Characterization and application of in situ curcumin/ZNP hydrogels for periodontitis treatment.

BACKGROUND: Periodontitis is a chronic inflammatory disease that occurs in tooth-supporting tissues. Controlling inflammation and alleviating periodontal tissue destruction are key factors in periodontal therapy. This study aimed to develop an in situ curcumin/zinc oxide (Cur/ZNP) hydrogel and investigate its characteristics and effectiveness in the treatment of periodontitis. METHODS: Antibacterial activity and cytotoxicity assays were performed in vitro. To evaluate the effect of the in situ Cur/ZNP hydrogel on periodontitis in vivo, an experimental periodontitis model was established in Sprague‒Dawley rats via silk ligature and inoculation of the maxillary first molar with Porphyromonas gingivalis. After one month of in situ treatment with the hydrogel, we examined the transcriptional responses of the gingiva to the Cur/ZNP hydrogel treatment and detected the alveolar bone level as well as the expression of osteocalcin (OCN) and osteoprotegerin (OPG) in the periodontal tissues of the rats. RESULTS: Cur/ZNPs had synergistic inhibitory effects on P. gingivalis and good biocompatibility. RNA sequencing of the gingiva showed that immune effector process-related genes were significantly induced by experimental periodontitis. Carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam1), which is involved in the negative regulation of bone resorption, was differentially regulated by the Cur/ZNP hydrogel but not by the Cur hydrogel or ZNP hydrogel. The Cur/ZNP hydrogel also had a stronger protective effect on alveolar bone resorption than both the Cur hydrogel and the ZNP hydrogel. CONCLUSION: The Cur/ZNP hydrogel effectively inhibited periodontal pathogenic bacteria and alleviated alveolar bone destruction while exhibiting favorable biocompatibility.

Multiple effects of dose-related GM-CSF on periodontal resorption in deep-frozen grafted teeth: A reverse study.

Autologous tooth grafting is a dental restorative modality based on periodontal ligament healing.Human periodontal ligament stem cells(PDLSCs) are involved in the formation and remodeling of periodontal tissue.Based on previous findings, the proliferation and differentiation of processing cryopreserved periodontal ligament stem cells (PDLSCs) exhibit similarities to those of fresh cells. However, there is evident absorption in the transplanted frozen tooth's roots and bones, with the underlying cause remaining unknown. Granulocyte macrophage colony-stimulating factor(GM-CSF) is named for its produce granulocyte and macrophage precursors from bone marrow precursors, and it also serves as one of the regulatory factors in inflammatory and osteoclast formation. This study aimed to investigate changes in GM-CSF expression in frozen PDLSCs (fhPDLSCs) and evaluate the impact of GM-CSF on PDLSCs with respect to cellular activity and osteogenic ability. The role of GM-CSF in periodontal absorption was further speculated by comparing with IL-1ß. The results revealed a significant increase in GM-CSF levels from fhPDLSCs compared to fresh cells, which exhibited an equivalent inflammatory stimulation effect as 1 ng/ml IL-1ß. Cell viability also increased with increasing concentrations of GM-CSF; however, the GM-CSF from fhPDLSCs was not sufficient to significantly trigger osteoclastic factors. Considering its interaction with IL-1ß and positive feedback mechanism, environments with high doses of GM-CSF derived from fhPDLSCs are more likely to activate osteoclastic responses.Therefore, for frozen tooth replantation, great attention should be paid to anti-inflammation and anti-infection.GM-CSF may serve as a potential therapeutic target for inhibiting periodontal resorption in delayed grafts.

Tim4 deficiency reduces CD301b+ macrophage and aggravates periodontitis bone loss.

