Rat Molars Research Articles

Gan-Lu-Yin (Kanroin), Traditional Chinese Herbal Extracts, Reduces Osteoclast Differentiation In Vitro and Prevents Alveolar Bone Resorption in Rat Experimental Periodontitis

Gan-Lu-Yin (GLY), a traditional Chinese herbal medicine, shows therapeutic effects on periodontitis, but that mechanism is not well known. This study aims to clarify the precise mechanism by investigating the inhibitory effects of GLY extracts on osteoclastogenesis in vitro and on bone resorption in periodontitis in vivo. RAW264.7 cells are cultured with soluble receptor activator of nuclear factor-kappa B (sRANKL) and GLY extracts (0.01–1.0 mg/mL), and stained for tartrate-resistant acid phosphatase (TRAP) to evaluate osteoclast differentiation. Experimental periodontitis is induced by placing a nylon ligature around the second maxillary molar in rats, and rats are administered GLY extracts (60 mg/kg) daily for 20 days. Their maxillae are collected on day 4 and 20, and the levels of alveolar bone resorption and osteoclast differentiation are estimated using micro-computed tomography (CT) and histological analysis, respectively. In RAW264.7 cells, GLY extracts significantly inhibit sRANKL-induced osteoclast differentiation at a concentration of more than 0.05 mg/mL. In experimental periodontitis, administering GLY extracts significantly decreases the number of TRAP-positive osteoclasts in the alveolar bone on day 4, and significantly inhibits the ligature-induced bone resorption on day 20. These results show that GLY extracts suppress bone resorption by inhibiting osteoclast differentiation in experimental periodontitis, suggesting that GLY extracts are potentially useful for oral care in periodontitis.

Effects of epigallocatechin gallate (EGCG) on the biological properties of human dental pulp stem cells and inflammatory pulp tissue

ObjectiveThis study aimed to investigate the effect of epigallocatechin gallate (EGCG) on the proliferation, mineralization, inflammation and hypoxia responses of human dental pulp stem cells (hDPSCs) in vitro and its effect on inflammatory pulp tissue in rats in vivo. DesignThe optimum concentration of EGCG was selected by creating a dose response curve. Expression of odontogenic/osteogenic-related genes and inflammatory cytokines after stimulation with Lipopolysaccharide (LPS) was detected by real-time PCR. Under hypoxic conditions, cell proliferation and expression of reactive oxygen species (ROS) and superoxide dismutase (SOD) were detected.In vivo, the maxillary first molars of SD rats were pulpotomized and stimulated with 5 mg/mL LPS for 30 min. Normal saline and EGCG were used to flush the pulp chamber. After 2 months, samples were removed for micro-CT scanning and HE staining. ResultsCCK-8 assay revealed that 10 μg/mL EGCG had no significant effect on the proliferation of hDPSCs. EGCG inhibited expression of IL-1β, IL-6, and TNF-α. Furthermore, EGCG rescued cell proliferation ability, increased SOD activity and reduced ROS expression under hypoxia.In vivo, reduced inflammatory cell accumulation was observed in the coronal pulp in the EGCG group, while in the control group, diffuse inflammatory cells were observed in the radicular pulp. ConclusionEGCG had no obvious effects on calcified nodule formation but significantly inhibited the inflammatory response of hDPSCs and inhibited apoptosis of hDPSCs caused by hypoxia injury. In vivo, EGCG exerts inhibitory effects on pulp tissue inflammation.

Histomorphometric and immunohistochemical study shows that tricalcium silicate cement associated with zirconium oxide or niobium oxide is a promising material in the periodontal tissue repair of rat molars with perforated pulp chamber floors.

