Mark W. Fear

Characterisation of the cell suspension harvested from the dermal epidermal junction using a ReCell® kit

BACKGROUND The use of non-cultured autologous cells to promote wound healing and in reconstructive procedures is increasing. One common method for preparing these cells is the use of the ReCell(®) device. However, despite its current clinical use, no characterisation of the cell suspension produced using a ReCell(®) device has been published. OBJECTIVE To characterise the ReCell suspension that is applied to wounds for cell type, viability, yield, stability and proliferative potential. METHODS The ReCell(®) device was used to harvest cells from a 2 cm(2) piece of split-thickness skin isolated using a dermatome. The resulting cell suspension was analysed for cell yield, cell type, viability over time, proliferative potential and reproducibility. RESULTS Average viable cell yield was 1.7×10(6)/cm(2) of tissue, with 75.5% of the total cell isolate viable. Total viable cell number was not significantly reduced after 4 h storage at 22°C or 4°C, and was stable for 24 h at 4°C. Proliferative potential was assessed using a colony forming assay, with 0.3% of viable cells isolated forming keratinocyte colonies. Predominantly the suspension contained keratinocytes (64.3±28.8%) and fibroblasts (30.3±14.0%), with a small population of melanocytes also identified (3.5±0.5%). Finally, the supernatant contained low total protein (0.92 mg/ml) and the supernatant had no significant effects on cell viability or growth when applied ex vivo. CONCLUSIONS These results suggest the ReCell(®) device provides a method for the preparation of a cell suspension with high viability and proliferative potential, containing viable melanocytes and no apparent toxic cell debris. Further work on the sustained viability of these cells in vivo, and in particular after application to the wound, will be important to better understand the potential of the ReCell(®) device in the clinic.

Bone marrow-derived cells in the healing burn wound—More than just inflammation

Scarring after severe burn is a result of changes in collagen deposition and fibroblast activity that result in repaired but not regenerated tissue. Re-epithelialisation of wounds and dermal cell repopulation has been thought to be driven by cells in the periphery of the wound. However, recent research demonstrated that cells originating from the bone marrow contribute to healing wounds in other tissues and also after incisional injury. We investigated the contribution of bone marrow-derived cells to long-term cell populations in scar tissue (primarily fibroblasts and keratinocytes) after severe burn. Wild-type mice were lethally irradiated and then the bone marrow reconstituted by injection of chimeric bone marrow cells expressing EGFP marker protein. Mice with chimeric bone marrow were then given a burn, either an 1-cm diameter injury (to mimic minor injury) or 2-cm diameter (to mimic moderate injury). Wounds were analysed at days 1, 3, 7, 14, 21, 28, 56 and 120 using FACS and immunohistochemistry to identify the percentage and cell type within the wound originating from the bone marrow. The inflammatory cell infiltrate at the early time-points was bone marrow in origin. At later time-points, we noted that over half of the fibroblast population was bone marrow-derived; we also observed that a small percentage of keratinocytes appeared to be bone marrow in origin. These findings support the theory that the bone marrow plays an important role in providing cells not only for inflammation but also dermal and epidermal cells during burn wound healing. This increases our understanding of cell origins in the healing wound, and has the potential to impact on clinical practice providing a potential mechanism for intervention away from conventional topical treatments and directed instead to systemic treatments affecting the bone marrow response.

Systemic Decreases in Cutaneous Innervation after Burn Injury

Innervation of the skin is important in order to maintain functional sensation and enable appropriate response to environmental stimuli. Injury to the skin may involve peripheral nerve damage. Previous studies have shown an initial loss of nerve fibers followed by an increase above normal fiber density, which is followed by apoptosis and ultimately reduced innervation and sensory function in scar tissue (Hermanson et al., 1987; Stella et al., Supp.(767) 1994; Altun et al., 2001; Ward et al., 2004; Nedelec et al., 2005). Although some studies have found an association between reduced nerve density and sensation (Stella et al., 1994; Ward et al., 2004), other studies have not (Griffin et al., 2001; Nedelec et al., 2005). The contradictory nature of these findings is at least in part due to small sample numbers, incomplete functional and anatomical assessment, and the variable timeframes between injury and analysis. Herein, to better understand the changes in cutaneous innervation and sensory function, we have analyzed neuroanatomy in a rat model of burn injury, and assessed neuroanatomy in patients with unilateral burn injuries at least 18 months post-injury, which is commonly defined as the end point for scar maturity (Nedelec et al., 2005). All animal experiments were approved by the institutional animal ethics committee and were performed in accordance with the NHMRC Australian code of practice for the care and use of animals for scientific purposes. The human study was carried out in accordance with the regulations outlined in the national statement on ethical conduct in research involving humans issued by the NHMRC and was approved by the Royal Perth Hospital ethics committee.

