【作者】 马文辉；

【导师】 张英泽；

【作者基本信息】 河北医科大学 ， 外科学， 2014， 博士

【摘要】 股骨颈骨折是一种临床上常见的骨折，约占全部骨折的3.58%，占股骨近端骨折的50%，其术后的骨折不愈合率和股骨头缺血坏死率较高，这也是其治疗上所面临的主要问题，日益受到临床医生的重视。迄今为止，虽然股骨颈骨折的治疗方法不断完善，但是，由于受多种因素的影响，它的治疗及结果等许多方面仍遗留有许多尚未解决的问题。我院采用一期游离髂骨移植结合三枚空心螺钉固定方法对股骨颈骨折进行早期干预治疗，取得了较好的效果。有限的临床经验证明，此方法具有手术操作简单、创伤小等优点；同时，一期植骨的稳定性好，提高了骨折愈合率，并有助于预防远期股骨头坏死，不失为临床治疗股骨颈骨折的一种有益选择。为了避免手术取骨所造成的损伤和并发症，我们进一步研究使用镁金属支架来代替游离植骨，这种材料能在体内逐渐降解，同时诱导周围新骨生成。为了更好地评价此种支架的腐蚀行为与生物力学特性，为其优化设计和临床应用提供理论依据和必要的参考，本课题系统研究了这种新型的经微弧氧化处理的镁材料在体外模拟生理环境中的降解行为，初步探索了镁离子对成骨细胞生物学行为的影响及其作用机制，并利用有限元分析法建立新型镁支架干预治疗股骨颈骨折的模型，系统研究了金属支架在正常步态周期中的应力变化规律，并与骨移植干预治疗的方法进行了对比分析。第一部分经微弧氧化处理的新型镁金属材料的体外降解行为和细胞相容性研究目的：通过在体外模拟生理环境中的电化学试验和浸泡试验，对经表面微弧氧化处理的新型镁材料的腐蚀行为进行系统的研究，并采用拉伸试验对材料在与模拟体液接触过程中的力学性能变化进行检测。此外，对材料在腐蚀过程中的细胞毒性进行评价。方法：将纯镁试样和经表面微弧氧化处理的镁试样分别浸泡于模拟体液中，用于浸泡的溶液量与试样表面积之比均为30ml/cm2。所有试样在试验前先称重并做记录。实验过程中，温度保持在37±0.5℃，液体的pH值维持在7.4左右。分别在浸泡后的6小时、1天、3天、7天、14天和30天取出试样，利用扫描电镜观察，结合能谱分析和XRD衍射分析对比研究两种试样的腐蚀形貌及其产物成分、降解速度变化和对溶液pH值的影响，并通过电子万能试验机对试样进行拉伸检测，计算材料的抗拉强度，比较不同试样的抗拉强度随腐蚀时间变化的情况。另外，分别以两种试样作为工作电极，利用电化学工作站对样本的电化学阻抗谱（EIS）和动电位极化曲线进行测试，绘制材料的Nyqiust曲线和极化曲线。最后，对Saos-2细胞进行体外培养，利用MTT法、免疫荧光染色技术检测不同材料浸提液对成骨细胞增殖活性和细胞凋亡的影响。使用SAS9.1统计软件对实验数据进行统计学处理，设P＜0.05为差异具有统计学意义。结果：在模拟体液中，经微弧氧化处理的镁金属的腐蚀速率远低于镁金属（P＜0.05），其对液体pH值的影响也小于镁金属，在72h内从最初的7.4快速上升并稳定在了8.3左右，而镁金属可使溶液的pH值达到约9.1的最高值。两种镁金属表面的腐蚀产物主体成分均为Mg(OH)2和羟基磷灰石。再者，微弧氧化处理可明显降低腐蚀过程中镁金属力学强度的损失，在SBF中浸泡30天后，材料的拉伸强度仍能保持原强度的90%。另外，阻抗频谱和动电位极化曲线也验证了经微弧氧化处理的镁金属的耐腐蚀性要高于镁金属。细胞毒性的分析结果表明，微弧氧化处理前后的镁金属均无明显细胞毒性，具有良好的细胞相容性。结论：微弧氧化处理可提高镁金属的耐腐蚀性，有利于维持其原有的力学性能，并且材料具有良好的细胞相容性，表面腐蚀产物羟基磷灰石的存在可提高材料的骨结合能力。第二部分镁离子对成骨细胞生物学行为的影响及其作用机制初探目的：采用人成骨细胞，研究镁离子对细胞增殖及其主要生物学行为的影响，并对其机制进行初步探讨。方法：对Saos-2细胞进行体外培养，将1mmol/L、2mmol/L和3mmol/L三种浓度MgSO4作用于成骨细胞，通过MTT法、免疫荧光染色技术检测不同因子对成骨细胞增殖活性和细胞凋亡的影响，并利用ELISA法测定不同因子对成骨细胞ALP活性及骨钙素合成等主要生物学特性的影响。另外，借助ELISA法，测定1mmol/L、2mmol/L和3mmol/L三种浓度MgSO4对成骨细胞Runx2和Fas表达的影响。使用SAS9.1统计软件对各实验的数据进行统计学比较，设P＜0.05为差异具有统计学意义。结果：不同浓度的镁离子均可促进成骨细胞的增殖（F=44.77，Р＜0.0001），这种刺激作用与镁离子的浓度和作用时间均呈正相关。荧光染色结果显示经镁离子作用后细胞的活性良好。并且，不同浓度的镁离子均可促进成骨细胞的碱性磷酸酶活性和骨钙素合成（Р＜0.05），这种刺激作用也与镁离子的浓度和作用时间均呈正相关。另外，不同浓度的镁离子，在不同时间点均可刺激成骨细胞RUNX2的表达，这种刺激作用与镁离子的浓度和作用时间呈正相关。而镁离子作用组的成骨细胞Fas表达量均明显低于正常对照组（F=16.97，Р＜0.0001），并且，Fas的表达量与镁离子的浓度呈负相关。结论：镁离子可促进成骨细胞的增殖以及ALP和BGP的合成，其机理可能与调节细胞Runx2和Fas的表达有关，一方面镁离子可通过促进细胞Runx2表达，来刺激成骨细胞的分化和成熟；另一方面，还可通过Fas途径抑制成骨细胞的凋亡，从而维持细胞的增殖与活性。第三部分镁金属支架干预治疗股骨颈骨折有限元模型的建立及其在正常步态周期中的力学分析研究目的：建立使用镁金属支架干预治疗股骨颈骨折的有限元模型，研究支架植入后，在正常步态周期中股骨颈部的应力变化规律，为这种新型金属支架的评价和优化提供有益的理论依据，进而指导其临床应用。方法：选取一名健康成年男性志愿者，对其髋关节及股骨中上段进行CT扫描，获得二维断层图像数据，使用Mimics10.01软件对图像进行处理、提取及重建，得到股骨三维模型，然后将模型导入到Ansys13.0有限元分析软件中，进行体网格划分，生成股骨三维有限元模型，并进一步模拟建立了使用金属支架干预治疗股骨颈骨折的有限元模型。设定边界和载荷条件，对模型进行生物力学分析。使用SAS9.1统计软件对实验数据进行统计学比较，设P＜0.05为差异具有统计学意义。结果：首先，建立了准确有效的股骨中上段的三维有限元模型以及金属支架干预治疗股骨颈骨折的有限元模型。进一步的分析表明，正常步态周期中骨折固定模型在股骨颈局部的Von Mises应力变化规律与正常股骨模型相似，并且两者的各期应力值以及位移之间均无显著差异（P＞0.05）。分析还表明，正常步态各期中，骨折固定模型在各个方向上的位移变化规律大致相同，均取决于髋关节作用力的大小，各轴向最大位移均出现在髋关节作用力最高的第二期。结论：本研究建立的股骨三维有限元模型准确有效，为后期的相关实验奠定了基础。另外，松质骨螺钉固定合并金属支架干预治疗股骨颈骨折不仅恢复了骨折部位应力传导的连续性，而且可获得较好的抗压、抗扭转和抗剪切稳定性，对其临床应用具有指导意义。第四部分镁金属支架和骨移植干预治疗股骨颈骨折生物力学的有限元对比研究目的：利用有限元法分析镁金属支架和骨移植在股骨颈骨折干预治疗中的应力分布规律，并与临床常用的单独三枚松质骨螺钉固定的方法进行比较。此外，还要对金属支架放置的角度进行研究。通过此实验有助于全面认识这种新型治疗方法的生物力学特点，为其临床应用提供理论指导。方法：建模的方法和过程以及材料属性同第三部分实验，分别建立镁金属支架和骨移植干预治疗股骨颈骨折的三维有限元模型，将金属支架或骨移植的植入角度分别设置为95°和127°。另外，还建立了单独三枚松质骨螺钉治疗股骨颈骨折的有限元模型。设定边界条件以及426N和2158N两种载荷，对模型进行生物力学分析。使用SAS9.1统计软件对实验数据进行统计学比较，设P＜0.05为差异具有统计学意义。结果：分析表明，使用金属支架或骨移植对股骨颈骨折进行干预治疗与单纯多枚螺钉固定相比，骨折局部所受的应力水平和产生的位移均无明显差异。另外，对于金属支架或骨移植而言，植入角度的改变并不会对骨折局部的应力产生显著影响，而且使用不同材料的模型之间所受应力和各轴向位移均无显著差异。结论：可见，松质骨螺钉固定合并金属支架干预治疗股骨颈骨折同样可获得良好的骨折稳定性，而支架的植入角度可根据情况进行适当地调整，再结合镁金属所固有的自身优势，所以它对于股骨颈骨折的干预治疗是一个值得推荐的选择。更多还原

