不同粒径重组人骨形态发生蛋白2/聚乳酸-聚羟基乙酸共聚物微球制备及体内外释放性能比较
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摘要
背景:应用生物可降解材料包载骨细胞生长因子制成微球缓释的技术,为生长因子的高效利用提供了可行性。目的:制备重组人骨形态发生蛋白2/聚乳酸-聚羟基乙酸共聚物纳米和微米微球,通过体内外释放实验比较两种微球的释放行为差异。方法:通过控制匀浆速度,采用乳化-溶剂挥发法制备重组人骨形态发生蛋白2/聚乳酸-聚羟基乙酸共聚物缓释纳米和微米微球。(1)体外缓释实验:将两种微球分别溶于PBS中70 d,采用ELISA法检测不同时间点上清液中重组人骨形态发生蛋白2的浓度;(2)体内缓释实验:将44只新西兰大白兔分为2组,分别在股骨转子缺损处植入重组人骨形态发生蛋白2/聚乳酸-聚羟基乙酸共聚物缓释纳米和微米微球,植入70 d内采用ELISA法检测股骨转子缺损处重组人骨形态发生蛋白2的浓度。结果与结论:(1)体外缓释实验:纳米微球前3 d存在突释情况,累计释放将近达到41%,随后呈持续平稳缓慢释放,70 d时达到83%左右;微米微球突释情况较纳米微球小,前3 d累计释放约为20%且持续释放时间长,70 d累计释放约为70%;(2)体内缓释实验:纳米微球前3 d存在突释情况,累计释放将近35%,随后呈持续平稳缓慢释放,70 d时达到72%左右;微米微球突释情况较纳米微球小,前3 d累计释放约为21%且持续释放时间长,70 d累计释放约为63%左右;(3)结果表明:重组人骨形态发生蛋白2/聚乳酸-聚羟基乙酸共聚物微米微球缓释释放时间与骨生长周期相适宜,更有利于临床治疗骨缺损修复。
BACKGROUND: The technology of biodegradable materials covering growth factors can be used to make sustained-release microspheres, which provides the feasibility for the efficient utilization of growth factors. OBJECTIVE: To prepare nano/micron-sized spheres using recombinant human bone morphogenetic protein 2/poly(lactic-co-glycolic acid)(rh BMP-2/PLGA) copolymer and to compare their release behaviors by in vivo and in vitro release experiments. METHODS: The rh BMP-2/PLGA nano/micron-sized spheres were prepared by emulsion solvent evaporation method to control the rate of pulp mixing.(1) In vitro release experiment: Prepared nano/micron-sized spheres were dissolved in PBS for 70 days, and then ELISA method was used to detect the rh BMP-2 concentration in the supernatant at different time.(2) In vivo release experiment: Forty-four New Zealand rabbits were divided into two groups, and rh BMP-2/PLGA nano/micron-sized spheres were respectively implanted into trochanteric defects. The concentration of rh BMP-2 in the defect site was detected by ELISA within 70 days after implantation. RESULTS AND CONCLUSION: In vitro sustained release experiment: There was a sudden release of nanospheres in the former 3 days, and the cumulative release nearly reached 41%, followed by a steady and slow release, and then the cumulative release was up to approximately 83% at 70 days. The initial release of micron-sized spheres was less than that of nanospheres, and the cumulative release was about 20% within the former 3 days and reached to 70% at 70 days. In vivo sustained release test: There was a sudden release of the nanospheres, the cumulative release was nearly 35%, followed by a steady and slow release, and then the cumulative release was up to approximately 72% at 70 days. The initial release of micron-sized spheres was less than that of nanospheres, and the cumulative release was about 21% in the former 3 days and increased to about 63% at 70 days. In both in vivo and in vitro release experiments, the release duration of micron-sized spheres was longer than that of nanospheres in the former 3 days. To conclude, the release time of rh BMP-2/PLGA micron-sized spheres fulfills the need of bone growth cycle, therefore, rh BMP-2/PLGA micron-sized spheres are more favorable than rh BMP-2/PLGA nanospheres for bone defect repair in clinical practice.
