低温成形组织工程支架修复脊髓损伤的实验研究
|
摘要
随着现代社会经济的发展,脊髓损伤的发生率也呈逐年上升的趋势。而组织工程技术的兴起,为治疗脊髓损伤带来新的希望。制造出性能优良、符合与正常组织、器官类似的支架是组织工程发展的关键。支架的制备方法很多,各有优缺点,快速成形的出现为支架的制备提供了新途径。作为新的快速成形工艺,低温沉积制造工艺不仅具备快速成形工艺所共有的特点,而且集成了热致相分离过程的优点,成为制备组织工程支架的新宠。
本文利用低温沉积制造工艺加工脊髓组织工程支架。首先根据脊髓组织工程队支架的性能要求,从多种生物材料中遴选出PLGA作为脊髓组织工程支架的成形材料。然后以脊髓生理结构为出发点,设计了一种将灰质和白质区域的孔隙率、孔隙尺寸及材料用一个隔离层加以区分的脊髓仿生支架,诱导移植到缺损部位后的细胞能定向分化,促进脊髓组织的再生。
低温沉积过程实质上就是由计算机三维模型转化为数控信息,驱动成形机在低温环境下将材料堆积成形的过程。支架的大孔结构通过喷头的喷射和扫描运动来实现,在这个过程中喷头温度和成形室温度直接影响到大孔的形状误差和粘接强度,所以对这两个温度的调控以及使喷丝速度与扫描速度匹配成为低温沉积过程中两个最重要的环节。本文从软件设定、浆料性质、速度匹配和温度调控四个方面,研究了材料配制、分层和利用成形机制造等一系列工艺过程中参数对成形结构的影响,制造出具有大孔结构和微孔结构的三维脊髓仿生支架。
通过对支架进行孔隙率的表征、降解率和亲水率的测定实验,表明用低温沉积制造的支架的平均孔隙率达87.43%,降解实验和支架的亲水性能都能很好的满足组织工程脊髓支架的要求。然后将培养的纯度达到95%以上的雪旺细胞种植在PLGA支架上,每天换培养液,到第三天后扫描电镜观察。观察显示,雪旺细胞在PLGA支架上粘附性良好,迅速生长、增殖,表明PLGA支架材料适合雪旺细胞的粘附和生长。最后建立脊髓损伤模型,将得到的雪旺细胞+PLGA支架移植到大鼠脊髓损伤部位,观察雪旺细胞的存活及动物的恢复情况。结果表明,在最初的一周内,各组大鼠在脊髓横断损伤后均有不同程度的截瘫表现,从第2周开始,各组动物的BBB评分开始恢复。
With the modern social and economic development, the incidence of spinal cord injury also showed increasing trend. The emergence of tissue engineering technology has brought new hope to the treatment of spinal cord injury. It is a key point to the development of tissue engineering technology that the scaffold is manufactured with excellent properties. There are many manufacturing methods, each of which has separate advantages and disadvantages. The development of rapid prototyping (RP) technology provides a new way of scaffold manufacturing. As a novel RP technique, Low-temperature Deposition Manufacturing (LDM) not only has the common characteristics of RP, but also has the advantage of thermally induced phase separation process. So it has become the new daring of making tissue engineering scaffolds.
In this thesis, the spinal cord tissue engineering scaffolds was modeled using LDM technique. Firstly, according to the performance requirements of spinal cord tissue engineering scaffold, PLGA was selected as the manufacturing material. Then based on the spinal cord physical structure, the spinal cord bionic scaffold was designed with an insulating layer to distinguish of porosity, pore size and material of gray matter and white matter, which could indu(?) oriented differentiation of cell after transplantation into the defect site and promote regeneration of the spinal cord tissues.
LDM is essentially a process that the computer-aided 3D model is transformed into numerical control (NC) information to drive the LDM machine to extrude and deposit the material in low temperature environment. Macro-porous structure of the scaffold was made by extrusion and scanning of the nozzle. In this process, the temperature of the nozzle and that in the modeling room has direct impact on the macro-porous shape errors and adhesive strength. So it is the most important part in LDM to regulate the two temperatures and match the extruding speed and scanning speed. The impact to the modeling results was mainly studied through four aspects in this thesis:parameters setting in the software, property of the slurry, match of the velocity and regulation of temperature, and then 3D spinal cord bionic scaffolds with macro-porous and micro-porous structure were formed.
