CN112933297A - 一种冻干递送外泌体的多级微纳结构骨修复支架 - Google Patents
一种冻干递送外泌体的多级微纳结构骨修复支架 Download PDFInfo
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Abstract
本发明涉及一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，所述方法包括以下步骤：a.通过细胞培养上清液超离得到外泌体浓缩液；b.将外泌体浓缩液吸附至具有大孔/微孔/介孔多级结构的MBG支架上并冷冻干燥。本发明的冻干递送外泌体的多级微纳结构骨修复支架，克服单纯MBG支架成骨诱导性能有限的问题，提高支架的生物活性和促成骨性能，具有符合骨修复治疗需要的外泌体缓释速率，从而提高支架材料的骨修复能力和新骨生长能力，实现骨组织完全再生/修复。所述修复支架中支架的微孔结构对外泌体形态和活性起保护作用，冻干递送外泌体的多级微纳结构骨修复支架具有显著提高的体内骨缺损修复效果。
Description
技术领域
本发明属于医学生物材料领域，具体涉及一种冻干递送外泌体的多级微纳结构骨修复支架及其制备方法。
背景技术
近年来大量研究发现细胞分泌的外泌体有促成骨分化、血管生成和骨质矿化的作用，有望成为新的骨再生治疗手段。
临床应用需考虑外泌体的长期储存、活性保持，以及进入骨缺损部位后如何缓释等问题。生物材料支架可作为载体发挥控释和活性保持的作用，提高所负载的生物活性物质的效率。外泌体溶液可在4℃稳定存放24小时(Protein&Cell 10(4)(2019)295-299)，-20℃保存6个月(International Journal of Molecular Sciences 15(3)(2014)4142-4157)，或-80℃长期保存(Current Protocols in Stem Cell Biology Chapter3(2006)Unit 322)。外泌体溶液所要求的特定低温储存条件可能成为其临床转化的限制，更稳定、低成本的储存形态有待开发。具有三维多级结构的支架已被证明可维持负载药物结构和活性(Acta Biomaterialia 42(2016)18-32)，理论上也可为外泌体的结构、活性和功能性提供保护作用。本专利采用具有大孔/微孔/介孔多级结构的介孔生物玻璃(MBG)支架作为外泌体冻干递送的载体，可应用于体内骨缺损修复。
目前仅有少量文献报道将外泌体负载于支架用于体内骨缺损修复：Zhang等(StemCell Research&Therapy 7(2016):136)和Qi等(International Journal of BiologicalSciences 12(7)(2016)836-849)将外泌体吸附于商业化的β-TCP支架表面，并在大鼠颅骨缺损修复模型中验证了其促成骨效果；Li等(Acs Applied Materials&Interfaces 10(6)(2018)5240-5254)将ASC-exo吸附于聚多巴胺包被的PLGA支架(PLGA/pDA)表面，可实现外泌体均匀缓慢释放，促进小鼠颅骨缺损的修复；Wei等(Acta Biomaterialia 86(2019)480-492)将外泌体装载于二氧化钛纳米管并证明了其体外促成骨活性。Liu等(Nanoscale 9(13)(2017)4430-4438)将外泌体包裹于改性的透明质酸水凝胶中实现缓释并应用于全层关节缺损修复。然而现有研究通过溶液吸附或水凝胶包埋将外泌体与支架相结合，无法长期保存，如何保障外泌体在支架上长期稳定存在是其推向临床应用必须解决的另一问题。Li等(中国组织工程研究(29)4593-4600)研究表明，外泌体有制成冻干粉长期保存的可能性，但需使用抗冻剂海藻糖维持其结构稳定性，该文献仅验证了外泌体冻干前后的生物安全性，未对其生物活性、功能性维持进行研究。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供以下技术方案：
