羟基磷灰石对人牙髓细胞内游离钙的影响

目的:检测羟基磷灰石(hydroxyapatite,HA)对体外培养的人牙髓细胞内游离Ca2+的作用.方法:DMEM培养液体外培养的人牙髓细胞随机分为3组,2个实验组培养液中分别加入Ca(OH) 2、HA共孵育24 h.钙荧光指示剂Fluo-3/AM负载后,3组均加入HA干预,在激光扫描共聚焦显微镜下观测细胞内Ca2+的变化.结果:瞬时加入HA后,3组细胞的钙离子荧光强度在30 s内均升高;空白对照组(A组)升高较为迅速,30 s后即开始缓慢下降;Ca(OH) 2组(B组)的升高幅度小于A组,60 s时达到最高(230.63±5.24);HA组(C组)升高变化较小,30 s时达到最高(161.25±3.81),随后小幅度下降.结论:HA可引起体外培养人牙髓细胞内[Ca2+]i的变化.     

1,25(OH)2D3激动髁突软骨细胞内Ca2+释放及其机制的研究

目的：探讨1，25(OH)2D3激动体外培养的兔髁突软骨细胞内Ca^2 的释放及其通道。方法：消化法培养2周新西兰白兔的髁突软骨细胞，分别进行20g／L肝素、1g／L普鲁卡因细胞内Ca^2 通道阻断处理，经钙荧光指示剂Fluo-3负载后，用激光扫描共聚焦显微镜测定l，25(OH)2D3刺激前后髁突软骨细胞内Ca^2 随时间变化情况。结果：10^-8mol／Ll，25(OH)2D3100μL刺激后对照组细胞内荧光强度随时间明显升高；普鲁卡因处理组变化与其相似，而肝素处理组在刺激前后细胞内荧光强度无明显的变化。结论：1，25(OH)2D3能激动髁突软骨细胞内三磷酸肌醇受体(IP3R)Ca^2 释放通道开放，使细胞内Ca^2 水平显著升高。     

TGF-β_1对人牙髓细胞内钙影响的激光共聚焦显微镜动态观察

目的 :探讨 TGF- β1 信号转导过程与人牙髓细胞内钙〔( Ca2 ) i〕的关系。方法 :体外培养人牙髓细胞经钙荧光指示剂 Fluo- 3负载后 ,用激光扫描共聚焦显微镜测定 TGF- β1 影响前后的人牙髓细胞内钙随时间变化情况。结果 :TGF- β1 ( 0 .1ng/ L)使人牙髓细胞内钙先升高后降低 ,最后维持在比加药前稍高的静息钙水平上。结论 :通过 TGF-β可引起牙髓细胞内 Ca2 水平的显著变化 ,证明 Ca2 参与了人牙髓细胞 TGF-β1 信号转导过程并参与将信号转入核内     

人牙乳头细胞分化过程中胞内Ca^2＋浓度变化和1，25（OH）2D3对Ca^2＋影响

目的：探讨人牙乳头细胞不同分化阶段和1,25（OH）2D3作用下细胞内Ca^2 的变化。方法：体外培养人牙乳头细胞,利用激光共聚焦显微镜观察细胞内Ca^2 浓度的变化。结果：具有某些成牙本质细胞表型的细胞（21d)胞内Ca^2 浓度比无分化表型的细胞（14d)高约2倍;1,25（OH）2D3使21d组细胞胞内Ca^2 明显升高,而14d组则无明显变化。结论：人牙乳头细胞在分化过程中胞内Ca^2 浓度上调;1,25（OH）2D3能提高牙乳头细胞静息内Ca^2 水平以达新的Ca^2 稳态,促进牙乳头细胞分化。     

人牙乳头细胞分化过程中胞内Ca~(2 )浓度变化和1,25(OH)_2D_3对Ca~(2 )影响

目的 :探讨人牙乳头细胞不同分化阶段和 1,2 5 (OH) 2 D3 作用下细胞内Ca2 的变化。方法 :体外培养人牙乳头细胞 ,利用激光共聚焦显微镜观察细胞内Ca2 浓度的变化。结果 :具有某些成牙本质细胞表型的细胞 (2 1d)胞内Ca2 浓度比无分化表型的细胞 (14d)高约 2倍 ;1,2 5 (OH) 2 D3 使 2 1d组细胞胞内Ca2 明显升高 ,而 14d组则无明显变化。结论 :人牙乳头细胞在分化过程中胞内Ca2 浓度上调 ;1,2 5 (OH) 2 D3 能提高牙乳头细胞静息内Ca2 水平以达新的Ca2 稳态 ,促进牙乳头细胞分化     

