Ligature-induced Periodontitis Model Research Articles

Alveolar Bone Loss in a Ligature-Induced Periodontitis Model in Rat Using Different Ligature Sizes.

Ligature-induced periodontitis model has been widely used as a preclinical stage for investigating new treatment modalities. However, the effect of different ligature sizes on alveolar bone loss has never been studied. Therefore, we examined alveolar bone loss in this rat model using different sizes of silk ligatures, as well as healing after ligature removal. Left maxillary second molars of Sprague-Dawley rats were ligated with 3-0, 4-0, or 5-0 silk ligatures (n = 4-5/group) for 14 days before harvested maxillae and gingival tissues. For subsequent experiment, animals were ligated for 14 days using the ligature size that induced the most alveolar bone loss before ligature removal and sacrificed at 0, 7 and 14 days (n = 5-6/group). All maxillae and gingival tissues were harvested to evaluate alveolar bone level, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) levels. Data was analyzed using SPSS Statistics 23.0 software (SPSS Inc., Chicago, Illinois, United States). Data from all experiments were tested for normality using Shapiro-Wilk test. Data between ligatured and nonligatured teeth were compared using Student's t-test or Wilcoxon signed-rank test. Differences among different ligature sizes were analyzed by analysis of variance followed by multiple comparisons with post-hoc test. A p-value less than 0.05 was considered statistically significant. The alveolar bone loss of ligated teeth was substantially higher than that of control after 14 days of ligation. While 3-0 and 4-0 resulted in significantly greater bone loss than 5-0 silk, the 3-0 group had the lowest rate of ligature loss. Therefore, alveolar bone healing postligature removal was investigated further using 3-0 silk. The results showed no significant bone level change at 2 weeks after ligature removal. In term of IL-1β and TNF-α levels, there was no statistically significant difference in IL-1β level between groups at any time point, while TNF-α was undetectable. These data showed that 3-0 silk was the most effective ligature size in promoting alveolar bone loss comparing with 4-0 and 5-0 silk. During the 2-week period following ligature removal, spontaneous bone healing was not observed.

Chenopodium Ambrosioides Linn Mitigates Bone Loss in Rats with Periodontitis.

Periodontitis is an inflammatory disease that causes bone loss. Some patients do not respond well to the classic treatment and need therapies that minimize bone loss, the main sequel of the disease. Chenopodium ambrosioides L. has stood out due to its anti-inflammatory and anti-oxidative activities. However, no study has yet investigated its effect on periodontitis. This study aimed to evaluate the bone protective effect of Chenopodium ambrosioides L. (CAL) extract on ligature-induced periodontitis model in rats. For this, a pre-clinical assay was performed, using male Wistar rats divided into 3 groups: Naive (N) (n=6), not submitted to any procedure; Saline (SAL) (n=6), submitted to ligature-induced periodontitis and receiving 2 ml/kg of 0.9% saline solution; and CAL extract, which was subdivided into 3 subgroups (n=6/subgroup) receiving the CAL at 3 (CAL3), 10 (CAL10) or 30 mg/kg (CAL30). All agents were given, by oral gavage, 30 min before periodontitis induction and daily until euthanasia (11th day). By then, maxillae were removed for macroscopic, histological, and histometric analyses. Kidneys, liver, and stomach were collected to evaluate the safety of CAL extract. High-performance liquid chromatography (HPLC) assay was used to investigate the flavonoid content in the extract. Chenopodium ambrosioides L. extract at 30mg/kg showed a reduction by 58% in bone loss marked by an increase (+35%) in the number of osteoblasts and a reduction (-51%) on the number of osteoclasts (p< 0.05). No significant alteration in the liver, kidney, or stomach was seen. Rutin was the main flavonoid found. In summary, it was observed that Chenopodium ambrosioides L. extract has shown important anti-inflammatory and bone anabolic and anti-resorptive properties without causing toxicity in the main organs. Rutin, as the main flavonoid of the extract, seems to be responsible for the beneficial effect of this agent.

Arrestin beta-2deficiency exacerbates periodontal inflammation by mediating activating transcription factor 6 activation and abnormal remodelling of the extracellular matrix.

To investigate the specific role of arrestin beta-2 (ARRB2) in the progression of periodontitis and the underlying mechanisms. Single-cell RNA sequencing data were used to analyse gene expression in periodontal tissues from healthy controls and patients with periodontitis. Real-time quantitative polymerase chain reaction, Western blotting and immunohistochemical staining were performed to detect the expression of ARRB2. Furthermore, a ligature-induced periodontitis model was created. Using radiographic and histological methods, RNA sequencing and luciferase assay, the role of ARRB2 in periodontitis and the underlying mechanisms were explored. Finally, the therapeutic effect of melatonin, an inhibitor of activating transcription factor 6 (ATF6), on periodontitis in mice was assessed in both in vivo and in vitro experiments. ARRB2 expression was up-regulated in inflammatory periodontal tissue. In the ligature-induced mouse model, Arrb2 knockout exacerbated alveolar bone loss (ABL) and extracellular matrix (ECM) degradation. ARRB2 exerted a negative regulatory effect on ATF6, an essential targeted gene. Melatonin ameliorated ABL and an imbalance in ECM remodelling in Arrb2-deficient periodontitis mice. ARRB2 mediates ECM remodelling via inhibition of the ATF6 signalling pathway, which ultimately exerts a protective effect on periodontal tissues.

