Mitochondrial biogenesis disorder and oxidative damage promote refractory apical periodontitis in rat and human.

AIM: To elucidate whether mitochondrial biogenesis disorder and damage from oxidative stress promote refractory apical periodontitis (RAP) in rat and human. METHODOLOGY: Twenty Enterococcus faecalis-induced RAPs were established in the maxillary first molars of male Wistar rats. Concurrently, 12 periapical lesion specimens from patients presenting with RAP were obtained by apicoectomy. Radiographic examination and histologic analysis were conducted to evaluate periapical bone tissue destruction and morphological changes. The expression of key regulators of mitochondrial biogenesis, PGC-1α and Nrf2, were detected by immunohistochemistry and double immunofluorescence staining, Western blot and real-time PCR were also assayed. Mitochondrial ROS (mtROS) was identified by MitoSOX staining. Mitochondrial function was detected by the quantification of ATP production, mitochondrial DNA (mtDNA) copy number and activities of mitochondrial respiratory chain complexes. Furthermore, mitochondrial oxidative stress was evaluated by the determination of 3-nitrotyrosine (3-NT), 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-deoxyguanosine (8-OHdG) expression levels, as well as malondialdehyde (MDA) expression and antioxidant capacity. Student's t-test was performed to determine significance between the groups; p < .05 was considered significant. RESULTS: In the maxilla, significantly more bone resorption, greater number of periapical apoptotic cells and Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were observed in the RAP group compared with the control group (p < .01). PGC-1α and Nrf2 were significantly reduced in rat and human RAP lesions compared to the control group (p < .01) at both the mRNA and protein levels. Double immunofluorescence analysis of PGC-1α or Nrf2 with TOMM20 also indicated that mitochondrial biogenesis was impaired in RAP group (p < .01). Additionally, mitochondrial dysfunction was observed in RAP group, as reflected by increased mtROS, decreased ATP production, reduced mtDNA copy number and complexes of the mitochondrial respiratory chain. Finally, the expression levels of mitochondrial oxidative stress markers, 3-NT, 4-HNE and 8-OHdG, were significantly increased in the RAP group (p < .01). Consistent with this, systemic oxidative damage was also present in the progression of RAP, including increased MDA expression and decreased antioxidant activity (p < .01). CONCLUSIONS: Mitochondrial biogenesis disorder and damage from oxidative stress contribute to the development of RAP.

Pre-treatment oral microbiome analysis and salivary Stephan curve kinetics in white spot lesion development in orthodontic patients wearing fixed appliances. A pilot study.

BACKGROUND: White spot lesions (WSLs) are a formidable challenge during orthodontic treatment, affecting patients regardless of oral hygiene. Multifactorial in nature, amongst potential contributors to their development are the microbiome and salivary pH. The aim of our pilot study is to determine if pre-treatment differences in salivary Stephan curve kinetics and salivary microbiome features correlate with WSL development in orthodontic patients with fixed appliances. We hypothesize that non-oral hygiene determined differences in saliva could be predictive of WSL formation in this patient population through analysis of salivary Stephan curve kinetics, and that these differences would further manifest as changes in the oral microbiome. METHODS: In this prospective cohort study, twenty patients with initial simplified oral hygiene index scores of "good" that were planning to undergo orthodontic treatment with self-ligating fixed appliances for at least 12 months were enrolled. At pre-treatment stage, saliva was collected for microbiome analysis, and at 15-minute intervals after a sucrose rinse over 45 min for Stephan curve kinetics. RESULTS: 50% of patients developed a mean 5.7 (SEM: 1.2) WSLs. There were no differences in saliva microbiome species richness, Shannon alpha diversity or beta diversity between the groups. Capnocytophaga sputigena exclusively and Prevotella melaninogenica predominantly were found in WSL patients, while Streptococcus australis was negatively correlated with WSL development. Streptococcus mitis and Streptococcus anginosus were primarily present in healthy patients. There was no evidence to support the primary hypothesis. CONCLUSIONS: While there were no differences in salivary pH or restitution kinetics following a sucrose challenge and no global microbial differences in WSL developers, our data showed change in salivary pH at 5 min associated with an abundance of acid-producing bacteria in saliva. The results suggest salivary pH modulation as a management strategy to inhibit the abundance of caries initiators. Our study may have uncovered the earliest predecessors to WSL/caries development.

