Number of teeth is associated with all-cause and disease-specific mortality.

BACKGROUND: Tooth loss has been shown to correlate with multiple systemic comorbidities. However, the associations between the number of remaining natural teeth (NoT) and all-cause mortality have not been explored extensively. We aimed to investigate whether having fewer NoT imposes a higher risk in mortality. We tested such hypotheses using three groups of NoT (20-28,10-19, and 0-9), edentulism and without functional dentition (NoT < 19). METHODS: The National Health and Nutrition Examination Survey in the United States (NHANES) (1999-2014) conducted dental examinations and provided linkage of mortality data. NHANES participants aged 20 years and older, without missing information of dental examination, age, gender, race, education, income, body-mass-index, smoking, physical activities, and existing systemic conditions [hypertension, total cardiovascular disease, diabetes, and stroke (N = 33,071; death = 3978), or with femoral neck bone mineral density measurement (N = 13,131; death = 1091)] were analyzed. Cox proportional hazard survival analyses were used to investigate risks of all-cause, heart disease, diabetes and cancer mortality associated with NoT in 3 groups, edentulism, or without functional dentition. RESULTS: Participants having fewer number of teeth had higher all-cause and disease-specific mortality. In fully-adjusted models, participants with NoT0-9 had the highest hazard ratio (HR) for all-cause mortality [HR(95%CI) = 1.46(1.25-1.71); p < .001], mortality from heart diseases [HR(95%CI) = 1.92(1.33-2.77); p < .001], from diabetes [HR(95%CI) = 1.67(1.05-2.66); p = 0.03], or cancer-related mortality [HR(95%CI) = 1.80(1.34-2.43); p < .001]. Risks for all-cause mortality were also higher among the edentulous [HR(95%CI) = 1.35(1.17-1.57); p < .001] or those without functional dentition [HR(95%CI) = 1.34(1.17-1.55); p < .001]. CONCLUSIONS: Having fewer NoT were associated with higher risks for all-cause mortality. More research is needed to explore possible biological implications and validate our findings.

Potential roles of miR-335-5p on pathogenesis of experimental periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is a prevalent oral disease responsible for tooth loss. MicroRNAs have been proven crucial in bone disorders over the past decades. Promotive effect on osteogenic activities by microRNA-335-5p (miR-335-5p) has been well demonstrated, but its role involved in the pathogenesis of periodontitis remains elusive. In this study, we established experimental periodontitis (EP) on transgenic mice overexpressing miR-335-5p (335-Tg) to investigate the novel effects of miR-335-5p on periodontal inflammation and bone loss. METHODS: Experimental periodontitis was established via ligation. The expression of inflammatory and osteoclastic genes was examined by quantitative real-time PCR (qPCR). Morphology of alveolar bone was analyzed by microcomputed tomography (µCT). Hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP), and Toll-like receptor 4 (TLR4) immunohistochemistry (IHC) staining were conducted for histological analysis. RESULTS: The expression of miR-335-5p decreased significantly in the periodontal tissues of EP. Compared to the WT-EP group, µCT analysis showed less bone loss in the 335-Tg-EP group accompanying with a decreased number of TRAP-positive osteoclasts. H&E and IHC staining exhibited attenuated inflammation and TLR4 expression in the 335-Tg-EP group. Furthermore, reduced expressions of IL-1ß, IL-6, TNF-α, and TLR4 were also detected in the 335-Tg-EP group. Overexpression of miR-335-5p in vivo weakened the periodontal bone destruction and inflammation compared with the WT-EP group. CONCLUSIONS: Our data exhibit novel roles of miR-335-5p in preventing bone loss and inflammation in experimental periodontitis.

Efficacy of collagen matrix seal and collagen sponge on ridge preservation in combination with bone allograft: A randomized controlled clinical trial.

