钛片表面粗糙度和氧化膜对成骨细胞增殖和分化的影响

目的：研究钛片表面粗糙度和氧化膜对成骨细胞增殖和分化的影响,为种植体表面处理提供理论依据。方法：采用粒度分别为108～130 μm(S1)、216～301 μm(S2)和356～411 μm(S3)的二氧化钛颗粒对纯钛钛片表面进行喷砂处理,钛浆喷涂(titanium-sprayed plasma, TPS)表面处理组由Straumman 公司提供,600目砂纸打磨组(S0)作为对照组,在钛片表面进行成骨细胞培养。采用表面轮廓测量仪测量其表面粗糙度,电子探针(electron microprobe)测定钛片表面氧化膜结构。分别在1、3、5及7 d时,采用四锉盐比色(MTT)法检测不同处理表面对成骨细胞增殖(OD值)的影响;通过碱性磷酸酶活性(ALP)及骨钙素分泌(OC)检测比较不同处理的表面对成骨细胞分化的影响。采用SPSS12.0软件包对数据进行单因素方差分析。结果：S0 、S1 、S2 、S3 和TPS组表面粗糙度由0.372 μm至5.239 μm递增;喷砂组钛片表面氧化膜结构完整、连续;粗糙表面比光滑表面更利于成骨细胞增殖和分化。喷砂表面粗糙度越高,越利于成骨细胞增殖和分化。S3组成骨细胞增殖和分化优于TPS组。结论：表面粗糙度较高的喷砂表面,更利于成骨细胞增殖和分化。     

亲水性纯钛表面对成骨细胞黏附行为的影响

目的:体外研究亲水性喷砂酸蚀钛表面对成骨细胞黏附、铺展行为和黏着斑激酶(FAK)表达的影响.方法:钛片表面分别采用大颗粒喷砂酸蚀表面处理(sandblasted,large-grit,acid-etched,SLA)及亲水性化学活化大颗粒喷砂酸蚀表面处理(chemically-modified hydrophilic SLA,modSLA),在其表面接种人成骨细胞,对细胞黏附率、细胞铺展情况以及黏着斑激酶(FAK)的表达进行检测.应用SAS6.0软件包对数据进行统计学分析.结果:成骨细胞在modSLA表面的早期黏附率(1h、3h)显著高于SLA表面(P<0.05);接种3h后,modSLA表面的成骨细胞呈现更多的肌动蛋白结构和明显的成骨细胞骨架结构,细胞铺展更加明显,modSLA组细胞形状因子均值显著低于SLA组(P<0.05);免疫荧光分析显示,6h modSLA表面细胞内FAK的荧光强度高于SLA组(P<0.05).结论:亲水性化学活化大颗粒喷砂酸蚀处理钛表面较大颗粒喷砂酸蚀处理钛表面能显著增强成骨细胞在材料表面的贴附,促进细胞骨架沿一定方向伸展,促进黏着斑激酶(FAK)的表达,从而增强细胞的黏附力.     

纯钛表面阳极氧化电压对成骨细胞早期黏附及伸展的影响

目的:通过成骨细胞离体培养试验,观察不同阳极氧化电压对成骨细胞早期黏附及伸展的影响。方法:分别在电压为140V、200V及260V,电流密度为70A/m2等条件下,在0.03mol/L甘油磷酸钙(Ca-GP)和0.15mol/L醋酸钙混合电解液中,对纯钛样本表面进行阳极氧化处理。对阳极氧化后样本的平均粗糙度进行测量,并在其表面进行人成骨细胞培养,对早期细胞黏附及伸展等情况进行研究。采用SPSS13.0forWindows统计分析软件中的单因素方差分析进行统计学处理。结果:纯钛表面平均粗糙度为0.17μm,经阳极氧化后,随电压升高,平均粗糙度分别为0.23μm、0.26μm及0.33μm,统计学分析表明有显著性差异(P<0.05)。经过2h细胞培养后,阳极氧化处理后,纯钛表面细胞骨架发生形态学改变,且随阳极氧化电压的增高,细胞形态不规则呈现升高的趋势。统计分析结果表明,260V电压阳极氧化组,细胞黏附数显著高于对照组(P<0.05)。结论:阳极氧化处理可改善纯钛表面特性,成骨细胞在其表面的黏附及伸展等早期细胞行为受阳极氧化电压的影响。     

