丝素蛋白多孔支架在大鼠脊髓血管化的实验研究
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摘要
目的观察丝素蛋白多孔支架（porous silk fibroin scaffolds,PSFSs）在大鼠脊髓损伤（spinal cord injury,SCI）部位的血管化，探讨组织工程方法修复SCI的作用机制，为丝素蛋白（silk fibroin,SF）用于组织工程修复中枢神经系统（central nervoussystem,CNS）损伤提供实验基础和理论依据。
方法（1）植入物的制备制备具有相近结构参数（孔径、孔隙率、孔隙间连通率）的PSFSs及聚乙烯醇（polyvinyl alcohol,PVA）海绵，进行扫描电镜观察；（2）动物模型的建立、实验分组及材料植入选取28只清洁级雌性Sprague Dawley（S-D）大鼠（体重250～300g），随机分为A、B两组（A为实验组，n=16；B为对照组，n=12）；采用手术方法建立T10平面脊髓左侧半横断损伤模型，A组植入PSFSs，B组植入PVA；术后单笼饲养、预防感染；（3）检测指标分别于术后4d、7d、10d、14d、21d、28d共6个时间点，每组各取2只大鼠灌注取材观察标本大体形态；行组织学、免疫组织化学（CD34）检测，观察局部炎症反应、材料变化情况及材料内微血管生成情况；并采用透射电镜观察A组术后14d、28d时微血管超微结构。
结果（1）PSFSs的初始形状为圆柱体，与大鼠脊髓外形相似，将其切割成适合的半圆形；扫描电镜图像示PSFSs具有合理的孔径及孔隙率，并具有较高的孔隙间连通率；PVA的结构参数与PSFSs相近；（2）成功地建立了大鼠半横断SCI模型，并总结出一套行之有效的术后护理方法；大鼠术后存活良好，并发症少，无样本丢失；（3）标本大体形态观察以材料为中心取长约10mm脊髓组织，见材料部位呈淡黄色、与周围脊髓组织融合良好，材料表面有少量结缔组织；（4）HE染色观察A组的炎症反应较B组轻、消退速度快，PSFSs降解较明显；植入术后7d PSFSs内可见管腔样结构形成、此时PVA内尚无管腔样结构形成；随时间推移，材料内管腔样结构增多；（5）免疫组织化学观察通过CD34染色观察材料内微血管生成情况、并计数微血管密度（microvessel density,MVD），A组在术后7d、10d、14d、21d、28d分别为1.4、3.6、10.6、8.6、8.8，对照组分别为0、2.2、4.8、4.6、4.0，差异具有统计学意义（P<0.05）；A组在术后7d即可观察到材料内有微血管形成，14d时达到峰值，随后有所下降并稳定于一定水平；B组7d时尚无微血管形成，14d时微血管数量较多，也呈现有所下降并趋于稳定的变化规律；（6）透射电镜观察术后14d，PSFSs内可见毛细血管形成，能看到完整的内皮细胞、呈扁平状，管腔中有红细胞，周细胞位于内皮细胞基底膜的外侧，环绕血管管腔；术后28d，材料内血管化进一步充分和完全，血管结构完整，成为有灌注的功能性血管。
结论PSFSs可以在大鼠SCI部位血管化，这是细胞存活和神经轴突再生的前提。SF有望成为组织工程的理想支架，用于修复CNS损伤。
Objective To observe the vascularization of porous silk fibrion scaffolds(PSFSs)following spinal cord injury(SCI) in rats, and to explore the mechanism of SCI repair intissue engineering so as to lay the foundation for silk fibroin(SF) of central nervoussystem(CNS) repair both theoretically and experimentally.
