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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 To compare the efficacy of calcium sodium phosphosilicate (CSPS) and arginine dentifrices on dentin permeability and acid tolerance. Material and Methods Sixty dentin discs were randomly assigned into 3 groups, then brushed for 1 min with CSPS, arginine, or fluoride (control) dentifrices. To test acid tolerance, each disc was soaked in 6% citric acid for 1 min. Dentin permeability was measured before, following brushing, and acid challenge. Ten discs per group were similarly treated and evaluated for tubule occlusion following a single dentifrice application, while other five discs per group were employed in an acid tolerance assay. Results The percentage reduction in dentin permeability was 39.26%, 32.27%, and 21.71% in the arginine, CSPS, and control groups, respectively. The differences in dentin permeability reduction between the arginine and CSPS groups following brushing and acid challenge were not significant (p = 0.398 and p = 0.211, respectively). The arginine dentifrice demonstrated a significant reduction in permeability compared with the control (p = 0.011). In addition, the occlusion exhibited by the arginine and CSPS dentifrices was more resistant to acid challenge compared with that of the control (p < 0.001). From SEM analysis, dentinal tubule occlusion was observed after a single application in all groups. Some open dentinal tubules were detected in the test groups, while almost all of the orifices were open in the fluoride group following acid challenge. Conclusions There is no significant difference between arginine and CSPS dentifrices in reducing dentin permeability following a single application and acid challenge. Following acid challenge, the reduced permeability generated by arginine and CSPS was more stable compared with the fluoride dentifrice.

Calprotectin, a complex of S100A8/S100A9, increases oral keratinocyte resistance to bacterial invasion. Calcium possibly induces conformational change of S100 proteins leading to signaling downstream effectors and S100A9 has the unique extended C-terminus, therefore, the calcium-binding and the C-terminal domains of S100A9 are suggested to determine calprotectin functions. The S100A9 structural motifs that confer calprotectin-induced resistance are yet unknown. To determine which the structural motifs of S100A9 in complex with S100A8 affect resistance to bacterial invasion, the S100A9 calcium-binding sites were ablated by site-directed mutagenesis and the C-terminus was truncated to delete the zinc-binding/arachidonic acid domain and phosphorylation site. Constructs to express the mutated S100A9 proteins with S100A8 were then transfected into KB cells and production of calprotectin complex was verified by reaction with the complex specific Mab 27E10. Stable mutants were tested for bacterial invasion by Listeria monocytogenes and Salmonella enterica serovar Typhimurium using an antibiotic protection assay and confirmed by double-immunofluorescence (IF) staining for invaded Listeria. Listeria binding to KB cells was quantified for up to 60 min, surface expression of calprotectin was estimated by IF and flow cytometry, and calprotectin mobilization to cytoskeleton was observed by IF. The result showed that S100A8 complexed with all mutants of S100A9. S100A9 C-terminus deletion transfectants resulted in fewer intracellular Listeria and Salmonella than the sham or intact S100A8/S100A9 cells (p<0.01). Ablated S100A9 C-terminus cells showed fewer invaded bacteria than positive control (p<0.05), while mutated S100A9 calcium-binding domain and the sham transfectants showed similar number of invaded bacteria. Listeria binding on KB cells and invasion appeared directly related. Surface calprotectin was undetectable, suggesting that binding only indirectly involved calprotectin. Furthermore, calprotectin mobilized to tubulin in a calcium-dependent manner, which was reversed by BAPTA-AM, an intracellular calcium chelator. In contrast, disruption of S100A9 calcium-binding domains inhibited calcium-dependent calprotectin mobilization. In conclusion, within KB cells, calprotectin mobilization to tubulin and epithelial resistance to invasion by bacteria appear to depend on the calcium-binding domains of S100A9.