To evaluate the periodontium response to tricalcium silicate (TCS) with zirconium oxide (ZrO2 ) or niobium oxide (Nb2 O5 ) used in the sealing of perforated pulp chamber floors in rat maxillary molars. In eighty rats, the perforations in right maxillary molars were filled with either TCS+ZrO2 , TCS+Nb2 O5 , White MTA (used as a gold standard material) or no repair material was placed (Sham Group, SG); the left molars of SG, were used as controls (CG). Sections of maxillary fragments following 7, 15, 30 and 60days were used to evaluate the volume densities of inflammatory cells (VvIC) and fibroblasts (VvFb), width of the periodontal space, amount of collagen, number of osteoclasts and number of IL-6-immunostained cells. The data were subjected to two-way ANOVA followed by Tukey's test (P≤0.05). At all periods, significant differences in VvIC were not detected among TCS+ZrO2, TCS+Nb2 O5 and MTA groups, which had values significantly lower (P<0.05) than the SG. Significant differences in the number of IL-6-immunolabelled cells were not observed among TCS+ZrO2 , TCS+Nb2 O5 and MTA groups (P>0.05) at 15, 30 and 60days. At 7, 15 and 30days, the number of osteoclast was significantly greater in TCS+ZrO2, TCS+Nb2 O5 and MTA (P<0.05) than in the CG; no significant difference was detected after 60days (P>0.05). The width of the periodontal space and amount of collagen in TCS+ZrO2 and TCS+Nb2 O5 groups were similar to the CG at 30 and 60days while SG specimens had a significant reduction (P<0.05) in the amount of collagen and significant increase (P<0.05) in the width of the periodontal space. TCS+ZrO2 and TCS+Nb2 O5 were associated with periodontium repair since these materials allowed the reestablishment of periodontal space width and collagen formation when used in the filling of uninfected perforations in the pulp chamber floor of maxillary rat molars. Furthermore, the significant reduction in the periodontal space of TCS+ZrO2 and TCS+Nb2 O5 specimens after 60days confirmed that the experimental materials were associated with a more rapid recovery of the injured tissues than MTA.

Hard tissue formation after direct pulp capping with osteostatin and MTA in vivo.

ObjectivesIn recent in vitro study, it was reported that osteostatin (OST) has an odontogenic effect and synergistic effect with mineral trioxide aggregate (MTA) in human dental pulp cells. Therefore, the aim of this study was to evaluate whether OST has a synergistic effect with MTA on hard tissue formation in vivo.Materials and MethodsThirty-two maxillary molars of Spraque-Dawley rats were used in this study. An occlusal cavity was prepared and the exposed pulps were randomly divided into 3 groups: group 1 (control; ProRoot MTA), group 2 (OST 100 μM + ProRoot MTA), group 3 (OST 10 mM + ProRoot MTA). Exposed pulps were capped with each material and cavities were restored with resin modified glass ionomer. The animals were sacrificed after 4 weeks. All harvested teeth were scanned with micro-computed tomography (CT). The samples were prepared and hard tissue formation was evaluated histologically. For immunohistochemical analysis, the specimens were sectioned and incubated with primary antibodies against dentin sialoprotein (DSP).ResultsIn the micro-CT analysis, it is revealed that OST with ProRoot MTA groups showed more mineralized bridge than the control (p < 0.05). In the H&E staining, it is showed that more quantity of the mineralized dentin bridge was formed in the OST with ProRoot MTA group compared to the control (p < 0.05). In all groups, DSP was expressed in newly formed reparative dentin area.ConclusionsOST can be a supplementary pulp capping material when used with MTA to make synergistic effect in hard tissue formation.

Expression of Matrix Metalloproteinases-8 and Myeloperoxidase in Pulp Tissue after Pulpotomy with Calcium Silicate Cements

Objective: To evaluate the pulp tissue of rat molars after pulpotomies with mineral trioxide aggregate (MTA), BiodentineTM (BDT) and calcium hydroxide (CH) mixed with sterile saline solution (24 hours, 72 hours, 7 days and 15 days), through correlating MPO activity with active neutrophils and MMP8 activity with tissue remodeling. Material and Methods: Thirty-eight Wistar rats were randomly distributed into groups (control, I (MTA gray), II (BDT), and III (CH)) and subdivided according to the study period of 24 hours, 72 hours, 7 days or 15 days after pulpotomy. MMP8 activity was assessed through fluorescence technique, and MPO activity was determined using the MPO assay. Results: A gradual decrease of MPO and MM8 activity occurred in the group MTA over the experimental periods (p<0.05). Groups BDT and CH exhibited an increase in the activity at 7 and 15 days (p<0.05). Conclusion: MTA demonstrated a decrease in the values of MPO e MMP8. BDT and CH showed high neutrophilic and collagenase activity over the experimental periods.

