新方法可获得更佳的组织工程小直径血管细胞外基质支架
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摘要
背景:获得适宜的支架材料是构建组织工程小直径血管的基础,前期研究发现,非离子型去垢剂的脱细胞效果差,联合应用离子型去垢剂能更有效去除细胞成分。目的:比较不同脱细胞方法制备猪颈动脉脱细胞支架的有效性,探索更适宜的细胞外基质支架制备方法。方法:取普通猪颈动脉30根,随机分为5组(n=6),其中A组不进行脱细胞处理;B组采用1%十二烷基硫酸钠+1%脱氧胆酸钠进行脱细胞处理;C组采用0.5%十二烷基硫酸钠+0.5%脱氧胆酸钠进行脱细胞处理;D组采用1%十二烷基硫酸钠+1%TritonX-100进行脱细胞处理;E组采用0.5%十二烷基硫酸钠+0.5%Triton X-100进行脱细胞处理。对各组样本分别行苏木精-伊红染色、五色套染、扫描电镜、免疫组织化学及生物机械力学检测。结果与结论:(1)苏木精-伊红染色、五色套染显示,B、C组脱细胞完全,D、E组于局部可见细胞残留;(2)扫描电镜显示,脱细胞各组胶原纤维束和弹性蛋白保存完整,B组孔隙结构的形态、尺寸优于C、D、E组;(3)免疫组织化学评估显示,脱细胞各组均有异种抗原α-1,3-Gal表达,B组α-1,3-Gal抗原表达低于C、D、E组;(4)生物机械力学检测显示,脱细胞各组爆破压力、缝线保持力和缝合保留强度较A组显著下降(P <0.05),脱细胞各组间比较无差异;脱细胞各组顺应性、长轴断裂伸长率、横轴最大应力、横轴断裂伸长率、横轴杨氏模量与对照组比较无差异(P> 0.05);脱细胞各组长轴最大应力和长轴杨氏模量较A组显著下降(P <0.05),其中B、C组长轴最大应力和长轴杨氏模量下降较D、E组明显(P<0.05);(5)结果表明,基于1%十二烷基硫酸钠+1%脱氧胆酸钠的方法可获得更佳的猪颈动脉细胞外基质支架,适用于构建组织工程小直径血管。
BACKGROUND: A proper scaffold is the basis for construction of tissue-engineered small diameter blood vessels. Preliminary study has shown non-ionic detergents with limited effects of decellularization, and combined application of ionic detergents exhibits more effective decellularization. OBJECTIVE: To compare the effectiveness of different preferable methods for preparing porcine carotid artery derived extracellular matrix scaffold, and to explore the optimal preparation method. METHODS: Porcine carotid arteries(n=30) were obtained and randomized into five groups(n=6 per group). Then porcine carotid arteries were decellularized, followed by treatment with 1% sodium dodecyl sulfate and 1% sodium deoxycholate(group B), 0.5% sodium dodecyl sulfate and 0.5% sodium deoxycholate(group C), 1% sodium dodecyl sulfate and 1% Triton X-100(group D), 0.5% sodium dodecyl sulfate and 0.5% Triton X-100(group E). Native procine carotid aitery served as control(group A). Tissue samples of each group underwent hematoxylin-eosin staining, and Movat's pentachrome staining, scanning electron microscope, immunohistochemistry and biomechanical test. RESULTS AND CONCLUSION: Hematoxylin-eosin staining and Movat's staining revealed that the cellular components were completely removed in the groups B and C; however, cellular residues were visualized in the groups D and E. Under the scanning electron microscope collagen and elastin fiber bundles were well preserved in the decellularization groups; however, the morphology and size of porous structure in the group B were superior to those in the groups C, D and E. Immunohistochemistry staining showed that the xenogeneic antigen, α-1,3-Gal, expressed in the decellularization groups; however, the expression of α-1,3-Gal was lower in the group B than the other groups. Biomechanical test indicated that burst pressure, suture-holding capacity and suture-holding strength were significantly decreased in the decellularization groups compared with group A(P < 0.05). However, there was no significant difference in the compliance, longitudinal elongation at brake, circumferential ultimate tensile stress, circumferential elongation at break and circumferential Young's modulus compared with control group(P > 0.05). Decellularization caused a significant decrease in the longitudinal ultimate tensile stress and longitudinal Young's modulus in the decellularization groups(P < 0.05). Furthermore, the longitudinal ultimate tensile stress and longitudinal Young's modulus in the groups B and C were significantly lower than those in the groups D and E(P < 0.05). Our results suggest that the novel decellularization method based on 1% sodium dodecyl sulfate and 1% sodium deoxycholate optimizes the extracellular matrix scaffold for the construction of tissue-engineered small-diameter blood vessel.
