Cellular cementum biology remains poorly understood, particularly the potential functions of cementocytes. We hypothesized that cementocytes would display morphological changes in response to the process of continuous new cementum formation. To test this proposition, we employed a mouse model of EIA to promote rapid new cementum formation. This model was validated by a combination of 3D high resolution micro-CT and 2D histomorphometry, confirming cellular cementum apposition on mesial and distal roots of molars removed from occlusion. In addition, cementocytes undergoing EIA displayed ultrastructural changes consistent with increased cell activity. We also applied the EIA model to Hyp mutant mice, a model for X-linked hypophosphatemia (XLH) that features altered phosphate metabolism and hypomineralized perilacunar halos contributing to an altered mechanical environment surrounding cementocytes. Intriguingly, compared with WT mice challenged by EIA, Hyp mouse molar super-eruption was not reduced, Hyp mice had greater cellular cementum apposition, albeit a larger proportion was cementoid, and Hyp cementocytes showed signs of metabolic activity. Hyp mouse cementocytes showed abnormal localization of markers DMP1 and OPN, and connexin GJA1 was found in both WT and Hyp cementocytes. This study adds additional evidence of cementocyte activation in association with cellular cementum formation in mice, prompting further studies to explore these functions.

4.1 Experimentally induced cellular cementum apposition

Cementum is present on human and mouse tooth roots primarily in two varieties. Acellular cementum is found on the cervical root and is critical for tooth attachment by anchoring PDL collagen fibers. The function of cellular cementum, found on apical roots and in furcation regions, is less clear, because there have been few human disorders or mouse models available to provide insights into this tissue. For several decades, the function of cellular cementum has been described as maintenance of post-eruption tooth occlusion by slow tissue apposition.14, 22, 23 Although difficult to prove over a lifetime of occlusion, this ability was in part demonstrated by a rodent EIA model wherein molars are removed from occlusion (also known as the unopposed molar, or super-eruption model) by extraction or grinding down their occlusal partner(s).12-14, 24-28 Removal of the molar from occlusion spurs additional cellular cementum (and apical alveolar bone) formation within a period of days to weeks. This experimental model has been previously studied by 2D histological and radiological approaches. In this study, we analyzed EIA with 3D high resolution micro-CT in combination with other modes of analysis, validating the model and presenting novel findings.

We document > 200 μm of cellular cementum apposition on mesial and distal roots of the super-erupting molar over 21 days, averaging nearly 10 μm/d. To date, it has been challenging to separate cellular cementum from dentin by micro-CT analysis, and therefore rarely performed. We developed a strategy to accomplish this10, 29-31 and extend this approach here by adapting cortical bone analysis algorithms32 to analyze several additional characteristics of cellular cementum subregions. EIA increases cellular cementum area and thickness in apical regions of cellular cementum. Using porosity measurements we adapted from cortical bone evaluation32 to evaluate cementocyte lacunae8 revealed significantly increased pore volume and average pore volume in apical (new cementum) regions on EIA versus control sides. Based on our approach, the EIA model produces a reliable increase in cellular cementum volume and new analytical approaches can reveal new insights into the underlying mechanisms.

4.2 A role for cementocytes in cementum biology

Increased production of cellular cementum as a result of EIA challenge supports the concept that apposition is regulated by mechanosensory response to changes in occlusal loading, and that neocementogenesis attempts to increase tooth height to restore occlusion. Although cementoblasts are presumed to promote acellular and cellular cementum formation during root development, functions of the resident cementocytes within cellular cementum remain unknown.6 Osteocytes entombed within lacunae extend dendritic processes into fluid-filled interconnected canaliculi in bone. Accumulated research has demonstrated osteocytes to be mechanoresponsive cells that respond to fluid flow in the lacunocanalicular system resulting from bone loading. By signaling to osteoblasts, osteoclasts, and other external cells, osteocytes regulate local bone remodeling.4, 5

