Osman, Zul Faizuddin
(2021)
Signalling pathways involved in IL-8 mediated odontogenic differentiation of shed cultured on human amniotic membrane with BMP-2.
PhD thesis, Universiti Sains Malaysia.
Abstract
Current knowledge about the treatment modalities using amniotic membrane
(AM) scaffold for dental regeneration is still limited. It has been previously shown
that bone morphogenetic protein-2 (BMP-2) growth factor assisted stem cells from
human exfoliated deciduous teeth (SHED) differentiation into odontoblast-like cells
via activation of interleukin-8 (IL-8) cytokine. Nevertheless, the fundamental
biology of IL-8 mediation in the process is yet to be understood. This study aims at
finding the roles and mechanisms of IL-8 immunomodulatory pathway during
odontoblast differentiation of SHED. In this study, SHED was cultured on AM
scaffold with BMP-2 treatment. Flow cytometry analysis, multiplex polymerase
chain reaction (PCR), and Western blot were conducted to determine the expression
of stem cell markers, the expression of inflammatory cytokines genes, the protein
expression of odontoblast markers, and molecules involved in the IL-8
immunomodulatory pathways. Finally, the ultrastructure of SHED and odontoblastlike
cells were also investigated via scanning electron microscopy (SEM) imaging.
The results presented in this thesis showed the potential of SHED to differentiate into
odontoblast-like cells on AM scaffold. SHED was characterised, and exhibited
expression profiles of various stem cell markers for the presence of mesenchymal
stem cell (MSC) markers (cluster of differentiation) CD44, CD73, CD90, CD105 and
the absence of hematopoietic markers CD34 and CD45 using flow cytometry analysis. The results also indicated that SHED revealed strong expression of MSC
surface markers CD90, CD44 and CD73 with 100% expression while CD105 has
84.1% expression. Under stimulation with BMP-2, it was demonstrated that proinflammatory
cytokines namely IL-8, IL-6, IL-1β, TGF-β and TNF-α were
expressed. Successful differentiation of SHED into odontoblast-like cells was
demonstrated by the expression of odontoblast markers such as dentin
sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), alkaline phosphatase
(ALP) and osteopontin (OPN). Exogenous IL-8 showed to enhance the expression of
odontogenic markers of DSPP, DMP1 and ALP. During the differentiation of SHED,
high expression of IL-8 protein stimulated the cascades of signalling pathway of
PI3K/Akt/NF-κB. This pathway is known to be responsible for regulating the
biological process of cell survival, proliferation and differentiation. Visualisation of
SHED ultrastructure further confirmed the differentiation process of SHED into
odontoblast-like cells. Emerging of phenotypic characteristics of odontoblast-like
cell with a columnar cell body and several processes as well as strong cell induced
mineralisation on the cell body suggesting morphology of mature odontoblast. In
conclusion, this study confirmed for the first time that complete differentiation
process of SHED into odontoblast-like cells occur with BMP-2 stimulation within 14
days via IL-8 signalling pathways mediation.
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