1，25（OH）2D3对培养人牙乳头细胞Calbindin—D28K表达和矿化能力的影响

目的：研究1,25（OH）2D3对培养人牙乳头细胞Calbindin-D28K（CaB）表达和矿化能力的影响。方法：用10^-8mol/L1,25(OH)2D3矿化液处理长期培养的第28天细胞24h,用放射免疫和免疫组化方法,观察CaB表达和碱性磷酸酶（ALP）、骨钙蛋白（OC）的变化。同位素示踪方法观察1,25(OH)2D3对胞外基质^45Ca^2 的影响。结果：加入1,25(OH)2D3后ALP活性约为对照组的4倍;OC含量约为对照组的1.5倍。CaB在培养28d牙乳头细胞及其胞外基质中表达,而在培养14d牙乳头细胞中CaB免疫反应阴性。1,25(OH)2D3可加强CaB表达,并使胞外基质对^45Ca^2 摄取量增加1.5倍。结论：1,25（OH）2D3有促进人牙乳头细胞矿化作用,表现在不仅能刺激OC、ALP的活性,而且可刺激具有分化表型的牙乳头细胞中CaB的合成,增加基质对Ca^2 的摄取。证实促进牙乳头细胞的钙转导是CaB在矿化过程中的作用之一,推测CaB被“俘获”在基质中时有诱导Ca^2 聚集和直接贮存的作用。     

Ca(OH)2和Hoshino制剂对猪牙乳头细胞增殖的影响

目的研究Ca(OH)₂和Hoshino制剂（甲硝唑、环丙沙星、米诺环素混合制剂）对体外培养的猪牙乳头细胞（procine dental papilla cells,pDPCs)增殖的影响,探讨临床使用牙髓血管再生治疗时根管消毒药物的选择。方法将第4代pDPCs分别在含不同浓度Ca(OH)₂（10%、1%、0.1%）和Hoshino制剂（0.5%、0.1%、0.05%）的培养基中培养,采用CCK-8法检测2种药物对细胞增殖活性的影响,采用SPSS19.0软件包对实验结果进行统计学分析。结果Ca(OH)₂对pDPCs细胞的增殖影响呈波动状,细胞培养9 d时,各组浓度Ca(OH)₂对细胞增殖无影响;Hoshino制剂可抑制pDPCs的增殖。结论实验中各浓度组Ca(OH)₂ 制剂对pDPCs 细胞的增殖无影响,而Hoshino制剂可抑制pDPCs 细胞的增殖。     

1,25(OH)_2D_3对培养人牙乳头细胞Calbindin-D28K表达和矿化能力的影响

目的 :研究 1,2 5 (OH) 2 D3 对培养人牙乳头细胞Calbindin -D2 8K(CaB)表达和矿化能力的影响。方法 :用 10 -8mol/L 1,2 5 (OH) 2 D3 矿化液处理长期培养的第 2 8天细胞 2 4h ,用放射免疫和免疫组化方法 ,观察CaB表达和碱性磷酸酶 (ALP)、骨钙蛋白 (OC)的变化。同位素示踪方法观察 1,2 5 (OH) 2 D3 对胞外基质4 5Ca2 的影响。结果 :加入1,2 5 (OH) 2 D3 后ALP活性约为对照组的 4倍 ;OC含量约为对照组的 1.5倍。CaB在培养 2 8d牙乳头细胞及其胞外基质中表达 ,而在培养 14d牙乳头细胞中CaB免疫反应阴性。 1,2 5 (OH) 2 D3 可加强CaB表达 ,并使胞外基质对4 5Ca2 摄取量增加 1.5倍。结论 :1,2 5 (OH) 2 D3 有促进人牙乳头细胞矿化作用 ,表现在不仅能刺激OC、ALP的活性 ,而且可刺激具有分化表型的牙乳头细胞中CaB的合成 ,增加基质对Ca2 的摄取。证实促进牙乳头细胞的钙转导是CaB在矿化过程中的作用之一 ,推测CaB被“俘获”在基质中时有诱导Ca2 聚集和直接贮存的作用。     

