丝素蛋白/纳米羟基磷灰石复合材料的仿生制备及促进脊柱融合的实验研究
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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。
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 biomaterial
Objective: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.
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。
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 biomaterial
Objective: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.
引文
1. Komori T. Regulation of bone development and maintenance by Runx2. Front Biosci 2008;13:898-903.
2. Adams GB, Scadden DT. The hematopoietic stem cell in its place. Nat Immunol 2006 Apr; 7(4):333-337.
3. Yin T, Li L. The stem cell niches in bone. J Clin Invest 2006 May; 116(5):1195-1201.
4. Huang X, Cho S, Spangrude GJ. Hematopoietic stem cells:generation and self-renewal. Cell Death Differ 2007 Nov;14(11):1851-1859.
5. Arai F, Hirao A, Ohmura M, et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004 Jul 23; 118(2):149-161.
6. Puri MC, Bernstein A. Requirement for the TIE family of receptor tyrosine kinases in adult but not fetal hematopoiesis. Proc Natl Acad Sci U S A 2003 Oct 28;100(22):12753-12758.
7. Borsato KS, Sasaki N. Measurement of partition of stress between mineral and collagen phases in bone using X-ray diffraction techniques. Journal of Biomechanics 1997;30(9):955-957.
8. Knott L, Bailey AJ. Collagen cross-links in mineralizing tissues:A review of their chemistry, function, and clinical relevance. Bone 1998;22(3):181-187.
9. Aigner T, Stove J. Collagens--major component of the physiological cartilage matrix, major target of cartilage degeneration, major tool in cartilage repair. Adv Drug Deliv Rev 2003 Nov 28;55(12):1569-1593.
10. Vaccaro AR, Chiba K, Heller JG, et al. Bone grafting alternatives in spinal surgery. Spine J 2002 May-Jun;2(3):206-215.
11. Boden SD. Overview of the biology of lumbar spine fusion and principles for selecting a bone graft substitute. Spine (Phila Pa 1976) 2002 Aug 15;27(16 Suppl 1):S26-31.
12. SD B, JH S, WC H. The use of an osteoinductive growth factor for lumbar spinal fusion:Part I. The biology of spinal fusion. Volvo Award in Basic Sciences Spine 1995;20:2626-32.
13. Lawrence JP, Waked W, Gillon TJ, et al. rhBMP-2 (ACS and CRM formulations) overcomes pseudarthrosis in a New Zealand white rabbit posterolateral fusion model. Spine (Phila Pa 1976) 2007 May15;32(11):1206-1213.
14. Boden SD. Bone repair and enhancement clinical trial design. Spine applications. Clin Orthop Relat Res 1998 Oct(355 Suppl):S336-346.
15. Urist MR. Bone:formation by autoinduction. Science 1965 Nov 12;150(698):893-899.
16. Oikarinen J. Experimental spinal fusion with decalcified bone matrix and deep-frozen allogeneic bone in rabbits. Clin Orthop Relat Res 1982 Jan-Feb(162):210-218.
17. Peterson B, Whang PG, Iglesias R, et al. Osteoinductivity of commercially available demineralized bone matrix. Preparations in a spine fusion model. J Bone Joint Surg Am 2004 Oct;86-A(10):2243-2250.
18. Bomback DA, Grauer JN, Lugo R, et al. Comparison of posterolateral lumbar fusion rates of Grafton Putty and OP-1 Putty in an athymic rat model. Spine (Phila Pa 1976) 2004 Aug 129(15) 1612 1617.
19. Martin GJ, Jr., Boden SD, Titus L, et al. New formulations of demineralized bone matrix as a more effective graft alternative in experimental posterolateral lumbar spine arthrodesis. Spine (Phila Pa 1976) 1999 Apr 1;24(7):637-645.
20. Thalgott JS, Giuffre JM, Klezl Z, et al. Anterior lumbar interbody fusion with titanium mesh cages, coralline hydroxyapatite, and demineralized bone matrix as part of a circumferential fusion. Spine J 2002 Jan-Feb;2(1):63-69.
21. Cammisa FP, Jr., Lowery G, Garfin SR, et al. Two-year fusion rate equivalency between Grafton DBM gel and autograft in posterolateral spine fusion:a prospective controlled trial employing a side-by-side comparison in the same patient. Spine (Phila Pa 1976) 2004 Mar 15;29(6):660-666.
22. Russell JL, Block JE. Clinical utility of demineralized bone matrix for osseous defects, arthrodesis, and reconstruction:impact of processing techniques and study methodology. Orthopedics 1999 May;22(5):524-531; quiz 532-523.
23. Zignani M, Le Minh T, Einmahl S, et al. Improved biocompatibility of a viscous bioerodible poly(ortho ester) by controlling the environmental pH during degradation. Biomaterials 2000 Sep;21(17):1773-1778.
24. Gorna K, Gogolewski S. Preparation, degradation, and calcification of biodegradable polyurethane foams for bone graft substitutes. J Biomed Mater Res A 2003 Dec 1;67(3):813-827.
25. Ponticiello MS, Schinagl RM, Kadiyala S, et al. Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J Biomed Mater Res 2000 Nov;52(2):246-255.
26. Troum S, Dalton ML, Jr. Osteogenesis in a rat model:use of bone marrow cells and biodegradable gelatin matrix carrier. J South Orthop Assoc 2001 Spring;10(1):37-43.
27. Yamamoto M, Ikada Y, Tabata Y. Controlled release of growth factors based on biodegradation of gelatin hydrogel. J Biomater Sci Polym Ed 2001;12(1):77-88.
28. Lahiji A, Sohrabi A, Hungerford DS, et al. Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes. J Biomed Mater Res 2000 Sep 15;51(4):586-595.
29. Scotchford CA, Cascone MG, Downes S, et al. Osteoblast responses to collagen-PVA bioartificial polymers in vitro:the effects of cross-linking method and collagen content. Biomaterials 1998 Jan-Feb;19(1-3):1-11.
30. Mizuno M, Shindo M, Kobayashi D, et al. Osteogenesis by bone marrow stromal cells maintained on type I collagen matrix gels in vivo. Bone 1997 Feb;20(2):101-107.
31. Lewandrowski KU, Gresser JD, Wise DL, et al. Osteoconductivity of an injectable and bioresorbable poly(propylene glycol-co-fumaric acid) bone cement. Biomaterials 2000 Feb;21(3):293-298.
32. Bradley M, Grieser F. Emulsion polymerization synthesis of cationic polymer latex in an ultrasonic field. J Colloid Interface Sci 2002 Jul 1;251(1):78-84.
33. GUO Q, THOMANN,#160, et al. Nanostructures, semicrytalline morphology, and nanoscale confinement effect on the crystallization kinetics in self-organized block copolymer/thermoset blends. Washington, DC, ETATS-UNIS:American Chemical Society,2003.
34. El-Ghannam A, Ducheyne P, Shapiro IM. Porous bioactive glass and hydroxyapatite ceramic affect bone cell function in vitro along different time lines. J Biomed Mater Res 1997 Aug;36(2):167-180.
35. Knabe C, Ostapowicz W, Radlanski RJ, et al. In vitro investigation of novel calcium phosphates using osteogenic cultures. J Mater Sci Mater Med 1998 Jun;9(6):337-345.
36. Webster TJ, Schadler LS, Siegel RW, et al. Mechanisms of enhanced osteoblast adhesion on nanophase alumina involve vitronectin. Tissue Eng 2001 Jun;7(3):291-301.
37. Du C, Cui FZ, Zhu XD, et al. Three-dimensional nano-HAp/collagen matrix loading with osteogenic cells in organ culture. J Biomed Mater Res 1999 Mar 15;44(4):407-415.
38. Pilliar RM, Filiaggi MJ, Wells JD, et al. Porous calcium polyphosphate scaffolds for bone substitute applications -- in vitro characterization. Biomaterials 2001 May;22(9):963-972.
39. Lin AS, Barrows TH, Cartmell SH, et al. Microarchitectural and mechanical characterization of oriented porous polymer scaffolds. Biomaterials 2003 Feb;24(3):481-489.
40. Li S, De Wijn JR, Li J, et al. Macroporous biphasic calcium phosphate scaffold with high permeability/porosity ratio. Tissue Eng 2003 Jun;9(3):535-548.
41. Hartgerink JD, Beniash E, Stupp SI. Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 2001 Nov 23;294(5547):1684-1688.
42. Okazaki M, Ohmae H, Hino T. Insolubilization of apatite-collagen composites by UV irradiation. Biomaterials 1989 Oct;10(8):564-568.
43. Clarke KI, Graves SE, Wong ATC, et al. Investigation into the formation and mechanical properties of a bioactive material based on collagen and calcium phosphate. Journal of Materials Science:Materials in Medicine 1993;4(2):107-110.
44. Doi Y, Horiguchi T, Moriwaki Y, et al. Formation of apatite-collagen complexes. J Biomed Mater Res 1996 May;31(1):43-49.
45. Hsu FY, Chueh SC, Wang YJ. Microspheres of hydroxyapatite/reconstituted collagen as supports for osteoblast cell growth. Biomaterials 1999 Oct;20(20):1931-1936.
