Assessing modified chitosan wound dressings to enhance wound healing in the porcine model

Abstract

Dressings enhancing wound healing can improve the outcome of wounds where tissue replacement is required, like for burns and ulcers. Treatment of these wounds is complex due to their depth and excessive tissue loss. Replacement of the lost tissue and delivery of growth factors could enhance healing and reduce scarring. The natural biomaterial; chitosan is reported to bind growth factors, with reduced wound healing times when used in dressings. This study aimed to modify chitosan into a wound dressing filler that would optimise growth factor delivery to full-thickness wounds and overall reduce healing times with minimum scarring. Lipophilic modified chitosan was chemically synthesised by addition of different percentages (10%, 20%, and 34%) of lauric acid residues into three lauroyl chitosan (LCs) derivatives (LCs10, LCs20, LCs34). Lauric acid was the fatty acid of choice due to its superior antimicrobial properties among the saturated fatty acids.1 The loading densities selected were based on commonly used concentration ranges as found in literature. The three derivatives were then characterised using Nuclear magnetic resonance (NMR) and Fourier-transform infrared (FT-IR) spectroscopy. Thereafter, swelling tests and water drop shape analysis followed to assess the physical characteristics of the derivatives. Cytotoxicity/proliferation assays using primary fibroblasts and sulphorhodamine-B for cell enumeration were performed followed by a preliminary skin sensitivity test. The acid phophatase assay was used to measure platelet adhesion while the enzyme-linked immunosorbent assay (ELISA) measured the release profile of platelet derived growth factor AB (PDGF-AB) over 24 hr. These assays assisted with determining which derivative had the optimum lauric acid loading density for wound healing. After determining the derivative with the optimum loading density, porcine collagen was extracted from skin and added to the selected LCs derivative at the ratio 1:4 to make a wound filler paste that would increase cellular ingrowth. Wound healing studies using LCs10 enriched with collagen fibres (Co/LCs10) alone and with platelet-rich plasma (Co/LCs10/PRP) as dressing material were performed using the porcine full skin thickness wound healing protocol. Finally, histological analysis of the cellular events taking place in the wounds at different stages of healing were done using the Haematoxylin and Eosin and the Masson’s Trichrome stains. Evidently, the FT-IR and NMR, displayed successful modification of chitosan with the lauric acid side chains with a visible aliphatic group in both spectra. Comparison of the LCs derivatives to the underivatized chitosan using the drop shape analysis, showed increased contact angles with increased hydrophobicity. It appeared that as the molar concentration of lauric acid increased, the contact angle also increased. In the swelling tests, LCs34 had the highest swelling capacity. Results from the in vitro assays showed that hydrophobic modification of chitosan reduced the adhesion capacity of platelets to chitosan as the lauric acid density on the underivatized chitosan increased. Cytotoxicity assays indicated that neither LCs nor chitosan were toxic to primary fibroblast cells, with the LCs34 significantly (43%) promoting fibroblast proliferation compared to the control. A preliminary skin sensitivity test comparing LCs34 to chitosan showed that LCs34 was compatible with human skin. From the ELISA study the LCs10 sample exhibited a sustained release of growth factors over 24 hr compared to both chitosan and collagen. Consequently, the LCs10 derivative was then selected for further analysis and for final analysis in the wound study. Sixteen full-thickness skin wounds were thereafter made along the dorsum of each of four pigs with two treatments and a control (Jelonet®) randomly applied as dressing material: Co/LCs10, Co/LCs10/PRP and the Jelonet® treatment. The differences in wound healing were observed with biopsies taken at 3-day intervals over 21 days. By the 12th day, all wounds had completely healed with little scarring. The Co/LCs10/PRP dressing significantly induced haemostasis, wound contraction and accelerated wound closure and healing from the wound bed. Results from histological examinations demonstrated advanced granulation tissue formation, collagen deposition and epithelialisation in the wounds treated with Co/LCs10/PRP. This study therefore revealed that hydrophobically modified chitosan at 10% loading density provided a wound dressing material that allowed sustained growth factor release. The Co/LCs10/PRP dressing also demonstrated that it was an improved wound dressing due to acceleration of wound healing, promotion of fibroblast proliferation with increased collagen deposition and minimal scarring. These materials may significantly reduce healing times of full-thickness wounds and should be studied further in in vivo models.