不同保存方法对纯钛表面理化性能以及生物活性的影响
|
摘要
研究背景：
尽管人工种植牙应用于临床已将近半个世纪,并且被广泛证实具有可靠的临床效果,但是仍然存在着以下问题：(1)种植体植入体内到临床负重仍需要较长的愈合时间,最少需要6-12周的时间,这一定程度上限制了种植修复的临床应用；(2)种植体与骨的结合率仍然较低,只有50～65%,不利于种植体的长期稳定。加快种植体-骨结合的速度不仅仅意味着修复时间的缩短,更重要的是使人工牙种植具有更高的安全性。研究表明,大部分的种植失败发生于种植体早期愈合的过程中,愈合时间越长,种植体受到各种风险因素的影响越大,种植失败的风险越高。因此,更快更好的骨结合仍然是我们追求的目标。
大量的研究表明,种植体与骨组织形成结合的速度和强度与种植体表面的生物活性密切相关,而生物活性又与种植体表面的理化性能紧密相连。生物活性(bioactivity)是植入材料能够与生物组织形成键合(Bone-bonding)的特性。即无论表面是否光滑,生物活性材料也能与生物组织产生直接的化学键性结合。
由于钛及钛合金的结构和性质与骨组织差异很大,加之表面钝态氧化膜的存在。通常不能像生物活性材料那样与骨组织发生化学键性结合。因此,一般认为钛金属为生物惰性材料,并努力寻求各种方法进行表面活化改性。钛的生物活化是指使钛金属经过表面处理后,能够在生理环境诱导下,使羟基磷灰石层在其表面形成,与骨组织实现直接的化学键结合。增加钛表面的生物活性有以下两种思路：(1)通过表面加成法在钛表面涂覆生物活性涂层(如羟基磷灰石、钙磷酸盐、生物大分子等)；(2)通过表面改性使原钝化态氧化膜转化为活化态氧化膜或其他活性膜(如钛酸盐,Ca+Ti, TiO2+Ca+P膜等)。目前,常用的一些表面处理技术包括喷砂酸蚀,微弧氧化,羟基磷灰石涂层等等。与光滑种植体表面相比,这些表面处理技术明显改善了种植体的表面活性,促进了种植体-骨的结合。其中,紫外线光催化技术是近几年的研究热点之一。1997年,Wang等首先发现了紫外线照射TiO2膜上可以获得高亲水性表面的现象,并在Nature上发表。此后,大量的学者对此进行了深入的研究。
本课题组通过紫外线光催化技术处理纯钛表面,成功获得了具有高表面能及高生物活性的表面,促进了蛋白质吸附、成骨细胞的黏附、增殖、分化和矿化。但进一步的研究结果表明,通过紫外线光催化技术获得的高表面能及高生物活性效应具有一定的时效性。经过一段时间的保存后,这种效应逐渐消失,钛片表面的生物活性明显降低。另外,在经过其他表面处理方式制备的钛表面上,也出现了钛表面生物活性随着保存时间的增加而下降的情况,即钛表面生物活性的老化现象(time-dependent degradation))。由此可见,钛表面生物活性的老化是钛基种植体普遍存在的现象。
目前,导致钛表面理化性能和生物活性随时间下降的原因尚未明确。有研究认为,这可能与钛表面在储存的过程中受到空气中碳氢化合物的污染,导致表面元素构成的改变及表面能下降有关。由于常规的种植体保存方式为常温常压保存于密封瓶里,可能会受到碳氢化合物的污染,因此,在储存的过程中钛表面理化性能及生物活性可能已经发生变化,这值得我们深入研究。
由于在实际应用中,临床医生不可能获得新制备的钛种植体,应用到临床上的种植体均经过或长或短的保存时间,可能出现了不同程度的生物活性的下降,因此,如何保持种植体的表面活性已成为一个重要的研究课题。本研究首先从保存方法的角度出发,研究不同保存方法对钛表面的理化性能及生物活性的影响,以尽可能保持钛表面的生物活性,使应用到临床上的种植体具有更佳骨结合效能。
研究目的：
1.探讨常规保存方法下,纯钛表面理化性能的时间性变化以及对生物活性影响。
2.探讨不同保存方法对纯钛表面理化性能及生物活性的影响,以及探讨其可能的机制。
研究方法：
1.钛片的制备及分组
将商用二级纯钛(TA2)切割成直径15mm,厚度1.5mm的钛片后,通过H2SO4(49%)/HCl (18%)酸蚀法制备出具有一定的粗糙度,表面形态较为均匀,可重复性较高,表面清洁度良好的钛表面用于本实验。
第一部分实验：探讨常温常压保存方法下,纯钛表面理化性能的时间相关性变化及对表面活性的影响。分组：组1：新制备钛片；组2：避光保存于密封盒内3天；组3：避光保存于密封盒内2周；组4：避光保存于密封盒内4周。制备完成的钛片用于表面理化性能检测、蛋白质吸附及细胞培养实验。
第二部分实验：探讨不同保存方法对纯钛表面理化性能及生物活性的影响。分组：组1：新制备钛片,即制备完成后即刻用于表面理化性能检测和细胞培养。组2：避光保存于密闭盒里。组3-5：制备完成后即刻保存于双蒸水(ddH2O)/氯化钠(NaCl)/氯化钙(CaCl2)溶液中。组2-5中的钛片根据实验设计,保存了3天,2周,4周不同时间,然后根据实验设计对钛片表面进行理化性能检测、蛋白质吸附及细胞培养实验。
以上实验均在清洁环境中完成。
2.钛片表面理化性能的检测：
通过扫描电镜观察、表面轮廓测量仪、X射线光电子能谱分析仪、水接触角分析仪等手段对不同处理组的钛片表面进行表面形貌、表面粗糙度、表面元素、表面亲水性进行检测。以评估不同组别钛片表面理化性能的差异。
3.蛋白吸附实验
使用牛血清白蛋白(BSA)作为模型蛋白,用于蛋白质吸附实验。BCA法在波长为562nm下测定OD值,根据标准曲线计算蛋白质吸附率(百分比)。
4.细胞培养实验
选取MG63成骨细胞系用于本实验,接种密度为2.4×101个/cm2。在不同的培养时间点,通过荧光染色剂染色,荧光显微镜观察,MTS试剂盒、ALP试剂盒、茜素红染色等方法对细胞的黏附、增殖、分化以及矿化情况进行分析,以评估不同组别钛片表面的细胞生物活性。
统计分析
采用Spss13.0统计软件对各组间样品的表面理化性能、蛋白质吸附、细胞黏附、细胞增殖和分化方面各项参数进行统计学分析。测定结果以x±s来表示,用析因设计分析处理组与不同时段是否存在交互效应,如存在交互效应,组间进一步采用单因素方差分析(one-way ANO VA),然后采用Levene's test检查方差齐性,方差齐时对样本均数进行两两比较的LSD检验,方差不齐时,采用近似F检验的Welch方法进行方差分析后,Dunnett'T3检验进行两两比较,假设检验为双侧检验,检验水准a=0.05,当P<0.05时,认为差异具有统计学意义,当P<0.01时,认为差异具有显著的统计学意义。
实验结果：
1.扫描电镜观察结果：各组的钛片均具有典型的酸蚀处理后的表面形态,呈较均匀的多孔状结构,孔径为1-3μm。表面轮廓仪分析证实,所有的表面具有相似的粗糙度,各项参数没有统计学差异。保存方式和保存时间对钛片的表面形态没有明显的影响。
2.X射线光电子能谱分析获得的新制备的钛片以及在其余4种不同方法中保存了3天,2周,4周不同时间的钛片表面的高分辨能谱图提示,所有样品的表面均可发现Ti、O、C和微量的N,但不同组别的元素比例差异较大。新钛片表面的碳元素含量最低,为26.0%。保存了3天后,在常温常压、ddH2O、NaCl溶液、CaCl2溶液下保存的钛表面C元素含量分别为36.3%、26.4%、26.7%、26.2%；保存了2周后,钛表面C元素含量分别为42.9%、29.8%、28.8%、28.2%；保存了4周后,钛表面C元素含量分别为48.2%、31.3%、30.7%、30.1%。不同组别间的Ti和O元素比例也体现出不同的变化。由此可见,在相同的保存时间点,保存在ddH2O、NaCl溶液、CaCl2溶液中的钛片表面碳元素显著低于常温常压下保存。但是需要注意的是,所有的保存方法都不能完全避免C元素的污染。
3.1μL ddH2O在各组不同钛片表面的静态水接触角表明,新制备的钛片表面表现出超亲水性的特性,当ddH2O接触到钛片表面时,便迅速向四周铺展,接近润湿整个钛片表面,静态水接触角为0°。当将钛片常温常压下保存时,随着保存时间的延长,钛表面由亲水性迅速向疏水性转变。在保存3天后,静态水接触角为变为59.70°；2周后,增加至90.27。；4周后,变为114.53。。而保存在ddH2O、NaCl溶液、CaCl2溶液中的钛片表面,在保存3天及2周后,静态水接触角仍然为0°,即便在第4周时,仍保持了超级亲水性(接触角<5°)的特性。
2μL ddH2O在钛片上的接触面积表明,在常温常压保存方式下,ddH2O在钛片上的接触面积也随着保存时间的延长而剧烈减少。但是要注意的是,在这三种保存方式下,ddH2O在钛片表面的润湿面积也随着保存时间的延长而逐渐减小,但仍明显优于常温常压下保存的钛片表面。
4.不同处理组钛片表面1h的蛋白质吸附率结果提示：在常温常压保存方式下,钛片表面蛋白质吸附能力随着保存时间延长下降的程度比保存在ddH2O、NaCl溶液中的钛片表面明显,而保存在CaCl2溶液中的钛片表面蛋白质吸附能力保持了稳定性,未随着保存时间的延长而明显下降,在3天、2周及4周的保存时间里,保存在CaCl2溶液中的钛片表面均表现出了最高的蛋白质吸附能力,在保存了3天钛片表面,蛋白质吸附能力甚至略高于新钛片组。
5.在细胞培养实验中所选择的30min、1h、2h及4h四个时间点中,所有处理组的钛片表面的细胞黏附量均随着培养时间的增加而增加。新制备的钛片表面的细胞黏附效果非常好,细胞接种30min后,约有40%的细胞成功黏附,而在接种4h后,大部分的细胞已经黏附到钛片表面。在常温常压下保存了3天,2周及4周的钛片表面,同一细胞培养时间点的细胞黏附率随着保存时间的增加而显著减少。在常温常压下保存了4周的钛片表面,细胞接种30min后,黏附率仅为新制备钛表面的5%,即便经过4h的培养,也仅有50%接种细胞黏附到钛片表面。通过比较保存在常温常压密封盒内、ddH2O、NaCl溶液、CaCl2溶液中4周的钛片表面在30min、1h、2h及4h细胞黏附情况,可以发现,保存在常温常压密封盒内细胞的黏附效果最差,显著低于其它三种保存方法。所有的保存方法中,在CaCl2溶液中保存的钛片表面在4个培养时间点均表现出最好的细胞黏附率,与新制备的钛片表面无统计学差异。
6.细胞接种4h后,在荧光显微镜下观察不同处理组钛片表面细胞骨架的形态,可见新制备的钛片表面大部分细胞伸展良好,细胞呈扁平状,胞浆丰富,大量的伪足向四周伸出。在常温常压下保存了3天的钛片表面上,仅见少部分伸展良好的细胞,其余大部分细胞胞体较小,呈圆形或椭圆形,未充分展开,在保存了2周和4周的钛片表面上,几乎所有的细胞胞体都呈圆形或椭圆形,未充分展开。在保存于ddH2O、NaCl溶液、CaCl2溶液中4周的钛片表面,细胞形态与新制备钛片表面上的细胞相近,大部分细胞伸展良好。
7.在细胞培养第1天和第3天,在常温常压下保存了3天,2周及4周的钛片表面,细胞的增殖活性均随着保存时间的增加而减少。通过比较保存在常温常压密封盒内、ddH2O、NaCl溶液、CaCl2溶液中4周的钛片表面的细胞增殖活性,可以得知,在细胞培养的第1天时,保存在ddH2O、NaCl溶液和CaCl2溶液中的钛片表面细胞增殖活性与新制备的钛片表面没有统计学差异,并且显著高于常温常压保存的钛片表面。在细胞培养的第3天时,保存在ddH2O、NaCl溶液和CaCl2溶液中的钛片表面细胞增殖活性依然高于常温常压下保存的钛片表面。其中,CaCl2组保存的钛片表面细胞增殖活性最好,显著高于保存在ddH2O及NaCl溶液中的钛片表面(p<0.05),但与新制备的钛片表面没有统计学差异。
8.碱性磷酸酶是细胞分化的早期标志。在细胞培养的第7天,对各组细胞培养样本进行碱性磷酸酶活性的检测。结果发现,各组细胞的碱性磷酸酶活性没有显著的差异。钛片的保存方法不会显著影响细胞的碱性磷酸酶活性。
细胞外基质矿化是通过茜素红染色来检测。实验证明,钛片表面细胞矿化能力受到保存方法的影响。常温常压保存的钛片表面细胞外基质矿化能力最低,显著低于其他组,而保存于CaCl2溶液中的钛片表面以及新制备的钛片表面的矿化能力最强。显著高于其它组。
结论：
1.空气中碳氢化合物的污染显著影响纯钛表面的理化性能和生物学性能。
2.常规的保存方法将纯钛种植体置于密封瓶里面,不能避免种植体受到空气中碳氢化合物的污染。
3.将种植体保存在液体中,能显著减少碳氢化合物的污染,维持钛表面的高表面能。与常规保存方法相比,更有利于蛋白质吸附和细胞的黏附、增殖和分化。
4.保存液的离子对种植体的表面理化性能和生物活性也有影响,二价阳离子Ca2+更有利于蛋白质吸附和细胞的黏附、增殖和分化。
Background:
Due to a bone-forming mechanism at the TiO2interface called "osseointegration", titanium is widely used as material for implants in reconstructive and restorative surgeries for osteoporotic fractures and edentulous jaws. For decades, dental implant has been widely used in restoration of missing tooth, and get some very good clinical results.Nevertheless, there are also many problems unsolved. The implant area bone-titanium contact percentage remains at50～65%, which is far below the ideal100%. In addition, the long healing time after implant surgery limit the clinical application of titanium implant. Much effort is needed to get faster and stronger integration between implant surface and host bone tissue.
The osseointegration of dental implants is dependent on their surface bioactivity, which are determining factors for rapid and intimate bone-implant contact. The concept of bioactivity means the ability of direct connection between the endogenous material surface and the host bone tissues with bone-bonding, and without intervening connective tissue, no matter it is rough surface or not.
Normally, titanium was regarded as a bioinert material because of the TiO2membrane outside the surface. In order to get better bioactivity, treatment of titanium surface has been a primary focus of implant science and technology. Various modification techniques, such as sandblasted and acid-etched(SLA), micro-arc oxidizing(MAO) and HA coating have been used to increase the bioactivity of titanium surface.
In recent years, the effects ultraviolet (UV) irradiation on titanium surface had earned consideration. In our previous study, we had reported ultraviolet (UV) irradiation can be used to decrease hydrocarbon contamination and obtain better bioactivity of titanium surface, which will increased the protein absorption and cell attachment, proliferation, differentiation and final osteointegration on it. But further study proved that these effects degrade over time. On other type of titanium surface, it has been also reported that the biological activity of old titanium surface was significantly lower than a new one.