The porosity, degradation rate and hydrophilic rate of the scaffold were measured. The results indicated that the average porosity of scaffold manufactured by LDM was 87.43% and degradation and hydrophilic can well meet the requirements of the spinal cord tissue engineering scaffolds. Then the cultured Schwann cells of purity of more than 95% were implanted in PLGA scaffold. The medium was changed every day and SCs were scanned after three days by electron microscopy. SEM examination confirmed that Schwann cell grow rapidly after inoculated in PLGA scaffolds, which shows that the PLGA scaffolds have excellent biocompatibility and bioactivity. Lastly, the spinal cord was set up and the complex of Schwann cells and PLGA scaffolds were transplanted into rats with spinal cord injury site. The result showed that each rat has different levels of paraplegia in the first week, but from the second week beginning, the BBB score of rats began to recover.
本文利用低温沉积制造工艺加工脊髓组织工程支架。首先根据脊髓组织工程队支架的性能要求,从多种生物材料中遴选出PLGA作为脊髓组织工程支架的成形材料。然后以脊髓生理结构为出发点,设计了一种将灰质和白质区域的孔隙率、孔隙尺寸及材料用一个隔离层加以区分的脊髓仿生支架,诱导移植到缺损部位后的细胞能定向分化,促进脊髓组织的再生。
低温沉积过程实质上就是由计算机三维模型转化为数控信息,驱动成形机在低温环境下将材料堆积成形的过程。支架的大孔结构通过喷头的喷射和扫描运动来实现,在这个过程中喷头温度和成形室温度直接影响到大孔的形状误差和粘接强度,所以对这两个温度的调控以及使喷丝速度与扫描速度匹配成为低温沉积过程中两个最重要的环节。本文从软件设定、浆料性质、速度匹配和温度调控四个方面,研究了材料配制、分层和利用成形机制造等一系列工艺过程中参数对成形结构的影响,制造出具有大孔结构和微孔结构的三维脊髓仿生支架。
通过对支架进行孔隙率的表征、降解率和亲水率的测定实验,表明用低温沉积制造的支架的平均孔隙率达87.43%,降解实验和支架的亲水性能都能很好的满足组织工程脊髓支架的要求。然后将培养的纯度达到95%以上的雪旺细胞种植在PLGA支架上,每天换培养液,到第三天后扫描电镜观察。观察显示,雪旺细胞在PLGA支架上粘附性良好,迅速生长、增殖,表明PLGA支架材料适合雪旺细胞的粘附和生长。最后建立脊髓损伤模型,将得到的雪旺细胞+PLGA支架移植到大鼠脊髓损伤部位,观察雪旺细胞的存活及动物的恢复情况。结果表明,在最初的一周内,各组大鼠在脊髓横断损伤后均有不同程度的截瘫表现,从第2周开始,各组动物的BBB评分开始恢复。
With the modern social and economic development, the incidence of spinal cord injury also showed increasing trend. The emergence of tissue engineering technology has brought new hope to the treatment of spinal cord injury. It is a key point to the development of tissue engineering technology that the scaffold is manufactured with excellent properties. There are many manufacturing methods, each of which has separate advantages and disadvantages. The development of rapid prototyping (RP) technology provides a new way of scaffold manufacturing. As a novel RP technique, Low-temperature Deposition Manufacturing (LDM) not only has the common characteristics of RP, but also has the advantage of thermally induced phase separation process. So it has become the new daring of making tissue engineering scaffolds.
In this thesis, the spinal cord tissue engineering scaffolds was modeled using LDM technique. Firstly, according to the performance requirements of spinal cord tissue engineering scaffold, PLGA was selected as the manufacturing material. Then based on the spinal cord physical structure, the spinal cord bionic scaffold was designed with an insulating layer to distinguish of porosity, pore size and material of gray matter and white matter, which could indu(?) oriented differentiation of cell after transplantation into the defect site and promote regeneration of the spinal cord tissues.
