Cementocyte alterations associated with experimentally induced cellular cementum apposition in Hyp mice

Abstract

Background: Cellular cementum, a mineralized tissue covering apical tooth roots, grows by apposition to maintain the tooth in its occlusal position. We hypothesized that resident cementocytes would show morphological changes in response to cementum apposition, possibly implicating a role in cementum biology.

Methods: Mandibular first molars were induced to super-erupt (EIA) by extraction of maxillary molars, promoting rapid new cementum formation. Tissue and cell responses were analyzed at 6 and/or 21 days post-procedure (dpp).

Results: High-resolution micro-computed tomography (micro-CT) and confocal laser scanning microscopy showed increased cellular cementum by 21 dpp. Transmission electron microscopy (TEM) revealed that cementocytes under EIA were 50% larger than control cells, supported by larger pore sizes detected by micro-CT. Cementocytes under EIA displayed ultrastructural changes consistent with increased activity, including increased cytoplasm and nuclear size. We applied EIA to Hyp mutant mice, where cementocytes have perilacunar hypomineralization defects, to test cell and tissue responses in an altered mechanoresponsive milieu. Hyp and WT molars displayed similar super-eruption, with Hyp molars exhibiting 28% increased cellular cementum area versus 22% in WT mice at 21 dpp. Compared to control, Hyp cementocytes featured well-defined, disperse euchromatin and a thick layer of peripherally condensed heterochromatin in nuclei, indicating cellular activity. Immunohistochemistry (IHC) for cementum markers revealed intense dentin matrix protein-1 expression and abnormal osteopontin deposition in Hyp mice. Both WT and Hyp cementocytes expressed gap junction protein, connexin 43.

Conclusion: This study provides new insights into the EIA model and cementocyte activity in association with new cementum formation.

Keywords: bone; extracellular matrix; hypophosphatemic rickets; periodontium; tooth eruption.

Figure 1.. Increased Cellular Cementum Formation is Induced by EIA.

Micro-CT renderings show (A, C) 3D and (B, D) 2D sagittal and coronal views of mandibular molars in control and experimentally induced apposition (EIA) groups (n=6/group). Cementum is color coded to separate acellular cementum (AC; light blue) and cellular cementum (CC) of the mesial (yellow) and distal root (green). CC is increased in EIA (red arrows) vs. control molars. (E) Compared to controls, EIA increased mesial and distal tooth length (**p<0.01), mesial and distal CC volumes (**p<0.01) and had no effect on CC mineral density (p>0.05 for both). Samples are color coded to match EIA and contralateral controls from the same mouse. (F, G) Representative control and EIA molars are shown in 2D coronal and sagittal transverse views. CC on root dentin (DE) is highlighted in yellow with pore spaces shown in blue. CC is divided by 100 μm increments indicated by hash marks that show the distance (μm) and are labelled as segments A-M (cervical to apical). Numbers of animals measured for each CC segment are indicated to the left of images (n=0-6). (H) Cortical bone algorithms were applied to each CC segment to measure CC thickness (Ct.Th), CC area (Ct.Ar), CC density, cortical porosity (Ct.Po), pore number (Po.N), pore volume (Po.V), average pore volume (AvgPo.V), and pore density (Po.Dn). Paired EIA and control segments were compared by 2-way ANOVA for paired samples, followed by post-hoc Bonferroni test. Statistical significance is indicated by *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. For apical segments greater than 1,000 μm from initiation of CC, there were not sufficient control samples to compare by statistical analysis and these are indicated by double dagger (‡).

Figure 2.. Increased Cementocyte Size and Altered Ultrastructure from EIA.

(A-D) Confocal laser scanning microscopy (CLSM) of fluorescein isothiocyanate (FITC)-stained hemi-mandibles (n=5 samples/group) showed cementocytes (Ccy) and canalicular networks within the cellular cementum (CC). (E) No difference in cellular CC area is observed at 6 dpp (p>0.05), however by 21 days, CC area increased in EIA vs. control side (**p<0.01). Numbers of Ccy increase in both EIA and control sides between 6 and 21 dpp (**p<0.01 for both), however no intergroup differences are observed on either day. Ccy density is decreased by EIA over time (**p<0.01). (F, G-J) Transmission electron microscopy (TEM) measurements (n=6 samples/group) indicate cell body area is not different between EIA and control sides at 6 dpp (p>0.05), however at 21 dpp, Ccy under EIA are 50% larger than control cells (**p<0.01). On the control side, Ccy area decreases by 25% between 6 and 21 dpp (*p<0.05) while EIA increases Ccy area between 6 and 21 dpp (*p<0.05). (G-J) Representative TEM images of Ccy on control and EIA sides. (K-N) Ccy present defined nuclei with an intact nuclear membrane (arrowhead) and relatively poorly developed rough endoplasmic reticulum (rER). Compared to control Ccy, those under EIA featured increased cytoplasmic and nuclei areas, more disperse euchromatin, and a thin peripherally condensed heterochromatin in nuclei. Larger pericellular spaces (red *) surrounding Ccy in lacunae are associated with cells under EIA.

