Synergistic effects of PDL stem cells/HUVECs in 3D cell sheet

Abstract

Objective: To investigate the expression of odontogenic differentiation markers and vascular network formation in 3-D cell sheet with different cell ratios of periodontal ligament stem cells (PDLSCs) and human umbilical vein endothelial cells (HUVECs).

Method:

Human PDLSCs were isolated and characterized by flow-cytometry, and HUVECs were used for construction of cell sheet. Both types of cells were seeded on temperature responsive culture dishes with PDLSCs alone, HUVECs alone, and various ratios of these cells (1:1, 2:1, 5:1 and 1:5) to obtain confluent cell sheet layers. The expression of odontogenic pathway markers including ALP, BSP, RUNX2 were analyzed at 3 and 7 days using RT-PCR. Further ALP protein quantification was done at 7 and 14days using alkaline phosphatase 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 (PDLSCs-PDLSCs-PDLSCs/PDLSCs-HUVECs-PDLSCs/Co-Culture 2:1 PDLSCs-HUVECs) were performed with H&E and immunofluorescence straining. Statistical analysis was done by t-test(p<0.05).

Result:

Significantly higher ALP gene expression was observed at 3days in 1:1 (PDLSCs:HUVECs) (2.52±0.67) and 5:1(4.05±1.07) co-culture groups compared with other groups(p<0.05), being consistent with ALP protein quantification. However, BSP and RUNX2 genes expressions were higher at 7days compared to that detected at 3 days. Significant calcium mineralization was detected and quantified by Alizarin red assay at 14days in 1:1(1323.55±6.54µm) and 5:1(994.67±4.15µm) co-cultures as compared with mono-culture cell sheets(p<0.05). H&E and CD31 immuno-staining illustrated the development of a layered cell sheet structure with endothelial cell islands within the constructed PDLSCs-HUVECs-PDLSCs and co-culture groups. Furthermore, HUVECs permeated into layered cell sheet, was suggestive of rudimentary vascular network initiation.

Conclusion: This study suggests that PDLSCs-HUVECs co-culture models exhibit significantly high levels of odontogenic markers with signs of initial vascualar formation. This novel 3-D cell sheet-based approach may be potentially beneficial for periodontal regenerative therapy.

This abstract is based on research that was funded entirely or partially by an outside source: Seed Funding Programme for Basic Research, The University of Hong Kong. 201111159191