LDM is essentially a process that the computer-aided 3D model is transformed into numerical control (NC) information to drive the LDM machine to extrude and deposit the material in low temperature environment. Macro-porous structure of the scaffold was made by extrusion and scanning of the nozzle. In this process, the temperature of the nozzle and that in the modeling room has direct impact on the macro-porous shape errors and adhesive strength. So it is the most important part in LDM to regulate the two temperatures and match the extruding speed and scanning speed. The impact to the modeling results was mainly studied through four aspects in this thesis:parameters setting in the software, property of the slurry, match of the velocity and regulation of temperature, and then 3D spinal cord bionic scaffolds with macro-porous and micro-porous structure were formed.
The porosity, degradation rate and hydrophilic rate of the scaffold were measured. The results indicated that the average porosity of scaffold manufactured by LDM was 87.43% and degradation and hydrophilic can well meet the requirements of the spinal cord tissue engineering scaffolds. Then the cultured Schwann cells of purity of more than 95% were implanted in PLGA scaffold. The medium was changed every day and SCs were scanned after three days by electron microscopy. SEM examination confirmed that Schwann cell grow rapidly after inoculated in PLGA scaffolds, which shows that the PLGA scaffolds have excellent biocompatibility and bioactivity. Lastly, the spinal cord was set up and the complex of Schwann cells and PLGA scaffolds were transplanted into rats with spinal cord injury site. The result showed that each rat has different levels of paraplegia in the first week, but from the second week beginning, the BBB score of rats began to recover.
引文
[1]张富强,王运赣.快速成型在生物医学中的应用[M].上海：人民军医出版社,2009.
[2]Langer R, Vacanti JP. Tissue engineering. Science [J],1993,260:920-926.
[3]Saltzman WM. Parkhurst MR, Parsons WP, et al. Three-dimensional cell cultures mimic tissues. AnnN Y Acad Sci [J].1992,665:259-265.
[4]杨志明.组织工程[M].北京.化学工业出版社.2001.123-124.
[5]Halberstadt C., Hardin R., Bezverkov K., et al. The in-vitro growth of a 3-demesional human dermal replacement using a single-pass perfusion system [J]. Biotechnol Bioeng,1994,43(8):740-746.
[6]Yang S., Leong K., Du Z., et al. The design of scaffolds for use in tissue engineering [J]. Part Ⅰ. Traditional factors. Tissue Eng,2001,7(6):679-689.
[7]Mikos A., Thorsen A., Czerwonka L., et al. Preparation and characterization of poly (L-lactic acid) foams [J]. Polymer,1994,35(5):1068-1077.
[8]Mooney D., Baldwin D., Suh N., et al. Novel approach to fabricate porous sponges of poly (D, L-lactic acid) without the use of organic solvents [J]. Biomaterials,1996,17(14):1417-1422.
[9]Nam Y, Park T. Biodegradable microcellular foams by modified thermally induced phase separation method [J]. Biomaterials,1999,20(19):1783-1790.
[10]田杰谟,李信勇.浸浆法制备生物多孔陶瓷[J].功能材料,2002,33(6)：656-660.
[11]刘建华,王国海等.脱细胞骨软骨支架的制备及形态学观察[J].实用医学杂志,2008,24(22)：3827-3829.
[12]孙明林.磷酸钙骨水泥/BMP复合人工骨的研制及相关研究[D].西安：第四军医大学全军骨科研究所,2001.
[13]蔡永梅.基于快速成型与逆向工程技术的快速磨具制造[D].新疆：新疆大学,2005
[14]Doshi J, Reneker DH. Electrospinning Process and Applications of Electrospun Fibers[J]. Journal of Electrostatics,1995,35(2-3):151-160.
[15]张玉朵.股骨三维重建与人工髋关节生物力学研究[D].天津：天津大学,2006.
[16]Sachs E., Cima M., Williams P., et al. Three-dimensional printing:rapid tooling and prototypes directly from a CAD model [J]. Eng Industry,1992,114:481-488.
[17]Lam CXF, Mo XM, Teoh SH, et al. Scaffold development using 3D printing with a starch-based polymer [J]. Mater Sci Eng C 2002,20(1-2):49-56.
[18]Peter Tek, Terry C. Chiganos, Javeed Shaikh Mohammed, et al. Rapid prototyping for neuroscience and neural engineering [J]. Journal of Neuroscience Methods,172 (2008):263-269.
