基于静电纺织技术构建血液生物膜的初探及生物相容性评价
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摘要
背景:已有研究表明纤维蛋白原可作为组织工程支架材料,若将血液中的纤维蛋白原提纯再利用,可制作出无免疫原性、生物相容性良好的支架材料。目的:探索静电纺丝技术制备血液生物膜的方法,评价支架的生物相容性。方法:将新鲜猪血液经分离、离心、纯化等步骤提纯出纤维蛋白原,分别经真空冷冻干燥机(A组)、低温喷雾干燥机(B组)、56℃烘箱干燥(C组)处理,采用静电纺织技术制作成纤维薄膜支架材料,检测3组支架材料的接触角,采用扫描电镜观察支架材料的三维结构。将3组电纺支架材料分别与骨髓间充质干细胞共培养,培养7 d后,扫描电镜观察细胞生长情况;培养1-4 d,Alamar Blue试剂盒检测B组支架材料表面细胞增殖。结果与结论:(1)扫描电镜显示,A组可见纤维结构,纤维粗细不均并有大量液滴状结构;B组可见排列有序的多层纤维结构,纤维直径、孔径大小接近一致,仅见少量液滴状结构;C组未见纤维结构,为大小不等的液滴状结构;(2)A-C组支架材料的接触角分别为(82±3)°、(67±5)°、(80±3)°,3组材料接触角都<90°,表明亲水性良好;(3)共培养7 d后,骨髓间充质干细胞均可黏附于3组支架材料上,其中B组材料表面的细胞分布较为均匀,细胞黏附生长于纤维表面,细胞核形态相对规则;A、C组可见少量细胞黏附生长于支架上,C组细胞量最少,细胞核形态不规则,两组均未见明显纤维结构;(4)Alamar Blue实验结果显示,随着培养时间的延长,B组支架材料表面的细胞呈增殖趋势,生长状态良好;(5)结果表明,采用静电纺织技术可制备出血液纤维蛋白原生物膜,其具有良好的生物相容性。
BACKGROUND: Previous studies have shown that fibrinogen can serve as tissue engineering scaffold material. Therefore, the reuse of fibrinogen in the autologous blood can make the scaffold material which has non-immunogenicity and good biocompatibility. OBJECTIVE: To explore the method of preparing blood biofilm using the electro-spinning technology and to evaluate the biocompatibility of the fibrinogen scaffold. METHODS: Fibrinogen was purified out of the fresh blood after the procedures of separation, centrifugation, and purification, and then subjected to vacuum freeze drying(group A), low temperature spray drying(group B), and oven drying at 56 oC(group C). It was finally made into fiber film using the electrospinning technology. Contact angle of the three sets of scaffold materials was detected, and the three-dimensional structure of the scaffolds was observed under scanning electron microscope. Bone marrow mesenchymal stem cells were co-cultured with the three sets of scaffolds in vitro. Cell growth was observed using scanning electron microscope at 7 days of culture, and cell proliferation in the group B was detected using Alamar Blue kit at 1-4 days of culture. RESULTS AND CONCLUSION:(1) Under the scanning electron microscope, fiber structure with uneven fiber thickness was observed, and a lot of droplet-like structures were also visible in the group A; the ordered multi-layer fiber structure with similar fiber diameter and pore size was observed in the group B, and only a small amount of droplet-like structures were detected; in the group C, there was no fiber structure, but the droplet-like structures of different sizes.(2) The contact angle of the scaffold material was(82±3)o in the group A,(67±5)o in the group B, and(80±3)o in the group C.(3) After 7 days of co-culture, the bone marrow mesenchymal stem cells adhered to the scaffold materials in the three groups. The cells on the surface of group B were evenly distributed, which were adherent to the fiber surface and had relative regular nuclei. Only a small amount of cells grew onto the scaffolds in the groups A and C, especially in the group C, in which the cells had irregular nuclei and no fiber structure was observed.(4) The cells in the group B exhibited an increasing proliferation tendency as shown by the results of Alamar Blue experiment, and the cell growth was in good status. All these findings indicate that it is feasible to prepare the blood fibrinogen biofilm with good biocompatibility using the electrospinning technology.