【Abstract】 The femoral neck fracture is a clinically common skeletal injury,accounting for about3.58%of all fractures and50%of the proximal femurfractures. Nonunion and avascular necrosis of femoral head are the mainproblems in treatment of femoral neck fracture. Up to now, considerableprogress has been made in the treatment of femoral neck fracture, but theguidelines for its management continue to be unsolved and are still evolving.We performed early intervention in femoral neck fracture with the method offree iliac bone graft combined with three cannulated screws fixation, andachieved satisfying clinical result. Based on the limited clinical experience, itwas demonstrated that this method had obvious advantages of simpleoperation, little truma, etc. Meanwhile, one stage bone grafting had reliablestability and improved the rate of fracture healing, helped to prevent theforward avascular necrosis of femoral head. Therefore, this method may be auseful option for the treatment of femoral neck fracture. However, the boneharvesting operation usually caused big surgical trauma and complications. Toavoid the disadvantages of the bone harvesting operation, we introduced anovel metal stent for early intervention in femoral neck fracture to replacebone graft. The novel metal stent is made of magnesium which can graduallydegrade in vivo and induce new bone formation around. In order to betterevaluate the degradation behavior and biomechanical properties of the novelmetal stent and to provide a theoretical basis for its further optimization andclinical application, the degradation behavior of MAO-Mg in simulated bodyfluid was studied systematically, the effects of magnesium ions on the mainbiological actions of osteoblast in vitro and the corresponding mechanismwere preliminarily discussed and disclosed. Additionally, the three-dimensional finite element model of the novel metal stent used for earlyintervention of femoral neck fracture was established to analyze the stresspatterns during a normal gait cycle, and compared with the method using bonegraft for early intervention of femoral neck fracture.Part1Study of degradation behavior and cytocompatibility for a novelMAO-treated magnesium materialObjective： The degradation behavior of MAO-Mg was studiedsystematically by immersion in simulated body fluid and electrochemical test,and the mechanical performance of MAO-Mg exposed in simulated body fluidwas investigated by tensile test. In addition, the cell toxicity of MAO-Mgduring the corrosion process was evaluated.Methods：Commercially available pure magnesium and MAO-coated Mgwere used in this study. All the samples were first weighed and recordedbefore the test. Immersion test was performed in a standard simulated bodyfluid (SBF) at a pH value of7.4and the temperature was maintained at37±0.5℃. The ratio of surface area to solution volume was1cm2:30ml. Sampleswere removed after6h,1,3,7,14and30days of immersion, rinsed withdistilled water and dried at room