AIM: To test whether the use of collagen matrix seal (CMS) results in similar hard and soft tissue remodelling to that with collagen sponge (CS) used as barriers 4 months following alveolar ridge preservation (ARP), in combination with freeze-dried bone allograft (FDBA). MATERIALS AND METHODS: Twenty-eight patients were randomly assigned to the two groups. Clinical and radiographic measurements were recorded with the same stent at baseline and 4 months for standardization. The flapless technique following a traumatic extraction was used for the two types of barriers. RESULTS: All patients completed the study, 14 in the CMS group and 14 in the CS group. Reduction in coronal ridge width (1.21 mm-14.91% CMS and 1.47 mm-20.40% CS) and vertical buccal bone resorption (0.30 mm CMS and 0.79 mm CS) were not significantly different. A slight increase in buccal gingival thickness at the coronal part was observed in both groups (0.9 mm CMS and 0.5 mm CS). CONCLUSIONS: Collagen matrix seal and CS, when combined with FDBA, significantly minimized ridge resorption in all dimensions and maintained buccal soft tissue thickness in sockets with a buccal plate loss of <2 mm in comparison to previously reported findings recorded after tooth extraction without ARP.

Fibronectin-binding protein TDE1579 affects cytotoxicity of Treponema denticola.

While FbpA, a family of bacterial fibronectin (FN) binding proteins has been studied in several gram-positive bacteria, the gram-negative Treponema denticola, an anaerobic periodontal pathogen, also has an overlooked fbp gene (tde1579). In this research, we confirm that recombinant Fbp protein (rFbp) of T. denticola binds human FN with a Kdapp of 1.5 × 10(-7) M and blocks the binding of T. denticola to FN in a concentration-dependent manner to a level of 42%. The fbp gene was expressed in T. denticola. To reveal the roles of fbp in T. denticola pathogenesis, an fbp isogenic mutant was constructed. The fbp mutant had 51% reduced binding ability to human gingival fibroblasts (hGF). When hGF were challenged with T. denticola, the fbp mutant caused less cell morphology change, had 50% reduced cytotoxicity to hGF, and had less influence on the growth of hGF cells.

Self-reported oral health is associated with systemic health outcomes and all-cause mortality.

BACKGROUND: Self-reported oral health questions (OHQs) are used commonly for epidemiologic surveillance of periodontal disease (PD). The authors' objective was to investigate how OHQs are associated with well-established systemic comorbidities of PD and their impact on all-cause mortality. The authors hypothesized that OHQs exhibit associations with systemic comorbidities similar to PD. METHODS: Two independent data sets were used to achieve these objectives: the Women's Health Study, a prospective cohort of women 45 years or older with self-reported information on PD, OHQs, cardiovascular disease, diabetes, and osteoporosis in various timeframes (continuous from 1992) and the National Health and Nutrition Examination Survey (NHANES), with data on OHQs and linked mortality (1999-2018). The authors applied multivariate logistic regression models and Cox proportional hazard regression survival analyses to test their hypotheses. RESULTS: The Women's Health Study participants who reported having PD until 2006 were more likely to later report deteriorating oral health, bone loss around their teeth, or periodontal treatment in 2018. Self-rated fair or poor oral health was independently associated with increased risk of cardiovascular disease (odds ratio, 1.39; 95% CI, 1.14 to 1.69; P < .001), diabetes (odds ratio, 1.21; 95% CI, 1.02 to 1.43; P = .028), and osteoporosis (odds ratio, 1.60; 95% CI, 1.38 to 1.84; P < .001). National Health and Nutrition Examination Survey participants who self-rated fair or poor oral health had higher risks of all-cause mortality (hazard ratio, 1.18; 95% CI, 1.02 to 1.37; P = .027). CONCLUSIONS: Self-reported oral health had a similar magnitude of associations with systemic comorbidities as established with PD previously. Moreover, self-rated fair or poor oral health, suboptimal dental visits, or infrequent flossing were associated with increased all-cause mortality. PRACTICAL IMPLICATIONS: These results support the use of OHQs in assessing systemic connections, especially when clinical dental access is limited. This clinical trial was registered at ClinicalTrials.gov. The registration number is NCT00000479.

Exosite interactions impact matrix metalloproteinase collagen specificities.

