SD大鼠牙髓增龄变化对Nd：YAG激光照射的应激反应观察

目的：观察增龄牙髓对Nd：YAG激光照射的应激反应变化。方法：观察幼年组（6周龄）、成年组（12周龄）、老年组（72周龄）雄性SD大鼠切牙经不同能量密度脉冲式Nd：YAG激光照射后6h牙髓的组织形态学变化,利用图像分析系统对不同年龄大鼠成牙本质细胞中诱导型热休克蛋白（HSP70）表达的平均光密度值进行分析比较。结果：激光能量密度为1433mJ／mm^2和1910mJ／mm^2时,老年组成牙本质细胞中诱导型HSP70的表达强度显著高于幼年和成年组（P〈0．05）;能量密度为2388mJ／mm^2时,老年组成牙本质细胞中诱导型HSP70的表达强度显著低于幼年和成年组（P〈0．05）,且老年组部分成牙本质细胞变性坏死。结论：老年大鼠牙髓对激光刺激的耐受性下降。     

极固宁TM对活髓基牙牙髓组织影响研究

目的 研究极固宁TM对金属烤瓷全冠活髓基牙牙体预备后1、2周牙髓组织形态学的影响。方法 选择2012年1月至2013年8月在福建医科大学附属口腔医院就诊患者15例,因正畸治疗需要而计划拔除40颗前磨牙（同一患者的同颌同名牙20对）,将每对同颌同名牙随机分为对照组和试验组。对照组：金属烤瓷全冠牙体预备后直接用氧化锌丁香油水门汀黏固暂时冠;试验组：在牙预备体上涂布极固宁TM后用氧化锌丁香油水门汀黏固暂时冠。分别于牙体预备后1、2周局麻下拔除同颌同名研究牙。常规组织病理切片,HE染色,显微镜下观察评价牙髓组织反应并测量颊侧髓角的剩余牙本质厚度。结果 金属烤瓷全冠牙体预备后1、2周时,试验组与对照组的牙预备体颊侧髓角的剩余牙本质厚度差异均无统计学意义（P > 0.05）。活髓基牙全冠牙体预备后1周,对照组的牙髓组织炎症反应比试验组严重（P < 0.05）;而牙体预备后2周,对照组与试验组的牙髓组织反应差异无统计学意义（P > 0.05）。不论是金属烤瓷全冠牙体预备后1周或2周,所有样本均未见第三期牙本质的形成。 结论 极固宁TM在金属烤瓷全冠牙体预备后1周时可降低活髓基牙牙髓组织的炎症反应。     

常规饲养大白鼠暴露牙髓的硬组织形成

作者以前的研究已表明:大白鼠切牙和磨牙牙髓,对实验性穿髓引起的外源性刺激反应不同。在牙髓暴露后切牙牙髓没有显著变化,而磨牙牙髓中有硬组织形成。在本研究中,对磨牙牙髓产生的硬组织进行了超微结构观察。本实验中形成的硬组织有两层不同结构,即骨样牙本质和正牙本质样结构。骨样牙本质结构见于靠近暴露面的牙髓,与这种新形成硬组织有关的细胞类似成骨细胞或造牙本质细胞。这些细胞含有发育很好的粗面内质网和高尔基氏器,并有活跃的蛋白质形     

LIM矿化蛋白1在大鼠牙髓损伤修复中的时空表达

目的:探讨LIM矿化蛋白1(LIM mineralization protein 1,LMP-1)在大鼠磨牙牙髓损伤修复中的表达.方法:建立大鼠磨牙牙髓损伤修复动物模型,用免疫组织化学染色方法观察LMP-1在大鼠牙髓损伤修复中的表达,并用Image-Pro Plus 6.0图像分析软件及单因素方差分析比较各组间的差异.结果:在正常大鼠牙髓组织中LMP-1表达阴性;在大鼠牙髓损伤后1d,LMP-1表达于成牙本质细胞及部分牙髓成纤维细胞;术后3 d,LMP-1表达于坏死牙髓下方的细胞增殖层;术后7d,LMP-1显著表达于增殖活跃的牙髓细胞及部分成牙本质细胞中.结论:LMP-1在牙髓损伤修复过程中呈时空特异性表达,可能参与成牙本质细胞及牙髓细胞的增殖、分化及修复性牙本质的形成.     

