钽涂层人工假体界面特性及生物学特性的研究
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摘要
理想的关节置换假体不仅要具有较高力学强度及低弹性模量,假体表面还应具有一定大小的微孔结构,以利于骨组织能够紧密地嵌合和长入假体的微孔中,增加假体术后的稳定作用;随着假体表面与骨结合的研究日益深入,假体表面处理方式也日趋多样化。
目前,生物陶瓷涂层材料及微孔金属涂层生物材料是假体表面处理的主要方向,分别以羟基磷灰石(Hydroxyapatite, HA,Ca10(PO4)6(OH)2)及钛(Titanium ,Ti)涂层为代表。它们是通过烧结或等离子喷涂的工艺将HA及Ti金属被覆在不锈钢或Ti金属基体表面,形成具有一定骨传导或骨诱导功能的生物涂层复合材料,克服了以往人体植入材料在机械强度、生物相容性、耐腐蚀等综合性能方面存在的不足,成为近年来比较理想的骨重建材料。但已有文献报道,由于HA属于人体可降解和吸收的材料,其涂层降解后会使材料基体与组织间产生间隙,逐渐导致假体松动。对于Ti涂层复合生物材料而言,虽然在表面机械强度、耐腐蚀以及假体晚期稳定性方面优于HA涂层生物材料,但在假体植入早期,Ti涂层与HA涂层材料在骨诱导功能方面还存在一定差距,临床长期的观察也发现Ti涂层关节假体同样存在涂层脱落而导致假体松动的现象,因此,寻找和研究既有良好的生物相容性、又能保证术后假体长期稳定的涂层材料仍是今后一段时间关节假体研究领域的主要方向之一。
自从1997年起,随着多孔钽材料作为小梁金属在临床矫形外科的广泛应用,各种钽(Tantalum,Ta)制生物材料的优势也逐渐展现出来,其良好的生物相容性、独特的物理及机械学特性为钽金属生物材料发展提供了广阔空间。
随着假体表面研究的进展以及多孔钽“外衬”部件在人工假体表面应用存在的局限性,使得研究者开始对钽金属是否可以作为假体涂层材料进行了新的思考。但作为一种新型生物材料涂层,Ta涂层是否具备生物材料涂层应有的材料学特性及良好的生物相容性?Ta涂层-基体结合强度、涂层的耐磨性是否能满足其作为人工假体涂层的要求?Ta涂层假体植入后的骨长入量是否会对假体后期的稳定性起到维持作用?这些问题便成为研究者关注的焦点。
本文通过三个部分对Ta涂层一般特性及生物学特性进行了进一步研究:第一部分为Ta涂层一般特性的研究;第二部分为Ta涂层生物学特性的研究;第三部分为Ta涂层人工假体生物力学行为及其涂层物理特性关系的初步研究。
一、Ta涂层一般特性的研究
1. Ta涂层界面物理特性的研究
目的:对Ta涂层界面结合强度、表面机械强度、涂层元素构成进行初步的研究和分析,为钽金属涂层材料的应用提供试验依据。方法:(1)采用大气等离子喷涂系统制备钽涂层试件,获得二维孔隙率为25~35%、涂层厚度为150μm的Ta涂层试件,通过扫描电子显微镜(SEM)观察、金相显微镜观察、能谱扫描(EDX)及X-射线衍射分析(XRD)等手段对Ta涂层的表面形貌、金相结构和化学构成进行分析;(2)界面力学检测:选用医用Ti-6Al-4V基体材料,经过喷砂及等离子喷涂工艺加工获得二维孔隙率为25~35%、涂层厚度为150μm的Ta涂层试件作为实验组,相同基体和规格的HA及Ti涂层的试件作为对照组;运用WS-2005涂层附着力自动划痕仪、HV-120维氏硬度仪分别对涂层与基体结合强度及涂层表面硬度进行评价。结果:(1)SEM观察发现,Ta涂层表面粗糙、孔隙形貌清晰;钽涂层组织致密,界面结合紧密,未发现内部分层和裂纹;(2)Ta涂层料表面硬度及涂层界面结合强度明显高于HA涂层和Ti涂层(P﹤0.01)。结论:Ta涂层界面结合紧密,表面孔隙结构稳定,与Ti涂层和HA涂层相比具有更高的结合强度和表面硬度,这对预防涂层脱落、维持假体表面结构及维持假体生物力学特性具有积极作用。
2. Ta涂层假体表面磨损机制的研究
目的:对Ta涂层假体表面的疲劳磨损机制及其生物磨损机制进行初步的研究和分析,为钽金属涂层假体的临床应用提供试验依据。方法:(1)实验组(A组)基体为医用316不锈钢(0Cr17Ni12Mo2),涂层厚度为400μm的Ta涂层试件,表面孔隙率为25-35%;对照组(B、C组)分别为316不锈钢基体HA与Ti涂层试件,试件标准与A组一致。采用MMW-2型微机控制摩擦磨损试验机及0Cr17Ni12Mo2对磨件进行涂层试件的摩擦磨损实验,对在一定载荷下的试件磨损外貌特征、磨损量进行观察、对比,采用SEM及XRD等方法对磨损实验前后涂层表面形貌、金相结构和化学构成进行分析对比;(2)采用以0Cr17Ni12Mo2为基体的Ta、HA及Ti涂层试件作为实验组(A组)及对照组(B、C组)进行试件-骨磨损试验,三种涂层厚度均为400μm、表面孔隙率为25-35%;对磨件为猪骨股骨近端骨骼制成;利用MMW-2型微机控制摩擦磨损试验机对一定载荷下的试件经过骨磨损后外貌特征、骨磨损量及试验中各组摩擦副间摩擦系数的变化进行分析,采用SEM及XRD等方法对磨损试验前后涂层表面形貌、金相结构和化学构成进行对比分析。结果:(1)各组涂层的滑动摩擦失效行为主要表现为疲劳剥落,316不锈钢基体Ta涂层表面耐磨性能明显高于HA及Ti涂层(P﹤0.01);(2)与金属对磨的磨损实验不同,在采用骨对磨件的磨损实验中三种涂层磨损试件的质量并没有明显的减少(P﹥0.05);随着磨损时间的延长,三种涂层试件对骨组织的摩擦系数均逐渐减少,最终摩擦系数均维持在0.40左右;同时,骨磨屑堆积在试样涂层表面孔隙里形成了一层平滑的固体润滑界面,在摩擦副间起到阻碍磨损的作用;结论:(1)Ta涂层磨损失效行为主要为疲劳剥落,其具有明显优于Ti涂层和HA涂层的耐磨损、抗疲劳作用,这对预防涂层脱落、维持假体表面结构及维持假体生物力学特性具有积极作用;(2)由于固体润滑界面形成及存在,使得骨-涂层摩擦不易对假体的Ta涂层造成破坏。
