喷砂酸蚀混合碱热处理后纯钛表面生物活性的研究

目的研究喷砂酸蚀混合碱热处理后的纯钛表面其生物活性。方法将经抛光(M)、喷砂酸蚀(SLA)、喷砂酸蚀混合碱热(AHH)处理后的纯钛试件分为3组。扫描电子显微镜(SEM)及能谱分析(EDS)对3组表面结构和表面化学元素及其含量进行分析;荧光显微镜下观察成纤维细胞在样品表面的粘附及铺展情况;并将各组试件浸泡模拟体液(SBF)中观察羟基磷灰石沉积情况。结果纯钛表面微纳复合结构中,微米孔直径3~5μm,纳米孔直径100~200 nm,同时在试件表面引入钙钠元素。成纤维细胞粘附:DAPI染色后细胞核呈蓝色荧光,罗丹明B染细胞骨架红色,SLA组、AHH组细胞铺展较M组好,呈空间铺展;且AHH组表面细胞数量明显多于其他两组。第1周时,AHH组表面沉积磷灰石明显可见,而未在M、SLA组检测到羟基磷灰石。结论喷砂酸蚀混合碱处理后的纯钛表面,表面活性好,有助于促进成纤维细胞早期粘附及其在空间上的铺展,同时又促进羟基磷灰石的沉积,进而体现了良好的生物性能,可以为种植体表面处理方法提供参考。     

铸钛的不同表面处理对钛-聚合瓷结合强度影响的实验研究

目的 研究铸钛的不同表面处理方法对钛-聚合瓷结合强度的影响。方法 将24个铸钛试件随机分为光 滑组、粗糙组、酸蚀光滑组和酸蚀粗糙组,每组6个。根据分组不同分别采用不同的表面处理方式,粗糙组进行喷 砂处理,酸蚀光滑组进行酸蚀处理,酸蚀粗糙组喷砂后进行酸蚀,光滑组表面不做处理。表面处理后的钛试件与 聚合瓷制备成钛-聚合瓷试件,测试其剪切结合强度,并在扫描电镜下观察钛表面形貌和剪切试验后钛与聚合瓷断 裂面的形貌。结果 光滑组、粗糙组、酸蚀光滑组和酸蚀粗糙组的剪切结合强度分别为（3.08±0.45）、（6.05±0.74）、（6.27±0.80）、（10.16±0.82）MPa。粗糙组、酸蚀光滑组和酸蚀粗糙组的剪切结合强度高于光滑组（P<0.01）,其中酸 蚀粗糙组的剪切结合强度最高,粗糙组和酸蚀光滑组间的剪切结合强度无统计学差异（P>0.05）。各组的钛表面形
貌和剪切试验后钛与聚合瓷断裂面的形貌均有一定的差异。结论 钛表面酸蚀处理和喷砂处理可提高钛-聚合瓷的剪切结合强度,喷砂后酸蚀处理是一种有效地提高钛-聚合瓷剪切结合强度的表面处理方法。     

喷砂酸蚀与双重酸蚀钛表面对成骨细胞行为影响的对比研究

目的 对比研究喷砂酸蚀与双重酸蚀钛表面对成骨细胞生物学行为的影响。 方法 在纯钛试件表面分别进行喷砂酸蚀与双重酸蚀处理。以光滑钛表面（Ti）为对照组,喷砂酸蚀钛表面（Ti-SLA）、双重酸蚀钛表面（Ti-DA）为实验组,通过扫描电镜（SEM）、表面接触角测试、X射线光电子能谱（XPS）观察分析三种钛表面的微形貌、润湿性和元素组成。将MC3T3-E1成骨细胞接种于三组试件表面,研究不同钛表面对成骨细胞生物学行为的影响。 结果 SEM观察显示Ti-DA组试件表面形成了较Ti-SLA组更均匀细密的微米级凹坑结构;各组试件的表面接触角无显著差异;XPS分析显示Ti-SLA组试件表面有微量铝元素残留;成骨细胞在三组试件表面的粘附、增殖无显著差异,而Ti-DA组显著促进成骨细胞的分化。 结论 与喷砂酸蚀钛表面相比,双重酸蚀钛表面的微米级凹坑结构更均匀细密,且无铝元素残留,能更有效地促进成骨细胞分化。     

