Type H Vessels Are Progressively Lost in a Mouse Model of Periodontitis: A 3D Tissue-clearing Analysis of Alveolar...

Type H vessels, a specialized subtype of bone-forming vasculature first identified in long bones, are now recognized as critical regulators of angiogenesis-osteogenesis coupling. Defined by high expression of CD31 and endomucin (EMCN), these vessels contribute to both physiological bone growth and pathological bone loss. Although recent studies have identified type H vessels in alveolar bone, their three-dimensional (3D) organization and responses to inflammation remain poorly understood. Here, we aimed to establish a reproducible methodology for visualizing and quantifying type H vessels in murine alveolar bones under both healthy and inflammatory conditions, utilizing a mouse model of periodontitis.

Experimental periodontitis was induced in C57BL/6 mice by placing 5-0 silk ligatures around the maxillary second molar for 0, 3, 7, or 14 days. For 3D vascular analysis, maxillary bone samples underwent tissue clearing with the X-CLARITY system to achieve optical transparency while preserving the antigenicity of the molecules of interest. Type H vessels were identified by dual immunofluorescence staining for CD31 and EMCN, followed by confocal microscopy. The captured digital images were reconstructed for quantification using Imaris software. Alveolar bone loss and microstructural changes were evaluated using micro-computed tomography (micro-CT). Gingival tissues were harvested to assess inflammatory or osteoblast-associated gene expression by qPCR. In parallel, bone marrow-derived endothelial cells (BMECs) were stimulated with recombinant TNF-α or rIL-1β, and changes in EMCN and CD31 expression were analyzed by qPCR and flow cytometry.

The clearing protocol yielded optically transparent maxillary alveolar bone while preserving tissue architecture and immunoreactivity. Confocal imaging revealed abundant CD31⁺EMCN⁺type H vessels in the control healthy periodontal tissue. Quantitative 3D analysis revealed a time-dependent reduction in type H vessel volume following ligature-induced periodontitis in mice. Strikingly, regions exhibiting diminished type H vasculature spatially coincided with areas of alveolar bone resorption identified by micro-CT and with elevated gingival Tnf and Il1b expression, indicating a link between vascular rarefaction and inflammatory bone loss. In parallel, the expression of osteogenic markers, including Alpl, Col1a1, and Bglap, was significantly decreased during murine periodontitis. In vitro, both recombinant TNF-α and IL-1β suppressed the expression of EMCN, a hallmark molecule of type H vessels, in BMECs.

This study provides the first 3D visualization and quantitative characterization of type H vessels in murine alveolar bone with or without periodontitis using a tissue-clearing-based approach. We demonstrate that inflammatory conditions associated with periodontitis cause marked loss of type H vasculature, thereby disrupting angiogenesis-osteogenesis coupling. These findings underscore the possible role of the vascular niche in periodontal disease pathogenesis and suggest that therapeutic preservation or restoration of type H vessels could represent a novel strategy to prevent inflammation-driven alveolar bone loss and enhance periodontal regeneration.