Application of eGFP to label human periodontal ligament stem cells in periodontal tissue engineering

Abstract Objectives To establish human periodontal ligament stem cells (hPDLSC) with high and stable expression of enhanced green fluorescent protein (eGFP) and to obtain an ideal vector expression system that suitable for gene therapy in periodontal tissue engineering. Methods hPDLSCs were transfected with eGFP for 48 h via different MOI (25, 50, 100, 200 and 400) by lentiviral vector, the transfection efficiency was evaluated by fluorescent microscopy and flow cytometry, and transfected hPDLSCs proliferation was evaluated by MTT. Pluripotent, differentiation capacity and ALP expression status were determined further. Osteoblast-associated genes expressions for osteogenesis were evaluated by quantitative-PCR. In addition, rat molar periodontal fenestration defect model was used for evaluating periodontal tissue engineering. Results The transfection efficiency after 48 h were 44.7%, 60.9%, 71.7%, 85.8%, and 86.9% respectively. There was no significant effect of transfection (at different MOI levels of 25, 50, 100, and 200) on the proliferation of hPDLSCs (designated as eGFP-hPDLSCs) compared with hPDLSCs ( P  > 0.05). However, proliferation of eGFP hPDLSCs at MOI 400 became slower ( P COL-1 , RUNX2 in hPDLSCS were higher than those in eGFP hPDLSCs ( P ALP and OPN in eGFP hPDLSCs were similar to those in hPDLSCs ( P  > 0.05). Newly regenerated bone formation was observed in the defect model used. Conclusions Among the transfection conditions, 48 h transfection at MOI 200 is optimal for labelling hPDLSCs with eGFP in a lentiviral vector. There is no change in capability of the eGFP hPDLSCs osteogenesis. The lentiviral vector with eGFP is an appropriate expression vector system and hPDLSCs are ideal seeding cells for gene therapy in periodontal tissue engineering.