影响成骨细胞在种植体表面黏附的因素

种植体与骨组织形成骨结合是种植体成功的前提。而成骨细胞在种植体表面的黏附作为成骨细胞与种植体作用的第一期,对于形成良好的骨结合界面至关重要。本文综述了影响成骨细胞在种植体表面黏附的因素。     

TiO2喷砂酸蚀处理对钛片表面氧化膜及成骨细胞生长影响的研究

目的研究纯钛钛片经喷砂及喷砂酸蚀处理后,表面氧化膜金相结构和化学成分的变化及对成骨细胞黏附和生长特性的影响。方法将直径为15 mm、厚度为1 mm的纯钛钛片分4组进行表面处理：1）机械打磨组（S0）;2）喷砂组（SB）;3）喷砂酸蚀1组（SLA1）;4）喷砂酸蚀2组（SLA2）。采用电子探针分析仪及X射线衍射仪检测4组钛片表面氧化膜的厚度、化学成分以及金相结构,扫描电镜观察其表面微观形态。而后将成骨细胞培养于4组钛片表面,采用MTT法分析比较4组钛片表面对成骨细胞黏附率以及增殖率的影响。结果与S0组相比,SB、SLA1、SLA2组的粗糙度明显增大（P<0.05）。SB、SLA1、SLA2组间表面平均粗糙度差异无统计学意义（P>0.05）。酸蚀处理使喷砂形成的氧化膜变薄,密度减低,且结构发生改变：原有的金红石型TiO2峰消失,锐钛矿型TiO2减少。在表面平均粗糙度相同条件下,SB组钛片表面氧化膜均匀致密,有利于成骨细胞早期的黏附和增殖。结论喷砂和喷砂酸蚀处理均增加了钛片表面的粗糙度,有利于成骨细胞的黏附和增殖,但酸蚀使TiO2喷砂表面的氧化膜层变薄,在平均粗糙度不变的情况下,单纯喷砂表面成骨细胞的黏附和增殖优于喷砂酸蚀处理表面。     

TiO2纳米管表面对成骨细胞与巨噬细胞黏附、形态及迁移的影响

目的:研究TiO₂纳米管对小鼠成骨细胞和巨噬细胞早期黏附、细胞形态及迁移运动的影响。方法:将小鼠成骨细胞(MC3T3-E1)和巨噬细胞(RAW264.7)分别接种于抛光(P)后的纯钛试样和抛光钛经20 V恒压阳极氧化制备出的纳米管形貌(NT)钛试样表面,扫描电镜观察钛试样的表面形貌以及细胞接种于各组钛试样3、6、12、24、48 h后的细胞形态,Hoechst染色后荧光显微镜统计各组钛试样在接种3、6、12、24 h后的细胞黏附数量,活细胞工作站观察细胞在不同形貌钛表面24 h的迁移运动轨迹。结果:NT组钛试样表面可见均匀排列的纳米管阵列,P组钛表面光滑。与P组相比,NT组表面成骨细胞黏附数量较多,细胞丝状伪足长且丰富,细胞迁移运动缓慢,而巨噬细胞的黏附数量和丝状伪足较少,细胞迁移速率更高。结论:钛表面纳米管形貌可促进成骨细胞黏附,抑制其迁移运动,而对巨噬细胞的影响则相反。     

