Burns Research > Laboratory Projects

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A Randomised Multi - Centred Trial to Evaluate Efficacy and Safety of Cultured Epithelial Autografts (CEA) in Combination with a Meshed (4:1) Split Skin Graft (SSG) after Debridement of a Burn Wound

P Maitz, Z Li, K Neuwendyk, J Harvey, J Vandervord, A Phoon, S Taggard, P Kennedy

Thin split thickness skin biopsies are taken under sterile conditions and transported to the Concord Skin Laboratory where the biopsies are processed. A piece of thin-split skin biopsy (about 4 cm2), taken from the available donor site under sterile condition, will be transported in biopsy transport medium to Skin Laboratory at Concord Hospital.  Keratinocytes will be isolated from the separated epidermis following enzymatic digestion. The cells will be seeded into two cell culture flasks and cultivated under established laboratory condition. The cells in one flask will be allowed to grow and differentiate into cultured epidermal autograft (CEA) sheet while the cells in another flask will be maintained at sub-confluent phase for preparing CEA suspension. Prior to surgery the patient will undergo a Laser Doppler on the burn that will be in the study to diagnose depth.

The suspension will be randomly allocated to syringe A or B with the control syringe containing only transport media. On the day of surgery, both CEA sheet and suspension will be harvested under sterile condition one hour before the surgery starts, labelled and then transported to the operating room in an esky with an ice brick.

On operation day (day 0) the burn wound will be debrided and a meshed SSG (4:1) will be applied and secured. Children will be in a separate study group and a suitable mesh size will be chosen by the surgeon. Four 10cm x 10cm window dressings (Surfasoft®) will then cover the SSG. Syringe A and B and a CEA sheet will then be applied to three of the windows and the forth will receive no additional treatment. The wounds will then be covered by a piece of Urgotul® (except the graft with the CEA sheet as Urgotul® is all ready in situ as the carrier dressing) and Surfasoft® secured dressings as per protocol. The dressings will remain intact for 5, days and assessed /redressed until healed. Scarring will then be monitored at 26 weeks and finally at 52 weeks.

A Clinical Evaluation of Efficacy and Safety of Cultured Epithelial Autograft (CEA) Suspension Applied to a Donor Site on a Burn Injured Patient.

P Maitz, P Kennedy, Z Li , S Taggart, T Leong, K Nieuwendyk. 

A patient with severe burn usually needs skin grafting, a surgical procedure that involves transplanting split skin grafts harvested from healthy donor site to wound area. The management of the donor site is, therefore, a very important issue in severe burn patient care. Rapid healing allows the repeat use of the same donor site in patients with large burns. But any delay in donor site healing could lead to complications such as infection and compromise the recovery process of burns patients. This study is designed to examine if the delivery of cultured autologous keratinocytes to donor site wound could facilitate or speed up its healing process.

Burn patients with a donor site ≥ 2% total body surface area will be recruited to join the trial subjecting to informed consent. The two donor sites of each patient will be divided into CEA group and control randomly. Participants will consent to a skin biopsy from which keratinocytes will be isolated and cultivated in Skin Laboratory at Concord  Hospital. On operation day, the cultured keratinocytes will be harvested and spray-delivered to the donor site in CEA group while control site wound receive control vehicle solution only. Evaluation of wound healing will occur by various methods including the measurement of evaporative water loss on different days post surgery and on each dressing change until the donor site has fully reepithelialized. Data will be analysed statistically to determine the effectiveness of cultured CEA suspension in donor site healing.

Skin Repair: Tissue Engineering using Synthetic Elastin

J Rnjak, Z Li, P Maitz, AS Weiss 

Synthetic human elastin is among a range of research_burns_proj3bioengineered materials aimed at mimicking native host connective tissue. Synthetic elastin scaffolds (Fig 1), produced by chemically cross-linking recombinant human tropoelastin, is a logical choice for a skin substitute matrix.

Synthetic human elastin has the potential to overcome difficulties associated with other matrices including animal- derived collagen or irradiated cadaver-derived dermis, as it is a human protein, and therefore not expected to be rejected. An additional benefit is that it is recombinant and therefore not extracted from humans, eliminating the risk of contamination, especially with agents that are difficult to eradicate such as latent viruses and prions.

The current project aims to grow human skin cells on synthetic human elastin scaffold (both sheets and elcectrospun 3D structure) in an attempt to develop an autologous skin substitute for treatment of burns injury.

Identifying the Diffusible Factor(s) Produced by Skin Cells Grown on Tropoelastin Scaffolds

J Almine, Z Li, P Maitz, AS Weiss 

The main aim of this project is to study the cell-scaffoldresearch_burns_proj4 interaction and identify the diffusible factor(s) produced by skin cells cultured on the scaffold, which promotes cell proliferation and possible keratinocyte differentiation.

Identifying the diffusible factor(s) responsible for the proliferation of keratinocytes and fibroblasts would be important progress in the treatment of burns and the development of a suitable skin graft. The treatment of burns patients involves the rapid coverage and closure of the wounds, which is dependent on cell proliferation and differentiation, ultimately re-establishing the epidermis and dermis. This process can be facilitated by the addition of a diffusible factor(s); consequently achieving rapid wound closure, reducing the chance of infection and re-forming skin with minimal scarring.

