Evidence Linking PPARG Genetic Variants with Periodontitis and Type 2 Diabetes Mellitus in a Brazilian Population.

The peroxisome proliferator-activated receptor gamma (PPARG) gene encodes a transcription factor involved in the regulation of complex metabolic and inflammatory diseases. We investigated whether single nucleotide polymorphisms (SNPs) and haplotypes of the PPARG gene could contribute with susceptibility to develop periodontitis alone or together with type 2 diabetes mellitus (T2DM). Moreover, we evaluated the gene-phenotype association by assessing the subjects' biochemical and periodontal parameters, and the expression of PPARG and other immune response-related genes. We examined 345 subjects with a healthy periodontium and without T2DM, 349 subjects with moderate or severe periodontitis but without T2DM, and 202 subjects with moderate or severe periodontitis and T2DM. PPARG SNPs rs12495364, rs1801282, rs1373640, and rs1151999 were investigated. Multiple logistic regressions adjusted for age, sex, and smoking status showed that individuals carrying rs1151999-GG had a 64% lower chance of developing periodontitis together with T2DM. The CCGT haplotype increased the risk of developing periodontitis together with T2DM. The rs1151999-GG and rs12495364-TC were associated with reduced risk of obesity, periodontitis, elevated triglycerides, and elevated glycated hemoglobin, but there was no association with gene expression. Polymorphisms of the PPARG gene were associated with developing periodontitis together with T2DM, and with obesity, lipid, glycemic, and periodontal characteristics.

Obesity Modifies the Proteomic Profile of the Periodontal Ligament.

This study aimed to assess the obesity effects on the proteomic profile of the periodontal ligament of rats submitted to obesity induction by a high-fat diet. Eight Holtzman rats were divided into control (n = 3) and obese (n = 5) groups. The maxillae were histologically processed for laser capture microdissection of the periodontal ligament of the first maxillary molars. Peptide mixtures were analyzed by LC-MS/MS. A total of 1379 proteins were identified in all groups. Among them, 335 (24.30%) were exclusively detected in the obese group, while 129 (9.35%) proteins were uniquely found in the control group. Out of the 110 (7.98%) differentially abundant proteins, 10 were more abundant and 100 had decreased abundance in the obese group. A gene ontology analysis showed some proteins related to obesity in the "extracellular exosome" term among differentially identified proteins in the gene ontology cellular component terms Prelp, Sec13, and Sod2. These three proteins were upregulated in the obese group (p < 0.05), as shown by proteomic and immunohistochemistry analyses. In summary, our study presents novel evidence that the proteomic profile of the periodontal ligament is altered in experimental obesity induction, providing a list of differentially abundant proteins associated with obesity, which indicates that the periodontal ligament is responsive to obesity.

Polymorphisms in risk genes of type 2 diabetes mellitus could be also markers of susceptibility to periodontitis.

OBJECTIVES: This study aimed to investigate polymorphisms in genes considered molecular biomarkers of type 2 diabetes mellitus (T2DM) to assess whether they are associated with periodontitis, and relating them to the periodontal status, glycemic and lipid profile of the subjects. DESIGN: We investigated individuals who underwent complete periodontal examination and biochemical evaluation. We categorized them into three groups: (i) periodontitis with T2DM (Periodontitis+T2DM group, n = 206); (ii) periodontitis without T2DM (Periodontitis group, n = 346); and (iii) healthy individuals without Periodontitis or T2DM (Healthy group, n = 345). We investigated three single nucleotide polymorphisms (SNPs) for AGER, RBMS1 and VEGFA genes. We applied multivariate logistic and multiple linear regression models for all groups and stratified the subjects by sex and smoking habits. RESULTS: Compared with RBMS1-rs7593730-CC+CT genotype carriers, RBMS1-rs7593730-TT carriers were more susceptible to periodontitis [odds ratio (OR) = 2.29; 95% confidence interval (CI) = 1.04-5.01; P-value = 0.033]. Among AGER-rs184003-CC carriers, never smokers had reduced risks of periodontitis and Periodontitis+T2DM than ever smokers. For either RBMS1-rs7593730-CC or VEGFA-rs9472138-CC carriers, never smokers had less susceptibility to develop periodontitis than ever smokers. Compared with AGER-rs184003-CC carriers, AGER-rs184003-AA carriers presented fewer remaining teeth. VEGFA-rs9472138-TT carriers showed a lower percentage of sites with characteristics of active periodontal disease (bleeding on pocket probing and interproximal clinical attachment level) compared with VEGFA-rs9472138-CC carriers. CONCLUSIONS: In the studied population, AGER rs184003, RBMS1 rs7593730, and VEGFA rs9472138, which are considered genetic markers for T2DM, were associated with periodontitis without T2DM or periodontitis together with T2DM.