Mineral trioxide aggregate-induced AMPK activation stimulates odontoblastic differentiation of human dental pulp cells.

To investigate the role of autophagy in MTA-induced odontoblastic differentiation of human dental pulp cells (HDPCs). In MTA-treated HDPCs, odontoblastic differentiation was assessed based on expression levels of dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP1), alkaline phosphatase activity (ALP) activity by ALP staining and the formation of mineralized nodule by Alizarin red S staining. Expression of microtubule-associated protein 1A/1B-light chain3 (LC3), adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signalling molecules and autophagy-related genes was analysed by Western blot analysis and Acridine orange staining was used to detect autophagic lysosome. For in vivo experiments, tooth cavity preparation models on rat molars were established and the expression of proteins-related odontogenesis and autophagy markers was observed by Immunohistochemistry and Western blot analysis. Kruskal-Wallis with Dunn's multiple comparison was used for statistical analysis. Mineral trioxide aggregate (MTA) promoted odontoblastic differentiation of HDPCs, accompanied by autophagy induction, including formation of autophagic lysosome and cleavage of LC3 to LC3II (P<0.05). Conversely, inhibition of autophagy through 3MA significantly attenuated the expression level of DSPP (P<0.05) and DMP1 (P<0.05) as well as formation of mineralized nodules (P<0.05), indicating the functional significance of autophagy in MTA-induced odontoblastic differentiation. Also, MTA increased the activity of AMPK (P<0.01), whereas inhibition of AMPK by compound C downregulated DSPP (P<0.01) and DMP1 (P<0.05), but increased the phosphorylation of mTOR (P<0.05), p70S6 (P<0.01) and Unc-51-like kinases 1 (ULK1) (ser757) (P<0.01), explaining the involvement of AMPK pathway in MTA-induced odontoblast differentiation. In vivo study, MTA treatment after tooth cavity preparation on rat molars upregulated DMP-1 and DSPP as well as autophagy-related proteins LC3II and p62, and enhanced the phosphorylation of AMPK. MTA induced odontoblastic differentiation and mineralization by modulating autophagy with AMPK activation in HDPCs. Autophagy regulation is a new insight on regenerative endodontic therapy using MTA treatment.

Effect of isotretinoin on induced tooth movement in rats

This study was performed to evaluate the effect of isotretinoin on tooth displacement and tissues related to induced tooth movement (ITM) in rats. Wistar rats were randomly divided into 4 groups: vegetable oil (O; n=40), 7.5mg/kg isotretinoin (I; n=40), vegetable oil+ITM (OM; n=44), and 7.5mg/kg isotretinoin and ITM (IM; n=39). After the daily application of the solutions for 30days, an orthodontic appliance was installed to mesially displace the maxillary first right molar (30cN) of rats in the OM and IM groups. The animals were killed 2, 7, 14, or 21days after placement of the devices. The animals in the O and I groups did not undergo ITM but were killed simultaneously. The animals were examined for tooth displacement, the neoformation of mature collagen, bone and root resorption, the presence of hyalinized areas, and trabecular bone modeling by microcomputed tomography. There was no difference in tooth displacement, the number of osteoclasts, the presence of hyalinized areas, or trabecular bone among the O, I, OM, and IM groups across the periods tested (P>0.05). A lower percentage of mature collagen was found in the IM group than in the OM group on day 7 (P<0.05). A lower frequency of root resorption was found in the IM group than in the OM group on days 2 and 21 (P<0.05). Isotretinoin at 7.5mg/kg decreased root resorption in rats subjected to ITM.

Local Vascularization during Orthodontic Tooth Movement in a Split Mouth Rat Model-A MRI Study.

