Human umbilical vein endothelial cells synergize osteo/odontogenic differentiation of periodontal ligament stem cells in 3D cell sheets.

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.