At present, the reasons for the decreasing of physicochemical properties and bioactivity of the titanium surface is not yet clear. Some papers had reported that titanium surface exposed to the air will adsorb hydrocarbons from air and these contaminations may change the surface composition and surface energy which affected the protein absorption and cell attachment, proliferation, differentiation and final osteointegration.
In clinical applications, it is impossible to get a new titanium implant immediately after processing. All the titanium implants go through different periods of aging process. Therefore, the preservation of the implant surface properties is important, as it relates to the actual performance of implant used in clinics.
Objective:
1. To investigate the time-dependent change of physicochemical properties and bioactivity of acid-etched titanium surface with reference to protein absorption and cell culture in vitro.
2. To investigate the effects of storage methods on time-related changes of titanium surface physicochemical properties, bioactivity and the potential mechanism.
Methods:
1. Specimen preparation
Commercially pure titanium discs (TA2, Baoji, Shanxi Province, China),15mm in diameter and1.5mm in thickness, were prepared, and treated with a mixture of18%HCl and49%H2SO4to get acid-etched surface, as experiment specimen.
Experiment1:
The experiment discs were divided into4groups. Group1:new titanium discs (control), the discs were used immediately after processing; Groups2～4:titanium discs placed in sealed containers and stored in a dark room for3days,2weeks,4weeks respectively.
Experiment2:
The experiment discs were divided into5groups. Group1:new titanium discs (control); Group2:traditional preservation method, titanium discs placed in sealed containers and stored in a dark room; Group3-5:titanium discs were submerged in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution immediately after processing. Group2-5were stored for3days,2weeks,4weeks before tested
2. Surface characterization
The surface morphology of the discs was examined with scanning electron microscopy (SEM)(S-3700N, Hitachi, JAPAN) and Optical Profiler (NT1100, Veeco, USA). X-ray photoelectron spectroscopy (XPS)(ESCALAB250, Thermo-VG Scientific, US) was used for determination of surface composition and elemental oxidation state of the titanium surfaces. The wettability of the discs was determined by means of an automatic contact angle measuring device (OCA15, Dataphysics, German).
3. Protein adsorption assay
Bovine serum albumin was used as model protein. The protein concentration was quantified by means of a microplate reader at562nm. The amount of protein adsorbed by the specimens was determined from the difference in protein concentration between the later and initial protein solution.
4. Osteoblastic cell culture
MG63osteoblast-like cells were used in these experiments. The cells were seeded onto the tested discs in24-well plates at a density of2.4×104cells/cm2. Initial attachment of cells was evaluated by measuring the amount of cells attached to titanium substrates after30min,1h,2h and4h of incubation. The adherent cells were fixed with4%paraformaldehyde and then stained with the fluorescent dye Hoechst (nucleolus, Blue color; Sigma). Cell adhesion was evaluated by counting the number of stained nuclei on each sheet.
After4h of incubation, specimens stained with fluorescent dye rhodamine phalloidin. Fluorescence microscope was used to examine cell morphology and cytoskeletal arrangement of the osteoblasts seeded onto titanium surfaces.
The proliferation of cells was quantified in terms of cell density at1and3culture days using MTS based colorimetry. The amount of formazan product was measured using a microplate reader at490nm.
After7days of culture, the alkaline phosphatase (ALP) activities of the samples were determined by a colorimetric assay using an ALP reagent SIGMA FAST p-Nitrophenyl phosphate (Sigma-Aldrich, Missouri, USA) containing p-nitrophenyl phosphate (p-NPP) as the substrate. The absorbance of p-nitrophenol formed was measured at405nm.
The mineralization capability of cultured osteoblasts was examined by Alizarin red staining. After incubation for14days.The cultures were stained with40mM Alizarin Red in distilled water for10min at room temperature. Subsequently, the cells were washed five times with ddH2O and images were acquired. For quantitative analysis, the stained cultures were dissolved in500μL10%Cetylpyridinium chloride in10mM sodium phosphate. Color intensity was measured by a microplate reader at620nm absorbance.
5. Statistical analysis
All the experiments described above were performed in triplicate. A one-way ANOVA was used to assess the statistical significance of the results between groups. All statistical analyses were performed using SPSS13.0software. P<0.05was considered statistically significant and P<0.01was considered to be highly statistically significant.
Result:
1. Scanning electron microscope (SEM) analysis and quantitative topographical analysis demonstrated no morphological differences among all surfaces as a result of storage method. The acid etched surface exhibited a porous microstructure with spherical pores. The micropore diameter was approximately1～3μm. Quantitative measurements of surface roughness showed no differences in any of the calculated roughness parameters among different groups, indicating that all the storage methods would not change the surface topography.
2. X-ray photoelectron spectroscopy (XPS) indicated the chemical composition varied with surface types. In tradition method, the atomic percentage of carbon continued to increase with storage time, from26.0%to48.2%, indicating more adsorption of CO2and other organic molecules from the atmosphere. At the same time, the composition of Ti and O decreased with storage time. The contents of Ti decreased from19.7%to14.1%, while O decreased from53.4%to36.9%. When stored in solution, the concentration of C on the titanium surface only increased slightly as the storage time increased, indicating storing the titanium discs in solution can protect titanium surface from the hydrocarbon contamination in atmosphere significantly.
3. Contact angle measurement indicated that the surface of new titanium surface exhibited superhydrophilic property with a contact angle of0.0°. But in tradition method, the surface property changed from being hydrophilic to hydrophobic as the storage time increased, and the contact angle changed from0.0°to113°. In contrast, the titanium discs stored in solution maintained the superhydrophilic (contact angle<5°) even after4weeks, but the area of2mL ddH2O spread over the titanium surface decreased with time. We hypothesis this phenomenon was caused by hydrocarbons contamination in the storage solution. This study showed that the rate of surface energy loss can be reduced by storing implant in solution, but cannot be eliminated.