[19]Landere R, Pfister A, Hubner U, et al. Fabrication of soft tissue engineering scaffolds by means of rapid prototyping techniques [J]. Journal of Materials Science,2002,37 (15):3107-3116.
[20]McWilliams J., Hysinger C., Beaman J. Design of a high temperature process for the selective laser sintering process [J]. In:Proceedings of solid free-form fabrication symposium. Austin, USA:1992. 110-117.
[21]Wiria FE, Leong KF, Chua CK, et al. Poly-epsilon-caprolactone/hydroxyapatite for tissue engineering scaffold fabrication via selective laser sintering[J]. Acta Biomater,2007,3(1):1-12.
[22]王星,吴任东,张磊等.基于尖笔直写的组织工程支架成形工艺研究[J].电加工与磨具,2008,5：73-76.
[23]LIU Feng, ZHANG Renji. Aerodynamically Assisted Tip-Pen Direct Writing [J]. TSINGHUA SCIENCE AND TECHNOLOGY,2009,14(S1):116-119.
[24]熊卓.骨组织工程支架的低温沉积制造及应用基础研究[D].北京：清华大学,2002,11.
[25]岳海源,汪玉良,脊髓损伤修复研究进展[J].国际骨科学杂志,2010,31(2)：113-115.
[26]Paulmurugan R, Masoud TF, Huang J, et al. Molecular imaging of drug-rnodulated protein-proton interactions in living subjects [J]. Cancer Res,2004,64(6):2113-2119.
[27]Svensson CI, Heffean MP, et al. The Rheb-mTOR pathway is unregulated in reactive astrocytes of the injured cord [J]. Neurosci,2009,29(4):1093-1104.
[28]Kim BG, Dai HN, et al. Modulation of dendritic spine remodeling in the motor cortex following spinal cord injury:effects of environmental enrichment and combinatorial treatment with transplants and neurptrophin-3[J]. Comp Neurol,2008,508(3):473-486.
[29]Lo WC, Hsu CH, et al. A novel cell-based therapy for contusion spinal cord injury using GDNF-delivering. NH3T3 cells with dual reporter genes monitored by molecular imaging [J]. Nucl Med, 2008,49(9):1512-1519.
[30]Wu J, Sun TS, et al. Ex vivo non-viral vector-mediated neurotrophin-3 gene transfer to olfactory ensheating gila:effects on atonal regeneration and functional recovery after imitation in rats with spinal cord injury[J]. Neurosci Bull,2008,24(2):57-65.
[31]姜涛,任先军.组织工程脊髓支架材料的研究进展[J].中国矫形外科杂志,2010,18(15)：1270-1273.
[32]Neves NM, Kouyumdziev A, Reis RL. The morphology, mechanical properties and ageing behavior of porous injection molded starch-based blends for tissue engineering scaffolding [J]. Mater Sci Eng C, 2005,25:195-200.
[33]Hatami M, Mehrjardi NZ, Kiani S, et al. Human embryonic stem cell-derived neural precursor transplants in collagen scaffolds promote recovery in injured rat spinal cord [J]. Cytotherapy,2009,11(5):618-630.
[34]Johnson PJ, Tatara A, Shiu A, et al. Controlled release of neurotrophin-3 and platelet derived growth factor from fibrin scaffolds containing neural progenitor cells enhances survival and differentiation into neuron in sub acute model of SCI[J]. Cell Transplant,2009,9:198.
[35]张雪娇,杨朝阳等.壳聚糖对大鼠脊髓损伤后小胶质细胞/巨噬细胞的影响[J].中国康复理论与实践,2009,15(4)：321-323.
[36]Tobias CA, Han SS, Shumsky JS, et al. Alginate encapsulated BDNF-producing fibroblast grafts permit recovery of function after spinal cord injury in the absence of immune suppression [J]. J Neurotrauma, 2005,22(1):138-156.
[37]Stokols Sh, Tuszynski M. Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury. Biomaterials [J].2006,27(3):443-451.
[38]Gupta D, Tator CH, Shoichet MS. Fast-gelling injectable blend of hyaluronan and methylcellulose for intrathecal, localized delivery to the injured spinal cord [J]. Biomaterials,2006,27(11):2370-2379.
