Tooth mobility restriction by multistranded and CAD/CAM retainers-an in vitro study.

OBJECTIVES: Orthodontic retainers should restrict physiological tooth mobility as little as possible. While this has been investigated for multistranded retainers, there is a lack of data for novel CAD/CAM retainers. To address this, the present study compared the restriction of physiological tooth mobility in multistranded retainers and different CAD/CAM retainers. MATERIAL/METHODS: One group of multistranded (n = 8) and five groups of CAD/CAM retainers (nickel-titanium (NiTi), titanium grade 5 (Ti5), polyetheretherketone (PEEK), zirconia (ZrO2), and cobalt-chromium (CoCr); each n = 8) bonded from canine to canine were investigated for their influence on vertical and horizontal tooth mobility using an in vitro model of a lower arch in a universal testing machine. Load-deflection curves were determined and statistically analysed. RESULTS: All retainers restricted tooth mobility to varying extents. The retainers had less of an influence on vertical tooth mobility, with less of a difference between retainers (14%-38% restriction). In contrast, significant (P ≤ 0.05) differences were observed between retainers in the restriction of horizontal tooth mobility. ZrO2 retainers had the greatest impact, restricting horizontal tooth mobility by 82% (68 ± 20 µm/100N), followed by CoCr (75%, 94 ± 26 µm/100N) and PEEK (73%, 103 ± 28 µm/100N) CAD/CAM retainers, which had comparable effects on horizontal tooth mobility. Ti5 (54%, 175 ± 66 µm/100N) and NiTi (34%, 248 ± 119 µm/100N) CAD/CAM retainers had less of an influence on horizontal tooth mobility, and were comparable to multistranded retainers (44%, 211 ± 77 µm/100N). LIMITATIONS: This is an in vitro study, so clinical studies are needed to draw clinical conclusions. CONCLUSIONS: Multistranded and CAD/CAM retainers have different effects on tooth mobility in vitro. These effects should be further explored in future in vivo studies.

Fit of anterior restorations made of 3D-printed and milled zirconia: An in-vitro study.

OBJECTIVES: To evaluate the fit of zirconia veneers made by either 3D printing or milling. METHODS: A typodont maxillary central incisor was prepared for a 0.5-mm-thick veneer and was reproduced 36 times from resin. Restorations were designed with a 20-µm-wide marginal and a 60-µm-wide internal cement gap, and were made from 3D-printed zirconia (LithaCon 3Y 210, Lithoz, n = 24) and milled zirconia (Cercon ht, DentsplySirona, n = 12). For milled zirconia, a drill compensation was needed to give the milling bur access to the intaglio surface. The restorations were cemented, cross-sectioned, and the cement gap size was analyzed by two raters. Inter-rater reliability was studied at 12 3D-printed veneers (intraclass correlation coefficient, ICC, mixed model, absolute agreement). Twelve remaining 3D-printed restorations were compared with 12 milled restorations regarding fit at three locations: marginally, labially, and at the incisal edge (Mann-Whitney U-tests, α<0.05). RESULTS: Inter-rater reliability was excellent, with an ICC single-measure coefficient of 0.944 (95%-confidence interval: [0.907; 0.966]). Gap sizes (mean ± SD / maximum) were 55 ± 9 / 143 µm at the margins, 68 ± 14 / 130 µm labially, and 78 ± 19 / 176 µm at the incisor edge for 3D-printed veneers. For milled veneers, gap sizes were 44 ± 11 / 141 µm at the margins, 85 ± 19 / 171 µm labially, and 391 ± 26 / 477 µm at the incisor edge. At the margins, the milled veneers outperformed the 3D-printed restorations (p = 0.011). The cement gap at the incisor edge was significantly smaller after 3D printing (p < 0.001). CONCLUSIONS: 3D-printed zirconia restorations showed clinically acceptable mean marginal gaps below 100 µm. Because drill compensation could be omitted with 3D printing, the fit at the sharp incisal edge was significantly tighter than with milling. CLINICAL SIGNIFICANCE: The fit of 3D-printed ceramic anterior restorations meets clinical standards. In addition, 3D printing is associated with a greater geometrical freedom than milling. With regard to fit this feature allows tighter adaptation even after minimally invasive preparation.

Neuronal Guidance Molecules in Bone Remodeling and Orthodontic Tooth Movement.

