Dental abnormalities in a mouse model for craniometaphyseal dysplasia

Abstract

Mice carrying a knock-in mutation (Phe377del) in the Ank gene replicate many skeletal characteristics of human craniometaphyseal dysplasia, including hyperostotic mandibles. Ank (KI/KI) mice have normal morphology of erupted molars and incisors but excessive cementum deposition with increased numbers of Ibsp- and Dmp1-positive cells on root surfaces. The cervical loops of adult Ank (KI/KI) lower incisors are at the level of the third molars, while they are close to the mandibular foramen in Ank (+/+) mice. Furthermore, Ank (KI/KI) incisors show decreased eruption rates, decreased proliferation of odontoblast precursors, and increased cell apoptosis in the stellate reticulum. However, their capability for continuous elongation is not compromised. Quantification of TRAP-positive cells in the apical ends of Ank (KI/KI) incisors revealed decreased osteoclast numbers and osteoclast surfaces. Bisphosphonate injections in Ank (+/+) mice replicate the Ank (KI/KI) incisor phenotype. These results and a comparison with the dental phenotype of Ank loss-of-function mouse models suggest that increased cementum thickness may be caused by decreased extracellular PPi levels and that the incisor phenotype is likely due to hyperostosis of mandibles, which distinguishes Ank (KI/KI) mice from the other Ank mouse models.

Conflict of interest statement

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Figures

Figure 1.

Thicker cementum deposition in AnkKI/KI mice. (A) Representative sections of 1st and 2nd molars of 10-week-old Ank+/+, Ank+/KI, AnkKI/KI, and Anknull/null mice stained with H&E. (B) Micro-CT images of 12-week-old Ank+/+ and AnkKI/KI molars. Red arrow indicates excessive cementum deposition of molar roots of AnkKI/KI mice. (C) Increased Ibsp- and Dmp1-positive cells on root surfaces of AnkKI/KI molars (4-week-old Ank+/+ and AnkKI/KI /Ibsp-Topaz/Dmp1-mCherry mice). Dentin (D); acellular cementum (AC); periodontal ligament (PDL); alveolar bone (B); cellular cementum (CC); cementoblasts (Cb); osteoblasts (Ob); osteocytes (Ost). Scale bar = 100 µm.

Figure 2.

Short lower incisors, abnormal position of the cervical loop, and decreased eruption rate in AnkKI/KI mice. (A) Radiographs of mandibles from 1-, 2-, 4-, and 20-week-old Ank+/+ and AnkKI/KI mice. (B) Comparison of 10-week-old Ank+/+, AnkKI/KI, and Anknull/null incisors by x-ray imaging and histology (H&E). Scale bar = 200 µm. (C) Cervical loop sections of 1-, 2-, and 20-week-old Ank+/+ and AnkKI/KI mice (H&E). Scale bar = 200 µm. (D) Histomorphometry of incisors from 13-week-old Ank+/+ and AnkKI/KI mice. Arrows represent the beginning of the first (calcein; green) and second labels (demeclocycline; yellow), respectively. Histograms show eruption rates and distance between the first and second labels in Ank+/+ (n = 5), Ank+/KI (n = 5), AnkKI/KI (n = 5), and Anknull/null (n = 3) mice. *p < 0.005. Mandibular foramen (MF), distal root of the second molar (2M), root of the third molar (3M), position of the cervical loop (CL). Scale bar = 500 µm.

Figure 3.

Decreased proliferation of odontoblast precursors and increased apoptosis in the stellate reticulum, but normal elongation of dental epithelium of AnkKI/KI lower incisors. (A) Sections of one-week-old Ank+/+ and AnkKI/KI cervical loops of incisors stained for BrdU. Graphs represent percentages of proliferative cells in the apical end of Ank+/+ (n = 3), Ank+/KI (n = 4), AnkKI/KI (n = 4), and Anknull/null (n = 3) mice. Odontoblast-lineage cells include pre-odontoblasts (PrO) and polarizing odontoblasts (PoO). Epithelial cells are stellate reticulum (SR), inner enamel epithelium (IE), and pre-ameloblasts (PA). *p < 0.05. Scale bar = 100 µm. (B) Sections of one-week-old Ank+/+ and AnkKI/KI cervical loops stained for TUNEL and percentage of apoptotic cells in the stellate reticulum (SR) of Ank+/+ (n = 5) and AnkKI/KI (n = 5) incisors. #p < 0.01. Scale bar = 50 µm. (C)Ank+/+ and AnkKI/KI incisor organ cultures. Dotted line indicates the location of cervical loop (CL). Scale bar = 200 µm. (D) Elongation rates of dental epithelium between Ank+/+ (n = 5) and AnkKI/KI (n = 5) incisor tooth germs are comparable.

Figure 4.

Abnormal bone remodeling in mandibles of AnkKI/KI mice. (A) Micro-CT images show excessive bone volume and decreased bone marrow spaces in AnkKI/KI mandibles at 1, 2, and 12 wks. (B) SEM images of bone at the apical end of the incisor (10-week-old Ank+/+ and AnkKI/KI mice). (C) TRAP staining of two-week-old Ank+/+ and AnkKI/KI mandibles. Arrows indicate TRAP-positive cells. Scale bar = 100 µm. (D) Bone volume is increased and osteoclast number decreased in AnkKI/KI mice. [BV/TV (%): bone volume/total volume. Oc.S/BS (%): osteoclast surface/bone surface. N.Oc/T.Ar (n/mm2): number of osteoclasts/tissue area. N.Oc: number of osteoclasts.] Ank+/+ (n = 5), AnkKI/KI (n = 6). *p < 0.01. (E) Alendronate injections in Ank+/+ mice replicate the failure of cervical loop migration similar to the AnkKI/KI phenotype. Six-week-old control (no injection) and alendronate-treated Ank+/+ mice. Mandibular foramen (MF), mandibular canal (MC), root of the third molar (3M), distal root of the second molar (2M), position of the cervical loop (CL).