Orthodontic tooth movement to therapeutically align malpositioned teeth is supposed to impact blood flow in the surrounding tissues. Here, we evaluated actual vascularization in the tension area of the periodontal ligament during experimental tooth movement in rats (N = 8) with magnetic resonance imaging (MRI). We inserted an elastic band between the left upper first and the second rat molar; the right side was not treated and served as control. After four days of tooth movement, we recorded T1-weighted morphologic and dynamic-contrast-enhanced MRI sequences with an animal-specific 7 Tesla MRI to assess of local vascularization. Furthermore, we quantified osteoclasts and monocytes in the periodontal ligament, which are crucial for orthodontic tooth movement, root resorptions as undesirable side effects, as well as the extent of tooth movement using paraffine histology and micro-CT analysis. Data were tested for normal distribution with Shapiro–Wilk tests followed by either a two-tailed paired t-test or a Wilcoxon matched-pairs signed rank test. Significant orthodontic tooth movement was induced within the four days of treatment, as evidenced by increased osteoclast and monocyte activity in the periodontal ligament as well as by µCT analysis. Contrast enhancement was increased at the orthodontically-treated side distally of the moved upper first left molar, indicating increased vascularization at the tension side of the periodontal ligament. Accordingly, we detected reduced time-to-peak and washout rates. Our study using MRI to directly assess local vascularization thus seems to confirm the hypothesis that perfusion is enhanced in tension zones of the periodontal ligament during orthodontic tooth movement.

The effects of calcitonin on post-orthodontic relapse in rats.

ObjectivesTo determine the effects of systematic calcitonin administration on post‐orthodontic relapse in rat model.Material and methodsThis experimental animal model involved 36 male Wister rats. The maxillary right first molars were moved mesially, using a modified orthodontic appliance delivered 50 gm, for 14 days, retained for 4 days and left to relapse for 10 days. The study group was divided into three subgroups in which a single injection of calcitonin (20 IU/Kg), three injections of calcitonin (20 IU/Kg), each every other day, and normal saline were administered subcutaneously after orthodontic tooth movement has finished. The relapse ratio, histomorphometric analysis including osteoblasts, osteoclasts numbers and bone area and immunohistochemical analysis including the expression of receptor activator of nuclear factor kappa Β (RANK), receptor activator of nuclear factor kappa Β ligand (RANKL) and osteoprotegerin (OPG) were measured and assessed.ResultsThe relapse ratio was significantly reduced in the three‐dose calcitonin group (28%) compared to the single dose calcitonin group (34%) and the control group (46%). This was accompanied by a nonsignificant increase in osteoblasts number and bone area in three‐dose calcitonin group and a nonsignificant reduction in osteoclast number. However, the immune histochemical expression of RANK, RANKL and OPG did not show statistically significant difference at the end of relapse period.ConclusionsSystemic administration of three doses of calcitonin may minimize the relapse ratio in experimentally moved rat molars.

The effect of topical application of meloxicam on inflamed dental pulp

Background/purposeEffective regulation of the inflammatory process is essential for pulp repair and regeneration. Meloxicam has anti-inflammatory activity in systemic administration. The purpose of this study is to observe effects of topically applied meloxicam on inflamed pulp and to explore its potential value in the treatment of pulpitis. Materials and methodsThe coronal pulp tissues of rat molars were stimulated with 10 mg/mL lipopolysaccharide (LPS group) and then treated with 500 μmol/L meloxicam (meloxicam group). The untreated pulp tissues were used as the control group. After 3 h of incubation in vitro, the gene expression of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in each group was detected by real-time RT-PCR. The pulp tissues of each group were randomly subcutaneously implanted into nude mice, and 500 μmol/L meloxicam was injected into the subcutaneous pocket of the meloxicam group. Haematoxylin eosin staining, Masson staining and immunohistochemical staining were performed on samples after 3 days and 4 weeks retrieval, respectively. ResultsCompared with the LPS group, the mRNA expression levels of TNF-α and IL-6 of the meloxicam group were significantly reduced in vitro. The inflammatory response and cyclooxygenase-2 expression of the meloxicam group were decreased, and osteodentin-like tissue was generated in the pulp cross section of the meloxicam group in vivo. ConclusionThe topical application of meloxicam inhibits the inflammatory response of inflamed pulp and further promotes the formation of osteodentin-like tissues but fails to induce the formation of the pulp–dentin complex. Topically applied meloxicam has the potential to regulate pulp inflammation.

