Proliferation and migration of keratinocytes and fibroblasts play an important role in cutaneous wound healing, while oral mucosal squamous cell proliferation and migration are crucial for oral wound healing. In this study, the phytochemical profile of Pluchea indica branch ethanolic extract was characterized. The bioactive compound of Pluchea indica branch ethanolic extract was identified and analyzed by the validated HPLC method. The nanoparticles of P. indica branch extract were formulated by solvent displacement method to increase the solubility and the colloidal stability of the extract. The stability of the nanoparticles was investigated by using the dynamic light scattering technique. Effects of P. indica crude extract and nanoparticles on cell viability, proliferation and migration of primary epidermal keratinocytes, human dermal fibroblasts, and oral mucosal keratinocyte cells were investigated by MTT assay and scratch assay, respectively. The results showed that P. indica branch extract contained a high content of total phenolic and total flavonoids. The HPLC analysis revealed that the main compound in the extract was 4,5-O-dicaffeoylquinic acid. The cell viability of the extract and nanoparticles decreased when cells were exposed to a high concentration of extract and nanoparticles. These results demonstrate that P. indica branch extract and extract nanoparticles at specific concentrations possess in vitro wound healing activity and they may be possibly used to treat different types of wounds including dermal and oral mucosal wounds.

Hydrogels are soft materials that contain high water content within their 3-dimensional structure. Such extremely hydrated environment allows hydrogels to recapitulate the structure of many native tissues inside the body. In biomedical application, hydrogels have been extensively used as biocompatible materials, drug delivery systems, and tissue-engineered scaffolds that can be designed to possess either permanent or slow-degradation properties. In this research, we applied gamma irradiation to develop transparent and conformal hydrogel sheets with sufficient mechanical strength from poly(vinyl alcohol) (PVA) and further modified the based PVA matrix with naturally-derived silk fibroin (SF) protein and silver nitrate (AgNO3) for wound healing purpose. The physical and mechanical properties of based PVA hydrogels formed at varied irradiation doses from 10 - 80 kGy were first characterized. The dose of 60 kGy was found to be optimal to process flexible and elastic PVA sheets with equilibrium degree of swelling of 1000 %, gel fraction of 90 %, and tensile strength of 19 kPa. To further enhance water absorption capacity, 10 - 40 % (w/w) silk fibroin was added to the based PVA matrix. Based on water absorption and gel fraction data, hydrogel sheets with 8PVA:2SF formulation was selected for antibacterial test. Disc diffusion assay showed that the incorporation of 0.4 mM AgNO3 in 8PVA:2SF hydrogel sheets could inhibit the growth of Staphyllococcus aureus and Pseudomonas aeruginosa. These results demonstrated that a prototypic, antibacterial hydrogel sheet dressing composed of both synthetic and natural polymers could be developed within a single-step by gamma irradiation technique.

Advanced wound dressings that can deliver potent antibacterial action are still much in need, especially for treating wound infections caused by drug-resistant bacteria. In this research, we utilized electron beam (EB) irradiation to develop antibacterial hydrogel sheet dressings from poly(vinyl alcohol) (PVA) and silver nanoparticles (AgNPs) in a two-step processing and evaluated their bactericidal efficacy, as well as the AgNP release. The effect of the irradiation dose on the swelling, gel fraction, network parameters, and mechanical properties of the hydrogels was first determined to establish the optimal doses for the two-step processing. The prototypic hydrogel sheets were then formed in the first EB irradiation and served as a matrix for the AgNP synthesis by the reduction of the silver nitrate precursors during the second EB irradiation. The diffusion assay showed that the minimal inhibition concentration (MIC) of the AgNP-load hydrogels was 0.25 and 0.5 mg/cm2 against Escherichia coli and Staphylococcus aureus, respectively. At these MIC levels, the released AgNPs increased sharply before reaching the maximum, ~950 and 1800 ppb, at 24 h as analyzed by atomic absorption. Therefore, we successfully demonstrated that this two-step processing by EB irradiation provides a convenient platform to fabricate AgNP-loaded hydrogel dressings that can be further developed for wound healing.

Cytotoxicity presents one of the required criteria in the biological evaluation of medical devices. In this study, the semi-direct contact test was used to evaluate the potential cytotoxicity of hydrogel wound dressings compared to the conventional extract test. Three types of hydrogel sheets were fabricated from poly(vinyl alcohol) (PVA) by gamma irradiation: Bare sheets, silver (Ag)-coated sheets, and Aloe vera (AV)-coated sheets. In the extract test, L929 cells were cultured in the extract derived from the elution of hydrogel samples. For the semi-direct contact test, the cells were cultured in situ with the hydrogel samples placed inside transwell inserts above the cell monolayers. At the endpoint of both tests, MTT assay was performed, and the cell viability was determined from the absorbance of formazan. Only the bare and AV-coated hydrogel sheets showed cell viability above the 70 % threshold that ensured the non-cytotoxicity by both tests. For Ag-coated sheets, less than 70 % cell viability occurred when Ag coating was ³ 0.2 mg/cm2. Interestingly, the formazan-depleted area underneath the Ag-coated sample could be clearly observed by the semi-direct contact test. The release of Ag in the form of nanoparticles was confirmed by UV-Vis absorption at 420 nm. In conclusion, the semi-direct contact test can serve as a reliable alternative to the conventional extract test in evaluating the potential cytotoxicity of hydrogel wound dressings. HIGHLIGHTS A semi-direct contact test with transwell inserts was investigated as an alternative approach to the conventional extract test in evaluating the cytotoxicity of hydrogel wound dressings Leachable substances from the hydrogel dressings permeated through the porous membrane of transwell inserts to the reach the cell monolayer underneath Cell viability results based on MTT assay obtained from semi-direct contact test were in agreement with the extract test Only the semi-direct contact test provided the visual assessment of localized toxicity caused by the release of silver nanoparticles from hydrogel sheets onto the cell monolayer GRAPHICAL ABSTRACT

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