重组胶原蛋白海绵的制备及性状表征
|
摘要
背景:重组胶原蛋白具有良好的亲水性及生物相容性,但其存在溶解性强及机械强度不足等缺点。化学交联可显著提高材料的韧性、力学强度及耐降解性。目的:制备重组胶原蛋白海绵,并对其理化性质及安全性进行评价。方法:采用微生物发酵法获得重组胶原蛋白,经戊二醛交联后,将其置于模具中冷冻干燥,获得多孔重组胶原蛋白海绵,检测交联后重组胶原海绵的吸水率与孔隙率,采用扫描电镜观察交联前后重组胶原海绵的表面形态,红外光谱仪对比交联前后重组胶原海绵结构的变化。采用交联重组胶原海绵浸提液培养L929细胞,培养第68小时,采用MTT法检测细胞相对增殖率,评估交联重组胶原海绵的细胞毒性。结果与结论:(1)交联后重组胶原海绵的吸水率为(2 903.83±47.90)%,平均孔隙率在85%以上;(2)扫描电镜显示,交联前,重组胶原海绵的孔隙呈蜂窝状,致密;交联后,重组胶原海绵的孔隙较大,呈板层结构,可看到板层间桥键连接,纵向可看到较大褶皱;(3)红外光谱显示,交联后的重组胶原海绵特征性化学结构特征无明显变化;(4)交联重组胶原海绵浸提液培养L929细胞的相对增殖率为84%,细胞毒性为1级,生物安全性良好;(5)结果表明采用戊二醛交联制备的重组胶原海绵,理化性质稳定,生物相容性良好。
BACKGROUND: Recombinant collagen has good hydrophilicity and biocompatibility, but it has the disadvantages of strong solubility and low mechanical strength. Chemical crosslinking can significantly improve the toughness, mechanical strength and degradation resistance of the material. OBJECTIVE: To prepare recombinant collagen sponge and to evaluate its physicochemical properties and safety. METHODS: The recombinant collagen was obtained via microbial fermentation and cross-linked with glutaraldehyde. The porous recombinant collagen sponge was finally obtained by freeze-drying in the mold. The water absorption, porosity and crosslinking agent residual of the recombinant collagen sponge were detected. The surface morphology of the recombinant collagen sponge before and after cross-linking was observed by scanning electron microscope. The changes in recombinant collagen structures before and after cross-linking were compared by infrared spectrometer. L929 cells were cultured with cross-linked recombinant collagen sponge extract. At 68 hours after culture, the relative proliferation rate of the cells was detected by MTT, and the cytotoxicity of cross-linked recombinant collagen sponge was evaluated. RESULTS AND CONCLUSION:(1) The water absorption of the cross-linked collagen sponge was(2 903.83±47.90)% and the average porosity was over 85%.(2) Under the scanning electron microscope, the pores of the collagen sponge were honeycomb and dense before cross-linking. After cross-linking, the pores of the collagen sponge were large and bridged bond linkage of laminar layer structure could be seen, and also the large folds could be seen in the longitudinal direction.(3) There was no obvious change in the characteristic chemical structure of the recombinant collagen sponge after cross-linking.(4) The relative proliferation rate of L929 cells was 84% after culture in cross-linked recombinant collagen sponge extract, and the cytotoxicity was level 1, indicating the biological safety was good. To conclude, the recombinant collagen sponge with glutaraldehyde cross-linking shows stable physicochemical properties and good biocompatibility.
