Dentoalveolar Defects in the Hyp Mouse Model of X-linked Hypophosphatemia

Abstract

Mutations in PHEX cause X-linked hypophosphatemia (XLH), a form of hypophosphatemic rickets. Hyp ( Phex mutant) mice recapitulate the XLH phenotype. Dental disorders are prevalent in individuals with XLH; however, underlying dentoalveolar defects remain incompletely understood. We analyzed Hyp mouse dentoalveolar defects at 42 and 90 d postnatal to comparatively define effects of XLH on dental formation and function. Phex mRNA was expressed by odontoblasts (dentin), osteocytes (bone), and cementocytes (cellular cementum) in wild-type (WT) mice. Enamel density was unaffected, though enamel volume was significantly reduced in Hyp mice. Dentin defects in Hyp molars were indicated histologically by wide predentin, thin dentin, and extensive interglobular dentin, confirming micro–computed tomography (micro-CT) findings of reduced dentin volume and density. Acellular cementum was thin and showed periodontal ligament detachment. Mechanical testing indicated dramatically altered periodontal mechanical properties in Hyp versus WT mice. Hyp mandibles demonstrated expanded alveolar bone with accumulation of osteoid, and micro-CT confirmed decreased bone volume fraction and alveolar bone density. Cellular cementum area was significantly increased in Hyp versus WT molars owing to accumulation of hypomineralized cementoid. Histology, scanning electron microscopy, and nanoindentation revealed hypomineralized “halos” surrounding Hyp cementocyte and osteocyte lacunae. Three-dimensional micro-CT analyses confirmed larger cementocyte/osteocyte lacunae and significantly reduced perilacunar mineral density. While long bone and alveolar bone osteocytes in Hyp mice overexpressed fibroblast growth factor 23 ( Fgf23), its expression in molars was much lower, with cementocyte Fgf23 expression particularly low. Expression and distribution of other selected markers were disturbed in Hyp versus WT long bone, alveolar bone, and cementum, including osteocyte/cementocyte marker dentin matrix protein 1 ( Dmp1). This study reports for the first time a quantitative analysis of the Hyp mouse dentoalveolar phenotype, including all mineralized tissues. Novel insights into cellular cementum provide evidence for a role for cementocytes in perilacunar mineralization and cementum biology.

Guidelines for Micro–Computed Tomography Analysis of Rodent Dentoalveolar Tissues

“…These also have their own capabilities and limitations that dictate to some degree the ease and efficiency of analysis. All analyses and images included in this report were accomplished using Scanco proprietary software for scan reconstructions (default settings) and AnalyzePro 1.0 medical imaging software (Analyze Direct, Overland Park, KS), a software system used in our studies (23,(44)(45)(46)(47)(48)(49).…”

“…4A1-3). In mouse studies where molars are erupted and amelogenesis has been completed, we typically threshold enamel at >1,600 mg HA/cm³ (44,45,48,49). After thresholding and confirming segmentation of enamel from dentin, enamel volume and average mineral density are easily calculated.…”

“…In a study to define the precise dentoalveolar pathology associated with XLH, Hyp mice were found to feature severe mineralization disorders in multiple dentoalveolar tissues (Fig. 5B), notably expanded pulp chambers and dentin defects (49). Molar dentin volume was decreased ~30-40%, while dentin mineral density was decreased ~4% in Hyp vs. control mice.…”

“…All analyses and images included in this report were accomplished using Scanco proprietary software for scan reconstructions (default settings) and AnalyzePro 1.0 medical imaging software (Analyze Direct, Overland Park, KS), a software system used in our studies. (23,(44)(45)(46)(47)(48)(49) CT images are created using X-rays directed and rotated around a sample (or a sample rotating in relation to the X-ray source), creating a series of projections. X-rays are a form of electromagnetic radiation with high photon energy, and image generation relies on the partial transmission of X-rays; ie, differential absorption of X-rays by tissues.…”

“…This ROI was selected to include the alveolar rise mesial to M1 and the interdental bone between M1 and M2, and this approach has been applied successfully to a wide range of mouse mandibles aged 2 weeks to over 1 year old. (23,44,45,(47)(48)(49)69,80,81) The ROI we describe here would suffice for many studies of alveolar bone but should be experimentally determined for the hypothesis being examined and the specific needs of the study. In publications, the ROI should be described in detail, including anatomical landmarks used and precise numbers of slices or μm for tissues like alveolar bone.…”