A peptide coating preventing the attachment of Porphyromonas gingivalis on the surfaces of dental implants.

OBJECTIVES: The aim of this study was to investigate whether a peptide-based coating can prevent the adhesion of Porphyromonas gingivalis, a key human pathogen associated with periodontitis and peri-implantitis. BACKGROUND: Nonsurgical and surgical interventions have been used for the treatment of peri-implantitis; however, the effectiveness of these approaches is usually unsatisfactory. The main reason is that dental plaque on the surface of the implant is difficult to remove due to its rough surface and thread design. Recently, a peptide-based coating for implant surfaces that can reject the adhesion of Escherichia coli and improve the attachment of host cells was developed. METHODS: A salivary pellicle was created on the surfaces of peptide-coated bare discs and verified with anti-human immunoglobulin G, A and M, and anti-fibrinogen. Early colonizers, Veillonella parvula and Streptococcus sobrinus, and the later colonizer, Porphyromonas gingivalis, were labelled with green and red fluorescent dyes, respectively, and seeded on the discs. Bacterial attachment was semi-quantified by fluorescence intensity. RESULTS: The salivary pellicle was evenly distributed on the discs, with or without the peptide coating, with an average thickness of 3.84 µm. A multi-species dental biofilm was created on the salivary pellicle. The peptide coating resulted in an approximate 25% reduction in the attachment of Veillonella parvula and Streptococcus sobrinus, and a 50% reduction in Porphyromonas gingivalis, when compared to control, uncoated implant discs. CONCLUSION: The novel peptide-based coating can inhibit the attachment of Porphyromonas gingivalis. It may have the potential to impede the development of peri-implantitis.

DNA methylation changes underlie the long-term association between periodontitis and atherosclerotic cardiovascular disease.

Periodontitis, the leading cause of adult tooth loss, has been identified as an independent risk factor for cardiovascular disease (CVD). Studies suggest that periodontitis, like other CVD risk factors, shows the persistence of increased CVD risk even after mitigation. We hypothesized that periodontitis induces epigenetic changes in hematopoietic stem cells in the bone marrow (BM), and such changes persist after the clinical elimination of the disease and underlie the increased CVD risk. We used a BM transplant approach to simulate the clinical elimination of periodontitis and the persistence of the hypothesized epigenetic reprogramming. Using the low-density lipoprotein receptor knockout (LDLRo ) atherosclerosis mouse model, BM donor mice were fed a high-fat diet to induce atherosclerosis and orally inoculated with Porphyromonas gingivalis (Pg), a keystone periodontal pathogen; the second group was sham-inoculated. Naïve LDLR o mice were irradiated and transplanted with BM from one of the two donor groups. Recipients of BM from Pg-inoculated donors developed significantly more atherosclerosis, accompanied by cytokine/chemokines that suggested BM progenitor cell mobilization and were associated with atherosclerosis and/or PD. Using whole-genome bisulfite sequencing, 375 differentially methylated regions (DMRs) and global hypomethylation in recipients of BM from Pg-inoculated donors were observed. Some DMRs pointed to the involvement of enzymes with major roles in DNA methylation and demethylation. In validation assays, we found a significant increase in the activity of ten-eleven translocase-2 and a decrease in the activity of DNA methyltransferases. Plasma S-adenosylhomocysteine levels were significantly higher, and the S-adenosylmethionine to S-adenosylhomocysteine ratio was decreased, both of which have been associated with CVD. These changes may be related to increased oxidative stress as a result of Pg infection. These data suggest a novel and paradigm-shifting mechanism in the long-term association between periodontitis and atherosclerotic CVD.