Effect of Bacterial Infection on Ghrelin Receptor Regulation in Periodontal Cells and Tissues.

The effect of bacterial infection on the expression of growth hormone secretagogue receptor (GHS-R) was investigated in periodontal cells and tissues, and the actions of ghrelin were evaluated. GHS-R was assessed in periodontal tissues of rats with and without periodontitis. Human gingival fibroblasts (HGFs) were exposed to Fusobacterium nucleatum in the presence and absence of ghrelin. GHS-R expression was determined by real-time PCR and immunocytochemistry. Furthermore, wound healing, cell viability, proliferation, and migration were evaluated. GHS-R expression was significantly higher at periodontitis sites as compared to healthy sites in rat tissues. F. nucleatum significantly increased the GHS-R expression and protein level in HGFs. Moreover, ghrelin significantly abrogated the stimulatory effects of F. nucleatum on CCL2 and IL-6 expressions in HGFs and did not affect cell viability and proliferation significantly. Ghrelin stimulated while F. nucleatum decreased wound closure, probably due to reduced cell migration. Our results show original evidence that bacterial infection upregulates GHS-R in rat periodontal tissues and HGFs. Moreover, our study shows that ghrelin inhibited the proinflammatory actions of F. nucleatum on HGFs without interfering with cell viability and proliferation, suggesting that ghrelin and its receptor may act as a protective molecule during bacterial infection on periodontal cells.

Interaction of periodontitis and orthodontic tooth movement-an in vitro and in vivo study.

OBJECTIVES: The aim of this in vitro and in vivo study was to investigate the interaction of periodontitis and orthodontic tooth movement on interleukin (IL)-6 and C-X-C motif chemokine 2 (CXCL2). MATERIALS AND METHODS: The effect of periodontitis and/or orthodontic tooth movement (OTM) on alveolar bone and gingival IL-6 and CXCL2 expressions was studied in rats by histology and RT-PCR, respectively. The animals were assigned to four groups (control, periodontitis, OTM, and combination of periodontitis and OTM). The IL-6 and CXCL2 levels were also studied in human gingival biopsies from periodontally healthy and periodontitis subjects by RT-PCR and immunohistochemistry. Additionally, the synthesis of IL-6 and CXCL2 in response to the periodontopathogen Fusobacterium nucleatum and/or mechanical strain was studied in periodontal fibroblasts by RT-PCR and ELISA. RESULTS: Periodontitis caused an increase in gingival levels of IL-6 and CXCL2 in the animal model. Moreover, orthodontic tooth movement further enhanced the bacteria-induced periodontal destruction and gingival IL-6 gene expression. Elevated IL-6 and CXCL2 gingival levels were also found in human periodontitis. Furthermore, mechanical strain increased the stimulatory effect of F. nucleatum on IL-6 protein in vitro. CONCLUSIONS: Our study suggests that orthodontic tooth movement can enhance bacteria-induced periodontal inflammation and thus destruction and that IL-6 may play a pivotal role in this process. CLINICAL RELEVANCE: Orthodontic tooth movement should only be performed after periodontal therapy. In case of periodontitis relapse, orthodontic therapy should be suspended until the periodontal inflammation has been successfully treated and thus the periodontal disease is controlled again.

Polymorphisms in Genes of Lipid Metabolism Are Associated with Type 2 Diabetes Mellitus and Periodontitis, as Comorbidities, and with the Subjects' Periodontal, Glycemic, and Lipid Profiles.

