In-office Bleaching Activated With Violet LED: Effect on Pulpal and Tooth Temperature and Pulp Viability.

OBJECTIVES: This study evaluated the influence of hydrogen peroxide (HP) with or without titanium dioxide nanotubes (TiO2) associated with violet LED (VL) regarding: a) the temperature change in the pulp chamber and facial surface; b) the decomposition of HP; and c) the cytotoxicity of the gels on pulp cells. METHODS AND MATERIALS: The experimental groups were: HP35 (35% HP/Whiteness HP, FGM); HP35+VL; HP35T (HP35+TiO2); HP35T+VL; HP7 (7.5% HP/White Class 7.5%, FGM); HP7+VL; HP7T (HP7+TiO2); and HP7T+VL. TiO2 was incorporated into the bleaching gels at 1%. Eighty bovine incisors were evaluated to determine temperature change in 8 experimental groups (n=10/group). A k-type thermocouple was used to evaluate the temperatures of the facial surface and in the pulp chamber, achieved by enabling endodontic access to the palatal surface, throughout the 30-minute session. HP decomposition (n=3) of gels was evaluated by using an automatic potentiometric titrator at the initial and 30-minute time points. Trans-enamel and trans-dentinal cell viability were assessed with a pulp chamber device as well as enamel and dentin discs (n=6), and the treatment extracts (culture medium + diffused components) were collected and applied to MDPC-23 odontoblast cells to evaluate cell viability according to the MTT test. RESULTS: A temperature increase in the pulp chamber was observed in the presence of VL at 30 minutes (p<0.05) (Mann-Whitney test). Also at 30 minutes, HP35 showed greater decomposition in the presence of VL rather than in its absence (p<0.05) (mixed linear models and the Tukey-Kramer test). HP7 provided greater cell viability than the groups treated with HP35 (p<0.05) (generalized linear models test). Cell viability was significantly lower for HP7 in the presence of VL (p<0.05). CONCLUSION: Pulpal temperature increased with VL (maximum of 1.9°C), but did not exceed the critical limit to cause pulp damage. Less concentrated HP resulted in higher cell viability, even when associated with VL.

Impact of Resin Composite Viscosity and Fill-technique on Internal Gap in Class I Restorations: An OCT Evaluation.

The aim of this in vitro study was to quantitatively evaluate the internal gap of resin composites of high-and low-viscosity used in single- and incremental-fill techniques in Class I cavities exposed to thermal cycling (TC) using optical coherence tomography (OCT). Cavities of 4-mm depth and 3-mm diameter were prepared in 36 third molars randomly distributed into four groups, according to viscosity of restorative resin-based composite (high or low viscosity, all from 3M Oral Care) and technique application (bulk or incremental fill) used (n=9): RC, high-viscosity, incremental-fill, resin-based composite (Filtek Z350 XT Universal Restorative); BF, high-viscosity, bulk-fill, resin-based composite (Filtek One Bulk Fill); LRC, lowviscosity, incremental-fill, resin-based composite (Filtek Z350 XT Flowable Universal Restorative); and LBF, low-viscosity, bulk-fill, resin-based composite (Filtek Flowable Restorative). Single Bond Universal Adhesive system (3M Oral Care) was used in all the experimental groups. The incremental-fill technique was used for RC and LRC groups (2-mm increments), and a single-layer technique was used for BF and LBF groups, as recommended by the manufacturer. The internal adaptation of the resin at all dentin walls was evaluated before and after TC (5000 cycles between 5°C and 55°C) using OCT images. Five images of each restored tooth were obtained. Images were analyzed using ImageJ software that measured the entire length of the gaps at the dentin-restoration interface. The length of gaps (µm) was analyzed using two-way repeated measures ANOVA and the Tukey tests (α=0.05). There was a significant interaction between material types and TC (p=0.006), and a significant difference among all material types (p<0.0001), before and after TC (p<0.0001). Increased internal gaps at the dentin-restoration interface were noticed after TC for all groups. RC presented the lowest value of internal gap before and after TC, while LBF showed the highest values of internal gap after TC. In conclusion, TC negatively affected the integrity of internal gap, whereas high-viscosity, incremental-fill, resin-based composite presented better performance in terms of internal adaptation than low-viscosity, bulk-fill materials in Class I cavities.