二、Ta涂层生物学特性的研究
1. Ta、HA、及Ti涂层假体细胞毒性对照研究
目的:通过对比研究评价Ta涂层假体的细胞毒性作用,初步了解和评价Ta涂层材料生物相容性,为Ta涂层假体临床应用的可行性提供实验依据。方法:通过对细胞相对增殖率(RGR)检测及细胞毒性测定对Ta涂层材料的生物相容性进行评价;方法:(1)Ta、Ha、Ti涂层材料及相应浸提液的制备;(2)L929细胞系的培养及传代;(3)利用Ta、Ha、Ti涂层材料浸提液对L929传代细胞进行培养,观察不同浸提液培养的细胞生长形态,并通过MTT比色法对各组涂层材料的细胞毒性进行分级;结果:(1)细胞形态学观察显示:Ta涂层组的细胞生长形态与HA和Ti涂层组一致,细胞生长形态良好,与空白阴性对照组无明显差别,而含有0.64%苯酚浸提液作为培养液培养的空白阳性对照组细胞,细胞广泛死亡;(2)MTT比色实验显示:Ta、Ha及Ti涂层组的细胞相对增殖率(RGR)无明显差别﹙P﹥0.05﹚,Ta涂层与其它涂层对照组及空白阴性对照组的细胞毒性没有显著差异,三种涂层的细胞毒性均为0级;结论:(1)Ta涂层假体对生长增殖中的细胞没有明显的毒性及抑制作用;(2)Ta涂层具有相对稳定的细胞相容性,与临床应用的Ti、HA涂层无明显差别。
2. Ta涂层假体植入后早期假体周围成骨量的对照研究
目的:通过对Ta、Ti涂层假体植入后周围成骨量的对比,对Ta涂层骨组织相容性进行评价;方法:(1)采用等离子喷涂工艺加工制备Ta、Ti涂层植入假体;(2)利用手术植入方法将不同涂层假体植入新西兰白兔的股骨髁内,术后分期通过假体表面肉眼观察、组织学评价、假体的表面SEM观察及EDX分析对假体周围骨长入情况进行观察分析;结果:(1)组织病理学观察发现假体植入后4 W、9W及15W时,Ta、Ti涂层与新生骨组织结合紧密而稳定,涂层无脱落;(2)与假体植入4W时组织病理学观察结果一致,假体植入4 W,SEM观察显示Ta、Ti涂层表面及周围均有明显的骨样组织形成,骨样组织结构清晰、组织分布均匀;(3)各植入期Ta、Ti涂层假体周围骨体积分数对比无明显差异(P﹥0.05);(4)假体植入4W假体表面EDX分析结果显示,Ta、Ti涂层假体表面新生组织的主要元素构成中含有、Ca、P等骨组织元素;结论:(1)Ta涂层假体具有较好的骨组织相容性及周围成骨效果,涂层-骨组织界面结合稳定,无涂层脱落或涂层与基体分层现象;(2)在假体植入早期,Ta涂层假体周围骨形成量与目前临床应用的Ti金属涂层假体无明显差别。
3. Ta涂层假体植入后早期稳定性的对照研究
目的:通过对Ta、Ti涂层假体及非涂层假体植入后假体周围成骨量及假体生物力学指标的对比分析,对Ta涂层假体植入后早期稳定性进行评价;方法:(1)采用等离子喷涂工艺加工制备Ta、Ti涂层植入假体;(2)采用手术植入方法将Ta、Ti涂层假体植入实验兔的股骨髁内,利用推出实验(pull-out testing)及组织病理学观察方法对术后各期假体轴向极限抗剪切强度及假体周围骨形成量进行对比;(3)假体拔出后周围组织HE染色及假体表面EDX分析对Ta涂层-基体界面抗剪切能力及假体植入后的稳定性进行分析;结果:(1)早期随着植入时间的延长,Ta、Ti涂层假体周围成骨量也在不断增加;术后4 W、9W及15W,Ta、Ti涂层假体周围骨体积分数对比无明显差异(P﹥0.05);(2)术后4、9及15W,Ta、Ti涂层假体间最大抗剪应力无显著差异(P﹥0.05);Ta涂层假体植入4W与16W时假体的最大抗剪应力之间有显著差异(P﹤0.05),同种材料植入8W时的最大抗剪应力与4W、16W时还不具有显著差异(P﹥0.05);(3)假体拔出后组织HE染色观察结果显示,非涂层假体周围为环状纤维组织,假体-组织界面平整,无异物存留;Ta、Ti涂层假体周围可见骨组织形成,假体拔出造成涂层-骨组织界面断裂、分离,但假体周围骨组织表面未见涂层存留物;(4)EDX分析结果显示,假体拔出后Ta、Ti涂层假体表面元素构成未发生改变;结论:(1)对复合涂层人工假体而言,假体周围成骨量并非假体植入后稳定性的唯一决定因素,假体表面孔隙率及涂层界面稳定也是影响假体植入后的稳定性重要因素;(2)与其它临床上使用的金属涂层假体相比,Ta涂层假体同样具有良好的周围组织相容性;(3)Ta涂层与基体具有良好的涂层稳定性,能够在较高剪切应力作用下维持假体涂层的完整性,这对于假体植入后的稳定性具有积极意义。
The ideal joint replacement prosthesis which coated with coating materials should possess not only high mechanical strength of whole prosthesis, but also porous structure at certain size on the surface of prosthesis, to facilitate the bone tissue adhering to and growing into the micropores on prosthetic surface to achieve the postoperative prosthetic stability. With deepening of the study on adhesion between bone and prosthetic surface, the treatment measures of prosthetic surface have been increasingly diversified.
