Local delivery of a CXCR3 antagonist decreases the progression of bone resorption induced by LPS injection in a murine model.

OBJECTIVES: This experimental study was carried out to investigate the effects of locally delivered nanoparticles (AMG-487 NP) containing a CXCR3 antagonist in inhibiting the progression of LPS-induced inflammation, osteoclastic activity, and bone resorption on a murine model. MATERIALS AND METHODS: Thirty, 7-week-old C57BL/6 J male mice were used. Inflammatory bone loss was induced by Porphyromonas gingivalis-lipopolysaccharide (P.g.-LPS) injections between the first and second maxillary molars, bilaterally, twice a week for 6 weeks (n = 20). AMG-487 NP were incorporated into a liposome carrier and locally delivered on sites where P.g.-LPS was injected. Control mice (n = 10) were injected with vehicle only. Experimental groups included (1) control, (2) LPS, and (3) LPS + NP. At the end of 1 and 6 weeks, mice were euthanized, maxillae harvested, fixed, and stored for further analysis. RESULTS: Volumetric bone loss analysis revealed, at 1 week, an increase in bone loss in the LPS group (47.9%) compared to control (27.4%) and LPS + NP (27.8%) groups. H&E staining demonstrated reduced inflammatory infiltrate in the LPS + NP group compared to LPS group. At 6 weeks, volumetric bone loss increased in all groups; however, treatment with the CXCR3 antagonist (LPS + NP) significantly reduced bone loss compared to the LPS group. CXCR3 antagonist treatment significantly reduced osteoclast numbers when compared to LPS group at 1 and 6 weeks. CONCLUSIONS: This study showed that local delivery of a CXCR antagonist, via nanoparticles, in a bone resorption model, induced by LPS injection, was effective in reducing inflammation, osteoclast numbers, and bone loss. CLINICAL RELEVANCE: CXCR3 blockade can be regarded as a novel target for therapeutic intervention of bone loss. It can be a safe and convenient method for periodontitis treatment or prevention applicable in clinical practice.

Ligature-induced peri-implantitis and periodontitis in mice.

AIM: Peri-implantitis (PI), inflammation around dental implants, shares characteristics with periodontitis (PD). However, PI is more difficult to control and treat, and detailed pathophysiology is unclear. We aimed to compare PI and PD progression utilizing a murine model. MATERIALS AND METHODS: Four-week-old male C57BL/6J mice had their left maxillary molars extracted. Implants were placed in healed extraction sockets and osseointegrated. Ligatures were tied around the implants and second molars. Controls did not receive ligatures. Mice were sacrificed 1 week, 1 and 3 months (n ≥ 5/group/time point) post-ligature placement. Bone loss analysis was performed. Histology was performed for: haematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-8 (MMP-8), nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), toluidine blue and calcein. RESULTS: PI showed statistically greater bone loss compared to PD at 1 and 3 months. At 3 months, 20% of implants in PI exfoliated; no natural teeth exfoliated in PD. H&E revealed that alveolar bone surrounding implants in PI appeared less dense compared to PD. PI presented with increased osteoclasts, MMP-8 and NF-κB, compared to PD. CONCLUSION: PI exhibited greater tissue and bone destruction compared to PD. Future studies will characterize the pathophysiological differences between the two conditions.

Nigella sativa oil mitigates xerostomia and preserves salivary function in radiotherapy-treated mice.