During orthodontic tooth movement, mechanically induced remodeling occurs in the alveolar bone due to the action of orthodontic forces. The number of factors identified to be involved in mechanically induced bone remodeling is growing steadily. With the uncovering of the functions of neuronal guidance molecules (NGMs) for skeletal development as well as for bone homeostasis, NGMs are now also among the potentially significant factors for the regulation of bone remodeling during orthodontic tooth movement. This narrative review attempts to summarize the functions of NGMs in bone homeostasis and provides insight into the currently sparse literature on the functions of these molecules during orthodontic tooth movement. Presently, four families of NGMs are known: Netrins, Slits, Semaphorins, ephrins and Eph receptors. A search of electronic databases revealed roles in bone homeostasis for representatives from all four NGM families. Functions during orthodontic tooth movement, however, were only identified for Semaphorins, ephrins and Eph receptors. For these, crucial prerequisites for participation in the regulation of orthodontically induced bone remodeling, such as expression in cells of the periodontal ligament and in the alveolar bone, as well as mechanical inducibility, were shown, which suggests that the importance of NGMs in orthodontic tooth movement may be underappreciated to date and further research might be warranted.

A Potential Role of Semaphorin 3A during Orthodontic Tooth Movement.

BACKGROUND: Induced tooth movement during orthodontic therapy requires mechano-induced bone remodeling. Besides various cytokines and growth-factors, neuronal guidance molecules gained attention for their roles in bone homeostasis and thus, potential roles during tooth movement. Several neuronal guidance molecules have been implicated in the regulation of bone remodeling. Amongst them, Semaphorin 3A is particular interesting as it concurrently induces osteoblast differentiation and disturbs osteoclast differentiation. METHODS: Mechano-regulation of Sema3A and its receptors PlexinA1 and Neuropilin (RT-qPCR, WB) was evaluated by applying compressive and tension forces to primary human periodontal fibroblasts (hPDLF) and alveolar bone osteoblasts (hOB). The association of the transcription factor Osterix (SP7) and SEMA3A was studied by RT-qPCR. Mechanisms involved in SEMA3A-mediated osteoblast differentiation were assessed by Rac1GTPase pull-downs, ß-catenin expression analyses (RT-qPCR) and nuclear translocation assays (IF). Osteogenic markers were analyzed by RT-qPCR. RESULTS: SEMA3A, PLXNA1 and NRP1 were differentially regulated by tension or compressive forces in hPDLF. Osterix (SP7) displayed the same pattern of regulation. Recombinant Sema3A induced the activation of Rac1GTPase, the nuclear translocation of ß-catenin and the expression of osteogenic marker genes. CONCLUSION: Sema3A, its receptors and Osterix are regulated by mechanical forces in hPDLF. SEMA3A upregulation was associated with Osterix (SP7) modulation. Sema3A-enhanced osteogenic marker gene expression in hOB might be dependent on a pathway involving Rac1GTPase and ß-catenin. Thus, Semaphorin 3A might contribute to bone remodeling during induced tooth movement.

OCT evaluation of orthodontic surface sealants: a 12-month follow-up randomized clinical trial.

OBJECTIVES: The aim of this single-center randomized controlled trial (NCT03753256) was to assess orthodontic surface sealant layer thickness and integrity in vivo during a 12-month follow-up by optical coherence tomography (OCT). MATERIALS AND METHODS: Using a split-mouth design, quadrants of 20 patients treated with fixed orthodontic appliances were included. Quadrants were randomly assigned to the sealants Pro Seal® (PS) or Opal® Seal™ (OS). OCT scans were performed immediately after the application of the sealants and after 3, 6, 9, and 12 months. Sealant layer thicknesses and their integrity were determined at 5 regions of interest (ROIs) known for high risks of demineralization. Sealant integrity loss was determined using a self-developed scale. RESULTS: A total of 16 patients successfully completed the study. The studied sealants showed significant differences in initial layer thickness. Mean layer thickness was significantly lower for PS (67.8 µm, (95% CI, 56.1-79.5)) than for OS (110.7 µm, (95% CI, 97.3-124.1)). Layer thickness loss was significant after 3 months for PS and after 6 months for OS. Sealant integrity was compromised in more than 50% of the ROIs already after 3 months for both sealants. CONCLUSIONS: Patients treated with fixed orthodontic surface sealants lost the integrity of the protective layer in more than 50% of cases after 3 months, and the layer thickness of the sealants was significantly reduced after 3-6 months. CLINICAL RELEVANCE: The protective effect against demineralization lesions of orthodontic sealants in patients treated with fixed appliances appears to be limited in time. Further preventive measures should be investigated. TRIAL REGISTRATION: ClinicalTrials.gov (NCT03753256).

Discoloration of surface sealants by plaque disclosing solution.