In the recent years, bioceramic and metallic coating has become the major choices for the surface treatment of prosthesis. Hydroxyapatite (HA, Ca10[PO4]6[OH]2) and titanium (Ti) coatings have represented the porous biological ceramic coating and the metal coating respectively. Most of the prostheses are made by coating HA or Ti onto the surface of stainless steel or Ti alloy substrates through plasma spraying or thermal sintering process. Such coatings provide a porous structure on prosthetic surface with the functions of bone induction and conduction to improve the biological connection between prosthesis and human tissues, overcomes the drawbacks of former prosthesis in comprehensive performance such as mechanical strength, biocompatibility, etc. and becomes the ideal material for bone reconstruction in recent years[1‐5].
However, with the increase in joint revision surgeries post arthroplasty, defects of HA- and Ti coated prosthesis have been gradually observed. It has been reported that the decohesion and degradation of HA coating and the migration of HA particles eventually lead to the prosthesis loosening. In addition, defects of HA-coated prosthesis in mechanical strength cause coating decohesion and fatigue fracture during the surgical procedure and under postoperative loading, to a certain extent, exacerbating the gap formation at prosthesis-coating and bone-prosthesis interfaces and facilitating the canal formation for HA particle migration. Following the gradual canal formation and the migration of degraded HA particles, prosthetic stability is severely affected.
Although Ti coated prosthesis has some advantages in surface mechanical strength, corrosion resistance and stability of prosthesis coating, but there is still a gap between Ti and HA coated prosthesis in bone induction and bone ingrowth at early stage of implantation. Long-term clinical observation also found that there are prosthesis loosening caused by coating decohesion and shedding in some cases after prosthetic replacement which applied Ti coated prosthesis.
With the diversifying in the methodology of processing prosthetic surface and the deepening in understanding the adhesion between the bone and the prosthetic surface, finding and researching coating materials that have good biocompatibility and can ensure the long-term postoperative prosthetic stability remain one of the main directions in the field of articular prosthesis study.
Since the middle of last century, Tantalum(Ta) biomaterial has been used in medical applications. The advantages of Ta as coating material become evident gradually after the application of porous Ta sheath components on the prosthetic surface. The good biocompatibility and superior chemical stability of Ta material in comparison to Ti and other metals provide a theoretical basis for its possible application as prosthetic coating. But as a a novel prosthetic coating, whether Ta materials possess good biocompatibility and other necessary physical properties such as becomes our focus of the present study. Since biocompatibility and stability are the keys determining whether a new prosthetic coating can be used in clinic, so this study has preliminarily investigated the physical characteristics, biocompatibility, and biomechanical behavior of Ta as coating material on the prosthetic surface, aiming to provide experimental basis for its future clinical application.
In this study we investigated the general physical characteristics, biocompatibility, and the relationship between biological behavior and physical characteristics of Ta coating prosthesis through three research parts. The first part is a research related to the general physical characteristics of Ta coating. The second part is a research about the biological behavior of Ta coating prosthesis. The third part is a research on the relationship between biological behavior and physical characteristics of Ta coating prosthesis.
Ⅰ.The general characteristics of Ta coated material.
1. The general physical characteristics of Ta coating interface.
Objective: Investigat the general physical characteristics of Ta coating interface, such as the adhesive strength of coating , mechanical strength of coating surface, the chemical composition of coating and provid an experimental data for clinical application of the Ta coating material. Methods:(1)The Ta-coated testing specimens (with 25~35% of two-dimensional [2D] porosity and a coating thickness of 150μm) was prepared by sand blasting and plasma spraying process, The morphous of coating surface, the metallographical structure and the coating’s chemical composition were investigated by the metallurgical microscope, the scanning electron microscope(SEM) observation, the energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction analysis (XRD). (2) Medical Ti-6Al-4V substrate was used to make Ta-coated interface mechanical testing specimens (with 25~35% of 2D porosity and a coating thickness of 150μm) by sand blasting and plasma spraying process. The same substrates and specifications were applied to the control with Ti and HA coating .biomechanical testing. The coating adhesive strength and the surface hardness of coating were tested using WS-2005 coating adhesion automatic scratch tester and HV-120 Vickers hardness tester. Results: (1) SEM observations show that there are obvious roughness and clear pore structure on Ta coating surface. The coating has a higher density and bonds closely to substrate. No internal delamination and cracks have found in Ta coating. (2) The Vickers surface hardness (HV) of Ta coating (423.57±7.41) is significantly higher (P < 0.01) than that of Ti coating (202.86±4.35) and HA coating (123.29±3.99); The coating adhesive strength for Ta coating to peel is statistically higher than that for Ti coating and HA coating (P < 0.01). Conclusions: There is a stable pore structure on the surface of Ta coating material. The interface of coating and substrate is bond closely. Ta coating has a higher coating-substrate adhesive strength and surface mechanical strength, hereby is resistant to the damages resulted from the repeated loading changes and the relatively high shear stress, which has significance for maintaining the prosthetic surface porosity and characteristics of biomechanics.
2.A study of the tribological properties of Ta coated biomaterials
Objective: To evaluate the tribological properties and the biotribological properties of Ta coated biomaterials for its future clinical application.