Loricrin and Cytokeratin Disorganisation in Severe Forms of Periodontitis.

OBJECTIVE: The aim of this research was to investigate the role of the cornified epithelium, the outermost layer of the oral mucosa, engineered to prevent water loss and microorganism invasion, in severe forms of periodontitis (stage III or IV, grade C). METHODS: Porphyromonas gingivalis, a major periodontal disease pathogen, can affect cornified epithelial protein expression through chronic activation of signal transducer and activator of transcription 6 (Stat6). We used a mouse model, Stat6VT, that mimics this to determine the effects of barrier defect on P gingivalis-induced inflammation, bone loss, and cornified epithelial protein expression, and compared histologic and immunohistologic findings with tissues obtained from human controls and patients with stage III and IV, grade C disease. Alveolar bone loss in mice was assessed using micro-computerised tomography, and soft tissue morphology was qualitatively and semi-quantitatively assessed by histologic examination for several proteins, including loricrin, filaggrin, cytokeratin 1, cytokeratin 14, a proliferation marker, a pan-leukocyte marker, as well as morphologic signs of inflammation. Relative cytokine levels were measured in mouse plasma by cytokine array. RESULTS: In the tissues from patients with periodontal disease, there were greater signs of inflammation (rete pegs, clear cells, inflammatory infiltrates) and a decrease and broadening of expression of loricrin and cytokeratin 1. Cytokeratin 14 expression was also broader and decreased in stage IV. P gingivalis-infected Stat6VT mice showed greater alveolar bone loss in 9 out of 16 examined sites, and similar patterns of disruption to human patients in expression of loricrin and cytokeratins 1 and 14. There were also increased numbers of leukocytes, decreased proliferation, and greater signs of inflammation compared with P gingivalis-infected control mice. CONCLUSIONS: Our study provides evidence that changes in epithelial organisation can exacerbate the effects of P gingivalis infection, with similarities to the most severe forms of human periodontitis.

Is There a Causal Link Between Periodontitis and Cardiovascular Disease? A Concise Review of Recent Findings.

There is substantial evidence in support of an association between periodontitis and cardiovascular disease. The most important open question related to this association is causality. This article revisits the question of causality by reviewing intervention studies and systematic reviews and meta analyses published in the last 3 years. Where are we now in answering this question? Whilst systematic reviews and epidemiological studies continue to support an association between the diseases, intervention studies fall short in determining causality. There is a dearth of good-quality, blinded randomised control trials with cardiovascular disease outcomes. Most studies use surrogate markers/biomarkers for endpoints, and this is problematic as they may not be reflective of cardiovascular disease status. This review further highlights another issue with surrogate markers/biomarkers: the potential for collider bias. Ethical considerations surrounding nontreatment have led to calls for a well-annotated database containing in-depth dental health data. Finally, a relatively new and important risk factor for cardiovascular disease, clonal haematopoiesis of indeterminate potential, is discussed. Clonal haematopoiesis of indeterminate potential increases cardiovascular risk by more than 40%, and inflammation is a contributing factor. The impact of periodontal disease on this emerging risk factor has yet to be explored. Although the question of causality in the association between periodontal disease and cardiovascular disease remains unanswered, the importance of good oral health in maintaining good heart health is reiterated.

A 2-plane micro-computed tomographic alveolar bone measurement approach in mice.