Impact of remnant healthy pulp and apical tissue on outcomes after simulated regenerative endodontic procedure in rat molars

When regenerative endodontic procedures (REPs) are performed on immature teeth diagnosed with pulp necrosis and apical periodontitis, various healing patterns occur. Furthermore, infected immature teeth with endodontic disorders often exhibit some remnant pulp and apical tissue. Therefore, this study investigated the impact of remnant healthy or fully functional pulp and apical tissue on healing patterns after REPs. Simulated REPs were performed on non-infected immature rat molars with different amounts of remnant pulp and apical tissue. Healing patterns in these teeth were assessed after 28 days. Teeth with 0.81–0.91 mm of remnant pulp healed with pulp-like tissue, dentin, and osteodentin-like dentin-associated mineralized tissue (OSD-DAMT); teeth with 0.60–0.63 mm of remnant pulp healed with pulp-like tissue and OSD-DAMT; teeth with 0.13–0.43 mm of remnant pulp healed with periodontal ligament (PDL)-like tissue, OSD-DAMT, and cementum-like dentin-associated mineralized tissue (CEM-DAMT); and teeth with disorganization of pulp and apical tissues at 0.15–0.38 mm beyond the root apex healed with PDL-like tissue, CEM-DAMT, and intracanal bone (IB). Loss of Hertwig’s epithelial root sheath was observed with IB formation. These results showed that four distinct healing patterns occurred after REPs, depending on the preoperative amount of remnant healthy pulp and apical tissue.

Methodological considerations for a model of endodontic treatment in Wistar rats

The use of correctly designed animal models is a fundamental step prior to clinical trials in humans. Although rats are easy to house and handle, and have molars that resemble those of humans, very few researchers use them as a model for root canal treatment, probably due to their small size and the lack of relevant data necessary to reproduce the model. Our aims were to describe the anatomic and histologic characteristics of the mandibular first molar of the Wistar rat and present a standardised model for its experimental endodontic treatment. Twenty female rats were used. The characteristics of the mesial and distal roots were described histologically and the quality of the results achieved following the treatment protocol presented herein was assessed by means of digital radiographs, microCT and histological sections. The age of 55 days was found to be the most adequate for performing this technique, but we consider the interval of 50 to 60 days to be suitable. Both canals are oval, although in opposite planes, and the furcating-facing walls present the minimum dentine thickness. It was essential to become familiar with these aspects in order to decide upon the most appropriate instrumentation and obturation techniques that would enable replication of this model in basic science research.

Glucagon-Like Peptide-1 Receptor Agonist Liraglutide Ameliorates the Development of Periodontitis

Periodontitis is one of the diabetic complications due to its high morbidity and severity in patients with diabetes. The prevention of periodontitis is especially important in diabetic patients because the relationship between diabetes and periodontitis is bidirectional. Here, we evaluated the impacts of glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on the amelioration of periodontitis. Five-wk-old Male Sprague–Dawley (SD) rats (n = 30) were divided into 3 groups: normal, periodontitis, and periodontitis with liraglutide treatment groups. Periodontitis was induced by ligature around the maxillary second molar in SD rats. Half of the rats were administered liraglutide for 2 weeks. Periodontitis was evaluated by histological staining, gene expressions of inflammatory cytokines in gingiva, and microcomputed tomography. Periodontitis increased inflammatory cell infiltration, macrophage accumulation, and gene expressions of tumor necrosis factor-α and inducible nitric oxide synthase in the gingiva, all of which were ameliorated by liraglutide. Liraglutide decreased M1 macrophages but did not affect M2 macrophages in periodontitis. Moreover, ligature-induced alveolar bone resorption was ameliorated by liraglutide. Liraglutide treatment also reduced osteoclasts on the alveolar bone surface. These results highlight the beyond glucose-lowering effects of liraglutide on the treatment of periodontitis.