白细胞介素-1β诱导牙髓细胞的金属蛋白酶-1和COX2的表达

目的 :探讨白细胞介素 1β对人牙髓细胞金属蛋白酶 1(matrixmetalloproteinase 1,MMP 1)、环氧合酶 2 (cyclooxygenase 2 ,COX2 )基因表达变化的生物学意义。方法 :培养人牙髓细胞 ,传代至第 4代 ,PT PCR检测人牙髓细胞是否表达IL 1β受体 ,进而用 1nmol/L人的重组IL 1β刺激人牙髓细胞 18h ,提取细胞总RNA ,反转录为cDNA ,半定量PCR检测IL 1β对人牙髓细胞的金属蛋白酶 1、COX2的mRNA的表达变化。结果 :人牙髓细胞表达IL 1β受体 ,而且用 1nmol/L人的重组IL 1β明显地刺激牙髓细胞的金属蛋白酶 1、COX2的mRNA表达。结论 :牙髓炎时牙髓组织内的IL 1β生成增多 ,会进一步引发金属蛋白酶 1、COX2基因异常表达相应增多 ,而金属蛋白酶 1导致炎症基质降解、COX2将引发炎性痛的病理改变。     

应用流式细胞术检测人牙髓细胞内活性氧的方法探讨

目的:建立用流式细胞术检测人牙髓细胞内活性氧变化的方法.方法:采用改良组织块法培养人牙髓细胞,牙髓细胞经不同浓度H2O2处理30 min后,以终浓度分别为10、20 μmol/L的2’,7’-二氯荧光素二乙酸酯(2’,7’-dichlorofluoresin diacetate,DCFH-DA)作为荧光探针,与牙髓细胞共同避光孵育20 min,使用流式细胞仪检测细胞内氧化性二氯荧光素(Dichlorofluorescein,DCF)的荧光强度.采用SPSS13.0软件包对数据进行统计学分析.结果:不同浓度探针装载率不同;不同浓度H2O2作用于细胞一定时间后,使用流式细胞术可检测到细胞内活性氧水平产生相应变化,且多次重复结果稳定.结论:选用终浓度为20 μmol/L的DCFH-DA作为探针装载率较高,利用流式细胞仪检测人牙髓细胞内活性氧的产生是一种可靠、稳定、简便的方法.     

氢氧化钙激动人牙髓细胞内钙释放及其通道的研究

目的：检测Ｃａ（ＯＨ）２激动体外培养的人牙髓细胞内Ｃａ＾２＋的释放和钙释放通道。方法：在ＲＰＭＩ１６４０培养液中分别加入Ｃａ（ＯＨ）２、Ｃａ（ＯＨ）２和肝素、Ｃａ（ＯＨ）２和普鲁卡因,培养第６代人牙髓细胞,用Ｆｌｕｏ－３荧光探针负载后,再次用Ｃａ（ＯＨ）２作用于牙髓细胞,用激光共聚焦显微镜检测胞内Ｃａ＾２＋。结果：含Ｃａ（ＯＨ）２、Ｃａ（ＯＨ）２和普鲁卡因的培养液培养的细胞内Ｃａ＾２＋浓度升高;含     

The effect of extracellular calcium ion on gene expression of bone-related proteins in human pulp cells

Calcium hydroxide is often used for induction of reparative dentin formation in endodontic treatment. However, little is known about the mechanism by which calcium hydroxide works. The calcium ion (Ca2+) is an important regulator of cell functions. In this study, we examined the effect of extracellular Ca2+ on gene expression of bone-related proteins in human cultured pulp cells in serum-free conditions. A Ca2+ level elevated by 0.7 mM induced an increase in mRNA expression of osteopontin and bone morphogenetic protein (BMP)-2. However, mRNA levels of BMP-4 and alkaline phosphatase decreased under the elevated Ca2+ culture condition. The same concentration of additional magnesium ions had little effect on expressions of the examined bone-related protein mRNAs. These findings suggest that Ca2+ in Ca(OH)2 specifically modulates osteopontin and BMP-2 levels during calcification in pulp.     

The comparison of a dentin adhesive with calcium hydroxide as a pulp-capping agent on the exposed pulps of human and sheep teeth.

