JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE) ›› 2017, Vol. 52 ›› Issue (5): 1-9.doi: 10.6040/j.issn.1671-9352.0.2017.112

    Next Articles

Preparation and characterization of a novel acellular porcine cornea stromata carrier scaffold

FAN Ting-jun, SHAN Ming, PANG Xin   

  1. Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong, China
  • Received:2017-03-21 Online:2017-05-20 Published:2017-05-15

Abstract: To obtain a satisfied carrier scaffold from xenogeneic corneal stroma for corneal tissue engineering, a novel technique using detergents of sodium deoxycholate(SD)and sodium orthovanadate(SO)was established to prepare an acellular porcine cornea stromata(aPCS)scaffold from fresh porcine cornea, and characterize its essential property for the first time in this study. The anterior lamella, 450 mm in thickness and sliced off from each cornea of fresh porcine eyeballs using a microkeratome, was decellularized with SD combined with SO, sodium dodecyl sulfate(SDS), and Triton X-100, and three kinds of aPCS scaffolds, ie SD-aPCS, SDS-aPCS and Triton-aPCS, were obtained, respectively. The physichemical property and histological structure of them was evaluated by light microscopy, spectrophotometer, paraffin section with hematoxylin-eosin(HE)staining, and frozen section with DAPI staining, and alcian blue staining, respectively. The ultrastructure of the SD-aPCS scaffold was verified by scanning electronic microscopy(SEM) 山 东 大 学 学 报 (理 学 版)第52卷 - 第5期樊廷俊,等:一种新型脱细胞猪角膜基质载体支架的制备及其鉴定研究 \=-and transmission electron microscopy(TEM), and their cytotoxicity and biocompatibility to non-transfected human corneal stromal(ntHCS)cells was evaluated by methyl thiazolyl tetrazolium(MTT), HE staining, DiI-fluorescence observation, and immunocytofluorescent staining. The results showed that the dry weight and water content of the three kinds of aPCS scaffolds had no significant difference, their innate cells were removed, and all of them were completely decellularized. Among them, the SD-aPCS scaffold had the highest transparency and glycosaminoglycan(GAG)content, followed by the SDS-aPCS and Triton-aPCS scaffold successively. In addition to the obvious disorder of the Triton-aPCS scaffold, the histological structure of SD-aPCS and SDS-aPCS scaffolds was very regular. Furthermore, the surface of Bowmens membrane of the SD-aPCS scaffold was smooth and no cracks were found, and the orientation and arrangement of collagen fiber were normal in ultrastructure. SD-aPCS scaffold had no cytotoxicity to ntHCS cells, and the cells injected into the scaffold could adhere tightly to collagen fibers, and spread and immigrated within scaffold lamella with the time of in vitro culture. These cells still maintained the positive expression pattern of the marker protein of vimentin, the cell junction proteins of integrin β1 and connextin-43, and the membrane transport protein of Na+-K+ pump. In conclusion, the SD-aPCS scaffold prepared by SD and SO detergents, with ideal physichemical property, histological structure and biocompatibility, could be used as an ideal scaffold in the in vitro construction of tissue engineered cornea and other related application researches.

Key words: scaffold, human corneal stromal cell, acellular porcine corneal stromata, sodium deoxycholate, biocompatibility

CLC Number: 