46. Du C, Cui FZ, Zhang W, et al. Formation of calcium phosphate/collagen composites through mineralization of collagen matrix. J Biomed Mater Res 2000 Jun 15;50(4):518-527.
47. Du C, Cui FZ, Feng QL, et al. Tissue response to nano-hydroxyapatite/collagen composite implants in marrow cavity. J Biomed Mater Res 1998 Dec 15;42(4):540-548.
48. Bennacef I, Tymciu S, Dhilly M, et al. Synthesis and biological evaluation of novel fluoro and iodo quinoline carboxamides as potential ligands of NK-3 receptors for in vivo imaging studies. Bioorg Med Chem 2004 Aug 15;12(16):4533-4541.
49. Chang MC, Ikoma T, Kikuchi M, et al. Preparation of a porous hydroxyapatite/collagen nanocomposite using glutaraldehyde as a crosslinkage agent. Journal of Materials Science Letters 2001;20(13):1199-1201.
50. Kikuchi M, Ikoma T, Itoh S, et al. Biomimetic synthesis of bone-like nanocomposites using the self-organization mechanism of hydroxyapatite and collagen. Composites Science and Technology 2004;64(6):819-825.
51. Rhee SH, Suetsugu Y, Tanaka J. Biomimetic configurational arrays of hydroxyapatite nanocrystals on bio-organics. Biomaterials 2001 Nov;22(21):2843-2847.
52. Kikuchi M, Itoh S, Ichinose S, et al. Self-organization mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological reaction in vivo. Biomaterials 2001 Jul;22(13):1705-1711.
53. Tampieri A, Celotti G, Landi E, et al. Biologically inspired synthesis of bone-like composite: self-assembled collagen fibers/hydroxyapatite nanocrystals. J Biomed Mater Res A 2003 Nov 1;67(2):618-625.
54. Silva CC, Pinheiro AG, Figueiro SD, et al. Piezoelectric properties of collagen-nanocrystalline hydroxyapatite composites. Journal of Materials Science 2002;37(10):2061-2070.
55. Saito T, Arsenault AL, Yamauchi M, et al. Mineral induction by immobilized phosphoproteins. Bone 1997 Oct;21(4):305-311.
56. Tong W, Eppell SJ. Control of surface mineralization using collagen fibrils. J Biomed Mater Res 2002 Sep 5;61(3):346-353.
57.陈际达.纳米羟基磷灰石/胶原复合材料制备方法研究.生物物理学报2001；17(4).
58. Wen HB, Cui FZ, Zhu XD. Microstructural features of non-union of human humeral shaft fracture. J Struct Biol 1997 Aug;119(3):239-246.
59. Cui FZ, Wen HB, Su XW, et al. Microstructures of external periosteal callus of repaired femoral fracture in children. J Struct Biol 1996 Nov-Dec;117(3):204-208.
60. Su X, Sun K, Cui FZ, et al. Organization of apatite crystals in human woven bone. Bone 2003 Feb;32(2):150-162.
61.廖素三.组织工程中胶原基纳米骨复合材料的研制.中国医学科学院学报2003;25(1).
62.黄先智.蚕丝蛋白在食品和化妆品中的应用.四川丝绸2003(2).
63.盛伟华.蚕丝蛋白材料对鼠胚表皮细胞毒性的实验研究.苏州大学学报(医学版)2005;25(4).
64. Cordewener FW, Bos RR, Rozema FR, et al. Poly(L-lactide) implants for repair of human orbital floor defects:clinical and magnetic resonance imaging evaluation of long-term results. J Oral Maxillofac Surg 1996 Jan;54(1):9-13; discussion 13-14.
65. Meinel L, Hofmann S, Karageorgiou V, et al. The inflammatory responses to silk films in vitro and in vivo. Biomaterials 2005 Jan;26(2):147-155.
66. Altman GH, Horan RL, Lu HH, et al. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 2002 Oct;23(20):4131-4141.
67.卢神州.羟基磷灰石/丝素蛋白纳米复合颗粒的制备.丝绸2006(2).
68. Wang L, Ning G-L, Senna M. Microstructure and gelation behavior of hydroxyapatite-based nanocomposite sol containing chemically modified silk fibroin. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2005;254(1-3):159-164.
69.赵勇.丝素蛋白膜仿生沉积磷灰石的实验研究.口腔医学研究2006;22(2).
1. Du C, Cui FZ, Zhang W, et al. Formation of calcium phosphate/collagen composites through mineralization of collagen matrix. J Biomed Mater Res 2000 Jun 15;50(4):518-527.
2. Kikuchi M, Itoh S, Ichinose S, et al. Self-organization mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological reaction in vivo. Biomaterials 2001 Jul;22(13):1705-1711.
3. Mann S. Molecular tectonics in biomineralization and biomimetic materials chemistry. Nature 1993;365:6.
4. Stupp SI, Braun PV. Molecular manipulation of microstructures:biomaterials, ceramics, and semiconductors. Science 1997 Aug 29;277(5330):1242-1248.
5. Muthukumar M, Ober CK, Thomas EL. Competing Interactions and Levels of Ordering in Self-Organizing Polymeric Materials. Science 1997 August 29,1997;277(5330):1225-1232.
6. Chang MC, Ko CC, Douglas WH. Preparation of hydroxyapatite-gelatin nanocomposite. Biomaterials 2003 Aug;24(17):2853-2862.
7. Kasuga T, Ota Y, Nogami M, et al. Preparation and mechanical properties of polylactic acid composites containing hydroxyapatite fibers. Biomaterials 2001 Jan;22(l):19-23.
8. Zhang JC, Lu HY, Lv GY, et al. The repair of critical-size defects with porous hydroxyapatite/polyamide nanocomposite:an experimental study in rabbit mandibles. Int J Oral Maxillofac Surg Feb 27.
9. Li Y, Cai Y, Kong X, et al. Anisotropic growth of hydroxyapatite on the silk fibroin films. Applied Surface Science 2008;255(5, Part 1):1681-1685.
10. Du C, Jin J, Li Y, et al. Novel silk fibroin/hydroxyapatite composite films:Structure and properties. Materials Science and Engineering:C 2009;29(1):62-68.
11. Korematsu A, Furuzono T, Yasuda S, et al. Nano-scaled hydroxyapatite/polymer composite Ⅲ. Coating of sintered hydroxyapatite particles on poly(4-methacryloyloxyethyl trimellitate anhydride)-grafted silk fibroin fibers. J Mater Sci Mater Med 2005 Jan;16(1):67-71.
12. Murugan R, Ramakrishna S. Bioresorbable composite bone paste using polysaccharide based nano hydroxyapatite. Biomaterials 2004;25(17):3829-3835.
13. Rhee SH, Tanaka J. Self-assembly phenomenon of hydroxyapatite nanocrystals on chondroitin sulfate. J Mater Sci Mater Med 2002 Jun;13(6):597-600.
14. Cao M, Wang Y, Guo C, et al. Preparation of ultrahigh-aspect-ratio hydroxyapatite nanofibers in reverse micelles under hydrothermal conditions. Langmuir 2004 May 25;20(11):4784-4786.
15. Zhang W, Liao SS, Cui FZ. Hierarchical Self-Assembly of Nano-Fibrils in Mineralized Collagen. Chemistry of Materials 2003;15(16):3221-3226.
16. Ethirajan A, Ziener U, Landfester K. Surface-Functionalized Polymeric Nanoparticles as Templates for Biomimetic Mineralization of Hydroxyapatite. Chemistry of Materials 2009;21(11):2218-2225.
1.张真.生物材料有效性和安全性评价的现状与趋势.生物医学工程学杂志2002;19(1).
2.杨晓芳.生物材料生物相容性评价研究进展.生物医学工程学杂志2001;18(1).
3. Rokusek D, Davitt C, Bandyopadhyay A, et al. Interaction of human osteoblasts with bioinert and bioactive ceramic substrates. J Biomed Mater Res A 2005 Dec 1;75(3):588-594.
4. Wei G, Ma PX. Structure and properties of nano-hydroxyapatite/polymer composite scaffolds for bone tissue engineering. Biomaterials 2004 Aug;25(19):4749-4757.
5. Kim SS, Sun Park M, Jeon O, et al. Poly(lactide-co-glycolide)/hydroxyapatite composite scaffolds for bone tissue engineering. Biomaterials 2006 Mar;27(8):1399-1409.
6. Tuzlakoglu K, Bolgen N, Salgado AJ, et al. Nano-and micro-fiber combined scaffolds:a new architecture for bone tissue engineering. J Mater Sci Mater Med 2005 Dec; 16(12):1099-1104.
7. Hee CK, Jonikas MA, Nicoll SB. Influence of three-dimensional scaffold on the expression of osteogenic differentiation markers by human dermal fibroblasts. Biomaterials 2006 Feb;27(6):875-884.
8. Murugan R, Ramakrishna S. Nano-featured scaffolds for tissue engineering:a review of spinning methodologies. Tissue Eng 2006 Mar;12(3):435-447.
9. Ginebra MP, Driessens FC, Planell JA. Effect of the particle size on the micro and nanostructural features of a calcium phosphate cement:a kinetic analysis. Biomaterials 2004 Aug;25(17):3453-3462.
10.Popat KC, Leary Swan EE, Mukhatyar V, et al. Influence of nanoporous alumina membranes on long-term osteoblast response. Biomaterials 2005 Aug;26(22):4516-4522.
