CN114081997A - 负载miR-93的矿化PLGA支架及其制备方法 - Google Patents
负载miR-93的矿化PLGA支架及其制备方法 Download PDFInfo
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- CN114081997A CN114081997A CN202111181486.XA CN202111181486A CN114081997A CN 114081997 A CN114081997 A CN 114081997A CN 202111181486 A CN202111181486 A CN 202111181486A CN 114081997 A CN114081997 A CN 114081997A
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Abstract
本发明涉及一种负载miR‑93的矿化PLGA支架及其制备方法，通过对制备方法的步骤和原料进行具体限定，从而可以有效的调控免疫应答改善移植物的成骨微环境，提升临界尺寸骨缺损修复水平。
Description
技术领域
本发明属于骨材料制备技术领域，具体涉及一种负载miR-93的矿化PLGA支架及其制备方法。
背景技术
骨缺损是临床常见难题。随着现代社会的不断发展，因创伤、感染、肿瘤及严重骨质疏松性骨折等导致的骨缺损日趋增多。据统计，美国每年因创伤进行骨移植治疗的患者高达100万例。我国每年因先天性疾病、交通伤及运动损伤等导致的骨缺损高达350万例。但当前临床治疗中，骨修复重建失败产生的二次手术和后续住院费用高达1.7～7.9万美元/例，治疗时间延长3～5倍，住院费用增加4～25倍。因此，骨缺损的修复重建成为当今骨科医生面临的巨大挑战。
在骨缺损修复过程中，免疫反应起着重要的启动和促进作用，当前骨组织工程材料大多存在“骨微环境免疫应答调控缺陷”问题。近年来，随着骨免疫在骨再生领域的研究不断深入，发现免疫细胞作为骨再生微环境的中心调控者，其创造的免疫微环境在骨再生的过程中发挥着至关重要的作用。因此，在生物骨材料的研发策略上倍加重视“免疫调节”性能。植入支架的结构决定了宿主反应的程度，支架的孔径和孔隙率在对成骨微环境及修复骨缺损都起着至关重要的作用。因此，制备具有合适孔隙结构的支架调节骨免疫反应，能营造适宜的成骨微环境促进骨缺损修复。
3D打印可以精准调控制备支架的结构。目前，3D打印技术主要包括熔融沉积成型技术(FDM)、立体平板印刷技术(SLA)、选区激光烧结技术(SLS)和激光快速成型技术(LRP)等。其中，FDM具有结构精细、力学性能好、方便开展等优点。
中国专利公开文本CN108853577A公开了一种3D打印Ti-PDA-PLGA微球骨缺损修复支架，由3D打印Ti支架、Ti支架表面的PDA涂层、PDA涂层上吸附的BMP-2以及携带VEGF的PLGA微球组成，采用聚多巴胺进行表面修饰，不仅简便而且明显改良了金属支架的亲水性及生物相容性，而聚多巴胺的吸附作用可以提升BMP-2的携带效率，并且能够实现BMP-2的缓释，而含VEGF-PLGA微球的引入，使得VEGF能够缓释的同时，两种不同释放模式互不影响，实现了缺损区长时间的高效成骨及成血管的进程。
中国专利公开文本CN110124107A公开了一种用于关节软骨修复的PLGA细胞支架及其制备方法和应用，PLGA细胞支架包括PLGA多孔支架以及种植于该PLGA多孔支架中的软骨细胞、骨髓间充质干细胞以及脂肪干细胞，多层软骨基质材料中包裹不同比例的软骨细胞、骨髓间充质干细胞(BM)、脂肪干细胞(SVF)，软骨细胞如同种子一样，起着中坚作用，SVF可以分泌II型胶原强化软骨细胞的作用，同时受BM细胞分泌的生长因子刺激而加快增殖速度，而且BM细胞加入提高了细胞粘附率。此外骨髓间充质干细胞、脂肪干细胞也能够在细胞因子诱导下，具有分化为软骨细胞的趋势。
