冻融联合灌注法优化大鼠肾脏脱细胞支架的制备
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摘要
本研究旨在探索优化肾脏脱细胞支架的制备方法,为肾脏组织工程及肾脏体外病理、毒理研究提供实验基础。取大鼠肾脏灌注PBS作为对照组(Control组),在不同流速下分别以十二烷基磺酸钠(Sodiumdodecyl sulfate,SDS)灌注(S组),Triton X-100联合SDS灌注(TS组),反复冻融后Triton X-100联合SDS灌注(FTS组),制备肾脏脱细胞支架,并测定其流体分布及脉管阻力。HE染色、DAPI染色、DNA定量检测脱细胞支架脱细胞程度,Masson染色、PAS染色、免疫组织化学染色检测脱细胞支架主要成分的保留和结构的完整,扫描电镜检测支架的超微结构,MTT法检测支架的细胞毒性,ELISA检测支架中生长因子的含量。结果显示,FTS组脱细胞用时较S组、TS组少,10 mL/min组支架脉管阻力较低,S组、TS组、FTS组流体分布与Control组存在差异。HE染色和DAPI染色显示各组支架未见细胞成分残留,DNA含量<50 ng/mg。Masson染色和PAS染色可见细胞外网状胶原及多糖,免疫组织化学染色见Ⅰ型胶原(CollagenⅠ)、Ⅳ型胶原蛋白(Collagen Ⅳ)、纤维连接蛋白(Fibronectin)、层粘连蛋白(Laminin)表达。扫描电镜见支架呈蜂窝状结构。MTT法检测支架细胞毒性分级在0–1级之间。ELISA检测提示FTS组VEGF、EGF、IGF-1、PDGF含量明显高于S组和TS组。综上,联合冻融和灌注法能够制备更为理想且有效的大鼠肾脏整器官脱细胞支架,为肾脏组织工程及肾脏体外病理、毒理学研究奠定基础。
We explored the improved method to prepare decellularized kidney scaffold and provide experimental basis for kidney tissue engineering and renal pathology and toxicology in vitro research. We perfused rat kidneys with PBS(group control) and prepared the decellularized kidney scaffolds with sodium dodecyl sulfate(SDS)(group S), Triton X-100 combined with SDS(group TS), and Triton X-100 combined with SDS after repeated freezing and thawing(group FTS) in different flow velocity. Meanwhile we measured their fluid distributions and vascular resistance. We examined the degree of decellularization of acellular scaffolds by HE, DAPI staining and DNA quantification. We examined the retention of main composition and structural integrity of decellularized scaffolds by Masson, PAS and immunohistochemical staining. We also detected the ultrastructure, cytotoxicity and the level of growth factor of the scaffolds by scanning electron microscope, MTT and ELISA, respectively. The results showed that the time of decellularization in group FTS was less than that in group S and TS. The vascular resistance of scaffolds decellularized at 10 mL/min flow velocity was lower. The fluid distribution in groups S, TS and FTS was different from that in control group. No residual cell was detected by HE and DAPI staining. DNA content was less than 50 ng/mg. Masson, PAS and immunohistochemical staining results showed that there was extracellular collagen, polysaccharide, type I collagen, type IV collagen, fibronectin and laminin in the decellularized scaffolds, and the scanning electron microscope result showed the scaffolds had the honeycomb structure. The cytotoxicity level of decellularized scaffolds was between grade 0 to 1. The level of VEGF, EGF, IGF-1 and PDGF-BB in group FTS were significantly higher than those in group S and TS. In concluding, combining freeze-thawing with perfusion can produce more ideal and effective whole organ decellularized scaffold of rat kidney, and make a foundation for the study of kidney tissue engineering and in vitro pathology and toxicology of kidney.
We explored the improved method to prepare decellularized kidney scaffold and provide experimental basis for kidney tissue engineering and renal pathology and toxicology in vitro research. We perfused rat kidneys with PBS(group control) and prepared the decellularized kidney scaffolds with sodium dodecyl sulfate(SDS)(group S), Triton X-100 combined with SDS(group TS), and Triton X-100 combined with SDS after repeated freezing and thawing(group FTS) in different flow velocity. Meanwhile we measured their fluid distributions and vascular resistance. We examined the degree of decellularization of acellular scaffolds by HE, DAPI staining and DNA quantification. We examined the retention of main composition and structural integrity of decellularized scaffolds by Masson, PAS and immunohistochemical staining. We also detected the ultrastructure, cytotoxicity and the level of growth factor of the scaffolds by scanning electron microscope, MTT and ELISA, respectively. The results showed that the time of decellularization in group FTS was less than that in group S and TS. The vascular resistance of scaffolds decellularized at 10 mL/min flow velocity was lower. The fluid distribution in groups S, TS and FTS was different from that in control group. No residual cell was detected by HE and DAPI staining. DNA content was less than 50 ng/mg. Masson, PAS and immunohistochemical staining results showed that there was extracellular collagen, polysaccharide, type I collagen, type IV collagen, fibronectin and laminin in the decellularized scaffolds, and the scanning electron microscope result showed the scaffolds had the honeycomb structure. The cytotoxicity level of decellularized scaffolds was between grade 0 to 1. The level of VEGF, EGF, IGF-1 and PDGF-BB in group FTS were significantly higher than those in group S and TS. In concluding, combining freeze-thawing with perfusion can produce more ideal and effective whole organ decellularized scaffold of rat kidney, and make a foundation for the study of kidney tissue engineering and in vitro pathology and toxicology of kidney.
