Role of NOD1/NOD2 receptors in Fusobacterium nucleatum mediated NETosis.

Polymorphonuclear neutrophils (PMNs) are indispensable in fighting infectious microbes by adopting various antimicrobial strategies including phagocytosis and neutrophil extracellular traps (NETs). Although the role and importance of PMNs in periodontal disease are well established, the specific molecular mechanisms involved in NET formation are yet to be characterized. In the present study, we sought to determine the role of periodontal pathogen on NET formation by utilizing Fusobacterium nucleatum. Our data demonstrates that F. nucleatum activates neutrophils and induces robust NETosis in a time-dependent manner via the upregulation of the Nucleotide oligomerization domain 1 (NOD1) and NOD2 receptors. Furthermore, CRISPR/Cas9 knockout of HL-60 cells and the use of ligands/inhibitors confirmed the involvement of NOD1 and NOD2 receptors in F. nucleatum-mediated NET formation. When treated with NOD1 and NOD2 inhibitors, we observed a significant downregulation of peptidylarginine deiminase 4 (PAD4) activity. In addition, neutrophils showed a significant increase and decrease of myeloperoxidase (MPO) and neutrophil elastase (NE) when treated with NOD1/NOD2 ligands and inhibitors, respectively. Taken together, CRISPR/Cas9 knockout of NOD1/NOD2 HL-60 cells and inhibitors of NOD signaling confirmed the role of NLRs in F. nucleatum-mediated NETosis. Our data demonstrates an important pathway linking NOD1 and NOD2 to NETosis by F. nucleatum, a prominent microbe in periodontal biofilms. This is the first study to elucidate the role of NOD-like receptors in NETosis and their downstream signaling network.

Reliability and Failure Modes of a Hybrid Ceramic Abutment Prototype.

PURPOSE: A ceramic and metal abutment prototype was fatigue tested to determine the probability of survival at various loads. MATERIALS AND METHODS: Lithium disilicate CAD-milled abutments (n = 24) were cemented to titanium sleeve inserts and then screw attached to titanium fixtures. The assembly was then embedded at a 30° angle in polymethylmethacrylate. Each (n = 24) was restored with a resin-cemented machined lithium disilicate all-ceramic central incisor crown. Single load (lingual-incisal contact) to failure was determined for three specimens. Fatigue testing (n = 21) was conducted employing the step-stress method with lingual mouth motion loading. Failures were recorded, and reliability calculations were performed using proprietary software. Probability Weibull curves were calculated with 90% confidence bounds. Fracture modes were classified with a stereomicroscope, and representative samples imaged with scanning electron microscopy. RESULTS: Fatigue results indicated that the limiting factor in the current design is the fatigue strength of the abutment screw, where screw fracture often leads to failure of the abutment metal sleeve and/or cracking in the implant fixture. Reliability for completion of a mission at 200 N load for 50K cycles was 0.38 (0.52% to 0.25 90% CI) and for 100K cycles was only 0.12 (0.26 to 0.05)-only 12% predicted to survive. These results are similar to those from previous studies on metal to metal abutment/fixture systems where screw failure is a limitation. No ceramic crown or ceramic abutment initiated fractures occurred, supporting the research hypothesis. The limiting factor in performance was the screw failure in the metal-to-metal connection between the prototyped abutment and the fixture, indicating that this configuration should function clinically with no abutment ceramic complications. CONCLUSION: The combined ceramic with titanium sleeve abutment prototype performance was limited by the fatigue degradation of the abutment screw. In fatigue, no ceramic crown or ceramic abutment components failed, supporting the research hypothesis with a reliability similar to that of all-metal abutment fixture systems. A lithium disilcate abutment with a Ti alloy sleeve in combination with an all-ceramic crown should be expected to function clinically in a satisfactory manner.

In Vivo Evaluation of Dual Acid-Etched and Grit-Blasted/Acid-Etched Implants With Identical Macrogeometry in High-Density Bone.