temperature. The surface morphology andcomposition of degradation products for the samples were analyzed byscanning electronic microscope (SEM), energy dispersive spectrometer (EDS)and X-ray diffraction analysis (XRD). The degradation rate, magnesium lossrate and the induced changes in pH were examined. Tension tests were carriedout with a universal testing machine to measure the tensile strength ofdifferent samples over time. In addition, electrochemical measurements wereperformed using a three electrode system, potentiodynamic polarization curvesand electrochemical impedance spectroscopy (EIS) of samples were measured,respectively. Lastly, Saos-2cells were cultured in Dulbecco’s modifiedEagle’s medium (DMEM), supplemented with15%fetal bovine serum (FBS).The effects on osteoblast proliferation and viability in the extraction mediumof the MAO-treated and untreated samples were measured by the MTT assay, and the osteoblastic apoptosis in different media was observed by fluorescentstaining. SAS software (version9.1) was used for the statistical analysis.Statistical significance was assigned to P＜0.05.Results：The corrosion rate of MAO-coated Mg in the SBF was muchlower than that of uncoated Mg, and MAO-coated Mg had lower influence onthe pH value variation of the SBF compared with uncoated Mg, the pH roserapidly from7.4to8.7and basically stabilized in the first72h, while the pHfor pure Mg reached9.1at last. The main components of the surface corrosionproducts on these metal samples were magnesium hydroxide [Mg(OH)2] andhydroxyapatite. Moreover, the MAO treatment slowed down the loss ofmechanical properties of the magnesium metal and maintained the strength ofthe sample at about90%after30days of immersion. The impedancespectroscopy and potentiodynamic polarization curves had also demonstratedthat the corrosion resistance ability of the MAO-coated Mg sample was higherthan that of uncoated magnesium. The cell toxicity experiments showed thattwo kinds of magnesium metal samples had not cytotoxic, and reached a levelof biosafety suitable for the cellular applications.Conclusion：The MAO coating could enhance the corrosion resistance oftreated Mg metal and help to keep its original mechanical properties. TheMAO-coated Mg material had good cytocompatibility, and the presence ofhydroxyapatite in the surface corrosion products could improve itsbiocompatibility and bone-binding capacity.Part2Research on the effects of magnesium ions on the biologicalbehaviors of osteoblast and the corresponding mechanismObjective：The effects of magnesium ions on the main biological actionsof osteoblast in vitro were were investigated and the correspondingmechanism were preliminarily discussed and disclosed.Methods：Saos-2cells were cultured in Dulbecco’s modified Eagle’smedium (DMEM), supplemented with15%fetal bovine serum (FBS). Theosteoblast was stimulated with MgSO4at a concentration of1,2,3mmol/L and then the effects of different treatment on osteoblast proliferation weremeasured by the MTT assay, and the osteoblastic apoptosis in different factorwas observed by fluorescent staining. The main biological characterizations ofosteoblast including ALP activation and BGP synthesis stimulated by differentfactors were surveyed with ELISA assay. Additionally, the