Changes in oral health during aging in a novel non-human primate model.

Oral health plays a significant role in the quality of life and overall well-being of the aging population. However, age-related changes in oral health are not well understood due to challenges with current animal models. In this study, we analyzed the oral health and microbiota of a short-lived non-human primate (i.e., marmoset), as a step towards establishing a surrogate for studying the changes that occur in oral health during human aging. We investigated the oral health of marmosets using cadaveric tissues in three different cohorts: young (aged ≤6 years), middle-aged, and older (>10 years) and assessed the gingival bacterial community using analyses of the V3-V4 variable region of 16S rRNA gene. The oldest cohort had a significantly higher number of dental caries, increased dental attrition/erosion, and deeper periodontal pocket depth scores. Oral microbiome analyses showed that older marmosets had a significantly greater abundance of Escherichia-Shigella and Propionibacterium, and a lower abundance of Agrobacterium/Rhizobium at the genus level. Alpha diversity of the microbiome between the three groups showed no significant differences; however, principal coordinate analysis and non-metric multidimensional scaling analysis revealed that samples from middle-aged and older marmosets were more closely clustered than the youngest cohort. In addition, linear discriminant analysis effect size (LEFSe) identified a higher abundance of Esherichia-Shigella as a potential pathogenic biomarker in older animals. Our findings confirm that changes in the oral microbiome are associated with a decline in oral health in aging marmosets. The current study suggests that the marmoset model recapitulates some of the changes in oral health associated with human aging and may provide opportunities for developing new preventive strategies or interventions which target these disease conditions.

Inclusion of postdoctoral trainees in a translational science training TL1 program was associated with greater diversification of research across the translational science continuum: a bibliometric analysis of TL1 trainee publications.

The NIH National Center for Advancing Translational Science (NCATS) was established to support translational research that spans the entire TS Continuum, with the goal of bridging the gap between preclinical biomedical research and real-world applications to advance treatments to patients more quickly. In 2018, the Translational Science Training (TST) TL1 Program at the University of Texas Health Science Center at San Antonio implemented new strategies to better include and encourage research more broadly across the TS Continuum, including the addition of postdoctoral scientists and a clinically trained Program Co-Director, expansion of team science and community engagement programming, and targeted trainee recruitment from schools of nursing, dentistry, and allied health, in addition to medicine. The objective of this bibliometric analysis was to determine if the program exhibited a more diverse mix of T-types after the adjustments made in 2018. The TST/TL1 Program experienced a shift in T-type, from mostly T0 (preclinical) to more T3/T4 (clinical implementation/public health) research, after new strategies were implemented. This supports the conclusion that strategic programmatic adjustments by an NCATS-funded predoctoral training program resulted in outcomes that better align with NCATS priorities to develop Trainees who contribute across the entire TS Continuum.

Comparison of bibliometrics for predoctoral Translational Science Training (TST) TL1 Program participants and nonparticipants, male and female participants, and participants from underrepresented and well-represented backgrounds.

Research education and training in Translational Science develops and sustains a workforce to efficiently advance studies designed to improve human health. We evaluated the effectiveness of a Translational Science Training (TST) TL1 Program. Participants had significantly better publications/year, citations/year, h-index, and m-quotient than nonparticipants. Female and male participants, and participants from underrepresented and well-represented backgrounds, performed similarly on all bibliometric assessments. Finally, TST/TL1 Program participants outperformed students from other PhD programs at our institution. This analysis suggests that the TST/TL1 Program has been effective for participants, including those who are female and from underrepresented backgrounds.

Endocytosis and Enamel Formation.

