Identification of novel candidate immune biomarkers linked to osteoclast activity during orthodontic tooth movement in a mouse model

Abstract Introduction This study aimed to identify key genes associated with osteoclast activity during orthodontic tooth movement (OTM) using bulk RNA sequencing and experimental validation. Methods An OTM model was established in mice by placing a nickel-titanium closed-coil spring between the maxillary first molar and incisors. Female C57BL/6 mice were assigned to Day 7, Day 14, and Control groups. Alveolar bone changes were assessed by micro-computed tomography (micro-CT) and hematoxylin and eosin (HE) staining. Osteoclasts were quantified by tartrate-resistant acid phosphatase (TRAP) staining. Total RNA from alveolar bone was analyzed by bulk RNA sequencing. Selected genes were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Immune cell quantification was performed by ImmuCellAI. Immune cell infiltration, and related gene expressions were evaluated by immunohistochemistry and immunofluorescence. Results A total of 227 and 152 upregulated differently expressed genes (DEGs) were identified on Day 7 and Day 14, respectively, many of which were related to immune response and osteoclastogenesis. Ten hub genes, including Il1b, Il6, Ccl3, Ccl2, Cxcl2, Mmp9, Timp1, Mmp3, Ccrl and Sele, were positively correlated with osteoclast-related genes (Lilr4b, Socs3, Ctsk, Fos, Il1b, Itgb3, Ccl3, Nmb, Ccr1, Spi1). Macrophage infiltration increased during OTM, and CCL3, CCL2, CXCL2, and CCR1 were colocalized with macrophages. Conclusion Our study highlights key immune genes and immune cell involvement in OTM. These findings may provide molecular and cellular targets for modulating osteoclast activity and orthodontic tooth movement.