脱细胞猪角膜基质支架的制备及其生物相容性
|
摘要
背景:研究发现多种生物材料可制备角膜基质支架且具有良好的生物相容性,而关于脱细胞猪角膜基质支架的制备和生物相容性研究不多。目的:制备脱细胞猪角膜基质支架,探讨其与人角膜基质细胞的生物相容性。方法:制备脱细胞猪角膜基质支架及其浸提液。取生长状态良好的人角膜基质细胞,分别以脱细胞猪角膜基质支架浸提液(实验组)、完全培养基培养(对照组)培养,培养后1,2,3 d,采用MTT法检测人角膜基质细胞的增殖;将人角膜基质细胞直接接种于脱细胞猪角膜基质支架上,接种1,3,7 d,采用免疫化学法测定人角膜基质细胞在支架内的生长情况。结果与结论:(1)随着时间的增加,实验组与对照组人角膜基质细胞数量不断增加,两组不同时间点的吸光度值比较差异无显著性意义;(2)人角膜基质细胞在脱细胞猪角膜基质支架上生长良好,细胞整联蛋白β1、波形蛋白、乙醛脱氢酶3A1呈阳性表达,CD34、CDK2和K-Ras蛋白也呈阳性表达;(3)结果表明,脱细胞猪角膜基质支架无细胞毒性,与人角膜基质细胞具有良好的生物相容性。
BACKGROUND: Studies have found that a variety of biological materials can be used for preparing corneal stroma scaffolds that have good biocompatibility, but research on preparation and biocompatibility of the acellular porcine corneal stroma scaffold is little.OBJECTIVE: To explore the preparation and biocompatibility of the acellular porcine corneal stroma scaffold. METHODS: Acellular porcine corneal stroma scaffold and its extract were prepared. Well-grown human corneal stromal cells were selected and cultured in the extract of acellular porcine corneal stroma scaffold(experimental group) or in the complete medium(control group), respectively. After 1, 2 and 3 days of culture, the proliferation ability of human corneal stromal cells was detected by MTT assay. In the meanwhile, human corneal cells were directly seeded onto the acellular porcine corneal stroma scaffold, and then the cell growth on the scaffold was detected using immunochemical method. RESULTS AND CONCLUSION: The number of human corneal stromal cells was in a rise with time in the two groups, and absorbance values had no significant difference between two groups at different time points of culture. Human corneal stromal cells grew well on the scaffold, and were positive for cell integrin β1, vimentin, aldehyde dehydrogenase 3A1, as well as CD34, CDK2 and K-Ras. These results show that the acellular porcine corneal stroma scaffold has no cytotoxicity, and has good biocompatibility.
BACKGROUND: Studies have found that a variety of biological materials can be used for preparing corneal stroma scaffolds that have good biocompatibility, but research on preparation and biocompatibility of the acellular porcine corneal stroma scaffold is little.OBJECTIVE: To explore the preparation and biocompatibility of the acellular porcine corneal stroma scaffold. METHODS: Acellular porcine corneal stroma scaffold and its extract were prepared. Well-grown human corneal stromal cells were selected and cultured in the extract of acellular porcine corneal stroma scaffold(experimental group) or in the complete medium(control group), respectively. After 1, 2 and 3 days of culture, the proliferation ability of human corneal stromal cells was detected by MTT assay. In the meanwhile, human corneal cells were directly seeded onto the acellular porcine corneal stroma scaffold, and then the cell growth on the scaffold was detected using immunochemical method. RESULTS AND CONCLUSION: The number of human corneal stromal cells was in a rise with time in the two groups, and absorbance values had no significant difference between two groups at different time points of culture. Human corneal stromal cells grew well on the scaffold, and were positive for cell integrin β1, vimentin, aldehyde dehydrogenase 3A1, as well as CD34, CDK2 and K-Ras. These results show that the acellular porcine corneal stroma scaffold has no cytotoxicity, and has good biocompatibility.
引文
[1]王斌,李长兵.深板层角膜移植治疗角膜病的临床观察[J].国际眼科杂志,2012,12(12):2394-2395.
[2]廖聪玲.应用国外捐献的供体角膜组织行穿透性角膜移植的前瞻性研究[D].大连医科大学,2013.
[3]Tahvildari M,Omoto M,Chen Y,et al.In Vivo Expansion of Regulatory T Cells by Low-Dose Interleukin-2 Treatment Increases Allograft Survival in Corneal Transplantation.Transplantation.2016;100(3):525-532.
