Ignite Creation Date:
2024-05-06 @ 11:57 AM
Last Modification Date:
2024-10-26 @ 12:52 PM
Study NCT ID:
NCT03635138
Status:
UNKNOWN
Last Update Posted:
2018-08-17
First Post:
2018-08-06
Brief Title:
Effect of the Incoportation of Copper and Zinc Nanoparticles Into Dental Adhesives
Sponsor:
University of Chile
Organization:
University of Chile
Study Overview
Official Title:
Effect of the Incoportation of Copper and Zinc Nanoparticles Into Dental Adhesives on Their Antimicrobial Properties and Dentinal Matrix Metalloproteinases Activity From Molecular Basis to Clinical Trials
Status:
UNKNOWN
Status Verified Date:
2018-08
Last Known Status:
NOT_YET_RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
1170575
Brief Summary:
The hypothesis is that Addition of copper or zinc nanoparticles to a dental adhesive confers antimicrobial and enzymatic degradation-resistant properties retaining its adhesion mechanical properties and biocompatibility To corroborate this hypothesis two groups of a dental adhesive doped with copper or zinc nanoparticles should be develop with a respective structural characterization by SEM-EDX AFM and FTIR This should be followed by a test of the antimicrobial activity of adhesive and a study of the influence of adhesive nanocomposites on matrix metalloproteases levels andor activity in vitro to determine some concentrations more relevant These would proceed to next stage With the selected adhesive doped concentrations should be evaluate mechanical properties of doped adhesives and assess the biocompatibility by assays in primary cultured gingival fibroblast and cells type odontoblasts Finally once the concentration of either Cu- or Zn-doped adhesives is known these will be evaluated with a clinical design phase in an in vivo model to study antimicrobial properties matrix metaloproteases levels andor activity We will also study biocompatibility of adhesive nanocomposites and mechanical properties to corroborate the in vitro and ex vivo properties determined There are results using copper nanoparticle on biomaterials that corroborates some properties such as antimicrobial activity against various species and copper release All the evidence suggests that at low concentrations of copper nanoparticles there are no significant effects on mechanical properties but with added antibacterial properties on the adhesive
Detailed Description:
General Objective To evaluate the influence of the incorporation of copper or zinc nanoparticles into a dental adhesive on its antimicrobial action and their influence on dentin matrix metalloproteases activity To analyze adhesive mechanical properties and biocompatibility Specifics Objectives
1 To develop and structurally characterize a dental adhesive doped with copper or zinc nanoparticles
11 Preparation of adhesive nanocomposites 12 Structural characterization of adhesive nanocomposites
2 To assess mechanical biochemical and functional effects of adhesive nanocomposites in vitro Assays will be addressed comparing addition of control adhesive or the adhesive doped with either copper or zinc on fresh recent extracted teethex vivo phase 21 To test the antimicrobial activity of adhesive 2 2 To study the influence of adhesive nanocomposites on matrix metalloproteases levels andor activity23 To evaluate mechanical properties of adhesive24 To assess biocompatibility Viability assays in primary cultured gingival fibroblast will be addressed
3 To evaluate in vivo model clinical phase antimicrobial properties matrix metalloproteases and cathepsin B levels andor activity biocompatibility of adhesive nanocomposites in vivo and mechanical properties Premolars indicated for extraction will be restored using either traditional adhesive or the adhesive nanocomposite After a month premolars will be extracted and analyzed as described
31 To determine surface antimicrobial properties of adhesive nanocomposites 32 To assess matrix metalloproteases and cathepsin B levels andor activity in dentin tissue macerated33 To determine biocompatibility Dental pulp will be obtained from extracted premolars and molecular markers of cell viability or cell damage will be addressed of adhesive nanocomposites34 To evaluate mechanical properties of adhesive
METHODS
1 To develop a dental adhesive doped with copper or zinc nanoparticles 11 Preparation of adhesive nanocomposites 1 CuNPadhesive nanocomposites will be prepared using the one-bottle adhesive system Prime Bond 21 Dentsply Caulk Milford DE This acetone-based adhesive contains a mixture of urethane dimethacrylate and acidic monomer dipentaerythritolpentaacrylatemonophosphate To prepare the adhesive nanocomposites appropriate amounts of nanoparticle powder CuNp and ZnNp Sigma Aldrich Santiago Chile will be weighed and added directly to the adhesive bottle under constant magnetic stirring The suspension will then be further dispersed in an ultrasonic bath at 37C for 10 minutes Adhesive nanocomposites will be prepared with CuNP content in the 0015 to 0045 wt range according to previous data from our laboratories The five concentrations of Cu and Zn-doped adhesives will be 00150 00075 and 000038 concentrations with antibacterial active properties based in our previous data Cu 1-2-3 and Zn 1-2-3
12 Structural characterization of adhesive nanocomposites 1 Morphology and composition of the nanocomposite adhesive materials will be characterized by SEM-EDX in backscattered electron mode to produce phase contrast and AFM microscopy The chemical structure of the polymer matrix and residual monomer contents will be analyzed by attenuated total reflection Fourier transform infrared and Raman spectroscopies The residual monomer will be analyzed considering the carbon-to-carbon double bond vibration of the acrylic monomer in the 1684 - 1636 cm-1 range
2 To assess antibacterial mechanical biochemical and functional effects of adhesive nanocomposites in vitro and ex vivo Assays will be addressed comparing addition of regular adhesive or the adhesive doped with either copper or zinc
21 Assaying antimicrobial properties of adhesive nanocomposite 2 211 Antibacterial activity Briefly 10-mm discs 2 mm in thickness will be fabricated using resin composite Filtek Supreme Ultra 3M St Paul MN and either left uncoated control or coated with the adhesive PrimeBond 21 and equivalent experimental adhesive blends of twelve groups and polymerized according to manufacturers recommendations for 10 seconds using an LED light curing unit Radii CalSDIAU at a power density of 1000 mWcm2 Amalgam discs Contour Kerr Orange CA will be fabricated as previously described and used as an additional positive control A total of fourteen study groups with eight specimens per group will be evaluated in this part of the experiment 1 control 2 amalgam metallic material used like positive control and 3 Cu 1 4 Cu 2 5 Cu 36 Zn17 Zn2 and 8 Zn3 To ensure removal of unpolymerized monomer the discs will be immersed in 10 mL sterile deionized water and agitated for 2 hours at 200 rpm and 378C after which the discs will be left to dry at room temperature for at least 24 hours The discs will be sterilized by incubation in 70 ethanol for 10 minutes and then aseptically dried for a minimum of 48 hours The surface of each disc will be inoculated with 100 mL of a defined viable concentration of 1 108 cellsmL anaerobically grown Streptococcus mutans ATCC1 25175 in brain heart infusion BHI 211059 Becton Dickinson and Company Franklin Lakes NJ broth To determine the viable cell concentration for each treatment group in colony forming units per mL CFUml the inoculated discs will be anaerobically after which the total number of recovered CFUs will be determined