Methods (1) Fabrication of the implants The implants including PSFSs and PVAwith similar pore sizes, porosity and interconnectivity were prepared.The surface structureof the implants was observed with scanning electron microscopy(SEM).(2) Establishmentof animal models,grouping,and transplantation of the implants28female SpragueDawley(S-D) rats, each with a body weight of250~300g, were selected and put into twogroups randomly (A:experimental group, n=16; B:control group,n=12). Following alaminectomy, all the animals received a lateral hemisection at the left of the spinal cord atthe T9–T10level. PSFSs was transplanted into the spinal cord of rats in group A andpolyvinyl alcohol(PVA) into the spinal cord of rats in group B. Then the rats were kept inseparate cages and cared to prevent infection.(3) Detection At6points of time，4d、7d、10d、14d、21d and28d postinjury，2rats from each group were killed following a perfusion.And the spinal cord containing the material was cut for histological andimmunohistological staining in order to observe the inflammatory response,variation,andthe formation of microvessels in the material. Furthermore,the unltrastructure ofmicrovessels formed in the material was detected with transmission electronmicroscopy(TEM) in group A at the time points of14d,28d postoperation.
Results (1) The initial shape of PSFSs was a cylinder, like the spinal cord of rats inappearance.PSFSs was cut to be Semicircular.SEM showed PSFSs with proper pore sizes,porosity,and high interconnectivity rate. The structural parameter of PVA was similarto that of PSFSs.(2) The SCI model of the hemisection in rats was successfully established.And a series of effective postoperative caring methods were summarized. All the ratssurvived after the operation, and with few complications.(3) Observation of the materialsA10mm spinal cord containing the material was cut, which was yellowish, well-integratedwith the surrounding spinal cord tissue and with a few connective tissue on the surface ofthe material.(4) Observation of HE staining HE staining showed that the inflammatoryresponse was more moderate, and the degradation of materials more obvious in group Athan that in group B.Lumen-like structure was seen in PSFSs at7d postoperation, but thisdid not occur in PVA. And the number of lumen-like structure increased gradually.(5)Immunohistochemical observation The formation of microvessels was observed byCD34staining,And the microvessels density(MVD) was quantified. The value of MVDwas1.4,3.6,10.6,8.6,8.8respectively at different points of time after the operation in groupA.On the contrary, it was0,2.2,4.8,4.6,4.0respectively in group B. The P value isstatistically significant(P<0.05). The microvessels formed as early as7d in group A, andthe peak appeared at14d postoperation. Then its quantity decreased to a stable level. Nomicrovessel was seen at7d in group B. But The trend of MVD in group B was similar tothat in group A.(6) Observation of TEM The ultrastructure of microvessels was observed byTEM. Endothelial cells around by Pericytes in PFSFs were detected at14d postoperation.Redcells were in the lumen.The ultrastructure of microvessels progressed at28d postoperation. Thisindicated the fomation of perfused, functional microvessels.
Conclusion PSFSs can be vascularized in SCI of rats, which is the precondition for cellsurvival and regeneration of neural axons. SF has the potential to be an ideal scaffold for centralnervous system(CNS) repair in tissue engineering.
方法（1）植入物的制备制备具有相近结构参数（孔径、孔隙率、孔隙间连通率）的PSFSs及聚乙烯醇（polyvinyl alcohol,PVA）海绵，进行扫描电镜观察；（2）动物模型的建立、实验分组及材料植入选取28只清洁级雌性Sprague Dawley（S-D）大鼠（体重250～300g），随机分为A、B两组（A为实验组，n=16；B为对照组，n=12）；采用手术方法建立T10平面脊髓左侧半横断损伤模型，A组植入PSFSs，B组植入PVA；术后单笼饲养、预防感染；（3）检测指标分别于术后4d、7d、10d、14d、21d、28d共6个时间点，每组各取2只大鼠灌注取材观察标本大体形态；行组织学、免疫组织化学（CD34）检测，观察局部炎症反应、材料变化情况及材料内微血管生成情况；并采用透射电镜观察A组术后14d、28d时微血管超微结构。
