宿主来源的基质金属蛋白酶对根部牙本质胶原的降解作用

目的观察根部牙本质中的基质金属蛋白酶（MMP）对牙本质胶原的降解作用.方法将脱矿的根部牙本质粉离心、冷冻干燥后分7组,每组6份,每份50.0 mg.各标本中分别加入1ml含不同成分的人工唾液,根据成分不同分为2 mmol/L4-乙酰氨基苯汞（APMA）组（MMP活化剂）;2、100、200 mmol/L乙二胺四乙酸（EDTA）组,0.2%、0.02%醋酸氯己定组（MMP抑制剂）;以空白人工唾液作对照组.37℃4 h后,用羟脯氨酸试剂盒测定各标本的胶原降解量.扫描电镜观察脱矿及脱矿后置于人工唾液中的根部标本表面结构变化.结果APMA组的胶原降解量最多,其次为对照组,两者间差异有统计学意义（P＜0.05）.各MMP抑制剂组的胶原降解量均显著少于APMA组和对照组,差异有统计学意义（P＜0.01）.扫描电镜结果表明,仅脱矿的根部表面胶原纤维较完整;而脱矿后置于人工唾液中的标本胶原纤维断裂,结构紊乱.结论脱矿过程中的低pH值能使根部牙本质中的MMP活化,在中性时可降解牙本质胶原.提示宿主来源的MMP可能是龋病进展过程中有机质破坏的重要原因之一.     

宿主基质金属蛋白酶在乳牙牙本质胶原降解中的作用

目的 研究乳牙牙本质中的基质金属蛋白酶在乳牙牙本质胶原降解中的作用.方法 因滞留拔除的正常乳牙制备牙本质粉,分为空白组、醋酸氯己定组和6-去甲基-6-脱氧-4-去二甲氨基四环素(CMT-3)组;每组6份标本,每份标本50.0mg.各组标本置于脱矿液中6h,人工唾液中18h,进行pH循环,共7次.醋酸氯己定组和CMT-3组的脱矿液和人工唾液中分别加入0.2％醋酸氯己定和0.02％ CMT-3.收集各组的脱矿液和人工唾液的上清液,用羟脯氨酸酶联免疫试剂盒分别检测各组脱矿液和人工唾液上清中的胶原降解量.结果 空白组的脱矿液和人工唾液上清中的胶原降解量均高于醋酸氯己定组和CMT-3组,差异有统计学意义(P＜0.05).醋酸氯己定组和CMT-3组的胶原降解量差异无统计学意义(P＞0.05).结论 乳牙牙本质中的基质金属蛋白酶活化后可降解牙本质胶原,使用基质金属蛋白酶抑制剂可抑制牙本质胶原的降解.     

基质金属蛋白酶8对牙本质树脂粘接的影响

目的 研究基质金属蛋白酶8(MMP-8)对牙本质树脂粘接的影响,探讨其抑制剂在粘接中的应用.方法 收集60颗口腔颌面外科拔除的人正常第三磨牙.其中20颗用于制备牙本质粉并随机平均分成4组,第一组不做处理,第二组磷酸酸蚀,第三组磷酸酸蚀后粘接剂处理,第四组磷酸酸蚀后先用基质金属蛋白酶8的特异性抑制剂(MMP-8 Inhibitor I)孵育;然后4组均粘接剂处理,提取蛋白质检测MMP-8的活性.另外40颗离体牙随机均分为对照组和MMP-8Inhibitor I处理组(实验组),制作牙本质树脂试件并储存在人工唾液中,分别在储存l天和3个月时进行微拉伸试验分析粘接强度的变化,电镜观察断面和混合层降解的情况.结果 MMP-8活性实验结果显示,第一组呈现少量酶活性:第二组活性大幅度提高;第三组活性下降;第四组未检测到MMP-8活性.微拉伸试验结果显示在相同的时间点,实验组的粘接强度高于对照组,差异有统计学意义.扫描电镜显示,3个月后实验组混合层的胶原纤维网比较完整,对照组的胶原纤维出现明显的降解.结论 MMP-8 Inhibitor I可以通过抑制牙本质中MMP-8的活性抑制混合层胶原纤维网的降解.     