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769-783 from type I-III collagens, the second inserted α1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784-792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k(cat)/K(m) and k(cat) for MMP-1. MMP-13 showed the opposite behavior with a decreased k(cat)/K(m) and k(cat) and a greatly improved K(m) in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k(cat)/K(m) and k(cat) for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K(m) and dramatically decreased k(cat), resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.

An In Vitro Pilot Study on the Effects of Silver Diamine Fluoride on Periodontal Pathogens and Three-Dimensional Scaffolds of Human Fibroblasts and Epithelial Cells.

Objective: The aims of this study were to investigate the antibacterial and cytotoxic effects of silver diamine fluoride (SDF) on periodontal pathogens and human skin constructs, respectively. Background: SDF has been proven to have bactericidal effects on cariogenic bacteria. No studies to date evaluated the bactericidal effects of SDF on periodontal pathogens nor its effect on epithelium and fibroblasts. Methods: Streptococcus mutans, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans were cultured in monospecies biofilms, exposed to increasing concentrations of SDF and inoculated on agar plates to assess viability. Human gingival fibroblasts in 2D cultures were exposed to 1 µL of 0.394% of SDF and viewed using real-time imaging. Finally, SDF was applied to human, 3D tissue scaffolds of fibroblasts and keratinocytes, and termed human skin equivalents (HSE). A clinical dose of 38% SDF was applied, and HSE were cultured for 12 hours, 1, 3, 5, and 10 days. The tissue was observed clinically and histologically with hematoxylin and eosin staining and TUNEL. Results: S. mutans and A. actinomycetemcomitans growth was completely inhibited using all dilutions of SDF, whereas P. gingivalis was still viable with 0.197% and 0.098% of SDF. Single-layer fibroblasts experienced immediate necrosis upon contact with SDF. Application of SDF to HSE showed maturation of a whitish lesion within 24 hours, followed by pigmented, crusted tissue after 3 days. Histological evaluation of treated tissues showed apoptotic cells in the epithelium and upper half of the connective tissue. Conclusion: Our data suggest that SDF has bactericidal properties against two periodontal pathogens: P. gingivalis and A. actinomycetemcomitans. SDF caused immediate necrosis of monolayer fibroblasts, but does not extend to the full extent of layered fibroblasts in HSE.

Candidate loci shared among periodontal disease, diabetes and bone density.

Introduction: While periodontal disease (PD) has been associated with type 2 diabetes (T2D) and osteoporosis, the underlying genetic mechanisms for these associations remain largely unknown. The aim of this study is to apply cross-trait genetic analyses to investigate the potentially shared biology among PD, T2D, and bone mineral density (BMD) by assessing pairwise genetic correlations and searching for shared polymorphisms. Methods: We applied cross-trait genetic analyses leveraging genome-wide association study (GWAS) summary statistics for: Periodontitis/loose teeth from the UKBB/GLIDE consortium (PerioLT, N=506594), T2D from the DIAGRAM consortium (Neff=228825), and BMD from the GEFOS consortium (N=426824). Among all three, pair-wise genetic correlations were estimated with linkage disequilibrium (LD) score regression. Multi-trait meta-analysis of GWAS (MTAG) and colocalization analyses were performed to discover shared genome-wide significant variants (pMTAG <5x10-8). For replication, we conducted independent genetic analyses in the Women's Genome Health Study (WGHS), a prospective cohort study of middle-aged women of whom 14711 provided self-reported periodontal disease diagnosis, oral health measures, and periodontal risk factor data including incident T2D. Results: Significant genetic correlations were identified between PerioLT/T2D (Rg=0.23; SE=0.04; p=7.4e-09) and T2D/BMD (Rg=0.09; SE=0.02; p=9.8e-06). Twenty-one independent pleiotropic variants were identified via MTAG (pMTAG<5x10-8 across all traits). Of these variants, genetic signals for PerioLT and T2D colocalized at one candidate variant (rs17522122; ProbH4 = 0.58), a 3'UTR variant of AKAP6. Colocalization between T2D/BMD and the original PerioLT GWAS p-values suggested 14 additional loci. In the independent WGHS sample, which includes responses to a validated oral health questionnaire for PD surveillance, the primary shared candidate (rs17522122) was associated with less frequent dental flossing [OR(95%CI)= 0.92 (0.87-0.98), p=0.007], a response that is correlated with worse PD status. Moreover, 4 additional candidate variants were indirectly supported by associations with less frequent dental flossing [rs75933965, 1.17(1.04-1.31), p=0.008], less frequent dental visits [rs77464186, 0.82(0.75-0.91), p=0.0002], less frequent dental prophylaxis [rs67111375, 0.91(0.83-0.99), p=0.03; rs77464186, 0.80(0.72-0.89), p=3.8e-05], or having bone loss around teeth [rs8047395, 1.09(1.03-1.15), p=0.005]. Discussion: This integrative approach identified one colocalized locus and 14 additional candidate loci that are shared between T2D and PD/oral health by comparing effects across PD, T2D and BMD. Future research is needed to independently validate our findings.