22 In vitro and ex vivo evaluation of MMPs activity 1-2 221 MMP activity inhibition assay in vitro Quantitative MMP-2 MMP-8 and MMP-9 activity in vitro will be determined by commercial MMP fluorometric assay kits SensoLyte assay kits AnaSpec San Jose CA USA for screening of the anti-MMP activity Cu and ZnNP doped adhesives at different concentrations and controls with no Cu or ZnNP adhesives will be added to the reaction buffer and the assay will be performed according manufacturers recommendations Upon cleavage into 2 separate fragments by MMP the fluorescence of 5-FAM will be monitored by a fluorescent microplate reader Sinergy TM
222 Determination of MMP activity and levels ex vivo dentin samples with samples obtained from restored teeth with doped adhesives and control Twenty-five n5 extracted caries-free human third molars will be collected after obtaining patients informed consent The teeth will be disinfected in 05 chloramine stored in distilled water and used within six months of extraction A small occlusal preparation with standardized dimensions of 3x3x5 mm will be made by a calibrated operator in the center of the tooth using a cylinder diamond bur 109018 Medium Grit 60730025 Dentsply NY USA and a high-speed hand piece while providing air-water spray After completing the cavity preparation it will be filled with resin composite Filtek Supreme 3M ESPE The experimental adhesives doped with Cu and Zn will be used according manufacturer instructions and the non-doped adhesive will be used as a control on a homologous upper premolar The resin composite Filtek Supreme 3M ESPE will be applied using the incremental technique The enamel will be completely removed with a slow-speed diamond saw Remet Bologna Italy under saline irrigation and the teeth will be sectioned to obtain 1-mm-thick coronal dentin wafers which will be subsequently pulverized into powder with a steel hammer Dentin powder will then be equally divided and demineralized at 4C for 24 hrs under stirring in one of the following water solutions 1 087 M acetic acid pH 23 2 026 M citric acid pH 23 or 3 05 M EDTA pH 64 or 4 05 M EGTA pH 64 Enzyme extraction of the demineralized dentin powder will be suspended in extraction buffer 50 mM Tris-HCl pH 60 containing 5 mM CaCl2 100 mM NaCl 01 Triton X-100 01 NONIDET P-40 01 mM ZnCl2 002 NaN3 and EDTA-free protease inhibitor cocktail Sigma Chemical St Louis MO USA The samples will then be ultrasonically treated at 40 W output for 3 bursts of 10 secs each at 4C Sonicator Ultrasonic Liquid Processor Model W-385 Heat Systems-Ultrasonic Inc Farmingdale NY USA The vials will be centrifuged at 18000 rpm for 30 min at 4C and the supernatants will be collected All the proteins present in the supernatants will be precipitated at 4C by the addition of powdered ammonium sulfate wv to achieve a final concentration of 85 and pH 70 The precipitate will be collected by centrifugation at 24000 rpm for 30 min at 4C redissolved in a 10-fold dilution in extraction buffer dialyzed through a 30-kDa membrane against extraction buffer overnight and stored at 4C until analyzed After this the dentin powder will be analyzed to assess MMP-2 MMP-9 and cathepsin B activity levels measured by gelatin zymography and to assess MMP-8 by collagen zymography as previously described56 Densitometry will be expressed as du Gel Logic 2200 pro Imaging System Carestream Health USA Briefly dentin powder samples will be subjected to electrophoresis under non-reducing denaturing conditions in SDSPAGE gels containing 1 mgmL of gelatin or 01 mgmL of collagen as substrates They will then be soaked twice in 25 Triton X-100 and incubated for 17 h in developing buffer 20 mM Tris pH 74 and 5 mM CaCl2 stained with Coomassie Brilliant Blue R-250 and destained with 10 acetic acid and 20 methanol solution Levels of MMP-2 MMP-8 MMP-9 cathepsin B and ICTP as a marker of dentin collagen hydrolysis will be determined either by a commercial ELISA Kit or multiplex immunoassays RD Systems Minneapolis MN USA for Luminex technology according to the manufacturers recommendations
23 Mechanical properties testing of adhesive nanocomposites 1-2 Bond strength 231 Tooth selection and preparation Seventy extracted caries-free human third molars will be collected after obtaining patients informed consents of dental clinic of the University of Chile 2000 third molar extractions per year ensuring the availability of these teeth that are normally discarded The teeth will be disinfected in 05 chloramine stored in distilled water and used within six months of extraction A flat dentine surface will be exposed after wet-grinding the occlusal enamel with a 180 SiC paper The exposed dentine surfaces will be further polished with a wet 600 SiC paper for 60 seconds to standardize the smear layer
232 Experimental design The teeth will be randomly assigned into twelve groups of five 6 groups per adhesive each of which will be treated with a different concentration of doped adhesive As control without NPs of system Prime Bond 21 Dentsply Caulk Milford DE will be used
233 Restorative procedure and specimen preparation The adhesive systems will be applied strictly in accordance with the respective manufacturers instructions
234 After bonding procedures All teeth will receive a micro hybrid composite restoration in two increments of 2 mm Each increment will be light polymerized for 40 seconds using an LED light curing unit at the 1200 mWcm2 setting Radii-cal SDI Limited Bayswater Victoria Australia Restored teeth will be stored in distilled water at 37C for 24 hours and then sectioned longitudinally in the mesio-distal and buccal-lingual directions across the bonded interface using a slow-speed diamond saw Isomet Buehler Ltd Lake Bluff IL to obtain 15 to 20 resin-dentine sticks each with a cross sectional area of approximately 08 mm2 when measured with a digital caliper Digimatic Caliper Mitutoyo Tokyo JapanEach specimen from each tooth will be evaluated for microtensile bond strength mTBS except for six randomly-selected resin-dentine bonded specimens which will be divided for measurement of the in situ degree of conversion DC and nanoleakage NL