上述专利公开文本均未考虑对支架进行矿化修饰以及microRNA-93负载。
影响支架调控微环境中免疫应答的理化性质主要包括成分、表面粗糙度、亲水性等。人工合成材料如聚乳酸(PLA)、聚羟基乙酸(PGA)、聚乳酸羟基乙酸(PLGA)和聚己内酯(PCL)等力学性能较好，但生物学性能欠佳。I型胶原为现有已知概念，为一种较粗的纤维束，分布广泛，主要存在于皮肤、肌腱、韧带及骨中，具有很强的抗张强度，约占人体胶原含量的90％。在支架中添加I型胶原蛋白和羟基磷灰石成分，形成仿生胶原矿化修饰，可以有效增强支架的骨免疫调节性能，同时，支架可以通过表面粗糙度和形貌调控免疫细胞，并且支架表面的亲水性可调节免疫细胞的粘附和激活，从而影响体内植入物在体内的骨整合。
局部缓释生物活性因子，是调控免疫应答的重要方法。miR-93即microRNA-93，是医学领域已知且已冠名的一类小的非编码RNA。miR-93是第一个被发现的miR-17microRNA簇，《Circulation》期刊的研究表明，MiR-93通过抑制干扰素调节因子9(IRF9)，下调巨噬细胞中免疫响应基因1(IRG1)和衣康酸的产生，进而促使巨噬细胞向M2亚型极化，为组织再生提供良好的免疫微环境。MiR-93可以通过诱导巨噬细胞向M2型极化，促进损伤组织再生。
发明内容
本发明提供了一种具有骨免疫调控特性的miR-93微球修饰的3D打印矿化PLGA(聚乳酸羟基乙酸)支架的制备方法，从而有效调控免疫应答改善移植物的成骨微环境，提升临界尺寸骨缺损修复水平，主要包括如下步骤：
步骤1，miR-93微球合成：通过快速膜乳化方法得到miR-93冻干微球。
步骤2，设定3D打印PLGA多孔支架的打印参数。
步骤3，3D打印：设置PLGA多孔支架具有均匀孔隙结构，通过型3D打印机分层有序打印出具有均匀孔隙结构的3D打印PLGA支架。
步骤4，等离子修饰：将步骤3得到的3D打印PLGA支架置于等离子体处理器中进行等离子体处理。
步骤5，miR-93微球负载与胶原浸润处理：将步骤1得到的miR-93冻干微球放入到I型胶原蛋白溶液中共混后得到共混液，然后将步骤4处理后的PLGA支架放入该共混液中浸润孵育，然后将处理后的PLGA支架取出漂洗、杀菌、保存。
步骤6，制备获得模拟体液。
步骤7，模拟体液矿化：在常温下将步骤5得到的PLGA支架浸泡于步骤6得到的模拟体液中进行孵育使样品表面构筑类骨磷灰石涂层，然后将样品取出，漂洗、杀菌、密封、保存，得到负载miR-93的矿化PLGA支架。
上述步骤具体如下：
步骤1，miR-93微球合成：将miR-93溶解于去离子水中，加入到1.8～2.2mmol/L的醋酸锌溶液中，用0.9～1.2mmol/L的NaOH将溶液pH调节为7.0后稀释，获得最终浓度为4.8～5.2mg/L的miR-93改良液，将该改良液作为内水相W1备用；然后将聚乙二醇溶于乙醇溶剂作为油相O，其中聚乙二醇和乙醇的比例为98～102mg：4.8～5.2ml；加入制备得到的内水相W1，其中内水相W1的加入量为乙醇溶剂加入量的18～22％，通过均质机采用5600～6100r/min的模式运转10～18s将内水相W1分散于油相O中，得到W1/O初乳，将所述W1/O初乳倒入到外水相W2中，所述外水相W2为含有聚乙烯醇的NaCl水溶液，采用磁力搅拌进行预复乳化，将预复乳化之后的混合物倒入到快速膜乳化装置中采用氮气压力将其反复压过微孔膜，得到直径均一的W1/O/W2副乳，将所述W1/O/W2副乳倒入到0.8～0.96％NaCl的水溶液中，该NaCl的水溶液使用量为内水相W1加入量的780～900倍，用磁力搅拌机去除油相中的乙醇，进而得到miR-93微球，固化后用去离子水离心洗涤，最后冻干制成粒径为30-50μm、载药率>50％、载药量为30％-50％的miR-93冻干微球。