Oxytocin alleviates periodontitis in adult rats.

The objective of the study was to evaluate the expression of oxytocin receptors in normal and inflamed gingiva, as well as the effects of systemic administration of oxytocin in bone loss and gum inflammatory mediators in a rat model of experimental periodontitis. Current evidence supports the hypothesis of a disbalance between the oral microbiota and the host's immune response in the pathogenesis of periodontitis. Increased complexity of the microbial biofilm present in the periodontal pocket leads to local production of nitrogen and oxygen-reactive species, cytokines, chemokines, and other proinflammatory mediators which contribute to periodontal tissue destruction and bone loss. Oxytocin has been suggested to participate in the modulation of immune and inflammatory processes. We have previously shown that oxytocin, nitric oxide, and endocannabinoid system interact providing a mechanism of regulation for systemic inflammation. Here, we aimed at investigating not only the presence and levels of expression of oxytocin receptors on healthy and inflamed gingiva, but also the effects of oxytocin treatment on alveolar bone loss, and systemic and gum expression of inflammatory mediators involved in periodontal tissue damage using ligature-induced periodontitis. Therefore, anti-inflammatory strategies oriented at modulating the host's immune response could be valuable adjuvants to the main treatment of periodontal disease. We used an animal model of ligature-induced periodontitis involving the placement of a linen thread (Barbour flax 100% linen suture, No. 50; size 2/0) ligature around the neck of first lower molars of adult male rats. The ligature was left in place during the entire experiment (7 days) until euthanasia. Animals with periodontitis received daily treatment with oxytocin (OXT, 1000 μg/kg, sc.) or vehicle and/or atosiban (3 mg/kg, sc.), an antagonist of oxytocin receptors. The distance between the cement-enamel junction and the alveolar bone crest was measured in stained hemimandibles in the long axis of both buccal and lingual surfaces of both inferior first molars using a caliper. TNF-α levels in plasma were determined using specific rat enzyme-linked immunosorbent assays (ELISA). OXT receptors, IL-6, IL-1β, and TNF-α expression were determined in gingival tissues by semiquantitative or real-time PCR. We show that oxytocin receptors are expressed in normal and inflamed gingival tissues in male rats. We also show that the systemic administration of oxytocin prevents the experimental periodontitis-induced increased gum expression of oxytocin receptors, TNF-α, IL-6, and IL-1β (p < .05). Furthermore, we observed a reduction in bone loss in rats treated with oxytocin in our model. Our results demonstrate that oxytocin is a novel and potent modulator of the gingival inflammatory process together with bone loss preventing effects in an experimental model of ligature-induced periodontitis.

N-acyl homoserine lactones lactonase est816 suppresses biofilm formation and periodontitis in rats mediated by Aggregatibacter actinomycetemcomitans

ABSTRACT Aims The current study aimed to explore the adjuvant therapeutic effect of N-acyl homoserine lactones (AHLs)-lactonase est816 on Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) biological behaviors and periodontitis progression. Methods The inhibitory properties of est816 were detected by live/dead bacterial staining, scanning electron microscope (SEM), crystal-violet staining and reverse-transcription quantitative PCR (RT-qPCR). Biocompatibility of est816 on human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGEs) was evaluated by CCK8 and ELISA. The ligature-induced periodontitis model was established in rats. Micro computed tomography and immunohistochemical and histological staining served to evaluate the effect of est816 on the prevention of periodontitis in vivo. Results est816 significantly attenuated biofilm formation, reduced the mRNA expression of cytolethal distending toxin, leukotoxin and poly-N-acetyl glucosamine (PNAG) and downregulated expressions of interleukin-6 and tumor necrosis factor-α with low cell toxicity. In vivo investigations revealed est816 decreased alveolar bone resorption, suppressed matrix metalloproteinase-9 expression and increased osteoprotegerin expression. Conclusion est816 inhibited A. actinomycetemcomitans biofilm formation and virulence release, resulting in anti-inflammation and soothing of periodontitis in rats, indicating that est816 could be investigated in further research on periodontal diseases.

Developmental endothelial locus-1 promotes osteogenic differentiation and alveolar bone regeneration in experimental periodontitis with type 2 diabetes mellitus.