235 Microtensile bond strength test Resin-dentine bonded sticks will be attached to a Geraldelis jig using cyanoacrylate adhesive and tested under tension Kratos Dinamometros Cotia SP Brazil at 05 mmmin until failure The mTBS values will be calculated by dividing the load at failure by the cross-sectional bonding area The failure mode of the specimens will be classified as cohesive failure exclusively within the dentine or resin composite adhesive failure at the resindentine interface or mixed failure at the resindentine interface including cohesive failure of the neighboring substrates Classification will be performed under a stereomicroscope SZ40 Olympus Tokyo Japan at 100x magnification Specimens with premature failures PFs will be included in the mean
236 Degree of conversion in situThree resin-dentine bonded sticks from each tooth will be wet-polished with 1500 2000 and 2500 SiC paper They will then be ultrasonically cleaned for 20 minutes and stored in water at 378C for 24 hours before measuring DC Micro-Raman spectroscopy analysis will be performed using Senterra equipment Bruker Optik GmbH Ettlingen Baden-Wurttemberg Germany Calibration of the micro-Raman spectrometer will be performed by resetting it to zero then measuring the coefficient of a silicon specimen Specimens will be analyzed using the following micro-Raman parameters 20 mW Neon laser at 532 nm spatial resolution at 3 mm spectral resolution at 5 cm-1 accumulation time at 30 seconds with 6 co-additions and magnification at 110x Olympus UK London UK to a 1-mm beam diameter Spectra will be taken at the dentine-adhesive interface at three different sites within the intertubular dentine for each resin-dentine bonded stick Spectra of unpolymerized adhesives will be used as references Post-processing of spectra will be performed using the dedicated Opus Spectroscopy Software version 65 The ratio of double-bond content of monomer to polymer in the adhesive will be calculated where R is the ratio of aliphatic and aromatic peak areas at 1639 cm-1 and 1609 cm-1 in polymerized and unpolymerized adhesives The in situ DC of all resin-dentine bonded sticks from the same tooth will be averaged for statistical purposes
237 Nanoleakage evaluation Three resin-bonded sticks from each tooth will be placed in ammoniacal silver nitrate prepared according to the protocol described by Tay et al57 and stored in darkness for 24 hours They will then be rinsed thoroughly in distilled water and immersed in photo developing solution for 8 hours under a fluorescent light to reduce silver ions to metallic silver grains within the voids along the bonded interface Specimens will be wet-polished with a 600 1000 1200 1500 2000 and 2500 SiC paper and with 1- and 025-mm diamond paste Buehler Ltd Lake Bluff IL using a polishing cloth They will be ultrasonically cleaned air dried mounted on stubs and coated with carbon-gold MED 010 Balzers Union Balzers Liechtenstein Resin-dentine interfaces will be analyzed in a field-emission scanning electron microscope operated in the backscattered mode LEO 435 VP LEO Electron Microscopy Ltd Cambridge UK Three images will be captured of each resin-dentine bonded stick The relative percentage of NL within the adhesive and hybrid layers in each specimen will be measured in all images using the Image J software58 by a blinded researcher Values from the same specimen will be averaged and the mean NL of all sticks from the same tooth will be average
24 Assess biocompatibility viability assays in primary cultured gingival fibroblast 241 Cell cultures Primary cultures of human fibroblasts will be obtained by the explant method from the retromolar tissue of third molars of three male adult individuals
Saos-2 human osteosarcoma cell line commonly used to assess biocompatibility between osteoblast and biomaterials will be cultured as described by Alcaide et al60
Cells will be seeded in a 96-well plate at a density of 3000 cells per well After 24h control culture medium will be replaced by 100 µl of different dilutions of the proper adhesive 10 01 1 001 0The plates will be incubated for 24h 48h and 72h in a 37C incubator 5CO2 previous to viability or cell death assays
242 Cell viability and cell cycle analysis Primary cultures of human fibroblasts will be obtained by the explant method from the retromolar tissue of third molars of three male adult individuals
Saos-2 human osteosarcoma cell line commonly used to assess biocompatibility between osteoblast and biomaterials will be cultured as described by Alcaide et al60
Cells will be seeded in a 96-well plate at a density of 3000 cells per well After 24h control culture medium will be replaced by 100 µl of different dilutions of the proper adhesive 10 01 1 001 0The plates will be incubated for 24h 48h and 72h in a 37C incubator 5CO2 previous to viability or cell death assays
243 Quantitation of viable early apoptotic late apoptotic and necrotic cells by fluorescence microscopy Protocol described by Kasibhatla et al will be used 61 Cells will be stained with an Acridine OrangeEthidium Bromide solution AOEB and will be examined by fluorescence microscopy AO will stain both live and dead cells EB will stain only cells that have lost membrane integrity Live cells will appear uniformly green Early apoptotic cells will stain green and contain bright green dots in the nuclei as a consequence of chromatin condensation and nuclear fragmentation Late apoptotic cells will also incorporate ethidium bromide and therefore stain orange but in contrast to necrotic cells the late apoptotic cells will show condensed and often fragmented nuclei Necrotic cells stain orange but have a nuclear morphology resembling that of viable cells with no condensed chromatin61
244 Detection of apoptosis by phosphatidylserine exposition DNA laddering and apoptotic mRNA and proteins quantitation The exposition of phosphatidylserine on the outside surface of the plasma membrane will be analyzed as early signal of apoptosis by staining with annexin V A5- allophycocyanin APC as described in Alcaide et al60 Fluorescence of the probe APC will be analyzed by flow cytometry
The apoptotic DNA ladder assay will be carried out using a DNA ladder Kit Roche Germany
mRNA and protein levels for tumor suppressor p53 pro-apoptotic bax caspase 3 and anti-apoptotic bcl-2 mediators will be addressed using quantitative PCR and immunoblot
245 Evaluation of autophagy To test putative effects of metal NPs on cell autophagy different autophagy markers will be addressed Autophagic flux and p62 level will be detected by western blot and autophagosomes will be visualized by fluorescence microscopy by counting LC3 positive puncta63
3 Evaluation of metalloprotease levels andor activity and biocompatibility of adhesive nanocomposites in vivo
Premolars indicated for extraction will be restored using either traditional adhesive or the adhesive nanocomposite After a month recall by telephonic contact and following the clinic examination the patients will be examined and premolars will be extracted and analyzed by blinds evaluators codified by numbers at all stages as described Extractions of premolars will be conducted by teachers of dentistry any effect post-procedure will be assumed and solved by the research team procedures for patients are free of charge and everything will be strictly adhered to the recommendations of the ethics committee of the faculty of dentistry of the university of Chile