[4]徐彬.组织工程人角膜上皮的体外重建、鉴定及其在新西兰兔角膜上皮移植中的作用研究[D].中国海洋大学,2012.
[5]陈根云,陈桂强.组织工程角膜内皮载体材料的研究进展[J].中国组织工程研究与临床康复,2008,12(6):1093-1096.
[6]SzabóDJ,Noer A,Nagymihály R,et al.Long-Term Cultures of Human Cornea Limbal Explants Form 3DStructures Ex Vivo-Implications for Tissue Engineering and Clinical Applications.PLo S One.2015;10(11):e0143053.
[7]随蓓蓓.胶原蛋白复合支架的制备及其与人角膜基质细胞的生物相容性研究[D].中国海洋大学,2013.
[8]Salim S,Ariani MD.In vitro and in vivo evaluation of carbonate apatite-collagen scaffolds with some cytokines for bonetissue engineering.J Indian Prosthodont Soc.2015;15(4):349-355.
[9]隋鲜鲜.甲壳素载体支架的生物相容性和对角膜上皮损伤的修复研究[D].中国海洋大学,2013.
[10]Pircher N,Fischhuber D,Carbajal L,et al.Preparation and Reinforcement of Dual-Porous Biocompatible Cellulose Scaffolds for Tissue Engineering.Macromol Mater Eng.2015;300(9):911-924.
[11]Feizi S,Montahai T,Moein H.Graft Biomechanics Following Three Corneal Transplantation Techniques.JOphthalmic Vis Res.2015;10(3):238-242.
[12]梁长森.角膜移植术后不同内皮型免疫排斥反应的活体共聚焦显微镜观察[D].济南大学,2013.
[13]杨贲.76例角膜病行部分穿透性角膜移植术的临床分析[D].吉林大学,2009.
[14]Hos D,D?rrie J,Schaft N,et al.Blockade of CCR7 leads to decreased dendritic cell migration to draining lymph nodes and promotes graft survival in low-risk corneal transplantation.Exp Eye Res.2015;146:1-6.
[15]Gain P,Jullienne R,He Z,et al.Global Survey of Corneal Transplantation and Eye Banking.JAMA Ophthalmol.2016;134(2):167-173.
[16]魏君.104例角膜病行穿透性角膜移植术后排斥反应的临床病例分析[D].吉林大学,2010.
[17]Miotto M,Gouveia RM,Connon CJ.Peptide Amphiphiles in Corneal Tissue Engineering.J Funct Biomater.2015;6(3):687-707.
[18]李善义,陈建苏.组织工程角膜内皮层移植的研究与应用[J].中国组织工程研究,2013,17(2):363-368.
[19]Levis HJ,Kureshi AK,Massie I,Tissue Engineering the Cornea:The Evolution of RAFT.J Funct Biomater.2015;6(1):50-65.
[20]尹澜,皮裕琍,郭青,等.组织工程角膜联合羊膜移植治疗眼表烧伤疗效观察[J].人民军医,2013,64(5):548-550.
[21]Lachaud CC,Soria F,Escacena N,et al.Erratum.Mesothelial Cells:A Cellular Surrogate for Tissue Engineering of Corneal Endothelium.Invest Ophthalmol Vis Sci.2015;56(3):1679.
[22]樊廷俊,刁金美.组织工程人角膜内皮的研究进展[J].山东大学学报(理学版),2014,64(1):1-7.
[23]刘多静,刘长勇,徐圆圆,等.胶原支架在组织工程角膜中的研究进展[J].生物医学工程与临床,2014,18(3):291-295.
[24]Vázquez N,Chacón M,Meanaá,et al.Keratin-chitosan membranes as scaffold for tissue engineering of human cornea.Histol Histopathol.2015;30(7):813-821.
[25]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.
[26]马群.壳聚糖基角膜内皮组织工程载体支架的制备及性能评价[D].中国海洋大学,2010.
[27]李强,孙正义.软骨组织工程支架材料研究的现状[J].中国组织工程研究与临床康复,2007,11(1):133-136.
[28]Markovic M,Van Hoorick J,H?lzl K,et al.Hybrid Tissue Engineering Scaffolds by Combination of Three-Dimensional Printing and Cell Photoencapsulation.J Nanotechnol Eng Med.2015;6(2):0210011-210017.