1. Valdes M, Palumbo M, Appel AJ, et al. Posterolateral intertransverse lumbar arthrodesis in the New Zealand White rabbit model:II. Operative technique. Spine J 2004 May-Jun;4(3):293-299.
2. Boden SD, Moskovitz PA, Morone MA, et al. Video-assisted lateral intertransverse process arthrodesis. Validation of a new minimally invasive lumbar spinal fusion technique in the rabbit and nonhuman primate (rhesus) models. Spine (Phila Pa 1976) 1996 Nov 15;21(22):2689-2697.
3. Lee T-C, Ho J-T, Hung K-S, et al. Bone Morphogenetic Protein Gene Therapy Using a Fibrin Scaffold for a Rabbit Spinal-Fusion Experiment. Neurosurgery 2006;58(2):373-380 310.1227/1201.NEU.0000199725.0000103186.F0000199726.
4. Curylo LJ, Johnstone B, Petersilge CA, et al. Augmentation of spinal arthrodesis with autologous bone marrow in a rabbit posterolateral spine fusion model. Spine (Phila Pa 1976) 1999 Mar 1;24(5):434-438; discussion 438-439.
5. Stanworth SJ, Newland AC. Stem cells:progress in research and edging towards the clinical setting. Clin Med 2001 Sep-Oct;1(5):378-382.
6.崔玉明.诱导骨髓基质细胞向软骨细胞分化的实验研究.中国临床康复2003;7(17).
7. Fleming JE, Jr., Cornell CN, Muschler GF. Bone cells and matrices in orthopedic tissue engineering. Orthop Clin North Am 2000 Jul;31(3):357-374.
8.周晓.组织工程骨修复骨缺损的研究进展.中国现代手术学杂志2006;10(4).
9. Grove JE, Bruscia E, Krause DS. Plasticity of bone marrow-derived stem cells. Stem Cells 2004;22(4):487-500.
10. Lee J, Elkahloun AG, Messina SA, et al. Cellular and genetic characterization of human adult bone marrow-derived neural stem-like cells:a potential antiglioma cellular vector. Cancer Res 2003 Dec 15;63(24):8877-8889.
11. Feiertag MA, Boden SD, Schimandle JH, et al. A rabbit model for nonunion of lumbar intertransverse process spine arthrodesis. Spine (Phila Pa 1976) 1996 Jan 1;21(1):27-31.
12. Boden SD. Overview of the biology of lumbar spine fusion and principles for selecting a bone graft substitute. Spine (Phila Pa 1976) 2002 Aug 15;27(16 Suppl 1):S26-31.
13. SD B, JH S, WC H. The use of an osteoinductive growth factor for lumbar spinal fusion:Part I. The biology of spinal fusion. Volvo Award in Basic Sciences Spine 1995;20:2626-32.
14. Toribatake Y, Hutton WC, Tomita K, et al. Vascularization of the fusion mass in a posterolateral intertransverse process fusion. Spine (Phila Pa 1976) 1998 May 15;23(10):1149-1154.
1. Altman GH, Diaz F, Jakuba C, et al. Silk-based biomaterials. Biomaterials 2003 Feb;24(3):401-416.
2. Sofia S, McCarthy MB, Gronowicz G, et al. Functionalized silk-based biomaterials for bone formation. J Biomed Mater Res 2001 Jan;54(1):139-148.
3. Altman GH, Horan RL, Lu HH, et al. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 2002 Oct;23(20):4131-4141.
4. Horan RL, Antle K, Collette AL, et al. In vitro degradation of silk fibroin. Biomaterials 2005 Jun;26(17):3385-3393.
5. Taddei P, Arai T, Boschi A, et al. In vitro study of the proteolytic degradation of Antheraea pernyi silk fibroin. Biomacromolecules 2006 Jan;7(1):259-267.
6. Takayuki A, Giuliano F, Riccardo I, et al. Biodegradation of Bombyx mori silk fibroin fibers and films. Journal of Applied Polymer Science,2004. p.2383-2390.
7. Li M, Ogiso M, Minoura N. Enzymatic degradation behavior of porous silk fibroin sheets. Biomaterials 2003 Jan;24(2):357-365.
8. Craig CL, Riekel C. Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders. Comp Biochem Physiol B Biochem Mol Biol 2002 Dec;133(4):493-507.
9. Valluzzi R, Winkler S, Wilson D, et al. Silk:molecular organization and control of assembly. Philosophical Transactions of the Royal Society of London Series B:Biological Sciences 2002 February 28,2002;357(1418):165-167.
10. PEREZ-RIGUEIRO,#160, Jos, et al. Similarities and differences in the supramolecular organization of silkworm and spider silk. Washington, DC, ETATS-UNIS:American Chemical Society, 2007.
11. Thiel BL, Guess KB, Viney C. Non-periodic lattice crystals in the hierarchical microstructure of spider (major ampullate) silk. Biopolymers 1997 Jun;41(7):703-719.
12. Santin M, Motta A, Freddi G, et al. In vitro evaluation of the inflammatory potential of the silk fibroin. Journal of Biomedical Materials Research 1999;46(3):382-389.
13. Panilaitis B, Altman GH, Chen J, et al. Macrophage responses to silk. Biomaterials 2003;24(18):3079-3085.
14. Chiarini A, Petrini P, Bozzini S, et al. Silk fibroin/poly(carbonate)-urethane as a substrate for cell growth:in vitro interactions with human cells. Biomaterials 2003 Feb;24(5):789-799.
15. Dal Pra I, Petrini P, Charini A, et al. Silk fibroin-coated three-dimensional polyurethane scaffolds for tissue engineering:interactions with normal human fibroblasts. Tissue Eng 2003 Dec;9(6):1113-1121.
16. Inouye K, Kurokawa M, Nishikawa S, et al. Use of Bombyx mori silk fibroin as a substratum for cultivation of animal cells. Journal of Biochemical and Biophysical Methods 1998;37(3):159-164.
17. Meinel L, Hofmann S, Karageorgiou V, et al. The inflammatory responses to silk films in vitro and in vivo. Biomaterials 2005 Jan;26(2):147-155.
18. Kardestuncer T, McCarthy MB, Karageorgiou V, et al. RGD-tethered silk substrate stimulates the differentiation of human tendon cells. Clin Orthop Relat Res 2006 Jul;448:234-239.
19. Minoura N, Aiba S, Higuchi M, et al. Attachment and growth of fibroblast cells on silk fibroin. Biochem Biophys Res Commun 1995 Mar 17;208(2):511-516.
20. Li C, Vepari C, Jin HJ, et al. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 2006 Jun;27(16):3115-3124.
21. Jin HJ, Chen J, Karageorgiou V, et al. Human bone marrow stromal cell responses on electrospun silk fibroin mats. Biomaterials 2004 Mar;25(6):1039-1047.
22. Unger RE, Wolf M, Peters K, et al. Growth of human cells on a non-woven silk fibroin net:a potential for use in tissue engineering. Biomaterials 2004;25(6):1069-1075.
23. Gosline JM, Guerette PA, Ortlepp CS, et al. The mechanical design of spider silks:from fibroin sequence to mechanical function. J Exp Biol 1999 Dec;202(Pt 23):3295-3303.
24. Sheu HS, Phyu KW, Jean YC, et al. Lattice deformation and thermal stability of crystals in spider silk. Int J Biol Macromol 2004 Oct;34(5):325-331.
25. Sirichaisit J, Brookes VL, Young RJ, et al. Analyis of structure/property relationships in silkworm (Bombyx mori) and spider dragline (Nephila edulis) silks using Raman spectroscopy. Biomacromolecules 2003 Mar-Apr;4(2):387-394.
26. Rising A, Nimmervoll H, Grip S, et al. Spider silk proteins-mechanical property and gene sequence. Zoolog Sci 2005 Mar;22(3):273-281.
27. Vollrath F. Strength and structure of spiders'silks. J Biotechnol 2000 Aug;74(2):67-83.
28. Zax DB, Armanios DE, Horak S, et al. Variation of mechanical properties with amino acid content in the silk of Nephila clavipes. Biomacromolecules 2004 May-Jun;5(3):732-738.
29. Riekel C, Madsen B, Knight D, et al. X-ray diffraction on spider silk during controlled extrusion under a synchrotron radiation X-ray beam. Biomacromolecules 2000 Winter;1(4):622-626.
30. Vollrath F, Madsen B, Shao Z. The effect of spinning conditions on the mechanics of a spider's dragline silk. Proc Biol Sci 2001 Nov 22;268(1483):2339-2346.
31. Liu Y, Shao Z, Vollrath F. Relationships between supercontraction and mechanical properties of spider silk. Nat Mater 2005 Dec;4(12):901-905.
32. Yang Y, Chen X, Shao Z, et al. Toughness of Spider Silk at High and Low Temperatures. Advanced Materials 2005;17(1):84-88.
33. Candelas G, Candelas T, Ortiz A, et al. Translational pauses during a spider fibroin synthesis. Biochem Biophys Res Commun 1983 Nov 15;116(3):1033-1038.
34. Vollrath F, Barth P, Basedow A, et al. Local tolerance to spider silks and protein polymers in vivo. In Vivo 2002 Jul-Aug;16(4):229-234.
