Influence of 2'-fucosyllactose and galacto-oligosaccharides on the growth and adhesion of Streptococcus mutans.

Human milk oligosaccharides, such as 2'-fucosyllactose (2'-FL), and galacto-oligosaccharides (GOS), a prebiotic carbohydrate mixture, are being increasingly added to infant formulas, necessitating the understanding of their impact on the oral microbiota. Here, for the first time, the effects of 2'-FL and GOS on the planktonic growth and adhesion characteristics of the caries-associated oral pathogen Streptococcus mutans were assessed, and the results were compared against the effects of xylitol, lactose and glucose. There were differences in S. mutans growth between 2'-FL and GOS. None of the three S. mutans strains grew with 2'-FL, while they all grew with GOS as well as lactose and glucose. Xylitol inhibited S. mutans growth. The adhesion of S. mutans CI 2366 to saliva-coated hydroxyapatite was reduced by 2'-FL and GOS. Exopolysaccharide-mediated adhesion of S. mutans DSM 20523 to a glass surface was decreased with 2'-FL, GOS and lactose, and the adhesion of strain CI 2366 strain was reduced only by GOS. Unlike GOS, 2'-FL did not support the growth of any S. mutans strain. Neither 2'-FL nor GOS enhanced the adhesive properties of the S. mutans strains, but they inhibited some of the tested strains. Thus, the cariogenic tendency may vary between infant formulas containing different types of oligosaccharides.

Matrix metalloproteinase-7 in periodontitis with type 2 diabetes mellitus.

BACKGROUND AND OBJECTIVE: Elevated levels of matrix metalloproteinase-7 (MMP7) have been observed in serum samples of subjects with type 2 diabetes mellitus (T2DM) and in gingival tissues of subjects with periodontitis. The aim of the present study was to collect in vivo and in silico evidence on the role of MMP7 in the interplay between T2DM and generalized periodontitis (GP). MATERIAL AND METHODS: The extent of MMP7 expression and localization were immunohistochemically analyzed in gingival tissues of patients with GP with T2DM (T2DM/GP, n = 11), systemically healthy patients with GP (n = 7), and systemically and periodontally healthy controls (n = 11). An in silico network model was built to determine the interactions between MMP7 and T2DM pathways. Regulation of neutrophil transmigration by MMP7 was analyzed in a knock-out mice model. RESULTS: In human gingival tissues, the proportion of cells with robust MMP7 expression was elevated in patients with T2DM/GP in comparison to controls (P = .014). According to the in silico analysis, "hydroxyl radical" and "hydrogen peroxide" compounds were among the most central nodes of the network, and were within the shortest paths connecting "glucose" to "MMP7." In MMP7 knock-out mice, an intense accumulation of neutrophils was observed in the gingival epithelium as compared to wild-type mice (P = .0001). CONCLUSION: Elevated MMP7 expression in gingival tissues of patients with T2DM/GP is related to the activation of reactive oxygen species by hyperglycemia. Suppression of MMP7 expression results in impaired neutrophil transmigration in gingiva.

Quorum-sensing molecule dihydroxy-2,3-pentanedione and its analogs as regulators of epithelial integrity.

BACKGROUND AND OBJECTIVE: Quorum-sensing molecules regulate the behavior of bacteria within biofilms and at the same time elicit an immune response in host tissues. Our aim was to investigate the regulatory role of dihydroxy-2,3-pentanedione (DPD), the precursor of universal autoinducer-2 (AI-2), and its analogs (ethyl-DPD, butyl-DPD and isobutyl-DPD) in the integrity of gingival epithelial cells. MATERIAL AND METHODS: Human gingival keratinocytes were incubated with four concentrations (10 µmol L-1 , 1 µmol L-1 , 100 nmol L-1 and 10 nmol L-1 ) of DPD and its analogs for 24 hours. The numbers of viable cells were determined using a proliferation kit, matrix metalloproteinase (MMP)-2 and -9 activities were determined by gelatin zymography, and expression of occludin protein and occludin mRNA were determined by western blotting and RT-qPCR, respectively. RESULTS: Increased cell proliferation was observed in gingival keratinocytes incubated with 100 nmol L-1 of butyl-DPD. MMP-9 activity was elevated in cells incubated with 10 µmol L-1 of ethyl-DPD. On the other hand, MMP-2 activity did not show any significant change when gingival keratinocytes were incubated with or without DPD or analogs. Western blot analyses demonstrated five forms (105, 61, 52.2, 44 and 37 kDa) of occludin. Incubation with 1 µmol L-1 and 100 nmol L-1 of DPD and with 10 nmol L-1 of ethyl-DPD increased dimeric (105 kDa) forms of occludin, while incubation with 100 nmol L-1 of isobutyl-DPD increased monomeric (61 kDa) forms. DPD and ethyl-DPD decreased, and 100 nmol L-1 of isobutyl-DPD and 10 nmol L-1 of butyl-DPD increased, the monomeric (52.2 kDa and 44 kDa) forms of occludin, whereas ethyl-DPD decreased and isobutyl-DPD increased, the low-molecular-weight (37 kDa) forms. According to RT-qPCR analysis, the exposure of gingival keratinocytes to 10 µmol L-1 of isobutyl-DPD up-regulated expression of occludin. CONCLUSION: The results indicate that isobutyl-DPD has the potential to enhance the integrity of the epithelium by stimulating the formation of occluding, without affecting the proliferation or gelatinolytic enzyme activities of the exposed cells. The modulatory effect of an AI-2 analog on the epithelial cell response is shown for the first time.