BACKGROUND: Type 2 diabetes mellitus (T2DM) and periodontitis (P) commonly occur as comorbidities, but the commonalities in the genetic makeup of affected individuals is largely unknown. Since dyslipidemia is a frequent condition in these individuals, we investigate the association of genomic variations in genes involved in lipid metabolism with periodontal, glycemic, lipid profiles, and the association with periodontitis and T2DM (as comorbidities). METHODS: Based on clinical periodontal examination and biochemical evaluation, 893 subjects were divided into T2DM+P (T2DM subjects also affected by periodontitis, n = 205), periodontitis (n = 345), and healthy (n = 343). Fourteen single-nucleotide polymorphisms (SNPs) were investigated: LDLR gene (rs5925 and rs688), APOB (rs676210, rs1042031, and rs693), ABCC8 (rs6544718 and 6544713), LPL (rs28524, rs3735964, and rs1370225), HNF1A (rs2650000), APOE (rs429358 and rs7412), and HNF4A (rs1800961). Multiple linear and logistic regressions (adjusted for covariates) were made for all populations and stratified by sex and smoking habits. RESULTS: Individuals carrying APOB-rs1042031-CT (mainly women and never smokers) had a lower risk of developing periodontitis and T2DM (T2DM+P); altogether, this genotype was related with healthier glycemic, lipid, and periodontal parameters. Significant disease-phenotype associations with gene-sex interaction were also found for carriers of APOB-rs1676210-AG, HNF4A-rs1800961-CT, ABCC8-rs6544718-CT, LPL-rs13702-CC, and LPL-rs285-CT. CONCLUSIONS: Polymorphisms in lipid metabolism genes are associated with susceptibility to T2DM-periodontitis comorbidities, demonstrating gene-sex interaction. The APOB-rs1042031 was the most relevant gene marker related to glucose and lipid metabolism profiles, as well as with obesity and periodontitis.

Experimental models of orthodontic tooth movement and their effects on periodontal tissues remodelling.

OBJECTIVES: The present study aimed to compare two different models of orthodontic tooth movement (OTM) in rats by evaluating tooth movement efficiency and periodontal tissues remodelling. DESIGN: Fifteen animals were randomly distributed into 3 groups: control group (untreated); ligature appliance (LA) as experimental OTM using a closed coil spring fixed around maxillary first molar by steel ligature; occlusal appliance (OA) as experimental OTM using a closed coil spring attached on the occlusal surface of the maxillary first molar. After 15 days, all animals were euthanized, and the maxilla of each animal was collected for qPCR, micro-computed tomography, and histological analyses. RESULTS: Interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha gene expressions were significantly upregulated in the animals of the LA group as compared to the other groups. No significant difference was observed in tooth displacement between both methods. The LA group presented higher linear bone loss and lower values of bone volume fraction, bone mineral density, trabecular number and increased values of trabecular separation compared to the other groups. The birefringent collagen content in the tension side of the periodontal ligament contained significantly lower collagen content in the LA group than in the control group. Furthermore, on the pressure side, the collagen content was significantly lower in the LA and OA groups than in the control group. CONCLUSIONS: The OA group presented little or no deleterious effect on periodontal tissues compared to the LA group, suggesting its use may be more reliable for OTM induction in rats for 15 days.

Polymorphisms and haplotypes in the Interleukin 17 Alfa gene: potential effect of smoking habits in the association with periodontitis and type 2 diabetes mellitus.

Few studies evaluate interrelationships between periodontitis (P) and Type 2 Diabetes Mellitus (T2DM). The aim of this study is to investigate the genetic susceptibility to periodontitis alone, or concomitant with T2DM (as comorbidities), analyzing single nucleotide polymorphisms (SNPs) in the Interleukin 17 alpha (IL17A) gene, considering the biochemical profile and smoking habits on the subjects' periodontal status. We investigated 879 individuals divided into: T2DM subjects also affected by severe or moderate periodontitis (T2DM-P, n = 199); non-diabetics with severe or moderate periodontitis (PERIODONTITIS, n = 342); and healthy subjects (HEALTHY, n = 338). Subjects underwent complete periodontal examination, history of smoking habits, glycemic and lipid biochemical evaluation. DNA from buccal cells was utilized to genotype the SNPs rs2275913, rs3819024 and rs10484879. The impact of the subjects' biochemical profile was analyzed in their periodontal status. Each SNP was analyzed independently, and as haplotypes, by multiple logistic regressions, adjusted for covariates, and also stratifying the groups by age, sex and smoking habits. Independently of the periodontitis degree, poorly-controlled T2DM subjects showed worse glycemic and lipid profile. Multiple logistic regressions demonstrated that smokers and former-smokers carrying the GG genotype of rs3819024 seemed to have higher risk for T2DM-Periodontitis (OR = 6.33; 95% CI = 1.26-31.77, p = 0.02), and mainly for T2DM alone (OR = 5.11; 95% CI = 1.37-19.06, p = 0.01), than never smokers. We found the potential effect of smoking habits in the association of IL17A-rs3819024-GG with diseased phenotypes. Because the observed wide confidence intervals, further studies enrolling larger populations, and SNPs' functional evaluations are needed to better understand our findings.

Association of type 2 diabetes mellitus and periodontal disease susceptibility with genome-wide association-identified risk variants in a Southeastern Brazilian population.