Teaching evidence-based practice at the University of Texas Health Science Center at San Antonio dental school.

The overarching goal of the Evidence-Based Practice Program at San Antonio is to provide our graduates with life-long learning skills that will enable them to keep up-to-date and equip them with the best possible patient care skills during their 30-40 years of practice. Students are taught to (1) ask focused clinical questions, (2) search the biomedical research literature (PubMed) for the most recent and highest level of evidence, (3) critically evaluate the evidence, and (4) make clinical judgments about the applicability of the evidence for their patients. Students must demonstrate competency with these "just-in-time" learning skills through writing concise one-page Critically Appraised Topics (CATs) on focused clinical questions. The school has established an online searchable library of these Critically Appraised Topics. This library provides students and faculty with rapid, up-to-date evidence-based answers to clinical questions. The long-range plan is to make this online library available to practitioners and the public.

Number of teeth is associated with hip fracture and femoral neck bone mineral density in the NHANES.

PURPOSE/INTRODUCTION: Tooth loss has been found to be associated with fractures and osteoporosis. However, the associations between number of teeth with bone mineral density as well as with hip fractures have not been explored in the same study setting. METHODS: Data from the cross-sectional National Health and Nutrition Examination Survey (2005-2010, 2013-2014, and 2017-2018) with completed femoral neck bone mineral density (BMD) measurements, osteoporosis questionnaires, and dentition examinations were analyzed. A total of 15,198 participants, with a mean age of 53.9 and diverse ethnicity, males (52%), and females (48%), were analyzed. Multivariate logistic regression analyses for self-reported hip fractures, self-reported osteoporosis, and measured low femoral BMD accounting for traditional risk factors were tested for the total number of natural teeth (NoT) present, or by NoT in the anterior or posterior segments. RESULTS: Subjects with fewer natural teeth present were more likely to report a hip fracture, osteoporosis, or having lower levels of femoral neck BMD. With one additional tooth present in the mouth, there was a decreased association with self-reported hip fracture [OR(95%CI) = 0.98(0.96-0.99); P = 0.005] or with less likelihood of having low femoral neck BMD [OR(95%CI) = 0.99(0.97-1.00); P = 0.007]. CONCLUSIONS: With the limitation of the cross-sectional study design, results should be interpreted cautiously, yet our analyses point to an association between a decreased number of natural teeth present and self-reported hip fractures or low femoral neck BMD. The number of teeth present could be potentially utilized for assessing risks of hip fracture and osteoporosis. Future research is needed to validate our findings.

Efficacy of an Er:YAG laser in the decontamination of dental implant surfaces: An in vitro study.