A novel de novo heterozygous ALPL nonsense mutation associated with adult hypophosphatasia.

Using genetic, clinical, biochemical, and radiographic assessment and bioinformatic approaches, we present an unusual case of adult HPP caused by a novel de novo heterozygous nonsense mutation in the alkaline phosphatase (ALPL). INTRODUCTION: Hypophosphatasia (HPP) is caused by genetic alterations of the ALPL gene, encoding the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Here, the purpose was to perform clinical and molecular investigation in a 36-year-old Caucasian woman suspected to present adult HPP. METHODS: Medical and dental histories were obtained for the proposita and family members, including biochemical, radiographic, and dental assessments. ALPL mutational analysis was performed by the Sanger sequencing method, and the functional impact prediction of the identified mutations was assessed by bioinformatic methods. RESULTS: We identified a novel heterozygous nonsense mutation in the ALPL gene (NM_000478.6:c.768G>A; W[TGG]>*[TGA]) associated with spontaneous vertebral fracture, severe back pain, musculoskeletal pain, low bone density, and short-rooted permanent teeth loss. Functional prediction analysis revealed that the Trp256Ter mutation led to a complete loss of TNSALP crown domain and extensive loss of other functional domains (calcium-binding domain, active site vicinity, and zinc-binding site) and over 60% loss of homodimer interface residues, suggesting that the mutant TNSALP molecules are nonfunctional and form unstable homodimers. Genotyping of the ALPL in the proposita's parents, sister, and niece revealed that in this case, HPP occurred due to a de novo mutation. CONCLUSION: The present study describes a novel genotype-phenotype and structure-function relationship for HPP, contributing to a better molecular comprehension of HPP etiology and pathophysiology.

Osteopontin regulates dentin and alveolar bone development and mineralization.

The periodontal complex is essential for tooth attachment and function and includes the mineralized tissues, cementum and alveolar bone, separated by the unmineralized periodontal ligament (PDL). To gain insights into factors regulating cementum-PDL and bone-PDL borders and protecting against ectopic calcification within the PDL, we employed a proteomic approach to analyze PDL tissue from progressive ankylosis knock-out (Ank-/-) mice, featuring reduced PPi, rapid cementogenesis, and excessive acellular cementum. Using this approach, we identified the matrix protein osteopontin (Spp1/OPN) as an elevated factor of interest in Ank-/- mouse molar PDL. We studied the role of OPN in dental and periodontal development and function. During tooth development in wild-type (WT) mice, Spp1 mRNA was transiently expressed by cementoblasts and strongly by alveolar bone osteoblasts. Developmental analysis from 14 to 240days postnatal (dpn) indicated normal histological structures in Spp1-/- comparable to WT control mice. Microcomputed tomography (micro-CT) analysis at 30 and 90dpn revealed significantly increased volumes and tissue mineral densities of Spp1-/- mouse dentin and alveolar bone, while pulp and PDL volumes were decreased and tissue densities were increased. However, acellular cementum growth was unaltered in Spp1-/- mice. Quantitative PCR of periodontal-derived mRNA failed to identify potential local compensators influencing cementum in Spp1-/- vs. WT mice at 26dpn. We genetically deleted Spp1 on the Ank-/- mouse background to determine whether increased Spp1/OPN was regulating periodontal tissues when the PDL space is challenged by hypercementosis in Ank-/- mice. Ank-/-; Spp1-/- double deficient mice did not exhibit greater hypercementosis than that in Ank-/- mice. Based on these data, we conclude that OPN has a non-redundant role regulating formation and mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. These findings may inform therapies targeted at controlling soft tissue calcification.

Periodontitis in Chédiak-Higashi Syndrome: An Altered Immunoinflammatory Response.