OBJECTIVE: Genome-wide association studies (GWAS) and literature have identified polymorphisms in the KCNJ11, HNF1A, IRS1, TCF7L2, CDKAL1, CDKN2B, RPSAP52, GPR45 HHEX, IL18, and RUNX2 genes associated with type 2 diabetes mellitus (T2DM) and/or periodontitis (P) in diverse populations, and we sought to evaluate them as genetic risk variants for these diseases in the Brazilian population. MATERIAL AND METHODS: Periodontal, glycemic, and lipid data were obtained from 931 individuals divided into: control (n = 334), periodontitis (P; n = 358), and periodontitis associated with T2DM (P + T2DM; n = 239). After genotyping, associations between polymorphisms and pathologies were tested by multiple logistic and linear regressions, adjusting for age, sex, and smoking habits. RESULTS: Considering the studied subjects, the increased risk to develop periodontitis in the periodontitis P + T2DM group was found for HNF1A-rs7957197-TA, CDKAL1-rs7754840-CG, RPSAP52-rs1531343-GC, TCF7L2-rs7903146-TT, and CDKN2B-rs7018475-GG. The association of these genetic variants for TCF7L2 and CDKN2B was confirmed for female, never smokers, and poorly controlled P + T2DM. CDKN2B-rs7018475 was associated with worse glycemic condition and periodontal parameters. CONCLUSION: These five reported genetic variants were associated in the studied Southeastern Brazilian population as genetic risk variants of periodontitis and T2DM associated to periodontitis as comorbidity. Gene-phenotype associations with sex and smoking habits and the CDKN2B-rs7018475 with the poor glycemic control and more severe periodontal conditions should be further investigated. CLINICAL RELEVANCE: Polymorphisms in the CDKAL1-rs7754840, HNF1A-rs7957197, RPSAP52-rs1531343, TCF7L2-rs7903146, and CDKN2B-rs7018475 might predispose to periodontitis and T2DM associated with periodontitis. These findings may be useful in public health genomics and future advanced clinical practice, since genetic carriage can be measured before disease onset, being of potential great benefit for treatment planning and prognosis in early disease stages.

Validation in a Brazilian population of gene markers of periodontitis previously investigated by GWAS and bioinformatic studies.

BACKGROUND: Bioinformatic tools and genome-wide association studies (GWAS) have led to comprehensive identification of single nucleotide polymorphisms (SNPs) associated with periodontitis in diverse populations. Here we aimed to detect and validate the association of seven SNPs as genetic markers of susceptibility to periodontitis in a Brazilian population. METHODS: This case-control study assessed complete periodontal parameters of 714 subjects with periodontal status classified as healthy/mild periodontitis (n = 356) and moderate/severe periodontitis (n = 358). Genotyping for rs187238, rs352140, rs1360573, rs2521634, rs3811046, rs3826782, and rs7762544 SNPs were evaluated. Genetic-phenotype associations, and sex or smoking effects of SNPs on periodontitis were tested using multiple logistic regressions adjusted for covariates. RESULTS: The rs2521634-AA (close to NPY gene) presented increased risk for severe periodontitis (OR = 2.34; 95% CI = 1.19-4.59). The rs3811046-GG (IL37 gene) demonstrated increased risk for moderate periodontitis (OR = 2.58; 95% CI = 1.28-5.18). Higher risk for moderate periodontitis was found in male with rs7762544-AG close to NCR2 gene. The rs352140-TT in the TLR9 gene proved to be associated with lower risk to severe periodontitis in men. The rs2521634-AA was associated with higher percentage of interproximal probing pocket depth (P = .004). CONCLUSIONS: This is the first evidence of validation in a Brazilian population of genetic markers of periodontitis previously investigated by GWAS and bioinformatics studies. SNPs in the NPY, IL37, and NCR2 genes were associated with susceptibility to moderate or severe periodontitis; whereas the TLR9 marker was associated with lower chance to develop severe periodontitis. Those SNPs had sex- and smoking-habit-specific effects on periodontitis; reinforcing the genetic profile predisposing to periodontitis.

CXCL5, CXCL8, and CXCL10 regulation by bacteria and mechanical forces in periodontium.