Objective: This study aimed to assess if Nigella sativa oil (NSO), a health supplement containing thymoquinone as a major component, can act as a protective agent in salivary gland stem cells following radiotherapy (RT) damage. Methods: Forty, 10-week-old, male C3H/HeJ mice were randomized to four experimental groups: sham RT + H2O gavage (control) (N = 4); 15 Gy RT + H2O gavage (N = 12); sham RT + NSO gavage (N = 12); and 15 Gy RT + NSO gavage (N = 12). Weight changes, saliva production, and salivary gland histopathologic staining were recorded for each group over the course of the experiment. Results: All mice in the sham RT + H2O gavage and sham RT + NSO gavage groups demonstrated 100% 60-day survival. RT + H2O compared to RT + NSO gavaged mice were significantly underweight by an average of 6.4 g (p < .001). Salivary output showed significant decline in RT + H2O gavaged mice at days 3 and 16, whereas salivary output in RT + NSO during these same time periods was comparable to the control. At day 60, all mice that survived recovered salivary function regardless of their treatment arm. Salivary specimens from the RT + NSO gavage group demonstrated early signs of recovery of Kr 5+ salivary gland stem cells in both submandibular and sublingual glands at day 16 with complete recovery by day 60, marked by strong histopathologic staining, whereas the RT + H2O gavage group did not recover as effectively. Conclusion: NSO may help preserve salivary function in mice treated with RT and may mitigate xerostomia by accelerating the recovery of salivary gland stem cells. Level of evidence: Not applicable.

Greater Telehealth Use Results in Increased Visit Frequency and Lower Physician Related-Distress in Adolescents and Young Adults With Type 1 Diabetes.

BACKGROUND: Type one diabetes (T1D) management is challenging for adolescents and young adults (AYAs) due to physiological changes, psychosocial challenges, and increasing independence, resulting in increased diabetes distress and hemoglobin A1c (HbA1c). Alternative care models that engage AYAs and improve diabetes-related health outcomes are needed. METHODS: A 15-month study evaluated an adaptation of the Colorado Young Adults with T1D (CoYoT1) Care model. CoYoT1 Care includes person-centered care, virtual peer groups, and physician training delivered via telehealth. AYAs (aged 16-25 years) were partially randomized to CoYoT1 or standard care, delivered via telehealth or in-person. As the study was ending, the COVID-19 pandemic forced all AYAs to transition to primarily telehealth appointments. This secondary analysis compares changes in clinic attendance, T1D-related distress, HbA1c, and device use between those who attended more than 50% of diabetes clinic visits via telehealth and those who attended more sessions in-person throughout the course of the study. RESULTS: Out of 68 AYA participants, individuals (n = 39, 57%) who attended most (>50%) study visits by telehealth completed more diabetes care visits (3.3 visits) than those (n = 29, 43%) who primarily attended visits in-person (2.5 visits; P = .007). AYAs who primarily attended visits via telehealth maintained stable physician-related distress, while those who attended more in-person visits reported increases in physician-related distress (P = .03). CONCLUSIONS: Greater usage of telehealth improved AYA engagement with their care, resulting in increased clinic attendance and reduced physician-related diabetes distress. A person-centered care model delivered via telehealth effectively meets the needs of AYAs with T1D.

Virtual Peer Groups Reduce HbA1c and Increase Continuous Glucose Monitor Use in Adolescents and Young Adults with Type 1 Diabetes.

Background: Adolescents and young adults (AYA) from diverse and marginalized backgrounds with type 1 diabetes (T1D) generally have higher hemoglobin A1c (HbA1c) levels and less frequent continuous glucose monitor (CGM) use than AYA from more privileged backgrounds. Further, scant data address the impact of virtual peer groups (VPG) on health-related outcomes for ethnically and racially diverse AYA with T1D. Methods: CoYoT1 to California was a 15-month randomized controlled trial for AYA aged 16-25 years. In this study, AYA were randomized to receive standard care (n = 28), or CoYoT1 care (n = 40), which consisted of person-centered provider visits and bimonthly VPG. VPG were AYA-driven discussions. AYA completed the Diabetes Distress Scale (DDS), Center for Epidemiologic Studies Depression (CES-D), and Diabetes Empowerment Scale-Short Form (DES-SF) scales at baseline and all study visits. Results: Participants were 50% Latinx and 75% publicly insured. Among CoYoT1 care participants, 19 attended at least 1 VPG session (VPG attendees) and 21 did not attend any VPG sessions. VPG attendees participated in 4.1 VPG sessions on average. VPG attendees had a relative reduction in HbA1C (treatment effect -1.08%, effect sizes values [ES] = -0.49, P = 0.04) and increase in CGM use (treatment effect +47%, ES = 1.00, P = 0.02) compared to standard care. VPG participation was not associated with statistically significant changes in DDS, CES-D, and DES-SF scores. Conclusions: In a 15-month randomized controlled trial, AYA with T1D who participated in VPG reported significant improvements in HbA1c and CGM use. Peer interactions may support unmet needs of AYA with T1D from diverse and marginalized backgrounds. ClinicalTrials.gov Identifier: NCT03793673.

