生物活性玻璃修饰聚对苯二甲酸乙二醇酯对骨愈合的影响

发布时间：2018-04-28 20:00

本文选题：前交叉韧带 + 聚对苯二甲酸乙二醇酯　；参考：《复旦大学》2012年硕士论文

【摘要】：前交叉韧带(anterior cruciate ligament, ACL)损伤是临床常见的运动损伤之一。目前临床上多采用ACL重建术治疗ACL损伤,从而避免内侧副韧带损伤(MCL)、半月板损伤、膝关节软骨损伤等一系列并发症。重建材料的选择大体上分为三种：自体肌腱、异体肌腱和人工韧带。它们或多或少存在着取材受限、取材部位的并发症、免疫排斥与疾病传播等缺点。而人工韧带则很大程度上规避了这些风险,因此人工韧带逐渐开始运用于临床。现在越来越多的临床医生开始接受一种新型人工韧带---[LARS(Ligament Advanced Reinforcement System)],因为它较好的力学性能和生物相容性,能缩短ACL重建术后愈合时间,能尽早实现患者重返日常生活。然而,越来越多的文章开始关注LARS人工韧带的失败案例,他们发现在LARS人工韧带与宿主骨之间存在阻碍愈合的纤维疤痕存在,这些很可能是由于LARS人工韧带原材料-—PET材料的生物相容性不高导致的。已经有很多文章开始关注于改进PET材料的生物相容性,作为首先接触到宿主的材料表面很大程度上决定了移植材料的生物学性能,因此通过表面修饰对现有材料进行改进。现有的材料学方面,如羟基化、磷酸化材料表面等得到了较为广泛的运用。早在上世纪70年代,Larry Hench就发现了生物玻璃这种人造骨移植材料,它是一种硅酸盐合成材料,其主要成分在于二氧化硅在混合物中的质量比低于60%。由于它能在体内形成羟基磷灰石层而广泛应用于被用作为骨性支架来研究Si02在混合物中的质量比在45-52%下能快速的与宿主骨发生化学连接,从而发挥其生物学性能,虽然质量比在55-60%下,较前反应稍慢,但其能保持较长的生物学活性。58S是生物玻璃家族中的一员,它比一般的生物玻璃有着更高的Ca/P比(CaO32.6%、SiO258.2%、P2059.2%)和缓释型,因此较多的运用在临床。本实验用生物玻璃58S涂层处理PET人工韧带,希望处理过的PET人工韧带能够有更好的生物活性,能够促进PET韧带与骨隧道之间的紧密连接,促进移植物与骨的愈合,从而提高PET人工韧带移植的成功率。我们用MC3T3-E1鼠成骨细胞和半关节模型来验证我们的假设。第一部分以明胶为载体的生物玻璃涂层复合材料的构建与测定目的构建并测定以明胶为载体的生物玻璃涂层复合材料。方法将LARS韧带骨道端(PET纤维)展平剪成24孔板大小(图1),同时制备PET膜片成24孔板大小,将上述材料浸泡在75%的酒精中4h去渍,等离子水清洗,在37℃烘箱下烘干,等离子处理材料表面后,置于按质量不同浓度配比(1:2,1:3,1：4,15)的58S与明胶的混合溶液中,磁力搅拌约5min,取出后烘干,随机选取涂层前和涂层后各一个样本,经离子溅射仪表面喷金处理后,用扫描电镜观察材料表面特征,选取最佳浓度配比的混合溶液作为涂层溶液进行涂层；同样选取涂层和未涂层组样本各一,运用X射线能谱仪分析涂层前后材料的组成元素变化。余下样本(n=48)经环氧乙烷消毒后备用。结果成功构建58S-PET复合材料,确定质量比为1：4的生物玻璃与明胶的混合溶液作为涂层溶液,扫描电镜观察材料表面特征能看见在原本光滑的PET材料表面附着了大量颗粒不等的物质,经X射线能谱仪分析结果显示,经58S涂层处理的PET纤维,其Si、Ca、P元素明显增高,符合58S的基本元素含量,佐证了在PET纤维上颗粒确为生物玻璃。余下样本依次经环氧乙烷消毒、DMEM培养液浸泡7天后用于成骨细胞培养。结论成功构建出58S-PET复合材料,有利于实验进一步开展。第二部分58S-PET复合材料体外成骨细胞增殖及活性实验目的观察并测定58S-PET复合材料相较未经处理的PET材料在体外成骨细胞中的效果。方法经上述处理好的PET纤维及膜片置于24孔培养板中,都加入MC3T3-E1细胞悬液(密度2×104/m1)。膜片组用倒置显微镜观察72小时后的涂层组(58S-PET)与未经涂层组(PET)之间的细胞形态变化,并在1,3,5天进行细胞计数。纤维组在第1,3,5天分别进行MTT、ALP测试其生物学活性及细胞增殖。结果细胞在培养72小时后两组细胞形态无明显差别,且实验组(58S-PET)细胞密度明显较未经处理的对照组(PET)高；第1,3,5天的细胞计数,细胞数量在实验组(58S-PET)和对照组(PET)均呈现增长趋势,但是在第5天实验组明显高于对照组(6.250±0.27,8.92±0.17;P0.05)；同样,在四唑盐比色法(MTT)和碱性磷酸酶(ALP)活性测定中,在涂层组在第3,5d均表现出明显的优势(P0.05) 结论58S-PET复合材料未表现出对于成骨细胞的毒性,且还能明显提高成骨细胞的数量和活性。第三部分58S-PET复合材料体内生物力学及组织学实验目的了解58S-PET复合材料在体内的生物力学及组织学效果。方法将24只雄性新西兰大白兔(体重2.7±0.3kg)并将其随机分成A、B两组：每组12只,造成双侧半关节移植物-骨愈合模型,其中A组植入经生物玻璃(58S)涂层处理的PET材料组,作为实验组(58S-PET)；B组植入未经处理组,作为对照组(PET)；为避免材料之间的相互影响,A、B两组双侧均植入同一材料。术后6,12周分别取材行生物力学及组织学检查。结果生物力学显示：两组材料植入后均表现出随时间推移最大拔出力均有显著提高,术后6周、12周两组间有显著性差异(p0.05),实验组均高于对照组。实验组6周(实验组61.70±6.95N)高于对照组(45.21±9.78N,p=0.03),随着材料植入术后时间的延长两组间最大拔出拉力呈现逐渐显著的趋势,实验组12周(实验组89.25±9.50N)显著高于对照组(71.38±6.26N,p=0.02)组织学观察：材料植入后第6周,实验组及对照组材料与宿主骨与骨界面模糊,有新骨形成,与宿主骨组织结合紧密,以实验组明显。第12周,两组植入材料已与宿主骨逐渐融合,新生骨进一步增多,移植物-骨愈合得到进一步增强。结论以58S作为涂层材料改进现有PET材料能改善宿主与移植物的愈合能力,缩短愈合时间。