OBJECTIVE: The aim of the present study was to evaluate the expressions of CXCL5, CXCL8, and CXCL10 in periodontal cells and tissues in response to microbial signals and/or biomechanical forces. METHODS: Human gingival biopsies from inflamed and healthy sites were used to examine the chemokine expressions and protein levels by real-time PCR and immunohistochemistry. The chemokines were also investigated in gingival biopsies from rats submitted to experimental periodontitis and/or tooth movement. Furthermore, chemokine levels were determined in human periodontal fibroblasts stimulated by the periodontopathogen Fusobacterium nucleatum and/or constant tensile forces (CTS) by real-time PCR and ELISA. Additionally, gene expressions were evaluated in periodontal fibroblasts exposed to F. nucleatum and/or CTS in the presence and absence of a MAPK inhibitor by real-time PCR. RESULTS: Increased CXCL5, CXCL8, and CXCL10 levels were observed in human and rat gingiva from sites of inflammation as compared with periodontal health. The rat experimental periodontitis caused a significant (p<0.05) increase in alveolar bone resorption, which was further enhanced when combined with tooth movement. In vitro, F. nucleatum caused a significant upregulation of CXCL5, CXCL8, and CXCL10 at 1 day. Once the cells were exposed simultaneously to F. nucleatum and CTS, the chemokines regulation was significantly enhanced. The transcriptional findings were also observed at protein level. Pre-incubation with the MEK1/2 inhibitor significantly (p<0.05) inhibited the stimulatory actions of F. nucleatum either alone or in combination with CTS on the expression levels of CXCL5, CXCL8, and CXCL10 at 1d. CONCLUSIONS: Our data provide original evidence that biomechanical strain further increases the stimulatory actions of periodontal bacteria on the expressions of these chemokines. Therefore, biomechanical loading in combination with periodontal infection may lead to stronger recruitment of immunoinflammatory cells to the periodontium, which might result in an aggravation of periodontal inflammation and destruction.

Using association rule mining to jointly detect clinical features and differentially expressed genes related to chronic inflammatory diseases.

OBJECTIVE: It is increasingly common to find patients affected by a combination of type 2 diabetes mellitus (T2DM), dyslipidemia (DLP) and periodontitis (PD), which are chronic inflammatory diseases. More studies able to capture unknown relationships among these diseases will contribute to raise biological and clinical evidence. The aim of this study was to apply association rule mining (ARM) to discover whether there are consistent patterns of clinical features (CFs) and differentially expressed genes (DEGs) relevant to these diseases. We intend to reinforce the evidence of the T2DM-DLP-PD-interplay and demonstrate the ARM ability to provide new insights into multivariate pattern discovery. METHODS: We utilized 29 clinical glycemic, lipid and periodontal parameters from 143 patients divided into five groups based upon diabetic, dyslipidemic and periodontal conditions (including a healthy-control group). At least 5 patients from each group were selected to assess the transcriptome by microarray. ARM was utilized to assess relevant association rules considering: (i) only CFs; and (ii) CFs+DEGs, such that the identified DEGs, specific to each group of patients, were submitted to gene expression validation by quantitative polymerase chain reaction (qPCR). RESULTS: We obtained 78 CF-rules and 161 CF+DEG-rules. Based on their clinical significance, Periodontists and Geneticist experts selected 11 CF-rules, and 5 CF+DEG-rules. From the five DEGs prospected by the rules, four of them were validated by qPCR as significantly different from the control group; and two of them validated the previous microarray findings. CONCLUSIONS: ARM was a powerful data analysis technique to identify multivariate patterns involving clinical and molecular profiles of patients affected by specific pathological panels. ARM proved to be an effective mining approach to analyze gene expression with the advantage of including patient's CFs. A combination of CFs and DEGs might be employed in modeling the patient's chance to develop complex diseases, such as those studied here.

Functional haplotype in the Interleukin8 (CXCL8) gene is associated with type 2 Diabetes Mellitus and Periodontitis in Brazilian population.

BACKGROUND AND AIM: Type 2 Diabetes Mellitus (T2DM) and Periodontitis (P) are prevalent multifactorial disorders worldwide, sharing a bidirectional relationship influenced by the genetic susceptibility of the host immune system. We investigated whether SNPs in the Interleukin 8 (IL8, alias CXCL8) gene could be associated with T2DM and Periodontitis. METHODS: Genomic DNA was obtained from 874 Brazilian individuals divided into: Healthy group (n = 307), Periodontitis group (n = 334), and individuals affected by both T2DM and Periodontitis (T2DM_P) group (n = 233). The SNPs -251(T>A) rs4073, +396(T>G) rs2227307 and +781(C>T) rs2227306 were genotyped by TaqMan®. Multiple logistic regressions were used to determine the degree of association between polymorphisms (and haplotypes) with periodontitis and T2DM adjusted for known confounders. RESULTS: The additive model revealed that the heterozygous AT(-251), GT(+396) and CT(+781) genotypes showed a lower risk for the diseased phenotypes, and carriers of the TTC/TTC haplotype were significantly susceptible to T2DM and Periodontitis concomitantly, as well to isolated Periodontitis (mainly the severe form). CONCLUSIONS: We concluded, for the first time, that these functional CXCL8 SNPs, and the homozygous TTC haplotype are relevant genetic factors for T2DM and Periodontitis as comorbidities, as well as for severe Periodontitis susceptibility in Brazilian population.

