Spatial Expression Patterns of the RANKL/RANK Axis and Inflammatory Markers in Remodeling-Associated Periodontal Tissues

Background: The remodeling of alveolar bone is governed by the Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL)/RANK/Osteoprotegerin (OPG) signaling axis, which is tightly regulated by the local immune microenvironment. However, previous studies utilizing whole-tissue homogenates have been unable to determine whether these molecular changes are spatially restricted to specific remodeling niches. This study aimed to characterize the site-specific molecular signature of the RANKL pathway and its association with inflammatory cytokines in periodontal tissues.Methods: A rat model of orthodontic tooth movement (OTM) was established. Laser Capture Microdissection (LCM) was employed to precisely isolate remodeling-associated periodontal tissues and physiological baseline tissues. mRNA expression of RANKL, RANK, OPG, and Nuclear Factor of Activated T-cells, cytoplasmic 1 (NFATc1) in these micro-dissected regions was quantified using qPCR. Whole-tissue lysates were analyzed by Western blotting for inflammatory cytokines (Tumor Necrosis Factor-alpha (TNF-α), interleukin (IL)-1β, IL-6). In vitro, human periodontal ligament cells (hPDLCs) were stimulated with TNF-α to investigate whether inflammatory stimulation is accompanied by changes in osteoclast-related molecular markers.Results: Histological and tartrate-resistant acid phosphatase (TRAP) staining indicated increased osteoclast-rich remodeling activity in selected regions. LCM-based analysis revealed spatially restricted upregulation of RANKL, RANK, and NFATc1, coupled with a disrupted RANKL/OPG ratio specifically within remodeling microenvironments, distinct from the surrounding tissue. Whole-tissue Western blotting showed concomitant increases in inflammatory cytokines and osteoclast-related proteins at the tissue level. TNF-α stimulation of hPDLCs increased the RANKL/OPG ratio and Macrophage Colony-Stimulating Factor (M-CSF) expression, suggesting that inflammatory stimulation may promote a molecular state permissive for osteoclast-related signaling.Conclusion: LCM-based analysis revealed that disruption of the local RANKL/OPG balance and upregulation of osteoclast-related molecules are spatially restricted to remodeling microenvironments rather than uniformly distributed across periodontal tissues. In vitro, TNF-α stimulation of hPDLCs dose-dependently shifted the molecular profile toward a state permissive for osteoclast-related signaling. These findings underscore the importance of spatial resolution in periodontal remodeling research and support a potential link between local inflammatory cues and altered osteoclast-related molecular signaling.