Enamel formation requires consecutive stages of development to achieve its characteristic extreme mineral hardness. Mineralization depends on the initial presence then removal of degraded enamel proteins from the matrix via endocytosis. The ameloblast membrane resides at the interface between matrix and cell. Enamel formation is controlled by ameloblasts that produce enamel in stages to build the enamel layer (secretory stage) and to reach final mineralization (maturation stage). Each stage has specific functional requirements for the ameloblasts. Ameloblasts adopt different cell morphologies during each stage. Protein trafficking including the secretion and endocytosis of enamel proteins is a fundamental task in ameloblasts. The sites of internalization of enamel proteins on the ameloblast membrane are specific for every stage. In this review, an overview of endocytosis and trafficking of vesicles in ameloblasts is presented. The pathways for internalization and routing of vesicles are described. Endocytosis is proposed as a mechanism to remove debris of degraded enamel protein and to obtain feedback from the matrix on the status of the maturing enamel.

Maturation stage enamel malformations in Amtn and Klk4 null mice.

Amelotin (AMTN) and kallikrein-4 (KLK4) are secreted proteins specialized for enamel biomineralization. We characterized enamel from wild-type, Amtn(-/-), Klk4(-/-), Amtn(+/-)Klk4(+/-) and Amtn(-/-)Klk4(-/-) mice to gain insights into AMTN and KLK4 functions during amelogenesis. All of the null mice were healthy and fertile. The mandibular incisors in Amtn(-/-), Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice were chalky-white and chipped. No abnormalities except in enamel were observed, and no significant differences were detected in enamel thickness or volume, or in rod decussation. Micro-computed tomography (µCT) maximum intensity projections localized the onset of enamel maturation in wild-type incisors distal to the first molar, but mesial to this position in Amtn(-/-), Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice, demonstrating a delay in enamel maturation in Amtn(-/-) incisors. Micro-CT detected significantly reduced enamel mineral density (2.5 and 2.4gHA/cm(3)) in the Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice respectively, compared with wild-type enamel (3.1gHA/cm(3)). Backscatter scanning electron microscopy showed that mineral density progressively diminished with enamel depth in the Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice. The Knoop hardness of the Amtn(-/-) outer enamel was significantly reduced relative to the wild-type and was not as hard as the middle or inner enamel. Klk4(-/-) enamel hardness was significantly reduced at all levels, but the outer enamel was significantly harder than the inner and middle enamel. Thus the hardness patterns of the Amtn(-/-) and Klk4(-/-) mice were distinctly different, while the Amtn(-/-)Klk4(-/-) outer enamel was not as hard as in the Amtn(-/-) and Klk4(-/-) mice. We conclude that AMTN and KLK4 function independently, but are both necessary for proper enamel maturation.

Correlation of ameloblastin with enamel mineral content.

In enamel formation, the deposition of minerals as crystallites starts when the mineralization front first forms at the start of the secretory stage. During maturation, the enamel layer accumulates significant amounts of new mineral as the crystallites grow in volume. Inversely related to mineral gain is loss of protein and water from the forming enamel. Both ameloblastin (Ambn) and enamelin are essential components for formation of a functional enamel layer. The aim of this study was to quantify the proportion of mineral and non-mineral material present in developing enamel relative to Ambn concentration using Ambn mutant mice mated with others overexpressing full-length Ambn from the mouse amelogenin promoter at lower (+), similar (++) or higher (+++) concentration than normal. Mandibular incisors (age: 7 weeks, n = 8) were imaged by micro-computed tomography and the enamel was analyzed from the apical region to the incisal edge in sequential 1.0 mm volumes of interest. Mineral density was determined using a series of hydroxyapatite (HA) phantoms to calibrate enamel density measurements. At the site where the mandibular incisor emerged into the oral cavity, the enamel volume, mineral weight, and mineral density were reduced when Tg Ambn was expressed at lower or higher levels than normal. While in wild-type the % mineral was >95%, it was negligible in Ambn-/-, 22.3% in Ambn-/-, Tg(+), 75.4% in Ambn-/-, Tg(++), and 45.2% in Ambn-/-, Tg(+++). These results document that the deposition of mineral and removal of non-mineral components are both very sensitive to expressed Ambn concentrations.

Restoring strength of incisors with veneers and full ceramic crowns.