31 In vivo evaluation of adhesive nanocomposites Seventy volunteers who require extraction of the upper right and left first premolars n 35 per group for orthodontic reasons will be selected from the Orthodontic Specialization Program in the clinic of the University of Chile there will be a recruitment period of 3 months with a co-investigator responsible for monitoring CB Inclusion criteria will be 1 treatment plans indicating premolar extractions for orthodontic reasons 2 presence of healthy intact non-carious non-restored and fully erupted treatment teeth and 3 patients having well-controlled health conditions that allow all procedures to be performed in the research with minimal risk Exclusion criteria will be 1 patients who do not agree to volunteer for the study and 2 patients not meeting all of the inclusion criteriaA small occlusal preparation will made by a calibrated operator in the center of the tooth using a cylinder diamond bur 63109018Medium Grit 60730025 DentsplyNYUSA and a high-speed hand piece while providing air-water spray with standardized dimensions of 3x3x5 mm After completing the cavity preparation it will be filled with resin composite Filtek Supreme 3M ESPE The experimental adhesives doped with Cu and Zn will be used according manufacturer instructions and the non-doped adhesive will be used as a control on the homologous upper premolar and design by simple randomization of parallel groups The resin composite Filtek Supreme 3M ESPE will be applied using the incremental technique and finally one coat of adhesive will be applied as a surface of composite restoration and polymerized for 10 sec The occlusion will be checked and the restorations will be finished and polished following manufacturer instructions Subsequently polishing and final finishing will used to apply a layer of adhesive on the final restoration which will be polymerized for 10 seconds Raddi Cal SDI AU One tooth at a time will be extracted one month after the restorations are made The patient will receive infiltrative and intraligamental anesthesia approximately 18 mL 2 Mepivacaine 36 mg with epinephrine at a ratio of 1100000 18 mg Then the teeth will be subjected to tests
32 Determine surface antimicrobial properties of adhesive nanocomposites To assess the antibacterial surface properties of doped adhesives one final layer of adhesives will be applied over the final surface of restorations in a in vivo model The procedure will be used to assess the number of CFU of SMutans on composite surfaces Main Outcome Prior to sampling the biofilm trays will be prepared to print the biofilm over the restorations For this application we will use disposable fluoride gel application trays Deepak Products Inc Miami USA each of which will be sterilized in a biosafety hood Esco Technologies Inc Harboro USA under ultraviolet light for 30 minutes Each tray will then be charged with 75 ml of TYCSB agar Casein yeast extract L-cysteine sucrose bacitracin Difco Laboratories Inc Detroit MI USA which is a selective medium for S mutans Subsequently to individualize the sample collection technique the trays will be cut to fit no more than 3 teeth Immediately after this the trays will be placed in sterile petri plates and stored in sealed plastic bags in a refrigerator Before use the trays will be placed in an incubatorstove ZDP-A2080 LabTech Co Namyangiu Korea for 24 h at 37 C as a quality control measure The third and fourth operators will perform the sampling which will be conducted between 1100-1300 to allow for biofilm reorganization following morning brushing The sampling will be performed by a blind operator gently pressing the tray for 20 s over the RC restoration followed by homologous RC restoration with and without Cu or Zn-doped adhesive
After the sample trays are stored in sterile petri plates they will be transported at 4 C and then incubated at 37 C in a microaerophilic jar candle CO2 10 for 48 h The count of Smutans will be performed by the fourth operator who will be blinded to the tray was used to print the RC restorations Later Gram staining will be performed to determine the micromorphology of the colonies The Smutans count will be expressed in colony forming units CFU of Smutans from the plates and trays with the TYCSB agar Select colonies that will be compatible with Smutans adhesion and morphological characteristics will be suspended in Todd-Hewitt broth Difco Laboratories Inc Detroit MI USA and incubated at 37 C for 48 h The colonies will then be subjected to biochemical tests to identify the species of S mutans and to distinguish it from S sobrinus The biochemical tests include raffinose fermentation melibiose fermentation and esculin hydrolysis tests A positive result for all three tests indicates the presence of S mutans
33 Assessment of metalloprotease levels andor activity in macerated dentin tissue will use a similar methodology to that in 242 331 Zymography for in situ to assess activities of MMPs 2 and 9 outcomeA method described by Mazzoni et al65 will be used on 20 n5 per group freshly extracted non-carious human upper premolars restored by a previous procedure After removal of the enamel and cementum 1-mm-thick disks of middledeep coronal dentin will be obtained A 10-mgmL stock solution of fluorescein-labeled gelatin was prepared by adding 10 mL of water to the vials containing the lyophilized substrate and storing at -20C until use The gelatin stock solution is diluted 18 with the dilution buffer NaCl 150 mM CaCl2 5 mM Tris-HCl 50 mM pH 80 and an anti-fading agent is added Mounting Medium with Dapi H-1200 Vectashield Vector Laboratories LTD Cambridgeshire UK A 50-μL quantity of the fluorescent gelatin mixture is placed on top of each slab and covered with a coverslip Slides are light-protected and incubated in humidified chambers at 37C For identification of the optimum incubation period fluorescent images are taken from 1 hr to 7 days Detailed description of the 3D analysis of in situ zymography with confocal microscopy Briefly hydrolysis of quenched fluorescein-conjugated gelatin substrate which is indicative of endogenous gelatinolytic enzyme activity was assessed by examination under a multi-photon confocal microscope ex 488nm and em lp530nm Zeiss LSM 780 Carl Zeiss Oberkochen Germany Optical sections of 85-μm-thick were acquired from different focal planes and the stacked images were analyzed quantified and processed with ZEN 2010 software Carl Zeiss
34 Determination of biocompatibility Dental pulp will be obtained from extracted premolars and molecular markers of cell viability outcomes or cell damage by adhesive nanocomposites will be addressed
Dental pulp will be obtained as described by Vaseemuddin66 from extracted premolars Total RNA will be separated and mRNA levels of specific apoptosis and autophagy markers will be measured by qPCR as previously described 244-245
35 Assessment of mechanical properties nanoleakage and degree conversion analysis to assess the bond stabilities outcome will be equal to methods in 21