PURPOSE: Based on the current evidence, the effect of implant macrogeometry has a significant influence on osseointegration. Thus, this study evaluated histomorphometrically and histologically the bone response to acid-etched in comparison to grit-blasted/acid-etched (GB) and machined control (C) surfaced implants possessing identical macrogeometry placed in high-density bone. MATERIALS AND METHODS: Implant surface topography of the 3 different surfaced implants has previously been characterized. The macrogeometry of the implants were conical, and healing chambers were created in the cortical regions. The 3 groups were placed in the external mandibular body of adult male sheep (n = 5). After 6 weeks in vivo, all samples were retrieved for histologic observation and histomorphometry (eg, bone-to-implant contact [BIC] and bone area fraction occupancy [BAFO]). RESULTS: No statistical difference was observed for BIC and for BAFO, although there was a tendency that the mean values for BAFO was higher for the textured surface groups. CONCLUSIONS: It is suggested that the effect of surface topography is minimal in high-density bone and osseointegration seemed to be macrogeometry dependent.

Surface characterization and in vivo evaluation of dual Acid-etched and grit-blasted/acid-etched implants in sheep.

PURPOSE: Dual acid-etching is widely used to modify dental implant topography and enhance early bone healing. This study evaluated the histomorphometric, biomechanical, and histological bone response to acid-etched (AA) in comparison with grit-blasted/acid-etched (GB) and machined control (C) implants within sites of relatively low-bone remodeling rates. MATERIALS AND METHODS: Implant surface topography was evaluated by scanning electron microscopy and optical interferometry (IFM). Six adult male sheep (n = 6) received 72 Ti-6Al-4V implants (n = 24 per surface) in both ilium (n = 12 per bone bilaterally). The implants remained for 3 and 6 weeks in vivo. The histomorphometric parameters bone-implant contact (BIC) and bone area fraction occupancy (BAFO) were evaluated. Biomechanical analysis consisted of torque-to-interface failure. RESULTS: IFM analysis showed the highest average surface roughness for GB and the highest density of summits and developed surface area percentage (P < 0.01) for AA. No difference was observed in BAFO for all groups in 3 and 6 weeks. Increased BIC and torque resistance were observed for AA implants at both time points after implantation. CONCLUSIONS: Overall, improved bone-to-implant response was observed for the AA implant surface.

Evaluation of surface roughness as a function of multiple blasting processing variables.

OBJECTIVES: This study evaluated the effect of implant surface blasting variables, such as blasting media size, velocity, and surface coverage and their two- and three-way interaction in surface roughness parameters. MATERIAL AND METHODS: Machined, grade IV titanium-alloy implants (n = 180) had their surfaces treated by a combination of 36 different blasting protocols according to the following variables: aluminum oxide blasting media particle size (50, 100, and 150 µm); velocity (75, 100, 125, and 150 m/s), and surface coverage (5, 15, 25 g/in.(2) ) (n = 5 per blasting protocol). A single 0.46 inch nozzle of the blaster was pointed at the threaded area and spaced 0.050 inches away. Surface topography (n = 5 measurements per implant) was assessed by scanning electron microscopy. Roughness parameters Sa, Sq, Sdr, and Sds were evaluated by optical interferometry. A GLM statistical model evaluated the effects of blasting variables on the surface parameters, and their two- and three-way interaction (P < 0.05). Statistical inferences for Sa and Sq were performed after a log(10) transformation to correct for data skewness. RESULTS: Prior to the log(10) transformation, Sa and Sq values for all processing groups ranged from ~0.5 to ~2.6 µm and from ~0.75 to 4 µm, respectively. Statistical inferences showed that Sa, Sq, and Sdr values were significantly dependent on blasting media, velocity, and surface coverage (all P < 0.001). Media × velocity, media × coverage, and media × velocity × coverage also significantly affected Sa, Sq, and Sdr values (P < 0.002). The highest levels were obtained with 100 µm blasting media, coverage for 5 g/in.(2) , and velocity of 100 m/s. No significant differences were observed for Sds (P > 0.15). CONCLUSIONS: The blasting variables produced different surface topography features and knowledge of their interaction could be used to tailor a desired implant surface configuration.