BACKGROUND: The technology of biodegradable materials covering growth factors can be used to make sustained-release microspheres, which provides the feasibility for the efficient utilization of growth factors. OBJECTIVE: To prepare nano/micron-sized spheres using recombinant human bone morphogenetic protein 2/poly(lactic-co-glycolic acid)(rh BMP-2/PLGA) copolymer and to compare their release behaviors by in vivo and in vitro release experiments. METHODS: The rh BMP-2/PLGA nano/micron-sized spheres were prepared by emulsion solvent evaporation method to control the rate of pulp mixing.(1) In vitro release experiment: Prepared nano/micron-sized spheres were dissolved in PBS for 70 days, and then ELISA method was used to detect the rh BMP-2 concentration in the supernatant at different time.(2) In vivo release experiment: Forty-four New Zealand rabbits were divided into two groups, and rh BMP-2/PLGA nano/micron-sized spheres were respectively implanted into trochanteric defects. The concentration of rh BMP-2 in the defect site was detected by ELISA within 70 days after implantation. RESULTS AND CONCLUSION: In vitro sustained release experiment: There was a sudden release of nanospheres in the former 3 days, and the cumulative release nearly reached 41%, followed by a steady and slow release, and then the cumulative release was up to approximately 83% at 70 days. The initial release of micron-sized spheres was less than that of nanospheres, and the cumulative release was about 20% within the former 3 days and reached to 70% at 70 days. In vivo sustained release test: There was a sudden release of the nanospheres, the cumulative release was nearly 35%, followed by a steady and slow release, and then the cumulative release was up to approximately 72% at 70 days. The initial release of micron-sized spheres was less than that of nanospheres, and the cumulative release was about 21% in the former 3 days and increased to about 63% at 70 days. In both in vivo and in vitro release experiments, the release duration of micron-sized spheres was longer than that of nanospheres in the former 3 days. To conclude, the release time of rh BMP-2/PLGA micron-sized spheres fulfills the need of bone growth cycle, therefore, rh BMP-2/PLGA micron-sized spheres are more favorable than rh BMP-2/PLGA nanospheres for bone defect repair in clinical practice.
引文
[1]卢晓郎,郑亦静,程涛.等.骨形态发生蛋白缓释微球结合富血小板凝胶对骨髓基质干细胞增殖分化的影响[J].中国组织工程研究,2016,20(47):7057-7063.
[2]Marsell R,Einhorn TA.The biology of fracture healing.Injury,2015;42(6):551-555.
[3]Knight DA,Rossi FM,Hackett TL.Mesenchymal stem cells for repair of the airway epithelium in asthma.Expert Rev Respir Med.2010;4(6):747-758.
[4]丁琛,刘浩.缓释重组人骨形态发生蛋白2仿生纳米纤维肝素/明胶复合支架的制备及表征[J].中国组织工程研究,2016,20(10):2384-2390.
[5]Haider A,Kim S,Huh MW,et al.BMP-2 Grafted n HA/PLGA Hybrid Nanofiber Scaffold Stimulates Osteoblastic Cells Growth.Biomed Res Int.2015;28(3):1909-1911.
[6]刘峰,孙健,李亚莉,等.骨形态发生蛋白2壳聚糖纳米缓释载体的制备及性能测定[J].上海口腔医学,2015,24(2):395-400.
[7]唐智明,熊龙,曾建华,等.中空羟基磷灰石复合重组人骨形态发生蛋白2微球体修复骨缺损[J].中国组织工程研究,2017,21(2):177-181.
[8]向柄彦,郭元.载药微球联合BMP-2治疗兔慢性骨髓炎的实验研究[J].重庆医学,2015,44(8):1032-1036
[9]巴格那,陈华荣,李婷,等.注射型纳米壳聚糖骨形态发生蛋白2复合体诱导牙周组织的再生[J].中国组织工程研究,2015,19(38):6184-6190.
[10]Zhao W,Li J,Jin K.Fabrication of functional PLGA-based electros pun scaffolds and their applications in biomedical engineering Mater.Sci Eng C Mater Biol Appl.2016;59:1181-1194.
[11]罗菲,卢来春,张纲.rh BMP-2-m PEG-PLA纳米缓释微球缓释凝胶的制备及体外释放实验研究[J].口腔医学,2014,31(1):35-38.
[12]费正奇,胡蕴玉,吴道澄,等.rh BMP-2/聚乳酸与聚乙醇酸共聚物载药微球的制备及成骨活性研究[J].生物医学工程与临床,2006,10(3):121-125.
[13]Cui HX,Guo J,Han Z.Biocompatibility of differently proportioned HA/PLGA/BMP-2 composite biomaterials in rabbits.Genet Mol Res.2015;14(4):13511-13518.
[14]安森博,汪龙,胡懿郃.聚乳酸-羟基乙酸共聚物载药微球性质及缓释行为的影响因素[J].中国医院药学杂志2016,36(13):1140-1144.
[15]周庆梅,孙健,李亚莉,等.生长因子缓释系统复合纳米支架料修复犬下颌骨缺损[J].中国组织工程研究,2013,17(21):3815-3822.
[16]黄鑫,孟国林,刘建,等.rh BMP-2壳聚糖微球的制备及体外检测[J].中国矫形外科杂志,2009,17(15):1172-1174.