4 Statistical analysis Statistical analysis will be performed using SPSS 230 software IBM New York NY USA Comparisons between quantitative variables microtensile bond strength nanoleakage degree of conversion count of CFU between two independent groups will be analyzed by unpaired t test ANOVA and pots-hoc test or respective non-parametric tests according to Shapiro-Wilk testing of data distribution Associations between sample determinations will be calculated by Pearsons or Spearmans correlation A power calculation was carried out using data from a previous study67 A sample size with a minimum of 35 patients per group and per each arm of study was needed to find a difference with statistical significance in a CFU final count main outcome considering a statistical power of 08 based to Vildosola et a study64 1-β Significance will be considered if the p value is 005 All statistical calculations and their respective powers be corroborated post-hoc according to the data to determine if it was necessary to increase the sample size in order to improve the statistical power of each comparison especially for comparison of data obtained from the analysis MMPs activity considered our primary outcome because in literature there are insufficient data to make a sample calculation a priori
1 To develop and structurally characterize a dental adhesive doped with copper or zinc nanoparticles
11 Preparation of adhesive nanocomposites 12 Structural characterization of adhesive nanocomposites
2 To assess mechanical biochemical and functional effects of adhesive nanocomposites in vitro Assays will be addressed comparing addition of control adhesive or the adhesive doped with either copper or zinc on fresh recent extracted teethex vivo phase 21 To test the antimicrobial activity of adhesive 2 2 To study the influence of adhesive nanocomposites on matrix metalloproteases levels andor activity23 To evaluate mechanical properties of adhesive24 To assess biocompatibility Viability assays in primary cultured gingival fibroblast will be addressed
3 To evaluate in vivo model clinical phase antimicrobial properties matrix metalloproteases and cathepsin B levels andor activity biocompatibility of adhesive nanocomposites in vivo and mechanical properties Premolars indicated for extraction will be restored using either traditional adhesive or the adhesive nanocomposite After a month premolars will be extracted and analyzed as described
31 To determine surface antimicrobial properties of adhesive nanocomposites 32 To assess matrix metalloproteases and cathepsin B levels andor activity in dentin tissue macerated33 To determine biocompatibility Dental pulp will be obtained from extracted premolars and molecular markers of cell viability or cell damage will be addressed of adhesive nanocomposites34 To evaluate mechanical properties of adhesive
METHODS
1 To develop a dental adhesive doped with copper or zinc nanoparticles 11 Preparation of adhesive nanocomposites 1 CuNPadhesive nanocomposites will be prepared using the one-bottle adhesive system Prime Bond 21 Dentsply Caulk Milford DE This acetone-based adhesive contains a mixture of urethane dimethacrylate and acidic monomer dipentaerythritolpentaacrylatemonophosphate To prepare the adhesive nanocomposites appropriate amounts of nanoparticle powder CuNp and ZnNp Sigma Aldrich Santiago Chile will be weighed and added directly to the adhesive bottle under constant magnetic stirring The suspension will then be further dispersed in an ultrasonic bath at 37C for 10 minutes Adhesive nanocomposites will be prepared with CuNP content in the 0015 to 0045 wt range according to previous data from our laboratories The five concentrations of Cu and Zn-doped adhesives will be 00150 00075 and 000038 concentrations with antibacterial active properties based in our previous data Cu 1-2-3 and Zn 1-2-3
12 Structural characterization of adhesive nanocomposites 1 Morphology and composition of the nanocomposite adhesive materials will be characterized by SEM-EDX in backscattered electron mode to produce phase contrast and AFM microscopy The chemical structure of the polymer matrix and residual monomer contents will be analyzed by attenuated total reflection Fourier transform infrared and Raman spectroscopies The residual monomer will be analyzed considering the carbon-to-carbon double bond vibration of the acrylic monomer in the 1684 - 1636 cm-1 range
2 To assess antibacterial mechanical biochemical and functional effects of adhesive nanocomposites in vitro and ex vivo Assays will be addressed comparing addition of regular adhesive or the adhesive doped with either copper or zinc
21 Assaying antimicrobial properties of adhesive nanocomposite 2 211 Antibacterial activity Briefly 10-mm discs 2 mm in thickness will be fabricated using resin composite Filtek Supreme Ultra 3M St Paul MN and either left uncoated control or coated with the adhesive PrimeBond 21 and equivalent experimental adhesive blends of twelve groups and polymerized according to manufacturers recommendations for 10 seconds using an LED light curing unit Radii CalSDIAU at a power density of 1000 mWcm2 Amalgam discs Contour Kerr Orange CA will be fabricated as previously described and used as an additional positive control A total of fourteen study groups with eight specimens per group will be evaluated in this part of the experiment 1 control 2 amalgam metallic material used like positive control and 3 Cu 1 4 Cu 2 5 Cu 36 Zn17 Zn2 and 8 Zn3 To ensure removal of unpolymerized monomer the discs will be immersed in 10 mL sterile deionized water and agitated for 2 hours at 200 rpm and 378C after which the discs will be left to dry at room temperature for at least 24 hours The discs will be sterilized by incubation in 70 ethanol for 10 minutes and then aseptically dried for a minimum of 48 hours The surface of each disc will be inoculated with 100 mL of a defined viable concentration of 1 108 cellsmL anaerobically grown Streptococcus mutans ATCC1 25175 in brain heart infusion BHI 211059 Becton Dickinson and Company Franklin Lakes NJ broth To determine the viable cell concentration for each treatment group in colony forming units per mL CFUml the inoculated discs will be anaerobically after which the total number of recovered CFUs will be determined
22 In vitro and ex vivo evaluation of MMPs activity 1-2 221 MMP activity inhibition assay in vitro Quantitative MMP-2 MMP-8 and MMP-9 activity in vitro will be determined by commercial MMP fluorometric assay kits SensoLyte assay kits AnaSpec San Jose CA USA for screening of the anti-MMP activity Cu and ZnNP doped adhesives at different concentrations and controls with no Cu or ZnNP adhesives will be added to the reaction buffer and the assay will be performed according manufacturers recommendations Upon cleavage into 2 separate fragments by MMP the fluorescence of 5-FAM will be monitored by a fluorescent microplate reader Sinergy TM