PURPOSE: The aim was to determine the in vitro fracture resistance of incisors restored with veneers and full ceramic crowns compared to unrestored teeth. MATERIALS AND METHODS: Seventy intact, extracted human maxillary central incisors were randomized and assigned to 7 groups (n = 10). The teeth in group 1 remained intact (control). The teeth in groups 2 to 6 were prepared and IPS Empress restorations were conditioned and bonded using an adhesive luting cement, Variolink II/Syntac (group 2: labial veneer with incisal overlap, group 3: 3/4 veneer with margin in enamel, group 4: 3/4 veneer with margin in dentin, group 5: crown with margin in enamel, group 6: crown with margin in dentin group 7: veneer on worn tooth. After finishing and polishing, specimens were stored in water and thermocycled for 2000 cycles between 5 degrees C and 55 degrees C. The maximal fracture load of the specimens (40-degree inclination) was determined using the universal testing machine (Zwick) at a constant crosshead speed (0.5 mm/min). The statistical analysis was performed using the Kruskal-Wallis test with Bonferroni correction (p < 0.05). Fracture surfaces were qualitatively analyzed by SEM. RESULTS: All restored teeth with cervical preparation margins in enamel showed a fracture load not significantly different from the intact teeth (control). Restored teeth with cervical preparation margins in dentin showed a significantly lower fracture load. All restorations showed a fracture load far above 400 N, serving as functional reference for anterior teeth. The failures were predominantly cohesive. CONCLUSION: For the restoration of tooth strength, defining the finishing lines of veneers and crowns in enamel is recommended. Restorations with finishing lines in dentin resulted in significant loss of strength. Three-quarter veneers with finishing lines in enamel are functionally equal to crowns with the advantage of conserving tooth structure.

Comparative clinical study of the effectiveness of three different bleaching methods.

OBJECTIVE: The current study assessed the efficacy of three current bleaching methods. METHODS: Seventy-five healthy subjects (45 female; 30 male) with anterior teeth, having a Vita Shade score of A2 or darker, participated in the study. The subjects were randomly assigned to one of three treatment groups: Group A: home-bleaching (illumine Home, 10% carbamide peroxide, trays, overnight, for two weeks), Group B: in-office bleaching (Illumine Office, 15% hydrogen peroxide, trays for 45 minutes, three times over three weeks), Group C: Whitestrips (strips, twice a day, 30 minutes each for two weeks). Following the screening visit, three weeks prior to the baseline examination, all subjects received a dental prophylaxis. The color of the teeth was determined using a colorimeter (ShadeEye NCC) and a custom-made stent at baseline (E0), immediately after completion of the bleaching (E3) and three months after treatment (E4). All subjects received oral hygiene instructions and a toothbrush and toothpaste for oral home care during the study period. The change of tooth color was determined for each treatment regimen between baseline and E3 and baseline and E4 and was statistically analyzed performing the Kruskal Wallis test and the Mann-Whitney-U test. The significance level was set atp < 0.01. RESULTS: The dropout rate was 0%. Mean (SD) deltaE* (overall color change) from baseline to immediately after treatment was 6.57 (2.13) for Group A, 5.77 (1.72) for Group B and 3.58 (1.57) for Group C. The mean (SD) tooth color change from baseline to three months after treatment deltaE* was: 4.98 (1.34) for Group A, 4.59 (1.42) for Group B and 2.99 (1.39) for Group C. Significant differences were found between home bleaching and Whitestrips, as well as between in-office bleaching and Whitestrips, but not between home-bleaching and in-office bleaching during the same time. CONCLUSION: Using an objective color measurement device, home bleaching and in-office bleaching were found to be superior to Whitestrips. Home bleaching and in-office bleaching were equally efficient for bleaching teeth and maintaining the results for up to three months.

Effect of a 5000 ppm fluoride toothpaste and a 250 ppm fluoride mouth rinse on the demineralisation of dentin surfaces.