Cementocytes share many features of osteocytes and may therefore mimic some osteocyte functions in cementum.6 Cementocytes become embedded in cementum ECM, reside in a lacunocanalicular system, and maintain gap junctions that make possible cell-cell communication.33-37 Therefore, a plausible mechanism exists for cementocytes to respond to altered tooth loading. Cementocytes present an in vivo and in vitro expression profile parallel to osteocytes, including key markers, dentin matrix protein 1 (DMP1), sclerostin (SOST), E11/gp38, tumor necrosis factor receptor superfamily member 11b (TNFRSF11B; osteoprotegerin/OPG), and tumor necrosis factor superfamily member 11 (TNFSF11, RANKL).6, 7, 38-40 Genetic ablation of Dmp1 causes abnormal osteocytes and defective bone mineralization, with parallel changes in cementocytes and cellular cementum.40 Knockout of Sost in mice relieves osteocyte inhibition of osteoblast Wnt signaling, promoting both increased bone and cellular cementum formation, suggesting common molecular regulation by osteocytes and cementocytes.41

We investigated evidence of cementocyte activity under EIA. Previously, we employed the EIA approach combined with proteomic analysis to identify 97 proteins solely in EIA versus control cementum.9 Factors altered by EIA included intracellular and cell membrane proteins, interpreted as indirect evidence of cementocyte activation.9 However, the proteomic approach could not definitively identify cementocytes as the source of the altered protein composition. Therefore, we expanded this line of inquiry with a multimodal approach to investigate changes in cementocytes under conditions of EIA. By CLSM, histomorphometry, micro-CT, and TEM analysis, cementocytes presented signs of cellular changes, suggesting activation of cells under challenge by EIA. The functions and mechanisms of increased cementocyte and lacunar size require further investigation.

To further test cementocyte functions, we challenged the Hyp mouse model of XLH by EIA. Unopposed Hyp mouse molars exhibited undiminished super-eruption compared with WT controls, and increased cellular cementum was measured on Hyp molars roots. These results were surprising because a previous study of unopposed molar eruption reported reduced cellular cementum apposition in Hyp versus control mice under challenge.25 The previous study employed fluorochrome labeling to measure rate of apposition, an approach that only indicates deposition of mineralized cementum. The impaired mineralization of Hyp mouse cementum and large proportion of cementoid likely impaired fluorochrome accumulation, therefore micro-CT measurement of molar relocation combined with histomorphometry measurement of cementum here provided a more comprehensive picture, revealing that super-eruption was not impaired in Hyp mice. Although Hyp mice displayed altered distribution of DMP1 and OPN in cellular cementum compared with WT, these key markers and potential mechanoresponsive factors were still present and presumably functional in Hyp versus WT mice. OPN has been shown to be important for translational movement of mouse molars, though was not essential for super-eruption.12 We localized connexin GJA1 in cementocytes in both WT and Hyp mouse cementum, providing new insights into gap junction function in cementocytes. GJA1 has been associated with mechanical response of osteocytes.21

Finite element analysis predicts an inverse relationship between stiffness of osteocyte perilacunar matrix and lacunar strain, indicating changes in the perilacunar environment likely change the strain signal sensed by osteocytes.42 In this case, decreased stiffness in Hyp mouse cementocyte perilacunar regions from hypomineralization defects would be predicted to increase strain, possibly promoting through undefined mechanisms increased cementum apposition. This may in part explain increased alveolar bone and cellular cementum production in conditions of hypomineralization.8 Challenge with EIA may exacerbate this situation, promoting an exaggerated response. Altered phosphate metabolism in Hyp mice may also contribute to cementocyte response to EIA, an aspect that deserved further study.

It remains unclear how occlusal loading and tension from PDL attachment may influence cementocytes and cellular cementum. A cementocyte cell line in vitro showed increased OPG/RANKL ratio under fluid flow conditions, whereas osteocytes decreased OPG/RANKL.7 It should be noted that the mechanisms underlying eruptive and post-eruptive tooth movement are complex, involve activities of multiple cell populations, depend on several biological processes, and remain only poorly understood at present. Therefore, apposition of cellular cementum is only one contributor to this process. Additional in vivo and in vitro studies will be key for improving understanding of cementocyte biology and potential use in therapeutic strategies. Further studies should explore specific changes in cementocytes, including gene and protein expression, and roles of cementocytes in other contexts, to provide additional insights on the functions of these cells.