Preventive contamination with Lactobacillus reuteri of the implant-abutment connection: an in vivo study.

The aim of this study is to preventively contaminate the abutment-fixture connection (AFC) with Lactobacillus reuteri(L. reuteri), to evaluate the presence of micro leaks in different types of implant-abutment connections, by measuring the concentration of (L. reuteri), in the sulcular fluid over time. This microorganism produces the Reuterine, an antibiotic which counteracts the development of other microbial species. Fourteen biphasic implants were placed on 10 patients (3 women; 7 men; mean age 55.9±16.54y). Eight implants had a flat top connection (internal Hex) while six implants had a tapered connection (conical plus octagonal). At the time of prosthetic finalization, before the healing screw was removed, the sulcular fluid was harvested, on each implant, by means of three sterile paper cones placed into the gingival sulcus and left in place for 20 sec before to be transferred to a sterile tube. Once the healing screw has been removed, first the implant connection has been gently dried with air jet for 10sec and then completely filled with (L. reuteri) DSM 17938 (Reuflor, Italchimici, Italy). Immediately after the prosthetic finalization the sulcular fluid was then harvested again, with the same procedure described before and repeated at 1 week and 1 month of follow up. The samples were then sent for subsequent DNA extraction and real-time PCR. Our results demonstrate that the concentration of (L. reuteri), in the sulcular fluid, does not persist over time in case of preventive contamination of the AFC, demonstrating no significative differences between flat top and tapered connections. Therefore, the use of the latter does not lead to a lower risk of Peri Implant Disease (PID). Long-term studies, involving a larger number of samples, are advisable to confirm these findings.

Screening of periodontopathic bacteria in Italian patients affected by periodontitis.

Periodontal disease (PD) is among the most common infectious diseases in the world, caused by pathogenic bacteria that trigger innate, inflammatory, and adaptive immune responses, leading to the destruction of supporting periodontal tissues and, if untreated, tooth loss. This study included 3593 patients, of them 1963 had a complete dataset and thus were analysed: 1088 (55%) were from Northern Italy, 749 (38%) from Central and 126 (7%) from Southern. Aggregatibacter actinomycetemcomitans, Porphyromonas gengivalis, Treponema denticola, and Tannerella forsythia, Campylobacter rectus, Fusobacterium nucleatum, and total bacterial load were investigated. There was a significant difference in geographic distribution as regard A. actinomycetemcomitans (p<0.001), C. rectus (p<0.001), F. nucleatum (p<0.001) and total bacterial load (p<0.001). No differences were detected as regard gender, whereas a significant higher F. nucleatum load was observed in younger patients.

Colonization of Legionella spp. In dental unit waterlines.

Legionella spp. are ubiquitous in aquatic habitats and water distribution systems, including dental unit waterlines. Surveys have shown that the percentage of samples taken at different dental sites that were positive for Legionella spp. were highly variable and ranged from 0% to 100%. Cultivation is the principal approach to evaluating bacterial contamination employed in the past, but applying this approach to testing for Legionella spp. may result in false-negative data or underestimated bacterial counts. PCR and direct fluorescent counts can detect viable non-cultivable bacteria, which are not counted by plating procedures. Legionella spp., commonly form such viable non-culturable cells and it is likely that they contribute to the difference between plate count results and those of PCR and fluorescent-antibody detection. However, studies have shown that Legionella is present in the municipal water source in spite of the current filtration and chlorination procedures. Once Legionella reaches the building water system, it settles down into a biofilm layer of stagnant water. By means of this layer, Legionella can protect itself from antimicrobial agents and then multiply. Dental unit waterlines may be contaminated with opportunistic bacteria. The water quality in the dental units should be controlled to eliminate opportunistic pathogens and to provide water for dental treatment that meets public health standards for potable water.

Pre-treatment with berberine enhances effect of 5-fluorouracil and cisplatin in HEP2 laryngeal cancer cell line.

Larynx squamous cell carcinoma represents one of the most common head and neck cancers in the world. Herbal drugs are popularly emerging as complementary and alternative therapies in cancer because of their cost effectiveness and minimal side effects. The present study was undertaken to explore the anti-tumor potential of berberine, an isoquinolone present in the extract of Tinospora cordifolia in HEP2 human laryngeal cancer cell line. Besides, it was aimed to investigate whether berberine could enhance the anti-cancer effect of 5-fluorouracil and cisplatin in HEP2. Our data seem to support a role for berberine in decreasing the expression of genes usually seen overexpressed in larynx squamous cell carcinoma and involved in pathways such as those of cell cycle and regulation, differentiation, and epithelial-mesenchymal transition. Moreover, a down regulation of these genes caused by cisplatin or 5-fluorouracil, treatment of election in laryngeal cancers was enhanced by a 4h pre-treatment with berberine.

