Combining nanofabrication with natural antimicrobials to control denture plaque

Management of fungal biofilms represents a significant challenge to oral healthcare. As a preventive approach, minimising adhesion between intra-oral devices and microorganisms would be an important step forward. Denture stomatitis (DS) is a multifactorial denture-associated inflammation of the oral mucosa where candidal biofilms are one of the contributing factors. Therefore, understanding candidal biofilms on dentures and finding novel strategies to control these biofilms are of significance. Interference with the adhesion step of biofilm formation is hypothetically effective strategy to control biofilms. To understand the relationship between denture candidal load, denture material type and C. albicans biofilm forming heterogeneity in DS, quantitative polymerase chain reaction (qPCR) molecular method and crystal violet (CV) assay were used. This study investigated two novel strategies to control C. albicans biofilms through interfering with adhesion: natural polyphenol curcumin (CUR) and modifying the topography of the denture material surface. Based on the optimised effective CUR concentrations, CUR adsorption to PMMA denture material was spectrophotometrically analysed. Based on these data, the effect of adsorbed CUR to PMMA and CUR pre-exposure on adhesion of C. albicans were assessed. The effect of CUR on Candida-Candida adhesion was investigated and the expression profile of selected adhesion and aggregation-associated genes was assessed using qPCR method. Micro/nano-fabricated polycarbonate and PMMA materials were replicated using injection and compression moulding techniques, respectively and were characterised using scanning electron microscopy (SEM). Adhesion of C. albicans on the micro and nano-scaled patterns was assessed using microscopic and qPCR molecular methods, respectively. The physical characteristics of the materials were assessed using theta tensiometer and a white light profiler. The data demonstrated that although C. albicans was detected in greater quantities in diseased individuals, it was not associated with increased biofilm biomass. Denture substrata were shown to influence biofilm biomass, with poly(methyl methacrylate) providing the most suitable environment for C. albicans to reside. Subsequent studies showed that CUR concentrations of 50 μg/ml could prevent adhesion to PMMA. This effect was enhanced by the CUR pre-treatment of yeast cells (>90% inhibition, p < 0.001). Investigation of the biological impact of CUR showed that it preferentially affected immature morphological forms (yeast and germlings), and actively promoted aggregation of the cells. Transcriptional analyses showed that CUR temporally modulated adhesion and aggregation associated genes. Finally, PMMA denture material was replicated to show nano features. These topographies influenced adhesion of C. albicans, depending on the candidal morphological form and the shape. Nano-pit spatial arrangements variably affect the adhesion of C. albicans, where SQ arrangement demonstrated a significant anti-adhesive capacity. Differential adhesin expression was observed on these surfaces, which were affected by the wettability and roughness of surfaces tested. In summary, C. albicans is an important determinant of denture disease, so preventing its adhesion and biofilm formation were worthwhile objectives. This thesis has shown that CUR molecules and SQ nano-pit topographies reduced C. albicans adhesion, demonstrating that chemical and physical inhibition strategies are useful. The data presented in this thesis showed the high potential of the novel strategies to be used against C. albicans biofilms, and encourages the further investigation of these approaches against polymicrobial denture biofilms

The anti-adhesive effect of curcumin on Candida albicans biofilms on denture materials

The use of natural compounds as an alternative source of antimicrobials has become a necessity given the growing concern over global antimicrobial resistance. Polyphenols, found in various edible plants, offers one potential solution to this. We aimed to investigate the possibility of using curcumin within the context of oral health as a way of inhibiting and preventing the harmful development of Candida albicans biofilms. We undertook a series of adsorption experiments with varying concentrations of curcumin, showing that 50 ug/ml could prevent adhesion. This effect could be further synergised by the curcumin pretreatment of yeast cells to obtain significantly greater inhibition (&gt;90, p&lt;0.001). Investigation of the biological impact of curcumin showed that it preferentially affected immature morphological forms (yeast and germlings), and actively promoted aggregation of the cells. Transcriptional analyses showed that key adhesins were down-regulated (ALS1 and ALS3), whereas aggregation related genes (ALS5 and AAF1) were up-regulated. Collectively, these data demonstrated that curcumin elicits anti-adhesive effects and that induces transcription of genes integrally involved in the processes related to biofilm formation. Curcumin and associated polyphenols therefore have the capacity to be developed for use in oral healthcare to augment existing preventative strategies for candidal biofilms on the denture surface

Candida albicans biofilm heterogeneity does not influence denture stomatitis but strongly influences denture cleansing capacity

Approximately 20  % of the UK population wear some form of denture prosthesis, resulting in denture stomatitis in half of these individuals. Candida albicans is primarily attributed as the causative agent, due to its biofilm -forming ability. Recently, there has been increasing evidence of C. albicans biofilm heterogeneity and the negative impact it can have clinically; however, this phenomenon has yet to be studied in relation to denture isolates. The aims of this study were to evaluate C. albicans biofilm formation of clinical denture isolates in a denture environment and to assess antimicrobial activity of common denture cleansers against these tenacious communities. C. albicans isolated from dentures of healthy and diseased individuals was quantified using real-time PCR and biofilm biomass assessed using crystal violet. Biofilm development on the denture substratum poly(methyl methacrylate), Molloplast B and Ufi-gel was determined. Biofilm formation was assessed using metabolic and biomass stains, following treatment with denture hygiene products. Although C. albicans was detected in greater quantities in diseased individuals, it was not associated with increased biofilm biomass. Denture substrata were shown to influence biofilm biomass, with poly(methyl methacrylate) providing the most suitable environment for C. albicans to reside. Of all denture hygiene products tested, Milton had the most effective antimicrobial activity, reducing biofilm biomass and viability the greatest. Overall, our results highlight the complex nature of denture- related disease, and disease development cannot always be attributed to a sole cause. It is the distinct combination of various factors that ultimately determines the pathogenic outcome

Nanoimprinting of biomedical polymers reduces candidal physical adhesion

Management of fungal biofilms represents a significant challenge to healthcare. As a preventive approach, minimising adhesion between indwelling medical devices and microorganisms would be an important step forward. This study investigated the anti-fouling capacity of engineered nanoscale topographies to the pathogenic yeast Candida albicans. Highly ordered arrays of nano-pit topographies were shown to significantly reduce the physical adherence capacity of C. albicans. This study shows a potential of nanoscale patterns to inhibit and prevent pathogenic biofilm formation on biomedical substrates