肝脏细胞外基质水凝胶的制备及表征
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摘要
背景:研究表明,肝脏细胞外基质水凝胶可促进肝脏特异性细胞的行为更加接近体内的状态,可增强肝细胞的活性及功能,促进内皮细胞的血管化和胆管上皮细胞的胆管化,但对于肝脏基质胶本身性质的研究较少。目的:采用温和去细胞技术制备肝脏细胞外基质水凝胶,并对水凝胶进行初步表征。方法:取新鲜冰冻的猪肝组织切片,常温下放入去离子水中搅拌4次;移入预热到37℃的0.02%胰酶/0.05%EDTA溶液中,37℃搅拌1h,去离子水清洗肝片,放入3%TritonX-100溶液中搅拌1.5h;去离子水冲洗肝片,放入4%脱氧胆酸钠溶液中搅拌1.5 h;过量去离子水冲洗肝片,获得去细胞肝脏支架;将肝脏支架移入0.1%过氧乙酸溶液中搅拌2.0-3.0 h,置入1×PBS溶液中搅拌15 min、去离子水中搅拌浸泡2次、1×PBS冲洗15 min;再通过冻干、液氮研磨、消化成肝脏基质溶液,经过后续的配平,多肽分子自组装成肝脏细胞外基质水凝胶。检测肝脏细胞外基质水凝胶的脱细胞程度、DNA含量、组成成分、浊度动力学及微观结构。结果与结论:①采用温和脱细胞技术得到了脱细胞肝脏支架,而且脱细胞较完全;②脱细胞肝脏支架的DNA含量较正常肝脏组织明显下降(P<0.001);③肝脏细胞外基质水凝胶前体溶液保留了许多猪肝细胞外基质成分,比如胶原蛋白、弹性蛋白、糖安聚糖及其前体等;④浊度动力学实验显示,随着肝脏细胞外基质凝胶质量浓度的增大,平台期的吸光度值升高;⑤扫描电镜显示,肝脏细胞外基质水凝胶呈现纤维网状多孔结构,纳米级细胞外基质纤维互相交错,并且随着肝脏细胞外基质水凝胶质量浓度的增加,纤维密度相对增加,直径无变化;⑥结果表明,采用温和去细胞技术制备的肝脏细胞外基质水凝胶,具有三维立体网络结构,为细胞的黏附、生长提供了结构基础。
BACKGROUND: Liver extracellular matrix hydrogel has been shown to promote the behavior of liver-specific cells closer to the body, enhance the activity and function of hepatocytes, and promote the vascularization of endothelial cells and the bile duct formation of bile duct epithelial cells, but the characteristics of liver matrigelis little known.OBJECTIVE: To prepare the liver extracellular matrix hydrogel by mild decellularization technique and to investigate its characterization preliminarily. METHODS: Fresh frozen pig liver tissue sections were taken and placed in deionized water at room temperature for 4 times, transferred to 0.02% trypsin/0.05% EDTA solution preheated at 37 °C, stirred at 37 °C for 1 hour, and washed with deionized water, put into 3% Triton X-100 solution and stirred for 1.5 hours. The liver piece was rinsed with deionized water, stirred in 4% sodium deoxycholate solution for 1.5 hours, and rinsed with excess deionized water to obtain the decellularized liver scaffold. The liver scaffold was transferred into 0.1% peracetic acid solution and stirred for 2.0-3.0 hours, placed in 1×PBS solution for 15 minutes, stirred in deionized water for twice, rinsed in 1×PBS for 15 minutes, and then lyophilized and liquid nitrogen-milled. Digested into a liver matrix solution, and after subsequent trimming, the polypeptide molecules self-assemble into a liver matrix hydrogel. The degree of decellularization, DNA content, composition, turbidity dynamics and microstructure of the liver matrix hydrogel were examined. RESULTS AND CONCLUSION:(1) The decellularized liver scaffold was obtained by mild decellularization technique, and the decellularization was complete.(2) The DNA content of the decellularized liver scaffold was significantly lower than that of normal liver tissue(P < 0.001).(3) The liver matrix hydrogel precursor solution retained many components of the pig liver extracellular matrix, such as collagen, elastin, glycan and its precursors.(4) The turbidity kinetics experiment showed that the absorbance value of the plateau increased with the increase of the mass concentration of the liver matrix gel.(5) Scanning electron microscope showed that the liver matrix hydrogel possessed a fibrous network porous structure, and the nano-scale extracellular matrix fibers were interlaced with each other. As the mass concentration of the liver matrix hydrogel increased, the fiber density increased relatively and the diameter revealed no change.(6) These results indicate that the liver matrix hydrogel is prepared by gentle decellularization technique and exhibits a three-dimensional network structure, which provides a structural basis for cell adhesion and growth.
