Transcriptomic profiling of tantalum metal implant osseointegration in osteopenic patients.

OBJECTIVES: The long-term success of dental implants is established by literature. Although clinically well defined, the complex genetic pathways underlying osseointegration have not yet been fully elucidated. Furthermore, patients with osteopenia/osteoporosis are considered to present as higher risk for implant failure. Porous tantalum trabecular metal (PTTM), an open-cell porous biomaterial, is suggested to present enhanced biocompatibility and osteoconductivity. The goal of this study was to evaluate the expression patterns of a panel of genes closely associated with osteogenesis and wound healing in osteopenic patients receiving either traditional titanium (Ti) or PTTM cylinders to assess the pathway of genes activation in the early phases of osseointegration. MATERIAL AND METHODS: Implant cylinders made of Ti and PTTM were placed in osteopenic volunteers. At 2- and 4 weeks of healing, one Ti and one PTTM cylinder were removed from each subject for RT-PCR analysis using osteogenesis PCR array. RESULTS: Compared to Ti, PTTM-associated bone displayed upregulation of bone matrix proteins, BMP/TGF tisuperfamily, soluble ligand and integrin receptors, growth factors, and collagen genes at one or both time points. Histologically, PTTM implants displayed more robust osteogenesis deposition and maturity when compared to Ti implants from the same patient. CONCLUSIONS: Our results indicate that PTTM properties could induce an earlier activation of genes associated with osteogenesis in osteopenic patients suggesting that PTTM implants may attenuate the relative risk of placing dental implants in this population.

Parathyroid hormone intermittent administration promotes delay on rat incisor eruption.

OBJECTIVE: This study evaluated the influence of parathyroid hormone (PTH) (1-34) intermittent administration on rat eruption rates of lower incisors under normo, hyper and hypofunctional conditions, Sharpey fibers insertion, and alveolar bone formation. MATERIALS AND METHODS: Wistar male rats received PTH (1-34) three times a week during the entire experimental period, 31days. Control animals received the same concentration of the vehicle solution during the same period. Three injections of alizarin were also performed. The experiment evaluated the eruptive rate, the alveolar bone formation and also the morphology, and the area density of Sharpey fibers. After the sacrifice, the mandibles were dissected and samples were prepared for fluorescence and scanning electron microscopy observations. RESULTS: PTH-treated animals showed significantly reduced eruption rates in all different functional conditions. Analysis evidenced that PTH-treated rats present an increase in bone formation and area density of the Sharpey fibers. CONCLUSION: We concluded that the PTH (1-34) intermittent administration reduced the eruptive process rates, through bone formation enhancement and increase in the area density of Sharpey fibers.

Association of Synergistetes and Cyclodipeptides with Periodontitis.

The purpose of this study was to evaluate the microbial community (MC) composition as it relates to salivary metabolites and periodontal clinical parameters in a 21-d biofilm-overgrowth model. Subjects (N = 168) were enrolled equally into 5 categories of periodontal status per the biofilm-gingival interface classification. Microbial species within subgingival plaque samples were identified by human microbiome identification microarray. Whole saliva was analyzed by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry for metabolite identification. Phylum was grouped into MCs according to principal component analysis. Generalized linear and regression models were used to examine the association among MC, species, periodontal clinical parameters, and salivary metabolome. Multiple comparisons were adjusted with the false discovery rate. The study population was distributed into 8 distinct MC profiles, designated MC-1 to MC-8. MC-2 explained 14% of the variance and was dominated by Synergistetes and Spirochaetes. It was the only community structure significantly associated with high probing depth (P = 0.02) and high bleeding on probing (P = 0.008). MC-2 was correlated with traditional periodontal pathogens and several newly identified putative periodontal pathogens: Fretibacterium fastidiosum, Fretibacterium sp. OT360/OT362, Filifactor alocis, Treponema lecithinolyticum, Eubacterium saphenum, Desulfobulbus sp./OT041, and Mogibacterium timidum. Synergistetes phylum was strongly associated with 2 novel metabolites-cyclo (-leu-pro) and cyclo (-phe-pro)-at 21 d of biofilm overgrowth (P = 0.02). In subjects with severe periodontitis (P2 and P3), cyclo (-leu-pro) and cyclo (-phe-pro) were significantly associated with increased changes in probing depth at 21 d of biofilm overgrowth (P ≤ 0.05). The analysis identified a MC dominated by Synergistetes, with classic and putative newly identified pathogens/pathobionts associated with clinical disease. The metabolomic discovery of 2 novel cyclodipeptides that have been reported to serve as quorum-sensing and/or bacteriocidal/bacteriostatic molecules, in association with Synergistetes, suggests a potential role in periodontal biofilm dysbiosis and periodontal disease that warrants further investigation.