BACKGROUND: Emergence of peri-implant diseases led to the development of various methods for implant surface decontamination. This study was designed to compare the efficacy of biofilm removal from implant-like titanium surfaces by an erbium-doped yttrium-aluminum-garnet (Er:YAG) laser, titanium brush, and carbon fiber curet. METHODS: Eight study subjects were recruited. A custom mouth appliance that held eight sandblasted and acid-etched titanium discs was fabricated for each subject. Subjects were asked to wear this appliance for 72 hours to allow for biofilm development. After retrieval, discs were removed and randomized to one of four treatment groups. The discs were stained with a two-component nucleic acid dye kit, and the residual biofilm was visualized under fluorescence microscopy. Quantification of residual biofilm was performed using an image analysis software and expressed as the percentage surface area. RESULTS: Fifty-nine titanium discs were randomized to the four treatment groups. The percentage of titanium disc area covered by residual biofilm was 74.0% ± 21.6%, 32.8% ± 24.0%, 11.8% ± 10.3%, and 20.1% ± 19.2% in the control, Er:YAG, titanium brush and carbon fiber curet groups, respectively (mean ± SD). The biofilm-covered area significantly decreased in each of the three treatment groups compared with control (P < 0.008). Comparisons between treatment groups did not reveal statistical significance. CONCLUSIONS: Er:YAG laser treatment is an effective method for reducing the bacterial biofilm on titanium discs. However, on a threadless titanium surface, Er:YAG laser does not exhibit a significantly greater efficacy in biofilm removal than commonly used titanium brushes or carbon fiber curets.

Identification and characterization of a novel adiponectin receptor agonist adipo anti-inflammation agonist and its anti-inflammatory effects in vitro and in vivo.

BACKGROUND AND PURPOSE: Adiponectin (APN) is an adipokine secreted from adipocytes that binds to APN receptors AdipoR1 and AdipoR2 and exerts an anti-inflammatory response through mechanisms not fully understood. There is a need to develop small molecules that activate AdipoR1 and AdipoR2 and to be used to inhibit the inflammatory response in lipopolysaccharide (LPS)-induced endotoxemia and other inflammatory disorders. EXPERIMENTAL APPROACH: We designed 10 new structural analogues of an AdipoR agonist, AdipoRon (APR), and assessed their anti-inflammatory properties. Bone marrow-derived macrophages (BMMs) and peritoneal macrophages (PEMs) were isolated from mice. Levels of pro-inflammatory cytokines were measured by reverse transcription and real-time quantitative polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and microarray in LPS-induced endotoxemia mice and diet-induced obesity (DIO) mice in which systemic inflammation prevails. Western blotting, immunohistochemistry (IHC), siRNA interference and immunoprecipitation were used to detect signalling pathways. KEY RESULTS: A novel APN receptor agonist named adipo anti-inflammation agonist (AdipoAI) strongly suppresses inflammation in DIO and endotoxemia mice, as well as in cultured macrophages. We also found that AdipoAI attenuated the association of AdipoR1 and APPL1 via myeloid differentiation marker 88 (MyD88) signalling, thus inhibiting activation of nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK) and c-Maf pathways and limiting the production of pro-inflammatory cytokines in LPS-induced macrophages. CONCLUSION AND IMPLICATIONS: AdipoAI is a promising alternative therapeutic approach to APN and APR to suppress inflammation in LPS-induced endotoxemia and other inflammatory disorders via distinct signalling pathways.

Volumetric Changes Following Lateral Guided Bone Regeneration.

Resorbable membranes are well described and employed for horizontal guided bone regeneration (GBR). However, the currently available literature does not provide information on the bone volumetric changes during the healing that follows GBR procedures and dental implant placement. Therefore, the aim of this pilot study was to initially analyze the volumetric bone changes after treating pristine edentulous mandibular defects with lateral GBR using freeze-dried bone allograft (FDBA) and collagen resorbable membrane. Six patients were selected for the analysis. Clinical changes in bone volume before and after GBR were measured. In addition, digital volumetric analysis of the augmented ridges was performed preoperatively, as well as 4 and 6 months after the GBR procedure. At the time of dental implant placement, bone cores were collected during the osteotomy for histologic analysis. Data on volume changes showed a mean of 297.5 ± 134 mm3 augmented bone volume at 4 months with 5% ± 3.78% resorption from 4 to ≥ 6 months. Histologic bone core analysis showed 44.9% plusmn; 5.1% mineralization in the area of augmentation. Within the limitations of this pilot study, resorbable membranes exhibited reliability for GBR in intercalated mandibular defects, providing sufficient bone volume gain at ≥ 6 months for implant stabilization and limited resorption during graft healing.

