颌骨缺损修复用多孔镁合金支架材料的生物安全性评价研究
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摘要
背景:研究已证实部分生物医用镁合金产品生物安全性及其他优势性能,但在其合金成分基础上改变制作工艺及产品形态制得的多孔支架材料生物安全性尚不明确。目的:探讨生物医用多孔镁合金支架材料的生物安全性能。方法:制取多孔镁合金支架材料浸提液,参照GB/T16886医疗器械生物学评价系列标准,进行体外细胞毒性实验、体外溶血实验、皮内反应实验、热源实验、急性全身毒性实验及遗传毒性实验,评估多孔镁合金支架材料浸提液对人骨髓间充质干细胞活性及血细胞胞膜结构完整性的影响,对动物皮内组织、体温、体质量、嗜多染红细胞染色体结构的影响;制备动物下颌骨缺损模型,将多孔镁合金材料植入缺损区,评估其对动物血清及尿液Mg~(2+)浓度、血细胞数目和重要脏器等的影响。实验已通过北京大学医学部动物伦理委员会审核批准,批号为LA2017217。结果与结论:(1)多孔镁合金支架材料浸提液可促进骨髓间充质干细胞的增殖,细胞毒性为0级;(2)多孔镁合金支架材料浸提液的溶血率为0.94%,皮内刺激反应为0分,符合医疗器械溶血实验(<5%)及皮内反应实验(最终记分≤1.0)相关要求;(3)注射多孔镁合金浸提液后,所有动物体温升高均≤0.4℃,升高总度数≤0.8℃,符合医疗器械热源实验相关要求;(4)多孔镁合金浸提液未引起动物全身毒性反应,未破坏嗜多染红细胞染色体结构;(5)多孔镁合金支架材料植入动物骨缺损1个月内,动物血清及尿液Mg~(2+)浓度无明显变化,红细胞、白细胞、血小板处在正常范围,未引起心、肝、肾、脾组织明显的病理性改变;(6)结果表明,多孔镁合金支架材料具备优良的生物安全性能。
BACKGROUND: Biosafety and other advantages of biomedical magnesium alloy products have been confirmed, but the biosafety of porous scaffold materials prepared by changing the manufacturing process and product morphology based on the alloy composition is unclear.OBJECTIVE: To investigate the biosafety properties of a biomedical porous magnesium alloy scaffold.METHODS: Extracts of porous magnesium alloy scaffold materials were prepared, and in vitro cytotoxicity experiment, in vitro hemolysis experiment, intrauthelial reaction experiment, pyrogen experiment, acute systemic toxicity experiment and genetic toxicity experiment were conducted according to GB/T16886 biological evaluation series of medical devices. The effects of extracts of porous magnesium alloy scaffold materials on the activity of human bone marrow mesenchymal stem cells and the structural integrity of blood cell membrane, and the effects on the animal endothelial tissue, body temperature, body mass and chromosomal structure of polychromatic red blood cells were evaluated.Animal mandibular defect model was prepared, and porous magnesium alloy was implanted into the defect area to evaluate its influence on serum and urine Mg~(2+) concentration, blood cell number and important organs. The study was approved by the Experimental Animal Ethics Committee of Peking University Health Science Center(approval number: LA2017217).RESULTS AND CONCLUSION:(1) The extracts of porous magnesium alloy scaffolds could promote the proliferation of bone marrow mesenchymal stem cells, and the cytotoxicity was 0.(2) The hemolysis rate of the extracts of porous magnesium alloy scaffold material was0.94%, and the response to intradermal stimulation was 0, which met the requirements of medical device hemolysis experiment(< 5%) and intradermal reaction experiment(final score ≤ 1.0).(3) After the injection of porous magnesium alloy extract, the increased degree of body temperature was ≤ 0.4 oC and elevated total degree ≤ 0.8 oC in all animals, which conformed to the requirements of the medical equipment of heat experiment.(4) The porous magnesium alloy extract caused no systemic toxicity or damaged chromosomal structure of polychromatic erythrocytes.(5) Within 1 month after implantation of porous magnesium alloy scaffold material into the bone defect, there was no significant change in serum and urine Mg~(2+) concentration, the red blood cells, white blood cells and platelets were in the normal range, and no obvious pathological changes were found in the heart, liver, kidney and spleen tissues.(6) These results imply that porous magnesium alloy scaffold has excellent biosafety property.