222 Determination of MMP activity and levels ex vivo dentin samples with samples obtained from restored teeth with doped adhesives and control Twenty-five n5 extracted caries-free human third molars will be collected after obtaining patients informed consent The teeth will be disinfected in 05 chloramine stored in distilled water and used within six months of extraction A small occlusal preparation with standardized dimensions of 3x3x5 mm will be made by a calibrated operator in the center of the tooth using a cylinder diamond bur 109018 Medium Grit 60730025 Dentsply NY USA and a high-speed hand piece while providing air-water spray After completing the cavity preparation it will be filled with resin composite Filtek Supreme 3M ESPE The experimental adhesives doped with Cu and Zn will be used according manufacturer instructions and the non-doped adhesive will be used as a control on a homologous upper premolar The resin composite Filtek Supreme 3M ESPE will be applied using the incremental technique The enamel will be completely removed with a slow-speed diamond saw Remet Bologna Italy under saline irrigation and the teeth will be sectioned to obtain 1-mm-thick coronal dentin wafers which will be subsequently pulverized into powder with a steel hammer Dentin powder will then be equally divided and demineralized at 4C for 24 hrs under stirring in one of the following water solutions 1 087 M acetic acid pH 23 2 026 M citric acid pH 23 or 3 05 M EDTA pH 64 or 4 05 M EGTA pH 64 Enzyme extraction of the demineralized dentin powder will be suspended in extraction buffer 50 mM Tris-HCl pH 60 containing 5 mM CaCl2 100 mM NaCl 01 Triton X-100 01 NONIDET P-40 01 mM ZnCl2 002 NaN3 and EDTA-free protease inhibitor cocktail Sigma Chemical St Louis MO USA The samples will then be ultrasonically treated at 40 W output for 3 bursts of 10 secs each at 4C Sonicator Ultrasonic Liquid Processor Model W-385 Heat Systems-Ultrasonic Inc Farmingdale NY USA The vials will be centrifuged at 18000 rpm for 30 min at 4C and the supernatants will be collected All the proteins present in the supernatants will be precipitated at 4C by the addition of powdered ammonium sulfate wv to achieve a final concentration of 85 and pH 70 The precipitate will be collected by centrifugation at 24000 rpm for 30 min at 4C redissolved in a 10-fold dilution in extraction buffer dialyzed through a 30-kDa membrane against extraction buffer overnight and stored at 4C until analyzed After this the dentin powder will be analyzed to assess MMP-2 MMP-9 and cathepsin B activity levels measured by gelatin zymography and to assess MMP-8 by collagen zymography as previously described56 Densitometry will be expressed as du Gel Logic 2200 pro Imaging System Carestream Health USA Briefly dentin powder samples will be subjected to electrophoresis under non-reducing denaturing conditions in SDSPAGE gels containing 1 mgmL of gelatin or 01 mgmL of collagen as substrates They will then be soaked twice in 25 Triton X-100 and incubated for 17 h in developing buffer 20 mM Tris pH 74 and 5 mM CaCl2 stained with Coomassie Brilliant Blue R-250 and destained with 10 acetic acid and 20 methanol solution Levels of MMP-2 MMP-8 MMP-9 cathepsin B and ICTP as a marker of dentin collagen hydrolysis will be determined either by a commercial ELISA Kit or multiplex immunoassays RD Systems Minneapolis MN USA for Luminex technology according to the manufacturers recommendations
23 Mechanical properties testing of adhesive nanocomposites 1-2 Bond strength 231 Tooth selection and preparation Seventy extracted caries-free human third molars will be collected after obtaining patients informed consents of dental clinic of the University of Chile 2000 third molar extractions per year ensuring the availability of these teeth that are normally discarded The teeth will be disinfected in 05 chloramine stored in distilled water and used within six months of extraction A flat dentine surface will be exposed after wet-grinding the occlusal enamel with a 180 SiC paper The exposed dentine surfaces will be further polished with a wet 600 SiC paper for 60 seconds to standardize the smear layer
232 Experimental design The teeth will be randomly assigned into twelve groups of five 6 groups per adhesive each of which will be treated with a different concentration of doped adhesive As control without NPs of system Prime Bond 21 Dentsply Caulk Milford DE will be used
233 Restorative procedure and specimen preparation The adhesive systems will be applied strictly in accordance with the respective manufacturers instructions
234 After bonding procedures All teeth will receive a micro hybrid composite restoration in two increments of 2 mm Each increment will be light polymerized for 40 seconds using an LED light curing unit at the 1200 mWcm2 setting Radii-cal SDI Limited Bayswater Victoria Australia Restored teeth will be stored in distilled water at 37C for 24 hours and then sectioned longitudinally in the mesio-distal and buccal-lingual directions across the bonded interface using a slow-speed diamond saw Isomet Buehler Ltd Lake Bluff IL to obtain 15 to 20 resin-dentine sticks each with a cross sectional area of approximately 08 mm2 when measured with a digital caliper Digimatic Caliper Mitutoyo Tokyo JapanEach specimen from each tooth will be evaluated for microtensile bond strength mTBS except for six randomly-selected resin-dentine bonded specimens which will be divided for measurement of the in situ degree of conversion DC and nanoleakage NL
235 Microtensile bond strength test Resin-dentine bonded sticks will be attached to a Geraldelis jig using cyanoacrylate adhesive and tested under tension Kratos Dinamometros Cotia SP Brazil at 05 mmmin until failure The mTBS values will be calculated by dividing the load at failure by the cross-sectional bonding area The failure mode of the specimens will be classified as cohesive failure exclusively within the dentine or resin composite adhesive failure at the resindentine interface or mixed failure at the resindentine interface including cohesive failure of the neighboring substrates Classification will be performed under a stereomicroscope SZ40 Olympus Tokyo Japan at 100x magnification Specimens with premature failures PFs will be included in the mean
236 Degree of conversion in situThree resin-dentine bonded sticks from each tooth will be wet-polished with 1500 2000 and 2500 SiC paper They will then be ultrasonically cleaned for 20 minutes and stored in water at 378C for 24 hours before measuring DC Micro-Raman spectroscopy analysis will be performed using Senterra equipment Bruker Optik GmbH Ettlingen Baden-Wurttemberg Germany Calibration of the micro-Raman spectrometer will be performed by resetting it to zero then measuring the coefficient of a silicon specimen Specimens will be analyzed using the following micro-Raman parameters 20 mW Neon laser at 532 nm spatial resolution at 3 mm spectral resolution at 5 cm-1 accumulation time at 30 seconds with 6 co-additions and magnification at 110x Olympus UK London UK to a 1-mm beam diameter Spectra will be taken at the dentine-adhesive interface at three different sites within the intertubular dentine for each resin-dentine bonded stick Spectra of unpolymerized adhesives will be used as references Post-processing of spectra will be performed using the dedicated Opus Spectroscopy Software version 65 The ratio of double-bond content of monomer to polymer in the adhesive will be calculated where R is the ratio of aliphatic and aromatic peak areas at 1639 cm-1 and 1609 cm-1 in polymerized and unpolymerized adhesives The in situ DC of all resin-dentine bonded sticks from the same tooth will be averaged for statistical purposes