基质金属蛋白酶-1对根面牙本质有机质降解作用的超微结构观察

目的　观察基质金属蛋白酶-1(MMP-1)对根面牙本质有机质的降解作用。方法　收集临床拔除的健康无 龋阻生牙,切取根颈1/3处约5 mm厚的牙体,制成牙本质组织块,随机分为4组。第1组用酸溶液脱矿处理21 d 后,放入MMP-1溶液中孵育7 d;第2组仅用酸溶液脱矿处理21 d;第3组仅用MMP-1溶液酶解7 d;第4组为正常牙 本质标本对照组,不作任何处理。将各标本切割制作样本,脱水干燥,喷金,扫描电镜观察。结果　酸和酶溶液共 同处理的标本牙本质硬组织脱矿明显,牙本质小管管腔失去原有形态,边界不清,周围暴露的胶原纤维断裂不连 续,排列杂乱不规则。酸或酶溶液单独处理组未发生明显的基质纤维降解现象。结论　内源性蛋白酶参与了根面 龋的发生发展过程,MMP-1能够明显降解脱矿后的牙本质有机质。     

化学交联剂对树脂牙本质粘接稳定性的影响

随着粘接剂的发展口腔粘接修复在临床得到广泛应用。但牙本质粘接耐久性的问题仍然不容乐观。由于口腔各种酶、细菌等多种因素会导致粘接界面胶原纤维暴露,而裸露的胶原纤维极易降解和疲劳破坏,从而影响牙本质粘接稳定性。基质金属蛋白酶介导的对胶原纤维的降解是粘接界面破坏的主要因素。抑制基质金属蛋白酶活性、增强胶原性能是延长粘接寿命的关键。为此,针对口腔牙本质粘接难点的问题,基于牙本质生物改性的概念,通过使用胶原交联剂的生物活性介导的仿生方法,改变牙本质生物理化性能来强化牙本质胶原纤维强度,增加粘接稳定性。本文主要介绍牙本质生物改性及其相关的基质成分,并对化学胶原交联剂碳化二亚胺及戊二醛进行了综述。     

基质金属蛋白酶2、8、9在乳牙牙本质中的表达及含量分析

目的观察具有胶原降解作用的基质金属蛋白酶（matrix metalloproteinase,MMPs）-2、8、9在乳牙牙本质中的表达及分布,并检测其在乳牙牙本质中的相对含量。方法选取因滞留拔除的无龋乳牙125颗,5颗固定、脱钙、切片后进行MMP-2、8、9免疫组化染色;120颗去除牙釉质、牙骨质和牙髓,制备成牙本质粉。用酶联免疫试剂盒分别测定脱矿上清液和酶提取上清液中的MMP-2、8、9的含量。结果 MMP-2、8、9在乳牙冠部牙本质全层均呈阳性表达。MMP-2、8、9在脱矿上清液中的含量分别为324.5±34.0μg/L,29.3±1.2μg/L和11.9±3.2μg/L;在酶提取上清液中的含量分别为325.9±19.9μg/L,31.1±0.9μg/L和8.5±1.5μg/L。结论基质金属蛋白酶MMP-2、8、9存在于乳牙冠部牙本质中。其中MMP-2的含量最多,其次为MMP-8,MMP-9的含量最少。     

脱矿牙本质三种干燥方法的场发射扫描电镜比较

目的:比较空气干燥、六甲基二硅胺烷(hexamethyldisilazane,HMDS)干燥、临界点干燥三种干燥方法对脱矿牙本质扫描电镜图像的影响。方法:冠部中层牙本质薄片磷酸酸蚀15sec后,经戊二醛固定、乙醇梯度脱水、干燥(三种干燥方法之一)处理后,喷金,场发射扫描电镜观察样品横断面和纵断面。结果:空气干燥组,胶原纤维之间的间隙消失,脱矿牙本质全层塌陷;HMDS干燥的胶原纤维之间间隙变小,脱矿牙本质表层塌陷;临界点干燥的胶原纤维塌陷变形最小,胶原之间的间隙大小均匀一致,脱矿牙本质全层为均匀多孔形貌。结论:临界点干燥较好的保存了牙本质胶原纤维的形貌,是脱矿牙本质扫描电镜最理想的干燥方法。     

人牙本质基质提取物磷蛋白的分析及其在再矿化中的作用

磷蛋白在牙齿发育期间牙本质的矿化及牙龋坏脱矿后再矿化中起重要作用。本文旨在探讨磷蛋白在牙本质基质再矿化中所起的作用。 材料和方法 选取8～16岁患者的根尖未成熟的牙齿,拔除后立即放入2.5M含蛋白酶抑制剂的NaCl溶液中。去除牙齿根周软组织、牙髓及牙冠,-70℃低温保存。切除后将根尖1/3磨成牙粉,用盐酸胍分离脱矿牙本质基质,再用溴化氰和胶原酶消化提取,提取物     

