The effect of lipopolysaccharide exposure on cutaneous wound repair in the rat

Gunnigle SM, Jones PF, Markham AF, Guillou PJ and Hart J.

Wound Repair Research Group, Molecular Medicine Unit, Clinical Sciences Building, St James’s University Hospital, Leeds, LS9 7TF, UK.

e-mail: stevegunnigle@hotmail.com

Background: Chronic wounds fail to heal within an expected time scale and are often characterised by persistent non-resolving inflammation. The underlying basis for this protracted inflammatory activity is not fully understood. With the skin’s barrier function compromised it is not surprising that chronic wound tissues are colonised, often at sub-infection levels, by one or more bacterial species. We suggest that the response elicited in the presence of certain inflammatory bacterial components may, in part, explain the protracted inflammation and slow repair demonstrated by chronic wounds.

Aim: To determine the effect of Pseudomonas aeruginosa derived lipopolysaccharide (LPS) on the repair of acute dermal wounds in the Wistar rats.

Methods: Excisional wounds created on the flank of large male Wistar rats were considered here. LPS was introduced by intradermal injection into the wound margins immediately after injury and 24 hours later. Three concentrations of LPS were considered namely 0.01, 0.1 and 1.0 mg per wound. Each experimental group contained six animals. Wound contraction was measured and compared between groups at 1, 3, 5, 7 and 9 days post wounding. Wound healing and the effect of differing doses of LPS was further examined by consideration of: macrophage recruitment, gelatinase activity (MMP-2 and -9) and the expression of certain inflammatory cytokines (IL-1ß and TNFa).

Results: The introduction of P. aeruginosa derived LPS was found to significantly delay wound contraction in a dose dependent fashion. The temporal scheme of macrophage infiltration was also altered; with LPS initially delaying recruitment and subsequently tending to promote retention of macrophages at the wound site. The effect of LPS on TNFa and IL-1b levels was complicated; the data did, however, suggest that LPS exposure resulted in elevated TNFa and IL-1b production. When protease levels were considered, LPS exposure tended to be without significant effect other than a small decrease in both MMP-2 and 9 levels.

Conclusion: In overview the data suggests that introduction of bacterial LPS into acute wounds: delays wound closure, modulates macrophage recruitment and gives rise to a more inflammatory cytokine profile. Such observations support the ‘bacterial contamination – chronic inflammation’ hypothesis, suggested above. The limited modulation in protease activity, may suggest that this aspect of the chronic wound environment is linked to some other controlling factor such as ischaemia or age.

This work has been supported by Johnson & Johnson Medical Ltd.

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Healing of denervated cutaneous wounds is accelerated by the topical application of NGF but not PDGF or TGF-b

James SE, Tereughi G and McGrouther DA

The Department of Plastic and Reconstructive Surgery, and The Blond McIndoe Laboratories, The Royal Free and University College Medical School, London, UK.

s.e.james@mcindoeucl.demon.co.uk

Neurogenic stimuli at the time of cutaneous wounding profoundly affect the cellular events involved in inflammation, proliferation and matrix deposition as well as cytokine and growth factor synthesis. We report the effect that growth factor treatments have on cutaneous wound healing in denervated skin using a modification of the rat groin free flap.

            Denervation is achieved by skeletalisation of the vascular pedicle and the subsequent temporary application of a vascular clamp; thereby denervating the skin flap and providing a model of die back neuropathy. Full thickness wounds measuring 1 cm x 1 cm were excised on the denervated flap and on the contralateral normally innervated dermatome. The wounds were treated topically with 50 m1 of either nerve growth factor (NGF 40 µg/ml), platelet derived growth factor (pDGF 50 µg/ml), transforming growth factor-ß (TGF-ß 1 µg/ml) or control carrier (0.1% bovine serum albumin in 4mM HCl). Their effect on the healing wounds was evaluated by wound closure rates and wound bed histology.

            Denervated wounds treated with PDGF or TGF-ß did not show a significant alteration in healing rates when compared to the control. Denervated wounds treated with topical NGF healed significantly more quickly than control wounds (day 5: NGF 0.304cm² ± 0.043 vs. Control 0.423cm² ± 0.026 wound area ± SEM p = 0.038), although the rate of wound closure in NGF treated wounds remains slightly slower than that found in untreated normal wounds. Acceleration of wound closure by NGF was also observed on normally innervated cutaneous wounds confirming previous findings. We conclude that topical application of NGF accelerates the closure of a denervated wound.

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Healing of superficial second-degree burn wounds is accelerated by stably in vitro KGF-transfected HaCaT-keratinocytes – experimental studies in pigs

Kopp J^1,2,3, Jiao XY^1,2,3, Ying K³, Dai FP^2,3, Stark GB^2,3 and Pallua N¹

¹ Dept of Plastic Surgery and Hand Surgery – Burn Center, University Medical Center, Aachen, Germany.  ² Department of Plastic and Handsurgery, University Medical Center, Freiburg, Germany
³ Burn Center, Changhai Hospital, Shanghai, P.R. China

Purpose. Epidermal regeneration is a complex process mainly influenced by growth factors. Keratinocyte Growth Factor (KGF) induces mitosis, proliferation and migration of keratinocytes. The purpose of this study was the establishment of a stably KGF-transfected, immortalized cell line, HaCaT­-keratinocytes, by liposome mediated gene transfer in vitro and to investigate their capability to accelerate wound healing processes in experi-mental superficial second degree burn wounds in pigs.

Materials and methods. HaCaT-Keratinocytes were transfected with KGF­ and LacZ-plasmid-LipofectAMINE-complexes and selected with G418. Transfection effectivity, gene integration and activity of the transgenic protein and marker gene was investigated by beta-Gal-staining, ELISA and PCR. Most effective KGF producing clones were tested by a colorimetric XTT­test, demonstrating a significant acceleration of cell proliferation and mitosis of human keratinocytes in an Air Liquid Interface (ALI) test system. After identification of the most effective clone, cells were incubated on a silicon­polyurethan-membrane forming a ‘Membrane Cell Graft’ (MCG). In three pigs 21 superficial second degree burn wounds each were created to investigate the wound healing capacity of the generated ‘polypeptide cell delivery system’ after grafting. Untrans-fected HaCaT-keratinocytes, membrane covered and untreated burn wounds served as control.

Results. In vitro results indicated that transgenic HaCaT-cells had a high stimulating efficiency in the ALI test system. Furthermore, the stable integration and structure of the transgene was demonstrated in a three month follow up. After grafting, histological and macroscopical investigations revealed that the in vivo application of transfected HaCaT-cells resulted in complete reepithelialization within five days while wounds covered with untransfected cells were healed after post grafting day seven. Untreated sites were covered by a thin neoepithelium after ten days. The results indicated that wound healing processes can be stimulated by paracrine secretion of transgenic proteins. Therefore the application of KGF-transfected HaCaT-keratinocytes could be useful in the treatment of partial thickness skin defects.