Establishment of 3d oral mucosa model using differentiated stem cells from human exfoliated deciduous teeth

Nor, Nurul Hafizah Mohd (2020) Establishment of 3d oral mucosa model using differentiated stem cells from human exfoliated deciduous teeth. PhD thesis, Universiti Sains Malaysia.

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Oral mucosa is a specialized type of tissue that lines the oral cavity. It consists of two main layers: stratified squamous epithelium and lamina propria. The epithelial layer is resided by the epithelial cells, while the lamina propria layer is majorly occupied by fibroblasts. As far as the in vitro oral mucosa is concerned, the construction of an oral mucosa model should be performed in full thickness architecture using both cells mentioned. Therefore, the present study aimed to differentiate stem cells from human exfoliated deciduous teeth (SHED) into fibroblastand epithelial-like cells to be subsequently used in the establishment of a 3D oral mucosa model. The differentiation of SHED was carried out by the involvement of growth factors, namely connective tissue growth factor (CTGF) for fibroblastic differentiation, whereas keratinocyte growth factor (KGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and insulin-like growth factor-2 (IGF-II) were employed in epithelial differentiation, respectively. The characterisation of the induced cells was done by morphological observation, proliferation rate, gene and protein expression analyses using semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR), immunofluorescence staining and flow cytometry. The collagen-glycosaminoglycan-chitosan (CGC) scaffold was constructed by combining collagen/chitosan/chondroitin sulphate/hyaluronic acid (100/12/5/1) thoroughly. The porous scaffold produced was characterized via their structural integrity, porosity, and density. The characterized differentiated cells were then co-cultured on CGC scaffold to generate a 3D oral mucosa model, which was later characterized via histological and immunofluorescence analyses. The results demonstrated the inductive effect of growth factors in both fibroblastic and epithelial differentiation of SHED. SHED derived-fibroblast-like cells are morphologically similar to SHED, while SHED derived-epithelial-like cells resembled native epithelial cells. Statistical analysis using one-way ANOVA of the proliferation assay showed a significant correlation (p<0.05) between the induced cells and growth factors involved. There were significant differences in gene and protein expressions between SHED and both differentiated cells. A white, porous lyophilized CGC scaffold produced was able to maintain its structural integrity and did not degrade throughout the whole experiments. The scaffold also exhibited good porosity and density. The co-culture system showed that the fibroblast- and epithelial-like cells derived from SHED were able to attach and proliferate when being seeded on CGC scaffold. The haematoxylin and eosin (H&E) staining of the established oral mucosa model also exhibited the infiltration and stratification of the fibroblast- and epithelial-like cells in some regions within CGC scaffolds. Also, the production of collagen could be observed via the Masson Trichrome staining. The immunofluorescence staining of the epithelial-like cells grown in the CGC scaffold also supported the presence of those cells. These findings hence provide a new understanding on the potential of SHED in the establishment of oral mucosa model for dental tissue regeneration.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Oral mucosa
Subjects: R Medicine > RK Dentistry
Divisions: Kampus Kesihatan (Health Campus) > Pusat Pengajian Sains Pergigian (School of Dental Sciences) > Thesis
Depositing User: Mr Abdul Hadi Mohammad
Date Deposited: 18 Nov 2020 03:28
Last Modified: 18 Nov 2020 03:28

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