不同种类交联剂对牙本质Ⅰ型胶原生物改性作用的研究进展

建立稳定的树脂-牙本质混合层是提高修复体粘接耐久性的有效方法。交联剂对牙本质的生物改性可增强胶原的力学性能及抗酶解性,抑制脱矿进程并促进牙本质再矿化,具有预防龋齿以及改善粘接剂性能的临床价值。本文分类阐述不同交联剂对牙本质Ⅰ型胶原的生物改性作用。     

Ⅰ型和Ⅲ型胶原在修复性牙本质形成中的免疫定位

目的：研究Ⅰ型和Ⅲ型胶原在修复性牙本质形成中的免疫定位和分布特征。方法：在大鼠第一磨牙制备单面洞，观察修复性牙本质形成，免疫组化ＳＡＢＣ法检测Ⅰ型胶原和Ⅲ型胶原的免疫反应。结果：在术后３ｄ，修复性牙本质尚未形成。Ⅰ型胶原和Ⅲ型胶原均分布于牙髓内，前期牙本质为弱阳性，术后１５ｄ，Ⅰ型胶原和Ⅲ型胶原在牙髓细胞内呈阳性染色。Ⅰ型胶原在前期牙本质中呈弱阳性，术后３０ｄ，Ⅰ型胶原和Ⅲ型胶原的旨阳性染色集中于     

酸蚀时间对牙本质Ⅰ型胶原降解的影响

目的 评价37%磷酸处理牙本质不同时间对牙本质Ⅰ型胶原降解的影响,以期为临床牙本质粘接操作提供实验依据.方法 37%磷酸酸蚀处理牙本质片0(空白对照组)、10、15、30、60 s,酶联免疫吸附法定量测定Ⅰ型胶原的降解量,场发射扫描电镜(field emission in-lens scanning electron microscope,FEISEM)观察胶原纤维的形态学变化.结果 酸蚀处理60 s时Ⅰ型胶原的降解量最多,为4.86(1.55)mg/g;其次为30 s组,为2.76(0.87)mg/g;再次为15 s组,为1.93(0.88)mg/g;10 s组胶原降解量较少,为0.95(0.38)mg/g;空白对照组胶原的降解量最少,为0.06(0.03)mg/g.两两比较显示,各组间差异均有统计学意义(P＜0.005).FEISEM显示,37%磷酸处理牙本质表面10 s,尽管已清除了玷污层,但牙本质小管口以及胶原纤维仍覆盖颗粒样物质.酸蚀15 s牙本质小管口开放,暴露清晰.酸蚀30 s暴露的胶原纤维表面比15 s组光滑,球状附着物减少;酸蚀60 s牙本质小管内主纤维数最减少,管内结构塌陷,次级纤维数量增加,存在纤维断裂的征象.结论 在实验时间0～60 s内,酸蚀15 s即可达到酸蚀目的 ,延长酸蚀时间,可引起更多的胶原纤维变性降解.

Abstract：

Objective To evaluate the effects of acid etching time on the degradation of type Ⅰcollagen in dentin. Methods Dentin was conditioned with 37% phosphoric acid for 10, 15, 30 and 60 s.There was no treatment for the control group. Quantity of collagen degradation in each group was determined by enzyme linked immunosorbent assay. Observations were carried out by means of a field emission in-lens scanning electron microscope (FEISEM). Results Samples conditioned with 37% phosphoric acid for 60 s showed the most degradation of collagen, which was 4. 86 (1.55) mg/g, followed by 30 s group and 15 s group, which were 2.76(0.87) mg/g and 1.93(0.88) mg/g, respectively. Group of 10 s was 0.95(0. 38) mg/g. The control group showed the least degradation of 0. 06(0. 03) mg/g. Significant differences in collagen degradation were found among groupo (P ＜ 0. 005). Smear layer were removed well but tubular orifices and collagen fibrils were covered by particles after dentin being etched with 37% phosphoric acid for 10 s, while open and clear tubular orifices were observed for 15 s group. Smoother surfaces of exposed collagen fibrils and fewer globular particles were found in 30 s group than in 15 s group. In the 60 s group,the number of major fibrils decreased while minor branching fibrils increased, which indicate that the intratubular structure collapsed and fibrils fractured. Conclusions Dentin conditioned with 37% phosphoric acid for 15 s can result in mineral dissolution without collagen structure damage. However, longer applications of 37% phosphoric acid within 60 s may increase collagen degradation.     