Insulin Response Genes in Different Stages of Periodontal Disease.

Bacterial infections are known to alter glucose metabolism within tissues via mechanisms of inflammation. We conducted this study to examine whether insulin response genes are differentially expressed in gingival tissues, comparing samples from experimental gingivitis and periodontitis subjects to those from healthy individuals. Total RNA was extracted from gingival biopsies from 26 participants: 8 periodontally healthy, 9 experimental gingivitis, and 9 periodontitis subjects. Gene expression patterns were evaluated with a polymerase chain reaction array panel to examine 84 candidate genes involved with glucose metabolism, insulin resistance, and obesity. Array data were evaluated with a t test adjusted by the false discover rate (P < 0.05), and ingenuity pathway analysis was performed for statistical testing of pathways. Although tissue samples were not sufficient to enable protein quantification, we confirmed the upregulation of the key gene using lipopolysaccharide-stimulated primary gingival epithelial cells by Western blot. The mRNA expression patterns of genes that are associated with insulin response and glucose metabolism are markedly different in experimental gingivitis subjects compared with healthy controls. Thirty-two genes are upregulated significantly by at least 2-fold, adjusted for false discover rate (P < 0.05). Periodontitis subjects show similar but attenuated changes in gene expression patterns, and no genes meet the significance criteria. Ingenuity pathway analysis demonstrates significant activation of the carbohydrate metabolism network in experimental gingivitis but not in periodontitis. G6PD protein increases in response to lipopolysaccharide stimulation in primary gingival epithelial cells, which is in the same direction as upregulated mRNA in tissues. Acute gingival inflammation may be associated with tissue metabolism changes, but these changes are not evident in chronic periodontitis. This study suggests that acute gingival inflammation may induce localized changes that modify tissue insulin/glucose metabolism.

Chronic periodontitis genome-wide association studies: gene-centric and gene set enrichment analyses.

Recent genome-wide association studies (GWAS) of chronic periodontitis (CP) offer rich data sources for the investigation of candidate genes, functional elements, and pathways. We used GWAS data of CP (n = 4,504) and periodontal pathogen colonization (n = 1,020) from a cohort of adult Americans of European descent participating in the Atherosclerosis Risk in Communities study and employed a MAGENTA approach (i.e., meta-analysis gene set enrichment of variant associations) to obtain gene-centric and gene set association results corrected for gene size, number of single-nucleotide polymorphisms, and local linkage disequilibrium characteristics based on the human genome build 18 (National Center for Biotechnology Information build 36). We used the Gene Ontology, Ingenuity, KEGG, Panther, Reactome, and Biocarta databases for gene set enrichment analyses. Six genes showed evidence of statistically significant association: 4 with severe CP (NIN, p = 1.6 × 10(-7); ABHD12B, p = 3.6 × 10(-7); WHAMM, p = 1.7 × 10(-6); AP3B2, p = 2.2 × 10(-6)) and 2 with high periodontal pathogen colonization (red complex-KCNK1, p = 3.4 × 10(-7); Porphyromonas gingivalis-DAB2IP, p = 1.0 × 10(-6)). Top-ranked genes for moderate CP were HGD (p = 1.4 × 10(-5)), ZNF675 (p = 1.5 × 10(-5)), TNFRSF10C (p = 2.0 × 10(-5)), and EMR1 (p = 2.0 × 10(-5)). Loci containing NIN, EMR1, KCNK1, and DAB2IP had showed suggestive evidence of association in the earlier single-nucleotide polymorphism-based analyses, whereas WHAMM and AP2B2 emerged as novel candidates. The top gene sets included severe CP ("endoplasmic reticulum membrane," "cytochrome P450," "microsome," and "oxidation reduction") and moderate CP ("regulation of gene expression," "zinc ion binding," "BMP signaling pathway," and "ruffle"). Gene-centric analyses offer a promising avenue for efficient interrogation of large-scale GWAS data. These results highlight genes in previously identified loci and new candidate genes and pathways possibly associated with CP, which will need to be validated via replication and mechanistic studies.