基质金属蛋白酶在牙本质龋中的作用

基质金属蛋白酶(MMPs)是一类蛋白水解酶,在体内可水解细胞外基质成分。在牙本质龋发生的过程中,致龋菌释放的酸使pH值下降,来自于唾液和牙本质的MMPs被激活,当唾液缓冲系统使pH值恢复中性后,MMPs可水解脱矿的牙本质。基质金属蛋白酶抑制剂如MMPs组织特异性抑制剂及从天然来源的物质中提取出的某些成分可有效抑制MMPs活性。本文就MMPs生物学特性、在牙本质龋中的作用以及其抑制剂的研究进展作一综述。     

人冠部牙本质中基质金属蛋白酶8的表达和分布

目的 研究正常人类基质金属蛋白酶8(MMP-8)在牙冠部牙本质中的表达和分布规律,探讨其在牙本质形成和改建中的作用.方法 对37颗正常人牙冠部的脱矿牙本质进行免疫组化染色,以Image pro-plus 6.0图像分析系统分层测量牙本质中免疫阳性区的光密度值.结果 人冠部牙本质全层中均可见MMP-8的免疫染色.在染色强度上,成牙本质细胞层最强,前期牙本质层、邻近釉牙本质界5μm区、成熟牙本质深中浅层依次降低.各层的平均光密度值依次为0.186/pix2、0.143/pix2、0.092/pix2、0.032/pix2、0.021/pix2和0.020/pix2.结论 人冠部牙本质全层中均含有MMP-8,其分布具有不均衡性,提示MMP-8在牙本质的形成、矿化和改建中具有重要的作用.     

Determination of Matrix Metalloproteinases in Human Radicular Dentin

Matrix metalloproteinases (MMPs) are present in sound coronal dentin and may play a role in collagen network degradation in bonded restorations. We investigated whether these enzymes can also be detected in root dentin. Crown and root sections of human teeth were powderized, and dentin proteins were extracted by using guanidine-HCl and EDTA. Extracts were analyzed by zymography and Western blotting for matrix metalloproteinases detection. Zymography revealed gelatinolytic activities in both crown and root dentin samples, corresponding to MMP-2 and MMP-9. MMP-2 was more evident in demineralized root dentin matrix, whereas MMP-9 was mostly extracted from the mineralized compartment of dentin and presented overall lower levels. Western blot analysis detected MMP-8 equally distributed in crown and root dentin. Because MMPs are also present in radicular dentin, their contribution to the degradation of resin-dentin bonds should be addressed in the development of restorative strategies for the root substrate.     

Collagen cross-linking and ultimate tensile strength in dentin

Several studies have indicated differences in bond strength of dental materials to crown and root dentin. To investigate the potential differences in matrix properties between these locations, we analyzed upper root and crown dentin in human third molars for ultimate tensile strength and collagen biochemistry. In both locations, tensile strength tested perpendicular to the direction of dentinal tubules (undemineralized crown = 140.4 +/- 48.6/root = 95.9 +/- 26.1; demineralized crown = 16.6 +/- 6.3/root = 29.0 +/- 12.4) was greater than that tested parallel to the tubular direction (undemineralized crown = 73.1 +/- 21.2/root = 63.2 +/- 22.6; demineralized crown = 9.0 +/- 3.9/root = 16.2 +/- 8.0). The demineralized specimens showed significantly greater tensile strength in root than in crown. Although the collagen content was comparable in both locations, two major collagen cross-links, dehydrodihydroxylysinonorleucine/its ketoamine and pyridinoline, were significantly higher in the root (by ~ 30 and ~ 55%, respectively) when compared with those in the crown. These results indicate that the profile of collagen cross-linking varies as a function of anatomical location in dentin and that the difference may partly explain the site-specific tensile strength.     