Gliclazide reduced oxidative stress, inflammation, and bone loss in an experimental periodontal disease model.

OBJECTIVE: The aim of this study was to evaluate the effects of gliclazide on oxidative stress, inflammation, and bone loss in an experimental periodontal disease model. MATERIAL AND METHODS: Male albino Wistar rats were divided into no ligature, ligature, and ligature with 1, 5, and 10 mg/kg gliclazide groups. Maxillae were fixed and scanned using micro-computed tomography to quantify linear and bone volume/tissue volume (BV/TV) and volumetric bone loss. Histopathological, immunohistochemical and immunofluorescence analyses were conducted to examine matrix metalloproteinase-2 (MMP-2), cyclooxygenase 2 (COX-2), cathepsin K, members of the receptor activator of the nuclear factor kappa-Β ligand (RANKL), receptor activator of nuclear factor kappa-Β (RANK), osteoprotegerin (OPG) pathway, macrophage migration inhibitory factor (MIF), superoxide dismutase-1 (SOD-1), glutathione peroxidase-1 (GPx-1), NFKB p 50 (Cytoplasm), NFKB p50 NLS (nuclear localization signal), PI3 kinase and AKT staining. Myeloperoxidase activity, malondialdehyde and glutathione levels, while interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by spectroscopic ultraviolet-visible analysis. A quantitative reverse transcription polymerase chain reaction was used to quantify the gene expression of the nuclear factor kappa B p50 subunit (NF-κB p50), phosphoinositide 3-kinase (PI3k), protein kinase B (AKT), and F4/80. RESULTS: Micro-computed tomography showed that the 1 mg/kg gliclazide treatment reduced linear bone loss compared to the ligature, 5 mg/kg gliclazide, and 10 mg/kg gliclazide treatments. All concentrations of gliclazide increased bone volume/tissue volume (BV/TV) compared to the ligature group. Treatment with 1 mg/kg gliclazide reduced myeloperoxidase activity, malondialdehyde, IL-1ß, and TNF-α levels (p≤0.05), and resulted in weak staining for COX-2, cathepsin k, MMP-2, RANK, RANKL, SOD-1, GPx-1,MIF and PI3k. In addition, down-regulation of NF-κB p50, PI3k, AKT, and F4/80 were observed, and OPG staining was strong after the 1 mg/kg gliclazide treatment. CONCLUSIONS: This treatment decreased neutrophil and macrophage migration, decreased the inflammatory response, and decreased bone loss in rats with ligature-induced periodontitis.

Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies.

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.

Genomewide Association Study Identifies Cxcl Family Members as Partial Mediators of LPS-Induced Periodontitis.