[17]Wen J,Kim GJ,Leong KW.Poly(D,L-lactide-co-ethyl ethylene phosphate)s as new drug carriers.J Control Release.2003;92(1-2):39-48.
[18]Chen L,Liu L,Li C,et al.A new growth factor controlled drug release system to promote healing of bone fractures:nanospheres of recombinant human bone morphogenetic-2 and polylacticacid.J Nanosci Nanotechnol.2011;11(4):3107-3114.
[19]Carreira AC,Lojudice FH,Halcsik E,et al.Bone morphogenetic proteins:facts,challenges,and future perspectives.J Dent Res.2014;93(4):335-345.
[20]Ji Y,Xu GP,Zhang ZP.BMP-2/PLGA delayed-release microspheres composite graft,selection of bone particulate diameters,and prevention of aseptic inflammation for bone tissue engineering.Ann Biomed Eng.2010;38(3):632-639.
[2]Marsell R,Einhorn TA.The biology of fracture healing.Injury,2015;42(6):551-555.
[3]Knight DA,Rossi FM,Hackett TL.Mesenchymal stem cells for repair of the airway epithelium in asthma.Expert Rev Respir Med.2010;4(6):747-758.
[4]丁琛,刘浩.缓释重组人骨形态发生蛋白2仿生纳米纤维肝素/明胶复合支架的制备及表征[J].中国组织工程研究,2016,20(10):2384-2390.
[5]Haider A,Kim S,Huh MW,et al.BMP-2 Grafted n HA/PLGA Hybrid Nanofiber Scaffold Stimulates Osteoblastic Cells Growth.Biomed Res Int.2015;28(3):1909-1911.
[6]刘峰,孙健,李亚莉,等.骨形态发生蛋白2壳聚糖纳米缓释载体的制备及性能测定[J].上海口腔医学,2015,24(2):395-400.
[7]唐智明,熊龙,曾建华,等.中空羟基磷灰石复合重组人骨形态发生蛋白2微球体修复骨缺损[J].中国组织工程研究,2017,21(2):177-181.
[8]向柄彦,郭元.载药微球联合BMP-2治疗兔慢性骨髓炎的实验研究[J].重庆医学,2015,44(8):1032-1036
[9]巴格那,陈华荣,李婷,等.注射型纳米壳聚糖骨形态发生蛋白2复合体诱导牙周组织的再生[J].中国组织工程研究,2015,19(38):6184-6190.
[10]Zhao W,Li J,Jin K.Fabrication of functional PLGA-based electros pun scaffolds and their applications in biomedical engineering Mater.Sci Eng C Mater Biol Appl.2016;59:1181-1194.
[11]罗菲,卢来春,张纲.rh BMP-2-m PEG-PLA纳米缓释微球缓释凝胶的制备及体外释放实验研究[J].口腔医学,2014,31(1):35-38.
[12]费正奇,胡蕴玉,吴道澄,等.rh BMP-2/聚乳酸与聚乙醇酸共聚物载药微球的制备及成骨活性研究[J].生物医学工程与临床,2006,10(3):121-125.
[13]Cui HX,Guo J,Han Z.Biocompatibility of differently proportioned HA/PLGA/BMP-2 composite biomaterials in rabbits.Genet Mol Res.2015;14(4):13511-13518.
[14]安森博,汪龙,胡懿郃.聚乳酸-羟基乙酸共聚物载药微球性质及缓释行为的影响因素[J].中国医院药学杂志2016,36(13):1140-1144.
[15]周庆梅,孙健,李亚莉,等.生长因子缓释系统复合纳米支架料修复犬下颌骨缺损[J].中国组织工程研究,2013,17(21):3815-3822.
[16]黄鑫,孟国林,刘建,等.rh BMP-2壳聚糖微球的制备及体外检测[J].中国矫形外科杂志,2009,17(15):1172-1174.
[17]Wen J,Kim GJ,Leong KW.Poly(D,L-lactide-co-ethyl ethylene phosphate)s as new drug carriers.J Control Release.2003;92(1-2):39-48.
[18]Chen L,Liu L,Li C,et al.A new growth factor controlled drug release system to promote healing of bone fractures:nanospheres of recombinant human bone morphogenetic-2 and polylacticacid.J Nanosci Nanotechnol.2011;11(4):3107-3114.
[19]Carreira AC,Lojudice FH,Halcsik E,et al.Bone morphogenetic proteins:facts,challenges,and future perspectives.J Dent Res.2014;93(4):335-345.
[20]Ji Y,Xu GP,Zhang ZP.BMP-2/PLGA delayed-release microspheres composite graft,selection of bone particulate diameters,and prevention of aseptic inflammation for bone tissue engineering.Ann Biomed Eng.2010;38(3):632-639.