PURPOSE: This study introduces a standardized 2-plane approach using 8 landmarks to assess alveolar bone levels in mice using micro-computed tomography. MATERIALS AND METHODS: Bone level differences were described as distance from the cemento-enamel junction (CEJ) to alveolar bone crest (ABC) and as percentages of vertical bone height and vertical bone loss, comparing mice infected with Porphyromonas gingivalis (Pg) to controls. Eight measurements were obtained per tooth: 2 in the sagittal plane (mesial and distal) and 6 in the coronal plane (mesiobuccal, middle-buccal, distobuccal, mesiolingual, middle-lingual, and distolingual). RESULTS: Significant differences in the CEJ-to-ABC distance between Pg-infected mice and controls were found in the coronal plane (middle-lingual, mesiobuccal, and distolingual for the first molar; and mesiobuccal, middle-buccal, and distolingual for the second molar). In the sagittal plane, the distal measurement of the second molar was different. The middle-buccal, mesiobuccal, and distolingual sites of the first and second molars showed vertical bone loss relative to controls; the second molar middle-lingual site was also different. In the sagittal plane, the mesial sites of the first and second molars and the distal site of the second molar showed loss. Significantly different vertical bone height percentages were found for the mesial and distal sites of the second molar (sagittal plane) and the middle-lingual and distolingual sites of the first molar(coronal plane). CONCLUSION: A reliable, standardized technique for linear periodontal assessments in mice is described. Alveolar bone loss occurred mostly on the lingual surface of the coronal plane, which is often omitted in studies.

Impact of a CD36 inhibitor on Porphyromonas gingivalis mediated atherosclerosis.

OBJECTIVE: To determine if AP5055 drug, an inhibitor of CD36, prevents the increase in Porphyromonas gingivalis (P. gingivalis) mediated atherosclerosis in low-density lipoprotein receptor knockout (LDLR KO) mice by targeting CD36. METHODS: Male LDLR KO mice were infected with P. gingivalis by oral lavage to induce periodontal disease and fed a western diet to induce atherosclerosis. Mice were treated with the CD36 inhibitor, AP5055 (1 mg/kg), or vehicle (1% DMSO). Aortae were dissected and stained with oil red-O for morphometric analysis; blood/plasma was collected to determine markers of inflammation by cytokine array and cholesterol levels. P. gingivalis-induced bone loss in mandibles was assessed using micro-CT. P. gingivalis lipopolysaccharide stimulated nuclear factor-kappa B (NF-κB) activity was measured using a reporter gene (secreted alkaline phosphatase) assay in AP5055 treated or untreated RAW-Blue macrophages. RESULTS: Isolated aortae showed a significant decrease in lesion area in the AP5055 treated group as compared to the control group. Mechanistically, in vitro analysis demonstrated that AP5055 inhibited NF-κB activity. Cytokine array showed a decrease in the expression of pro-inflammatory cytokines and decreased levels of plasma cholesterol in AP5055 treated mice. Micro-CT measurements of bone loss were not significant between the two groups. CONCLUSION: CD36 inhibitor AP5055 abrogates atherosclerotic lesion burden associated with periodontal disease, accompanied by a reduction in markers of inflammation. These experiments may support the development of drugs targeting CD36 for human disease.

Aggressive periodontitis: The unsolved mystery.

Aggressive periodontal disease is an oral health mystery. Our current understanding of this disease is that specific bacteria invade the oral cavity and the host reacts with an inflammatory response leading to mass destruction of the alveolar bone. Aggressive periodontal disease is typically observed in a population under the age of 30 and occurs so rapidly that it is difficult to treat. Unfortunately, the consequence of this disease frequently involves tooth extractions. As a result, the aftermath is chewing disability and damage to self-esteem due to an altered self-image. Furthermore, patients are encumbered by frequent dental appointments which have an economic impact in regards to both personal financial strain and absent days in the workplace. Aggressive periodontal disease has a tremendous effect on patients' overall quality of life and needs to be investigated more extensively in order to develop methods for earlier definitive diagnosis and effective treatments. One of the mysteries of aggressive periodontal disease is the relatively nominal amount of plaque present on the tooth surface in relation to the large amount of bone loss. There seems to be a hidden factor that lies between the response by the patient's immune system and the bacterial threat that is present. A better mechanistic understanding of this disease is essential to provide meaningful care and better outcomes for patients.

Apical periodontitis and atherosclerosis: Is there a link? Review of the literature and potential mechanism of linkage.