This study sought to explore the role of developmental endothelial locus-1 (DEL-1) in osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and investigate the therapeutic effect of DEL-1 in ligature-induced experimental periodontitis with type 2 diabetes mellitus (T2DM). T2DM is a significant risk factor for periodontitis. Treatment modalities for periodontitis with T2DM are being explored. DEL-1 is a versatile protein that can modulate the different stages of inflammatory diseases including periodontitis. The direct effect of DEL-1 on osteogenic differentiation of PDLSCs in periodontitis with T2DM is poorly understood. Primary hPDLSCs were isolated from periodontal ligament tissue and identified by flow cytometry. In osteogenesis experiments, alkaline phosphatase (ALP), Alizarin Red staining and western blot were used to assess the osteogenic effect of DEL-1 on hPDLSCs in high glucose and inflammation environments. The mouse model of ligature-induced experimental periodontitis was established. H&E and Masson's trichrome staining were used to assess the change of periodontal tissue after local periodontal injection of DEL-1. Immunohistochemical staining was used to evaluate osteogenic-related protein expression. hPDLSCs expressed mesenchymal stem cell (MSC)-specific surface markers and were negative for hematopoietic cell surface markers. hPDLSCs had the potential for multidirectional differentiation. DEL-1 could enhance the osteogenic differentiation of hPDLSCs in high glucose and inflammation environments, although it did not return to the control level. Histological staining showed that DEL-1 contributed to alveolar bone regeneration and osteogenic-related protein expression, but the degree of improvement in T2DM mice was lower than in non-T2DM mice. In summary, we demonstrated that DEL-1 could promote osteogenic differentiation of hPDLSCs in high glucose and inflammation environment and rescue alveolar bone loss in experimental periodontitis with T2DM, which could provide a novel therapeutic target for periodontitis with T2DM.

Integrated microbiome and metabolomics revealed the protective effect of baicalin on alveolar bone inflammatory resorption in aging

BackgroundWith the growing aging population and longer life expectancy, periodontitis and tooth loss have become major health concerns. The gut microbiota, as a key regulator in bone homeostasis, has gathered immense interest. Baicalin, a flavonoid compound extracted from Scutellaria baicalensis Georgi, has shown antioxidant and anti-inflammatory activities. PurposeThis study investigated, for the first time, the protective mechanism of baicalin against alveolar bone inflammatory resorption in aging mice by regulating intestinal flora and metabolites, as well as intestinal barrier function. MethodsA ligature-induced periodontitis model was established in d-galactose (D-gal)-induced aging mice, and baicalin was administered at different dosages for 13 weeks. Body weight was measured weekly. The antioxidant and anti-inflammatory activity of baicalin were evaluated using serum superoxide dismutase (SOD), malonaldehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) levels. The immune capability was assessed by thymus and spleen indices. Histopathological changes were observed in the heart, liver, ileum, and periodontal tissues. Alveolar bone absorption of maxillary second molars was examined, and osteoclasts were counted by tartrate-resistant acid phosphatase (TRAP) staining. Furthermore, fecal samples were analyzed using 16S rRNA sequencing and non-targeted metabolomics to identify differences in intestinal bacterial composition and metabolites. ResultsBaicalin exhibited anti-aging properties, as evidenced by increased SOD activity and decreased levels of MDA, IL-6, and TNF-α in serum compared to the control group. Baicalin also ameliorated alveolar bone loss in the d-gal-induced aging-periodontitis group (p < 0.05). Furthermore, baicalin restored ileal permeability by up-regulating the expression of ZO-1 and occludin in aging-periodontitis groups (p < 0.05). Alpha diversity analysis indicated that baicalin-treated mice harbored a higher diversity of gut microbe. PCoA and ANOSIM results revealed significant dissimilarity between groups. The Firmicutes/Bacteroidetes (F/B) ratio, which decreased in periodontitis mice, was restored by baicalin treatment. Additionally, medium-dosage baicalin promoted the production of beneficial flavonoids, and enriched short-chain fatty acids (SCFAs)-producing bacteria. ConclusionIntestinal homeostasis is a potential avenue for treating age-related alveolar bone loss. Baicalin exerts anti-inflammatory, antioxidant, and osteo-protective properties by regulating the gut microbiota and metabolites.

Mice Lacking PLAP-1/Asporin Show Alteration of Periodontal Ligament Structures and Acceleration of Bone Loss in Periodontitis.

