【作者】 丁涛；

【导师】 杨惠林；

【作者基本信息】 苏州大学 ， 骨外科学， 2010， 博士

【摘要】 第一部分丝素蛋白/磷酸钙骨水泥的制备、抗稀散性及体外凝血实验研究【目的】制备丝素蛋白/磷酸钙骨水泥(silk fibroin/calcium phosphate cement,SF/CPC)复合人工骨材料,对骨水泥在液体中的抗稀散性进行测试,试验不同SF添加质量分数对稳定性的影响,同时测试PMMA、CPC和SF/CPC对离体动物血浆凝血指标的影响,为下一部分的动物体内实验提供参照。【方法】将家蚕丝纤维脱胶、溶解、透析、喷雾干燥后制得丝素蛋白粉末。通过液相沉淀法制备磷酸四钙,将其和无水磷酸氢钙等摩尔比混合,加入4 wt%羟基磷灰石晶种。配制质量分数分别从0.5 wt%、1.0 wt%、1.5 wt%、2.0 wt%、2.5 wt%到3.0 wt%的丝素复合磷酸钙骨水泥6组,按液固比0.4ml/g与固化液混合,制备6种SF/CPC,未添加SF的CPC组作为空白对照。观察各种材料在液体中的抗稀散性,定量测定骨水泥残余质量百分比。将固化后的PMMA、CPC和SF/CPC骨水泥材料颗粒加入新鲜猪血浆,测定材料对凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血酶时间(TT)、纤维蛋白原定量(Fig)及D-二聚体(D-D)等凝血指标的影响。【结果】由于SF的添加,CPC在液体中的抗稀散性明显提高,随着SF质量分数的提高,SF/CPC的骨水泥残余率逐渐上升,至2.5wt%-3.0wt%时基本达到100%。三种骨水泥作用动物血浆后仅CPC组的PT略有减少,其它凝血指标未见明显改变。【结论】丝素蛋白的添加可以明显改善磷酸钙骨水泥在液体中易崩解的特性,SF/CPC的抗稀散性与添加SF的质量分数有同增关系,兼顾稳定性与注射性,选择2.5wt%的SF/CPC进行后续的研究。骨水泥材料对体外血浆的凝血指标影响很小,仅观察到CPC使PT有轻微的缩短,丝素的添加可进一步提高CPC的血液相容性。第二部分丝素蛋白/磷酸钙骨水泥在骨水泥肺栓塞动物模型中的测试研究【目的】建立骨水泥肺栓塞动物模型,研究PMMA、CPC及SF/CPC三种材料造成肺栓塞后,对心血管系统的血流动力学、肺氧合及抗凝血酶活性的影响,探讨丝素改良骨水泥材料降低心血管系统并发症的可行性。【方法】实验动物麻醉后气管插管,呼吸机保持正常的呼气末潮气量和动脉CO2浓度。下肢股动脉置入测压管,供监测有创动脉血压和采集血标本;经颈内静脉置入肺动脉漂浮导管测量肺动脉压及中心静脉压。动物行开胸手术,经肺动脉干注入PMMA、CPC或SF/CPC骨水泥,持续监测血流动力学参数,包括平均肺动脉压(mean pulmonary arterial pressure,MPAP)、平均动脉压(mean arterial pressure,MABP)、中心静脉压(central venous pressure,CVP)和心率(heart rate,HR) ;测定预设时间点的动脉血PH值、动脉血氧分压(PaO2)和动脉血二氧化碳分压(PaCO2)以及注射前后抗凝血酶Ⅲ活性(ATⅢ)。处死后肺标本行CT检查,立体三维重建血管内骨水泥铸形。【结果】CPC对心血管系统的影响最为严重,平均肺动脉压的升高最大时为21.33±5.44mmHg(注射后50分钟),平均动脉压下降最大时为-27.15±11.79mmHg(注射后50分钟),在注入骨水泥10分钟时出现心率的瞬时下降(-27.67±6.14次/分)。PMMA组及SF/CPC组的变化较轻微,与CPC组比较差异有统计学意义。注射骨水泥后CPC组血气指标改变明显,PH值下降,变化最大时-0.13±0.02(注射后60分钟),动脉血二氧化碳分压(PaCO2)升高,变化最大时15.9±7.67mmHg(注射后60分钟),注射后10分钟动脉血氧分压(PaO2)即已有明显下降(-23±1.26mmHg); PMMA及SF/CPC对肺栓塞动物动脉血气的影响较小。AT-III在三组均有下降,CPC组降幅最大,与另两组间差异有统计学意义。【结论】注射型骨水泥直接注入动物肺动脉干可建立椎体成形术中骨水泥血管渗漏致肺栓塞模型,对PMMA、CPC及丝素复合CPC三种材料进行的测试比较结果证明,骨水泥的抗稀散性对肺栓塞后心血管并发症严重程度起主要影响作用。丝素的复合使CPC在血液中的抗稀散性明显提高,有效地减少了栓子特别是微栓子的产生,能明显减轻骨水泥栓塞后对血流动力学、呼吸功能的影响,降低激活凝血系统的危险,从而降低急性心肺功能障碍的发生率和死亡率。第三部分丝素蛋白/磷酸钙骨水泥对藻酸盐凝胶培养系统中椎间盘细胞活性、基质多糖及胶原代谢影响的实验研究【目的】研究PMMA、CPC和SF/CPC三种骨水泥材料浸出培养液对藻酸盐凝胶培养系统中椎间盘细胞活性、基质多糖和胶原代谢的影响。【方法】兔椎间盘标本取出后,在藻酸盐凝胶系统中分别使用常规培养液(对照组)、PMMA、CPC及SF/CPC材料浸出培养液培养,至1周、2周及3周时,对各组标本行Hoechst33342和PI染色检测细胞坏死率,免疫组化检测I型胶原、Ⅱ型胶原的表达,番红O(Safranin O)染色检测基质蛋白多糖含量。在荧光显微镜下计数Hoechst33342和PI染色切片中强红色荧光+强蓝色荧光重叠的细胞数占总细胞数的百分比;利用Image-pro plus 6.0软件对免疫组化I型胶原、Ⅱ型胶原染色切片中棕黄色阳性沉淀及番红O染色切片中番红色部分作平均光密度(IOD/Area)分析。【结果】各周PMMA组及CPC组坏死细胞率均高于对照组及SF/CPC组,第一周时SF/CPC组坏死细胞率明显低于PMMA组和CPC组(P<0.05),第三周时SF/CPC组坏死细胞率与PMMA组无统计学差异,但明显低于CPC组(P<0.05)。纤维环外层Ⅰ型胶原光密度测定结果显示,第一周时PMMA组光密度值明显低于其他组,第二周、第三周时CPC组及SF/CPC组较对照组有升高。纤维环内层及髓核区II型胶原光密度测定结果显示,第二周时,PMMA及CPC组明显低于SF/CPC组和对照组,SF/CPC组近似于对照组。Safranin O染色光密度结果显示,对照组平均光密度值随时间呈下降趋势,PMMA组基质多糖含量下降最早且最为明显。【结论】PMMA对藻酸盐凝胶系统中培养的椎间盘细胞活性、基质胶原及多糖代谢影响最为明显,SF/CPC较CPC对椎间盘组织有更好的生物相容性,对椎间盘代谢的干扰最小。椎体成形术所用骨水泥材料可加速椎间盘的退变,影响程度与材料的类型有关,选择适当的填充材料可降低此并发症的发生率。第四部分丝素蛋白/磷酸钙骨水泥对椎间盘基因表达影响的实验研究【目的】研究骨水泥材料对椎间盘退变相关基因表达的影响,从基因水平分析椎体成形术后椎间盘退变的机理,为预防和治疗措施提供靶基因及作用机制等理论基础。【方法】建立藻酸盐凝胶系统对兔椎间盘进行体外培养,分别以常规培养液(对照组)、PMMA、CPC及SF/CPC材料的浸出培养液培养,至1周、2周时提取各组标本RNA行PCR扩增和产物检测以及SYBR? Green I荧光定量PCR检测,对AGC1、COLⅠ、COLⅡ、BMP-2、IL-1β、TIMP-1、MMP-3、MMP-2、SOX-9、TGF-β1、Cox-2在各组的表达水平进行比较,通过内标基因GAPDH和对照组基因对目的基因进行校正,2-△△CT法估算目的基因的相对表达量。