4. The protein adsorption rates on titanium discs stored in various protocols with different storage times are presented as a percentage relative to the total amount incubated for1h. At different storage time point, protein adsorption rate of CaCl2group was highest. At3days and4weeks storage time, protein adsorption rate of CaCl2group even higher than new surface. While the surface stored in tradition method showed the lowest protein absorption rate, and decreased with storage time significantly. The surface property was stable when the discs were stored in solution, protein absorption rate did not significantly changed with storage time. In addition, an intriguing phenomenon is that the surface of titanium discs submerged in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution showed different protein absorption.
5. In this experiment, on all surfaces, cell attachment increased with incubation time. The new surface showed very good cell attachment results. At30min incubation time, the adherent cell number on new surface was about40%of the total cell numbers seeded on the discs, and after4hours incubation, almost all the cells seeded on the disc had attached to the surface.When the titanium discs were stored in tradition method, the adherent cell numbers on titanium surface decreased with the storage time. The cell number on the surface of4-week old was only about5%of that of new surface after30min incubation. Even after4hours incubation, only about50%of total cells attached to the surface of4-week old titanium surface storage in tradition method. At each incubation time adopted in this study, all the surface stored in solution showed better cell attachment than tradition method.CaCl2group was the best one, showed no statistical difference in the cell adhesion with new surface, and the tradition method was worse.
6. After4h of incubation, Fluorescence microscope was used to examine cell morphology and cytoskeletal arrangement of the osteoblasts seeded onto titanium surfaces. The cells seeded on the new titanium surface were noticeably larger and spread better, exhibiting a flattened morphology that was elongated omnidirectionally with numerous lamellipodia. When the titanium disc were stored in tradition method, on3-day old surface, we also found some cells had spread into a triangle and flat shape, but most of them maintained a round shape. The cells seeded on the surface of2-week old and4-week old showed a rather round morphology with sparse filopodial extensions, but no significant morphological difference was observed between them. On the4-week old surface stored in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution, the cells were noticeably larger and spreaded better on these surfaces, showed no significant morphological difference with new surface.
7. When the titanium discs were stored in tradition method, at both incubation time points, the cell proliferation decreased with the storage time. After1day incubation, on the4-week old surface stored in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution, the cell density were noticeably larger than the4-week old surface stored in tradition method. Even after3day incubation, this trend still existed. In addition, CaCl2group showed significantly higher proliferative activity than ddHaO group and NaCl group, but showed no significant morphological difference with new surface.
8. After seven days of culture, the ALP activities of the samples were determined by a colorimetric assay using an ALP reagent SIGMA FASTp-nitrophenyl phosphate (p-NNP)(Sigma-Aldrich, Missouri,USA) containing p-NPP as the substrate. The ALP activity of osteoblasts on different titanium surfaces are not obviously affected by the storage method, there was no difference among the tested titanium discs.After incubation for14days, the mineralization capability of cultured osteoblasts was examined by alizarin red staining. Matrix mineralization is dramatically affected by the storage methods. New surface and CaCl2group induce highest mineralization and no obvious difference in matrix mineralization can be found between these two groups. But the titanium surface stored in tradition method induced lower mineralization than other surface.
Conclusion:
1. Hydrocarbons contamination on titanium surface significantly affect the bioactivity of acid-etched surfaces, including protein adsorption and osteoblast cell response.
2. Traditional methods of storing an implant in a sealed container cannot protect the surface from hydrocarbons contamination.
3. The present study has revealed that the storage method significantly affected titanium surface bioactivity. Traditional storage method shows the worse effect on maintaining titanium surface bioactivity, which degrade with storage time significantly. Storing the titanium discs in solution can protect the surface from the hydrocarbon contamination of surrounding environment and enhance surface energy.
4. The charge of ions in the storage solution may play an important role in affecting the bioactivity evaluation of titanium surface. Bivalence cation plays a positive role in helping the interactivity between protein/cell and titanium surface.
尽管人工种植牙应用于临床已将近半个世纪,并且被广泛证实具有可靠的临床效果,但是仍然存在着以下问题：(1)种植体植入体内到临床负重仍需要较长的愈合时间,最少需要6-12周的时间,这一定程度上限制了种植修复的临床应用；(2)种植体与骨的结合率仍然较低,只有50～65%,不利于种植体的长期稳定。加快种植体-骨结合的速度不仅仅意味着修复时间的缩短,更重要的是使人工牙种植具有更高的安全性。研究表明,大部分的种植失败发生于种植体早期愈合的过程中,愈合时间越长,种植体受到各种风险因素的影响越大,种植失败的风险越高。因此,更快更好的骨结合仍然是我们追求的目标。
大量的研究表明,种植体与骨组织形成结合的速度和强度与种植体表面的生物活性密切相关,而生物活性又与种植体表面的理化性能紧密相连。生物活性(bioactivity)是植入材料能够与生物组织形成键合(Bone-bonding)的特性。即无论表面是否光滑,生物活性材料也能与生物组织产生直接的化学键性结合。
由于钛及钛合金的结构和性质与骨组织差异很大,加之表面钝态氧化膜的存在。通常不能像生物活性材料那样与骨组织发生化学键性结合。因此,一般认为钛金属为生物惰性材料,并努力寻求各种方法进行表面活化改性。钛的生物活化是指使钛金属经过表面处理后,能够在生理环境诱导下,使羟基磷灰石层在其表面形成,与骨组织实现直接的化学键结合。增加钛表面的生物活性有以下两种思路：(1)通过表面加成法在钛表面涂覆生物活性涂层(如羟基磷灰石、钙磷酸盐、生物大分子等)；(2)通过表面改性使原钝化态氧化膜转化为活化态氧化膜或其他活性膜(如钛酸盐,Ca+Ti, TiO2+Ca+P膜等)。目前,常用的一些表面处理技术包括喷砂酸蚀,微弧氧化,羟基磷灰石涂层等等。与光滑种植体表面相比,这些表面处理技术明显改善了种植体的表面活性,促进了种植体-骨的结合。其中,紫外线光催化技术是近几年的研究热点之一。1997年,Wang等首先发现了紫外线照射TiO2膜上可以获得高亲水性表面的现象,并在Nature上发表。此后,大量的学者对此进行了深入的研究。
本课题组通过紫外线光催化技术处理纯钛表面,成功获得了具有高表面能及高生物活性的表面,促进了蛋白质吸附、成骨细胞的黏附、增殖、分化和矿化。但进一步的研究结果表明,通过紫外线光催化技术获得的高表面能及高生物活性效应具有一定的时效性。经过一段时间的保存后,这种效应逐渐消失,钛片表面的生物活性明显降低。另外,在经过其他表面处理方式制备的钛表面上,也出现了钛表面生物活性随着保存时间的增加而下降的情况,即钛表面生物活性的老化现象(time-dependent degradation))。由此可见,钛表面生物活性的老化是钛基种植体普遍存在的现象。
目前,导致钛表面理化性能和生物活性随时间下降的原因尚未明确。有研究认为,这可能与钛表面在储存的过程中受到空气中碳氢化合物的污染,导致表面元素构成的改变及表面能下降有关。由于常规的种植体保存方式为常温常压保存于密封瓶里,可能会受到碳氢化合物的污染,因此,在储存的过程中钛表面理化性能及生物活性可能已经发生变化,这值得我们深入研究。
由于在实际应用中,临床医生不可能获得新制备的钛种植体,应用到临床上的种植体均经过或长或短的保存时间,可能出现了不同程度的生物活性的下降,因此,如何保持种植体的表面活性已成为一个重要的研究课题。本研究首先从保存方法的角度出发,研究不同保存方法对钛表面的理化性能及生物活性的影响,以尽可能保持钛表面的生物活性,使应用到临床上的种植体具有更佳骨结合效能。
研究目的：
1.探讨常规保存方法下,纯钛表面理化性能的时间性变化以及对生物活性影响。
2.探讨不同保存方法对纯钛表面理化性能及生物活性的影响,以及探讨其可能的机制。
研究方法：
1.钛片的制备及分组
将商用二级纯钛(TA2)切割成直径15mm,厚度1.5mm的钛片后,通过H2SO4(49%)/HCl (18%)酸蚀法制备出具有一定的粗糙度,表面形态较为均匀,可重复性较高,表面清洁度良好的钛表面用于本实验。
第一部分实验：探讨常温常压保存方法下,纯钛表面理化性能的时间相关性变化及对表面活性的影响。分组：组1：新制备钛片；组2：避光保存于密封盒内3天；组3：避光保存于密封盒内2周；组4：避光保存于密封盒内4周。制备完成的钛片用于表面理化性能检测、蛋白质吸附及细胞培养实验。
第二部分实验：探讨不同保存方法对纯钛表面理化性能及生物活性的影响。分组：组1：新制备钛片,即制备完成后即刻用于表面理化性能检测和细胞培养。组2：避光保存于密闭盒里。组3-5：制备完成后即刻保存于双蒸水(ddH2O)/氯化钠(NaCl)/氯化钙(CaCl2)溶液中。组2-5中的钛片根据实验设计,保存了3天,2周,4周不同时间,然后根据实验设计对钛片表面进行理化性能检测、蛋白质吸附及细胞培养实验。
以上实验均在清洁环境中完成。
2.钛片表面理化性能的检测：
通过扫描电镜观察、表面轮廓测量仪、X射线光电子能谱分析仪、水接触角分析仪等手段对不同处理组的钛片表面进行表面形貌、表面粗糙度、表面元素、表面亲水性进行检测。以评估不同组别钛片表面理化性能的差异。
3.蛋白吸附实验
使用牛血清白蛋白(BSA)作为模型蛋白,用于蛋白质吸附实验。BCA法在波长为562nm下测定OD值,根据标准曲线计算蛋白质吸附率(百分比)。
4.细胞培养实验
选取MG63成骨细胞系用于本实验,接种密度为2.4×101个/cm2。在不同的培养时间点,通过荧光染色剂染色,荧光显微镜观察,MTS试剂盒、ALP试剂盒、茜素红染色等方法对细胞的黏附、增殖、分化以及矿化情况进行分析,以评估不同组别钛片表面的细胞生物活性。
统计分析