[Abstract]:Anterior cruciate ligament (ACL) injury is one of the most common sports injuries. At present, ACL reconstruction is used in the treatment of ACL injury, which avoids the complications of medial collateral ligament injury (MCL), meniscus injury, and knee cartilage injury. The selection of reconstruction materials is generally divided into three kinds: autologous tendon, Allogeneic tendons and artificial ligaments. They have more or less defects in material limitation, complications of taking parts, immune rejection and spread of disease. And artificial ligaments largely avoid these risks, so artificial ligaments are gradually starting to apply to the clinic. More and more clinicians are now beginning to accept a new type of artificial toughening. With ---[LARS (Ligament Advanced Reinforcement System)], because of its good mechanical properties and biocompatibility, it can shorten the healing time after ACL reconstruction and make the patient return to daily life as soon as possible.
However, more and more articles begin to pay attention to the failure cases of LARS artificial ligaments. They found that there is a fibrous scar that hinders healing between the LARS artificial ligaments and the host bone. These are probably due to the poor biocompatibility of the raw material of the LARS artificial ligament - the material of the PET. Many articles have begun to focus on the improvement. The biocompatibility of PET material, which is the first contact to the surface of the host material, largely determines the biological properties of the transplanted materials. Therefore, the existing materials are improved by surface modification. The existing materials, such as hydroxylation and phosphorylated materials, have been widely used.
As early as the 70s of last century, Larry Hench discovered the artificial bone graft of Bioglass. It is a silicate synthetic material. Its main component is that the mass ratio of silica in the mixture is less than 60%. because it can form a hydroxyapatite layer in the body, and it should be used as a bone scaffold to study Si02 in the mixture. The mass of the compound can be linked to the host bone quickly with the host bone, and thus exerts its biological performance. Although the mass is slower than that under 55-60%, the longer biological activity of.58S is a member of the Bioglass family, which has a higher Ca/P ratio than the normal Bioglass (CaO32.6%, SiO258.2%). P2059.2% and slow release, so it is more widely used in clinic.
This experiment treated the PET artificial ligament with the 58S coating of Bioglass. It is hoped that the treated PET artificial ligament can have better biological activity, can promote the close connection between the PET ligament and the bone tunnel, promote the healing of the graft and bone, and thus improve the success rate of the transplantation of the PET artificial ligament. We use the MC3T3-E1 mouse osteoblasts and the half joint model. Type to verify our hypothesis.