expressions ofRunx2and Fas of the osteoblast under the stimulation of magnesium ions at1,2,3mmol/L were investigated by ELISA assay. SAS software (version9.1)was used for the statistical analysis on experimental data. Statisticalsignificance was assigned to P＜0.05.Results：The proliferation index (OD value) of the osteoblasts exposed tomagnesium ions at the concentrations of1,2and3mmol/L for24,48and72hours were all significantly higher than those of the blank control group(F=44.77，Р＜0.0001), and the OD value was positively associated with theconcentration of magnesium ions and the time of exposure. The result offluorescent staining showed the osteoblast had good activity under the actionof magnesium ions. In addition, the ALP activity and osteocalcin level ofosteoblasts treated with magnesium ions were stimulated in thedose-dependent and time-dependent manner. Moreover, the expression ofRunx2of the osteoblast under the stimulation of magnesium ions increasedsignificantly, and the expression quantity was positively with theconcentrations of magnesium ions. But the expression of Fas treated withmagnesium ions were significantly lower than that of normal control group(F=16.97，Р＜0.0001), and its expression quantity was inversely associatedwith the concentration of magnesium ions.Conclusion：The magnesium ions could induce the proliferation of theosteoblasts, and stimulate their ALP activity and osteocalcin synthesis, themechanism may be associated with the regulation of expressions of Runx2andFas. The magnesium ions could promote the differentiation and mature of theosteoblast by stimulating the expression of Runx2, on the other hand, itinhibited the osteoblast apoptosis by decreasing the expression of Fas. Part3The establishment of three-dimensional finite element model of thenovel metal stent used for early intervention of femoral neckfracture and the stress analysis during a normal gait cycleObjective：To establish the three-dimensional finite element model of thenovel metal stent used for early intervention of femoral neck fracture, toanalyze the stress patterns during a normal gait cycle, in order to help us tobetter evaluate the design and biomechanical properties of the novel metalstent and to direct its clinical application.Methods：A normal adult male volunteer was selected for the test. Hiship and proximal femurs were scanned by CT to obtain the two-dimensionalcomputer tomography image data, and reconstruct the three-dimensionalmodel of femur using Mimics10.01software. And then, the model wasimported to the Ansys13.0software and meshed to establish thethree-dimensional finite element model of the proximal femur, furthersimulated and established the three-dimensional finite element model of thenovel metal stent used for early intervention of femoral neck fracture. Next,the boundary and loading condition were set to perform the biomechanicalanalysis on these models. SAS software (version9.1) was used for thestatistical analysis on data. Statistical significance was assigned to P＜0.05.Results：Firstly, the precise and effective finite element models of theproximal femur and the novel metal stent were established in this study. Andthen the further analysis demonstrated that the model of femoral neck facturefixation had the similar