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Human umbilical vein endothelial cells synergize osteo/odontogenic differentiation of periodontal ligament stem cells in 3 D cell sheets
Author(s) -
Prgeeth Pandula P. K. C.,
Samaranayake L. P.,
Jin L. J.,
Zhang C. F.
Publication year - 2014
Publication title -
journal of periodontal research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.31
H-Index - 83
eISSN - 1600-0765
pISSN - 0022-3484
DOI - 10.1111/jre.12107
Subject(s) - periodontal ligament stem cells , bone sialoprotein , runx2 , chemistry , umbilical vein , stem cell , alkaline phosphatase , microbiology and biotechnology , periodontal fiber , pathology , biology , medicine , dentistry , biochemistry , in vitro , osteocalcin , enzyme
Background and Objective To investigate the expression of osteo/odontogenic differentiation markers and vascular network formation in a 3D cell sheet with varying cell ratios of periodontal ligament stem cells ( PDLSC s) and human umbilical vein endothelial cells ( HUVEC s). Material and Methods Human PDLSC s were isolated and characterized by flow cytometry, and co‐cultured with HUVEC s for the construction of cell sheets. Both types of cells were seeded on temperature‐responsive culture dishes with PDLSC s alone, HUVEC s alone and various ratios of the latter cells (1 : 1, 2 : 1, 5 : 1 and 1 : 5) to obtain confluent cell sheets. The expressions of osteo/odontogenic pathway markers, including alkaline phosphatase (ALP), bone sialoprotein (BSP) and runt‐related transcription factor 2 (RUNX2) , were analyzed at 3 and 7 d using RT ‐ PCR . Further ALP protein quantification was performed at 7 and 14 d using ALP assay. The calcium nodule formation was assessed qualitatively and quantitatively by alizarin red assay. Histological evaluations of three cell sheet constructs treated with different combinations ( PDLSC – PDLSC – PDLSC / PDLSC – HUVEC – PDLSC /co‐culture–co‐culture–co‐culture) were performed with hematoxylin and eosin and immunofluorescence staining. Statistical analysis was performed using t ‐test ( p < 0.05). Results Significantly higher ALP gene expression was observed at 3 d in 1 : 1 ( PDLSC – HUVEC ) (2.52 ± 0.67) and 5 : 1 (4.05 ± 1.07) co‐culture groups compared with other groups ( p < 0.05); this was consistent with ALP protein quantification. However, the expression of BSP and RUNX2 genes was higher at 7 d compared to 3 d. Significant calcium mineralization was detected as quantified by alizarin red assay at 14 d in 1 : 1 (1323.55 ± 6.54 μm) and 5 : 1 (994.67 ± 4.15 μm) co‐cultures as compared with monoculture cell sheets ( p < 0.05). Hematoxylin and eosin and CD 31 immunostaining clearly exemplified the development of a layered cell sheet structure with endothelial cell islands within the constructed PDLSC – HUVEC – PDLSC and co‐culture groups. Furthermore, HUVEC s invaded the layered cell sheet, suggestive of rudimentary vascular network initiation. Conclusion This study suggests that the PDLSC – HUVEC co‐culture, cell sheet, model exhibits significantly high levels of osteo/odontogenic markers with signs of initial vascular formation. This novel 3D cell sheet‐based approach may be potentially beneficial for periodontal regenerative therapy.