OBJECTIVE: This study evaluated the biocompatibility of a one-step dentin bonding agent (Prime&Bond 2.1) in pulp capping compared with calcium hydroxide [Ca(OH)2]. METHOD AND MATERIALS: Thirty sheep teeth and 20 intact human premolars were used. After cavity preparation, pulp exposure was achieved with a bur (#390). Adhesive pulp capping was performed in 25 teeth (15 sheep and 10 human). In the control group (12 sheep and 10 human teeth), pulps were capped with Ca(OH)2 and all of the cavities in both groups were sealed with resin composite. Three of the sheep teeth were used as intact controls. Teeth were extracted 7 or 90 days following treatment and prepared for histological examination and bacterial detection. RESULTS: At 7 days, severe inflammatory responses underlying the bonding agent and in the coronal pulp were observed with soft tissue disorganization in both human and sheep teeth capped with Prime&Bond 2.1. All of the teeth capped with Ca(OH)2 exhibited mild inflammatory reactions limited with the perforation area. After 90 days with the bonding agent, in 3 of 9 sheep teeth, chronic inflammatory reactions were significant, while slight pulpal reactions were observed in the others and dentin bridge formation in all of the sheep teeth was found. However, in human pulps, persistent, unresolved inflammation with the lack of dentin bridge formation was observed. In the Ca(OH)2 group, pulp repair with dentin bridging was found in all of the teeth, both sheep and human. No correlation was found between the presence of inflammation and bacterial staining using Spearman rank correlation test (P > .05). CONCLUSION: Prime&Bond 2.1 facilitates enhanced pulp healing and bridge formation in sheep teeth, but in human teeth it was not as successful as Ca(OH)2 as a pulp capping agent.     

Direct capping of human pulps with a dentin bonding system and calcium hydroxide: an immunohistochemical analysis

Pulp capping is a treatment where a protective agent is applied to an exposed pulp to allow the maintenance of its vitality and function. The present study analyzed the immunohistochemical expression of fibronectin and type III collagen in human dental pulps submitted to direct pulp capping with calcium hydroxide [Ca(OH)2] or the Single Bond adhesive system (SBAS). The results demonstrated that both proteins were not expressed in the SBAS group, although in the group capped with Ca(OH)2 a diffuse labeling in the extracellular matrix was initially observed, followed by a late expression in the odontoblast-like layer and beneath the dentin bridge. It seems that application of adhesive systems in direct contact with healthy pulps will not lead to expression of proteins that are believed to be essential for pulpal repair. Moreover, Ca(OH)2 showed good biocompatibility properties with the dental pulp tissue, inducing the expression of reparative molecules, and therefore remains the material of choice for the treatment of accidental pulp exposures.     

Comparative evaluation of diffused calcium and hydroxyl ion release from three different Indirect pulp capping agents in permanent teeth – An in vitro study

BackgroundIndirect pulp capping therapy has gained increased popularity in paediatric dentistry since it is less invasive, and is of low cost. The aim of the present study was to evaluate and to compare the diffusion of calcium (Ca2+) and hydroxyl (OH–) ions through coronal dentin into pulp after indirect pulp capping in vitro using TheraCal LC, ProRoot MTA and Calcimol LC.Materials and methodsTotal of 60 human caries-free maxillary first premolars were selected for the study. Samples were divided into 4 groups with 15 in each group: Group 1 TheraCal LC; Group 2 ProRoot MTA; Group 3 Calcimol LC; Group 4 Control Group. Indirect pulpcapping on the coronal RDT (remaining dentine thickness) system was performed using pulp-capping materials, such as TheraCal LC, ProRoot MTA and Calcimol LC, on the respective samples. The control group was completely filled with composite. Ca2+ ions (ppm) and OH– ions (pH) were analysed in deionized water using a multimeter connected to a calcium probe (calcium ion electrode) and pH metre connected to a temperature-compensated pH probe after 3 h, 24 h, 7 days, 14 days, 28 days and 60 days.ResultsCalcium release was significantly higher (P < 0.05) in the TheraCal LC group than in the other groups. Slightly alkaline pH values were observed in all the groups except for the control.ConclusionTheraCal is a new light-curable pulp capping material that initially releases high Ca2+ ions and creates an environmental pH close to physiological pH after 60 days.     

