Saudi Cultural Missions Theses & Dissertations
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Item Restricted In vitro and in vivo models to study the effect of biofilms and antimicrobial wound dressings on wound healing(The University of Manchester, 2024-06-25) Baqader, Sajwa; Thomason, Helen; Humphrey, Gavin; McBain, AndrewBackground: Biofilms have been strongly implicated in delayed wound healing. Reduced oxygen and low microbial growth rates within wound biofilms are a recognized driver of tolerance towards antimicrobial agents, and poor treatment outcomes. There is however uncertainty about the contribution of biofilm to delayed healing and the mechanisms involved. This emphasises the need to examine the differences in the wound healing process between the planktonic and biofilm-infected wounds through preclinical models. Improved knowledge in this area may lead to the development of improved treatment strategies. Antimicrobial chemicals, especially silver-based ones, are frequently employed in wound dressings, but little is known about their efficacy and there are few reliably predictive preclinical wound models. This doctoral thesis focuses on visualising the variations in metabolic activity in bacterial cells during biofilm development and studying the differences in the wound healing process between planktonic and biofilm-infected wounds. This thesis also evaluates the antibacterial efficacy of innovative wound dressings that incorporate silver oxynitrate, which produces highly reactive Ag2+ and Ag3+ ions, using in vitro and in vivo models. Methods: Initially, a 96 well plate-based assay was used to determine the bactericidal activity of dressings containing silver oxynitrate and untreated controls against planktonic S. aureus in both log- and stationary phases. A colony biofilm model was then used to assess the efficacy of dressings containing silver oxynitrate in eradicating biofilms over 1, 3, and 7 days. Next, an in vivo excisional wound model either uninfected or infected with 104 S. aureus in planktonic form, or as preformed biofilms was established, and used to assess effects of dressings containing silver oxynitrate compared to non-antimicrobial controls and uninfected wound controls. Bromodeoxyuridine (BrdU), a nucleotide analogue that incorporates into nascent DNA in actively dividing cells was used to visualise proliferating. Biofilms were exposed to BrdU for 4 h before harvesting at 1, 3 and 7 days. Wound healing was characterised by quantification of wound width, area, and re-epithelialisation from histological sections. Inflammation was determined based on neutrophil/macrophage marker immunohistochemistry. Wound biofilms were characterised by anti- BrdU immunofluorescence, fluorescence in-situ hybridisation specific prob, viability mapping, tissue Gram stain, quantitative bacterial culture, and scanning electron microscopy. Results: Strong BrdU staining was observed corresponding to active growth of 1 day S. aureus biofilm and slow metabolic rates occurred following 3 days old biofilms. No BrdU signal was detected in the 7 days old biofilms of S. aureus. Over 7 days, wound width and areas increased significantly in wounds infected with preformed biofilm and in planktonic infected wounds compared to uninfected wounds. A significant reduction in the reepithelialisation percentage was observed in wounds infected with preformed biofilms and infected planktonic wounds. In contrast, uninfected wounds exhibited a small wound width, area, and comparatively high reepithelialisation percentage. The number of neutrophils and macrophages was significantly higher in wounds infected with preformed biofilm than wounds that infected with planktonic bacteria. S. aureus abundance on surface of both planktonic and biofilm infected wounds was markedly increased after seven days. Dressings containing silver oxynitrate significantly reduced planktonic bacterial cell counts more during log phase than stationary phase. Furthermore, this dressing effectively decreased number of bacterial cells in colony biofilm following a 7-day exposure. The dressings containing silver oxynitrate also significantly reduced S. aureus viable counts, wound width, area, inflammation, and increased reepithelialisation more in planktonic infected wounds than biofilm infected wounds over 7 days. SEM imaging revealed that infected wounds treated with silver oxynitrate-incorporating dressings contained fewer bacteria and biofilm extracellular polymeric substance was disrupted. Conclusion: The in vitro colony biofilm method of labelling metabolically active S. aureus with BrdU over 7 days demonstrated the bacterial cells dormancy. The murine wound model provides evidence of the impact of S. aureus in planktonic and biofilm form in delaying healing process. The use of dressings containing silver oxynitrate demonstrated a greater impact on the treatment of infected murine wounds in both states, with greater efficacy against planktonic infected wounds. The in vitro and in vivo models mentioned above can be used to evaluate effectiveness of other wound treatments obtain better wound management and patient outcomes.19 0Item Restricted The antibacterial efficacy of isolated bacteriophages against periodontal biofilms: A systematic review of in vitro studies(Saudi Digital Library, 2023-08-07) Binsaif, Nasser; Ramage, GordonBackground and aim: Periodontitis is a widespread, irreversible disease that threatens the oral health and quality of life of adult individuals. Antibiotics have been used to systemically treat periodontitis in combination with mechanical therapy, but they are increasingly resisted by the human microbiome. Phage therapy has been proposed to exhibit antibacterial activity, consequently, replace the use of antibiotic drugs. This study aimed to identify and summarise the current in vitro evidence of phage therapy’s effectiveness against periodontal biofilm. Methods: The PubMed, Web of Science, and Ovid (Embase) databases were searched to identify in vitro literature that investigated phage therapy as a treatment for periodontal biofilm using search terms referring to periodontal biofilm and bacteriophages. The literature was screened by reading the titles and abstracts to identify studies to be read fully, and inclusion and exclusion criteria were also applied. Thereafter, the included studies were processed to extract relevant study characteristics and general outcomes before performing quality assessments. Results: Ten publications were found to meet the inclusion criteria. All of these studies indicated that bacteriophages exhibited significant antibacterial activity against monospecies biofilms comprised of the micro-organisms F. nucleatum, E. faecalis, S. mutans, or A. actinomycetemcomitans. Regarding bacteriophage resistance, only one study reported resistance of E. faecalis biofilm after 24 hours, and the remaining studies did not report resistance activity. Discussion and conclusion: Recent in vitro studies suggest the use of phage therapy as a novel systemic periodontal treatment. However, a specific phage for use against P. gingivalis remains undetected. Moreover, the antibacterial efficacy of phage therapy against complex periodontal biofilms is still unclear.70 0Item Restricted The Effect of Simvastatin Scaffolds on human Periodontal Ligament Cells as a Potential Endodontic Regenerative Technique(2023) Almohimeed, Khawlah; Crawford, Aileen; Martin, NicolasRegenerative endodontic procedures have received considerable critical attention in the field of restorative and paediatric dentistry. Vital pulp is the best filling material a tooth can have, thus, maintaining pulp vitality is crucial in this respect. This is a preliminary study is to investigate the dose and timely release of 2% simvastatin from PLGA scaffolds as well as assess viability of human periodontal ligament cells (hPDL). The release of simvastatin in DMEM was much higher than PBS in the first 24 hours. Due to limitations in accessing the lab during Covid-19 Pandemic, spectrophotometer readings have only been obtained for a 96-hour incubation period on the Simvastatin PLGA scaffolds. HPDL showed viability after 96-hour incubation in 2% simvastatin PLGA scaffolds. the conclusion was that our newly fabricated 2% simvastatin PLGA scaffolds showed biocompatibility. Within the limitations of this study, we can conclude that using the newly fabricated 2% simvastatin PLGA scaffolds-maintained cell viability for 96 hours although it was lower than desired. Further research is needed in this respect especially that the present study contains preliminary data.26 0