Pocket depth and bleeding on probing and their associations with dental, lifestyle, socioeconomic and blood variables: a cross-sectional, multicenter feasibility study of the German National Cohort.

BACKGROUND: To investigate the periodontal disease status in a multi-center cross-sectional study in Germany. Associations of dental, socio-economic, blood and biomedical variables with periodontal outcome parameters were evaluated. METHODS: From 4 different centers N = 311 persons were included, drawn randomly from the registration offices. Maximal pocket depth (PD) was used as primary indicator for periodontitis. It was classified as: no/mild ≤3 mm, moderate 4-5 mm, severe ≥6 mm. Associations between socioeconomic (household income, education), lifestyle, and biomedical factors and PD or bleeding on probing (BOP) per site ("Yes"/"No") was analyzed with logistic regression analysis. RESULTS: Mean age of subjects was 46.4 (range 20-77) years. A significantly higher risk of deeper pockets for smokers (OR = 2.4, current vs. never smoker) or persons with higher BMI (OR = 1.6, BMI increase by 5) was found. Severity of periodontitis was significantly associated with caries lesions (p = 0.01), bridges (p < .0001), crowns (p < .0001), leukocytes (p = 0.04), HbA1c (p < .0001) and MCV (p = 0.04). PD was positively correlated with BOP. No significant associations with BOP were found in regression analysis. CONCLUSIONS: Earlier findings for BMI and smoking with severity of PD were confirmed. Dental variables might be influenced by potential confounding factors e.g. dental hygiene. For blood parameters interactions with unknown systemic diseases may exist.

The impact of Porphyromonas gingivalis lipids on apoptosis of primary human chondrocytes.

The role of oral bacterial infections including periodontal disease in the pathogenesis of rheumatoid arthritis (RA) has gained increasing interest. Among the major periodontal pathogens, Porphyromonas gingivalis has been mostly associated with RA pathogenesis. The aim of this study was to analyze the effect of P. gingivalis total lipid (TL) fraction and dihydroceramides, as potent virulence factors, on human primary chondrocytes. Primary chondrocyte cultures were incubated with P. gingivalis phosphoglycerol dihydroceramide (PG DHC) lipids, the TL fraction or phosphoethanolamine dihydroceramide. Cell morphology changes were determined by phase contrast light microscopy. Early and late apoptosis cell analysis was performed by Annexin-V, active caspases, and 7-Aminoactinomycin D staining, and examined by flow cytometry, and cell necrosis was evaluated by lactate dehydrogenase release. Procaspase-3 activation was determined by Western blot analysis. Microscopic analysis showed altered cell morphology and cell shrinkage following incubation with P. gingivalis TLs and PG DHC lipids. Flow cytometry demonstrated an increase of Annexin-V positive and active caspases positive chondrocytes after incubation with TL and PG DHC fractions but not after phosphoethanolamine dihydroceramide (control lipid) treatment or in untreated control cells. Furthermore, Western blot analysis showed an early cleavage of procaspase-3 after 1 hr. Significant lactate dehydrogenase release following incubation with P. gingivalis lipids was demonstrated. The present data demonstrate that P. gingivalis lipids promote apoptosis in primary human chondrocytes, and thereby may contribute to the joint damage seen in the pathogenesis of RA.

Lysyl oxidase propeptide inhibits FGF-2-induced signaling and proliferation of osteoblasts.

