四种烤瓷基底冠金属对人牙龈成纤维细胞分泌前列腺素E2和环加氧酶2的影响

目的 观察4种烤瓷基底冠金属浸提液对人牙龈成纤维细胞分泌前列腺素E2和环加氧酶2的影响.方法 制备镍铬合金、钴铬合金、纯钛、金钯合金金属浸提液,以无金属浸泡的达尔伯克改良伊格尔培养基培养液作为空白对照,培养人牙龈成纤维细胞,分别培养1、6、12、24h,收集上清液,酶联免疫吸附测定法测定前列腺素E2的表达水平;制作细胞爬片,用免疫荧光法观察各组环加氧酶2的表达.结果 镍铬合金、钴铬合金组6、12、24h前列腺素E2表达水平(镍铬:45.568±0.926、60.538±0.988、73.754±0.507;钴铬:40.496±0.693、53.216±0.327、65.470±1.086)高于相应空白对照组(31.122±0.642、31.230±0.634、30.980±0.746),差异均有统计学意义(P＜0.05);纯钛、金钯合金组前列腺素E2表达水平(钛:31.564±0.719、31.998±0.856、32.066±0.513;金钯:31.540±0.821、31.136±0.518、31.340±0.443)与相应空白对照组差异均无统计学意义(P＞0.05).镍铬合金、钴铬合金组环加氧酶2的染色深而均匀;纯钛、金钯合金组与空白对照组染色浅且不均匀.结论 镍铬合金、钴铬合金使人牙龈成纤维细胞分泌前列腺素E2和环加氧酶2增加,而纯钛、金钯合金不影响前列腺素E2和环加氧酶2的分泌.     

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目的评价2种自制含钛镍铬烤瓷合金的细胞毒性。方法本研究于2009年5—12月在中国医科大学中心实验室完成。分别将2种自制含钛镍铬烤瓷合金（2、3号合金）、纯钛、普通镍铬合金及铅的浸提液与体外培养的细胞接触后,进行MTT实验来评定材料的细胞毒性。结果2、3号合金毒性为1级,钛的毒性为0级,镍铬毒性为1级,铅在细胞培养72h后毒性级由1级增至2级。结论自制的含钛镍铬烤瓷合金符合临床应用要求,生物相容性好于目前,I盏床常用的普通镍铬合金。     

4种修复金属材料对炎性牙周组织炎症因子表达的影响

目的：评估4种口腔修复金属材料对牙周炎症状态下牙龈组织炎症因子表达的影响。方法收集牙周炎患者的炎症牙龈组织10份，原代培养人牙龈成纤维细胞，以DMEM培养的细胞作为空白对照（空白对照组），实验组采用钴铬合金（钴铬合金组）、钛合金（钛合金组）、纯钛（钝钛组）及金钯合金（金钯合金组）4种金属浸提液加入培养基。ELISA和RT?PCR法检测相关炎症因子的蛋白和mRNA表达情况。结果 ELISA和RT?PCR结果显示，与空白对照组相比，钴铬合金组和钛合金组白细胞介素?6、白细胞介素?1β、肿瘤坏死因子?α的蛋白和mRNA表达水平均较高（P<0.01），而纯钛组和金钯合金组与空白对照组相比差异无统计学意义（P>0.05）。结论对炎症牙龈成纤维细胞，纯钛和金钯合金比钴铬合金和钛合金具有更好的生物相容性。     

牙齿外伤钛固定夹板浸提液对人牙龈成纤维细胞生物学性能的影响

目的:观察牙齿外伤钛固定夹板(titanium trauma splint,TTS)浸提液对人牙龈成纤维细胞(human gingival fibroblasts,HGF)生物学性能的影响,为临床应用提供理论依据。方法:分离、培养鉴定人牙龈成纤维细胞,将牙齿外伤钛固定夹板浸提液加入人牙龈成纤维细胞培养基中,通过倒置显微镜观察细胞形态学改变、MTT法细胞毒性实验和细胞凋亡实验,观察牙齿外伤钛固定夹板浸提液对人牙龈成纤维细胞生物学性能的影响。结果:牙齿外伤钛固定夹板浸提液对人牙龈成纤维细胞的形态无明显改变,其增殖和凋亡的影响较正常人牙龈成纤维细胞相比无明显细胞毒性,不引起细胞凋亡。结论:牙齿外伤钛固定夹板具有良好的生物安全性,有一定的临床应用前景。     