一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于包括以下步骤：
a.通过细胞培养上清液超离得到外泌体浓缩液；
b.将外泌体浓缩液吸附至具有大孔/微孔/介孔多级结构的MBG支架上并冷冻干燥。
优选的，上述制备方法中步骤b为将具有大孔/微孔/介孔多级结构的介孔生物玻璃(MBG)支架灭菌，将外泌体浓缩液滴加至MBG支架上，静置使其在支架内部均匀分散并吸附入表面微孔内，冻干，得到冻干递送外泌体的多级微纳结构骨修复支架。
优选的，上述制备方法中外泌体浓缩液以50-500μL/cm3的比例滴加至MBG支架上。
优选的，上述制备方法中具有大孔/微孔/介孔多级结构的介孔生物玻璃(MBG)支架是通过溶剂挥发诱导自组装的溶胶凝胶法结合多模板法制备所得。
优选的，上述制备方法中具有大孔/微孔/介孔多级结构的介孔生物玻璃(MBG)支架的制备方法为将6重量份MBG溶胶和1-3重量份的高分子微球(微孔模板剂)均匀混合后，将混合物以0.4-1g/cm3的质量体积比浇注在聚氨酯海绵(大孔模板)上，60℃烘干、550℃煅烧，得到具有大孔/微孔/介孔多级结构的MBG支架。
优选的，上述制备方法中MBG溶胶的制备方法为以正硅酸乙酯、四水硝酸钙、磷酸三乙酯为原料，F127为介孔模板剂，将介孔模板剂8重量份加入80重量份的乙醇中，盐酸调节pH小于2，待介孔模板剂充分溶解后，加入四水硝酸钙1.52重量份、磷酸三乙酯0.46重量份和正硅酸乙酯10.4重量份，40±1℃下搅拌反应24h后蒸发浓缩得到粘稠的MBG溶胶。
优选的，上述制备方法中，外泌体浓缩液通过细胞培养上清液过滤和超速离心所得。
优选的，上述制备方法中，外泌体浓缩液的制备方法为提取并培养细胞，当细胞融合度达到80-90％时，将细胞培养基替换为含2-10％无外泌体血清的条件培养基，继续培养24-48小时后，收集上清液，用0.22μm的滤器去除悬浮细胞和细胞碎片，然后依次以10000×g速率离心30分钟、100,000×g离心90分钟，去除上清液并将外泌体沉淀重悬于50μL PBS中。
优选的，上述制备方法中，所述的细胞选自不同种属的间充质干细胞(骨髓来源、脂肪来源等)、巨噬细胞、中性粒细胞、内皮细胞等贴壁培养细胞；条件培养基选自成骨分化诱导条件培养基、成脂分化诱导条件培养基、包含M-CSF或其他生长因子的条件培养基等。
本发明还提供以下技术方案，一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于，包括如下步骤：
a.提取并培养rBMSCs/rASCs至P3～P5、融合达到90％后，PBS冲洗并将培基更换为包含2％无外泌体血清的成骨诱导培基(OI，含100nM地塞米松、10mMβ-甘油磷酸钠和0.05mM抗坏血酸)或普通培基(Ctrl)。培养48小时后，收集上清液培养基，使用0.22μm过滤器过滤上清液去除悬浮细胞，在4℃下10000×g离心30分钟去除细胞碎片，然后100000×g离心90分钟获得外泌体并重悬于50μL PBS中，储存于-80℃以备进一步研究。根据不同干细胞来源和培养基条件，将所得外泌体分为4组：BMSC-OI-exo、BMSC-ctrl-exo、ASC-OI-exo和ASC-ctrl-exo。
