Preparation and characterization of liver extracellular matrix hydrogel

doi:10.3969/j.issn.2095-4344.1682

Abstract

Abstract:

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.

Key words: biomaterial, decellularized scaffold, hydrogel, turbidity kinetics, mass spectrum, microstructure, extracellular matrix, cells

CLC Number:

Cao Yulun, Cheng Yuan, He Guolin, Li Yang, Peng Qing, Gao Yi, Pan Mingxin. Preparation and characterization of liver extracellular matrix hydrogel[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(18): 2847-2851.

Figures/Tables 6

References

[1]Spang MT,Christman KL.Extracellular matrix hydrogel therapies: In vivo applications and development. Acta Biomaterialia.2018;68:1-14.

[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.