绿茶提取物表没食子儿茶素没食子酸酯对酸蚀症牙本质胶原降解作用的初步研究

目的观察绿茶提取物表没食子儿茶素没食子酸酯（EGCG）对酸蚀症暴露的牙本质胶原降解作用的影响。方法选取酸蚀症志愿者64名,随机分成两组。制作个别托盘,托盘内注入羧甲基纤维素钠（CMC）凝胶（对照组）或添加了EGCG的CMC凝胶（试验组）。要求志愿者于夜间睡时佩戴,清晨取下。试验前1 d,以及试验后第1、2、3、4周收集培养液,用放射免疫试剂盒法测量Ⅰ型胶原羧基端肽（ICTP）的质量浓度。结果凝胶种类和使用时间对ICTP的质量浓度均有影响;EGCG能快速、有效地降低ICTP的表达水平,与对照组相比,差异有统计学意义（P<0.05）。结论EGCG能有效地减少脱矿牙本质胶原的降解,可以提高牙本质抗酸蚀的能力。     

钟状期牙胚来源的猪牙乳头细胞培养

目的 观察新生猪牙胚的发育情况并培养猪牙乳头细胞,对传代细胞的生物学特性进行研究.方法 选择3日龄新生猪,分离牙胚,通过组织学、免疫组化等研究牙胚的发育情况.分离牙乳头组织,酶消化法培养牙乳头细胞并鉴定;观察细胞生长特性,通过牙本质涎蛋白、Ⅰ型胶原、Ⅲ型胶原免疫组化染色比较体内外细胞的生物学性状.结果 新生猪磨牙尚未萌出,其中第二磨牙为典型的钟状期牙胚.分离培养的牙乳头细胞在体外生长良好,经鉴定细胞来源于间充质组织,免疫组化表明,牙本质涎蛋白和Ⅰ型胶原表达阳性,Ⅲ型胶原表达阴性.结论 新生猪可以提供处于钟状期的牙胚,通过酶消化法可成功培养猪牙乳头细胞,第3代细胞的生物学特性与体内细胞有一定相似性,可用于进一步深入研究.     

半胱氨酸组织蛋白酶对牙本质粘接耐久性的影响

半胱氨酸组织蛋白酶是组织蛋白酶家族的主要成员.牙本质酸蚀脱矿后裸露的胶原纤维被内源性蛋白酶降解是影响牙本质粘接耐久性的主要因素之一.除基质金属蛋白酶外,半胱氨酸组织蛋白酶也在粘接过程中被激活并参与混合层中胶原的破坏.本文就半胱氨酸组织蛋白酶与基质金属蛋白酶的相互作用、在牙本质粘接中的作用以及半胱氨酸组织蛋白酶抑制剂对提高牙本质粘接耐久性的作用的研究进展作一综述.     

氧化海藻酸钠预处理提高树脂牙本质粘接的耐久性

目的研究氧化海藻酸钠（ADA）改性脱矿牙本质胶原的交联度和显微形貌,及其对牙本质基质金属蛋白酶（MMP）的抑制作用和树脂牙本质粘接强度的影响。 方法制备ADA,进行傅里叶红外线光谱分析（FTIR）。采用0.5%、1%、2%、3%和4%的ADA处理脱矿牙本质粉末（DDP）1、2、30 min,5%戊二醛溶液作为阳性对照,茚三酮比色法检测胶原交联度。制备（1.0 ± 0.1）mm厚牙本质片,35%磷酸溶液酸蚀后ADA处理1 min,保持表面干燥或湿润,场发射扫描电镜（FESEM）观察其显微形貌。Sensolyte Generic MMP assay kit试剂盒检测不同浓度ADA对MMP-9的抑制作用。制备树脂牙本质粘接样本,检测ADA预处理试件冷热循环前后的微拉伸强度。使用SPSS 22.0对交联度实验进行多因素方差分析和Tukey检验,MMP-9抑制实验进行秩和检验和Bonferroni检验,微拉伸实验进行单因素方差分析和LSD检验。 结果FTIR显示海藻酸钠氧化后生成醛基形成ADA。DDP经ADA处理后,胶原交联度随处理浓度及处理时间呈依赖性增加,不同浓度组间（F = 1329.423,P<0.001）及不同时间组间（F = 1142.93,P<0.001）差异均有统计学意义。35%磷酸溶液脱矿牙本质表面经处理后,除阴性对照和0.5% ADA干燥处理组胶原纤维塌陷外,其余组均保持蓬松状态。0.5% ~ 4% ADA对MMP-9的抑制程度为93.5% ~ 100%,不同浓度组间差异有统计学意义（F = 13.786,P<0.001）。ADA预处理后,2% ADA组粘接样本冷热循环前后的粘接强度最高,分别为（28.2 ± 4.2）、（18.3 ± 3.7）MPa,差异有统计学意义（F_前 = 5.544,P_前<0.001;F_后 = 5.181,P_后<0.001）MPa。 结论2% ADA处理脱矿牙本质可提高Ⅰ型胶原纤维的交联度,使胶原纤维网保持蓬松状态,同时能抑制MMP-9活性,提高树脂牙本质粘接即刻及老化后的微拉伸强度,有利于改善树脂牙本质的粘接耐久性。     

