Human Hertwig's Epithelial Root Sheath Cells In Association With Chitosan Scaffold And Tgfβ1 Modulate The Cementoblastic Differentiation Of Stem Cells From Human Extracted Deciduous Teeth
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Date
2016-01
Authors
Farea Ali, Manal Abdulrahman
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Destruction of the periodontium as a result of furcal perforation may lead to loss of
the tooth. Regeneration of cementum is essential to allow the reconstruction of the
lost attachment. Multipotent dental pulp stem cells derived from human exfoliated
deciduous teeth (SHED) represent a promising cell source for tissue engineering.
Hertwig's epithelial root sheath (HERS) cells have the ability to form cementum-like
tissue and differentiate into cementoblast-like cells. On the other hand, transforming
growth factor-β1 (TGFβ1) plays a key role in controlling cell proliferation and
differentiation. Chitosan has been chosen as a scaffold in the current study because it
supports cells attachment, proliferation, osteoblastic differentiation and hard tissue
regeneration. To the best of our knowledge, the ability of SHED to differentiate into
cementoblast-like cells has not been investigated in the dental field yet. The aim of
this study was to evaluate the the ability of SHED co-cultured with HERS cells in
presence of chitosan-TGFβ1 to differentiate into cementoblast-like cells. To this end,
HERS cells were isolated using selective digestion method and characterized by
immunofluorescence staining, flow cytometry and sqRT-PCR. Thereafter, SHED
seeded on chitosan scaffold and co-cultured with HERS cells in the presence of
TGFβ1. Eight groups were assigned for the downstream analysis: SHED (S),
SHED+chitosan (SC), SHED+TGFβ1 (ST), SHED+chitosan+TGFβ1 (SCT),
SHED+HERS (SH), SHED+HERS+chitosan (SHC), SHED+HERS+TGFβ1 (SHT),
SHED+HERS+chitosan+TGFβ1 (SHCT). SHED proliferation was assessed by
PrestoBlue assay. Live/dead assay was then performed and SHED attachment to
chitosan scaffold was examined by scanning electron microscope (SEM). For
cemento/osteogenic differentiation analysis, morphological appearance, alkaline
phosphatase (ALP) activity, mineralization behaviour and gene/protein expression of
cemento/osteoblast phenotype of SHED were evaluated. In addition, the
inflammatory response of SHED was analysed. Results of the present study showed
that HERS cells had typical epithelial-like cells morphology and expressed
epithelial-like markers. SHED remained viable and attached well to the chitosan
structure. HERS cells in association with chitosan-TGFβ1 significantly enhanced the
proliferation and cemento/osteogenic differentiation of SHED, which was
demonstrated by high ALP activity, strong mineral deposition and up-regulation of
cementum/bone-related gene and protein expressions (i.e. ALP, collagen type I, bone
sialoprotein, osteocalcin and cementum attachment protein). Low levels of
inflammatory genes expression were detected. In conclusion, HERS cells have been
successfully isolated using selective digestion method. Our co-culture system
confirmed the synergistic effect of HERS cells in a combination with chitosan-
TGFβ1 to induce SHED differentiation along the cemento/osteoblastic lineage; which
possesses a novel therapeutic strategy for endodontic furcation perforation repair and
periodontal tissue engineering.