Elsevier

Burns

Volume 33, Issue 6, September 2007, Pages 693-700
Burns

The use of a non-cultured autologous cell suspension and Integra® dermal regeneration template to repair full-thickness skin wounds in a porcine model: A one-step process

https://doi.org/10.1016/j.burns.2006.10.388Get rights and content

Abstract

Integra® is a skin substitute used for dermal reconstruction. Current clinical practice consists of two procedures, first applying Integra® to the wound and then replacing the silicone pseudo-epidermis with an epidermal autograft 3 weeks later. This two-step repair limits the clinical use of the product. An effective one-step procedure could reduce the time taken to repair and decrease the number of procedures for use of Integra®.

This study examined the effects of simultaneous application of a non-cultured autologous suspension of cells, isolated using the ReCell® autologous cell harvesting device, in combination with Integra®, to achieve a one-step skin repair.

In two female Yorkshire swine, 10 full-thickness wounds were created. Wounds were treated with Integra® seeded with cell suspension and compared to controls of Integra® alone and cell suspension alone. Weekly macroscopic and histological assessment demonstrated that the wounds treated simultaneously with Integra® and non-cultured autologous cells had enhanced epithelialization at an early time-point compared to controls.

Wounds treated simultaneously with Integra® and cell suspension demonstrate that cells remain viable, migrate through the Integra® template and self-organize into differentiated epidermis. The results indicate that combining Integra® with autologous cells facilitates one-step skin reconstruction of a full-thickness skin wound.

Introduction

Epithelial autografts are a well-established modality for skin replacement, and it is widely recognized that the epidermis can be reconstructed from both cultured and non-cultured autologous cell populations [1], [2]. Although intact epidermis alone provides an effective barrier to water loss and bacterial invasion [3], dermal reconstruction is essential to optimize the functional and aesthetic outcome [4], [5]. Consequently, the development of an artificial dermis that can be used as a replacement to autografts is an essential modality for optimal full-thickness wound repair [6], [7].

The Integra® dermal regeneration template (Integra Lifesciences Inc., USA) is a bi-laminar skin replacement used in the reconstruction of the dermis following large acute burn injuries where donor autograft tissue is insufficient [8]. Integra® is also being increasingly used for dermal replacement in post-scarring reconstructive procedures [9]. Integra® comprises a layer of cross-linked bovine collagen and shark glycosaminoglycans that facilitates the formation of a reticular dermis [10], [11], [12]. A secondary layer is composed of a synthetic polysilicoxane polymer (silicone) layer that acts as a temporary pseudo-epidermis.

The Integra® is inserted on a clean, viable, excised wound bed. The highly porous, cross-linked collagen–glycosaminoglycans copolymer matrix induces rapid absorption of exudate followed by progressive fibroblast infiltration. The architecture of the scaffold is such that cells migrating into the template express a reticular dermal phenotype [13], [14], [15]. Over a period of 2–3 weeks, the fibroblasts proliferate and migrate along the interstices of the matrix and begin producing host collagen, whilst vascularisation of the neodermis also occurs [13], [14], [15].

In the current use of Integra®, a second stage surgical procedure is required after approximately 21 days. At this point the neodermis has formed, and the temporary silicone pseudo-epidermis is replaced with an epidermal autograft. Typically, a split-thickness graft between 0.006 and 0.007 in. is meshed (up to 1:3) and applied directly onto the dermal template. Pre-confluent epidermal autograft suspensions have been applied over the split-thickness graft to enhance epithelialization and minimize mesh pattern scarring [16]. Whilst the two-step procedure is effective, the relatively long time to heal suggests there is a need for a simple one-step procedure that reduces the time to heal, the number of interventions and improves the outcome.

