磷酸锌矿化胶原膜对MG63细胞生物学行为的影响

目的：研究磷酸锌矿化胶原膜对人成骨样细胞MG63增殖及分化的影响。方法：制备磷酸锌矿化胶原膜并在其上接种MG63成骨细胞株,扫描电镜观察MG63细胞在胶原膜表面的生长情况,采用CCK一8及碱性磷酸酶（ALP）试剂盒检测MG63细胞的增殖及分化能力。结果：扫描电镜下可见胶原膜表面有大量晶体样沉积物,在其上培养1d后MG63细胞粘附于胶原膜表面,开始向各方向伸出伪足,与胶原膜相互交联。与阴性对照组相比,磷酸锌矿化胶原膜于第3天明显促进MG63细胞增殖,于第1、5、7d无显著影响;于第1d可提高碱性磷酸酶活性,促进MG63细胞分化,于第7、14d无显著影响。结论：磷酸锌矿化胶原膜具有良好的生物相容性,可促进MG63成骨细胞的早期增殖及分化。     

二氧化钛纳米管的制备及其对成骨细胞增殖和碱性磷酸酶活性的影响

目的 探讨阳极氧化过程中不同工艺条件对TiO_2纳米管形貌的影响,并对TiO_2纳米管对成骨细胞增殖和碱性磷酸酶相对活性的影响进行初步评定.方法 采用阳极氧化法在钛基底表面制备TiO_2纳米管,通过控制阳极氧化电压(5、15、20和25 V)、作用时间(1.5和3 h)以及阳极化后冲洗工艺(是否行超声处理),得到不同结构的TiO_2纳米管,扫描电镜观察TiO_2纳米管管径和管长.以未行阳极氧化的钛试件为对照组,以行阳极氧化(阳极氧化电压为15 V,作用时间为3 h,反应后行超声清洗)的钛试件为纳米管组;在两组钛试件表面培养成骨细胞,用扫描电镜观察接种2 h后的细胞形态;用甲基噻唑基四唑(MTT)检测培养24、48、96 h后细胞的增殖情况;用碱性磷酸酶半定量测试培养7、14、21 d后细胞的碱性磷酸酶活性.结果 阳极氧化的电压可以影响TiO_2纳米管的管径和管长.细胞在纳米管表面的铺展范围大于对照组.培养96 h后纳米管组吸光度值为(0.62±0.02).对照组为(0.55±0.03),两组差异有统计学意义(P＜0.05).21 d后纳米管组碱性磷酸酶相对活性[(130.8±5.1)A405/mg]与对照组[(109.6±4.5)A_(405)/mg]的差异有统计学意义(P＜0.05).结论 阳极氧化的工艺条件,特别是电压可以控制TiO_2纳米管形貌,TiO_2纳米管结构可促进成骨细胞的早期黏附、增殖和碱性磷酸酶的活性.     

电解蚀刻法处理的钛及钛合金表面的对比研究

目的 通过成骨细胞的体外培养,初步探讨钛及钛合金微-纳米三维形貌对成骨细胞生物学行为的影响。方法采用电解蚀刻法在纯钛及钛合金表面构建出不同尺寸的微-纳米三维形貌,并观察其三维结构表面对成骨细胞黏附、增殖、细胞形态、碱性磷酸酶（ALP）活性的影响。结果在成骨细胞的黏附和增殖方面,纯钛组和钛合金组表面均高于纯钛机械抛光组。纯钛组表面细胞胞体饱满,伸出大量伪足,并可见大量功能颗粒。ALP活性显著高于钛合金和纯钛机械抛光组表面。结论通过电解蚀刻法在纯钛和钛合金表面可形成不同直径和深度的碗形巢样及纳米结构;两个表面即30~50 μm和5~8 μm的表面和光滑表面相比,都明显促进了细胞的附着;30~50 μm的纯钛表面更有利于促进细胞的增殖和分化。     

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。     

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。     

新型钛合金阳极氧化后对成骨细胞增殖、分化的影响

目的研究新型医用钛合金Ti-24Nb-4Zr-7.9Sn(TNZS)经过阳极氧化(AD)技术处理后对成骨细胞增殖和分化的影响。方法将人成骨样MG63细胞接种于Ti6Al4V、TNZS、AD-TNZS表面,采用MTT法检测成骨细胞的增殖情况,考马斯亮蓝G250染色法检测细胞总蛋白质含量,对硝基苯磷酸盐法检测碱性磷酸酶(ALP)活性。结果人成骨样MG63细胞在Ti6Al4V、TNZS以及AD-TNZS表面均能良好生长,细胞增殖率、细胞总蛋白含量未见明显差异(P>0.05);但培养至4、7d时,AD-TNZS组细胞ALP活性明显高于其他组(P<0.05)。结论阳极氧化处理的TNZS钛合金可促进成骨细胞的分化。     