PURPOSE: Surface sealants are widely used as a prevention strategy and are indicated for young patients with insufficient oral hygiene who also need plaque removal by professional tooth cleaning. The aim of this study was to evaluate discoloration of surface sealants by plaque disclosing solutions and to test to what extent this discoloration can be reduced again by professional tooth cleaning. METHODS: In all, 96 extracted lesion-free human teeth were randomly assigned to treatment with either Pro Seal® (PS; Opal Orthodontics, South Jordan, UT, USA) or Opal®Seal™ (OS; Reliance Orthodontic Products, Itasca, IL, USA). Color evaluations after application of the plaque disclosing solution Mira-2-Ton® (Hager & Werken, Duisburg, Germany) were performed using a clinical spectrophotometer. Staining and polishing were repeated once. Color differences (∆E) above 3.77 were regarded as clinically relevant. RESULTS: All sealants showed high, clinically relevant ∆E values after the first staining. Polishing led to significantly decreased ∆E values on PS-treated teeth; however, the median ∆E value remained above the clinically relevant threshold. Polishing on OS-treated teeth only slightly reduced ∆E values. After professional tooth cleaning both PS and OS showed clinically relevant ∆E values. CONCLUSION: Surface sealants show clinically relevant discoloration after exposure to plaque disclosing solution under in vitro conditions. Such discolorations could not be removed by professional tooth cleaning. Thus, in clinical practice, plaque disclosing solutions might cause esthetic deficits in surface sealant-treated teeth. The impact of plaque disclosing solutions under clinical conditions (e.g., in the presence of saliva and by various aspects of a person's nutrition) should be investigated in clinical studies.

Abrasion of Pro Seal® and Opal® Seal™ by professional tooth cleaning protocols: results from an in vitro study and a randomized controlled trial.

BACKGROUND: The integrity of orthodontic surface sealants after professional tooth cleaning (PTC) has previously only been evaluated in vitro. Recently, we have shown that optical coherence tomography (OCT) can successfully be used for the longitudinal assessments of sealant thickness in vitro and in vivo. OBJECTIVES: Thus, the aim of the present study was to assess the sealant thickness after PTC in vitro and in vivo by OCT. TRIAL DESIGN: Single-centre four-arm parallel-group randomized controlled trial. METHODS: Ninety-six extracted human teeth were randomly assigned to the surface sealants Pro Seal® (PS) and Opal® Seal™ (OS) and to PTC protocols: (1) polishing with brush and prophy paste (Cleanic®) or (2) erythritol air-polishing. Sealant thickness was assessed by OCT immediately after application (baseline), after thermocycling and after polishing for totals of 5, 10, 15, 30, 60, 90, and 120 seconds. Additionally, a clinical trial was conducted. Therefore, using a split-mouth design, quadrants of 20 patients and PTC protocols were randomized by an external randomization centre using computer generated tables to assign the surface sealants and PTC protocols. Sealant thicknesses were analysed at baseline, before and after PTC. Due to the optical properties of sealants, a complete blinding was not feasible. RESULTS: In vitro both sealants revealed significant layer thickness losses after both PTC protocols. PS lost 0.77 µm/s [95% CI (confidence interval): 0.67, 0.87] from air-polishing and 0.43 µm/s (95% CI: 0.37, 0.49) from polishing with brush while OS lost 0.44 µm/s (95% CI: 0.32, 0.55) from air-polishing and 0.79 µm/s (95% CI: 0.68, 0.89) from polishing with brush of layer thickness. Sealant thickness loss of was significantly higher after erythritol air-polishing for PS and after polishing with brush for OS. The results of a concurrent randomized controlled trial (RCT) were comparable to those achieved in the in vitro part of this study. LIMITATIONS: Long-term surface sealant abrasion should be validated by additional RCTs. CONCLUSIONS: For PTC on surface sealant treated teeth, low abrasive protocols should be used. Air-polishing should be avoided on PS protected teeth and polishing with brush on OS treated teeth. TRIAL REGISTRATION: ClinicalTrials.gov NCT03753256.

Demineralization detection in orthodontics using an ophthalmic optical coherence tomography device equipped with a multicolor fluorescence module.