Periodontitis is a common chronic inflammatory disease that causes the periodontal bone destruction and may ultimately result in tooth loss. With the progression of periodontitis, the osteoimmunology microenvironment in periodontitis is damaged and leads to the formation of pathological alveolar bone resorption. CD301b+ macrophages are specific to the osteoimmunology microenvironment, and are emerging as vital booster for conducting bone regeneration. However, the key upstream targets of CD301b+ macrophages and their potential mechanism in periodontitis remain elusive. In this study, we concentrated on the role of Tim4, a latent upstream regulator of CD301b+ macrophages. We first demonstrated that the transcription level of Timd4 (gene name of Tim4) in CD301b+ macrophages was significantly upregulated compared to CD301b- macrophages via high-throughput RNA sequencing. Moreover, several Tim4-related functions such as apoptotic cell clearance, phagocytosis and engulfment were positively regulated by CD301b+ macrophages. The single-cell RNA sequencing analysis subsequently discovered that Cd301b and Timd4 were specifically co-expressed in macrophages. The following flow cytometric analysis indicated that Tim4 positive expression rates in total macrophages shared highly synchronized dynamic changes with the proportions of CD301b+ macrophages as periodontitis progressed. Furthermore, the deficiency of Tim4 in mice decreased CD301b+ macrophages and eventually magnified alveolar bone resorption in periodontitis. Additionally, Tim4 controlled the p38 MAPK signaling pathway to ultimately mediate CD301b+ macrophages phenotype. In a word, Tim4 might regulate CD301b+ macrophages through p38 MAPK signaling pathway in periodontitis, which provided new insights into periodontitis immunoregulation as well as help to develop innovative therapeutic targets and treatment strategies for periodontitis.

Dual Role of IGF2BP2 in Osteoimmunomodulation during Periodontitis.

Periodontitis is a complex disease characterized by distinct inflammatory stages, with a peak of inflammation in the early phase and less prominent inflammation in the advanced phase. The insulin-like growth factor 2-binding proteins 2 (IGF2BP2) has recently been identified as a new m6A reader that protects m6A-modified messenger RNAs (mRNAs) from decay, thus participating in multiple biological processes. However, its role in periodontitis remains unexplored. Here, we investigated the role of IGF2BP2 in inflammation and osteoclast differentiation using a ligature-induced periodontitis model. Our findings revealed that IGF2BP2 responded to bacterial-induced inflammatory stimuli and exhibited differential expression patterns in early and advanced periodontitis stages, suggesting its dual role in regulating this disease. Depletion of Igf2bp2 contributed to increased release of inflammatory cytokines, thereby exacerbating periodontitis after 3 d of ligature while suppressing osteoclast differentiation and ameliorating periodontitis after 14 d of ligature. Mechanistically, we demonstrated that IGF2BP2 directly interacted with Cd5l and Cd36 mRNA via RNA immunoprecipitation assay. Overexpression of CD36 or recombinant CD5L rescued the osteoclast differentiation ability of Igf2bp2-null cells upon lipopolysaccharide stimulus, and thus the downregulation of Cd36 and Cd5l effectively reversed periodontitis in the advanced stage. Altogether, this study deepens our understanding of the potential mechanistic link among the dysregulated m6A reader IGF2BP2, immunomodulation, and osteoclastogenesis during different stages of periodontitis.

Transferrin receptor 2 mitigates periodontitis-driven alveolar bone loss.

Periodontitis is associated with significant alveolar bone loss. Patients with iron overload suffer more frequently from periodontitis, however, the underlying mechanisms remain largely elusive. Here, we investigated the role of transferrin receptor 2 (Tfr2), one of the main regulators of iron homeostasis, in the pathogenesis of periodontitis and the dental phenotype under basal conditions in mice. As Tfr2 suppresses osteoclastogenesis, we hypothesized that deficiency of Tfr2 may exacerbate periodontitis-induced bone loss. Mice lacking Tfr2 (Tfr2-/- ) and wild-type (Tfr2+/+ ) littermates were challenged with experimental periodontitis. Mandibles and maxillae were collected for microcomputed tomography and histology analyses. Osteoclast cultures from Tfr2+/+ and Tfr2-/- mice were established and analyzed for differentiation efficiency, by performing messenger RNA expression and protein signaling pathways. After 8 days, Tfr2-deficient mice revealed a more severe course of periodontitis paralleled by higher immune cell infiltration and a higher histological inflammation index than Tfr2+/+ mice. Moreover, Tfr2-deficient mice lost more alveolar bone compared to Tfr2+/+ littermates, an effect that was only partially iron-dependent. Histological analysis revealed a higher number of osteoclasts in the alveolar bone of Tfr2-deficient mice. In line, Tfr2-deficient osteoclastic differentiation ex vivo was faster and more efficient as reflected by a higher number of osteoclasts, a higher expression of osteoclast markers, and an increased resorptive activity. Mechanistically, Tfr2-deficient osteoclasts showed a higher p38-MAPK signaling and inhibition of p38-MAPK signaling in Tfr2-deficient cells reverted osteoclast formation to Tfr2+/+ levels. Taken together, our data indicate that Tfr2 modulates the inflammatory response in periodontitis thereby mitigating effects on alveolar bone loss.