采用Spss13.0统计软件对各组间样品的表面理化性能、蛋白质吸附、细胞黏附、细胞增殖和分化方面各项参数进行统计学分析。测定结果以x±s来表示,用析因设计分析处理组与不同时段是否存在交互效应,如存在交互效应,组间进一步采用单因素方差分析(one-way ANO VA),然后采用Levene's test检查方差齐性,方差齐时对样本均数进行两两比较的LSD检验,方差不齐时,采用近似F检验的Welch方法进行方差分析后,Dunnett'T3检验进行两两比较,假设检验为双侧检验,检验水准a=0.05,当P<0.05时,认为差异具有统计学意义,当P<0.01时,认为差异具有显著的统计学意义。
实验结果：
1.扫描电镜观察结果：各组的钛片均具有典型的酸蚀处理后的表面形态,呈较均匀的多孔状结构,孔径为1-3μm。表面轮廓仪分析证实,所有的表面具有相似的粗糙度,各项参数没有统计学差异。保存方式和保存时间对钛片的表面形态没有明显的影响。
2.X射线光电子能谱分析获得的新制备的钛片以及在其余4种不同方法中保存了3天,2周,4周不同时间的钛片表面的高分辨能谱图提示,所有样品的表面均可发现Ti、O、C和微量的N,但不同组别的元素比例差异较大。新钛片表面的碳元素含量最低,为26.0%。保存了3天后,在常温常压、ddH2O、NaCl溶液、CaCl2溶液下保存的钛表面C元素含量分别为36.3%、26.4%、26.7%、26.2%；保存了2周后,钛表面C元素含量分别为42.9%、29.8%、28.8%、28.2%；保存了4周后,钛表面C元素含量分别为48.2%、31.3%、30.7%、30.1%。不同组别间的Ti和O元素比例也体现出不同的变化。由此可见,在相同的保存时间点,保存在ddH2O、NaCl溶液、CaCl2溶液中的钛片表面碳元素显著低于常温常压下保存。但是需要注意的是,所有的保存方法都不能完全避免C元素的污染。
3.1μL ddH2O在各组不同钛片表面的静态水接触角表明,新制备的钛片表面表现出超亲水性的特性,当ddH2O接触到钛片表面时,便迅速向四周铺展,接近润湿整个钛片表面,静态水接触角为0°。当将钛片常温常压下保存时,随着保存时间的延长,钛表面由亲水性迅速向疏水性转变。在保存3天后,静态水接触角为变为59.70°；2周后,增加至90.27。；4周后,变为114.53。。而保存在ddH2O、NaCl溶液、CaCl2溶液中的钛片表面,在保存3天及2周后,静态水接触角仍然为0°,即便在第4周时,仍保持了超级亲水性(接触角<5°)的特性。
2μL ddH2O在钛片上的接触面积表明,在常温常压保存方式下,ddH2O在钛片上的接触面积也随着保存时间的延长而剧烈减少。但是要注意的是,在这三种保存方式下,ddH2O在钛片表面的润湿面积也随着保存时间的延长而逐渐减小,但仍明显优于常温常压下保存的钛片表面。
4.不同处理组钛片表面1h的蛋白质吸附率结果提示：在常温常压保存方式下,钛片表面蛋白质吸附能力随着保存时间延长下降的程度比保存在ddH2O、NaCl溶液中的钛片表面明显,而保存在CaCl2溶液中的钛片表面蛋白质吸附能力保持了稳定性,未随着保存时间的延长而明显下降,在3天、2周及4周的保存时间里,保存在CaCl2溶液中的钛片表面均表现出了最高的蛋白质吸附能力,在保存了3天钛片表面,蛋白质吸附能力甚至略高于新钛片组。
5.在细胞培养实验中所选择的30min、1h、2h及4h四个时间点中,所有处理组的钛片表面的细胞黏附量均随着培养时间的增加而增加。新制备的钛片表面的细胞黏附效果非常好,细胞接种30min后,约有40%的细胞成功黏附,而在接种4h后,大部分的细胞已经黏附到钛片表面。在常温常压下保存了3天,2周及4周的钛片表面,同一细胞培养时间点的细胞黏附率随着保存时间的增加而显著减少。在常温常压下保存了4周的钛片表面,细胞接种30min后,黏附率仅为新制备钛表面的5%,即便经过4h的培养,也仅有50%接种细胞黏附到钛片表面。通过比较保存在常温常压密封盒内、ddH2O、NaCl溶液、CaCl2溶液中4周的钛片表面在30min、1h、2h及4h细胞黏附情况,可以发现,保存在常温常压密封盒内细胞的黏附效果最差,显著低于其它三种保存方法。所有的保存方法中,在CaCl2溶液中保存的钛片表面在4个培养时间点均表现出最好的细胞黏附率,与新制备的钛片表面无统计学差异。
6.细胞接种4h后,在荧光显微镜下观察不同处理组钛片表面细胞骨架的形态,可见新制备的钛片表面大部分细胞伸展良好,细胞呈扁平状,胞浆丰富,大量的伪足向四周伸出。在常温常压下保存了3天的钛片表面上,仅见少部分伸展良好的细胞,其余大部分细胞胞体较小,呈圆形或椭圆形,未充分展开,在保存了2周和4周的钛片表面上,几乎所有的细胞胞体都呈圆形或椭圆形,未充分展开。在保存于ddH2O、NaCl溶液、CaCl2溶液中4周的钛片表面,细胞形态与新制备钛片表面上的细胞相近,大部分细胞伸展良好。
7.在细胞培养第1天和第3天,在常温常压下保存了3天,2周及4周的钛片表面,细胞的增殖活性均随着保存时间的增加而减少。通过比较保存在常温常压密封盒内、ddH2O、NaCl溶液、CaCl2溶液中4周的钛片表面的细胞增殖活性,可以得知,在细胞培养的第1天时,保存在ddH2O、NaCl溶液和CaCl2溶液中的钛片表面细胞增殖活性与新制备的钛片表面没有统计学差异,并且显著高于常温常压保存的钛片表面。在细胞培养的第3天时,保存在ddH2O、NaCl溶液和CaCl2溶液中的钛片表面细胞增殖活性依然高于常温常压下保存的钛片表面。其中,CaCl2组保存的钛片表面细胞增殖活性最好,显著高于保存在ddH2O及NaCl溶液中的钛片表面(p<0.05),但与新制备的钛片表面没有统计学差异。
8.碱性磷酸酶是细胞分化的早期标志。在细胞培养的第7天,对各组细胞培养样本进行碱性磷酸酶活性的检测。结果发现,各组细胞的碱性磷酸酶活性没有显著的差异。钛片的保存方法不会显著影响细胞的碱性磷酸酶活性。
细胞外基质矿化是通过茜素红染色来检测。实验证明,钛片表面细胞矿化能力受到保存方法的影响。常温常压保存的钛片表面细胞外基质矿化能力最低,显著低于其他组,而保存于CaCl2溶液中的钛片表面以及新制备的钛片表面的矿化能力最强。显著高于其它组。
结论：
1.空气中碳氢化合物的污染显著影响纯钛表面的理化性能和生物学性能。
2.常规的保存方法将纯钛种植体置于密封瓶里面,不能避免种植体受到空气中碳氢化合物的污染。
3.将种植体保存在液体中,能显著减少碳氢化合物的污染,维持钛表面的高表面能。与常规保存方法相比,更有利于蛋白质吸附和细胞的黏附、增殖和分化。
4.保存液的离子对种植体的表面理化性能和生物活性也有影响,二价阳离子Ca2+更有利于蛋白质吸附和细胞的黏附、增殖和分化。
Background:
Due to a bone-forming mechanism at the TiO2interface called "osseointegration", titanium is widely used as material for implants in reconstructive and restorative surgeries for osteoporotic fractures and edentulous jaws. For decades, dental implant has been widely used in restoration of missing tooth, and get some very good clinical results.Nevertheless, there are also many problems unsolved. The implant area bone-titanium contact percentage remains at50～65%, which is far below the ideal100%. In addition, the long healing time after implant surgery limit the clinical application of titanium implant. Much effort is needed to get faster and stronger integration between implant surface and host bone tissue.
The osseointegration of dental implants is dependent on their surface bioactivity, which are determining factors for rapid and intimate bone-implant contact. The concept of bioactivity means the ability of direct connection between the endogenous material surface and the host bone tissues with bone-bonding, and without intervening connective tissue, no matter it is rough surface or not.
Normally, titanium was regarded as a bioinert material because of the TiO2membrane outside the surface. In order to get better bioactivity, treatment of titanium surface has been a primary focus of implant science and technology. Various modification techniques, such as sandblasted and acid-etched(SLA), micro-arc oxidizing(MAO) and HA coating have been used to increase the bioactivity of titanium surface.
In recent years, the effects ultraviolet (UV) irradiation on titanium surface had earned consideration. In our previous study, we had reported ultraviolet (UV) irradiation can be used to decrease hydrocarbon contamination and obtain better bioactivity of titanium surface, which will increased the protein absorption and cell attachment, proliferation, differentiation and final osteointegration on it. But further study proved that these effects degrade over time. On other type of titanium surface, it has been also reported that the biological activity of old titanium surface was significantly lower than a new one.
At present, the reasons for the decreasing of physicochemical properties and bioactivity of the titanium surface is not yet clear. Some papers had reported that titanium surface exposed to the air will adsorb hydrocarbons from air and these contaminations may change the surface composition and surface energy which affected the protein absorption and cell attachment, proliferation, differentiation and final osteointegration.
In clinical applications, it is impossible to get a new titanium implant immediately after processing. All the titanium implants go through different periods of aging process. Therefore, the preservation of the implant surface properties is important, as it relates to the actual performance of implant used in clinics.
Objective:
1. To investigate the time-dependent change of physicochemical properties and bioactivity of acid-etched titanium surface with reference to protein absorption and cell culture in vitro.
2. To investigate the effects of storage methods on time-related changes of titanium surface physicochemical properties, bioactivity and the potential mechanism.
Methods:
1. Specimen preparation
Commercially pure titanium discs (TA2, Baoji, Shanxi Province, China),15mm in diameter and1.5mm in thickness, were prepared, and treated with a mixture of18%HCl and49%H2SO4to get acid-etched surface, as experiment specimen.
Experiment1:
The experiment discs were divided into4groups. Group1:new titanium discs (control), the discs were used immediately after processing; Groups2～4:titanium discs placed in sealed containers and stored in a dark room for3days,2weeks,4weeks respectively.
Experiment2:
The experiment discs were divided into5groups. Group1:new titanium discs (control); Group2:traditional preservation method, titanium discs placed in sealed containers and stored in a dark room; Group3-5:titanium discs were submerged in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution immediately after processing. Group2-5were stored for3days,2weeks,4weeks before tested
2. Surface characterization
The surface morphology of the discs was examined with scanning electron microscopy (SEM)(S-3700N, Hitachi, JAPAN) and Optical Profiler (NT1100, Veeco, USA). X-ray photoelectron spectroscopy (XPS)(ESCALAB250, Thermo-VG Scientific, US) was used for determination of surface composition and elemental oxidation state of the titanium surfaces. The wettability of the discs was determined by means of an automatic contact angle measuring device (OCA15, Dataphysics, German).
3. Protein adsorption assay
Bovine serum albumin was used as model protein. The protein concentration was quantified by means of a microplate reader at562nm. The amount of protein adsorbed by the specimens was determined from the difference in protein concentration between the later and initial protein solution.