Zymographic analysis and characterization of MMP-2 and -9 forms in human sound dentin

The role and function of dentin matrix metalloproteinases (MMPs) are not well-understood, but they may play a key role in dentinal caries and the degradation of resin-bonded dentin matrices. To test the null hypothesis that MMP-9 is not found in dentin matrix, we used gelatin zymography to extract and isolate all molecular forms of gelatinolytic MMPs in demineralized mature sound dentin powder obtained from extracted human molars, characterizing and identifying the enzymes by Western blotting. Gelatinolytic MMPs were detected in extracts of demineralized dentin matrix and identified as MMP-2 and MMP-9. Acidic extracts (pH 2.3) yielded 3-8 times more MMP activity than did EDTA (pH 7.4). Their activation may contribute to dentin matrix degradation, which occurs during caries progression and following resin bonding. Inhibition of MMP-2 and -9 proteolytic activity may slow caries progression and increase the durability of resin-dentin bonds.     

Biomimetic remineralization of artificial caries dentin lesion using Ca/P-PILP

ObjectiveTo assess the ultrastructural change of demineralized dentin collagen during calcium phosphate polymer-induced liquid precursor (Ca/P-PILP) mediated remineralization process and to evaluate the biomimetic remineralization potential of high concentration Ca/P-PILP at demineralized artificial caries dentin lesion, additionally to investigate the bond interfacial integrity as well as the bonding strength of the biomimetic remineralized artificial caries dentin lesion.MethodsDemineralized dentin collagen of 5 μm thick was biomimetically remineralized with low, medium concentration Ca/P-PILP for 10 days and high concentration Ca/P-PILP for 10, 15, 20 days. Artificial caries dentin lesion at a thickness of 150 ± 50 μm was biomimetically remineralized with high concentration Ca/P-PILP for 20 days. The biomimetic remineralization of demineralized dentin collagen was observed by scanning electron microscopy (SEM). The biomimetic remineralization intensity and depth of artificial caries dentin lesion was assessed by Electron Probe Micro Analyzer (EPMA). The bonding interfacial integrity between remineralized artificial caries dentin and composite resin was observed by Swept-source optical coherence tomography (SS-OCT) and the bonding strength of remineralized artificial caries dentin was evaluated by micro-tensile bond strength analysis (μTBS).ResultsSolely PAA-PASP solution and solely saturated Ca/P solution can’t achieve dentin collagen remineralization. Increased concentration of Ca/P-PILP and prolonged remineralization time can enhance the biomimetic remineralization intensity of demineralized dentin collagen. After treating with high concentration Ca/P-PILP, a 150 ± 50 μm thick layer of demineralized artificial caries dentin lesion was not fully remineralized, and the biomimetic remineralization intensity reached up to 88.0%. Furthermore, a better bonding interfacial integrity with less microgap and increased bond strength at both baseline level and aging level were observed when artificial caries dentin lesion was biomimetically remineralized with high concentration Ca/P-PILP.SignificanceBiomimetic remineralization of demineralized caries dentin lesion promotes its clinical properties for resin composited adhesive restoration.     

Host-derived proteinases and degradation of dentine collagen in situ

Dentine root caries is a process of demineralization and degradation of the organic matrix by proteinases. In this in situ study, the presence and activity of the matrix metalloproteinases 1, 2 and 9 (MMP-1, MMP-2, MMP-9) in saliva and in completely demineralized dentine specimens were investigated. Furthermore, the activity of cathepsin B was determined in saliva. A correlation between these enzymes and the level of degraded collagen was investigated. Demineralized dentine specimens were mounted in the partial prosthesis of 17 volunteers. Saliva samples were taken at 0, 2 and 4 weeks. After 4 weeks, the enzymes were extracted from the dentine specimens and the collagen loss was assessed. The collagen loss varied between 0 and 40.3%. Zymography of the saliva and the dentine extract samples showed that (pro-)MMP-2 and (pro-)MMP-9 were present. The levels of active MMPs were assessed, using fluorogenic MMP-specific substrates. All but 3 of the 51 saliva samples showed MMP-1 activity ranging from 1.5 to 101.1 relative fluorescence units (RFU)/s. Forty-eight saliva samples showed gelatinolytic MMP-2/MMP-9 activity (1.7-141.1 RFU/s). MMP-1 activity was shown in all dentine extracts varying between 3.5 and 295.0 RFU/s. From the dentine extracts, 15 showed MMP-2/MMP-9 activity (0.2-13.7 RFU/s). The MMP activity from both saliva and dentine extracts did not correlate with the collagen loss. The activity of salivary cathepsin B varied from 4.8 to 42.2 arbitrary units/min. A positive correlation was found between salivary MMP activity and cathepsin B activity. This study revealed that gelatinolytic enzyme activity was present both in saliva and dentine collagen. No correlation could be observed, however, between the level of enzyme activity and the collagen loss of the dentine specimens.     