BACKGROUND: A proper scaffold is the basis for construction of tissue-engineered small diameter blood vessels. Preliminary study has shown non-ionic detergents with limited effects of decellularization, and combined application of ionic detergents exhibits more effective decellularization. OBJECTIVE: To compare the effectiveness of different preferable methods for preparing porcine carotid artery derived extracellular matrix scaffold, and to explore the optimal preparation method. METHODS: Porcine carotid arteries(n=30) were obtained and randomized into five groups(n=6 per group). Then porcine carotid arteries were decellularized, followed by treatment with 1% sodium dodecyl sulfate and 1% sodium deoxycholate(group B), 0.5% sodium dodecyl sulfate and 0.5% sodium deoxycholate(group C), 1% sodium dodecyl sulfate and 1% Triton X-100(group D), 0.5% sodium dodecyl sulfate and 0.5% Triton X-100(group E). Native procine carotid aitery served as control(group A). Tissue samples of each group underwent hematoxylin-eosin staining, and Movat's pentachrome staining, scanning electron microscope, immunohistochemistry and biomechanical test. RESULTS AND CONCLUSION: Hematoxylin-eosin staining and Movat's staining revealed that the cellular components were completely removed in the groups B and C; however, cellular residues were visualized in the groups D and E. Under the scanning electron microscope collagen and elastin fiber bundles were well preserved in the decellularization groups; however, the morphology and size of porous structure in the group B were superior to those in the groups C, D and E. Immunohistochemistry staining showed that the xenogeneic antigen, α-1,3-Gal, expressed in the decellularization groups; however, the expression of α-1,3-Gal was lower in the group B than the other groups. Biomechanical test indicated that burst pressure, suture-holding capacity and suture-holding strength were significantly decreased in the decellularization groups compared with group A(P < 0.05). However, there was no significant difference in the compliance, longitudinal elongation at brake, circumferential ultimate tensile stress, circumferential elongation at break and circumferential Young's modulus compared with control group(P > 0.05). Decellularization caused a significant decrease in the longitudinal ultimate tensile stress and longitudinal Young's modulus in the decellularization groups(P < 0.05). Furthermore, the longitudinal ultimate tensile stress and longitudinal Young's modulus in the groups B and C were significantly lower than those in the groups D and E(P < 0.05). Our results suggest that the novel decellularization method based on 1% sodium dodecyl sulfate and 1% sodium deoxycholate optimizes the extracellular matrix scaffold for the construction of tissue-engineered small-diameter blood vessel.
引文
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[15]Wilczek P,Lesiak A,Niemiec-Cyganek A,et al.Biomechanical properties of hybrid heart valve prosthesis utilizing the pigs that do not express the galactose-alpha-1,3-galactose(alpha-Gal)antigen derived tissue and tissue engineering technique.J Mater Sci Mater Med.2015;26(1):5329.
[16]B?er U,Lohrenz A,Klingenberg M,et al.The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts.Biomaterials.2011;32(36):9730-9737.
[17]B?eer U,Buettner FF,Klingenberg M,et al.Immunogenicity of intensively decellularized equine carotid arteries is conferred by the extracellular matrix protein collagen type VI.PLoS One.2014;9(8):e105964.
[18]Naso F,Gandaglia A,Iop L,et al.First quantitative assay of alpha-Gal in soft tissues:presence and distribution of the epitope before and after cell removal from xenogeneic heart valves.Acta Biomater.2011;7(4):1728-1734.
[19]Naso F,Gandaglia A,Iop L,et al.Alpha-Gal detectors in xenotransplantation research:a word of caution.Xenotransplantation.2012;19(4):215-220.
[20]员海超,蒲春晓,魏强,等.组织工程细胞外基质材料研究进展[J].中国修复重建外科杂志,2012,6(10):1251-1254.
[21]Faulk DM,Carruthers CA,Warner HJ,et al.The effect of detergents on the basement membrane complex of a biologic scaffold material.Acta Biomater.2014;10(1):183-193.
[22]Venkatasubramanian RT,Wolkers WF,Shenoi MM,et al.Freeze-thaw induced biomechanical changes in arteries:role of collagen matrix and smooth muscle cells.Ann Biomed Eng.2010;38(3):694-706.
[23]Crapo PM,Wang Y.Physiologic compliance in engineered small-diameter arterial constructs based on an elastomeric substrate.Biomaterials.2010;31(7):1626-1635.