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

BACKGROUND: With 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. PURPOSE: This 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. METHODS: A 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. RESULTS: Baicalin 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. CONCLUSION: Intestinal 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.

Protective Effect of Bovine Milk Extracellular Vesicles on Alveolar Bone Loss.

SCOPE: Bovine milk extracellular vesicles (MEVs) have demonstrated therapeutic potential in regulating bone cell activity. However, the outcome of their use on alveolar bone loss has not yet been demonstrated. METHODS AND RESULTS: This study evaluates the effect of oral administration of MEVs on ovariectomized (OVX) mice. There is a reduced height of the alveolar bone crest in OVX mice by MEVs treatment, but the alveolar bone parameters are not altered. OVX mice are then submitted to a force-induced bone remodeling model by orthodontic tooth movement (OTM). MEVs-treated mice have markedly less bone remodeling movement, unlike the untreated OVX mice. Also, OVX mice treated with MEVs show an increased number of osteoblasts and osteocytes associated with higher sclerostin expression and reduce osteoclasts in the alveolar bone. Although the treatment with MEVs in OVX mice does not show differences in root structure in OTM, few odontoclasts are observed in the dental roots of OVX-treated mice. Compared to untreated mice, maxillary and systemic RANKL/OPG ratios are reduced in OVX mice treated with MEVs. CONCLUSION: Treatment with MEVs results in positive bone cell balance in the alveolar bone and dental roots, indicating its beneficial potential in treating alveolar bone loss in the nutritional context.

Omics-Based Mathematical Modeling Unveils Pathogenesis of Periodontitis in an Experimental Murine Model.

Periodontitis is a multifactorial disease that progresses via dynamic interaction between bacterial and host-derived genetic factors. The recent trend of omics analyses has discovered many periodontitis-related risk factors. However, how much the individual factor affects the pathogenesis of periodontitis is still unknown. This article aims to identify multiple key factors related to the pathogenesis of periodontitis and quantitatively predict the influence of each factor on alveolar bone resorption by omics analysis and mathematical modeling. First, we induced periodontitis in mice (n = 3 or 4 at each time point) by tooth ligation. Next, we assessed alveolar bone resorption by micro-computed tomography, alterations in the gene expression by RNA sequencing, and the microbiome of the gingivae by 16S ribosomal RNA sequencing during disease pathogenesis. Omics data analysis identified key players (bacteria and molecules) involved in the pathogenesis of periodontitis. We then constructed a mathematical model of the pathogenesis of periodontitis by employing ordinary differential equations that described the dynamic regulatory interplay between the key players and predicted the alveolar bone integrity as output. Finally, we estimated the model parameters using our dynamic experimental data and validated the model prediction of influence on alveolar bone resorption by in vivo experiments. The model predictions and experimental results revealed that monocyte recruitment induced by bacteria-mediated Toll-like receptor activation was the principal reaction regulating alveolar bone resorption in a periodontitis condition. On the other hand, osteoblast-mediated osteoclast differentiation had less impact on bone integrity in a periodontitis condition.

Osteoprotective effect by interleukin-4 (IL-4) on lipoprotein-induced periodontitis.

