【作者】 陈晓庆；

【导师】 杨惠林；

【作者基本信息】 苏州大学 ， 骨外科学， 2009， 博士

【摘要】 第一部分丝素蛋白/磷酸钙骨水泥的制备及其理化性能研究【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,观察丝素蛋白含量对复合材料抗压强度、凝固时间及注射性能的影响,找出含合适比例SF的SF/CPC,满足临床实验研究要求。【方法】将家蚕丝纤维脱胶,溶于CaCl₂·CH₃CH₂OH·H₂O（摩尔比1:2:8）三元溶液,透析后喷雾干燥,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。设定6个组别,分别在磷酸钙固相粉末中加入0.5 wt%、1.0 wt%、1.5 wt%、2.0 wt%、2.5 wt%、3.0 wt%质量比例的丝素蛋白,按液固比0.4ml/g与固化液混合,制备6种SF/CPC。以单纯磷酸钙为实验对照组,测定6种复合材料的抗压强度、凝固时间及注射性能。扫描电镜观察材料断面形貌,X-射线衍射仪分析材料成分。【结果】随着SF量的增加,SF/CPC的抗压强度呈现上升的趋势,凝固时间逐步缩短,注射系数也随之下降。扫描电镜发现,SF呈网状,贯穿于CPC晶体间,并将CPC晶体紧密连接。X-射线衍射分析表明,24h后SF/CPC的水化反应基本结束,复合材料最终的成分为羟基磷灰石。【结论】固相中含2.5 wt%SF的SF/CPC拥有良好的抗压强度和凝固时间,并保留了良好的注射性能。SF的加入并不影响CPC的水化产物。因此我们选择这种材料进行后续的体外细胞相容性研究。第二部分丝素蛋白/磷酸钙骨水泥的生物相容性研究【目的】体外分离培养兔骨髓基质干细胞（bone marrow stromal cells,BMSCs）,扩增后与丝素蛋白/磷酸钙骨水泥（SF/CPC）进行体外复合培养,检测SF/CPC的细胞相容性。【方法】抽取新西兰白兔的骨髓5ml,通过密度梯度离心法获取BMSCs,体外贴壁培养、扩增、传代,倒置显微镜下观察原代及传代细胞形态、数量生长情况,描绘生长曲线。将第3代BMSCs,接种到预湿的CPC及SF/CPC材料上复合培养,以单纯BMSCs相同条件下培养作为对照。通过相差显微镜、扫描电镜等方法观察细胞在材料中的生长情况。以MTT法检测材料对细胞增殖活性的影响,以材料浸提液的细胞毒性试验评估材料是否有细胞毒性。【结果】BMSCs在培养皿中贴壁生长、增殖,形态良好,生长旺盛。扫描电镜及光镜观察发现,细胞材料复合后,BMSCs在CPC及SF/CPC上能够良好地粘附和增殖。MTT法测定OD值证实,BMSCs细胞活性不受材料影响,与对照组相比,差异无统计学意义（P＞0.05）。材料浸提液细胞毒性试验显示,细胞毒性分级均为0～Ⅰ级,两种材料浸提液对细胞生长基本无毒性作用。【结论】可以通过体外分离纯化兔BMSCs,扩增后细胞能保持较高的活性,适于作为材料相容性的检测细胞。CPC与SF/CPC对BMSCs的生长和活性无影响,具有良好的细胞相容性,具有体内植入的可行性。第三部分丝素蛋白/磷酸钙骨水泥强化绵羊骨缺损椎体的体外实验研究【目的】制备一种椎体骨缺损模型,评价骨水泥增强后的力学性能,并为体内植入修复椎体骨缺损打下基础。【方法】48个新鲜成年绵羊腰椎单椎体标本,随机分为8组,每组6椎,分别测定骨密度。取4组标本,利用直径分别为φ2.0mm,φ4.0mm,φ6.0mm,φ8.0mm的钻头垂直于椎体矢状面中点钻入椎体侧方,深度为10.0mm。对制备的骨缺损椎体模型进行生物力学测试,测量单椎体的抗压强度和刚度,并与第5组的正常椎体进行比较。根据以上生物力学测试结果,选择一固定直径,将剩下的6、7、8组制成骨缺损模型,然后三组分别注入CPC、SF/CPC及PMMA骨水泥,模拟体液环境固化24小时后进行生物力学测试,以第5组作为对照组进行比较。【结果】不同直径的椎体骨缺损模型中,随着缺损直径增大,其抗压强度与刚度呈逐渐下降趋势。φ6.0mm时差异有统计学意义。填充24小时后,SF/CPC组及PMMA组的抗压强度和刚度与正常椎体组相比没有区别（P＞0.05）,而CPC组的抗压强度及刚度均明显低于正常椎体组（P＜0.05）【结论】缺损直径为φ6mm,深度为10mm绵羊椎体骨缺损是一种合适的椎体骨缺损模型,可用来判断椎体强化剂的体外性能。SF/CPC能有效即时强化骨缺损椎体。由于该模型保持了椎体外形的完整,可以用于进一步的体内实验。第四部分丝素蛋白/磷酸钙骨水泥强化绵羊骨缺损椎体的体内实验研究【目的】探索SF/CPC作为可注射性支撑人工骨的可行性,期望为临床治疗椎体骨缺损提供新的强化剂。【方法】12只成年绵羊,侧方腹膜后入路,在L3、L4、L5椎体建立骨缺损模型,随机顺序植入CPC、SF/CPC、PMMA三种材料。于术后第1、6月分别处死6只绵羊,随机选择其中的2只,将其标本行不脱钙切片,苦味酸-品红染色后对材料-骨界面进行组织学观察,并对CPC、SF/CPC两组的新骨形成和水泥残留进行定量分析。另外4只以L6椎体作为对照组,进行生物力学检测,测量单椎体的抗压强度和刚度。【结果】组织学观察显示:1月时,CPC及SF/CPC组材料和骨形成直接结合,吸收成骨较为表浅;6月时,CPC组的吸收成骨仍仅限于材料表面,而SF/CPC组骨长入明显、材料吸收加快。PMMA和骨之间的结合疏松,部分界面之间存在膜性结构,材料表面无新骨形成,6月和1月相比变化不明显。组织学定量分析显示:6月时,SF/CPC组的新骨生成和水泥残留和CPC组相比差异有统计学差异（P＜0.05）。生物力学测试表明:6月时,CPC组和SF/CPC组椎体的抗压强度和刚度均较1月时有提升,而PMMA组则下降。SF/CPC、PMMA组椎体在两个时间点的抗压强度和刚度均和正常椎体无明显区别（P＞0.05）。【结论】SF/CPC具有良好的生物活性和骨传导性以及相对较快的降解-成骨速度,并在此过程中很好的维持了椎体的力学性能,有望取代PMMA成为一种新的活性椎体强化剂。更多还原

