Nitric acid passivation does not affect in vitro biocompatibility of titanium

PURPOSE: In general, both chemical composition and surface features of implants affect cell response. The aim of this study was to evaluate the effect of titanium (Ti) passivation on the response of rat bone marrow cells, considering cell attachment, cell morphology, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bonelike nodule formation. MATERIALS AND METHODS: Cells were cultured on both commercially pure titanium (cpTi) and titanium-aluminium-vanadium alloy (Ti-6Al-4V) discs, either passivated or not. For attachment evaluation, cells were cultured for 4 and 24 hours. Cell morphology was evaluated after 4 days. After 7, 14, and 21 days, cell proliferation, total protein content, and ALP activity were evaluated. Bonelike nodule formation was evaluated after 21 days. Data were compared by analysis of variance and the Duncan multiple range test. RESULTS: Cell attachment, cell morphology, cell proliferation, total protein content, ALP activity, and bonelike nodule formation all were unaffected by Ti composition or passivation. DISCUSSION AND CONCLUSION: Although the protocol for passivation used here could interfere with the pattern of ions released from Ti-6Al-4V and cpTi surfaces, the present study did not show any effect of this surface treatment on in vitro biocompatibility of Ti as evaluated by osteoblast attachment, proliferation, and differentiation.     

Response of rat bone marrow cells to commercially pure titanium submitted to different surface treatments

OBJECTIVES: Alterations in the commercially pure titanium (cpTi) surface may be undertaken to improve its biological properties. The aim of this study is to investigate the biocompatibility of cpTi submitted to different surface treatments. METHODS: The cpTi surfaces were prepared so that machined and blasted surfaces, either acid etched or not, were compared using rat bone marrow cells cultured to differentiated into osteoblast. For attachment evaluation, cells were cultured for 4 and 24h. Cell morphology was evaluated after 3 days. After 7, 14, and 21 days cell proliferation was evaluated. Total protein content and alkaline phosphatase (ALP) activity were evaluated after 14 and 21 days. For bone-like nodule formation, cells were cultured for 21 days. Data were compared by analysis of variance. RESULTS: Cell attachment, cell morphology, cell proliferation, and ALP activity were not affected by surface treatments. Total protein content was reduced by blasted and acid etched surface. Bone-like nodule formation was significantly reduced by blasted, acid etched, and a combination of both blasted and acid etched surfaces. CONCLUSIONS: Based on these results, it can be suggested that cpTi surfaces that were submitted only to machining treatment favor the final event of osteoblastic differentiation of the rat bone marrow cells, evidenced by increased bone-like nodule formation.     

Surface properties and biocompatibility of acid-etched titanium

The purpose of this study was to evaluate the effects of acid-etched titanium on the biological responses of osteoblast-like MC3T3-E1 cells. Four types of treatments (polishing, sandblasting, concentrated H2SO4 etching, and concentrated H2SO4 etching with vacuum firing) were carried out on the surfaces of commercially pure titanium (cpTi) disks. MC3T3-E1 cells were then cultured on the treated cpTi surfaces. Through surface roughness measurement and SEM analysis, it was found that the acid-etched surfaces showed higher roughness values than the sandblasted ones. Scanning electron microscope analysis showed that the cells on the disks treated with acid-etching and acid-etching with vacuum firing spread as well as the sandblasted ones. There were no significant differences in cell proliferation and collagen production on cpTi among the four different surface treatments. Based on the results of this study, it was concluded that etching with concentrated sulfuric acid was a simple and effective way to roughen the surface of titanium without compromising its biocompatibility.     

In vitro corrosion behavior of bioceramic, metallic, and bioceramic-metallic coated stainless steel dental implants.