Designer self-assembling hydrogel scaffolds can impact skin cell proliferation and migration

There is a need to develop economical, efficient and widely available therapeutic approaches to enhance the rate of skin wound healing. The optimal outcome of wound healing is restoration to the pre-wound quality of health. In this study we investigate the cellular response to biological stimuli using functionalized nanofibers from the self-assembling peptide, RADA16. We demonstrate that adding different functional motifs to the RADA16 base peptide can influence the rate of proliferation and migration of keratinocytes and dermal fibroblasts. Relative to unmodified RADA16; the Collagen I motif significantly promotes cell migration, and reduces proliferation.

Changes in cutaneous innervation in patients with chronic pain after burns

BACKGROUND Chronic pain is a common occurrence for burn patients and has significant impact on quality of life. However, the etiology is not well understood. Understanding the mechanisms underlying the restoration of sensory function and the development of chronic pain after burn is critical to improving long-term outcomes. OBJECTIVE To determine whether cutaneous innervation in burn patients with chronic pain is altered when compared to patients without chronic pain. METHODS Twelve patients with unilateral injury and who reported chronic pain were recruited. Each patient underwent sensory function testing and both scar and matched site uninjured skin biopsy. Biopsies were analyzed for total nerve density and nociceptive C-fiber density using immunohistochemistry. Results were compared to a control group of 33 patients with unilateral injury and no reported long-term pain. RESULTS Sensory function was significantly diminished in scar compared to uninjured tissue in both study groups, but chronic pain patients did not have significantly diminished function when compared to control. Total nerve density was not significantly different between scar and uninjured sites in either group, or between groups. However, the density of nociceptive nerve fibers was significantly elevated in both uninjured (p=0.0193) and scar sites (p=0.0316) of the patients with chronic pain when compared to the control group. CONCLUSIONS This data suggests that differences in cutaneous innervation may contribute to chronic pain after burn. There also appears to be a systemic difference in cutaneous innervation extending to distal uninjured sites. Therefore efforts to affect cutaneous reinnervation after burn may lead to less patients experiencing chronic pain.

AP-1 inhibitory peptides are neuroprotective following acute glutamate excitotoxicity in primary cortical neuronal cultures

J. Neurochem. (2010) 112, 258–270.

Exogenous metallothionein-IIA promotes accelerated healing after a burn wound

Severe injury to the epidermal barrier often results in scarring and life‐long functional deficits, the outcome worsening with a number of factors including time taken to heal. We have investigated the potential of exogenous metallothionein IIA (Zn7‐MT‐IIA), a naturally occurring small cysteine‐rich protein, to accelerate healing of burn wounds in a mouse model. Endogenous MT‐I/II expression increased in basal keratinocytes concurrent with reepithelialization after a burn injury, indicating a role for MT‐I/II in wound healing. In vitro assays of a human keratinocyte cell line indicated that, compared with saline controls, exogenous Zn7‐MT‐IIA significantly increased cell viability by up to 30% (p<0.05), decreased apoptosis by 13% (p<0.05) and promoted keratinocyte migration by up to 14% (p<0.05), all properties that may be desirable to promote rapid wound repair. Further in vitro assays using immortalized and primary fibroblasts indicated that Zn7‐MT‐IIA did not affect fibroblast motility or contraction (p>0.05). Topical administration of exogenous Zn7‐MT‐IIA (2 μg/mL) in vivo, immediately postburn accelerated healing, promoted faster reepithelialization (3 days: phosphate‐buffered saline (PBS), 8.9±0.3 mm diameter vs. MT‐I/II, 7.1±0.7 mm; 7 days: PBS 5.8±0.98 mm vs. MT‐I/II, 3.6±1.0 mm, p<0.05) and reduced epidermal thickness (MT‐I/II: 45±4 μm vs. PBS: 101±19 μm, p<0.05) compared with controls. Our data suggest that exogenous Zn7‐MT‐IIA may prove a valuable therapeutic for patients with burns and other skin injuries.

