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Adhesive with Calcium Phosphate and its Phase TransformationObjectives: To study the process of calcium phase transition in the calcium phase adhesive and the interaction between organic and inorganic phases of calcium phosphate adhesive were studied.Materials and Methods: 1.In ethanol solution,calcium phosphate nanoclusters were prepared in which triethylamine(TEA)served as stabilizers,calcium phosphate nanoclusters gel was obtained by centrifugation and re-dispered into the prepared adhesive system by high-speed centrifugation,and then centrifuge again to obtain calcium phosphate adhesive composite.The samples were characterized by scanning electron microscopy(SEM),transmission electron microscopy(TEM),and elemental analysis(EDS mapping).The mineral content in the calcium phosphate adhesive was determined bythermogravimetric analyzer(TGA,at a heating rate of 10 °C min-1 in flowing air).2.64 calcium phosphate adhesive in sized of 1 * 1cm films with a thickness of about 0.1mm were prepared and light-cured,afterthat soaked into 10 m L artificial saliva,and incubetd in a 37? incubator for 1,2,4,7 days before analysid.They were characterized by attenuated total reflection Fourier infrared spectroscopy(ATR-FTIR),and X-ray diffraction(XRD)analysis.FTIR spectroscopy was used to calculate the split function(SF)to evaluate the degree of crystallization of calcium phosphate.The morphologies were determined by transmission eletron micoscopy(TEM).3.0.01 g of uncured calcium phosphate adhesive were placed at the bottom of a corning costar,added with 1m L of artificial saliva,and incubated in a 37? incubator.10?L supernatant of samples were applied for cryo-electron microscope observation at 0,24 h,48h after sonication for 10 min.The solution was dissolved in 100 ?L of absolute ethanol,dropped on a copper mesh,vitrified at the temperature of liquid nitrogen,and the morphologies of the particles in the uncured adhesive was observed by a cryo-electron microscope eqquiped with a selected area electron diffraction(SAED)detector.4.Hydroxyapatite(HAp)nano-rods with morphology similar to enamel rods were prepared by hydrothermal method,mixing calcium phosphate adhesive with HAp nano-rods,high-speed centrifugation and dropped onto a marked transmission electron microscopy grid,and then photo-curing,placing in a 37?100% humidity incubator,without direct contacting with the solution.After 1,2,4,7,28 days,the position of a rod of HAp were recorded and the same position were examined by using a high-resolution TEM(HRTEM)with Fourier transform FFT analyzes.5.The interaction between the monomer molecules and the calcium phosphate particles was analyzed by FTIR spectroscopy.Results:1.Calcium phosphate adhesive film was a uniform,continuous and homogenous solid structure,and the inorganic content was about 16%.The scanning electron microscope showed that the calcium phosphate adhesive continuous,and the particle size was about 20 nm.The EDS mapping results showed that the C,O,Ca,and P elements were homogenously distributed in the calcium phosphate adhesive.The TEM electron microscope results showed the calcium phosphate particles were in size of about 10-20 nm,continuous distributed in the adhesive.2.The FTIR results showed that the phosphate stretching peak(?3)of 570 cm-1 gradually splited into two peaks of 560 cm-1 and 600 cm-1 after 1-7 days incubation in artificial saliva.The splitting index SF develops approximately "S",indicating that the phase change in 0-7 days first increased after one day of incubation.then became slower,suggesting the phase transition of the crystal was gradually completed.XRD resluts showed that the calcium phosphate adhesive showed one amorphous peak around 20°,and showed characteristic peaks of HAp 2? = 25.8°(002),31.5°(211),and 34°(202)within 2-7 days.TEM results showed that the instant calcium phosphate molecules were spherical and turn into dense needle-like crystal structure at 7 days.These two morphologies could be observed at a same sample without obvious boundaries.3.cryo-TEM showed that when the uncured calcium phosphate adhesive was cultured in artificial saliva,needle-like