Lipoproteins are immunostimulatory bacterial components suggested to participate in inflammation-induced bone loss in periodontal disease through stimulation of osteoclast differentiation. Toll-like receptor 2 activation by Pam2CSK4 (PAM2), known to mimic bacterial lipoproteins, was previously shown to enhance periodontal bone resorption in mice. The anti-inflammatory cytokine interleukin-4 (IL-4) is a known inhibitor of RANKL-induced bone resorption in vitro. Here, we have investigated whether IL-4 could decrease PAM2-induced periodontal bone loss and osteoclastogenesis in vivo. In a model of periodontitis induced by gingival injections of PAM2 in mice, concomitant injections of IL-4 reduced bone loss. Histologically, IL-4 reduced the recruitment of inflammatory cells and the formation of TRAP+ osteoclasts stimulated by PAM2. Mouse bone marrow macrophages (BMMs) and neonatal calvarial osteoblasts were used to assess the effect of IL-4 on PAM2-induced osteoclastogenesis in vitro. In RANKL-primed BMMs stimulated by PAM2 Nfatc1, Ctsk, and Acp5 gene expression was up-regulated and resulted in robust formation of TRAP+ multinucleated osteoclasts, effects which were impaired by IL-4. These effects were mediated by impairment in PAM2-induced c-fos expression. In primary calvarial osteoblast cultures, IL-4 decreased PAM2-induced Tnfsf11 (encoding RANKL) mRNA and enhanced Tnfrsf11b (encoding OPG) expression. Our data demonstrate that the osteoprotective effect by IL-4 on lipoprotein-induced periodontal disease occurs through the inhibition of osteoclastogenesis by three mechanisms, one by acting directly on osteoclast progenitors, another by acting indirectly through decreasing the expression of osteoclast-regulating cytokines in osteoblasts and a third by decreasing inflammation.

Occlusal Force Maintains Alveolar Bone Homeostasis via Type H Angiogenesis.

Physiologically, teeth and periodontal tissues are exposed to occlusal forces throughout their lifetime. Following occlusal unloading, unbalanced bone remodeling manifests as a net alveolar bone (AB) loss. This phenomenon is termed alveolar bone disuse osteoporosis (ABDO), the underlying mechanism of which remains unclear. Type H vessels, a novel capillary subtype tightly coupled with osteogenesis, reportedly have a role in skeletal remodeling; however, their role in ABDO is not well studied. In the present study, we aimed to explore the pathogenesis of and therapies for ABDO. The study revealed that type H endothelium highly positive for CD31 and endomucin was identified in the periodontal ligament (PDL) but rarely in the AB of the mice. In hypofunctional PDL, the density of type H vasculature and coupled osterix+ (OSX+) osteoprogenitors declined significantly. In addition, the angiogenic factor Slit guidance ligand 3 (SLIT3) was downregulated in the disused PDL, and periodontal injection of the recombinant SLIT3 protein partially ameliorated type H vessel dysfunction and AB loss in ABDO mice. With regard to the molecular mechanism, a mechanosensory signaling circuit, PIEZO1/Ca2+/HIF-1α/SLIT3, was validated by applying cyclic compression to 3-dimensional-cultured PDL cells using the Flexcell FX-5000 compression system. In summary, PDL plays a pivotal role in mechanotransduction by translating physical forces into the intracellular signaling axis PIEZO1/Ca2+/HIF-1α/SLIT3, which promotes type H angiogenesis and OSX+ cell-related osteogenensis, thereby contributing to AB homeostasis. Our findings advance the understanding of PDL in AB disorders. Further therapies targeting SLIT3 may provide new insights into preventing bone loss in ABDO.

Blue Light-Emitting Diode Irradiation Without a Photosensitizer Alters Oral Microbiome Composition of Ligature-Induced Periodontitis in Mice.