OBJECTIVES: The most common metals and alloys used in dentistry may be exposed to a process of corrosion in vivo that make them cytotoxic. The biocompatibility of dental alloys is primarily related to their corrosion behavior. The aim of this work was to evaluate the corrosion behavior and thus the biocompatibility of the uncoated and coated stainless steels and compare the effect of type of coatings on corrosion behavior. METHODS: Three types of coatings, hydroxyapatite (HA), titanium (Ti), and a double-layer HA/Ti on AISI 316L stainless steel were made. HA coating was produced using plasma-spraying technique and Ti coating was made using physical vapor deposition process. In order to perform a novel double-layer composite coating, a top layer of HA was plasma-sprayed over a physical vapor deposited Ti layer on AISI 316L stainless steel. Structural characterization techniques including XRD, SEM and EDX were used to investigate the microstructure, morphology and crystallinity of the coatings. Electrochemical potentiodynamic tests were performed in physiological solutions in order to determine and compare the corrosion behavior of the coated and uncoated specimens as an indication of biocompatibility. RESULTS: Double-layer HA/Ti coating on AISI 316L SS had a positive effect on improvement of corrosion behavior. The decrease in corrosion current densities was significant for these coated specimens and was much lower than the values obtained for uncoated and single HA coated specimens. Ti coating on AISI 316L SS also has a beneficial effect on corrosion behavior. The results were compared with the results of corrosion behavior of HA coated commercially pure titanium (cpTi) and uncoated cpTi. SIGNIFICANCE: These results demonstrated that the double-layer HA/Ti coated 316L SS can be used as an endodontic implant and two goals including improvement of corrosion resistance and bone osteointegration can be obtained simultaneously.     

Rat bone marrow cell response to titanium and titanium alloy with different surface roughness

In general, cell response is affected by both chemical composition and surface roughness of implant materials. The aim of this study was to evaluate the effect of titanium (Ti) chemical composition and surface roughness on the response of rat bone marrow cells, examining cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on both commercially pure titanium (cpTi) and titanium-6-aluminum-4-vanadium alloy (Ti-A) discs with four different average roughnesses (Ra). For attachment evaluation, cells were cultured for 2 h. After 14 days, cell proliferation, total protein content, and ALP activity were evaluated. Bone-like nodule formation was evaluated after 21 days. Data were compared by anova and Duncan's multiple range test when appropriate. Cell attachment and total protein content were affected by neither Ti chemical composition (P = 0.201, and P = 0.639, respectively) or surface roughness (P = 0.972, and P = 0.660, respectively). Proliferation, ALP activity, and bone-like nodule formation were affected only by Ti chemical composition (P = 0.0001, P = 0.064, and P = 0.0001, respectively). These results suggest that cpTi would optimize osteoblastic differentiation by rat bone marrow cells, including reduced cell proliferation, and increased ALP activity and bone-like nodule formation, while surface roughness, within the Ra parameters used, would not affect significantly the rat bone marrow cell response.     

一步微弧氧化法处理钛合金的体外生物相容性研究

目的　研究一步微弧氧化法处理钛合金（Ti6Al4V）的体外生物学性能,为下一步临床应用微弧氧化法处理钛合金种植体提供前期理论基础。方法　通过一步微弧氧化法在Ti6Al4V表面生成膜层并表征膜层形态（实验组）,设置对照组为未处理的Ti6Al4V和喷砂酸蚀处理的Ti6Al4V,3组分别与成骨细胞共同培养,测定成骨细胞的增殖、碱性磷酸酶（alkaline phosphatase,ALP）活性以及Ⅰ型胶原蛋白（collagen Ⅰ,COL-Ⅰ）、骨钙素（osteocalcin,OC）的mRNA相对表达情况。结果　微弧氧化处理后Ti6Al4V表面含有大量的陶瓷烧结颗粒,以羟基磷灰石（hydroxyapatite,HA）为主要成分。3组试样的体外实验结果表明,经微弧氧化处理的Ti6Al4V表面的细胞增殖高于其他两组,细胞内的ALP活性、COL-Ⅰ、OC的mRNA相对表达也均高于其他两组。结论　通过微弧氧化法在Ti6Al4V表面生成的陶瓷膜对成骨细胞的增殖分化有促进作用,有良好的生物相容性。     

