HUVECs Synergize Osteo/Odontogenic Differentiation Of PDLSCs In 3D Cell Sheets

Abstract

Objective: To investigate the expression of osteo/odontogenic differentiation markers and vascular network formation in a 3-D cell sheet with varying cell ratios of periodontal ligament stem cells (PDLSCs) and human umbilical vein endothelial cells (HUVECs). Method: Human PDLSCs were isolated and characterized and co-cultured with HUVECs. 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). The expressions of osteo/odontogenic pathway markers ALP, BSP and RUNX2 were analyzed at 3 and 7 days using RT-PCR. Further, ALP protein quantification was performed at 7 and 14 days using alkaline phosphatase assay. The calcium nodule formation was assessed qualitatively and quantitatively by alizarin red assay. Histological evaluations of cell sheet constructs with combinations (PDLSCs-PDLSCs-PDLSCs/PDLSCs-HUVECs-PDLSCs/Co-Culture-Co-culture-Co-culture) were observed with scanning electron microscope (SEM), H&E and immunofluorescence staining. Cell viability was assessed with the live/dead assay. Statistical analysis was performed using 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, the expression of BSP and RUNX2 genes was higher at 7days compared to 3days. Significant calcium mineralization was detected as 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). SEM, H&E and CD31 immuno-staining exemplified the development of a layered cell sheet structure with endothelial cell islands within the constructed PDLSCs-HUVECs-PDLSCs and co-culture groups. HUVECs invaded the layered cell sheet, suggestive of rudimentary vascular network initiation. After 7 days, majority of the cells were viable in all 3 groups.

Conclusion: PDLSCs-HUVECs co-culture cell sheet model exhibits significantly high levels of osteo/odontogenic markers with signs of initial vascular 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: This work is supported by seed funding programme for Basic Research,The University of Hong Kong. 201111159191