Periodontal ligament-associated protein 1 (PLAP-1), also known as Asporin, is an extracellular matrix protein expressed in the periodontal ligament and plays a crucial role in periodontal tissue homeostasis. Our previous research demonstrated that PLAP-1 may inhibit TLR2/4-mediated inflammatory responses, thereby exerting a protective function against periodontitis. However, the precise roles of PLAP-1 in the periodontal ligament (PDL) and its relationship to periodontitis have not been fully explored. In this study, we employed PLAP-1 knockout mice to investigate its roles and contributions to PDL tissue and function in a ligature-induced periodontitis model. Mandibular bone samples were collected from 10-week-old male C57BL/6 (WT) and PLAP-1 knockout (KO) mice. These samples were analyzed through micro-computed tomography (μCT) scanning, hematoxylin and eosin (HE) staining, picrosirius red staining, and fluorescence immunostaining using antibodies targeting extracellular matrix proteins. Additionally, the structure of the PDL collagen fibrils was examined using transmission electron microscopy (TEM). We also conducted tooth extraction and ligature-induced periodontitis models using both wild-type and PLAP-1 KO mice. PLAP-1 KO mice did not exhibit any changes in alveolar bone resorption up to the age of 10 weeks, but they did display an enlarged PDL space, as confirmed by μCT and histological analyses. Fluorescence immunostaining revealed increased expression of extracellular matrix proteins, including Col3, BGN, and DCN, in the PDL tissues of PLAP-1 KO mice. TEM analysis demonstrated an increase in collagen diameter within the PDL of PLAP-1 KO mice. In line with these findings, the maximum stress required for tooth extraction was significantly lower in PLAP-1 KO mice in the tooth extraction model compared to WT mice (13.89 N ± 1.34 and 16.51 N ± 1.31, respectively). In the ligature-induced periodontitis model, PLAP-1 knockout resulted in highly severe alveolar bone resorption, with a higher number of collagen fiber bundle tears and significantly more osteoclasts in the periodontium. Our results demonstrate that mice lacking PLAP-1/Asporin show alteration of periodontal ligament structures and acceleration of bone loss in periodontitis. This underscores the significant role of PLAP-1 in maintaining collagen fibrils in the PDL and suggests the potential of PLAP-1 as a therapeutic target for periodontal diseases.

LncRNA MALAT1/microRNA-30b axis regulates macrophage polarization and function.

Macrophages (Mφ) are long-lived myeloid cells that can polarize towards the proinflammatory M1 or proresolving M2 phenotype to control diverse biological processes such as inflammation, tissue damage, and regeneration. Noncoding RNA are a class of nonprotein-coding transcriptome with numerous interdependent biological roles; however, their functional interaction in the regulation of Mφ polarization and immune responses remain unclear. Here, we show antagonistic relationship between lncRNA (MALAT1) and microRNA (miR-30b) in shaping macrophage polarization and immune functions. MALAT1 expression displays a time-dependent induction during Mφ differentiation and, upon challenge with TLR4 agonist (E. coli LPS). MALAT1 knockdown promoted the expression of M2Mφ markers without affecting M1Mφ markers, suggesting that MALAT1 favors the M1 phenotype by suppressing M2 differentiation. Compared to the control, MALAT1 knockdown resulted in reduced antigen uptake and processing, bacterial phagocytosis, and bactericidal activity, strongly supporting its critical role in regulating innate immune functions in Mφ. Consistent with this, MALAT1 knockdown showed impaired cytokine secretion upon challenge with LPS. Importantly, MALAT1 exhibit an antagonistic expression pattern with all five members of the miR-30 family during M2 Mφ differentiation. Dual-luciferase assays validated a novel sequence on MALAT1 that interacts with miR-30b, a microRNA that promotes the M2 phenotype. Phagocytosis and antigen processing assays unequivocally demonstrated that MALAT1 and miR-30b are functionally antagonistic. Concurrent MALAT1 knockdown and miR-30b overexpression exhibited the most significant attenuation in both assays. In human subjects with periodontal disease and murine model of ligature-induced periodontitis, we observed higher levels of MALAT1, M1Mφ markers and downregulation of miR-30b expression in gingival tissues suggesting a pro-inflammatory function of MALAT1 in vivo. Overall, we unraveled the role of MALAT1 in Mφ polarization and delineated the underlying mechanism of its regulation by involving MALAT-1-driven miR-30b sequestration.

Procyanidin B2 enhances anti-inflammatory responses of periodontal ligament cells by inhibiting the dominant negative pro-inflammatory isoforms of peroxisome proliferator-activated receptor γ

Background/purposePeriodontal breakdown in periodontitis is exacerbated by pro-inflammatory responses of periodontal stromal cells such as periodontal ligament fibroblasts (PDLFs). Procyanidin B2 (PB2) is a ligand of the peroxisome proliferator-activated receptor (PPARγ). Herein, we investigated the expression of PPARγ isoforms in PDLFs and periodontal tissue, and examined the effects of PB2 on PPARγ isoform-dependent antiinflammatory responses. Materials and methodsPPARγ isoforms were examined by PCR. PPARγ isoform-dependent inflammatory functions and anti-inflammatory effects of PB2 in PDLFs were evaluated based on IL-6 expression. Co-immunoprecipitation analysis of fixed chromatin-tethered protein (CoIPfctp) was conducted to investigate the association of each PPARγ isoform with the NF-κB-transcriptional complex. The effects of PB2 on periodontitis progression were evaluated using a ligature-induced murine periodontitis model. ResultsThree isoforms of PPARγ were expressed in PDLFs and periodontal tissues, consisting of the main full-length isoform (PPARγ) and two dominant negative isoforms that lack the ligand binding domain, namely the ubiquitously-expressed isoform (PPARγ-UBI) and unknown isoform (PPARγ-PDL). PPARγ and PPARγ-UBI were predominantly expressed. CoIP-fctp revealed that PPARγ-UBI was selectively associated with NF-κB p65, a key transcriptional factor of IL-6 expression. PB2 suppressed LPS-induced-IL-6 expression exacerbated by the over-expression of PPARγ-UBI. In the murine periodontitis model, topical application of PB2 significantly mitigated alveolar bone loss. ConclusionThese results suggest that the anti-inflammatory effects of PB2 in periodontal tissues/cells are distinct, and these effects arise from the inhibition of PPARγ-UBI; hence, the application of PB2 and modification of the splicing event in three PPARγ isoforms have therapeutic potential for preventing periodontitis.