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??Objective??To study the in vitro biocompatibility of titannium alloy treated with micro-arc oxidation??MAO????in order to provide the theoretical basis for the next application of MAO in the treatment of titanium alloy implants. Methods??The biofilm layer containing hydroxyapatite ??HA?? was formed on the surface of Ti6Al4V by one-step  MAO ??experiment group??. The control groups were the untreated Ti6Al4V and the sandblasted Ti6Al4V. The three groups were co-cultured with osteoblasts to determine the proliferation??alkaline phosphatase ??ALP?? activity and mRNA expression of collagen ?? ??COL-?? and osteocalcin ??OC??. Results??The surface after the treatment was "crater" structure with a large number of HA. The in vitro results of three groups of materials showed that the cell proliferation rate of the Ti6Al4V treated with MAO was higher than that of the other two groups??and the ALP activity and COL-??and OC mRNA expression of cells were higher than those in the other two groups. Conclusion??The biofilm layer formed on the surface of Ti6Al4V by  MAO  has a beneficial effect on the adhesion and proliferation of cells??which promotes the differentiation  and shows good biocompatibility.     

种植体表面TiO2/HA梯度涂层的生物相容性研究

目的：通过观察大鼠成骨细胞（osteoblast,OB）在微弧氧化（micro-arc oxidation,MAO）和水热处理形成的TiO2/HA梯度涂层表面的粘附和增殖等情况,评价微弧氧化陶瓷膜表面的生物相容性及微弧氧化-水热处理工艺应用于钛植入材料表面处理的可行性。方法：将纯钛试件共60枚分为微弧氧化处理组（M组）、微弧氧化-水热处理组（M-H组）和纯钛对照组。采用扫描电镜（scanning electron microscopy,SEM）观察表面形貌并用X线衍射（X-ray diffraction,XRD）对其进行成分分析。然后对不同时间点OB细胞在各组材料表面的附着、生长、增殖情况以及碱性磷酸酶（alkaline phosphatase,ALP）活性进行检测,并对实验结果进行统计分析。结果：微弧氧化处理后试件表面呈现多孔状,主要为锐钛矿型TiO2,也包含金红石型TiO2。经过后续水热处理,从扫描电镜照片可以看到试件表面析出一层白色柱形结晶体,同时XRD谱线出现了羟基磷灰石的衍射峰。试件与OB细胞共培养后可见细胞在材料周围生长良好,培养5d后,改性两组的材料表面细胞增殖比纯钛组及空白对照组明显增多。培养7d后,M组和M-H组细胞ALP活性大幅提高,各组间比较均有统计学差异。结论：MAO及水热处理后的TiO2/HA梯度涂层表面能有效提高OB细胞的早期粘附、增殖及ALP活性。     

Effect of cpTi surface roughness on human bone marrow cell attachment,proliferation, and differentiation

There is general agreement that rough surfaces improve both biologic and biomechanical responses to titanium (Ti) implants. The aim of this investigation was to study the effect of Ti surface roughness on the response of human bone marrow cell culture evaluating: cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on commercially pure titanium (cpTi) discs with fourdifferent average roughnesses (Ra). For attachment evaluation, cells were cultured for 4 h. After 21 days, cell proliferation, total protein content, and ALP activity were evaluated. For bone-like nodule formation, cells were cultured for 28 days. Data were compared by ANOVA and Duncan's multiple range test. Cell attachment was not affected by surface roughness. For cells cultured on Ti with Ra ranging from 0.80 microm to 1.90 microm, proliferation was reduced while total protein content, and ALP activity were increased. There was a non-statistically significant increase of bone-like nodule formation on a surface with Ra near 0.80 microm. These results suggest that for Ti an Ra ranging from 0.80 microm to 1.90 microm would optimize both intermediary and final cellular responses but not affect the initial response, and a smoother surface would not favor any evaluated response.     