BACKGROUND: Biosafety and other advantages of biomedical magnesium alloy products have been confirmed, but the biosafety of porous scaffold materials prepared by changing the manufacturing process and product morphology based on the alloy composition is unclear.OBJECTIVE: To investigate the biosafety properties of a biomedical porous magnesium alloy scaffold.METHODS: Extracts of porous magnesium alloy scaffold materials were prepared, and in vitro cytotoxicity experiment, in vitro hemolysis experiment, intrauthelial reaction experiment, pyrogen experiment, acute systemic toxicity experiment and genetic toxicity experiment were conducted according to GB/T16886 biological evaluation series of medical devices. The effects of extracts of porous magnesium alloy scaffold materials on the activity of human bone marrow mesenchymal stem cells and the structural integrity of blood cell membrane, and the effects on the animal endothelial tissue, body temperature, body mass and chromosomal structure of polychromatic red blood cells were evaluated.Animal mandibular defect model was prepared, and porous magnesium alloy was implanted into the defect area to evaluate its influence on serum and urine Mg~(2+) concentration, blood cell number and important organs. The study was approved by the Experimental Animal Ethics Committee of Peking University Health Science Center(approval number: LA2017217).RESULTS AND CONCLUSION:(1) The extracts of porous magnesium alloy scaffolds could promote the proliferation of bone marrow mesenchymal stem cells, and the cytotoxicity was 0.(2) The hemolysis rate of the extracts of porous magnesium alloy scaffold material was0.94%, and the response to intradermal stimulation was 0, which met the requirements of medical device hemolysis experiment(< 5%) and intradermal reaction experiment(final score ≤ 1.0).(3) After the injection of porous magnesium alloy extract, the increased degree of body temperature was ≤ 0.4 oC and elevated total degree ≤ 0.8 oC in all animals, which conformed to the requirements of the medical equipment of heat experiment.(4) The porous magnesium alloy extract caused no systemic toxicity or damaged chromosomal structure of polychromatic erythrocytes.(5) Within 1 month after implantation of porous magnesium alloy scaffold material into the bone defect, there was no significant change in serum and urine Mg~(2+) concentration, the red blood cells, white blood cells and platelets were in the normal range, and no obvious pathological changes were found in the heart, liver, kidney and spleen tissues.(6) These results imply that porous magnesium alloy scaffold has excellent biosafety property.
引文
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[22]朱兆金.骨科新型医用可降解植入材料JDBM镁合金的生物毒性、髓内针及植入物感染细菌生物膜的基础研究[D].苏州:苏州大学,2013.
[23]刘天甲,顾硕,周璐,等.可降解镁合金颅骨固定系统在动物实验中的安全性及有效性[J].中国组织工程研究,2018,22(30):4876-4881.
[24]Qin H,Zhao Y,An Z,et al.Enhanced antibacterial properties,biocompatibility,and corrosion resistance of degradable Mg-Nd-Zn-Zr alloy.Biomaterials.2015;53:211-220.
[25]邵惠锋,贺永,傅建中.增材制造可降解人工骨的研究进展-从外形定制到性能定制[J].浙江大学学报(工学版),2018,52(6):1035-1057.
[26]倪昕晔,熊信柏,尤瑞金,等.碳/碳复合材料表面掺镁羟基磷灰石生物涂层的体外性能[J].中国组织工程研究,2017,21(34):5443-5448.
[27]Zhao SF,Jiang QH,Peel S,et al.Effects of magnesium-substituted nanohydroxyapatite coating on implant osseointegration.Clin Oral Implants Res.2013;24Suppl A100:34-41.
[28]王平,张会敏,李刚.鹿角多肽对骨髓间充质干细胞的影响[J].中华中医药杂志,2018,33(12):5644-5647.
[29]Wang Y,Zhu Z,He Y,et al.In vivo degradation behavior and biocompatibility of Mg-Nd-Zn-Zr alloy at early stage.Int J Mol Med.2012;29(2):178-184.
[30]章晓波,毛琳,袁广银,等.心血管支架用Mg-Nd-Zn-Zr生物可降解镁合金的性能研究[J].稀有金属材料与工程,2013,42(6):1300-1305.
[31]洪岩松,杨柯,张广道,等.可降解镁合金的动物体内骨诱导作用[J].金属学报,2008,44(9):1035-1041.
[2]厉国定,胡晓亮,倪明,等.生物可吸收材料在骨折内固定治疗的临床研究[J].生物骨科材料与临床研究,2011,8(1):34-35,38.
[3]Niu J,Yuan G,Liao Y,et al.Enhanced biocorrosion resistance and biocompatibility of degradable Mg-Nd-Zn-Zr alloy by brushite coating.Mater Sci Eng C Mater Biol Appl.2013;33(8):4833-4841.
[4]Mao L,Shen L,Chen J,et al.Enhanced bioactivity of Mg-Nd-Zn-Zr alloy achieved with nanoscale MgF2 surface for vascular stent application.ACS Appl Mater Interfaces.2015;7(9):5320-5330.
[5]袁广银,章晓波,牛佳林,等.新型可降解生物医用镁合金JDBM的研究进展[J].中国有色金属学报,2011,21(10):2476-2488.
[6]Chen Y,Zhao S,Liu B,et al.Corrosion-controlling and osteo-compatible Mg ion-integrated phytic acid(Mg-PA)coating on magnesium substrate for biodegradable implants application.ACS Appl Mater Interfaces.2014;6(22):19531-19543.