Objective: This study investigated the suppressive effects of blue light-emitting diode (LED) irradiation on bone resorption and changes in the oral microbiome of mice with ligature-induced periodontitis. Background: Wavelength of blue light has antimicrobial effects; however, whether blue LED irradiation alone inhibits the progression of periodontitis remains unclear. Methods: Nine-week-old male mice ligated ligature around the right maxillary second molar was divided into ligation alone (Li) and ligation with blue LED irradiation (LiBL) groups. The LiBL group underwent blue LED (wavelength, 455 nm) irradiation four times in a week at 150 mW/cm2 without a photosensitizer on the gingival tissue around the ligated tooth at a distance of 5 mm for 5 min. The total energy density per day was 45 J/cm2. Bone resorption was evaluated using micro-computed tomography at 8 days. Differences in the oral microbiome composition of the collected ligatures between the Li and LiBL groups were analyzed using next-generation sequencing based on the 16S rRNA gene from the ligatures. Results: Blue LED irradiation did not suppress bone resorption caused by ligature-induced periodontitis. However, in the LiBL group, the α-diversity, number of observed features, and Chao1 were significantly decreased. The relative abundances in phylum Myxococcota and Bacteroidota were underrepresented, and the genera Staphylococcus, Lactococcus, and Lactobacillus were significantly overrepresented by blue LED exposure. Metagenomic function prediction indicated an increase in the downregulated pathways related to microbial energy metabolism after irradiation. The co-occurrence network was altered to a simpler structure in the LiBL group, and the number of core genera decreased. Conclusions: Blue LED irradiation altered the composition and network of the oral microbiome of ligature-induced periodontitis in mice.

Avenanthramide-C Shows Potential to Alleviate Gingival Inflammation and Alveolar Bone Loss in Experimental Periodontitis.

Periodontal disease is a chronic inflammatory disease that leads to the gradual destruction of the supporting structures of the teeth including gums, periodontal ligaments, alveolar bone, and root cementum. Recently, interests in alleviating symptoms of periodontitis (PD) using natural compounds is increasing. Avenanthramide-C (Avn-C) is a polyphenol found only in oats. It is known to exhibit various biological properties. To date, the effect of Avn-C on PD pathogenesis has not been confirmed. Therefore, this study aimed to verify the protective effects of Avn-C on periodontal inflammation and subsequent alveolar bone erosion in vitro and in vivo. Upregulated expression of catabolic factors, such as matrix metalloproteinase 1 (MMP1), MMP3, interleukin (IL)-6, IL-8, and COX2 induced by lipopolysaccharide and proinflammatory cytokines, IL-1ß, and tumor necrosis factor α (TNF-α), was dramatically decreased by Avn-C treatment in human gingival fibroblasts and periodontal ligament cells. Moreover, alveolar bone erosion in the ligature-induced PD mouse model was ameliorated by intra-gingival injection of Avn-C. Molecular mechanism studies revealed that the inhibitory effects of Avn-C on the upregulation of catabolic factors were mediated via ERK (extracellular signal-regulated kinase) and NF-κB pathway that was activated by IL-1ß or p38 MAPK and JNK signaling that was activated by TNF-α, respectively. Based on this study, we recommend that Avn-C may be a new natural compound that can be applied to PD treatment.

Artesunate ameliorates ligature-induced periodontitis by attenuating NLRP3 inflammasome-mediated osteoclastogenesis and enhancing osteogenic differentiation.

Periodontitis, arguably the greatest common infective chronic inflammatory disease, is characterized by an imbalance of the host immune system and excessive osteoclastogenesis activity with severe alveolar bone loss. Nevertheless, in consideration of the harmful effects of repeated treatment, more sensible intervention drugs for periodontitis need to be developed. Artesunate (ART), derived from Artemisia annua L., has shown remarkable pharmacokinetic and clinical value, as well as anti-inflammatory and immunomodulatory effects in various immune and chronic diseases due to its endoperoxide group. However, the role of ART in mediating periodontitis-induced alveolar bone resorption has not been examined. In this study, ART treatment effectively ameliorated ligature-induced periodontitis via attenuating osteoclast formation in a dose-dependent manner. Mechanistically, RNA-seq revealed that ART dramatically reduced the enrichment of NLRP3 inflammasome-related genes. Concordant with our study, MCC950, a specific inhibitor of NLRP3 inflammasome, also greatly restrained osteoclastogenesis, suggesting that ART suppressed osteoclast formation by blocking NLRP3 inflammasome activation. In addition to regulating osteoclastogenesis, ART significantly enhanced osteogenic differentiation by alleviating the expression of cytokines in inflammatory conditions. Our data shed light on the probably potential mechanism of ART treatment for the intervention of periodontitis.

HIF-1α regulates osteoclastogenesis and alveolar bone resorption in periodontitis via ANGPTL4.