Human papilloma virus in the tonsillar microbiota of an Afghan population group.

Cancer of the oral cavity is known to have a diverse aetiology that includes infectious agents. Human papilloma virus has been found to be associated with several types of human cancer, inclusive of cervical, vulvar, vaginal, penile, anal, and cancer of tonsil. The aim of this manuscript is to investigate the presence of human papilloma virus in tonsillar microbiota of an Afghan population group. A sample of the tonsillar microbiota was collected by oral swab paper stick from 80 healthy donors. The sample was investigated for the presence of high-risk human papillomavirus types 16, 18, 31 and 45 by real time PCR. Eight samples produced some positive endpoint signals for human papillomaviruses. The human papillomavirus 31 was the unique papillomavirus detected; its calculated prevalence rate was 0.10 (C.I. 0.05-0.19). However, the viral load was always very low, in the order of 10-3 viral genomes per cell. The high prevalence of high-risk human papillomavirus in healthy population suggest a need for further investigation on virus spreading and supports the development of vaccination strategies.

Effect of biostimulation on oral fibroblast: a pilot study.

Bio-stimulation is a technique in aesthetic medicine in which different drugs such as nucleotides, antioxidants and glucosaminoglycans precursors are injected in the dermis to improving the anabolic function of dermal fibroblasts, i.e., protein synthesis, replication and production of extracellular matrix components. It can be achieved with multiple intra-dermal injections, using two protocols: 1) Polydeoxyribonucleotide (PDRN) plus glucosamine sulphate (Gluc); 2) N-acetylcysteine (NAC) and amino acids (Aa) (named Bio- NAC procedure). Since the role of drugs used in biostimulation on human dermal fibroblasts is not completely understood, the aim of this study is to evaluate the effect of these substances in primary cell cultures by using RT-PCR and a panel of specific genes (ELN, DSP, FN1, FBN1, ITGA1, ITGA2, ITGA5, ITGB1, COL1A1,COL3A1) to detect their effect on cell metabolism and extracellular matrix components. Both the treatments were responsible for Elastine and Desmoplakin genes activation. Only NAC plus Aa treatment enhance the expression of other genes related to tissue growth and elasticity like FBN1, ITGA1 and ITGB1. All the other genes investigated (FN1, ITGA5, ITGA2, COL1A1, COL3A1) were down-regulated by both treatments. Since the precise role of these proteins in tissue integrity and aging is not known, this study confirms the usefulness of biostimulation therapies in enhancing some of the genes responsible of cellular wellbeing. This study could be useful to consider the possibility of injective biostimulation in oral cavity, clinical applications in oral healing and in gingival atrophy as well.

Biorevitalization: an in vitro study on gingival fibroblast.

Bio-revitalization is a therapy commonly used in aesthetic medicine to improve skin quality by di¬rectly integrating hyaluronic acid alone or added to other molecules (i.e. vitamins) through intradermal injections. These injections are not aimed to fill roughness but to achieve extracellular matrix optimi¬zation. The injective medical devices used in aesthetic medicine differ for hyaluronic acid content and for the presence of additional molecules that characterize the formulation of a particular company. The aim of the present study is to compare HA with different compounds in regard to their effects on cultured fibroblasts over time by using RT-PCR and a panel of genes (ELN, DSP, FN1, FBN1, ITGA1, ITGA2, ITGA5, ITGB1, COL1A1, COL3A1) involved in connective integrity. Bio-revitalization is able to activate genes involved in tissue integrity. The reported data add new insight in the comprehension of molecular mechanism related to BR. These preliminary data have to be developed through additional experiments. However, an injective therapy seems to be effective in gingival fibroblast stimulation.

Focus on periodontal disease and colorectal carcinoma.

Diagnosis of focal disease, the theory that the human oral microbial (HOM) could affect the onset and development of systemic diseases, was very popular in the past, but the lack of scientific evidence has led to the abandonment of this idea. Interestingly, increasing evidence over the past 3 or so decades suggests that HOM can indeed serve as a reservoir for systemic dissemination of pathogenic bacteria and their toxins in distant body sites, favouring the developments of malignant tumours. Malignant tumours are complex communities of oncogenically transformed cells with aberrant genomes, associated non-neoplastic cells including immune and stromal cells, and sometimes HOM, including bacteria and viruses. Recent data suggest that HOM and periodontal disease play an active role in the pathogenesis of colorectal cancer, in fact HOM has been found within the colorectal cancer microenvironment, and the composition of the HOM was different from that of adjacent non-neoplastic tissue. An association of fusobacterium nucleatum with the colonic mucosa of colorectal cancer has been proven. Several questions thus arise. Is periodontal disease a risk factor for colorectal carcinoma? Given the connectivity of the digestive tract, could fusubacterium nucleatum or other HOM be involved in additional gastrointestinal disorders? Furthermore, based on the "mobility" of Fusubacterium nucleatum and the omnipresence of cadherins, could this organism be involved in cancers beyond the gastrointestinal tract? Answers to these questions will shed new lights on the role of the HOM in onset of diseases.