种植体表面粗糙度对成骨细胞增殖及ALP含量的影响

目的:何种粗糙度的种植体能产生最快、最强的骨结合尚有争议,本研究将成骨细胞接种到不同粗糙度的钛盘表面,观察成骨细胞生长、增殖及碱性磷酸酶(alkalinephosphatase,ALP)分泌情况,探讨最适合的种植体表面粗糙度范围。方法:纯钛圆盘试件48个,分为4组,前三组喷砂加双重酸蚀粗化处理,获得表面粗糙度为(1.00±0.20)、(1.67±0.08)、(2.40±0.20)μm的三组试件。未处理的试件粗糙度为(0.12±0.03)μm,作为对照组。将成骨细胞接种到4组试件表面,观察成骨细胞形态、增殖以及ALP含量的变化,并作统计分析。结果:成骨细胞在粗糙度1.00μm的钛盘表面呈单层生长;粗糙度1.67μm的钛盘表面细胞呈立方形,有较多伪足,细胞数量及ALP含量最多;粗糙度2.40μm的试件细胞生长不规则,细胞数量及ALP含量最低。结论:粗糙的种植体表面比光滑表面更有利于成骨细胞的黏附、增殖及表达。其中粗糙度为1.00～2.40μm时成骨细胞增殖和表达最显著。     

不同TiO2纳米管表面对成骨细胞黏附的研究

目的:研究MC3T3-E1成骨细胞在不同直径和长度TiO2纳米管上的黏附/生长情况.方法:选取固定长度约500 nm,直径约30 nm/80 nm的TiO2纳米管,以及固定直径约80 nm,厚度约500 nm/1.5μm的TiO2纳米管材料,体外研究其成骨细胞的黏附生长情况.结果:细胞形态和细胞计数显示成骨细胞在直径为...     

钛种植体表面粗糙度对细菌黏附的影响

目的：研究钛种植体的不同表面粗糙度对变形链球菌及血链球菌黏附的影响.方法：用光电3-D表面测量系统测定两种纯钛片机械切割表面和大颗粒喷砂酸蚀表面的表面粗糙度.将钛片与变形链球菌和血链球菌共同培养,培养时间分别为4h,1d和5d.通过菌落形成单位（CFU）计数法及结晶紫染色法,比较不同时间点两种细菌在两种粗糙度钛片上的黏附量.结果：机械切割表面和大颗粒喷砂酸蚀表面钛片的表面粗糙度Ra值分别为1.25 μm和4.25 μm.CFU计数显示,在不同的培养时间点,两组钛片上的变形链球菌及血链球菌活菌黏附数量相近,差异无统计学意义（P＞0.05）.结晶紫染色显示,在不同的培养时间点,大颗粒喷砂酸蚀组钛片上血链球菌的细菌黏附总量均多于机械切割组钛片,差异有统计学意义（P ＜0.05）.在培养早期（4h）,大颗粒喷砂酸蚀组钛片上变形链球菌的细菌黏附总量大于机械切割组钛片;但在培养后期（1 d,5 d）两组钛片上变形链球菌的细菌黏附总量相近,差异无统计学意义（P＞0.05）.结论：钛片不同表面粗糙度对变形链球菌和血链球菌的活菌黏附数量无影响,但粗糙表面上黏附的细菌及基质总量大于中度粗糙表面.对于变形链球菌而言,粗糙度对细菌及基质黏附总量的影响随着生物膜的成熟而消失.     

牙周韧带细胞及成骨样细胞在纯钛表面附着的形态学比较

目的：从形态学上比较牙周韧带细胞及成骨样细胞在纯钛表面的附着及增殖情况。方法：在相同的培养条件下，商用纯钛表面分别接种牙周韧带细胞和成骨样细胞，采用倒置显微镜及扫描电镜进行形态学观察。结果：两种细胞在纯钛表面均附着、生长良好，7天后细胞即连接成片，并在钛片边缘存在“细胞聚集”现象，呈现复层生长。扫描电镜显示两种细胞附着早期呈“伪足”样伸展，附着后期形成大量的细胞外基质成份。两种细胞在细胞形态、“伪足”形态及钛片周边聚集形态上均存在明显差异。结论：牙周韧带细胞和成骨样细胞与纯钛均有良好的生物相容性，在体外培养过程中，呈现不同细胞形态、“伪足”形态及钛片周边聚集形态，提示两种细胞在分化过程中可能存在差异。     