[29]随蓓蓓.组织工程载体支架材料研究进展[J].科协论坛(下半月),2013,28(5):52-53.
[30]Kim IG,Ko J,Lee HR,et al.Mesenchymal cells condensation-inducible mesh scaffolds for cartilage tissue engineering.Biomaterials.2016;85:18-29.
[31]隋鲜鲜,韩宝芹,刘万顺,等.组织工程甲壳素载体支架对角膜上皮损伤修复作用的研究[J].功能材料,2013,16:2313-2319.
[32]鞠成群.脱细胞角膜基质联合内皮样细胞构建生物工程角膜后板层的实验研究[D].山东大学,2012.
[33]Ma XY,Zhang Y,Zhu D,et al.Corneal Stroma Regeneration with Acellular Corneal Stroma Sheets and Keratocytes in a Rabbit Model.PLo S One.2015;10(7):e0132705.
[34]朱婧.胚胎干细胞源角膜缘干细胞联合脱细胞角膜缘基质修复眼表的研究[D].山东大学,2013.
[35]苗莹.基于脱细胞猪角膜基质的组织工程人角膜基质的体外重建及其动物移植研究[D].中国海洋大学,2013.
[36]Feng Y,Wang W.In vivo confocal microscopic observation of lamellar corneal transplantation in the rabbit using xenogenic acellular corneal scaffolds as a substitute.Chin Med J(Engl).2015;128(7):933-940.
[37]Alio del Barrio JL,Chiesa M,Garagorri N,et al.Acellular human corneal matrix sheets seeded with human adipose-derived mesenchymal stem cells integrate functionally in an experimental animal model.Exp Eye Res.2015;132:91-100.
[38]庞鵾鹏.以异种脱细胞角膜基质构建组织工程兔角膜前板层的实验研究[D].山东大学,2011.
[39]张菊.以脱细胞猪角膜基质为支架体外培养人角膜上皮细胞与成纤维细胞的实验研究[D].山东大学,2015.
[40]Zhu J,Zhang K,Sun Y,et al.Reconstruction of functional ocular surface by acellular porcine cornea matrix scaffold and limbal stem cells derived from human embryonic stem cells.Tissue Eng Part A.2013;19(21-22):2412-2425.
[41]汲婧,刘子源,张晶,等.脱细胞真皮基质在角膜中植入性的生物力学研究[J].力学学报,2014,46(1):145-154.
[42]Liu XN,Zhu XP,Wu J,et al.Acellular ostrich corneal stroma used as scaffold for construction of tissue-engineered cornea.Int J Ophthalmol.2016;9(3):325-331.
[43]Zhang J,Zhang CW,Du LQ,et al.Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro.Int J Ophthalmol.2016;9(1):1-8.
[44]Xue C,Xia Y,Chen Y,et al.Treatment of large corneal perforations with acellular multilayer of corneal stromal lenticules harvested from femtosecond laser lenticule extraction.Zhonghua Yan Ke Za Zhi.2015;51(9):655-659.
[45]Shao Y,Tang J,Zhou Y,et al.A novel method in preparation of acellularporcine corneal stroma tissue for lamellar keratoplasty.Am J Transl Res.2015;7(12):2612-2629.
[46]Wee SW,Choi SU,Kim JC.Deep anterior lamellar keratoplasty using irradiated acellular cornea with amniotic membrane transplantation for intractable ocular surface diseases.Korean J Ophthalmol.2015;29(2):79-85.
[47]Alio del Barrio JL,Chiesa M Garagorri N,et al.Acellular human corneal matrix sheets seeded with human adipose-derived mesenchymal stem cells integrate functionally in an experimental animal model.Exp Eye Res.2015;132:91-100.
[48]Diao JM,Pang X,Qiu Y,et al.Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.Exp Eye Res.2015;132:216-224.
[49]Liu Z,Zhou Q,Zhu J,et al.Using genipin-crosslinked acellular porcine corneal stroma for cosmetic corneal lens implants.Biomaterials.2012;33(30):7336-7346.
[50]Hahn N,Dietz CT,Kühl S,et al.KLEIP deficiency in mice causes progressive corneal neovascular dystrophy.Invest Ophthalmol Vis Sci.2012;53(6):3260-3268.
[2]廖聪玲.应用国外捐献的供体角膜组织行穿透性角膜移植的前瞻性研究[D].大连医科大学,2013.