Pro-lysyl oxidase is secreted as a 50-kDa proenzyme and is then cleaved to a 30-kDa mature enzyme (lysyl oxidase (LOX)) and an 18-kDa propeptide (lysyl oxidase propeptide (LOX-PP)). The presence of LOX-PP in the cell layers of phenotypically normal osteoblast cultures led us to investigate the effects of LOX-PP on osteoblast differentiation. Data indicate that LOX-PP inhibits terminal mineralization in primary calvaria osteoblast cultures when added at early stages of differentiation, with no effects seen when present at later stages. LOX-PP was found to inhibit serum- and FGF-2-stimulated DNA synthesis and FGF-2-stimulated cell growth. Enzyme-linked immunosorbent assay and Western blot analyses show that LOX-PP inhibits FGF-2-induced ERK1/2 phosphorylation, signaling events that mediate the FGF-2-induced proliferative response. LOX-PP inhibits FGF-2-stimulated phosphorylation of FRS2alpha and FGF-2-stimulated DNA synthesis, even after inhibition of sulfation of heparan sulfate proteoglycans. These data point to a LOX-PP target at or near the level of fibroblast growth factor receptor binding or activation. Ligand binding assays on osteoblast cell layers with (125)I-FGF-2 demonstrate a concentration-dependent inhibition of FGF-2 binding to osteoblasts by LOX-PP. In vitro binding assays with recombinant fibroblast growth factor receptor protein revealed that LOX-PP inhibits FGF-2 binding in an uncompetitive manner. We propose a working model for the respective roles of LOX enzyme and LOX-PP in osteoblast phenotype development in which LOX-PP may act to inhibit the proliferative response possibly to allow cells to exit from the cell cycle and progress to the next stages of differentiation.

Epigenetic adaptations of the masticatory mucosa to periodontal inflammation.

BACKGROUND: In mucosal barrier interfaces, flexible responses of gene expression to long-term environmental changes allow adaptation and fine-tuning for the balance of host defense and uncontrolled not-resolving inflammation. Epigenetic modifications of the chromatin confer plasticity to the genetic information and give insight into how tissues use the genetic information to adapt to environmental factors. The oral mucosa is particularly exposed to environmental stressors such as a variable microbiota. Likewise, persistent oral inflammation is the most important intrinsic risk factor for the oral inflammatory disease periodontitis and has strong potential to alter DNA-methylation patterns. The aim of the current study was to identify epigenetic changes of the oral masticatory mucosa in response to long-term inflammation that resulted in periodontitis. METHODS AND RESULTS: Genome-wide CpG methylation of both inflamed and clinically uninflamed solid gingival tissue biopsies of 60 periodontitis cases was analyzed using the Infinium MethylationEPIC BeadChip. We validated and performed cell-type deconvolution for infiltrated immune cells using the EpiDish algorithm. Effect sizes of DMPs in gingival epithelial and fibroblast cells were estimated and adjusted for confounding factors using our recently developed "intercept-method". In the current EWAS, we identified various genes that showed significantly different methylation between periodontitis-inflamed and uninflamed oral mucosa in periodontitis patients. The strongest differences were observed for genes with roles in wound healing (ROBO2, PTP4A3), cell adhesion (LPXN) and innate immune response (CCL26, DNAJC1, BPI). Enrichment analyses implied a role of epigenetic changes for vesicle trafficking gene sets. CONCLUSIONS: Our results imply specific adaptations of the oral mucosa to a persistent inflammatory environment that involve wound repair, barrier integrity, and innate immune defense.

Advanced glycation end products stimulate osteoblast apoptosis via the MAP kinase and cytosolic apoptotic pathways.

We have previously shown that diabetes significantly enhances apoptosis of osteoblastic cells in vivo and that the enhanced apoptosis contributes to diabetes impaired new bone formation. A potential mechanism is enhanced apoptosis stimulated by advanced glycation end products (AGEs). To investigate this further, an advanced glycation product, carboxymethyl lysine modified collagen (CML-collagen), was injected in vivo and stimulated a 5-fold increase in calvarial periosteal cell apoptosis compared to unmodified collagen. It also induced apoptosis in primary cultures of human or neonatal rat osteoblastic cells or MC3T3-E1 cells in vitro. Moreover, the apoptotic effect was largely mediated through RAGE receptor. CML-collagen increased p38 and JNK activity 3.2- and 4.4-fold, respectively. Inhibition of p38 and JNK reduced CML-collagen stimulated apoptosis by 45% and 59% and by 90% when used together (P<0.05). The predominant apoptotic pathway induced by CML-collagen involved caspase-8 activation of caspase-3 and was independent of NF-kappaB activation. When osteoblastic cells were exposed to a long-term low dose incubation with CML-collagen, there was a higher degree of apoptosis compared to short-term incubation. In more differentiated osteoblastic cultures, apoptosis was enhanced even further. These results indicate that advanced glycation end products, which accumulate in diabetic and aged individuals, may promote apoptosis of osteoblastic cells and contribute to deficient bone formation.

