纳米羟基磷灰石/胶原/聚乳酸新型可吸收材料的短期生物安全性
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摘要
背景:课题组前期制备出具有良好生物活性的纳米羟基磷灰石/胶原/聚乳酸[nano-hydroxyapatite/collagen/poly(L-lactic)acid,n HAC/PLA]复合材料。目的:评估纳米羟基磷灰石/胶原/聚乳酸材料的生物安全性。方法:取含聚乳酸分别为100,150,200g/L的n HAC/PLA材料,制备浸提液,分别记为n HAC/PLA1、nHAC/PLA2、n HAC/PLA3。①细胞毒性实验:采用n HAC/PLA1、n HAC/PLA2、nHAC/PLA3浸提液、细胞培养基培养L929细胞,采用MTT比色法评价材料的细胞毒性;②急性全身毒性实验:取C57小鼠(郑州大学动物实验中心提供),尾静脉分别注射n HAC/PLA1、n HAC/PLA2、n HAC/PLA3浸提液与生理盐水,注射后24,48,72 h,观察动物一般情况及体质量变化;③血清毒性实验:取SD大鼠(郑州大学动物实验中心提供),分别胃饲n HAC/PLA1、n HAC/PLA2、n HAC/PLA3浸提液与生理盐水,7 d后,采用酶联免疫法检测大鼠血清中白细胞介素1蛋白水平;④致敏实验:将25只白色豚鼠(郑州大学动物实验中心提供)随机分为5组:实验组采用n HAC/PLA1、n HAC/PLA2、n HAC/PLA3浸提液,阴性对照组采用生理盐水,阳性对照组采用体积分数5%甲醛溶液,经过皮内诱导、局部诱导和激发阶段后24,48,72 h,观察各组激发部位皮肤情况;⑤溶血实验:向nHAC/PLA1、nHAC/PLA2、nHAC/PLA3浸提液、生理盐水、蒸馏水中分别加入兔抗凝血,检测溶血率。实验方案经郑州大学伦理委员会审查批准。结果与结论:纳米羟基磷灰石/胶原/聚乳酸材料的体外细胞毒性为0级,急性全身毒性、血清毒性、致敏性和溶血实验结果均为阴性,表明纳米羟基磷灰石/胶原/聚乳酸材料均具有良好的生物安全性。
BACKGROUND: A novel nano-hydroxyapatite/collagen/poly(lactic acid) composite was manufactured in our preliminary experiments.OBJECTIVE: To evaluate the biosafety of nano-hydroxyapatite/collagen/poly(lactic acid) composite.METHODS: Nano-hydroxyapatite/collagen/poly(lactic acid) composite was prepared into three kinds of leaching liquor containing 100, 150 and 200 g/L poly(lactic acid), namely, nHAC/PLA1, nHAC/PLA2, nHAC/PLA3.(1) Cytotoxicity test: L929 cells were cultured with nHAC/PLA1,nHAC/PLA2, nHAC/PLA3 leaching liquor and culture medium and cell toxicity was evaluated using MTT assay.(2) Acute systemic toxicity test:nHAC/PLA1, nHAC/PLA2, nHAC/PLA3 leaching liquor and normal saline were injected into C57 mice(Laboratory Animal Center of Zhengzhou University, China) via the tail vein. At 24, 48, 72 hours after injection, the general situation of mice was observed and body weight was recorded.(3) Serum toxicity test: SD rats(Laboratory Animal Center of Zhengzhou University) were intragastrically administered nHAC/PLA1, nHAC/PLA2, nHAC/PLA3 leaching liquor and normal saline. Seven days later, serum level of interleukin-1 protein was detected by enzyme-linked immunosorbent assay.(4) Skin sensitization test: 25 guinea pigs(Laboratory Animal Center of Zhengzhou University) were randomly treated with nHAC/PLA1, nHAC/PLA2, nHAC/PLA3 leaching liquor(experimental groups), normal saline(negative control group),and 5% formaldehyde(positive control group). At 24, 48 and 72 hours after intracutaneous induction, local induction, and provocation, skin sensitization reactions, including erythema and edema, were observed in each group.(5) Hemolysis test: anti-rabbit antibodies were added into the leaching liquor of test samples, saline and distilled water to determine the rate of hemolysis. The study was approved by Ethics Committee of Zhengzhou University, China.RESULTS AND CONCLUSION: The in vitro cytotoxicity of nano-hydroxyapatite/collagen/poly(lactic acid) composite was graded 0. Negative results were obtained in acute systemic toxicity test, serum toxicity test, skin sensitization test, acute hemolytic test, and hemolysis test of nano-hydroxyapatite/collagen/poly(lactic acid) composite. These results suggest good biosafety of nano-hydroxyapatite/collagen/poly(lactic acid) composite.