  • Q813
[1] GEORGE A J, LARKIN D F. Corneal transplantation: the forgotten graft[J]. Am J Transplant, 2004, 4(5):678-685.
[2] 周庆军, 谢立信. 组织工程角膜的基础研究和临床应用现状[J]. 中华细胞与干细胞杂志(电子版), 2014, 4(1):1-4. ZHOU Qingjun, XIE Lixin. Tissue-engineered cornea: new progresses and challenges[J]. Chin J Cell Stem Cell(Electronic Edition), 2014, 4(1):1-4.
[3] SHAH A, BRUGNANO J, SUN S, et al. The development of a tissue-engineered cornea: biomaterials and culture methods[J]. Pediatr Res, 2008, 63(5):535-544.
[4] 赵文卓, 樊廷俊, 胡修忠, 等. 组织工程人角膜基质体外重建的研究进展[J]. 山东大学学报(理学版), 2011, 49(8):62-66. ZHAO Wenzhuo, FAN Tingjun, HU Xiuzhong, et al. Research advances on in vitro reconstruction of tissue-engineered human corneal stroma[J]. Journal of Shandong University(Natural Science), 2011, 49(8):62-66.
[5] CRAPO P M, GILBERT T W, BADYLAK S F. An overview of tissue and whole organ decellularization processes[J]. Biomaterials, 2011, 32(12):3233-3243.
[6] WILSON S L, SIDNEY L E, DUNPHY S E, et al. Keeping an eye on decellularized corneas: a review of methods, characterization and applications[J]. J Funct Biomater, 2013, 4(3):114-161.
[7] CHOI H J, KIM M K, LEE H J, et al. Efficacy of pig-to-rhesus lamellar corneal xenotransplantation[J]. Invest Ophthalmol Vis Sci, 2011, 52(9):6643-6650.
[8] WU Zheng, ZHOU Yang, LI Naiyang, et al. The use of phospholipase A2 to prepare acellular porcine corneal stroma as a tissue engineering scaffold[J]. Biomaterials, 2009, 30(21):3513-3522.
[9] PANG Kunpeng, DU Liqun, WU Xinyi. A rabbit anterior cornea replacement derived from acellular porcine cornea matrix, epithelial cells and keratocytes[J]. Biomaterials, 2010, 31(28):7257-7265.
[10] AMANO S, SHIMOMURA N, YOKOO S, et al. Decellularizing corneal stroma using N2 gas[J]. Mol Vis, 2008, 14(104/105):878-882.
[11] PONCE MARQUEZ S, MARTYINEZ V S, MCINTOSH AMBROSE W, et al. Decellularization of bovine corneas for tissue engineering applications[J]. Acta Biomater, 2009, 5(6):1839-1847.
[12] DU Liqun, WU Xinyi, PANG Kunpeng, et al. Histological evaluation and biomechanical characterization of an acellular porcine cornea scaffold[J]. Br J Ophthalmol, 2010, 95(3):410-414.
[13] LYNCH A P, AHEARNE M. Strategies for developing decellularized corneal scaffolds[J]. Exp Eye Res, 2013, 108(3):42-47.
[14] FAN Tingjun, HU Xiuzhong, ZHAO Jun, et al. Establishment of an untransfected human corneal stromal cell line and its biocompatibility to acellular porcine corneal stroma[J]. Int J Ophthalmol, 2012, 5(3):286-292.
[15] XU Yonggen, XU Yongsheng, HUANG Chen, et al. Development of a rabbit corneal equivalent using an acellular corneal matrix of a porcine substrate[J]. Mol Vis, 2008, 14(253-255):2180-2189.
[16] DIAO Jinmei, PANG Xin, QIU Yue, et al. Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata[J]. Exp Eye Res, 2015, 132:16-224.
[17] SHAN M, FAN T J. Cytotoxicity of carteolol to human corneal epithelial cells by inducing apoptosis via triggering the Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway[J]. Toxicol In Vitro, 2016, 35:36-42.
[18] XU CC, CHAN RW, TIRUNAGARI N. A biodegradable, acellular xenogeneic scaffold for regeneration of the vocal fold lamina propria[J]. Tissue Eng, 2007, 13(3):551-566.
[19] XU Bin, HU Xiuzhong, ZHAO Jun, et al. Establishment of an untransfected human corneal stromal cell line and its biocompatibility to acellular porcine corneal stroma[J]. Int J Ophthalmol, 2012, 5(3):286-292.
[20] BERGMANSON J P, HORNE J, DOUGHTY M J, et al. Assessment of the number of lamellae in the central region of the normal human corneal stroma at the resolution of the transmission electron microscope[J]. Eye Contact Lens, 2005, 31(6):281-287.
[21] CHEN S, BIRK D. The regulatory roles of small leucine-rich proteoglycans in extracellular matrix assembly[J]. FEBS J, 2013, 280(10):2120-2137.
[22] BAYYOUD T, THALER S, HOFMANN J, et al. Decellularized bovine corneal posterior lamellae as carrier matrix for cultivated human corneal endothelial cells[J]. Curr Eye Res, 2012, 37(3):179-186.
[23] GONZALEZ-ANDRADES M, DE LA CRUZ CARDONA J, IONESCU A M, et al. Generation of bioengineered corneas with decellularized xenografts and human keratocytes[J]. Invest Ophthalmol Vis Sci, 2011, 52(1):215-222.
[24] MAUERER R, EBERT S, LANGMANN T. High glucose, unsaturated and saturated fatty acids differentially regulate expression of ATP-binding cassette transporters ABCA1 and ABCG1 in human macrophages[J]. Exp Mol Med, 2009, 41(2):126-132.
[25] DAXER A, MISOF K, GRABNER B, et al. Collagen fibrils in the human corneal stroma: structure and aging[J]. Invest Ophthalmol Vis Sci, 1998, 39(3):644-648.
[26] GLAESER R M, JUBB J S, HENDERSON R. Structural comparison of native and deoxycholate-treated purple membrane[J]. Biophys J, 1985, 48(5):775-780.
[27] SEGREST J P, WILKINSON T M, SHENG L. Isolation of glycophorin with deoxycholate[J]. Biochim Biophys Acta, 1979, 554(2):533-537.
[1] YUAN Qing-xian, GAO Li-lan, LI Rui-xin, LIU Ying-jie, LIN Xiang-long, ZHANG Xi-zheng. Silk fibroin-type Ⅱ collagen cartilage scaffold fabricated by 3D printing technology [J]. JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE), 2018, 53(3): 82-87.
[2] YUAN Xiao-long, XU Bin, FAN Ting-jun. Fabrication of a three-dimensional fish collagen scaffold and its biocompatibility characterization [J]. JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE), 2016, 51(1): 36-42.
[3] FAN Ting-jun, DIAO Jin-mei. Research advances of tissue-engineered human corneal endothelium [J]. JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE), 2014, 49(1): 1-7.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!