Effect of calcium fluoride on the activity of dentin matrix-bound enzymes

ObjectiveMatrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) are two distinct enzymatic pathways responsible for the degradation of collagen fibrils in demineralized dentin. NaF and KF have been shown to inhibit salivary MMP-2, -9 and CCs. This study investigated the inhibitory effect of calcium fluoride (CaF₂) on the dentin matrix-bound MMPs and CCs.DesignPhosphoric acid (10%)-demineralized dentin beams (1 × 2×6 mm) were incubated at 37 °C in an 1 ml of artificial saliva (AS, control), or AS with 6, 12, 24, 48, 120. 179 and 238 mM F containing CaF₂ (n = 10/group) for 1, 7 and 21 days. All groups were further incubated in AS only for 6 months. Total MMP activity, dry mass loss, CTX and hydroxyproline (HYP) analyses were performed after each incubation. The beams were examined under scanning electron microscopy (SEM). MMP-2 and MMP-9 activities were screened with gelatin zymography. Data were analyzed by using ANOVA and Tukey HSD tests (p = .05).ResultsThe total MMP activity was similar for all groups after 21 days and 6 months. After 21 days, the cumulative mass loss and CTX levels were lower compared to control for the CaF₂ ≥48 and CaF₂≥120 mM, respectively (p < .05). After 6 months, no significant difference was detected in the dry mass loss and CTX compared to the control (p > .05), whereas HYP level was higher with F 24 and 238 mM groups. CaF₂-like minerals were observed on the beams under SEM. There was no gelatinase inhibition in zymography.ConclusionCaF₂ does not prevent the degradation of demineralized dentin matrices due to the catalytic activity of MMPs and CCs.     

The influence of chemically-induced modifications of root surfaces on cell migration, attachment, and orientation

Surface demineralization of tooth root surfaces has been shown to improve re-attachment of cells and to promote tissue reconstruction following periodontal surgery. Exposure of collagen fibers has been thought to facilitate migration, attachment, and orientation of fibroblasts on the root surface. However, using an in vitro assay, we have found that both attachment and orientation of human gingival fibroblasts on demineralized dentin surfaces are further improved following digestion of the exposed collagen with bacterial collagenase. In contrast, pronase and trypsin digestion of the surface collagen had no significant effect, whereas heat denaturation had an inhibitory effect. Dissociative extraction of the demineralized dentin slices with 4 mol/L guanidine hydrochloride (GuHCl) also improved attachment and orientation, and when undemineralized dentin was subjected to dissociative extraction, cell attachment was comparable and orientation superior to that on demineralized surfaces. These studies indicate that demineralization is not a prerequisite for facilitating attachment, and that enhanced attachment and orientation of cells are not dependent upon a collagenous substratum.     

Remineralization potential of fully demineralized dentin infiltrated with silica and hydroxyapatite nanoparticles

Objective

This study investigates the potential of a novel guided tissue regeneration strategy, using fully demineralized dentin infiltrated with silica and hydroxyapatite (HA) nanoparticles (NPs), to remineralize dentin collagen that is completely devoid of native hydroxyapatite.

Methods

Dentin blocks were fully demineralized with 4 N formic acid and subsequently infiltrated with silica and HA NPs. The remineralizing potential of infiltrated dentin was assessed following a twelve week exposure to an artificial saliva solution by means of TEM, EDS and micro-CT. Measurements were taken at baseline and repeated at regular intervals for the duration of the study to quantify the P and Ca levels, the mineral volume percentage and mineral separation of the infiltrated dentin specimens compared to sound dentin and non-infiltrated controls.

Results

Infiltration of demineralized dentin with nano-HA restored up to 55% of the P and Ca levels at baseline. A local increase in the concentration of calcium phosphate compounds over a period of twelve weeks resulted in a higher concentration in P and Ca levels within the infiltrated specimens when compared to the non-infiltrated controls. Remineralization of demineralized dentin with silica NPs by immersion in artificial saliva was the most effective strategy, restoring 20% of the P levels of sound dentin. Micro-CT data showed a 16% recovery of the mineral volume in dentin infiltrated with silica NPs and a significant decrease in the mineral separation to levels comparable to sound dentin.

Significance

Demineralized dentin infiltrated with silica NPs appears to encourage heterogeneous mineralization of the dentin collagen matrix following exposure to an artificial saliva solution.