BACKGROUND: A novel nano-hydroxyapatite/collagen/poly(lactic acid) composite was manufactured in our preliminary experiments.OBJECTIVE: To evaluate the biosafety of nano-hydroxyapatite/collagen/poly(lactic acid) composite.METHODS: Nano-hydroxyapatite/collagen/poly(lactic acid) composite was prepared into three kinds of leaching liquor containing 100, 150 and 200 g/L poly(lactic acid), namely, nHAC/PLA1, nHAC/PLA2, nHAC/PLA3.(1) Cytotoxicity test: L929 cells were cultured with nHAC/PLA1,nHAC/PLA2, nHAC/PLA3 leaching liquor and culture medium and cell toxicity was evaluated using MTT assay.(2) Acute systemic toxicity test:nHAC/PLA1, nHAC/PLA2, nHAC/PLA3 leaching liquor and normal saline were injected into C57 mice(Laboratory Animal Center of Zhengzhou University, China) via the tail vein. At 24, 48, 72 hours after injection, the general situation of mice was observed and body weight was recorded.(3) Serum toxicity test: SD rats(Laboratory Animal Center of Zhengzhou University) were intragastrically administered nHAC/PLA1, nHAC/PLA2, nHAC/PLA3 leaching liquor and normal saline. Seven days later, serum level of interleukin-1 protein was detected by enzyme-linked immunosorbent assay.(4) Skin sensitization test: 25 guinea pigs(Laboratory Animal Center of Zhengzhou University) were randomly treated with nHAC/PLA1, nHAC/PLA2, nHAC/PLA3 leaching liquor(experimental groups), normal saline(negative control group),and 5% formaldehyde(positive control group). At 24, 48 and 72 hours after intracutaneous induction, local induction, and provocation, skin sensitization reactions, including erythema and edema, were observed in each group.(5) Hemolysis test: anti-rabbit antibodies were added into the leaching liquor of test samples, saline and distilled water to determine the rate of hemolysis. The study was approved by Ethics Committee of Zhengzhou University, China.RESULTS AND CONCLUSION: The in vitro cytotoxicity of nano-hydroxyapatite/collagen/poly(lactic acid) composite was graded 0. Negative results were obtained in acute systemic toxicity test, serum toxicity test, skin sensitization test, acute hemolytic test, and hemolysis test of nano-hydroxyapatite/collagen/poly(lactic acid) composite. These results suggest good biosafety of nano-hydroxyapatite/collagen/poly(lactic acid) composite.
引文
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[3]Hanna LA,Basalious EB,Elgazayerly ON.Respirable controlled release polymeric colloid(RCRPC)of bosentan for the management of pulmonary hypertension:in vitro aerosolization,histological examination and in vivo pulmonary absorption.Drug Deliv.2017;24(1):188-198.
[4]章越,丁陈陈,温露,等.核壳型聚乳酸-羟基乙酸共聚物磁性纳米系统用于中药复方多组分的时空递释[J].药学学报,2018,53(12):1968-1975.
[5]Libonati F,Nair AK,Vergani L,et al.Fracture mechanics of hydroxyapatite single crystalsunder geometric confinement.JMech Behav Biomed Mater.2013;20:184-191.
[6]Venkatesan J,Kim SK.Nano-Hydroxyapatite Composite Biomaterials for Bone TissueEngineering-A Review.J Biomed Nanotechnol.2014;10(10):3124-3140.
[7]Liao SS,Cui FZ,Zhang W,et al.Hierarchically biomimetic bone scaffold materials:Nano-HA/collagen/PLA composite.JBiomed Mater Res B Appl Biomater.2004;69(2):158-165.
[8]Bhuiyan DB,Middleton JC,Tannenbaum R,et al.Bone regeneration from human mesenchymal stem cells on porous hydroxyapatite-PLGA-collagen bioactive polymer scaffolds.Biomed Mater Eng.2017;28(6):671-685.