黏结剂直接盖髓体外实验研究

目的:采用器官培养模型方法,评价自酸蚀黏结剂直接盖髓后的牙髓反应.方法:取临床上因正畸拔除的健康完整新鲜前磨牙30个,随机分为3组,制备<牙合>面洞,机械露髓,分别用自酸蚀黏结剂Xeno Ⅲ和氢氧化钙Dycal盖髓,复合树脂充填窝洞,余1组不做任何处理作正常对照,然后将牙齿纵向切割成2mm厚的牙片,DMEM体外培养3、7、14 d,分别进行HE检测和免疫组化检测.结果:HE显示术后3 d各组均表现为成牙本质细胞排列紊乱、毛细血管扩张;术后7、14 d牙髓逐渐恢复正常,3组均无牙本质桥形成,对照组釉质、牙本质、牙髓呈有序结构.免疫组化结果显示:术后3 d两组成牙本质细胞、露髓孔下方部分牙髓组织中DSPP均呈阳性表达,7、14 d表达同正常对照组.结论:自酸蚀黏结剂Xeno Ⅲ和氢氧化钙在短期内对牙髓组织刺激反应相似.     

TGF-β1在氟中毒大鼠切牙牙髓中的表达

目的:研究过量氟对大鼠牙髓细胞TGF-β1表达的影响。方法:20只Wister大鼠,随机分为对照组和实验组。对照组用等量蒸馏水灌胃,实验组以20mg.kg-1.d-1氟化钠水灌胃。8周后处死动物,利用磨片、HE染色、免疫组化染色技术观察过量氟对大鼠切牙形态及TGF-β1表达的影响。采用SPSS10.0软件对数据进行t检验。结果:实验组大鼠切牙釉质牙本质生长线明显,球间牙本质增多,牙髓细胞及髓腔内侧牙本质TGF-β1表达显著弱于对照组,差异有显著性(P<0.01)。结论:过量氟可抑制牙髓细胞表达TGF-β1,影响牙体硬组织的结构。     

咬合创伤后牙本质形成相关细胞的形态学变化

目的：探讨大鼠咬合创伤牙齿牙本质形成相关细胞以及牙本质的变化。方法：制备大鼠下颌第一磨牙咬合创伤,观察7d、15d、30d、90d时间点牙本质的形成与吸收,牙髓组织中与牙本质形成相关的细胞的形态和数目变化。结果：实验性咬合创伤的早期,牙髓组织中成牙本质细胞和多细胞层细胞增生活跃,牙本质形成增多现象,增生区域的牙体硬组织表面为坚硬组织破坏和牙本质暴露。后期可以出现细胞萎缩现象,牙本质吸收明显。结论：咬合创伤影响牙本质的形成功能,这种影响可能与相应区域牙本质是否暴露无关。     

即刻牙本质封闭技术的研究进展

大量牙体缺损时一般使用修复体修复,但活髓牙的修复体修复过程中牙体预备导致大量的牙本质暴露,在暂时修复期间细菌的毒素及外界的冷热刺激可能会导致敏感。临床上缓解活髓牙牙体预备后敏感的方法主要有药物、激光或两者的联合应用,无论哪种方法其原理都是封闭牙本质小管以隔绝外界的刺激^[1]。     

