Abstract
Blood vessels are essential for efficient gas exchange, providing nutrition and removing cellular metabolites. A stable vasculature is essential for the optimum functioning of a tooth and for maintaining the pulp's vitality. The vasculature participates in regulating tooth development and delivering vital growth factors to the dental apparatus. The function of blood vessels goes beyond gas exchange as studies have discovered newer roles of endothelial cells in providing patterning information during organogenesis. To improve current knowledge about molar vascularisation, the project aims to 1) Understand the blood vessel migration during embryonic and post-natal tooth development, 2) Identify molecular signals that are involved in regulating endothelial cell migration during molar formation, 3) Study the function of endothelial cells in providing morphogenetic cues during tooth development. Maintenance of a viable vasculature in a bioengineered tooth is a clinical hindrance in the field of Regenerative Dentistry. The project will provide key information to help design effective revascularization therapies for bio-engineered teeth.Chapter 3 investigates the spatiotemporal distribution of the vasculature during murine molar development. Endothelial cell surface markers were used to identify endothelial cell expression at different stages of tooth development. The vasculature analysis starts at bud stage E13.5 and continues till post-natal day 2. Endothelial cells surround the dental tooth bud and migrate into the dental mesenchyme at E14.5. Interestingly, the central part of the dental mesenchyme is
devoid of vasculature. I show that within a short period, by E15.5, abundant endothelial cells colonize the dental mesenchyme and appear to target the presumptive cusp tips. A combination of angiogenesis and vasculogenesis was observed during odontogenesis to form the blood vessel network during tooth development. Importantly, non-differentiated mesoderm-derived cells were responsible for the vascularisation of the dental papilla shown by the Mesp1cre/tdtomato mice model. During the late bell stage morpho-differentiation of the inner enamel epithelium initiates where cells become tall/columnar. Notably, endothelial cells appeared to be attracted to thinner regions of the inner enamel epithelium and maintained a distance from thicker dental epithelium. Blood vessel migration through the dental mesenchyme and outer enamel epithelium was conserved across mice and humans. Mice and human endothelial cells expressed similar markers such as CD31, CD34 and endomucin.
During late bell stage disruption in laminin expression and tight junction loss facilitated blood vessel invasion into the stellate reticulum. Endothelial cells moved in and perforated the stratum intermedium layer while blood vessels from the mesenchyme traversed the newly differentiated odontoblast layer.
Chapter 4 identifies the molecular signalling cues that regulate endothelial cell migration during tooth development. To answer this, we utilized small molecule Wnt inhibitor to conduct loss of function experiments. Inducing genetic deletions to study the function of individual genes through Cre-LoxP mediated recombination makes the mouse an excellent model system for studying developmental processes. To target the Wnt pathway, we utilized K14creWntless knock-out mice models. The downregulation of endothelial cells and failure to undergo vascular remodelling suggested that Wnts participate in regulating vascularisation during tooth development. Furthemore, downregulation of Vegf in the K14creWntless mutants suggested that Wnts maintain Vegf expression during odontogenesis. The project uses the K14creFgfr2fl/fl model to investigate the relationship between Fgfs and Vegf during vascularisation of the molar. Lef-1 activity in the mutant inner enamel epithelium and residual Vegf signalling appeared to be sufficient to drive blood vessels inside the dental mesenchyme.
Chapter 5 further investigates molecular regulation of blood vessel through the dental epithelium during late bell stage of tooth development. For this aim we analysed the K14creSmoothenedfl/fl mice model and studied the epithelial changes that might be linked to blood vessel invasion through epithelium. Interestingly, in the absence of Hh signalling in the epithelium blood vessels failed to move in due to the outer enamel epithelium remaining intact.
Chapter 6 explores the role of endothelial cells in controlling murine molar patterning. The project uses a small molecule Vegf pathway inhibitor SU5416. Blocking Vegf caused epithelial disruption and failure of odontoblast and ameloblast differentiation. To continue the in-vivo investigations, the genetic knock-out model Cdh5creVegfr2fl/fl was used. Genetic ablation of Vegfr2 from Cdh5+ve endothelial cells disrupted third molar morphogenesis. Ameloblast and odontoblast differentiation was blocked, confirmed by a lack of Amelogenin and Dspp expression, respectively.
| Date of Award | 1 Feb 2024 |
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| Original language | English |
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| Supervisor | Abigail Tucker (Supervisor) & Maisa Seppala (Supervisor) |