钛酸钡涂层修饰的多孔Ti6Al4V支架在超声介导下修复大段骨缺损的体内外生物学评价

发布时间：2018-05-17 18:42

  本文选题：大段骨缺损 + 钛酸钡　； 参考：《第四军医大学》2017年博士论文

【摘要】：研究背景当今社会中,由于感染、创伤、骨肿瘤骨病切除等往往造成骨组织的缺损,对于常规的骨缺损,经过积极治疗后常常能够获得较理想的治疗效果,但是对于大段骨组织的缺失(等于或者大于长骨直径的1.5倍),即使经过积极治疗也难以获得良好的治疗效果,尤其是负重部位的大段骨缺损,更是困扰着医学研究人员。虽然目前已经有较多人工骨移植材料被研发,但是它们或多或少存在一定的缺陷,例如移植材料的强度有限或者虽具有较好的强度但是缺乏活性,甚至既具有了好的强度也有了不错的生物活性但是仍然难以满足临床上的应用。近年来,多孔钛合金技术的发展给大段骨缺损的治疗带来了新的治疗思路,多孔钛合金具有良好的生物相容性、耐腐蚀性、高的机械强度,同时其整体弹性模量可以根据骨的弹性模量通过调节孔径大小、连通径大小以及孔隙率的高低来做出调整。而且,多孔结构不仅能够作为营养物质运输的有效通道,而且为新生骨组织的长入也提供了良好的空间,因此将多孔钛合金作为治疗负重部位大段骨缺损的基底材料不失为一种不错的选择。但是,多孔钛合金属于生物惰性材料,其植入体内后,不能很好地诱导骨组织长入,骨与材料界面难以达到理想的整合效果。因为机体内一切生命活动都有赖于微弱生物电活动,因此提高多孔钛合金的生物电活性或许能够改善其成骨活性。目前,压电陶瓷是一种良好的力-电转换材料,在其受到外界力的作用并发生形变时(即使是极其微小的形变)在其周围会产生微弱的电流;而当其处在电场中时,其亦会发生形变,从而实现力-电之间的互相转换。因此,如果能将压电陶瓷作为涂层制备于多孔钛合金表面,其在力的作用下能够很好的改善多孔钛合金的生物电活性。目前,医学领域中对压电陶瓷已有了大量研究,但是只有钛酸钡压电陶瓷研究和应用的最为广泛,而且其作为涂层的制备工艺也更为成熟。钛酸钡压电陶瓷具有良好的生物相容性和压电特性,作为涂层的制备方法包括等离子喷涂、火焰喷涂等,但是这些方法所制备的涂层厚度和分布多不够均匀,涂层结合多不牢固。水热合成法通过化学合成的方法,能在多孔钛合金表面原位生成厚薄均匀的钛酸钡涂层,并且结合稳定牢固。低强度脉冲超声波已经被美国食品和药品管理局(FDA)批准用于治疗临床上新鲜骨折以及骨不连等,证明低强度脉冲超声波本身能够促进骨组织的再生。另外,低强度脉冲超声波作为一种微弱的机械力,其作用于压电陶瓷后,能够使其发生形变从而产生压电效应,压电效应所产生的微电流进一步刺激新骨的形成。因此,本实验将钛酸钡压电陶瓷涂层制备于多孔Ti6Al4V支架表面后,并将低强度脉冲超声波作用于涂层修饰后的支架,观察和评估了体外的细胞学效应和体内修复大段骨缺损的效果。研究目的将钛酸钡压电陶瓷涂层制备于多孔Ti6Al4V支架表面,对修饰后支架的自身特性和表面特性与单纯多孔Ti6Al4V支架进行对比;检测低强度脉冲超声波作用于修饰前支架和修饰后支架对于体外细胞生物学特性的影响;检测低强度脉冲超声波作用于修饰前支架和修饰后支架对于体内大段骨缺损治疗效果,包括骨量增长的多少和骨与支架的整合程度。研究方法1)通过电子束熔融技术制备两种不同规格的多孔Ti6Al4V支架,即体外细胞实验所用支架直径12mm,高2mm和体内动物实验所用支架直径5mm,高13mm,孔径大小设计为700μm,孔隙率为70%,横梁直径为380μm。通过水热合成法在支架表面制备钛酸钡压电陶瓷涂层,并利用扫描电镜、普通能谱分析和X射线光电子能谱分析检测涂层是否制备成功。通过水接触角和表面粗糙度比较涂层修饰前后,多孔Ti6Al4V支架表面特性的变化。通过Micro-CT扫描比较了修饰前后多孔Ti6Al4V支架的孔径、连通径、孔隙率以及横梁大小的变化。通过力学测试评价涂层修饰前后,多孔Ti6Al4V支架弹性模量和强度的变化。2)将低强度脉冲超声波作用于涂层修饰前后的多孔Ti6Al4V支架,并将兔骨髓间充质干细胞种植于支架表面。根据超声波条件的加载与否,实验被分为BaTiO_3/pTi+LIPUS组、BaTiO_3/pTi组、LIPUS+pTi组和pTi组四组。并在培养4天和7天时利用CCK-8检测了细胞的增殖活性;在4天时利用SEM,在4天和7天时利用荧光染色观察了细胞的基本形态和黏附活性;在4天时利用流式细胞仪,在4天和7天时利用细胞死活荧光染色分析了细胞的死亡和凋亡情况;在7天和14天时,分别利用碱性磷酸酶活性和成骨相关基因的PCR结果评估了细胞的成骨分化情况。3)建立新西兰大白兔桡骨中段大段骨缺损的模型,并将钛酸钡压电陶瓷涂层修饰前后的多孔Ti6Al4V支架分别移植于骨缺损处,术后给予低强度脉冲超声波刺激。根据是否加载超声刺激,实验同样被分为BaTiO_3/pTi+LIPUS组、BaTiO_3/pTi组、LIPUS+pTi组和pTi组四组。在术后6周和12周分别取材,通过影像学X线平片和Micro-CT扫描分析了材料内部骨组织的再生情况,通过荧光标记计算了骨矿化沉积率的快慢,通过组织学切片VG染色进一步分析了新生骨组织的生长状况和骨-支架界面的骨整合情况,通过生物力学检测了最大拔出力。研究结果1)通过SEM、SEM自带的能谱分析仪和XPS检测,发现钛酸钡压电陶瓷涂层被成功制备于多孔Ti6Al4V支架表面;水接触角结果显示,钛酸钡压电陶瓷涂层改善了多孔Ti6Al4V支架表面的亲水性;表面粗糙度结果表明,钛酸钡涂层修饰后的支架有了更好的表面粗糙度;Micro-CT扫描结果表明,涂层修饰前后支架的孔径、连通径、孔隙率和横梁直径均无统计学意义上的变化;力学测试结果显示,涂层修饰前后支架的弹性模量和力学强度均无明显变化。2)CCK-8结果表明,在4天和7天时,BaTiO_3/pTi+LIPUS组与其它三组比较有了更好的细胞增殖活性(*p0.05 vs.pTi组,#p0.05 vs.BaTiO_3/pTi组,+p0.05 vs.LIPUS+pTi组),LIPUS+pTi组和BaTiO_3/pTi组的细胞增殖活性高于pTi组(*p0.05)。扫描电镜发现BaTiO_3/pTi+LIPUS组细胞形态不仅更为饱满,伸展状态良好,而且在支架表面形成的细胞层,LIPUS+pTi组和BaTiO_3/pTi组的细胞状态要好于pTi组。荧光染色后,细胞总面积与细胞核面积之比显示在4天和7天时,BaTiO_3/pTi+LIPUS组的值均高于LIPUS+pTi组(P0.05)、BaTiO_3/pTi组(P0.05)和pTi组(P0.05),而LIPUS+pTi组和BaTiO_3/pTi组的值仅在7天时在统计学上高于pTi组(P0.05)。