35. Scheller J, Henggeler D, Viviani A, et al. Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation. Transgenic Res 2004 Feb;13(1):51-57.
36. Kuettner KE. Biochemistry of articular cartilage in health and disease. Clin Biochem 1992 Jun;25(3):155-163.
37. Mow VC, Ratcliffe A, Robin Poole A. Cartilage and diarthrodial joints as paradigms for hierarchical materials and structures. Biomaterials 1992;13(2):67-97.
38. Minas T, Nehrer S. Current concepts in the treatment of articular cartilage defects. Orthopedics 1997Jun;20(6):525-538.
39. Hofmann S, Knecht S, Langer R, et al. Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells. Tissue Eng 2006 Oct;12(10):2729-2738.
40. Marolt D, Augst A, Freed LE, et al. Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors. Biomaterials 2006;27(36):6138-6149.
41. Meinel L, Karageorgiou V, Hofmann S, et al. Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds. Journal of Biomedical Materials Research Part A 2004;71A(1):25-34.
42. Wang Y, Kim UJ, Blasioli DJ, et al. In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells. Biomaterials 2005 Dec;26(34):7082-7094.
43. Aoki H, Tomita N, Morita Y, et al. Culture of chondrocytes in fibroin-hydrogel sponge. Biomed Mater Eng 2003;13(4):309-316.
44. Morita Y, Tomita N, Aoki H, et al. Visco-elastic properties of cartilage tissue regenerated with fibroin sponge. Biomed Mater Eng 2002;12(3):291-298.
45. Wang Y, Blasioli DJ, Kim HJ, et al. Cartilage tissue engineering with silk scaffolds and human articular chondrocytes. Biomaterials 2006 Sep;27(25):4434-4442.
46. Gellynck.K, Verdonk.P, K.F A, et al. Chondrocyte Growth in Porous Spider Silk 3D-Scaffolds. European Cells and Materials 2005;10(suppl 2):45.
47. Sikavitsas VI, Temenoff JS, Mikos AG. Biomaterials and bone mechanotransduction. Biomaterials 2001 Oct;22(19):2581-2593.
48. Hofmann S, Hagenmuller H, Koch AM, et al. Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. Biomaterials 2007 Feb;28(6):1152-1162.
49. Kim K-H, Jeong L, Park H-N, et al. Biological efficacy of silk fibroin nanofiber membranes for guided bone regeneration. Journal of Biotechnology 2005;120(3):327-339.
50. Kirker-Head C, Karageorgiou V, Hofmann S, et al. BMP-silk composite matrices heal critically sized femoral defects. Bone 2007 Aug;41(2):247-255.
51. Meinel L, Betz O, Fajardo R, et al. Silk based biomaterials to heal critical sized femur defects. Bone 2006 Oct;39(4):922-931.
52. Meinel L, Hofmann S, Betz O, et al. Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials:comparison of adenovirus mediated gene transfer and protein delivery of BMP-2. Biomaterials 2006 Oct;27(28):4993-5002.
53. Karageorgiou V, Meinel L, Hofmann S, et al. Bone morphogenetic protein-2 decorated silk fibroin films induce osteogenic differentiation of human bone marrow stromal cells. J Biomed Mater Res A 2004 Dec 1;71(3):528-537.
54. Furuzono T, Kishida A, Tanaka J. Nano-scaled hydroxyapatite/polymer composite I. Coating of sintered hydroxyapatite particles on poly(gamma-methacryloxypropyl trimethoxysilane)grafted silk fibroin fibers through chemical bonding. J Mater Sci Mater Med 2004 Jan;15(1):19-23.
55. Tanaka T, Hirose M, Kotobuki N, et al. Nano-scaled hydroxyapatite/silk fibroin sheets support osteogenic differentiation of rat bone marrow mesenchymal cells. Materials Science and Engineering:C 2007;27(4):817-823.
56. Fini M, Motta A, Torricelli P, et al. The healing of confined critical size cancellous defects in the presence of silk fibroin hydrogel. Biomaterials 2005 Jun;26(17):3527-3536.
57. Kim HJ, Kim UJ, Vunjak-Novakovic G, et al. Influence of macroporous protein scaffolds on bone tissue engineering from bone marrow stem cells. Biomaterials 2005 Jul;26(21):4442-4452.
58. Bai J, Ma T, Chu W, et al. Regenerated spider silk as a new biomaterial for MEMS. Biomed Microdevices 2006 Dec;8(4):317-323.
59. Bini E, Foo CW, Huang J, et al. RGD-functionalized bioengineered spider dragline silk biomaterial. Biomacromolecules 2006 Nov;7(11):3139-3145.
60. Doroski DM, Brink KS, Temenoff JS. Techniques for biological characterization of tissue-engineered tendon and ligament. Biomaterials 2007 Jan;28(2):187-202.
61. Horan RL, Collette AL, Lee C, et al. Yarn design for functional tissue engineering. J Biomech 2006;39(12):2232-2240.
62. Chen J, Altman GH, Karageorgiou V, et al. Human bone marrow stromal cell and ligament fibroblast responses on RGD-modified silk fibers. J Biomed Mater Res A 2003 Nov 1;67(2):559-570.
63. Chen J, Horan RL, Bramono D, et al. Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering. Tissue Eng 2006 Nov;12(11):3085-3095.
64. Moreau JE, Chen J, Horan RL, et al. Sequential growth factor application in bone marrow stromal cell ligament engineering. Tissue Eng 2005 Nov-Dec; 11 (11-12):1887-1897.
65. Yoon JH, Halper J. Tendon proteoglycans:biochemistry and function. J Musculoskelet Neuronal Interact 2005 Mar;5(1):22-34.
66. Takezawa T, Ozaki K, Takabayashi C. Reconstruction of a hard connective tissue utilizing a pressed silk sheet and type-I collagen as the scaffold for fibroblasts. Tissue Eng 2007 Jun;13(6):1357-1366.
67. Liu H, Ge Z, Wang Y, et al. Modification of sericin-free silk fibers for ligament tissue engineering application. J Biomed Mater Res B Appl Biomater 2007 Jul;82(1):129-138.
68. Rathore O, Sogah DY. Self-assembly of beta-sheets into nanostructures by poly(alanine) segments incorporated in multiblock copolymers inspired by spider silk. J Am Chem Soc 2001 Jun 6;123(22):5231-5239.
69. Zhou C, Leng B, Yao J, et al. Synthesis and characterization of multiblock copolymers based on spider dragline silk proteins. Biomacromolecules 2006 Aug;7(8):2415-2419.
70. GRIP S, RISING A, NIMMERVOLL H, et al. Transient Expression of a Major Ampullate Spidroin 1 Gene Fragment from Euprosthenops sp. in Mammalian Cells. Cancer Genomics-Proteomics 2006 March 2006;3(2):83-87.
71. Lazaris A, Arcidiacono S, Huang Y, et al. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells. Science 2002 Jan 18;295(5554):472-476.
72. Huemmerich D, Scheibel T, Vollrath F, et al. Novel assembly properties of recombinant spider dragline silk proteins. Curr Biol 2004 Nov 23;14(22):2070-2074.
73. Lee KS, Kim BY, Je YH, et al. Molecular cloning and expression of the C-terminus of spider flagelliform silk protein from Araneus ventricosus. J Biosci 2007 Jun;32(4):705-712.
74. Fahnestock SR, Irwin SL. Synthetic spider dragline silk proteins and their production in Escherichia coli. Appl Microbiol Biotechnol 1997 Jan;47(1):23-32.
75. Schmidt M, Romer L, Strehle M, et al. Conquering isoleucine auxotrophy of Escherichia coli BLR(DE3) to recombinantly produce spider silk proteins in minimal media. Biotechnol Lett 2007 Nov;29(11):1741-1744.
76. Stark M, Grip S, Rising A, et al. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007 May;8(5):1695-1701.
77. Fahnestock SR, Bedzyk LA. Production of synthetic spider dragline silk protein in Pichia pastoris. Appl Microbiol Biotechnol 1997 Jan;47(1):33-39.
78. Hood EE, Jilka JM. Plant-based production of xenogenic proteins. Curr Opin Biotechnol 1999 Aug;10(4):382-386.
79. Scheller J, Guhrs KH, Grosse F, et al. Production of spider silk proteins in tobacco and potato. Nat Biotechnol 2001 Jun;19(6):573-577.
80. Teule F, Furin W, Cooper A, et al. Modifications of spider silk sequences in an attempt to control the mechanical properties of the synthetic fibers. Journal of Materials Science 2007;42(21):8974-8985.
81. Metwalli E, Slotta U, Darko C, et al. Structural changes of thin films from recombinant spider silk proteins upon post-treatment. Applied Physics A:Materials Science & Processing 2007;89(3):655-661.
82. Li G, Zhou P, Shao Z, et al. The natural silk spinning process. A nucleation-dependent aggregation mechanism? Eur J Biochem 2001 Dec;268(24):6600-6606.
83. Lundmark K, Westermark GT, et al. Protein fibrils in nature can enhance amyloid protein A amyloidosis in mice:Cross-seeding as a disease mechanism. Proceedings of the National Academy of Sciences of the United States of America 2005 April 26,2005;102(17):6098-6102.