结果（1）PSFSs的初始形状为圆柱体，与大鼠脊髓外形相似，将其切割成适合的半圆形；扫描电镜图像示PSFSs具有合理的孔径及孔隙率，并具有较高的孔隙间连通率；PVA的结构参数与PSFSs相近；（2）成功地建立了大鼠半横断SCI模型，并总结出一套行之有效的术后护理方法；大鼠术后存活良好，并发症少，无样本丢失；（3）标本大体形态观察以材料为中心取长约10mm脊髓组织，见材料部位呈淡黄色、与周围脊髓组织融合良好，材料表面有少量结缔组织；（4）HE染色观察A组的炎症反应较B组轻、消退速度快，PSFSs降解较明显；植入术后7d PSFSs内可见管腔样结构形成、此时PVA内尚无管腔样结构形成；随时间推移，材料内管腔样结构增多；（5）免疫组织化学观察通过CD34染色观察材料内微血管生成情况、并计数微血管密度（microvessel density,MVD），A组在术后7d、10d、14d、21d、28d分别为1.4、3.6、10.6、8.6、8.8，对照组分别为0、2.2、4.8、4.6、4.0，差异具有统计学意义（P<0.05）；A组在术后7d即可观察到材料内有微血管形成，14d时达到峰值，随后有所下降并稳定于一定水平；B组7d时尚无微血管形成，14d时微血管数量较多，也呈现有所下降并趋于稳定的变化规律；（6）透射电镜观察术后14d，PSFSs内可见毛细血管形成，能看到完整的内皮细胞、呈扁平状，管腔中有红细胞，周细胞位于内皮细胞基底膜的外侧，环绕血管管腔；术后28d，材料内血管化进一步充分和完全，血管结构完整，成为有灌注的功能性血管。
结论PSFSs可以在大鼠SCI部位血管化，这是细胞存活和神经轴突再生的前提。SF有望成为组织工程的理想支架，用于修复CNS损伤。
Objective To observe the vascularization of porous silk fibrion scaffolds(PSFSs)following spinal cord injury(SCI) in rats, and to explore the mechanism of SCI repair intissue engineering so as to lay the foundation for silk fibroin(SF) of central nervoussystem(CNS) repair both theoretically and experimentally.
Methods (1) Fabrication of the implants The implants including PSFSs and PVAwith similar pore sizes, porosity and interconnectivity were prepared.The surface structureof the implants was observed with scanning electron microscopy(SEM).(2) Establishmentof animal models,grouping,and transplantation of the implants28female SpragueDawley(S-D) rats, each with a body weight of250~300g, were selected and put into twogroups randomly (A:experimental group, n=16; B:control group,n=12). Following alaminectomy, all the animals received a lateral hemisection at the left of the spinal cord atthe T9–T10level. PSFSs was transplanted into the spinal cord of rats in group A andpolyvinyl alcohol(PVA) into the spinal cord of rats in group B. Then the rats were kept inseparate cages and cared to prevent infection.(3) Detection At6points of time，4d、7d、10d、14d、21d and28d postinjury，2rats from each group were killed following a perfusion.And the spinal cord containing the material was cut for histological andimmunohistological staining in order to observe the inflammatory response,variation,andthe formation of microvessels in the material. Furthermore,the unltrastructure ofmicrovessels formed in the material was detected with transmission electronmicroscopy(TEM) in group A at the time points of14d,28d postoperation.
Results (1) The initial shape of PSFSs was a cylinder, like the spinal cord of rats inappearance.PSFSs was cut to be Semicircular.SEM showed PSFSs with proper pore sizes,porosity,and high interconnectivity rate. The structural parameter of PVA was similarto that of PSFSs.(2) The SCI model of the hemisection in rats was successfully established.And a series of effective postoperative caring methods were summarized. All the ratssurvived after the operation, and with few complications.(3) Observation of the materialsA10mm spinal cord containing the material was cut, which was yellowish, well-integratedwith the surrounding spinal cord tissue and with a few connective tissue on the surface ofthe material.(4) Observation of HE staining HE staining showed that the inflammatoryresponse was more moderate, and the degradation of materials more obvious in group Athan that in group B.Lumen-like structure was seen in PSFSs at7d postoperation, but thisdid not occur in PVA. And the number of lumen-like structure increased gradually.(5)Immunohistochemical observation The formation of microvessels was observed byCD34staining,And the microvessels density(MVD) was quantified. The value of MVDwas1.4,3.6,10.6,8.6,8.8respectively at different points of time after the operation in groupA.On the contrary, it was0,2.2,4.8,4.6,4.0respectively in group B. The P value isstatistically significant(P<0.05). The microvessels formed as early as7d in group A, andthe peak appeared at14d postoperation. Then its quantity decreased to a stable level. Nomicrovessel was seen at7d in group B. But The trend of MVD in group B was similar tothat in group A.(6) Observation of TEM The ultrastructure of microvessels was observed byTEM. Endothelial cells around by Pericytes in PFSFs were detected at14d postoperation.Redcells were in the lumen.The ultrastructure of microvessels progressed at28d postoperation. Thisindicated the fomation of perfused, functional microvessels.
