【作者】 范纯泉；

【导师】 李家顺； 叶晓健；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 脊柱融合术是脊柱外科最常见的手术之一,主要用于治疗由于创伤、退变、畸形、肿瘤等疾病导致的脊柱不稳。虽然内固定的使用为脊柱提供了即刻稳定性,确保了植骨融合所需的生物力学环境,但脊柱的长期稳定及生物学愈合还必须要依靠骨性融合。在目前临床应用的脊柱融合材料中,自体骨移植通常被认为是“金标准”,但是自体骨来源有限,而且增加了手术部位,并伴有取骨区疼痛、神经损伤、骨折、感染等并发症,异体骨有免疫源性,而且存在传染疾病的潜在风险。因此,需要寻找理想的人工骨移植修复材料来促进脊柱融合。现有的人工骨修复材料：金属材料,高分子材料、生物陶瓷材料等存在各自优缺点,单一材料都不能满足临床的需求,所以,目前研究的方向是利用不同材料的优点,克服各自不足,制备具有良好性能的复合生物材料。丝素蛋白是一类由蚕丝和蜘蛛丝中提取的蛋白,与骨骼中的胶原蛋白相似,良好的生物相容性,生物安全性,优越的力学性能,并且丝素蛋白表面容易化学共价修饰粘附位点和细胞因子,具有一定的生物降解性能,降解产物无毒副作用,对周围组织有营养与修复作用,同时材料的来源广阔,因此是一种具有广阔应用前景的生物材料,它作为临床外科缝线已经有悠久的历史,目前在载药系统以及组织工程方面的应用研究很多,在骨科领域,包括骨支架,软骨支架以及人工肌腱和韧带等方面都有相关研究。近年来国外有报道丝素蛋白能够作为无机物矿化的模板,诱导羟基磷灰石的晶体生长,其与纳米羟基磷灰石的复合可以模拟天然骨组织中的有机无机成分,可以弥补单纯纳米羟基磷灰石力学性能的不足,因此丝素蛋白与纳米羟基磷灰石复合的生物材料有望成为理想的骨移植修复材料。本实验以丝素蛋白和钙磷溶液为原料,通过共沉淀法仿生合成了丝素蛋白／纳米羟基磷灰石类骨质复合材料,实现复合材料在结构和组成上与天然骨组织的仿生,然后对该复合材料的成骨细胞相容性进行评价,并进一步通过丝素蛋白／纳米羟基磷灰石复合材料与自体骨髓基质细胞复合用于兔腰椎后外侧融合模型观察其骨融合效果,为探索一种可应用于促进脊柱融合,满足临床需求的的骨修复材料奠定实验基础。实验第一部分：丝素蛋白／纳米羟基磷灰石复合材料的仿生制备及表征目的：模拟骨组织的结构和成分,仿生合成丝素蛋白／纳米羟基磷灰石复合材料。方法：以氯化钙和磷酸氢二铵为原料,丝素蛋白作为调控纳米羟基磷灰石晶体生长的模板,通过共沉淀法合成了丝素蛋白／纳米羟基磷灰石类骨质复合材料。由于骨组织有机/无机为30/70比例,选用nHA含量为70%时制备的丝素蛋白／纳米羟基磷灰石复合材料作为研究对象。通过透射电镜(TEM)、X射线衍射(XRD)、傅立叶傅立叶变换红外光谱仪(FTIR)、热重分析仪(TGA)分析复合材料的晶相组成、微观形貌、化学结构和热稳定性。结果：通过FTIR、XRD、TEM、TGA及电子衍射等检测结果显示,制备的SF/nHA复合生物材料中丝素蛋白含量为32%,模拟了天然骨组织中的有机成分比例；钙磷比为1.66,与标准羟基磷灰石的钙磷比1.67相近,获得的磷灰石晶体为含有碳酸根的低结晶度纳米羟基磷灰石,复合材料中的HA晶体长度约200-500nm左右,宽度在20-30nm,与天然骨组织中的磷灰石晶体相似；获得的SF/nHA复合生物材料中,nHA的钙离子与丝素蛋白的羰基通过强烈的化学键合作用形成了热稳定的复合物；另外,研究结果表明,丝素蛋白作为定向诱导HA晶体生长的模板,调控纳米羟基磷灰石晶体沿C轴择优取向生长,生长方向与丝素蛋白纤维长轴平行,与天然骨组织中磷灰石晶体在胶原纤维表面沉积的结构一致。结论：通过共沉淀技术成功仿生合成了在组成和结构上均与天然骨组织结构类似的丝素蛋白／纳米羟基磷灰石类骨质复合材料。实验第二部分：丝素蛋白／纳米羟基磷灰石复合材料的成骨细胞相容性研究目的：评价丝素蛋白／纳米羟基磷灰石复合材料与骨组织功能细胞-成骨细胞的相容性。方法：采用体外细胞培养技术,将MC3T3-E1成骨细胞接种在复合材料上进行共培养,用倒置显微镜及扫描电镜观察细胞在材料上的粘附及增殖情况,用茜素红染色、Von Kossa染色等检测成骨细胞分泌细胞外钙基质的能力。结果：丝素蛋白／纳米羟基磷灰石复合材料可以促进MC3T3-E1成骨细胞的粘附、增殖,促进成骨细胞分泌细胞外基质和形成钙结节。结论：丝素蛋白／纳米羟基磷灰石复合材料具有良好的成骨细胞相容性。实验第三部分：丝素蛋白／纳米羟基磷灰石复合材料复合自体骨髓基质细胞促进兔脊柱后外侧融合的实验研究。目的：了解丝素蛋白／纳米羟基磷灰石促进兔脊柱融合的能力。方法：抽取兔子股骨大粗隆下骨髓,体外分离纯化得到兔骨髓基质细胞,进行成骨方向诱导分化,行细胞形态学观察、细胞表面抗原鉴定及碱性磷酸酶染色和Von Kossa染色评价细胞成骨情况。将丝素蛋白／纳米羟基磷灰石复合材料与骨髓基质细胞复合后应用于新西兰兔腰椎后外侧脊柱融合模型,通过大体观察、影像学观察及组织学观察、手法扪诊检查评价成骨及脊柱融合情况。结果：1、BMSCs在培养皿中贴壁生长、增殖,条件培养液诱导后表现出明显的成骨活性,细胞增殖良好,体外矿化结节Von Kossa染色阳性、碱性磷酸酶染色阳性,证实其有成骨潜能。2、术后12周取材。大体观察可见SF/nHA侧骨组织再生明显,新生骨体积大,X线、手法扪诊均提示SF/nHA侧促进脊柱融合的作用与自体骨相似,组织学分析提示SF/nHA复合材料逐渐转化为骨组织。结论：1、可以通过体外分离纯化兔BMSCs,在一定条件下能够向成骨方向诱导分化,并能使细胞保持较高的成骨活性,适于作为材料相容性的检测细胞及构建组织工程化骨的种子细胞。2、SF/nHA复合材料复合自体骨髓基质细胞植入新西兰兔腰椎后外侧,能够获得良好的脊柱融合效果,其脊柱融合效果与自体移植骨相似。更多还原

【Abstract】 Spinal fusion is one of the most common spinal surgery primarily for treatment of spinal instability caused by trauma,degeneration,deformity,cancer and other spine diseases.internal fixation is used to provide immediate stability of the spine, providing the necessary biomechanical fusion environment,however,The long-term stability and biological healing of the spine must rely on bony fusion. In the current clinical use of spinal fusion materials, autogenous bone grafting is generally considered the "gold standard", the major disadvantages of autograft are the limited amount of graft material available as the potential morbidity of the harvest complications including prolonged pain at the donor site,nerve damage,fracture and infection.bone allograft has also been used,its disadvantages include immune response and potential