4. Osteoblastic cell culture
MG63osteoblast-like cells were used in these experiments. The cells were seeded onto the tested discs in24-well plates at a density of2.4×104cells/cm2. Initial attachment of cells was evaluated by measuring the amount of cells attached to titanium substrates after30min,1h,2h and4h of incubation. The adherent cells were fixed with4%paraformaldehyde and then stained with the fluorescent dye Hoechst (nucleolus, Blue color; Sigma). Cell adhesion was evaluated by counting the number of stained nuclei on each sheet.
After4h of incubation, specimens stained with fluorescent dye rhodamine phalloidin. Fluorescence microscope was used to examine cell morphology and cytoskeletal arrangement of the osteoblasts seeded onto titanium surfaces.
The proliferation of cells was quantified in terms of cell density at1and3culture days using MTS based colorimetry. The amount of formazan product was measured using a microplate reader at490nm.
After7days of culture, the alkaline phosphatase (ALP) activities of the samples were determined by a colorimetric assay using an ALP reagent SIGMA FAST p-Nitrophenyl phosphate (Sigma-Aldrich, Missouri, USA) containing p-nitrophenyl phosphate (p-NPP) as the substrate. The absorbance of p-nitrophenol formed was measured at405nm.
The mineralization capability of cultured osteoblasts was examined by Alizarin red staining. After incubation for14days.The cultures were stained with40mM Alizarin Red in distilled water for10min at room temperature. Subsequently, the cells were washed five times with ddH2O and images were acquired. For quantitative analysis, the stained cultures were dissolved in500μL10%Cetylpyridinium chloride in10mM sodium phosphate. Color intensity was measured by a microplate reader at620nm absorbance.
5. Statistical analysis
All the experiments described above were performed in triplicate. A one-way ANOVA was used to assess the statistical significance of the results between groups. All statistical analyses were performed using SPSS13.0software. P<0.05was considered statistically significant and P<0.01was considered to be highly statistically significant.
Result:
1. Scanning electron microscope (SEM) analysis and quantitative topographical analysis demonstrated no morphological differences among all surfaces as a result of storage method. The acid etched surface exhibited a porous microstructure with spherical pores. The micropore diameter was approximately1～3μm. Quantitative measurements of surface roughness showed no differences in any of the calculated roughness parameters among different groups, indicating that all the storage methods would not change the surface topography.
2. X-ray photoelectron spectroscopy (XPS) indicated the chemical composition varied with surface types. In tradition method, the atomic percentage of carbon continued to increase with storage time, from26.0%to48.2%, indicating more adsorption of CO2and other organic molecules from the atmosphere. At the same time, the composition of Ti and O decreased with storage time. The contents of Ti decreased from19.7%to14.1%, while O decreased from53.4%to36.9%. When stored in solution, the concentration of C on the titanium surface only increased slightly as the storage time increased, indicating storing the titanium discs in solution can protect titanium surface from the hydrocarbon contamination in atmosphere significantly.
3. Contact angle measurement indicated that the surface of new titanium surface exhibited superhydrophilic property with a contact angle of0.0°. But in tradition method, the surface property changed from being hydrophilic to hydrophobic as the storage time increased, and the contact angle changed from0.0°to113°. In contrast, the titanium discs stored in solution maintained the superhydrophilic (contact angle<5°) even after4weeks, but the area of2mL ddH2O spread over the titanium surface decreased with time. We hypothesis this phenomenon was caused by hydrocarbons contamination in the storage solution. This study showed that the rate of surface energy loss can be reduced by storing implant in solution, but cannot be eliminated.
4. The protein adsorption rates on titanium discs stored in various protocols with different storage times are presented as a percentage relative to the total amount incubated for1h. At different storage time point, protein adsorption rate of CaCl2group was highest. At3days and4weeks storage time, protein adsorption rate of CaCl2group even higher than new surface. While the surface stored in tradition method showed the lowest protein absorption rate, and decreased with storage time significantly. The surface property was stable when the discs were stored in solution, protein absorption rate did not significantly changed with storage time. In addition, an intriguing phenomenon is that the surface of titanium discs submerged in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution showed different protein absorption.
5. In this experiment, on all surfaces, cell attachment increased with incubation time. The new surface showed very good cell attachment results. At30min incubation time, the adherent cell number on new surface was about40%of the total cell numbers seeded on the discs, and after4hours incubation, almost all the cells seeded on the disc had attached to the surface.When the titanium discs were stored in tradition method, the adherent cell numbers on titanium surface decreased with the storage time. The cell number on the surface of4-week old was only about5%of that of new surface after30min incubation. Even after4hours incubation, only about50%of total cells attached to the surface of4-week old titanium surface storage in tradition method. At each incubation time adopted in this study, all the surface stored in solution showed better cell attachment than tradition method.CaCl2group was the best one, showed no statistical difference in the cell adhesion with new surface, and the tradition method was worse.
6. After4h of incubation, Fluorescence microscope was used to examine cell morphology and cytoskeletal arrangement of the osteoblasts seeded onto titanium surfaces. The cells seeded on the new titanium surface were noticeably larger and spread better, exhibiting a flattened morphology that was elongated omnidirectionally with numerous lamellipodia. When the titanium disc were stored in tradition method, on3-day old surface, we also found some cells had spread into a triangle and flat shape, but most of them maintained a round shape. The cells seeded on the surface of2-week old and4-week old showed a rather round morphology with sparse filopodial extensions, but no significant morphological difference was observed between them. On the4-week old surface stored in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution, the cells were noticeably larger and spreaded better on these surfaces, showed no significant morphological difference with new surface.
7. When the titanium discs were stored in tradition method, at both incubation time points, the cell proliferation decreased with the storage time. After1day incubation, on the4-week old surface stored in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution, the cell density were noticeably larger than the4-week old surface stored in tradition method. Even after3day incubation, this trend still existed. In addition, CaCl2group showed significantly higher proliferative activity than ddHaO group and NaCl group, but showed no significant morphological difference with new surface.
8. After seven days of culture, the ALP activities of the samples were determined by a colorimetric assay using an ALP reagent SIGMA FASTp-nitrophenyl phosphate (p-NNP)(Sigma-Aldrich, Missouri,USA) containing p-NPP as the substrate. The ALP activity of osteoblasts on different titanium surfaces are not obviously affected by the storage method, there was no difference among the tested titanium discs.After incubation for14days, the mineralization capability of cultured osteoblasts was examined by alizarin red staining. Matrix mineralization is dramatically affected by the storage methods. New surface and CaCl2group induce highest mineralization and no obvious difference in matrix mineralization can be found between these two groups. But the titanium surface stored in tradition method induced lower mineralization than other surface.
Conclusion:
1. Hydrocarbons contamination on titanium surface significantly affect the bioactivity of acid-etched surfaces, including protein adsorption and osteoblast cell response.
2. Traditional methods of storing an implant in a sealed container cannot protect the surface from hydrocarbons contamination.
3. The present study has revealed that the storage method significantly affected titanium surface bioactivity. Traditional storage method shows the worse effect on maintaining titanium surface bioactivity, which degrade with storage time significantly. Storing the titanium discs in solution can protect the surface from the hydrocarbon contamination of surrounding environment and enhance surface energy.
4. The charge of ions in the storage solution may play an important role in affecting the bioactivity evaluation of titanium surface. Bivalence cation plays a positive role in helping the interactivity between protein/cell and titanium surface.
引文
[1]Ogawa T, Nishimura I. Different bone integration profiles of turned and acid-etched implants associated with modulated expression of extracellular matrix genes[J]. Int J Oral Maxillofac Implants,2003,18(2):200-210.
[2]Weinlaender M, Kenney E B, Lekovic V, et al. Histomorphometry of bone apposition around three types of endosseous dental implants [J]. Int J Oral Maxillofac Implants,1992,7(4):491-496.
[3]Raghavendra S, Wood M C, Taylor T D. Early wound healing around endosseous implants:a review of the literature[J]. Int J Oral Maxillofac Implants,2005,20(3):425-431.
[4]Hench L L, Wilson J. Surface-active biomaterials[J]. Science, 1984,226(4675):630-636.
[5]Kieswetter K, Schwartz Z, Dean D D, et al. The role of implant surface characteristics in the healing of bone[J]. Crit Rev Oral Biol Med, 1996,7(4):329-345.
[6]Meyer A E, Baier R E, Natiella J R, et al. Investigation of tissue/implant interactions during the first two hours of implantation[J]. J Oral Implantol, 1988,14(3):363-379.
[7]Puleo D A, Nanci A. Understanding and controlling the bone-implant interface[J]. Biomaterials,1999,20(23-24):2311-2321.
[8]Amor N, Geris L, Vander S J, et al. Computational modelling of biomaterial surface interactions with blood platelets and osteoblastic cells for the prediction of contact osteogenesis[J]. Acta Biomater,2011,7(2):779-790.
[9]Wrobel E, Witkowska-Zimny M, Przybylski J. Biological mechanisms of implant osseointegration[J]. Ortop Traumatol Rehabil,2010,12(5):401-409.
[10]葛泉波,何淑兰,毛津淑,等.生物材料与细胞相互作用及表面修饰[J].化学通报(印刷版),2005,68(1)：43-48.
[11]冯波,翁杰,屈树新,等.医用钛表面生物活性化研究[J].功能材料, 2005(1):23-27.
[12]Orsini G, Assenza B, Scarano A, et al. Surface analysis of machined versus sandblasted and acid-etched titanium implants[J]. Int J Oral Maxillofac Implants,2000,15(6):779-784.
[13]Cochran D L, Buser D, Ten B C, et al. The use of reduced healing times on ITI implants with a sandblasted and acid-etched (SLA) surface:early results from clinical trials on ITI SLA implants [J]. Clin Oral Implants Res, 2002,13(2):144-153.
[14]Rong W, Kazuhito H, Fujishima A. Light-induced amphiphilic surfaces[J]. Nature,388:431-432.
[15]Hori N, Ueno T, Suzuki T, et al. Ultraviolet light treatment for the restoration of age-related degradation of titanium bioactivity. [J]. Int J Oral Maxillofac Implants,2010,25(1):49-62.
[16]Att W, Hori N, Takeuchi M, et al. Time-dependent degradation of titanium osteoconductivity:an implication of biological aging of implant materials.[J]. Biomaterials,2009,30(29):5352-5363.
[17]Att W, Hori N, Iwasa F, et al. The effect of UV-photofunctionalization on the time-related bioactivity of titanium and chromium-cobalt alloy s[J]. Biomaterials,2009,30(26):4268-4276.
[18]Han Y, Chen D, Sun J, et al. UV-enhanced bioactivity and cell response of micro-arc oxidized titania coatings[J]. Acta Biomater,2008,4(5):1518-1529.
