钛表面贻贝蛋白包被对人真皮成纤维细胞粘附和增殖的影响

目的:观察人真皮成纤维细胞在两种贻贝足丝蛋白提取物Usun-afix和BD CELL-TAKTM试剂包被钛表面的粘附和增殖状况,研究贻贝蛋白包被法是否能够用于经皮种植体表面处理。方法:分别制备光滑钛片和两种贻贝蛋白包被的钛片样本,选取P3代人真皮成纤维细胞接种到3种培养基底上:①不做处理的光滑钛表面(对照组);②Usun-afix包被的钛表面;③BD CELL-TAKTM包被的钛表面。MTT法检测1、3、5 d细胞在3种钛表面的增殖数量;利用DiI-AcLDL荧光染色结合MTT法检测15、30 min和1、2、4 h时间点细胞的粘附状况。结果:培养1、3、5 d,成纤维细胞在3种表面活性均较好,3组之间无明显统计学差异(P>0.05);DiI-AcLDL荧光染色显示,在培养15、30 min和1 h各时间点,Usun-afix和BD CELL-TAKTM包被钛表面粘附细胞数目明显多于光滑钛表面,在1、2、4 h各时间点,两包被组与对照组相比有更好的细胞伸展形态;不同时间点MTT检测显示,两包被组在接种15、30 min和1、2 h各时间点的吸光度值均明显高于对照组(P<0.05),在4 h时,虽两包被组OD值比对照组略高,但无统计学差异(P>0.05);两包被组在各个时间点的促粘附效果相似,差异均无统计学意义(P>0.05)。结论:成纤维细胞接种早期(<4 h),可更容易粘附到两种贻贝足丝蛋白提取物Usun-afix和BD CELL-TAKTM试剂包被的钛表面,而贻贝蛋白对成纤维细胞的增殖能力没有明显的促进效果。     

minTBP-1-PRGDN双靶向融合肽对成骨细胞增殖影响的研究

目的：探讨融合肽minTBP-1-PRGDN作为钛种植体表面涂层,对钛片表面成骨细胞增殖的影响。方法：MTT法检测MC3T3-E1成骨细胞在不同涂层的钛片表面粘附24h、48h、72h后增殖数量的差异;金相显微镜观察各组细胞的生长形态及密度;Real-timePCR定量比较细胞增殖相关基因cyclin D1的表达差异。结果：各组中成骨细胞均随着时间延长而出现增殖,24h时minTBP-PRGDN组细胞增殖数量明显高于空白对照组（P〈0.05）;72h时,minTBP-PRGDN组的cyclin D1表达水平最高。结论：融合肽minTBP-PRGDN能有效促进钛表面成骨细胞的增殖。     

喷砂酸蚀对超细晶纯钛表面MC3T3-E1细胞粘附与增殖的影响

目的:研究喷砂酸蚀对超细晶纯钛表面MC3T3-E1细胞粘附与增殖的影响。方法:将超细晶纯钛棒和纯钛棒切割为直径6 mm,厚度3 mm钛片试件,试验组为喷砂酸蚀超细晶纯钛组,对照组分别为未处理的超细晶纯钛组和喷砂酸蚀纯钛组。在对其表面形貌特征和亲水性进行检测后,在试件表面接种MC3T3-E1细胞,观察细胞的初期粘附情况,测定细胞密度,存活和生长状态。结果:喷砂酸蚀超细晶纯钛后,其表面形成大量微小的弹坑状凹陷,且表面具有良好的亲水性。接种细胞后,喷砂酸蚀超细晶纯钛初期粘附优于对照组;细胞密度在培养中后期优于对照组;而细胞活性在培养中期优于两对照组,培养后期3组间无明显差异。结论:喷砂酸蚀超细晶纯钛的表面性能得到改善,能诱导MC3T3-E1细胞在其表面粘附和增殖,可作为纯钛种植体种植体的替代材料。     