黏着斑蛋白的表达显示,在4天和7天时,Ba TiO_3/pTi+LIPUS组均有较高的表达(*p0.05 vs.pTi组,#p0.05 vs.Ba TiO_3/pTi组,+p0.05 vs.LIPUS+pTi组),LIPUS+pTi组的表达均高于pTi组(P0.05),BaTiO_3/pTi组只在7天时高于pTi组(P0.05)。流式细胞仪细胞凋亡指数和细胞死活染色结果结果显示,在4天和7天时,BaTiO_3/pTi+LIPUS组(*p0.05,#p0.05,+p0.05)LIPUS+pTi组(*p0.05)BaTiO_3/pTi组(*p0.05)pTi组。碱性磷酸酶活性结果显示,在7天和14天时,BaTiO_3/pTi+LIPUS组(*p0.05,#p0.05,+p0.05)LIPUS+pTi组(*p0.05)BaTiO_3/pTi组(*p0.05)pTi组。成骨基因RNA表达结果显示,在7天和14天时,对于ALP、RUNX2和COL-1基因的表达来说,Ba TiO_3/pTi+LIPUS组(*p0.05,#p0.05,+p0.05)LIPUS+pTi组(*p0.05)BaTiO_3/pTi组(*p0.05)pTi组,但是,对于OPN基因表达来说,Ba TiO_3/pTi组在7天和14天时均大于其他三组(*p0.05,#p0.05,+p0.05),而LIPUS+pTi组和BaTiO_3/pTi组仅在14天时大于pTi组(P0.05)。3)影像学检查发现,在6周和12周时,新生骨组织长入的量,BaTiO_3/pTi+LIPUS组(*p0.05,#p0.05,+p0.05)LIPUS+pTi组(*p0.05)BaTiO_3/pTi组(*p0.05)pTi组。骨矿化沉积率的计算结果表明,对于新骨生长速度来说,BaTiO_3/pTi+LIPUS组(*p0.05,#p0.05,+p0.05)LIPUS+pTi组(*p0.05)BaTiO_3/pTi组(*p0.05)pTi组。组织学VG染色结果与影像学和荧光标记结果相一致,BaTiO_3/pTi+LIPUS组(*p0.05,#p0.05,+p0.05)LIPUS+pTi组(*p0.05)BaTiO_3/pTi组(*p0.05)pTi组,另外,BaTiO_3/pTi+LIPUS组、LIPUS+pTi组、Ba TiO_3/pTi组这三组骨整合均较pTi组好。最大拔出力的结果与影像学和组织学保持一致,BaTiO_3/pTi+LIPUS组(*p0.05,#p0.05,+p0.05)LIPUS+pTi组(*p0.05)BaTiO_3/pTi组(*p0.05)pTi组。研究结论1)成功在多孔Ti6Al4V支架表面制备了钛酸钡压电陶瓷涂层,而且该涂层在改善多孔Ti6Al4V支架表面的亲水性和表面粗糙度的基础上,并未影响支架本身的特性,包括孔径、连通径、孔隙率、弹性模量、力学强度。2)在体外研究中,钛酸钡压电陶瓷涂层作为独立因素能够改善多孔Ti6Al4V支架上细胞的形态、黏附、增殖和分化;低强度脉冲超声波同样可以作为独立的因素改善多孔Ti6Al4V支架上细胞的生物学表现;当低强度脉冲超声波作用于钛酸钡压电陶瓷涂层时,其改善孔Ti6Al4V支架上细胞生物行为学的能力进一步被提高。3)在体内研究中,钛酸钡压电陶瓷涂层能够作为独立因素提高多孔Ti6Al4V支架内新生骨组织的长入并且促进骨与支架之间的整合;低强度脉冲超声波同样可以作为独立因素改善多孔Ti6Al4V支架内新生骨组织的生成量和骨整合能力;钛酸钡压电陶瓷涂层在低强度脉冲超声波的作用下,其促进骨再生的能力和骨与支架整合的能力进一步被加强,相较于单纯多孔Ti6Al4V支架来说,骨组织的生成量增加了10%-20%。4)多孔Ti6Al4V支架、钛酸钡压电陶瓷涂层、低强度脉冲超声波的结合能显著提高负重部位大段骨缺损的修复效能,为临床应用提供了理论基础。但是,其修复机制尚未完全明了,有待进一步的探究。
[Abstract]:Research background today, infection, trauma, bone tumor resection often cause bone tissue defects. For conventional bone defects, after active treatment, they often get better therapeutic effects, but the loss of large bone tissue is equal to or greater than 1.5 times the diameter of the long bone, even after active treatment. It is difficult to obtain good therapeutic effects, especially large segment bone defects in the weight bearing area, which is more perplexing medical researchers. Although many artificial bone graft materials have been developed, there are more or less defects, such as the limited strength of the graft material, the better strength but the lack of activity, and the lack of activity. In recent years, the development of porous titanium alloy technology has brought new treatment ideas for the treatment of large bone defects. Porous titanium alloys have good biocompatibility, corrosion resistance, high mechanical strength, and the overall elastic modulus of the porous titanium alloy. The volume can be adjusted according to the modulus of elasticity of the bone, the size of the diameter and the porosity of the bone. Moreover, the porous structure can not only serve as an effective channel for the transport of nutrients, but also provide a good space for the