牙本质非胶原蛋白诱导牙髓-牙本质复合体反应的动物实验观察

目的：观察提取的猪牙本质非胶原蛋白对修复性牙本质形成的诱导活性。方法：用提取的猪EDTA可溶性牙本质非胶原蛋白作盖髓剂,对Wistar大鼠的健康第一磨牙进行直接盖髓实验,采用氢氧化钙（Dycal）和空白组为对照,通过组织学实验观察牙髓-牙本质复合体的反应。结果：盖髓术后7 d,各组之间软、硬组织反应的差异无统计学意义。术后14 d,NCPs组和Dycal组的修复反应均明显优于空白对照组（P〈0.01）,且NCPs组对牙髓的刺激小于Dycal组（P〈0.05）。结论：NCPs对牙髓刺激性小,可以诱导牙髓细胞分化为成牙本质细胞样细胞,形成修复性牙本质,其诱导活性至少与常规盖髓剂相当。     

骨唾蛋白和骨连接蛋白在正常年轻恒牙及龋患牙中分布的研究

目的 :旨在研究骨唾蛋白 (bonesialoprotein ,BSP)和骨连接蛋白 (osteonectin ,ON)在正常年轻恒牙和龋患牙中的表达 ,以了解这两种蛋白在牙本质形成过程中的作用。方法 :制备 5 μm厚连续切片 ,用于BSP和ON的免疫组化定位研究 ,并通过图像分析 ,将这两种蛋白在牙髓中央和牙髓周围组织的表达强度进行比较。结果 :BSP和ON均在正常恒牙的成牙本质细胞及前期牙本质中表达 ,且在牙髓中央和牙髓周围组织的表达强度有显著性差异 ,在龋患牙中BSP牙髓外层着色依然显著高于中央区 ,且BSP抗体在第 2、3期牙本质交界处呈强阳性反应。ON则在龋患牙中普遍表达 ,在第 3期牙本质中表达不明显。结论 :BSP和ON可能参与第 2期牙本质的形成。第 3期牙本质形成可能与BSP有关 ,与ON无关     

Upregulation of transient receptor potential ankyrin 1 (TRPA1) but not transient receptor potential vanilloid 1 (TRPV1) during primary tooth carious progression

ObjectiveTo quantify the changes in Transient Receptor Potential Ankyrin 1 (TRPA1) and Transient Receptor Potential Vanilloid 1 (TRPV1) expression throughout the process of inflammation induced by caries.MethodsForty primary teeth were obtained from children requiring dental extractions under local or general anesthesia. The teeth were grouped according to three stages reflecting the progression of dental caries: nine with intact dentin, 15 with exposed dentin (but not to the extent of the pulp), and 16 with exposed pulp. Immunofluorescence was used to validate the presence of dental pulp inflammation by demonstrating a decrease in NF-κB nuclear translocation. The expression levels of TRPA1 and TRPV1 were quantified in the pulp horn and the subodontoblastic and midcoronal regions of the pulp.ResultsThe percentage of cells with NF-κB nuclear translocation was highest for teeth with intact dentin and decreased progressively during the progression of caries. TRPA1 expression was lowest in intact teeth and gradually increased as caries advanced. TRPV1 expression was similar in teeth with intact dentin, exposed dentin, and exposed pulp.ConclusionThe differences in TRPA1 and TRPV1 expression in response to caries suggest that these receptors play unique roles in the immune response during the progression of caries and that the pathophysiology of inflammation in the dental pulp varies between the early and late stages of caries.     

层粘连蛋白在修复性牙本质形成中的免疫定位

目的 研究层粘连蛋白（laminin,LN）在牙髓修复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,分别观察3d、15d和30d后修复性牙本质的形成情况。以免疫组化技术检测LN的免疫反应。结果 术后3d,修复性牙本质尚未形成。牙髓内成牙本质细胞呈阳性染色,前期牙本质为弱阳性。术后15d,可见修复性牙本质形成和成牙本质细胞样细胞的出现,成牙本质细胞样细胞和牙髓细胞免疫染色呈阳性。术后30d,已形成的修复性牙本质内LN染色阳性,免疫活性特别集中于修复性牙本质与牙髓交界处,成牙本质细胞样细胞染色呈强阳性。结论 LN的分布特征提示,在修复性牙本质形成过程中,LN可能是牙髓细胞附着的一个有利因素。     