System-level analyses of keystone genes required for mammalian tooth development.

When a null mutation of a gene causes a complete developmental arrest, the gene is typically considered essential for life. Yet, in most cases, null mutations have more subtle effects on the phenotype. Here we used the phenotypic severity of mutations as a tool to examine system‐level dynamics of gene expression. We classify genes required for the normal development of the mouse molar into different categories that range from essential to subtle modification of the phenotype. Collectively, we call these the developmental keystone genes. Transcriptome profiling using microarray and RNAseq analyses of patterning stage mouse molars show highly elevated expression levels for genes essential for the progression of tooth development, a result reminiscent of essential genes in single‐cell organisms. Elevated expression levels of progression genes were also detected in developing rat molars, suggesting evolutionary conservation of this system‐level dynamics. Single‐cell RNAseq analyses of developing mouse molars reveal that even though the size of the expression domain, measured in the number of cells, is the main driver of organ‐level expression, progression genes show high cell‐level transcript abundances. Progression genes are also upregulated within their pathways, which themselves are highly expressed. In contrast, a high proportion of the genes required for normal tooth patterning are secreted ligands that are expressed in fewer cells than their receptors and intracellular components. Overall, even though expression patterns of individual genes can be highly different, conserved system‐level principles of gene expression can be detected using phenotypically defined gene categories.

The blockade of kappa opioid receptors exacerbates alveolar bone resorption in rats

ObjectivesBone resorption associated to chronic diseases, such as arthritis and periodontitis, results from exacerbated immuno-inflammatory host response that leads to tissue breakdown. The significance of opioid pathways as endogenous modulators of inflammatory events has already been described. Thus, the aim of this work is to determine whether some of the main three opioid receptors are endogenously activated to prevent bone loss during experimentally-induced alveolar bone resorption. DesignThis study used an experimental model of alveolar bone resorption induced by ligature in rats. A silk thread was placed around the 2nd maxillary molar of male Wistar rats. In the 3rd, 4th and 5th day after ligation the rats received a local injection of different concentrations of opioid antagonists Cyprodime, Naltrindole, or Nor-binaltorphimine, which specifically block mü, delta and kappa opioid receptors, respectively. In the 7th experimental day, rats were euthanized and their maxillae collected for evaluation of alveolar bone and fiber attachment loss, morphometric counting of osteoclasts and osteoblasts, as well as the levels of cytokines IL-1β, IFN-γ, and IL-6 by ELISA. ResultsSelective antagonism of kappa opioid receptors, but not mü and delta, exacerbated alveolar bone resorption induced by ligature in rats. The increased bone loss associated with higher number of osteoclasts surrounding alveolar bone, although osteoblasts’ counting remained unchanged. The concentrations of IL-1β and IL-6 in periodontal tissues were also significantly higher in the rats treated with the kappa antagonist. ConclusionInhibiting kappa opioid receptors exacerbates alveolar bone resorption.

Treatment with functionalized designer self-assembling peptide hydrogels promotes healing of experimental periodontal defects.

It has been reported that self-assembling peptide (SAP) hydrogels with functionalized motifs enhance proliferation and migration of host cells. How these designer SAP hydrogels perform in the treatment of periodontal defects remains unknown. This study aimed to test the potential of local application of designer SAP hydrogels with two different functionalized motifs in the treatment of experimental periodontal defects. In vitro, viability/proliferation of rat periodontal ligament-derived cells (PDLCs) cultured on an SAP hydrogel RADA16 and RADA16 with functionalized motifs, PRG (integrin binding sequence) and PDS (laminin cell adhesion motif), was assessed. Cell morphology was analyzed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). In vivo, standardized periodontal defects were made mesially in the maxillary first molars of Wistar rats. Defects received RADA16, PRG, PDS or left unfilled. At 2 or 4weeks postoperatively, healing was assessed by microcomputed tomography, histological and immunohistochemical methods. Viability/proliferation of PDLCs was significantly greater on PRG than on RADA16 or PDS at 72hours. rPDLCs in the PRG group showed enhanced elongations and cell protrusions. In vivo, at 4weeks, bone volume fractions in the PRG and PDS groups were significantly greater than the RADA16 group. Histologically, bone formation was more clearly observed in the PRG and PDS groups compared with the RADA16 group. At 4weeks, epithelial downgrowth in the hydrogel groups was significantly reduced compared to the Unfilled group. In Azan-Mallory staining, PDL-like bundles ran in oblique direction in the hydrogel groups. At 2weeks, in the area near the root, proliferating cell nuclear antigen (PCNA)-positive cells were detected significantly more in the PRG group than other groups. At 4weeks, in the middle part of the defect, a significantly greater level of vascular endothelial growth factor (VEGF)-positive cells and α-smooth muscle actin (SMA)-positive blood vessels were observed in the PRG group than in other groups. The results indicate that local application of the functionalized designer SAP hydrogels, especially PRG, promotes periodontal healing by increasing cell proliferation and angiogenesis.

