Large critical-size bone defects in the oral and craniofacial region are difficult to regenerate. We evaluated the effectiveness of mRNA encoding bone morphogenic protein-2 (BMP-2) in enhancing bone regeneration using a rat calvarial defect model. Two delivery approaches were investigated: (1) in vivo application of BMP-2 mRNA encapsulated in lipid nanoparticles incorporated in a scaffold, and (2) application of ex vivo BMP-2 mRNA-transfected rat bone marrow mesenchymal stem cells (rBMSCs), loaded on a scaffold and implanted into calvarial defects. The direct application of BMP-2 mRNA encapsulated in lipid nanoparticles improved bone regeneration as indicated by micro-computed tomography analysis. The enhancement was even more pronounced with ex vivo transfected rBMSCs. rBMSCs transfected with FGF-2 mRNA did not improve bone regeneration, either alone or combined with BMP-2 mRNA-transfected rBMSCs. Similarly, PDGF-BB mRNA-transfected rBMSCs failed to enhance bone regeneration alone and notably suppressed BMP-2 mRNA-transfected rBMSCs’ effects. Interestingly, BMP-2 mRNA-transfected rat fibroblasts showed comparable bone regeneration to transfected rBMSCs. Osteogenic differentiation was absent in BMP-2 mRNA-transfected rBMSCs, implying that they may primarily serve as a source of translated BMP-2 for bone regeneration rather than undergoing osteogenic differentiation. These findings highlight the translational potential of BMP-2 mRNA for bone regeneration, particularly in oral and craniofacial applications.

Objectives Ligature-induced periodontitis model has been widely used as a preclinical stage for investigating new treatment modalities. However, the effect of different ligature sizes on alveolar bone loss has never been studied. Therefore, we examined alveolar bone loss in this rat model using different sizes of silk ligatures, as well as healing after ligature removal. Materials and Methods Left maxillary second molars of Sprague-Dawley rats were ligated with 3-0, 4-0, or 5-0 silk ligatures (n = 4–5/group) for 14 days before harvested maxillae and gingival tissues. For subsequent experiment, animals were ligated for 14 days using the ligature size that induced the most alveolar bone loss before ligature removal and sacrificed at 0, 7 and 14 days (n = 5–6/group). All maxillae and gingival tissues were harvested to evaluate alveolar bone level, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) levels. Statistical Analysis Data was analyzed using SPSS Statistics 23.0 software (SPSS Inc., Chicago, Illinois, United States). Data from all experiments were tested for normality using Shapiro–Wilk test. Data between ligatured and nonligatured teeth were compared using Student's t-test or Wilcoxon signed-rank test. Differences among different ligature sizes were analyzed by analysis of variance followed by multiple comparisons with post-hoc test. A p-value less than 0.05 was considered statistically significant. Results The alveolar bone loss of ligated teeth was substantially higher than that of control after 14 days of ligation. While 3-0 and 4-0 resulted in significantly greater bone loss than 5-0 silk, the 3-0 group had the lowest rate of ligature loss. Therefore, alveolar bone healing postligature removal was investigated further using 3-0 silk. The results showed no significant bone level change at 2 weeks after ligature removal. In term of IL-1β and TNF-α levels, there was no statistically significant difference in IL-1β level between groups at any time point, while TNF-α was undetectable. Conclusion These data showed that 3-0 silk was the most effective ligature size in promoting alveolar bone loss comparing with 4-0 and 5-0 silk. During the 2-week period following ligature removal, spontaneous bone healing was not observed.