risk of infectious transmission.therefore,we are looking for the ideal bone grafting biomaterial to promote spinal fusion.the bone substitutes currently available have specific disadvantages and none of them is entirely suitable for clinical application including metals,polymer and ceramics.so,current researth is focusing on how to combine the advantages of various material,overcome their shortcoming,to preparation of composite biomaterials with excellent performance.Silk fibroin has excellent biocompatibility, biodegradability and unique mechanical properties,and can induce hydroxyapatite crystal nucleation and growth as the template of inorganic mineralization.silk fibroin/nano-hydroxyapatite composite biomaterial simulates the composition and structure of nature bone tissue,improve nano-hydroxyapatites insufficient mechanical properties,and so is expected to be an ideal bone graft biomaterial.In this experiment, biomimetic synthesis of silk fibroin/nano-hydroxyapatite like-bone composite biomaterial is accomplished by coprecipitation method using silk fibroin,CaC12 and (NH4)2HP04 as raw materials,mimicing the organic and inorganic constituents of natural tissue.after that,the biological safety and biocompatibility with osteoblasts are evaluated, and the degradation performance and ectopic bone formation capacity of silk fibroin/nano-hydroxyapatite composite biomaterial is studied by putting it into bags of rabbit muscle.further,we evaluate the effect of silk fibroin/nano-hydroxyapatite material compound autogenous bone marrow stromal cells on the enhangcement of rabbit spinal fusion,in order to explore an ideal bone repair material to meet clinical need.Part 1:Biomimetic synthesis and characterization of silk fibroin/nano-hydroxyapatite composite biomaterialObjective:Simulating the composition and structure of nature bone tissue,biomimetic synthesis of silk fibroin/nano-hydroxyapatite composite.Method:silk fibroin/nano-hydroxyapetite composite biomaterial is synthesized by the coprecipitation method using silk fibroin,CaC12 and (NH4)2HP04 as raw materials.as organic/inorganic components ratio in nature bone is 30/70,composites biomaterial with 70% nano-hydroxyapatite are selected for further study.the microstructure,phase composition,chemical structure and Thermal stability are determined by scanning electron microscopy(SEM),transmission electron microscopy(TEM),X-ray diffraction(XRD), Fourier Fourier transform infrared spectrometer (FTIR), thermal gravimetric analysis (TGA) and so on.Results:The as-prepared SF/nHA composites contain silk fibroin 32%,simulating the proportion of organic ingradients in natural bone tissue,with calcium phosphorus ratio of 1.66,similar to the standard calcium phosphate ratio in hydroxyapatite. Characterization results show that the inorganic component in the composite is poorly crystalline HA containing carbonate ions. The typical HA crystallites possess 200-500 nm in length and around 20-30 nm in width. The SF is incorporated into the composite by strong chemical interactions between HA and SF, probably take place via the chemical bonding between calcium ions and the carbonyl groups of SF. the silk fibroin will induce self-organized orientation of HA nanocrystal as the template, the growth of HA crystal will progress