[3]Tahvildari M,Omoto M,Chen Y,et al.In Vivo Expansion of Regulatory T Cells by Low-Dose Interleukin-2 Treatment Increases Allograft Survival in Corneal Transplantation.Transplantation.2016;100(3):525-532.
[4]徐彬.组织工程人角膜上皮的体外重建、鉴定及其在新西兰兔角膜上皮移植中的作用研究[D].中国海洋大学,2012.
[5]陈根云,陈桂强.组织工程角膜内皮载体材料的研究进展[J].中国组织工程研究与临床康复,2008,12(6):1093-1096.
[6]SzabóDJ,Noer A,Nagymihály R,et al.Long-Term Cultures of Human Cornea Limbal Explants Form 3DStructures Ex Vivo-Implications for Tissue Engineering and Clinical Applications.PLo S One.2015;10(11):e0143053.
[7]随蓓蓓.胶原蛋白复合支架的制备及其与人角膜基质细胞的生物相容性研究[D].中国海洋大学,2013.
[8]Salim S,Ariani MD.In vitro and in vivo evaluation of carbonate apatite-collagen scaffolds with some cytokines for bonetissue engineering.J Indian Prosthodont Soc.2015;15(4):349-355.
[9]隋鲜鲜.甲壳素载体支架的生物相容性和对角膜上皮损伤的修复研究[D].中国海洋大学,2013.
[10]Pircher N,Fischhuber D,Carbajal L,et al.Preparation and Reinforcement of Dual-Porous Biocompatible Cellulose Scaffolds for Tissue Engineering.Macromol Mater Eng.2015;300(9):911-924.
[11]Feizi S,Montahai T,Moein H.Graft Biomechanics Following Three Corneal Transplantation Techniques.JOphthalmic Vis Res.2015;10(3):238-242.
[12]梁长森.角膜移植术后不同内皮型免疫排斥反应的活体共聚焦显微镜观察[D].济南大学,2013.
[13]杨贲.76例角膜病行部分穿透性角膜移植术的临床分析[D].吉林大学,2009.
[14]Hos D,D?rrie J,Schaft N,et al.Blockade of CCR7 leads to decreased dendritic cell migration to draining lymph nodes and promotes graft survival in low-risk corneal transplantation.Exp Eye Res.2015;146:1-6.
[15]Gain P,Jullienne R,He Z,et al.Global Survey of Corneal Transplantation and Eye Banking.JAMA Ophthalmol.2016;134(2):167-173.
[16]魏君.104例角膜病行穿透性角膜移植术后排斥反应的临床病例分析[D].吉林大学,2010.
[17]Miotto M,Gouveia RM,Connon CJ.Peptide Amphiphiles in Corneal Tissue Engineering.J Funct Biomater.2015;6(3):687-707.
[18]李善义,陈建苏.组织工程角膜内皮层移植的研究与应用[J].中国组织工程研究,2013,17(2):363-368.
[19]Levis HJ,Kureshi AK,Massie I,Tissue Engineering the Cornea:The Evolution of RAFT.J Funct Biomater.2015;6(1):50-65.
[20]尹澜,皮裕琍,郭青,等.组织工程角膜联合羊膜移植治疗眼表烧伤疗效观察[J].人民军医,2013,64(5):548-550.
[21]Lachaud CC,Soria F,Escacena N,et al.Erratum.Mesothelial Cells:A Cellular Surrogate for Tissue Engineering of Corneal Endothelium.Invest Ophthalmol Vis Sci.2015;56(3):1679.
[22]樊廷俊,刁金美.组织工程人角膜内皮的研究进展[J].山东大学学报(理学版),2014,64(1):1-7.
[23]刘多静,刘长勇,徐圆圆,等.胶原支架在组织工程角膜中的研究进展[J].生物医学工程与临床,2014,18(3):291-295.
[24]Vázquez N,Chacón M,Meanaá,et al.Keratin-chitosan membranes as scaffold for tissue engineering of human cornea.Histol Histopathol.2015;30(7):813-821.
[25]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.
[26]马群.壳聚糖基角膜内皮组织工程载体支架的制备及性能评价[D].中国海洋大学,2010.
[27]李强,孙正义.软骨组织工程支架材料研究的现状[J].中国组织工程研究与临床康复,2007,11(1):133-136.