Methods(:1)medical 316 stainless steel (0Cr17Ni12Mo2) materials were used as the base material for both the experimental group(group A)and the control group. The base material was pretreated with sand blasting, and Tantalum coating was prepared by plasma spraying system (Sulzer & Metco, USA). The system forms Ta coating with 25-35% 2D porosity and a coating thickness of 400μm. For the control groups (Groups B and C), base materials of stainless steel were coated with HA and Ti, respectively with the same specifications as in Group Ta). 0Cr17Ni12Mo2 rods were used as grinding materials, The test was done with a MMW-2 type computer-controlled abrasive wear test machine. The Wear morphous of coating surface and the amount of wear were observed and analyzed. In the meantime, the metallographical structure and the coating’s chemical composition were investigated by the metallurgical microscope observation and the energy-dispersive X-ray spectroscopy (EDX).(2)Ta-, HA- and Ti-coated test pieces with 316 stainless steel as substrates were used as the experimental group (group A)and the control group(Groups B and C) respectively. All coating with 25-30% 2D porosity and a coating thickness of 400μm. The pig femur were used as grinding materials, processed in accordance with standard wear test specimen preparation. The test was done with a MMW-2 type computer-controlled abrasive wear test machine. The Wear morphous of coating surface, the wear amount of test peices, the coating-bone friction coefficient, and the impact of bone-coating friction under load were observed and analyzed. In the meantime, the metallographical structure and the chemical composition on surface of test pieces were investigated by the metallurgical microscope observation and the energy-dispersive X-ray spectroscopy (EDX) respectively. Results: (1) The failure mechanism of Ta coating was mainly fatigue spalling. Compared with HA and Ti coatings, Ta surface coating suffered the least weight loss, and its surface wear resistance was higher than that of Ti coating and HA coating (P <0.01). (2) There was no significant reduction in the mass of the metal test pieces in metal-bone wear experiment. With time of testing, the friction coefficients gradually decreased, and finally settled around 0.40, and bone debris gradually deposited and accumulated in the porous surface of the coatings, forming a smooth film of bone debris which prevent further damage on bone grinding materials. Conclusions: (1) The failure mechanism of Ta coating was mainly fatigue spalling. Ta coating has good abrasion wear resistance and fatigue resistance, and has a superior mechanical characteristics than HA and Ti coatings for biological materials (prosthesis). (2) The bone-coating friction does not damage Ta coating on the prosthesis due to the formation and maintenance of a solid lubrication interface.
Ⅱ.The biological characteristics of Ta coated material.
1. A study of the cytotoxicity and biocompatibility of Ta coating prosthesis.
Objective: To preliminary assess the cytotoxicity and biocompatibility of Ta coated biomaterials for its future clinical application. Methods:(1)Ta-, HA- and Ti-coated test pieces with Medical Ti alloy(Ti-6Al-4V)material as substrates were prepared for experiment of the cytotoxicity, and the leaching liquor of these test pieces were prepared for cell culture respectively(.2)The L929 cell were cultured and subcultured for the further cytotoxicity experiment.(3)The L929 cell were subcultured using the different leaching liquor of test pieces as culture solution, and the cytotoxicity grade of test pieces were evaluated through the MTT colorimetric test for cell culture respectively. Results: There are no significantly difference in the Relevant Growth Rate (RGR) and cytotoxicity among the Ta coating, HA coating and Ti coating (P >0.05), the cytotoxicity grade of Ta-coated test piece was 0 grade. Conclusion: Ta coating material has a good biocompatibility and toxicological characteristics for the human body.
2. A study of periprosthetic osteogenesis on Ta coated prostheses.
Objective: To observate the periprosthetic osteogenesis of Ta coated prostheses in vivo at early stage after its implantation, so as to provide evidences for the application of Ta coated biomaterials. Methods:(1)Ta coated prostheses were prepared for experiment of implantation. In the meantime, Ti coated prostheses was prepared as control group (2) Prostheses were implanted the femurs epicondyle of the New Zealand white rabbits, and histopathological observation, histomorphometric analysis, SEM observation, EDX analysis were employed to evaluate the periprosthetic osteogenesis of prostheses. Results: (1) observation of histopathology show that At Early Stage (4W) after the implantation, there are significant bone formation around the the Ta and Ti coated implants, and new bone tissues bond closely and stably to these all coatings. With prolonged time after the implantation, thickness of new bone tissues and number of trabecular bones around the two coatings increase continuously. 9W and 15W after the implantation, new bone tissues bond closely and stably to Ta- and Ti-coated implants. (2) Histomorphometric analysis of the bone tissue show that Ta- and Ti-coated implants has an obvious trends in periprosthetic osteogenesis At Early Stage after the implantation, though there are no significantly difference in periprosthetic osteogenesis among Ta- and Ti-coated implants(P﹥0.05). (3) SEM observation of the new tissues on prosthetic surface shows the attachment of a thin layer of new bony tissues to the surface of Ta- and Ti-coated prosthesis with regular, uniformed, and clear trabecular structure. (4) EDX analysis reveals that major component elements of the new tissues on surfaces of the two coatings are C, O, Ca, and P. Conclusion: Just like other metal-coated prostheses which have used in the recent clinic orthopedics, Ta coating has a good comprehensive performance on the stability and periprosthetic osteogenesis after its implantation.
Ⅲ.The stability of Ta-coated prosthesis in vivo at early stage of implantation.
Objective: To evaluate the stability Ta-coated prosthesis in vivo at early stage of implantation and provide evidences for the application of Ta coated prosthesis. Methods:(1)Ta coated prostheses were prepared as experimental group, Ti coated prostheses as a control group.(2)Prostheses were implanted the femurs epicondyle of the New Zealand white rabbits, and biomechanical testing (pull-out testing), histopathological observation, histomorphometric analysis, EDX analysis were employed to evaluate the ultimate shear strength of prostheses and the amount of osteogenesis around prostheses, both which effect the stability of HA-coated prosthesis in vivo. Results: (1) At Early Stage after the implantation, there are no significantly difference in periprosthetic osteogenesis among Ta- and Ti-coated implants(P >0.05). (2) There are no significantly difference in the ultimate shear strength between Ta- and Ti-coated implants (P >0.05). In the meanwhile, the ultimate shear strength of all kinds of coated prosthesis at 16 weeks is significant higher than that at 4 weeks after implantation. (3)Histological observation and EDX analysis after pull-out testing show that the periprosthetic bone tissues are evident on the surface of prostheses coating at early stages after implantation. Push-out test fractures the prosthesis and bone interface around Ta- and Ti-coated implants, and no coating residues can be observed on fractured bone section. (4) According to EDX analysis of the prosthetic surface elements before and after push-out test. It was found that the ultimate shear stress is not able to cause Ta and Ti coating damaged. Conclusion:(1) The periprosthetic bone ingrowth and the bone formation is not only factor that ensure stability of coating prostheses after its implantation. The adhesive strength and porosity of coating are also important factors for the stability of coating prosthesis in vivo. (2)Ta-coated prosthesis exhibits good biocompatibility with the bone tissue which is comparable with the current clinically metal-coated prosthesis. (3)Ta coating has a higher coating-substrate adhesive strength and hereby is resistant to the damages resulted from the repeated loading changes and the relatively high shear stress, which promotes the postoperative prosthetic stability and prolongs the life of prosthesis in vivo.