[19]Aita H, Hori N, Takeuchi M, et al. The effect of ultraviolet functionalization of titanium on integration with bone.[J]. Biomaterials,2009,30(6):1015-1025.
[20]Li S, Ni J, Liu X, et al. Surface characteristics and biocompatibility of sandblasted and acid-etched titanium surface modified by ultraviolet irradiation: an in vitro study [J]. J Biomed Mater Res B Appl Biomater, 2012,100(6):1587-1598.
[21]高岩,周磊,蒋颖,等.两种波段紫外线对微弧氧化纯钛表面理化性质及体外生物活性影响的研究[J].中华口腔医学杂志,2012,47(6)：359-363.
[22]Hori N, Att W, Ueno T, et al. Age-dependent degradation of the protein adsorption capacity of titanium. [J]. J Dent Res,2009,88(7):663-667.
[23]Kasemo B, Lausmaa J. Biomaterial and implant surfaces:on the role of cleanliness, contamination, and preparation procedures [J]. J Biomed Mater Res,1988,22(A2 Suppl):145-158.
[24]Kilpadi D V, Lemons J E, Liu J, et al. Cleaning and heat-treatment effects on unalloyed titanium implant surfaces[J]. Int J Oral Maxillofac Implants, 2000,15(2):219-230.
[25]Serro A P, Saramago B. Influence of sterilization on the mineralization of titanium implants induced by incubation in various biological model fluids[J]. Biomaterials,2003,24(26):4749-4760.
[26]Massaro C, Rotolo P, De Riccardis F, et al. Comparative investigation of the surface properties of commercial titanium dental implants. Part I:chemical composition[J]. J Mater Sci Mater Med,2002,13(6):535-548.
[27]Buser D, Broggini N, Wieland M, et al. Enhanced bone apposition to a chemically modified SLA titanium surface[J]. J Dent Res,2004,83(7):529-533.
[28]Darvell B W, Samman N, Luk W K, et al. Contamination of titanium castings by aluminium oxide blasting[J]. J Dent,1995,23(5):319-322.
[29]Esposito M, Hirsch J M, Lekholm U, et al. Biological factors contributing to failures of osseointegrated oral implants. (Ⅱ). Etiopathogenesis[J]. Eur J Oral Sci,1998,106(3):721-764.
[30]易树,尹光福,郑昌琼.生物材料表面界面特性与其血液相容性的关系[J].中国口腔种植学杂志,2003,8(2)：83-88.
[31]Anselme K. Osteoblast adhesion on biomaterials[J]. Biomaterials, 2000,21(7):667-681.
[32]Park J H, Olivares-Navarrete R, Baier R E, et al. Effect of cleaning and sterilization on titanium implant surface properties and cellular response [J]. Acta Biomater,2011.
[33]Zhao L, Mei S, Wang W, et al. The role of sterilization in the cytocompatibility of titaniananotubes[J]. Biomaterials,2010,31(8):2055-2063.
[34]Mendes G C C, Brand T R S, Silva C L M. Ethylene oxide sterilization of medical devices:A review[J]. American Journal of Infection Control, 2007,35(9):574-581.
[35]An Y H, Alvi F I, Kang Q, et al. Effects of sterilization on implant mechanical property and biocompatibility[J]. Int J Artif Organs,2005,28(11):1126-1137.
[36]Zhao G, Schwartz Z, Wieland M, et al. High surface energy enhances cell response to titanium substrate microstructure[J]. J Biomed Mater Res A, 2005,74(1):49-58.
[37]Zhang Y, Andrukhov O, Berner S, et al. Osteogenic properties of hydrophilic and hydrophobic titanium surfaces evaluated with osteoblast-like cells (MG63) in coculture with human umbilical vein endothelial cells (HUVEC)[J]. Dent Mater,2010,26(11):1043-1051.
[38]van Kooten T G, Schakenraad J M, van der Mei H C, et al. Influence of substratum wettability on the strength of adhesion of human fibroblasts[J]. Biomaterials,1992,13(13):897-904.
[39]Lemons J E. Dental implant retrieval analyses[J]. J Dent Educ, 1988,52(12):748-756.
[40]Iwanaga S, Akiyama Y, Kikuchi A, et al. Fabrication of a cell array on ultrathin hydrophilic polymer gels utilising electron beam irradiation and UV excimer laser ablation[J]. Biomaterials,2005,26(26):5395-5404.
[41]Billiau A, Edy V G, Heremans H, et al. Human interferon:mass production in a newly established cell line, MG-63[J]. Antimicrob Agents Chemother, 1977,12(1):11-15.
[42]Takeuchi Y, Fukumoto S, Matsumoto T. Relationship between actions of transforming growth factor (TGF)-beta and cell surface expression of its receptors in clonal osteoblastic cells[J]. J Cell Physiol,1995,162(3):315-321.
[43]Macdonald D E, Rapuano B E, Deo N, et al. Thermal and chemical modification of titanium-aluminum-vanadium implant materials:effects on surface properties, glycoprotein adsorption, and MG63 cell attachment[J]. Biomaterials,2004,25(16):3135-3146.
[44]Martin J Y, Schwartz Z, Hummert T W, et al. Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63).[J]. J Biomed Mater Res,1995,29(3):389-401.
[45]Benya P D, Shaffer J D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels[J]. Cell, 1982,30(1):215-224.
[46]Mata A, Su X, Fleischman A J, et al. Osteoblast attachment to a textured surface in the absence of exogenous adhesion proteins [J]. IEEE Trans Nanobioscience,2003,2(4):287-294.
[47]Subbiahdoss G, Kuijer R, Grijpma D W, et al. Microbial biofilm growth vs. tissue integration:"the race for the surface" experimentally studied[J]. Acta Biomater,2009,5(5):1399-1404.
[48]Hallab N J, Bundy K J, O'Connor K, et al. Evaluation of metallic and polymeric biomaterial surface energy and surface roughness characteristics for directed cell adhesion[J]. Tissue Eng,2001,7(1):55-71.
[49]van Kooten T G, Spijker H T, Busscher H J. Plasma-treated polystyrene surfaces:model surfaces for studying cell-biomaterial interactions[J]. Biomaterials,2004,25(10):1735-1747.
[50]Mata A, Su X, Fleischman A J, et al. Osteoblast attachment to a textured surface in the absence of exogenous adhesion proteins[J]. IEEE Trans Nanobioscience,2003,2(4):287-294.
[51]Tsai J A, Lagumdzija A, Stark A, et al. Albumin-bound lipids induce free cytoplasmic calcium oscillations in human osteoblast-like cells [J]. Cell Biochem Funct,2007,25(3):245-249.
[52]Pols H A, Schilte H P, Nijweide P J, et al. The influence of albumin on vitamin D metabolism in fetal chick osteoblast-like cells [J]. Biochem Biophys Res Commun,1984,125(1):265-272.
[53]Macdonald D E, Rapuano B E, Schniepp H C. Surface oxide net charge of a titanium alloy:comparison between effects of treatment with heat or radiofrequency plasma glow discharge[J]. Colloids Surf B Biointerfaces, 2011,82(1):173-181.
[54]Rapuano B E, Macdonald D E. Surface oxide net charge of a titanium alloy: modulation of fibronectin-activated attachment and spreading of osteogenic cells[J]. Colloids Surf B Biointerfaces,2011,82(1):95-103.
[55]Hori N, Ueno T, Minamikawa H, et al. Electrostatic control of protein adsorption on UV-photofunctionalized titanium. [J]. Acta Biomater, 2010,6(10):4175-4180.
[56]Branemark P I, Adell R, Breine U, et al. Intra-osseous anchorage of dental prostheses. I. Experimental studies[J]. Scand J Plast Reconstr Surg, 1969,3(2):81-100.
[57]Davies J E. In vitro modeling of the bone/implant interface[J]. Anat Rec, 1996,245(2):426-445.
[58]宋巍,陈红.蛋白质在纳米拓扑结构材料表面的吸附[J].科学通报,2007,52(23)：2701-2704.
[59]Cai K, Bossert J, Jandt K D. Does the nanometre scale topography of titanium influence protein adsorption and cell proliferation?[J]. Colloids Surf B Biointerfaces,2006,49(2):136-144.
[60]Rechendorff K, Hovgaard M B, Foss M, et al. Enhancement of protein adsorption induced by surface roughness [J]. Langmuir, 2006,22(26):10885-10888.
[61]宋巍.聚合物表面拓扑结构对蛋白质吸附、细胞黏附的影响[D].武汉理工大学,2008.
[62]Riedel M, Muller B, Wintermantel E. Protein adsorption and monocyte activation on germanium nanopyramids[J]. Biomaterials, 2001,22(16):2307-2316.
[63]Lundqvist M, Sethson I, Jonsson B H. Protein adsorption onto silica nanoparticles:conformational changes depend on the particles' curvature and the protein stability[J]. Langmuir,2004,20(24):10639-10647.
[64]Lincks J, Boyan B D, Blanchard C R, et al. Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition[J]. Biomaterials,1998,19(23):2219-2232.
[65]Boyan B D, Sylvia V L, Liu Y, et al. Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2[J]. Biomaterials, 1999,20(23-24):2305-2310.
[66]Deligianni D D, Katsala N, Ladas S, et al. Effect of surface roughness of the titanium alloy Ti-6A1-4V on human bone marrow cell response and on protein adsorption[J]. Biomaterials,2001,22(11):1241-1251.
[67]Marchisio M, Di Carmine M, Pagone R, et al. Implant surface roughness influences osteoclast proliferation and differentiation[J]. J Biomed Mater Res B Appl Biomater,2005,75(2):251-256.
[68]Buser D, Schenk R K, Steinemann S, et al. Influence of surface characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs[J]. J Biomed Mater Res,1991,25(7):889-902.
[69]Han C H, Johansson C B, Wennerberg A, et al. Quantitative and qualitative investigations of surface enlarged titanium and titanium alloy implants[J]. Clin Oral Implants Res,1998,9(1):1-10.
[70]Nasatzky E, Gultchin J, Schwartz Z. [The role of surface roughness in promoting osteointegration][J]. Refuat Hapeh Vehashinayim,2003,20(3):8-19, 98.
[71]Martin J Y, Schwartz Z, Hummert T W, et al. Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63)[J]. J Biomed Mater Res,1995,29(3):389-401.
[72]Schwartz Z, Martin J Y, Dean D D, et al. Effect of titanium surface roughness on chondrocyte proliferation, matrix production, and differentiation depends on the state of cell maturation[J]. J Biomed Mater Res,1996,30(2):145-155.
[73]Silva T S, Machado D C, Viezzer C, et al. Effect of titanium surface roughness on human bone marrow cell proliferation and differentiation:an experimental study[J]. Acta Cir Bras,2009,24(3):200-205.