237 Nanoleakage evaluation Three resin-bonded sticks from each tooth will be placed in ammoniacal silver nitrate prepared according to the protocol described by Tay et al57 and stored in darkness for 24 hours They will then be rinsed thoroughly in distilled water and immersed in photo developing solution for 8 hours under a fluorescent light to reduce silver ions to metallic silver grains within the voids along the bonded interface Specimens will be wet-polished with a 600 1000 1200 1500 2000 and 2500 SiC paper and with 1- and 025-mm diamond paste Buehler Ltd Lake Bluff IL using a polishing cloth They will be ultrasonically cleaned air dried mounted on stubs and coated with carbon-gold MED 010 Balzers Union Balzers Liechtenstein Resin-dentine interfaces will be analyzed in a field-emission scanning electron microscope operated in the backscattered mode LEO 435 VP LEO Electron Microscopy Ltd Cambridge UK Three images will be captured of each resin-dentine bonded stick The relative percentage of NL within the adhesive and hybrid layers in each specimen will be measured in all images using the Image J software58 by a blinded researcher Values from the same specimen will be averaged and the mean NL of all sticks from the same tooth will be average
24 Assess biocompatibility viability assays in primary cultured gingival fibroblast 241 Cell cultures Primary cultures of human fibroblasts will be obtained by the explant method from the retromolar tissue of third molars of three male adult individuals
Saos-2 human osteosarcoma cell line commonly used to assess biocompatibility between osteoblast and biomaterials will be cultured as described by Alcaide et al60
Cells will be seeded in a 96-well plate at a density of 3000 cells per well After 24h control culture medium will be replaced by 100 µl of different dilutions of the proper adhesive 10 01 1 001 0The plates will be incubated for 24h 48h and 72h in a 37C incubator 5CO2 previous to viability or cell death assays
242 Cell viability and cell cycle analysis Primary cultures of human fibroblasts will be obtained by the explant method from the retromolar tissue of third molars of three male adult individuals
Saos-2 human osteosarcoma cell line commonly used to assess biocompatibility between osteoblast and biomaterials will be cultured as described by Alcaide et al60
Cells will be seeded in a 96-well plate at a density of 3000 cells per well After 24h control culture medium will be replaced by 100 µl of different dilutions of the proper adhesive 10 01 1 001 0The plates will be incubated for 24h 48h and 72h in a 37C incubator 5CO2 previous to viability or cell death assays
243 Quantitation of viable early apoptotic late apoptotic and necrotic cells by fluorescence microscopy Protocol described by Kasibhatla et al will be used 61 Cells will be stained with an Acridine OrangeEthidium Bromide solution AOEB and will be examined by fluorescence microscopy AO will stain both live and dead cells EB will stain only cells that have lost membrane integrity Live cells will appear uniformly green Early apoptotic cells will stain green and contain bright green dots in the nuclei as a consequence of chromatin condensation and nuclear fragmentation Late apoptotic cells will also incorporate ethidium bromide and therefore stain orange but in contrast to necrotic cells the late apoptotic cells will show condensed and often fragmented nuclei Necrotic cells stain orange but have a nuclear morphology resembling that of viable cells with no condensed chromatin61
244 Detection of apoptosis by phosphatidylserine exposition DNA laddering and apoptotic mRNA and proteins quantitation The exposition of phosphatidylserine on the outside surface of the plasma membrane will be analyzed as early signal of apoptosis by staining with annexin V A5- allophycocyanin APC as described in Alcaide et al60 Fluorescence of the probe APC will be analyzed by flow cytometry
The apoptotic DNA ladder assay will be carried out using a DNA ladder Kit Roche Germany
mRNA and protein levels for tumor suppressor p53 pro-apoptotic bax caspase 3 and anti-apoptotic bcl-2 mediators will be addressed using quantitative PCR and immunoblot
245 Evaluation of autophagy To test putative effects of metal NPs on cell autophagy different autophagy markers will be addressed Autophagic flux and p62 level will be detected by western blot and autophagosomes will be visualized by fluorescence microscopy by counting LC3 positive puncta63
3 Evaluation of metalloprotease levels andor activity and biocompatibility of adhesive nanocomposites in vivo
Premolars indicated for extraction will be restored using either traditional adhesive or the adhesive nanocomposite After a month recall by telephonic contact and following the clinic examination the patients will be examined and premolars will be extracted and analyzed by blinds evaluators codified by numbers at all stages as described Extractions of premolars will be conducted by teachers of dentistry any effect post-procedure will be assumed and solved by the research team procedures for patients are free of charge and everything will be strictly adhered to the recommendations of the ethics committee of the faculty of dentistry of the university of Chile
31 In vivo evaluation of adhesive nanocomposites Seventy volunteers who require extraction of the upper right and left first premolars n 35 per group for orthodontic reasons will be selected from the Orthodontic Specialization Program in the clinic of the University of Chile there will be a recruitment period of 3 months with a co-investigator responsible for monitoring CB Inclusion criteria will be 1 treatment plans indicating premolar extractions for orthodontic reasons 2 presence of healthy intact non-carious non-restored and fully erupted treatment teeth and 3 patients having well-controlled health conditions that allow all procedures to be performed in the research with minimal risk Exclusion criteria will be 1 patients who do not agree to volunteer for the study and 2 patients not meeting all of the inclusion criteriaA small occlusal preparation will made by a calibrated operator in the center of the tooth using a cylinder diamond bur 63109018Medium Grit 60730025 DentsplyNYUSA and a high-speed hand piece while providing air-water spray with standardized dimensions of 3x3x5 mm After completing the cavity preparation it will be filled with resin composite Filtek Supreme 3M ESPE The experimental adhesives doped with Cu and Zn will be used according manufacturer instructions and the non-doped adhesive will be used as a control on the homologous upper premolar and design by simple randomization of parallel groups The resin composite Filtek Supreme 3M ESPE will be applied using the incremental technique and finally one coat of adhesive will be applied as a surface of composite restoration and polymerized for 10 sec The occlusion will be checked and the restorations will be finished and polished following manufacturer instructions Subsequently polishing and final finishing will used to apply a layer of adhesive on the final restoration which will be polymerized for 10 seconds Raddi Cal SDI AU One tooth at a time will be extracted one month after the restorations are made The patient will receive infiltrative and intraligamental anesthesia approximately 18 mL 2 Mepivacaine 36 mg with epinephrine at a ratio of 1100000 18 mg Then the teeth will be subjected to tests