Macrophage-related gingival transcriptomic patterns and microbiome alterations in experimental periodontitis in nonhuman primates.

This study examined the microbiome features specifically related to host macrophage polarization in health, initiation and progression of periodontitis, and in resolution samples using a nonhuman primate model of ligature-induced periodontitis. The oral microbiome is a complex of bacterial phyla, genera, and species acquired early in life into the individual autochthonous oral ecology. The microbiome changes overtime in response to both intrinsic and extrinsic stressors, and transitions to a dysbiotic ecology at sites of periodontal lesions. Comparisons were made between the microbial and host features in young (≤7 years) and adult (≥12 years) cohorts of animals. Footprints of macrophage-related genes in the gingival tissues were evaluated using expression profiles including M0, M1, and M2 related genes. Within the gingival tissues, similar macrophage-related gene patterns were observed with significant increases with disease initiation and continued elevation throughout disease in both age groups. Approximately, 70% of the taxa were similar in relative abundance between the two groups; however, the adults showed a large number of OTUs that were significantly altered compared with the younger animals. Developing a correlation map identified three major node levels of interactions that comprised approximately ⅓ of the Operational Taxonomic Units (OTUs) that dominated the microbiomes across the samples. Also noted was a much greater frequency of significant correlations of individual OTUs with the macrophage phenotype markers, compared with disease and resolution samples in both age groups, with a greater frequency in the younger group. Moreover, these correlations were assigned to differentially expressed genes representing M0, M1, and M2-related phenotypes. A cluster analyses across the macrophage-related transcriptome and the OTUs demonstrated multiple somewhat distinct bacterial consortia, incorporating both commensal and putative pathogens, linked to the gene responses that differed in health, disease, and resolution samples. Finally, there were minimal alterations in the OTUs in individual clusters with specific macrophage-related responses in the younger group, while in the adult samples substantial variations were noted with genes from all macrophage phenotypes. The results confirmed important features that could reflect macrophage polarization in periodontal lesions, and provided some initial data supporting specific members of the oral microbiome feature prominently related to specific gene response patterns consistent with macrophages in the gingival tissues.

The effect of the Litcubanine A on the treatment of murine experimental periodontitis by inhibiting monocyte-macrophage chemotaxis and osteoclast differentiation.

Periodontal disease is an inflammatory disease of periodontal tissues that is closely connected with systemic diseases. During periodontitis, the inappropriate recruitment and activation of monocytes-macrophages causes an increase in osteoclast activity and disrupts bone homeostasis. Therefore, it is a promising therapeutic strategy to treat periodontitis by regulating the functions of monocytes-macrophages. Litcubanine A (LA) is an isoquinoline alkaloid extracted from the traditional Chinese medicine Litsea cubeba, which was proven to have reproducible anti-inflammatory effects, but its regulatory role on bone homeostasis in periodontitis is still not clear. In this study, zebrafish experiments and a mouse ligature-induced periodontitis model were performed, and histological analysis was used to investigate the effect of LA on macrophage chemotaxis under the inflammatory environment. Real-time PCR was used to detect the regulatory effect of LA (100 nM ~ 100 μM) on the chemotaxis function of macrophages induced by LPS. Apoptosis assay and flow cytometry were used to elucidate the influence of LA on macrophage apoptosis and proliferation. To further clarify the regulatory role of LA on macrophage osteoclast differentiation, real-time PCR, histological analysis, western blot, and micro-computed tomography (micro-CT) were performed in vivo and in vitro to verify the impact of LA on bone homeostasis. Compared with the control group, the chemotaxis function of macrophage was significantly attenuated by LA in vivo. LA could significantly inhibit the expression of genes encoding the chemokine receptors Ccr1 and Cxcr4, and its ligand chemokine Cxcl12 in macrophages, and suppresses the differentiation of osteoclastic precursors to osteoclasts through the MAPK signaling pathway. There were significantly lower osteoclast differentiation and bone loss in the LA group compared with the control in the ligature-induced periodontitis model. LA is a promising candidate for the treatment of periodontitis through its reproducible functions of inhibiting monocyte-macrophage chemotaxis and osteoclast differentiation.

