Atypical Mesenchymal Stromal Cell Responses to Topographic Modifications of Titanium Biomaterials Indicate Cytoskeletal- and Genetic Plasticity-Based Heterogeneity of Cells.

Titanium (Ti) is widely used as a biomaterial for endosseous implants due to its relatively inert surface oxide layer that enables implanted devices the ability of assembling tissue reparative components that culminate in osseointegration. Topographic modifications in the form of micro- and nanoscaled structures significantly promote osseointegration and enhance the osteogenic differentiation of adult mesenchymal stromal cells (MSCs). While the biological mechanisms central to the differential responses of tissues and cells to Ti surface modifications remain unknown, adhesion and morphological adaptation are amongst the earliest events at the cell-biomaterial interface that are highly influenced by surface topography and profoundly impact the regulation of stem cell fate determination. This study correlated the effects of Ti topographic modifications on adhesion and morphological adaptation of human MSCs with phenotypic change. The results showed that modified Ti topographies precluded the adhesion of a subset of MSCs while incurring distinct morphological constraints on adherent cells. These effects anomalously corresponded with a differential expression of stem cell pluripotency and Wnt signalling-associated markers on both modified surfaces while additionally differing between hydrophobic and hydrophilic surface modifications-though extent of osteogenic differentiation induced by both modified topographies yielded similarly significant higher levels of cellular mineralisation in contrast to polished Ti. These results suggest that in the absence of deposited proteins and soluble factors, both modified topographies incur the selective adhesion of a subpopulation of progenitors with relatively higher cytoskeletal plasticity. While the presence of deposited proteins and soluble factors does not significantly affect adherence of cells, nanotopographic modifications enhance expression of pluripotency markers in proliferative conditions, which are conversely overridden by both modified topographies in osteogenic inductive conditions. Further deciphering the mechanisms underlying cellular selectivity and Ti topographic responsiveness will improve our understanding of stem cell heterogeneity and advance the potential of MSCs in regenerative medicine.

Mitochondrial oxidative stress, endothelial function and metabolic control in patients with type II diabetes and periodontitis: A randomised controlled clinical trial.

BACKGROUND: Periodontitis (PD) and type 2 diabetes (T2D) are characterized by increased mitochondrial oxidative stress production (mtROS), which has been associated with a greater risk of cardiovascular diseases (CVD). Intensive PD treatment (IPT) can significantly improve endothelial function and metabolic control, although the mechanisms remain unclear. We explored whether, in patients with PD and T2D, changes of mtROS are associated with improvement of endothelial function and metabolic control after IPT. METHODS: 51 patients with T2D and PD were enrolled in a single-blind controlled trial and randomised to either intensive (n = 27) or standard (CPT, n = 24) PD treatment. Levels of mtROS in peripheral blood mononuclear cells (PBMC) were measured using a FACS-based assay at baseline and 24 h, 1 week, 2 and 6 months after PD treatment. Inflammatory cytokines, CVD risk factors, metabolic control and endothelial function were assessed at baseline and 6 months after intervention. RESULTS: After 6 months from PD treatment, the IPT group had lower mtROS (in both the whole PBMC and lymphocytes), circulating levels of HbA1c, glucose, INF-γ, TNF-α (p < 0.05 for all), and improved endothelial function (p < 0.05) compared to the CPT group. There was an association between higher mtROS and lower endothelial function at baseline (r = -0.39; p = 0.01) and, in the IPT group, changes of mtROS were associated with changes of endothelial function (r = 0.41; p < 0.05). CONCLUSIONS: Reduced mtROS is associated with improved endothelial function and accompanied by better metabolic control in patients with T2D and PD. mtROS could represent a novel therapeutic target to prevent CVD in T2D.

Association between short leukocyte telomere length, endotoxemia, and severe periodontitis in people with diabetes: a cross-sectional survey.