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[11]SO 10993-12:2012 Biological evaluation of medical device,Part 12:Sample preparation and reference materials.
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[14]ISO 10993-11:2010 Biological evaluation of medical device,Part 11:Tests for systemic toxicity.
[15]沈晴昳,李国强,李恺.新型复合磷酸钙镁基水门汀的生物相容性[J].中国组织工程研究,2018,22(2):241-247.
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[17]ISO/TR 7405-1984(E).Haemolysis test(in vitro test directly on materials).
[18]Yeon YK,Park HS,Lee JM,et al.New concept of 3D printed bone clip(polylactic acid/hydroxyapatite/silk composite)for internal fixation of bone fractures.J Biomater Sci Polym Ed.2018;29(7-9):894-906.
[19]Wang X,Zhang GL,Qi F,et al.Enhanced bone regeneration using an insulin-loaded nano-hydroxyapatite/collagen/PLGAcomposite scaffold.Int J Nanomedicine.2017;13:117-127.
[20]史建国,王卫宁,李春吾,等.脂肪干细胞复合生物可降解材料体外构建组织工程尿道支架[J].中国组织工程研究,2019,23(13):1989-1994.
[21]Chienb BW,Ibrahim NA,Wan MZWY,et al.Plasticized poly(lactic acid)with low molecular weight poly(ethylene glycol):mechanical,thermal,and morphology properties.J Appl Polym Sci.2013;130(6):4576-4580.
[22]Rasal RM,Janorkar AV.Poly(lactic acid)modifications.Prog Polym Sci.2010;35(3):338-356.
[23]Lee BK,Yun Y,Park K.PLA micro-and nano-particles.Adv Drug Deliv Rev.2016;107(15):176-191.
[24]Du C,Cui FZ,Zhang W,et al.Formation of calcium phosphate/collagen composites through mineralization of collagen matrix.J Biomed Mater Res.2000;50(4):518-527.
[25]Hao HP.Biological Evaluation of Medical Devices Implementation Guide.Beijing:Standards Press of China,2000.
[26]De SC,Hebling J,Scheffel DL,et al.Methods to evaluate and strategies to improve the biocompatibility of dental materials and operative techniques.Dent Mater.2014;30(7):769-784.
[27]Murni NS,Dambatta MS,Yeap SK,et al.Cytotoxicity evaluation of biodegradable Zn-3Mg alloy toward normal human osteoblast cells.Mater Sci Eng C Mater Biol Appl.2015;49:560-566.
[28]Cui X,Liang T,Liu C,et al.Correlation of particle properties with cytotoxicity and cellular uptake of hydroxyapatite nanoparticles in human gastric cancer cells.Mater Sci Eng CMater Biol Appl.2016;67:453-460.
[29]Ong THD,Yu N,Meenashisundaram GK,et al.Insight into cytotoxicity of Mg nanocomposites using MTT assay technique.Mater Sci Eng C Mater Biol Appl.2017;78:647-652.
[30]Heaney ML,Golde DW.Soluble cytokine receptors.Blood.1996;87(3):847-857.
[2]Félix Lanao RP,Jonker AM,Wolke JG,et al.Physicochemical properties and applications of poly(lactic-co-glycolic acid)for use in bone regeneration.Tissue Eng Part B Rev.2013;19(4):380-390.
[3]Hanna LA,Basalious EB,Elgazayerly ON.Respirable controlled release polymeric colloid(RCRPC)of bosentan for the management of pulmonary hypertension:in vitro aerosolization,histological examination and in vivo pulmonary absorption.Drug Deliv.2017;24(1):188-198.
[4]章越,丁陈陈,温露,等.核壳型聚乳酸-羟基乙酸共聚物磁性纳米系统用于中药复方多组分的时空递释[J].药学学报,2018,53(12):1968-1975.
[5]Libonati F,Nair AK,Vergani L,et al.Fracture mechanics of hydroxyapatite single crystalsunder geometric confinement.JMech Behav Biomed Mater.2013;20:184-191.
[6]Venkatesan J,Kim SK.Nano-Hydroxyapatite Composite Biomaterials for Bone TissueEngineering-A Review.J Biomed Nanotechnol.2014;10(10):3124-3140.