微/纳米化载锶涂层对骨髓间充质干细胞生物活性影响的研究

目的:评价微/纳米化载锶涂层对骨髓间充质干细胞(BMMSCs)生物活性的影响。方法:将纯钛片分为3组,A组:光滑组(未经任何处理,n=24);B组:氢氟酸(HF)酸蚀组(n=24);C组:HF酸蚀+磁控溅射组(n=27)。SEM观察钛片表面形貌;X射线能谱(EDS)分析其表面元素含量;表面接触角检测钛片表面亲水性;离子释放试验检测C组的锶离子释放情况。在3组钛片表面分别接种BMMSCs,观察BMMSCs早期粘附能力;MTT检测细胞增殖能力;ALP活性评估细胞成骨分化能力。结果:3组钛片经不同方法处理后,B组形成微米级表面形貌;C组形成微/纳米表面形貌,并载入了锶元素,且锶元素可以离子形式释放;B、C组的亲水性、细胞粘附及增殖能力均高于A组,且B组高于C组;C组表面细胞的ALP活性显著高于B组。结论:微/纳米化载锶涂层有助于促进BMMSCs的增殖和成骨分化能力。     

骨髓基质细胞在钙磷涂层纯钛表面早期粘附的实验研究

目的：探讨表面经仿生法涂层纯钛片对骨髓基质细胞粘附特性的影响，方法：取三代兔骨髓基质细胞，接种于经仿生法钙磷涂层纯钛片表面和商用纯钛片表面，MTT法比较接种为0．5h,1h,2h,4h各时间点细胞在不同表面的粘附情况，并以荧光染色揭示细胞在不同钛片表面上的形态学差异。结果：荧光染色研究结果发现，2小时时粘附于涂层钛片的细胞体积更大，更不规则的多角形，多个伪足向四周伸展，而且粘附于仿生法钙磷涂层纯钛片表面的相对细胞数从1小时开始显著高于商用纯钛片表面相对细胞数，2小时点为细胞粘附的最高峰，结论：仿生法钙磷涂层能促进细胞早期粘附，是研究种植体表面改性的一种值得进一步探讨的实验室方法。     

钛种植体表面微形态对成骨细胞生长影响的体外研究

目的:研究钛种植体表面微形态对成骨细胞生长的影响。方法:将原代培养的成骨细胞与三种不同表面处理的钛片(机械打磨组G、喷砂组SB、钛浆喷涂组TPS)共同培养,采用扫描电镜、MTT法、碱性磷酸酶活性(ALP)及骨钙素分泌(OC)的检测来观察不同表面微形态对成骨细胞粘附、增殖、分化的影响。结果:成骨细胞在不同钛片表面粘附生长,SB组、TPS组表面细胞呈分化表型。SB组、TPS组细胞增殖率高于G组(P <0 .0 5 )。第1d、5d、10d ,SB组、TPS组ALP的活性高于对照组(P <0 .0 5 ) ;G组第1d、3d、5dALP分泌与对照组比较,差异无显著性(P >0 .0 5 )。第3d、5d、10dSB组、TPS组OC分泌量与对照组相比有显著性差异(P <0 .0 5 )。结论:粗糙表面(SB组、TPS组)比光滑表面(G组)更有利于成骨细胞的粘附、增殖,能促进成骨细胞向成熟的表型分化。     

成骨细胞在微弧氧化处理后纯钛表面的附着、增殖及ALP活性

目的 :对表面微弧氧化 (microarcoxidation ,MAO)处理后的纯钛材料进行成骨细胞生物相容性检测 ,评价改进的MAO工艺应用于钛植入材料表面处理的可能性。方法 :纯钛材料经过 2种MAO处理后 (MAO 1和MAO 2工艺 ) ,采用MC 3T3细胞系对不同时间点成骨细胞在材料表面的附着率、生长增殖情况以及ALP活性进行检测 ,以未经处理光滑纯钛表面作为对照 ,以SPSS对实验结果进行统计分析。结果 :早期 (0 .5h、1h)细胞附着率差异有统计学意义 ,MAO 1组 >MAO 2组 >纯钛组 ;2h后 ,MAO 1组与MAO 2组无差异 ,2组细胞附着率都显著高于纯钛组。细胞增殖及ALP活性测试中 ,MAO 1处理组在各时间点都显著高于另外 2组。结论 :MAO处理后的纯钛对成骨细胞的生物相容性优于未处理组 ,改进的MAO 1处理工艺较一般工艺可以更有效提高成骨细胞的早期粘附、增殖及ALP活性。     