【Abstract】 PartⅠ:Preparation of Silk Fibroin/Calcium Phosphate Cement（SF/CPC） Composite and Study on the Physicochemical Properties【Objective】To prepare a novel bone substitute of silk fibroin/calcium phosphate cement（SF/CPC） composite for bone substitute,and to explore the influences of silk fibroin on the compressive strength,setting time and injectability and select a cement composite with a proper ratio of silk fibroin for further clinical experiments.【Methods】The loosened refined silk fiber were dissolved in the CaCl₂·CH₃CH₂OH·H₂O ternary solution at a mole ratio of 1:2:8 to get a pure SF solution then prepared into SF powder using spray dryer.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.Compressive strength,setting time and injectability were measured among the six groups of cement composite as well as CPC group.The structural characteristics of all groups were observed under scanning electron microscope（SEM）,final products were analyzed using x-ray diffraction analyzer（XRD）.【Results】The compressive strength increased along with the mass fraction of SF powder in the solid phase,while the trend of setting time was opposite.Injectability was also impaired by SF powder;however,it was still higher than 85%with a 2.5 wt%SF/CPC. SEM observation showed SF penetrated throughout CPC crystals,which were tightly connected.The XRD displayed that hydration reaction of SF/CPC almost ended after 24 hours and the final product of SF/CPC was poor crystalline hydroxyapatite.【Conclusions】SF/CPC with a SF mass fraction of 2.5 wt%in its solid phase have a high compressive strength and low setting time,together with an acceptable injectability of more than 85%.The final products of the cement composite were unchanged.It could be suitable for bone substitute application.Thus,we will choose this kind of SF/CPC for the next cytocompatibility study.PartⅡ:Studies on Cellular Compatibility of the SF/CPC【Objective】To evaluate SF/CPC cellular compatibility by using co-culturing of bone marrow stromal cells（BMSCs） from rabbit’s marrow and SF/CPC.【Methods】Primarily cultured BMSCs were isolate from a rabbit’s bone marrow by density gradient centrifugation.The third passage BMSCs were seeded into CPC and SF/CPC for 3 to 5 days.BMSCs alone were cultured at the same condition to act as controls.The cellular morphology and function（attachment,proliferation and differentiation） were assessed separately by means of phase contrast microscope,SEM and MTT assay.The material leaching liquor was used to test cell toxicity.【Results】BMSCs could adhere to CPC and SF/CPC,and proliferate and grow on the surface of the composites normally.The cellular activity and function were not affected by the materials,and no statistical difference was found between the two groups and the control group（P＞0.05）.Cell toxicity test discovered these materials had no toxic effect on BMSCs.