The Complement-Targeted Inhibitor Mini-FH Protects against Experimental Periodontitis via Both C3-Dependent and C3-Independent Mechanisms.

A minimized version of complement factor H (FH), designated mini-FH, was previously engineered combining the N-terminal regulatory domains (short consensus repeat [SCR]1-4) and C-terminal host-surface recognition domains (SCR19-20) of the parent molecule. Mini-FH conferred enhanced protection, as compared with FH, in an ex vivo model of paroxysmal nocturnal hemoglobinuria driven by alternative pathway dysregulation. In the current study, we tested whether and how mini-FH could block another complement-mediated disease, namely periodontitis. In a mouse model of ligature-induced periodontitis (LIP), mini-FH inhibited periodontal inflammation and bone loss in wild-type mice. Although LIP-subjected C3-deficient mice are protected relative to wild-type littermates and exhibit only modest bone loss, mini-FH strikingly inhibited bone loss even in C3-deficient mice. However, mini-FH failed to inhibit ligature-induced bone loss in mice doubly deficient in C3 and CD11b. These findings indicate that mini-FH can inhibit experimental periodontitis even in a manner that is independent of its complement regulatory activity and is mediated by complement receptor 3 (CD11b/CD18). Consistent with this notion, a complement receptor 3-interacting recombinant FH segment that lacks complement regulatory activity (specifically encompassing SCRs 19 and 20; FH19-20) was also able to suppress bone loss in LIP-subjected C3-deficient mice. In conclusion, mini-FH appears to be a promising candidate therapeutic for periodontitis by virtue of its ability to suppress bone loss via mechanisms that both include and go beyond its complement regulatory activity.

Sex effects on gingival transcriptomic patterns during initiation, progression, and resolution of periodontitis.

The prevalence and severity of periodontitis demonstrates altered population distribution with age, sex, and race and ethnicity. While males exhibit greater frequency of disease, particularly with aging, the underlying basis for this observation remains obscure. This study used a nonhuman primate (Macaca mulatta) model of experimental ligature-induced periodontitis in adult animals to evaluate gingival transcriptomic differences stratified based upon sex of the animal. The 18 animals represented humans ages 40-80 years, with gingival tissue samples obtained at baseline, 0.5 months (initiation), 1 and 3 months (progression), and at 5 months that were 60 days after ligature removal for clinical disease resolution. Microarray analysis was used to quantify gene expression profiles in the gingival tissues. The results demonstrated clear gene expression differences in healthy (baseline) tissues between the sexes, with elevations in females associated with immune responses and elevation in males related to tissue structural genes. With disease initiation, fewer genes differed between the sexes, while these differences were significantly increased in progressing disease and resolution, particularly in male animals. Overexpressed biological processes showed tissue structural/functional genes at initiation, with host response pathways altered during disease progression. Resolution samples generally demonstrated biological processes of cellular metabolism that differed from baseline healthy samples. The transcriptomic findings support sex as a biological variable in periodontitis using a nonhuman primate model of experimental periodontitis.

The Interconnection Between Periodontitis and Inflammatory Bowel Disease

Abstract Objective Periodontitis, an oral infection affecting ~50% of Americans, is associated with the pathogenesis of inflammatory bowel disease (IBD), however, the contributory mechanisms remain ill defined. Previous GWAS studies have identified SNPs in the Interferon Regulatory Factor 8 (IRF8) gene as risk factors for IBD. The objective of this study was to explore the relationship between periodontitis and IBD using Irf8 knockout (KO) mice. Methods Periodontitis was induced in wild-type (WT) and Irf8 KO mice using the well-established oral ligature-induced periodontitis (LIP) model. The effects of LIP on oral bacterial colonization in the mouth and gut were measured by 16S rRNA and shotgun metagenomics sequencing. The influence of LIP on oral bacteria-mediated immune cell infiltration in the colonic mucosa was measured by flow cytometry, RT-qPCR, and ELISA. Results LIP promoted expansion of native oral pathogens and accumulation of the same species in the mouth and gut of Irf8 KO mice, which was associated with colitis development in Irf8 KO mice when compared to WT mice. LIP Irf8 KO mice exhibited ~2-fold increased infiltration of Th17 cells and ~2–4-fold decrease infiltration of Th1 and Treg cells in the colonic mucosa, which led to over production of IL17 and decreased production of IFN-γ and IL10. Similarly, IL-1β-producing cells were found to be higher in the colonic mucosa of LIP Irf8 KO vs WT mice. Resolution of periodontitis correlated with reduced intestinal load of oral bacteria and reversal of colitis in Irf8 KO mice. Conclusion Our study provides new perspectives on the pathophysiology of IBD. Irf8 deficiency along with dysbiotic oral and gut microbiota promote colitis in mice by activating IL17 and IL-1β signaling pathways.

Novel Herbal Therapeutic YH23537 Improves Clinical Parameters in Ligature-Induced Periodontal Disease Model in Beagle Dogs.