过氧化氢及热处理的纯钛钛片对小鼠MC3T3-E1粘附、增殖和分化影响的研究

目的探讨过氧化氢及热处理的纯钛钛片对小鼠MC3T3-E1粘附、增殖和分化能力的影响。方法喷砂、双酸处理的纯钛钛片设为对照组,而喷砂、酸蚀、过氧化氢及热处理的纯钛钛片设为实验组。采用扫描电镜(SEM)和X射线衍射仪(XRD)对2组钛片的表面进行表征。接着在2组钛片表面培养MC3T3-E1,检测其早期粘附、增殖及分化的能力。结果SEM和XRD检测结果显示对照组钛片表面为粗糙的网状孔洞结构,而实验组钛片同样为粗糙的网状孔洞形貌,表面还覆盖有锐钛矿晶体结构;实验组钛片明显促进了成骨细胞的早期粘附(P<0.05),而且培养在实验组钛片表面的细胞增殖得更快(P<0.05)。同时,在实验组钛片表面生长的成骨细胞表达了更高的碱性磷酸酶及骨钙素(无论是蛋白水平还是基因水平)。结论喷砂、酸蚀及过氧化氢热处理纯钛种植体可能有利于与周围骨组织的结合。     

钛表面不同处理对成骨细胞生长影响的实验研究

目的：探讨纯钛表面不同处理对成骨细胞生长的影响。方法：分离、切取日本大耳白兔胫骨骨膜,应用植块法培养兔骨膜原代成骨细胞,应用碱性磷酸酶（ALP）染色,钙结节染色,进行成骨细胞鉴定。将原代培养的成骨细胞与不同处理的纯钛片（抛光处理、喷砂处理）紫外灯光照处理后联合培养。采用扫描电镜、碱性磷酸酶活性（ALP）检测,MTT检测,观察不同处理表面的微型态对成骨细胞黏附、增殖的影响。结果：扫描电镜观察成骨细胞平铺在抛光处理的钛片的表面,没有伪足伸出;在喷砂处理的钛片表面上成骨细胞伪足伸入孔洞内,有伪足伸出。喷砂组ALP活性明显高于抛光组。结论：粗糙钛表面比光滑钛表面更有利于成骨细胞的黏附、增值;紫外灯光照钛片对成骨细胞的黏附、增殖无不利影响。     

人血管内皮细胞在不同钛金属表面的生物学表现

目的：研究人血管内皮细胞在不同粗糙度和亲水性的钛金属表面的增殖与血管化、炎症相关因子的基因表达。材料与方法：将人脐带静脉血管内皮细胞（HUVEC）置于直径15mm厚度1mm的商业纯钛金属表面培养。钛金属根据不同粗糙度和亲水性分为4组：疏水光滑表面（P）、疏水粗糙表面（PT）、亲水光滑表面（modP）与亲水粗糙表面（modPT）。在24、48与72小时,采用直接计数与CCK-8试验评价细胞增殖。在120小时,采用real-timePCR评价血管化、炎症相关因子,vWF、TM、EPCR、E-selectin及ICAM-1的基因表达。结果：所有时间点,HUVEC在modP和modPT表面的增殖均显著低于P和PT表面。在72小时,HUVEC在PT和modPT表面的增殖分别低于P和modP表面。在modPT表面,所有因子的表达均显著高于modP表面。PT表面的vWF、EPCR、ICAM-1的表达显著高于P表面。modPT表面的EPCR和E-selectin的表达显著高于PT表面。结论：本研究提示粗糙和亲水的钛金属表面抑制血管内皮细胞的增殖,加强血管化和炎症相关因子的基因表达。     