步骤2，设定3D打印PLGA多孔支架的打印参数：底层填充厚度0.8～1.2mm，填充密度18～22％，打印速度28～32mm/s，喷头温度208～212℃，热床温度48～53℃，打印材料挤出量98～100％，喷嘴孔径0.2～0.5mm。
步骤3，3D打印：设置PLGA多孔支架具有均匀孔隙结构，且设置其孔隙率为80％-85％，支架孔径为200-350μm，将熔融的PLGA多孔支架在208～212℃通过熔融沉积成型3D打印机分层有序打印出具有均匀孔隙结构的3D打印PLGA支架。
步骤4，等离子修饰：将步骤3得到的3D打印PLGA支架置于等离子体处理器中进行等离子体处理，等离子体处理器内抽真空至10Pa以下，充入空气调节压力至230～250Pa，待等离子体处理器腔内压力稳定后，控制频率为13.3～13.68MHz，放电功率为19～21W，处理时间为28～33min，产生辉光放电等离子体对样本进行处理，最后将所获得的改性PLGA支架通过钴60消毒55～65min后密封，然后在3～5℃的条件下保存。
步骤5，miR-93微球负载与胶原浸润处理：将步骤1得到的miR-93冻干微球放入到1.8～2.2mg/L的I型胶原蛋白溶液中共混后得到共混液，所述miR-93冻干微球与I型胶原蛋白溶液的共混比例为(8～12mg)：(95～102ml)，然后将步骤4处理后的PLGA支架放入该共混液中浸润22～26h孵育，温度为3～5℃，然后将处理后的PLGA支架取出用二次水(即第二次蒸馏过的水)轻柔漂洗2～5次，紫外线杀菌60min，在3～5℃条件下保存。
步骤6，模拟体液的制备：将蒸馏水放入烧杯，在磁力搅拌下加热至35～38℃，然后依次加入NaCl，NaHCO3，KCl，K2HPO4·3H2O，MgCl2·6H2O，CaCl2，Na2SO4，每一个物质完全溶解后再放入下一个；其中蒸馏水：NaCl：NaHCO3：KCl：K2HPO4·3H2O：MgCl2·6H2O：CaCl2：Na2SO4为(780～810mL)：(7.988～7.999g)：(0.33～0.36g)：(0.220～0.228g)：(0.226～0.230g)：(0.300～0.308g)：(0.270～0.281g)：(0.069～0.073g)，待所有物质溶解完全后，加入三羟甲基氨基甲烷-盐酸缓冲液调节pH值，所述三羟甲基氨基甲烷-盐酸缓冲液即每50mmol/L(CH2OH)3CNH2与0.1M的HCl缓冲调节pH值至7.38～7.42，温度为36.2～36.8℃，定容至900～1100mL，即获得浓度为0.9～1.1的模拟体液。
步骤7具体为，模拟体液矿化：在常温下将步骤5得到的PLGA支架浸泡于步骤6得到的浓度为0.9～1.1的模拟体液中进行孵育使样品表面构筑类骨磷灰石涂层，孵育时间为22～25h，然后将样品取出，用二次水轻柔漂洗2～5次,紫外线杀菌55～65min后密封，在3～5℃条件下保存，得到负载miR-93的矿化PLGA支架。
作为优选，步骤1中，miR-93溶解于去离子水中，加入到1.8～2.2mmol/L的醋酸锌溶液，溶解有miR-93的去离子水与醋酸锌溶液两者的比例为(1.8～2.3)：1。
作为优选，步骤2中，设定的3D打印PLGA多孔支架的打印参数具体为：底层填充厚度1mm，填充密度20％，打印速度30mm/s，喷头温度210℃，热床温度50℃，打印材料挤出量100％，喷嘴孔径0.3mm。
作为优选，步骤4中等离子体处理器内抽真空至10Pa以下，充入空气调节压力至240Pa，待等离子体处理器腔内压力稳定后，控制频率为13.56MHz，放电功率为20W；步骤4中的处理时间为30min，处理气体为空气；处理之后将改性PLGA支架通过钴60消毒60min后密封，然后在4℃的条件下保存。
