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。     

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.     

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     

Ⅰ型牙本质发育不全伴成骨不全1例

成骨不全(osteogenesis imperfecta, OI)又称脆骨病,是一组结缔组织遗传性疾病,以骨骼脆弱和频繁骨折为主要特征。其出生时的发病率为1∶10000～1∶20000。OI具有多种继发性特征,如蓝色巩膜、牙本质发育不全(dentinogenesis imperfecta, DGI)、听力损失等。本文报告了1例24岁男孩,其主诉为牙齿折断伴疼痛。根据病史采集、临床检查、影像学检查、和基因学检查,我们得出了OI伴Ⅰ型牙本质发育不全的结论。     

牙本质发育不良Ⅰ型及其分型治疗

牙本质发育不良Ⅰ型（DD-Ⅰ）是一种罕见的遗传性牙本质形成障碍疾病,乳恒牙均可受累。该病在临床上表现为牙冠外形色泽正常,牙齿松动明显,可伴有自发性牙槽脓肿或囊肿等。影像学检查则可见牙髓腔消失或呈“新月形”牙髓残余,根短钝或无牙根等表现。关于DD-Ⅰ的发病机制已为大多数学者所研究,其临床治疗通常具有挑战性,本文对近年来DD-Ⅰ的临床分型及表现、致病基因、组织学特点、治疗相关研究进行综述,以期为临床诊治该病提供一定的指导。     

一例牙本质发育不良Ⅰ型患者的临床特点及其种植治疗

牙本质发育不良Ⅰ型（dentin dysplasia type Ⅰ,DD-Ⅰ）是一种伴有牙本质形成障碍、可遗传的罕见病。其特征是牙冠正常,但牙根发育异常,出现短、钝化和畸形的牙根;在患者年轻时即出现牙松动,可伴牙槽脓肿;影像学上表现为闭塞的牙髓腔。典型的DD-Ⅰ是常染色体显性遗传,家系中患者常很早即出现多颗牙丧失,有的30多岁即可表现为无牙颌。本文报告了一例DD-Ⅰ病例,通过总结其临床表现、影像学及组织学特点以及相关治疗,以期为DD-Ⅰ临床诊治提供指导。     

髁突骨上组织改建中Ⅰ、Ⅱ型胶原的免疫组织化学研究

目的：研究髁突骨上组织改建中Ⅰ、Ⅱ型胶原的变化特征,增加对颞颌关节改建的进一步认识。方法：用免疫组织化学的方法检测拔除成年家兔左侧下凳磨牙后２周、１月和３月时Ⅰ、Ⅱ型的分布及含量的变化。结果：术后２周Ⅰ、Ⅱ型胶原的表达均明显减少,１月和３月其表达有所回升,并且１型胶原出现不均衡分布,拔牙侧与非拔牙侧相比,前者１型胶原的表达较后者稍强,而２型胶原的表达则较弱。结论：成年家兔颞颌关节的改建能力有限,超     

波形蛋白标记观察人恒前磨牙成牙本质细胞突的分布

目的 :利用免疫酶组化染色技术 ,对人恒前磨牙成牙本质细胞突内的波形蛋白进行定位研究。方法 :将牙齿拔除后立即磨成薄片 ,10 0ml/L中性福尔马林液固定 72h ,10 0ml/L硝酸脱钙。石蜡包埋 ,制取6 μm厚的组织切片。采用SABC法进行免疫组化染色。 结果 :阳性染色的波形蛋白结构在牙本质小管内呈连续较直的长条状。在冠部至釉牙本质界 ,在根部达牙本质小管末端。其分布趋势是从牙本质内层到外层逐渐减少 ,阳性表达程度逐渐减弱。结论 :成牙本质细胞突贯通牙本质全层达牙本质小管末端。     

