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
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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.
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