areas appeared on the spherical particles after 24 hours,and the spherical contours disappeared the crystals turned into complete short rods after 48 hours,SAED results showed the HAp typical 002,211 and 004 rings.4.High-resolution displayed the calcium pho composite was aged at 37 °C for a maximum of 4 weeks in a humid chamber after which the ACP particles totally transformed into apatite.Lattice fringe FFT results showed that the interplanar spacing was 0.34 nm and 0.28 nm,suggesting hydroxyapatite crystals.5.After calcium phosphate was added to the adhesive,the characteristic peak of P-O stretching vibration locates at 1050 cm-1 shifted to 1015 cm-1,implying the formation of N-H...O network between adhesive monomer and calcium phosphate particles.It plays a role in stabilizing calcium phosphate transformation.Conclusions: The calcium phosphate adhesive can be adsorbed on the HAp rod,and the phase transition occurs through the first generation of crystalline domains on the surface to the total crystalization.The monomer in the adhesive can slow down the phase transition of calcium phosphate through the interaction of side chains with calcium phosphate and the formation of network-limiting effects after curing.After the calcium phosphate adhesive adheres to the enamel surface,the crystals obtained by phase transition were hierarchical strcuture similar to the enamel crystals,which is expected to be developed as an enamel repair material.Part ? The Restoration of Enamel Using Calcium Phosphate AdhesiveObjectives: This study was to investigate the remineralization effect of calcium phosphate adhesive on enamel lesion.Materials and methods: 1.calcium phosphate adhesive were prepared as the same in Part1.2.Tweleve human third molars teeth were collected.The white lesions on enamel disks(4mm × 4 mm × 1 mm)were created using a lactic acid layer(0.1M.p H=4.6)gelfor 21 days at 37?.The artificial demineralization samples were characterized by digital photo camera,scanning electron microscope(SEM)and elemental surface scanning(EDS mapping)after 0.1M HCl erosion for 10 S.Then 12 enamel disks were randomly divided into the following two groups(n=6):(1)Control group: white lesion enamel without treatment were incubated in the artifiacl salivasolution.(2)The prepared sample was put into artificial saliva(10 ml / piece)and placed in a 37 ° C water bath incubator,and the mineralization solution was changed every day.After 28 days,the samples were taken out and analyzed by transmission electron microscopy(TEM),selected area electron diffraction(SAED),and elemental analysis(EDS mapping).3 Nano-indentation test: The enamel flakes treated in accordance with the above method were evaluated for the mechanical strength of natural enamel,white enamel,and repaired enamel using a nanoindentation.Results: The SEM results showed that after 28 days of gel demineralization,a chalky demineralized layer was formed on the enamel surface.A demineralized layer with a thickness of about 70 ?m was observed.Elemental mapping results showed the absence of calcium and phosphorus elements in the interrod substance.The calcium phosphate adhesive applied on the enamel formed a dense calcium phosphate adhesive layer,in which the calcium phosphate particles are spherical,about 20-50 nm in size,and have a strong affinity with the enamel crystals.TEM and SEM results showed that the morphology of ACP particles was spherical and non-crystallined.After 28 days of mineralization,TEM graphs showed a dense crystal-hybrid layer on the enamel surface,corresponding SAED results showing 002,211,and 004 diffraction rings,inditcating the exit of hydroxyapatite crystal.Elemental mapping showed that Ca and P elementsare abundent and homogeneously distributed in the crystal-adhesive hybrid.The HRTEM image shows the interplanar spacing of the particles were 0.34 nm and 0.28 nm,consistent with the HAp lattice planes of 002 and 211,suggesting that the particles were hydroxyapatite crystals.The results of the nanoindentation test are: the hardness of natural enamel was 2.230 ± 0.03 Gpa and the elastic modulus was 58.10 ± 0.69 GPa;those of white enamel and restored enamel was 0.331 ± 