Biomechanical evaluation of undersized drilling on implant biomechanical stability at early implantation times.

PURPOSE: The present study evaluated the effect of different drilling dimensions (undersized, regular, and oversized) in the insertion and removal torques of dental implants in a beagle dog model. METHODS: Six beagle dogs were acquired and subjected to bilateral surgeries in the radii 1 and 3 weeks before euthanasia. During surgery, 3 implants, 4 mm in diameter by 10 mm in length, were placed in bone sites drilled to 3.2 mm, 3.5 mm, and 3.8 mm in final diameter. The insertion and removal torque was recorded for all samples. Statistical analysis was performed by paired t tests for repeated measures and by t tests assuming unequal variances (all at the 95% level of significance). RESULTS: Overall, the insertion torque and removal torque levels obtained were inversely proportional to the drilling dimension, with a significant difference detected between the 3.2 mm and 3.5 mm relative to the 3.8 mm groups (P < 0.03). Although insertion torque-removal torque paired observations was statistically maintained for the 3.5 mm and 3.8 mm groups, a significant decrease in removal torque values relative to insertion torque levels was observed for the 3.2 mm group. A different pattern of healing and interfacial remodeling was observed for the different groups. CONCLUSIONS: Different drilling dimensions resulted in variations in insertion torque values (primary stability) and stability maintenance over the first weeks of bone healing.

Histomorphometry and bone mechanical property evolution around different implant systems at early healing stages: an experimental study in dogs.

PURPOSE: Commercial implants differ at macro-, micro-, and nanolevels, which makes it difficult to distinguish their effect on osseointegration. The aim of this study was to evaluate the early integration of 5 commercially available implants (Astra OsseoSpeed, Straumann SLA, Intra-Lock Blossom Ossean, Nobel Active, and OsseoFix) by histomorphometry and nanoindentation. MATERIALS AND METHODS: Implants were installed in the tibiae of 18 beagle dogs. Samples were retrieved at 1, 3, and 6 weeks (n = 6 for each time point) and were histologically and nanomechanically evaluated. RESULTS: The results presented that both time (P < 0.01) and implant system and time interaction (P < 0.02) significantly affected the bone-to-implant contact (BIC). At 1 week, the different groups presented statistically different outcomes. No significant changes in BIC were noted thereafter. There were no significant differences in rank elastic modulus (E) or in rank hardness (H) for time (E: P > 0.80; H: P > 0.75) or implant system (E: P > 0.90; H: P > 0.85). CONCLUSIONS: The effect of different implant designs on osseointegration was evident especially at early stages of bone healing.

Effect of drilling dimension on implant placement torque and early osseointegration stages: an experimental study in dogs.

PURPOSE: Primary stability has been regarded as a key factor to ensure uneventful osseointegration of dental implants. Such stability is often achieved by placing implants in undersized drilled bone. The present study evaluated the effect of drilling dimensions in insertion torque and early implant osseointegration stages in a beagle dog model. MATERIALS AND METHODS: Six beagle dogs were acquired and subjected to bilateral surgeries in the radii 1 and 3 weeks before death. During surgery, 3 implants, 4 mm in diameter by 10 mm in length, were placed in bone sites drilled to 3.2 mm, 3.5 mm, and 3.8 mm in diameter. The insertion torque was recorded for all samples. After death, the implants in bone were nondecalcified processed and morphologically and morphometrically (bone-to-implant contact and bone area fraction occupancy) evaluated. Statistical analyses were performed using the Kruskal-Wallis test followed by Dunn's post hoc test for multiple comparisons at the 95% level of significance. RESULTS: The insertion torque levels obtained were inversely proportional to the drilling dimension, with a significant difference detected between the 3.2-mm and 3.8-mm groups (P = .003). Despite a significant increase in the bone-to-implant contact over time in vivo for all groups (P = .007), no effect for the drilling dimension was observed. Additionally, no effect of the drilling dimension and time was observed for the bone area fraction occupancy parameter (P = .31). The initial healing pathways differed between implants placed in bone drilled to different dimensions. CONCLUSIONS: Although different degrees of torque were observed with different drilling dimensions and these resulted in different healing patterns, no differences in the histometrically evaluated parameters were observed.