Periodontitis (PD) is characterized by bacterial infection and inflammation of tooth-supporting structures and can lead to tooth loss. PD affects ∼47% of the US population over age 30 years and has a heritability of about 50%. Although the host immunoinflammatory response and genetic background play a role, little is known of the underlying genetic factors. We examined natural genetic variation in lipopolysaccharide (LPS)-induced PD across a panel of inbred mouse strains, the hybrid mouse diversity panel (HMDP). We observed a strain-dependent sixfold difference in LPS-induced bone loss across the HMDP with a heritability of 53%. We performed a genomewide association study (GWAS) using FAST-LMM, which corrects for population structure, and identified loci significantly associated with PD. We examined candidate genes at a locus on chromosome 5, which suggested a relationship between LPS-induced bone loss and, together with expression data, identified Cxcl family members as associated with PD. We observed an increase in Cxcl10 protein, as well as immune cells and pro-inflammatory cytokines in C57BL/6J (high bone loss strain) but not in A/J (low bone loss strain) after LPS injections. Genetic deletion of CXCR3 (Cxcl9 and10 receptor) demonstrated a ∼50% reduction in bone loss and reduced osteoclasts after LPS injections. Furthermore, WT mice treated with AMG-487 (a CXCR3 antagonist) showed a ∼45% reduction in bone loss and decreased osteoclasts after LPS injections. We conclude that CXCR3 is a strong candidate for modulating the host response in individuals susceptible to PD. © 2018 American Society for Bone and Mineral Research.

Early intervention of peri-implantitis and periodontitis using a mouse model.

BACKGROUND: Peri-implantitis is an inflammatory response to bacterial biofilm resulting in bone loss and can ultimately lead to implant failure. Because of the lack of predictable treatments available, a thorough understanding of peri-implantitis's pathogenesis is essential. The objective of this study is to evaluate and compare the response of acute induced peri-implantitis and periodontitis lesions after insult removal. METHODS: Implants were placed in one-month-old C57BL/6J male mice eight weeks post extraction of their left maxillary molars. Once osseointegrated, ligatures were placed around the implants and contralateral second molars of the experimental groups. Controls did not receive ligatures. After one week, half of the ligatures were removed, creating the ligature-retained and ligature-removed groups. Mice were sacrificed at two time points, 5 and 14 days, from ligature removal. The specimens were analyzed via micro-computed tomography and histology. RESULTS: By 5 and 14 days after ligature removal, the periodontitis group experienced significant bone gain, whereas the peri-implantitis group did not. Histologically, all implant groups exhibited higher levels of cellular infiltrate than any of the tooth groups. Osteoclast numbers increased in peri-implantitis and periodontitis ligature-retained groups and decreased following insult removal. Collagen was overall more disorganized in peri-implantitis than periodontitis for all groups. Peri-implantitis experimental groups revealed greater matrix metalloproteinase-8 and NF-kB levels than periodontitis. CONCLUSIONS: Implants respond slower and less favorably to insult removal than teeth. Future research is needed to characterize detailed peri-implantitis disease pathophysiology.

Comparing the Healing Potential of Late-Stage Periodontitis and Peri-Implantitis.

Peri-implantitis is defined as an inflammatory disease affecting the tissues around osseointegrated functioning implants. Unfortunately, detailed peri-implantitis pathogenesis is not well understood and current treatments lack predictability. Compare the healing potential of late-stage ligature-induced periodontitis and peri-implantitis after ligature removal. Four-week-old C57BL/6J male mice had their left maxillary molars extracted. After 8 weeks, implants were placed in healed sockets and allowed to osseointegrate. Mice were separated into control (no ligature) and experimental (ligature) groups. In the experimental group, ligatures were placed around the implant and the contralateral second molar. Four weeks later, the ligature group was randomly divided into ligature-retained and ligature-removed groups. Mice were sacrificed at 2 time points: 1 and 2 weeks after ligature removal. The samples were analyzed by microcomputed tomography (micro-CT) and histology. Ligature-induced significant bone loss in peri-implantitis and periodontitis were compared with respective controls. At the 2-week time point, bone formation was observed in the ligature-removed groups compared with respective controls; however, more bone was regained in periodontitis ligature-removed compared with the peri-implantitis ligature-removed group. Histologically, the peri-implantitis ligature-retained group had higher inflammatory levels and a higher number of osteoclasts compared with the periodontitis ligature-retained group. Moreover, in the peri-implantitis ligature-retained group, collagen appeared less organized compared with the periodontitis ligature-retained group at both time points; although collagen tended to reorganize following ligature removal in both conditions. Peri-implantitis does not respond to treatment as well as periodontitis. Future work includes understanding peri-implantitis pathogenesis and developing predictable treatment protocols.