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目的评价自制含钛镍铬烤瓷合金的细胞毒性。方法于2006年5月至2007年1月,在中国医科大学基础部第五实验室将自制含钛镍铬烤瓷合金材料的浸提液与体外培养的细胞接触后,进行MTT实验来评定材料的细胞毒性,并与临床常用的镍铬合金、纯钛的生物学性能相比较。结果自制含钛镍铬烤瓷合金组对细胞增殖的抑制作用大于纯钛组而小于镍铬合金组,但统计学分析结果显示:5种金属材料与阴性对照组的吸光度值之间的差异均无统计学意义(P>0.05),与阳性对照组之间的吸光度值之间的差异有统计学意义(P<0.05)。结论自制含钛镍铬烤瓷合金仅具有极微弱的细胞毒性,符合口腔科材料临床应用的要求,为其在临床烤瓷合金中的应用提供了生物学依据。     

人纤维增生性牙龈成纤维细胞自分泌TGF-β对其细胞外基质合成的影响

遗传性牙龈纤维瘤病(HGF)是以牙龈组织的纤维性增生为主要特征的一种家族遗传性疾病。体外实验结果表明,HGF来源的牙龈成纤维细胞比正常人细胞能产生更多的Ⅰ型胶原和FN等细胞外基质分子。转化生长因子β(TGF-β)是一族具有重要生物学功能的细胞因子,至少有5种类型,分别为TGF-β_(1-5),能刺激成纤维细胞增生和FN以及胶原的产生,促进损伤的修复过程。本实验目的在于测定HGF的牙龈成纤维细胞是否存在自分泌TGF-β,以及细胞产FN和Ⅰ型胶原是否受自分泌TGF-β的调节。     

热休克蛋白在白细胞介素-1β诱导人牙龈成纤维细胞损伤中的表达

目的：观察热休克蛋白（HSP）27、70和90在白细胞介素（IL）-1β诱导的人牙龈成纤维细胞（HGF）损伤过程中的表达。方法???原代培养人HGF，以蛋白质印迹技术检测不同质量浓度的IL-1β诱导后HSP27、HSP70、HSP90在HGF中的表达，运用细胞划痕实验观察HGF的体外迁移能力变化。结果???经IL-1β诱导后，HGF中HSP70和HSP90的表达量无明显变化，HSP27的表达量明显上调并随IL-1β诱导质量浓度的增加而增加。同时细胞划痕实验显示，随着HSP27表达量的增加，HGF的迁移能力明显降低。结论HSP在经IL-1β诱导的人HGF损伤过程中的表达情况各不相同，其中HSP27在IL-1β诱导的HGF炎症损伤中的表达升高，其表达量与HGF体外愈合能力呈负相关关系。HSP27在上皮炎症损伤中起着关键性的作用，但具体作用机制还有待进一步的研究。     

镍铬合金烤瓷牙龈沟液IL-8含量的性别差异

目的：研究男性和女性镍铬合金烤瓷牙修复后不同时期龈沟液IL-8含量水平,了解镍铬合金在不同时期对牙龈刺激程度的性别差异。方法：采用双抗体夹心酶联免疫吸附法（ABC-ELISA）测定男女各30例镍铬合金烤瓷牙制备前、修复后1周、修复后3个月和修复后6个月烤瓷牙龈沟液IL-8总量和含量及龈沟液量。结果：两组不同时期的IL-8总量和龈沟液量之间差异有统计学意义（P〈0.01）;IL-8总量、龈沟液量和IL-8含量修复3月时女性组高于男性组（P〈0.05）。结论：IL-8可作为评价龈组织刺激程度的指标,镍铬合金对牙龈有刺激性,女性更为明显。     

3种牙科金属材料对L929细胞的毒性及对凋亡相关基因与蛋白表达的影响

目的:研究3种牙科金属材料(金合金、纯钛、镍铬合金)浸提液对小鼠成纤维细胞L929的细胞毒性及凋亡相关基因与蛋白表达的影响,探讨3种牙科金属材料引起细胞凋亡的可能通路.方法:以金合金(A组)、纯钛(B组)、镍铬合金(C组)的浸提液(每组8个样本)培养小鼠成纤维细胞L929,以含10％胎牛血清的RPMI 1640培养基作为阴性对照(D组),以铜合金作为阳性对照(E组).采用MTT法在细胞水平评价3种牙科金属材料的细胞毒性,采用RT-PCR在分子水平上检测3种牙科金属材料浸提液对L929细胞凋亡相关基因caspase-3、-8、-9 mRNA及蛋白表达的影响.采用SPSS11.5软件包对数据进行统计学分析.结果:各实验组细胞毒性均为0级.镍铬合金组caspase-3mRNA与caspase-9mRNA的表达与其他实验组、阴性对照及阳性对照组间差异显著,镍铬合金组caspase-3蛋白与caspase-9蛋白的表达与其他实验组及阴性对照组间差异显著,各组caspase-8mRNA及蛋白的表达无显著差异.结论:金合金与纯钛浸提液对小鼠L929细胞凋亡相关基因及蛋白的表达无显著诱导作用;镍铬合金诱导小鼠L929细胞凋亡相关基因caspase-3mRNA、caspase-9mRNA及蛋白表达增加,可能通过线粒体通路诱导细胞凋亡.     

