人牙本质源MMPs的鉴定及其与牙本质粘接持久性的相关性研究
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摘要
口腔粘接技术(Dental bonding technology)的发展使得各种牙体缺损修复成为可能，因为兼顾微创修复和美学修复的优点，已成为口腔医学领域一个重要的研究内容。牙釉质或牙本质与口腔修复材料之间形成牢固、长期有效的粘接力，是口腔粘接修复成功的关键。因为牙本质结构、成分的特殊性，其粘接成为口腔粘接领域的一个难点及热点。过去20年中，粘接材料及粘接操作方法都经历着日新月异的发展，现有粘接修复体在短期内可获得良好的粘接效果，有报道称可以达到50~70MPa。但是在口腔复杂环境中行使功能后，树脂粘接修复体会逐步出现修复体边缘着色、继发龋，甚至修复体脱落。研究表明体内牙本质粘接界面的粘接强度会在3~5年后显著降低，体外实验牙本质粘接强度甚至会在6个月后就发生显著的降低，即发生了牙本质粘接界面的退变。修复体一旦发生粘接界面的退变，就会引起修复体边缘适合性降低或固位丧失，最终导致粘接修复的失败。因此，粘接耐久性缺陷已成为制约口腔粘接技术发展、亟待解决的重要科学问题。
研究发现，牙本质内含有一种内源性的蛋白水解酶——基质金属蛋白酶(Matrix Metalloproteinases, MMPs)，这种酶具有降解所有细胞外基质的功能。牙本质有机基质中约90%的成分为Ⅰ型胶原纤维，牙本质源的基质金属蛋白酶可能会降解牙本质粘接界面的Ⅰ型胶原纤维，参与牙本质粘接界面的退变过程。牙本质内含有多种MMPs，每种MMP都有特异性的降解底物以及作用方式，目前对于MMPs在牙本质中的分布情况，以及其在粘接界面退变过程中的作用和作用机制尚缺乏足够的研究。
本研究的目的在于通过研究牙本质源MMPs在不同种类牙本质基质中的分布及其与牙本质粘接持久性的相关性，探讨参与牙本质粘接界面退变过程中牙本质源MMPs的种类及作用，为寻求可靠、有效地抑制牙本质粘接界面退变的方法，提高其粘接持久性奠定理论基础。
1.主要研究方法：
1)采用免疫组织化学染色和液相芯片技术检测正常及硬化牙本质中的MMPs的种类和分布情况；
2)采用冷热循环和人工唾液长期浸泡两种方法来模拟老化条件，检测老化处理后牙本质粘接界面的退变情况及MMPs的含量变化；
3)借助微剪切粘接强度测试粘接强度，FE-SEM结合EDX分析检测粘接界面的纳米渗漏情况，Ⅰ型胶原吡啶交联终肽反应检测粘接界面胶原的降解及MMPs的活性，探讨不同粘接系统的牙本质粘接持久性及牙本质源MMPs与牙本质粘接持久性的关系。
2.主要研究结论：
1)五种MMPs：MMP-1、MMP-2、MMP-3、MMP-8和MMP-9均存在于正常的人牙本质及磨损牙齿的硬化牙本质中。
2)在正常人冠方牙本质中，牙本质源MMPs：MMP-1、MMP-2、MMP-3、MMP-8和MMP-9的分布趋势均为由近髓腔处向外侧釉牙本质界处递减，在同一层次的牙本质中，五种牙本质源MMPs的含量由高到低为MMP-8﹥MMP-2﹥MMP-9﹥MMP-3﹥MMP-1。
3)随着年龄的增长，人牙本质源MMP-2和MMP-3有降低的趋势。
4)牙本质源MMP-8和MMP-9在人磨损牙齿的硬化牙本质中含量显著高于正常牙本质，说明其与非龋坏硬化牙本质的形成密切相关。
5)老化处理后，深层牙本质的粘接强度下降情况及胶原降解程度均比浅层牙本质显著，提示深层牙本质粘接界面的退变情况比浅层牙本质严重，这可能与牙本质源MMPs的分布差异有关。
6)老化处理后，全酸蚀粘接剂Single Bond2和自酸蚀粘接剂S3Bond粘接的牙本质界面均产生了粘接退变，牙本质源MMPs可能参与了SingleBond2组粘接界面的退变，其在S3Bond组的粘接界面退变中的作用仍无法肯定；粘接剂处理后牙本质中的胶原酶MMP-8，明胶酶MMP-2、MMP-9含量会随着老化时间的延长显著降低，提示其可能参与了牙本质粘接界面的退变。
7)使用MMP-8抑制剂可以提高牙本质粘接界面的耐久性，其作用与广谱MMPs抑制剂洗必泰溶液无显著性差异，提示胶原酶MMP-8可能在牙本质粘接界面退变过程中发挥主导作用。
The development of dental bonding technology makes variety of teeth defectsrestoration become possible. Because of the advantages of minimally invasiverestoration and aesthetic restoration, it has become an important researchingcontent in dentistry. Solid, long-term adhesion between enamel or dentin anddental prosthetic material is the key to the success of dental adhesive restoration.Because of the particularity of the dentin structure and composition, adhesionbecomes one of the difficult points and hot spots in the field of dental adhesion.In the past20years, the adhesive materials and bonding methods are undergoingrapid development. And the immediate bonding strength of the currently existingdentin bonding materials has significantly improved, which is reported up to50-70MPa. But after used in the complex oral environment, prosthetic restorationwould gradually appear coloration of the prosthesis edge, secondary caries, andeven the prosthesis off. Studies showed that in vivo dentin bonding strength ofthe adhesive interface decreased significantly in3to5years, and in vitroexperiments dentin bonding strength even decreased significantly after sixmonths, which meant degeneration in the dentin adhesive interface occurred.Once degeneration occurred, it would cause reduction of fitness of restorationmargin and loss of retention, which ultimately led to failure of adhesiverestoration. Therefore, the defects of bonding durability had become an importantscientific problem to be solved, which would constrain the development of oralbonding technology.
