In vivo evaluation of a nanoantibiotic-integrated collagen-chitosan scaffold for bone regeneration in a critical-size rat defect model

Background: Bone loss resulting from periodontal disease, trauma, or anatomical factors is a prevalent challenge in periodontology. This study evaluates the effectiveness of a biodegradable collagen-chitosan scaffold loaded with metronidazole nanoantibiotics to enhance bone regeneration in vivo using a Sprague-Dawley rat skull defect model. The scaffold was surgically placed in critical-size defects, with groups either receiving scaffolds loaded with metronidazole nanoparticles (MNP), empty scaffolds, or no scaffold. The aim of the research is to offer an alternative to conventional non-biodegradable scaffolds, which often require additional surgical removal. Materials and Methods: A sample size of 18 rats was determined based on the Resource Equation Method, ensuring an adequate sample while anticipating a 20% attrition rate, in line with ethical guidelines for animal testing (3Rs: replacement, reduction, and refinement). Male Sprague-Dawley rats (8 weeks old, 250-300g) were divided into three groups: a scaffold loaded with metronidazole nanoparticles (MNP), an empty scaffold, and a scaffold-free control. Anaesthesia was administered using intraperitoneal injections of ketamine (80 mg/kg) and xylazine (10 mg/kg). A 5 mm defect was surgically created in the skull using a trephine bur, followed by placement of the respective scaffold treatment. The surgical site was closed, and post-operative monitoring focused on pain and healing indicators over a four-week period four weeks post-surgery; X-ray imaging was employed to assess bone healing at the defect site. Radiographic images were analysed using Image J software, measuring the percentage of new bone formation as a function of the original defect size. Bone regeneration was quantified by calculating the area of defect closure based on ROI measurements. Following radiographic evaluation, histological analysis was conducted on decalcified tissue sections, stained with hematoxylin and eosin (H&E), and examined microscopically to evaluate new bone morphology. Results: Results demonstrated that the MNP-loaded scaffold group showed significantly higher rates of organisation than the empty scaffold and control groups. The histological analysis confirmed a denser bone regeneration and compact bone formation compared to both within the defect area, suggesting that the MNP-infused scaffold enhances cellular activity and tissue integration. This study highlights the potential of a biodegradable, antibiotic-loaded scaffold to support bone regeneration in critical defects, providing a sustainable solution that reduces the need for secondary surgeries and may be suitable for clinical translation in periodontal applications.