Salivary Gland and Tooth Abnormalities Massively Increase Caries Susceptibility in A Mouse Model of Cleft Lip/Palate
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Objectives: Cleft lip and palate (CLP) is one of the most common birth defects and has been found to be associated with multiple dentofacial anomalies, including hypoplastic teeth, and defects of the orbicularis oris muscle. In view of this associated spectrum of anomalies, numerous studies have investigated the risk of caries development in the CLP population, with the vast majority reporting an increase in caries incidence and severity. However, the cause of the observed increase in caries is largely unknown, making it difficult to manage and significantly prevent. Conditionally targeted mice for the cleft gene, Irf6 (Irf6 Null-E) have been established by deleting the gene only in the oral epithelium after the development of the lip has been completed, thus permitting investigation of the gene’s other roles in the oral phenotypes. Therefore, these mice provide an ideal system with which to investigate the susceptibility and risk factors for caries progression. The overall goal of this research is to assess the function of Irf6 in oral epithelia during morphogenesis of teeth and salivary glands, and in particular how it later impacts tooth structure, salivary function and caries susceptibility. I hypothesize that mice lacking the expression of Irf6 in oral epithelia will have a higher caries incidence compared to matched controls because of abnormal crown morphology and decreased enamel mineralization, as well as altered salivary gland function. Methods: A dental epithelium-specific Irf6 knockout (Irf6 Null-E) mouse was generated using a Pitx2-Cre driver line. Dental development was analyzed by microcomputed tomography, and immuno-histochemistry. Salivary glands were assessed functionally using pilocarpine and histologically postmortem. We then employed an established caries induction protocol that involved infection of the oral cavity with S. mutans, a cariogenic bacterium, followed by feeding with a high-sucrose diet for eight weeks. Bacteria were then recovered from the animals by sonication of the lower jaw in normal saline, bacterial numbers counted to investigate the extent of oral cavity colonization, and the incidence and severity of caries scored under a dissecting microscope and following microCT imaging. Results: I here found, as other previously did (Chu, 2015), that Irf6 Null-E mice recapitulate the dental phenotype observed in patient with cleft lip and palate, such as hypodontia, supernumerary teeth, and misshapen teeth (i.e., flat crown and root taurodontism), as well as enamel mineralization defects that were associated with delayed polarization of ameloblasts. This thesis demonstrated that Irf6 ablation drastically alters the structure of major salivary glands. Histologically, salivary glands from Irf6 null mice exhibited persistent dysplastic changes, disorganized structure that were associated with reduced and disorganized expression of adhesion proteins, altered salivary cell polarization, increased salivary cell proliferation, and reduced acinar cell differentiation. As a consequence, salivary gland hypofunction including low flow rate and reduced buffering capacity, as well as changes in mucin distribution, were observed in the null mice. A striking increase in caries was observed in Irf6 Null mice compared to controls (P<0.0001), and this was associated with a significant increase in both total cultivable flora and the S. mutans population (P <0.001). Conclusions: In addition to altered tooth morphology and decreased enamel mineralization, abnormal salivary gland function may be significantly increasing bacterial colonization and caries susceptibility and severity in the CLP population. For children with IRF6 mutations, implementation of early oral health strategies may be warranted.
- Dentistry