Attachment of human marrow stromal cells to titanium surfaces

The attachment of human bone marrow stromal cells to titanium alloy (Ti6Al4V) surfaces was investigated. Titanium disks were polished and modified by surface roughening and by passivation in nitric add. Cell attachment to titanium surfaces and tissue culture plastic (TCP) was determined by tetrazolium bromide (MTT) assay at 2, 6, 24, and 48 hours after seeding. Cell proliferation was determined by thymidine incorporation. Attachment on titanium surfaces was 75.6% to 94.9% of attachment on TCP control. The difference between cell attachment on the TCP compared with smooth or rough titanium was statistically significant (P < .05). However, no statistically significant difference was found between attachment to TCP and passivated titanium. Cell proliferation on titanium surfaces after 24 hours was approximately 70% of proliferation on TCP. There was a statistically significant difference (P < .05) between proliferation on tissue culture and smooth and passivated titanium but not on rough titanium. These results indicate that titanium provides a surface that is conducive to cell attachment and that passivating titanium improves cell attachment, approaching levels seen with TCP, a surface specifically developed to enhance cell attachment. Increasing surface roughness results in improved cell proliferation on titanium.     

兔骨髓基质细胞在不同纯钛表面的早期粘附

目的：比较兔骨髓基质细胞在3种不同化学蚀刻纯钛表面的早期粘附情况。方法：分别用HNO3、热H2SO4／H2O2、热H2SO4／HCl处理纯钛片30min。采用扫描电子显微镜、X射线光电子能谱对试样表面形貌及成分进行分析。取兔骨髓基质细胞接种于钛片表面，培养30、60、120min，采用荧光显微镜和四唑盐比色法对细胞粘附进行观察和分析。结果：HNO3组表面形貌光滑，平整；H2SO4／HCl、H2SO4／H2O2组表面粗糙。3组钛片表面的主要成分为钛、氧和碳。细胞在H2SO4／HCl、HNO3组表面粘附伸展良好，在H2SO4／H2O2组表面伸展较差。结论：细胞在H2SO4／H2O2组表面的粘附不及H2SO4／HCl组，甚至不如HNO3组。     

载锌纳米线修饰钛表面的血管内皮细胞生物学行为研究

目的 探究载锌纳米线修饰钛表面对血管内皮细胞生物学行为的影响。方法 纯钛试件抛光清洗后,通过酸蚀和碱热处理,在其表面制备纳米线(NW-Ti)和载锌纳米线(Zn-NW-Ti)结构。以光滑钛表面(cp-Ti)为对照组,纳米线、载锌纳米线修饰钛表面为实验组。通过扫描电镜、X射线光电子能谱(XPS)、接触角测量仪分析各组钛表面的微形貌、元素组成和亲水性;采用锌离子检测试剂盒检测载锌纳米线修饰钛表面释放的锌离子浓度;将人脐静脉内皮细胞系细胞(HUVECs)接种于各组试件表面,通过细胞粘附、增殖、迁移及实时荧光定量PCR实验,研究不同钛表面对人脐静脉内皮细胞行为的影响。结果 载锌纳米线修饰钛表面为形貌均一的纳米线状拓扑结构,并含有微量锌元素,其释放的锌离子浓度约为1 mg/L。与其余两组相比,载锌纳米线修饰钛表面能促进人脐静脉内皮细胞的粘附、增殖和迁移,并上调血管生成相关因子HIF-1α、VEGF-A的表达水平。结论 载锌纳米线修饰钛表面能促进血管内皮细胞粘附、增殖、迁移及相关功能基因的表达。     

Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfaces

Objective: The aim of this study is to analyze the morphology and proliferation of human osteoblastic cells in vitro on five commercially available titanium surfaces. Materials and methods: Human primary cells of the osteoblastic lineage were obtained from bone explants. The cells were plated on polished (T1), machined (T2), sand‐blasted/acid‐etched (T3), sand‐blasted/acid‐etched, modified with hydrogen peroxide rinse (T4), and plasma‐sprayed titanium (T5) disks. Cell morphology was studied after 6, 24, 72 h, 7 and 14 days of culture by scanning electron microscopy. The formation and distribution of focal adhesions was investigated by immunocytochemical staining at 3, 6 and 24 h. Cell growth was measured by an MTT assay after 3, 7 and 9 days of culture. Moreover, the production of osteocalcin and osteoprotegerin (OPG) was evaluated in the supernatants by ELISA. Results: Morphological analysis revealed that substrate topography profoundly affected cells' shape and their anchoring structures. Large lamellipodia were formed on polished and machined surfaces, while thin filopodia were more frequently observed on T3 and T4 samples. Moreover, cells formed stronger focal adhesions on T3 and T4 surfaces, and cell proliferation was higher on rough surfaces. Osteocalcin production was higher on the T4 surface, whereas OPG steadily increased on every surface. Conclusions: Taken together, these data show that all the surfaces allowed cell attachment, adhesion and proliferation, but T4 and T5 surfaces appeared to be a better substrate for the adhesion, proliferation and differentiation of cells of the osteoblastic lineage. To cite this article:
Passeri G, Cacchioli A, Ravanetti F, Galli C, Elezi E, Macaluso GM. Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfaces.
Clin. Oral Impl. Res. 21 , 2010; 756–765.
doi: 10.1111/j.1600‐0501.2009.01906.x     

紫外线照射对两种钛种植体表面形貌体外炎症反应的影响

目的:紫外线照射的抛光试件和TiO2纳米管试件对巨噬细胞增殖和细胞因子分泌的影响.方法:抛光组和纳米管组试件避光保存后,各取一半经紫外线照射获得抛光+紫外线组和纳米管+紫外线组试件.测量各组试件表面接触角;在各组试件上培养RAW264.7巨噬细胞,另设空白对照组.检测培养4、24和72h时巨噬细胞黏附和增殖情况;液相芯...     

微纳复合结构对成纤维细胞黏附增殖的影响

目的：探讨经不同方法处理后的纯钛表面对成纤维细胞黏附增殖的影响。方法：将36个试件分平均为3组：机械抛光组（A组）;喷砂酸蚀组（B组）;喷砂酸蚀碱热组（C组）,每组均12个试件。通过扫描电子显微镜（SEM）观察分析3组试件表面微观结构和细胞在试件表面的铺展情况,激光共聚焦显微镜（CLSM）检测各试件表面的粗糙度;运用CCK-8试剂盒在450 nm波长下检测各试件对成纤维细胞（ L929）黏附与增殖的吸光度值（ OD值）。结果：A组表面光滑,试件表面成纤维细胞骨架大多呈梭形铺展,伸展较差;B组和C组表面粗糙,且C组表面可见微纳复合结构,试件表面成纤维细胞骨架呈三维空间向铺展,表面黏附成纤维细胞数量明显多于A组和B组。观察第1,3,5 d试件表面细胞增殖情况,可见粗糙表面较光滑表面更利于成纤维细胞的增殖。结论：喷砂酸蚀碱热方法处理后的纯钛表面形成微米-纳米复合孔洞,表面活性好,促进成纤维细胞早期黏附及表面铺展,且不抑制细胞的增殖。     

两种生长因子对人牙周膜成纤维细胞在钛金属表面附着和生长的影响

目的：研究两种生长因子对牙周膜成纤维细胞在然金属表面附着和生长的影响。方法：将纯钛、钛75试件入在12孔培养板内，取生长良好的第五代人牙周膜成纤维细胞（PDLF）接种在试件表面，分别在接种后4h、12h、24h、72h进行贴壁细胞地数。结果：接种后4h、12h、24h、72h、bFGF组纯钛、钛75表面细胞附着数与空白对照组的差异均有显著性（P＜0．05），rhBMP－2组纯钛、然75表面细胞附着数在初期（24h）与空白对组无显著性差异（P＞0．05）。72h时与空白对照组差异有显著性（P＜0．05），表明bFGF促进细胞附着和生长作用显著，而rhBMP－2促进细胞生长作用较促附着作用明显。结果：PDLF在钛金属表面的附着和生长可被生长因子所增强，但不同的生长因子对细胞附着和生长的生物学效应不尽相同。     