84. Kenney JM, Knight D, Wise MJ, et al. Amyloidogenic nature of spider silk. Eur J Biochem 2002 Aug;269(16):4159-4163.
2. Adams GB, Scadden DT. The hematopoietic stem cell in its place. Nat Immunol 2006 Apr; 7(4):333-337.
3. Yin T, Li L. The stem cell niches in bone. J Clin Invest 2006 May; 116(5):1195-1201.
4. Huang X, Cho S, Spangrude GJ. Hematopoietic stem cells:generation and self-renewal. Cell Death Differ 2007 Nov;14(11):1851-1859.
5. Arai F, Hirao A, Ohmura M, et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004 Jul 23; 118(2):149-161.
6. Puri MC, Bernstein A. Requirement for the TIE family of receptor tyrosine kinases in adult but not fetal hematopoiesis. Proc Natl Acad Sci U S A 2003 Oct 28;100(22):12753-12758.
7. Borsato KS, Sasaki N. Measurement of partition of stress between mineral and collagen phases in bone using X-ray diffraction techniques. Journal of Biomechanics 1997;30(9):955-957.
8. Knott L, Bailey AJ. Collagen cross-links in mineralizing tissues:A review of their chemistry, function, and clinical relevance. Bone 1998;22(3):181-187.
9. Aigner T, Stove J. Collagens--major component of the physiological cartilage matrix, major target of cartilage degeneration, major tool in cartilage repair. Adv Drug Deliv Rev 2003 Nov 28;55(12):1569-1593.
10. Vaccaro AR, Chiba K, Heller JG, et al. Bone grafting alternatives in spinal surgery. Spine J 2002 May-Jun;2(3):206-215.
11. Boden SD. Overview of the biology of lumbar spine fusion and principles for selecting a bone graft substitute. Spine (Phila Pa 1976) 2002 Aug 15;27(16 Suppl 1):S26-31.
12. SD B, JH S, WC H. The use of an osteoinductive growth factor for lumbar spinal fusion:Part I. The biology of spinal fusion. Volvo Award in Basic Sciences Spine 1995;20:2626-32.
13. Lawrence JP, Waked W, Gillon TJ, et al. rhBMP-2 (ACS and CRM formulations) overcomes pseudarthrosis in a New Zealand white rabbit posterolateral fusion model. Spine (Phila Pa 1976) 2007 May15;32(11):1206-1213.
14. Boden SD. Bone repair and enhancement clinical trial design. Spine applications. Clin Orthop Relat Res 1998 Oct(355 Suppl):S336-346.
15. Urist MR. Bone:formation by autoinduction. Science 1965 Nov 12;150(698):893-899.
16. Oikarinen J. Experimental spinal fusion with decalcified bone matrix and deep-frozen allogeneic bone in rabbits. Clin Orthop Relat Res 1982 Jan-Feb(162):210-218.
17. Peterson B, Whang PG, Iglesias R, et al. Osteoinductivity of commercially available demineralized bone matrix. Preparations in a spine fusion model. J Bone Joint Surg Am 2004 Oct;86-A(10):2243-2250.
18. Bomback DA, Grauer JN, Lugo R, et al. Comparison of posterolateral lumbar fusion rates of Grafton Putty and OP-1 Putty in an athymic rat model. Spine (Phila Pa 1976) 2004 Aug 129(15) 1612 1617.
19. Martin GJ, Jr., Boden SD, Titus L, et al. New formulations of demineralized bone matrix as a more effective graft alternative in experimental posterolateral lumbar spine arthrodesis. Spine (Phila Pa 1976) 1999 Apr 1;24(7):637-645.
20. Thalgott JS, Giuffre JM, Klezl Z, et al. Anterior lumbar interbody fusion with titanium mesh cages, coralline hydroxyapatite, and demineralized bone matrix as part of a circumferential fusion. Spine J 2002 Jan-Feb;2(1):63-69.
21. Cammisa FP, Jr., Lowery G, Garfin SR, et al. Two-year fusion rate equivalency between Grafton DBM gel and autograft in posterolateral spine fusion:a prospective controlled trial employing a side-by-side comparison in the same patient. Spine (Phila Pa 1976) 2004 Mar 15;29(6):660-666.
22. Russell JL, Block JE. Clinical utility of demineralized bone matrix for osseous defects, arthrodesis, and reconstruction:impact of processing techniques and study methodology. Orthopedics 1999 May;22(5):524-531; quiz 532-523.
23. Zignani M, Le Minh T, Einmahl S, et al. Improved biocompatibility of a viscous bioerodible poly(ortho ester) by controlling the environmental pH during degradation. Biomaterials 2000 Sep;21(17):1773-1778.
24. Gorna K, Gogolewski S. Preparation, degradation, and calcification of biodegradable polyurethane foams for bone graft substitutes. J Biomed Mater Res A 2003 Dec 1;67(3):813-827.
25. Ponticiello MS, Schinagl RM, Kadiyala S, et al. Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J Biomed Mater Res 2000 Nov;52(2):246-255.
26. Troum S, Dalton ML, Jr. Osteogenesis in a rat model:use of bone marrow cells and biodegradable gelatin matrix carrier. J South Orthop Assoc 2001 Spring;10(1):37-43.
27. Yamamoto M, Ikada Y, Tabata Y. Controlled release of growth factors based on biodegradation of gelatin hydrogel. J Biomater Sci Polym Ed 2001;12(1):77-88.
28. Lahiji A, Sohrabi A, Hungerford DS, et al. Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes. J Biomed Mater Res 2000 Sep 15;51(4):586-595.
29. Scotchford CA, Cascone MG, Downes S, et al. Osteoblast responses to collagen-PVA bioartificial polymers in vitro:the effects of cross-linking method and collagen content. Biomaterials 1998 Jan-Feb;19(1-3):1-11.
30. Mizuno M, Shindo M, Kobayashi D, et al. Osteogenesis by bone marrow stromal cells maintained on type I collagen matrix gels in vivo. Bone 1997 Feb;20(2):101-107.
31. Lewandrowski KU, Gresser JD, Wise DL, et al. Osteoconductivity of an injectable and bioresorbable poly(propylene glycol-co-fumaric acid) bone cement. Biomaterials 2000 Feb;21(3):293-298.
32. Bradley M, Grieser F. Emulsion polymerization synthesis of cationic polymer latex in an ultrasonic field. J Colloid Interface Sci 2002 Jul 1;251(1):78-84.
33. GUO Q, THOMANN,#160, et al. Nanostructures, semicrytalline morphology, and nanoscale confinement effect on the crystallization kinetics in self-organized block copolymer/thermoset blends. Washington, DC, ETATS-UNIS:American Chemical Society,2003.
34. El-Ghannam A, Ducheyne P, Shapiro IM. Porous bioactive glass and hydroxyapatite ceramic affect bone cell function in vitro along different time lines. J Biomed Mater Res 1997 Aug;36(2):167-180.
35. Knabe C, Ostapowicz W, Radlanski RJ, et al. In vitro investigation of novel calcium phosphates using osteogenic cultures. J Mater Sci Mater Med 1998 Jun;9(6):337-345.
36. Webster TJ, Schadler LS, Siegel RW, et al. Mechanisms of enhanced osteoblast adhesion on nanophase alumina involve vitronectin. Tissue Eng 2001 Jun;7(3):291-301.
37. Du C, Cui FZ, Zhu XD, et al. Three-dimensional nano-HAp/collagen matrix loading with osteogenic cells in organ culture. J Biomed Mater Res 1999 Mar 15;44(4):407-415.
38. Pilliar RM, Filiaggi MJ, Wells JD, et al. Porous calcium polyphosphate scaffolds for bone substitute applications -- in vitro characterization. Biomaterials 2001 May;22(9):963-972.
39. Lin AS, Barrows TH, Cartmell SH, et al. Microarchitectural and mechanical characterization of oriented porous polymer scaffolds. Biomaterials 2003 Feb;24(3):481-489.
40. Li S, De Wijn JR, Li J, et al. Macroporous biphasic calcium phosphate scaffold with high permeability/porosity ratio. Tissue Eng 2003 Jun;9(3):535-548.
41. Hartgerink JD, Beniash E, Stupp SI. Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 2001 Nov 23;294(5547):1684-1688.
42. Okazaki M, Ohmae H, Hino T. Insolubilization of apatite-collagen composites by UV irradiation. Biomaterials 1989 Oct;10(8):564-568.
43. Clarke KI, Graves SE, Wong ATC, et al. Investigation into the formation and mechanical properties of a bioactive material based on collagen and calcium phosphate. Journal of Materials Science:Materials in Medicine 1993;4(2):107-110.
44. Doi Y, Horiguchi T, Moriwaki Y, et al. Formation of apatite-collagen complexes. J Biomed Mater Res 1996 May;31(1):43-49.
45. Hsu FY, Chueh SC, Wang YJ. Microspheres of hydroxyapatite/reconstituted collagen as supports for osteoblast cell growth. Biomaterials 1999 Oct;20(20):1931-1936.
46. Du C, Cui FZ, Zhang W, et al. Formation of calcium phosphate/collagen composites through mineralization of collagen matrix. J Biomed Mater Res 2000 Jun 15;50(4):518-527.