引文
[1]Zhang JS,Wang ZB,Lin KZ,et al.In vivo regeneration of renal vessels post whole decellularized kidneys transplantation.Oncotarget,2015,6(38):40433-40442.
[2]Perico N,Remuzzi G.Chronic kidney disease:a research and public health priority.Nephrol Dial Transplant,2012,27(Suppl 3):iii19-iii26.
[3]Madariaga MLL,Ott HC.Bioengineering kidneys for transplantation.Semin Nephrol,2014,34(4):384-393.
[4]Bonandrini B,Figliuzzi M,Papadimou E,et al.Recellularization of well-preserved acellular kidney scaffold using embryonic stem cells.Tissue Eng Part A,2014,20(9/10):1486-1498.
[5]Sundaram S,Niklason LE.Smooth muscle and other cell sources for human blood vessel engineering.Cells Tissues Organs,2012,195(1/2):15-25.
[6]Gao QD,Zhu XL,Xiang JX,et al.Strategies to choose scaffold materials for tissue engineering.Chin J Biotech,2016,32(2):172-184(in Chinese).高庆东,祝旭龙,向俊西,等.基于组织工程研究的可降解支架材料选择策略.生物工程学报,2016,32(2):172-184.
[7]Peloso A,Ferrario J,Maiga B,et al.Creation and implantation of acellular rat renal ECM-based scaffolds.Organogenesis,2015,11(2):58-74.
[8]Poornejad N,Momtahan N,Salehi ASM,et al.Efficient decellularization of whole porcine kidneys improves reseeded cell behavior.Biomed Mater,2016,11(2):025003.
[9]Conconi MT,de Coppi P,Di Liddo R,et al.Tracheal matrices,obtained by a detergent-enzymatic method,support in vitro the adhesion of chondrocytes and tracheal epithelial cells.Transpl Int,2005,18(6):727-734.
[10]Baiguera S,Jungebluth P,Burns A,et al.Tissue engineered human tracheas for in vivo implantation.Biomaterials,2010,31(34):8931-8938.
[11]Ott HC,Matthiesen TS,Goh SK,et al.Perfusion-decellularized matrix:using nature's platform to engineer a bioartificial heart.Nat Med,2008,14(2):213-221.
[12]Hansen KJ,Favreau JT,Guyette JP,et al.Functional effects of delivering human mesenchymal stem cell-seeded biological sutures to an infarcted heart.Biores Open Access,2016,5(1):249-260.
[13]Uygun BE,Soto-Gutierrez A,Yagi H,et al.Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.Nat Med,2010,16(7):814-820.
[14]Petersen TH,Calle EA,Zhao LP,et al.Tissue-engineered lungs for in vivo implantation.Science,2010,329(5991):538-541.
[15]Nakayama KH,Batchelder CA,Lee CI,et al.Decellularized rhesus monkey kidney as a three-dimensional scaffold for renal tissue engineering.Tissue Eng Part A,2010,16(7):2207-2216.
[16]Andersen KK,Oliveira CL,Larsen KL,et al.The role of decorated SDS micelles in sub-CMC protein denaturation and association.J Mol Biol,2009,391(1):207-226.
[17]Michaux C,Pouyez J,Wouters J,et al.Protecting role of cosolvents in protein denaturation by SDS:a structural study.BMC Struct Biol,2008,8:29.
[18]Poornejad N,Frost TS,Scott DR,et al.Freezing/thawing without cryoprotectant damages native but not decellularized porcine renal tissue.Organogenesis,2015,11(1):30-45.
[19]Crapo PM,Gilbert TW,Badylak SF.An overview of tissue and whole organ decellularization processes.Biomaterials,2011,32(12):3233-3243.
[20]Frantz C,Stewart KM,Weaver VM.The extracellular matrix at a glance.J Cell Sci,2010,123(Pt 24):4195-4200.
[21]Brown B,Lindberg K,Reing J,et al.The basement membrane component of biologic scaffolds derived from extracellular matrix.Tiss Eng,2006,12(3):519-526.
[22]Zepeda-Orozco D,Wen HM,Hamilton BA,et al.EGF regulation of proximal tubule cell proliferation and VEGF-A secretion.Physiol Rep,2017,5(18):e13453.
[23]Gonzalez-Rodriguez E,Gaeggeler HP,Rossier BC.IGF-1 vs insulin:respective roles in modulating sodium transport via the PI-3 kinase/Sgk1 pathway in a cortical collecting duct cell line.Kidney Int,2007,71(2):116-125.
[24]Salva E,Turan S?,Akbu?a J.Inhibition of Glomerular Mesangial cell proliferation by siPDGF-B-and siPDGFR-β-containing chitosan nanoplexes.AAPS PharmSciTech,2017,18(4):1031-1042.