[74]Gotfredsen K, Wennerberg A, Johansson C, et al. Anchorage of TiO2-blasted, HA-coated, and machined implants:an experimental study with rabbits[J]. J Biomed Mater Res,1995,29(10):1223-1231.
[75]Wennerberg A, Albrektsson T, Andersson B, et al. A histomorphometric and removal torque study of screw-shaped titanium implants with three different surface topographies[J]. Clin Oral Implants Res,1995,6(1):24-30.
[76]Becker W, Becker B E, Ricci A, et al. A prospective multicenter clinical trial comparing one-and two-stage titanium screw-shaped fixtures with one-stage plasma-sprayed solid-screw fixtures [J]. Clin Implant Dent Relat Res, 2000,2(3):159-165.
[77]Albrektsson T, Wennerberg A. The impact of oral implants-past and future, 1966-2042[J]. J Can Dent Assoc,2005,71(5):327.
[78]Han Y, Hong S, Xu K. Structure and in vitro bioactivity of titania-based films by micro-arc oxidation [J]. Surface and Coatings Technology, 2003,168(2-3):249-258.
[79]Rohanizadeh R, Al-Sadeq M, Legeros R Z. Preparation of different forms of titanium oxide on titanium surface:effects on apatite deposition[J]. J Biomed Mater Res A,2004,71(2):343-352.
[80]Chen H, Brook M A, Chen Y, et al. Surface properties of PEO-silicone composites:reducing protein adsorption[J]. J Biomater Sci Polym Ed, 2005,16(4):531-548.
[81]Jin Z, Feng W, Zhu S, et al. Protein-resistant materials via surface-initiated atom transfer radical polymerization of 2-methacryloyloxyethyl phosphorylcholine[J]. J Biomater Sci Polym Ed,2010,21(10):1331-1344.
[82]胡小洋,陈红,张燕霞,等.聚乙二醇及其衍生物改性生物医用材料表面的血液相容性[J].高分子材料科学与工程,2007(6)：127-131.
[83]Hambleton J, Schwartz Z, Khare A, et al. Culture surfaces coated with various implant materials affect chondrocyte growth and metabolism[J]. J Orthop Res, 1994,12(4):542-552.
[84]Lu H, Zhou L, Wan L, et al. Effects of storage methods on time-related changes of titanium surface properties and cellular response[J]. Biomed Mater, 2012,7(5):55002.
[85]严洪海,赵士若,陈关福.金属表面的蛋白吸附的机理研究现状[J].中国口 腔种植学杂志,1997,2(4)：189-193.
[86]Bonfield T L, Colton E, Anderson J M. Plasma protein adsorbed biomedical polymers:activation of human monocytes and induction of interleukin 1[J]. J Biomed Mater Res,1989,23(6):535-548.
[87]Hersel U, Dahmen C, Kessler H. RGD modified polymers:biomaterials for stimulated cell adhesion and beyond[J]. Biomaterials,2003,24(24):4385-4415.
[88]Vieira E P, Rocha S, Carmo P M, et al. Adsorption and diffusion of plasma proteins on hydrophilic and hydrophobic surfaces:effect of trifluoroethanol on protein structure[J]. Langmuir,2009,25(17):9879-9886.
[89]Sawase T, Jimbo R, Baba K, et al. Photo-induced hydrophilicity enhances initial cell behavior and early bone apposition[J]. Clin Oral Implants Res, 2008,19(5):491-496.
[90]Feng L, Andrade J D. Protein adsorption on low temperature isotropic carbon: V. How is it related to its blood compatibility?[J]. J Biomater Sci Polym Ed, 1995,7(5):439-452.
[91]Feng L, Andrade J D. Protein adsorption on low-temperature isotropic carbon:I. Protein conformational change probed by differential scanning calorimetry[J]. J Biomed Mater Res,1994,28(6):735-743.
[2]Weinlaender M, Kenney E B, Lekovic V, et al. Histomorphometry of bone apposition around three types of endosseous dental implants [J]. Int J Oral Maxillofac Implants,1992,7(4):491-496.
[3]Raghavendra S, Wood M C, Taylor T D. Early wound healing around endosseous implants:a review of the literature[J]. Int J Oral Maxillofac Implants,2005,20(3):425-431.
[4]Hench L L, Wilson J. Surface-active biomaterials[J]. Science, 1984,226(4675):630-636.
[5]Kieswetter K, Schwartz Z, Dean D D, et al. The role of implant surface characteristics in the healing of bone[J]. Crit Rev Oral Biol Med, 1996,7(4):329-345.
[6]Meyer A E, Baier R E, Natiella J R, et al. Investigation of tissue/implant interactions during the first two hours of implantation[J]. J Oral Implantol, 1988,14(3):363-379.
[7]Puleo D A, Nanci A. Understanding and controlling the bone-implant interface[J]. Biomaterials,1999,20(23-24):2311-2321.
[8]Amor N, Geris L, Vander S J, et al. Computational modelling of biomaterial surface interactions with blood platelets and osteoblastic cells for the prediction of contact osteogenesis[J]. Acta Biomater,2011,7(2):779-790.
[9]Wrobel E, Witkowska-Zimny M, Przybylski J. Biological mechanisms of implant osseointegration[J]. Ortop Traumatol Rehabil,2010,12(5):401-409.
[10]葛泉波,何淑兰,毛津淑,等.生物材料与细胞相互作用及表面修饰[J].化学通报(印刷版),2005,68(1)：43-48.
[11]冯波,翁杰,屈树新,等.医用钛表面生物活性化研究[J].功能材料, 2005(1):23-27.
[12]Orsini G, Assenza B, Scarano A, et al. Surface analysis of machined versus sandblasted and acid-etched titanium implants[J]. Int J Oral Maxillofac Implants,2000,15(6):779-784.
[13]Cochran D L, Buser D, Ten B C, et al. The use of reduced healing times on ITI implants with a sandblasted and acid-etched (SLA) surface:early results from clinical trials on ITI SLA implants [J]. Clin Oral Implants Res, 2002,13(2):144-153.
[14]Rong W, Kazuhito H, Fujishima A. Light-induced amphiphilic surfaces[J]. Nature,388:431-432.
[15]Hori N, Ueno T, Suzuki T, et al. Ultraviolet light treatment for the restoration of age-related degradation of titanium bioactivity. [J]. Int J Oral Maxillofac Implants,2010,25(1):49-62.
[16]Att W, Hori N, Takeuchi M, et al. Time-dependent degradation of titanium osteoconductivity:an implication of biological aging of implant materials.[J]. Biomaterials,2009,30(29):5352-5363.
[17]Att W, Hori N, Iwasa F, et al. The effect of UV-photofunctionalization on the time-related bioactivity of titanium and chromium-cobalt alloy s[J]. Biomaterials,2009,30(26):4268-4276.
[18]Han Y, Chen D, Sun J, et al. UV-enhanced bioactivity and cell response of micro-arc oxidized titania coatings[J]. Acta Biomater,2008,4(5):1518-1529.
[19]Aita H, Hori N, Takeuchi M, et al. The effect of ultraviolet functionalization of titanium on integration with bone.[J]. Biomaterials,2009,30(6):1015-1025.
[20]Li S, Ni J, Liu X, et al. Surface characteristics and biocompatibility of sandblasted and acid-etched titanium surface modified by ultraviolet irradiation: an in vitro study [J]. J Biomed Mater Res B Appl Biomater, 2012,100(6):1587-1598.
[21]高岩,周磊,蒋颖,等.两种波段紫外线对微弧氧化纯钛表面理化性质及体外生物活性影响的研究[J].中华口腔医学杂志,2012,47(6)：359-363.
[22]Hori N, Att W, Ueno T, et al. Age-dependent degradation of the protein adsorption capacity of titanium. [J]. J Dent Res,2009,88(7):663-667.
[23]Kasemo B, Lausmaa J. Biomaterial and implant surfaces:on the role of cleanliness, contamination, and preparation procedures [J]. J Biomed Mater Res,1988,22(A2 Suppl):145-158.
[24]Kilpadi D V, Lemons J E, Liu J, et al. Cleaning and heat-treatment effects on unalloyed titanium implant surfaces[J]. Int J Oral Maxillofac Implants, 2000,15(2):219-230.
[25]Serro A P, Saramago B. Influence of sterilization on the mineralization of titanium implants induced by incubation in various biological model fluids[J]. Biomaterials,2003,24(26):4749-4760.
[26]Massaro C, Rotolo P, De Riccardis F, et al. Comparative investigation of the surface properties of commercial titanium dental implants. Part I:chemical composition[J]. J Mater Sci Mater Med,2002,13(6):535-548.
[27]Buser D, Broggini N, Wieland M, et al. Enhanced bone apposition to a chemically modified SLA titanium surface[J]. J Dent Res,2004,83(7):529-533.
[28]Darvell B W, Samman N, Luk W K, et al. Contamination of titanium castings by aluminium oxide blasting[J]. J Dent,1995,23(5):319-322.
[29]Esposito M, Hirsch J M, Lekholm U, et al. Biological factors contributing to failures of osseointegrated oral implants. (Ⅱ). Etiopathogenesis[J]. Eur J Oral Sci,1998,106(3):721-764.
[30]易树,尹光福,郑昌琼.生物材料表面界面特性与其血液相容性的关系[J].中国口腔种植学杂志,2003,8(2)：83-88.
[31]Anselme K. Osteoblast adhesion on biomaterials[J]. Biomaterials, 2000,21(7):667-681.
[32]Park J H, Olivares-Navarrete R, Baier R E, et al. Effect of cleaning and sterilization on titanium implant surface properties and cellular response [J]. Acta Biomater,2011.
[33]Zhao L, Mei S, Wang W, et al. The role of sterilization in the cytocompatibility of titaniananotubes[J]. Biomaterials,2010,31(8):2055-2063.
[34]Mendes G C C, Brand T R S, Silva C L M. Ethylene oxide sterilization of medical devices:A review[J]. American Journal of Infection Control, 2007,35(9):574-581.
[35]An Y H, Alvi F I, Kang Q, et al. Effects of sterilization on implant mechanical property and biocompatibility[J]. Int J Artif Organs,2005,28(11):1126-1137.
[36]Zhao G, Schwartz Z, Wieland M, et al. High surface energy enhances cell response to titanium substrate microstructure[J]. J Biomed Mater Res A, 2005,74(1):49-58.
[37]Zhang Y, Andrukhov O, Berner S, et al. Osteogenic properties of hydrophilic and hydrophobic titanium surfaces evaluated with osteoblast-like cells (MG63) in coculture with human umbilical vein endothelial cells (HUVEC)[J]. Dent Mater,2010,26(11):1043-1051.
