【作者】 田甜；

【导师】 张凌琳；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2021， 博士

【摘要】 龋病是最常见的慢性疾病之一。随着牙釉质龋的发展,龋损沿釉牙本质界向侧方扩散,造成牙本质中的锥形破坏,最终突破牙本质屏障,导致牙髓损伤。目前,牙本质龋的临床常用修复方法是使用复合树脂进行粘接修复,但复合树脂粘接修复存在树脂老化、牙本质胶原酶降解以及边缘微渗漏等一系列问题,导致3年成功率往往不足50%。因此,促进牙本质龋损修复并提高牙本质粘接效果的材料研发十分必要。近年来,再矿化是修复牙本质龋损、改善牙本质粘接性能的研究方向之一。本课题组前期设计合成了釉原蛋白来源的功能多肽QP5,该多肽有效稳定无定型磷酸钙(Amorphous calcium phosphate,ACP)、抑制羟基磷灰石(Hydroxyapatite,HA)成核,促进人工早期牙釉质龋再矿化。多肽类药物在临床应用中存在有效浓度下降快,作用时间短等问题,课题组进一步制备了以壳聚糖为载体的QP5水凝胶并将其应用于促进釉质早期龋的再矿化中。然而,QP5能否对脱矿牙本质同样发挥促矿化作用,其牙本质应用形式如何,均有待进一步实验研究。本研究拟将N,N,N-三甲基壳聚糖(N,N,N-Trimethyl chitosan,TMC)作为载体负载釉原蛋白功能多肽QP5,制备成复合纳米粒,探究釉原蛋白功能多肽-壳聚糖纳米粒(Amelogenin-functional peptide-chitosan nanoparticles,TMC-QP5/NPs)对脱矿牙本质再矿化的作用,以及该再矿化作用对牙本质粘接效果的影响。目的:本研究拟利用离子交联法制备TMC-QP5/NPs复合纳米粒,检测其基本理化性质、载药性能、体外释药性能、稳定性以及细胞毒性,探究TMC-QP5/NPs复合纳米粒对脱矿牙本质的再矿化及该再矿化作用对牙本质粘接性能的影响,为TMC-QP5/NPs复合纳米粒在脱矿牙本质的再矿化修复、牙本质的粘接改良方面的应用奠定基础。方法:1、以TMC为载体,三聚磷酸钠(Sodium tripolyphosphate,TPP)为交联剂,通过离子交联法、优化制备工艺来制备并筛选TMC-QP5/NPs;利用动态光散射法测量其水合粒径、多分散指数和Zeta电位;利用高效液相色谱检测其包封率、载药量,进行体外36小时释药曲线研究。通过扫描电镜、透射电镜和原子力显微镜分析形貌表征;利用傅里叶变换红外光谱分析化学结构;通过差示扫描量热分析评估其热稳定性能;通过对粒径、多分散指数的7天动态监测评估其储存稳定性;通过细胞毒性实验检测TMC-QP5/NPs对人牙髓细胞的毒性。2、构建脱矿牙本质模型,设置TMC-QP5/NPs、TMC、QP5、Na F和2-[4-(羟乙基)-1-哌嗪基]乙磺酸(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,HEPES)五个实验组,进行体外再矿化实验。通过显微硬度恢复率评价再矿化后牙本质表面力学性能变化;通过扫描电镜和原子力显微镜观察再矿化后牙本质表面形貌和粗糙度;利用傅里叶变换红外光谱、X-射线衍射分析再矿化产物的结构及成分变化;通过基质金属蛋白酶(Matrix metalloproteinases,MMPs)活性抑制实验评价各组材料对牙本质内源性MMPs活性的抑制作用。3、将再矿化后的牙本质进行复合树脂粘接,构建体外树脂-牙本质粘接模型,通过微渗漏实验评估各组材料的促再矿化作用对树脂-牙本质粘接界面封闭性能的影响;通过剪切粘接强度实验和断裂类型分析,评估各组材料的促再矿化作用对树脂-牙本质粘接界面机械性能的影响。结果:1、通过制备工艺参数筛选,得到尺寸均一,分散性良好的球状TMC-QP5/NPs,其粒径为276.76±7.07nm,多分散指数为0.245±0.04,Zeta电位为17.9±0.61m V;TMC-QP5/NPs的包封率为69.63±2.22%,载药量为13.21±0.73%,在前4小时突释,随后持续缓慢地释放QP5;傅里叶变换红外光谱结果提示TMC与TPP发生交联,成功负载QP5;差示扫描量热分析证明TMC-QP5/NPs的耐热稳定性良好;其粒径和多分散指数在7天内的增幅小,储存稳定性良好;浓度为0.1%～0.4%的TMC-QP5/NPs对人牙髓细胞没有明显细胞毒性。2、经TMC-QP5/NPs矿化后的牙本质表面显微硬度恢复率达54.04%,优于TMC和对照组;TMC-QP5/NPs、QP5和Na F能诱导脱矿牙本质胶原纤维矿物沉积;TMC-QP5/NPs相比QP5,更能降低脱矿牙本质的表面粗糙度;牙本质表面的再矿化产物为HA;TMC-QP5/NPs能有效抑制牙本质内源性MMPs的活性,该作用明显优于QP5和Na F。3、TMC-QP5/NPs的促矿化作用有效减少了树脂-牙本质粘接界面的微渗漏,提高粘接界面封闭性能的作用强于QP5,弱于Na F;经TMC-QP5/NPs矿化后的牙本质粘接界面剪切粘接强度为32.01±2.09MPa、内聚破坏比例为33.3%,均高于QP5和Na F组,证明其粘接界面的机械性能更好。结论:本研究使用离子交联法制备的TMC-QP5/NPs复合体带正电荷,是小粒径、尺寸均一、分散性佳的球状纳米颗粒,具有良好的稳定性,无明显细胞毒性,能在体外持续缓慢地释放QP5。TMC-QP5/NPs可促进脱矿牙本质的再矿化,该再矿化作用有利于提高树脂-牙本质粘接界面的封闭性能与机械性能,TMC-QP5/NPs有望成为良好的牙本质龋修复粘接材料。更多还原

【Abstract】 Dental caries is one of the most common chronic diseases.With the development of enamel caries,the caries damage spreads laterally along the enamel-dentin boundary,causing conical destruction in the dentin and eventually breaking the dentin barrier leading to pulp damage.At present,the common clinical restoration method for dentin caries is to use composite resin for bonded restoration,but composite resin bonded restoration has a series of problems such as resin aging、dentin collagenase degradation and marginal microleakage,resulting in a 3-year success rate often less than 50%.Therefore,it is necessary to develop materials that promote the repair of dentin caries and improve the dentin bonding effect.In recent years,remineralization is one of the research directions to repair dentin caries and improve dentin bonding performance.Our group has designed