OBJECTIVE Shortened leukocyte telomere length (LTL) and diagnosis of periodontitis are associated with an increased risk of complications and mortality in diabetes. This study investigated the association between LTL, endotoxemia, and severity of periodontitis in a large cohort of people with diabetes. RESEARCH DESIGN AND METHODS Six hundred thirty individuals (371 with type 2 and 259 with type 1 diabetes) were recruited from the University College Hospital in London, U.K. During a baseline visit, blood was collected for standard biochemical tests and DNA extraction, while a dental examination was performed to determine diagnosis and extent of periodontitis. LTL was measured by real-time PCR, and endotoxemia was assessed by the limulus amoebocyte lysate method. RESULTS Two hundred fifty-five individuals were diagnosed with gingivitis, 327 with periodontitis (114 with moderate and 213 with severe disease), and 48 with edentulous. Diagnosis of periodontitis was associated with shorter LTL (P = 0.04). A negative association between LTL and endotoxemia was found in the severe periodontitis and type 2 diabetes groups (P = 0.01 for both). Shorter LTL was associated with increased extent of periodontitis (P = 0.01) and increased insulin resistance (homeostatic model assessment). Multiple adjustments for biochemical, anthropometric, and medication-use variables did not affect the results. CONCLUSIONS LTL is associated with endotoxemia and diagnosis of periodontitis in people with diabetes. LTL shortening might represent a novel biological pathway accounting for previous epidemiological data that documented higher prevalence of diabetes and its complications in people with periodontitis and vice versa.

Association between COX-2 rs 6681231 genotype and interleukin-6 in periodontal connective tissue. A pilot study.

OBJECTIVES: The aim of this pilot study was to investigate associations between IL-6 and COX-2 expression in gingival biopsies and both clinical diagnosis and genotypes in the IL-6 and COX-2 genes. DESIGN: A case-control study included 41 gingival biopsies obtained from Caucasian patients grouped according to clinical diagnosis of gingival health (n = 10), gingivitis (n = 15) or chronic periodontitis (n = 16). Immunohistochemistry analyses were performed to determine COX-2 expression in lamina propria, IL-6 expression in lamina propria and gingival epithelium and level of inflammatory cell infiltrate. Individual DNA was extracted and genotyped by real-time PCR for IL6 SNPs rs 2069827 and rs 2069825 and for COX-2 rs 6681231. RESULTS: The percentage of cellular COX-2 expression was associated with the extent of periodontal disease (Arbes index p = 0.026) and inflammatory infiltrate (p<0.0001). No association was observed between IL6 haplotypes and cells positive to IL-6 or COX-2 in gingival tissues. The COX-2 rs 6681231 was associated with cells positive to IL-6 in the connective tissue (p = 0.032). CONCLUSIONS: COX-2 expression in gingival tissues may be a marker of periodontal disease severity. COX-2 rs 6681231 may be associated with IL-6 local production in gingival tissues.

No association between A actinomycetemcomitans or P gingivalis and chronic or aggressive periodontitis diagnosis.

OBJECTIVES: Only a few studies have attempted to detect differences in microbiologic profiles of patients with chronic periodontitis (CP) and aggressive periodontitis (AgP). The aim of this analysis was to assess if clinical diagnosis or other subject factors showed association with the presence of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in a cohort of periodontitis patients. METHOD AND MATERIALS: Statistical analysis for association between bacterial detection and clinical diagnosis was performed on a total of 267 consecutive periodontitis cases diagnosed with either CP (n = 183) or AgP (n = 84). All subjects had microbiologic samples collected from the four deepest pockets and analyzed by nested polymerase chain reaction. RESULTS: A actinomycetemcomitans was detected in 54% and 48% of CP and AgP subjects, respectively. A slightly higher detection of P gingivalis was found in CP (67% ) compared with AgP (52%) cases. The detection of P gingivalis was associated with older age (P = .002), less disease severity (P = .015), and IL6-1480 genotypes (P = .026), while A actinomycetemcomitans was associated with IL6-1480 genotypes (P = .001). CONCLUSION: Detection of known periodontopathogenic bacteria is not able to discriminate different forms of periodontitis.

Oxidative stress, chronic inflammation, and telomere length in patients with periodontitis.

The aim of this study was to determine leukocyte telomere length (LTL) in individuals with periodontitis and controls, exploring its relationship with systemic inflammation and oxidative stress. Five hundred sixty-three participants were recruited for this case-control study: 356 subjects with and 207 subjects without periodontitis. LTL was measured by a qPCR technique from leukocytes' DNA. Global measures of oxidative stress (reactive oxygen metabolites) and biological antioxidant potential in plasma were performed together with high-sensitivity assays for C-reactive protein (CRP). Leukocyte counts and lipid profiles were performed using standard biochemistry. Cases had higher levels of CRP (2.1±3.7mg/L vs 1.3±5.4mg/L, P<0.001) and reactive oxygen metabolites (378.1±121.1 U Carr vs 277.4±108.6 U Carr, P<0.001) compared to controls. Overall, cases had shorter LTL with respect to controls (1.23±0.42 vs 1.12±0.31T/S ratio, P=0.006), independent of age, gender, ethnicity, and smoking habit. When divided by subgroup of periodontal diagnosis (chronic, n=285; aggressive, n=71), only chronic cases displayed shorter LTL (P=0.01). LTL was negatively correlated with age (P=0.001; R=-0.2), oxidative stress (P=0.008; R=-0.2), and severity of periodontitis (P=0.003; R=-0.2) in both the whole population and the subgroups (cases and controls). We conclude that shorter telomere lengths are associated with a diagnosis of periodontitis and their measures correlate with the oxidative stress and severity of disease.