[39]钱玉强,沈忆新等.嗅鞘细胞与丝素蛋白纳米纤维生物相容性研究[J].中国修复重建外科杂志,2009,11(23)：1365-1370
[40]Hurtado A, Moon LD, Maquet V, et al. Poly (D, L-lactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete a bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord[J]. Biomaterials.2006,27(3):430-442.
[41]Vacanti MP, Leonard JL, Dore B, et al. Tissue-engineered spinal cord [J]. Transplant Proc,2001, 33(1-2):592-598.
[42]Laverty PH, Leskovar A, Breur GJ, et al. A preliminary study of intravenous surfactants in paraplegic dogs:polymer therapy in canine clinical SCI [J]. J Neurotrauma,2004,21(12):1767-1777.
[43]Novikov LN, Novicova LN, Mosahebi A, et al. A novel biodegradable implant for neuronal rescue and regeneration after spinal cord injury. Biomaterials.2002,23(16):3369-3376.
[44]Tan PS, Teoh SH. Effect of stiffness of polycaprolactone (PCL) membrane on cell proliferation [J]. Mater Sci Eng C,2007,27:304-308.
[45]宋玉林,郑启新等.含1KVAV两亲性肽自组装凝胶二维诱导神经干细胞的分化[J].中国组织工程研究与临床康复,2009,139(34)：6667-6670.
[46]张之虎.脊髓损伤的诊断治疗和康复[D].北京：工人出版社,1986,8.
[47]CAI Jianhui, SHI Tingchun, et al. The research of tissue engineering scaffold about spinal cord injury [J]. Applied Mechanics Materials,2010,33:3838-389.
[48]蔡建辉.脊髓损伤修复组织工程支架研制[D].杭州：杭州电子科技火学,2010,11
[49]索海瑞,岳秀艳等.组织工程支架的低温沉积制造工艺参数研究[J].机电工程,2009,26(3)：57-60
[50]向虎,等.骨组织工程材料PLG A/TCP的温度黏度性能[J].清华大学学报(自然科学版),2004,44(8)：1100-1103.
[51]Fei Yang, Wenjin Cui, Zhuo Xiong, etc. Poly (L, L-lactide-co-glycolide)/tricalcium phosphate composite scaffold and its various changes during degradation in vitro [J]. Polymer Degradation and Stability, 2006(91):3065-3073.
[52]张翠兰,王志,李凭力,等.热致相分离法制备聚丙稀微孔膜[J].膜科学与技术,2000,20(6)：36-40.
[53]骆峰,张军,王晓琳,等.热诱导相分离法制备高分子微孔膜的原理与进展[J].南京化工大学学报,2001,23(4)：91-96.
[54]叶超群,孙天胜.嗅鞘细胞和雪旺细胞混合移植对大鼠脊髓损伤修复的影响[J].中国脊柱脊髓杂志,2009,19(9)：676-681.
[55]Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats [J]. Neurotrauma,1995,12:1-21.
[2]Langer R, Vacanti JP. Tissue engineering. Science [J],1993,260:920-926.
[3]Saltzman WM. Parkhurst MR, Parsons WP, et al. Three-dimensional cell cultures mimic tissues. AnnN Y Acad Sci [J].1992,665:259-265.
[4]杨志明.组织工程[M].北京.化学工业出版社.2001.123-124.
[5]Halberstadt C., Hardin R., Bezverkov K., et al. The in-vitro growth of a 3-demesional human dermal replacement using a single-pass perfusion system [J]. Biotechnol Bioeng,1994,43(8):740-746.
[6]Yang S., Leong K., Du Z., et al. The design of scaffolds for use in tissue engineering [J]. Part Ⅰ. Traditional factors. Tissue Eng,2001,7(6):679-689.
[7]Mikos A., Thorsen A., Czerwonka L., et al. Preparation and characterization of poly (L-lactic acid) foams [J]. Polymer,1994,35(5):1068-1077.
[8]Mooney D., Baldwin D., Suh N., et al. Novel approach to fabricate porous sponges of poly (D, L-lactic acid) without the use of organic solvents [J]. Biomaterials,1996,17(14):1417-1422.
[9]Nam Y, Park T. Biodegradable microcellular foams by modified thermally induced phase separation method [J]. Biomaterials,1999,20(19):1783-1790.
[10]田杰谟,李信勇.浸浆法制备生物多孔陶瓷[J].功能材料,2002,33(6)：656-660.