Atherosclerosis is a progressive narrowing of arteries that may lead to occlusion as a consequence of lipid deposition. It underlies coronary heart disease, as well as myocardial and cerebral infarctions. Recent attention has been directed towards the potential contribution of chronic inflammatory processes that may amplify vascular inflammation in atherosclerosis, as it is recognized as a chronic inflammatory disease. In this category are two of the most prevalent oral diseases: periodontal disease and apical periodontitis (AP). There is increasing epidemiologic evidence for a positive association between periodontal disease and cardiovascular disease (CVD) as well as between AP and CVD. A review of the literature, as well as a potential mechanism for the linkage between AP and atherosclerosis, are presented in this article.

CD36/SR-B2-TLR2 Dependent Pathways Enhance Porphyromonas gingivalis Mediated Atherosclerosis in the Ldlr KO Mouse Model.

There is strong epidemiological association between periodontal disease and cardiovascular disease but underlying mechanisms remain ill-defined. Because the human periodontal disease pathogen, Porphyromonas gingivalis (Pg), interacts with innate immune receptors Toll-like Receptor (TLR) 2 and CD36/scavenger receptor-B2 (SR-B2), we studied how CD36/SR-B2 and TLR pathways promote Pg-mediated atherosclerosis. Western diet fed low density lipoprotein receptor knockout (Ldlr°) mice infected orally with Pg had a significant increase in lesion burden compared with uninfected controls.This increase was entirely CD36/SR-B2-dependent, as there was no significant change in lesion burden between infected and uninfected Cd36o/Ldlro mice [corrected]. Western diet feeding promoted enhanced CD36/SR-B2-dependent IL1ß generation and foam cell formation as a result of Pg lipopolysaccharide (PgLPS) exposure. CD36/SR-B2 and TLR2 were necessary for inflammasome activation and optimal IL1ß generation, but also resulted in LPS induced lethality (pyroptosis). Modified forms of LDL inhibited Pg-mediated IL1ß generation in a CD36/SR-B2-dependent manner and prevented pyroptosis, but promoted foam cell formation. Our data show that Pg infection in the oral cavity can lead to significant TLR2-CD36/SR-B2 dependent IL1ß release. In the vessel wall, macrophages encountering systemic release of IL1ß, PgLPS and modified LDL have increased lipid uptake, foam cell formation, and release of IL1ß, but because pyroptosis is inhibited, this enables macrophage survival and promotes increased plaque development. These studies may explain increased lesion burden as a result of periodontal disease, and suggest strategies for development of therapeutics.

A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions.

The macrophage scavenger receptor CD36 plays an important role in binding and uptake of oxidized forms of low-density lipoprotein (LDL), foam cell formation, and lesion development during atherosclerosis. The structural basis of CD36-lipoprotein ligand recognition is an area of intense interest. In a companion article we reported the characterization of a structurally conserved family of oxidized choline glycerophospholipids (oxPC(CD36)) that serve as novel high affinity ligands for cells stably transfected with CD36, mediating recognition of multiple oxidized forms of LDL (Podrez, E. A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M., Finton, P., Shan, L., Gugiu, B., Fox, P. L., Hoff, H. F., Salomon, R. G., and Hazen, S. L. (July 8, 2002) J. Biol. Chem. 277, 10.1074/jbc.M203318200). Here we use macrophages from wild-type and CD36 null mice to demonstrate that CD36 is the major receptor on macrophages mediating recognition of oxPC(CD36) species when presented (+/- plasma) in pure form, within PC bilayers in small unilamellar vesicles, and within liposomes generated from lipid extracts of native LDL. We also show that oxPC(CD36) promote CD36-dependent recognition when present at only a few molecules per particle, resulting in macrophage binding, uptake, metabolism, cholesterol accumulation, and foam cell formation. Finally, using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS), we demonstrate that oxPC(CD36) are generated in vivo and are enriched in atherosclerotic lesions. Collectively, our data suggest that formation of this novel family of oxidized phospholipids participates in CD36-mediated recognition of oxidized lipoproteins and foam cell formation in vivo.