Lactobacillus acidophilus NCFM affects colonic mucosal opioid receptor expression in patients with functional abdominal pain - a randomised clinical study.

BACKGROUND: In a recent double-blinded clinical trial, the probiotic combination of Lactobacillus acidophilus NCFM (L-NCFM) and B-LBi07 reduced bloating symptoms in patients with functional bowel disorders; an effect more evident in those who reported abdominal pain. In mice, L-NCFM but not B-LBi07 induced colonic mu-opioid receptor (MOR) and cannabinoid receptor 2 (CB2) expression, and reduced visceral sensitivity. AIMS: To determine if L-NCFM was the active component in the clinical trial and to investigate the mechanism of action in humans with mild to moderate abdominal pain. METHODS: Caucasian women (n = 20) 18-70 years with mild to moderate abdominal pain were enrolled in a double-blind, two-armed, single-centre study. Patients were given either L-NCFM alone or in combination with B-LBi07 for 21 days at a total dose of 2 × 10(10) CFU b.d. Colonic biopsies were collected during unsedated, unprepped flexible sigmoidoscopy before and at the end of probiotic consumption. mRNA and immunostaining were then performed on these biopsies. Patients kept symptom diaries for the 7 days prior to starting probiotic therapy and for the last 7 days of therapy. RESULTS: L-NCFM alone, but not with B-LBi07, induced colonic MOR mRNA and protein expression, as well as downstream signalling, as measured by enterocyte STAT3-phosphorylation. In contrast, CB2 expression was decreased. Both treatment groups trended towards improvement in symptoms, but the study was insufficiently powered to draw meaningful conclusions. CONCLUSIONS: Lactobacillus acidophilus NCFM modulates mu-opioid receptor expression and activity, while the combination of L-NCFM and B-LBi07 does not. This study provides a possible mechanism for action by which probiotics modulates pain sensation in humans (Clinical Trial Number: NCT01064661).

Impact of tooth loss on oral and systemic health.

Periodontitis is a primarily bacterial infection that is common in dentate individuals, while denture stomatitis is a predominantly fungal infection that is common among denture wearers. Both infections may increase a patient's risk for chronic systemic infection dissemination, and may in turn increase the risk of chronic, inflammatory-based systemic diseases. Systemic diseases for which chronic oral infections are believed to confer attributable risk include atherosclerotic and coronary disease, stroke, chronic obstructive pulmonary disease, diabetes, and hypertension. It appears that invasive oral pathogens trigger a systemic inflammatory response via mediators released by the cardiovascular system and liver, putting the patient at increased risk for these diseases. Data comparing gene expression between denture wearers with and without denture stomatitis (and associated Candida albicans infections) has demonstrated unique up- and down-regulation patterns for a number of genes. It appears that down-regulated genes (whose functions are thereby diminished) are associated with reduced epithelial barrier integrity. By contrast, there appears to be an association between up-regulated genes (which have enhanced function) and inflammatory responses that facilitate the ability of C. albicans to bind with and penetrate the oral mucosa. Molecular biological approaches suggest that future therapeutic development could target reducing either the local inflammatory processor, the binding and attachment of C. albicans to the oral mucosa, or both. Ongoing investigations are attempting to incorporate interventions into matrices, to provide a local and sustained presence to therapeutic interventions.

Toll-like receptor 4 mediates intrauterine growth restriction after systemic Campylobacter rectus infection in mice.