[38]van Kooten T G, Schakenraad J M, van der Mei H C, et al. Influence of substratum wettability on the strength of adhesion of human fibroblasts[J]. Biomaterials,1992,13(13):897-904.
[39]Lemons J E. Dental implant retrieval analyses[J]. J Dent Educ, 1988,52(12):748-756.
[40]Iwanaga S, Akiyama Y, Kikuchi A, et al. Fabrication of a cell array on ultrathin hydrophilic polymer gels utilising electron beam irradiation and UV excimer laser ablation[J]. Biomaterials,2005,26(26):5395-5404.
[41]Billiau A, Edy V G, Heremans H, et al. Human interferon:mass production in a newly established cell line, MG-63[J]. Antimicrob Agents Chemother, 1977,12(1):11-15.
[42]Takeuchi Y, Fukumoto S, Matsumoto T. Relationship between actions of transforming growth factor (TGF)-beta and cell surface expression of its receptors in clonal osteoblastic cells[J]. J Cell Physiol,1995,162(3):315-321.
[43]Macdonald D E, Rapuano B E, Deo N, et al. Thermal and chemical modification of titanium-aluminum-vanadium implant materials:effects on surface properties, glycoprotein adsorption, and MG63 cell attachment[J]. Biomaterials,2004,25(16):3135-3146.
[44]Martin J Y, Schwartz Z, Hummert T W, et al. Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63).[J]. J Biomed Mater Res,1995,29(3):389-401.
[45]Benya P D, Shaffer J D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels[J]. Cell, 1982,30(1):215-224.
[46]Mata A, Su X, Fleischman A J, et al. Osteoblast attachment to a textured surface in the absence of exogenous adhesion proteins [J]. IEEE Trans Nanobioscience,2003,2(4):287-294.
[47]Subbiahdoss G, Kuijer R, Grijpma D W, et al. Microbial biofilm growth vs. tissue integration:"the race for the surface" experimentally studied[J]. Acta Biomater,2009,5(5):1399-1404.
[48]Hallab N J, Bundy K J, O'Connor K, et al. Evaluation of metallic and polymeric biomaterial surface energy and surface roughness characteristics for directed cell adhesion[J]. Tissue Eng,2001,7(1):55-71.
[49]van Kooten T G, Spijker H T, Busscher H J. Plasma-treated polystyrene surfaces:model surfaces for studying cell-biomaterial interactions[J]. Biomaterials,2004,25(10):1735-1747.
[50]Mata A, Su X, Fleischman A J, et al. Osteoblast attachment to a textured surface in the absence of exogenous adhesion proteins[J]. IEEE Trans Nanobioscience,2003,2(4):287-294.
[51]Tsai J A, Lagumdzija A, Stark A, et al. Albumin-bound lipids induce free cytoplasmic calcium oscillations in human osteoblast-like cells [J]. Cell Biochem Funct,2007,25(3):245-249.
[52]Pols H A, Schilte H P, Nijweide P J, et al. The influence of albumin on vitamin D metabolism in fetal chick osteoblast-like cells [J]. Biochem Biophys Res Commun,1984,125(1):265-272.
[53]Macdonald D E, Rapuano B E, Schniepp H C. Surface oxide net charge of a titanium alloy:comparison between effects of treatment with heat or radiofrequency plasma glow discharge[J]. Colloids Surf B Biointerfaces, 2011,82(1):173-181.
[54]Rapuano B E, Macdonald D E. Surface oxide net charge of a titanium alloy: modulation of fibronectin-activated attachment and spreading of osteogenic cells[J]. Colloids Surf B Biointerfaces,2011,82(1):95-103.
[55]Hori N, Ueno T, Minamikawa H, et al. Electrostatic control of protein adsorption on UV-photofunctionalized titanium. [J]. Acta Biomater, 2010,6(10):4175-4180.
[56]Branemark P I, Adell R, Breine U, et al. Intra-osseous anchorage of dental prostheses. I. Experimental studies[J]. Scand J Plast Reconstr Surg, 1969,3(2):81-100.
[57]Davies J E. In vitro modeling of the bone/implant interface[J]. Anat Rec, 1996,245(2):426-445.
[58]宋巍,陈红.蛋白质在纳米拓扑结构材料表面的吸附[J].科学通报,2007,52(23)：2701-2704.
[59]Cai K, Bossert J, Jandt K D. Does the nanometre scale topography of titanium influence protein adsorption and cell proliferation?[J]. Colloids Surf B Biointerfaces,2006,49(2):136-144.
[60]Rechendorff K, Hovgaard M B, Foss M, et al. Enhancement of protein adsorption induced by surface roughness [J]. Langmuir, 2006,22(26):10885-10888.
[61]宋巍.聚合物表面拓扑结构对蛋白质吸附、细胞黏附的影响[D].武汉理工大学,2008.
[62]Riedel M, Muller B, Wintermantel E. Protein adsorption and monocyte activation on germanium nanopyramids[J]. Biomaterials, 2001,22(16):2307-2316.
[63]Lundqvist M, Sethson I, Jonsson B H. Protein adsorption onto silica nanoparticles:conformational changes depend on the particles' curvature and the protein stability[J]. Langmuir,2004,20(24):10639-10647.
[64]Lincks J, Boyan B D, Blanchard C R, et al. Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition[J]. Biomaterials,1998,19(23):2219-2232.
[65]Boyan B D, Sylvia V L, Liu Y, et al. Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2[J]. Biomaterials, 1999,20(23-24):2305-2310.
[66]Deligianni D D, Katsala N, Ladas S, et al. Effect of surface roughness of the titanium alloy Ti-6A1-4V on human bone marrow cell response and on protein adsorption[J]. Biomaterials,2001,22(11):1241-1251.
[67]Marchisio M, Di Carmine M, Pagone R, et al. Implant surface roughness influences osteoclast proliferation and differentiation[J]. J Biomed Mater Res B Appl Biomater,2005,75(2):251-256.
[68]Buser D, Schenk R K, Steinemann S, et al. Influence of surface characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs[J]. J Biomed Mater Res,1991,25(7):889-902.
[69]Han C H, Johansson C B, Wennerberg A, et al. Quantitative and qualitative investigations of surface enlarged titanium and titanium alloy implants[J]. Clin Oral Implants Res,1998,9(1):1-10.
[70]Nasatzky E, Gultchin J, Schwartz Z. [The role of surface roughness in promoting osteointegration][J]. Refuat Hapeh Vehashinayim,2003,20(3):8-19, 98.
[71]Martin J Y, Schwartz Z, Hummert T W, et al. Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63)[J]. J Biomed Mater Res,1995,29(3):389-401.
[72]Schwartz Z, Martin J Y, Dean D D, et al. Effect of titanium surface roughness on chondrocyte proliferation, matrix production, and differentiation depends on the state of cell maturation[J]. J Biomed Mater Res,1996,30(2):145-155.
[73]Silva T S, Machado D C, Viezzer C, et al. Effect of titanium surface roughness on human bone marrow cell proliferation and differentiation:an experimental study[J]. Acta Cir Bras,2009,24(3):200-205.
[74]Gotfredsen K, Wennerberg A, Johansson C, et al. Anchorage of TiO2-blasted, HA-coated, and machined implants:an experimental study with rabbits[J]. J Biomed Mater Res,1995,29(10):1223-1231.
[75]Wennerberg A, Albrektsson T, Andersson B, et al. A histomorphometric and removal torque study of screw-shaped titanium implants with three different surface topographies[J]. Clin Oral Implants Res,1995,6(1):24-30.
[76]Becker W, Becker B E, Ricci A, et al. A prospective multicenter clinical trial comparing one-and two-stage titanium screw-shaped fixtures with one-stage plasma-sprayed solid-screw fixtures [J]. Clin Implant Dent Relat Res, 2000,2(3):159-165.
[77]Albrektsson T, Wennerberg A. The impact of oral implants-past and future, 1966-2042[J]. J Can Dent Assoc,2005,71(5):327.
[78]Han Y, Hong S, Xu K. Structure and in vitro bioactivity of titania-based films by micro-arc oxidation [J]. Surface and Coatings Technology, 2003,168(2-3):249-258.
[79]Rohanizadeh R, Al-Sadeq M, Legeros R Z. Preparation of different forms of titanium oxide on titanium surface:effects on apatite deposition[J]. J Biomed Mater Res A,2004,71(2):343-352.
[80]Chen H, Brook M A, Chen Y, et al. Surface properties of PEO-silicone composites:reducing protein adsorption[J]. J Biomater Sci Polym Ed, 2005,16(4):531-548.
[81]Jin Z, Feng W, Zhu S, et al. Protein-resistant materials via surface-initiated atom transfer radical polymerization of 2-methacryloyloxyethyl phosphorylcholine[J]. J Biomater Sci Polym Ed,2010,21(10):1331-1344.
[82]胡小洋,陈红,张燕霞,等.聚乙二醇及其衍生物改性生物医用材料表面的血液相容性[J].高分子材料科学与工程,2007(6)：127-131.
[83]Hambleton J, Schwartz Z, Khare A, et al. Culture surfaces coated with various implant materials affect chondrocyte growth and metabolism[J]. J Orthop Res, 1994,12(4):542-552.
[84]Lu H, Zhou L, Wan L, et al. Effects of storage methods on time-related changes of titanium surface properties and cellular response[J]. Biomed Mater, 2012,7(5):55002.
[85]严洪海,赵士若,陈关福.金属表面的蛋白吸附的机理研究现状[J].中国口 腔种植学杂志,1997,2(4)：189-193.
[86]Bonfield T L, Colton E, Anderson J M. Plasma protein adsorbed biomedical polymers:activation of human monocytes and induction of interleukin 1[J]. J Biomed Mater Res,1989,23(6):535-548.
[87]Hersel U, Dahmen C, Kessler H. RGD modified polymers:biomaterials for stimulated cell adhesion and beyond[J]. Biomaterials,2003,24(24):4385-4415.
[88]Vieira E P, Rocha S, Carmo P M, et al. Adsorption and diffusion of plasma proteins on hydrophilic and hydrophobic surfaces:effect of trifluoroethanol on protein structure[J]. Langmuir,2009,25(17):9879-9886.
[89]Sawase T, Jimbo R, Baba K, et al. Photo-induced hydrophilicity enhances initial cell behavior and early bone apposition[J]. Clin Oral Implants Res, 2008,19(5):491-496.
[90]Feng L, Andrade J D. Protein adsorption on low temperature isotropic carbon: V. How is it related to its blood compatibility?[J]. J Biomater Sci Polym Ed, 1995,7(5):439-452.
[91]Feng L, Andrade J D. Protein adsorption on low-temperature isotropic carbon:I. Protein conformational change probed by differential scanning calorimetry[J]. J Biomed Mater Res,1994,28(6):735-743.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |