Natalie L. Giles

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.

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.

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 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.

Secreted Frizzled related protein-4 (sFRP4) promotes epidermal differentiation and apoptosis

The skin provides vital protection from infection and dehydration. Maintenance of the skin is through a constant program of proliferation, differentiation and apoptosis of epidermal cells, whereby proliferating cells in the basal layer differentiating to form the keratinized, anucleated stratum corneum. The WNT signalling pathway is known to be important in the skin. WNT signalling has been shown to be important both in epidermal development and in the maintenance and cycling of hair follicles and epidermal stem cells. However, the precise role for this pathway in epidermal differentiation remains unknown. We investigated the role of the WNT signalling inhibitor sFRP4 in epidermal differentiation. sFRP4 is expressed in both normal skin and keratinocytes in culture. Expression of sFRP4 mRNA and protein increases with keratinocyte differentiation and apoptosis, whilst exposure of keratinocytes to exogenous sFRP4 promotes apoptosis and expression of the terminal differentiation marker Involucrin. These data suggest sFRP4 promotes epidermal differentiation.

Characterization of trifluralin binding with recombinant tubulin from Trypanosoma brucei.

The binding kinetics of five novel trifluralin analogs with recombinant α- and β-tubulin proteins from Trypanosoma brucei rhodesiense was determined. Native tubulin from rats was used to determine the extent of binding of each analog to mammalian tubulin. The results of this study clearly demonstrate two important characteristics of the binding of these trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian tubulin irrespective of the chemical composition of the trifluralin analog tested. Secondly, they have a stronger affinity for trypanosomal α-tubulin compared with trypanosomal β-tubulin. In addition, compounds 1007, 1008, 1016, and 1017 have strong binding affinities for α-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin.