吉林大学学报(医学版)  2019, Vol. 45 Issue (02): 439-444     DOI: 10.13481/j.1671-587x.20190241

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赵一安, 杨舒婷, 李亚龙, 刘国民, 孙淑芬, 罗云纲
ZHAO Yian, YANG Shuting, LI Yalong, LIU Guomin, SUN Shufen, LUO Yungang
静电喷雾法制备聚乳酸-羟基乙酸共聚物/羟基磷灰石微载体的工艺及其效果评价
Preparation process of polylactic acid-glycolic acid copolymer/hydroxyapatite microcarrier by electrostatic spraying method and its effect evaluation
吉林大学学报(医学版), 2019, 45(02): 439-444
Journal of Jilin University (Medicine Edition), 2019, 45(02): 439-444
10.13481/j.1671-587x.20190241

文章历史

收稿日期: 2018-06-25
静电喷雾法制备聚乳酸-羟基乙酸共聚物/羟基磷灰石微载体的工艺及其效果评价
赵一安1 , 杨舒婷2 , 李亚龙3 , 刘国民3 , 孙淑芬4 , 罗云纲1     
1. 吉林大学第二医院口腔科, 吉林 长春 130041;
2. 吉林大学口腔医院修复科, 吉林 长春 130021;
3. 吉林大学第二医院骨科, 吉林 长春 130041;
4. 吉林大学口腔医院牙体牙髓科, 吉林 长春 130021
[摘要]: 目的: 探讨静电喷雾法制备聚乳酸-羟基乙酸共聚物/羟基磷灰石(PLGA/HA)微载体的工艺过程,阐明PLGA/HA微载体的优势表征。方法: 采用HA(20 nm,99.9%)和PLGA(LA/GA=50/50,Mw30k),利用静电喷雾法制备PLGA/HA微载体,观察不同HA质量浓度(1%、3%、5%)、不同电压(4.0、4.5、>5.0kV)对微载体形貌的影响,获得最佳制球参数。以PLGA微载体为PLGA组,PLGA+1% HA为1% PLGA/HA组,PLGA+3% HA为3% PLGA/HA组,PLGA+5% HA为5% PLGA/HA组,单纯细胞作为空白对照组。采用扫描电镜(SEM)、细胞增殖实验、细胞荧光染色实验和傅里叶红外光谱(FTIR)等检测PLGA/HA微载体表征。结果: SEM观察,PLGA/HA微载体颗粒均匀,均为椭圆形或圆形,无异常形态球,表面光滑,无锐利边缘,球体之间无粘连、无聚集,不同浓度配比微载体之间无明显差别。细胞增殖实验检测,黏附细胞数量5% PLGA/HA组> 3% PLGA/HA组> 1% PLGA/HA组> PLGA组>空白对照组(P < 0.05),细胞数量随HA浓度增加而升高,5% PLGA/HA组微载体对细胞亲和性最佳,微载体无细胞毒性。细胞荧光染色实验检测,MC3T3-E1细胞在微载体上黏附状态良好。FTIR检测显示HA特征吸收峰,表明复合微载体中含有PLGA与HA。结论: 成功利用静电喷雾技术建立PLGA/HA微载体的制备工艺,该方法操作简单便捷,制球效果优良,在骨组织工程方面有广泛应用前景。
关键词: 静电喷雾    微载体    聚乳酸-羟基乙酸共聚物    羟基磷灰石    骨组织工程    
Preparation process of polylactic acid-glycolic acid copolymer/hydroxyapatite microcarrier by electrostatic spraying method and its effect evaluation
ZHAO Yian1 , YANG Shuting2 , LI Yalong3 , LIU Guomin3 , SUN Shufen4 , LUO Yungang1     
1. Department of Stomatology, Second Hospital, Jilin University, Changchun 130041, China;
2. Department of Prosthodontics, Stomatology Hospital, Jilin University, Changchun 130021, China;
3. Department of Orthopedics, Second Hospital, Jilin University, Changchun 130041, China;
4. Department of Endodontics and Operative Dentistry, Stomatology Hospital, Jilin University, Changchun 130021, China
[ABSTRACT]: Objective: To investigate the preparation process of polylactic acid-glycolic acid copolymer/hydroxyapatite (PLGA/HA) microcarriers by electrostatic spraying method, and to elucidate the superiority of PLGA/HA microcarriers. Methods: The PLGA/HA microcarriers were prepared by electrostatic spraying method using nano-HA (20 nm, 99.9%) and PLGA (LA/GA=50/50, Mw30k), and the influence of different concentrations (1%, 3%, 5%) and different voltages (4.0, 4.5, > 5.0 kV) of HA in the morphology of the microcarriers was investigated and the best ball making parameters were obtained.The PLGA microcarriers were used as PLGA group, PLGA+1%HA as 1% PLGA/HA group, PLGA+3% HA as 3% PLGA/HA group, PLGA+5%HA as 5%PLGA/HA group, and the simple cells were used as blank control group.The characteristics of PLGA/HA microcarriers were detected by scanning electron microscope (SEM), cell proliferation test, cell fluorescence staining experiment, and Fourier transform infrared spectroscopy (FTIR). Results: The SEM results showed that the microcarrier particles were uniform, all of them were elliptical or circular, without abnormal shape spheres, with smooth surface, without sharp edges, adhesion between the spheres and aggregation, and there were no significant differences between the different concentrations of microcarriers.The cell proliferation test results showed that the order of adhesion cells was 5%PLGA/HA group > 3%PLGA/HA group > 1% PLGA/HA group > PLGA group > blank control group (P < 0.05); the number of cells was increased with the increasing of HA concentration; the microcarriers in 5%PLGA/HA group had the best cell affinity and the microcarriers had no cytotoxity.The cell fluorescence staining experiment showed that the MC3T3-E1 cells adhered well on the microcarriers.The FTIR analysis results showed that HA characteristic absorption peak was observed, indicating that the composite microcarrier contained PLGA and HA. Conclusion: The preparation process of PLGA/HA microcarriers is successfully established by electrostatic spraying method.The method is simple and convenient to operate, and has excellent ball-making effect.It has broadly application prospects in bone tissue engineering.
KEYWORDS: electrostatic spraying     microcarriers     polylactic acid-glycolic acid copolymer     hydroxyapatite     bone tissue engineering    

牙周疾病治疗后牙周组织的完全再生是一个尚未解决的问题[1]。目前临床所应用的牙周组织再生术中使用的骨组织支架材料大多有其局限性,如支架材料具有抗原性、降解速率难以调控和强度较差等,并不能完全满足临床需要[2-3]。以细胞微载体为骨组织工程支架材料是目前有关研究的重要方向[4]。微载体培养能够加速牙周膜基质细胞的增殖,使成骨相关基因表达上调和牙骨质蛋白23、骨涎蛋白、骨桥蛋白及骨膜素上调[5]。有研究[6]通过运用聚乳酸-羟基乙酸共聚物微球顺序释放骨生长肽(osteogenic growth peptide, OGP)和骨形态发生蛋白2(bone morphogenetic protein-2,BMP-2)模式来促进骨髓间充质干细胞(mesnchymal stem cells, BMSCs)的成骨分化。聚乳酸-羟基乙酸共聚物(polylactic-co-glycolic acid, PLGA)因其良好的生物相容性与降解性能可作为原料制备微载体,并取得较多研究进展[7]。但PLGA缺乏天然识别位点,既不能促进细胞附着,也不具备骨诱导能力[8]。羟基磷灰石(hydroxyapatite, HA)与人体骨组织结构相近,且具有优良的组织相容性和生物传导性,是理想的骨缺损修复材料[9],但其降解性能较差[10]。复合PLGA/HA体系可同时获得二者的优势性能,构建出兼备成骨活性与降解性能的载体材料。静电喷雾技术是一种利用电流体动力学原理将聚合物溶液或溶体制成微、纳米级粒子或纤维物质的方法[11]。沈文等[12]利用静电喷雾法制备聚乳酸(polylactic acid,PLA)负载阿维菌素(avermectin, AVM)的载药微球,粒径可达5~20 μm。本实验利用静电喷雾技术,通过调整电压、接受距离和推进速度等参数制备微载体。利用静电发生器在喷头与接收液之间形成高压静电场,工作液流经喷头时被带上电荷,在静电力与重力的作用下,液滴做定向运动,最终在接收液中固定。可以通过调节工作液的推进速度、喷头距离接收液面的高度和电压等控制微载体的形态、尺寸。该方法制备的微载体颗粒均匀一致、结构形态可控,还可避免传统方法如乳液溶剂挥发法[13]和喷雾干燥法[14]等大量使用有机溶剂和乳剂并难以去除及应用局限等问题,在药物缓释和骨组织工程支架等领域具有广泛的应用前景。本实验应用高压静电喷雾技术,以PLGA/HA为工作液、60%乙醇为接收液制备微载体,并观察该方法制备的微载体的特征,为细胞微载体构建提供参考。

1 材料与方法 1.1 主要试剂和仪器

N-甲基吡咯烷酮(NMPA,AR>99.0%)(上海阿拉丁生化科技股份有限公司),DMEM培养基(美国Hyclone公司),FBS血清(美国Gibco公司), PLGA(LA/GA=50/50,长春圣博玛生物材料有限公司),HA(20 nm,北京德科岛金科技有限公司), 无水乙醇(北京化工厂)。电子天平(福州华志科学仪器有限公司),磁力搅拌器和恒温干燥箱(上海精宏实验设备有限公司),静电发生器(上海孚蕊哲静电科技有限公司),CKX41SF型激光共聚焦显微镜(laser scanning confocal microscope, LSCM)(日本Olympus公司)。

1.2 PLGA/HA基液的配制

称取0.5 g PLGA混入10 mL NMPA溶剂中,配制质量浓度为5%的PLGA溶液,70℃水浴条件下,磁力搅拌12 h,加入质量浓度为3%的HA粉末,70℃水浴条件下,磁力搅拌12h,再次混匀。

1.3 PLGA/HA微载体制备

连接静电发生器与自动推进装置(图 1),将接收液置于喷头正下方,喷头距离接收液面12 cm,用5 mL注射器抽取配制好的工作液置于推进器中,选择27G针头(D=0.15 mm),调节参数,推进速度0.47 m·h-1, 电压4.5 kV,喷头距离接收液面12 cm,接入自动搅拌装置,制备同时搅拌接收液,使微载体体系分散。

图 1 静电喷雾法装置示意图 Fig. 1 Diagram of electrospinning device
1.4 PLGA/HA微载体表征

扫描电镜(scanning electron microscope, SEM)观察静电喷雾法制备的3% HA浓度的PLGA/HA复合微载体的表面形态, 鬼笔环肽(FITC Phalloidin)和4’, 6-二脒基-2-苯基吲哚(4’, 6-diamidino-2-phenylindole,DAPI)染色观察微载体表面MC3T3-E1细胞分布情况,傅里叶变换红外光谱仪(fourier transform infrared spectroscope, FTIR)对微载体进行定性分析。

1.5 MC3T3-E1细胞复苏和培养

取出-80℃冻存的MC3T3-E1细胞,快速置于37℃水浴,并不断摇动,使液体快速融化,1 min后取出,加入5 mL完全培养基,吹打均匀后,1500r·min-1离心5 min,吸除上清液,将细胞沉淀吹入完全培养基,于5%CO2、37℃培养箱中孵育,至细胞融合达80%。

1.6 细胞增殖实验检测微载体细胞亲和性和细胞毒性

以PLGA微载体为PLGA组,PGLA+1%HA为1%PGLA/HA组,PGLA+3%HA为3%PGLA/HA组, PGLA+5%HA为5%PGLA/HA组,单纯细胞为空白对照组。将各组微载体与MC3T3-E1细胞共培养。取各组适量微载体平铺覆盖12孔板板底70%~80%,每孔3复孔,并加入2×104个MC3T3-E1细胞,于培养箱中孵育,72 h后首次换液,之后每隔48 h换液1次。共培养4和7d后吸弃上清,PBS冲洗3次,加入无血清培养基和CCK-8溶液,培养箱孵育2h,吸取上清,转移至96孔板,酶标仪检测吸光度(A)值,A值与活细胞数量成正比,以A值代表细胞数量,取平均值,绘制柱状图。

1.7 荧光染色观察MC3T3-E1细胞在PLGA/HA微载体上的生长和分布

将各组微载体与MC3T3-E1细胞共培养至细胞融合80%,去除培养基,PBS冲洗,加入4%多聚甲醛固定10 min,再行PBS冲洗3次,分别进行FITC和4’, 6-DAPI染色,PBS冲洗3次,染色过程注意避光,LSCM观察MC3T3-E1细胞在PLGA/HA微载体上的生长状态和分布情况。

1.8 统计学分析

采用GraphPad Prism7统计软件进行统计学分析。各组细胞增殖实验中A值以x±s表示,多组间样本均数比较采用单因素方差分析。以P<0.05为差异有统计学意义。

2 结果 2.1 不同电压下微载体形貌

电压在4.0~4.5 kV范围内微载体形态趋于稳定,呈球形,粒径在4.5 kV时趋于稳定,电压大于5 kV时微球形态不规则。见表 1

表 1 不同电压下微载体的形貌 Tab. 1 Morphology of microcarriers under different voltages
HA
(η/%)
Voltage
(V/kV)
Particle size
(d/μm)
Microsphere appearance
3 4.0 200-350 Sphere
3 4.5 350-380 Sphere
3 >5.0 50-400 Irregular sphere/ Hemisphere
2.2 倒置显微镜下微载体形态

倒置显微镜下见微载体边缘光滑,大小形态一致,微载体之间无粘连,各组微载体形态表现未见差异。见图 2

A:1%PLGA/HA group; B:3%PLGA/HA group; C:5%PLGA/HA group. 图 2 倒置显微镜观察各组PLGA/HA微载体形态表现(Bar=200 μm) Fig. 2 Morphology of PLGA/HA microcarriers in various groups detected by inverted microscope(Bar=200 μm)
2.3 细胞增殖实验检测各组微载体的细胞亲和性和细胞毒性

PLGA组、1%PLGA/HA组、3%PLGA/HA组和5%PLGA/HA组细胞数量均高于空白对照组(P < 0.05), 随着HA质量浓度升高,微载体对细胞的促进增殖作用越明显。在培养7d时,5%PLGA/HA组细胞数量明显高于PLGA组(P < 0.01)。见图 3

图 3 各组细胞增殖实验结果 Fig. 3 Cell proliferation test results in various groups
2.4 荧光染色观察各组MC3T3-E1细胞生长

FITC与DAPI染色后细胞在PLGA/HA微载体上的生长状态与分布情况:蓝色荧光为DAPI染色细胞核,绿色荧光为FITC染色肌动蛋白微丝,MC3T3-E1细胞生长状态良好,均匀黏附分布在PLGA/HA微载体表面。3%PLGA/HA组和5%PLGA/HA组被染色细胞数量均高于1%PLGA/HA组。见图 4(插页五)。

A, B:1%PLGA/HA; CD:3%PLGA/HA; E, F:5%PLGA/HA; A, C, E:DAPI staining; BD, F:FITC staining. 图 4 LSCM观察各组MC3T3-E1细胞的生长(Bar=200 μm) Fig. 4 Growth of MC3T3-E1 cells in various groups observed by LSCM(Bar=200 μm)
2.5 SEM观察各组微载体表面形态和超微结构