【结果】第一周时PMMA组AGC1的表达量低于对照组,CPC组及SF/CPC组均高于对照组,第二周时SF/CPC组表达量仍保持较高水平;各骨水泥组COL1的表达量在第一周时较对照组均有反应性的调高,第二周回落仅SF/CPC组表达量仍能保持与对照组接近;各骨水泥组COL2基因表达在第一周时均未测出,第二周时虽可测出但均极低;第一周时PMMA组及CPC组MMP-2、MMP-3的表达量均高于对照组,第二周时CPC组表达量下降,SF/CPC组表达则始终低于对照组;第一周时PMMA组和CPC组的TIMP-1的表达量增高,第二周时各组表达量较第一周时均有下降;各骨水泥组IL-1β的表达在第一周及第二周均高于对照组,第二周时SF/CPC组的表达低于PMMA组和CPC组;CPC组COX-2的表达量高于其他组;第一周时大部分骨水泥组TGF-β1、BMP-2及SOX9表达量高于对照组,第二周时均有不同程度的下降,TGF-β1和SOX9的表达低于对照组。【结论】骨水泥材料对椎间盘退变相关基因表达量有影响,能促使体外培养的椎间盘退变,但椎间盘组织本身的保护和修复机制也有相应的反应性激活。不同类型的材料对特定基因表达的影响存在差异,PMMA和CPC使COL1基因表达短暂反应性增高后降至正常对照以下,而SF/CPC作用下COL1基因的表达仍能保持一定水平, PMMA对AGC1的表达抑制作用强,SF/CPC未引起基质降解相关基因MMP-2和MMP-3的表达上升,对炎症因子基因表达增高的刺激作用较PMMA和CPC组小。在影响基因表达水平上,SF/CPC促椎间盘退变的作用最小。更多还原

【Abstract】 PartⅠ: Preparation of Silk Fibroin/Calcium Phosphate Cement (SF/CPC) Composite, Study of Washout resistance and coagulation test in vitro【Objective】To prepare a novel bone substitute of silk fibroin/calcium phosphate cement (SF/CPC) composite, and to explore the influences of silk fibroin and its fraction on the washout resistance of CPC in fluids. The effects of cements including PMMA, CPC and SF/CPC on the plasmatic phase of coagulation were assayed for further animal experiments.【Methods】The loosened refined silk fiber were dissolved, dialyzed and spray dried to extract SF powder. Tetracalcium phosphate (TTCP) was prepared with liquid phase precipitation method, then mixed with dicalcium phosphate anhydrous (DCPA) at a mole ratio of 1:1, hydroxyapatite (HA) was also added with 4 wt%. Six groups were set according to the concentrations of SF powder in the solid phase of CPC: 0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt% respectively. Then the solid phase blended with the liquid phase with a ratio of 0.4ml/g to prepare the cement composite. CPC free from SF served as control. Washout behaviour was observed and percent remaining cement was measured to determine the stability of samples in the wet environment. After cements curing, particles were contacted with the fresh porcine plasma, after which the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen quantitation (Fig) and D-dipolymer were assayed.【Results】With the addition of SF, the washout resistance of CPC in fluids were highly developed. Percentage of remaining cement increased with the mass fraction of SF and reached approximately 100% in SF/CPC composites with 2.5 wt% and 3.0 wt% of SF. Only a slight decrease of PT value in CPC group occurred after the plasma in contact with three kinds of cements, without any significant change in other coagulation parameters.【Conclusions】The incorporation of SF remarkably improved the anti-decay properties of CPC in an aqueous environment. The washout resistance ability increased with the mass fraction of SF. In consideration of both stability and injectability, SF/CPC containing 2.5 wt% SF served as SF/CPC object in the following research. PMMA, CPC and SF/CPC barely influence the coagulation parameters in vitro. Only a slight decrease of PT value was observed in CPC group compared to control. The incorporation of SF further develops the blood compatibility of CPC.PartⅡ: Test of SF/CPC using an animal pulmonary cement embolism model【Objective】To develop an animal pulmonary cement embolism model, by which the effect of cements on the hemodynamic change, respiratory function and the antithrombin activity were measured after the pulmonary embolism caused by cement injection. The efficiency of SF reducing the risk of cardiovascular complication was evaluated.