微米-纳米微结构纯钛表面的细胞及分子生物学研究

目的:研究兼具微米和纳米形貌的纯钛表面微结构对成骨细胞生物学行为的影响。方法:通过电化学方法在纯钛表面形成直径约30-50μm,深度约10-20μm的均匀"碗形凹"样结构,内含直径约8-10μm微米凹和2-4μm的微米孔,以及大量的纳米凹、纳米孔和纳米台阶等微结构。将成骨细胞分别接种于微米-纳米纯钛表面、喷砂加酸蚀表面及机械表面,后两组作为对照,进行体外共同培养。采用MTT法评价细胞在第1d、3d、5d和7d的细胞增殖率;计算细胞在接种1h、2h、6h、12h和24h后的贴壁率;采用场发射扫描电镜观察第1d、3d、5d细胞形态变化,并对第1d、3d、5d和7d时细胞碱性磷酸酶功能活性进行检测。采用RT-PCR方法测定成骨细胞的骨相关基因COLLI、OPN、OCN、RUNX2的表达。结果:成骨细胞在微米-纳米纯钛表面的细胞增殖率、贴壁率、碱性磷酸酶活性检测值均高于对照组,差异有显著性。扫描电镜观察细胞在微米-纳米纯钛表面呈多边形,充分伸展,形成扁宽的伪足牢固附着于微米及纳米微结构表面;喷砂加酸蚀表面主要为梭形;机械表面成骨细胞呈球形及梭形。电解蚀刻表面的骨相关基因COLLI、OPN、OCN、RUNX2表达明显高于喷砂加酸蚀表面及机械表面,差异有显著性。结论:兼具微米-纳米微结构的纯钛表面能促进体外成骨细胞的粘附、伸展、增殖、分化功能及骨相关基因COLLI、OPN、OCN、RUNX2的表达,对表面成骨具有积极的影响。     

不同纳米颗粒二氧化钛的细胞相容性研究

目的:探索二氧化钛发挥最佳生物学效应的纳米晶尺寸。方法:采用MTT法观察成骨细胞在材料表面的增殖情况,细胞培养第5d时对碱性磷酸酶活性进行检测,并用SEM观察第3d时细胞在材料表面的附着形态。结果:成骨细胞在20nm组材料表面的细胞增殖数显著高于其它各组,并具有更好的细胞伸展形态;而200nm组材料碱性磷酸酶表达最高。结论:不同纳米尺寸二氧化钛的细胞相容性存在差别。     

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。     

Effects of fluoride-modified titanium surfaces on osteoblast proliferation and gene expression

PURPOSE: The objective of this study was to test the hypothesis that fluoride-modified titanium surfaces would enhance osteoblast differentiation. Osteoblast growth on a moderately rough etched fluoride-modified titanium surface (alteration in cellular differentiation) was compared to osteoblast growth on the same surface grit-blasted with titanium dioxide. The potential role of nanometer-level alterations on cell shape and subsequent differentiation was then compared. MATERIALS AND METHODS: Human embryonic palatal mesenchymal (HEPM) cultures were incubated on the respective surfaces for 1, 3, and 7 days, followed by analysis for cell proliferation, alkaline phosphatase (ALP) -specific activity, and mRNA steady-state expression for bone-related genes (ALP, type I collagen, osteocalcin, bone sialoprotein [BSP] II, Cbfa1, and osterix) by real-time polymerase chain reaction (PCR). RESULTS: The different surfaces did not alter the mRNA expression for ALP, type I collagen, osterix, osteocalcin, or BSP II. However, Cbfa1 expression on the fluoride-modified titanium surface was significantly higher (P < .001) at 1 week. The number of cells on this surface was 20% lower than the number of cells on the surface TiO2-blasted with 25-microm particles but not significantly different from the number of cells on the surface TiO2-blasted with 125-microm particles. Cells grown on all the titanium surfaces expressed similar levels of ALP activity. CONCLUSIONS: The results indicated that a fluoride-modified surface topography, in synergy with surface roughness, may have a greater influence on the level of expression of Cbfa1 (a key regulator for osteogenesis) than the unmodified titanium surfaces studied.     