growth of the new bone tissue. Therefore, porous titanium alloy is used as a large part of the treatment of weight bearing parts. The base material of bone defect is a good choice. However, porous titanium alloy is a biological inert material. After implantation, it can not induce bone tissue to grow well. The bone and material interface can not achieve the ideal integration effect. Because all life activities in the body depend on weak bioelectrical activity, so the porous titanium is improved. The bioelectrical activity of an alloy may improve its osteogenic activity. At present, a piezoelectric ceramic is a good force - electric conversion material, which produces a weak current around it when it is subjected to external forces and is deformed (even extremely small); and when it is in an electric field, it will also be deformed, thus realizing force - Therefore, if the piezoelectric ceramic can be prepared as a coating on the surface of the porous titanium alloy, it can improve the bioelectrical activity of the porous titanium alloy very well. At present, a lot of research has been done on the piezoelectric ceramics in the medical field, but only barium titanate piezoelectric ceramics are the most widely used in the field of research and application. The preparation technology of the coating is also more mature. Barium titanate piezoelectric ceramics have good biocompatibility and piezoelectric properties. The preparation methods of the coating include plasma spraying, flame spraying, etc. but the thickness and distribution of the coating are not uniform enough and the coating is not solid. The hydrothermal synthesis method is passed. The method of chemical synthesis can produce a thick and uniform barium titanate coating on the surface of porous titanium alloy, and it is stable and stable. Low intensity pulsed ultrasound has been approved by the US Food and Drug Administration (FDA) for clinical fresh fractures and bone nonunion. It is proved that low intensity pulse ultrasound can promote bone group itself. In addition, the low strength pulse ultrasonic as a weak mechanical force, which acts on the piezoelectric ceramic, can cause the deformation to produce the piezoelectric effect. The micro current produced by the piezoelectric effect further stimulates the formation of the new bone. Therefore, this experiment has prepared the barium titanate piezoelectric ceramic coating on the surface of the porous Ti6Al4V scaffold. The effect of the in vitro cytological effect and the repair of large bone defects in the body was observed and evaluated in vitro by using low intensity pulsed ultrasound as a scaffold. The purpose of this study was to prepare barium titanate piezoelectric ceramic coating on the surface of porous Ti6Al4V scaffold. The self and surface properties of the modified scaffold and the simple porous Ti6Al4V branch were studied. The effects of