OBJECTIVES: Demineralizations such as white spot lesions are among the most prevalent side effects during orthodontic treatment. Fluorescence devices, including quantitative light-induced fluorescence (QLF), exploiting the intrinsic fluorescence of enamel and teeth and most recently optical coherence tomography (OCT) were introduced for early demineralization detection. In addition to near-infrared OCT scanning, multicolor modules allow for imaging with different laser wavelengths and the detection of reflective- and fluorescent light. The aim of this study was to evaluate a modified multicolor ophthalmic OCT device for the detection of early carious lesions in vitro and in vivo. MATERIALS AND METHODS: Twenty-seven extracted lesion free human teeth were randomly assigned to three different demineralization protocols. Carious lesion detection was performed using macrophotography, OCT, and reflectance/fluorescence imaging using green laser and blue laser light. In addition, teeth of 5 orthodontic patients were OCT scanned, and fluorescence imaging using blue laser light was performed to assess demineralization after orthodontic therapy. RESULTS: Both in vitro and in vivo, OCT allowed for precise determination of lesion depth and enamel loss. Fluorescence imaging using blue laser light was most sensitive for the detection of early demineralization in vitro and in vivo. However, established and severe demineralizations were also reliably detected by macrophotography in vitro and in vivo. CONCLUSION: Demineralization can be detected with high sensitivity using blue fluorescence imaging with multicolor OCT devices. CLINICAL RELEVANCE: In the future, OCT fluorescence imaging might be considered for longitudinal monitoring of dental hard tissue during orthodontic treatment in clinical trials.

Comparison of crevicular fluid cytokine levels after the application of surface sealants : A randomized trial.

PURPOSE: Surface sealants are widely used in orthodontic practice to avoid enamel decalcifications in patients treated with fixed orthodontic appliances. While their clinical benefit was tested in several studies, their biocompatibility has been evaluated to a lesser extent. Therefore, the aim of this randomized prospective study was to evaluate possible adverse biological effects of three commonly used surface sealants and a bonding primer on gingival tissues by analysing cytokines in crevicular fluid of orthodontic patients after the application of surface sealants. METHODS: A single centre parallel trial was conducted. Using a split-mouth design quadrants of 15 patients requiring orthodontic treatment with fixed appliances were randomized to one of three commonly used surface sealants (Pro Seal®, Opal®Seal™, Protecto®CaF2Nano) and a bonding primer (Transbond™ XT). Interleukin (IL)-8 and IL-10 levels in crevicular fluids of the individual quadrants were assessed at four different time points (before application, and at 30, 60 and 90 min after application). RESULTS: In all, 60 quadrants of 15 patients were randomized (Pro Seal® n = 15, Opal®Seal™ n = 15, Protecto®CaF2Nano n = 15, Transbond™ XT n = 15) and analysed. No significant changes for IL-8 or IL-10 levels in crevicular fluid after the application of surface sealants or bonding primer were detected. However, interpatient variability was high. No further clinical side effects were detected. CONCLUSIONS: Commonly used pre-bonding surface sealants (Pro Seal®, Opal®Seal™) do not appear to have a significant impact on inflammatory cytokines levels of crevicular fluid and do not appear to contribute to sensitization or hypersensitivity events.

Assessing abrasion of orthodontic surface sealants using a modified ophthalmic optical coherence tomography device.

OBJECTIVE: Optical coherence tomography (OCT) is a clinical standard in ophthalmology. Currently, its application in dentistry is gaining increasing interest. In this study, we tested the possibility to use a modified commercially available spectral domain OCT (SD-OCT) to assess the layer thickness of orthodontic surface sealants. MATERIALS AND METHODS: Reference samples of surface sealants for calibration and repeatability testing were measured using a micrometer screw. SD-OCT measurements were compared with micro-CT and light microscopic analyses. After validating the calibration of the SD-OCT, surface sealant layer thickness after aging (thermo cycling) and simulation of professional tooth cleaning (PTC) was assessed using the SD-OCT on 45 extracted teeth assigned to three test groups (n = 15 each): Light Bond™ Sealant, Pro Seal®, and Opal® Seal. RESULTS: SD-OCT showed excellent repeatability and accuracy for measurements of surface sealant layer thickness. Compared with micro-CT, SD-OCT showed better accordance with the reference measurements. The analysis of surface sealants after thermo cycling and PTC revealed poor resistance of Light Bond after only aging and demonstrated substantial wear of all sealants after aging and PTC. CONCLUSION: Imaging using commercially available ophthalmic SD-OCT might represent a suitable non-invasive methodology for longitudinal assessments of surface sealant layer thickness in vitro and in vivo. CLINICAL RELEVANCE: SD-OCT might be a suitable non-invasive method for longitudinal assessments of surface sealant durability in clinical trials.

Biological evaluation of subgingivally placed direct resin composite materials.