Chédiak-Higashi syndrome (CHS), a rare autosomal recessive disorder caused by mutations in the lysosomal trafficking regulator gene (LYST), is associated with aggressive periodontitis. It is suggested that LYST mutations affect the toll-like receptor (TLR)-mediated immunoinflammatory response, leading to frequent infections. This study sought to determine the periodontal status of patients with classic (severe) and atypical (milder) forms of CHS and the immunoregulatory functions of gingival fibroblasts in CHS patients. In contrast to aged-matched healthy controls, atypical (n = 4) and classic (n = 3) CHS patients presented with mild chronic periodontitis with no evidence of gingival ulceration, severe tooth mobility, or premature exfoliation of teeth. As a standard of care, all classic CHS patients had undergone bone marrow transplantation (BMT). Primary gingival fibroblasts obtained from atypical and BMT classic CHS patients displayed higher protein expression of TLR-2 (1.81-fold and 1.56-fold, respectively) and decreased expression of TLR-4 (-2.5-fold and -3.85-fold, respectively) at baseline when compared with healthy control gingival fibroblasts. When challenged with whole bacterial extract of Fusobacterium nucleatum, both atypical and classic CHS gingival fibroblasts failed to up-regulate TLR-2 and TLR-4 expression when compared with their respective untreated groups and control cells. Cytokine multiplex analysis following F. nucleatum challenge showed that atypical CHS gingival fibroblasts featured significantly increased cytokine expression (interleukin [IL]-2, IL-4, IL-5, IL-6, IL-10, IL-12, interferon-γ, tumor necrosis factor-α), whereas classic CHS cells featured similar/decreased cytokine expression when compared with treated control cells. Collectively, these results suggest that LYST mutations in CHS patients affect TLR-2 and TLR-4 expression/function, leading to dysregulated immunoinflammatory response, which in turn may influence the periodontal phenotype noted in CHS patients. Furthermore, our results suggest that atypical CHS patients and classic CHS patients who undergo BMT early in life are less susceptible to aggressive periodontitis and that hematopoietic cells play a critical role in mitigating the risk of aggressive periodontitis in CHS. Knowledge Transfer Statement: Results from this study can be used to create awareness among clinicians and researchers that not all CHS patients exhibit historically reported aggressive periodontitis, especially if they have atypical CHS disease or have received bone marrow transplantation. LYST mutations in CHS patients may affect TLR-2 and TLR-4 expression/function leading to dysregulated immunoinflammatory response, which in turn may influence the periodontal phenotype noted in CHS patients.

Impact of smoking on experimental gingivitis. A clinical, microbiological and immunological prospective study.

OBJECTIVE: The present study assessed the effect of smoking on clinical, microbiological and immunological parameters in an experimental gingivitis model. MATERIAL AND METHODS: Twenty-four healthy dental students were divided into two groups: smokers (n = 10); and nonsmokers (n = 14). Stents were used to prevent biofilm removal during brushing. Visible plaque index (VPI) and gingival bleeding index (GBI) were determined 5- on day -7 (running phase), baseline, 21 d (experimental gingivitis) and 28 d (resolution phase). Supragingival biofilm and gingival crevicular fluid were collected and assayed by checkerboard DNA-DNA hybridization and a multiplex analysis, respectively. Intragroup comparison was performed by Friedman and Dunn's multiple comparison tests, whereas the Mann-Whitney U-test was applied for intergroup analyses. RESULTS: Cessation of oral hygiene resulted in a significant increase in VPI, GBI and gingival crevicular fluid volume in both groups, which returned to baseline levels 7 d after oral hygiene was resumed. Smokers presented lower GBI than did nonsmokers (p < 0.05) at day 21. Smokers had higher total bacterial counts and higher proportions of red- and orange complex bacteria, as well as lower proportions of Actinomyces spp., and of purple- and yellow-complex bacteria (p < 0.05). Furthermore, the levels of key immune-regulatory cytokines, including interleukin (IL)-8, IL-17 and interferon-γ, were higher in smokers than in nonsmokers (p < 0.05). CONCLUSION: Smokers and nonsmokers developed gingival inflammation after supragingival biofilm accumulation, but smokers had less bleeding, higher proportions of periodontal pathogens and distinct host-response patterns during the course of experimental gingivitis.

PTH and Vitamin D Repress DMP1 in Cementoblasts.