[28]Markovic M,Van Hoorick J,H?lzl K,et al.Hybrid Tissue Engineering Scaffolds by Combination of Three-Dimensional Printing and Cell Photoencapsulation.J Nanotechnol Eng Med.2015;6(2):0210011-210017.
[29]随蓓蓓.组织工程载体支架材料研究进展[J].科协论坛(下半月),2013,28(5):52-53.
[30]Kim IG,Ko J,Lee HR,et al.Mesenchymal cells condensation-inducible mesh scaffolds for cartilage tissue engineering.Biomaterials.2016;85:18-29.
[31]隋鲜鲜,韩宝芹,刘万顺,等.组织工程甲壳素载体支架对角膜上皮损伤修复作用的研究[J].功能材料,2013,16:2313-2319.
[32]鞠成群.脱细胞角膜基质联合内皮样细胞构建生物工程角膜后板层的实验研究[D].山东大学,2012.
[33]Ma XY,Zhang Y,Zhu D,et al.Corneal Stroma Regeneration with Acellular Corneal Stroma Sheets and Keratocytes in a Rabbit Model.PLo S One.2015;10(7):e0132705.
[34]朱婧.胚胎干细胞源角膜缘干细胞联合脱细胞角膜缘基质修复眼表的研究[D].山东大学,2013.
[35]苗莹.基于脱细胞猪角膜基质的组织工程人角膜基质的体外重建及其动物移植研究[D].中国海洋大学,2013.
[36]Feng Y,Wang W.In vivo confocal microscopic observation of lamellar corneal transplantation in the rabbit using xenogenic acellular corneal scaffolds as a substitute.Chin Med J(Engl).2015;128(7):933-940.
[37]Alio del Barrio JL,Chiesa M,Garagorri N,et al.Acellular human corneal matrix sheets seeded with human adipose-derived mesenchymal stem cells integrate functionally in an experimental animal model.Exp Eye Res.2015;132:91-100.
[38]庞鵾鹏.以异种脱细胞角膜基质构建组织工程兔角膜前板层的实验研究[D].山东大学,2011.
[39]张菊.以脱细胞猪角膜基质为支架体外培养人角膜上皮细胞与成纤维细胞的实验研究[D].山东大学,2015.
[40]Zhu J,Zhang K,Sun Y,et al.Reconstruction of functional ocular surface by acellular porcine cornea matrix scaffold and limbal stem cells derived from human embryonic stem cells.Tissue Eng Part A.2013;19(21-22):2412-2425.
[41]汲婧,刘子源,张晶,等.脱细胞真皮基质在角膜中植入性的生物力学研究[J].力学学报,2014,46(1):145-154.
[42]Liu XN,Zhu XP,Wu J,et al.Acellular ostrich corneal stroma used as scaffold for construction of tissue-engineered cornea.Int J Ophthalmol.2016;9(3):325-331.
[43]Zhang J,Zhang CW,Du LQ,et al.Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro.Int J Ophthalmol.2016;9(1):1-8.
[44]Xue C,Xia Y,Chen Y,et al.Treatment of large corneal perforations with acellular multilayer of corneal stromal lenticules harvested from femtosecond laser lenticule extraction.Zhonghua Yan Ke Za Zhi.2015;51(9):655-659.
[45]Shao Y,Tang J,Zhou Y,et al.A novel method in preparation of acellularporcine corneal stroma tissue for lamellar keratoplasty.Am J Transl Res.2015;7(12):2612-2629.
[46]Wee SW,Choi SU,Kim JC.Deep anterior lamellar keratoplasty using irradiated acellular cornea with amniotic membrane transplantation for intractable ocular surface diseases.Korean J Ophthalmol.2015;29(2):79-85.
[47]Alio del Barrio JL,Chiesa M Garagorri N,et al.Acellular human corneal matrix sheets seeded with human adipose-derived mesenchymal stem cells integrate functionally in an experimental animal model.Exp Eye Res.2015;132:91-100.
[48]Diao JM,Pang X,Qiu Y,et al.Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.Exp Eye Res.2015;132:216-224.
[49]Liu Z,Zhou Q,Zhu J,et al.Using genipin-crosslinked acellular porcine corneal stroma for cosmetic corneal lens implants.Biomaterials.2012;33(30):7336-7346.
[50]Hahn N,Dietz CT,Kühl S,et al.KLEIP deficiency in mice causes progressive corneal neovascular dystrophy.Invest Ophthalmol Vis Sci.2012;53(6):3260-3268.