A Preliminary investigation of the reinnervation and return of sensory function in burn patients treated with INTEGRA

BACKGROUND Loss of sensory function in scar after burn is common, although the basis for this loss is not clear. Additionally, little is known about the effects of different treatment modalities on sensory function and neuroanatomical outcomes in burn patients. Here, we investigated the effects of the use of the INTEGRA(®) dermal scaffold on neuroanatomy and sensory function in acute burn patients. HYPOTHESIS AND OBJECTIVES We hypothesized that the use of artificial dermal templates would inhibit or reduce reinnervation after excision, since regrowth of nerves requires complex molecular interactions. Therefore the primary objective of this study was to identify whether there is regrowth of nerve fibres in the INTEGRA(®) dermal scaffold. The secondary objective was to identify whether the INTEGRA(®) dermal scaffold reduced nerve regrowth or limited sensory function outcomes in acute burn patients. METHODS Five patients treated with INTEGRA(®), cultured epithelial autograft spray (prepared using ReCell(®) (CEA)) and split skin graft (SSG) were assessed for sensory function in scar and uninjured contralateral control skin. Neuroanatomy of scar and control sites was assessed using immunohistochemistry for PGP9.5, CGRP and substance P neuronal markers. Nerve density and sensory function was also assessed in a comparative group (n=8) treated with CEA and SSG only. RESULTS Neuroanatomy was not significantly different in the INTEGRA(®) patients when compared to the CEA/SSG group only. The patients treated with INTEGRA(®) had worse sensory function than those with CEA/SSG only. CONCLUSIONS Peripheral nerves do reinnervate the INTEGRA(®) dermal scaffold. There is no statistically significant reduction in reinnervation observed when compared to a control group. It is possible that the use of artificial dermal constructs, while permissive for nerve regrowth, limit functionality when compared to nerves that regrow through dermal tissue. Further research to understand the causes of this, and into enhancing reinnervation in dermal scaffolds may improve sensory outcome in the most severely burned patients.

Understanding the long-term impacts of burn on the cardiovascular system

BACKGROUND Whilest the most obvious impact of burn is on the skin, systemic responses also occur after burn that lead to wide-spread changes to the body, including the heart. The aim of this study was to assess if burn in mid-aged and older adults is associated with increased long-term admissions and death due to diseases of the circulatory system. METHODS A population-based longitudinal study using linked hospital morbidity and death data from Western Australia was undertaken of adults aged at least 45 years when hospitalized for a first burn (n=6004) in 1980-2012 and a frequency matched non-injury comparison cohort, randomly selected from Western Australias electoral roll (n=22,673). Crude admission rates and cumulative length of stay for circulatory diseases were calculated. Negative binomial and Cox proportional hazards regression modelling were used to generate incidence rate ratios (IRR) and hazard ratios (HR), respectively. HR was used as a measure of the mortality rate ratio (MRR). RESULTS After adjustment for demographic factors and pre-existing health status, the burn cohort had 1.46 times (95% confidence interval (CI): 1.36-1.56) as many admissions and almost three times the number of days in hospital with a circulatory system diagnosis (IRR, 95%CI: 2.90, 2.60-3.25) than the uninjured cohort for circulatory diseases. The burn cohort had higher admission rates for ischaemic heart disease (IRR, 95%CI: 1.21, 1.07-1.36), heart failure (IRR, 95%CI: 2.29, 1.85-2.82) and cerebrovascular disease (IRR, 95%CI: 1.57, 1.33-1.84). The burn cohort was found to have increased long-term mortality caused by circulatory system diseases (MRR, 95%CI: 1.11, 1.02-1.20). CONCLUSIONS Findings of increased hospital admission rates, prolonged length of hospital stay and increased long-term mortality related to circulatory system diseases in the burn cohort provide evidence to support that burn has long-lasting systemic impacts on the heart and circulation.

A peptide inhibitor of c‐Jun promotes wound healing in a mouse full‐thickness burn model

Significant damage to tissue surrounding burn injuries occurs after the removal of the thermal source. This damage is caused by a combination of both necrotic and apoptotic cell death in the zone of stasis. Preserving the zone of stasis can reduce the wound size and thereby improve wound healing. We tested whether a peptide previously identified to inhibit necrotic and apoptotic cell death in neurons through c‐Jun inhibition could enhance wound healing. We first tested the effects of this peptide on a keratinocyte and fibroblast cell line in culture. The peptide promoted proliferation of keratinocytes but had no effect on fibroblast proliferation, while the peptide also inhibited ultraviolet‐induced apoptosis of keratinocytes. We finally tested the peptide in vivo, using a mouse model of burn injury. Wounds that were treated with the peptide reepithelialized faster than controls, while cell death surrounding the wound site was markedly reduced 24 hours postinjury, suggesting that the prevention of apoptosis as well as the proliferative effects of this peptide contribute to the wound healing process. Our data implicate c‐Jun in multiple processes during wound repair and demonstrate that treatment of burn injuries using inhibitors of c‐Jun dimerization at the time of injury can promote wound healing.