Currently, available medicine does not satisfy the clinical unmet needs of periodontal disease. Therefore, novel drugs with improved efficacy profiles are needed. We previously demonstrated that YH14642, water extracts of Notoginseng Radix and Rehmanniae Radix Preparata, improved probing depths in double-blind phase II clinical trial. However, it still has hurdles for commercialization due to the low efficiency of active compound extraction. To resolve this issue, we developed YH23537 through process optimization to extract active compounds efficiently while still achieving the chemical profile of YH14642. In this study, we investigated the therapeutic effects of YH23537 compared with YH14642 using a canine model of ligature-induced periodontitis. Human gingival fibroblast (hGF) cells were treated with various concentrations of YH23537 or YH14642 with lipopolysaccharide (LPS) for 24 hr. IL-6 and IL-8 levels in the conditioned media were determined using Luminex. Sixteen 3-year-old male beagle dogs had their teeth scaled and polished using a piezo-type ultrasonic scaler under general anesthesia and brushed once daily for the following 2 weeks. Two weeks after the scaling procedure, the left upper second premolar (PM2), third premolar (PM3), and fourth premolar (PM4) as well as the left lower PM3, PM4, and first molar (M1) were ligated with silk-wire twisted ligatures. The dogs were fed with soft moistened food to induce periodontitis for 8 weeks, and the ligatures were then removed. YH23537 and YH14642 were administered for 4 weeks, and clinical periodontal parameters such as plaque index (PI), gingival index (GI), probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BoP) were determined before and 1, 2, 3, and 4 weeks after treatment. YH23537 inhibited IL-6 and IL-8 secretion in a dose-dependent manner in hGF cells stimulated with LPS. The IC50 values for YH23537 were 43 and 54 μg/ml for IL-6 and IL-8, respectively, while the values for YH14642 were 104 and 117 μg/ml, respectively. In the animal study, clinical parameters including GI, PD, CAL, and BoP were significantly increased after 8 weeks of ligature-induced periodontitis. The YH23537 300 and YH23537 900 mg groups had significant improvements in CAL from 1 to 4 weeks after treatment in comparison to the placebo group. GR values in the YH23537 900 mg group were decreased throughout the treatment period. GI values were also reduced significantly after 4-week treatment with 300 and 900 mg of YH23537. YH23537 at 300 mg doses showed comparable efficacy for CAL and GR with 1,000 mg of YH14642. YH23537 showed therapeutic efficacy against periodontitis in dogs, mediated by anti-inflammatory effects. These findings indicate that YH23537 has the potential for further development as a new drug for patients suffering from periodontal disease.

Preventive Effects of Chlorogenic Acid on Alveolar Bone Loss in Ligature-Induced Periodontitis in Mice

Chlorogenic acid (CGA) is a polyphenol that is present in coffee beans, many vegetables, and fruits. Since CGA has been reported to exert antioxidant and anti-inflammatory effects, it is expected to protect against periodontitis. In the present study, we used a ligature-induced experimental periodontitis model and investigated the beneficial effects of CGA against alveolar bone resorption caused by experimental periodontitis. To examine the inhibitory effects of CGA on bone loss, a ligature was wrapped around the maxillary right second molar, and CGA was intraperitoneally injected once a day for 2 weeks. In another experiment to investigate the restorative effects of CGA on bone loss, a ligature was wrapped around the maxillary right second molar for 2 weeks, it was then removed, and CGA was intraperitoneally injected once a day for 2 weeks. At the end of the experiments, the maxillae were removed, and CT images were taken. Alveolar bone loss was measured as the distance from the cement–enamel junction to the alveolar crest. The statistical analysis was performed using GraphPad Prism6 (Dunn’s multiple comparison test). The results revealed that the ratio of the buccal alveolar bone loss (vs. the bone loss on the nonligated side) induced by ligation was significantly decreased by the administration of CGA (5 mg/kg) for 2 weeks. Moreover, the bone loss ratio on the buccal and palatal sides after 2 weeks of ligation was significantly decreased by the 2-week administration of CGA (5 mg/kg). The present results revealed that CGA exerted preventive effects against alveolar bone loss caused by experimental periodontitis.

Ligature-Induced Periodontitis Drives Colorectal Cancer: An Experimental Model in Mice