BACKGROUND: Previous studies have shown that fibrinogen can serve as tissue engineering scaffold material. Therefore, the reuse of fibrinogen in the autologous blood can make the scaffold material which has non-immunogenicity and good biocompatibility. OBJECTIVE: To explore the method of preparing blood biofilm using the electro-spinning technology and to evaluate the biocompatibility of the fibrinogen scaffold. METHODS: Fibrinogen was purified out of the fresh blood after the procedures of separation, centrifugation, and purification, and then subjected to vacuum freeze drying(group A), low temperature spray drying(group B), and oven drying at 56 oC(group C). It was finally made into fiber film using the electrospinning technology. Contact angle of the three sets of scaffold materials was detected, and the three-dimensional structure of the scaffolds was observed under scanning electron microscope. Bone marrow mesenchymal stem cells were co-cultured with the three sets of scaffolds in vitro. Cell growth was observed using scanning electron microscope at 7 days of culture, and cell proliferation in the group B was detected using Alamar Blue kit at 1-4 days of culture. RESULTS AND CONCLUSION:(1) Under the scanning electron microscope, fiber structure with uneven fiber thickness was observed, and a lot of droplet-like structures were also visible in the group A; the ordered multi-layer fiber structure with similar fiber diameter and pore size was observed in the group B, and only a small amount of droplet-like structures were detected; in the group C, there was no fiber structure, but the droplet-like structures of different sizes.(2) The contact angle of the scaffold material was(82±3)o in the group A,(67±5)o in the group B, and(80±3)o in the group C.(3) After 7 days of co-culture, the bone marrow mesenchymal stem cells adhered to the scaffold materials in the three groups. The cells on the surface of group B were evenly distributed, which were adherent to the fiber surface and had relative regular nuclei. Only a small amount of cells grew onto the scaffolds in the groups A and C, especially in the group C, in which the cells had irregular nuclei and no fiber structure was observed.(4) The cells in the group B exhibited an increasing proliferation tendency as shown by the results of Alamar Blue experiment, and the cell growth was in good status. All these findings indicate that it is feasible to prepare the blood fibrinogen biofilm with good biocompatibility using the electrospinning technology.
引文
[1]Neal RA,McClugage SG,Link MC,et al.Laminin nanofiber meshes that mimic morpholo-gical properties and bioactivity of basement membranes.Tissue Eng.2009;15(1):11-21.
[2]赵敏丽,隋刚,杨小平,等.电纺丝PLLA/HA复合纤维支架的制备及体外降解性能研究[J].中国生物医学工程学报,2006,25(4):476-480.
[3]Wang S,Zhang Y,Wang H,et al.Fabrication and properties of the electrospun polylactide/silk fibroin-gelatin composite tubular scaffold.Biomacromolecules.2009;10(8):2240-2244.
[4]Herrick S,Blanc-Brude O,Gray A,et al.Fibrinogen Int J Biochem Cell Biol.1999;31(7):741-746.
[5]Ayres CE,Jha BS,Sell SA,et al.Nanotechnology in the design of soft tissue scaffolds:innovations in structure and function.Wiley Interdiscip Rev Nanomed Nanobiotechnol.2010;2(1):20-34.
[6]Spotnitz WD.Commercial fibrin sealants in surgical care.Am J Surg.2001;182(2):8S-14S.
[7]Balasubramanian P,Prabhakaran MP,Kai D.Human cardiomyocyte interaction with electros-pun fibrinogen/gelatin nanofibers for myocardial regeneration.J Biomater Sci Polym Ed.2013;24(14):1660-1675.
[8]Linnes MP,Ratner BD,Giachelli CM.A fibrinogen-based precision microporous scaffold for tissue engineering.Biomaterials.2007;28(35):5298-5306.
[9]Mc Manus MC,Boland ED,Simpson DG,et al.Electrospun fibrinogen:feasibility as a tissue engineering scaffold in a rat cell culture model.JBiomed Mater Res A.2007;81(2):299-309.
[10]Mc Manus M,Boland E,Sell S,et al.Electrospun nanofibre fibrinogen for urinary tract tissue reconstruction.Biomed Mater.2007;2(4):257-262.
[11]Gugutkov D,Gustavsson J,Cantini M,et al.Electrospun fibrinogen-PLA nanofibres for vas-cular tissue engineering.J Tissue Eng Regen Med.2017;11(10):2774-2784.
[12]Sell SA,McClure MJ,Garg K,et al.Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering.Adv Drug Deliv Rev.2009;61(12):1007-1019.
[13]Ye Q,Zünd G,Benedikt P,et al.Fibrin gel as a three dimensional matrix in cardiovascular tissue engineering.Eur J Cardiothorac Surg.2000;17(5):587-591.
[14]Sell SA,McClure MJ,Garg K,et al.Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering.Adv Drug Deliv Rev.2009;61(12):1007-1019.
[15]Kondo M,Niwa T,Okamoto H,et al.Particle characterization of poorly water-soluble drugs using a spray freeze dryingtechnique.Chem Pharm Bull(Tokyo).2009;57(7):657-662.