低能量激光疗法联合Dycal用于大鼠磨牙直接盖髓术的体内研究

目的:观察低能量激光疗法(LLLT)联合Dycal用于大鼠磨牙直接盖髓对牙髓炎、修复性牙本质形成及IL-1β、TRPA1表达的影响。方法:64只大鼠随机均分成对照组(NC)、低能量激光组(LLLT)、Dycal组(Dy)、联合组(LLLT+Dy)(n=16)。直接盖髓术后3、7、14、28 d取标本,行HE及免疫组化(IHC)染色观察。结果:LLLT组与LLLT+Dy组炎症反应在术后14、28 d都存在显著性差异(P<0.016 7);修复性牙本质桥形成在术后14 d, LLLT+Dy组与LLLT组、Dy组存在显著性差异(P<0.016 7);术后28 d, LLLT+Dy组与LLLT组存在显著性差异(P<0.016 7)。IL-1β、TRPA1阳性MOD值在术后7 d存在显著性差异,Dy组>LLLT组>LLLT+Dy组>NC组(P<0.05),术后14 d存在显著性差异,LLLT组、Dy组均>LLLT+Dy组(P<0.05)。结论:低能量激光联合Dycal可能通过减少IL-1β、TRPA1的表达来减轻牙髓炎症、促进修复性牙本质的形...     

PIEZO1 Ion Channels Mediate Mechanotransduction in Odontoblasts

IntroductionOdontoblasts, terminally differentiated dentin-forming cells with their processes that penetrate into dentin, have been considered potential sensory cells. Current research suggests that odontoblasts sense external stimuli and transmit pain signals. PIEZO1, as a specific mechanically activated ion channel, may play an important role in mechanical transduction in odontoblasts. In this study, we devoted to investigating the functions and underlying molecular mechanisms of PIEZO1 ion channels in odontoblast mechanotransduction.MethodsHuman dental pulp stem cells were cultured in vitro and induced to differentiate into odontoblast-like cells (OLCs). The expression of PIEZO1 protein in pulp, dental pulp stem cells, and OLCs was detected by immunohistochemistry or immunofluorescence. The mechanical sensitivity of OLCs was detected by a constructed fluid shear stress model and examined by calcium fluorescence intensity. A single-cell mechanical stimulation model was used to detect the PIEZO1 electrophysiological properties of OLCs. Yoda1 (a PIEZO1-specific agonist), GsMTx4 (a PIEZO1 antagonist), and non–calcium ion extracellular solution were utilized to confirm PIEZO1 mechanotransduction in OLCs in both fluid shear stress and single-cell mechanical stimulation assays. The amount of ATP released by OLCs was measured under stimulation with Yoda1 and GsMTx4. Rat trigeminal ganglion neurons were cultured in vitro and detected by whole-cell patch-clamp recording under ATP stimulation.ResultsPIEZO1 ion channels were positively expressed in OLCs and odontoblastic bodies and processes but weakly expressed in dental pulp cells. After the treatment of OLCs with shearing stress or Yoda1, the fluorescence intensity of intracellular calcium ions increased rapidly but did not noticeably change after treatment with GsMTx4 or the non–calcium ion extracellular solution. When single-cell mechanical stimuli were applied to OLCs, the evoked inward currents were recorded by patch-clamp electrophysiology. The inward currents increased and current inactivation became slower after Yoda1 treatment, but these currents almost completely disappeared after the addition of GsMTx4. The amount of ATP released by OLCs increased significantly after Yoda1 stimulation, while GsMTx4 reversed the release of ATP. Whole-cell patch-clamp detection showed that ATP evoked slow inward currents and increased the frequency of action potentials of trigeminal ganglion neurons.ConclusionsTaken together, these findings indicated that odontoblasts evoked a fast inward current via PIEZO1 ion channels after the application of external mechanical stimuli and released ATP to transmit signals to adjacent cells. Thus, PIEZO1 ion channels in odontoblasts mediate mechanotransduction under various pathophysiological conditions in dentin.     

