Murine IRF8 Mutation Offers New Insight into Osteoclast and Root Resorption.

Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature-inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8's role in osteoclastogenesis.

Bone Sialoprotein Is Critical for Alveolar Bone Healing in Mice.

Bone sialoprotein (BSP) is an extracellular matrix (ECM) protein associated with mineralized tissues, particularly bone and cementum. BSP includes functional domains implicated in collagen binding, hydroxyapatite nucleation, and cell signaling, although its function(s) in osteoblast and osteoclast differentiation and function remain incompletely understood. Genetic ablation of BSP in Ibsp knockout (Ibsp-/-) mice results in developmental bone mineralization and remodeling defects, with alveolar bone more severely affected than the femurs and tibias of the postcranial skeleton. The role of BSP in alveolar bone healing has not been studied. We hypothesized that BSP ablation would cause defective alveolar bone healing. We employed a maxillary first molar extraction socket healing model in 42-d postnatalIbsp-/- and wild-type (WT) control mice. Tissues were collected at 0, 7, 14, 21, and 56 d postprocedure (dpp) for analysis by micro-computed tomography (microCT), histology, in situ hybridization (ISH), immunohistochemistry (IHC), and quantitative polymerase chain reaction (qPCR) array. As expected, alveolar bone healing progressed in WT mice with increasing bone volume fraction (BV/TV), bone mineral density (BMD), and tissue mineral density (TMD), transitioning from woven to mature bone from 7 to 56 dpp. Ibsp messenger RNA (mRNA) and BSP protein were strongly expressed during alveolar bone healing in parallel with other osteogenic markers. Compared to WT, Ibsp-/- mice exhibited 50% to 70% reduced BV/TV and BMD at all time points, 7% reduced TMD at 21 dpp, abnormally increased Col1a1 and Alpl mRNA expression, and persistent presence of woven bone and increased bone marrow in healing sockets. qPCR revealed substantially dysregulated gene expression in alveolar bone of Ibsp-/- versus WT mice, with significantly disrupted expression of 45% of tested genes in functional groups, including markers for osteoblasts, osteoclasts, mineralization, ECM, cell signaling, and inflammation. We conclude that BSP is a critical and nonredundant factor for alveolar bone healing, and its absence disrupts multiple major pathways involved in appropriate healing.

Loss of Discoidin Domain Receptor 1 Predisposes Mice to Periodontal Breakdown.

The discoidin domain receptors, DDR1 and DDR2, are nonintegrin collagen receptors and tyrosine kinases. DDRs regulate cell functions, and their extracellular domains affect collagen fibrillogenesis and mineralization. Based on the collagenous nature of dentoalveolar tissues, we hypothesized that DDR1 plays an important role in dentoalveolar development and function. Radiography, micro-computed tomography (micro-CT), histology, histomorphometry, in situ hybridization (ISH), immunohistochemistry (IHC), and transmission electron microscopy (TEM) were used to analyze Ddr1 knockout (Ddr1-/-) mice and wild-type (WT) controls at 1, 2, and 9 mo, and ISH and quantitative polymerase chain reaction (qPCR) were employed to assess Ddr1/DDR1 messenger RNA expression in mouse and human tissues. Radiographic images showed normal molars but abnormal mandibular condyles, as well as alveolar bone loss in Ddr1-/- mice versus WT controls at 9 mo. Histological, histomorphometric, micro-CT, and TEM analyses indicated no differences in enamel or dentin Ddr1-/- versus WT molars. Total volumes (TVs) and bone volumes (BVs) of subchondral and ramus bone of Ddr1-/- versus WT condyles were increased and bone volume fraction (BV/TV) was reduced at 1 and 9 mo. There were no differences in alveolar bone volume at 1 mo, but at 9 mo, severe periodontal defects and significant alveolar bone loss (14%; P < 0.0001) were evident in Ddr1-/- versus WT mandibles. Histology, ISH, and IHC revealed disrupted junctional epithelium, connective tissue destruction, bacterial invasion, increased neutrophil infiltration, upregulation of cytokines including macrophage colony-stimulating factor, and 3-fold increased osteoclast numbers (P < 0.05) in Ddr1-/- versus WT periodontia at 9 mo. In normal mouse tissues, ISH and qPCR revealed Ddr1 expression in basal cell layers of the oral epithelia and in immune cells. We confirmed a similar expression pattern in human oral epithelium by ISH and qPCR. We propose that DDR1 plays an important role in periodontal homeostasis and that absence of DDR1 predisposes mice to periodontal breakdown.