目前,生物陶瓷涂层材料及微孔金属涂层生物材料是假体表面处理的主要方向,分别以羟基磷灰石(Hydroxyapatite, HA,Ca10(PO4)6(OH)2)及钛(Titanium ,Ti)涂层为代表。它们是通过烧结或等离子喷涂的工艺将HA及Ti金属被覆在不锈钢或Ti金属基体表面,形成具有一定骨传导或骨诱导功能的生物涂层复合材料,克服了以往人体植入材料在机械强度、生物相容性、耐腐蚀等综合性能方面存在的不足,成为近年来比较理想的骨重建材料。但已有文献报道,由于HA属于人体可降解和吸收的材料,其涂层降解后会使材料基体与组织间产生间隙,逐渐导致假体松动。对于Ti涂层复合生物材料而言,虽然在表面机械强度、耐腐蚀以及假体晚期稳定性方面优于HA涂层生物材料,但在假体植入早期,Ti涂层与HA涂层材料在骨诱导功能方面还存在一定差距,临床长期的观察也发现Ti涂层关节假体同样存在涂层脱落而导致假体松动的现象,因此,寻找和研究既有良好的生物相容性、又能保证术后假体长期稳定的涂层材料仍是今后一段时间关节假体研究领域的主要方向之一。
自从1997年起,随着多孔钽材料作为小梁金属在临床矫形外科的广泛应用,各种钽(Tantalum,Ta)制生物材料的优势也逐渐展现出来,其良好的生物相容性、独特的物理及机械学特性为钽金属生物材料发展提供了广阔空间。
随着假体表面研究的进展以及多孔钽“外衬”部件在人工假体表面应用存在的局限性,使得研究者开始对钽金属是否可以作为假体涂层材料进行了新的思考。但作为一种新型生物材料涂层,Ta涂层是否具备生物材料涂层应有的材料学特性及良好的生物相容性?Ta涂层-基体结合强度、涂层的耐磨性是否能满足其作为人工假体涂层的要求?Ta涂层假体植入后的骨长入量是否会对假体后期的稳定性起到维持作用?这些问题便成为研究者关注的焦点。
本文通过三个部分对Ta涂层一般特性及生物学特性进行了进一步研究:第一部分为Ta涂层一般特性的研究;第二部分为Ta涂层生物学特性的研究;第三部分为Ta涂层人工假体生物力学行为及其涂层物理特性关系的初步研究。
一、Ta涂层一般特性的研究
1. Ta涂层界面物理特性的研究
目的:对Ta涂层界面结合强度、表面机械强度、涂层元素构成进行初步的研究和分析,为钽金属涂层材料的应用提供试验依据。方法:(1)采用大气等离子喷涂系统制备钽涂层试件,获得二维孔隙率为25~35%、涂层厚度为150μm的Ta涂层试件,通过扫描电子显微镜(SEM)观察、金相显微镜观察、能谱扫描(EDX)及X-射线衍射分析(XRD)等手段对Ta涂层的表面形貌、金相结构和化学构成进行分析;(2)界面力学检测:选用医用Ti-6Al-4V基体材料,经过喷砂及等离子喷涂工艺加工获得二维孔隙率为25~35%、涂层厚度为150μm的Ta涂层试件作为实验组,相同基体和规格的HA及Ti涂层的试件作为对照组;运用WS-2005涂层附着力自动划痕仪、HV-120维氏硬度仪分别对涂层与基体结合强度及涂层表面硬度进行评价。结果:(1)SEM观察发现,Ta涂层表面粗糙、孔隙形貌清晰;钽涂层组织致密,界面结合紧密,未发现内部分层和裂纹;(2)Ta涂层料表面硬度及涂层界面结合强度明显高于HA涂层和Ti涂层(P﹤0.01)。结论:Ta涂层界面结合紧密,表面孔隙结构稳定,与Ti涂层和HA涂层相比具有更高的结合强度和表面硬度,这对预防涂层脱落、维持假体表面结构及维持假体生物力学特性具有积极作用。
2. Ta涂层假体表面磨损机制的研究
目的:对Ta涂层假体表面的疲劳磨损机制及其生物磨损机制进行初步的研究和分析,为钽金属涂层假体的临床应用提供试验依据。方法:(1)实验组(A组)基体为医用316不锈钢(0Cr17Ni12Mo2),涂层厚度为400μm的Ta涂层试件,表面孔隙率为25-35%;对照组(B、C组)分别为316不锈钢基体HA与Ti涂层试件,试件标准与A组一致。采用MMW-2型微机控制摩擦磨损试验机及0Cr17Ni12Mo2对磨件进行涂层试件的摩擦磨损实验,对在一定载荷下的试件磨损外貌特征、磨损量进行观察、对比,采用SEM及XRD等方法对磨损实验前后涂层表面形貌、金相结构和化学构成进行分析对比;(2)采用以0Cr17Ni12Mo2为基体的Ta、HA及Ti涂层试件作为实验组(A组)及对照组(B、C组)进行试件-骨磨损试验,三种涂层厚度均为400μm、表面孔隙率为25-35%;对磨件为猪骨股骨近端骨骼制成;利用MMW-2型微机控制摩擦磨损试验机对一定载荷下的试件经过骨磨损后外貌特征、骨磨损量及试验中各组摩擦副间摩擦系数的变化进行分析,采用SEM及XRD等方法对磨损试验前后涂层表面形貌、金相结构和化学构成进行对比分析。结果:(1)各组涂层的滑动摩擦失效行为主要表现为疲劳剥落,316不锈钢基体Ta涂层表面耐磨性能明显高于HA及Ti涂层(P﹤0.01);(2)与金属对磨的磨损实验不同,在采用骨对磨件的磨损实验中三种涂层磨损试件的质量并没有明显的减少(P﹥0.05);随着磨损时间的延长,三种涂层试件对骨组织的摩擦系数均逐渐减少,最终摩擦系数均维持在0.40左右;同时,骨磨屑堆积在试样涂层表面孔隙里形成了一层平滑的固体润滑界面,在摩擦副间起到阻碍磨损的作用;结论:(1)Ta涂层磨损失效行为主要为疲劳剥落,其具有明显优于Ti涂层和HA涂层的耐磨损、抗疲劳作用,这对预防涂层脱落、维持假体表面结构及维持假体生物力学特性具有积极作用;(2)由于固体润滑界面形成及存在,使得骨-涂层摩擦不易对假体的Ta涂层造成破坏。
二、Ta涂层生物学特性的研究
1. Ta、HA、及Ti涂层假体细胞毒性对照研究