Genetic polymorphisms in the Interleukins IL1B, IL4, and IL6 are associated with concomitant periodontitis and type 2 diabetes mellitus in Brazilian patients.

OBJECTIVE: To assess whether single nucleotide polymorphisms (SNPs) in the IL10, IL1A, IL1B, IL4, TNFA, IL6, OPG, RANK, and RANKL genes, "classically" related with periodontitis, could be associated with susceptibility to T2DM, and also with both diseases concomitantly. BACKGROUND: There are common pathogenic mechanisms in type 2 diabetes mellitus (T2DM) and periodontitis, but the knowledge of the genetic aspect of this is limited. In patients affected by concomitant T2DM and periodontitis, whose incidence is increasing, there is scarce information regarding the gene-phenotype association, including whether there are genes able to influence both diseases as comorbidities. METHODS: Periodontal clinical parameters and biochemical profile (Insulin, Fasting Glycemia, HbA1c, Triglycerides, Total Cholesterol, HDL-cholesterol, and LDL-cholesterol) data were obtained from 894 individuals divided into following three groups: Healthy (H; n = 347), Periodontitis (P; n = 348), and Periodontitis + T2DM (P + T2DM; n = 199). DNA from oral epithelial cells was collected for genotyping. Associations between SNPs and pathologies were tested by multiple logistic regression models, adjusting for age, sex, and smoking habits. We also investigated whether there are sex or smoking effects of each SNP in these phenotypes. RESULTS: The rs1143634-GA (IL1B) SNP showed significantly less likely to develop P + T2DM for all population and mainly for women (adjusted OR = 0.37, 95% CI = 0.16-0.88), while women carrying the rs224320 CT (IL4) were more susceptible to develop P + T2DM (adjusted OR = 1.81, 95% CI = 1.04-3.15). Men carrying the rs1800795-CC (IL6) genotype were less likely to develop T2DM (adjusted OR = 0.12, 95% CI = 0.02-0.70, P = .01). CONCLUSIONS: Some SNPs in the IL1B, IL4, and IL6 genes demonstrated sex-influenced association with concomitant periodontitis and T2DM, increasing the evidence of a common genetic component between these diseases and contributing with the understanding of their common pathogenic mechanisms.

Resistin Is Increased in Periodontal Cells and Tissues: In Vitro and In Vivo Studies.

Resistin, a proinflammatory adipokine, is elevated in many inflammatory diseases. However, little is known about its performance in periodontitis. The present study is aimed at evaluating resistin expression and synthesis in periodontal cells and tissues under inflammatory/microbial stress in addition to its effects on the periodontium. In vivo, 24 male rats were randomly divided into two groups: control and ligature-induced periodontal disease. After 6 and 12 days, animals were sacrificed to analyze gene expression of adipokines, bone loss, inflammation, and resistin synthesis. In vitro, human periodontal ligament (PDL) fibroblasts were used to evaluate the expression of resistin after inflammatory stimuli. In addition, PDL fibroblasts were exposed to resistin to evaluate its role on soft and hard tissue metabolism markers. The periodontitis group demonstrated significant bone loss, an increase in the number of inflammatory cells and vascular structures, an increase in resistin expression and synthesis, and a decrease in the expression of adiponectin, leptin, and its functional receptor. PDL fibroblasts showed a significant increase in resistin expression and synthesis in response to the inflammatory stimulus by IL-1ß. Resistin induced an increase in cytokine expression and a decrease in the regulation of some hard tissue and matrix formation genes in PDL fibroblasts. These data indicate that resistin is produced by periodontal cells and tissues, and this effect is enhanced by inflammatory stimuli. Moreover, resistin seems to interfere with soft and hard tissue metabolism during periodontitis by reducing markers related to matrix formation and bone tissue.

CXCL1, CCL2, and CCL5 modulation by microbial and biomechanical signals in periodontal cells and tissues-in vitro and in vivo studies.