TLR3-Dependent Activation of TLR2 Endogenous Ligands via the MyD88 Signaling Pathway Augments the Innate Immune Response.

The role of the adaptor molecule MyD88 is thought to be independent of Toll-like receptor 3 (TLR3) signaling. In this report, we demonstrate a previously unknown role of MyD88 in TLR3 signaling in inducing endogenous ligands of TLR2 to elicit innate immune responses. Of the various TLR ligands examined, the TLR3-specific ligand polyinosinic:polycytidylic acid (poly I:C), significantly induced TNF production and the upregulation of other TLR transcripts, in particular, TLR2. Accordingly, TLR3 stimulation also led to a significant upregulation of endogenous TLR2 ligands mainly, HMGB1 and Hsp60. By contrast, the silencing of TLR3 significantly downregulated MyD88 and TLR2 gene expression and pro-inflammatory IL1ß, TNF, and IL8 secretion. The silencing of MyD88 similarly led to the downregulation of TLR2, IL1ß, TNF and IL8, thus suggesting MyD88 to somehow act downstream of TLR3. Corroborating in vitro data, Myd88-/- knockout mice downregulated TNF, CXCL1; and phospho-p65 and phospho-IRF3 nuclear localization, upon poly I:C treatment in a mouse model of skin infection. Taken together, we identified a previously unknown role for MyD88 in the TLR3 signaling pathway, underlying the importance of TLRs and adapter protein interplay in modulating endogenous TLR ligands culminating in pro-inflammatory cytokine regulation.

Influence of atmospheric pressure plasma treatment on mechanical proprieties of enamel and sealant bond strength.

OBJECTIVES: To define the effect of APP treatments on the mechanical properties of enamel and on its ability to promote sealant bonding to unetched enamel. METHODS: Human molar teeth were sectioned exposing flat enamel regions at the buccal and lingual surfaces. The specimens were divided into two substrate groups (etched and unetched) and distributed over three surface treatments (i) 5 slm Argon APP treatment, NaOH surface treatment, and (iii) compressed air application (control). The Enamel surfaces were characterized by SEM, IFM, and Goniometer instruments. For the mechanical tests nanoindentation and microshear bond strength were employed. Initial data evaluation comprised normality verification (SPS S software) and variance checking and the appropriated statistical analysis model employed. For all statistical inferences, significance was set at 0.05. RESULTS: SE was significantly higher for the etched and unetched group treated with Plasma relative to the NaOH and control groups. Nanoindentation testing determined that Rank hardness was significantly higher in the control and Plasma group relative to NaOH for the etched group. Rank Elastic Modulus was significantly higher on Control groups relative to NaOH and Plasma groups for the etched substrate. No difference was detected between treatments for the unetched group. For the µSBS test, we observed that APP treatment on etched and unetched enamel increased bonds significantly (p < 0.001). CONCLUSIONS: This study demonstrated that APP increased SE, surface wettability and bond strength between enamel and sealants potentially serving as a substitute for conventional acid etching procedures or as an adjuvant for self-etch sealants.

The in vivo effect of P-15 coating on early osseointegration.

The aim of this study was to evaluate mechanically and morphologically the effect of a specific peptide sequence P-15, when incorporated into implant surfaces. Three types of implants were used for the study: Group A: commercially pure titanium implant (blasted and acid etched) + electrochemical thin calcium phosphate deposition, Group B: commercially pure titanium implant (blasted and acid etched) + electrochemical thin calcium phosphate deposition + P-15 incorporation, and as control, Group C: commercially pure titanium implant (blasted and acid etched). After a topographical characterization, transcortical osteotomies were made, and all implant groups (102 implants per group) were randomly placed bilaterally in the tibiae of adult beagle dogs (n = 24). At, 1, 2, and 4 weeks post-surgery, the animals were sacrificed and the samples were retrieved for removal torque tests, for nano indentation, and for histomorphometrical analysis. The results (mean ± 95% CI) showed that Group B (34.4 ± 8.7%) presented statistically higher bone-to-implant contact than the other groups (A = 23.9 ± 7.8%; C = 21.7 ± 8.3%) at 1 week, indicating an enhanced osteogenesis due to the peptide incorporation. The results suggested that the incorporation of P-15 to implant surfaces increased its bioactivity and the effects were notable especially in the early stages of the healing process.

