title: Assessing the bioactive profile of anti-fungal loaded Calcium sulphate
Creators: name: Butcher, Mark Craig
Publisher: name: University of Glasgow
Year: 2020
Subjects: subject: Calcium sulphate, biomaterials, stimulan, Candida albicans, fungal, biofilm, wound, infection.

description: Biomaterials, natural or synthetic materials that have been adapted for clinical use, have been a historically important tool in the development of care regimen for many years. In order to be considered successful, the material must be strong, fit to purpose and biocompatible. Calcium sulphate (CS), a naturally occurring compound, has often been used clinically as a bone cement or void filling biomaterial, precisely due to its biocompatibility, rigidity, and capacity to be fully resorbed. A significant danger to the success of biomaterials as biological implants is that of microbial invasion and adhesion to the surface material which drives infection of the surrounding area, and this can require many lengthy and costly procedures to correct. To address this issue, the resorptive properties of calcium sulphate have often been studied to examine their potential for release of antibacterial compounds to help combat local infection. However, there has been little said for those infections driven by fungal organisms. It was hypothesised that antifungal-loaded CS beads would be successful in eluting antifungal agents, consistently, over a period of 7-days, and would significantly inhibit both planktonic and sessile fungal cells of clinically relevant organisms. Firstly, it was the aim to determine what, if any, concentration of drug was eluted from CS beads and if it was sufficient to provide a minimum inhibitory concentration against clinically relevant fungi. Next, it was an aim to investigate how the CS beads might inhibit pre-grown and developing biofilms. Finally, it was an aim to investigate what, if any, effect beads may have on fungal cells incubated alongside host tissue. To close the loop and explore future directions in the field, the literature relating to wound biofilms was interrogated to provide a quantitative analysis of terms relating to the topic, as well as highlight the relevance of this study to the field. A panel of 16 fungal species were selected and challenged, planktonically, over 7 days, through minimum inhibitory concentration (MIC) tests to determine eluted dose and longevity from antifungal CS beads. Pre-grown biofilms were incubated alongside CS beads to determine capacity for biofilm inhibition, this was visualised by light and scanning electron micrography (SEM) and quantified through biomass and viability assays, and live/dead quantitative PCR. The inhibition of the developing biofilm was also examined through application of a novel hydrogel model via colony count, live/dead PCR and SEM. Candida auris was examined within a co-culture system alongside antifungal CS beads and analysed via qPCR and for gene expression of inflammatory markers. Finally, key terms and trends relating to wound biofilms were examined via bibliometric analysis using the Bilbioshiny® Shiny app for RStudio® Antifungal CS beads were determined to elute concentrations of antifungal drugs far exceeding MIC level, consistently, over a 7-day period inhibiting several key fungal pathogens. Inhibition of pre-grown fungal biofilms was also successful, with significant reduction of C. albicans cells across all treatments and timepoints (P&lt;0.0001) with re-growth for some organisms visible at later timepoints. Developing biofilms were also determined to show susceptibility to antifungal challenge to loaded CS beads across all organisms, with amphotericin B (AMB) loaded CS significantly inhibiting growth of C. auris #8978 (P&lt;0.0001) and #8973 (P&lt;0.0005). Co-culture of C. auris, CS and reconstructed epithelium (RE) produced the same profile of inhibition as previous investigations, while the inflammatory profile of each drug highlighted an increased inflammation response to caspofungin (CSP) beads. Finally, our analysis of the literature provided insight into the development of interest in the field of wound biofilms while also highlighting the growing interest in novel measures to address antimicrobial resistance and an observable lack of literature relating to fungal driven infection. This study shows that antifungal loaded CS successfully release supra-MIC levels of antifungal compounds that could find success as both a prophylactic deterrent of biofilm formation, and as a local treatment option in circumstances where systemic drugs may not be sufficient or biologically available. Initial investigations relating to co-culture work have highlighted a need for further examination of CS beads in the context of immunological response, particularly when considering the potentially toxic effects of some antifungal compounds.