Influence of periodontal therapy on the regulation of soluble cell adhesion molecule expression in aggressive periodontitis patients.

BACKGROUND: Inflammatory periodontal disease is associated with an increased risk of cardiovascular disease. Circulating cell adhesion molecules (CAM) (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1], and E-selectin) have been suggested as potential candidate markers of endothelial dysfunction, which contribute to the pathogenesis of cardiovascular diseases. The regulation of CAM in subjects with severe periodontitis and the influence of periodontal intervention on systemic CAM levels are not clear. The aim of this study was to determine whether intensive periodontal therapy reduces serum levels of CAM in patients with generalized aggressive periodontitis. METHODS: Blood samples were collected at six treatment time points from 21 patients with previously untreated generalized aggressive periodontitis (mean age: 34.6 +/- 4.3 years). Patients received subgingival scaling and root planing and antibiotic therapy and were monitored over a 6-month recall period. Serum levels of soluble ICAM-1 (sICAM-1), VCAM-1 (sVCAM-1), and E-selectin (sE-selectin) were measured by enzyme-linked immunosorbent assay. RESULTS: sE-selectin plasma levels decreased significantly (P <0.01) during periodontal therapy. Mean plasma levels were 65.95 ng/ml before treatment and 44.71 ng/ml 6 months after antibiotic therapy. sICAM-1 and sVCAM-2 serum levels were unaffected by therapeutic intervention. CONCLUSIONS: Periodontal therapy reduces plasma sE-selectin levels. Whether this leads to a reduction in risk of future cardiovascular events in patients with aggressive periodontal disease warrants further studies.

Enamel matrix derivative induces connective tissue growth factor expression in human osteoblastic cells.

BACKGROUND: Enamel matrix derivative (EMD) stimulates the production of transforming growth factor-beta (TGF-beta), which has been suggested to play a role in mediating the effects of EMD in periodontal tissue regeneration. Connective tissue growth factor (CTGF) is a mediator of TGF-beta and promotes cell development. The interaction between EMD and CTGF is unknown. This study explored the effects of EMD on CTGF expression in human osteoblastic cells and whether the interaction is modulated by the TGF-beta signaling pathway. Also, the roles of CTGF in cell proliferation, cell cycle progression, and mineralized nodule formation of EMD-induced osteoblastic cultures were examined. METHODS: Human osteoblastic cells (Saos-2) were treated with 25 to 100 microg/ml EMD with or without the addition of TGF-beta inhibitor. CTGF mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR), and CTGF protein levels were assayed by Western blot analysis. In addition, cell cycle progression and DNA synthesis were determined by flow cytometry and 5-bromo-2'-deoxyuridine (BrdU) incorporation following EMD treatment with or without CTGF antibody. Mineralization was examined by alizarin red staining and quantified by elution with cetylpyridinium chloride. RESULTS: Western blot and RT-PCR analysis demonstrated a dose-dependent increase of CTGF expression by EMD. EMD-induced CTGF expression was reduced significantly in the presence of TGF-beta inhibitor. Cell cycle and BrdU analysis revealed an increase in cell proliferation following EMD treatment, which was due to an increase in the percentage of cells in the G2/M phase of the cell cycle. No significant effect was found when anti-CTGF antibody was added. Conversely, mineralization was inhibited significantly in EMD-treated cultures in the presence of anti-CTGF antibody. CONCLUSIONS: EMD stimulates CTGF expression, and the interaction is modulated via TGF-beta in osteoblastic cells. Also, CTGF affects EMD-induced osteoblastic mineralization but not cell proliferation. To our knowledge, these results provide novel insight into EMD-CTGF interaction, two biomodifiers that have therapeutic relevance to tissue engineering and regeneration.