low intensity pulsed ultrasound on the biological characteristics of the cells in vitro, and the effect of low intensity pulsed ultrasound on the treatment of large bone defects in the body, including the amount of bone mass growth and the integration of bone and scaffold, was detected by the effect of low intensity pulsed ultrasound on the biological characteristics of the cells in vitro. Degree. Method 1) two kinds of porous Ti6Al4V scaffolds with different specifications were prepared by electron beam melting technique, that is, the diameter of scaffold used in vitro cell experiment 12mm, high 2mm and the diameter of scaffold used in the animal experiment in vivo, 5mm, high 13mm, the diameter of 700 u m, the porosity of 70%, and the diameter of the transverse beam of 380 mu on the surface of the scaffold. Barium titanate piezoelectric ceramic coating was prepared. The coating was successfully prepared by scanning electron microscopy, ordinary energy spectrum analysis and X ray photoelectron spectroscopy. The surface properties of porous Ti6Al4V scaffolds were compared before and after the coating modified by water contact angle and surface roughness. The porous Ti6Al4V scaffolds before and after the modification were compared by Micro-CT scanning. Pore size, connection diameter, porosity and change of beam size. Through mechanical tests, the elastic modulus and strength of the porous Ti6Al4V scaffold were evaluated before and after the coating modified.2). The low intensity pulsed ultrasound was applied to the porous Ti6Al4V scaffold before and after the coating, and the rabbit bone marrow mesenchymal stem cells were planted on the surface of the scaffold. The experiment was divided into four groups, BaTiO_3/pTi+LIPUS group, BaTiO_3/pTi group, LIPUS+pTi group and pTi group. CCK-8 was used to detect cell proliferation activity by CCK-8 for 4 days and 7 days. The basic morphology and adhesion activity of the cells were observed by SEM at 4 days and 4 and 7 days by fluorescence staining, and the flow cytometry was used at 4 days. Cell death and apoptosis were analyzed by cell dead fluorescence staining at 4 and 7 days. At 7 and 14 days, PCR results of alkaline phosphatase activity and osteogenic related genes were used to evaluate the osteogenic differentiation of cells.3). A model of large bone defect in the middle segment of the radius of New Zealand white rabbit was established and barium titanate piezoceramic was used. The porous Ti6Al4V scaffold before and after the coating was transplanted to the bone defect and was given low intensity pulse ultrasonic stimulation after operation. The experiment was also divided into four groups, group BaTiO_3/pTi+LIPUS, group BaTiO_3/pTi, group LIPUS+pTi and group pTi according to whether the ultrasonic stimulation was loaded. The regeneration of bone tissue in the material was analyzed by scanning, and the rate of