Conclusion PSFSs can be vascularized in SCI of rats, which is the precondition for cellsurvival and regeneration of neural axons. SF has the potential to be an ideal scaffold for centralnervous system(CNS) repair in tissue engineering.
引文
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[1]周建军,吴国材,冯华.脊髓损伤的动物模型进展[J].脊柱外科杂志，2004,2(3):173-175.
[2]周婷婷,郑志红.脊髓损伤动物模型的构建与应用[J].实验动物与比较医学，2008,28(6):411-415.
[3] Kaptanoglu E,Tuncel M,Palaoglu S,et a1.Comparison of the effects of melatonin andmethylprednisolone in experimental spinal cord injury[J].J Neurosurg,2000,93(1Suppl):77-84.
[4] Talac R,Friedman JA,Moore MJ,et al.Animal models of spinal cord injury forevaluation of tissue engineering treatment strategies[J].Biomater-ials,2004;25(9):1505-1510.
[5] Yan HB, J in DD, Lu KW, et al.Establishment of s table rat models of complete spinalcord transection[J].Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu2007;11(6):1091-1094(China)
[6]刘锐,游思维,刘惠玲,等.构建还原论式大鼠脊髓全横断损伤模型的性别影响[J].中国临床康复,2005,9(17):95-97.
[7]乔晓峰,杨建华,朱光宇,等.脊髓损伤模型的制备及其评价[J].中国全科医学,2009,12(7B):1279-1281.
[8]高天君,刘少君,侯树勋.脑源性神经营养因子基因转染嗅鞘细胞移植修复脊髓损伤的观察[J].解放军医学杂志,2005,30(11):1003-1005,1011.
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[13] Koichi I, Toshiki Y, Haruhiko K, et al. Transplantation of olfactory mucosa followingspinal cord injury promotes recovery in rats[J]. Neuroreport,2008;19(13):1249-1252.
[14] Marc D.Kubasak,Devin L,et al.OEG implantation and step training enhancehindlimb-stepping ability in adult spinal transected rats[J]. Brain (2008),131,264-276.
[15] Mu oz-Quiles C,Santos-Benito FF,Llamusí MB,et al.Chronic spinal injury repairby olfactory bulb ensheathing glia and feasibility for autologous therpy[J].JNeuropathol Exp Neurol.2009;68(12):1294-1308.
[16]王广志,刘明娜.接受嗅鞘细胞移植脊髓损伤大鼠电生理及后肢功能变化[J].中国组织工程研究与临床康复,2010,14(1):112-115.
[17]丁鹏,薛黎萍,宋晓斌.骨髓基质细胞移植促进脊髓全横断损伤区神经元轴突的再生变化[J].中国组织工程研究与临床康复,2009,13(49):9631-9636.
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[20]林杨元,王玮.大鼠脊髓横断伤后护理对生存质量及功能评分的影响[J].局解手术学杂志,2006,15(6):361-362.
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[1]周建军,吴国材,冯华.脊髓损伤的动物模型进展[J].脊柱外科杂志，2004,2(3):173-175.
[2]周婷婷,郑志红.脊髓损伤动物模型的构建与应用[J].实验动物与比较医学，2008,28(6):411-415.
[3] Kaptanoglu E,Tuncel M,Palaoglu S,et a1.Comparison of the effects of melatonin andmethylprednisolone in experimental spinal cord injury[J].J Neurosurg,2000,93(1Suppl):77-84.
[4] Talac R,Friedman JA,Moore MJ,et al.Animal models of spinal cord injury forevaluation of tissue engineering treatment strategies[J].Biomater-ials,2004;25(9):1505-1510.