Effects of metformin on inflammation, oxidative stress, and bone loss in a rat model of periodontitis.

AIM: To evaluate the effects of metformin (Met) on inflammation, oxidative stress, and bone loss in a rat model of ligature-induced periodontitis. MATERIALS & METHODS: Male albino Wistar rats were divided randomly into five groups of twenty-one rats each, and given the following treatments for 10 days: (1) no ligature + water, (2) ligature + water, (3) ligature + 50 mg/kg Met, (4) ligature + 100 mg/kg Met, and (5) ligature + 200 mg/kg Met. Water or Met was administered orally. Maxillae were fixed and scanned using Micro-computed Tomography (µCT) to quantitate linear and bone volume/tissue volume (BV/TV) volumetric bone loss. Histopathological characteristics were assessed through immunohistochemical staining for MMP-9, COX-2, the RANKL/RANK/OPG pathway, SOD-1, and GPx-1. Additionally, confocal microscopy was used to analyze osteocalcin fluorescence. UV-VIS analysis was used to examine the levels of malondialdehyde, glutathione, IL-1ß and TNF-α from gingival tissues. Quantitative RT-PCR reaction was used to gene expression of AMPK, NF-κB (p65), and Hmgb1 from gingival tissues. Significance among groups were analysed using a one-way ANOVA. A p-value of p<0.05 indicated a significant difference. RESULTS: Treatment with 50 mg/kg Met significantly reduced concentrations of malondialdehyde, IL-1ß, and TNF-α (p < 0.05). Additionally, weak staining was observed for COX-2, MMP-9, RANK, RANKL, SOD-1, and GPx-1 after 50 mg/kg Met. OPG and Osteocalcin showed strong staining in the same group. Radiographically, linear measurements showed a statistically significant reduction in bone loss after 50 mg/kg Met compared to the ligature and Met 200 mg/kg groups. The same pattern was observed volumetrically in BV/TV and decreased osteoclast number (p<0.05). RT-PCR showed increased AMPK expression and decreased expression of NF-κB (p65) and HMGB1 after 50 mg/kg Met. CONCLUSIONS: Metformin, at a concentration of 50 mg/kg, decreases the inflammatory response, oxidative stress and bone loss in ligature-induced periodontitis in rats.

A Murine Model of Lipopolysaccharide-Induced Peri-Implant Mucositis and Peri-Implantitis.

Dental implants are a widely used treatment option for tooth replacement. However, they are susceptible to inflammatory diseases such as peri-implant mucositis and peri-implantitis, which are highly prevalent and may lead to implant loss. Unfortunately, the understanding of the pathogenesis of peri-implant mucositis and peri-implantitis is fragmented and incomplete. Therefore, the availability of a reproducible animal model to study these inflammatory diseases would facilitate the dissection of their pathogenic mechanisms. The objective of this study is to propose a murine model of experimental peri-implant mucositis and peri-implantitis. Screw-shaped titanium implants were placed in the upper healed edentulous alveolar ridges of C57BL/6J mice 8 weeks after tooth extraction. Following 4 weeks of osseointegration, Porphyromonas gingivalis -lipolysaccharide (LPS) injections were delivered to the peri-implant soft tissues for 6 weeks. No-injections and vehicle injections were utilized as controls. Peri-implant mucositis and peri-implantitis were assessed clinically, radiographically (microcomputerized tomograph [CT]), and histologically following LPS-treatment. LPS-injections resulted in a significant increase in soft tissue edema around the head of the implants as compared to the control groups. Micro-CT analysis revealed significantly greater bone loss in the LPS-treated implants. Histological analysis of the specimens demonstrated that the LPS-group had increased soft tissue vascularity, which harbored a dense mixed inflammatory cell infiltrate, and the bone exhibited noticeable osteoclast activity. The induction of peri-implant mucositis and peri-implantitis in mice via localized delivery of bacterial LPS has been demonstrated. We anticipate that this model will contribute to the development of more effective preventive and therapeutic approaches for these 2 conditions.

