Development and characterisation of metronidazole-loaded collagen-chitosan scaffolds for periodontal bone regeneration : in vitro and in vivo studies

Abstract

Thousands of surgical procedures are performed daily to repair or replace tissue damaged by disease or trauma, with scaffolds playing a critical role in supporting bone regeneration. However, the clinical application of a scaffold remains challenging, particularly in the treatment of periodontal disease, where bone loss and microbial infection significantly impede the healing process. Tissue engineering aims to overcome these limitations by developing biodegradable scaffolds that not only support tissue regrowth but also deliver therapeutic agents to the defect site. These scaffolds act as temporary templates that guide regeneration while simultaneously preventing infection. Recent advances in nano-assisted drug delivery have enabled localised and sustained drug release, thereby reducing systemic side effects and minimising the risk of antibiotic resistance. This study aimed to develop a biodegradable collagen–chitosan scaffold loaded with metronidazole nanoparticles (CCMNP) for periodontal bone regeneration. The scaffold was fabricated by blending chitosan and collagen at a 70:30 ratio, with MNP incorporated at various concentrations (0–40% w/v). Physical crosslinking was achieved using dehydrothermal treatment. Characterisation of the scaffolds was conducted using field emission scanning electron microscopy (FESEM) to assess morphology, pore structure, and pore size. In vitro studies were conducted to assess antibacterial activity, biocompatibility, and cell adhesion. Antibacterial efficacy was tested against Porphyromonas gingivalis and Fusobacterium nucleatum using the disc diffusion method. Biocompatibility was evaluated using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with human gingival fibroblasts (HGF-1), and cell adhesion was visualised using FESEM. Based on the in vitro results, the scaffold exhibiting the optimal characteristic was selected for in vivo evaluation. The selected scaffold was implanted into critical-size calvarial bone defects in a rat model. Bone regeneration was assessed after a four-week healing period using histological staining and morphometric analysis. The findings revealed that the 30% w/v MNP-loaded scaffold exhibited desirable physical characteristics, including appropriate pore size and controlled biodegradability. It showed a significant inhibitory effect against the tested periodontal pathogens and promoted the proliferation, viability, and adhesion of HGF-1 cells. In vivo analysis demonstrated enhanced new bone formation at the defect site compared to controls, confirming the scaffold’s regenerative potential. In conclusion, the CC scaffold loaded with 30% w/v MNP demonstrated promising results for periodontal bone regeneration. It offers a targeted, dual action approach by combining antimicrobial protection with structural support for tissue healing. This strategy provides an alternative to conventional systemic antibiotic therapies and contributes to the advancement of scaffold-based regenerative techniques in tissue engineering and dental medicine.