BACKGROUND: Recombinant collagen has good hydrophilicity and biocompatibility, but it has the disadvantages of strong solubility and low mechanical strength. Chemical crosslinking can significantly improve the toughness, mechanical strength and degradation resistance of the material. OBJECTIVE: To prepare recombinant collagen sponge and to evaluate its physicochemical properties and safety. METHODS: The recombinant collagen was obtained via microbial fermentation and cross-linked with glutaraldehyde. The porous recombinant collagen sponge was finally obtained by freeze-drying in the mold. The water absorption, porosity and crosslinking agent residual of the recombinant collagen sponge were detected. The surface morphology of the recombinant collagen sponge before and after cross-linking was observed by scanning electron microscope. The changes in recombinant collagen structures before and after cross-linking were compared by infrared spectrometer. L929 cells were cultured with cross-linked recombinant collagen sponge extract. At 68 hours after culture, the relative proliferation rate of the cells was detected by MTT, and the cytotoxicity of cross-linked recombinant collagen sponge was evaluated. RESULTS AND CONCLUSION:(1) The water absorption of the cross-linked collagen sponge was(2 903.83±47.90)% and the average porosity was over 85%.(2) Under the scanning electron microscope, the pores of the collagen sponge were honeycomb and dense before cross-linking. After cross-linking, the pores of the collagen sponge were large and bridged bond linkage of laminar layer structure could be seen, and also the large folds could be seen in the longitudinal direction.(3) There was no obvious change in the characteristic chemical structure of the recombinant collagen sponge after cross-linking.(4) The relative proliferation rate of L929 cells was 84% after culture in cross-linked recombinant collagen sponge extract, and the cytotoxicity was level 1, indicating the biological safety was good. To conclude, the recombinant collagen sponge with glutaraldehyde cross-linking shows stable physicochemical properties and good biocompatibility.
引文
[1]高长有,马列.医用高分子材料[M].北京:化学工业出版社,2006:22-26.
[2]Deepthi S,Nivedhitha SM,Deepti KJ,et al.Layered chitosan-collagen hydrogel/aligned PLLA nanofiber construct for flexor tendon regeneration.Carbohydr Polym.2016;153:492-500.
[3]Panwar P,Lamour G,Mackenzie NCW,et al.Changes in structuralmechanical properties and degradability of collagen during aging-associated modifications.J Biol Chem.2015;290(38):23291-23306.
[4]Walters BD,Stegemann JP.Strategies for directing the structure and function of three-dimensional collagen biomaterials across length scales.Acta Biomaterialia.2014;10(4):1488-1501.
[5]Maynes R.Structure and function of collagen types.Elsevier,2012.
[6]Usha R,Sreeram KJ,Rajaram A,et al.Stabilization of collagen with EDC/NHS in the presence of l-lysine:A comprehensive study.Colloids Surf B Biointerfaces.2012;90:83-90.
[7]王晓军,惠俊峰,米钰,等.重组类人胶原蛋白的分离纯化[J].中国生物制品学杂志,2003,16(4):212-214.
[8]Ramachandran G.Biochemistry of collagen.Springer Science&Business Media,2013.
[9]Jia L,Duan Z,Fan D,et al.Human-like collagen/nanohydroxyapatite scaffolds for the culture of chondrocytes.Mater Sci Eng C Mater Biol Appl.2013;33(2):727-734.
[10]柴雅琳,何会霞,肖建喜.重组胶原蛋白-羟基磷灰石复合材料的制备与表征[J].皮革科学与工程,2017,27(4):36-42.
[11]Chang L,Liu XL,Di Fan D,et al.The efficiency of magnetic hyperthermia and in vivo histocompatibility for human-like collagen protein-coated magnetic nanoparticles.Int J Nanomedicine.2016;11:1175-1185.
[12]陈琳,吕洋,管利东,等.生物支架材料胶原膜交联前后的特性分析[J].中国修复重建外科杂志,2008,22(2):183-187.
[13]Murphy CM,Matsiko A,Haugh MG,et al.Mesenchymal stem cell fate is regulated by the composition and mechanical properties of collagen-glycosaminoglycan scaffolds.J MechBehav Biomed Mater.2012;11:53-62.
[14]何静.胶原蛋白在生物医药工程中的应用研究进展[J].河南科技,2015,564(5):104-105.
[15]Hu Y,Liu L,Gu ZP,et al.Modification of collagen with a natural derived cross-linker,alginate dialdehyde.Carbohydr Polym.2014;102(1):324-332.