【Methods】After general anesthesia, animals were endotracheal intubated and mechanically ventilated by a respirator. A normal tidal volume and a normal arterial carbon dioxide were maintained during the experiment. Fluid-filled catheter was inserted into abdominal aorta via the left femoral artery for the monitoring of arterial blood pressure and blood sampling. A pulmonary catheter was placed into the pulmonary artery through the right jugular vein for the measurement of mean pulmonary arterial pressure and central venous pressure. The pulmonary trunk was exposed via a median sternotomy. Cement was injected into the main pulmonary artery via a venous catheter. The following hemodynamics were measured and analyzed: mean arterial blood pressure (MABP, mmHg), mean central venous pressure (MCVP, mmHg), mean pulmonary arterial pressure (MPVP, mmHg) and heart rate (HR, beats/min). Blood samples were drawn at each time point for blood gases analyzing, and pre- and post-injection for measurement of AT III. Postmortem lungs were subject to computer tomography (CT) examination along with three-dimensional reconstructions of cement casts.【Results】CPC was showed to have the most severe influence upon cardiovascular system, with a maximum elevation of 21.33±5.44mmHg in PAP, a maximum decrease of -27.15±11.79mmHg in MABP. At the 10 min after injection, a transient decrease (-27.7±6.17 beats/min) of HR was observed in CPC group. The changes in PMMA and SF/CPC group were relatively slight with a statistically significant difference from CPC groups. Blood gases revealed significant hypercarbia, acidemia and hypoxemia in CPC group after injection, while these impairments of respiratory function were mild in PMMA and SF/CPC group. There were a largest change of -0.13±0.02 in PH value (60 min after injection), 15.9±7.67mmHg in partial pressure of arterial carbon dioxide (60 min after injection) and -23±1.26mmHg in arterial oxygen tension (10 min after injection) in CPC group. Although all dropped, the decline degree of AT III activity level was the most in CPC group with statistically significant (p<0.05) difference between other two groups.【Conclusions】The pulmonary cement embolism model in animals were successfully developed by directly injecting cements into the pulmonary arterial trunk. The results indicated that, the washout resistance properties of cements significantly influenced the severity of cardiovascular complications post pulmonary embolism. The incorporation of SF evidently improved the stability of CPC that effectively reduced the formation of embolus especially the micro-embolus. The effect on