47. Du C, Cui FZ, Feng QL, et al. Tissue response to nano-hydroxyapatite/collagen composite implants in marrow cavity. J Biomed Mater Res 1998 Dec 15;42(4):540-548.
48. Bennacef I, Tymciu S, Dhilly M, et al. Synthesis and biological evaluation of novel fluoro and iodo quinoline carboxamides as potential ligands of NK-3 receptors for in vivo imaging studies. Bioorg Med Chem 2004 Aug 15;12(16):4533-4541.
49. Chang MC, Ikoma T, Kikuchi M, et al. Preparation of a porous hydroxyapatite/collagen nanocomposite using glutaraldehyde as a crosslinkage agent. Journal of Materials Science Letters 2001;20(13):1199-1201.
50. Kikuchi M, Ikoma T, Itoh S, et al. Biomimetic synthesis of bone-like nanocomposites using the self-organization mechanism of hydroxyapatite and collagen. Composites Science and Technology 2004;64(6):819-825.
51. Rhee SH, Suetsugu Y, Tanaka J. Biomimetic configurational arrays of hydroxyapatite nanocrystals on bio-organics. Biomaterials 2001 Nov;22(21):2843-2847.
52. Kikuchi M, Itoh S, Ichinose S, et al. Self-organization mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological reaction in vivo. Biomaterials 2001 Jul;22(13):1705-1711.
53. Tampieri A, Celotti G, Landi E, et al. Biologically inspired synthesis of bone-like composite: self-assembled collagen fibers/hydroxyapatite nanocrystals. J Biomed Mater Res A 2003 Nov 1;67(2):618-625.
54. Silva CC, Pinheiro AG, Figueiro SD, et al. Piezoelectric properties of collagen-nanocrystalline hydroxyapatite composites. Journal of Materials Science 2002;37(10):2061-2070.
55. Saito T, Arsenault AL, Yamauchi M, et al. Mineral induction by immobilized phosphoproteins. Bone 1997 Oct;21(4):305-311.
56. Tong W, Eppell SJ. Control of surface mineralization using collagen fibrils. J Biomed Mater Res 2002 Sep 5;61(3):346-353.
57.陈际达.纳米羟基磷灰石/胶原复合材料制备方法研究.生物物理学报2001；17(4).
58. Wen HB, Cui FZ, Zhu XD. Microstructural features of non-union of human humeral shaft fracture. J Struct Biol 1997 Aug;119(3):239-246.
59. Cui FZ, Wen HB, Su XW, et al. Microstructures of external periosteal callus of repaired femoral fracture in children. J Struct Biol 1996 Nov-Dec;117(3):204-208.
60. Su X, Sun K, Cui FZ, et al. Organization of apatite crystals in human woven bone. Bone 2003 Feb;32(2):150-162.
61.廖素三.组织工程中胶原基纳米骨复合材料的研制.中国医学科学院学报2003;25(1).
62.黄先智.蚕丝蛋白在食品和化妆品中的应用.四川丝绸2003(2).
63.盛伟华.蚕丝蛋白材料对鼠胚表皮细胞毒性的实验研究.苏州大学学报(医学版)2005;25(4).
64. Cordewener FW, Bos RR, Rozema FR, et al. Poly(L-lactide) implants for repair of human orbital floor defects:clinical and magnetic resonance imaging evaluation of long-term results. J Oral Maxillofac Surg 1996 Jan;54(1):9-13; discussion 13-14.
65. Meinel L, Hofmann S, Karageorgiou V, et al. The inflammatory responses to silk films in vitro and in vivo. Biomaterials 2005 Jan;26(2):147-155.
66. Altman GH, Horan RL, Lu HH, et al. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 2002 Oct;23(20):4131-4141.
67.卢神州.羟基磷灰石/丝素蛋白纳米复合颗粒的制备.丝绸2006(2).
68. Wang L, Ning G-L, Senna M. Microstructure and gelation behavior of hydroxyapatite-based nanocomposite sol containing chemically modified silk fibroin. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2005;254(1-3):159-164.
69.赵勇.丝素蛋白膜仿生沉积磷灰石的实验研究.口腔医学研究2006;22(2).
1. Du C, Cui FZ, Zhang W, et al. Formation of calcium phosphate/collagen composites through mineralization of collagen matrix. J Biomed Mater Res 2000 Jun 15;50(4):518-527.
2. Kikuchi M, Itoh S, Ichinose S, et al. Self-organization mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological reaction in vivo. Biomaterials 2001 Jul;22(13):1705-1711.
3. Mann S. Molecular tectonics in biomineralization and biomimetic materials chemistry. Nature 1993;365:6.
4. Stupp SI, Braun PV. Molecular manipulation of microstructures:biomaterials, ceramics, and semiconductors. Science 1997 Aug 29;277(5330):1242-1248.
5. Muthukumar M, Ober CK, Thomas EL. Competing Interactions and Levels of Ordering in Self-Organizing Polymeric Materials. Science 1997 August 29,1997;277(5330):1225-1232.
6. Chang MC, Ko CC, Douglas WH. Preparation of hydroxyapatite-gelatin nanocomposite. Biomaterials 2003 Aug;24(17):2853-2862.
7. Kasuga T, Ota Y, Nogami M, et al. Preparation and mechanical properties of polylactic acid composites containing hydroxyapatite fibers. Biomaterials 2001 Jan;22(l):19-23.
8. Zhang JC, Lu HY, Lv GY, et al. The repair of critical-size defects with porous hydroxyapatite/polyamide nanocomposite:an experimental study in rabbit mandibles. Int J Oral Maxillofac Surg Feb 27.
9. Li Y, Cai Y, Kong X, et al. Anisotropic growth of hydroxyapatite on the silk fibroin films. Applied Surface Science 2008;255(5, Part 1):1681-1685.
10. Du C, Jin J, Li Y, et al. Novel silk fibroin/hydroxyapatite composite films:Structure and properties. Materials Science and Engineering:C 2009;29(1):62-68.
11. Korematsu A, Furuzono T, Yasuda S, et al. Nano-scaled hydroxyapatite/polymer composite Ⅲ. Coating of sintered hydroxyapatite particles on poly(4-methacryloyloxyethyl trimellitate anhydride)-grafted silk fibroin fibers. J Mater Sci Mater Med 2005 Jan;16(1):67-71.
12. Murugan R, Ramakrishna S. Bioresorbable composite bone paste using polysaccharide based nano hydroxyapatite. Biomaterials 2004;25(17):3829-3835.
13. Rhee SH, Tanaka J. Self-assembly phenomenon of hydroxyapatite nanocrystals on chondroitin sulfate. J Mater Sci Mater Med 2002 Jun;13(6):597-600.
14. Cao M, Wang Y, Guo C, et al. Preparation of ultrahigh-aspect-ratio hydroxyapatite nanofibers in reverse micelles under hydrothermal conditions. Langmuir 2004 May 25;20(11):4784-4786.
15. Zhang W, Liao SS, Cui FZ. Hierarchical Self-Assembly of Nano-Fibrils in Mineralized Collagen. Chemistry of Materials 2003;15(16):3221-3226.
16. Ethirajan A, Ziener U, Landfester K. Surface-Functionalized Polymeric Nanoparticles as Templates for Biomimetic Mineralization of Hydroxyapatite. Chemistry of Materials 2009;21(11):2218-2225.
1.张真.生物材料有效性和安全性评价的现状与趋势.生物医学工程学杂志2002;19(1).
2.杨晓芳.生物材料生物相容性评价研究进展.生物医学工程学杂志2001;18(1).
3. Rokusek D, Davitt C, Bandyopadhyay A, et al. Interaction of human osteoblasts with bioinert and bioactive ceramic substrates. J Biomed Mater Res A 2005 Dec 1;75(3):588-594.
4. Wei G, Ma PX. Structure and properties of nano-hydroxyapatite/polymer composite scaffolds for bone tissue engineering. Biomaterials 2004 Aug;25(19):4749-4757.
5. Kim SS, Sun Park M, Jeon O, et al. Poly(lactide-co-glycolide)/hydroxyapatite composite scaffolds for bone tissue engineering. Biomaterials 2006 Mar;27(8):1399-1409.
6. Tuzlakoglu K, Bolgen N, Salgado AJ, et al. Nano-and micro-fiber combined scaffolds:a new architecture for bone tissue engineering. J Mater Sci Mater Med 2005 Dec; 16(12):1099-1104.
7. Hee CK, Jonikas MA, Nicoll SB. Influence of three-dimensional scaffold on the expression of osteogenic differentiation markers by human dermal fibroblasts. Biomaterials 2006 Feb;27(6):875-884.
8. Murugan R, Ramakrishna S. Nano-featured scaffolds for tissue engineering:a review of spinning methodologies. Tissue Eng 2006 Mar;12(3):435-447.
9. Ginebra MP, Driessens FC, Planell JA. Effect of the particle size on the micro and nanostructural features of a calcium phosphate cement:a kinetic analysis. Biomaterials 2004 Aug;25(17):3453-3462.
10.Popat KC, Leary Swan EE, Mukhatyar V, et al. Influence of nanoporous alumina membranes on long-term osteoblast response. Biomaterials 2005 Aug;26(22):4516-4522.