The role of TLR4/MyD88/NF-κB pathway in periodontitis-induced liver inflammation of rats.

ObjectivesThe aim of this study was to clarify the immune mechanism of hepatic injury induced by periodontitis using a rat model.MethodsTwenty‐four SPF male Wistar rats were randomly divided into two groups: control group (CG) and periodontitis group (PG). In order to induce experimental periodontitis, we tied the wire ligature around bilateral maxillary first molar of rats. After 8 weeks, the following indicators were valued: gingival index, tooth mobility, probing pocket depth; indexes about oxidative stress and circulating biomarkers; bone retraction by micro‐CT analysis; Toll‐like receptor 4 (TLR4), myeloid differential protein‐88 (MyD88), and nuclear factor kappa B (NF‐κB) by qRT‐PCR and Western blotting; tumor necrosis factor α (TNF‐α), and interleukin‐6 (IL‐6) by qRT‐PCR and immunohistochemical staining; inflammation of periodontal and hepatic tissues by histopathological observation.ResultsPeriodontal indicators and micro‐CT results showed the raised levels of inflammatory response and bone retraction in PG compared with CG. The mRNA and protein levels of TLR4, MyD88, NF‐κB, TNF‐α, and IL‐6 have indicated high values in PG versus CG. Histopathological analysis revealed a correlation between periodontitis and hepatic injury.ConclusionTLR4/MyD88/NF‐κB pathway may play a role in periodontitis‐induced liver inflammation of rats.

Mineral trioxide aggregate suppresses pro-inflammatory cytokine expression via the calcineurin/nuclear factor of activated T cells/early growth response 2 pathway in lipopolysaccharide-stimulated macrophages.

To elucidate mechanisms by which mineral trioxide aggregate (MTA) suppresses pro-inflammatory cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Mineral trioxide aggregate extracts were prepared by immersing set ProRoot MTA in culture medium. RAW264.7 cells were cultured in the presence of LPS and MTA extracts. mRNA expression levels of interleukin (IL)-1α, IL-6, early growth response 2 (Egr2), suppressor of cytokine signalling 3 (Socs3) and IL-10 were quantified with reverse transcription-quantitative polymerase chain reaction. Phosphorylation of nuclear factor-kappa B (NF-κB) p65 in RAW264.7 cells was analysed by Western blotting. Intracellular calcium imaging was performed with Fluo-4 AM. The activity of nuclear factor of activated T cells (NFAT) was determined by luciferase assays. Enforced expression and silencing of Egr2 in RAW264.7 cells were carried out using an expression vector and specific RNAi, respectively. In vivo kinetics of Egr2+ cells in MTA-treated rat molar pulp tissues were examined using immunohistochemistry. Data were analysed by one-way analysis of variance, followed by the Tukey-Kramer test (P<0.05). Exposure to MTA extracts resulted in reduced mRNA expression levels of IL-1α and IL-6, as well as reduced expression of phosphorylated NF-κB, in LPS-stimulated RAW264.7 cells. Exposure to MTA extracts induced Ca2+ influx, which was blocked by NPS2143, an antagonist of calcium-sensing receptor (CaSR); Ca2+ influx then triggered activation of calcineurin/NFAT signalling and enhanced mRNA expression of Egr2. Enforced expression of Egr2 in RAW264.7 cells promoted the expression of both IL-10 and Socs3. In vivo application of MTA onto rat molar pulp tissue resulted in the appearance of Egr2-expressing cells that coexpressed CD163, a typical M2 macrophage marker. Mineral trioxide aggregate extracts induced downregulation of IL-1α and IL-6 in LPS-stimulated RAW264.7 cells via CaSR-induced activation of calcineurin/NFAT/Egr2 signalling and subsequent upregulation of IL-10 and Socs3.