OBJECTIVES: This study was established to investigate whether the chemokines CXCL1, CCL2, and CCL5 are produced in periodontal cells and tissues and, if so, whether their levels are regulated by microbial and/or mechanical signals. MATERIALS AND METHODS: The chemokine expression and protein levels in gingival biopsies from patients with and without periodontitis were analyzed by RT-PCR and immunohistochemistry. The chemokines were also analyzed in gingival biopsies from rats subjected to experimental periodontitis and/or orthodontic tooth movement. Additionally, chemokine levels were determined in periodontal fibroblasts exposed to the periodontopathogen Fusobacterium nucleatum and mechanical forces by RT-PCR and ELISA. RESULTS: Higher CXCL1, CCL2, and CCL5 levels were found in human and rat gingiva from sites of periodontitis as compared with periodontally healthy sites. In the rat experimental periodontitis model, the bacteria-induced upregulation of these chemokines was significantly counteracted by orthodontic forces. In vitro, F. nucleatum caused a significant upregulation of all chemokines at 1 day. When the cells were subjected simultaneously to F. nucleatum and mechanical forces, the upregulation of chemokines was significantly inhibited. The transcriptional findings were paralleled at protein level. CONCLUSIONS: This study provides original evidence in vitro and in vivo that the chemokines CXCL1, CCL2, and CCL5 are regulated by both microbial and mechanical signals in periodontal cells and tissues. Furthermore, our study revealed that biomechanical forces can counteract the stimulatory actions of F. nucleatum on these chemokines. CLINICAL RELEVANCE: Mechanical loading might aggravate periodontal infection by compromising the recruitment of immunoinflammatory cells.

Regulation of somatostatin receptor 2 by proinflammatory, microbial and obesity-related signals in periodontal cells and tissues.

BACKGROUND: Periodontitis is a chronic disease characterized by a progressive and irreversible destruction of the tooth-supporting tissues, including gingiva and periodontal ligament (PDL). Microorganisms, such as Fusobacterium nucleatum, evoke an inflammatory host response, which leads to increased levels of inflammatory mediators, such as interleukin (IL)-1ß. Periodontitis has been linked to obesity, and adipokines have been suggested to represent a pathomechanistic link. The hormone somatostatin (SST) exerts antiproliferative, antiangiogenetic, proapoptotic, anti-nociceptive and other effects through binding to its receptors, such as SSTR2. Therefore, the objective of the present study was to examine the regulation of SSTR2 in periodontal cells and tissues under inflammatory, microbial and obesity-related conditions. METHODS: In-vitro, human PDL fibroblasts were exposed to IL-1ß, F. nucleatum, leptin or visfatin. The SSTR2 regulation was assessed by real-time PCR and immunocytochemistry. In-vivo, the SSTR2 expression was analyzed in gingival biopsies of periodontally diseased and healthy subjects by real-time PCR and immunohistochemistry. Additionally, the SSTR2 expression was determined in gingival biopsies of rats with ligature-induced periodontitis, rats with diet-induced obesity, and periodontally and systemically healthy control animals. For statistical analyses, the Mann-Whitney-U test and ANOVA with post-hoc tests were applied (p < 0.05). RESULTS: Exposure of PDL cells to IL-1ß and F. nucleatum caused a significant SSTR2 upregulation by 2.6-fold and 6.4-fold, respectively. Additionally, leptin and visfatin increased significantly the SSTR2 gene expression by 3.0-fold and 2.8-fold, respectively. These stimulatory effects were also observed at protein level. SSTR2 expressions in human gingival biopsies from sites of periodontitis were significantly higher than those in healthy biopsies. Similarly, SSTR2 expression levels were significantly enhanced at periodontally-diseased sites in rat experimental periodontitis. Finally, the SSTR2 expression was significantly upregulated in gingival biopsies of obese rats as compared to normal weight control animals. CONCLUSIONS: Our study provides original insights into the SSTR2 regulation in cells and tissues of the periodontium. We demonstrate for the first time that proinflammatory, microbial and obesity-associated molecules result in an SSTR2 upregulation. Since SST has been shown to be antiproliferative, antiangiogenetic, and proapoptotic, our study suggests that SSTR2 might play a critical role in the aetiopathogenesis of periodontitis.

Physicochemical, morphological, and biological analyses of Ti-15Mo alloy surface modified by laser beam irradiation.