OBJECTIVE: The mechanism of alveolar bone resorption caused by periodontitis is not fully understood. We sought to investigate whether microenvironmental changes of local hypoxia are involved in these processes. METHODS: In this study, periodontitis models of control mice and knockout of Hypoxia Induced Factor 1α (HIF-1α) harboring Cathepsin K (CTSK) Cre mice were constructed to study the effect of osteoclasts affected by hypoxic environment on alveolar bone resorption. RAW264.7 cells were subsequently induced by CoCl2 to observe the effects of HIF-1α and Angiopoietin-like Protein 4 (ANGPTL4) on osteoblast differentiation and fusion. RESULTS: The degree of alveolar bone resorption in the periodontitis tissues was lesser in mice with conditional knockout of HIF-1α in osteoclasts than in wild-type mice. We also observed that HIF-1α conditional knockout mice had fewer osteoclasts on the alveolar bone surface than control mice. HIF-1α increases the expression of ANGPTL4 and promotes the differentiation of RAW264.7 cells into osteoblasts and cell fusion under chemically simulated hypoxic conditions. CONCLUSION: HIF-1α regulates osteoclastogenesis and participates in bone resorption in periodontitis through ANGPTL4.

Modulatory influence of melatonin on apical periodontitis in Wistar rats fed a high-fat diet.

OBJECTIVE: This study aimed to evaluate the influence of high-fat diet (HFD) and melatonin (MEL) treatment on the progression of inflammation and alveolar bone resorption (ABR) in rats with AP. DESIGN: Forty male Wistar rats were divided into four groups: apical periodontitis (AP), HFDAP, APMEL and HFDAPMEL. The animals were fed an HFD or standard diet for 107 days. On the 7th day, the rats were subjected to AP, and after 70 days, the rats in the MEL groups were treated with MEL for 30 days. Post treatment, the animals were euthanized, and their jaws were retrieved for evaluation of bone resorption, intensity of the inflammatory response, and immunohistochemical analysis including tartrate-resistant acid phosphatase (TRAP) and interleukin-1ß (IL-1ß) levels and tumor necrosis factor (TNF)-α expression. RESULTS: The APMEL group showed reduction in the inflammatory infiltrate and IL-1ß expression relation to HFDAP, while the TNF-α levels did not differ among the groups. The HFDAP group showed an increase in the ABR. MEL reduced the TRAP levels in the APMEL and HFDAPMEL groups. CONCLUSIONS: while MEL could reduce TRAP levels in the APMEL and HFDAPMEL groups, the reduction in the HFDAPMEL group was smaller than that in the APMEL group, demonstrating that the interaction between AP and HFD decreased the anti-resorptive effects of MEL.

Reactive oxygen species-mediated endoplasmic reticulum stress contributes to osteocyte death induced by orthodontic compressive force.

During orthodontic tooth movement (OTM), osteocytes, the most mechanosensitive cells in alveolar bone, suffer the heavy orthodontic force and initiate alveolar bone resorption on the compression side. However, the inherent mechanisms of compressive force-induced osteocyte death are not fully understood. In this study, we established an OTM model on Sprague-Dawley rats by inserting coil springs to investigate osteocyte damage on the compression side of alveolar bone. We then applied compressive force on the MLO-Y4 osteocyte-like cell line in vitro to explore whether the reactive oxygen species (ROS)-mediated endoplasmic reticulum stress (ERS) pathway is involved in compressive force-induced osteocyte death. We found that the orthodontic force caused apparent alveolar bone loss, osteocyte death, and elevated serum sclerostin and receptor activator of NF-κB ligand (RANKL) levels in rats. In vitro, compressive force inhibited cell viability but increased the LDH leakage and loss of mitochondrial membrane potential in MLO-Y4 cells. Simultaneously, it activated protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2 (eIF2α), and their downstream pro-apoptotic ERS signaling proteins and caused significant osteocyte apoptosis, which can be blocked by ERS inhibitor salubrinal. Moreover, the compressive force elevated intracellular ROS levels, while the ROS scavenger N-acetyl-L-cysteine (NAC) alleviated ERS and apoptosis in loaded osteocytes. These results suggest that the orthodontic compressive force induced osteocyte apoptosis via the ROS-mediated ERS pathway. This study first proposes the ERS pathway as a new potential pathway for regulating the rate of OTM based on osteocyte death. RESEARCH HIGHLIGHTS: The orthodontic force increases osteocyte death in rat alveolar bone. The compressive force causes osteocyte apoptosis by the endoplasmic reticulum stress (ERS) pathway in vitro. The ROS scavenger NAC blocked compressive force-induced ERS and osteocyte apoptosis.