Placement of composite resin restorations in deep subgingival cavities can damage surrounding soft tissues. In addition, commonly used resin-based composites (RBCs) might interfere with wound healing and periodontal health. To clarify cellular interactions with RBCs, we used an MTT assay to investigate adhesion of primary human gingival fibroblasts and human osteoblasts (hFOB 1.19) on five RBC materials with and without surface modifications (alumina blasting with 50- or 110-µm Al2O3). In addition, high-performance liquid chromatography (HPLC) was used to determine release of resin monomers from RBCs after 1 h, 1 day, and 7 days. As compared with tissue culture plastics (the control), cellular adhesion was significantly lower (P < 0.001) for human gingival fibroblasts and osteoblasts. Only minor, nonsignificant differences between individual RBCs were identified. HPLC analyses identified the release of three bifunctional methacrylates bisphenol A glycerolate dimethacrylate, triethylene glycol dimethacrylate, and diurethane dimethacrylate from RBCs and showed that monomer release increased between 1 h and 1 day but remained low. The present findings suggest that surface adhesion in the subgingival area is limited for the tested RBCs. Although residual monomer release was low for all tested RBCs, it might be sufficient to adversely affect cell adhesion.

Surgery-first approach in orthognathic surgery: Psychological and biological aspects - A prospective cohort study.

INTRODUCTION: The aim of this pilot study was to investigate psychological and biological changes after application of a surgery-first orthognathic treatment approach. METHODS: A prospective cohort study of 9 patients (6 women and 3 men; mean age 26.7 years) suffering from skeletal Class II and III deformities was conducted. Skeletal changes from pre-to post-treatment were analyzed based on data acquired by use of cone-beam computed tomography (CBCT). Psychological changes were analyzed using the orthognathic quality of life (OQLQ) questionnaire, Sense of Coherence 29-item scale (SOC-29) and longitudinal day-to-day questionnaire. For biological evaluation, concentrations of IL-1 ß, IL-6, TGF ß 1-3, MMP-2 and VEGF were assessed in crevicular fluid by bead-based multiplex assays at one preoperative and various postoperative time points. RESULTS: A significant improvement (P = 0.015) in quality of life, as measured with the OQLQ, was observed between baseline and 3 months post-surgery. The most affected dimensions were: facial aesthetics (p = 0.022), oral function (p = 0.051) and social aspects (p = 0.057). Sense of coherence (SOC) significantly improved after treatment by 9 points (P = 0.029). Despite the significant improvement in OQLQ and SOC during the course of the study, the personal experience of appearance varied distinctly in course and intensity. In accordance with the temporal pattern of fracture healing, the analysis of crevicular fluid revealed an increase in pro-resorptive factors (IL-1 ß, IL-6 and MMP-2) at early postoperative time points, while remodeling factors (members of the TGF-ß superfamily) were detected at later postoperative time points. CONCLUSIONS: Orthognathic treatment using the surgery-first approach has a positive impact on patient's psychosocial status. Accelerated tooth movement after surgery might, to a certain extent, be due to elevated levels of bone remodeling factors with overlapping functions during fracture healing and tooth movement.

In vitro evaluation of different dental materials used for the treatment of extensive cervical root defects using human periodontal cells.

INTRODUCTION: Repair materials for extensive cervical root defects may come in direct contact with periodontal tissues. This in vitro study compared the effects of four calcium silicate cements (CSC), one resin-modified glass ionomer cement, and one glass carbomer cement on primary human gingival fibroblasts (HGF), alveolar osteoblasts (HAO), and a human osteoblast cell line (hFOB 1.19). METHODS: HGF, HAO, and hFOB were seeded on discoid test specimens. Relative numbers of viable cells were quantitatively assessed after 1 and 24 h for cytotoxicity/adhesion assays and after 4, 24, 48, and 72 h for proliferation assays. Data were statistically analyzed using non-parametric tests (α = 0.05). RESULTS: Relative to the control (100 %), CSC allowed for mean numbers of 71-81 % viable HGF and 80-82 % viable HAO. Then, 64 % of HGF and 56 % of HAO were assessed on GC Fuji II LC. Mean numbers of viable cells were 59-64 % HGF and 67-68 % HAO for GCP Glass Fill specimens. Cells exposed to CSC over 24 h remained viable and even increased in number. Both cell types adhered almost equally well to CSC and GC Fuji II LC. GCP Glass Fill continued to decrease cell viability and adhesion. CSC-based materials and GC Fuji II LC allowed for HGF and hFOB proliferation; however, none of the tested materials specifically stimulated cell proliferation. CONCLUSIONS: CSC characterized by low cytotoxicity. GC Fuji II LC shows moderate cytotoxic effects. ProRoot MTA, Harvard MTA, Biodentine, EndoSequence putty, and GC Fuji II LC allow HGF and HAO to adhere and HGF and hFOB to proliferate. GCP Glass Fill decreases cell viability, adhesion, and proliferation. CLINICAL RELEVANCE: CSC remain the paramount biologic choice for the repair of extensive cervical root defects. GC Fuji II LC might be considered in addition to CSC when the defect comprises supracrestal areas and the restoration requires superior aesthetic and mechanical characteristics.