Diagnosing periodontal and dental indicators with horizontal and vertical bitewing radiographs.

OBJECTIVE: The aim of this study is to compare information provided by the 2 orientations of bitewing radiographs, horizontal (HBW) and vertical (VBW) taken in a dental school. METHODS AND MATERIALS: Radiographic records were reviewed at Tufts University School of Dental Medicine (TUSDM) for patients showing posterior bone loss who had both HBW and VBW. 320 records were reviewed with 6 criteria: visibility of crestal bone from the distal of the cuspids to the distal of the most posterior tooth, visibility of horizontal or angular bone loss, the crestal density of bone, visibility of interproximal contact areas, visibility of the entire anatomical crown, and visibility of furcations. RESULTS: Significantly higher number of VBW compared with HBW (P < 0.0001) showed the levels of alveolar bone loss (52.81% vs. 3.75%), the type of loss (angular or horizontal) (50.94% vs. 3.75%), the crestal bone density (28.75% vs. 0.63%), the contact areas (20.63% vs. 14.38%), and the furcations (43.44% vs. 1.25%). A greater number of HBW showed the entire anatomical crown compared with VBW. No significant difference was detected in the number of radiographs taken per HBW and VBW set. CONCLUSION: For patients with alveolar bone loss, VBW are superior to HBW when assessing bone levels, density, morphology, tooth furcations, and evaluating interproximal contact areas for caries. It is recommended that the vertical bitewing technique be taught as a standard in dental, dental hygiene, and dental assisting schools for adult patients showing evidence of posterior interdental bone loss.

Nuclear magnetic resonance mapping and functional confirmation of the collagen binding sites of matrix metalloproteinase-2.

Interactions of matrix metalloproteinase-2 (MMP-2) with native and denatured forms of several types of collagen are mediated by the collagen binding domain (CBD). CBD positions substrates relative to the catalytic site and is essential for their cleavage. Our previous studies identified a CBD binding site on the alpha1(I) collagen chain. The corresponding synthetic collagen peptide P713 bound CBD with high affinity and was used in this study to identify specific collagen binding residues by NMR analysis of (15)N-labeled CBD complexed with P713. Results obtained showed that P713 caused chemical shift perturbations of several surface-exposed CBD backbone amide resonances in a concentration-dependent manner. The 10 residues that underwent the largest chemical shift perturbations (R(252) in module 1, R(296), F(297), Y(302), E(321), Y(323), and Y(329) in module 2, and R(368), W(374), and Y(381) in module 3) were investigated by site-specific substitution with alanine. The structural integrity of the CBD variants was also analyzed by one-dimensional (1)H NMR. Surface plasmon resonance and microwell protein binding assays of control and CBD variants showed that residues in all three CBD modules contributed to collagen binding. Single-residue substitutions altered the affinity for peptide P713, as well as native and denatured type I collagen, with the greatest effects observed for residues in modules 2 and 3. Additional alanine substitutions involving residues in two or three modules simultaneously further reduced the level of binding of CBD to native and denatured type I collagen and demonstrated that all three modules contribute to substrate binding. These results have localized and confirmed the key collagen binding site residues in the three fibronectin type II-like modules of MMP-2.

Cellular effects of enamel matrix derivative are associated with different molecular weight fractions following separation by size-exclusion chromatography.