MTT法检测钛酸钡涂层对小鼠成纤维细胞L929增殖率的影响

目的:探索纯钛烤瓷冠中钛酸钡涂层对小鼠成纤维细胞L929形态及增殖率的影响,并评价其细胞毒性分级.方法:以含钛酸钡涂层的钛瓷试件、常规钛瓷试件的浸提液体外培养小鼠成纤维细胞1、3、5d,并以含10％胎牛血清的RPMI 1640培养基为阴性对照组,1％苯酚溶液为阳性对照组,采用MTT法检测试件浸提液对小鼠成纤维细胞L929增殖率的影响.结果:含钛酸钡涂层的钛瓷结合试件浸提液培养的小鼠成纤维细胞L929形态正常,生长旺盛;钛酸钡涂层组吸光度值大于阴性对照组(P＜0.05),含钛酸钡涂层的纯钛试件浸提液的细胞毒性分级为0级.结论:含钛酸钡涂层的钛瓷结合试件有良好的细胞相容性.     

In vitro IL-1beta release from gingival fibroblasts in response to pure metals, dental alloys and ceramic

Little information is available on the immunological basis for side-effects of dental materials. The objective of this study is to evaluate effects of pure metals, dental alloys and ceramic on cell viability and interleukin-1 beta (IL-1beta) release in three-dimensional human gingival fibroblast cultures as an indicator of their biological performance in gingival tissues. The gingival fibroblast cultures were exposed to test specimens fabricated from nickel, iron, molybdenum, copper, indium, gold, Ni-Cr-Mo alloy (Remanium CS), Au-Pt-In alloy (Pontostar) and a dental ceramic (In-ceram). Cell viability was determined by the MTT method 24 and 48 h after exposure. Assays for IL-1beta were carried out by ELISA. Statistical analysis was performed applying the non-parametric Mann-Whitney pairwise test. Dental ceramic and gold did not influence cell viability after 24 and 48 h. Cell viability was determined after 24 and 48 h to nickel (79-77%), iron (92-90%), molybdenum (86-83%), copper (48-36%), indium (90-90%), Remanium CS (83-80%), Pontostar (94-91%) compared with control cultures. Dental ceramic, Pontostar and gold had no significant influence on IL-1beta secretion. The highest amounts of IL-1beta (10-fold) levels were determined in cell cultures exposed to copper. Indium, molybdenum and iron induced twofold IL-1beta levels compared with untreated control cultures. These results support that some metals may alter immune responses and thereby contribute to a variety of dental pathological conditions and three-dimensional cell culture models for gingival fibroblasts appear to be suitable for in vitro studies.     

含钛镍铬合金的腐蚀性能研究

目的:研究经烤瓷烧结程序处理后的Ni-Cr-Ti合金在人工唾液中的腐蚀性能。方法:采用电感耦合等离子体光谱分析法(ICP-AES)检测Ni-Cr-Ti合金和Ni-Cr合金在人工唾液中浸泡3、15、90d的离子析出量。结果:Ni-Cr-Ti合金在各时间段在人工唾液中析出的金属离子总量较Ni-Cr合金低,差异有显著性(P<0.05)。Ni-Cr-Ti合金和Ni-Cr合金析出的金属离子总量均随时间延长而增高,具有时间相关性。结论:Ni-Cr-Ti合金耐腐蚀性较Ni-Cr合金强,在人工唾液中析出的金属离子较Ni-Cr合金少。但Ni-Cr-Ti合金在人工唾液中仍有相当数量的Ni、Be离子析出,建议临床上应用于前牙时,应选择肩台瓷技术修复。     

Prostaglandin E2 secretion from gingival fibroblasts treated with interleukin-1β: effects of lipid extracts from Porphyromonas gingivalis or calculus