and synthesized QP5,a functional peptide of enamelogenic protein origin,which effectively stabilizes Amorphous calcium phosphate(ACP),inhibits Hydroxyapatite(HA)nucleation,and promotes remineralization of artificial early tooth enamel caries.Since peptide drugs have problems in clinical application such as rapid decrease in effective concentration and short duration of action,the group further prepared QP5 hydrogel with chitosan as carrier and applied it to promote remineralization of enamel early caries.However,whether QP5 has the same pro-mineralization effects on demineralized dentin and what form of dentin application it can take are subject to further experimental studies.In this study,N,N,N-Trimethyl chitosan(TMC)was used as a carrier to prepare composite nanoparticles loaded with amelogeninfunctional peptide QP5,and to investigate the effects of amelogenin-functional peptide-chitosan nanoparticles(TMC-QP5/NPs)on remineralization of demineralized dentin,and the effect of this remineralization on dentin bonding.Objective:In this study,we propose to prepare TMC-QP5/NPs composite nanoparticles by ionic cross-linking method,and examine their basic physicochemical properties,drug loading properties,in vitro drug release properties,stability and cytotoxicity were examined,to investigate the remineralization of demineralized dentin by TMC-QP5/NPs composite nanoparticles and the effect of this remineralization on the bonding properties of dentin,and to lay the foundation for the application of TMC-QP5/NPs for remineralization restoration of demineralized dentin and bonding improvement of dentin.Methods:1.Using TMC as the carrier and Sodium tripolyphosphate(TPP)as the cross-linking agent,TMC-QP5/NPs were prepared and screened by ionic cross-linking method and optimized preparation process;their hydrated particle size,polydispersity index and zeta potential were measured by dynamic light scattering method;their encapsulation rate and drug loading capacity were detected by high performance liquid chromatography,and the In vitro 36-hour drug release profile study was performed.The observation of morphology by scanning electron microscopy,transmission electron microscope and atomic force microscope.The chemical structure was analyzed by Fourier transform infrared(FTIR)spectroscopy;the thermal stability was assessed by differential scanning calorimetry;the storage stability was evaluated by 7-day dynamic monitoring of particle size and polydispersity index;and the toxicity of TMC-QP5/NPs to human dental pulp cells was tested by cytotoxic experiments.2.Construct the demineralized dentin model,TMC-QP5/NPs、TMC、QP5、Na F and 2-[4-(hydroxyethyl)-1-piperazinethanesulfonic acid(HEPES)were set up as five experimental groups,to perform in vitro remineralization experiments.The changes in the mechanical properties of the dentin surface after remineralization were evaluated by Percentage of surface microhardness recovery;the morphology and roughness of the dentin surface after remineralization were observed by scanning electron microscopy and atomic force microscopy;the structural and compositional changes of the remineralization products were analyzed by Fourier transform infrared spectroscopy and X-ray