应用流式细胞技术分析Ca（OH）2及HA对牙髓细胞周期的影响

目的：采用流式细胞技术分析盖髓剂氢氧化钙[Ca(OH2)]及羟基磷灰石（HA）对牙髓细胞周期的影响。方法：将香猪恒牙牙髓机械暴露后,行氢氧化钙和羟基磷灰石盖髓术,分别于术后2,4,8,12周拔牙,采取牙须,胶原酶消化后流式细胞仪检测。将组方图存入磁盘进行计算机分析处理,得到G0／G1,G2,S及M细胞百分比。结果氢氧化钙和羟基磷灰石均能使牙髓细胞的G0／G1期细胞减少,S期细胞积累,结论：提示氢氧化钙及羟基磷灰石可能有促进G0／G1期细胞进入S期,增加DNA合成的作用。     

纤维蛋白粘接剂盖髓的动物实验研究

目的：观察纤维蛋白粘接剂（ＦＳ）对人工暴露的牙髓组织愈合的影响。方法：采用ＦＳ行狗牙直接盖髓术,常规制备牙－颌骨联合切片,光学显微镜观察牙髓创面的愈合及牙本质的修复。结果：ＦＳ盖髓后牙髓无出血,炎性反应轻。氢氧化钙盖髓后牙髓出血多,炎性反应较重。ＦＳ组术后４２ｄ出现骨样牙本质桥修复,氢氧化钙组术后２８ｄ出现骨样牙本质桥修复,术后６３ｄ,ＦＳ组６例中４例有牙本质桥形成,氢氧化钙组６例均有牙本质桥形成。结论：ＦＳ虽无牙本质诱导活性但因其良好的保护及促进牙髓组织创面愈合能力仍不失为一种较为理想的盖髓剂。     

The effect of two different calcium hydroxide combinations on root dentine microhardness

AIM: To evaluate the effect of a calcium hydroxide and glycerine mix and a calcium hydroxide and water mix on the microhardness of human root dentine. METHODOLOGY: Eleven freshly extracted maxillary canine and central incisor teeth were used. The teeth were sectioned transversally to produce a total of 22 dentine discs from the middle-third of the root. The specimens were divided into two groups of 11 discs each. Dentine samples were treated with either a Ca(OH)2-glycerine combination or a Ca(OH)2-distilled water combination for 1, 3 and 7 days. Dentine microhardness was measured with a Knoop indenter with a load of 100 g for 15 s before and during the experimental period. Each root disc received a series of three indentations around the pulp space, 1 mm from canal wall. RESULTS: Statistical analysis showed that both combinations significantly decreased dentine microhardness after 3 and 7 days (P < 0.01). The reduction in dentine microhardness following the use of a Ca(OH)2-glycerine combination was significantly greater than that after a Ca(OH)2-distilled water combination after 3 and 7 days (P < 0.01). CONCLUSION: The use of Ca(OH)2 combinations for intracanal dressing softens dentine.     

Immunolocalization of fibronectin during reparative dentinogenesis in rat molor teeth after pulp capping with mineral trioxide aggregate or calcium hydroxide

The aim of this study was to evaluate the immunolocalization of fibronectin during reparative dentinogenesis in rat teeth after pulp capping with mineral trioxide aggregate (MTA) or calcium hydroxide (Ca(OH)2). The pulps of 72 upper and lower first molar teeth from 18 male Wistar rats were experimentally exposed. The pulps were capped with MTA or (Ca(OH)2); final restoration followed with zinc oxide and eugenol cement. The animals were euthanized at, respectively, one, three, seven and fourteen days postoperatively. At day one, all groups showed varying degrees of inflammation, from mild to severe. There was no positive reaction for fibronectin at day one. After three days, a partial acute pulpitis was observed in the Ca(OH)2 group. There was less inflammation in the MTA group (p<0.05), and a layer of fibrin barrier was observed along the pulp walls of the MTA material. The layer of fibrodentin formation showed positive reaction for fibronectin. At seven days, the Ca(OH)2 group showed mild inflammation and demonstrated more immunostaining for fibronectin than the Ca(OH)2 group (p<0.05) at three days. Pulps capped with MTA at seven days showed thicker fibrin barrier formation than the MTA group at three days and more immunostaining for fibronectin in whole groups (p<0.05). At fourteen days, there was no positive reaction for fibronectin in either the MTA or Ca(OH)2 group. It seems MTA showed better biocompability properties with the dental pulp tissue, inducing the expression of reparative molecule fibronectin compared with Ca(OH)2. Therefore, MTA may be a better choice for pulp capping procedures.