壳聚糖修饰的羟基磷灰石纳米粒载体介导BMP-2转染成骨细胞的研究

目的:研究壳聚糖(chitosan,CS)修饰的羟基磷灰石(hydroxyapatite,HA)纳米粒载体携带骨形态发生蛋白2(Bone morphogenetic protein2,BMP2)基因转染成骨细胞,及转染后对细胞增殖与分化的影响。方法:采用MTT法检测HA-CS纳米粒载体对成骨细胞的毒性反应;用HA-CS纳米粒载体转染技术,将BMP-2目的基因导入成骨细胞,评估转染效率;检测细胞增殖情况及碱性磷酸酶(alkaline phoaphatase,ALP)活性。结果:MTT结果提示HA-CS纳米粒混悬液浓度在500mg/L以下对成骨细胞无毒性作用;其转染效率优于没经修饰的HA;转染后促进成骨细胞增殖并增加了ALP活性。结论:HA-CS纳米粒在一定浓度下对成骨细胞无毒性作用,作为输送载体可以安全地将目的基因BMP-2导入成骨细胞,并促进了成骨细胞的增殖与分化。     

Behavior of two osteoblast-like cell lines cultured on machined or rough titanium surfaces

Background: Two osteosarcoma-derived cell lines have been extensively used to investigate the biological events occurring on titanium surfaces: MG63 and Saos-2. However, the behavior of the two lines on different titanium surfaces has never been compared.
Aim: The aim of the present study was to compare the behavior of MG63 and Saos-2 cells on two different titanium surfaces, machined and rough (sandblasting and acid-etched). We compared cell proliferation and morphology, alkaline phosphatase (ALP) activity and secretion of osteocalcin (OC).
Results: The most pronounced difference between the two cell lines was that ALP activity in the Saos-2 cells was 10-fold higher than in the MG63 cells. The proliferation rate of the MG63 cells was much higher than that of the Saos-2 cells at all the tested cell concentrations. MG-63 cells, but not Saos-2 cells, grown on rough surface titanium proliferated more rapidly than cells grown on machined surfaces. Morphological analysis revealed that Saos-2 cells and cells grown on the rougher surface, displayed a more mature phenotype. The level of OC secreted by the Saos-2 cells, but not the MG63 cells, were higher on the rough surface than on the machined surface.
Conclusions: This study shows that Saos-2 cells exhibit a more mature osteoblast phenotype, compared with that of MG63 cells, rendering them a good candidate for an in vitro model of osseointegration.     

Differentiation and cytokine synthesis of human alveolar osteoblasts compared to osteoblast-like cells (MG63) in response to titanium surfaces.

OBJECTIVES: The aim of this study was to investigate the influence of different implant surface topographies and chemistries on the expression of differentiation/proliferation markers on MG63 cells and primary human alveolar osteoblasts. METHODS: Hydrophobic acid-etched (A) and hydrophobic coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and hydrophilic coarse-grit-blasted (modSLA) surfaces were produced. Thereby, modA and modSLA surfaces were rinsed under nitrogen protection and stored in a sealed glass tube containing isotonic NaCl solution at pH 4-6. Tissue culture plates without specimens served as controls. The behavior of MG63 cells and primary human alveolar osteoblasts (AOB) grown on all surfaces was compared through determination of alkaline phosphatase (ALP) activity, cell proliferation ((3)H-thymidin incorporation, MTT colorimetric assay) and expression of osteocalcin (OC), osteoprotegerin (OPG), transforming growth factor-beta1 (TGF-beta(1)) and vascular endothelial growth factor (VEGF), detected with commercial available test kits. RESULTS: Proliferation of MG63 and primary cells was highest on controls, followed by A surfaces, modA and SLA surfaces being almost on the same level and lowest on modSLA surfaces. modSLA surfaces exhibited highest ALP and OC production, followed by SLA, modA and A surfaces. Proliferation and OC production were comparable for MG63 cells and AOB. OPG, TGF-beta(1) and VEGF produced on primary cells showed a slightly different rank order on different surfaces compared to MG63 cells. modSLA still showed the highest production of OPG, TGF-beta(1) and VEGF, but was followed by modA, SLA and A. Statistical significance was checked by ANOVA (p<0.0035). SIGNIFICANCE: MG63 cells and primary human alveolar osteoblasts showed similar proliferation and differentiation characteristics on different titanium surfaces. Only modA surfaces showed enhanced expression of OPG, TGF-beta(1) and VEGF on MG63 cells compared to primary human alveolar osteoblasts. Overall, the lowest proliferation rates and the highest expressions of differentiation markers and growth factor productions were observed on modSLA.     