Antimicrobial Effects of Mastic Extract Against Oral and Periodontal Pathogens.

BACKGROUND: Various antimicrobial agents are widely used in the therapy of oral inflammatory diseases. However, their side effects and the appearance of drug resistance justify research on natural antimicrobial agents to target oral pathogens that are safe for the host. In the present study, antimicrobial properties of mastic extract on commensal and pathogenic oral bacteria, as well as its possible cytotoxic effect toward cells of epithelial and mesenchymal origin, were evaluated and compared with the common antimicrobial agents hydrogen peroxide (H2O2) and chlorhexidine digluconate (CHX). METHODS: Oral and periodontal pathogens (Porphyromonas gingivalis, Streptococcus mutans [Sm], Streptococcus oralis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia, and Prevotella nigrescens) were treated with different concentrations of mastic extract, 3% H2O2, and 0.2% CHX, and evaluated with an agar diffusion test. The cytotoxic effect of mastic extract was tested on four cell lines of epithelial and mesenchymal origin (HaCaT, SaOS-2, MC3T3-E1, periodontal ligament [PDL] cells) by neutral red and 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. RESULTS: Mastic extract led to significantly (P ≤0.016) increased inhibition of the tested periodontal pathogens compared with H2O2. No effect of mastic extract was observed on Sm. Mastic extract showed beneficial effects on cell viability because viability values of tested cells were significantly (P ≤0.016) lower for cells treated with CHX and H2O2 compared with mastic extract-treated cells after stimulation for 2, 4, and 6 hours. CONCLUSION: The present data demonstrate mastic extract's inhibition of periodontal pathogens, as well as beneficial effects on cell viability, compared with H2О2, suggesting that it could be considered an alternative antibacterial agent in the prevention of periodontal disease.

Plant-derived pectin nanocoatings to prevent inflammatory cellular response of osteoblasts following Porphyromonas gingivalis infection.

BACKGROUND: Bioengineered plant-derived Rhamnogalacturonan-Is (RG-Is) from pectins are potential candidates for surface nanocoating of medical devices. It has recently been reported that RG-I nanocoatings may prevent bacterial infection and improve the biocompatibility of implants. The aim of the study was to evaluate in vitro impact of bioengineered RG-I nanocoatings on osteogenic capacity and proinflammatory cytokine response of murine osteoblasts following Porphyromonas gingivalis infection. METHODS: Murine MC3T3-E1 osteoblasts and isolated primary calvarial osteoblasts from C57BL/6J (B6J osteoblasts) mice were infected with P. gingivalis and incubated on tissue culture polystyrene plates with or without nanocoatings of unmodified RG-Is isolated from potato pulps (PU) or dearabinanated RG-Is (PA). To investigate a behavior of infected osteoblasts cultured on RG-Is cell morphology, proliferation, metabolic activity, mineralization and osteogenic and pro-inflammatory gene expression were examined. RESULTS: Following P. gingivalis infection, PA, but not PU, significantly promoted MC3T3-E1 and BJ6 osteoblasts proliferation, metabolic activity, and calcium deposition. Moreover, Il-1b, Il-6, TNF-α, and Rankl gene expressions were downregulated in cells cultured on PU and to a higher extent on PA as compared to the corresponding control, whereas Runx, Alpl, Col1a1, and Bglap gene expressions were upregulated vice versa. CONCLUSION: Our data clearly showed that pectin RG-Is nanocoating with high content of galactan (PA) reduces the osteoblastic response to P. gingivalis infection in vitro and may, therefore, reduce a risk of inflammation especially in immunocompromised patients with rheumatoid or periodontal disorders.

Cross-Sectional Association Between Number of Teeth and High-Sensitivity C-Reactive Protein Among Middle-Aged Germans.