0.01 Gpa,5.87 ± 1.1 GPa;1.483 ± 0.04 Gpa,and 28.588 ± 0.65 Gpa respectively.Conclusion: The calcium phosphate adhesive forms a dense crystalline adhesive layer on the enamel surface,and the repair layer improves the hardness and modulus of the enamel white spots.Part ? Mineralizing Adhesive to Seal and Mineralize Dentin,Dentinal Tubules and Odontoblast ProcessesObjectives: This study proposed the therapeutic management of demineralized dentin surfaces using a mineralizing adhesive to seal and remineralize dentin,dentinal tubules and odontoblast processes,as well as its in vitro cytotoxicity.Materials and Methods: 1.An experimental self-etch adhesive and a mineralizing adhesive consisting of the adhesive and 20 wt% of poly-aspartic acid-stabilized amorphous calcium phosphate(PAsp-ACP)nanoparticles were prepared,which were characterized by X-ray diffraction(XRD),Fourier transform infrared spectroscopy(FTIR),transmissionelectron microscopy(TEM)and scanning electron microscopy(SEM)2.After 60 acid-etched mid-coronal dentin discs were treated with distilled water(control),a desensitizing agent(Gluma),the experimental self-etch adhesive and the mineralizing adhesive,halve of the samples(n=8)were subjected to an additional abrasive and acidic challenges once a week: the dentin surfaces were gently brushed for 20 strokes for 1 min using a soft bristle toothbrush with approximately 20 wt% of a toothpaste slurry and then thoroughly rinsed with deionized water.Subsequently,the specimens were immersed in 6 wt % of a citric acid solution(p H=1.5)for 1 min and thoroughly rinsed with deionized water.Dentin permeability was measured and their mineralization was evaluated by Raman,FTIR,XRD,TEM,and selected area electron diffraction(SAED),irrespective of abrasive and acidic challenges.3.Human dental pulp stem cells(h DPSCs)were employed to evaluate the cytotoxicity of the experimental adhesive and the mineralizing adhesive.The experimental adhesive and the mineralizing adhesive were light-cured respectively,before they were supplemented with 100 m L /1g of 10% fetal bovine serum(FBS,Gibco,USA)and incubated at 37 °C for 24 hrs to obtain leach solutions.100 ?L of one of concentrations of the adhesive extracts or the mineralizing adhesive extracts(0,10,100,1000,2000 ?g/m L)was added into a 24-well plate,each containing 500 ?L of cell suspension with a density of 5 × 104 cells/well.the resin volume divided by the total solution volume for the three solutions was 0.12,0.24,and 0.47 mm3/m L,respectively.After 1,3,and 7 days of incubation,Cell Counting Kit-8(Dojindo Laboratories)was employed to determine the cell proliferation rate using a microplate reader(Bio Tek Eon)at 450 nm,which expresses as a relative cell viability(%)compared with that of the control group(0 mm3/m L).HDPSCs were cultured in the same way as Cytotoxicity Test for 14 days.von Kossa staining was performed to examine the calcium deposition by cells and the results of the von Kossa staining were imaged by a camera attached to a phase?contrast microscope.Results: 1.The TEM graphs and element mapping of the mineralizing adhesive reveals that the Ca and P elements are homogeneously distributed in the mineralizing adhesive.Both FTIR and XRD spectra suggest that the ACP nanoparticles still remained in an amorphous state.2.The dentine permeability was calculated(Lp)and data were analyzed by Kruskal-Wallis test followed by post-hoc pairwise comparisons with Bonferroni correction(? = 5%).The permeability was significantly reduced in both Group E and A compared with Group C and G(p<0.05),but no difference between Group A and E was found(p>0.05).In abrasion and acid challenge subgroups,Group A showed a significantly reduction than other three groups(p<0.05).Both Group E and A can form resin tags leading to dentinal tubule occlusion,as detected by SEM.NACP-loaded adhesives formed a mineral-rich layer over the surface of dentin and exhibited the highest abrasion and acid-resistant stability among four groups.TEM and SEM graphs reveals that the application of self-etch mineralizing adhesives resulted in efficient dentinal tubule occlusion,abrasion and acid-resistant stability.3.Raman spectra and