[11]刘建华,王国海等.脱细胞骨软骨支架的制备及形态学观察[J].实用医学杂志,2008,24(22)：3827-3829.
[12]孙明林.磷酸钙骨水泥/BMP复合人工骨的研制及相关研究[D].西安：第四军医大学全军骨科研究所,2001.
[13]蔡永梅.基于快速成型与逆向工程技术的快速磨具制造[D].新疆：新疆大学,2005
[14]Doshi J, Reneker DH. Electrospinning Process and Applications of Electrospun Fibers[J]. Journal of Electrostatics,1995,35(2-3):151-160.
[15]张玉朵.股骨三维重建与人工髋关节生物力学研究[D].天津：天津大学,2006.
[16]Sachs E., Cima M., Williams P., et al. Three-dimensional printing:rapid tooling and prototypes directly from a CAD model [J]. Eng Industry,1992,114:481-488.
[17]Lam CXF, Mo XM, Teoh SH, et al. Scaffold development using 3D printing with a starch-based polymer [J]. Mater Sci Eng C 2002,20(1-2):49-56.
[18]Peter Tek, Terry C. Chiganos, Javeed Shaikh Mohammed, et al. Rapid prototyping for neuroscience and neural engineering [J]. Journal of Neuroscience Methods,172 (2008):263-269.
[19]Landere R, Pfister A, Hubner U, et al. Fabrication of soft tissue engineering scaffolds by means of rapid prototyping techniques [J]. Journal of Materials Science,2002,37 (15):3107-3116.
[20]McWilliams J., Hysinger C., Beaman J. Design of a high temperature process for the selective laser sintering process [J]. In:Proceedings of solid free-form fabrication symposium. Austin, USA:1992. 110-117.
[21]Wiria FE, Leong KF, Chua CK, et al. Poly-epsilon-caprolactone/hydroxyapatite for tissue engineering scaffold fabrication via selective laser sintering[J]. Acta Biomater,2007,3(1):1-12.
[22]王星,吴任东,张磊等.基于尖笔直写的组织工程支架成形工艺研究[J].电加工与磨具,2008,5：73-76.
[23]LIU Feng, ZHANG Renji. Aerodynamically Assisted Tip-Pen Direct Writing [J]. TSINGHUA SCIENCE AND TECHNOLOGY,2009,14(S1):116-119.
[24]熊卓.骨组织工程支架的低温沉积制造及应用基础研究[D].北京：清华大学,2002,11.
[25]岳海源,汪玉良,脊髓损伤修复研究进展[J].国际骨科学杂志,2010,31(2)：113-115.
[26]Paulmurugan R, Masoud TF, Huang J, et al. Molecular imaging of drug-rnodulated protein-proton interactions in living subjects [J]. Cancer Res,2004,64(6):2113-2119.
[27]Svensson CI, Heffean MP, et al. The Rheb-mTOR pathway is unregulated in reactive astrocytes of the injured cord [J]. Neurosci,2009,29(4):1093-1104.
[28]Kim BG, Dai HN, et al. Modulation of dendritic spine remodeling in the motor cortex following spinal cord injury:effects of environmental enrichment and combinatorial treatment with transplants and neurptrophin-3[J]. Comp Neurol,2008,508(3):473-486.
[29]Lo WC, Hsu CH, et al. A novel cell-based therapy for contusion spinal cord injury using GDNF-delivering. NH3T3 cells with dual reporter genes monitored by molecular imaging [J]. Nucl Med, 2008,49(9):1512-1519.
[30]Wu J, Sun TS, et al. Ex vivo non-viral vector-mediated neurotrophin-3 gene transfer to olfactory ensheating gila:effects on atonal regeneration and functional recovery after imitation in rats with spinal cord injury[J]. Neurosci Bull,2008,24(2):57-65.
[31]姜涛,任先军.组织工程脊髓支架材料的研究进展[J].中国矫形外科杂志,2010,18(15)：1270-1273.
[32]Neves NM, Kouyumdziev A, Reis RL. The morphology, mechanical properties and ageing behavior of porous injection molded starch-based blends for tissue engineering scaffolding [J]. Mater Sci Eng C, 2005,25:195-200.