BACKGROUND: The association between number of teeth and low-grade systemic inflammation deserves consideration within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam since the association between number of teeth and myocardial infarction has been established. METHODS: Two subsamples (n = 2,439 and 728) were randomly selected from EPIC-Potsdam. Participants provided information on number of natural teeth, anthropometry, lifestyle factors, and illness-related factors. High-sensitivity C-reactive protein (hsCRP) was measured from serum. Adjusted means of hsCRP across five categories of numbers of teeth in each subsample and in the combined sample were determined, and linear trends were checked. Non-linear associations were investigated with restricted cubic spline (RCS) regression. RESULTS: In the first subsample, the full multivariable-adjusted model showed that participants with 28 to 32, 24 to 27, 18 to 23, 1 to 17, and 0 teeth had mean hsCRP values of 1.32, 1.39, 1.54, 1.38, and 1.48 mg/L, respectively; in the second subsample, mean hsCRP values were 1.64, 1.67, 1.73, 1.47, and 1.87 mg/L; combined hsCRP values were 1.49, 1.53, 1.64, 1.44, and 1.65 mg/L. No linear trend was observed in these models, and RCS regression showed no non-linear association. CONCLUSION: This study shows that number of teeth has a weak association with hsCRP, if any, thereby excluding this marker of low-grade systemic inflammation as a possible explanation for the association between number of teeth and myocardial infarction.

Increased Periodontal Attachment Loss in Patients With Systemic Sclerosis.

BACKGROUND: Patients with inflammatory rheumatic diseases and periodontitis share common pathogenetic characteristics, such as proinflammatory traits causative for tissue degradation and loss of function. The aim of the present case control study is to investigate the association between systemic sclerosis (SSc) and periodontitis. METHODS: The association between SSc and periodontitis was examined in 58 SSc patients and 52 control patients, matched for age and sex. The periodontal examination included periodontal attachment loss (AL), probing depth, bleeding on probing, plaque index (PI), and gingival index (GI). Potential risk factors of periodontitis were assessed through patients' questionnaires. RESULTS: In unadjusted analyses, patients with SSc had a significant 0.61 mm higher AL (95% confidence interval [CI] 0.24 to 0.97; P = 0.002) when compared with controls. In a stepwise logistic regression, including SSc status, age, sex, education, smoking, alcohol consumption, and body mass index, only SSc status, age, and sex remained significantly associated with periodontitis. Adjusted for age and sex, patients with SSc had a 0.52 mm higher AL compared with controls (95% CI 0.16 to 0.88; P = 0.005). The strength of the association of SSc with AL remained statistically significant after additional adjustment for PI (0.44 mm; 95% CI 0.02 to 0.86; P = 0.04) or GI (0.61 mm; 95% CI 0.24 to 0.97; P = 0.001). CONCLUSIONS: This study demonstrates higher AL in patients with SSc, which remained significant after adjustment. The study indicates a possible relationship between SSc and periodontitis.

Association between Number of Teeth and Chronic Systemic Diseases: A Cohort Study Followed for 13 Years.

BACKGROUND: There is growing evidence of an association between oral health, specifically dental status, and chronic systemic diseases. However, varying measures of dental status across different populations and low study sample has made comparison of studies and conclusion of findings unclear. Our aim is to examine whether the number of teeth as a measure of dental status is associated with incident chronic diseases in a cohort setting. METHODS: We conducted a cohort study among 24,313 middle-aged Germans followed up for 13 years. Data on number of teeth as a measure of dental status were obtained through self-reports. Outcomes were clinically-verified incident non-fatal myocardial infarction, stroke, type 2 diabetes mellitus, and cancer. Hazard ratio (HR) and 95% confidence intervals (CI) were obtained from Cox regression models. RESULTS: Increasing number of teeth is inversely related to risk of myocardial infarction (HR: 0.97; 95% CI: 0.96, 0.99). The full multivariate model of teeth groups showed a strong linear trend for myocardial infarction, a less strong trend for stroke, and no relation with type 2 diabetes mellitus and cancer in a competing risk model. Participants with 18-23 teeth and those without teeth were at 76% (95%CI: 1.04, 3) and 2.93 times (95%CI: 1.61, 5.18) higher risk of myocardial infarction compared to those with nearly all teeth (28-32 teeth). CONCLUSIONS: Number of teeth is specifically associated with myocardial infarction and not with other chronic disease indicating that dental status further strengthens the link between oral health and cardiovascular diseases.

