration after traumatic lesion of the adult zebrafish brain occurs efficiently from radial glia-type stem/progenitor cells.     

Gingival Fibroblasts Display Reduced Adhesion and Spreading on Extracellular Matrix: A Possible Basis for Scarless Tissue Repair?

Unlike skin, oral gingiva do not scar in response to injury. The basis of this difference is likely to be revealed by comparing the responses of dermal and gingival fibroblasts to fibrogenic stimuli. Previously, we showed that, compared to dermal fibroblasts, gingival fibroblasts are less responsive to the potent pro-fibrotic cytokine TGFβ, due to a reduced production of endothelin-1 (ET-1). In this report, we show that, compared to dermal fibroblasts, human gingival fibroblasts show reduced expression of pro-adhesive mRNAs and proteins including integrins α2 and α4 and focal adhesion kinase (FAK). Consistent with these observations, gingival fibroblasts are less able to adhere to and spread on both fibronectin and type I collagen. Moreover, the enhanced production of ET-1 mRNA and protein in dermal fibroblasts is reduced by the FAK/src inhibitor PP2. Given our previous observations suggesting that fibrotic fibroblasts display elevated adhesive properties, our data suggest that scarring potential may be based, at least in part, on differences in adhesive properties among fibroblasts resident in connective tissue. Controlling adhesive properties may be of benefit in controlling scarring in response to tissue injury.     

Peptides released by ameboid microglia regulate astroglial proliferation

Peptides that stimulate astroglial proliferation are produced in traumatized adult rat brain by 10 d after injury. These same peptides are released by ameboid microglia activated in vitro. Our findings suggest that astroglial scarring is regulated in part by the release of factors from ameboid microglia near the site of brain injury.     

CD44-dependent inflammation,fibrogenesis, and collagenolysis regulates extracellular matrix remodeling and tensile strength during cutaneous wound healing

Cutaneous wound healing consists of three main phases: inflammation, re-epithelialization, and tissue remodeling. During normal wound healing, these processes are tightly regulated to allow restoration of skin function and biomechanics. In many instances, healing leads to an excess accumulation of fibrillar collagen (the principal protein found in the extracellular matrix - ECM), and the formation of scar tissue, which has compromised biomechanics, tested using ramp to failure tests, compared to normal skin (Corr and Hart, 2013 [1]). Alterations in collagen accumulation and architecture have been attributed to the reduced tensile strength found in scar tissue (Brenda et al., 1999; Eleswarapu et al., 2011). Defining mechanisms that govern cellular functionality and ECM remodeling are vital to understanding normal versus pathological healing and developing approaches to prevent scarring. CD44 is a cell surface adhesion receptor expressed on nearly all cell types present in dermis. Although CD44 has been implicated in an array of inflammatory and fibrotic processes such as leukocyte recruitment, T-cell extravasation, and hyaluronic acid (the principal glycosaminoglycan found in the ECM) metabolism, the role of CD44 in cutaneous wound healing and scarring remains unknown. We demonstrate that in an excisional biopsy punch wound healing model, CD44-null mice have increased inflammatory and reduced fibrogenic responses during early phases of wound healing. At wound closure, CD44-null mice exhibit reduced collagen degradation leading to increased accumulation of fibrillar collagen, which persists after wound closure leading to reduced tensile strength resulting in a more severe scarring phenotype compared to WT mice. These data indicate that CD44 plays a previously unknown role in fibrillar collagen accumulation and wound healing during the injury response.     

LIQUID NITROGEN IN THE TREATMENT OF SKIN DISEASES

Liquid nitrogen is a satisfactory freezing agent in the treatment of such skin diseases as warts, keratoses, superficial hemangiomas, leukoplakia, keloids, superficial scarring and dermatitis venenata. It is available and easily applied. Its use is described and the results of treatment in 154 cases of warts are presented.     

Deep Tissue Injury in Development of Pressure Ulcers: A Decrease of Inflammasome Activation and Changes in Human Skin Morphology in Response to Aging and Mechanical Load

Molecular mechanisms leading to pressure ulcer development are scarce in spite of high mortality of patients. Development of pressure ulcers that is initially observed as deep tissue injury is multifactorial. We postulate that biomechanical forces and inflammasome activation, together with ischemia and aging, may play a role in pressure ulcer development. To test this we used a newly-developed bio-mechanical model in which ischemic young and aged human skin was subjected to a constant physiological compressive stress (load) of 300 kPa (determined by pressure plate analyses of a person in a reclining position) for 0.5–4 hours. Collagen orientation was assessed using polarized light, whereas inflammasome proteins were quantified by immunoblotting. Loaded skin showed marked changes in morphology and NLRP3 inflammasome protein expression. Sub-epidermal separations and altered orientation of collagen fibers were observed in aged skin at earlier time points. Aged skin showed significant decreases in the levels of NLRP3 inflammasome proteins. Loading did not alter NLRP3 inflammasome proteins expression in aged skin, whereas it significantly increased their levels in young skin. We conclude that aging contributes to rapid morphological changes and decrease in inflammasome proteins in response to tissue damage, suggesting that a decline in the innate inflammatory response in elderly skin could contribute to pressure ulcer pathogenesis. Observed morphological changes suggest that tissue damage upon loading may not be entirely preventable. Furthermore, newly developed model described here may be very useful in understanding the mechanisms of deep tissue injury that may lead towards development of pressure ulcers.     

Functional and structural evaluation of the vasculature of skin flaps after ischemia and reperfusion

Free radicals and other toxic oxygen species play a role in the pathogenesis of ischemic organ damage. The abdominal skin flap has been used as a model to study the effects of superoxide dismutase on the survival of ischemic skin. We have evaluated the evolution of functional and structural injury to the vasculature after ischemic injury in superoxide dismutase-treated and control skin flaps. Ischemia was induced by creating abdominal skin flaps and occluding either the venous or both the venous and arterial blood supplies. Superoxide dismutase was administered immediately after the occlusion was released. At 1 hour of reflow, erythrocyte stasis, platelet deposition, neutrophil adherence, and injury to the endothelium of the large vessels and of the microvasculature were evident. The blood flow in the ischemic skin was only 3 percent of normal. Superoxide dismutase caused no change in the ultrastructure of the vasculature and a marginal decrease in vascular permeability in the ischemic skin at 1 hour of reflow. Increased fluorescent staining of the skin was evident after 24 hours of reflow in the superoxide dismutase-treated flaps. These findings indicate that injury to vascular endothelium by ischemia and reperfusion plays a role in the evolution of skin necrosis.