Campylobacter rectus is associated with fetal exposure and low birthweight in humans. C. rectus also invades placental tissues and induces fetal intrauterine growth restriction (IUGR) in mice, along with overexpression of Toll-like receptors (TLR4), suggesting that TLR4 may mediate placental immunity and IUGR in mice. To test this hypothesis we examined the effect of in vitro TLR4 neutralization on trophoblastic proinflammatory activity and studied the IUGR phenotype in a congenic TLR4-mutant mouse strain after in vivo C. rectus infection. Human trophoblasts were pretreated with TLR4 neutralizing antibodies and infected with C. rectus; proinflammatory cytokine production was assessed by cytokine multiplex assays. Neutralizing TLR4 antibodies significantly impaired the production of proinflammatory cytokines in trophoblastic cells after infection in a dose-dependent manner. We used a subcutaneous chamber model to provide a C. rectus challenge in BALB/cAnPt (TLR4(Lps-d) ) and wild-type (WT) females. Females were mated with WT or TLR4(Lps-d) males once/week; pregnant mice were infected at (E)7.5 and sacrificed at (E)16.5 to establish IUGR phenotypes. Maternal C. rectus infection significantly decreased fetal weight/length in infected WT when compared with sham WT controls (P < 0.05, analysis of variance). However, infected TLR4(Lps-d -/-) mice did not show statistically significant differences in fetal weight and length when compared with WT controls (P > 0.05). Furthermore, heterozygous TLR4(Lps-d +/-) fetuses showed IUGR phenotype rescue. We conclude that TLR4 is an important mediator of trophoblastic proinflammatory responses and TLR4-deficient fetuses do not develop IUGR phenotypes after C. rectus infection, suggesting that placental cytokine activation is likely to be mediated by TLR4 during low birthweight/preterm birth pathogenesis.

Genome-wide association study of periodontal pathogen colonization.

Pathological shifts of the human microbiome are characteristic of many diseases, including chronic periodontitis. To date, there is limited evidence on host genetic risk loci associated with periodontal pathogen colonization. We conducted a genome-wide association (GWA) study among 1,020 white participants of the Atherosclerosis Risk in Communities Study, whose periodontal diagnosis ranged from healthy to severe chronic periodontitis, and for whom "checkerboard" DNA-DNA hybridization quantification of 8 periodontal pathogens was performed. We examined 3 traits: "high red" and "high orange" bacterial complexes, and "high" Aggregatibacter actinomycetemcomitans (Aa) colonization. Genotyping was performed on the Affymetrix 6.0 platform. Imputation to 2.5 million markers was based on HapMap II-CEU, and a multiple-test correction was applied (genome-wide threshold of p < 5 × 10(-8)). We detected no genome-wide significant signals. However, 13 loci, including KCNK1, FBXO38, UHRF2, IL33, RUNX2, TRPS1, CAMTA1, and VAMP3, provided suggestive evidence (p < 5 × 10(-6)) of association. All associations reported for "red" and "orange" complex microbiota, but not for Aa, had the same effect direction in a second sample of 123 African-American participants. None of these polymorphisms was associated with periodontitis diagnosis. Investigations replicating these findings may lead to an improved understanding of the complex nature of host-microbiome interactions that characterizes states of health and disease.

MicroRNA modulation in obesity and periodontitis.

The aim of this pilot investigation was to determine if microRNA expression differed in the presence or absence of obesity, comparing gingival biopsies obtained from patients with or without periodontal disease. Total RNA was extracted from gingival biopsy samples collected from 20 patients: 10 non-obese patients (BMI < 30 kg/m(2)) and 10 obese patients (BMI > 30 kg/m(2)), each group with 5 periodontally healthy sites and 5 chronic periodontitis sites. MicroRNA expression patterns were assessed with a quantitative microRNA PCR array to survey 88 candidate microRNA species. Four microRNA databases were used to identify potential relevant mRNA target genes of differentially expressed microRNAs. Two microRNA species (miR-18a, miR-30e) were up-regulated among obese individuals with a healthy periodontium. Two microRNA species (miR-30e, miR-106b) were up-regulated in non-obese individuals with periodontal disease. In the presence of periodontal disease and obesity, 9 of 11 listed microRNAs were significantly up-regulated (miR-15a, miR-18a, miR-22, miR-30d, miR-30e, miR-103, miR-106b, miR-130a, miR-142-3p, miR-185, and miR-210). Predicted targets include 69 different mRNAs from genes that comprise cytokines, chemokines, specific collagens, and regulators of glucose and lipid metabolism. The expression of specific microRNA species in obesity, which could also target and post-transcriptionally modulate cytokine mRNA, provides new insight into possible mechanisms of how risk factors might modify periodontal inflammation and may represent novel therapeutic targets.