[16]Tang YP,Yang XL,Hang BJ,et al.Efficient production ofhydroxylated human-like collagen via the co-expression of three key genes in Escherichia coli Origami(DE3).Appl Biochem Biotechnol.2016;178(7):1458-1470.
[17]Chen MM,Huang YQ,Guo H,et al.Preparation,characterization,and potential biomedical application of composite sponges based on collagen from silver carp skin.J Appl Polym Sci.2014;131(21):1-8.
[18]Inzana JA,Olvera D,Fuller SM,et al.3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration.Biomaterials.2014;35(13):4026-4034.
[19]迟妍妍,乐尧金,刘旭昭,等.胶原蛋白海绵的生物特性及体内降解吸收[J].中国组织工程研究,2014,18(34):5515-5519.
[20]Antoine EE,Vlachos PP,Rylander MN.Review of collagen I hydrogels for bioengineered tissue microenvironments:characterization of mechanics,structure,and transport.Tissue Eng Part B Rev.2014;20(6):683-696.
[21]Olsen D,Yang CL,Bodo M,et al.Recombinant collagen and gelatin for drug delivery.Adv Drug Del Rev.2003,55(12):1547-1567.
[22]刘振锋,吕卫金,戴圣佳,等.胶原蛋白的提取、改性及应用研究进展[J].食品与药品,2014,16(6):443-447.
[23]刘辉辉,郭善禹.皮肤组织损伤修复的研究进展[J].外科理论与实践,2013,18(2):188-192.
[24]Zhang X,Yang Y,Yao J,et al.Strong collagen hydrogels by oxidized dextran modification.ACS Sustain Chem Eng.2014;2(5):1318-1324.
[25]Gong ZJ,Zhang S,Wang K,et al.Chimeric flaps pedicled with the lateral circumflex femoral artery for individualised reconstruction of through-and-through oral and maxillofacial defects.Br J Oral Maxillofac Surg.2015;53(2):148-152.
[26]Sheu MT,Huang JC,Yeh GC,et al.Characterization of collagen gel solutions and collagen matrices for cell culture.Biomaterials.2001;22(13):1713-1719.
[27]Chen JT,Zhao YP,Xu Z,et al.Study on the FTIR of Recombinant Collagen and Bovine Type I Collagen.Chin J Mater Res.2008;22(3):119-121.
[28]蔡洁.新型交联剂的合成及交联改性胶原的研究[D].广州:华南理工大学,2014.
[29]程玮璐,李慧,贺金梅,等.可降解复合止血胶原蛋白海绵的制备及生物应用性能研究[J].化学与黏合,2016,38(4):231-234.
[30]GB/T16886.1-2011医疗器械风险管理过程中的评价与实验.
[31]Zhao L,Xian L,Zhao J,et al.A novel smart injectable hydrogel prepared by microbial transglutaminase and human-like collagen:Its characterization and biocompatibility.Mater Sci Eng C.2016;68:317-326.
[32]曹谊林,刘伟,张文杰,等.组织工程研究进展[J].上海交通大学学报(医学版),2012,32(9):1241-1250.
[33]邹勇,刘文杰.胶原在止血材料中的应用研究进展[J].明胶科学与技术,2010,30(1):1-4.
[34]Jayalakshmi K,Pasiyappazham R,Vairamani S,et al.Isolation and partial characterization of collagen from outer skin of Sepia pharaonis(Ehrenberg,1831)from Puducherry coast.Biochem Biophys Rep.2017;10:39-45.
[35]Chan EC,Peshavariya HM,Liu GS,et al.Nox4 modulates collagen production stimulated by transforming growth factorβ1 in vivo and in vitro.Biochem Biophys Res Commun.2013;430(3):918-925.
[36]刘新华,但年华,李正军,等.胶原基复合医用海绵的研究进展[J].西部皮革,2012,35(8):13-18.
[37]Si NP,Hye JL,Kwang HL,et al.Biological characterization of EDC-crosslinked collagen-hyaluronic acid matrix in dermal tissue restoration.Biomaterials.2003;24:1631-1641.