Effect of Aqueous Storage on Original and Repair Bond Strength and Residual Monomer Release of Fiberreinforced Composites.

PURPOSE: To evaluate the effects of aqueous storage on shear bond strength (SBS) and monomer release of fiberreinforced composites (FRCs). MATERIALS AND METHODS: Four unidirectional FRCs were tested, including one semi-interpenetrating polymer network (IPN) (ES, everStick) and three cross-linked polymer (CLP) FRCs (GT, GrandTec; TF, TenderFiber; DP, Dentapreg). The SBS of samples of original resin to fresh FRC with an intact oxygen inhibition layer (n = 30/group) and repair resin to FRC after surface treatment (n = 30/group) was evaluated after 6 and 12 months of storage in artificial saliva. Monomer release of polymerized resin-coated and uncoated FRCs was detected by high-performance liquid chromatography after immersion for 1 h, 1 day, and 7 days. RESULTS: After 6 months, a significant decrease in SBS was seen with ES-repair (p < 0.0001). After 12 months, significant decreases were seen with ES-original (p < 0.0001), ES-repair (p < 0.0001), and TF-repair (p = 0.0003). A significant reduction was also found for GT-original (p = 0.0254) and GT-repair (p = 0.0176). At 6 and 12 months, GT showed the highest SBS values, with DP-repair being statistically similar to GT at 12 months. For UDMA and bis- GMA, the greatest amounts of release were seen in uncoated specimens, followed by flowable resin-coated and viscous resin-coated specimens. CONCLUSION: Matrix composition, interfacial bonding, and resin coverage seem to account for differences in the aging behavior of FRCs. The semi-IPN material is likely to suffer most from the challenging oral conditions. CLP FRCs might be more stable over the long term. Coverage of FRCs with viscous resin is highly recommended to reduce residual monomer release.

Inhibition of AXUD1 attenuates compression-dependent apoptosis of cementoblasts.

OBJECTIVES: Root resorptions are common undesirable side effects of orthodontic treatment. In most patients, these defects are repaired by cementoblasts. However, in 1-5 % of patients, the repair fails. The repair mechanism is not well understood. Apoptosis of cementoblasts might contribute to an impaired repair of root resorptions induced by orthodontic forces. MATERIALS AND METHODS: To gain insight into putative molecular pathways leading to compression-induced apoptosis of human primary cementoblasts (HPCBs), three independent cell populations were subjected to compressive loading at 5, 20, and 30 g/cm2 for 1, 6, and 10 h. The mRNA expression of AXUD1, a novel pro-apoptotic gene, was monitored by quantitative reverse transcription PCR (qRT-PCR). To identify a possible function in compression-dependent apoptosis, AXUD1 was silenced in cementoblasts using an siRNA approach. Apoptosis of cementoblasts was measured by annexin V staining and flow cytometry. The phosphorylation of c-Jun-N-terminal kinases (JNKs) was investigated by Western blotting. RESULTS: AXUD1 was significantly induced in a time- and force-dependent manner. The rate of apoptotic HPCBs increased by 20-40 % after 10 h of compression (30 g/cm2). Phosphorylation of JNKs was detected after 10 h at 30 g/cm2. SiRNA-mediated knockdown of AXUD1 led to decreased phosphorylation of JNKs and reduced apoptosis rates in compressed HPCBs. CONCLUSIONS: Compression-induced apoptosis of HPCBs is mediated by AXUD1 via a JNK-dependent pathway. CLINICAL RELEVANCE: AXUD1-dependent apoptosis of human cementoblasts might contribute to an impaired repair of root resorptions during orthodontic tooth movement. Further studies are needed to develop treatment strategies aiming to minimize root resorption during orthodontic tooth movement.

A Prospective Clinical Pilot Study on the Level of Matrix Metalloproteinase-9 in Dental Pulpal Blood as a Marker for the State of Inflammation in the Pulp Tissue.