trend for Von Mises stress to the model of normalfemur during a normal gait cycle, and the values of stress and displacementbetween the two models had no significant difference. It was also found thatthe magnitudes of the axial displacement in the model of femoral neck facturefixation increased with the hip joint load, and the maximal displacement waspresented in the second phase of the normal gait cycle.Conclusion：It was showed that the finite element models of the proximalfemur established in this study was objective and effective, and which laid the foundation for the further related studies. In addition, the method of the novelmetal stent implantation combined with three cannulated screws for earlyintervention of femoral neck fracture could not only recover the stressconduction of femoral neck but also better resist compression, torsion, andshear and increase the stability of fracture. Thus this study had a certainguiding significance to the clinical application of the novel metal stent andprovided a theoretical basis for its further optimization.Part4The comparative analysis of a novel metal stent against bone graftused for early intervention of femoral neck fracture. A study offinite element analysisObjective：The purpose of this study was to evaluate the stress patterns ofthe novel metal stent and bone graft used in the early intervention of femoralneck fracture, and to compare with the method of three cannulated screwsfixation commonly used in clinic. Moreover, the implantation angle of thenovel metal stent or bone graft was analyzed. The study contributed tocomprehensively understanding on biomechanical properties of the novelmethod and providing a theoretical guidance for its further clinical application.Methods：Finite element models of the novel metal stent and bone graftused in the early intervention of femoral neck fracture were created accordingto the same way mentioned in the third test. The implantation angle of thenovel metal stent or bone graft was set95°and127°, respectively. Moreover,the finite element models of three cannulated screws fixation for femoral neckfracture was also established. The boundary condition and two loads of426Nand2158N were set to perform the biomechanical analysis on these models.SAS software (version9.1) was used for the statistical analysis on data.Statistical significance was assigned to P＜0.05.Results：The finite element analysis demonstrated that the levels of stressand displacement of femoral neck had no significant difference among threekinds of models. In case of the novel metal stent or bone graft, the change ofthe implantation angle from95°to127°did not affect on the stress of femoral neck. And the stress and the axial displacement between the model ofthe novel metal stent and the model of bone graft had also no significantdifference.Conclusion：It was found that the novel metal stent could also be usedfor the early intervention of femoral neck fracture with good stability offracture compared to using bone graft. The implantation angle of the metalstent could be adjusted as necessary. Based on results of this study and its owninherent advantages, it was concluded that the novel metal stent could be areliable option for the early intervention of femoral neck fracture.更多还原