BACKGROUND: Enamel matrix derivative (EMD) was shown to enhance soft tissue healing and regeneration of the periodontium; however, the mechanisms of this action are unknown. It is assumed that amelogenin, the most abundant protein in EMD, is the protein primarily responsible for the effects of EMD. The purpose of this study was to fractionate EMD and associate its specific cellular effects with different molecular weight fractions following size-exclusion chromatography. METHODS: Freshly dissolved EMD was fractionated by gel filtration, and forty-five 7-ml fractions were collected, desalted, lyophilized, and resuspended. These fractions were analyzed for their effects on the differentiation of osteoprogenitor cells (C2C12) and the proliferation and differentiation of human microvascular endothelial cells (HMVECs). Alkaline phosphatase activity (C2C12) was measured as a marker for osteogenic differentiation before and after preincubation of the fractions with the bone morphogenetic protein (BMP) decoy receptor, noggin. Angiogenesis (HMVEC) was evaluated as a marker for endothelial cell differentiation. Enzymographic assays used polyacrylamide gels copolymerized with denatured type I collagen to determine gelatinolytic activities in each fraction. RESULTS: EMD fractionated into three major protein peaks following size exclusion chromatography with cross-linked dextran particle matrix. Peak I was associated with the column void volume, whereas peak III eluted near the salt volume. Peak II eluted between these two peaks. Proliferation and angiogenic activities were associated with peaks II and III for the microvascular cells. The differentiation of osteoprogenitor cells, indicated by alkaline phosphatase activity, was induced by EMD components present in peak I and the leading edge of peak II. The additional observation that this differentiation was inhibited by prior treatment of the fractions with noggin suggested the activity was induced by BMP rather than amelogenin or other unknown proteins. Gelatinolytic activities were detected in the early fractions of peaks I and II of gel-fractionated EMD. CONCLUSIONS: The cellular activities stimulated by EMD are not associated with a single molecular weight species. The fact that noggin abolishes C2C12 alkaline phosphatase activity suggests that effects on osteoprogenitor cell differentiation are the result of a BMP-like protein(s), whereas effects on proliferation and angiogenesis are associated with lower molecular weight species present in peaks II and III. Finally, unheated EMD displays gelatinolytic activities that are also detectable following size-exclusion separation of its constituents. The masses of these activities were consistent with those reported for latent and active matrix metalloproteinase-20.

A Comparison between Primary and Secondary Flap Coverage in Ridge Preservation Procedures: A Pilot Randomized Controlled Clinical Trial.

AIMS: To assess the bone dimensional changes after extraction and alveolar ridge preservation (ARP) using primary coverage (closed flap technique, CFT) or healing by secondary intention (open flap technique, OFT). MATERIALS AND METHODS: Ten patients (split mouth design) were planned for extraction and ARP. All sites received ARP with freeze-dried bone allograft (FDBA) and nonresorbable membrane after extraction. Clinical standardized measurements were used to assess the dimensional alterations of the alveolar ridge. RESULTS: All patients completed the study, and a total of 20 sites were randomized to CFT or OFT group. Center height (mean difference of 8.1 mm, SD =1.9 CFT, and 7.5 mm, SD= 1.8 OFT) and buccal height (mean difference of 0.8 mm, SD =1.0 CFT, and 0.3 mm, SD= 1.1 OFT) were significantly different within the same group. However, there was no statistically significant difference between groups. In the OFT group, the keratinized tissue width was higher and the pain VAS scores at 24 hours were lower compared with the CFT (p = 0.004 and p = 0.006, respectively). CONCLUSIONS: Leaving the flap open did not have any effects on the dimensional changes of bone height or width. However, there was a wider band of keratinized tissue and less pain with the CFT compared with the OFT. The study protocol was registered at ClinicalTrials.gov, Identifier NCT03136913.

Association between time since quitting smoking and periodontitis in former smokers in the National Health and Nutrition Examination Surveys (NHANES) 2009 to 2012.