【Conclusions】BMSCs could be isolated,cultured and proliferated with active function in vitro.CPC and SF/CPC have a good cellular biocompatibility and can be used as a bone substitute.PartⅢ:Study on sheep vertebral body defect model augmentation with SF/CPC in vitro【Objective】To develop a sheep vertebral body defect model for screening vertebral augmentation filler materials and to evaluate the augmentation effect of SF/CPC in this model in vitro. 【Methods】Forty-eight lumbar vertebrae harvested from ten fresh skeletally mature female sheep cadavers were evenly assigned to eight groups,6 vertebrae each.Bone mineral density（BMD） was examined each group.Four groups of vertebrae were drilled in the perpendicular direction to center of the sagittal plane of the vertebra,with the different drills,φ2.0mm,φ4.0mm,φ6.0mm,φ8.0mm to form a cylindrical bone defect.The depth was 10.0mm,keeping the contralateral cortex intact.The fifth group served as control.All vertebrae were compressed in a material testing system to determine the strength and stiffness.Based on the above test,a certain defect was selected as the vertebral body defect model.The three left groups were prepared into this model,and then augmented by CPC,SF/CPC,PMMA.The strength and stiffness of the three groups were evaluated after setting for 24h in simulated body fluid.【Results】There was no difference in BMD among the eight groups of vertebrae.It was found that the strength and stiffness of the vertebrae with bone defect decreased as the diameter of the bone defect increasing.The compressive strength and stiffness of the vertebrae withφ6.0mm defect was significantly lower in comparison with the control group（P＜0.05）.After augmentation,there was no difference among SF/CPC,PMMA and intact groups（P＞0.05）,while CPC group had a lower value compared with intact group in compressive strength and stiffness（P＜0.05）.【Conclusion】The sheep vertebrae with aφ6.0mm and 10.0mm in depth defect is a suitable model to screen vertebral augmentation filler materials.SF/CPC has a prompt augmentation effect in this model.This model keeps the vertebral figuration intact,so it can be used in further studies in vivo.PartⅣ:Studies on sheep vertebral defect model augmentation with SF/CPC in vivo【Objective】To determine the feasibility of SF/CPC composites as an injectable bone subsitute and investigate the possibility on clinical use as a new vertebral augmentation filler material.【Methods】Twelve adult sheep were operated on left retroperitoneum approach and cavities were made on L3,L4 or L5.CPC,SF/CPC,and PMMA was transplanted to the cavities at random order.Six sheep were killed at 1- and 6-month-time point postoperation, respectively.Two specimens were subjected to un-decalcified sections and stained with更多还原