实验性牙龈炎Ⅰ型胶原的变化

目的观察实验性牙龈炎早期炎症牙龈组织Ⅰ型胶原的变化。方法以正畸钢丝拴结豚鼠切牙牙颈部，取临床牙周炎患者牙周袋内菌斑种入豚鼠受试牙龈沟，连续5天，受试牙龈局部红肿、龈沟加深，形成实验性牙龈炎动物模型。取其切牙连同周围牙同组织，常规石蜡切片，免疫组化PAP法检查牙龈Ⅰ型胶原，同时HE染色。结果炎性牙龈Ⅰ型胶原较正常着色明显减弱，着色缺乏连续性。结论早期炎症已有胶原受损，其损伤可能主要由炎症反应所致。     

PAH对氧化锆前体在胶原纤维内沉积的催化效应

目的：分析PAH对氧化钇稳定的氧化锆前体在胶原纤维内沉积过程中的作用。方法：在氧化钇稳定的氧化锆前体溶液或胶原支架中分别不加或加入PAH,通过颗粒度、TEM、SEM、XRD的检测,比较PAH对氧化锆前体沉积在胶原纤维内的催化作用。结果：加入PAH的颗粒度较未加入前增高。 TEM、SEM显示只有加PAH的YSZ才能沉积在I型胶原内。XRD结果显示高温烧结使胶原中的氧化锆由无定形态转变为四方晶体。结论：PAH对氧化钇稳定的氧化锆起催化作用,且PAH能促进YSZ沉积在胶原纤维内。     

Effect of Odanacatib on the release of NTX (Amino Terminal Telopeptide) from LPS contaminated type I dentin collagen

ObjectiveTo evaluated the Odanacatib inhibitor treatment on lipopolysaccharide (LPS) contamination effect on cathepsin-K mediated dentin degradation by analysis of type I collagen C- and N-termini telopeptides.MethodsPulverized and disks of human dentin were demineralized and LPS contaminated, or stored in deionized water (DW) for 12 h. Samples were challenged with lactic acid (LA). Aliquots of dentin powder were treated with 1 mL Odanacatib or stored in DW for 30 min. Dentin collagen degradation was determined by sub-product release of C-terminal (ICTP and CTX) and N-terminal (NTX) telopeptides, normalized to total protein (tp) concentration (n = 3). Dentin matrix was evaluated for gravimetric (n = 8) and ultrastructural changes. Data were analyzed by Student t-test, one-way ANOVA and Tukey’s test (α = 5 %).ResultsLA incubation significantly increased telopeptide release compared with DW (p < 0.05). In untreated groups, significantly higher CTXtp, NTXtp telopeptide rates were observed for LA+LPS samples compared with DW (p < 0.01). Odanacatib significantly reduced ICTPtp, CTXtp, and NTXtp telopeptide release for LPS, LA, and LA+LPS conditions. In untreated groups, LPS and LA+LPS challenge significantly increased dentin weight loss (p = 0.02). Within each storage condition, Odanacatib treatment did not affect weight change (p > 0.05) of dentin disks.SignificanceThis study showed that LPS contamination resulted in significantly higher rates of NTX than CTX from dentin matrix. Odanacatib significantly reduced telopeptide release rates of LPS contaminated dentin matrix.     

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.     

聚甲基丙烯酸β-羟乙酯对牙本质基质金属蛋白酶活性的影响

目的研究聚甲基丙烯酸β-羟乙酯（PHEMA）对牙本质中基质金属蛋白酶（MMP）的影响。 方法通过本体聚合将树脂单体甲基丙烯酸β-羟乙酯（HEMA）制备成PHEMA,同时制备人牙本质粉,并随机平均分成6组,对照组不做处理,实验组以不同高度的PHEMA试件处理,提取牙本质蛋白,用Ⅰ型胶原吡啶交联终肽（ICTP）试剂盒分析样本中ICTP的浓度,应用SPSS 17.0软件包对实验数据进行统计学分析,采用单因素方差（One-Way ANOVA）分别分析PHEMA和时间这两个处理因素对的ICTP影响,选择LSD-t检验进行组间两两比较,明胶酶谱法检测PHEMA对牙本质中MMP-2、MMP-8、MMP-9活性的影响,通过Image J图像分析软件,采用灰度法进行图像分析。 结果ICTP浓度与MMP活性之间存在正相关关系。PHEMA可有效抑制ICTP的浓度（F = 26.792,P = 0.001）,且ICTP浓度随着时间延长而降低。PHEMA可有效抑制MMP-2（F = 15.317,P = 0.0009）、MMP-8（F = 6.475,P = 0.004）的活性,且抑制作用随接触面积的增大而增强;PHEMA对MMP-9（F = 1.093,P = 0.413）未出现明显的活性抑制作用。 结论PHEMA可有效抑制牙本质中MMP-2、MMP-8的活性,且抑制作用随接触面积的增大而增强。     