Periodontitis is a prevalent inflammatory oral disease associated with an increased risk of colorectal cancer. Experimental animal models are critical tools to investigate the effects and mechanisms of periodontitis on colorectal cancer. Several murine periodontitis models have been used in research, including oral gavage, periodontal pathogen injection, and ligature models. The role of experimental periodontitis caused by silk ligation in colorectal cancer remains unclear. In this study, we used an experimental periodontitis model on a colitis-associated colorectal cancer model and a spontaneous model, respectively. We observed the promotion of colorectal cancer in ligature-induced periodontitis mice compared to those control mice in 2 different models, as assessed by tumor number, tumor size, and tumor load. Since bacterial dysbiosis is an important feature of periodontitis, we next analyzed the oral and gut microbiomes using 16S ribosomal RNA gene sequencing. We found that the experimental periodontitis model reshaped the microbial community in the oral cavity and gut. In addition, we found a higher extent of programmed death 1 (PD-1)–positive CD8+ T-cell infiltration in tumor samples of the periodontitis group than in controls by immunofluorescence staining. Regarding the potential molecular mechanism, we transplanted the fecal microbiota of the periodontitis patient into mice and observed a tumor-promoting effect in the periodontitis group, assessed by tumor volume and tumor weight, together with a low level of INF-γ+ CD8+ T-cell infiltration in subcutaneous tumor mice. Taken together, we show that ligature-induced periodontitis model promotes colorectal cancer by microbiota remodeling and suppression of the immune response.

A comprehensive multiparameter flow cytometry panel for immune profiling and functional studies of frozen tissue, bone marrow, and spleen

Flow cytometry (FC) is a highly informative technology that can provide valuable information about immune phenotype monitoring and immune cell states. However, there is a paucity of comprehensive panels developed and validated for use on frozen samples. Here, we developed a 17-plex flow cytometry panel to detect subtypes, frequencies, and functions of different immune cells that can be leveraged to study the different cellular characteristics in different disease models, physiological, and pathological conditions. This panel identifies surface markers to characterize T cells (CD8+, CD4+), natural killer (NK) cells and their subtypes (immature, cytotoxic, exhausted, activated),natural killer T (NKT) cells, neutrophils, macrophages (M1 (pro-inflammatory) and M2 (anti-inflammatory)), monocytes and their subtypes (classical and non-classical), dendritic cells (DC) and their subtypes (DC1, DC2), and eosinophils. The panel was designed to include only surface markers to avoid the necessity for fixation and permeabilization steps. This panel was optimized using cryopreserved cells.Immunophenotyping of spleen and bone marrow using the proposed panel was efficient in correctly differentiating the immune cell subtypes in inflammatory model of ligature-induced periodontitis, in which we found increased percentage of NKT cells, activated and mature/cytotoxic NK cells in the bone marrow of affected mice. This panel enables in-depth immunophenotyping of murine immune cells in bone marrow, spleen, tumors, and other non-immune tissues of mice. It could be a tool for systematic analysis of immune cell profiling in inflammatory conditions, systemic diseases, and tumor microenvironments.

Regenerative Potential of Bone Morphogenetic Protein 7-Engineered Mesenchymal Stem Cells in Ligature-Induced Periodontitis.

Periodontitis is an oral disease caused by bacterial infection that has stages according to the severity of tissue destruction. The advanced stage of periodontitis presents irreversible destruction of soft and hard tissues, which finally results in loss of teeth. When conventional treatment modalities show limited results, tissue regeneration therapy is required in patients with advanced periodontitis. In the present study, we aimed to evaluate the effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) delivering bone morphogenetic protein 7 (BMP7) on tissue regeneration in a periodontitis model. BMP7 is a member of the BMP family that shows bone-forming ability; however, BMPs rapid clearing and degradation and unproven efficacy make it difficult to apply it in clinical dentistry. To overcome this, we established BMP7-expressing engineered BM-MSCs (BMP7-eBMSCs) that showed superior osteogenic differentiation potential when subcutaneously transplanted with a biphasic calcium phosphate scaffold into immunocompromised mice. Furthermore, the efficacy of BMP7-eBMSC transplantation for periodontal tissue regeneration was evaluated in a rat ligature-induced periodontitis model. Upon measuring two-dimensional and three-dimensional amounts of regenerated alveolar bone using microcomputed tomography, the amounts were found to be significantly higher in the BMP7-eBMSC transplantation group than in the eBMSC transplantation group. Most importantly, fibrous periodontal ligament (PDL) tissue regeneration was also achieved upon BMP7-eBMSC transplantation, which was evaluated by calculating the modified relative connective tissue attachment. The amount of connective tissue attachment in the BMP7-eBMSC transplantation group was significantly higher than that in the ligature-induced periodontitis group, although the increase was comparable between the BMP7-eBMSC and human PDL stem cell transplantation groups. Taken together, our results suggested that sustainable release of BMP7 induces periodontal tissue regeneration and that transplantation of BMP7-eBMSCs is a feasible treatment option for periodontal regeneration. Impact Statement Periodontitis is the second most common human dental disease affecting chronic systemic diseases. Despite the tremendous efforts trying to cure the damaged periodontal tissues using tissue engineering technologies, a definitive regenerative method has not been in consensus. Researchers are seeking more feasible and abundant source of mesenchymal stem cells (MSCs), and furthermore, how to use reliable growth factors under more efficient control are the issues to be solved. In this study, we aimed to evaluate the effect of bone morphogenetic protein 7 (BMP7) gene delivering bone marrow-derived MSCs on periodontal tissue regeneration to evaluate the efficacy of BMP7 and engineered BMSCs for periodontal tissue regeneration.