SEM观察结果:微载体颗粒均匀,均为椭圆形或圆形,无异常形态球,表面光滑,无锐利边缘,球体之间无粘连、无聚集,各组微载体表面形态之间无明显差别。见图 5

A, B:1%PLGA/HA group; C, D:3%PLGA/HA group; E, F:5%PLGA/HA group; A, C, E:Bar=500 μm; B, D, F:Bar=100 μm. 图 5 SEM观察各组PLGA/HA微载体表面形态 Fig. 5 Surface morphology of PLGA/HA microcarriers in various groups observed by SEM
2.6 FTIR分析微载体的吸收峰

PLGA特征吸收位于2 990-1与2 940 cm-1(-CH3),1753 cm-1(C=O),1183与1083 cm-1(C-O)。在500~600 cm-1(P-O)处观察到HA特征吸收峰,且吸收峰高度5%PLGA/HA组>3%PLGA/HA组>1%PLGA/HA组, 表明HA混入PLGA溶液中, 制得PLGA/HA复合微载体。见图 6

The arrow denoted the characteristic absorption peak of HA. 图 6 各组PLGA/HA微载体FTIR分析结果 Fig. 6 FTIR analysis result of PLGA/HA microcarriers in various groups
3 讨论

PLGA的优异性能是其生物降解性可以通过改变乳酸(lactic acid, LA)和乙醇酸(glycolic acid, GA)的比例来控制[15],被广泛应用于医疗领域。微球的形状、直径、粒度分布、表面形貌和化学成分等可能影响细胞的黏附和增殖,对于微球在组织工程中的成功应用至关重要[16-17]。纳米HA在骨组织工程中的应用日渐成熟。研究[18]显示:PLGA和HA纳米复合材料在支架形式中有优异的生物降解性,而PLGA/HA微载体在体内也具有相似的生物降解性[19]。本实验采用静电喷雾技术制备PLGA/HA微载体,优化微载体制备工艺,相对于乳液溶剂挥发法和相分离法其操作更加简单便捷。该方法在高出球率的前提下获得表面均匀、大小一致的细胞微载体,对细胞微载体的制备工艺进行了改良。本研究结果显示:PLGA/HA微载体表面细胞生长及黏附状态良好,能够有效地支持细胞的黏附与扩增,具有细胞亲和力,无细胞毒性。PLGA/HA微载体相对于PLGA微载体对细胞的增殖促进作用更强,ZHANG等[20]在研究中也证实了PLGA/HA支架相对于PLGA支架有更优异的生物性能;随着HA浓度的增加。细胞增殖量也随之增加。HE等[21]也证实高浓度HA表现出比低浓度HA复合支架更好的生物相容性及骨形成,为后续研究更高HA浓度的微载体对细胞的增殖促进作用提供了研究思路。

与乳化溶剂挥发法和相分离法等比较,本实验应用静电喷雾法制备的微载体具有更多优势性能。陈红等[22]在对比实验中得出结论:乳化溶剂挥发法制得的微球分散性差,大小不均,粘连程度高。本实验所制得微载体分散、无粘连,且颗粒均匀一致。乳化溶剂挥发法制得的微球不利于细胞黏附,油相与水相比也会影响微球的粒径[23]。本实验证实静电喷雾法制得微球粒径均匀一致,细胞黏附状态良好。喷雾干燥法在高温条件下制球,在材料选择上有较多局限,而且不利于加入生物活性物质[24]。静电喷雾法制球条件温和,适于添加生物活性物质,制球材料选择更为广泛。

综上所述,本实验成功运用静电喷雾法制得PLGA/HA微载体并进行测试,可作为后续实施体内实验的基础。本研究结果为细胞微载体相关研究与制备方法提供参考,该种微载体在骨组织工程支架材料领域具有广泛应用潜力。

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