[5] Yan HB, J in DD, Lu KW, et al.Establishment of s table rat models of complete spinalcord transection[J].Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu2007;11(6):1091-1094(China)
[6]刘锐,游思维,刘惠玲,等.构建还原论式大鼠脊髓全横断损伤模型的性别影响[J].中国临床康复,2005,9(17):95-97.
[7]乔晓峰,杨建华,朱光宇,等.脊髓损伤模型的制备及其评价[J].中国全科医学,2009,12(7B):1279-1281.
[8]高天君,刘少君,侯树勋.脑源性神经营养因子基因转染嗅鞘细胞移植修复脊髓损伤的观察[J].解放军医学杂志,2005,30(11):1003-1005,1011.
[9]王增涛,主编.Wistar大鼠解剖图谱.山东:科学技术出版社,2009.2-3.
[10] Yeo SJ, Huang SN, OPark SW, et al. Development of rat model of grade contusivespinal cord injury using a pneumatic impact device[J]. J Korean Med Sci.2004;19(4):574.
[11] Giglio CA,Defino HL,da-Silva CA,et a1.Behavioral and physiological methods forearly quantitative assessment of spinal cord injury and prognosis in rats[J]．BrazilianJouml of Medical and Biological Research，2006，39：1613．1623．
[12] Davies JE,Huang C,Proschel C,et al.Astrocytes derived from glial-restrictedprecursors promote spinal cord repair[J].Journal of biology,2006,5(3):7.
[13] Koichi I, Toshiki Y, Haruhiko K, et al. Transplantation of olfactory mucosa followingspinal cord injury promotes recovery in rats[J]. Neuroreport,2008;19(13):1249-1252.
[14] Marc D.Kubasak,Devin L,et al.OEG implantation and step training enhancehindlimb-stepping ability in adult spinal transected rats[J]. Brain (2008),131,264-276.
[15] Mu oz-Quiles C,Santos-Benito FF,Llamusí MB,et al.Chronic spinal injury repairby olfactory bulb ensheathing glia and feasibility for autologous therpy[J].JNeuropathol Exp Neurol.2009;68(12):1294-1308.
[16]王广志,刘明娜.接受嗅鞘细胞移植脊髓损伤大鼠电生理及后肢功能变化[J].中国组织工程研究与临床康复,2010,14(1):112-115.
[17]丁鹏,薛黎萍,宋晓斌.骨髓基质细胞移植促进脊髓全横断损伤区神经元轴突的再生变化[J].中国组织工程研究与临床康复,2009,13(49):9631-9636.
[18] Fernando Fidel Santos-Beaito,et al.Long-Term Care of Paraplegic Laboratorymammals[J].Journal of Neurotrauma.2006;23(3/4):521-536.
[19]陈秉耀,游思维,王颖.大鼠脊髓全横断术后护理措施[J].实验动物科学与管理,2000,17(4):55-57.
[20]林杨元,王玮.大鼠脊髓横断伤后护理对生存质量及功能评分的影响[J].局解手术学杂志,2006,15(6):361-362.
[21] Guillermo GA, Rubén LV，Joaquim F,et al. Acute transplantation of olfactoryensheathing cells or schwann cells promotes recovery after spinal cord injury in therat[J]. Journal of Neuroscience Research,2004;75(5):632-641.
[22]陈杰,马进,丁兆平,等.一种模拟失重影响的大鼠尾部悬吊模型[J].空间科学学报,1993,13:159-162.
[23]徐帆,陈建敏,张晓.脊髓损伤动物模型的制备及功能评价[J].创伤外科杂志,2007;9(1):91-93.
[24] Basso DM,Beattie MS,Bresbahan JC.Graded histological and locomotor outcomesafter spinal cord injury using the NYU weight-drop device versus transection[J].ExpNeurol,1996;139(2):244-256.
[25] Kiehn O,Butt SJ.Physiological,anatomical and genetic identification of CPG neuronsin the developing mammalian spinal cord[J].Prog Neurobiol,2003,70(4):347-361.
[26]陈向荣,游思维,金大地.BBB评分评估脊髓损伤大鼠后肢运动功能的探讨[J].中国脊柱脊髓杂志,2004,14(9):547-549.
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