Mannose-binding lectin gene polymorphism in relation to periodontal infection.

BACKGROUND AND OBJECTIVE: Mannose-binding lectin (MBL) plays an important role in innate immunity. MBL deficiency is usually caused by mutations in exon 1 of the MBL structural gene (MBL2). Our aim was to investigate MBL2 polymorphisms and their relation to salivary levels of periodontal inflammatory/tissue destruction markers and two major periodontitis-associated bacteria. MATERIAL AND METHODS: Salivary samples from 222 subjects were available for genotyping by pyrosequencing. The subjects between 40 and 60 years of age and having a minimum of 20 teeth were divided into three periodontal groups: 80 had generalized periodontitis, 65 had localized periodontitis and 77 were periodontitis-free. A comparison between their MBL2 genotypes and salivary detection rates and levels of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis as well as interleukin -1ß, matrix metalloproteinase -8, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 was performed. RESULTS: The frequencies of the MBL2 wild-type (A/A), heterozygote variants (A/O) and homozygote variants (O/O) were 69.4%, 26.6% and 4%, respectively. In A. actinomycetemcomitans-positive subjects having homozygote or heterozygote MBL2 variants, the salivary concentrations of IL-1ß (p = 0.010) were elevated and those of TIMP-1 (p = 0.001) were decreased. In addition their matrix metalloproteinase -8/TIMP-1 ratio was higher (p < 0.001) and they had more pocket teeth (p = 0.012) than subjects negative for A. actinomycetemcomitans. CONCLUSION: Our findings indicate that the carriage of A. actinomycetemcomitans may facilitate extended periodontal inflammation and destruction in subjects with a variant form of human MBL2.

Alveolar bone loss in relation to toll-like receptor 4 and 9 genotypes and Porphyromonas gingivalis carriage.

Toll-like receptors (TLRs) are highly developed sensors to detect microbe-associated molecular patterns. Functional polymorphisms of the genes TLR4 and TLR9 were found to be associated with alveolar bone loss in a Porphyromonas gingivalis-induced periodontitis model in mice. Our aim was to examine whether such an association can be detected in a group of Finnish adults. Polymorphisms of TLR4 Asp299Gly (rs4986790) and TLR9 rs187084 (1486 T/C) were genotyped by pyrosequencing and PCR from the saliva samples of 223 adults (age range 40-60 years). Alveolar bone loss, measured from panoramic radiographs, were compared between TLR genotype groups according to subjects' salivary carriage of P. gingivalis, measured using a single copy gene-based real-time PCR. The frequencies of TLR4 wild type and heterozygote variants were 87.4 % and 12.6 %, respectively, while those of TLR9 wild type, heterozygote, and homozygote variants were 25.6 %, 39.1 %, and 35.3 %, respectively. In the TLR4 heterozygote group, P. gingivalis-positive subjects had more alveolar bone loss than P. gingivalis-negative subjects (p = 0.027), while no difference was observed in the wild type group. P. gingivalis-negative individuals with TLR9 heterozygotes exhibited significantly less alveolar bone loss compared to those with TLR9 wild type (p = 0.007). Polymorphisms of TLR4 in P. gingivalis carriers seem to expose to alveolar bone loss. Polymorphisms of TLR9 can be protective against alveolar bone loss in the absence of P. gingivalis.

Periodontal health knowledge and smoking are associated with periodontal treatment need according to tooth brushing levels.