作为优选，步骤5中，I型胶原蛋白溶液的浓度为2mg/mL，浸润时间为24h孵育，温度4℃，然后将样品取出用二次水轻柔漂洗3次，紫外线60min消毒后密封，在4℃条件下保存。
作为优选，步骤7中，将步骤5得到的PLGA支架浸泡于步骤6得到浓度为1.0的SBF中进行孵育使样品表面构筑类骨磷灰石涂层，孵育时间为24h，然后将样品取出，用二次水轻柔漂洗3次,紫外线60min消毒后密封，在4℃条件下保存。
一种负载miR-93的矿化PLGA支架，该支架为多孔结构，孔径200-350μm、孔隙率高，有利于营养物质的传递和细胞的长入，支架经过等离子体修饰、胶原和负载、SBF孵育类羟基磷灰石涂层后，材料表面显著粗糙出现沟壑状形态且出现点状结晶结构，利用细胞的粘附和体液的浸润。该支架具有一定的力学性能，可以满足植入后临时的力学支撑。此外，该支架负载的miR-93微球粒径为30-50μm、载药率>50％、载药量30％-50％，植入前5天释放量约为40-45％，显示出爆发式释放的特征。在最初的爆燃释放后，剩余的微球被降解释放。大约90％会在30天内从微球中释放。
作为优选，所述负载miR-93的矿化PLGA支架采用前述的制备方法制备得到。
作为优选，所述负载miR-93微球的矿化PLGA支架的表面矿化颗粒粒径为20-50nm。
本发明的技术效果在于：
本发明通过合理设置3D打印材料的矿化修饰，通过合理调节等离子修饰、胶原浸润处理、模拟体液矿化的参数，从而为制备具有良好骨免疫和成骨微环境调节作用的3D打印支架奠定基础，表面矿化颗粒粒径为20-50nm。
本发明通过合理设定微球合成的快速膜乳化方法，从而可以构建粒径均一可控、药物负载安全有效的载药微球。
本发明将3D打印与体外矿化技术两种技术进行有效的结合并且通过合理设置各个步骤的参数，从而使得制备的负载miR-93的3D打印矿化支架的性能良好，其药物缓释>30天。
通过在矿化3D打印支架的基础上，构建miR-93缓释骨材料复合体系，大大的增强了支架的免疫调控性能，降低了微环境免疫炎症反应，诱导成骨微环境促进新骨生成。通过体外缓释性能检测、体外生物活性及体内修复效能研究，评价本发明的复合支架的组成、结构、理化性质及活性因子对骨缺损修复的作用明显，对骨免疫微环境调控性能优良。
本发明设置的负载miR-93的矿化PLGA支架，通过合理设置多孔结构，使得其孔隙率高，有利于营养物质的传递和细胞的长入，支架经过等离子体修饰、胶原和负载、SBF孵育类羟基磷灰石涂层后，利用细胞的粘附和体液的浸润。该支架具有所需的力学性能，可以满足植入后临时的力学支撑。通过合理设置负载的miR-93微球，使其粒径为30-50μm、载药率>50％、载药量30％-50％，从而满足植入前5天释放量约为40-45％，显示出爆发式释放的特征。在最初的爆燃释放后，剩余的微球被降解释放。大约90％会在30天内从微球中释放。
附图说明
图1为本发明3D打印及矿化修饰PLGA支架示意图。
图2为本发明缓释微球制备的示意图。
图3为本发明PLGA支架结构示意图。
图4为本发明缓释微球支架微观结构示意图。
图5为本发明细胞试验示意图。
图6为本发明动物试验结果数据图。
具体实施方式
结合附图进行进一步说明：
实施例1：
将20mg miR-93溶解于去离子水中，加入2mmol/L醋酸锌溶液，上述两者比例为2：1，用1mmol/L NaOH将溶液PH调节为7.0后稀释，获得miR-93最终浓度为5mg/L，将其作为内水相W1备用。将100mg聚乙二醇溶于5ml乙醇溶剂作为油相，加入1ml内水相W1，通过均质机采用6000r/min模式运转15s将W1分散于油相O中，得到W1/O初乳，将其倒入外水相W2中，所述外水相W2为含有聚乙烯醇的NaCl的水溶液中，采用磁力搅拌进行预复乳化，将其倒入快速膜乳化装置中以适当氮气压力将其反复压过微孔膜，得到直径均一的W1/O/W2副乳，将其倒入800mL 0.9％NaCl水溶液中，用磁力搅拌机去除油相中乙醇，进而得到miR-93微球，固化后用去离子水离心洗涤，最后冻干制成微球粒径为30-50μm、载药率>50％、载药量30％-50％。通过快速膜乳化技术，在此基础上构建粒径均一可控、药物负载安全有效的载药微球，