Complex lipids of Porphyromonas gingivalis have been identified in lipid extracts from calculus-contaminated root surfaces and in diseased gingival tissues. However, little is known about the biological effects of these complex lipids on host cells. The purpose of this study was to evaluate the effects of P. gingivalis or calculus lipids on prostaglandin secretion from gingival fibroblasts. Lipids were extracted from paired subgingival plaque and teeth samples, and calculus-contaminated root surfaces before and after scaling and root planing, in order to determine the relevant levels of lipid extracts for the treatment of gingival fibroblasts in culture. Primary cultures of gingival fibroblasts were exposed to lipid extracts from either P. gingivalis or calculus/teeth for a period of 7 days. Control and lipid-treated cultures were exposed to human recombinant interleukin-1β for 48 h and prostaglandin secretion from interleukin-1β-treated fibroblasts was compared with control and lipid-treated fibroblasts without interleukin-1β treatment. These experiments demonstrated that P. gingivalis lipids or calculus-tooth lipids potentiate interleukin-1β-mediated prostaglandin secretory responses from gingival fibroblasts. Additionally, P. gingivalis or calculus-tooth lipid extracts were readily taken up by gingival fibroblasts as measured by bacterial fatty acid recovery in lipid extracts of cultured fibroblasts. These results indicate that bacterial lipid penetration into gingival tissues in combination with a chronic inflammatory response may substantially potentiate prostaglandin secretion from gingival fibroblasts, thereby promoting tissue destructive processes associated with adult periodontitis.     

EFFECT OF TEMPERATURE VARIATION ON THE CYTOTOXICITY OF CAST DENTAL ALLOYS AND COMMERCIALLY PURE TITANIUM

Cell culture system has been used to evaluate alloy cytotoxicity under different environments, testing the extracts, but the effect of temperature variation on the cytotoxicity of dental alloys has not been analyzed. Objective: The aim of the present study was to investigate if temperature variation could affect dental alloy cytotoxicity, testing alloy extracts in an epithelial cell culture system. Material and methods: Discs of Ni-Cr, Co-Cr-Mo, Ni-Cr-Ti, Ti-6Al-4V and commercially pure titanium (cp Ti) were cast by arc melting, under argon atmosphere, injected by vacuum-pressure. Discs were immersed in artificial saliva and subjected to different temperatures: 37°C and thermocycling (37°C/5°C/37°C/55°C/37°C). After thermocycling, extracts were put in a subconfluent culture during 6 h, and the number of cells and their viability were used to evaluate cytotoxicity in these temperatures. For each alloy, data from temperature conditions were compared by Student''s t-test (α=0.05). Results: The cytotoxicity tests with alloy/metal extracts showed that Ni-Cr, Co-Cr-Mo, Ti-6Al-4V and cp Ti extracts (p>0.05) did not affect cell number or cell viability, while Ni-Cr-Ti (p<0.05) extract decreased cell number and viability when the alloy was subjected to thermocycling. Conclusion: Within the limitations of the present study, the Ni-Cr-Ti alloy had cell number and viability decreased when subjected to temperature variation, while the other alloys/metal extracts did not show these results.     

The effects of folic acid application on IL-1beta levels of human gingival fibroblasts stimulated by phenytoin and TNFalpha in vitro: a preliminary study

Folic acid (FA), that is required for the integrity of gingival tissues, was found to decrease in patients using phenytoin (PHT). Interleukin-1 beta (IL-1beta) was reported to enhance the extracellular matrix synthesis in fibroblasts. The purpose of this study was to assess the role of FA supplementation on PHT-induced overgrowth by investigating its effect on IL-1beta production of human gingival fibroblasts induced by tumor necrosis factor alfa (TNFalpha) in cell culture. PHT (20 microg/ml), FA (20 or 40 ng/ml) + PHT, PHT + TNF (10 ng/ml), FA (20 or 40 ng/ml) + PHT + TNF, or only culture medium (control) was added to 24-well plates containing fibroblasts. After an incubation period of 72 h, culture medium and cells were harvested separately. Then, IL-1beta levels in cell lysate were measured using enzyme-linked immunosorbent assay. The cellular IL-1beta level in the PHT group was 1 pg/ml. In PHT + 20 or 40 ng/ml FA-added cultures, the results obtained respectively were 0.8 and 0.7 pg/ml, whereas the control group value was 0.7 pg/ml. IL-1beta level was 4 pg/ml in the cultures that PHT and TNFalpha were applied simultaneously (P < 0.05). When PHT and either 20 or 40 ng/ml FA were simultaneously added into TNFalpha-induced cultures, the IL-1beta levels were 1.8 and 1.3 pg/ml, respectively. IL-1beta level in gingival tissues might play a role in PHT-induced overgrowth by increasing in the gingival tissues, and FA application might play a role in decreasing gingival tissues. However, further studies are needed for a more complete understanding of PHT-induced gingival overgrowth at the cellular level.     