The first part is the construction and determination of the Bioglass coating composite with gelatin as carrier.
Objective to construct and determine the Bioglass coating composite with gelatin as carrier.
Methods the size of the LARS ligament end (PET fiber) was flattened into 24 orifice plates (Figure 1), and the size of the 24 hole plate was prepared by the PET diaphragm. The above material was soaked in 75% alcohol, 4H was soaked, plasma water was washed, dried at 37 centigrade oven, and after plasma treatment on the surface of the material, 58S and gelatin with different mass ratio (1:2,1:3,1:4,15) were placed on the surface of the material with different mass concentration (1:2,1:3,1:4,15). In the mixed solution, the magnetic force is stirred for about 5min, and then dried, and one sample is randomly selected before and after the coating. After spraying gold on the surface of the ion sputtering instrument, the surface characteristics of the material are observed by scanning electron microscope, and the mixed solution of the best concentration ratio is selected as the coating solution. The samples of the coating and the uncoated group are also selected. First, the changes in the composition of the material before and after coating were analyzed by X ray energy spectrometer. The remaining samples (n=48) were sterilized by ethylene oxide.
Results the 58S-PET composite was successfully constructed and the mixed solution of bio glass and gelatin with a mass ratio of 1:4 was determined as a coating solution. The surface characteristics of the material could be observed on the surface of the original smooth PET material by scanning electron microscope. The results of PET fiber treated by 58S coating showed that the PET fiber was treated by 58S coating. The Si, Ca, and P elements were significantly higher in dimension, which conformed to the basic elements content of 58S. It was confirmed that the particles on PET fiber were truly Bioglass. The remaining samples were sterilized by ethylene oxide in turn and DMEM culture solution was soaked for 7 days to be used for osteoblast culture.
Conclusion 58S-PET composites were successfully constructed, which is conducive to further development of the experiment.
The second part is about the proliferation and activity of 58S-PET composite osteoblasts in vitro.
Objective To observe and determine the effect of 58S-PET composite material on osteoblasts in vitro compared with untreated PET material.
Methods the treated PET fiber and film were placed in the 24 hole culture plate and added MC3T3-E1 cell suspension (density 2 x 104/m1). The cell morphology changes between the coating group (58S-PET) and the uncoated group (PET) were observed by inverted microscope for 72 hours after 72 hours, and the cell count was carried out on 1,3,5 days. The fiber group was respectively entered on the day of 1,3,5. MTT, ALP test its biological activity and cell proliferation.
Results there was no significant difference in cell morphology between the two groups after 72 hours of culture, and the cell density of the experimental group (58S-PET) was significantly higher than that of the untreated control group (PET). The cell count on day 1,3,5, the number of cells in the experimental group (58S-PET) and the control group (PET) showed a growing trend, but in the fifth day group, the experimental group was significantly higher than the control group (6.250 + 0.2). 7,8.92 + 0.17; P0.05); similarly, in four zolium salt Colorimetry (MTT) and alkaline phosphatase (ALP) activity determination, the coating group showed obvious advantages in 3,5d (P0.05).
Conclusion 58S-PET composite does not show toxicity to osteoblasts, and can significantly increase the number and activity of osteoblasts.
The third part is the biomechanics and histology experiment of 58S-PET composites.
Objective to understand the biomechanical and histological effects of 58S-PET composites in vivo.
Methods 24 male New Zealand white rabbits (2.7 0.3KG) were randomly divided into A, B two groups, 12 rats in each group, resulting in bilateral half joint graft bone healing model, and group A was implanted into the PET material group treated with biological glass (58S) coating, as the experimental group (58S-PET); B group was implanted in the untreated group as the control group (PET); to avoid the control group (PET). A and B two groups were implanted with the same material on both sides. Biomechanical and histological examinations were taken at 6,12 weeks after operation.
The results of biomechanics showed that the maximum pulling force of the two groups were significantly improved after the implantation of the two groups, and there were significant differences between the two groups after 6 weeks and 12 weeks after the operation. The experimental group was higher than the control group. The experimental group was 6 weeks (61.70 + 6.95N) higher than the control group (45.21 + 9.78N, p=0.03), with the delay of the time after the material implantation. The maximum pulling pull between the two groups showed a gradual and significant trend. The experimental group was significantly higher than that of the control group (71.38 + 6.26N, p=0.02) in the experimental group (71.38 + 6.26N, p=0.02). The material and the host bone and bone interface were blurred in the experimental group and the control group after sixth weeks of implantation. Twelfth weeks later, the two groups of implant materials were gradually fused with the host bone, and the new bone was further increased, and the graft bone healing was further enhanced.
Conclusion using 58S as the coating material to improve the existing PET materials can improve the healing ability of host and graft and shorten the healing time.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R873

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