Effect of cryopreservation of teeth on the structural integrity of dentin

The autotransplantation of teeth after cryopreservation has become an increasingly viable method for whole tooth replacement. While the immediate success rates are quite high, damage introduced by cryopreservation within the dentin or enamel could be detrimental to the durability of these teeth.

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.     

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.     

Stabilization of dentin matrix after cross-linking treatments,in vitro

Objectives

To evaluate the effect of EDC on elastic modulus (E), MMPs activity, hydroxyproline (HYP) release and thermal denaturation temperature of demineralized dentin collagen.

Methods

Dentin beams were obtained from human molars and completely demineralized in 10 wt% H₃PO₄ for 18 h. The initial E and MMP activity were determined with three-point bending and microcolorimetric assay, respectively. Extra demineralized beams were dehydrated and the initial dry mass (DM) was determined. All the beams were distributed into groups (n = 10) and treated for 30 s or 60 s with: water, 0.5 M, 1 M or 2 M EDC or 10% glutaraldehyde (GA). After treatment, the new E and MMP activity were redetermined. The beams submitted to DM measurements were storage for 1 week in artificial saliva, after that the mass loss and HYP release were evaluated. The collagen thermal denaturation temperature (TDT) was determined by DSC analysis. Data for E, MMP activity and HYP release were submitted to Wilcoxon and Kruskal–Wallis or Mann–Whitney tests. Mass loss and TDT data were submitted to ANOVA and Tukey tests at the 5% of significance.

Results

EDC was able to significantly increase collagen stiffness in 60 s. 10% GA groups obtained the highest E values after both 30 and 60 s. All cross-linking agents decreased MMP activity and HYP release and increased TDT temperature. Significant differences were identified among EDC groups after 30 or 60 s of cross-linking, 1 M or 2 M EDC showed the lowest MMP activity.

Significance

Cross-linking agents are capable of preventing dentin collagen degradation. EDC treatment may be clinically useful to increase resin-dentin stability.     

Alternative model for cathepsin K activation in human dentin

ObjectiveTo evaluate the protease activity in dentin matrices subjected to lactic acid (LA) in comparison to polyacrylic acid (PAA) challenge model at cathepsin K (CT-K) optimum pH 5.5 to assess effectiveness of inhibitors in dentin collagen degradation.MethodsDentin disks measuring 0.5 mm prepared from human molars were completely demineralized in 10% H₃PO₄. Demineralized dentin disks were challenged with 0.1 M LA, 1.1 mM PAA, artificial saliva (AS), or deionized water (C) for 24 h or 7-days. Dentin collagen properties were tested by measurement of %dry mass change, and ultimate tensile strength (UTS). Degradation of dentin type I collagen was measured by telopeptide assays measuring the sub-product release of C-terminal cross-linked telopeptides (ICTP) and C-terminal peptide (CTX) in the incubation media in relation to total protein concentration, which correlates with matrix metalloproteinases (MMPs) and CT-K activities.ResultsGravimetric analysis showed statistically significant difference between C and other groups (p < 0.04) at 24 h. LA specimens showed significantly higher weight loss from 24 h to 7-days (p = 0.02). UTS revealed statistically significant difference between AS and LA at 24 h and 7-days. UTS at 24 h and 7-days for C and AS had significantly higher mean values compared to LA and PAA. Telopeptide assays reported that CTX_tp results showed that LA at 24 h had significantly higher mean values compared to C and AS.SignificanceLA has the ability to activate endogenous CT-K in dentin as measured by the release of CTX (CT-K specific telopeptide). This LA based model has the potential application for further investigations on the activity and possible inhibitors of CT-K in human dentin.