将构建好的3D打印PLGA多孔支架模型STL文件导入打印软件Allcct生成代码后传输至CR-30403D打印机，设定基本打印参数：底层填充厚度1mm，填充密度20％，打印速度30mm/s,喷头温度210℃，热床温度50℃，打印材料挤出量100％，喷嘴孔径0.3mm。将熔融的PLGA在210℃通过3D打印机微机控制数控系统，分层有序打印出具有均匀孔隙结构的3D打印PLGA支架，其中PLGA支架中多孔的孔隙率为82％，支架孔径为280μm。
负载miR-93的3D打印矿化支架的表面功能化处理：(1)等离子修饰：采用等离子体系统处理，处理时间为30min，处理气体为空气，最后将改性PLGA支架通过钴60消毒60min后密封，在4℃条件下保存使用；(2)miR-93微球负载与胶原浸润处理：将空气等离子体预处理后的PLGA支架放入2mg/mL的I型胶原/miR-93微球共混液中浸润24h孵育，温度4℃，将样品取出用二次水轻柔漂洗三次，紫外线消毒60min后密封，在4℃条件下保存使用；(3)模拟体液矿化：将miR-93微球负载与胶原浸润处理后的样品取出用二次水轻柔漂洗三次后，将其浸泡于36.5℃的浓度1.0的SBF中进行孵育使样品表面构筑类骨磷灰石涂层，孵育时间为24h。到预定孵育时间后，将样品取出，用二次水轻柔漂洗三次,紫外线消毒60min后密封，在4℃条件下保存使用。
利用扫描电镜检测、傅里叶红外检测、X线光电子能谱、接触角分析等检测对材料的理化性质，运用万能力学试验仪等分析其力学特性，通过细胞体外实验，利用MTT、激光共聚焦等实验，评价其细胞亲和性、细胞毒性、免疫调控及成骨性能。傅里叶红外光谱分析结果曲线表明，等离子体修饰后PLGA支架表面的官能团发生改变，出现如C＝O和-COO的活性官能团。扫描电镜图像显示支架均为均匀多孔结构，具有相同的孔径，随着观察倍数的放大，材料表面显著粗糙出现沟壑状形态且出现点状结晶结构，微球负载于支架之间。X射线光电子能谱分析检测，材料表面-C-O-，C＝O、-COOH键显著增加，材料表面元素中出现了大量的钙、磷、镁等元素，结果证实了胶原负载与类羟基磷灰石涂层修饰成功。排体积法测得支架孔隙率均为80％以上。亲水性检测结果显示液滴不但向内部扩散同时向四周扩散，可发现扩散面积显著增大。生物力学三点弯实验显示支架最大载荷70-80N、最大强度18-22MPa、弹性载荷35-45N、弹性强度10-18MPa。MTT检测显示，细胞与支架培养后均显示出良好的生长状态，且随着时间的推移细胞显著增殖。碱性磷酸酶检测检测显示共培养21天时，ALP活性随培养时间显著增加。茜素红染色和定量分析发现，共培养21天后支架染色最为显著，且钙盐形成含量随培养时间增加而增加。扫描电子显微镜检查显示MC3T3-E1细胞在修饰后PLGA支架上伸出许多伪足与材料表面相互接触，并且出现多个细胞粘附团聚。激光共聚焦显示支架可有效促进巨噬细胞的M2极化。ELISA检测显示免疫调控CD86、CXCL、CCL19显著降低，CD163、CD206、MRC1、CCL13显著增加。成骨细胞分化相关基因检测显示ALP基因、OPN基因、Col-1基因、OCN基因和RunX2基因表达均显著增加。
对比例1：
将构建好的3D打印PLGA多孔支架模型STL文件导入打印软件Allcct生成代码后传输至CR-30403D打印机，设定基本打印参数：底层填充厚度1mm，填充密度20％，打印速度30mm/s,喷头温度210℃，热床温度50℃，打印材料挤出量100％，喷嘴孔径0.3mm。将熔融的PLGA在210℃通过3D打印机微机控制数控系统按照设定路径，分层有序打印出具有均匀孔隙结构的3D打印PLGA支架。