hemodynamic changes and respiratory function were well reduced by the addition of SF, as well as a decreased risk of stimulation of clotting system. The rate of acute cardiovascular deterioration and mortality were therefore decreased.PartⅢ: The influence of SF/CPC on cell viability, metabolism of proteoglycan and collagen of the intervertebral disc cultured in an alginate gel system【Objective】To investigate the influence of SF/CPC, CPC and PMMA on cell viability, metabolism of proteoglycan and collagen of the intervertebral disc cultured in the alginate gel system.【Methods】The discs were taken from rabbits and cultured in alginate gel system with either ordinary complete medium (control group) or complete medium in which cements samples had been extracted. At 1w, 2w and 3w, cell viability was measured by Hoechst 33342–PI cocktail nuclear staining; the expressions of type I, II collagen were detected using immunohistochemistry; proteoglycan contant measurement were done using Safranin O staining. After Hoechst 33342–PI cocktail nuclear staining, the number of cells with both bright blue and bright red were counted under fluorescence microscope in separate fields of view per slide and expressed as percentage of the total cells counted. Integrated option density (IOD) of brown DAB deposits staining type I, II collagen were measured quantitatively using Image Pro Plus software; Integrated option density of orange-red part in safranin O stained sections were also analyzed.【Results】The percentage of dead cells in PMMA group and CPC group were significantly higher than that of control and SF/CPC group. The population of dead cells in SF/CPC was lower than that either in PMMA on in CPC group, which was statistically significant at 1w (week). The difference was not significant on the population of dead cells between SF/CPC group and PMMA group, but between SF/CPC group and CPC group at 3w (P<0.05). The percentage of dead cells was and 2w (P<0.05). The results of the IOD measurement of type I collagen staining in the outer layer of the anulus fibrosus showed that, the value in PMMA group at 1w was prominently lower that other groups; at 2w and 3w, the values in CPC and SF/CPC group were higher than control. The results of the IOD measurement of type II collagen staining in the inner layer of the anulus fibrosus and nucleus pulposus field showed that, the values of PMMA and CPC group were markedly lower than SF/CPC group; at 3w, there was some restore in CPC group, the value of SF/CPC group was still equal to the control. The results of the IOD measurement of Safranin O staining showed that, even in the control group the IOD value kept declining, the decline rate was the most in PMMA group.