BACKGROUND: The purpose of this study was to test the null hypothesis that there is no difference between the effect of (1) a 5000 ppm fluoride toothpaste, and (2) a 250 ppm fluoride mouth rinse on demineralized human dentin surfaces, against the alternative hypothesis of a difference. FINDINGS: Dentin specimens were obtained from the cervical regions of 45 extracted human third molars. Half the surface of each specimen was sealed with a self-etching adhesive system and served as the reference surface. The dentin specimens were randomly assigned to one of the three groups, 5000 ppm fluoride toothpaste (Duraphat), 250 ppm fluoride mouth rinse (Meridol) and distilled water (negative control).An intraoral appliance was made for one volunteer. In each test cycle, 15 specimens were inserted in the appliance and worn for 24 hours a day, over a period of three weeks.Once daily, the appliance was immersed in the agent being tested; either toothpaste slurry, mouth rinse or distilled water for 60 seconds.Demineralization was assessed in terms of lesion depth (microm) and mineral loss (vol. % x microm) by transversal microradiography. Data analysis was accomplished using Kolmogorov-Smirnov test and ANOVA (SPSS 12.0).Statistically significant differences for mineral loss and lesion depth were found between the toothpaste and the mouth rinse as well as between the toothpaste and the control group, but not between the mouth rinse and the control group. CONCLUSION: Within the limitations of this study, the results suggest that treatment of demineralised dentin with a toothpaste containing 5000 ppm fluoride may considerably reduce mineral loss and lesion depth on exposed dentin.

Role of Bcl2 in osteoclastogenesis and PTH anabolic actions in bone.

INTRODUCTION: B-cell leukemia/lymphoma 2 (Bcl2) is a proto-oncogene best known for its ability to suppress cell death. However, the role of Bcl2 in the skeletal system is unknown. Bcl2 has been hypothesized to play an important anti-apoptotic role in osteoblasts during anabolic actions of PTH. Although rational, this has not been validated in vivo; hence, the impact of Bcl2 in bone remains unknown. MATERIALS AND METHODS: The bone phenotype of Bcl2 homozygous mutant (Bcl2(-/-)) mice was analyzed with histomorphometry and muCT. Calvarial osteoblasts were isolated and evaluated for their cellular activity. Osteoclastogenesis was induced from bone marrow cells using RANKL and macrophage-colony stimulating factor (M-CSF), and their differentiation was analyzed. PTH(1-34) (50 microg/kg) or vehicle was administered daily to Bcl2(+/+) and Bcl2(-/-) mice (4 days old) for 9 days to clarify the influence of Bcl2 ablation on PTH anabolic actions. Western blotting and real-time PCR were performed to detect Bcl2 expression in calvarial osteoblasts in response to PTH ex vivo. RESULTS: There were reduced numbers of osteoclasts in Bcl2(-/-) mice, with a resultant increase in bone mass. Bcl2(-/-) bone marrow-derived osteoclasts ex vivo were significantly larger in size and short-lived compared with wildtype, suggesting a pro-apoptotic nature of Bcl2(-/-) osteoclasts. In contrast, osteoblasts were entirely normal in their proliferation, differentiation, and mineralization. Intermittent administration of PTH increased bone mass similarly in Bcl2(+/+) and Bcl2(-/-) mice. Finally, Western blotting and real-time PCR showed that Bcl2 levels were not induced in response to PTH in calvarial osteoblasts. CONCLUSIONS: Bcl2 is critical in osteoclasts but not osteoblasts. Osteoclast suppression is at least in part responsible for increased bone mass of Bcl2(-/-) mice, and Bcl2 is dispensable in PTH anabolic actions during bone growth.

Microbiota of exposed root surfaces after fluoride, chlorhexidine, and periodontal maintenance therapy: a 3-year evaluation.