[38]Mirazul I,Vytautas C,Chaoliang,et al,Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels.Acta Biomaterialia.2015;12:70-80.
[39]Fagerholm P,Lagali NS,Ong JA,et al.Stable corneal regeneration four years after implantation of a cell-free recombinant human collagen scaffold.Biomaterials.2014;35(8):2420-2427.
[40]刘文广,May Griffith,Frank Li.基于重组人胶原构建角膜替代物[J].高分子通报,2010,23(8):000001-6.
[41]马慧军,贾赤宇,魏蓉,等.胶原蛋白凝胶促进皮肤创面修复的临床研究[J].中国美容医学,2011,20(2):235-236.
[2]Deepthi S,Nivedhitha SM,Deepti KJ,et al.Layered chitosan-collagen hydrogel/aligned PLLA nanofiber construct for flexor tendon regeneration.Carbohydr Polym.2016;153:492-500.
[3]Panwar P,Lamour G,Mackenzie NCW,et al.Changes in structuralmechanical properties and degradability of collagen during aging-associated modifications.J Biol Chem.2015;290(38):23291-23306.
[4]Walters BD,Stegemann JP.Strategies for directing the structure and function of three-dimensional collagen biomaterials across length scales.Acta Biomaterialia.2014;10(4):1488-1501.
[5]Maynes R.Structure and function of collagen types.Elsevier,2012.
[6]Usha R,Sreeram KJ,Rajaram A,et al.Stabilization of collagen with EDC/NHS in the presence of l-lysine:A comprehensive study.Colloids Surf B Biointerfaces.2012;90:83-90.
[7]王晓军,惠俊峰,米钰,等.重组类人胶原蛋白的分离纯化[J].中国生物制品学杂志,2003,16(4):212-214.
[8]Ramachandran G.Biochemistry of collagen.Springer Science&Business Media,2013.
[9]Jia L,Duan Z,Fan D,et al.Human-like collagen/nanohydroxyapatite scaffolds for the culture of chondrocytes.Mater Sci Eng C Mater Biol Appl.2013;33(2):727-734.
[10]柴雅琳,何会霞,肖建喜.重组胶原蛋白-羟基磷灰石复合材料的制备与表征[J].皮革科学与工程,2017,27(4):36-42.
[11]Chang L,Liu XL,Di Fan D,et al.The efficiency of magnetic hyperthermia and in vivo histocompatibility for human-like collagen protein-coated magnetic nanoparticles.Int J Nanomedicine.2016;11:1175-1185.
[12]陈琳,吕洋,管利东,等.生物支架材料胶原膜交联前后的特性分析[J].中国修复重建外科杂志,2008,22(2):183-187.
[13]Murphy CM,Matsiko A,Haugh MG,et al.Mesenchymal stem cell fate is regulated by the composition and mechanical properties of collagen-glycosaminoglycan scaffolds.J MechBehav Biomed Mater.2012;11:53-62.
[14]何静.胶原蛋白在生物医药工程中的应用研究进展[J].河南科技,2015,564(5):104-105.
[15]Hu Y,Liu L,Gu ZP,et al.Modification of collagen with a natural derived cross-linker,alginate dialdehyde.Carbohydr Polym.2014;102(1):324-332.
[16]Tang YP,Yang XL,Hang BJ,et al.Efficient production ofhydroxylated human-like collagen via the co-expression of three key genes in Escherichia coli Origami(DE3).Appl Biochem Biotechnol.2016;178(7):1458-1470.
[17]Chen MM,Huang YQ,Guo H,et al.Preparation,characterization,and potential biomedical application of composite sponges based on collagen from silver carp skin.J Appl Polym Sci.2014;131(21):1-8.
[18]Inzana JA,Olvera D,Fuller SM,et al.3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration.Biomaterials.2014;35(13):4026-4034.
[19]迟妍妍,乐尧金,刘旭昭,等.胶原蛋白海绵的生物特性及体内降解吸收[J].中国组织工程研究,2014,18(34):5515-5519.