OBJECTIVE: The aim of this study is to determine whether periodontal health knowledge is associated with frequency of tooth brushing and periodontal treatment need. METHODS: Four hundred and two subjects participated in the study. Data on sociodemographic variables (age, gender, marital status, income, and education), general health, smoking behaviour tooth cleaning habits and knowledge on periodontal health/disease were collected with a questionnaire. Periodontal treatment need was examined using the Community Periodontal Index of Treatment Needs (CPITN). According to the CPITN scores, the treatment needs were grouped as minimum (CPITN = 0), low-level (CPITN = 1-2), or high-level (CPITN = 3-4). RESULTS: Statistical differences were found between the frequency of tooth brushing and smoking status, marital status, periodontal health knowledge and periodontal treatment needs. Gender (females), place of residence (urban areas), education and periodontal health knowledge had positive relationship with tooth brushing frequency, while smoking and periodontal treatment need had negative relationship. When multivariate logistic regression analysis was applied, age, marriage and poor periodontal knowledge were associated with increased low-level periodontal treatment needs, and age, marriage and smoking were associated with increased high-level periodontal treatment need. CONCLUSION: In the limits of this study, we suggest that gender, smoking habits, marital status, place of residence, education and periodontal health knowledge are determining factors related to tooth brushing frequency. Periodontal knowledge and smoking are associated with periodontal treatment needs.

Longitudinal study of salivary proteinases during pregnancy and postpartum.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinases (MMPs) and their regulators are connected to periodontal inflammation and destruction. However, the presence and role of the salivary MMPs in pregnancy-related gingivitis are not well known. Our longitudinal study aimed to monitor salivary proteinase levels and possible changes, and relate them to periodontal status during pregnancy and postpartum. MATERIAL AND METHODS: Salivary samples were collected from 30 periodontally healthy pregnant women five times (once during each trimester, 4-6 wk after delivery and after lactation) and, as their controls, from 24 non-pregnant women three times (during successive months). Periodontal examination included visible plaque index, bleeding on probing, probing pocket depth and clinical attachment level measurements. Matrix metalloproteinase-8 levels were measured by immunofluorometric assay, and MMP-2 and MMP-9 levels and molecular forms by gelatin zymography. Salivary elastase, myeloperoxidase and tissue inhibitor of matrix metalloproteinase-1 levels were measured by ELISA. RESULTS: Elastase concentrations maintained stable during the follow-up, while myeloperoxidase concentrations increased significantly after delivery. During pregnancy, MMP-8 concentrations were significantly lower than postpartum concentrations, being lowest during the second trimester and highest after delivery, and varying inversely to pregnancy gingivitis, observed as elevated percentages of bleeding on probing and probing pocket depth during the second and third trimester. In pregnant women, the highest MMP-2 and MMP-9 levels were found in saliva after lactation. In the control group, both clinical and enzymological findings remained stable during the follow-up period. CONCLUSION: Our results suggest that hormonal changes during pregnancy induce or enhance susceptibility to gingivitis, while salivary proteinase and myeloperoxidase levels are reduced.

Prevotella intermedia ATCC 25611 targets host cell lamellipodia in epithelial cell adhesion and invasion.

INTRODUCTION: The Prevotella intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, and Prevotella pallens, are phylogenetically closely related and potentially connected with oral and gastrointestinal tract disease pathogenesis. The aim of the present study was to examine whether these species differ in their capabilities of adhesion to and invasion of epithelial cells. METHODS: Adhesion and invasion were assayed by standard antibiotic/culture assays and fluorescent microscopy techniques. The effect of Prevotella strains on epithelial cell viability was measured using a commercial cell proliferation assay. RESULTS: The strains P. intermedia ATCC 25611 and P. nigrescens ATCC 33263 adhered to epithelial cells, the adhesion numbers of P. intermedia being twice as high as those of P. nigrescens. These strains invaded epithelial cells but invasion was weak. The adhesion of P. intermedia was specifically targeted to epithelial cell lamellipodia. The number of adhered P. intermedia cells increased or decreased when the formation of lamellipodia was stimulated or inhibited, respectively. None of the tested strains showed toxic effects on epithelial cells; a clinical P. intermedia strain even increased the number of viable cells by about 20%. CONCLUSION: The results suggest that among the P. intermedia group bacteria, P. intermedia and P. nigrescens type strains can adhere to and invade epithelial cells, the capability of P. intermedia ATCC 25611(T) being highest in this context. This strain proved to have a special affinity in binding to epithelial cell lamellipodia.