INTRODUCTION: Differentiation between reversible pulpitis (savable pulp) and irreversible inflammation of the pulp tissue (nonsavable pulp) based only on clinical and radiographic diagnoses has proven to be difficult. Pulp exposure allows for the collection of pulpal blood to quantitatively determine the level of inflammation markers or proteolytic enzymes, even with small samples. Pulpitis is associated with the invasion of neutrophil granulocytes and their release of matrix metalloproteinase-9 (MMP-9). METHODS: Forty-four patients (aged 18-74 years, mean = 35 years), each with 1 tooth with carious pulp exposure presenting with different stages of pulpitis, were included in this prospective, 2-center clinical study; 26 patients presented with irreversible pulpitis (groups 3 and 4), 10 with reversible pulpitis (group 2), and 8 with completely asymptomatic teeth with deep carious lesions (group 1). Six of the 26 patients with teeth diagnosed with irreversible pulpitis had not taken any nonsteroidal anti-inflammatory drugs and were evaluated as a separate group (group 4). Partial pulpotomy and blood sample collection from the pulp chamber were performed. The total levels of MMP-9 and tissue inhibitor of metalloproteinase-1 were assessed by fluorometric and colorimetric enzyme-linked immunosorbent assays, respectively. The Mann-Whitney U test and Spearman rank correlations were used to compare the MMP-9 levels with different stages of pulpal inflammation; significance was set at .05. RESULTS: The MMP-9 levels in the asymptomatic teeth (group 1) were significantly different from those in the teeth with reversible pulpitis (group 2, P = .006) or irreversible pulpitis (group 4, P < .001). A statistically significant difference was also observed between the MMP-9 levels in group 1 and group 3 (P < .001) in which the patients had taken nonsteroidal anti-inflammatory drugs. CONCLUSIONS: These findings indicate that the MMP-9 levels in pulpal blood samples could be a useful ancillary diagnostic tool for distinguishing different stages of pulp tissue inflammation.

Compression of human primary cementoblasts leads to apoptosis: A possible cause of dental root resorption?

OBJECTIVES: One of the most common side effects of orthodontic treatment is root resorption on the pressure side of tooth movement. This is usually repaired by cementoblasts, but 1-5 % of patients eventually experiences a marked reduction in root length because no repair has occurred. The reason why cementoblasts should lose their repair function in such cases is not well understood. There is evidence from genome-wide expression analysis (Illumina HumanHT-12 v4 Expression BeadChip Kit; > 30,000 genes) that apoptotic processes are upregulated after the compression of cementoblasts, which is particularly true of the pro-apoptotic gene AXUD1. METHODS: Human primary cementoblasts (HPCBs) from two individuals were subjected to compressive loading at 30 g/cm(2) for 1/6/10 h. The cells were then evaluated for apoptosis by flow cytometry, for mRNA expression of putative genes (AXUD1, AXIN1, AXIN2) by quantitative PCR, and for involvement of c-Jun-N-terminal kinases (JNKs) in the regulation of AXUD1 via western blotting. In addition, platelet-derived growth factor receptor-ß (PDGFRß) was selectively inhibited by SU16f to analyze the effect of PDGFRß-dependent signal transduction on AXUD1 and AXIN1 expression. RESULTS: The percentage of apoptotic HPCBs rose after only 6 h of compressive loading, and 18-20 % of cells were apoptotic after 10 h. Microarray data revealed significant upregulation of the pro-apoptotic gene AXUD1 after 6 h and quantitative PCR significant AXUD1 upregulation after 6 and 10 h of compression. AXIN1 and AXIN2 expression in HPCBs was significantly increased after compressive loading. Our tests also revealed that PDGFRß signaling inhibition by SU16f augmented the expression of AXIN1 and AXUD1 in HPCBs under compression. CONCLUSION: Increased apoptosis of compressed HPCBs might help explain why cementoblasts, rather than invariably repairing all cases of root resorption, sometimes allow the original root length to shorten. The pathway hypothesized to lead to cementoblast apoptosis involves PDGF signaling, with this signal transduction's inhibition augmenting the expression of pro-apoptotic genes. Thus activating PDGF signaling may modify the signaling pathway for the apoptosis of cementoblasts, which would reveal a protective role of PDGF for these cells. Further studies are needed to develop strategies of treatment capable of minimizing root resorption.

Biological evaluation of enamel sealants in an organotypic model of the human gingiva.