Skin Cell Culture on Hollow Fibre-Collagen Scaffold

C Shu, M Lord, C McFarland, P Maitz, Z Li 

Cultured skin substitutes are usually grown on the rigid surface of tissue culture flasks under laboratory conditions. This does not reflect the natural process of human skin development in the dynamic in vivo environment, whereby mechanical loads such as bending, folding, stretching and twisting are continually imposed on the developing tissue. To address this, we have developed a hollow fibre-collagen scaffold system for development of cultured skin grafts. Fibres extracted from plasmapheresis cartridges are incorporated into the scaffold design to allow complete nutrient diffusion to support cell growth. The collagen-hollow fibre scaffold structure has the advantage of flexibility and direct delivery of nutrients through  diffusion, which more closely resembles in vivo conditions than a conventional tissue culture flask. This flexible scaffold potentially allows the mechanical manipulation of the three-dimensional cell culture (Fig 2), and may stimulate realistic sheet skin structure formation. We have been investigating the cell growth conditions and to examine cell behaviour in the scaffold system in attempt to develop a skin substitute for clinical use using this system.

Skin Tissue Engineering Using a Biodegradable Polymer

C Shu, M Lord, C McFarland, P Maitz, Z Li 

Y Wang, D Martínez Tobón, A Taylor, P Maitz, Z Li 

Engineered skin substitutes, resembling natural research_burns_proj6human skin structure and containing living skin cells, would provide excellent alternatives for severe burn wound management.

The aim of this study is to construct a bio-active, hybrid scaffold that is biodegradable, biocompatible and porous in structure to support skin cell growth. This project is designed to develop a composite using collagen, and a FDA-approved biodegradable polyester, polycaprolactone. The scaffold will be used to generate 3D skin substitute under laboratory condition. More importantly, the scaffolds will be made bio-active containing protein factors to facilitate wound healing. Porous bio-scaffolds are developed by lyophilization technique (Fig 3) or fabricated by electrospun nano-fibres (Fig 4).

At this stage, work is focused on characterizing structural features of scaffold including pore size, optimizing skin cell growth in the scaffold. Human skin cells including fibroblasts and keratinocytes obtained from a small skin biopsy and expanded in the laboratory are seeded into the scaffold to grow. The constructs are currently under investigation to determine skin cell proliferation and differentiation and the expression of growth factors and other proteins crucial to wound healing. Long term goals include animal studies and eventually clinical trials.

Efficacy and Safety of Engineered Skin Substitute and Dressing Materials on Skin Wound Healing: A Mouse Model Study

Y Wang, K Neuwiendyk, J Rnjak, P Maitz and Z Li 

Lack of autologous skin graft is always a major issue in treating patients with large and deep burns injuries. Clinically, it is still quite often to observe delayed wound healing, which could lead to wound infection, scar development, deterioration of patient’s well-being and even death.

Cultured autologous skin cells or substitutes are emerging as an important alternative for wound coverage and closure. The advance in biotechnology has enabled us to grow different types of skin cells and skin substitutes by skin tissue engineering technology in our laboratory. Skin tissue engineering involves using different biomaterials such as recombinant collagen and elastin or bio-compatible polymers as porous scaffolds to support skin cell attachment, growth and differentiation into skin tissue. Various wound dressing material and dressing regimes are also designed in our laboratory in an attempt to provide favourable growth condition for cultured skin cells and to speed up the wound healing process. Wound healing is a very complicate process in which host factors and metabolisms play critical role. Although the engineered skin looks structurally similar to normal human skin containing epidermal and dermal layers, the bio-safety and efficacy of engineered skin and wound dressing products will need to be tested in an animal model before proceeding to further clinical trial.

The aims of this study are therefore to establish a mouse model to assess the role of engineered skin products or dressings in wound healing. The animal host response of each mouse as the recipient of skin products or dressing materials will also be examined at cellular and molecular levels. This study will provide significant information on the efficacy and safety of laboratory-developed bio-scaffold, skin substitutes and dressing materials.

Quality Assessment of Meshed Skin Graft Temporally Stored at 4°C

Z Li, P Maitz, C Overend, J Ledgard, K Kennedy. 

Excess split-skin autografts harvested and meshed during a surgical session are often stored at 4oC short-term for later burn surgery or graft failure. The quality of the stored skin is obviously critical to the success of skin graft and needs to be assured.

The current procedure in skin graft storage in Australian hospitals involves wrapping the meshed autograft on a piece of saline-moistened gauze; transfer it into a sterile specimen jar and stored in a 4oC fridge for further use within two weeks. Some studies have demonstrated that the viability of the stored skin tissue could last for several weeks depending on the storage condition although it decreases through this period. However, the viability assay in previous studies was usually achieved by Trypan blue staining of the skin cells, a method which does not truly reflect the growth or cloning ability. In this study, we have been carrying out a time-course assessment of the stored skin tissue using both cell viability assay and skin cell culture techniques. We have also checked the microbiological status of the stored skin grafts, and tried to optimize the storage conditions of skin graft in different media.

Biofilm and Infection of Burn Wound

P Kennedy, S Brammah, E Wills 

One of the most significant problems in burn care is research_burns_proj9that of infection. Following a burn injury the defensive mechanisms of the skin are impaired or destroyed and colonization by micro-organisms rapidly occurs. Many of the micro-organisms commonly found on the burns wound are known to produce biofilms, a collection of organisms attached to a surface and sounded by matrix containing polysaccharides known as extracellular polymeric substances (EPS). Biofilms are the cause of significant morbidity and mortality in relation to implanted medical devices and septic complications associated with indwelling intravenous catheters. The organisms within biofilms are well known to develop resistance to antibiotics and to the immune system. It is estimated that two third of all chronic disease are biofilm related. Biofilm 5formation (Fig 5 and Fig 6) in burn wounds has not been thoroughly examined. This study will help to understand the mechanisms of bacterial wound invasion and burn wound sepsis, and therefore help the management of burn wound.

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