To date there are no published studies on the transcriptome characteristics of primary molar ankylosis. The present study addresses this knowledge gap and has uncovered 432 DE genes, representing 2.3% of the total genes expressed by the furcal bone. From these genes, biological pathways relating to inflammatory response and epithelial cell turnover were predicted to be affected in ankylosis. In contrast with animal models of ankylosis, these findings open up new gateways for studying the pathogenesis of human molar ankylosis.
With respect to methodology, RNA-seq was selected over microarrays for its advantage in providing direct digital readout of expressed genes without reliance on pre-existing gene knowledge. Compared to hybridization approaches, RNA-seq has low background signals (from DNA contamination) and greater sensitivity for discovering low copy transcripts.19 Most importantly, RNA-seq is highly accurate for quantifying transcript abundance across technical and biological replicates, without the need for subsequent verification using quantitative PCR.20
To maximize signal strength, the experimental design needed to minimize clinical differences between the two sample groups. For this reason, molars with successors were excluded to prevent contaminating genes originating from the erupting tooth follicle. The two sample groups were designed to be as similar as possible, differentiated only by the presence or absence of infraocclusion without presumption of ankylosis. This is because studies have demonstrated ankylosis to be invariably present in infraoccluded primary molars with missing successors,16 but the same cannot be said for non-infraoccluded primary molars. Although the vast majority of the latter are non-ankylosed, histological ankylosis has occasionally been found.21 Therefore, non-infraoccluded teeth cannot be assumed to be non-ankylosed, especially since normal percussion sound and normality mobility do not reliably exclude ankylosis.22 In the current study, tissue scarcity precluded a parallel histological analysis, although even this would be of limited value given that histology cannot detect transcriptional activity leading to future clinical ankylosis.
The results showed that primary molar infraocclusion may be regarded as a phenotype with a molecular correlate involving at least 432 DE genes. However, since non-infraocclusion may represent a temporary absence of ankylosis in a dynamic disease, the differential gene outputs between the two groups may be viewed as a continuum, rather than as a distinct separation, between two different states of the pathology.
The above caveat notwithstanding, the pattern of gene expression was clearly different between the two groups, as demonstrated by hierarchical clustering (Fig. 1b) and PCA (Fig. 1c). Not only was there a clear separation between infraocclusion and non-infraocclusion, but both hierarchical clustering and PCA showed greater variation in the infraoccluded group compared to the non-infraoccluded group, which showed tighter clustering. This variation is a common finding in pathological conditions, since the pathology is often due to variable dysregulation of normally tightly regulated biological functions. For this reason, infraoccluded samples that looked like outliers (e.g. sample D5 in Figs. 1b, c) were not excluded, as they may represent the biological variability of ankylosis.
The number of DE genes (432) between the two groups represented only a small proportion (2.3%) of the total number of genes detected by RNA-seq. This small number appeared to reflect a strong degree of biological and molecular homogeneity between the two conditions (i.e. infraoccluded versus non-infraoccluded). In other transcriptome-wide studies using RNA-seq, for example comparing various types of cancers, the percentage of DE genes usually ranged from 30–70%, which reflects divergent tissue types with heterogeneous gene outputs.23 In contrast, exposure of a single cell type (Acinetobacter baumannii) to a single provocation (ethanol) elicited only 2% DE genes with RNA-seq.24 This supports the view that the current experimental design has maximized biological similarities between the sample groups, with the main difference being the presence, or not, of infraocclusion. It follows that the DE genes are likely to represent gene outputs associated with the infraocclusion phenotype.
Tooth ankylosis is a disruption in tooth eruption associated with loss of periodontal ligament (PDL) integrity. Given that genes causing disease are likely to be ontogenetically active during organ development,25 we reasoned that DE genes should also be expressed in developing tooth structures. Using immunohistochemical validation, we showed that all selected proteins were expressed in developing tooth structures. Although the spatial distribution of these proteins cannot speak to their etiology in tooth ankylosis, it would reaffirm the notion that pathological conditions frequently recapitulate their ontogenetic history during development. Of special note is the dental follicle (immunostained in five out of six proteins) that is comprised of pluripotent ectomesenchymal cells that subsequently give rise to the periodontal ligament, bone and cementum; all of which are involved in the ankylotic process. The detection of CK14 and PKP1 in the inner enamel epithelium deserves special mention given its developmental role leading to the epithelial rests of Malassez (ERM), a structure that has been implicated in ankylotic pathogenesis. The ERM play an important role in maintaining PDL space to prevent ankylosis.26 For example, experimental reduction of ERM distribution in rat teeth via denervation of the inferior alveolar nerve resulted in narrowing of the PDL space and ankylosis after 6 weeks. Subsequently, regeneration of ERM 10 weeks after denervation corresponded with widening of the PDL space.27 Other studies have observed that absence of the ERM in regenerated PDL is associated with narrower PDL space,28 and ERM are always present in the vital PDL areas of replanted teeth.29 These findings suggest that normal, so-called “quiescent” ERM may be involved in the maintenance of PDL space, and consequently, prevention of ankylosis. Therefore, ERM dysregulation may result in loss of its ability to prevent ankylosis.