钛基底表面处理对钛-树脂粘接强度的影响

目的：探讨不同钛基底表面处理对钛与树脂粘接强度的影响。方法将纯钛试件随机平均分组：光滑组（A组）、酸蚀组（B组）、喷砂组（C组）、喷砂+酸蚀组（D组）、微弧氧化组（E组）、氮化钛涂层组（F组）。根据以上分组,对试件进行相应的表面处理,用粗糙度仪进行钛表面粗糙度测量,后与Cemerage冠桥树脂结合,扫描电子显微镜（SEM）观察试件表面形貌,能谱分析仪（EDS）对表面元素进行分析,万能试验机检测试件与Cemerage冠桥树脂的粘接强度。结果各组的粗糙度分别为A组（0.370±0.039）μm;B组（1.456±0.044）μm;C组（2.044±0.019）μm;D组（1.970±0.047）μm,E组（0.683±0.023）μm;F组（2.195±0.066）μm。各组的剪切强度分别为A组（5.84±0.30）MPa;B组（10.22±0.63）MPa;C组（10.78±0.45）MPa;D组（12.24±0.46）MPa;E组（13.82±0.61）MPa;F组（16.81±0.74）MPa。结论钛基底表面喷砂结合酸蚀,微弧氧化及氮化钛涂层处理均可有效提高树脂与钛的粘接强度。     

不同表面处理钛片对成骨细胞骨架影响的研究

目的研究喷砂、酸蚀、碱热处理的钛片表面对成骨细胞F- actin骨架的影响。方法将纯钛钛片根据处理方法不同分为6组：机械打磨组（ G组）、喷砂组（ SB组）、酸蚀组（ SLA组）、光滑碱热组（ AH1组）、喷砂碱热组（ AH2组）、喷砂酸蚀碱热组（ AH3组）。在各组钛片表面培养成骨细胞1、2、4、12 h后,用Phalloidin- TRITC染色,在激光共聚焦显微镜下观察6组钛片表面成骨细胞F- actin骨架的变化。结果成骨细胞接种于6组钛片表面1 h后,各组表面肌动蛋白纤维丝不能清晰看见,SB、AH2、AH3、SLA组肌动蛋白呈环状,纤维分布于粗糙表面隆起部分的边缘。接种2 h后,各组肌动蛋白纤维开始铺展,SB组微丝束有方向性的排列;G组细胞的肌动蛋白纤维成束,排列于细胞周围,有沿沟纹方向铺展趋势;AH1组细胞肌动蛋白纤维于核周围的环绕核呈平行环状排列,周边纤维呈放射状分布;AH2、AH3组纤维呈网状排列,周边纤维汇集呈指状突起,伸入周围孔洞或附着于表面隆起部分的边缘。接种4 h后,G、AH1组肌动蛋白纤维开始定向平行排列;与细胞长轴一致。接种12 h后,各组表面肌动蛋白纤维完全铺展,互相平行排列,与细胞长轴一致,并横跨整个细胞,止于细胞周缘。SB、AH3、SLA组细胞肌动蛋白纤维汇集成束,伸入周围孔洞或附着于表面隆起部分的边缘,将细胞悬挂于凹窝上方。结论成骨细胞接种于6组钛片后,在不同的表面上肌动蛋白的重组有一定的顺序和形态。SB表面最利于细胞骨架排列及伸展。     