选择性激光烧结多孔钛种植体及其性能研究

目的 分析选择性激光烧结（SLS）多孔钛种植体的机械性能及生物相容性,探讨其与壳聚糖（CS）/羟磷灰石（HA）复合涂层结合后的促骨结合作用。方法 制备Ti6Al4V试件,部分试件表面进行CS/HA涂层处理;对试件进行扫描电子显微镜观察和机械性能检测;体外培养MC3T3-E1细胞,进行活/死细胞染色、甲基噻唑基四唑（MTT）及碱性磷酸酶（ALP）水平检测;将柱状螺纹种植体植入兔股骨髁部,分析其体内生物学性能。结果 试件准弹性梯度随孔隙率增大而减小,孔隙率为30%时与皮质骨接近,70%时与松质骨接近;试件具有良好的生物相容性。复合CS/HA涂层后可促进MC3T3-E1细胞增殖、分化,有利于骨组织长入孔隙,形成良好的骨结合。结论 多孔钛种植体具有良好的机械性能和生物相容性,与CS/HA涂层结合后具有骨诱导性,利于形成稳定的骨结合。     

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。     

Response of osteoblast‐like SAOS‐2 cells to zirconia ceramics with different surface topographies

Objectives: Zirconia is a suitable biomaterial for use in medicine (stomatology, orthopaedics) due to its good biocompatibility and outstanding mechanical properties. This study compares the effect of (i) zirconia to the widely used titanium and (ii) zirconia with two different surface topographies (sandblasted and sandblasted/etched) on the adhesion, proliferation and differentiation of SAOS‐2 osteoblasts. Methods: SAOS‐2 cells were cultured on either sandblasted or sandblasted/etched zirconia and compared with sandblasted/etched titanium. 2 and 24 h after plating, cell morphology was investigated by scanning electron microscope (SEM) and fluorescence imaging. At 24 and 48 h, cell number‐relevant parameters were determined. Alkaline phosphatase (ALP) activity and mineral accumulation were measured at days 8, 11, 15 and day 22 of culture, respectively. Results: SEM and fluorescence images revealed a faster spreading as well as higher number of adherent cells after 24 h incubation on zirconia compared with titanium. Also, the cellular metabolic activity after 24 h and the proliferation rate after 48 h is higher with zirconia compared with titanium. Zirconia had a more pronounced effect compared with titanium on the differentiation of SAOS‐2 cells: ALP activity, an early differentiation marker increased earlier and mineralization, a late differentiation marker was increased. Only minor differences were found between zirconia with two different surface topographies; etched zirconia promoted slightly greater the differentiation of SAOS‐2 cells. Conclusions: These data indicate that zirconia mediates a pronounced stronger effect on the adhesion, proliferation and differentiation compared with titanium; and that topographical differences of zirconia have minor effects on osteoblast biology. To cite this article:
Hempel U, Hefti T, Kalbacova M, Wolf‐Brandstetter C, Dieter P, Schlottig F. Response of osteoblast‐like SAOS‐2 cells to zirconia ceramics with different surface topographies.
Clin. Oral Impl. Res. 21 , 2010; 174–181.
doi: 10.1111/j.1600‐0501.2009.01797.x     

2 种新型骨植入钛合金的细胞生物相容性研究

目的:研究国内2种新研制骨植入钛合金Ti1、Ti2对成骨细胞生物学行为的影响。方法:采用SD乳鼠体外原代分离培养的成骨细胞,将细胞分别接种于新型钛合金表面建立体外共同培养。采用MTT比色试验检测第3天成骨细胞的增殖百分率,采用扫描电镜(SEM)观察细胞形态学变化,并且对培养第5天时细胞碱性磷酸酶(ALP)功能活性进行检测。结果:成骨细胞在新合金表面增殖百分率、碱性磷酸酶活性检测值均高于对照组,细胞增殖百分率及碱性磷酸酶吸光度值与对照组间统计学分析无显著性差异(P>0.05)。扫描电镜观察细胞在新合金表面伸展状况良好、黏附牢固,并具有成骨细胞典型形态特征。结论:2种新型钛合金对成骨细胞学行为无不良影响。     

聚多巴胺仿生掺锶羟基磷灰石涂层的制备及对骨髓间充质干细胞生物学特性的影响

目的:通过修饰聚多巴胺并结合仿生矿化法在钛合金表面制备掺锶羟基磷灰石涂层,体外评价该复合材料对大鼠骨髓间充质干细胞生物学特性的调控作用.方法:采用两步法制备掺锶羟基磷灰石涂层.采用扫描电镜、X射线光电子能谱、X射线衍射图谱、拉曼光谱和接触角测量等材料表征证实钛合金表面聚多巴胺仿生掺锶羟基磷灰石涂层的形成.将大鼠骨髓间充...     