diffraction;the inhibitory effect of each group of materials on the activity of endogenous Matrix metalloproteinases(MMPs)in dentin was evaluated by matrix metalloproteinase activity inhibition assay.3.The remineralized dentin was subjected to composite resin bonding,and an in vitro resin-dentin bonding model was constructed to evaluate the effect of the remineralization action of each group of materials on the sealing properties of the resin-dentin bonding interface through microleakage experiments;the effect of the remineralization action of each group of materials on the mechanical properties of the resin-dentin bonding interface was evaluated through shear bond strength experiments and fracture type analysis.Results:1.The spherical TMC-QP5/NPs with uniform size and good dispersion were obtained by screening the preparation process parameters with a particle size of276.76 ± 7.07 nm,polydispersity index of 0.245 ± 0.04 and zeta potential of 17.9 ±0.61 m V;The encapsulation rate of TMC-QP5/NPs was 69.63±2.22%,and the drug loading was 13.21±0.73%,with a sudden release in the first 4 h,followed by a continuous slow release of QP5;Fourier transform infrared spectroscopy results suggested that TMC cross-linked with TPP and successfully loaded QP5;differential scanning calorimetric analysis demonstrated the good thermal stability of TMC-QP5/NPs;their The particle size and polydispersity index showed a small increase in 7 days and good storage stability;TMC-QP5/NPs at concentrations of0.1%～0.4% were not significantly cytotoxic to human dental pulp cells.2.The recovery rate of microhardness of dentin surface after mineralization by TMC-QP5/NPs reached 54.04%,which was better than that of TMC and control;TMC-QP5/NPs,QP5 and Na F could induce collagen fiber mineral deposition of demineralized dentin;TMC-QP5/NPs could reduce the surface roughness of demineralized dentin more than QP5;The remineralization product of dentin surface was HA;TMC-QP5/NPs could effectively inhibit the activity of dentin endogenous MMPs,which was significantly better than QP5 and Na F.3.The pro-mineralization effect of TMC-QP5/NPs effectively reduced the microleakage at the resin-dentin bonding interface,and improved the sealing performance of the bonding interface,stronger than QP5 and weaker than Na F;the shear bond strength of the dentin bonding interface after mineralization by TMC-QP5/NPs was 32.01±2.09 MPa,and the cohesion damage ratio was 33.3%,which were higher than QP5 and Na F group,which proved that the mechanical properties of the bonding interface were better.Conclusion:In this study,TMC-QP5/NPs complexes prepared by ionic cross-linking were positively charged,spherical nanoparticles with small particle size,homogeneous size and good dispersion,with good stability and no significant cytotoxicity,and could release QP5 continuously and slowly in vitro.TMC-QP5/NPs could promote the remineralization of demineralized dentin,and this remineralization was beneficial to improve the resin-dentin adhesive interface This remineralization was beneficial to improve the sealing and mechanical properties of the resin-dentin bonding interface,and TMC-QP5/NPs was expected to be a good bonding material for dentin caries restoration.更多还原