Figure 3.. Cellular Cementum Apposition in Hyp Mice with EIA.

(A, B) Representative micro-CT renderings show 2D and 3D sagittal and coronal views of mandibular molars in WT and Hyp mice undergoing experimentally induced apposition (EIA) at 0 or 21 days post-procedure (dpp). Overlays include 0 dpp (red) and 21 dpp (green) to illustrate occlusal movement of molars in both WT and Hyp mice, where yellow indicates overlap of the two time points. (C, D) Measurements of eruption at both mesial and distal locations indicate significantly increased eruption of 150-200 μm in both WT and Hyp mouse molars at 21 vs. 0 dpp (n=4-6/group). Intragroup differences were compared by independent samples t-test (**p<0.01; ***p<0.001; ****p<0.0001). (E-H) H&E staining shows cellular cementum (CC) in control and EIA WT and Hyp mouse molars (n=3-6 samples/group). The DE-CC border is indicated by black dotted lines. Hyp CC exhibits a layer of normal appearing cementum near the DE-CC border and unstained/less stained hypomineralized cementoid (red stars) closer to the PDL. Increased CC (red arrows) is observed in both WT and Hyp molars under EIA, compared to controls. (I) Both WT and Hyp mouse molars show increased mineralized cellular cementum area in 21 vs. 0 dpp. (J) No changes in cementoid area are noted for either WT or Hyp mice between 0 and 21 dpp. (K) For total cellular cementum area combining mineralized and cementoid cementum, WT mice increase cellular cementum 22% from 0 to 21 dpp and Hyp mice increase cellular cementum 28% from 0 to 21 dpp. Intragroup differences were compared by independent samples t-test (*p<0.05).

Figure 4.. Altered Cementocyte Ultrastructure in Hyp Mouse Cementocytes Under EIA.

Representative TEM images of WT and Hyp cementocytes (Ccy) within cellular cementum (CC) and cementoblasts (Cb) under EIA. Lacunar borders (Lac) for Ccy are indicated. (A-D) Ccy under EIA present large nuclei (Nu), rough endoplasmic reticulum (rER), and mitochondria (Mt). Hyp Ccy are surrounded by hypomineralized halos where yellow dotted lines indicate borders and yellow * indicate hypomineralized ECM surrounding cells. Compared to WT, Hyp Ccy feature well-defined and dispersed euchromatin (E) and a thicker layer of peripherally condensed heterochromatin (H) in Nu. Cementoblasts presented a well-defined Nu with an intact nuclear membrane and prominent nucleoli. (E-H) Cb in both groups exhibited ultrastructural features consistent with metabolically active cells, including abundant cytoplasm, rER, and Mt. Compared to WT, Hyp mouse Cb presented more abundant cytoplasm and well-developed rER.

Figure 5.. Altered Expression of ECM Markers in Hyp Mice Undergoing EIA.

(A, B) In both WT and Hyp mice undergoing EIA, highest DMP1 immunostaining is associated with cellular cementum (CC) and alveolar bone (AB). Hyp mouse cementocytes (Ccy) show more intense DMP1 localization vs. WT cementocytes (insets par scaled at 40X). (C, D) In both WT and Hyp mice, highest OPN immunostaining is localized in CC, dentin (DE), and AB. OPN distribution is abnormal in Hyp vs. WT CC, with OPN-rich and poor areas. Hyp mouse Ccy show unusual OPN deposition associated with hypomineralized perilacunar halos (black arrowheads in inset in panel D; insets par scaled at 40X). (E, F) GJA1 is expressed by a small number of cementocytes in both WT and Hyp molars undergoing EIA (insets par scaled at 40X).