Porphyromonas gingivalis Suppresses Differentiation and Increases Apoptosis of Osteoblasts From New Zealand Obese Mice.

BACKGROUND: Metabolic syndrome (MetS), a complex cluster of risk factors for chronic diseases such as cardiovascular disease, is observed to be increasingly associated with periodontal disease. However, the fundamental contribution of periodontal bacteria to periodontal bone loss in patients with MetS remains unclear. The aim of the present study is to analyze the effect of Porphyromonas gingivalis on differentiation of primary osteoblasts from New Zealand obese (NZO) mice, a model for MetS, compared with C57 Black 6 JAX (C57BL/6J) mice osteoblasts. METHODS: Primary calvarial osteoblasts, isolated from 3-day-old NZO and control C57BL/6J mice, were stimulated with P. gingivalis. Proliferation was quantified by 5-bromo-2'-deoxyuridine incorporation. Cell cycle and early and late apoptosis were measured by flow cytometry. Gene expression was determined by real-time polymerase chain reaction (RT-PCR). RESULTS: Twelve hours after P. gingivalis stimulation, NZO osteoblasts showed significantly decreased proliferation (P ≤0.01) with increased G2 cell cycle phase compared with normal osteoblasts. Flow cytometry analysis demonstrated significant (P ≤0.01) increase of early apoptotic cells (annexin V positive) and late apoptosis (caspase 3 activity) in NZO cells compared with control cells at 3 and 6 hours after stimulation. No significant lactate dehydrogenase release was found after P. gingivalis stimulation. RT-PCR data showed significantly suppressed expression (P ≤0.01) of collagen 1, osteocalcin, and Runt-related transcription factor 2 in NZO cells compared with normal osteoblasts. CONCLUSIONS: The present data demonstrate that P. gingivalis downregulates proliferation and promotes apoptosis in primary NZO osteoblasts, unlike C57BL/6J osteoblasts. Also, suppressed osteoblastic marker expression in NZO cells may contribute to pathogenesis of periodontitis, suggesting a similar process in patients with MetS.

Effect of untreated and treated temporomandibular joint arthritis on mandibular volume development in growing rabbits.

OBJECTIVES: The goal of this work was to investigate the volume development of the mandible in growing rabbits with bilaterally induced temporomandibular joint (TMJ) arthritis that was either left untreated or treated with the tumor necrosis factor-alpha (TNF-α) antagonist etanercept. METHODS: A total of 18 New Zealand White rabbits aged 8 weeks were randomized to three groups of 6 animals each. Two of these groups were used as arthritis groups by sensitizing the 12 animals to ovalbumin (OA) at 10 weeks, followed by intraarticular OA injections to induce bilateral TMJ arthritis and repeating these injections every 3 weeks to maintain the inflammation. One of the two arthritis groups was treated by weekly subcutaneous etanercept injections, whereas the other group was left untreated. The remaining 6 animals served as controls. Maxillofacial CT scans were obtained at 3-week intervals (from week 10 of the rabbits' lives to the end of the experiment at 22 weeks) to volumetrically track the development of the mandibles after segmentation. RESULTS: The mandibles did not grow at a continuous rate, but the rate of development was found to decrease in all groups over the course of the study (weeks 10-22). The most extensive volume increases were noted during weeks 10-13. Severe growth deficiencies, especially of the condylar processes, were observed in the arthritis group not receiving treatment. The arthritis group treated with etanercept showed better rates of growth without, however, reaching the normal range of the control group. CONCLUSION: Antigen-induced TMJ arthritis was found to involve severe problems of growth similar to those in juvenile idiopathic arthritis. Etanercept can improve the volume development but does not reestablish an entirely normal rate of growth.