[24]Murase Y,Narita Y,Kagami H,et al.Evaluation of compliance and stiffness of decellularized tissues as scaffolds for tissue-engineered small caliber vascular grafts using intravascular ultrasound.ASAIO J.2006;52(4):450-455.
[2]Seifu DG,Purnama A,Mequanint K,et al.Small-diameter vascular tissue engineering.Nat Rev Cardiol.2013;10(7):410-421.
[3]Desai M,Seifalian AM,Hamilton G.Role of prosthetic conduits in coronary artery bypass grafting.Eur JCardiothorac Surg.2011;40(2):394-398.
[4]Huang AH,Niklason LE.Engineering of arteries in vitro.Cell Mol Life Sci.2014;71(11):2103-2118.
[5]Quint C,Kondo Y,Manson RJ,et al.Decellularized tissue-engineered blood vessel as an arterial conduit.Proc Natl Acad Sci U S A.2011;108(22):9214-9219.
[6]Cebotari S,Tudorache I,Ciubotaru A,et al.Use of fresh decellularized allografts for pulmonary valve replacement may reduce the reoperation rate in children and young adults:early report.Circulation.2011;124(11 Suppl):S115-S123.
[7]Badylak SF,Weiss DJ,Caplan A,et al.Engineered whole organs and complex tissues.Lancet.2012;379(9819):943-952.
[8]蒲磊,吴剑,孟明耀,等.不同脱细胞方案制备主动脉细胞外基质支架的比较研究[Z].2014:28,1413-1421.
[9]Bouten CV,Dankers PY,Driessen-Mol A,et al.Substrates for cardiovascular tissue engineering.Adv Drug Deliv Rev.2011;63(4-5):221-241.
[10]Conklin BS,Richter ER,Kreutziger KL,et al.Development and evaluation of a novel decellularized vascular xenograft.Med Eng Phys.2002;24(3):173-183.
[11]Gilbert T,Sellaro T,Badylak S.Decellularization of tissues and organs.Biomaterials.2006;27(19):3675-3683.
[12]Knight RL,Wilcox HE,Korossis SA,et al.The use of acellular matrices for the tissue engineering of cardiac valves.Proc Inst Mech Eng H.2008;222(1):129-143.
[13]Schoen FJ,Levy RJ.Calcification of tissue heart valve substitutes:progress toward understanding and prevention.Ann Thorac Surg.2005;79(3):1072-1080.
[14]Keane TJ,Londono R,Turner NJ,et al.Consequences of ineffective decellularization of biologic scaffolds on the host response.Biomaterials.2012;33(6):1771-1781.
[15]Wilczek P,Lesiak A,Niemiec-Cyganek A,et al.Biomechanical properties of hybrid heart valve prosthesis utilizing the pigs that do not express the galactose-alpha-1,3-galactose(alpha-Gal)antigen derived tissue and tissue engineering technique.J Mater Sci Mater Med.2015;26(1):5329.
[16]B?er U,Lohrenz A,Klingenberg M,et al.The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts.Biomaterials.2011;32(36):9730-9737.
[17]B?eer U,Buettner FF,Klingenberg M,et al.Immunogenicity of intensively decellularized equine carotid arteries is conferred by the extracellular matrix protein collagen type VI.PLoS One.2014;9(8):e105964.
[18]Naso F,Gandaglia A,Iop L,et al.First quantitative assay of alpha-Gal in soft tissues:presence and distribution of the epitope before and after cell removal from xenogeneic heart valves.Acta Biomater.2011;7(4):1728-1734.
[19]Naso F,Gandaglia A,Iop L,et al.Alpha-Gal detectors in xenotransplantation research:a word of caution.Xenotransplantation.2012;19(4):215-220.
[20]员海超,蒲春晓,魏强,等.组织工程细胞外基质材料研究进展[J].中国修复重建外科杂志,2012,6(10):1251-1254.
[21]Faulk DM,Carruthers CA,Warner HJ,et al.The effect of detergents on the basement membrane complex of a biologic scaffold material.Acta Biomater.2014;10(1):183-193.
[22]Venkatasubramanian RT,Wolkers WF,Shenoi MM,et al.Freeze-thaw induced biomechanical changes in arteries:role of collagen matrix and smooth muscle cells.Ann Biomed Eng.2010;38(3):694-706.
[23]Crapo PM,Wang Y.Physiologic compliance in engineered small-diameter arterial constructs based on an elastomeric substrate.Biomaterials.2010;31(7):1626-1635.
[24]Murase Y,Narita Y,Kagami H,et al.Evaluation of compliance and stiffness of decellularized tissues as scaffolds for tissue-engineered small caliber vascular grafts using intravascular ultrasound.ASAIO J.2006;52(4):450-455.
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