Corrosion resistance and biocompatibility of a new porous surface for titanium implants

Alterations of the commercially pure titanium (cpTi) surface may be undertaken to improve its biological properties. The aim of this study was to investigate the biocompatibility of cpTi when submitted to a new, porous titanium, surface treatment (porous Ti). Five types of surface treatments, namely sintered microspheres porous titanium (porous Ti), titanium plasma spray (TPS), hydroxyapatite (HA), sandblasted and acid etched (SBAE), and resorbable blast medium, sandblasted with hydroxyapatite (RBM) were made. In the experimental methods, the corrosion potentials were measured over time, and then a linear sweep voltammetric analysis measured the polarization resistances and corrosion currents. For biocompatibility evaluation, MG63 osteoblast-like cells were used. Cell morphology, cell proliferation, total protein content, and alkaline phosphatase (ALP) activity were evaluated after 2 h, and after 2, 4 and 7 d. Porous Ti and SBAE showed a better corrosion resistance, with a weak corrosion current and a high polarization resistance, than the other surfaces. Cell attachment, cell morphology, cell proliferation, and ALP synthesis were influenced by the surface treatments, with a significant increase observed of the activity of osteoblast cells on the porous coating (porous Ti). Based on these results, it is suggested that the porous Ti surface has a significantly better biocompatibility than the other surface treatments and an excellent electrochemical performance.     

成骨细胞中蛋白激酶A的作用

目的 研究蛋白激酶A在大鼠成骨细胞分化成骨过程中的作用。方法 纯化培养大鼠成骨细胞后，以cAMP,PKI,BMP-2,PKI-BMP-2(PKI预刺激1h后加BMP－2）共4种干预方式，分别作用细胞10，30，60min，及24h，通过MTT试验观察细胞增殖状况，并检测细胞内ALP活性，观察细胞的分化程度。结果 各种干预因素对细胞的增殖（MTT）无统计学上的显著影响。但是，BMP－2和c-AMP使细胞的ALP活性明显增高（P＜0．05）；PKI作用1h后，使ALP活性下降（P＜0．05）。BMP－2可以部分逆转PKI的抑制作用。结论 PKA参与大鼠成骨细胞功能的分化成熟。BMP－2可以减轻抑制PKA后引起的ALP活性的下降。     

浓缩生长因子提取液对钛片表面MC3T3-E1细胞增殖分化的影响

目的 探讨浓缩生长因子提取液（CGFe）对钛片表面MC3T3-E1细胞增殖分化的影响。方法 实验组使用含CGFe的α-MEM培养液（含10%胎牛血清）培养MC3T3-E1细胞,对照组则使用不含CGFe的α-MEM培养液（含10%胎牛血清）培养细胞。采用MTT法测定培养1、3、5 d时的细胞增殖情况,碱性磷酸酶（ALP）活性测定法检测培养3、5 d时的细胞分化情况;通过扫描电子显微镜（SEM）观察培养12 h时细胞在钛片表面的形态;通过荧光实时定量聚合酶链反应（PCR）测定细胞培养3、7 d时核心结合蛋白因子2（Runx2）和成骨细胞特异性转录因子Osterix（Osx）基因的相对表达量。结果 MTT结果显示：随培养时间延长,细胞数量逐渐增加;各时间点实验组细胞的吸光度值均明显高于对照组（P<0.05）。ALP活性随着培养时间延长而增加,两个时间点实验组的吸光度值均明显高于对照组（P<0.05）。SEM观察：培养12 h时,实验组细胞在钛片表面的形态较对照组更加伸展。PCR结果显示：随着培养时间延长,两组细胞Runx2和Osx基因的表达量逐渐增加,两个时间点实验组的表达量均明显高于对照组（P<0.05）。结论 CGFe能有效地促进MC3T3-E1细胞的增殖、分化及在钛片表面的伸展。     

阳极氧化TiO2纳米管碱热处理对成骨细胞行为的影响

目的：研究碱热处理对阳极氧化法制备的TiO2纳米管形貌的影响,并对TiO2纳米管碱热处理后对成骨细胞行为的影响进行研究。方法：采用碱热法对钛纳米管进行表面改性,采用SEM对纳米管改性后的形貌进行观察,并以未做处理的TiO2纳米管作为对照组,实验组为不同碱热处理的TiO2纳米管,观察成骨细胞在其表面的生长情况。结果：碱热处理条件可以影响到TiO2纳米管的表面形貌,成骨细胞在未处理的TiO2纳米管表面增殖最好（48 h和72 h）,但成骨细胞在碱热处理后钛纳米管表面ALP活性最高（2周）。结论：碱热处理条件可以影响TiO2纳米管形貌,TiO2纳米管碱热处理后可以促进成骨细胞的分化。