bone mineralization deposition was calculated by fluorescence labeling. The growth status of the new bone tissue and the bone integration of the bone scaffold interface were further analyzed by the histological section VG staining. The maximum pulling force was detected by the biomechanics. The results of the study were 1) by SEM and SEM. The energy spectrum analyzer and XPS test showed that barium titanate piezoelectric ceramic coating was successfully prepared on the surface of porous Ti6Al4V stent, and the water contact angle showed that barium titanate piezoelectric ceramic coating improved the hydrophilicity of the porous Ti6Al4V stent surface, and the surface roughness results showed that the scaffold after the barium titanate coating had better surface roughness. The results of Micro-CT scan showed that the diameter, diameter, porosity and diameter of the scaffold were no significant changes before and after the coating. The results of mechanical test showed that the modulus of elasticity and mechanical strength of the scaffolds were not significantly changed by.2) CCK-8 results showed that in 4 and 7 days, the BaTiO_3/pTi+LIPUS group and the other three were the other three. The group had better cell proliferation activity (group *p0.05 vs.pTi, #p0.05 vs.BaTiO_3/pTi group, +p0.05 vs.LIPUS+pTi group), and the cell proliferation activity of LIPUS+pTi group and BaTiO_3/pTi group was higher than that of group pTi (*p0.05). The cell morphology of BaTiO_3/pTi+LIPUS group was not only more full, well stretched, but also formed on the surface of the scaffold. Cell layer, LIPUS+pTi group and BaTiO_3/pTi group were better than group pTi. After fluorescent staining, the ratio of total area to cell nuclear area was higher than that of group LIPUS+pTi (P0.05), BaTiO_3/pTi group (P0.05) and pTi group (P0.05) at 4 days and 7 days, while the value of LIPUS+pTi group and BaTiO_3/pTi group was only 7 days. The expression of macula protein was higher than that of pTi group (P0.05). The expression of macula protein showed that at 4 and 7 days, the Ba TiO_3/pTi+LIPUS group had higher expression (*p0.05 vs.pTi group, #p0.05 vs.Ba TiO_3/pTi group, +p0.05 vs.LIPUS+pTi group), and the expression of LIPUS+pTi group was higher than that of the group. The results of apoptotic index and cell death staining showed that at 4 and 7 days, group BaTiO_3/pTi+LIPUS (*p0.05, #p0.05, +p0.05) group LIPUS+pTi (*p0.05) BaTiO_3/pTi group (*p0.05) pTi group. The results of alkaline phosphatase activity showed that the BaTiO_3/pTi+ LIPUS group was in the 7 and 14 days. 5) pTi group. The expression of osteogenic gene RNA showed that at the 7 and 14 days, the expression of ALP, RUNX2 and COL-1 genes, Ba TiO_3/pTi+LIPUS group (*p0.05, #p0.05, +p0.05) LIPUS+pTi group (*p0.05) group, but, for 7 and 14 days, the group was larger than the other three groups. .05, +p0.05), and group LIPUS+pTi and