[33]Hatami M, Mehrjardi NZ, Kiani S, et al. Human embryonic stem cell-derived neural precursor transplants in collagen scaffolds promote recovery in injured rat spinal cord [J]. Cytotherapy,2009,11(5):618-630.
[34]Johnson PJ, Tatara A, Shiu A, et al. Controlled release of neurotrophin-3 and platelet derived growth factor from fibrin scaffolds containing neural progenitor cells enhances survival and differentiation into neuron in sub acute model of SCI[J]. Cell Transplant,2009,9:198.
[35]张雪娇,杨朝阳等.壳聚糖对大鼠脊髓损伤后小胶质细胞/巨噬细胞的影响[J].中国康复理论与实践,2009,15(4)：321-323.
[36]Tobias CA, Han SS, Shumsky JS, et al. Alginate encapsulated BDNF-producing fibroblast grafts permit recovery of function after spinal cord injury in the absence of immune suppression [J]. J Neurotrauma, 2005,22(1):138-156.
[37]Stokols Sh, Tuszynski M. Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury. Biomaterials [J].2006,27(3):443-451.
[38]Gupta D, Tator CH, Shoichet MS. Fast-gelling injectable blend of hyaluronan and methylcellulose for intrathecal, localized delivery to the injured spinal cord [J]. Biomaterials,2006,27(11):2370-2379.
[39]钱玉强,沈忆新等.嗅鞘细胞与丝素蛋白纳米纤维生物相容性研究[J].中国修复重建外科杂志,2009,11(23)：1365-1370
[40]Hurtado A, Moon LD, Maquet V, et al. Poly (D, L-lactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete a bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord[J]. Biomaterials.2006,27(3):430-442.
[41]Vacanti MP, Leonard JL, Dore B, et al. Tissue-engineered spinal cord [J]. Transplant Proc,2001, 33(1-2):592-598.
[42]Laverty PH, Leskovar A, Breur GJ, et al. A preliminary study of intravenous surfactants in paraplegic dogs:polymer therapy in canine clinical SCI [J]. J Neurotrauma,2004,21(12):1767-1777.
[43]Novikov LN, Novicova LN, Mosahebi A, et al. A novel biodegradable implant for neuronal rescue and regeneration after spinal cord injury. Biomaterials.2002,23(16):3369-3376.
[44]Tan PS, Teoh SH. Effect of stiffness of polycaprolactone (PCL) membrane on cell proliferation [J]. Mater Sci Eng C,2007,27:304-308.
[45]宋玉林,郑启新等.含1KVAV两亲性肽自组装凝胶二维诱导神经干细胞的分化[J].中国组织工程研究与临床康复,2009,139(34)：6667-6670.
[46]张之虎.脊髓损伤的诊断治疗和康复[D].北京：工人出版社,1986,8.
[47]CAI Jianhui, SHI Tingchun, et al. The research of tissue engineering scaffold about spinal cord injury [J]. Applied Mechanics Materials,2010,33:3838-389.
[48]蔡建辉.脊髓损伤修复组织工程支架研制[D].杭州：杭州电子科技火学,2010,11
[49]索海瑞,岳秀艳等.组织工程支架的低温沉积制造工艺参数研究[J].机电工程,2009,26(3)：57-60
[50]向虎,等.骨组织工程材料PLG A/TCP的温度黏度性能[J].清华大学学报(自然科学版),2004,44(8)：1100-1103.
[51]Fei Yang, Wenjin Cui, Zhuo Xiong, etc. Poly (L, L-lactide-co-glycolide)/tricalcium phosphate composite scaffold and its various changes during degradation in vitro [J]. Polymer Degradation and Stability, 2006(91):3065-3073.
[52]张翠兰,王志,李凭力,等.热致相分离法制备聚丙稀微孔膜[J].膜科学与技术,2000,20(6)：36-40.
[53]骆峰,张军,王晓琳,等.热诱导相分离法制备高分子微孔膜的原理与进展[J].南京化工大学学报,2001,23(4)：91-96.
[54]叶超群,孙天胜.嗅鞘细胞和雪旺细胞混合移植对大鼠脊髓损伤修复的影响[J].中国脊柱脊髓杂志,2009,19(9)：676-681.
[55]Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats [J]. Neurotrauma,1995,12:1-21.