五种烤瓷合金金瓷结合强度的比较

目的：比较镍铬合金、含钛的镍铬合金、钴铬合金、钯银合金和纯钛的金瓷结合强度。方法：执行ISO9693标准,采用三点弯曲试验分别测定镍铬合金、含钛的镍铬合金、钻铬合金、钯银合金、纯钛在常规热处理条件下的金瓷结合强度。结果：金瓷结合强度分别为：镍铬合金（37．82±2．72）Mpa;含钛的镍铬合金（39．23±2．45）Mpa;钴铬合金（39．06±3．41）Npa;钯银合金（47．98±3．74）Npa;纯钛（32．61±5．62）Mpa。镍铬合金、含钛的镍铬合金、钻铬合金组间差异无统计学意义（P〉0．05）,这3种合金与纯钛、钯银合金组间差异都有统计学意义（P〈0．05）,纯钛与钯银合金组间差异也有统计学意义（P〈0．05）。结论：①镍铬合金、含钛的镍铬合金、钴铬合金金瓷结合强度相近,都大于纯钛且小于钯银合金的金瓷结合强度。②镍铬合金、含钛的镍铬合金、钻铬合金、钯银合金和纯钛的金瓷结合强度都大于25Mpa,按ISO9693标准均可应用于临床。     

Nicotine and lipopolysaccharide affect cytokine expression from gingival fibroblasts

BACKGROUND: This in vitro study investigated the influence of nicotine, lipopolysaccharide (LPS), and a combination of both agents on cytokine expression from human gingival fibroblasts (HGFs). METHODS: HGFs were exposed for 48 hours to 250 microg/ml nicotine, 1 microg/ml Porphyromonas gingivalis LPS, or both. The expression of multiple cytokines was detected in the HGFs conditioned media using cytokine protein arrays. RESULTS: The untreated HGFs expressed several cytokines, which included relatively high levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1). They also expressed low levels of growth-regulated oncogene (GRO), IL-3, and IL-10. Nicotine had the greatest effect on the expression of GRO-alpha, IL-7, IL-10, and IL-15 compared to the untreated control. P. gingivalis LPS had the greatest effect on the expression of GRO-alpha; IL-7; IL-10; and RANTES (regulated on activation, normal T-cell expressed, and presumably secreted) compared to the untreated control. The combination of both agents had the biggest impact on the expression of GRO-alpha, IL-7, IL-10, IL-15, RANTES, and interferon-gamma (IFN-gamma) compared to the untreated control. CONCLUSION: HGFs exposed to nicotine, P. gingivalis LPS, or a combination of both agents increased the expression of multiple cytokines.     

Prostaglandin E production and viability of cells cultured in contact with freshly mixed endodontic materials

AIM: To determine whether commonly used endodontic sealers could either induce or increase the release of prostaglandin E2 (PGE2) when in contact with cell types found in the periapical tissues. METHODOLOGY: Freshly mixed samples of Roth 801 sealer, Sealapex and ProRoot mineral trioxide aggregate (MTA) were placed in contact with cultured macrophages and fibroblasts for 24 h. The supernatant from the cultures was assayed for PGE2 using enzyme-linked immunosorbent assay. Cell viability counts were made. As a positive control, similar cultures were also exposed to lipopolysaccharide and the supernatant analysed for PGE2. Data were compared by anova. RESULTS: The three materials examined in these experiments did not stimulate increased PGE2 release from either of the cell lines. In control cultures, lipopolysaccharide increased PGE2 release from macrophages but not from fibroblasts. Viability counts revealed that, whilst Roth 801 sealer caused some cell death in both fibroblasts and macrophages, Sealapex led to cell death only in the macrophage cultures. ProRoot MTA did not lead to statistically significant cell death in either culture. CONCLUSIONS: Under 24-h culture conditions, the three freshly mixed test materials did not increase directly either production or release of PGE2 from either macrophages or gingival fibroblasts. Roth 801 decreased cell viability counts for both fibroblasts and macrophages. Sealapex decreases macrophage viability. ProRoot MTA did not affect viability in either cell line.