Abstract
Periodontal disease is a biofilm (bacteria)-induced inflammatory disease that leads to the progressive destruction of both soft and hard periodontal tissues. Current clinical treatment includes non-surgical approaches, with or without adjunctive therapies such as antibacterial agents, as well as surgical approaches. Mānuka oil, derived from Leptospermum scoparium, a native plant in New Zealand, shows strong anti-bacterial activity mainly due to its β-triketones compounds.
In this study, mānuka oil and pure β-triketones were encapsulated in chitosan nanospheres using a microfluidics platform via ionic gelation with sodium tripolyphosphate (TPP). Nanospheres were characterised with dynamic light scattering (DLS), showing an average size of 216.0 nm (PDI: 0.260) for control chitosan nanospheres, 253.0 nm (PDI: 0.421) for mānuka oil-loaded chitosan nanospheres and 196.5 nm (PDI: 0.446) for β-triketone-loaded chitosan nanospheres. Fourier-transform infrared spectroscopy (FTIR) spectra confirmed the successful synthesis of nanospheres, and fluorescence-labelling with fluorescein isothiocyanate (FITC) enabled their visualisation. Encapsulation efficiency was 23.42% for mānuka oil-loaded nanospheres and was 62.13% for β-triketone-loaded nanospheres, with both showing sustained release over 24 hours. Antimicrobial properties of nanospheres were evaluated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against oral-related bacteria, including Staphylococcus aureus (S. aureus), Streptococcus mutans (S. mutans), Fusobacterium nucleatum (F. nucleatum) subspecies polymorphum and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans). Biocompatibility of the nanospheres was assessed on human mesenchymal stem cells by Prestoblue® assay.
Nanospheres were further incorporated into carboxymethylcellulose (CMC) gels to enable local delivery for treatment of periodontal diseases. To achieve the comparable viscosity and injectability with current commercial products, three types of CMC with different viscosities were tested. Final formulation comprised 4.5% medium-viscosity CMC gel incorporating 50 or 100 mg/ml nanospheres. The structure of nanosphere-gel composites was confirmed with Scanning Electron Microscopy (SEM), and gel retention was assessed in an in vitro periodontal pocket model. Antimicrobial properties were evaluated against S. mutants, F. nucleatum and A. actinomycetemcomitans using a well diffusion assay, and the biocompatibility was assessed on human gingival fibroblasts.
Therapeutic efficacy in periodontal treatment was assessed in vivo using a rat ligature-induced periodontitis model. For sterilisation, CMC and nanosphere powder underwent either gamma irradiation or UV light exposure. Gamma irradiation reduced gel viscosity and changed nanoparticle size and Polydispersity Index (PDI). No significant effects were observed after exposure to UV light. Sterilised nanosphere-gel composites were applied for 7 days after tooth ligature placement. Micro-CT analysis showed reduced alveolar bone loss in rats treated with 4.5% CMC gel containing 100 mg/ml mānuka oil-loaded nanospheres than in either the untreated control or the CURAPROX Perio Plus gel (0.5% chlorhexidine) group. Immunohistochemistry and histomorphometric analysis confirmed that this formulation attenuated periodontitis-induced bone loss, osteoclast activity, and inflammatory response compared with both the untreated control and CURAPROX-treated groups.
In conclusion, this study developed mānuka oil and pure β-triketone-loaded nanospheres, which were subsequently integrated into a CMC biogel for local drug delivery within periodontal pockets to reduce inflammatory response and prevent bone loss in treating periodontal disease.
This work was supported by the New Zealand Ministry of Business, Innovation and Employment (PROP-76129-ENDSI-UOO).