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- Title
Human umbilical vein endothelial cells synergize osteo/odontogenic differentiation of periodontal ligament stem cells in 3 D cell sheets.
- Authors
Prgeeth Pandula, P. K. C.; Samaranayake, L. P.; Jin, L. J.; Zhang, C. F.
- Abstract
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 ( PDLSCs) and human umbilical vein endothelial cells ( HUVECs). Material and Methods Human PDLSCs were isolated and characterized by flow cytometry, and co-cultured with HUVECs for the construction of cell sheets. Both types of cells were seeded on temperature-responsive culture dishes with PDLSCs alone, HUVECs 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 CD31 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, HUVECs 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.
- Subjects
CHINA; STEM cells; THERAPEUTIC use of biochemical markers; CELL differentiation; PERIODONTAL ligament; ACADEMIC medical centers; FLOW cytometry; NEOVASCULARIZATION; POLYMERASE chain reaction; T-test (Statistics); REVERSE transcriptase polymerase chain reaction; UMBILICAL veins; PHYSIOLOGY
- Publication
Journal of Periodontal Research, 2014, Vol 49, Issue 3, p299
- ISSN
0022-3484
- Publication type
Article
- DOI
10.1111/jre.12107