BACKGROUND: Liver extracellular matrix hydrogel has been shown to promote the behavior of liver-specific cells closer to the body, enhance the activity and function of hepatocytes, and promote the vascularization of endothelial cells and the bile duct formation of bile duct epithelial cells, but the characteristics of liver matrigelis little known.OBJECTIVE: To prepare the liver extracellular matrix hydrogel by mild decellularization technique and to investigate its characterization preliminarily. METHODS: Fresh frozen pig liver tissue sections were taken and placed in deionized water at room temperature for 4 times, transferred to 0.02% trypsin/0.05% EDTA solution preheated at 37 °C, stirred at 37 °C for 1 hour, and washed with deionized water, put into 3% Triton X-100 solution and stirred for 1.5 hours. The liver piece was rinsed with deionized water, stirred in 4% sodium deoxycholate solution for 1.5 hours, and rinsed with excess deionized water to obtain the decellularized liver scaffold. The liver scaffold was transferred into 0.1% peracetic acid solution and stirred for 2.0-3.0 hours, placed in 1×PBS solution for 15 minutes, stirred in deionized water for twice, rinsed in 1×PBS for 15 minutes, and then lyophilized and liquid nitrogen-milled. Digested into a liver matrix solution, and after subsequent trimming, the polypeptide molecules self-assemble into a liver matrix hydrogel. The degree of decellularization, DNA content, composition, turbidity dynamics and microstructure of the liver matrix hydrogel were examined. RESULTS AND CONCLUSION:(1) The decellularized liver scaffold was obtained by mild decellularization technique, and the decellularization was complete.(2) The DNA content of the decellularized liver scaffold was significantly lower than that of normal liver tissue(P < 0.001).(3) The liver matrix hydrogel precursor solution retained many components of the pig liver extracellular matrix, such as collagen, elastin, glycan and its precursors.(4) The turbidity kinetics experiment showed that the absorbance value of the plateau increased with the increase of the mass concentration of the liver matrix gel.(5) Scanning electron microscope showed that the liver matrix hydrogel possessed a fibrous network porous structure, and the nano-scale extracellular matrix fibers were interlaced with each other. As the mass concentration of the liver matrix hydrogel increased, the fiber density increased relatively and the diameter revealed no change.(6) These results indicate that the liver matrix hydrogel is prepared by gentle decellularization technique and exhibits a three-dimensional network structure, which provides a structural basis for cell adhesion and growth.
引文
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[22]Aamodt JM,Grainger DW.Extracellular matrix-based biomaterial scaffolds and the host response.Biomaterials.2016;86:68-82.
[23]Lin P,Chan WC,Badylak SF,et al.Assessing porcine liver-derived biomatrix for hepatic tissue engineering.Tissue Eng.2004;10(7-8):1046-1053.
[24]Gilbert TW,Wognum S,Joyce EM,et al.Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix.Biomaterials.2008;29(36):4775-4782.
[25]Singelyn JM,Dequach JA,Seif-Naraghi SB,et al.Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering.Biomaterials.2009;30(29):5409-5416.
[26]Agmon G,Christman KL.Controlling stem cell behavior with decellularized extracellular matrix scaffolds.Curr Opin Solid State Mater Sci.2016;20(4):193-201.
[27]Faulk DM,Wildemann JD,Badylak SF.Decellularization and cell seeding of whole liver biologic scaffolds composed of extracellular matrix.J Clin Exp Hepatol.2015;5(1):69-80.
[28]Ji R,Zhang N,You N,et al.The differentiation of MSCs into functional hepatocyte-like cells in a liver biomatrix scaffold and their transplantation into liver-fibrotic mice.Biomaterials.2012;33(35):8995-9008.
[29]Londono R,Gorantla VS,Badylak SF.Emerging Implications for Extracellular Matrix-Based Technologies in Vascularized Composite Allotransplantation.Stem Cells Int.2016;2016:1541823.
[30]O'Neill JD,Freytes DO,Anandappa AJ,et al.The regulation of growth and metabolism of kidney stem cells with regional specificity using extracellular matrix derived from kidney.Biomaterials.2013;34(38):9830-9841.
[2]Gibbs DMR,Black CRM,Dawson JI,et al.A review of hydrogel use in fracture healing and bone regeneration.J Tissue Eng Regen Med.2016;10(3):187-198.
[3]Saldin LT,Cramer MC,Velankar SS,et al.Extracellular matrix hydrogels from decellularized tissues:Structure and function.Acta Biomaterialia.2017;49:1-15.
[4]Badylak S,Freytes D,Gilbert T.Extracellular matrix as a biological scaffold material:Structure and function.Acta Biomaterialia.2009;5(1):1-13.