纯钛微纳米复合形貌对成骨细胞生物学行为的影响

目的对比研究纯钛表面不同微纳米图案对成骨细胞生物学活性的影响,探讨微米与纳米结构在影响细胞行为当中的不同作用。 方法制备4组纯钛微纳米复合表面形貌：喷砂（S）、喷砂-酸蚀（SLA）、喷砂-碱热（SAH）及喷砂-阳极氧化（SAN）。检测各组材料表面理化性能,扫描电镜观察各组材料表面形貌及细胞黏附形态,CCK-8法测定细胞在材料表面增殖能力,碱性磷酸酶（ALP）活性检测细胞在材料表面分化能力。利用单因素方差分析进行统计分析,最小有意义差异（LSD）t检验对同时间点不同组的CCK-8及ALP结果进行比较。 结果（1）粗糙度结果示：SLA组粗糙度大于其余3组,差异有统计学意义（F_Ra = 38.449,P_Ra<0.001;F_Rq = 29.564,P_Rq<0.001）。（2）扫描电镜示：S组仅形成弹坑状一级微米结构,黏附细胞伪足短小,SLA、SAH及SAN组分别修饰沟壑状、网状及管状二级纳米多孔图案,细胞于SAH、SAN组表面伪足更为伸展,其中SAH组表面细胞伪足生长进入孔隙形成机械锁结。（3）CCK-8结果示：第5天,SAH和SAN组细胞增殖A值（1.546和1.528）显著高于S组（1.31）,差异有统计学意义（F = 3.229,P = 0.042）;第7天时,SAH和SAN组细胞增殖A值（2.646和2.57）显著高于S组（2.24）,差异有统计学意义（F = 3.51,P = 0.035）。（4）细胞分化检测示：接种7 d后,SAH组ALP活性（77.656）显著高于S、SLA及SAN组（53.132、51.052和62.207）,差异有统计学意义（F = 29.734,P<0.001）;接种14 d后,SAH与SAN组ALP活性（104.107和109.963）显著高于S与SLA两组（82.885和73.303）,差异有统计学意义（F = 46.052,P<0.001）。 结论钛片表面微纳米图案影响细胞伪足形态,促进细胞增殖及ALP活性,其中多孔形貌显著增加细胞活性,碱热处理表面早期ALP活性显著增加,且形成纳米网比纳米管更有利于形成机械锁结。     

四种纳米粒径钛片生物相容性检测

目的研究4种纳米粒径钛片的生物相容性。方法分别在常温、100℃、250℃和380℃,采用直流磁控溅射法,形成4种纳米粒径表面的钛片。在24孔培养板上,将SD（Sprague-Dawley）乳鼠第3代成骨细胞接种于4种纳米粒径的钛片和未处理的钛片表面,分别为常温组、100℃组、250℃组、380℃组、非处理组;另设1个空白对照组,细胞培养板内不放入钛片。四甲基偶氮唑盐比色法测定6组细胞的增殖情况。结果培养后第1、4、7天,250℃组和380℃组材料表面细胞持续增殖,其中250℃组最高;培养后第7天,4个温度处理组细胞光密度值与非处理组的差异均有统计学意义（P〈0.05）。结论纳米化纯钛材料表面可促进成骨细胞的增殖。     