The ecological catastrophe of oral diseases: a possible link between periodontitis and protozoa.

Periodontal disease (PD) is one of the prevalent diseases in the adult population. The ethiology of PD has never been completely understood, however, loss of balance between the host immune system and the microbial virulence of PD pathogens may be considered the trigger of PD. In fact, the immune system, activated by microbiological agents, attacks the host and not the biofilm bacteria, causing the destruction of periodontal tissue, alveolar bone and loss of teeth. Parasites may play an important role in the pathology of PD. The first studied and the most common parasite in the oral cavity is Entamoeba gingivalis. A possible link between E. gingivalis and PD has never been demonstrated completely, however E. gingivalis is infrequently found in people without PD. In addition, there is evidence that E. gingivalis could favour the onset and progression of PD. In conclusion, we can assert that E. gingivalis and PD may be correlated. This relationship can open new therapeutical approaches for treating PD, particularly in cases refractory to therapy.

Infectogenomics: lack of association between VDR, IL6, IL10 polymorphisms and “red complex” bacterial load in a group of Italian adults with chronic periodontal disease.

Periodontitis is a multifactorial disease that, if untreated, may cause teeth loss. Clinicians and researchers have reported that genetic factors influence the clinical manifestations of periodontal disease (PD), modulating both inflammation of the mucous membranes and loss of alveolar bone. The acquisition of new nowledge about genetic susceptibility of PD, would directly impact on prognosis and treatment of the disease. In addition, a better understanding of PD pathogenesis could improve the diagnostic tools for the prevention and therapies for modulation of immune responses and treatment of PD. In this study, we evaluated genetic polymorphisms of VRD, IL6 and IL10 and amounts of periodontal pathogens in Italian adults affected by PD. We included 326 cases classified according the criteria of the American Academy of Periodontology. No significant differences in bacterial load were found in patients carrying PD susceptibility alleles of IL6, IL10 and VDR genes. In conclusion, no interaction between genetic factors and amount of periodontal pathogens in periodontal pockets were found in PD patients.

EFFECT OF SOMATOSTATIN ON DENTAL PULP STEM CELLS.

Dental pulp stem cells (DPSCs) are multipotent stem cells with the potential to differentiate into various cell types. For this reason, they have been proposed as an alternative source for mesenchymal stem cells. Somatostatin is a peptide hormone with an inhibitory effect on several endogenous hormones. The aim of our study is to investigate whether somatostatin can promote or inhibit differentiation of DPSCs in osteoblasts and bone tissue. DPSCs were extracted from third molars of healthy subjects, and were treated with somatostatin at the concentration of 100 ng/ml for 24 and 48 h. Gene expression in treated DPSCs was compared with untreated cells (control) in order to check the effect of somatostatin on stem cell differentiation. After 24 h of treatment many genes investigated were down-regulated in treated DPSCs vs untreated DPSCs. Significantly up-regulated gene (Fold change > 2) was the Bone Morphogenetic Protein BMP4. On the contrary somatostatin induced the over-expression of bone related genes after 48 h of treatment (i.e. BMPR1B and BMPR2). TGFB family genes and their receptors were also significantly up-regulated after 48 h of treatment. Somatostatin demonstrated to promote the self-renewal of DPSCs: in our experiments somatostatin mainly acted on TGFB family genes. Further studies are needed to explore this new way of creating bone tissue.

CDH1 POLYMORPHISMS AND LOW EXPRESSION OF E-CADHERIN AND ß-CATENIN IN COLORECTAL CANCER PATIENTS.