[5]Zhang Y,He Y,Bharadwaj S,et al.Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype.Biomaterials.2009;30(23-24):4021-4028.
[6]Freytes DO,Martin J,Velankar SS,et al.Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.Biomaterials.2008;29(11):1630-1637.
[7]Lewis PL,Su J,Yan M,et al.Complex bile duct network formation within liver decellularized extracellular matrix hydrogels.Sci Rep.2018;8(1):12220.
[8]Agarwal T,Narayan R,Maji S,et al.Decellularized Caprine liver extracellular matrix as a 2D substrate coating and 3D hydrogel platform for vascularized liver tissue engineering.J Tissue Eng Regen Med.2018;12(3):e1678-e1690.
[9]Loneker AE,Faulk DM,Hussey GS,et al.Solubilized liver extracellular matrix maintains primary rat hepatocyte phenotype in-vitro.J Biomed Mater Res A.2016;104(7):1846-1847.
[10]Sellaro TL,Ranade A,Faulk DM,et al.Maintenance of human hepatocyte function in vitro by liver-derived extracellular matrix gels.Tissue Eng Part A.2010;16(3):1075-1082.
[11]Keane TJ,Londono R,Carey RM,et al.Preparation and characterization of a biologic scaffold from esophageal mucosa.Biomaterials.2013;34(28):6729-6737.
[12]Meng F,Modo M,Badylak SF.Biologic scaffold for CNS repair.Regen Med.2014;9(3):367-383.
[13]Lee JS,Shin J,Park H,et al.Liver extracellular matrix providing dual functions of two-dimensional substrate coating and three-dimensional injectable hydrogel platform for liver tissue engineering.Biomacromolecules.2013;15(1):206-218.
[14]Wolf MT,Daly KA,Brennan-Pierce EP,et al.A hydrogel derived from decellularized dermal extracellular matrix.Biomaterials.2012;33(29):7028-7038.
[15]Gilbert TW,Sellaro TL,Badylak SF.Decellularization of tissues and organs.Biomaterials.2006;27(19):3675-3683.
[16]Ng SL,Narayanan K,Gao S,et al.Lineage restricted progenitors for the repopulation of decellularized heart.Biomaterials.2011;32(30):7571-7580.
[17]马金辉,于洁,乔叶薷,等.两种去污剂在制备脱细胞肺支架中的对比[J].中国组织工程研究,2018,22(2):248-253.
[18]郑方平,毛凯丽,赵应征.脑去细胞生物支架的制备与鉴定[J].中国生物医学工程学报,2018,37(2):202-207.
[19]Crapo PM,Gilbert TW,Badylak SF.An overview of tissue and whole organ decellularization processes.Biomaterials.2011;32(12):3233-3243.
[20]Mazza G,Al-Akkad W,Rombouts K,et al.Liver tissue engineering:From implantable tissue to whole organ engineering.Hepatol Commun.2018;2(2):131-141.
[21]Zhang X,Dong J.Direct comparison of different coating matrix on the hepatic differentiation from adipose-derived stem cells.Biochem Biophys Res Commun.2015;456(4):938-944.
[22]Aamodt JM,Grainger DW.Extracellular matrix-based biomaterial scaffolds and the host response.Biomaterials.2016;86:68-82.
[23]Lin P,Chan WC,Badylak SF,et al.Assessing porcine liver-derived biomatrix for hepatic tissue engineering.Tissue Eng.2004;10(7-8):1046-1053.
[24]Gilbert TW,Wognum S,Joyce EM,et al.Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix.Biomaterials.2008;29(36):4775-4782.
[25]Singelyn JM,Dequach JA,Seif-Naraghi SB,et al.Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering.Biomaterials.2009;30(29):5409-5416.
[26]Agmon G,Christman KL.Controlling stem cell behavior with decellularized extracellular matrix scaffolds.Curr Opin Solid State Mater Sci.2016;20(4):193-201.
[27]Faulk DM,Wildemann JD,Badylak SF.Decellularization and cell seeding of whole liver biologic scaffolds composed of extracellular matrix.J Clin Exp Hepatol.2015;5(1):69-80.
[28]Ji R,Zhang N,You N,et al.The differentiation of MSCs into functional hepatocyte-like cells in a liver biomatrix scaffold and their transplantation into liver-fibrotic mice.Biomaterials.2012;33(35):8995-9008.
[29]Londono R,Gorantla VS,Badylak SF.Emerging Implications for Extracellular Matrix-Based Technologies in Vascularized Composite Allotransplantation.Stem Cells Int.2016;2016:1541823.
[30]O'Neill JD,Freytes DO,Anandappa AJ,et al.The regulation of growth and metabolism of kidney stem cells with regional specificity using extracellular matrix derived from kidney.Biomaterials.2013;34(38):9830-9841.