I型和Ⅲ型胶原在修复性牙本质形成中的免疫定位

目的 :研究 I型和 型胶原在修复性牙本质形成中的免疫定位和分布特征。方法 :在大鼠第一磨牙制备单面洞 ,观察修复性牙本质形成。免疫组化 SABC法检测 I型胶原和 型胶原的免疫反应。结果 :在术后 3d,修复性牙本质尚未形成。I型胶原和 型胶原均分布于牙髓内 ,前期牙本质为弱阳性。术后 15 d,I型胶原和 型胶原在牙髓细胞内呈阳性染色。I型胶原在前期牙本质中呈弱阳性。术后 30 d,I型胶原和 型胶原的强阳性染色集中于已形成的修复性牙本质下的牙髓内 ,成牙本质细胞样细胞染色呈强阳性。结论 :形成修复性牙本质的成牙本质细胞样细胞合成、分泌 I型胶原和 型胶原。 I型胶原是修复性牙本质的主要基质成分     

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。     

牙本质中可溶性磷蛋白对再矿化的抑制作用

目的 研究去除牙本质中可溶性磷蛋白后对进一步再矿化的影响，了解磷蛋白在牙本质矿化中的作用机制。方法 用EDTA脱矿法去除牙本质中的固有可溶性磷蛋白，获得脱矿的牙本质籽晶，进行再矿化实验，并与以正常牙本质和乙酸脱矿法（可保留可溶性磷蛋白）获得的牙本质再矿化过程对照。实验应用等组分晶体生长体系，记录再矿化过程添加液何种随时间的增加量，以计算再矿化速率。结果 经EDTA脱矿0.5h及2h的牙本质籽晶的再矿化速率比正常牙本质粉明显增快，在200min时观察到的速率增加可超过100%，而脱矿5h和10h以及所有乙酸脱矿牙本质籽晶的再矿化速率均较正常未脱矿籽晶慢。结论 牙本质固有的可溶性磷蛋白具有抑制脱矿牙本质再矿化的作用。     

Immunohistochemical analysis of collagen fibrils within the hybrid layer: a FEISEM study

This study evaluated the immunohistochemical labeling pattern of dentin collagen fibrils within hybrid layers created by different bonding systems using high resolution SEM. Four different adhesive materials, including self-etching and total-etching systems, were examined: Prime & Bond NT, OptiBond SOLO Plus, Single Bond and Clearfil Protect Bond. All materials were applied to the dentin of extracted human third molars. After cutting the bonded specimens transversely, an anti-collagen type I antibody was incubated on the surface of the dentin-adhesive interface and gold-conjugated secondary antibody was applied to reveal collagen labeling under high resolution SEM. The hybrid layers showed a significant number of collagen fibrils embedded in the resin matrix. The presence of exposed dentin collagen fibrils, as determined by positive labeling with an anti-type I collagen monoclonal antibody, is considered an indication of the presence of incompletely embedded fibrils and, thus, the quality of dentin matrix hybridization. The hybrid layers produced by total-etching systems showed higher labeling compared to those produced by the self-etching system. Positive collagen labeling was also found along the resin tags produced by total-etching adhesive systems.     

High resolution SEM evaluation of dentin etched with maleic and citric acid.

OBJECTIVES: This study evaluated the ultra-morphological effects of maleic and citric acid on human dentin by means of a field emission in-lens scanning electron microscope (FEISEM). Both acids were tested on human dentin at pH 0.7 and 1.4 in aqueous solutions. METHODS: Each of 12 dentin disks were divided into four groups and exposed to either maleic acid at pH 0.7, maleic acid at pH 1.4, citric acid at pH 0.7 and citric acid at pH 1.4. All samples were then fixed and dehydrated in a critical point drying apparatus. Observations were carried out by means of a FEISEM (JEOL 890) after coating with a carbon-platinum film. RESULTS: Both acids removed smear layer and partially removed smear plugs. Details of fine structures measuring from 5 to 15 nm were shown on the intertubular demineralized dentin. Maleic acid at pH 0.7 showed the highest depth of demineralization of all the tested samples; citric acid, showed a higher depth of demineralization values when tested at pH 1.4 than at pH 0.7. SIGNIFICANCE: The FEISEM reveals ultra-structural aspects of the demineralization process of the dentin tissue of the both acids tested. Differences related to the pH of the acids were found. Images obtained at high magnification clarify the dentin collagen structure of both peritubular and intertubular dentin. Small periodic structures associated with collagen fibrils were also imagined.     