目的:通过对比研究评价Ta涂层假体的细胞毒性作用,初步了解和评价Ta涂层材料生物相容性,为Ta涂层假体临床应用的可行性提供实验依据。方法:通过对细胞相对增殖率(RGR)检测及细胞毒性测定对Ta涂层材料的生物相容性进行评价;方法:(1)Ta、Ha、Ti涂层材料及相应浸提液的制备;(2)L929细胞系的培养及传代;(3)利用Ta、Ha、Ti涂层材料浸提液对L929传代细胞进行培养,观察不同浸提液培养的细胞生长形态,并通过MTT比色法对各组涂层材料的细胞毒性进行分级;结果:(1)细胞形态学观察显示:Ta涂层组的细胞生长形态与HA和Ti涂层组一致,细胞生长形态良好,与空白阴性对照组无明显差别,而含有0.64%苯酚浸提液作为培养液培养的空白阳性对照组细胞,细胞广泛死亡;(2)MTT比色实验显示:Ta、Ha及Ti涂层组的细胞相对增殖率(RGR)无明显差别﹙P﹥0.05﹚,Ta涂层与其它涂层对照组及空白阴性对照组的细胞毒性没有显著差异,三种涂层的细胞毒性均为0级;结论:(1)Ta涂层假体对生长增殖中的细胞没有明显的毒性及抑制作用;(2)Ta涂层具有相对稳定的细胞相容性,与临床应用的Ti、HA涂层无明显差别。
2. Ta涂层假体植入后早期假体周围成骨量的对照研究
目的:通过对Ta、Ti涂层假体植入后周围成骨量的对比,对Ta涂层骨组织相容性进行评价;方法:(1)采用等离子喷涂工艺加工制备Ta、Ti涂层植入假体;(2)利用手术植入方法将不同涂层假体植入新西兰白兔的股骨髁内,术后分期通过假体表面肉眼观察、组织学评价、假体的表面SEM观察及EDX分析对假体周围骨长入情况进行观察分析;结果:(1)组织病理学观察发现假体植入后4 W、9W及15W时,Ta、Ti涂层与新生骨组织结合紧密而稳定,涂层无脱落;(2)与假体植入4W时组织病理学观察结果一致,假体植入4 W,SEM观察显示Ta、Ti涂层表面及周围均有明显的骨样组织形成,骨样组织结构清晰、组织分布均匀;(3)各植入期Ta、Ti涂层假体周围骨体积分数对比无明显差异(P﹥0.05);(4)假体植入4W假体表面EDX分析结果显示,Ta、Ti涂层假体表面新生组织的主要元素构成中含有、Ca、P等骨组织元素;结论:(1)Ta涂层假体具有较好的骨组织相容性及周围成骨效果,涂层-骨组织界面结合稳定,无涂层脱落或涂层与基体分层现象;(2)在假体植入早期,Ta涂层假体周围骨形成量与目前临床应用的Ti金属涂层假体无明显差别。
3. Ta涂层假体植入后早期稳定性的对照研究
目的:通过对Ta、Ti涂层假体及非涂层假体植入后假体周围成骨量及假体生物力学指标的对比分析,对Ta涂层假体植入后早期稳定性进行评价;方法:(1)采用等离子喷涂工艺加工制备Ta、Ti涂层植入假体;(2)采用手术植入方法将Ta、Ti涂层假体植入实验兔的股骨髁内,利用推出实验(pull-out testing)及组织病理学观察方法对术后各期假体轴向极限抗剪切强度及假体周围骨形成量进行对比;(3)假体拔出后周围组织HE染色及假体表面EDX分析对Ta涂层-基体界面抗剪切能力及假体植入后的稳定性进行分析;结果:(1)早期随着植入时间的延长,Ta、Ti涂层假体周围成骨量也在不断增加;术后4 W、9W及15W,Ta、Ti涂层假体周围骨体积分数对比无明显差异(P﹥0.05);(2)术后4、9及15W,Ta、Ti涂层假体间最大抗剪应力无显著差异(P﹥0.05);Ta涂层假体植入4W与16W时假体的最大抗剪应力之间有显著差异(P﹤0.05),同种材料植入8W时的最大抗剪应力与4W、16W时还不具有显著差异(P﹥0.05);(3)假体拔出后组织HE染色观察结果显示,非涂层假体周围为环状纤维组织,假体-组织界面平整,无异物存留;Ta、Ti涂层假体周围可见骨组织形成,假体拔出造成涂层-骨组织界面断裂、分离,但假体周围骨组织表面未见涂层存留物;(4)EDX分析结果显示,假体拔出后Ta、Ti涂层假体表面元素构成未发生改变;结论:(1)对复合涂层人工假体而言,假体周围成骨量并非假体植入后稳定性的唯一决定因素,假体表面孔隙率及涂层界面稳定也是影响假体植入后的稳定性重要因素;(2)与其它临床上使用的金属涂层假体相比,Ta涂层假体同样具有良好的周围组织相容性;(3)Ta涂层与基体具有良好的涂层稳定性,能够在较高剪切应力作用下维持假体涂层的完整性,这对于假体植入后的稳定性具有积极意义。
The ideal joint replacement prosthesis which coated with coating materials should possess not only high mechanical strength of whole prosthesis, but also porous structure at certain size on the surface of prosthesis, to facilitate the bone tissue adhering to and growing into the micropores on prosthetic surface to achieve the postoperative prosthetic stability. With deepening of the study on adhesion between bone and prosthetic surface, the treatment measures of prosthetic surface have been increasingly diversified.