3D打印支架的表面功能化处理：(1)等离子修饰：采用等离子体系统处理，处理时间为30min，处理气体为空气，最后将改性PLGA支架通过钴60消毒60min后密封，在4℃条件下保存使用；(2)胶原浸润处理：将空气等离子体预处理后的PLGA支架放入2mg/mL的I型胶原中浸润24h孵育，温度4℃，将样品取出用二次水轻柔漂洗三次，紫外线消毒60min后密封，在4℃条件下保存使用；(3)模拟体液矿化：将胶原浸润处理后的PLGA支架取出用二次水轻柔漂洗三次后，将其浸泡于36.5℃的浓度1.0的SBF中进行孵育使样品表面构筑类骨磷灰石涂层，孵育时间为24h。到预定孵育时间后，将样品取出，用二次水轻柔漂洗三次,紫外线消毒60min后密封，在4℃条件下保存使用。
骨缺损动物模型构建及分组对比试验：选择同一种群新西兰白兔32只，雌雄不限，随机分为4组并标记，每组各8只。构建桡骨中段缺损模型(缺损长度1.2cm)。A组为空白对照组，造缺损，不植入任何材料，用于观察正常情况下桡骨缺损区域新生骨生长变化情况及新生骨的生成速度；B组为植入对比例1的复合孔径3D打印支架组；C组为植入本发明实施例1的miR-93微球复合孔径3D打印支架组。术中切开皮肤，切透骨膜，暴露桡骨，保留骨膜。采用摆锯制备长度1.2cm大小的骨缺损，植入骨材料固定后，覆盖骨膜，缝合皮肤。
在相同饲养条件，每月行大体观察及影像学检查。大体观察包括动物健康状态、手术切口愈合情况、有无感染及并发症、以及头围测量。影像学检查主要采用X射线、常规CT和Micro-CT分析骨愈合情况、骨密度变化以及新骨生长情况。术后6个月对每组进行组织学观察评价。组织学评价主要采用HE染色、Masson三色法、甲苯胺蓝染色法评价材料新骨形成的情况，采用骨形态计量分析定量计算。每组选取一定标本进行力学测试，检测新骨的抗压强度和弹性模量。A组可见桡骨骨缺损处未有新骨形成，直至术后8周后在断端出现骨痂且随着的时间推移，断端骨痂生长向中心迁移，但到24周时依旧留有巨大骨缺损。B组可在术后4周观察到骨折断端由新生毛刺状低亮影向缺损处长入，至8周时缺损处已完全被低亮影像填充，证实存在新生骨痂填充。12周时已见缺损周围形成高亮条带状影且与骨折断端相连接，基本实现内外侧皮质骨相连，16周可见该缺损处骨痂密度增高且均匀并接近周围正常骨组织。20周已基本形成骨重塑。C组术后4周即可观察到骨痂形成并充满缺损处，密度低于周围骨组织介于周围软组织谜底，且密度不均匀呈斑片状模糊低亮影。术后8周可见骨折周围高亮皮质骨形成且与骨折断端基本连接，缺损处呈现高亮影且密度趋于均一。至术后12周时整体骨缺损区域新生骨密度影与周围骨组织接近，骨痂已开始塑性且出现髓腔再通。16周后已基本完成骨的重塑，骨缺损处骨密度与周围骨组织密度无异，周围皮质骨与断端形成良好连接。Micro-CT检查显示A组未有骨形成缺损处仍然存在。B组可见较多的支架中存在新骨形成和长入，特别是支架表面处骨形成增多。C组支架内及支架上已长满新骨，断端形成骨性连接且支架周围形成一层皮质骨包绕。C组较B组可以显著发现因新骨形成最大，从而导致骨小梁形成数量最多、厚度最大且骨小梁间距最短。HE染色显示A组骨缺损区域由纤维软组织填充，部分存在点状新生骨向髓腔内生长。B组支架外部及深部内也存在新生骨组织，新生骨包围支架生长且接触端支架降解后形成不规则形态。C组新骨形成最多，且新生骨组织多为编织骨，并且部分编织骨完成了重塑改建，与断端皮质骨相连形成整体的皮质骨样结构。此外可见支架内部充满新生骨组织沿着支架爬行生长连接成片。
Claims (10)
1.一种负载miR-93的矿化PLGA支架的制备方法，其特征在于，包括如下步骤：
步骤1，miR-93微球合成：通过快速膜乳化方法得到miR-93冻干微球；