The effect of brain-derived neurotrophic factor on periodontal furcation defects.

This study aimed to observe the regenerative effect of brain-derived neurotrophic factor (BDNF) in a non-human primate furcation defect model. Class II furcation defects were created in the first and second molars of 8 non-human primates to simulate a clinical situation. The defect was filled with either, Group A: BDNF (500 µg/ml) in high-molecular weight-hyaluronic acid (HMW-HA), Group B: BDNF (50 µg/ml) in HMW-HA, Group C: HMW-HA acid only, Group D: empty defect, or Group E: BDNF (500 µg/ml) in saline. The healing status for all groups was observed at different time-points with micro computed tomography. The animals were euthanized after 11 weeks, and the tooth-bone specimens were subjected to histologic processing. The results showed that all groups seemed to successfully regenerate the alveolar buccal bone, however, only Group A regenerated the entire periodontal tissue, i.e., alveolar bone, cementum and periodontal ligament. It is suggested that the use of BDNF in combination with a scaffold such as the hyaluronic acid in periodontal furcation defects may be an effective treatment option.

Evaluation of osteogenic cell culture and osteogenic/peripheral blood mononuclear human cell co-culture on modified titanium surfaces.

This study aimed to determine the effect of a bioactive ceramic coating on titanium in the nanothickness range on human osteogenic cells, peripheral blood mononuclear cells (PBMC) and on osteogenic cells co-cultured with PBMC without exogenous stimuli. Cell viability, proliferation, adhesion, cytokine release (IL1ß, TGFß1, IL10 and IL17) and intracellular stain for osteopontin and alkaline phosphatase were assessed. Morphologic evaluation showed smaller and less spread cell aspects in co-culture relative to osteogenic cell culture. Cell viability, proliferation and adhesion kinetics were differently influenced by surface texture/chemistry in culture versus co-culture. Cytokine release was also influenced by the interaction between mononuclear and osteogenic cells (mediators released by mononuclear cells acted on osteogenic cells and vice versa). In general, 'multi-cell type' interactions played a more remarkable role than the surface roughness or chemistry utilized on the in vitro cellular events related to initial stages of bone formation.

Implant biomechanical stability variation at early implantation times in vivo: an experimental study in dogs.

PURPOSE: To demonstrate the degree of stability decrease and subsequent increase of dental implants at early implantation times in a beagle model. MATERIALS AND METHODS: The mandibular premolars and first molars of eight beagle dogs were extracted and the ridges allowed to heal for 8 weeks. Thirty-two (n = 16 each group) implants were placed bilaterally, and remained in vivo for 1 and 3 weeks. The implants with comparable dimensions were divided as follows: group 1, Straumann Bone Level with SLActive surface; group 2, Nobel Speedy Replace RP with TiUnite surface. During insertion and following sacrifice, the implants were torqued to determine insertion and interface failure values. Histologic sections were prepared for microscopy. Statistical analysis was performed using Kruskal-Wallis and multiple paired and non-paired t tests considering unequal variances at a 95% level of significance. RESULTS: High insertion torque values were observed along with a significant decrease at 1 week in vivo (P = .003). At 3 weeks, the biomechanical fixation levels increased and were comparable to the insertion torque value. Histology showed that interfacial bone remodeling and initial woven bone formation was observed around both implant groups at 1 and 3 weeks. CONCLUSIONS: As time elapsed early after implantation, the biomechanical stability of dental implants initially decreased and subsequently increased.