32 Determine surface antimicrobial properties of adhesive nanocomposites To assess the antibacterial surface properties of doped adhesives one final layer of adhesives will be applied over the final surface of restorations in a in vivo model The procedure will be used to assess the number of CFU of SMutans on composite surfaces Main Outcome Prior to sampling the biofilm trays will be prepared to print the biofilm over the restorations For this application we will use disposable fluoride gel application trays Deepak Products Inc Miami USA each of which will be sterilized in a biosafety hood Esco Technologies Inc Harboro USA under ultraviolet light for 30 minutes Each tray will then be charged with 75 ml of TYCSB agar Casein yeast extract L-cysteine sucrose bacitracin Difco Laboratories Inc Detroit MI USA which is a selective medium for S mutans Subsequently to individualize the sample collection technique the trays will be cut to fit no more than 3 teeth Immediately after this the trays will be placed in sterile petri plates and stored in sealed plastic bags in a refrigerator Before use the trays will be placed in an incubatorstove ZDP-A2080 LabTech Co Namyangiu Korea for 24 h at 37 C as a quality control measure The third and fourth operators will perform the sampling which will be conducted between 1100-1300 to allow for biofilm reorganization following morning brushing The sampling will be performed by a blind operator gently pressing the tray for 20 s over the RC restoration followed by homologous RC restoration with and without Cu or Zn-doped adhesive
After the sample trays are stored in sterile petri plates they will be transported at 4 C and then incubated at 37 C in a microaerophilic jar candle CO2 10 for 48 h The count of Smutans will be performed by the fourth operator who will be blinded to the tray was used to print the RC restorations Later Gram staining will be performed to determine the micromorphology of the colonies The Smutans count will be expressed in colony forming units CFU of Smutans from the plates and trays with the TYCSB agar Select colonies that will be compatible with Smutans adhesion and morphological characteristics will be suspended in Todd-Hewitt broth Difco Laboratories Inc Detroit MI USA and incubated at 37 C for 48 h The colonies will then be subjected to biochemical tests to identify the species of S mutans and to distinguish it from S sobrinus The biochemical tests include raffinose fermentation melibiose fermentation and esculin hydrolysis tests A positive result for all three tests indicates the presence of S mutans
33 Assessment of metalloprotease levels andor activity in macerated dentin tissue will use a similar methodology to that in 242 331 Zymography for in situ to assess activities of MMPs 2 and 9 outcomeA method described by Mazzoni et al65 will be used on 20 n5 per group freshly extracted non-carious human upper premolars restored by a previous procedure After removal of the enamel and cementum 1-mm-thick disks of middledeep coronal dentin will be obtained A 10-mgmL stock solution of fluorescein-labeled gelatin was prepared by adding 10 mL of water to the vials containing the lyophilized substrate and storing at -20C until use The gelatin stock solution is diluted 18 with the dilution buffer NaCl 150 mM CaCl2 5 mM Tris-HCl 50 mM pH 80 and an anti-fading agent is added Mounting Medium with Dapi H-1200 Vectashield Vector Laboratories LTD Cambridgeshire UK A 50-μL quantity of the fluorescent gelatin mixture is placed on top of each slab and covered with a coverslip Slides are light-protected and incubated in humidified chambers at 37C For identification of the optimum incubation period fluorescent images are taken from 1 hr to 7 days Detailed description of the 3D analysis of in situ zymography with confocal microscopy Briefly hydrolysis of quenched fluorescein-conjugated gelatin substrate which is indicative of endogenous gelatinolytic enzyme activity was assessed by examination under a multi-photon confocal microscope ex 488nm and em lp530nm Zeiss LSM 780 Carl Zeiss Oberkochen Germany Optical sections of 85-μm-thick were acquired from different focal planes and the stacked images were analyzed quantified and processed with ZEN 2010 software Carl Zeiss
34 Determination of biocompatibility Dental pulp will be obtained from extracted premolars and molecular markers of cell viability outcomes or cell damage by adhesive nanocomposites will be addressed
Dental pulp will be obtained as described by Vaseemuddin66 from extracted premolars Total RNA will be separated and mRNA levels of specific apoptosis and autophagy markers will be measured by qPCR as previously described 244-245
35 Assessment of mechanical properties nanoleakage and degree conversion analysis to assess the bond stabilities outcome will be equal to methods in 21
4 Statistical analysis Statistical analysis will be performed using SPSS 230 software IBM New York NY USA Comparisons between quantitative variables microtensile bond strength nanoleakage degree of conversion count of CFU between two independent groups will be analyzed by unpaired t test ANOVA and pots-hoc test or respective non-parametric tests according to Shapiro-Wilk testing of data distribution Associations between sample determinations will be calculated by Pearsons or Spearmans correlation A power calculation was carried out using data from a previous study67 A sample size with a minimum of 35 patients per group and per each arm of study was needed to find a difference with statistical significance in a CFU final count main outcome considering a statistical power of 08 based to Vildosola et a study64 1-β Significance will be considered if the p value is 005 All statistical calculations and their respective powers be corroborated post-hoc according to the data to determine if it was necessary to increase the sample size in order to improve the statistical power of each comparison especially for comparison of data obtained from the analysis MMPs activity considered our primary outcome because in literature there are insufficient data to make a sample calculation a priori
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None