多孔磷酸三钙-羟基磷灰石对人牙龈成纤维细胞黏附行为的影响

目的　研究多孔磷酸三钙 羟基磷灰石 (tricalcium phosphate hydroxyapatite ,TCP HA)复合材料对人牙龈成纤维细胞黏附行为的影响。方法　应用离子束辅助沉积法在纯钛试件表面制备多孔TCP HA复合涂层材料和羟基磷灰石 (hydroxyapatite,HA)涂层材料 ,定量对比人牙龈成纤维细胞在涂层和未涂层材料表面初期黏附、增殖、细胞铺展面积、细胞外基质和黏着斑形成的情况。结果　TCP HA和HA涂层材料表面黏附的细胞数、细胞铺展面积高于纯钛未涂层组 ,差异有显著性(P <0 0 5 ) ,黏着斑的形成早于未涂层组 ;TCP HA表面黏附的细胞数和Ⅰ型胶原的形成都高于纯钛未涂层组和HA涂层组。在培养 2 4h后TCP HA组表面黏附细胞数为 198 1± 2 7 7,Ⅰ型胶原形成的荧光强度为 15 4 10± 31 5 6 ,同纯钛组表面的细胞数 ( 12 5 1± 2 9 9)和Ⅰ型胶原荧光强度( 132 6 3± 35 2 6 )相比 ,差异有显著性 (P <0 0 5 )。结论　与纯钛未涂层和HA涂层材料相比 ,多孔TCP HA复合涂层材料更有利于人牙龈成纤维细胞的初期黏附 ,具有更良好的生物相容性     

Bonding strength and durability of alkaline-treated titanium to veneering resin

The shear bonding strengths of a veneering resin to polished, sandblasted, and retention bead-cast commercially pure titanium (cpTi) plates with and without alkaline treatment were measured before and after thermal cycling. The bonding strengths to polished cpTi with and without alkaline treatment decreased remarkably with thermal cycling (p<0.01). The bonding strength to sandblasted cpTi with alkaline treatment at 5,000 thermal cycles showed no significant differences from those before thermal cycling (p>0.05), and those at 20,000 thermal cycles showed values which were quite small (p<0.01). On the other hand, there were no significant differences in the bonding strengths of veneering resin to retention bead-cast cpTi in all conditions (p>0.05). These results suggested that although alkaline treatment is a simple and effective surface modification technique for titanium improving adhesion to resin due to formation of tight-fine rutile particles, it does not provide sufficient bonding durability for long-period restorations.     

水热合成法制备羟基磷灰石生物涂层的体内及体外实验研究

目的对水热合成法制备羟基磷灰石生物涂层材料的细胞相容性及动物体内植入体与骨界面的状况进行研究.方法水热合成法制备羟基磷灰石涂层,采用细胞培养和动物实验评估涂层材料的生物相容性.结果成骨细胞在涂层材料表面具有良好的细胞增殖率,碱性磷酸酶活性逐渐增加.植入动物体内1月后即有骨组织与涂层材料结合,3月后骨结合量增加.涂层材料在体内稳定,未见溶解脱落.结论水热合成法制备的羟基磷灰石生物涂层材料具有良好的生物效应.     

种植体用钛合金酸碱热处理后的表面性能及诱导能力

目的研究酸蚀加碱热处理后种植体用钛合金（H6A14V）表面微观结构的变化及在模拟体液中诱导钙磷沉积的能力。方法对钛合金表面分别采用打磨抛光、大颗粒喷砂酸蚀（SLA）、碱热、酸蚀加碱热处理,测试其表面理化性质变化,然后对其进行模拟体液（SBF）浸泡实验,采用带能谱分析的扫描电镜（SEM）及X射线衍射（XRD）观察分析沉积物。结果经过酸蚀加碱热处理的钛合金表面形成了多级孔洞的微观粗糙结构;经SBF浸泡实验后,其表面出现羟基磷灰石沉积,钙磷原子数百分比高于其他实验组（P〈0．05）。结论酸蚀加碱热处理的钛合金表面同时具有良好的表面形态和生物活性,在模拟体液中浸泡可诱导羟基磷灰石形成,有利于骨结合的早期形成。