Human dental pulp fibroblasts express the "cold-sensing" transient receptor potential channels TRPA1 and TRPM8

Introduction

Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts.

Methods

Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca²⁺ microfluorimetry.

Results

Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca²⁺ microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca²⁺]_i) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists.

Conclusions

Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth.     

纤维粘连蛋白在牙髓修复中作用的免疫组织化学研究

目的:研究纤维粘连蛋白在牙髓修复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,分别观察3 d、15 d和30 d后修复性牙本质形成情况。免疫组织化学技术检测纤维粘连蛋白的免疫反应。结果:术后3 d, 修复性牙本质尚未形成。牙髓内成牙本质细胞呈弱阳性染色,前期牙本质也为弱阳性。术后15 d,可见修复性牙本质形成,成牙本质细胞样细胞和牙髓成纤维细胞免疫染色呈阳性。术后30 d,已形成的修复性牙本质内纤维粘连蛋白染色为强阳性,特别集中于修复性牙本质与牙髓的交界面,成牙本质细胞样细胞染色呈强阳性。结论:纤维粘连蛋白可能在修复性牙本质形成中起重要调节作用。     

牙髓干细胞修复再生同质异体大鼠牙髓牙本质复合体的研究

目的:观察第3代同质异体牙髓干细胞(dental pulp stem cell,DPSCs) 定植于冠髓被摘除的大鼠髓腔后,再生修复牙髓牙本质复合体的能力。方法:18只SD大鼠随机分为实验组和对照组,两组均全麻下摘除大鼠上颌双侧第一磨牙冠髓,实验组同期髓腔定植体外培养的同质异体大鼠牙髓干细胞,对照组冠髓摘除后不进行任何处理,术后均使用光固化树脂充填封闭髓腔。两组动物均于术后2、4、6周处死,取其上颌骨进行大体观察,X线观察,组织学观察,牙本质的厚度测量。结果:实验组4周时,肉眼可见牙髓充血减少;6周时,X线显示牙本质厚度增加,根尖孔闭合;6周时,光镜下显示明显新生牙本质形成,成牙本质细胞栅栏状排列;牙本质厚度测量结果显示,实验组牙本质厚度2周时增厚,4周增厚更明显,持续至6周,优于对照组(P<0.05)。结论:牙髓干细胞髓腔内定植,可以促进牙髓牙本质复合体修复再生,具有潜在的临床应用价值。     

The effect of capsaicin on substance P expression in pulp tissue inflammation

AIM: To evaluate the effect of capsaicin on substance P (SP) expression during induced inflammation in rat pulp tissue. METHODOLOGY: Radioimmunoanalysis was used to measure SP levels in 36 mandibular molar pulps taken from six Wistar rats. Twelve samples were obtained from healthy pulps and used as negative control group. Another 12 samples were obtained after inducing inflammation with mechanical pulp exposure; these were used as the positive control group. Capsaicin was infiltrated into the inferior dental nerve in the experimental group and 12 samples were obtained after mechanical pulp exposure. RESULTS: The lowest SP expression was found in mechanically exposed pulps where capsaicin pretreatment had been carried out (0.028 ng mL(-1)), followed by healthy pulps (0.302 ng mL(-1)). The highest SP expression was found in mechanically exposed pulps with no capsaicin pretreatment (124 ng mL(-1)). The Kruskal-Wallis test showed statistically significant differences between the groups (P < 0.001). CONCLUSION: Inferior dental nerve infiltration with capsaicin reduces SP expression in dental pulp tissue in rats.