TiO_2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。     

聚合后热压处理对不同表面处理的铸钛与聚合瓷结合强度的影响

目的 研究聚合后热压处理对不同表面处理的铸钛与聚合瓷结合强度的影响。方法 将30个金属试件随机分为5组：光滑组（A组）、喷砂组（B组）、酸蚀喷砂组（C组）、喷砂热压组（D组）、酸蚀喷砂热压组（E组）,根据分组对试件进行相应处理并涂布聚合瓷,测试其剪切结合强度及表面显微硬度,并对钛与聚合瓷断裂面的形貌进行扫描电镜观察。结果 A、B、C、D、E组的剪切结合强度分别为（5.92±0.54）、（10.25±0.55）、（14.97±0.88）、（14.41±0.63）、（19.95±0.52） MPa,除C组与D组间无统计学差异外,其余两组之间均有统计学差异（P<0.01）。B、C、D和E组的钛表面可见部分聚合瓷残留于金属表面。D、E组的显微硬度值高于B、C组（P<0.05）。结论 聚合后热压处理可以显著提高钛与Ceramage聚合瓷的结合强度及聚合瓷的表面显微硬度。     

不同粗化纯钛表面对人成骨样细胞生物学行为的影响

目的研究不同粗化纯钛表面对人成骨样细胞生物学行为的影响。方法实验分4纰,对照组为打磨抛光的光滑纯钛片;A、B、C组分别为粒径100～200μm、200～300μm、300～400μm的A12O3颗粒喷砂酸蚀处理后的纯钛片。用场发射扫描电镜观察4组钛片表面形貌,表面轮廓仪测定钛片表面粗糙度（Ra值）。将人成骨肉瘤细胞系MG63接种于4组钛片上,激光共聚焦显微镜观察不同时fHJ段荧光染色后的细胞骨架。结果4组钛片扫描电镜观察结果显示对照组具有一致的平行沟纹,喷砂酸蚀组具有典型的喷眇酸蚀表面微形貌。表面轮廓仪测得对照组、A组、B组及C组的Ra值分别为0．27±0．01、1．69±0．05、2．08±0．11及2．55±0．20。激光共聚焦显微镜观察显示了4组纯钛片表面上MG63不同的细胞骨架解聚和重排的过程。结论不同粗化和喷砂酸蚀表面钛片影响人成骨样细胞粘附、伸展及分化过程中细胞骨架的变化,粒径200～300μmA1O3喷砂的酸蚀纯钛表面更利于细胞骨架的解聚和重排。     

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.     

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。     

Different titanium surface treatment influences human mandibular osteoblast response

BACKGROUND: Six titanium disks with six different surface treatments were examined: SS: smooth (polished) surface; TPS: plasma spray; C100: sand blasting by aluminum oxide (Al2O3) diameter 100 microm and acid etching; C150: sand blasting by Al2O3 diameter 150 microm and acid etching; B60: sand blasting by zirconium oxide (ZrO2) diameter 60 microm and acid etching; and B120: sand blasting by ZrO2 diameter 120 microm and acid etching. METHODS: The surface characteristics were determined by scanning electron microscopy (SEM) observation and a roughness tester. Raman spectroscopy was used to determine the presence of residual substances on the samples. Cells were seeded onto the disk and after 24 hours, 6 days, and 12 days were observed under SEM and growth curves generated with a cell counter. Some samples were used to determine alkaline phosphatase activity (ALP), using a colorimetric assay. RESULTS: SEM observation revealed drastic differences in surface microtopography, with a higher cell density on sand-blasted and acid-etched (SLA) samples than SS and TPS, and more regularly aligned cells on B60 and B120 surfaces than on the others. The growth curves showed a greater adhesion of cells on the etched/blasted surfaces compared to the SS and TPS surfaces. The number of cells increased on all the SLA samples, especially B60, throughout the experiment. At the same time, there was considerable ALP activity on the B60 sample, while it remained at extremely low levels on SS and TPS surfaces. Raman analyses revealed Al2O3 debris on C100 and C150, partly explaining the poorer performances of these two surface treatments, since this substance was shown to be toxic for cultured osteoblasts. CONCLUSIONS: Surface treatments influence the growth and the metabolic activity of cultured osteoblasts, and B60 seems to be the most favorable surface inducing a more pronounced proliferation of cells together with a high differentiation degree.     