导电高分子聚吡咯纯钛表面改性对成骨细胞生长的影响

目的 :研究电场刺激下纯钛表面聚吡咯涂层对成骨细胞的黏附、增殖的影响。方法 :10 0mV阳极电刺激下 ,观察接种 4、8、12、16、2 0、2 4h成骨细胞的黏附数量以及 2 4h、72h的MTT吸光度值 ,SEM观察成骨细胞的生长情况。结果 :电刺激组 (Ti+电刺激 ,Ti+Ppy +电刺激 )的成骨细胞贴附密度在 4、8、12、16h均高于无电刺激组 (Ti、Ti+Ppy) (P <0 .0 5 ) ;聚吡咯涂层电刺激组的成骨细胞贴附密度 (Ti +Ppy +电刺激 )在4h、8h高于单纯电刺激组 (Ti+电刺激 ) (P <0 .0 5 ) ;72h后钛表面聚吡咯涂层加电刺激组 (Ti+Ppy +电刺激 )的吸光度高于其它三组 (Ti、Ti+Ppy、Ti+电刺激 ) (P <0 .0 1)。 结论 :聚吡咯涂层具有良好的生物相容性 ,并且在阳极电刺激下可以显著的促进成骨细胞的早期贴附和增殖     

Interaction of chlorhexidine with smooth and rough types of titanium surfaces

BACKGROUND: Chlorhexidine (CHX) digluconate exerts plaque inhibitory efficacy in the natural dentition environment due to a superior degree of persistence at the tooth surface. The purpose of the present study was to assess the interaction of CHX with titanium surfaces to estimate its antiplaque potential in the peri-implant environment. METHODS: Saliva-coated machined smooth (S) and sand-blasted acid-etched rough (R) titanium disks were soaked in either 0.1% or 0.2% CHX solution. After 24 hours, CHX amounts that were adsorbed, washed out, and desorbed from the titanium surfaces were determined spectrophotometrically at 230 nm. The antibacterial activity of CHX-treated titanium disks was assessed by measuring bacterial inhibition zones on Streptococcus mutans lawns. RESULTS: Titanium disks adsorbed 3% to 8% of the available CHX, which was significantly higher with 0.2% CHX (P<0.001) than with 0.1% CHX and two-fold higher on the R titanium disks compared to S titanium surface (P<0.001). After rinsing with water, 2.2% of the adsorbed CHX was washed out. Over 24 hours, S- and R-type disks released 1.1% and 0.6% of the adsorbed agent, respectively. Larger bacterial inhibition zones were obtained with 0.2% CHX and in R disks compared to S disks. CONCLUSIONS: CHX displayed persistence at the titanium surface. The adsorption level and bacterial growth inhibition were affected by CHX concentration and titanium surface characteristics, with higher levels of adsorption and antibacterial activity with 0.2% CHX and rough titanium surface. The slow CHX release rate suggests persistence of this agent at the titanium-pellicle surface, which can provide a long-term antiplaque effect.     

Immunolocalization of proteins specific for adhaerens junctions in human gingival epithelial cells grown on differently processed titanium surfaces

The localization of desmoplakins 1 and 2 (DP 1&2), components of desmosomes, vinculin, and actin, was studied in gingival epithelial cells grown on cell culture glass and on titanium plates with various surface topography. The results showed that epithelial cells attached and spread more readily on smooth than on rough, sandblasted titanium surfaces. Moreover, the cells appeared to develop more granular DP 1&2 immunoreactivity at their ventral surfaces when grown on smooth or etched titanium as compared to glass. In cells grown on sandblasted titanium surfaces, DP 1&2-specific immunoreactivity was primarily located at cell-cell contacts. Cells grown on smooth titanium surfaces harbored a fine network of actin filaments with apparent cell-to-cell organization. Vinculin was confined to cell-cell contact areas. No vinculin-containing focal adhesions could be detected, suggesting that the cells adhere either by means of close contacts, extracellular matrix contacts, or by means of hemidesmosomes. The findings suggest that smooth of finely grooved titanium surfaces could be optimal in maintaining the adhesion and specialized phenotype of gingival epithelial cells.