Elsevier

Burns

Volume 36, Issue 3, May 2010, Pages e10-e20
Burns

Case report
Sprayed cultured autologous keratinocytes used alone or in combination with meshed autografts to accelerate wound closure in difficult-to-heal burns patients

https://doi.org/10.1016/j.burns.2008.11.011Get rights and content

Introduction

Extensive deep burns present a significant challenge to the burns surgeon. The limited availability of skin donor sites in such patients means that the time to achieve complete skin cover is prolonged and consequently increases the risk of complications such as sepsis. Research has shown that prompt excision of burned tissue [1] and early wound cover [2], [3] will lead to improved survival, more rapid recovery and reduction in scarring [4].

Skin graft expansion is an essential tool in major burns. However, as expansion ratios increase so do the difficulties in handling the skin graft. Furthermore, higher expansion ratios result in larger areas of exposed, unhealed tissue (interstices) which are then dependent on epithelial migration from the meshed skin graft margins to close the wound. Lattice meshed grafts [5] have become the mainstay in burns treatment, but at expansion ratios of greater than 1:4 they become difficult to handle, have unpredictable take rates with increased hypertrophic scarring [6] as well as leading to increased contracture of the wound area [7], [8]. It has also been demonstrated that the actual expansion may be significantly less than expected [9], [10], [11].

‘Meek’ meshing of skin graft is particularly useful in situations where higher expansion ratios are required. The Meek micrografting technique was initially described in 1958 [12] and although its subsequent use was eclipsed by the use of lattice meshed skin, recent revisions to the technique have led to an increase in its use [8], [11], [13], [14], [15]. Expansion ratios of 1:9 are easily achieved and this is combined with easier handling of the skin when compared with widely meshed lattice graft. Large areas can be covered with this technique with more accurate expansion, but the time to complete healing may still be prolonged because of the significant area between the islands of autograft tissue. It is also thought that ‘take’ rates are not as badly affected by microbial infections when compared with lattice grafts [8], [10], [13], [14], [15].

Cultured autologous keratinocytes, developed by Rheinwald and Green in the mid-70s [16] have been used extensively to help treat severe burns [17], [18], [19], [21], [22], [23] and chronic ulcers [24]. Initially the cultured cells were applied to the wound bed as a mature sheet, and were able to form a neo-dermis even when grafted onto muscle facia [20]. However, long-term survival and stability were a problem in some instances, resulting in varying degrees of success [25], [26], [27], [28], reviewed further in [29].

The unpredictability of successful ‘take’ rates may be due to the fact that the sheets can be very fragile and difficult to handle. It has been proposed that on their transport from the laboratory to the patient they could be prone to breaking up [30]. Histological analysis of CEA sheets has also been undertaken [31], [32], [33], in order to establish the possible cause of this fragility. These studies demonstrated delayed Rete ridge and anchoring fibril formation leading to a delayed maturation of the basement membrane. Further studies revealed an alteration in expression of integrins by which attachment of the CEA sheet is mediated [34], [35], [36]. The release of the epithelial sheet from the plastic culture flask by enzyme digestion may be responsible for the destruction or alteration in the integrins and basement membrane proteins [37] necessary for a successful attachment to the wound bed. The time delay between harvest and application may also be a factor [38].

We postulate that the condition of the cell sheet prior to removal from the plastic flask also influences its survival on the wound bed. It is important to have viable basal cells in the cell sheet as these are the cells which are going to attach and grow when placed on the wound bed. Within the cell culture flask the upper layers of the developing cell sheet will start to keratinise and provide a barrier between the liquid nutrients of the growth medium and the basal cells. This barrier may become more pronounced as the cell layers increase in number and as such, the thicker cell sheets, which may be easier to remove from the flask and easier to handle clinically, may have fewer viable basal cells and thus give rise to poorer take rates. It is proposed that these fully formed sheets contain, within the basal layer, increasing numbers of dying cells as well as reducing numbers of viable dividing cells as they become further isolated from the surface growth medium. This may be responsible for the unpredictability of take rates when compared with that of conventional split thickness skin grafts [20], [26], [28], [39], [40], [41]. The successful use of immature, but difficult to handle sheets to re-epithelialise a wound pre-treated with the dermal substitute Integra [42] would confirm this hypothesis.

It is thought that by administering the cultured cells to the wound in a pre-confluent state may avoid some of the above problems [30]. There have been many different methods of delivery of these cells described (reviewed in [29], [43]). We have used the sprayed method in our clinical practice which has been shown to have reasonable clinical success in the treatment of dermal burns at other centres [19], [44], [45].

In this article we report the use of sprayed, cultured autologous keratinocytes in combination with widely meshed autografts (both Meek and lattice techniques) and report here three cases with significant full thickness burns. The patients had 8% (elderly), 31% (non-healing) and 90% TBSA burns. We also report 2 patients treated with cultured cells alone; a 56% TBSA (non-healing) and a 15% TBSA female ballerina who wished to avoid unnecessary donor-site scars.

Section snippets

Cell culture

Skin biopsies were harvested from a non-burned area away from the site of injury, and at the same time as a scheduled procedure carried out under a general anaesthetic. In order to ensure sterility of the tissue, the harvest site was prepared by washing with 70% aqueous chlorhexidine gluconate (Hibiscrub, ICI), rinsed with sterile saline, washed with 5% povidine iodine and then left for a minimum of 5 min. The iodine was washed off with sterile saline, followed by a final wash with 70% alcohol

Histology

Minimal sized tissue biopsies were taken for cryosectioning and were prepared and cut by standard techniques. The 15-micron cut sections were visualised either by standard Haematoxylin and Eosin (H&E) staining for general morphology or standard immuno-histochemical staining with fluorescein-tagged antibodies to cytokeratin 14 for epithelial cell identification.

Case 1: Lattice mesh + cultured cells on an elderly patient

An 80-year-old woman sustained 8% full thickness flame burns circumferentially to both thighs after dropping a lit cigarette onto a polyester nightdress. She suffered from advanced secondary progressive multiple sclerosis and dementia. Exposure time was prolonged and subsequent first aid was delayed. On admission her burns were debrided and cryopreserved human allograft was applied.

At day 5 after burn a small area (∼4 cm2) of thin split-thickness skin was taken for cell culture as previously

Discussion

The ultimate aim for the treatment of any large burn is rapid cover of the entire wound with the patient's own skin. However, as burn wound size increases the amount of available donor site diminishes while the risks of sepsis increase. Sepsis increases the risk of graft failure thereby further challenging the burns surgeon.

It has been well documented that widely meshing skin allows greater coverage of the wound but still leaves a significant area of wound in the interstices to epithelialise.

Acknowledgements

We are grateful to Mr Keith Wood (Blond McIndoe Centre–retired) for the preparation and staining of the histological sections; Prof Irene Leigh (The Royal London Hospital) for the supply of the monoclonal antibody to cytokeratin 14; and Coster Aerosols Ltd for supplying the spray nozzles.

This work was funded by the Blond McIndoe Research Foundation and the East Grinstead Medical Research Trust together with The Band Trust.(SEJ); The Mark Hanna Fellowship (RVS & MJR); Archibald McIndoe's Guinea

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  • Cited by (0)

    1

    Current address: Brighton and Sussex Medical School Research Centre, Falmer, Brighton, Sussex., UK.

    2

    Current address: School of Pharmacy & Biomolecular Sciences, University of Brighton, Moulsecoomb, Sussex. BN2 4GJ, UK.

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