1. Valdes M, Palumbo M, Appel AJ, et al. Posterolateral intertransverse lumbar arthrodesis in the New Zealand White rabbit model:II. Operative technique. Spine J 2004 May-Jun;4(3):293-299.
2. Boden SD, Moskovitz PA, Morone MA, et al. Video-assisted lateral intertransverse process arthrodesis. Validation of a new minimally invasive lumbar spinal fusion technique in the rabbit and nonhuman primate (rhesus) models. Spine (Phila Pa 1976) 1996 Nov 15;21(22):2689-2697.
3. Lee T-C, Ho J-T, Hung K-S, et al. Bone Morphogenetic Protein Gene Therapy Using a Fibrin Scaffold for a Rabbit Spinal-Fusion Experiment. Neurosurgery 2006;58(2):373-380 310.1227/1201.NEU.0000199725.0000103186.F0000199726.
4. Curylo LJ, Johnstone B, Petersilge CA, et al. Augmentation of spinal arthrodesis with autologous bone marrow in a rabbit posterolateral spine fusion model. Spine (Phila Pa 1976) 1999 Mar 1;24(5):434-438; discussion 438-439.
5. Stanworth SJ, Newland AC. Stem cells:progress in research and edging towards the clinical setting. Clin Med 2001 Sep-Oct;1(5):378-382.
6.崔玉明.诱导骨髓基质细胞向软骨细胞分化的实验研究.中国临床康复2003;7(17).
7. Fleming JE, Jr., Cornell CN, Muschler GF. Bone cells and matrices in orthopedic tissue engineering. Orthop Clin North Am 2000 Jul;31(3):357-374.
8.周晓.组织工程骨修复骨缺损的研究进展.中国现代手术学杂志2006;10(4).
9. Grove JE, Bruscia E, Krause DS. Plasticity of bone marrow-derived stem cells. Stem Cells 2004;22(4):487-500.
10. Lee J, Elkahloun AG, Messina SA, et al. Cellular and genetic characterization of human adult bone marrow-derived neural stem-like cells:a potential antiglioma cellular vector. Cancer Res 2003 Dec 15;63(24):8877-8889.
11. Feiertag MA, Boden SD, Schimandle JH, et al. A rabbit model for nonunion of lumbar intertransverse process spine arthrodesis. Spine (Phila Pa 1976) 1996 Jan 1;21(1):27-31.
12. Boden SD. Overview of the biology of lumbar spine fusion and principles for selecting a bone graft substitute. Spine (Phila Pa 1976) 2002 Aug 15;27(16 Suppl 1):S26-31.
13. SD B, JH S, WC H. The use of an osteoinductive growth factor for lumbar spinal fusion:Part I. The biology of spinal fusion. Volvo Award in Basic Sciences Spine 1995;20:2626-32.
14. Toribatake Y, Hutton WC, Tomita K, et al. Vascularization of the fusion mass in a posterolateral intertransverse process fusion. Spine (Phila Pa 1976) 1998 May 15;23(10):1149-1154.
1. Altman GH, Diaz F, Jakuba C, et al. Silk-based biomaterials. Biomaterials 2003 Feb;24(3):401-416.
2. Sofia S, McCarthy MB, Gronowicz G, et al. Functionalized silk-based biomaterials for bone formation. J Biomed Mater Res 2001 Jan;54(1):139-148.
3. Altman GH, Horan RL, Lu HH, et al. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 2002 Oct;23(20):4131-4141.
4. Horan RL, Antle K, Collette AL, et al. In vitro degradation of silk fibroin. Biomaterials 2005 Jun;26(17):3385-3393.
5. Taddei P, Arai T, Boschi A, et al. In vitro study of the proteolytic degradation of Antheraea pernyi silk fibroin. Biomacromolecules 2006 Jan;7(1):259-267.
6. Takayuki A, Giuliano F, Riccardo I, et al. Biodegradation of Bombyx mori silk fibroin fibers and films. Journal of Applied Polymer Science,2004. p.2383-2390.
7. Li M, Ogiso M, Minoura N. Enzymatic degradation behavior of porous silk fibroin sheets. Biomaterials 2003 Jan;24(2):357-365.
8. Craig CL, Riekel C. Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders. Comp Biochem Physiol B Biochem Mol Biol 2002 Dec;133(4):493-507.
9. Valluzzi R, Winkler S, Wilson D, et al. Silk:molecular organization and control of assembly. Philosophical Transactions of the Royal Society of London Series B:Biological Sciences 2002 February 28,2002;357(1418):165-167.
10. PEREZ-RIGUEIRO,#160, Jos, et al. Similarities and differences in the supramolecular organization of silkworm and spider silk. Washington, DC, ETATS-UNIS:American Chemical Society, 2007.
11. Thiel BL, Guess KB, Viney C. Non-periodic lattice crystals in the hierarchical microstructure of spider (major ampullate) silk. Biopolymers 1997 Jun;41(7):703-719.
12. Santin M, Motta A, Freddi G, et al. In vitro evaluation of the inflammatory potential of the silk fibroin. Journal of Biomedical Materials Research 1999;46(3):382-389.
13. Panilaitis B, Altman GH, Chen J, et al. Macrophage responses to silk. Biomaterials 2003;24(18):3079-3085.
14. Chiarini A, Petrini P, Bozzini S, et al. Silk fibroin/poly(carbonate)-urethane as a substrate for cell growth:in vitro interactions with human cells. Biomaterials 2003 Feb;24(5):789-799.
15. Dal Pra I, Petrini P, Charini A, et al. Silk fibroin-coated three-dimensional polyurethane scaffolds for tissue engineering:interactions with normal human fibroblasts. Tissue Eng 2003 Dec;9(6):1113-1121.
16. Inouye K, Kurokawa M, Nishikawa S, et al. Use of Bombyx mori silk fibroin as a substratum for cultivation of animal cells. Journal of Biochemical and Biophysical Methods 1998;37(3):159-164.
17. Meinel L, Hofmann S, Karageorgiou V, et al. The inflammatory responses to silk films in vitro and in vivo. Biomaterials 2005 Jan;26(2):147-155.
18. Kardestuncer T, McCarthy MB, Karageorgiou V, et al. RGD-tethered silk substrate stimulates the differentiation of human tendon cells. Clin Orthop Relat Res 2006 Jul;448:234-239.
19. Minoura N, Aiba S, Higuchi M, et al. Attachment and growth of fibroblast cells on silk fibroin. Biochem Biophys Res Commun 1995 Mar 17;208(2):511-516.
20. Li C, Vepari C, Jin HJ, et al. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 2006 Jun;27(16):3115-3124.
21. Jin HJ, Chen J, Karageorgiou V, et al. Human bone marrow stromal cell responses on electrospun silk fibroin mats. Biomaterials 2004 Mar;25(6):1039-1047.
22. Unger RE, Wolf M, Peters K, et al. Growth of human cells on a non-woven silk fibroin net:a potential for use in tissue engineering. Biomaterials 2004;25(6):1069-1075.
23. Gosline JM, Guerette PA, Ortlepp CS, et al. The mechanical design of spider silks:from fibroin sequence to mechanical function. J Exp Biol 1999 Dec;202(Pt 23):3295-3303.
24. Sheu HS, Phyu KW, Jean YC, et al. Lattice deformation and thermal stability of crystals in spider silk. Int J Biol Macromol 2004 Oct;34(5):325-331.
25. Sirichaisit J, Brookes VL, Young RJ, et al. Analyis of structure/property relationships in silkworm (Bombyx mori) and spider dragline (Nephila edulis) silks using Raman spectroscopy. Biomacromolecules 2003 Mar-Apr;4(2):387-394.
26. Rising A, Nimmervoll H, Grip S, et al. Spider silk proteins-mechanical property and gene sequence. Zoolog Sci 2005 Mar;22(3):273-281.
27. Vollrath F. Strength and structure of spiders'silks. J Biotechnol 2000 Aug;74(2):67-83.
28. Zax DB, Armanios DE, Horak S, et al. Variation of mechanical properties with amino acid content in the silk of Nephila clavipes. Biomacromolecules 2004 May-Jun;5(3):732-738.
29. Riekel C, Madsen B, Knight D, et al. X-ray diffraction on spider silk during controlled extrusion under a synchrotron radiation X-ray beam. Biomacromolecules 2000 Winter;1(4):622-626.
30. Vollrath F, Madsen B, Shao Z. The effect of spinning conditions on the mechanics of a spider's dragline silk. Proc Biol Sci 2001 Nov 22;268(1483):2339-2346.
31. Liu Y, Shao Z, Vollrath F. Relationships between supercontraction and mechanical properties of spider silk. Nat Mater 2005 Dec;4(12):901-905.
32. Yang Y, Chen X, Shao Z, et al. Toughness of Spider Silk at High and Low Temperatures. Advanced Materials 2005;17(1):84-88.
33. Candelas G, Candelas T, Ortiz A, et al. Translational pauses during a spider fibroin synthesis. Biochem Biophys Res Commun 1983 Nov 15;116(3):1033-1038.
34. Vollrath F, Barth P, Basedow A, et al. Local tolerance to spider silks and protein polymers in vivo. In Vivo 2002 Jul-Aug;16(4):229-234.
35. Scheller J, Henggeler D, Viviani A, et al. Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation. Transgenic Res 2004 Feb;13(1):51-57.