mapping reveal that the dentin surfaces treated with the mineralizing adhesive(Group D)exhibit the highest ratio of the mineral/matrix(961 cm-1/1660 cm-1)among the all groups,regardless of abrasive and acidic challenges.XRD patterns reveal that mineralizing adhesive(Group D)exhibit the highest degree of crystallinity.TEM results reveals the remineralization in peri-and inter-tubular dentin as well as odontoblast process.4.The neat experimental adhesive and the mineralizing adhesive demonstrated excellent cell viability even at a high concentration(0.47 mm3/m L).After 14 days of h DPSC culture,a higher level of calcium deposition was observed in Group Dcompared to the other groups after von Kossa staining.Conclusion: The mineralizing adhesive composed of a self-etch adhesive containing PAsp-ACP nanoparticles could produce a hybrid mineralization with good biocompability,which consists of the light-cured mineralizing adhesive layer,the remineralized dentin and odontoblast processes,as well as the interiorly-mineralized resin tags.The mineralizing adhesive not only significantly reduced the dentin permeability but also to some extent resisted the abrasive and acidic challenges.These results demonstrate that the mineralizing adhesive can be used to seal and mineralize dentin surfaces,dentinal tubules and odontoblast processes for the therapeutic management of demineralized dentin surfaces.Part ? a Pilot Study to learn the remineralization of dentin bonding interface with self-etch adhesiveObjectives: This study was in aim of providing methodology guidance to learn the remineralization of bonding interface discern from incomplete demineralization using a collagen-grid sandwich model.Materials and methods: 48 non-carious human third molars were collected under written informed consent as well as approved by the Institutional Ethics Committee(#201708).The mid-coronal dentin of each tooth was exposed using a slow-speed saw and polished with 600-grit Si C paper under running water.They were randomly divided into 3 groups according to the different dentin surface treatments(N=16):(1)Control(one-step self-etch approach): The polished dentin surfaces were polishes as abovementionedas not further treated before the application of dentin adhesive;(2)Prime-and-rinse approach: The polished dentin surface was treated with thea experimental novel primer for 15 s and water-sprayed for 30 s before the application of dentin adhesive;(3)Prime-and-nonrinse approach(reverse two-step self-etch approach): The dentin surface was treated with the experimentala novel primer for 15 s and dried with strong air flow for 5 s.During prirme-and-rinse procedure,the rinsed-off liquid was collected and lyophilized.The solid materialsprecipitants were determined by XRD.During adhesive procedure,2 Ni grids with reconstituted collagen fibrils each tooth were sandwiched between adhesive and composition resin.Subsequently,the teeth were incubated in ddw for 28 days.After retrieval,each grid was examined by TEM and with selected area electron diffraction.elements distribution in Collagen was studied by energy dispersive X-ray spectroscopy.Results: XRD patterns reveal typical peaks of assigned to MDP-Ca salt at 2?= 4.84°,peaks of DCPD at 11.8°,and a broad diffraction peak of MDP at around 20°.Contrasting with the collagen embedded in the interface of control groups and prime-and-rinse groups,the collagen in the nonrinse interface exhibited weak mineralization.TEM image provides a typical(002)diffraction ring,despite with extremely low intensity.The characteristic morphology change includes that fibers become thicker with some tiny crystals orderly align in some areas,which are weak intrafibrillar remineralization crystals.The chemical composition of collagen in nonrinse interface in was further confirmed by the element mappings of calcium and phosphate within the collagen fibrils.Conclusion: Residual calcium salt preserved in bonding interface of prime-and-nonrinse can induce intra-filbrillar mineralization.This study also demonstrate that collagen model is a powerful tool for examination of interface remineralization. |
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