[25]Batchelder CA,Martinez ML,Tarantal AF.Natural scaffolds for renal differentiation of human embryonic stem cells for kidney tissue engineering.PLoS ONE,2015,10(12):e0143849.
[2]Perico N,Remuzzi G.Chronic kidney disease:a research and public health priority.Nephrol Dial Transplant,2012,27(Suppl 3):iii19-iii26.
[3]Madariaga MLL,Ott HC.Bioengineering kidneys for transplantation.Semin Nephrol,2014,34(4):384-393.
[4]Bonandrini B,Figliuzzi M,Papadimou E,et al.Recellularization of well-preserved acellular kidney scaffold using embryonic stem cells.Tissue Eng Part A,2014,20(9/10):1486-1498.
[5]Sundaram S,Niklason LE.Smooth muscle and other cell sources for human blood vessel engineering.Cells Tissues Organs,2012,195(1/2):15-25.
[6]Gao QD,Zhu XL,Xiang JX,et al.Strategies to choose scaffold materials for tissue engineering.Chin J Biotech,2016,32(2):172-184(in Chinese).高庆东,祝旭龙,向俊西,等.基于组织工程研究的可降解支架材料选择策略.生物工程学报,2016,32(2):172-184.
[7]Peloso A,Ferrario J,Maiga B,et al.Creation and implantation of acellular rat renal ECM-based scaffolds.Organogenesis,2015,11(2):58-74.
[8]Poornejad N,Momtahan N,Salehi ASM,et al.Efficient decellularization of whole porcine kidneys improves reseeded cell behavior.Biomed Mater,2016,11(2):025003.
[9]Conconi MT,de Coppi P,Di Liddo R,et al.Tracheal matrices,obtained by a detergent-enzymatic method,support in vitro the adhesion of chondrocytes and tracheal epithelial cells.Transpl Int,2005,18(6):727-734.
[10]Baiguera S,Jungebluth P,Burns A,et al.Tissue engineered human tracheas for in vivo implantation.Biomaterials,2010,31(34):8931-8938.
[11]Ott HC,Matthiesen TS,Goh SK,et al.Perfusion-decellularized matrix:using nature's platform to engineer a bioartificial heart.Nat Med,2008,14(2):213-221.
[12]Hansen KJ,Favreau JT,Guyette JP,et al.Functional effects of delivering human mesenchymal stem cell-seeded biological sutures to an infarcted heart.Biores Open Access,2016,5(1):249-260.
[13]Uygun BE,Soto-Gutierrez A,Yagi H,et al.Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.Nat Med,2010,16(7):814-820.
[14]Petersen TH,Calle EA,Zhao LP,et al.Tissue-engineered lungs for in vivo implantation.Science,2010,329(5991):538-541.
[15]Nakayama KH,Batchelder CA,Lee CI,et al.Decellularized rhesus monkey kidney as a three-dimensional scaffold for renal tissue engineering.Tissue Eng Part A,2010,16(7):2207-2216.
[16]Andersen KK,Oliveira CL,Larsen KL,et al.The role of decorated SDS micelles in sub-CMC protein denaturation and association.J Mol Biol,2009,391(1):207-226.
[17]Michaux C,Pouyez J,Wouters J,et al.Protecting role of cosolvents in protein denaturation by SDS:a structural study.BMC Struct Biol,2008,8:29.
[18]Poornejad N,Frost TS,Scott DR,et al.Freezing/thawing without cryoprotectant damages native but not decellularized porcine renal tissue.Organogenesis,2015,11(1):30-45.
[19]Crapo PM,Gilbert TW,Badylak SF.An overview of tissue and whole organ decellularization processes.Biomaterials,2011,32(12):3233-3243.
[20]Frantz C,Stewart KM,Weaver VM.The extracellular matrix at a glance.J Cell Sci,2010,123(Pt 24):4195-4200.
[21]Brown B,Lindberg K,Reing J,et al.The basement membrane component of biologic scaffolds derived from extracellular matrix.Tiss Eng,2006,12(3):519-526.
[22]Zepeda-Orozco D,Wen HM,Hamilton BA,et al.EGF regulation of proximal tubule cell proliferation and VEGF-A secretion.Physiol Rep,2017,5(18):e13453.
[23]Gonzalez-Rodriguez E,Gaeggeler HP,Rossier BC.IGF-1 vs insulin:respective roles in modulating sodium transport via the PI-3 kinase/Sgk1 pathway in a cortical collecting duct cell line.Kidney Int,2007,71(2):116-125.
[24]Salva E,Turan S?,Akbu?a J.Inhibition of Glomerular Mesangial cell proliferation by siPDGF-B-and siPDGFR-β-containing chitosan nanoplexes.AAPS PharmSciTech,2017,18(4):1031-1042.
[25]Batchelder CA,Martinez ML,Tarantal AF.Natural scaffolds for renal differentiation of human embryonic stem cells for kidney tissue engineering.PLoS ONE,2015,10(12):e0143849.
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