Glipizide Alleviates Periodontitis Pathogenicity via Inhibition of Angiogenesis, Osteoclastogenesis and M1/M2 Macrophage Ratio in Periodontal Tissue.

New consensus indicates type 2 diabetes mellitus (T2DM) and periodontitis as comorbidity and may share common pathways of disease progression. Sulfonylureas have been reported to improve the periodontal status in periodontitis patients. Glipizide, a sulfonylurea widely used in the treatment of T2DM, has also been reported to inhibit inflammation and angiogenesis. The effect of glipizide on the pathogenicity of periodontitis, however, has not been studied. We developed ligature-induced periodontitis in mice and treated them with different concentrations of glipizide and then analyzed the level of periodontal tissue inflammation, alveolar bone resorption, and osteoclast differentiation. Inflammatory cell infiltration and angiogenesis were analyzed using immunohistochemistry, RT-qPCR, and ELISA. Transwell assay and Western bolt analyzed macrophage migration and polarization. 16S rRNA sequencing analyzed the effect of glipizide on the oral microbial flora. mRNA sequencing of bone marrow-derived macrophages (BMMs) stimulated by P. gingivalis lipopolysaccharide (Pg-LPS) after treatment with glipizide was analyzed. Glipizide decreases alveolar bone resorption, periodontal tissue degradation, and the number of osteoclasts in periodontal tissue affected by periodontitis (PAPT). Glipizide-treated periodontitis mice showed reduced micro-vessel density and leukocyte/macrophage infiltration in PAPT. Glipizide significantly inhibited osteoclast differentiation in vitro experiments. Glipizide treatment did not affect the oral microbiome of periodontitis mice. mRNA sequencing and KEGG analysis showed that glipizide activated PI3K/AKT signaling in LPS-stimulated BMMs. Glipizide inhibited the LPS-induced migration of BMMs but promoted M2/M1 macrophage ratio in LPS-induced BMMs via activation of PI3K/AKT signaling. In conclusion, glipizide inhibits angiogenesis, macrophage inflammatory phenotype, and osteoclastogenesis to alleviate periodontitis pathogenicity suggesting its' possible application in the treatment of periodontitis and diabetes comorbidity.

The adiponectin receptor agonist AdipoAI attenuates periodontitis in diabetic rats by inhibiting gingival fibroblast-induced macrophage migration.

BACKGROUND AND PURPOSE: Low-grade inflammation, a common feature of both diabetes and periodontitis, partly accounts for the complexity and refractoriness of diabetes-associated periodontitis. Adiponectin (APN), the most abundant adipokine in human blood, has been widely reported to have anti-inflammatory functions. Herein, we investigated the ability of an APN receptor agonist, AdipoAI, to alleviate diabetes-associated periodontitis. Furthermore, we revealed the possible mechanism underlying its anti-inflammatory effects. EXPERIMENTAL APPROACH: The maxillary first molar of Zucker diabetic fatty (ZDF) rats was ligated to construct a diabetes-associated periodontitis model, and rats were administered AdipoAI by gavage. We examined diabetes-related indexes, pathological changes in insulin target organs, alveolar bone resorption and systemic and local inflammation. In vitro, transwell assays were used to evaluate monocyte/macrophage migration induced by human gingival fibroblasts (hGFs) with/without AdipoAI treatment. Additionally, we examined chemokine expression levels in hGFs and hGF-induced monocyte/macrophage migration upon siRNA knockdown of Adiponectin receptor expression. Expression of Adipo1/Adipo2 receptors and inflammation-related signalling pathways were examined by IHC and WB, followed by confirmation with an NF-κB P65 inhibitor (BAY 11-7082). KEY RESULTS: AdipoAI lowered fasting blood glucose and serum insulin in ZDF rats and alleviated inflammation in insulin target tissues. Locally, AdipoAI reduced alveolar bone absorption and gingival inflammation. Mechanistically, AdipoAI inhibited hGF-induced monocyte/macrophage migration by reducing CCL2 secretion. In hGFs, AdipoAI attenuated LPS-induced activation of NF-κB P65 and CCL2 expression, which was dependent on the Adipo receptor 1. CONCLUSION AND IMPLICATIONS: AdipoAI, with its ability to alleviate inflammatory damage in tissues, is a candidate for diabetes-associated periodontitis treatment.