Interleukin-6 polymorphisms are associated with pathogenic bacteria in subjects with periodontitis.

BACKGROUND: Growing evidence suggests that individual genetic susceptibility may influence the host's response to infections. Previously, we showed that a common variation in the interleukin (IL)-6 gene was associated with increased odds of detection of common periodontal pathogens from individuals with aggressive periodontitis. The aim of this study was to investigate the association between IL-6 polymorphisms and periodontopathogenic bacteria in a larger, ethnically mixed population of subjects with periodontitis. METHODS: Genomic DNA was extracted from 107 subjects diagnosed with severe forms of periodontitis to study a cluster of polymorphisms in inflammatory genes, including IL-6. The presence of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, and Tannerella forsythia (previously T. forsythensis) in their subgingival biofilm was determined by polymerase chain reaction analysis. Serum IL-6 and C-reactive protein (CRP) concentrations were analyzed by enzyme-linked immunosorbent assay. RESULTS: Multiple logistic regression analysis revealed that the IL-6 -6106 polymorphism was associated with the detection of A. actinomycetemcomitans (P = 0.009; odds ratio [OR] = 3.5; 95% confidence interval [CI]: 1.38 to 9.16) and the concomitant detection of A. actinomycetemcomitans and P. gingivalis (P = 0.015; OR = 3.6; 95% CI: 1.28 to 10.04). The IL-6 -174 polymorphism was associated with increased odds of the concomitant detection of A. actinomycetemcomitans and P. gingivalis (P = 0.042; OR = 2.8; 95% CI: 1.04 to 7.75). Haplotype analysis of all five IL-6 polymorphisms confirmed an association with the detection of A. actinomycetemcomitans (P = 0.046). The IL-6 -6106 polymorphism was also associated with CRP serum levels at multivariate analysis (P = 0.024). CONCLUSIONS: These findings confirm the hypothesis that complex interactions between the microbiota and host genome are at the basis of susceptibility to periodontitis. Periodontal disease may represent a useful model to study the pathways and mechanisms of host-pathogen interactions in inflammatory diseases.

Vitamin D receptor polymorphism (-1056 Taq-I) interacts with smoking for the presence and progression of periodontitis.

AIM: The aim of this analysis was to investigate the relationship between a vitamin D receptor (VDR) polymorphism and the diagnosis and progression of periodontitis. MATERIAL AND METHODS: Data were derived from two different studies, including 231 subjects with healthy periodontium, 224 aggressive periodontitis and 79 chronic periodontitis (CP) patients in a case-control investigation. Sixty-one of these CP patients also took part in an observational study with a 1-year follow-up, in which progression of periodontitis was determined at the subject level. All 534 subjects provided a blood sample from which genomic DNA was extracted to study VDR -1056 TaqI polymorphism. RESULTS: The interaction between smoking and VDR polymorphism was associated with the diagnosis of periodontitis in Caucasians [p=0.001, odds ratio (OR)=1.33, 95% confidence intervals (CI)=1.12-1.57] and all subjects (p=0.033, OR=1.60, 95% CI=1.04-2.48). In the longitudinal study, subjects were divided into two clusters at 1 year according to the median number of progressing sites (Delta cumulative attachment loss >2 mm). Logistic regression analysis revealed that the interaction between VDR Taq-I polymorphism and smoking showed limited evidence of association with the "severe progression" cluster (p=0.033, OR=15.24, 95% CI=1.24-187.42). CONCLUSIONS: Vitamin D receptor Taq-I TT polymorphism was moderately associated with both the presence and the progression of periodontitis in smokers, while no association was detected in non-smoking individuals. VDR genetic factors may interact with smoking in the pathogenesis of periodontitis.

Association between interleukin-6 promoter haplotypes and aggressive periodontitis.