OBJECTIVES: Various sealant materials have been suggested to decrease decalcification during orthodontic treatment. However, only a few in vitro studies on the cytotoxicity of resinous pit and fissure sealants have been published, and to the best of our knowledge no similar studies are available for the enamel sealants used in orthodontics. Therefore, we aimed to characterize the possible adverse effects of enamel sealants, especially on the gingival epithelium. METHODS: Organotypic cultures of the human gingival mucosa were used to assess the possible impact of six enamel sealants. Differentiation and apoptosis were determined by immunofluorescent staining. The pro-inflammatory cytokines IL-1ß and IL-6 were quantified by ELISA. Cytotoxicity was measured using MTS assays in monolayer cultures of human gingival fibroblasts. Leaching of monomers from enamel sealants was quantified using HPLC. RESULTS: The differentiation of the organotypic gingival mucosa remained unaffected. All under-cured and several standard-cured sealants (Light Bond™ Sealant, Light Bond™ Filled Sealant, and L.E.D. Pro Seal®) significantly induced apoptosis in the organotypic model. Light Bond™ Sealant, Light Bond™ Filled Sealant, and L.E.D. Pro Seal® caused a significant induction of pro-inflammatory cytokines. Reducing curing time had an influence on cytotoxicity in monolayer cultures of primary human oral cells. All resin-based sealants leached monomers. SIGNIFICANCE: Enamel sealants might exert adverse effects on the gingival epithelium. Due to the vicinity of the enamel sealant to the gingival epithelium, and the large surface area of applied sealants, these materials should be carefully applied and sufficiently cured.

Gene expression profile of compressed primary human cementoblasts before and after IL-1ß stimulation.

OBJECTIVES: Root resorptions due to a reduced repair function of cementoblasts are common side effects during orthodontic tooth movement (OTM). The mechanisms, which lead to an impaired cementoblast function, are not fully understood. Therefore, we aimed to investigate changes in the gene expression of cementoblasts during mechanical stimulus under inflammatory conditions. MATERIALS AND METHODS: Human primary cementoblasts (HPCB) were exposed to compression for 6 h or stimulation with IL-1ß for 96 h and subsequent 6 h compression. Genome-wide expression analysis was performed using microarray analysis. Prominent gene expression alterations (COX2, AXUD1, FOSB, CCL2, IFI6, and PTGES) were verified by quantitative RT-PCR (qRT-PCR) in two HPCB populations. A caspase 3/7 activity assay was used to determine caspase-3 and caspase-7 activity in stressed cells. RESULTS: Gene expression cluster analysis revealed apoptosis as an important process induced under both conditions. Apoptosis (pro- and anti-apoptotic) related gene expression was most relevant after pro-inflammatory stimulation and compression. qRT-PCR analysis confirmed significant up-regulation of COX2, AXUD1, and FOSB in both HPCB populations after compression, while selected genes significantly increased after pro-inflammatory stimulation and compression. Compression of cementoblasts increased caspase. The combination of pro-inflammatory stimulation and compression led to a slightly smaller increase of caspase activity. CONCLUSIONS: Gene ontology analysis showed that compressed HPCB up-regulate genes that are associated with apoptosis. Combining compression with a pro-inflammatory stimulus (IL-1ß) augmented the positive regulation of apoptosis-related pathways. The induction of apoptosis related gene expression (pro- and anti-apoptotic genes) in cementoblasts suggests an involvement of apoptosis in cementoblast regulation during OTM. CLINICAL RELEVANCE: As apoptosis is induced in HPCB after compression and inflammation, it is conceivable that HPCB cell death might contribute to root resorptions due to a loss of repair activity of cementoblasts. Further studies should be conducted to clarify the implication of the identified genes on root resorptions in order to develop therapeutic strategies to prevent a shortening of roots.

Cytotoxicity of coated and uncoated fibre-reinforced composites.

OBJECTIVE: Currently, there are many fibre-reinforced composites (FRCs) available which differ in the type and volume fraction of fibres, pre-treatment of fibres and matrix composition. The aims of this in vitro investigation were to determine whether there is a difference in biocompatibility of FRCs and if coating FRCs with resin composites influences their cytotoxic potential. MATERIALS AND METHODS: Five different FRC materials were tested which were either uncoated or coated with flowable or viscous resin composite. Artificial saliva extracts were prepared according to USP-XXIII and ISO-10993 to determine cytotoxicity by testing cell viability and growth of primary human gingival fibroblasts (HGF) using MTT assay, LIVE/DEAD(®) assay and cell proliferation assay. The influence of eluates on fibres of the cytoskeleton was investigated by vimentin, tubulin and actinin immunostainings. A two-way ANOVA followed by Scheffe's post-hoc test, which included the factors FRC material and coating procedure, was performed to assess cytotoxicity. RESULTS: All extracts of FRC materials displayed minor cytotoxic potential on HGF cell viability, cell proliferation and integrity of the cytoskeleton. The type of FRC material significantly influenced cell viability (MTT assay) (p < 0.0001), whereas neither the presence of a coating nor the type of coating material resulted in altered cell viability. Distribution and organization of cytosolic fibres was not affected after HGF exposure to eluates. CONCLUSIONS: There is a lack of knowledge about the leaching behaviour of commonly available fully pre-impregnated FRCs and their interactions with coating materials. The coating of FRCs with resin composite materials did not impact biocompatibility.