Nanotherapeutics for regeneration of degenerated tissue infected by bacteria through the multiple delivery of bioactive ions and growth factor with antibacterial/angiogenic and osteogenic/odontogenic capacity

Therapeutic options are quite limited in clinics for the successful repair of infected/degenerated tissues. Although the prevalent treatment is the complete removal of the whole infected tissue, this leads to a loss of tissue function and serious complications. Herein the dental pulp infection, as one of the most common dental problems, was selected as a clinically relevant case to regenerate using a multifunctional nanotherapeutic approach. For this, a mesoporous bioactive glass nano-delivery system incorporating silicate, calcium, and copper as well as loading epidermal growth factor (EGF) was designed to provide antibacterial/pro-angiogenic and osteo/odontogenic multiple therapeutic effects. Amine-functionalized Cu-doped bioactive glass nanospheres (Cu-BGn) were prepared to be 50–60 nm in size, mesoporous, positive-charged and bone-bioactive. The Cu-BGn could release bioactive ions (copper, calcium and silicate ions) with therapeutically-effective doses. The Cu-BGn treatment to human umbilical vein endothelial cells (HUVEC) led to significant enhancement of the migration, tubule formation and expression of angiogenic gene (e.g. vascular endothelial growth factor, VEGF). Furthermore, the EGF-loaded Cu-BGn (EGF@Cu-BGn) showed pro-angiogenic effects with antibacterial activity against E. faecalis, a pathogen commonly involved in the pulp infection. Of note, under the co-culture condition of HUVEC with E. faecalis, the secretion of VEGF was up-regulated. In addition, the osteo/odontogenic stimulation of the EGF@Cu-BGn was evidenced with human dental pulp stem cells. The local administration of the EGF@Cu-BGn in a rat molar tooth defect infected with E. faecalis revealed significant in vivo regenerative capacity, highlighting the nanotherapeutic uses of the multifunctional nanoparticles for regenerating infected/damaged hard tissues.

Pulpal upregulation of connexin 43 during pulpitis.

Connexins are building blocks of membranous channels that form gap junctions and hemichannels. These channels are essential portals for information exchange and coordination during inflammation. Pathologic levels of these conduits may result in excessive inflammation and collateral destruction. This study aimed to analyse temporospatial levels of connexin 43 (Cx43) during pulpitis in extracted human teeth and in a rodent model. A specific interest was directed at the pulpal stroma as it is conserved during vital pulp therapy. Pulpal tissues were attained from human extracted teeth of various pulpal inflammatory stages and fixed for cryosections. Pulpal exposures were created in bilateral maxillary molars in Sprague-Dawley rats. Rats were sacrificed at days 1 to 5 post-exposure. Immunofluorescence histology was performed to detect Cx43, markers for inflammation, and cell death. Immunofluorescent levels in the pulpal stroma at 3 sites (wound/near/far) were matched to pulpal condition (human) or days post-exposure (rodent). Cx43 upregulation was observed with increased severity of pulpitis both in humans and rodent model. The upregulation appeared to be global and included distant regions. Elevated levels of neutrophils were present in advanced pulpitis. Apoptosis and necroptosis seem to be upregulated in human samples as Cx43 levels rose. We observed a disseminated upregulation of Cx43 throughout the pulpal stroma as inflammation became advanced. This observation may facilitate cell death signal transfer or represent overt levels of purinergic signalling that leads to pro-inflammatory conditions. Cx43 downregulation may represent a potential therapeutic approach to enable resolution of pulpal inflammation.