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.
Abstract
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.
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