Characterisation and Regulation of the Tooth-Bone Interface during Tooth Development

Student thesis: Doctoral ThesisDoctor of Philosophy


The tooth is closely related to the periodontium in which it sits, with a soft tissue interface forming between the alveolar bone and hard tissues of the tooth. This is known as the tooth-bone interface (TBI). In functional teeth, the TBI houses the periodontal ligament, while during development the TBI creates a space into which the tooth can grow. This project aims to provide an understanding of how the formation of the tooth and bone are coordinated during development and characterise the underlying factors and mechanisms that prevent bone formation and invasion at the interface between the tooth and bone.
Using murine mandibular first molar (M1) TRAP stained histological sections, osteoclasts were found to be closely associated with the border of the developing bone, lining the TBI, but not within the TBI itself. Slice culture was used to follow tooth development in explant culture as it provided an excellent opportunity for manipulation and lineage tracing. DiI labelling experiments showed the contribution of two sources of cells in the formation of alveolar bone namely, dental follicle cells from around the tooth and, bone cells from the margins of the dentary. Isolation experiments were used to investigate the impact of the tooth on the bone and bone on the tooth. Isolation of E14.5 mandibular first molar (M1) tooth germ from the surrounding mesenchyme and alveolar bone resulted in tooth germ expansion while removing the tooth epithelium did not change the normal layout of osteoclasts at E14.5. The effect of manipulating the BMP signalling pathway on the differentiation of cells in the TBI during tooth development was studied. A local reduction in the TBI was observed next to the BMP-4 beads whereas a local widening in the TBI was observed when Noggin beads were implanted. The effect of manipulating the RANK-RANKL signalling pathway was investigated next. In situ hybridisation revealed the presence of OPG, RANK, and RANKL in the alveolar bone but OPG and RANKL only in the dental epithelium. Addition of exogenous RANKL to tooth explants in culture resulted in a statistically significant increase in osteoclast numbers and a widening of the TBI. On the other hand, the results obtained after exogenous OPG addition were regarded as inconsistent due to high variability. However, correlation of the difference in bone growth within a cultured tooth germ with the presence of osteoclasts showed absence of osteoclasts in areas of bone encroachment and the opposite, presence of osteoclasts, in areas devoid of bone. The TBI then was analysed in c-Fos mutants, a knockout mouse known to have a defect in osteoclastogenesis, resulting in lack of osteoclast production. Genotyping showed that the c-Fos mutant embryos were displaying the expected Mendelian ratio, but almost all the homozygotes died after birth, and the heterozygotes viability was found to be compromised. Micro-CT analysis of a 3 week old c-Fos homozygote showed a strong osteopetrotic phenotype. Defects in the midline diastema, tooth impaction, and lack of roots were also observed. The TBI showed signs of bone invasion, encroaching on the M1. TRAP assay revealed few positive-stained mononucleated cells, which were probably macrophages.
In conclusion this thesis demonstrates that the formation and maintenance of the TBI appears to be a finely regulated two-pronged process with control of osteoclast differentiation used to remove the bone (osteoclastogenesis), combined with inhibition of bone induction (osteogenesis). Together these two processes create a bone-free zone around the tooth. By changing either of these processes the TBI is disrupted and tooth development is altered.
Date of Award2018
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorAbigail Tucker (Supervisor) & Fraser McDonald (Supervisor)

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