【Conclusion】PMMA significantly inhibits the cell viability, metabolism of proteoglycan and collagen of the intervertebral disc cultured in the alginate gel system. Compared to CPC, SF/CPC presents a better biocompatibility with intervertebral disc. The bone cements used in vertebroplasty might accelerate the degeneration of disc with different degree depending on the cement type. Appropriate material should be selected to eliminate the incidence of this complication.PartⅣ: Influence of SF/CPC on the genes expression of intervertebral disc【Objective】To study the influence of SF/CPC, CPC and PMMA on the degeneration related genes expression of intervertebral disc. To explore the mechanism of the degeneration of intervertebral disc after vertebroplasty at a gene level, which will apply the theoretical basis on target genes and interaction mechanisms for the prevention and treatment.【Methods】The discs were taken from rabbits and cultured in alginate gel system with complete medium in which cements samples had been extracted. At 1w and 2w, the mRNA were isolated and a reverse transcription–competitive polymerase chain reaction (RT–PCR) technique was used to measure expression of products, along with the quantitative real-time RT-PCR using SYBR? Green I fluorescent dye to quantitatively detect and compare the expression of AGC1、CollagenⅡ、CollagenⅠ、BMP-2、IL-1β、TIMP-1、MMP-3、MMP-2、SOX-9、TGF-β1、Cox-2 between groups. The housekeeper gene GAPDH was used to calibrate the target gene and the relative expression of genes were calculated using 2-△△CT method.【Results】At 1w, the expression of AGC1 in PMMA group was lower than control while were higher in CPC and SF/CPC group. The expression of AGC1 kept elevated in SF/CPC group at 2w. At 1w, the expression of COL1 in all cements group increased reactively. At 2w all felt back except which in SF/CPC group still keeping similar to control. COL2 did not express in detectable amounts in all cements group at 1w and although detectable at 2w was extremely low. At 1w, the expression of MMP-2 and MMP-3 in PMMA and CPC groups were both lower than control and at 2w were expressed in reduced amounts in CPC group, while was expressed in lower amount in SF/CPC group. TIMP-1 expression was stimulated at 1w and declined at 2w in all cements groups. IL-1βoverexpressed in all cements group all the time while the value of SF/CPC group was lower than others at 2w. CPC group showed a higher expression of COX-2 Compared with others. The expression of TGF-β1, BMP-2 and SOX9 were higher than control at 1w and decline back in different degree with the expression of TGF-β1 and SOX9 being lower than control.【Conclusion】The bone cements materials have effect on the degeneration related genes expression of intervertebral disc. The effect may accelerate the degeneration process of disc cultured in vitro, while also stimulates the self protective and self healing function. The cements differ from each other on the expression of certain gene. The expression of COL1 drop after a transient reactive increase in PMMA and CPC groups while keep at a fair level in SF/CPC group. AGC1 is sensitive to PMMA characterizing with a down regulation. The expression of matrix degradation relative genes such as MMP-2 and MMP-3 are not elevated by SF/CPC, which also has a less influence on the same elevating of proinflammatory genes with PMMA and CPC group. At the gene level, SF/CPC has the lowest effect on the degeneration of disc.更多还原