[20]Antoine EE,Vlachos PP,Rylander MN.Review of collagen I hydrogels for bioengineered tissue microenvironments:characterization of mechanics,structure,and transport.Tissue Eng Part B Rev.2014;20(6):683-696.
[21]Olsen D,Yang CL,Bodo M,et al.Recombinant collagen and gelatin for drug delivery.Adv Drug Del Rev.2003,55(12):1547-1567.
[22]刘振锋,吕卫金,戴圣佳,等.胶原蛋白的提取、改性及应用研究进展[J].食品与药品,2014,16(6):443-447.
[23]刘辉辉,郭善禹.皮肤组织损伤修复的研究进展[J].外科理论与实践,2013,18(2):188-192.
[24]Zhang X,Yang Y,Yao J,et al.Strong collagen hydrogels by oxidized dextran modification.ACS Sustain Chem Eng.2014;2(5):1318-1324.
[25]Gong ZJ,Zhang S,Wang K,et al.Chimeric flaps pedicled with the lateral circumflex femoral artery for individualised reconstruction of through-and-through oral and maxillofacial defects.Br J Oral Maxillofac Surg.2015;53(2):148-152.
[26]Sheu MT,Huang JC,Yeh GC,et al.Characterization of collagen gel solutions and collagen matrices for cell culture.Biomaterials.2001;22(13):1713-1719.
[27]Chen JT,Zhao YP,Xu Z,et al.Study on the FTIR of Recombinant Collagen and Bovine Type I Collagen.Chin J Mater Res.2008;22(3):119-121.
[28]蔡洁.新型交联剂的合成及交联改性胶原的研究[D].广州:华南理工大学,2014.
[29]程玮璐,李慧,贺金梅,等.可降解复合止血胶原蛋白海绵的制备及生物应用性能研究[J].化学与黏合,2016,38(4):231-234.
[30]GB/T16886.1-2011医疗器械风险管理过程中的评价与实验.
[31]Zhao L,Xian L,Zhao J,et al.A novel smart injectable hydrogel prepared by microbial transglutaminase and human-like collagen:Its characterization and biocompatibility.Mater Sci Eng C.2016;68:317-326.
[32]曹谊林,刘伟,张文杰,等.组织工程研究进展[J].上海交通大学学报(医学版),2012,32(9):1241-1250.
[33]邹勇,刘文杰.胶原在止血材料中的应用研究进展[J].明胶科学与技术,2010,30(1):1-4.
[34]Jayalakshmi K,Pasiyappazham R,Vairamani S,et al.Isolation and partial characterization of collagen from outer skin of Sepia pharaonis(Ehrenberg,1831)from Puducherry coast.Biochem Biophys Rep.2017;10:39-45.
[35]Chan EC,Peshavariya HM,Liu GS,et al.Nox4 modulates collagen production stimulated by transforming growth factorβ1 in vivo and in vitro.Biochem Biophys Res Commun.2013;430(3):918-925.
[36]刘新华,但年华,李正军,等.胶原基复合医用海绵的研究进展[J].西部皮革,2012,35(8):13-18.
[37]Si NP,Hye JL,Kwang HL,et al.Biological characterization of EDC-crosslinked collagen-hyaluronic acid matrix in dermal tissue restoration.Biomaterials.2003;24:1631-1641.
[38]Mirazul I,Vytautas C,Chaoliang,et al,Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels.Acta Biomaterialia.2015;12:70-80.
[39]Fagerholm P,Lagali NS,Ong JA,et al.Stable corneal regeneration four years after implantation of a cell-free recombinant human collagen scaffold.Biomaterials.2014;35(8):2420-2427.
[40]刘文广,May Griffith,Frank Li.基于重组人胶原构建角膜替代物[J].高分子通报,2010,23(8):000001-6.
[41]马慧军,贾赤宇,魏蓉,等.胶原蛋白凝胶促进皮肤创面修复的临床研究[J].中国美容医学,2011,20(2):235-236.