步骤2，设定3D打印PLGA多孔支架的打印参数；
步骤3，3D打印：设置PLGA多孔支架具有均匀孔隙结构，通过型3D打印机分层有序打印出具有均匀孔隙结构的3D打印PLGA支架；
步骤4，等离子修饰：将步骤3得到的3D打印PLGA支架置于等离子体处理器中进行等离子体处理；
步骤5，miR-93微球负载与胶原浸润处理：将步骤1得到的miR-93冻干微球放入到I型胶原蛋白溶液中共混后得到共混液，然后将步骤4处理后的PLGA支架放入该共混液中浸润孵育，然后将处理后的PLGA支架取出漂洗、杀菌、保存；
步骤6，制备获得模拟体液；
步骤7，模拟体液矿化：在常温下将步骤5得到的PLGA支架浸泡于步骤6得到的模拟体液中进行孵育使样品表面构筑类骨磷灰石涂层，然后将样品取出，漂洗、杀菌、密封、保存，得到负载miR-93的矿化PLGA支架。
2.根据权利要求1所述的负载miR-93的矿化PLGA支架的制备方法，其特征在于，
步骤1具体为，miR-93微球合成：将miR-93溶解于去离子水中，加入到1.8～2.2mmol/L的醋酸锌溶液中，用0.9～1.2mmol/L的NaOH将溶液pH调节为7.0后稀释，获得最终浓度为4.8～5.2mg/L的miR-93改良液，将该改良液作为内水相W1备用；然后将聚乙二醇溶于乙醇溶剂作为油相O，其中聚乙二醇和乙醇的比例为98～102mg：4.8～5.2ml；加入制备得到的内水相W1，其中内水相W1的加入量为乙醇溶剂加入量的18～22％，通过均质机采用5600～6100r/min的模式运转10～18s将内水相W1分散于油相O中，得到W1/O初乳，将所述W1/O初乳倒入到外水相W2中，所述外水相W2为含有聚乙烯醇的NaCl水溶液，采用磁力搅拌进行预复乳化，将预复乳化之后的混合物倒入到快速膜乳化装置中采用氮气压力将其反复压过微孔膜，得到直径均一的W1/O/W2副乳，将所述W1/O/W2副乳倒入到0.8～0.96％NaCl的水溶液中，该NaCl的水溶液使用量为内水相W1加入量的780～900倍，用磁力搅拌机去除油相中的乙醇，进而得到miR-93微球，固化后用去离子水离心洗涤，最后冻干制成粒径为30-50μm、载药率>50％、载药量为30％-50％的miR-93冻干微球；
步骤2具体为，设定3D打印PLGA多孔支架的打印参数：底层填充厚度0.8～1.2mm，填充密度18～22％，打印速度28～32mm/s，喷头温度208～212℃，热床温度48～53℃，打印材料挤出量98～100％，喷嘴孔径0.2～0.5mm；
步骤3具体为，3D打印：设置PLGA多孔支架具有均匀孔隙结构，且设置其孔隙率为80％-85％，支架孔径为200-350μm，将熔融的PLGA多孔支架在208～212℃通过熔融沉积成型3D打印机分层有序打印出具有均匀孔隙结构的3D打印PLGA支架；
步骤4具体为，等离子修饰：将步骤3得到的3D打印PLGA支架置于等离子体处理器中进行等离子体处理，等离子体处理器内抽真空至10Pa以下，充入空气调节压力至230～250Pa，待等离子体处理器腔内压力稳定后，控制频率为13.3～13.68MHz，放电功率为19～21W，处理时间为28～33min，产生辉光放电等离子体对样本进行处理，最后将所获得的改性PLGA支架通过钴60消毒55～65min后密封，然后在3～5℃的条件下保存；
步骤5具体为，miR-93微球负载与胶原浸润处理：将步骤1得到的miR-93冻干微球放入到1.8～2.2mg/L的I型胶原蛋白溶液中共混后得到共混液，所述miR-93冻干微球与I型胶原蛋白溶液的共混比例为(8～12mg)：(95～102ml)，然后将步骤4处理后的PLGA支架放入该共混液中浸润22～26h孵育，温度为3～5℃，然后将处理后的PLGA支架取出用二次水轻柔漂洗2～5次，紫外线杀菌58-62min，在3～5℃条件下保存；