喷砂酸蚀对钛铌锆锡合金表面成骨细胞粘附、增殖和分化的影响

目的:研究喷砂酸蚀(SLA)对钛及钛铌锆锡合金(Ti-24Nb-4Zr-7.9Sn,TNZS)表面形貌的影响,观察合金的形貌学特征,评价其生物相容性。方法:将试样分为钛机械打磨并抛光组(Ti组),钛铌锆锡机械打磨并抛光组(TNZS组),钛喷砂酸蚀组(Ti-SLA组)和钛铌锆锡喷砂酸蚀组(TNZS-SLA组),共4组。通过扫描电镜观察各组试样的表面形貌,3D激光共聚焦显微镜和接触角测量仪测量各组试样表面的粗糙度与亲水性。接种MC3T3-E1小鼠前成骨细胞于各组试样表面,检测细胞在试样表面的粘附、增殖与矿化的能力,评估其生物相容性。结果:SLA处理后在材料表面形成纳米级及微米级的凹坑,产生均匀分布的粗糙结构,经过处理后材料仍保持亲水性。细胞在TNZS组上短期粘附明显高于其它组(P<0.05),TNZS-SLA组细胞增殖、分化能力均明显高于其它组(P<0.05)。结论:喷砂酸蚀后材料表面相对于光滑材料表面能更有效的促进成骨细胞在其表面增殖、分化,经喷砂酸蚀的钛铌锆锡合金具有良好的细胞相容性。     

Bone marrow mesenchymal stem cell response to nano-structured oxidized and turned titanium surfaces

Objectives: The aim of this study was to analyse the topographic features of a novel nano‐structured oxidized titanium implant surface and to evaluate its effect on the response of human bone marrow mesenchymal stem cells (BM‐MSC) compared with a traditional turned surface. Methods: The 10 × 10 × 1 mm turned (control) and oxidized (test) titanium samples (P.H.I. s.r.l.) were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) and characterized by height, spatial and hybrid roughness parameters at different dimensional ranges of analysis. Primary cultures of BM‐MSC were seeded on titanium samples and cell morphology, adhesion, proliferation and osteogenic differentiation, in terms of alkaline phosphatase activity, osteocalcin synthesis and extracellular matrix mineralization, were evaluated. Results: At SEM and AFM analyses turned samples were grooved, whereas oxidized surfaces showed a more complex micro‐ and nano‐scaled texture, with higher values of roughness parameters. Cell adhesion and osteogenic parameters were greater on oxidized (P<0.05 at least) vs. turned surfaces, whereas the cell proliferation rate was similar on both samples. Conclusions: Although both control and test samples were in the range of average roughness proper of smooth surfaces, they exhibited significantly different topographic properties in terms of height, spatial and, mostly, of hybrid parameters. This different micro‐ and nano‐structure resulted in an enhanced adhesion and differentiation of cells plated onto the oxidized surfaces.     

微弧氧化钛基种植材料对成骨细胞早期黏附的影响

目的：检测纯钛种植体材料微弧氧化（microarc oxidation，MAO）表面改性后的成骨细胞生物相容性，探讨微弧氧化技术在钛种植体表面改性中的价值。方法：在纯钛种植体材料表面用微弧氧化法制备羟基磷灰石陶瓷薄膜，将MAO改性钛种植体材料作为实验组Ⅰ，将纯钛表面阳极氧化改性处理的种植体材料作为实验组Ⅱ．并设立对照组Ⅰ（纯钛种植体材料）和对照组Ⅱ（即细胞直接生长在培养板上），分别进行扫描电镜（SEM）、能谱分析（EDS）、X射线衍射图谱分析（XRD）等检测，比较成骨细胞的黏附水平，对数据采用SPSS11．0统计软件包进行单因素方差分析。结果：MAO改性后生成粗糙、多孔的陶瓷薄膜层，与处理前电解液成分相比，钙磷比无显著改变。MAO改性钛组黏附的细胞密度显著高于其他组（P〈0．01），而对照组Ⅰ、Ⅱ的细胞密度无显著差异（P〉0．05）。结论：与阳极氧化表面改性材料相比，该钛基微弧氧化薄膜层能够显著促进成骨细胞的附着，具有良好的细胞相容性。     

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.     

