title: The endodontic biofilm: effects of chitosan as a novel antimicrobial agent
Creators: name: Abusrewil, Sumaya Mabrouk
Publisher: name: University of Glasgow
Year: 2023

description: Background and objectives: Endodontic infection or root canal infection, as it is commonly referred to, is a biofilm disease that is difficult to completely irradicate with current treatment protocols, and as such, persistent microorganisms may lead to ongoing or recurrent disease. Root canal treatment is founded on the ability to eradicate microbial infection and prevent reinfection of the highly complex root canal space. There is a growing realisation that endodontic infections are polymicrobial and may contain Candida spp. Despite this understanding, the development of new endodontic therapeutics and models of pathogenesis remains limited to mono-species biofilm models, which are bacterially focused. The main aims of this thesis were firstly to develop and optimise an interkingdom endodontic biofilm model comprised of microbial species frequently identified in endodontic infections and to use this model to test antibiofilm actives. Secondly, to evaluate the antibiofilm efficacy of Mineral Trioxide Aggregate and Biodentine™ calcium silicate cements, used in the management of endodontic diseases, and how modification with chitosan may impact on their antimicrobial properties. Thirdly, to investigate the effect of chitosan incorporation on some of the physico mechanical and biological properties of Biodentine. Materials and methods: Biofilms containing Fusobacterium nucleatum (ATCC 10953), Porphyromonas gingivalis (ATCC 33277), Streptococcus gordonii (ATCC 35105) and Candida albicans (SC5314) were established. Biofilms were optimised in different growth conditions, using quantitative polymerase chain reaction and qualitative microbiology techniques. The in vitro biofilm model was treated with chlorhexidine, ethylenediaminetetraacetic acid and chitosan solutions. This was reaffirmed on a biological substrate (bovine dentine), to further validate this model and the antimicrobial effectiveness of chitosan. To evaluate the antibiofilm efficacy of calcium silicate materials, the regrowth of mono-species (C. albicans), three multispecies (F. nucleatum, P. gingivalis and S. gordonii) and four multispecies (F. nucleatum, P. gingivalis, S. gordonii and C. albicans) biofilms on ProRoot MTA and Biodentine discs were explored using livedead qPCR. These were compared to regrowth on bovine dentine discs. The effect on regrowth of biofilms was assessed following the addition of 2.5 wt% and 5 wt% of chitosan medium molecular weight powder to each calcium silicate cement. Subsequently, the setting time, disintegration, radiopacity, compressive strength, microhardness and biocompatibility of the new composite of Biodentine modified with chitosan were assessed. Next, the regrowth of the four multispecies (F. nucleatum, P. gingivalis, S. gordonii and C. albicans) biofilms on Biodentine discs were explored following the addition of 0.5 wt% and 1 wt% of chitosan powder of either high, medium or low molecular weights. This was compared to regrowth on the unmodified formula of Biodentine. Finally, the physico-mechanical and biological properties of the new composite of Biodentine modified with chitosan low molecular weight were evaluated. Results: Assessment of antimicrobial activity of CHX, EDTA and solubilised chitosan showed significant effectiveness of each antimicrobial agent. Chitosan was similarly effective at preventing biofilm regrowth on bovine dentine. In comparison to a dentine substrate, ProRoot MTA and Biodentine did not show an ability to inhibit biofilm regrowth. The addition of chitosan powder to MTA imparted no antimicrobial enhancement. In contrast, a dose dependent reduction in multispecies biofilm regrowth was determined upon the addition of chitosan to Biodentine. Interestingly, the antibiofilm effect of chitosan increases with the decreased chitosan molecular weight. Importantly, interkingdom interactions were noted, whereby the inclusion of C. albicans to the biofilm enhanced bacterial tolerance in the presence of chitosan and conversely bacterial presence reduced C. albicans tolerance. However, incorporation of 2.5 wt% and 5 wt% chitosan MMw compromises most of the material properties of Biodentine. In contrast, the addition of 0.5 wt% and 1 wt% chitosan LMw showed no detrimental effects on physical and biological properties of Biodentine material, however, significant reductions were noted in mechanical properties of Biodentine when chitosan was incorporated. Nevertheless, the new composite would still be applicable where other root filling materials might be considered, when the material strength and hardness are not critical issues. Conclusion: This thesis describes a robust and reproducible multispecies interkingdom biofilm model that can be employed to assess efficacy of novel endodontic therapeutics. This work demonstrates the potential to enhance the antimicrobial properties of Biodentine, when modified with chitosan microparticles, opening the door for exploration of antimicrobial strategies for prevention and management of endodontic infections. The findings also highlight the importance of using appropriate biofilm model systems when exploring antimicrobial properties of materials in vitro, so that interspecies and interkingdom interactions that modify tolerance are not overlooked.