牙周韧带细胞在纯钛表面的附着特征

目的　观察牙周韧带细胞在商用纯钛表面的附着特征。方法　MTT法分析牙周韧带细胞在商用纯钛表面的早期粘附 ,采用落射荧光观察细胞的粘附形态。结果　牙周韧带细胞在纯钛表面的早期粘附 2h达到饱和态 ,且呈现不同的粘附时相。细胞之间逐渐以丝状结构联接 ,丝状结构与钛片的打磨走向一致 ,最后融合成片。结论　牙周韧带细胞在纯钛表面的粘附具有规律性 ,纯钛的表面处理方法对细胞的生长极性有明显影响     

纯钛表面牙周韧带细胞牙骨质附着蛋白的表达

目的以牙骨质附着蛋白为分化指标，分析牙周韧带细胞在纯钛表面的分化趋势，并探讨诱导矿化对牙周韧带细胞牙骨质附着蛋白表达的影响。方法第三代牙周韧带细胞在商用纯钛表面接种后，分别进行常规培养和矿化液诱导矿化培养，通过免疫荧光原位检测牙骨质附着蛋白的表达，并比较诱导矿化对牙骨质附着蛋白表达的影响。结果牙周韧带细胞在纯钛表面初期呈条纹状生长，常规培养和诱导矿化培养务件下均能表达牙骨质附着蛋白，诱导矿化对牙骨质蛋白的表达无明显影响。结论在常规培养务件和诱导矿化培养条件下，纯钛表面生长的牙周韧带细胞能高度表达牙骨质附着蛋白，提示纯钛表面体外培养的牙周韧带细胞具有形成牙骨质的分化趋势，从而可为种植体周构建牙周韧带结构提供在种植体侧的锚着点。     

表面粗糙度对水与唾液在纯钛及PMMA上润湿作用的影响

目的:评价全口义齿基托材料及其表面粗糙度对义齿固位的影响。方法:测定水与唾液在表面粗糙度不同的纯钛和聚甲基丙烯酸甲酯(PMMA)上的前进接触角和后退接触角,通过两者的差值θh反映水与唾液对它们的润湿效果。结果:1、随着表面粗糙度增加,唾液在纯钛上的θh逐渐降低,但当Ra≥1.0,Rz≥1.5时,θh突然增大;而PMMA组,当Ra<1.2,Rz<2时,粗糙度对θh无明显影响,当Ra≥1.2,Rz≥2时θh也显著增加,多数情况下,唾液在相同粗糙度的纯钛上的θh大于在PMMA上的θh。2、水在光滑的PMMA和纯钛上的前进接触角比唾液的大。结论:唾液对纯钛的润湿效果,可以通过高度磨光或形成较为粗糙的表面(Ra≥1.0,Rz≥1.5)得到提高,多数情况下这种润湿效果好于PMMA。提示从润湿效果看,对于不能高度研磨和抛光的义齿组织面,全口义齿上颌腭托选择纯钛并形成较为粗糙的表面可能更有利于固位。     

二种阳极氧化电解液对钛表面细胞相容性的影响

目的:研究纯钛表面阳极氧化过程中电解液成份对其细胞相容性的影响。方法:在电流密度为70A/m2及200伏电压等条件下分别在0.03M甘油磷酸钙(Ca-GP)与0.15M醋酸钙混合液(混合组)及0.2M磷酸溶液中对纯钛样本表面进行阳极氧化处理。在阳极氧化及纯钛样本表面进行人类成骨细胞培养,以观察细胞毒性、细胞增殖及分化等细胞行为的改变。结果:细胞毒性试验结果表明,本研究中所用的电解液不会对阳极氧化后纯钛样本产生细胞毒性。所有样本表面经过1d、2d或4d细胞增殖培养后磷酸组与纯钛组之间没有显著性差异,而在0.03M甘油磷酸钙(Ca-GP)与0.15M醋酸钙混合液中进行阳极氧化处理后的纯钛样本,经过2d及4d成骨细胞培养后细胞增殖数量较纯钛组及磷酸组显著增高。细胞分化试验结果表明,除在4d时混合液组碱性磷酸酶活性较纯钛组及磷酸组显著性降低外,其余各组间无显著性差异。结论:对纯钛表面的阳极氧化处理过程中,电解液成份可对阳极氧化后纯钛表面的细胞相容性产生影响。     