BACKGROUND: Interleukin-6 (IL-6) polymorphisms have been shown to affect IL-6 promoter activity. This study investigated the possible role of IL-6 genetic polymorphisms and haplotypes in the predisposition to aggressive periodontitis (AgP). MATERIAL AND METHODS: A case-control association study on 224 AgP patients and 231 healthy controls was performed in order to detect differences in genotype distributions of five single nucleotide polymorphisms (SNPs) located in the promoter region of the IL-6 gene. RESULTS: The IL-6 -1363 polymorphism was associated with a diagnosis of AgP in subjects of all ethnicities (p=0.006, adjusted logistic regression). The -1480 SNP was associated with LAgP in subjects of all ethnicities (p=0.003). The -1480 and -6106 polymorphisms were associated with Localized AgP in Caucasians (n=24) (p=0.007 and 0.010, respectively). Haplotypes determined by the -1363 and -1480 polymorphisms were also associated with LAgP (p=0.001) in Caucasians. CONCLUSIONS: This study supports the hypothesis of a link between IL-6 genetic factors and AgP and highlights the importance of two IL-6 polymorphisms (-1363 and -1480) in modulating disease phenotype and susceptibility.

Differential regulation of circulating levels of molecular chaperones in patients undergoing treatment for periodontal disease.

BACKGROUND: Evidence is emerging that molecular chaperones, in addition to their intracellular protein folding actions, can act as intercellular signaling proteins with an ability to modulate leukocyte function. Recent evidence has also shown that these proteins can exist in the circulation and may be involved in disease pathogenesis. We have used periodontitis and its treatment as a model of inflammation in the human to determine its effects on levels of circulating HSP10, HSP60 and BiP. METHODOLOGY/PRINCIPAL FINDINGS: A group of periodontal patients and matched controls were examined at the beginning of the study and then at 1 day and 6 months following periodontal or control therapy. Plasma levels of HSP10, HSP60 and BiP were measured by immunoassay and related to other plasma measures of inflammation. Periodontal patients had significantly less circulating levels of HSP10 or BiP compared with the controls. In contrast, more periodontal patients had intermediate levels of HSP60. Treatment of the periodontitis caused an increase in plasma levels of HSP10 although it had no effect on BiP. Treatment had no influence of HSP60 levels. Plasma HSP10 levels after therapy correlated with markers of periodontal clinical improvement. CONCLUSIONS/SIGNIFICANCE: Circulating levels of molecular chaperones are influenced by local inflammation. HSP10 is known to be an anti-inflammatory factor. The marked decrease of this circulating protein in active inflammation and its recovery post-treatment suggests that it may have a role in controlling periodontal inflammation.

Treatment of periodontitis and endothelial function.

BACKGROUND: Systemic inflammation may impair vascular function, and epidemiologic data suggest a possible link between periodontitis and cardiovascular disease. METHODS: We randomly assigned 120 patients with severe periodontitis to community-based periodontal care (59 patients) or intensive periodontal treatment (61). Endothelial function, as assessed by measurement of the diameter of the brachial artery during flow (flow-mediated dilatation), and inflammatory biomarkers and markers of coagulation and endothelial activation were evaluated before treatment and 1, 7, 30, 60, and 180 days after treatment. RESULTS: Twenty-four hours after treatment, flow-mediated dilatation was significantly lower in the intensive-treatment group than in the control-treatment group (absolute difference, 1.4%; 95% confidence interval [CI], 0.5 to 2.3; P=0.002), and levels of C-reactive protein, interleukin-6, and the endothelial-activation markers soluble E-selectin and von Willebrand factor were significantly higher (P<0.05 for all comparisons). However, flow-mediated dilatation was greater and the plasma levels of soluble E-selectin were lower in the intensive-treatment group than in the control-treatment group 60 days after therapy (absolute difference in flow-mediated dilatation, 0.9%; 95% CI, 0.1 to 1.7; P=0.02) and 180 days after therapy (difference, 2.0%; 95% CI, 1.2 to 2.8; P<0.001). The degree of improvement was associated with improvement in measures of periodontal disease (r=0.29 by Spearman rank correlation, P=0.003). There were no serious adverse effects in either of the two groups, and no cardiovascular events occurred. CONCLUSIONS: Intensive periodontal treatment resulted in acute, short-term systemic inflammation and endothelial dysfunction. However, 6 months after therapy, the benefits in oral health were associated with improvement in endothelial function.

Periodontal infections cause changes in traditional and novel cardiovascular risk factors: results from a randomized controlled clinical trial.