In the recent years, bioceramic and metallic coating has become the major choices for the surface treatment of prosthesis. Hydroxyapatite (HA, Ca10[PO4]6[OH]2) and titanium (Ti) coatings have represented the porous biological ceramic coating and the metal coating respectively. Most of the prostheses are made by coating HA or Ti onto the surface of stainless steel or Ti alloy substrates through plasma spraying or thermal sintering process. Such coatings provide a porous structure on prosthetic surface with the functions of bone induction and conduction to improve the biological connection between prosthesis and human tissues, overcomes the drawbacks of former prosthesis in comprehensive performance such as mechanical strength, biocompatibility, etc. and becomes the ideal material for bone reconstruction in recent years[1‐5].
However, with the increase in joint revision surgeries post arthroplasty, defects of HA- and Ti coated prosthesis have been gradually observed. It has been reported that the decohesion and degradation of HA coating and the migration of HA particles eventually lead to the prosthesis loosening. In addition, defects of HA-coated prosthesis in mechanical strength cause coating decohesion and fatigue fracture during the surgical procedure and under postoperative loading, to a certain extent, exacerbating the gap formation at prosthesis-coating and bone-prosthesis interfaces and facilitating the canal formation for HA particle migration. Following the gradual canal formation and the migration of degraded HA particles, prosthetic stability is severely affected.
Although Ti coated prosthesis has some advantages in surface mechanical strength, corrosion resistance and stability of prosthesis coating, but there is still a gap between Ti and HA coated prosthesis in bone induction and bone ingrowth at early stage of implantation. Long-term clinical observation also found that there are prosthesis loosening caused by coating decohesion and shedding in some cases after prosthetic replacement which applied Ti coated prosthesis.
With the diversifying in the methodology of processing prosthetic surface and the deepening in understanding the adhesion between the bone and the prosthetic surface, finding and researching coating materials that have good biocompatibility and can ensure the long-term postoperative prosthetic stability remain one of the main directions in the field of articular prosthesis study.
Since the middle of last century, Tantalum(Ta) biomaterial has been used in medical applications. The advantages of Ta as coating material become evident gradually after the application of porous Ta sheath components on the prosthetic surface. The good biocompatibility and superior chemical stability of Ta material in comparison to Ti and other metals provide a theoretical basis for its possible application as prosthetic coating. But as a a novel prosthetic coating, whether Ta materials possess good biocompatibility and other necessary physical properties such as becomes our focus of the present study. Since biocompatibility and stability are the keys determining whether a new prosthetic coating can be used in clinic, so this study has preliminarily investigated the physical characteristics, biocompatibility, and biomechanical behavior of Ta as coating material on the prosthetic surface, aiming to provide experimental basis for its future clinical application.
In this study we investigated the general physical characteristics, biocompatibility, and the relationship between biological behavior and physical characteristics of Ta coating prosthesis through three research parts. The first part is a research related to the general physical characteristics of Ta coating. The second part is a research about the biological behavior of Ta coating prosthesis. The third part is a research on the relationship between biological behavior and physical characteristics of Ta coating prosthesis.
Ⅰ.The general characteristics of Ta coated material.
1. The general physical characteristics of Ta coating interface.
Objective: Investigat the general physical characteristics of Ta coating interface, such as the adhesive strength of coating , mechanical strength of coating surface, the chemical composition of coating and provid an experimental data for clinical application of the Ta coating material. Methods:(1)The Ta-coated testing specimens (with 25~35% of two-dimensional [2D] porosity and a coating thickness of 150μm) was prepared by sand blasting and plasma spraying process, The morphous of coating surface, the metallographical structure and the coating’s chemical composition were investigated by the metallurgical microscope, the scanning electron microscope(SEM) observation, the energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction analysis (XRD). (2) Medical Ti-6Al-4V substrate was used to make Ta-coated interface mechanical testing specimens (with 25~35% of 2D porosity and a coating thickness of 150μm) by sand blasting and plasma spraying process. The same substrates and specifications were applied to the control with Ti and HA coating .biomechanical testing. The coating adhesive strength and the surface hardness of coating were tested using WS-2005 coating adhesion automatic scratch tester and HV-120 Vickers hardness tester. Results: (1) SEM observations show that there are obvious roughness and clear pore structure on Ta coating surface. The coating has a higher density and bonds closely to substrate. No internal delamination and cracks have found in Ta coating. (2) The Vickers surface hardness (HV) of Ta coating (423.57±7.41) is significantly higher (P < 0.01) than that of Ti coating (202.86±4.35) and HA coating (123.29±3.99); The coating adhesive strength for Ta coating to peel is statistically higher than that for Ti coating and HA coating (P < 0.01). Conclusions: There is a stable pore structure on the surface of Ta coating material. The interface of coating and substrate is bond closely. Ta coating has a higher coating-substrate adhesive strength and surface mechanical strength, hereby is resistant to the damages resulted from the repeated loading changes and the relatively high shear stress, which has significance for maintaining the prosthetic surface porosity and characteristics of biomechanics.
2.A study of the tribological properties of Ta coated biomaterials
Objective: To evaluate the tribological properties and the biotribological properties of Ta coated biomaterials for its future clinical application.