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     

Comparison of osteoblast spreading on microstructured dental implant surfaces and cell behaviour in an explant model of osseointegration. A scanning electron microscopic study

OBJECTIVES: To compare interactions between rat calvarial osteoblasts and titanium dental implants with different microstructured surfaces. MATERIAL AND METHODS: Seven commercially available implants were used. Surfaces included plasma-sprayed, grit-blasted and/or acid-etched, smooth-machined and anodised titanium. Two methods were used to compare cell behaviour: (1) A cell-spreading assay in which percentages of cells at four different stages of attachment were identified by scanning electron microscopy and quantified within a 30 min attachment period. (2) Implants were placed in 'pocket culture' within nylon mesh sacs in contact with explanted calvarial bone fragments for 2 and 4 weeks. RESULTS: Surfaces combining grit blasting and acid etching, of microporous topography, showed significantly enhanced rates of cell spreading in comparison with the others. Differential cell morphology was observed in both suspension assays and pocket cultures. In the latter, cells migrated onto all surfaces. Multicellular layers with extracellular matrix (ECM) were present between the layers and on the material surfaces after 2 weeks. After 4 weeks, cell layers were more consolidated, and microstructures were obscured by layers of cells and ECM. Mineralised tissue was seen in association with ECM on grit-blasted surfaces of rough and smooth microtopography. CONCLUSIONS: The two methods provided complementary information: a rough surface of porous microstructure may enhance the rate of cell spreading. Differentiation and calcification occurred on surfaces of both rough and smooth microstructure.     

In vitro preliminary study of osteoblast response to surface roughness of titanium discs and topical application of melatonin

Objectives: To observe human osteoblast behavior cultured in vitro on titanium discs (Ti) in relation to surface roughness and melatonin application. Study Design: Human osteoblasts (MG-63) were cultured on 60 Ti6Al4V discs divided into three groups: Group I: discs treated with dual acid etching; Group II dual acid etching and blasting with calcium phosphate particles; Group III (control) machined discs. Surface roughness and topography of the discs were examined with scanning electron microscope (SEM) and confocal laser scanning electron microscope( CLSM). Osteoblast adhesion, proliferation and cell morphology were determined by means of fluorescence microscopy with Image-Pro Plus software and SEM. Results: Group II presented the roughest discs, while the least rough were Group III. Cell adhesion was greatest in Group II. The addition of melatonin improved cell proliferation. Conclusions: 1. Surface treatments (dual acid etching, calcium phosphate impaction) increase surface roughness in comparison with machined titanium. 2. Greater surface roughness tends to favor cell adhesion after 24-hour cell culture. 3. The addition of melatonin tends to favor osteoblast proliferation. Key words:Osteoblasts, titanium, roughness, melatonin, dental implants, osseointegration.     

Focal adhesion formation of primary human gingival fibroblast on hydrothermally and in-sol-made TiO2-coated titanium

Optimal cell adhesion of the gingival fibroblasts to dental implants is important for maintaining good implant integration. The aim of this study was to discover, if the nanoporous TiO₂-coating on titanium alloy substrates is able to increase the cell adhesion of the human gingival fibroblasts (HGF). The study consisted of three differently produced titanium groups: hydrothermally produced TiO₂-coating (HT), novel TiO₂-coating made in sol (SOL), and noncoated control group. Primary HGF cells were initiated from gingival biopsies from patients having a third molar extraction. HGF were cultivated on titanium discs for 2 and 24 h to determine the initial attachment with confocal microscope. The cell spreading and adhesion protein signals were measured. In addition, expression of adhesion proteins vinculin, paxillin, and focal adhesion kinase (FAK) were measured after 3 days of cultivation by using Western Blotting. Higher protein levels of paxillin, vinculin, and FAK were induced on both coated discs compared to noncoated discs. The difference was statistically significant (p < 0.05) concerning expression of paxillin. The cell spreading was significantly larger on SOL discs after 2 and 24 h when comparing to noncoated controls. The confocal microscope analyses revealed significantly higher adhesion protein signals on both HT- and SOL-coated titanium compared to control group. This study showed, that both methods to produce TiO₂-coatings are able to increase HGF adhesion protein expression and cell spreading on titanium surface. Accordingly, the coatings can potentially improve the gingival attachment to titanium implant surfaces.     

不同钛表面形貌对人牙龈成纤维细胞生物学行为的影响

目的:良好的种植体颈部软组织封闭是种植体远期成功的重要因素之一。生物封闭的的主要决定因素之一是种植体颈部的表面形貌。本实验的目的是通过比较三种不同的纯钛表面形貌结构对人牙龈成纤维细胞的生物学行为的影响,寻找一种有利于牙龈胶原纤维附着的较理想的种植体颈部表面形貌,为种植体颈部设计提供指导。方法:本研究采用机械加工处理、电化学腐蚀、电化学腐蚀加酸蚀的方法在纯钛金属表面形成三组不同的表面形貌结构;用激光共聚焦显微镜检测其表面粗糙度;扫描电镜观察其表面微观形态并用X射线能谱色散谱仪检测其表面成分;将其与人牙龈成纤维细胞共同培养,MTT法检测表面细胞的增殖情况;细胞计数仪进行表面细胞计数;扫描电镜观察表面细胞的形状及排列。结果:机械加工组表面较光滑,呈浅的等向排列的微沟纹,表面粗糙度Sa为0.8783±0.2578μm;电化学腐蚀组表面略粗糙,呈圆形或椭圆形浅碟状凹,直径约10～15μm,分布均匀,凹内含有散在的小孔,小孔的直径约为1-5μm,表面粗糙度Sa为1.7530±0.3711μm;电化学腐蚀加酸蚀组表面略粗糙,呈圆形或椭圆形浅碟状凹,直径约10～15μm,分布均匀,凹内含有散在的小孔,小孔的直径约为1-5μm,表面见均匀的半球形纳米突起形成,直径约50～100nm,表面粗糙度Sa为1.6763±0.3440μm。表面成分检测显示三组均未有任何污染物在钛表面。牙龈成纤维细胞在各组钛片表面生长良好,形态正常,各组钛片对于细胞没有明显的毒性,生长曲线与正常细胞的生长曲线规律一致。机械加工组表面细胞沿着材料表面微沟纹平行排列,细胞扁平,伸展较差,细胞伸出的伪足较短;电化学腐蚀组表面细胞自由分布,细胞丰满,伸展良好,细胞伸出的伪足较长,有的伪足伸入到邻近凹内;电化学腐蚀加酸蚀组表面细胞丰满,伸展良好,伸出大量伪足,伪足很长,呈细丝状或带状,可伸入到相隔较远的凹孔内,呈悬空样,也可相互交织成网状。结论:电化学腐蚀加酸蚀可在纯钛表面形成均匀分布的直径为10-20μm的凹及直径为1-5μm的孔,凹及孔的表面均匀布满了直径为50-100nm的半球状突起。电化学腐蚀加酸蚀表面促成纤维细胞粘附作用最强。本实验中形成的微米凹、孔形态及半球形纳米形态均有利于成纤维细胞的粘附。电化学腐蚀加酸蚀方法形成的表面是一种较为理想的种植体颈部表面,可以为种植体颈部设计提供指导。