Intranuclear Delivery of Nuclear Factor-Kappa B p65 in a Rat Model of Tooth Replantation

Abstract

After avulsion and replantation, teeth are at risk of bone and root resorption. The present study aimed to demonstrate that the intra-nuclear transducible form of transcription modulation domain of p65 (nt-p65-TMD) can suppress osteoclast differentiation in vitro, and reduce bone resorption in a rat model of tooth replantation. Cell viability and nitric oxide release were evaluated in RAW264.7 cells using CCK-8 assay and Griess reaction kit. Osteoclast differentiation was evaluated using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and tartrate-resistant acid phosphatase (TRAP) staining. Thirty-two maxillary rat molars were extracted and stored in saline (n = 10) or 10 µM nt-p65-TMD solution (n = 22) before replantation. After 4 weeks, specimens were scored according to the inflammatory pattern using micro-computed tomography (CT) imaging and histological analyses. nt-p65-TMD treatment resulted in significant reduction of nitric oxide release and osteoclast differentiation as studied using PCR and TRAP staining. Further, micro-CT analysis revealed a significant decrease in bone resorption in the nt-p65-TMD treatment group (p < 0.05). Histological analysis of nt-p65-TMD treatment group showed that not only bone and root resorption, but also inflammation of the periodontal ligament and epithelial insertion was significantly reduced. These findings suggest that nt-p65-TMD has the unique capabilities of regulating bone remodeling after tooth replantation.

Keywords: intra-nucleus delivery; nuclear factor kappa B; osteoclast; tooth replantation.

Figure 1

(A) Structure of nt-p65-TMD. (B) Transduction efficiency of nt-p65-TMD. Dose-dependent intracellular transduction efficiency of nt-p65-TMD into RAW264.7 cells. nt-p65-TMD was detected by immunoblot analysis with anti-FLAG antibody. β-actin was used as loading control. (C) Cell viability of nt-p65-TMD in RAW264.7 cells. Cells were treated with different concentrations of nt-p65-TMD for 24 h, and cell viability was assessed using CCK-8 assay kit. LY294002 treatment groups were considered positive controls. The data are expressed as mean ± standard deviation. Cell viability differed significantly for the 2 and 5 µM nt-p65-TMD treatment groups compared to the control group. Data show mean ± standard deviation values of three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared to the control value (100%). (D) NO release of nt-p65-TMD in RAW264.7 cells. Cells were incubated in the presence of different concentrations of nt-p65-TMD and 0.1 µg/mL LPS for 20 h. Then, the culture supernatant was analyzed for NO. Data show mean ± standard deviation values of three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared to LPS-stimulation value (100%).

Figure 2

Osteoclast differentiation of nt-p65-TMD in RAW264.7 cells (A–F) tartrate-resistant acid phosphatase (TRAP) staining of nt-p65-TMD in RAW264.7 cells. Cells were pretreated with different concentrations of nt-p65-TMD for 2 h followed by treatment with 10 ng/mL receptor activator of nuclear factor-kB ligand (RANKL) for 3 days. Subsequently, RAW264.7 cells were fixed and stained to detect TRAP. The osteoclasts were stained red. Scale bars = 50 µm (G–J) Changes in the expression of the osteoclast-related genes of nt-p65-TMD in RAW264.7 cells. Cells were pretreated with different concentrations of nt-p65-TMD for 2 h followed by treatment with 10 ng/mL RANKL for 3 days. RNA was isolated from RAW264.7 cells and cDNA was synthesized. Expression of tartrate-resistant acid phosphatase (TRAP), TNF receptor associated factor 6 (TRAF6), CathepsinK (CTCK), and nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) was evaluated using quantitative RT-PCR relative to RANKL treatment group (normalized to 100%). Data were obtained from five separate experiments, with all samples run in duplicate. The data are expressed as mean ± standard deviation values. The expression of the genes differed significantly; t-test and Mann–Whitney U test, * p < 0.05, ** p < 0.01.

Figure 3

Two-dimensional horizontal microcomputed tomography images of rat maxillary first molars 4 weeks after replantation. (A,D) Control: The teeth without extraction and replantation. Both bone and root resorption was not observed. (B,E) Saline group: 0.9% physiological saline was applied to the extracted teeth surface and alveolar socket. Severe, extensive bone (white asterisk) and root resorption (arrow) were observed. (C,F) nt-p65-TMD group: 10 µM p65-TMD solution was applied to the surface of the extracted teeth and alveolar socket. A relatively short range of bone resorption (white asterisk) was observed compared to that in the saline group. (G–J) Evaluated scores, represented as mean ± standard deviation, for (G) bone resorption, (H) root resorption, (I) ankylosis, and (J) pulp mineralization in the saline and nt-p65-TMD groups. Based on the scores of the four categories, only bone resorption showed a significant decrease in the nt-p65-TMD group; Mann–Whitney U test, * p < 0.05.

Figure 4

Histological analysis of replanted rat maxillary first molars 4 weeks after replantation. (A,D) Control group: The teeth without extraction and replantation. Inflammatory root resorption and bone resorption was not observed. (B,E) Saline group: The extracted teeth were replanted after storing in 0.9% physiological saline for 5 min. A large number of inflammatory cells were observed, and the apical third was lost by severe inflammatory root resorption. (C,F) nt-p65-TMD group: Localized root resorption and partial recovery of periodontal ligament and fibroblasts were observed (G–J) Evaluated scored, expressed as mean ± standard deviation, for (G) bone resorption, (H) root resorption, (I) inflammation at the epithelial insertion, and (J) inflammation in the periodontal ligament in the saline and nt-p65-TMD groups. Hematoxylin-eosin staining, Scale bars = 1000 µm (applies to A–C) and 500 µm (applies to D–F); Mann–Whitney U test, * p < 0.05, ** p < 0.01 (applies to G–J).