Determining optimal surface roughness of TiO(2) blasted titanium implant material for attachment, proliferation and differentiation of cells derived from human mandibular alveolar bone

In the complex process of bone formation at the implant-tissue interface, implant surface roughness is an important factor modulating osteoblastic function. In this study, primary cultures of osteoblast-like cells, derived from human mandibular bone, were used. The aim was to examine the effect of varying surface roughness of titanium implant material on cellular attachment, proliferation and differentiation. A recognized method of increasing surface roughness and enlarging the surface area of titanium implants is by blasting with titanium dioxide particles: the four specimen types in the study comprised surfaces which were machine-turned only, or blasted after turning, with 63-90 microm, 106-180 microm, or 180-300 microm TiO(2) particles, respectively. The specimens were analyzed by scanning electron microscopy and confocal laser scanning. The turned samples had the smoothest surfaces: average height deviation (S(a)) of 0.20 microm. The roughest were those blasted with 180-300 microm particles, S(a) value 1.38 microm. Blasting with intermediate particle sizes yielded S(a) values of 0.72 microm and 1.30 microm, respectively. Cell profile areas were measured using a semiautomatic interactive image analyzer. Figures were expressed as percentage of attachment. DNA synthesis was estimated by measuring the amount of [(3)H]-thymidine incorporation into trichloroacetic acid (TCA) insoluble cell precipitates. The specific activity of alkaline phosphatase was assayed using p-nitrophenylphosphate as a substrate. The ability of the cells to synthesize osteocalcin was investigated in serum-free culture medium using the ELSA-OST-NAT immunoradiometric kit. After 3 h of culture, the percentage of cellular attachment did not differ significantly between specimens blasted with 180-300 micromparticles and the turned specimens. All blasted surfaces showed significantly higher [(3)H]-thymidine incorporation than the turned surfaces (P<0.05), with the highest on the surfaces blasted with 180-300 microm particles. Osteocalcin synthesis by the cells in response to stimulation by 1,25(OH)2D3, was also significantly greater (P<0.05) on the surfaces blasted with TiO(2) particles. However, analysis of alkaline phosphatase activity disclosed no significant differences among the four surface modifications. It is concluded that in this cellular model, the proliferation and differentiation of cells derived from human mandibular bone is enhanced by surface roughness of the titanium implant. However, increasing the size of the blasting particles to 300 microm does not further increase the initial attachment of the cells compared to turned surfaces and those blasted with 63-90 microm particles.     

多孔海绵状纯钛种植材料对成骨细胞生长的影响

目的：探讨具有连续多孔立体结构纯钛种植材料对成骨细胞生长的影响。方法：在体外对3种不同孔隙直径的多孔钛材料进行成骨细胞复合培养,在相差倒置显微镜下观察细胞在材料表面及孔隙中的生长情况,利用MTT法评价不同孔隙直径对细胞生长的影响。结果：成骨细胞在试件表面生长良好,成功长入连续孔隙之中,并以多突触的方式生长,在钛珠之间形成细胞桥;细胞增殖度检测显示多孔立体结构对成骨细胞的生长有一定的促进作用,200μTI孔径的作用最明显。结论：多孔立体结构能促进成骨细胞在纯钛种植材料表面和孔隙中的生长。     