BACKGROUND: Fluoride and chlorhexidine (CHX) are state-of-the-art preventive measures for remineralizing teeth and for preventing plaque accumulation. The aim of this study was to examine the effects of fluoride and CHX varnishes on root caries and microbiota located on root surfaces. METHODS: Thirty-three patients from a periodontal maintenance program, having at least one tooth with gingival recession in each quadrant, participated in this study. One tooth per quadrant was assigned randomly to the control group or to one of the test groups that were treated with fluoride varnish, 1% CHX, or 40% CHX. The varnish treatment and the tooth cleaning were repeated every 3 months. Clinical examinations were performed at baseline and once a year for 3 years. Caries status and oral hygiene indices were evaluated clinically. The total cultivable microbiota and percentage of Mutans streptococci (MS), Actinomyces (ACC), and lactobacilli (LB) were analyzed. RESULTS: Oral hygiene was improved greatly during the course of the study. The percentage of MS, ACC, and LB of the total cultivable microbiota revealed a statistically significant reduction between baseline and final examination for each of the four groups. CONCLUSION: Professional tooth cleaning alone at 3-month intervals might be as effective in reducing MS, ACC, and LB as adjunctive treatment with fluoride or chlorhexidine.

Effect of a 40% chlorhexidine varnish on demineralization of dentin surfaces in situ.

PURPOSE: To evaluate the effect of varnish with different chlorhexidine concentrations on the demineralization of dentin surfaces in situ. METHODS: An intraoral model was used to study the ability of chlorhexidine to prevent demineralization. Dentin specimens from extracted human teeth were treated with chlorhexidine varnish and exposed to the oral environment of 47 subjects. The dentin specimens were prepared from the cervical regions of 47 third molars, sterilized by irradiation with 60 GY and mounted in intraoral appliances worn by the subjects. Before the delivery of the appliance, the specimens and participants were treated once with one of the three different varnishes (according to the manufacturer's instructions). All participants were randomly assigned to one of the three groups: (1) EC 40 (n=16), (2) Cervitec (n=15), or (3) ChemFil Varnish (control group, n=16). The appliances were worn day and night for 3 weeks. At mealtimes, the appliance was stored in 10% sucrose solution. After exposure to demineralization, the dentin surface was evaluated by microradiography to assess the depth of the lesion (microm) and the loss of mineral (Vol% microm). Data analysis was accomplished using one-way ANOVA plus LSD testing (P< 0.05). RESULTS: Both chlorhexidine varnishes EC 40 and Cervitec resulted in significantly decreased mineral loss and lesion depth in dentin than the control. In addition, EC 40 treated specimens demonstrated significantly reduced lesion depth compared to Cervitec and the control (109.22 microm vs. 139.23 microm and 178.21 microm).

Enamel formation and amelogenesis imperfecta.

Dental enamel is the epithelial-derived hard tissue covering the crowns of teeth. It is the most highly mineralized and hardest tissue in the body. Dental enamel is acellular and has no physiological means of repair outside of the protective and remineralization potential provided by saliva. Enamel is comprised of highly organized hydroxyapatite crystals that form in a defined extracellular space, the contents of which are supplied and regulated by ameloblasts. The entire process is under genetic instruction. The genetic control of amelogenesis is poorly understood, but requires the activities of multiple components that are uniquely important for dental enamel formation. Amelogenesis imperfecta (AI) is a collective designation for the variety of inherited conditions displaying isolated enamel malformations, but the designation is also used to indicate the presence of an enamel phenotype in syndromes. Recently, genetic studies have demonstrated the importance of genes encoding enamel matrix proteins in the etiology of isolated AI. Here we review the essential elements of dental enamel formation and the results of genetic analyses that have identified disease-causing mutations in genes encoding enamel matrix proteins. In addition, we provide a fresh perspective on the roles matrix proteins play in catalyzing the biomineralization of dental enamel.

Porcine SPARC: isolation from dentin, cDNA sequence, and computer model.