Methods(:1)medical 316 stainless steel (0Cr17Ni12Mo2) materials were used as the base material for both the experimental group(group A)and the control group. The base material was pretreated with sand blasting, and Tantalum coating was prepared by plasma spraying system (Sulzer & Metco, USA). The system forms Ta coating with 25-35% 2D porosity and a coating thickness of 400μm. For the control groups (Groups B and C), base materials of stainless steel were coated with HA and Ti, respectively with the same specifications as in Group Ta). 0Cr17Ni12Mo2 rods were used as grinding materials, The test was done with a MMW-2 type computer-controlled abrasive wear test machine. The Wear morphous of coating surface and the amount of wear were observed and analyzed. In the meantime, the metallographical structure and the coating’s chemical composition were investigated by the metallurgical microscope observation and the energy-dispersive X-ray spectroscopy (EDX).(2)Ta-, HA- and Ti-coated test pieces with 316 stainless steel as substrates were used as the experimental group (group A)and the control group(Groups B and C) respectively. All coating with 25-30% 2D porosity and a coating thickness of 400μm. The pig femur were used as grinding materials, processed in accordance with standard wear test specimen preparation. The test was done with a MMW-2 type computer-controlled abrasive wear test machine. The Wear morphous of coating surface, the wear amount of test peices, the coating-bone friction coefficient, and the impact of bone-coating friction under load were observed and analyzed. In the meantime, the metallographical structure and the chemical composition on surface of test pieces were investigated by the metallurgical microscope observation and the energy-dispersive X-ray spectroscopy (EDX) respectively. Results: (1) The failure mechanism of Ta coating was mainly fatigue spalling. Compared with HA and Ti coatings, Ta surface coating suffered the least weight loss, and its surface wear resistance was higher than that of Ti coating and HA coating (P <0.01). (2) There was no significant reduction in the mass of the metal test pieces in metal-bone wear experiment. With time of testing, the friction coefficients gradually decreased, and finally settled around 0.40, and bone debris gradually deposited and accumulated in the porous surface of the coatings, forming a smooth film of bone debris which prevent further damage on bone grinding materials. Conclusions: (1) The failure mechanism of Ta coating was mainly fatigue spalling. Ta coating has good abrasion wear resistance and fatigue resistance, and has a superior mechanical characteristics than HA and Ti coatings for biological materials (prosthesis). (2) The bone-coating friction does not damage Ta coating on the prosthesis due to the formation and maintenance of a solid lubrication interface.
Ⅱ.The biological characteristics of Ta coated material.
1. A study of the cytotoxicity and biocompatibility of Ta coating prosthesis.
Objective: To preliminary assess the cytotoxicity and biocompatibility of Ta coated biomaterials for its future clinical application. Methods:(1)Ta-, HA- and Ti-coated test pieces with Medical Ti alloy(Ti-6Al-4V)material as substrates were prepared for experiment of the cytotoxicity, and the leaching liquor of these test pieces were prepared for cell culture respectively(.2)The L929 cell were cultured and subcultured for the further cytotoxicity experiment.(3)The L929 cell were subcultured using the different leaching liquor of test pieces as culture solution, and the cytotoxicity grade of test pieces were evaluated through the MTT colorimetric test for cell culture respectively. Results: There are no significantly difference in the Relevant Growth Rate (RGR) and cytotoxicity among the Ta coating, HA coating and Ti coating (P >0.05), the cytotoxicity grade of Ta-coated test piece was 0 grade. Conclusion: Ta coating material has a good biocompatibility and toxicological characteristics for the human body.
2. A study of periprosthetic osteogenesis on Ta coated prostheses.
Objective: To observate the periprosthetic osteogenesis of Ta coated prostheses in vivo at early stage after its implantation, so as to provide evidences for the application of Ta coated biomaterials. Methods:(1)Ta coated prostheses were prepared for experiment of implantation. In the meantime, Ti coated prostheses was prepared as control group (2) Prostheses were implanted the femurs epicondyle of the New Zealand white rabbits, and histopathological observation, histomorphometric analysis, SEM observation, EDX analysis were employed to evaluate the periprosthetic osteogenesis of prostheses. Results: (1) observation of histopathology show that At Early Stage (4W) after the implantation, there are significant bone formation around the the Ta and Ti coated implants, and new bone tissues bond closely and stably to these all coatings. With prolonged time after the implantation, thickness of new bone tissues and number of trabecular bones around the two coatings increase continuously. 9W and 15W after the implantation, new bone tissues bond closely and stably to Ta- and Ti-coated implants. (2) Histomorphometric analysis of the bone tissue show that Ta- and Ti-coated implants has an obvious trends in periprosthetic osteogenesis At Early Stage after the implantation, though there are no significantly difference in periprosthetic osteogenesis among Ta- and Ti-coated implants(P﹥0.05). (3) SEM observation of the new tissues on prosthetic surface shows the attachment of a thin layer of new bony tissues to the surface of Ta- and Ti-coated prosthesis with regular, uniformed, and clear trabecular structure. (4) EDX analysis reveals that major component elements of the new tissues on surfaces of the two coatings are C, O, Ca, and P. Conclusion: Just like other metal-coated prostheses which have used in the recent clinic orthopedics, Ta coating has a good comprehensive performance on the stability and periprosthetic osteogenesis after its implantation.
Ⅲ.The stability of Ta-coated prosthesis in vivo at early stage of implantation.
Objective: To evaluate the stability Ta-coated prosthesis in vivo at early stage of implantation and provide evidences for the application of Ta coated prosthesis. Methods:(1)Ta coated prostheses were prepared as experimental group, Ti coated prostheses as a control group.(2)Prostheses were implanted the femurs epicondyle of the New Zealand white rabbits, and biomechanical testing (pull-out testing), histopathological observation, histomorphometric analysis, EDX analysis were employed to evaluate the ultimate shear strength of prostheses and the amount of osteogenesis around prostheses, both which effect the stability of HA-coated prosthesis in vivo. Results: (1) At Early Stage after the implantation, there are no significantly difference in periprosthetic osteogenesis among Ta- and Ti-coated implants(P >0.05). (2) There are no significantly difference in the ultimate shear strength between Ta- and Ti-coated implants (P >0.05). In the meanwhile, the ultimate shear strength of all kinds of coated prosthesis at 16 weeks is significant higher than that at 4 weeks after implantation. (3)Histological observation and EDX analysis after pull-out testing show that the periprosthetic bone tissues are evident on the surface of prostheses coating at early stages after implantation. Push-out test fractures the prosthesis and bone interface around Ta- and Ti-coated implants, and no coating residues can be observed on fractured bone section. (4) According to EDX analysis of the prosthetic surface elements before and after push-out test. It was found that the ultimate shear stress is not able to cause Ta and Ti coating damaged. Conclusion:(1) The periprosthetic bone ingrowth and the bone formation is not only factor that ensure stability of coating prostheses after its implantation. The adhesive strength and porosity of coating are also important factors for the stability of coating prosthesis in vivo. (2)Ta-coated prosthesis exhibits good biocompatibility with the bone tissue which is comparable with the current clinically metal-coated prosthesis. (3)Ta coating has a higher coating-substrate adhesive strength and hereby is resistant to the damages resulted from the repeated loading changes and the relatively high shear stress, which promotes the postoperative prosthetic stability and prolongs the life of prosthesis in vivo.
引文
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