A number of pre-clinical investigations attempted to address developing a one-step procedure for use with Integra®[17], [18], [19], [20]. Whilst the few cases of using cultured autologous keratinocyte sheets reported appear to have low efficiencies of take [21], the use of cultured keratinocytes seeded directly into the Integra® template has been shown to result in migration and proliferation of keratinocytes to re-form an intact epidermis on the surface of the Integra®[17], [20]. This was initially demonstrated using cells centrifuged into the Integra®. However, more recent studies have simplified this approach by directly seeding cells to the underside of the Integra® or directly to the wound bed and also successfully demonstrated re-organization and re-epithelialization [18]. The use of cultured cells has been shown to be effective in decreasing time to heal and regenerating a thickened epithelial layer when used in a one-step procedure, but comparable results using non-cultured cells were less successful, with less rapid epithelialization, most likely due to the limited numbers of proliferating cells within the non-cultured population when compared to the cultured population [17]. However, the use of cultured cells is not always suitable for a one-step procedure in an acute injury. The timeframe for surgical intervention is variable, and the compromise of culturing cells for less time results in low cell yields, whilst delaying surgery for optimal culturing may compromise the results. In addition, the spontaneous re-organization of cultured cells may not be as good as that of non-cultured cells [17]. The problem remains of identifying a method, easily applicable to the clinic, for preparing non-cultured cells for simultaneous application with the Integra® template. This would allow rapid preparation of a single-step substrate for full-thickness repair.

This study uses ReCell®, a single-use disposable device that harvests cells from the DEJ for application as a non-cultured autologous epidermal cell suspension. The resulting cell suspension is a mixed population containing all the cell types present at the DEJ and primarily keratinocytes from the basal layer. The epidermal cell suspension is prepared in theatre for immediate use by harvesting a thin (0.006 in.) split-thickness skin biopsy measuring up to 2 cm × 2 cm and processed by means of enzymatic and physical disruption. The resulting cell population produced by the device can be used to cover a surface area up to 320 cm2 from a 4 cm2 split-skin graft biopsy and includes keratinocytes, melanocytes and Langerhans cells in ratios previously reported to be present at the DEJ (data not shown). The cells are suspended in Compound Sodium Lactate for delivery to the Integra®.

We hypothesized that using cells harvested with the ReCell® device and applied simultaneously with an Integra® template would spontaneously organize into a differentiated epidermis. We also hypothesized that this procedure would result in faster epithelialization than the normal two-step procedure, overcoming some of the problems previously associated with non-cultured cells [17] due to the high percentage of basal layer keratinocytes within the harvested population, and providing an improved one-step procedure for Integra® based dermal and epidermal reconstruction.

Section snippets

Materials and methods

This study was carried out in accordance with the regulations outlined in the USDA Animal Welfare Act (9 CFR, Parts 1–3) and the conditions specified in The Guide for Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press). The study received ethical approval from Pluris's Institutional Animal Care and Use Committee.

Two female Yorkshire domestic swine, both of similar age and weighing between 25 and 30 kg were selected for the study (n = 2). Swine were chosen because of

Gross wound observations

All wounds appeared equivalent at time of surgery (day 0, Fig. 2). By day 14, significant differences were observed between those wounds treated with both Integra® and autologous cells and the control groups (Fig. 3). Re-epithelialization had progressed well in wounds treated with Integra® and autologous cells (groups A and B). In the control wounds treated with Integra® alone, there was little evidence of re-epithelialization at this stage, whilst those treated with cells alone appeared to

Discussion

Currently, a two-stage procedure is the standard protocol for full-thickness repair using the Integra® dermal template. This has the disadvantages of both the long healing time and the requirement for two procedures. Whilst previous studies have addressed the development of a simplified one-step process, these have either been more difficult to apply in a clinical situation, involving centrifugation steps [17], [20], or required the use of cultured cells [18], increasing the time required for

Conflicts of interest statement

Both Fiona Wood and Marie Stoner are co-founders and directors of Clinical Cell Culture, the manufacturer of the ReCell® device used in this study and co-sponsor of the study.

Acknowledgements

This study was conducted in general compliance with the US FDA Good Laboratory Practice Regulations (21 CFR, Part 58) by Pluris Research Inc. (Franklin Tennessee, USA) with sponsorship provided by Integra® Lifesciences Inc. (San Diego, USA). Clinical Cell Culture (Perth, Australia) provided ReCell® devices to Pluris for use during the study.

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