BaTiO_3/pTi only 14 days longer than pTi group (P0.05).3) imaging examination found that the amount of new bone tissue in the 6 and 12 weeks, BaTiO_3/pTi+LIPUS group (*p0.05, #p0.05, +p0.05) LIPUS+pTi group. Speed, group BaTiO_3/pTi+LIPUS (*p0.05, #p0.05, +p0.05) group LIPUS+pTi (*p0.05) BaTiO_3/pTi group (*p0.05) pTi group. Histology VG staining results are consistent with the results of imaging and fluorescence labeling. The three groups of bone integration in group Ba TiO_3/pTi were better than those in group pTi. The results of maximum pulling force were consistent with imaging and histology. Group BaTiO_3/pTi+LIPUS (*p0.05, #p0.05, +p0.05) LIPUS+pTi group (*p0.05) BaTiO_3/pTi group (*p0.05) pTi group. Conclusion 1) barium titanate piezoelectric ceramic coating was successfully prepared on the surface of porous scaffold. On the basis of improving the hydrophilicity and surface roughness of the surface of the porous Ti6Al4V scaffold, the coating does not affect the characteristics of the scaffold itself, including the pore size, the diameter, the porosity, the modulus of elasticity and the mechanical strength.2) in the study. The barium titanate piezoelectric ceramic coating can improve the morphology and adhesion of the cells on the porous Ti6Al4V scaffold as an independent factor. Proliferation and differentiation; low intensity pulsed ultrasound can also be an independent factor to improve the biological performance of cells on the porous Ti6Al4V scaffold; when low intensity pulsed ultrasound acts on barium titanate piezoelectric ceramic coating, the energy of cell biobehavior on the porous Ti6Al4V scaffold is further improved by.3) in the body study, titanic acid Barium piezoelectric ceramic coating can increase the growth of the new bone tissue in the porous Ti6Al4V scaffold and promote the integration of the bone and the scaffold as an independent factor. The low intensity pulse ultrasound can also improve the formation and the bone integration of the new bone tissue in the porous Ti6Al4V scaffold as an independent factor; barium titanate piezoelectric ceramic coating is low Under the action of intensity pulse ultrasound, the ability to promote bone regeneration and the ability to integrate the bone with the scaffold is further strengthened. Compared with the simple porous Ti6Al4V scaffold, the formation of bone tissue is increased by 10%-20%.4) porous Ti6Al4V scaffold, barium titanate piezoelectric ceramic coating, and the combination of low intensity pulsed ultrasonic wave can significantly increase the weight negative part. The repair efficiency of large segmental bone defects provides a theoretical basis for clinical application. However, its repair mechanism is not yet fully understood and needs further investigation.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R68;R318.08
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本文编号：1902437