Effects of citric acid and EDTA conditioning on exposed root dentin: An immunohistochemical analysis of collagen and proteoglycans

OBJECTIVE: Preservation of structural and biochemical properties of the root dentin matrix is crucial to favor healing and regenerative periodontal processes. Aim of this study was to evaluate the biochemical characteristics of collagen and chondroitin sulphate of root dentin surfaces exposed by periodontal disease after acid conditioning by means of an immunohistochemical technique. DESIGN: Human teeth scheduled for extraction due to periodontal reason were submitted to: (A) scaling and root planning; (B) ultrasonic instrumentation; (C) no instrumentation. Teeth were then exposed to: (1) 10% citric acid; (2) 17% EDTA; (3) no etching. A double immunolabeling technique was performed to identify type-I collagen and proteoglycans and analyzed under FEI-SEM. RESULTS: Use of 10% citric acid revealed intense labeling for collagen fibrils and proteoglycans; lower labeling was found after EDTA conditioning. Unetched specimens showed residual smear layer on the dentin surface resulting in no evident surface labeling. CONCLUSIONS: This study supports the hypothesis that manual or ultrasonic instrumentation alone is not able to expose the sound dentin matrix, whereas a subsequent acidic conditioning exposes collagen fibrils and associated proteoglycans. The immunohistochemical technique revealed that despite their acidity, both citric acid and EDTA were able to preserve the structural and biochemical properties of the exposed dentin matrix.     

Effect of dentinal pretreatments on coronal dentin primary carious lesions: a field emission SEM study

The aim of the study was the morphological analysis of coronal dentin caries and the modifications induced by different pretreatments with phosphoric acid or sodium hypochlorite. Carious dentin specimens were obtained from human molars affected by carious lesions. Specimens were divided in four groups and submitted to: (1) untreated; (2) 35% phosphoric acid for 15 s; (3) 35% phosphoric acid for 15 s and 5% sodium hypochlorite for 2 min; (4) sodium hypochlorite for 5 min. Specimens were observed under high-resolution SEM. Different areas were identified within the carious lesion: a deeper, inner region revealing closed highly mineralized tubules, and a more superficial outer layer showing an increasing demineralization rate toward the surface of the lesion. Phosphoric acid followed by NaOCl treatment removed all collagen fibrils from greatly altered carious outer-dentin layer. The 5-min treatment with sodium hypochlorite affected both inner and outer dentin, removing all collagen fibrils and increasing the porosities of deeper intertubular hypermineralized dentin. FEISEM analysis confirmed that only inner carious dentin after phosphoric acid treatment may be considered a suitable substrate for dentinal bonding system. On the contrary, the outer dentin is an unstable substrate for any type of bonding systems and must be avoided/removed from any surface before conditioner application.     

Reduced antigenicity of type I collagen and proteoglycans in sclerotic dentin

Antigenic alterations to the dentin organic matrix may be detected by an immunohistochemical approach. We hypothesized that alterations in the antigenicity of type I collagen and proteoglycans occur in sclerotic dentin under caries lesions. Transverse sections were prepared from carious teeth in the sclerotic zone and normal hard dentin. A double-immunolabeling technique was performed on these sections, with anti-type I collagen and anti-chondroitin 4/6 sulfate monoclonal primary antibodies. We used gold-conjugated secondary antibodies to visualize the distribution of intact collagen fibrils and proteoglycans by high-resolution SEM. For sclerotic dentin, labeling densities were 19.57 +/- 3.01/microm2 for collagen and 9.84 +/- 2.62/microm2 for proteoglycans. For normal hard dentin, values were 35.20 +/- 2.73/microm2 and 17.03 +/- 1.98/microm2, respectively. Distribution of intact collagen fibrils and proteoglycans in sclerotic dentin was significantly lower than in normal hard dentin. Reductions in antigenicity from the organic matrix of sclerotic dentin under caries lesions raise concern about the potential of intrafibrillar remineralization.     