The study showed that dentin contained endogenous proteolytic enzymes-Matrix Metalloproteinases, MMPs. The enzyme could degrade all extracellularmatrix. About90%of the composition of dentin organic matrix is type Ⅰcollagen fibers. And the dentinal matrix metalloproteinases may degrade type Icollagen fibers in the dentin adhesive interface, to participate in the degenerativeprocess of the dentin adhesive interface. The dentin contains a variety of MMPs,and each MMP has a specific degradation substrate and the mode of function.Now the distribution of MMPs in dentin, as well as the role in and the mechanismof degeneration process in the bonding interface is still lack of sufficient research.
The purpose of this study is to research on the distribution of dentinal MMPs indifferent types dentin matrix and its correlation with the durability of dentinadhesion, and to explore the types and the role of the dentinal MMPs involved indentin interface degeneration. It also searches for a reliable and effective way toinhibit degeneration in dentin interface, and lay the theoretical foundation ofimprovement the adhesion durability.
1. Methods
1) Using immunohistochemical staining and liquid chip to detect the type anddistribution of MMPs in normal and sclerotic dentin.
2) Using thermal cycling and long-term soak in artificial saliva to simulateaging conditions, in order to detect degeneration in the aging treated dentininterface and variation in the content of MMPs.
3) To evaluate the bonding strength with the micro-shear bonding strength test,to detect and analyze nanoleakage in adhesive interface by FE-SEMcombined with EDX, and to detect degradation of collagen and activity ofMMPs in the bonding interface through Cross-linked carboxyterminaltelopeptide of type I collagen reaction, in order to investigate of different adhesive systems and the relationship between dentinal MMPs and dentinadhesive durability.
2. Results
1) Five kinds of MMPs of MMP-1, MMP-2and MMP-3, MMP-8and MMP-9both existed in normal dentin and sclerotic dentin.
2) In the normal coronal dentin, the distribution of dentinal MMPs: MMP-1,MMP-2and MMP-3, MMP-8and MMP-9both decreased from near pulpcavity to dento enamel junction. In the same layer of dentin, the contents ofthe five dentinal MMPs decreased as MMP-8> MMP-2> MMP-9> MMP-3>MMP-1.
3) Along with the increase of age, the human dentinal MMP-2and MMP-3decreased.
4) The contents of dentinal MMP-8and MMP-9in the sclerotic dentin weresignificantly higher than those in normal dentin, which suggests dentinalMMP-8and MMP-9may closely relate to the formation of non-carioussclerotic dentin.
5) After aging treatment, the decreasement of bonding strength and thedegradation of collagen in deep-dentin were both more significant than thosein superficial dentin. It suggested that the degeneration of dentin interface indeep dentin was more serious than that in superficial dentin, which mayrelate to the differences of dentinal MMPs distribution.