Epithelial-mesenchymal transition (EMT) process has a central role in tumor progression and metastases. Loss of cell-to-cell adhesiveness is a key step in EMT. In particular, E-cadherin and ß-catenin, components of the adherens junctions, play a strategic role. Accumulation of ß-catenin at cytoplasmic level following adherens junctions disruption, induces its translocation into the nucleus, where it binds to members of the TCF/LEF family of transcription factors. In particular, Lymphoid Enhancer-Binding factor 1 (LEF1) product can target genes involved in EMT. The aim of the present study was to evaluate the influence of CDH1 and CTNNB1 genes, coding for E-cadherin and ß-catenin respectively and LEF1 in a sample study of 140 Italian patients affected by colorectal cancer. An association study between four single nucleotide polymorphisms (rs11865026, rs11642413, rs13689, and rs10431923) of CDH1 and the disease did not provide statistically significant results. The gene expression analysis carried out for CDH1, CTNNB1 and LEF1 in 54 paired specimens from 27 patients provided evidence of a reduced expression of the first two in cancer tissues. We believe there may be a sort of cross regulation between the products of these two genes which closely interact in EMT activation and that such hypothesis should be further investigated in a greater number of cases.

QUANTITATIVE ANALYSIS OF PERIODONTAL PATHOGENS IN PERIODONTITIS AND GINGIVITIS.

Periodontal tissues surround the teeth and provide their attachment. Periodontal diseases include a mild and reversible form named gingivitis and periodontitis that is the main cause of tooth loss in adults. Gingivitis, that affects gums and coronal junctional epithelium, as well as periodontitis, that is characterized by loss of connective tissue attachment, are caused by a persistent inflammatory response promoted by alteration of periodontal biofilm. The aim of the study was to test whether the prevalence or relative amount of each species was associated with a particular clinical condition. Periodontal evaluation of 539 unrelated patients was performed by the Periodontal Screening and Recording (PSR) system. Subgingival samples were obtained from the site with the worst PSR score. A selection of eleven bacterial species was evaluated by quantitative real time PCR. Some bacterial species were found to be associated with all phases of periodontal disease, such as Tannerella forsythia, Treponema denticola, and Treponema lecithinolyticum, while other species were more specifically associated with periodontitis, such as Porphyromonas endodontalis and Porphyromonas gingivalis, or with gingivitis, such as Capnocytophaga ochracea and Campylobacter rectus. Quantitative and qualitative analyses helps to better understand the microbial changes associated with different stages of periodontal disease.

E-CADHERIN CODING GENE (CDH1) AND NONSYNDROMIC CLEFT LIP WITH OR WITHOUT CLEFT PALATE: IS THERE ANY ASSOCIATION?

Epithelial to Mesenchymal Transition (EMT) is an important process involved in cancer, embryogenesis and organ development. Its role in nonsyndromic cleft lip with or without cleft palate (NSCL/P) has been extensively investigated and successfully linked to the disease. In this study, we focused on a gene, CDH1, encoding for E-cadherin, a key protein in EMT. We carried out an association study on an Italian sample group, genotyping four single nucleotide variations within the CDH1 gene, in order to verify the potential role of this gene in NSCL/P etiology. Neither the haplotype nor the family-based association test revealed any association between the genotyped SNPs and the pathology. Our results demonstrate that, in our Italian sample study, the analyzed single nucleotide polymorphisms are not associated to NSCL/P.

INTERLEUKIN-6 GENE POLYMORPHISM MODULATES THE RISK OF PERIODONTAL DISEASES.

Gingivitis and periodontitis are the two main periodontal diseases. Both are characterized by inflammation of the tissues surrounding the teeth but while tissue damages observed in gingivitis are mild and reversible, destruction caused by periodontitis is deeper and irreversible. Periodontal diseases and levels of degeneration of tissues surrounding teeth depend on several interacting endogenous and exogenous factors. Polymorphisms of genes encoding molecules that modulate the immune response and tissue homeostasis are the main causes of individual susceptibility to periodontal diseases. The aim of this study was to investigate IL6, IL10 and VDR gene polymorphisms in a large number of subjects affected by either gingivitis or chronic periodontitis. The sample included 750 Italian patients. We found that the rs1800795 SNP located in the IL6 gene promoter was strongly associated with the occurrence of both gingivitis and periodontitis. Indeed, homozygous individuals with variant allele appeared less-susceptible to both gingivitis OR=0.47 (95% C.I. 0.27-0.82) and periodontitis OR=0.36 (95% C.I. 0.21-0.64). No evidence of association between periodontal diseases and IL10 or VDR polymorphisms was obtained. This data confirmed the role of IL6 in susceptibility to periodontitis among the Italian population. The evidence that IL6 polymorphisms are also involved in gingivitis has implications in periodontal disease pathogenesis and reduces the appeal of IL6 as a periodontitis biomarker.

Evidence of LEF1 fetal-maternal interaction in cleft lip with or without cleft palate in a consistent Italian sample study.

