Regulation of regenerative periodontal healing by NAMPT

Abstract

Periodontitis is an inflammatory disease characterized by destruction of the tooth-supporting tissues. Obese individuals have an increased risk of periodontitis, and elevated circulating levels of nicotinamide phosphoribosyltransferase (NAMPT) may be a pathomechanistic link between both diseases. Recently, increased levels of NAMPT have also been found in patients with periodontitis, irrespective of the presence of obesity. This in vitro study sought to examine the effects of NAMPT on the regenerative capacity of human periodontal ligament (PDL) cells and, thereby, periodontal healing. PDL cells treated with enamel matrix derivative (EMD), which was used to mimic regenerative healing conditions in vitro, were grown in the presence and absence of NAMPT for up to 14 d. EMD stimulated significantly (P < 0.05) the expression of growth factors and their receptors, matrix molecules, osteogenesis-associated factors, and wound closure and calcium accumulation. In the presence of NAMPT, all these stimulatory effects were significantly (P < 0.05) reduced. In conclusion, the beneficial effects of EMD on a number of PDL cell functions critical for periodontal regeneration are counteracted by NAMPT. Enhanced levels of NAMPT, as found in obesity and periodontal inflammation, may compromise the regenerative capacity of PDL cells and, thereby, periodontal healing in the presence of EMD.

Figure 1

Study design flowchart. EMD: enamel matrix derivative; NAMPT: nicotinamide phosphoribosyltransferase; SMAD: sma- and mad-related protein; WB: western blot; IF: immunofluorescence; VEGF: vascular endothelial growth factor; TGFβ1: transforming growth factor β1; COL1: collagen type I; POSTN: periostin; RUNX2: runt-related transcription factor 2; BMPR1A, 1B, or 2: bone morphogenetic protein receptor 1A, 1B, or 2; TGFβR2: TGFβ receptor 2; qPCR: quantitative polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay.

Figure 2

Effect of EMD on the VEGF (a), TGFβ1 (b), COL1 (e), and POSTN (f) mRNA expressions in the presence and absence of NAMPT (100 ng/mL) at 1 d and 3 d. Untreated cells served as control. All experiments were performed in triplicate and repeated at least twice. Mean ± SEM (n = 18); *significant (P < 0.05) difference between groups. Effect of various concentrations (0, 30, 100, and 300 ng/mL) of NAMPT on the VEGF (c) and TGFβ1 (d) mRNA expressions in EMD-treated cells at 1 d. All experiments were performed in triplicate and repeated at least twice. Mean ± SEM (n = 9).

Figure 3

Effect of EMD on the RUNX2 (a) mRNA expression in the presence and absence of NAMPT (100 ng/mL) at 1 d and 3 d. Untreated cells served as control. All experiments were performed in triplicate and repeated at least twice. Mean ± SEM (n = 18); *significant (P < 0.05) difference between groups. Effect of EMD on the calcium accumulation in the presence and absence of NAMPT (100 ng/mL) in cultures of PDL cells ((b) and (c)). Untreated cells served as control. The calcium accumulation was analysed by alizarin red S staining, visualized by microscopy (c), and quantified by elution with cetylpyridinium chloride (b) at 14 d. All experiments were performed in triplicate and repeated at least twice. Mean ± SEM (n = 12); *significant (P < 0.05) difference between groups. Images from one representative donor are shown.

Figure 4

Effect of various concentrations (0, 30, 100, and 300 ng/mL) of NAMPT on the EMD-induced PDL cell wound fill rate (a). The wound closure, that is, the percentage of fill of the initially cell free zones created by wounding, were analysed over 5 d. All experiments were performed in triplicate and repeated at least twice. Mean ± SEM (n = 12); *significantly (P < 0.05) different from EMD-treated cells in the absence of NAMPT. Effect of EMD on the expression of BMPR1A, BMPR1B, BMPR2 and TGFβR2 in the presence and absence of NAMPT (100 ng/mL) at 1 d (b). Untreated cells served as control. All experiments were performed in triplicate and repeated at least twice. Mean ± SEM (n = 18); *significant (P < 0.05) difference between groups.

Figure 5

Effect of EMD on the nuclear translocation of SMAD1/5/8 in the presence and absence of NAMPT (100 ng/mL) at 60 min, as determined by immunofluorescence (a). Untreated cells served as control. Stimulation of SMAD1/5/8 phosphorylation by EMD over 60 min, as analyzed by immunoblotting (b). Effect of EMD on SMAD1/5/8 phosphorylation in the presence and absence of NAMPT (100 ng/mL) at 60 min, as examined by immunoblotting (c). All experiments were performed in triplicate and repeated at least twice. Images and blots from one representative donor are shown.