Perform a physicochemical and morphological characterization of a Ti-15Mo alloy surface modified by laser beam irradiation and to evaluate in vitro the morphological response and proliferation of osteoblastic cells seeded onto this alloy. Disks were made of two different metals, Ti-15Mo alloy and cpTi, used as control. A total of four groups were evaluated: polished cpTi (cpTi-pol), laser-irradiated cpTi (cpTi-L), polished Ti-15Mo alloy (Ti-15Mo-pol), and laser-irradiated Ti-15Mo alloy (Ti-15Mo-L). Before and after laser irradiation of the surfaces, physicochemical and morphological analyses were performed: scanning electron microscopy (FEG-SEM), energy-dispersive spectroscopy (EDX), and X-ray diffraction (XRD). The wettability of the samples was evaluated by contact angle measurement. Murine preosteoblastic cells MC3T3-E1 were cultured onto the experimental disks for cell proliferation, morphology, and spreading analyses. Laser groups presented irregular-shaped cavities on its surface and a typical microstructured surface with large depressions (FEG-SEM). The contact angle for both laser groups was 0°, whereas for the polished groups was ≈ 77 and ≈ 78 for cpTi-pol and Ti-15Mo-pol, respectively. Cell proliferation analysis demonstrated a higher metabolic activity in the laser groups (p < 0.05). From the fluorescence microscopy, Ti-15Mo-L surface seems to induce greater cellular differentiation compared to the cpTi-L surface. The preliminary biological in vitro analyses suggested possible advantages of laser surface treatment in the Ti-15Mo alloy regarding cell proliferation and maturation.

Regulation of tyrosine hydroxylase in periodontal fibroblasts and tissues by obesity-associated stimuli.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the synthesis of catecholamines and has been connected to aggravated progression of periodontal disease under chronic stress. Obesity is known to increase the risk of periodontitis and adipokines have been suggested to be a pathomechanistic link. This study examines if obesity-associated stimuli have regulatory effects on TH levels in periodontal cells and tissues. Human periodontal ligament fibroblasts were cultured in the presence of leptin or visfatin for up to 2 days. Untreated cells served as control. TH regulation was analyzed by real-time PCR, immunocytochemistry and ELISA. TH gene expression in periodontal tissues of normal-weight and obese rodents was determined. Examination of gingival biopsies from rats and patients with and without periodontal disease was performed by real-time PCR or immunohistochemistry. For statistics, ANOVA and post hoc tests were applied (p < 0.05). In vitro, TH gene expression and protein levels were increased by leptin and visfatin. In vivo, TH gene expression was upregulated in periodontal tissues of obese rodents as compared to normal-weight animals. Additionally, increased TH gene expression was found in rat gingival biopsies with experimental periodontitis. Human gingival biopsies from sites of periodontitis confirmed the animal data by demonstrating elevated TH levels at periodontally diseased sites. This study provides original evidence that obesity-associated stimuli induce a TH upregulation in periodontal cells and tissues. Since TH levels were also increased at periodontitis sites, our in vitro and animal findings suggest that this enzyme could represent a pathomechanism whereby obesity contributes to periodontitis.

ST2 regulates bone loss in a site-dependent and estrogen-dependent manner.

Interleukin-33 (IL-33) and its receptor, ST2, are implicated in bone remodeling. The lack of estrogen after menopause results in an accelerated bone loss. Here we investigated the role of ST2 in the bone loss induced by estrogen deficiency. ST2-deficient mice (ST2-/- ) and their littermates (wildtype [WT]) were ovariectomized (OVX), while ovary-intact mice were used as controls. Bone sites were analyzed by microcomputed tomography, histomorphometry, and quantitative real-time polymerase chain reaction (qPCR). Deletion of IL-33 or ST2 resulted in a similar bone loss in the femur and maxilla. Ovariectomy in WT mice caused bone loss in the same areas. The lack of ST2 in OVX mice did not alter bone remodeling in the femur but prevented bone loss in the maxilla. Consistently, ovariectomy increased the IL-33 messenger RNA (mRNA) levels in the maxilla but not in the femur. Under mechanical stimulation, ovariectomy and ST2 deletion independently increased bone remodeling induced by orthodontic tooth movement, which was also associated with a greater number of osteoclasts and a reduced number of osteoblasts in the maxillary bone. ST2-/- OVX mice, however, displayed twice as many osteoblasts as that of WT OVX mice. Ovariectomy and ST2 deletion differently altered the cytokine mRNA levels in the maxilla. Remarkably, interleukin-10 expression was decreased in both WT OVX and ST2-/- mice, and this reduction was completely restored in ST2-/- OVX mice. The results demonstrate that estrogen and IL33/ST2 independently protect against bone loss. However, the ovariectomy-induced bone loss is IL-33/ST2-dependent in the maxilla but not in the femur, indicating a bimodal and site-specific role of ST2 in bone remodeling.