Comparison of the properties of human CD146+ and CD146- periodontal ligament cells in response to stimulation with tumour necrosis factor α

Abstract

Objectives: Periodontal ligament stem cells (PDLSCs) can be used in periodontal regeneration. Tumour necrosis factor-alpha (TNF-α) participates in the regulation of cell proliferation, apoptosis, differentiation, and migration. However, whether TNF-α can affect the biological features of PDLSCs is still unclear. The objective of this study was to illustrate the biological effects (proliferation, apoptosis, osteogenesis and migration) of TNF-α on human CD146 positive periodontal ligament cells (CD146+PLDCs) and CD146 negative periodontal ligament cells (CD146-PDLCs).

Methods: CD146±PDLCs were isolated from human PDLCs and analyzed using a fluorescence-activated cell sorter. The biological effects of TNF-α on CD146±PDLCs were evaluated by CCK-8 assay (proliferation), DAPI staining (apoptosis), alizarin red staining and alkaline phosphatase activities assay (osteogenesis), and wounding assay and transwell assay (migration).

Results: CD146+PDLCs, which expressed MSC surface markers CD105, CD90, CD73, CD44, and Stro-1, showed higher proliferative and osteogenic potential than CD146-PDLCs. TNF-α at a dose of 2.5ng/ml was found to enhance both proliferation and osteogenesis in CD146+PDLCs. At 5ng/ml, TNF-α promoted proliferation, osteogenesis, and apoptosis in CD146+PDLCs and enhanced osteogenesis in CD146-PDLCs. At 10ng/ml, TNF-α only aggravated apoptosis in CD146+PDLCs. The migratory ability of both CD146+PDLCs and CD146-PDLCs was not altered by TNF-α.

Conclusions: CD146+PDLCs were subpopulation of MSC. It showed greater proliferative and osteogenic potential than CD146-PDLCs. At low concentration, TNF-α was beneficial to CD146+PDLCs on proliferation and osteogenesis, and at high concentration it was detrimental. CD146-PDLCs were found to be less sensitive to TNF-α.

Keywords: 3,3′-diaminobenzidine; 4′,6-diamidino-2-phenylindole; AP-1; Cell movement; Cell survival; DAB; DAPI; FADD; FAK; Fas-associated protein with death domain; GCF; MSC; NF-κB; Osteogenesis; PDLCs; PDLSCs; Periodontal ligament cells; TNF receptor type 1-associated death domain protein; TNF receptor-associated factor 2; TNF-α; TNF-α receptor; TNFR; TRADD; TRAF2; Tumour necrosis factor-alpha; VCAM-1; activator protein-1; cFLIP; cIAPs; cellular FADD-like interleukin-1 beta-converting enzyme inhibitory protein; cellular inhibitor of apoptosis; focal adhesion kinase; gingival crevicular fluid; mTNF; membranous TNF-α; mesenchymal stem cells; nuclear factor kappa B; periodontal ligament cells; periodontal ligament stem cells; sTNF; soluble TNF-α; tumour necrosis factor-α; vascular cell adhesion protein 1.