spontaneously with preferred orientation (along its c-axis),which.is similar to the apatite crystals deposited on the surface of collagen fibers in natural bone tissue.Conclusion:Like-bone silk fibroin/nano-hydroxyapatites composite biomaterial is synthesized by the coprecipitation method,the composition and structure is similar to the nature bone tissue.Part 2:A study on the biocompatibility of SF/nHA composite biomaterial cocultureed with rat osteoblasts.Objective:to evaluate the biocompatibility of SF/nHA composite biomaterial with bone tissue functional cells-osteoblasts.Methods:MC3T3-E1 osteoblasts were seeded on the SF/nHA composite and co-cultured.the adherent cell number and morphological changes of oateoblasts were observed by scanning electron microscope.the adhesion and proliferation were determined by inverted microscope and scanning electron microscope,the cells were also stained for matrix minerlization.Results:the adherent number of MC3T3-E1 osteoblasts on the sueface of SF/nHA composites are higher than that of control,and the osteoblasts are more easily to adhere and spread on SF/nHA,the proliferation mineralization of steoblasts was improved also.Conlusion:biomimetic synthesized SF/nHA composite biomaterial has excellent biocompatibility for osteoblasts.Part 3:the study on the enhancement of rabbit Posterolateral spinal fusion with silk fibroin/nano-hydroxyapatite composite biomaterial compound autogenous bone marrow stromal cells. Objective:To evaluate the effect of the SF/nHA composite biomaterial on the enhancement of posterolateral spinal fusion in rabbit. Methods:Obtain bone marrow from Rabbit beneath the greater femur trochanter, Isolate and purify BMSCs in vitro and then induce BMSCs in osteogenic differentiation.observe cell’s morphology,identify cell surface antigen,evaluate cell’s ossification by alkali phosphatase stain and Von Kossa stain. SF/nHA composite biomaterial compound BMSCs were applied to New-Zealand rabbit Posterolateral lumbar spine fusion model. Gross and imaging observation,histological examination and manual palpation of the fusion masses were performed to evaluate the ability of enhancement of spinal fusion. Results:1.BMSCs cultured in the culture dish has good performance of adhesion,growth,proliferation,and showed good significant osteogenic activity after osteogenic induction..the results of Von Kossa stain and alkali phosphatase stain indicated BMSCs has osteogenesis potential.2. The rabbits were sacrificed at 12 weeks after the fusion surgery, and the samples were obtained.gross observation showed that there is obsvious new bone regeneration on the silk fibroin/nano-hydroxyapatite composite biomaterial sides similar to the autografted sides.X-ray,and palpation also evidenced the results.hietological examination demonstrated that SF/nHA composite biomaterial transformed into bone tissue gradually. Conclusions:1.purified rabbit BMSCs in vitro can been induced and differentiated into osteoblasts with high osteogenic activity,can satisfy the requirement of seeding cell for bone tissue engineering research and biocompatibility study.2.slik fibroin/nano-hydroxyapatite composite biomaterial used for rabbit posterolateral lumbar spinal fusion can effectively enhance bony fusion, similar to autografts.更多还原