36. Kuettner KE. Biochemistry of articular cartilage in health and disease. Clin Biochem 1992 Jun;25(3):155-163.
37. Mow VC, Ratcliffe A, Robin Poole A. Cartilage and diarthrodial joints as paradigms for hierarchical materials and structures. Biomaterials 1992;13(2):67-97.
38. Minas T, Nehrer S. Current concepts in the treatment of articular cartilage defects. Orthopedics 1997Jun;20(6):525-538.
39. Hofmann S, Knecht S, Langer R, et al. Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells. Tissue Eng 2006 Oct;12(10):2729-2738.
40. Marolt D, Augst A, Freed LE, et al. Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors. Biomaterials 2006;27(36):6138-6149.
41. Meinel L, Karageorgiou V, Hofmann S, et al. Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds. Journal of Biomedical Materials Research Part A 2004;71A(1):25-34.
42. Wang Y, Kim UJ, Blasioli DJ, et al. In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells. Biomaterials 2005 Dec;26(34):7082-7094.
43. Aoki H, Tomita N, Morita Y, et al. Culture of chondrocytes in fibroin-hydrogel sponge. Biomed Mater Eng 2003;13(4):309-316.
44. Morita Y, Tomita N, Aoki H, et al. Visco-elastic properties of cartilage tissue regenerated with fibroin sponge. Biomed Mater Eng 2002;12(3):291-298.
45. Wang Y, Blasioli DJ, Kim HJ, et al. Cartilage tissue engineering with silk scaffolds and human articular chondrocytes. Biomaterials 2006 Sep;27(25):4434-4442.
46. Gellynck.K, Verdonk.P, K.F A, et al. Chondrocyte Growth in Porous Spider Silk 3D-Scaffolds. European Cells and Materials 2005;10(suppl 2):45.
47. Sikavitsas VI, Temenoff JS, Mikos AG. Biomaterials and bone mechanotransduction. Biomaterials 2001 Oct;22(19):2581-2593.
48. Hofmann S, Hagenmuller H, Koch AM, et al. Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. Biomaterials 2007 Feb;28(6):1152-1162.
49. Kim K-H, Jeong L, Park H-N, et al. Biological efficacy of silk fibroin nanofiber membranes for guided bone regeneration. Journal of Biotechnology 2005;120(3):327-339.
50. Kirker-Head C, Karageorgiou V, Hofmann S, et al. BMP-silk composite matrices heal critically sized femoral defects. Bone 2007 Aug;41(2):247-255.
51. Meinel L, Betz O, Fajardo R, et al. Silk based biomaterials to heal critical sized femur defects. Bone 2006 Oct;39(4):922-931.
52. Meinel L, Hofmann S, Betz O, et al. Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials:comparison of adenovirus mediated gene transfer and protein delivery of BMP-2. Biomaterials 2006 Oct;27(28):4993-5002.
53. Karageorgiou V, Meinel L, Hofmann S, et al. Bone morphogenetic protein-2 decorated silk fibroin films induce osteogenic differentiation of human bone marrow stromal cells. J Biomed Mater Res A 2004 Dec 1;71(3):528-537.
54. Furuzono T, Kishida A, Tanaka J. Nano-scaled hydroxyapatite/polymer composite I. Coating of sintered hydroxyapatite particles on poly(gamma-methacryloxypropyl trimethoxysilane)grafted silk fibroin fibers through chemical bonding. J Mater Sci Mater Med 2004 Jan;15(1):19-23.
55. Tanaka T, Hirose M, Kotobuki N, et al. Nano-scaled hydroxyapatite/silk fibroin sheets support osteogenic differentiation of rat bone marrow mesenchymal cells. Materials Science and Engineering:C 2007;27(4):817-823.
56. Fini M, Motta A, Torricelli P, et al. The healing of confined critical size cancellous defects in the presence of silk fibroin hydrogel. Biomaterials 2005 Jun;26(17):3527-3536.
57. Kim HJ, Kim UJ, Vunjak-Novakovic G, et al. Influence of macroporous protein scaffolds on bone tissue engineering from bone marrow stem cells. Biomaterials 2005 Jul;26(21):4442-4452.
58. Bai J, Ma T, Chu W, et al. Regenerated spider silk as a new biomaterial for MEMS. Biomed Microdevices 2006 Dec;8(4):317-323.
59. Bini E, Foo CW, Huang J, et al. RGD-functionalized bioengineered spider dragline silk biomaterial. Biomacromolecules 2006 Nov;7(11):3139-3145.
60. Doroski DM, Brink KS, Temenoff JS. Techniques for biological characterization of tissue-engineered tendon and ligament. Biomaterials 2007 Jan;28(2):187-202.
61. Horan RL, Collette AL, Lee C, et al. Yarn design for functional tissue engineering. J Biomech 2006;39(12):2232-2240.
62. Chen J, Altman GH, Karageorgiou V, et al. Human bone marrow stromal cell and ligament fibroblast responses on RGD-modified silk fibers. J Biomed Mater Res A 2003 Nov 1;67(2):559-570.
63. Chen J, Horan RL, Bramono D, et al. Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering. Tissue Eng 2006 Nov;12(11):3085-3095.
64. Moreau JE, Chen J, Horan RL, et al. Sequential growth factor application in bone marrow stromal cell ligament engineering. Tissue Eng 2005 Nov-Dec; 11 (11-12):1887-1897.
65. Yoon JH, Halper J. Tendon proteoglycans:biochemistry and function. J Musculoskelet Neuronal Interact 2005 Mar;5(1):22-34.
66. Takezawa T, Ozaki K, Takabayashi C. Reconstruction of a hard connective tissue utilizing a pressed silk sheet and type-I collagen as the scaffold for fibroblasts. Tissue Eng 2007 Jun;13(6):1357-1366.
67. Liu H, Ge Z, Wang Y, et al. Modification of sericin-free silk fibers for ligament tissue engineering application. J Biomed Mater Res B Appl Biomater 2007 Jul;82(1):129-138.
68. Rathore O, Sogah DY. Self-assembly of beta-sheets into nanostructures by poly(alanine) segments incorporated in multiblock copolymers inspired by spider silk. J Am Chem Soc 2001 Jun 6;123(22):5231-5239.
69. Zhou C, Leng B, Yao J, et al. Synthesis and characterization of multiblock copolymers based on spider dragline silk proteins. Biomacromolecules 2006 Aug;7(8):2415-2419.
70. GRIP S, RISING A, NIMMERVOLL H, et al. Transient Expression of a Major Ampullate Spidroin 1 Gene Fragment from Euprosthenops sp. in Mammalian Cells. Cancer Genomics-Proteomics 2006 March 2006;3(2):83-87.
71. Lazaris A, Arcidiacono S, Huang Y, et al. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells. Science 2002 Jan 18;295(5554):472-476.
72. Huemmerich D, Scheibel T, Vollrath F, et al. Novel assembly properties of recombinant spider dragline silk proteins. Curr Biol 2004 Nov 23;14(22):2070-2074.
73. Lee KS, Kim BY, Je YH, et al. Molecular cloning and expression of the C-terminus of spider flagelliform silk protein from Araneus ventricosus. J Biosci 2007 Jun;32(4):705-712.
74. Fahnestock SR, Irwin SL. Synthetic spider dragline silk proteins and their production in Escherichia coli. Appl Microbiol Biotechnol 1997 Jan;47(1):23-32.
75. Schmidt M, Romer L, Strehle M, et al. Conquering isoleucine auxotrophy of Escherichia coli BLR(DE3) to recombinantly produce spider silk proteins in minimal media. Biotechnol Lett 2007 Nov;29(11):1741-1744.
76. Stark M, Grip S, Rising A, et al. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007 May;8(5):1695-1701.
77. Fahnestock SR, Bedzyk LA. Production of synthetic spider dragline silk protein in Pichia pastoris. Appl Microbiol Biotechnol 1997 Jan;47(1):33-39.
78. Hood EE, Jilka JM. Plant-based production of xenogenic proteins. Curr Opin Biotechnol 1999 Aug;10(4):382-386.
79. Scheller J, Guhrs KH, Grosse F, et al. Production of spider silk proteins in tobacco and potato. Nat Biotechnol 2001 Jun;19(6):573-577.
80. Teule F, Furin W, Cooper A, et al. Modifications of spider silk sequences in an attempt to control the mechanical properties of the synthetic fibers. Journal of Materials Science 2007;42(21):8974-8985.
81. Metwalli E, Slotta U, Darko C, et al. Structural changes of thin films from recombinant spider silk proteins upon post-treatment. Applied Physics A:Materials Science & Processing 2007;89(3):655-661.
82. Li G, Zhou P, Shao Z, et al. The natural silk spinning process. A nucleation-dependent aggregation mechanism? Eur J Biochem 2001 Dec;268(24):6600-6606.
83. Lundmark K, Westermark GT, et al. Protein fibrils in nature can enhance amyloid protein A amyloidosis in mice:Cross-seeding as a disease mechanism. Proceedings of the National Academy of Sciences of the United States of America 2005 April 26,2005;102(17):6098-6102.
84. Kenney JM, Knight D, Wise MJ, et al. Amyloidogenic nature of spider silk. Eur J Biochem 2002 Aug;269(16):4159-4163.