| Part I The application of polymer-stabilized "seawater-like" mineralizing precursor medium on biomimetic mineralization of demineralized dentineObjective:To fabricate the mineralizing precursor medium and evaluate its effectiveness on in vitro dentinal tubule occlusion,acid-resistant stability and monitor the dynamic component change of dentine surface,thus assessing its future clinical application on dentine hypersensitivity.Experimental I Mineralization of demineralized dentine via the application of mineralizing precursor mediumMaterials and Methods:Human healthy third molars were obtained and 1.0 ± 0.1 mm thickness dentine slices were sectioned parallel to the occlusal surface of the tooth above the cemento-enamel junction using a low-speed water cooled diamond saw.After consecutively polished by silicon-carbide papers with grit 1000-5000 under constant water irrigation,the samples were partially demineralized in EDTA solution(0.5 M,pH 7.4)for 5 min,thus simulating a sensitive tooth model,followed by ultrasonic cleaning.The supersaturated "seawater-like" mineralizing precursor medium was synthesized via air diffusion method based on a reversible reaction(CaCO3+CO2+H2O(?)Ca(HCO3)2),followed by the addition of poly(acrylic acid)(PAA)and Mg2+.Then the In vitro dentinal tubules occlusion effect and acid-resistant stability on sensitive tooth model were monitored by FE-SEM,dentine permeability measurement and microhardness evaluation at different time points of remineralization and post-treatment with 6%citric acid challenge.Results:The results demonstrated that the application of "seawater-like" mineralizing precursor medium effectively occluded dentinal tubules,reduced dentine permeability,increased surface microhardness and provided certain acid-resistant stability.Experimental II Dynamic component change of dentine surface via the application of mineralizing precursor mediumMaterials and Methods:Human healthy third molars were obtained and 1.0 ± 0.1 mm thickness dentine slices were sectioned parallel to the occlusal surface of the tooth above the cemento-enamel junction using a low-speed water cooled diamond saw.After consecutively polished by silicon-carbide papers with grit 1000-5000 under constant water irrigation,the samples were partially demineralized in EDTA solution(0.5 M,pH 7.4)for 5 min,thus simulating a sensitive tooth model,followed by ultrasonic cleaning.The supersaturated "seawater-like" mineralizing precursor medium was synthesized via air diffusion method based on a reversible reaction(CaCO3+CO2+H2O(?)Ca(HCO3)2),followed by the addition of poly(acrylic acid)(PAA)and Mg2+.Then the dynamic component change on dentine surface were monitored by XRD,ATR-IR,Raman at different time points of remineralization and post-treatment with 6%citric acid challenge.Results:The results verified the presence of ACC as an intermediate phase and that the newly-generated minerals were mainly calcite,which provided certain acid-resistant stability.Meanwhile,the residual phosphate crystals in the demineralized dentine samples also participated in the mineralization process.Part II The exploration of mineralization mechanism of the polymer-stabilized "seawater-like" mineralizing precursor mediumObjective:To establish the single-layer collagen model and evaluate the intrafibrillar mineralization ability of the mineralizing precursor medium,thus offering a clue to uncover its mineralizing mechanism.Experimental III The reconstitution of single-layer collagen modelMaterials and Methods:Lyophilized type I collagen powder was dissolved into 100mM aqueous acetic acid solution with a final concentration of 0.02mg/ml.Subsequently,200-mesh Ni TEM grid was laid on a 50μL drop of above collagen solution and maintained in humidity chamber containing ammonia vapor to adjust the PH around 8,followed by incubated in room temperature for 3d.As a cross-linking solution,0.3M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC)was then dropped on the grids surface and vacuum-dried.After the grids were stained by 1%uranyl acetate for 5min,TEM and SAED observation was applied to confirm the successful self-assembly of collagen fibrils onto the grids.Results:The TEM and SAED results confirmed the successful self-assembly of collagen fibril with a typical cross striation pattern with alternating dark and light bands,which represents the broad "gap zones" and "overlap zones",respectively.Experimental Ⅳ The intrafibrillar mineralization ability of the mineralizing precursor mediumMaterials and Methods:After the successfully confirmation of the self-assembly of collagen fibrils onto the TEM grids with 1%uranyl acetate.The coated grids without staining were transferred into the mineralizing precursor medium obtained in experimental I and floated upside-down on the top at 37℃.Finally,they were retrieved after 30min,2d and 3d for SEM,TEM and selected area electron diffraction(SAED)analysis to evaluate its intrafibrillar mineralization.Results:The incorporation of mineralizing precursor medium displayed effectively intrafibrillar mineralization ability,which carried out crystal epitaxial growth within and outside the collagen fibrils simultaneously,forming the calcite polycrystal strand.It could shed some light for the interpretation about the impressive dentinal occlusion effect and acid-resistant stability.Part Ⅲ In vitro cellular biological response of the polymer-stabilized "seawater-like" mineralizing precursor mediumObjective:To observe the cell morphology,skeleton and proliferation during mineralizing process with the mineralizing precursor mediumExperimental V In vitro cellular biological response of the mineralizing precursor mediumMaterials and Methods:The supersaturated "seawater-like" mineralizing precursor medium was synthesized and sterile filtered.Then they were serially diluted for use by a-MEM medium supplemented with 10%fetal bovine serum,1%penicillin and 1%streptomycin sulphate,with volume ratio of 10%,20%and 40%.Meanwhile,the human dental pulp stem cells(HDPSCs)was isolated from healthy pulp tissues of young premolars for orthodontic extraction based on the outgrowth method.The third to five passage cells were utilized to evaluate the In vitro cellular biological response of the mineralizing precursor medium by FE-SEM observation,immunofluorescence assay,Cell Counting Kit-8(CCK-8)assay and BCA(bicinchoninic acid)protein assay.Results:The results illustrated that 10%and 20%Ca2+-Mg2+-PAA group is harmless but rewarding for the cell attachment and growth.Conclusion:1、The application of "seawater-like" mineralizing precursor medium effectively occluded dentinal tubules,reduced dentine permeability,raised surface microhardness,provided certain acid-resistant stability,which is promising for the further application of dentine hypersensitivity.2、The amorphous calcium carbonate(ACC)acted as an intermediate phase during the mineralization process and that the newly-generated minerals were mainly calcite,which provided certain acid-resistant stability.3、The dynamic procedure of hierarchical intrafibrillar nanocrystalline assembly were observed,which offered a clue to uncover its mineralizing mechanism.4、The mineralizing precursor medium in a certain range of concentration possessed favorable in-vitro biocompatibility.5、The "bottom-up" concept for nacre structure duplication has been applied to biomimetic remineralization of human dentine successfully,revealing great potential for supplying dentists a therapeutic regimen to counter dentine hypersensitivity in the future. |
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