Abstract
The apical migration of junctional epithelium is a key event in the progression of periodontitis. In addition one of the key factors which prevents periodontal regeneration is the downgrowth of the gingival epithelium during healing. There is evidence that the growth of the epithelium is controlled by its underlying connective tissue and that the superficial connective tissue (SCT) supports epithelial growth and differentiation, whilst the deeper connective tissues (DCT) does not support epithelial growth. However, differences between these apparently similar connective tissues have not been defined and the factors which they produce to control the epithelial growth are not known. Therefore, the aims of this PhD project were to characterise differences between gingival fibroblasts from SCTs and periodontal ligament fibroblasts from DCTs and to investigate the molecular mechanisms by which DCTs may prevent epithelial downgrowth.To investigate fibroblast phenotypes within the dentogingival tissues a panel of markers known to differentiate dermal fibroblasts were tested on mouse dentogingival tissues by immunohistochemistry. The results identified Sca1 expression as a key marker which differentiates gingival fibroblasts from periodontal ligament fibroblasts. Further characterisation using Periostin and Asporin confirmed that these extracellular matrix markers were expressed in the periodontal ligament tissues only.
The effects of these different fibroblast subsets in determining epithelial phenotype were subsequently studied through the construction of 3D organotypic cultures which consisted of H400 epithelial cells overlying type I collagen gel embedded with either human periodontal ligament fibroblasts (HPDLF) or human gingival fibroblasts (HGF). The results showed that that HGF consistently resulted in the formation of a multi-layered epithelium whilst HPDLF did not support epithelial growth.
Further characterisation of the epithelial phenotype was carried out following transfer of HGF constructs onto HPDLF collagen matrices and measuring the extent of epithelial growth (migration and proliferation) onto the HPDLF collagen bed. The recombination uniquely models the anatomical relationships within the dentogingival junction. The data showed that there was a lack of epithelial migration and proliferation following contact with HPDLF. Additionally, epithelial phenotype was characterised via multiphoton fluorescence microscopy in response to wounding of HGF and HPDLF constructs using a multiphoton laser. The data showed that HGF constructs had the greatest increase in cell numbers and fastest rate of wound closure over 24 hours. Taken together, these characterisation studies suggest that only HGF supported epithelial migration and proliferation, whereas there was a lack of both cellular processes following epithelial contact with HPDLF.
Lastly, the study suggests the involvement of Wnt signalling in the fibroblast regulation of dentogingival epithelia. The mRNA expression of candidate Wnt agonists and antagonists were assessed via qPCR for their differential expression in HGF and HPDLF. In particular, the Wnt antagonist SFRP4 was identified as being abundantly expressed in HPDLF, where the expression of HPDLF was 32 times that of HGF. Transient gene silencing of SFRP4 by siRNA in HPDLF constructs showed that a downregulation of SFRP4 permits epithelial growth. This suggests that a downregulation of Wnt signalling would prevent the apical migration of the junctional epithelium thereby promoting periodontal health.
The findings from this PhD study support the role of specific fibroblasts populations in the regulation of different epithelial phenotypes. In addition, the study suggests a molecular basis for the regulation of the junctional epithelium.
| Date of Award | 1 May 2019 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Francis Hughes (Supervisor) & Mandeep Ghuman (Supervisor) |