[7]Wang Y,Ouyang Y,Pang X,et al.Effects of degradable MG-ND-ZN-ZR alloy on osteoblastic cell function.Int JImmunopathol Pharmacol.2012;25(3):597-606.
[8]Guan X,Xiong M,Zeng F,et al.Enhancement of osteogenesis and biodegradation control by brushite coating on Mg-Nd-Zn-Zr alloy for mandibular bone repair.ACS Appl Mater Interfaces.2014;6(23):21525-21533.
[9]Yoshizawa S,Brown A,Barchowsky A,et al.Role of magnesium ions on osteogenic response in bone marrow stromal cells.Connect Tissue Res.2014;55 Suppl 1:155-159.
[10]孔祥东,郝永强,王磊,等.新型医用可降解镁合金(JDBM)螺钉的生物安全性研究[J].组织工程与重建外科杂志,2015,11(3):124-127.
[11]Jia G,Hou Y,Chen C,et al.Precise fabrication of open porous Mg scaffolds using NaCl templates:Relationship between space holder particles,pore characteristics and mechanical behavior.Mater Design.2018;140:106-113.
[12]奚廷斐.医疗器械生物学评价标准[M].北京:中国标准出版社,2012.
[13]陈硕,蒋电明,何彬,等.新型自组装肽纳米纤维支架材料的生物安全性评价[J].中国现代医学杂志,2017,27(1):16-23.
[14]Yu Y,Lu H,Sun J.Long-term in vivo evolution of high-purity Mg screw degradation-Local and systemic effects of Mg degradation products.Acta Biomater.2018;71:215-224.
[15]Windhagen H,Radtke K,Weizbauer A,et al.Biodegradable magnesium-based screw clinically equivalent to titanium screw in hallux valgus surgery:short term results of the first prospective,randomized,controlled clinical pilot study.Biomed Eng Online.2013;12:62.
[16]Lee JW,Han HS,Han KJ,et al.Long-term clinical study and multiscale analysis of in vivo biodegradation mechanism of Mg alloy.Proc Natl Acad Sci U S A.2016;113(3):716-721.
[17]曹耿华,毕衍泽,陈民芳,等.骨折内固定用新型Mg-Zn-Ca/HA复合材料的成分、组织结构与性能[J].稀有金属材料与工程,2013,42(8):1632-1637.
[18]Jiang H,Wang J,Chen M,et al.Biological activity evaluation of magnesium fluoride coated Mg-Zn-Zr alloy in vivo.Mater Sci Eng C Mater Biol Appl.2017;75:1068-1074.
[19]曲立杰,李慕勤,刘苗,等.超声-微弧氧化医用镁合金体外降解性研究[Z].西安:96-100.
[20]Zhao C,Hou P,Ni J,et al.Ag-Incorporated FHA Coating on Pure Mg:Degradation and in Vitro Antibacterial Properties.ACS Appl Mater Interfaces.2016;8(8):5093-5103.
[21]马越,程翔,曹尤雅,等.牙科用微弧氧化Zr-Cu-Al-Ag合金的血液相容性评价[J].现代口腔医学杂志,2016,30(6):352-355.
[22]朱兆金.骨科新型医用可降解植入材料JDBM镁合金的生物毒性、髓内针及植入物感染细菌生物膜的基础研究[D].苏州:苏州大学,2013.
[23]刘天甲,顾硕,周璐,等.可降解镁合金颅骨固定系统在动物实验中的安全性及有效性[J].中国组织工程研究,2018,22(30):4876-4881.
[24]Qin H,Zhao Y,An Z,et al.Enhanced antibacterial properties,biocompatibility,and corrosion resistance of degradable Mg-Nd-Zn-Zr alloy.Biomaterials.2015;53:211-220.
[25]邵惠锋,贺永,傅建中.增材制造可降解人工骨的研究进展-从外形定制到性能定制[J].浙江大学学报(工学版),2018,52(6):1035-1057.
[26]倪昕晔,熊信柏,尤瑞金,等.碳/碳复合材料表面掺镁羟基磷灰石生物涂层的体外性能[J].中国组织工程研究,2017,21(34):5443-5448.
[27]Zhao SF,Jiang QH,Peel S,et al.Effects of magnesium-substituted nanohydroxyapatite coating on implant osseointegration.Clin Oral Implants Res.2013;24Suppl A100:34-41.
[28]王平,张会敏,李刚.鹿角多肽对骨髓间充质干细胞的影响[J].中华中医药杂志,2018,33(12):5644-5647.
[29]Wang Y,Zhu Z,He Y,et al.In vivo degradation behavior and biocompatibility of Mg-Nd-Zn-Zr alloy at early stage.Int J Mol Med.2012;29(2):178-184.
[30]章晓波,毛琳,袁广银,等.心血管支架用Mg-Nd-Zn-Zr生物可降解镁合金的性能研究[J].稀有金属材料与工程,2013,42(6):1300-1305.
[31]洪岩松,杨柯,张广道,等.可降解镁合金的动物体内骨诱导作用[J].金属学报,2008,44(9):1035-1041.
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