6) After aging treatment, adhesive degeneration exited both in the dentininterface of the total etch adhesive Single Bond2and self-etching adhesiveS3Bond adhesive. Dentinal MMPs may involve in the degeneration ofadhesive interface of the Single Bond2group. But their roles in thedegeneration of the adhesive interface of the S3Bond group were still not sure. After treatment of adhesive, the contents of collagenase MMP-8andgelatinase MMP-2, MMP-9decreased significantly along with the aging time,suggesting that they may be involved in the degeneration of the dentinadhesive interface.
7) Using MMP-8inhibitors could increase adhesion durability of dentinadhesion interface, and there were not significant differences betweenMMP-8inhibitors and broad-spectrum MMPs inhibitor chlorhexidinesolution, which suggested that the collagenase MMP-8may play a leadingrole in the degeneration in the dentin adhesive interface.
研究发现，牙本质内含有一种内源性的蛋白水解酶——基质金属蛋白酶(Matrix Metalloproteinases, MMPs)，这种酶具有降解所有细胞外基质的功能。牙本质有机基质中约90%的成分为Ⅰ型胶原纤维，牙本质源的基质金属蛋白酶可能会降解牙本质粘接界面的Ⅰ型胶原纤维，参与牙本质粘接界面的退变过程。牙本质内含有多种MMPs，每种MMP都有特异性的降解底物以及作用方式，目前对于MMPs在牙本质中的分布情况，以及其在粘接界面退变过程中的作用和作用机制尚缺乏足够的研究。
本研究的目的在于通过研究牙本质源MMPs在不同种类牙本质基质中的分布及其与牙本质粘接持久性的相关性，探讨参与牙本质粘接界面退变过程中牙本质源MMPs的种类及作用，为寻求可靠、有效地抑制牙本质粘接界面退变的方法，提高其粘接持久性奠定理论基础。
1.主要研究方法：
1)采用免疫组织化学染色和液相芯片技术检测正常及硬化牙本质中的MMPs的种类和分布情况；
2)采用冷热循环和人工唾液长期浸泡两种方法来模拟老化条件，检测老化处理后牙本质粘接界面的退变情况及MMPs的含量变化；
3)借助微剪切粘接强度测试粘接强度，FE-SEM结合EDX分析检测粘接界面的纳米渗漏情况，Ⅰ型胶原吡啶交联终肽反应检测粘接界面胶原的降解及MMPs的活性，探讨不同粘接系统的牙本质粘接持久性及牙本质源MMPs与牙本质粘接持久性的关系。
2.主要研究结论：
1)五种MMPs：MMP-1、MMP-2、MMP-3、MMP-8和MMP-9均存在于正常的人牙本质及磨损牙齿的硬化牙本质中。
2)在正常人冠方牙本质中，牙本质源MMPs：MMP-1、MMP-2、MMP-3、MMP-8和MMP-9的分布趋势均为由近髓腔处向外侧釉牙本质界处递减，在同一层次的牙本质中，五种牙本质源MMPs的含量由高到低为MMP-8﹥MMP-2﹥MMP-9﹥MMP-3﹥MMP-1。
3)随着年龄的增长，人牙本质源MMP-2和MMP-3有降低的趋势。
4)牙本质源MMP-8和MMP-9在人磨损牙齿的硬化牙本质中含量显著高于正常牙本质，说明其与非龋坏硬化牙本质的形成密切相关。
5)老化处理后，深层牙本质的粘接强度下降情况及胶原降解程度均比浅层牙本质显著，提示深层牙本质粘接界面的退变情况比浅层牙本质严重，这可能与牙本质源MMPs的分布差异有关。
6)老化处理后，全酸蚀粘接剂Single Bond2和自酸蚀粘接剂S3Bond粘接的牙本质界面均产生了粘接退变，牙本质源MMPs可能参与了SingleBond2组粘接界面的退变，其在S3Bond组的粘接界面退变中的作用仍无法肯定；粘接剂处理后牙本质中的胶原酶MMP-8，明胶酶MMP-2、MMP-9含量会随着老化时间的延长显著降低，提示其可能参与了牙本质粘接界面的退变。
7)使用MMP-8抑制剂可以提高牙本质粘接界面的耐久性，其作用与广谱MMPs抑制剂洗必泰溶液无显著性差异，提示胶原酶MMP-8可能在牙本质粘接界面退变过程中发挥主导作用。
The development of dental bonding technology makes variety of teeth defectsrestoration become possible. Because of the advantages of minimally invasiverestoration and aesthetic restoration, it has become an important researchingcontent in dentistry. Solid, long-term adhesion between enamel or dentin anddental prosthetic material is the key to the success of dental adhesive restoration.Because of the particularity of the dentin structure and composition, adhesionbecomes one of the difficult points and hot spots in the field of dental adhesion.In the past20years, the adhesive materials and bonding methods are undergoingrapid development. And the immediate bonding strength of the currently existingdentin bonding materials has significantly improved, which is reported up to50-70MPa. But after used in the complex oral environment, prosthetic restorationwould gradually appear coloration of the prosthesis edge, secondary caries, andeven the prosthesis off. Studies showed that in vivo dentin bonding strength ofthe adhesive interface decreased significantly in3to5years, and in vitroexperiments dentin bonding strength even decreased significantly after sixmonths, which meant degeneration in the dentin adhesive interface occurred.Once degeneration occurred, it would cause reduction of fitness of restorationmargin and loss of retention, which ultimately led to failure of adhesiverestoration. Therefore, the defects of bonding durability had become an importantscientific problem to be solved, which would constrain the development of oralbonding technology.