Argon-based atmospheric pressure plasma enhances early bone response to rough titanium surfaces.

This study investigated the effect of an Argon-based atmospheric pressure plasma (APP) surface treatment operated chairside at atmospheric pressure conditions applied immediately prior to dental implant placement in a canine model. Surfaces investigated comprised: rough titanium surface (Ti) and rough titanium surface + Argon-based APP (Ti-Plasma). Surface energy was characterized by the Owens-Wendt-Rabel-Kaelble method and chemistry by X-ray photoelectron spectroscopy (XPS). Six adult beagles dogs received two plateau-root form implants (n = 1 each surface) in each radii, providing implants that remained 1 and 3 weeks in vivo. Histometric parameters assessed were bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO). Statistical analysis was performed by Kruskall-Wallis (95% level of significance) and Dunn's post-hoc test. The XPS analysis showed peaks of Ti, C, and O for the Ti and Ti- Plasma surfaces. Both surfaces presented carbon primarily as hydrocarbon (C-C, C-H) with lower levels of oxidized carbon forms. The Ti-Plasma presented large increase in the Ti (+11%) and O (+16%) elements for the Ti- Plasma group along with a decrease of 23% in surface-adsorbed C content. At 1 week no difference was found in histometric parameters between groups. At 3 weeks significantly higher BIC (>300%) and mean BAFO (>30%) were observed for Ti-Plasma treated surfaces. From a morphologic standpoint, improved interaction between connective tissue was observed at 1 week, likely leading to more uniform and higher bone formation at 3 weeks for the Ti-Plasma treated implants was observed.

Assessment of a chair-side argon-based non-thermal plasma treatment on the surface characteristics and integration of dental implants with textured surfaces.

The biomechanical effects of a non-thermal plasma (NTP) treatment, suitable for use in a dental office, on the surface character and integration of a textured dental implant surface in a beagle dog model were evaluated. The experiment compared a control treatment, which presented an alumina-blasted/acid-etched (AB/AE) surface, to two experimental treatments, in which the same AB/AE surface also received NTP treatment for a period of 20 or 60 s per implant quadrant (PLASMA 20' and PLASMA 60' groups, respectively). The surface of each specimen was characterized by electron microscopy and optical interferometry, and surface energy and surface chemistry were determined prior to and after plasma treatment. Two implants of each type were then placed at six bilateral locations in 6 dogs, and allowed to heal for 2 or 4 weeks. Following sacrifice, removal torque was evaluated as a function of animal, implant surface and time in vivo in a mixed model ANOVA. Compared to the CONTROL group, PLASMA 20' and 60' groups presented substantially higher surface energy levels, lower amounts of adsorbed C species and significantly higher torque levels (p=.001). Result indicated that the NTP treatment increased the surface energy and the biomechanical fixation of textured-surface dental implants at early times in vivo.

The effect of different implant macrogeometries and surface treatment in early biomechanical fixation: an experimental study in dogs.

Implant surface characterization and biomechanical testing were made to evaluate the effect of different surface treatments along with different implant bulk configurations expressed as biomechanical fixation at early implantation times. Three implant surfaces, namely bioactive ceramic electrodeposition (ED), alumina-blasted/acid etched (AB/AE), and resorbable blasting media (RBM) were fabricated in three implant macrogeometries (cylindrical, small chamber, and large chamber). All combinations between surface and bulk configurations were placed in the radii of beagle dogs (n=18), which were euthanized 14 and 40 days after surgery (n=9 animals per time in vivo). The implants were subjected to torque to interface fracture. Effects of time, surface, and macrogeometry on torque to interface fracture were evaluated by a GLM at 95% level of significance. The results showed a significant increase in torque as time elapsed in vivo (p<0.001), and that the ED surface presented significantly higher values compared to AB/AE and RBM (p<0.001) at both times. The small chamber only presented a significantly higher biomechanical fixation compared to other geometries at 40 days in vivo (p=0.02). Biomechanical fixation at 14 and 40 days was affected by implant surface treatment, whereas implant design only affected results at 40 days in vivo.