自动研磨对纯钛铸件表面粗糙度的影响

目的　探讨不同自动研磨条件对纯钛铸件表面研磨效果的影响。方法　分别使用４ 种形态的磨粒,在转速分别为２５０ｒｐｍ 、４５０ｒｐｍ 、和６５０ｒｐｍ 条件下, 对１０ ｍ ｍ ×１０ｍ ｍ ×１ ．４ ｍ ｍ 纯钛铸件分别自动研磨１０ｍｉｎ 、３０ｍｉｎ 、６０ ｍｉｎ 和９０ ｍｉｎ , 使用电子天平和表面粗糙度测定仪分别测定各组试件研磨前后的重量和表面粗糙度（ Ｒａ值） , 并计算出研磨减重率（ Ｗｔ％ ） 。结果　随着自动研磨机的转速增高、研磨时间延长, 钛铸件的研磨量增大, Ｒａ 值随之下降。有较锐边角的磨粒研磨效率高, 适用于粗研磨; 不定形磨粒研磨效率稍差, 但表面粗糙度改善效果好, 可用于中研磨; 而球形磨粒适用于最后抛光。自动研磨后纯钛铸件 Ｒａ 值可降至０ ．３１５５μｍ 。结论　自动研磨机可用于纯钛义齿铸件的研磨、抛光。     

Effect of magnetic field on the fibronectin adsorption, cell attachment and proliferation on titanium surface

OBJECTIVES: We studied the effect of various static magnetic fields (SMFs) on the adsorption of specific recombinant fibronectin (FN) peptide (hFNIII9-10) on the titanium surface. Furthermore, the responses of human osteosarcoma TE-85 cells in the SMF were observed. MATERIAL AND METHODS: Various magnetic fields--1, 2, 3, 5, 7, 10 mT--were established by controlling the distance from Nd-Fe-B magnet to the disks. For FN adsorption experiment, machined titanium disks were incubated in 1 microM hFNIII9-10 at 37 degrees C overnight under magnetic field. The adsorbed hFNIII9-10 was measured as optical density (OD). For attachment study, TE-85 cells were incubated for 2 h on the hFNIII9-10 coated machined titanium disks and OD values were measured. As for proliferation study, titanium disks were incubated for 48 h after washing unattached cells in 2 h. The amount of proliferated TE-85 cell was also measured as OD value. Attachments of TE-85 cells under various intensities of magnetic field were observed using a scanning electron microscope. RESULTS: The amount of adsorbed hFNIII9-10 showed no significant difference between control (0 mT) and six experimental groups (1, 2, 3, 5, 7, 10 mT). However, TE-85 cells attached significantly higher in groups of 1, 2, 5, 10 mT than in control group (P=0). Cell attachment in groups of 3, 7 mT showed no significant difference with that of control group. TE-85 cells were observed to attach through filopodia. Especially in 1 mT, flattened cells were predominant. In proliferation assay, 1 mT stimulated TE-85 cells showed significantly higher proliferation than those in 2, 3 and 7 mT (P=0). CONCLUSION: Magnetic fields under 10 mT did not influence FN adsorption on the titanium surface. However, a significant effect was found on cell attachment and proliferation.     

Effect of three distinct treatments of titanium surface on osteoblast attachment, proliferation, and differentiation

Cell-titanium interactions are crucial to the clinical success of bone and dental implants. The physico-chemical characteristics of the substrates surface influence osteoblast proliferation, differentiation, and activity as well. The osteoblast behavior was analyzed on three different titanium surfaces: ground with an abrasive 600 grit SiC paper, blasted with alumina particles (65 microm diameter) and alumina blasted followed by a double chemical etch (4% HF+4% HF/8% H2O2). Scanning electron microscopy (SEM) and profilometry showed distinct microtopographies. Ground samples showed parallel-groove orientation. The Al2O3-blasted surface presented the roughest microtopography with aluminum-rich particles incrusted in the titanium surface. Osteoblasts cells from femora of Balb/c mice were seeded onto the substrates tested. Cell morphology and initial attachment were evaluated by SEM. Osteoblasts adhered to and spread on all samples tested. However, on rough surfaces, osteoblasts did not spread completely and acquired a polygonal morphology. Besides, the cell proliferation rate was diminished at the beginning of incubation on rough surfaces. Our results suggest a delay, rather than an impairment, in osteoblast viability and alkaline phosphatase activity when cells are cultured on rough surfaces, inducing a distinct osteoblast phenotype, rather than blocking its activity. At least in the culture conditions used in this work, alumina particles did not affect osteoblast behavior.