The study showed that dentin contained endogenous proteolytic enzymes-Matrix Metalloproteinases, MMPs. The enzyme could degrade all extracellularmatrix. About90%of the composition of dentin organic matrix is type Ⅰcollagen fibers. And the dentinal matrix metalloproteinases may degrade type Icollagen fibers in the dentin adhesive interface, to participate in the degenerativeprocess of the dentin adhesive interface. The dentin contains a variety of MMPs,and each MMP has a specific degradation substrate and the mode of function.Now the distribution of MMPs in dentin, as well as the role in and the mechanismof degeneration process in the bonding interface is still lack of sufficient research.
The purpose of this study is to research on the distribution of dentinal MMPs indifferent types dentin matrix and its correlation with the durability of dentinadhesion, and to explore the types and the role of the dentinal MMPs involved indentin interface degeneration. It also searches for a reliable and effective way toinhibit degeneration in dentin interface, and lay the theoretical foundation ofimprovement the adhesion durability.
1. Methods
1) Using immunohistochemical staining and liquid chip to detect the type anddistribution of MMPs in normal and sclerotic dentin.
2) Using thermal cycling and long-term soak in artificial saliva to simulateaging conditions, in order to detect degeneration in the aging treated dentininterface and variation in the content of MMPs.
3) To evaluate the bonding strength with the micro-shear bonding strength test,to detect and analyze nanoleakage in adhesive interface by FE-SEMcombined with EDX, and to detect degradation of collagen and activity ofMMPs in the bonding interface through Cross-linked carboxyterminaltelopeptide of type I collagen reaction, in order to investigate of different adhesive systems and the relationship between dentinal MMPs and dentinadhesive durability.
2. Results
1) Five kinds of MMPs of MMP-1, MMP-2and MMP-3, MMP-8and MMP-9both existed in normal dentin and sclerotic dentin.
2) In the normal coronal dentin, the distribution of dentinal MMPs: MMP-1,MMP-2and MMP-3, MMP-8and MMP-9both decreased from near pulpcavity to dento enamel junction. In the same layer of dentin, the contents ofthe five dentinal MMPs decreased as MMP-8> MMP-2> MMP-9> MMP-3>MMP-1.
3) Along with the increase of age, the human dentinal MMP-2and MMP-3decreased.
4) The contents of dentinal MMP-8and MMP-9in the sclerotic dentin weresignificantly higher than those in normal dentin, which suggests dentinalMMP-8and MMP-9may closely relate to the formation of non-carioussclerotic dentin.
5) After aging treatment, the decreasement of bonding strength and thedegradation of collagen in deep-dentin were both more significant than thosein superficial dentin. It suggested that the degeneration of dentin interface indeep dentin was more serious than that in superficial dentin, which mayrelate to the differences of dentinal MMPs distribution.
6) After aging treatment, adhesive degeneration exited both in the dentininterface of the total etch adhesive Single Bond2and self-etching adhesiveS3Bond adhesive. Dentinal MMPs may involve in the degeneration ofadhesive interface of the Single Bond2group. But their roles in thedegeneration of the adhesive interface of the S3Bond group were still not sure. After treatment of adhesive, the contents of collagenase MMP-8andgelatinase MMP-2, MMP-9decreased significantly along with the aging time,suggesting that they may be involved in the degeneration of the dentinadhesive interface.
7) Using MMP-8inhibitors could increase adhesion durability of dentinadhesion interface, and there were not significant differences betweenMMP-8inhibitors and broad-spectrum MMPs inhibitor chlorhexidinesolution, which suggested that the collagenase MMP-8may play a leadingrole in the degeneration in the dentin adhesive interface.
引文
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