[16]Carlisle CR,Coulais C,Namboothiry M,et al.The mechanical properties of individual,electrospun fibrinogen fibers.Biomaterials.2009;30(6):1205-1213.
[17]Mc Manus MC,Boland ED,Simpson DG,et al.Electrospun fibrinogen:feasibility as a tissue engineering scaffold in a rat cell culture model.JBiomed Mater Res A.2007;81(2):299-309.
[18]Zhang Y,Ye C,Wang G,et al.Kidney-targeted transplantation of mesenchymal stem cells by ultrasound-targeted microbubble destruction promotes kidney repair in diabetic nephropathy rats.Biomed Res Int.2013;2013:526367.
[19]张丽文,罗瑞明,李亚蕾,等.食品真空冷冻联合干燥技术研究进展[J].中国调味品,2017,42(3):152-156.
[20]史宏灿,徐志飞,秦雄,等.生物材料人工气管的设计与动物实验研究[J].第二军医大学学报,2002,6(10):1142-1145.
[2]赵敏丽,隋刚,杨小平,等.电纺丝PLLA/HA复合纤维支架的制备及体外降解性能研究[J].中国生物医学工程学报,2006,25(4):476-480.
[3]Wang S,Zhang Y,Wang H,et al.Fabrication and properties of the electrospun polylactide/silk fibroin-gelatin composite tubular scaffold.Biomacromolecules.2009;10(8):2240-2244.
[4]Herrick S,Blanc-Brude O,Gray A,et al.Fibrinogen Int J Biochem Cell Biol.1999;31(7):741-746.
[5]Ayres CE,Jha BS,Sell SA,et al.Nanotechnology in the design of soft tissue scaffolds:innovations in structure and function.Wiley Interdiscip Rev Nanomed Nanobiotechnol.2010;2(1):20-34.
[6]Spotnitz WD.Commercial fibrin sealants in surgical care.Am J Surg.2001;182(2):8S-14S.
[7]Balasubramanian P,Prabhakaran MP,Kai D.Human cardiomyocyte interaction with electros-pun fibrinogen/gelatin nanofibers for myocardial regeneration.J Biomater Sci Polym Ed.2013;24(14):1660-1675.
[8]Linnes MP,Ratner BD,Giachelli CM.A fibrinogen-based precision microporous scaffold for tissue engineering.Biomaterials.2007;28(35):5298-5306.
[9]Mc Manus MC,Boland ED,Simpson DG,et al.Electrospun fibrinogen:feasibility as a tissue engineering scaffold in a rat cell culture model.JBiomed Mater Res A.2007;81(2):299-309.
[10]Mc Manus M,Boland E,Sell S,et al.Electrospun nanofibre fibrinogen for urinary tract tissue reconstruction.Biomed Mater.2007;2(4):257-262.
[11]Gugutkov D,Gustavsson J,Cantini M,et al.Electrospun fibrinogen-PLA nanofibres for vas-cular tissue engineering.J Tissue Eng Regen Med.2017;11(10):2774-2784.
[12]Sell SA,McClure MJ,Garg K,et al.Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering.Adv Drug Deliv Rev.2009;61(12):1007-1019.
[13]Ye Q,Zünd G,Benedikt P,et al.Fibrin gel as a three dimensional matrix in cardiovascular tissue engineering.Eur J Cardiothorac Surg.2000;17(5):587-591.
[14]Sell SA,McClure MJ,Garg K,et al.Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering.Adv Drug Deliv Rev.2009;61(12):1007-1019.
[15]Kondo M,Niwa T,Okamoto H,et al.Particle characterization of poorly water-soluble drugs using a spray freeze dryingtechnique.Chem Pharm Bull(Tokyo).2009;57(7):657-662.
[16]Carlisle CR,Coulais C,Namboothiry M,et al.The mechanical properties of individual,electrospun fibrinogen fibers.Biomaterials.2009;30(6):1205-1213.
[17]Mc Manus MC,Boland ED,Simpson DG,et al.Electrospun fibrinogen:feasibility as a tissue engineering scaffold in a rat cell culture model.JBiomed Mater Res A.2007;81(2):299-309.
[18]Zhang Y,Ye C,Wang G,et al.Kidney-targeted transplantation of mesenchymal stem cells by ultrasound-targeted microbubble destruction promotes kidney repair in diabetic nephropathy rats.Biomed Res Int.2013;2013:526367.
[19]张丽文,罗瑞明,李亚蕾,等.食品真空冷冻联合干燥技术研究进展[J].中国调味品,2017,42(3):152-156.
[20]史宏灿,徐志飞,秦雄,等.生物材料人工气管的设计与动物实验研究[J].第二军医大学学报,2002,6(10):1142-1145.
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