Study of odontoclast dysregulation in feline tooth resorption

Abstract

Feline tooth resorption (TR) is a common and painful disease characterised by loss of mineralised tissues of the tooth. Due to the progressive nature of the disease the only available treatment is to extract affected teeth. Odontoclasts are specialised cells that resorb teeth and they share many similarities with osteoclasts, the bone resorbing cells. The main physiological role of these cells is to resorb deciduous teeth to allow for the permanent tooth replacement. In some cats these cells become dysregulated and attack the permanent teeth later in life. However, the aetiology of tooth resorption is still unclear. The aim of this PhD project was to identify transcriptomic changes between TR negative and TR positive teeth, set up a TR model to study odontoclast dysregulation in vitro and to use this system to test a novel therapeutic target for the treatment of TR. In this study, a successful RNA isolation procedure for feline teeth collected in a clinical setting and at post-mortem was developed. To elucidate the role of inflammation and vitamin D involvement in TR, quantitative PCR was performed but there was no statistically significant difference in the expression of any inflammatory cytokines, vitamin D receptor or receptor activator of nuclear factor kappa-B ligand (RANKL) between TR negative and TR positive teeth. Feline osteoclasts were successfully generated from feline bone marrow in vitro. I also found that primary feline periodontal ligament cells could induce osteoclast formation. Osteoclast precursors from TR positive cats formed higher numbers of osteoclasts when co-cultured with periodontal ligament cells compared to TR negative cats. Transcriptome analysis by RNA sequencing identified many differentially expressed genes between TR negative and TR positive teeth from the same cat. I also found that up-regulated genes in TR positive teeth, from the literature, are known to be associated with osteoclast differentiation and calcium signalling pathways. Apart from the genes related to osteoclast biology, I also identified up-regulated genes involved in muscle and tooth development, which suggests TR positive teeth undergo repair or bone formation in response to tooth resorption. Based on RNA sequencing data and quantitative PCR, I was able to identify a list of genes which may be involved in odontoclast dysregulation in TR. Among those, matrix metalloproteinase 9 (MMP9) was of great interest due to its potential role in osteoclast biology. High MMP9 expressing odontoclasts were found in tooth sections undergoing tooth resorption by immunohistochemistry. In functional studies, MMP9 inhibitor reduced both osteoclast formation and resorption activity in vitro. Furthermore, feline MMP9 siRNA inhibited osteoclast differentiation but showed little effect on dentine resorption activity. These results confirm the hypothesis that the transcriptome of TR positive teeth was locally different from TR negative teeth. The results revealed an upregulation of genes in pathways associated with osteoclast formation in TR positive teeth. Further testing of candidate genes as potential therapeutic targets in feline TR could be performed using the feline tooth resorption model system developed in this study.