[7]Liao SS,Cui FZ,Zhang W,et al.Hierarchically biomimetic bone scaffold materials:Nano-HA/collagen/PLA composite.JBiomed Mater Res B Appl Biomater.2004;69(2):158-165.
[8]Bhuiyan DB,Middleton JC,Tannenbaum R,et al.Bone regeneration from human mesenchymal stem cells on porous hydroxyapatite-PLGA-collagen bioactive polymer scaffolds.Biomed Mater Eng.2017;28(6):671-685.
[9]ISO 10993-5:2014 Biological evaluation of medical devices,Part 5:Tests for in vitro cytotoxicity.
[10]ISO 10993-1:2010 Biological evaluation of medical device,Part 1:Guidance on selection of tests.
[11]SO 10993-12:2012 Biological evaluation of medical device,Part 12:Sample preparation and reference materials.
[12]杨杨,季娟娟,彭蓓,等.MTT法评价4种铸造合金的细胞毒性研究[J].临床口腔医学杂志,2008,24(11):649-651.
[13]Nakamura M,Kawahara H,Kataoka Y,et al.Biocompatibility of dental amalgams in vitro during 52 week period.Shika Rikōgaku Zasshi.1980;21(55):228-244.
[14]ISO 10993-11:2010 Biological evaluation of medical device,Part 11:Tests for systemic toxicity.
[15]沈晴昳,李国强,李恺.新型复合磷酸钙镁基水门汀的生物相容性[J].中国组织工程研究,2018,22(2):241-247.
[16]张桥.卫生毒理性研究[M].北京:人民卫生出版社,2001:236-246.
[17]ISO/TR 7405-1984(E).Haemolysis test(in vitro test directly on materials).
[18]Yeon YK,Park HS,Lee JM,et al.New concept of 3D printed bone clip(polylactic acid/hydroxyapatite/silk composite)for internal fixation of bone fractures.J Biomater Sci Polym Ed.2018;29(7-9):894-906.
[19]Wang X,Zhang GL,Qi F,et al.Enhanced bone regeneration using an insulin-loaded nano-hydroxyapatite/collagen/PLGAcomposite scaffold.Int J Nanomedicine.2017;13:117-127.
[20]史建国,王卫宁,李春吾,等.脂肪干细胞复合生物可降解材料体外构建组织工程尿道支架[J].中国组织工程研究,2019,23(13):1989-1994.
[21]Chienb BW,Ibrahim NA,Wan MZWY,et al.Plasticized poly(lactic acid)with low molecular weight poly(ethylene glycol):mechanical,thermal,and morphology properties.J Appl Polym Sci.2013;130(6):4576-4580.
[22]Rasal RM,Janorkar AV.Poly(lactic acid)modifications.Prog Polym Sci.2010;35(3):338-356.
[23]Lee BK,Yun Y,Park K.PLA micro-and nano-particles.Adv Drug Deliv Rev.2016;107(15):176-191.
[24]Du C,Cui FZ,Zhang W,et al.Formation of calcium phosphate/collagen composites through mineralization of collagen matrix.J Biomed Mater Res.2000;50(4):518-527.
[25]Hao HP.Biological Evaluation of Medical Devices Implementation Guide.Beijing:Standards Press of China,2000.
[26]De SC,Hebling J,Scheffel DL,et al.Methods to evaluate and strategies to improve the biocompatibility of dental materials and operative techniques.Dent Mater.2014;30(7):769-784.
[27]Murni NS,Dambatta MS,Yeap SK,et al.Cytotoxicity evaluation of biodegradable Zn-3Mg alloy toward normal human osteoblast cells.Mater Sci Eng C Mater Biol Appl.2015;49:560-566.
[28]Cui X,Liang T,Liu C,et al.Correlation of particle properties with cytotoxicity and cellular uptake of hydroxyapatite nanoparticles in human gastric cancer cells.Mater Sci Eng CMater Biol Appl.2016;67:453-460.
[29]Ong THD,Yu N,Meenashisundaram GK,et al.Insight into cytotoxicity of Mg nanocomposites using MTT assay technique.Mater Sci Eng C Mater Biol Appl.2017;78:647-652.
[30]Heaney ML,Golde DW.Soluble cytokine receptors.Blood.1996;87(3):847-857.