Grape seed proanthocyanidins increase collagen biodegradation resistance in the dentin/adhesive interface when included in an adhesive

Objectives

Contemporary methods of dentin bonding could create hybrid layers (HLs) containing voids and exposed, demineralised collagen fibres. Proanthocyanidins (PA) have been shown to cross-link and strengthen demineralised dentin collagen, but their effects on collagen degradation within the HL have not been widely studied. The purpose of this study was to compare the morphological differences of HLs created by BisGMA/HEMA model adhesives with and without the addition of grape seed extract PA under conditions of enzymatic collagen degradation.

Methods

Model adhesives formulated with and without 5% PA were bonded to the acid etched dentin. 5-μm-thick sections cut from the bonded specimens were stained with Goldner's trichrome. The specimens were then exposed to 0.1% collagenase solution for 0, 1, or 6 days. Following collagenase treatment, the specimens were analysed with SEM/TEM.

Results

Staining did not reveal a difference in the HLs created with the two adhesives. SEM showed the presence of intact collagen fibrils in all collagenase treatment conditions for specimens bonded with adhesive containing PA. These integral collagen fibrils were not observed in the specimens bonded with adhesive without PA after the same collagenase treatment. TEM confirmed that the specimens containing PA still showed normal collagen fibril organisation and dimensions after treatment with collagenase solution. In contrast, disorganised collagen fibrils in the interfacial zone lacked the typical cross-banding of normal collagen after collagenase treatment for specimens without PA.

Conclusions

The presence of grape seed extract PA in dental adhesives may inhibit the biodegradation of unprotected collagen fibrils within the HL.     

Histomorphologic Characterization of Noncarious and Caries-Affected Dentin/Adhesive Interfaces

PURPOSE: The purpose of this study was to compare the dentin/adhesive interfacial characteristics when bonding to noncarious as well as caries-affected dentin. MATERIALS AND METHODS: Seven extracted, unerupted, third molars were sectioned into halves. Artificial caries was created on one-half of each tooth, leaving the other half as a control. Dentin surfaces were treated with UNO adhesive according to the manufacturer's instructions for the wet-bonding technique and under environmental conditions present in the oral cavity. Dentin/adhesive interface sections of each half-tooth were stained with Goldner's trichrome, a classic bone stain, and examined using light microscopy. The width of exposed collagen was measured directly from photomicrographs, and adhesive penetration was analyzed qualitatively. RESULTS: The degree and extent to which the adhesive encapsulated the demineralized dentin matrix were reflected in the color difference in the stained sections with the noncarious dentin sections showing a degree of collagen encapsulation superior to that of the caries-affected dentin sections. The overall mean widths of exposed collagen were significantly (p < or = .05) greater at the caries-affected dentin/adhesive interface, 8.6 (1.7) microm, as compared with those at the noncarious dentin/adhesive interface, 6.0 (1.5) microm. CONCLUSIONS: The morphologic characteristics of the caries-affected dentin/interface suggest an increase in the exposed collagen zone and a decrease in the quality of the adhesive infiltration when compared with noncarious dentin. The evidence suggests that dentin substrate characteristics have a significant effect on the dentin/adhesive interface structure.     

Long-term culture of human periodontal ligament cells with autologous root discs

Recent attempts at gaining an understanding of the factors necessary for periodontal reattachment have employed an in vitro system in which connective tissue cells are cultured with dental root discs. The present study was undertaken to optimize culture conditions. Periodontal ligament (PDL) cells and root discs were derived from 2 wisdom teeth extracted from each of 3 patients. Pairs of planed root discs (0.5 mm thick) were gently placed in dishes containing autologous PDL-cells and separated by a 0.5 mm wide gap. After 10 to 13 weeks in culture the specimens were processed for light- and electron microscopy and the collagen fibril diameter was determined. In the interdental space, all cultures contained an aggregate of cells and collagen fibrils. Adherent to root discs with exposed dentin was a loose capsule of cells and collagen fibrils oriented parallel to the disc surface. Adjacent to cellular cementum a discontinuous, dense, short fiber fringe was found along the cultureJroot interface. There was some intermingling between cemental and culture collagen fibrils. The fibril diameter of cemental collagen (59.4 ± 5.0 nm) and culture collagen (31.8 ± 3.5 nm) was very different. Thus, autologous PDL-cell/disc-cultures can be maintained for prolonged periods of time, resulting in the formation of discontinuous patches of condensed collagen fibrils radiating from some portions of the decalcified cemental surface.