Stimulation of epithelial cell matrix metalloproteinase (MMP-2, -9, -13) and interleukin-8 secretion by fusobacteria.

BACKGROUND/AIMS: Bacterial pathogens involved in periodontal diseases exert their destructive effects primarily by stimulating the host cells to increase their secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs). This study aimed to determine the epithelial cell matrix metalloproteinase and interleukin-8 (IL-8) secretion upon exposure to fusobacteria. METHODS: Eight different oral and non-oral Fusobacterium strains were incubated with HaCaT epithelial cells. Gelatin zymography and Western blot analysis were performed to detect collagenase 3 (MMP-13), gelatinase A (MMP-2), gelatinase B (MMP-9), and IL-8 secretion by epithelial cells. RESULTS: All Fusobacterium strains, especially Fusobacterium necrophorum ATCC 25286, Fusobacterium nucleatum ATCC 25586, and Fusobacterium varium ATCC 51644, increased MMP-9 and MMP-13 secretion. Fusobacterium simiae ATCC 33568, and to a lesser extent F. nucleatum and F. necrophorum, increased epithelial MMP-2 secretion. F. nucleatum and F. necrophorum also increased IL-8 secretion. F. varium ATCC 27725, a strain that only weakly stimulated MMP production, strongly increased the IL-8 production, suggesting that their expression is differently regulated. CONCLUSION: We conclude that the pathogenic potential of fusobacteria may partly result from their ability to stimulate secretion of MMP-9, MMP-13, and IL-8 from epithelial cells.

Palatal mucosa necrosis because of accidental sodium hypochlorite injection instead of anaesthetic solution.

AIM: A case is reported in which sodium hypochlorite (NaOCl) was inadvertently injected into the palatal mucosa instead of local anaesthetic solution. SUMMARY: An adult male was referred to the University clinic 15 days after an inadvertent NaOCl injection into the palatal mucosa. Soft tissue necrosis was evident, without obvious bony involvement. Tissues healed without scarring within 30 days. No surgical intervention was required. KEY LEARNING POINTS: NaOCl is highly irritant when extruded into vital tissues.--Dentists should be careful to avoid the misuse of NaOCl, and should check the nature of any agent before injecting it into patients.--NaOCl should not be dispensed in a way that could allow it to be mistaken for local anaesthetic solutions.--The well-perfused tissues of the oral cavity have considerable healing ability.

Assessment of mandibular bone mineral density in patients with type 2 diabetes mellitus.

OBJECTIVES: To assess mandibular bone mineral density in patients with type 2 diabetes mellitus using panoramic radiographs. METHODS: Nineteen patients with type 2 diabetes mellitus and 17 control subjects participated in the study. Bone mineral density measurements were performed on the panoramic radiographs with the help of a five-step copper stepwedge phantom, attached to each film cassette, which was calibrated before hand by dual energy X-ray absorptiometry (DXA). The analysis was performed on digitized images by dedicated software (Scion Image). The accuracy of the measurement technique was also evaluated by cross-calibration with a DXA phantom. RESULTS: Mean mandibular bone mineral density (g cm(-2)) was 1.53+/-0.27 in women and 1.52+/-0.29 in men with type 2 diabetes. In age and gender matched control subjects the values were 1.56+/-0.28 and 1.46+/-0.23 in women and in men, respectively. No statistically significant difference was observed between groups in mandibular bone mineral density. CONCLUSIONS: The present study showed that bone mineral density of the mandible does not seem to be affected in patients with type 2 diabetes mellitus. We also observed that a panoramic radiograph could serve for accurate mandibular bone density determination, when calibrated well with the DXA method.

Gingival fibromatosis, short stature, border-line IQ, facial dysmorphism and hepatomegaly.

Gingival fibromatosis, short stature, border-line IQ, facial dysmorphism and hepatomegaly: Gingival fibromatosis is a rare and benign disorder. The enlarged gingivae are firm and may interfere with speech, closure of the lips, and mastication. We report a thirteen years old girl, with gingival fibromatosis referred to the periodontics clinics. Full mouth gingivectomy and gingivoplasty were performed. Medical investigation showed short stature, low-borderline IQ, facial dysmorphism, and hepatomegaly. Histological analysis revealed hyperplasia in the epithelial area and fibrotic appearance of gingival connective tissue with dense collagen fibre clusters. Pedigree analysis confirmed that mode of inheritance is autosomal recessive. According to the combination of clinical findings, this case report may represent a previously unreported syndrome.