BACKGROUND: Chronic infections, such as periodontitis, are associated with increased risk of systemic diseases driven by a persistent low-grade systemic inflammation and metabolic changes. Severity of periodontitis has also been associated with increased systolic blood pressure (BP). However, the issue remains poorly investigated. We aimed to estimate the effect of periodontal therapy on traditional and novel cardiovascular risk factors in systemically healthy individuals who have periodontitis. METHODS: We enrolled 40 otherwise healthy patients with severe chronic generalized periodontitis in a 6-month pilot intervention trial. Individuals were randomized either to a standard course of periodontal therapy (subgingival scaling and root planing) or an intensive one (including the adjunctive use of a locally delivered antimicrobial, IPT). RESULTS: Compared to control, IPT produced significant reductions in a cluster of inflammatory markers at 1 (P = .0406) and 2 (P = .0060) months together with an improvement in lipid markers at 2 (P = .0320) and 6 (P = .0432) months after therapy. Intensive periodontal therapy produced greater reductions in IL-6 at 1 (0.4 +/- 0.2 ng/L difference, 95% CI 0.03-0.9, P = .0284) and 2 months (0.3 +/- 0.2 ng/L difference, 95% CI 0.1-0.8, P = .0284), together with decreases in C-reactive protein (0.4 +/- 0.2 mg/L difference, 95% CI 0.01-0.8, P = .0438) and total cholesterol (0.3 +/- 0.1 mmol/L difference, 95% CI 0.04-0.6, P = .0254). Moreover, a 7 +/- 3-mm Hg decrease in systolic BP was observed at 2 months in the IPT group (95% CI 1-12, P = .0211), and this difference was greater in current smokers (14 +/- 5 mm Hg 95% CI 3-25, P = 0.0124). Intensive periodontal therapy subjects exhibited a 1.53% +/- 1.20% (95% CI 1.05-2.24, P = .0290) and 2.00% +/- 1.42% (95% CI 0.98-4.09, P = .0568) decreases in cardiovascular risk scores (Framingham) at 2 and 6 months, respectively, when compared to those in the standard group. CONCLUSIONS: Our findings suggest that intensive periodontal treatment reduces systemic inflammatory markers and systolic BP, and improves lipid profiles with subsequent changes in cardiovascular risk when compared to standard therapy.

The immunosuppressant and hyperplasia-inducing drug cyclosporin A regulates the cell cycle and cyclin B1 gene expression in gingival fibroblasts in vitro.

Gingival overgrowth is a frequent and adverse side-effect caused by certain immunosuppressant, anti-convulsant and calcium channel-blocking drugs. Although the precise mechanism is not yet known, the enhanced proliferation of gingival fibroblasts observed in this disease could be caused at least partly by the effects of the drugs on the cell cycle and cyclin expression in these cells. In the present study, flow cytometry analysis of the effects of the immunosuppressant drug cyclosporin A showed that it enhanced cell-cycle progression of gingival fibroblasts in vitro and also up-regulated the expression of cyclin B1. In addition, reverse transcriptase/polymerase chain reaction analysis of gingival overgrowth tissues showed markedly elevated transcription of the cyclin B1 gene. Thus, the increase in cell growth that occurs in drug-induced gingival overgrowth may be mediated by over-expression of cyclin B1.

Gene expression profiles of periodontal ligament cells treated with enamel matrix proteins in vitro: analysis using cDNA arrays.

BACKGROUND: A number of procedures have been used to achieve periodontal regeneration. Recently, enamel matrix derivative (EMD) has been the subject of significant basic and clinical investigations. The precise molecular events involved in EMD modulation of periodontal wound healing are not completely understood; however, cDNA microarray technology may enable rapid and accurate examination of EMD-mediated changes in gene expression in periodontal ligament (PDL) cells in vitro. The present study was undertaken to explore the selective effects of EMD on the activities of 268 cytokine, growth factor, and receptor genes in PDL. METHODS: PDL cells were cultured in the absence and presence of EMD at a concentration of 100 microg/ml for 4 days. RNA was extracted and used to generate labeled cDNA probes. These were hybridized to cDNA arrays comprising 268 genes and exposed to x-ray films. Autoradiographs were digitized and analyzed. RESULTS: Forty-six percent (125 of 268) of the tested genes were found to be expressed by the PDL cells. Of these 125 genes, 38 were differentially expressed by PDL cells which had been cultured in the presence of EMD. Among the 38, 12 were found to be downregulated, notably mostly inflammatory genes, whereas 26 genes demonstrated upregulation, many of these coding for growth factors and growth factor receptors. CONCLUSIONS: The present study has shown that EMD down-regulates the expression of genes involved in the early inflammatory phases of wound healing while simultaneously upregulating genes encoding growth and repair-promoting molecules. This may partly explain the apparent efficacy of EMD application in periodontal regeneration.