Gliclazide reduced oxidative stress, inflammation, and bone loss in an experimental periodontal disease model

Abstract Objective The aim of this study was to evaluate the effects of gliclazide on oxidative stress, inflammation, and bone loss in an experimental periodontal disease model. Material and Methods Male albino Wistar rats were divided into no ligature, ligature, and ligature with 1, 5, and 10 mg/kg gliclazide groups. Maxillae were fixed and scanned using micro-computed tomography to quantify linear and bone volume/tissue volume (BV/TV) and volumetric bone loss. Histopathological, immunohistochemical and immunofluorescence analyses were conducted to examine matrix metalloproteinase-2 (MMP-2), cyclooxygenase 2 (COX-2), cathepsin K, members of the receptor activator of the nuclear factor kappa-Β ligand (RANKL), receptor activator of nuclear factor kappa-Β (RANK), osteoprotegerin (OPG) pathway, macrophage migration inhibitory factor (MIF), superoxide dismutase-1 (SOD-1), glutathione peroxidase-1 (GPx-1), NFKB p 50 (Cytoplasm), NFKB p50 NLS (nuclear localization signal), PI3 kinase and AKT staining. Myeloperoxidase activity, malondialdehyde and glutathione levels, while interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by spectroscopic ultraviolet-visible analysis. A quantitative reverse transcription polymerase chain reaction was used to quantify the gene expression of the nuclear factor kappa B p50 subunit (NF-κB p50), phosphoinositide 3-kinase (PI3k), protein kinase B (AKT), and F4/80. Results Micro-computed tomography showed that the 1 mg/kg gliclazide treatment reduced linear bone loss compared to the ligature, 5 mg/kg gliclazide, and 10 mg/kg gliclazide treatments. All concentrations of gliclazide increased bone volume/tissue volume (BV/TV) compared to the ligature group. Treatment with 1 mg/kg gliclazide reduced myeloperoxidase activity, malondialdehyde, IL-1β, and TNF-α levels (p≤0.05), and resulted in weak staining for COX-2, cathepsin k, MMP-2, RANK, RANKL, SOD-1, GPx-1,MIF and PI3k. In addition, down-regulation of NF-κB p50, PI3k, AKT, and F4/80 were observed, and OPG staining was strong after the 1 mg/kg gliclazide treatment. Conclusions This treatment decreased neutrophil and macrophage migration, decreased the inflammatory response, and decreased bone loss in rats with ligature-induced periodontitis.

Dental diagnostics: molecular analysis of oral biofilms.

Dental biofilms are complex, multi-species bacterial communities that colonize the mouth in the form of plaque and are known to cause dental caries and periodontal disease. Biofilms are unique from planktonic bacteria in that they are mutualistic communities with a 3-dimensional structure and complex nutritional and communication pathways. The homeostasis within the biofilm colony can be disrupted, causing a shift in the bacterial composition of the colony and resulting in proliferation of pathogenic species. Because of this dynamic lifestyle, traditional microbiological techniques are inadequate for the study of biofilms. Many of the bacteria present in the oral cavity are viable but not culturable, which severely limits laboratory analysis. However, with the advent of new molecular techniques, the microbial makeup of oral biofilms can be better identified. Some of these techniques include DNA-DNA hybridization, 16S rRNA gene sequencing, denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism, denaturing high-performance liquid chromatography and pyrosequencing. This review provides an overview of biofilm formation and examines the major molecular techniques currently used in oral biofilm analysis. Future applications of the molecular analysis of oral biofilms in the diagnosis and treatment of caries and periodontal disease are also discussed.