步骤6具体为，模拟体液的制备：将蒸馏水放入烧杯，在磁力搅拌下加热至35～38℃，然后依次加入NaCl，NaHCO3，KCl，K2HPO4·3H2O，MgCl2·6H2O，CaCl2，Na2SO4，每一个物质完全溶解后再放入下一个；其中蒸馏水：NaCl：NaHCO3：KCl：K2HPO4·3H2O：MgCl2·6H2O：CaCl2：Na2SO4为(780～810mL)：(7.988～7.999g)：(0.33～0.36g)：(0.220～0.228g)：(0.226～0.230g)：(0.300～0.308g)：(0.270～0.281g)：(0.069～0.073g)，待所有物质溶解完全后，加入三羟甲基氨基甲烷-盐酸缓冲液调节pH值，所述三羟甲基氨基甲烷-盐酸缓冲液即每50mmol/L(CH2OH)3CNH2与0.1M的HCl缓冲调节pH值至7.38～7.42，温度为36.2～36.8℃，定容至900～1100mL，即获得浓度为0.9～1.1的模拟体液；
步骤7具体为，模拟体液矿化：在常温下将步骤5得到的PLGA支架浸泡于步骤6得到的浓度为0.9～1.1的模拟体液中进行孵育使样品表面构筑类骨磷灰石涂层，孵育时间为22～25h，然后将样品取出，用二次水轻柔漂洗2～5次,紫外线杀菌55～65min后密封，在3～5℃条件下保存，得到负载miR-93的矿化PLGA支架。
3.根据权利要求2所述的负载miR-93的矿化PLGA支架的制备方法，其特征在于，
步骤1中，miR-93溶解于去离子水中，加入到1.8～2.2mmol/L的醋酸锌溶液，溶解有miR-93的去离子水与醋酸锌溶液两者的比例为(1.8～2.3)：1。
4.根据权利要求2所述的负载miR-93的矿化PLGA支架的制备方法，其特征在于，
步骤2中，设定的3D打印PLGA多孔支架的打印参数具体为：底层填充厚度1mm，填充密度20％，打印速度30mm/s，喷头温度210℃，热床温度50℃，打印材料挤出量100％，喷嘴孔径0.3mm。
5.根据权利要求2所述的负载miR-93的矿化PLGA支架的制备方法，其特征在于，
步骤4中等离子体处理器内抽真空至10Pa以下，充入空气调节压力至240Pa，待等离子体处理器腔内压力稳定后，控制频率为13.56MHz，放电功率为20W；
步骤4中的处理时间为30min，处理气体为空气；处理之后将改性PLGA支架通过钴60消毒约60min后密封，然后在4℃的条件下保存。
6.根据权利要求2所述的负载miR-93的矿化PLGA支架的制备方法，其特征在于，
步骤5中，I型胶原蛋白溶液的浓度为2mg/mL，浸润时间为24h孵育，温度4℃，然后将样品取出用二次水轻柔漂洗3次，紫外线60min消毒后密封，在4℃条件下保存。
7.根据权利要求2所述的负载miR-93的矿化PLGA支架的制备方法，其特征在于，
步骤7中，将步骤5得到的PLGA支架浸泡于步骤6得到浓度为1.0的SBF中进行孵育使样品表面构筑类骨磷灰石涂层，孵育时间为24h，然后将样品取出，用二次水轻柔漂洗3次,紫外线60min消毒后密封，在4℃条件下保存。
8.一种负载miR-93的矿化PLGA支架，其特征在于，所述负载miR-93的矿化PLGA支架为多孔结构，孔径为200-350μm，表面粗糙具有沟壑状形态且具有点状结晶结构，其中，负载的miR-93微球粒径为30-50μm、载药率>50％、载药量为30％-50％。
9.根据权利要求8所述的负载miR-93的矿化PLGA支架，其特征在于，所述负载miR-93的矿化PLGA支架采用权利要求1-7任一项的制备方法制备得到。
10.根据权利要求8和9所述的负载miR-93的矿化PLGA支架，其特征在于，所述负载miR-93微球的矿化PLGA支架的表面矿化颗粒粒径为20-50nm。
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