Extracellular Release of Citrullinated Vimentin Directly Acts on Osteoclasts to Promote Bone Resorption in a Mouse Model of Periodontitis.

Elevated osteoclast (OC)-mediated bone resorption, a common pathological feature between periodontitis and rheumatoid arthritis (RA), implicates a possible mutually shared pathogenesis. The autoantibody to citrullinated vimentin (CV), a representative biomarker of RA, is reported to promote osteoclastogenesis (OC-genesis). However, its effect on OC-genesis in the context of periodontitis remains to be elucidated. In an in vitro experiment, the addition of exogenous CV upregulated the development of Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear OCs from mouse bone marrow cells and increased the formation of resorption pits. However, Cl-amidine, an irreversible pan-peptidyl arginine deiminase (PAD) inhibitor, suppressed the production and secretion of CV from RANKL-stimulated OC precursors, suggesting that the citrullination of vimentin occurs in OC precursors. On the other hand, the anti-vimentin neutralizing antibody suppressed in vitro Receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced OC-genesis. The CV-induced upregulation of OC-genesis was abrogated by the Protein kinase C (PKC)-δ inhibitor Rottlerin, accompanied by the downmodulation of OC-genesis-related genes, including Osteoclast stimulatory transmembrane protein (OC-STAMP), TRAP and Matrix Metallopeptidase 9 (MMP9) as well as extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP)-kinase phosphorylation. Elevated levels of soluble CV and vimentin-bearing mononuclear cells were found in the bone resorption lesions of periodontitis induced in mice in the absence of an anti-CV antibody. Finally, local injection of anti-vimentin neutralizing antibody suppressed the periodontal bone loss induced in mice. Collectively, these results indicated that the extracellular release of CV promoted OC-genesis and bone resorption in periodontitis.

Pirfenidone Inhibits Alveolar Bone Loss in Ligature-Induced Periodontitis by Suppressing the NF-κB Signaling Pathway in Mice.

There has been increasing interest in adjunctive use of anti-inflammatory drugs to control periodontitis. This study was performed to examine the effects of pirfenidone (PFD) on alveolar bone loss in ligature-induced periodontitis in mice and identify the relevant mechanisms. Experimental periodontitis was established by ligating the unilateral maxillary second molar for 7 days in mice (n = 8 per group), and PFD was administered daily via intraperitoneal injection. The micro-computed tomography and histology analyses were performed to determine changes in the alveolar bone following the PFD administration. For in vitro analysis, bone marrow macrophages (BMMs) were isolated from mice and cultured with PFD in the presence of RANKL or LPS. The effectiveness of PFD on osteoclastogenesis, inflammatory cytokine expression, and NF-κB activation was determined with RT-PCR, Western blot, and immunofluorescence analyses. PFD treatment significantly inhibited the ligature-induced alveolar bone loss, with decreases in TRAP-positive osteoclasts and expression of inflammatory cytokines in mice. In cultured BMM cells, PFD also inhibited RANKL-induced osteoclast differentiation and LPS-induced proinflammatory cytokine (IL-1ß, IL-6, TNF-a) expression via suppressing the NF-κB signal pathway. These results suggest that PFD can suppress periodontitis progression by inhibiting osteoclastogenesis and inflammatory cytokine production via inhibiting the NF-κB signal pathway, and it may be a promising candidate for controlling periodontitis.