Genes encoding the major enamel matrix proteins and non-collagenous proteins of bone and dentin are members of the secretory calcium-binding phosphoprotein (SCPP) family, which originated from ancestral SPARC (secreted protein, acidic and rich in cysteine; BM-40/osteonectin). To better understand the role of SPARC in mineralizing systems, we isolated SPARC from developing pig teeth, deduced its primary structure from the cDNA sequence, and determined its quaternary structure by homology modelling with reference to human SPARC crystal structures. The guanidine/EDTA extract from porcine dentin was fractionated by anion-exchange and size-exclusion chromatography. Stains-all positive bands at 38 and 35 kDa gave the N-terminal sequences APQQEALPDETEV and DFEKNYNMYIFPV, which corresponded to the SPARC N terminus and an internal region of the protein. Porcine SPARC contains 300 amino acids, including the 17-amino acid signal peptide, and shares 96.2% amino acid sequence identity with human SPARC. Without post-translational modifications, the 283-amino acid secreted protein has a molecular mass of 32.3 kDa. The three-dimensional model revealed that porcine SPARC contains a single N-linked glycosylation at N113, seven intramolecular disulfide bridges, and assembles into dimers. SPARC is composed of three structural/functional domains: an acidic Ca2+-binding, a follistatin-like, and an extracellular calcium-binding domain.

Bisphosphonate modulates cementoblast behavior in vitro.

BACKGROUND: Cementum formation is deemed to be instrumental for the successful regeneration of periodontal tissues, and thus events and modifiers of cementum formation and mineralization need to be determined. This study aimed to determine whether the bisphosphonate 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) altered the behavior of immortalized cementoblasts (osteocalcin-cementoblasts [OCCM]). METHODS: OCCM from transgenic mice were exposed to HEBP at concentrations ranging from 0.01 to 10.0 microM. The assays performed included the count of cell number for proliferation, Northern blot analysis for gene expression (up to 10 days for core binding factor alpha-1 [Cbfa1], bone sialoprotein [BSP], osteocalcin [OCN], and osteopontin [OPN], markers for cementoblast/osteoblast maturation/mineralization), von Kossa stain and alizarin red S stain for mineralization, and enzyme assay (p-nitrophenol phosphate cleavage) for alkaline phosphatase (ALP) activity. RESULTS: Mineral nodule formation was inhibited at the higher doses of HEBP (1.0 and 10.0 microM) only. At early stages (1, 3, and 6 days), gene expression assays revealed only subtle changes in treated cells versus untreated cells, but by day 10, groups treated with lower doses (0.01 and 0.1 microM) were markedly different at the gene expression level. OCN was significantly downregulated (70%) at the lowest dose, with less pronounced effects at higher doses. In concurrence, the master switch gene for osteoblasts, Cbfa1, was also downregulated at the lower doses. Inversely, OPN mRNA was enhanced at the lower doses. ALP activity was not altered by HEBP. CONCLUSION: Bisphosphonate alters cementoblast function in vitro through the regulation of gene expression and mineral formation.

Biological foundation for periodontitis as a potential risk factor for atherosclerosis.

OBJECTIVES: Links between periodontal diseases and systemic diseases have been well documented by epidemiological studies. Recently, research has shifted to elucidating the biologic mechanism for a causal relationship. One focus of interest is atherosclerosis, the underlying event of cardiovascular diseases due to its serious health impact. However, it is still not clear whether periodontopathic pathogens are truly etiologic agents or ubiquitous bystanders. This article reviews the current understanding about the molecular biological interactions between periodontal disease and atherosclerosis and the biological plausibility of periodontitis as a potential risk factor for cardiovascular disease. MATERIALS AND METHODS: The current literature regarding periodontal diseases and atherosclerosis and coronary vascular disease was searched using the Medline and PubMed databases. RESULTS: In vitro experiments and animal models are appropriate tools to investigate the biological interactions between periodontal disease and atherosclerosis at the cell molecular level. The concepts linking both pathologies refer to inflammatory response, immune responses, and hemostasis. In particular, Porphyromonas gingivalis appears to have unique, versatile pathogenic properties. Whether or not these findings from isolated cells or animal models are applicable in humans with genetic and environmental variations is yet to be determined. Likewise, the benefit from periodontal therapy on the development of atherosclerosis is unclear. Approaches targeting inflammatory and immune responses of periodontitis and atherosclerosis simultaneously are very intriguing. CONCLUSION: An emerging concept suggests that a pathogenic burden from different sources might overcome an individual threshold culminating in clinical sequela. P. gingivalis contributes directly and indirectly to atherosclerosis.