Effects of enamel matrix derivative and transforming growth factor-ß1 on connective tissue growth factor in human periodontal ligament fibroblasts.

BACKGROUND: Enamel matrix derivative (EMD) is suggested to stimulate transforming growth factor-ß (TGF-ß) production. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-ß. This study explores the effects of EMD and TGF-ß1 on CTGF in periodontal ligament (PDL) fibroblasts and their interactions in PDL proliferation and development. METHODS: Human PDL cells were stimulated with EMD. To explore the effects of EMD and TGF-ß1 on CTGF expression, cells were treated with and without TGF-ß inhibitor, and CTGF protein levels were assayed by Western blot analysis. To study the role of CTGF in PDL development, cells were treated with CTGF inhibitor. DNA synthesis was analyzed by bromodeoxyuridine enzyme-linked immunosorbent assay. Reverse-transcription polymerase chain reaction was performed to examine messenger RNA expression of PDL osteoblastic differentiation markers: type I collagen, alkaline phosphatase, and osteocalcin. RESULTS: EMD induced a concentration-dependent increase of CTGF protein expression in PDL cells. EMD- and TGF-ß1-stimulated CTGF expression was significantly reduced in the presence of TGF-ß inhibitor. CTGF inhibition downregulated both EMD- and TGF-ß1-induced DNA synthesis. The effect of CTGF and EMD on osteoblastic mRNA expression in PDL cells is not obvious. CONCLUSIONS: EMD stimulates CTGF expression in human PDL cells, a process modulated by the TGF-ß pathway. CTGF can affect EMD- and TGF-ß1-induced PDL cell proliferation, but its effects on PDL with regard to osteoblastic differentiation remain inconclusive. The results provide novel insights into EMD-CTGF interaction in PDL cells.

Effects of enamel matrix derivative on proliferation and differentiation of primary osteoblasts.

OBJECTIVE: The aim of this study was to investigate the effects of enamel matrix derivative (EMD) on proliferation, protein synthesis, and mineralization in primary mouse osteoblasts. STUDY DESIGN: Osteoblasts were obtained from mouse calvaria by enzymatic digestion and grown in monolayer together with EMD (2-100 microg/ml). Metabolic activity and cell proliferation were determined by tetrazolium salt assay (MTT) and by 5-bromo-2'-deoxyuridine (BrdU) incorporation. For differentiation studies, a 3-dimensional organoid culture system was used. Osteoblastic differentiation was estimated by alkaline phosphatase (ALP) activity and calcium content. Collagen synthesis was assessed by [(3)H]-proline incorporation. Morphologic observations were made by electron microscopy. RESULTS: EMD treatments increased metabolic cell activity and BrdU incorporation. In the organoid cultures, ALP activity and calcium accumulation were enhanced by EMD treatment, but [(3)H]-proline incorporation was not. Morphologically, an increased deposition of mineralized nodules was found. CONCLUSIONS: EMD treatment enhanced cellular activities of primary osteoblasts and might support the regeneration of periodontal bony defects.

The association between rheumatoid arthritis and periodontal disease.

Chronic, plaque-associated inflammation of the gingiva and the periodontium are among the most common oral diseases. Periodontitis (PD) is characterized by the inflammatory destruction of the periodontal attachment and alveolar bone, and its clinical appearance can be influenced by congenital as well as acquired factors. The existence of a rheumatic or other inflammatory systemic disease may promote PD in both its emergence and progress. However, there is evidence that PD maintains systemic diseases. Nevertheless, many mechanisms in the pathogenesis have not yet been examined sufficiently, so that a final explanatory model is still under discussion, and we hereby present arguments in favor of this. In this review, we also discuss in detail the fact that oral bacterial infections and inflammation seem to be linked directly to the etiopathogenesis of rheumatoid arthritis (RA). There are findings that support the hypothesis that oral infections play a role in RA pathogenesis. Of special importance are the impact of periodontal pathogens, such as Porphyromonas gingivalis on citrullination, and the association of PD in RA patients with seropositivity toward rheumatoid factor and the anti-cyclic citrullinated peptide antibody.