A complex feedback mechanism between parathyroid hormone (PTH), 1,25(OH)2D3 (1,25D), and fibroblast growth factor 23 (FGF-23) maintains mineral homeostasis, in part by regulating calcium and phosphate absorption/reabsorption. Previously, we showed that 1,25D regulates mineral homeostasis by repressing dentin matrix protein 1 (DMP1) via the vitamin D receptor pathway. Similar to 1,25D, PTH may modulate DMP1, but the underlying mechanism remains unknown. Immortalized murine cementoblasts (OCCM.30), similar to osteoblasts and known to express DMP1, were treated with PTH (1-34). Real-time quantitative polymerase chain reaction (PCR) and Western blot revealed that PTH decreased DMP1 gene transcription (85%) and protein expression (30%), respectively. PTH mediated the downregulation of DMP1 via the cAMP/protein kinase A (PKA) pathway. Immunohistochemistry confirmed the decreased localization of DMP1 in vivo in cellular cementum and alveolar bone of mice treated with a single dose (50 µg/kg) of PTH (1-34). RNA-seq was employed to further identify patterns of gene expression shared by PTH and 1,25D in regulating DMP1, as well as other factors involved in mineral homeostasis. PTH and 1,25D mutually upregulated 36 genes and mutually downregulated 27 genes by ≥2-fold expression (P ≤ 0.05). Many identified genes were linked with the regulation of bone/tooth homeostasis, cell growth and differentiation, calcium signaling, and DMP1 transcription. Validation of RNA-seq results via PCR array confirmed a similar gene expression pattern in response to PTH and 1,25D treatment. Collectively, these results suggest that PTH and 1,25D share complementary effects in maintaining mineral homeostasis by mutual regulation of genes/proteins associated with calcium and phosphate metabolism while also exerting distinct roles on factors modulating mineral metabolism. Furthermore, PTH may modulate phosphate homeostasis by downregulating DMP1 expression via the cAMP/PKA pathway. Targeting genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health.

Mineralization defects in cementum and craniofacial bone from loss of bone sialoprotein.

Bone sialoprotein (BSP) is a multifunctional extracellular matrix protein found in mineralized tissues, including bone, cartilage, tooth root cementum (both acellular and cellular types), and dentin. In order to define the role BSP plays in the process of biomineralization of these tissues, we analyzed cementogenesis, dentinogenesis, and osteogenesis (intramembranous and endochondral) in craniofacial bone in Bsp null mice and wild-type (WT) controls over a developmental period (1-60 days post natal; dpn) by histology, immunohistochemistry, undecalcified histochemistry, microcomputed tomography (microCT), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and quantitative PCR (qPCR). Regions of intramembranous ossification in the alveolus, mandible, and calvaria presented delayed mineralization and osteoid accumulation, assessed by von Kossa and Goldner's trichrome stains at 1 and 14 dpn. Moreover, Bsp(-/-) mice featured increased cranial suture size at the early time point, 1 dpn. Immunostaining and PCR demonstrated that osteoblast markers, osterix, alkaline phosphatase, and osteopontin were unchanged in Bsp null mandibles compared to WT. Bsp(-/-) mouse molars featured a lack of functional acellular cementum formation by histology, SEM, and TEM, and subsequent loss of Sharpey's collagen fiber insertion into the tooth root structure. Bsp(-/-) mouse alveolar and mandibular bone featured equivalent or fewer osteoclasts at early ages (1 and 14 dpn), however, increased RANKL immunostaining and mRNA, and significantly increased number of osteoclast-like cells (2-5 fold) were found at later ages (26 and 60 dpn), corresponding to periodontal breakdown and severe alveolar bone resorption observed following molar teeth entering occlusion. Dentin formation was unperturbed in Bsp(-/-) mouse molars, with no delay in mineralization, no alteration in dentin dimensions, and no differences in odontoblast markers analyzed. No defects were identified in endochondral ossification in the cranial base, and craniofacial morphology was unaffected in Bsp(-/-) mice. These analyses confirm a critical role for BSP in processes of cementogenesis and intramembranous ossification of craniofacial bone, whereas endochondral ossification in the cranial base was minimally affected and dentinogenesis was normal in Bsp(-/-) molar teeth. Dissimilar effects of loss of BSP on mineralization of dental and craniofacial tissues suggest local differences in the role of BSP and/or yet to be defined interactions with site-specific factors.