Familial studies suggest that ankylosis is not a monogenic disease.5 Likewise, the present study did not identify a dominant gene for ankylosis. A pathological condition such as ankylosis requires the coordinated expression of multiple genes, often linked across multiple signalling pathways, as revealed in the current study. For this reason, the differential expression of one gene alone would be neither expected nor meaningful. On the other hand, the differential expression of multiple genes involved in a common biological pathway provides confidence that this pathway is being affected in infraocclusion. Pathway analysis using GSEA showed that certain pathways, in particular those associated with epithelial cell development and the inflammatory response, were over-represented in the infraoccluded group. Parallel analysis using IPA confirmed an increased activation of the inflammatory response and increased epithelial cell proliferation and differentiation in the infraoccluded group.
On closer inspection of the genes supporting epithelial change, keratins in particular were among the most highly expressed genes. Keratins form the intermediate filaments of epithelial cells and are known to reflect the tissue type as well as changes in differentiation states. The keratins in this study were consistent with those expressed in human ERM, and in particular, ERM undergoing proliferation and differentiation in response to inflammation30,31,32,33 (Table 2). The increased expression of desmosomal cadherins, desmoglein 2 (DSG2) and desmocollin 2 (DSC2), and the cytoplasmic desmosome-associated protein, plakophilin 1 (PKP1), are likewise consistent with human ERM.32 Collectively, the results suggest that there is a change in the ERM state during ankylosis, which may be associated with an increased inflammatory response.
The above is concordant with established evidence that ankylosis is highly associated with root resorption, which is driven by pro-inflammatory mediators.34,35 Ankylosis in primary molars appears as an area of previous root resorption repaired by bone extending from the surrounding alveolus.16,21 In this way, ankylotic sites are constantly remodelling and relocating, closely following the progression of root resorption in an apical to cervical direction.16 Interestingly, root resorption, and presumably the accompanying inflammation, is greater in ankylosed, compared to non-ankylosed, primary teeth,36,37,38 further highlighting an association between ankylosis and inflammation.
The ebb and flow of the ankylotic process is reflected in multiple niches of hard tissue resorption and remineralization. Thus it was interesting to note that several mineralization-associated genes were found to be differentially expressed. These include decreased expression of bone-degrading genes (e.g. MMP2, MMP1439), increased expression of pro-mineralization genes (e.g. C4orf26,40 ODAM,41,42 BMP5, ASPN43), and decreased expression of anti-mineralization genes (POSTN,44 AXIN2,45 NFATC246). A study of mutant mice bred for elevated Wnt signalling via β-catenin stabilization in osteocytes and cementocytes showed PDL space mineralization, failure of eruption, and dental ankylosis.12 In this study, it is possible that the decreased expression of AXIN2, which negatively regulates Wnt signalling via β-catenin degradation, similarly acts to increase Wnt signalling.45 Given that the differential expression pattern of these mineralization-associated genes seems to indicate a net anabolic effect, ankylosis may be associated with dysregulation of mineralization, as has been observed in animal models.12,13,14
Due to the case-control nature of this study, the results can only demonstrate an association, not a causal relationship, between ankylosis and the list of DE genes. The DE genes may well be the result, rather than the cause, of ankylosis, given that the condition was pre-existing before tissue sampling. Despite this limitation, the results of the present study would be valuable to future studies. This is because the behaviour of human primary molar ankylosis is currently not replicable in animal models, and any histological or transcriptomic analysis of human teeth precludes that tooth from longitudinal follow-up. Therefore, the aim of the study was to establish the differences in gene expression of ankylosed teeth, from which to build future experiments to explore a causal relationship, which could involve testing candidate genes in animal models, or modulating pathways highlighted to be important in ankylosis.
In conclusion, this case-control study found that primary molar ankylosis with infraocclusion was characterized by the differential expression of genes consistent with an increased inflammatory response and increased epithelial cell proliferation and differentiation. Independent validation of six DE genes showed their protein expression in developing tooth structures, especially the dental follicle. A hypothesis was generated that an increased inflammatory response and ERM dysregulation form part of the pathogenesis of primary molar ankylosis. This provides the foundation for hypothesis testing in future experimental models.