BACKGROUND: The aims of this study were to analyze the periodontal conditions among non-smokers, former smokers and current smokers in the two National Health and Nutrition Examination Surveys (NHANES) acquired between 2009 to 2012 and determine the association between time since quitting smoking and periodontal status. METHODS: Smoking status and periodontal examination data from NHANES 2009 to 2010 and 2011 to 2012 were analyzed. Respondents included in the analysis were aged ≥18 years, had undergone a complete NHANES Oral Health - Periodontal Exam with all measurements recorded as required for the periodontal classification algorithm, and had complete data from the NHANES Smoking - Cigarette Use questionnaire. Logistic regression was conducted with time since quitting as the exposure and presence of periodontitis as the outcome, and included adjustment for confounders. RESULTS: Smoking status was significantly associated with periodontal status (Chi-square; P < 0.0001). The rate of periodontitis was highest among smokers (35%), compared with former smokers (19%) and never smokers (13%). Among former smokers, after adjusting for confounders, each additional year since quitting smoking was associated with a significant reduction in the odds ratio (OR) for periodontitis by 3.9% (OR for each year 0.961, 95% confidence interval 0.948 to 0.975). CONCLUSIONS: Among former smokers, a longer time since quitting smoking was associated with a lower likelihood of periodontitis. Consequently, dental practitioners have a public health mandate to help their patients quit smoking. Future research should determine the best strategies for facilitating smoking cessation in dental patients.

Advanced glycation of type I collagen and fibronectin modifies periodontal cell behavior.

BACKGROUND: Advanced glycation end products (AGEs) have been linked to pathogenic mechanisms of diabetes mellitus. However, little is known about the contribution of protein glycation to periodontal disease in patients with diabetes. Therefore, this study investigated whether glycation of type I collagen (COLI) and fibronectin (FN) modified the behavior of human gingival fibroblasts (hGFs) and periodontal ligament fibroblasts (hPDLs). METHODS: Procedures for rapid in vitro glycation of COLI and FN used methylglyoxal (MG). Formation of AGEs was analyzed by changes in protein migration using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with antibodies specific for MG-glycated proteins. Experiments then characterized the effects of glycated FN and COLI on the behavior of hGFs and hPDLs. RESULTS: MG glycated COLI and FN in <6 hours. Confirming the specificity of the reactions, antibodies specific for MG-induced AGEs reacted with glycated FN and COLI but not with control proteins. In cell culture experiments, glycated FN was significantly less efficient in supporting the attachment of hGFs and hPDLs (P <0.05). Moreover, the morphologic parameters, including length, area, perimeter, and shape factor, were altered (P <0.001) for cells on both glycated proteins. Finally, cell migration was reduced on glycated FN and COLI (P <0.001). CONCLUSIONS: MG treatment efficiently glycated COLI and FN, providing a new tool to study the effects of diabetes on periodontal disease. The substantial effects of glycated COLI and FN on hGF and hPDL behavior indicated that protein glycation contributed to the pathogenesis and altered periodontal wound healing observed in patients with diabetes.

Fibronectin fragmentation is a feature of periodontal disease sites and diabetic foot and leg wounds and modifies cell behavior.

BACKGROUND: Fibronectin (FN) undergoes fragmentation in periodontal disease sites and in poorly healing diabetic wounds. The biologic effects of FN fragments on wound healing remain unresolved. This study characterized the pattern of FN fragmentation and its effects on cellular behavior compared to intact FN. METHODS: Polyclonal antibodies were raised against FN and three defined recombinant segments of FN and used to analyze gingival crevicular fluid from periodontal disease sites in systemically healthy subjects and in subjects with diabetes, as well as chronic leg and foot wound exudates from subjects with diabetes. Subsequently, the behavior of human gingival fibroblasts (hGFs) and HT1080 reference cells were analyzed by measuring cell attachment, migration, and chemotaxis in the presence of intact FN or recombinant FN fragments. RESULTS: FN fragmentation was evident in fluids from periodontal disease sites and diabetic leg and foot wounds. However, no fragmentation pattern distinguished systemically healthy subjects from subjects with diabetes. hGFs and HT1080 cells required significantly higher concentrations of FN fragments to achieve attachment comparable to intact FN. Cells cultured on FN fragments also were morphologically different from cells cultured on full-length FN. Migration was reduced for hGFs cultured on FN fragments relative to full-length FN. In contrast, FN fragments increased HT1080 fibrosarcoma cell migration over intact FN. CONCLUSIONS: FN fragmentation is a prominent feature of periodontal and chronic leg and foot wounds in diabetes. Furthermore, cell culture assays confirmed the hypothesis that exposure to defined FN fragments significantly alters cell behavior.