Epithelial mesenchymal transformation is considered a cardinal process in orofacial development. Several molecular players appear to be involved in this delicate mechanism; the activation of LEF1 transcription factor by transforming growth factor beta 3 seems to be a key step for the correct flow of events. The failure of orofacial processes during embryonic development may provoke cleft lip and/or cleft palate malformations. The scope of the present investigation was to verify whether genetic variants at LEF1 could influence the risk of orofacial clefting. The approach was a family based association study involving a total of 512 Italian patients and their parents, 401 having cleft lip with or without cleft palate (CL/P) and 111 with cleft palate only (CPO). Haplotype association analysis provided moderate evidence of an association with clefting (p 0.01). A log-linear likelihood-based method was used to verify maternal and foetal-maternal association. An association between the maternal genotype and the occurrence of CL/P was observed at two polymorphic loci, at rs10022956 (P = 0.0049) and rs10025431 (P = 0.0065) respectively, while a foetal-maternal effect modulating the risk of clefting was found at locus rs10025431 (P = 0.0071). These data further corroborate the importance of the mother's genotype with regard to susceptibility to malformations and early-onset diseases.

Study of the 12q13 region in nonsyndromic cleft lip with or without cleft palate.

The 12q13 region has been suggested as a candidate locus for orofacial cleft by different investigators. In the present study we tested the region for linkage with non syndromic cleft lip with or without cleft palate in a collection of 39 Italian multigenerational families, using microsatellite markers. No evidence of linkage was detected between the marker map and NSCLP under different mode of inheritance nor with a nonparametric method. Formal level of linkage exclusion, were obtained for each point of the map. Genetic heterogeneity and the different impact of the candidate locus among populations could explain conflicting results obtained in different studies.

No association between polymorphisms in cubilin, a gene of the homocysteine metabolism and the risk of non-syndromic cleft lip with or without cleft palate.

Epidemiological studies have correlated lower maternal periconceptional levels of plasma folate and cobalamin with increased risk of delivering offspring presenting congenital malformations such as cleft lip with or without cleft palate (CL/P) or neural tube defects. A number of genetic studies aimed at correlating these biochemical levels or the occurrence of malformations with specific genetic defects or polymorphisms have been successfully performed. The cubilin gene (CUBN) codes for a carrier that plays a crucial role in cobalamin cell internalization. CUBN polymorphisms were previously found to be associated with spina bifida occurrence. In this work, a family-based association study was performed to test CUBN involvement in CL/P. A sample of 391 CL/P triads was investigated with three single nucleotide polymorphisms mapping on the cubilin gene. Association tests indicated no significant association between CL/P and marker alleles or marker haplotypes. No evidence of maternal effect and imprinting were obtained. These data suggest that CUBN is not involved in CL/P onset in the investigated Italian population.

Study of ABCB1 multidrug resistance protein in a common orofacial malformation.

The onset of embryonic malformations is greatly determined by the intrauterine environment, conditioned by maternal lifestyle, diet, drugs and medication intake, in addition to both foetal and maternal genotypes. Maternal C677T MTHFR genotype has been identified as important factor in cleft lip with or without cleft palate (CL/P) etiology. In the present study we evaluated the possible interaction between maternal methylenetetrahydrofolate reductase (MTHFR) and foetal ABCB1 genotypes. ABCB1 gene codes for a drug-transport pump in charge to protect the cell by extruding a variety of harmful exogens, but with a reduced activity in a folate-restricted condition. Maternal 677T genotype is translated in a reduced folate availability for the developing embryo who consequently may becomes more exposed to external insults. A family based association analysis was performed to test the effect of ABCB1 polymorphisms in clefting, in the whole sample and in the stratified sample accordingly to maternal MTHFR genotype. No evidence of association between ABCB1 polymorphisms and CL/P was detected. This suggests that ABCB1 or ABCB1-MTHFR feto-maternal interaction could have no effect in orofacial clefting or could play a role in a limited number of cases.

Evidence of an involvement of TFAP2A gene in non-syndromic cleft lip with or without cleft palate: an Italian study.

Unraveling of factors involved in multifactorial diseases is a great challenge. Different approaches can be contemplate and applied to a variety of congenital malformations. In the present investigation TFAP2A has been considered a good candidate gene for nonsyndromic cleft lip with or without cleft palate (NSCLP) aetiology, basing on a sum of considerations. TFAP2A has been seen involved in orofacial development in mice; it is located in the NSCLP candidate region 6p24; it codes for a transcription factor which regulates expression of IRF6, a gene implied in NSCLP; finally, it is embroiled in the branchiooculofacial syndrome, that includes clefting as feature. A family based association analysis was performed with a sample study of 405 NSCLP triads. Evidence of association was obtained with both single marker and haplotype analyses, thus providing a support for TFAP2A in NSCLP aetiology.