Chitosan reinforced with kenaf nanocrystalline cellulose hydrogel as an effective carrier for platelet lysate to accelerate wound healing

Abstract

A full-thickness wound involves the loss of both epidermal and dermal layers. Healing of larger and deeper wounds may take a long time and can result in serious complications. The benchmark treatment for major skin loss is split-skin grafting but it is associated with certain shortcomings like limited availability of healthy skin, formation of scarring, and risk of infection. Platelet-rich plasma (PRP) is well known to play a crucial role in skin wound healing by providing the high number of platelets, growth factors and cytokines necessary for effective wound closure. Despite its extensive use, there are still inconsistency over its clinical efficacy, biological variabilities, high cost and most importantly short half-life of growth factors (GF) present in PRP. The main objective of this study is to investigate the potential delivery system consists of chitosan/nanocrystalline cellulose (CS/NCC) hydrogel as matrix to deliver autologous platelet lysate (PL) for immediate treatment of full-thickness wound. NCC was synthesized from raw kenaf bast fibres and incorporated into the CS hydrogel. The physicochemical properties, in vitro cytocompatibility, cell proliferation, wound scratch assay, PL release, and CS stabilizing effect of the hydrogel composites were analysed. The study of swelling ratio (>1000%) and moisture loss (60–90%) showed the excellent water retention capacity of the CS-NCC-PL hydrogels as compared with the commercial product. In vitro PL release study (flux = 0.165 mg/cm2/h) indicated that NCC act as a nanofiller and allowed sustained release of PL compared with the CS hydrogel. The CS also showed the protective effect on growth factor (GF) present in PL, thereby promoting rapid wound healing. The CS-NCC hydrogels also augmented human dermal fibroblast (HDFs) proliferation and wound closure in vitro. Gene expression of 10 important wound healing genes (MMP1, MMP2, MMP13, TIMP1, TIMP2, Col1, Col3, Fibronectin, Elastin and TGF-ß) were measured using qPCR. PL supplemented hydrogel upregulated the mRNA expression of extracellular matrix (ECM) genes. In-vivo study was conducted in full-thickness wound model and found that CS-NCC-PL hydrogel dressing closed 81.8% of wound over the duration of 14 days. Histological assessment of the wound showed that the hydrogel involves in reepithelization, angiogenesis and collagen deposition. Based on the hydroxyproline content in wound biopsies, on day 14 CS-NCC-PL group treated wound exhibited a significantly increased (p<0.05) in hydroxyproline (60.62 ± 11.46 μg/100mg) as compared to PL (44.25 ± 2.93 μg/100mg,), Saline (46.17 ± 5 μg /100 mg) and CS-NCC (44.06 ± 2.6 μg /100 mg). From immunohistochemistry (IHC) staining findings, the expression of α-SMA is higher on day 7 suggesting presence of higher number of myofibroblasts responsible to collagen deposition in CS-NCC-PL group, followed by PL treatment. Same results were obtained with the marker CD31 used to check the angiogenesis, whereby it’s expression peaked on day 7 and subsequently declined on Day 14 as the wound approaching the remodeling phase. As a conclusion, CS-NCC-PL hydrogel wound dressing containing bioactive GF is successfully developed and shows great promise in treating full-thickness wound