Etiologic factors of hyposalivation and consequences for oral health.

Hyposalivation is represented by a reduced salivary flow rate and can be caused by etiologic factors such as systemic diseases and intake of various medications or by radiotherapy following head and neck cancer. The aim of this review was to compile data about the qualitative and quantitative changes of salivary components during hyposalivation, and to summarize their consequences for oral health. A Medline/PubMed/Scopus search was conducted to identify and summarize articles published in English and German that reported on etiology of hyposalivation and changes in the salivary composition due to hyposalivation of different origins. The search revealed 94 articles, 71 of which were original articles. Apart from the reduction of the salivary flow rate, the quality of saliva is strongly altered because of systemic diseases, medications, and radiotherapy, including increased viscosity and pH shift to more acidic values and changes in salivary protein compositions. Furthermore, hyposalivation may be accompanied by pronounced shifts in specific microbial components, in particular toward a highly acidogenic microflora. Moreover, therapy of hyposalivation is often restricted to palliative treatment (ie, saliva substitutes or gels). To prevent tooth tissue demineralization, clinicians should consider saliva substitutes that are supersaturated with calcium and phosphates and contain fluoride.

Intracellular distribution of the lysyl oxidase propeptide in osteoblastic cells.

Lysyl oxidase plays a critical role in the formation of the extracellular matrix, and its activity is required for the normal maturation and cross-linking of collagen and elastin. An 18-kDa lysyl oxidase propeptide (LOPP) is generated from 50-kDa prolysyl oxidase by extracellular proteolytic cleavage during the biosynthesis of active 30-kDa lysyl oxidase enzyme. The fate and the functions of the LOPP are largely unknown, although intact LOPP was previously observed in osteoblast cultures. We investigated the spatial localization of molecular forms of lysyl oxidase, including LOPP in proliferating and differentiating osteoblasts, by using confocal immunofluorescence microscopy and Western blots of cytoplasmic and nuclear extracts. In the present study, a stage-dependent intracellular distribution of LOPP in the osteoblastic cell was observed. In proliferating osteoblasts, LOPP epitopes were principally associated with the Golgi and endoplasmic reticulum, and mature lysyl oxidase epitopes were found principally in the nucleus and perinuclear region. In differentiating cells, LOPP and mature lysyl oxidase immunostaining showed clear colocalization with the microtubule network. The subcellular distribution of LOPP and its temporal and physical association with microtubules were confirmed by Western blot and far Western blot studies. We also report that N-glycosylated and nonglycosylated LOPP are present in MC3T3-E1 cell cultures. We conclude that LOPP has a stage-dependent intracellular distribution in osteoblastic cells. Future studies are needed to investigate whether the LOPP associations with microtubules or the osteoblast nucleus have functional effects for osteoblast differentiation and bone formation.

Effects of an enamel matrix derivative on human osteoblasts and PDL cells grown in organoid cultures.

OBJECTIVE: The objective of this study was to investigate cellular effects of enamel matrix derivative (EMD) in human derived, primary osteoblasts and periodontal ligament (PDL) cells grown in organoid cultures. STUDY DESIGN: Cell replication was assessed by BrdU-incorporation. [(3)H]-proline incorporation was measured to determine the synthesis of proline-containing proteins, such as collagen. In addition, calcium accumulation and alkaline-phosphatase-activity were quantified. Electron microscopy for morphological analysis was performed. RESULTS: Our results showed that EMD enhances BrdU-incorporation in PDL cells and osteoblasts. Also, in osteoblast organoid cultures [3H]-proline incorporation was 3-fold increased (P < .01). Extensive matrix deposition was noted in osteoblast cultures by electron microscopy. In osteoblasts, high levels of calcium accumulation and alkaline-phosphatase-activity were found. However, EMD did not promote mineralization. CONCLUSION: Our results indicate that under organoid culture conditions EMD is able to promote the synthesis of proline-containing proteins such as collagen but not matrix mineralization of primary human osteoblastic cells.