Characterization and evaluation of ascorbic acid-induced cell sheet formation in human periodontal ligament stem cells: An in vitro study

Objectives: Periodontal ligament-derived stem cells (PDLSCs) are regarded as a viable option for periodontal regeneration using cell sheet technology. The objective of the present in vitro study was to characterize human PDLSCs based on their phenotypic and biological properties and to evaluate the ascorbic acid (AA or vitamin C)-induced cell sheet by analyzing the molecular markers. Methods: PDLSCs were established from premolars, and their morphology, viability, proliferation, phenotypic marker expression, and ability to differentiate into osteocytes and adipocytes were analyzed. PDLSCs were then induced to form cell sheets using 100 mu M AA, and gene expression was examined by real-time polymerase chain reaction. Results: PDLSCs showed fibroblastic morphology with >95% viability. The cells were highly proliferative and positive for surface antigens CD29, CD73, and CD90 but negative for CD34 and CD45. They were capable of differentiating into osteocytes and adipocytes. Induction with 100 tM AA transformed PDLSCs into two-to three-layered cell sheets. There was no significant upregulation in ALP and RUNX2 expression in the AA-induced cell sheet. However, the expression levels of late osteoblast differentiation marker (bone gamma-carboxy glutamate protein); cementogenic markers (cementum attachment protein and CP23), and genes encoding extracellular matrix (ECM) proteins [collagen type 1 alpha 1 and integrin beta 1) were higher in AA-induced cell sheets by PDLSCs. Conclusions: The stimulating effect of AA on cell sheet formation by PDLSCs was confirmed by the expression of typical markers involved in osteogenesis/cementogenesis and ECM secretion, which makes this procedure a prospective option for periodontal tissue regeneration applications. (C) 2021 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.