b.40℃水浴下，依次加入300g无水乙醇，6mL 1mol/L HCl和24g F127，剧烈搅拌至完全溶解、溶液呈澄清状态；依次加入4.56g四水硝酸钙和1.29mL磷酸三乙酯，滴加31.2g正硅酸乙酯，保持40℃水浴并剧烈搅拌反应24小时，将产物60℃旋蒸得到粘稠的MBG溶胶。将PU海绵剪成直径5mm的圆片作为支架大孔模板，将6gMBG溶胶和2.5g高分子微球均匀混合、灌注于PU海绵并反复挤压至均匀分布于海绵上。在烘箱中60℃烘干72小时，然后转入马弗炉550℃煅烧6小时，得到具有大孔/微孔/介孔多级结构的MBG支架。
c.将MBG支架进行高压灭菌器灭菌后，将50uL BMSC-OI-exo浓缩液滴加于MBG支架，在4℃静置4小时使外泌体完全吸附到支架的表面微孔中。然后，将支架冻干过夜得到负载外泌体的MBG支架(exo-MBG)。
本发明还提供以下技术方案，一种冻干递送外泌体的多级微纳结构骨修复支架，其特征在于，由前述任一项所述的方法制备得到。
有益效果，本发明的冻干递送外泌体的多级微纳结构骨修复支架，克服单纯MBG支架成骨诱导性能有限的问题，通过将冻干的方式递送外泌体从而提高支架的生物活性和促成骨性能，具有符合骨修复治疗需要的外泌体缓释速率，从而提高支架材料的骨修复能力和新骨生长能力，实现骨组织完全再生/修复。所述修复支架中支架的微孔结构对外泌体形态和活性起保护作用，冻干递送外泌体的多级微纳结构骨修复支架具有显著提高的体内骨缺损修复效果。
附图说明：
图1：(A)流式测定干细胞表面标志抗原(CD29+/CD45-/CD90+)鉴定提取的rASC和rBMSC；(B)TEM拍摄的外泌体形貌；(C)NTA测定的外泌体粒径分布曲线及其在溶液中的布朗运动图像；(D)Western Blot鉴定外泌体标志物(CD9/CD63)。
图2：(A)梯度浓度的各组外泌体作用4天后，靶细胞BMSC的ALP活性；(B)最佳浓度各组外泌体作用7天后，靶细胞BMSC的ALP染色；(C)最佳浓度的各组外泌体作用21天后，靶细胞BMSC的矿化染色；(D)最佳浓度各组外泌体作用4天后，靶细胞BMSC的成骨相关基因表达。
图3：(A)SEM和TEM拍摄的多级微纳结构MBG支架的大孔、微孔和介孔形貌；MBG的(B)EDS图谱，(C)广角XRD图谱，(D)小角XRD图谱，(E)氮气吸附脱附曲线，(F)介孔孔径分布图，(G)支架抗压强度曲线。
图4：(A)直接滴加并冻干于SEM样品台的外泌体形貌；(B)冻干负载于多级结构MBG支架的外泌体形貌；(C)冻干负载于多级结构MBG支架的外泌体缓释曲线；(D)外泌体BMSC-OI-exo、直接冻干后的外泌体、冻干负载于多级结构MBG支架的外泌体的NTA粒径分布曲线；(E)外泌体BMSC-OI-exo、直接冻干后的外泌体、冻干负载于多级结构MBG支架的外泌体、MBG支架、负载外泌体的MBG支架的成骨诱导性能对比。
图5：(A)负载BMSC-OI-exo的MBG支架用于大鼠颅骨缺损修复；(B)术后12周micro-CT三维重建和截面图；(C)顺序荧光染色及其定量分析；(D)硬组织切片VG染色和组织切片HE染色、Masson三色染色。
具体实施方式
下面结合具体实施例来进一步描述本发明，但实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1外泌体的提取和鉴定
(1)细胞提取及鉴定
大鼠BMSC提取：对4周龄、体重为80-100g的雄性SD大鼠实施三倍剂量1％戊巴比妥钠腹腔注射处死后，在75％乙醇中浸泡10分钟，在无菌条件下取出其股骨和胫骨，用手术剪刀去掉其两端的骨骺，暴露骨髓腔两端。用10mL规格的注射器吸取包含10％FBS、1％青霉素/链霉素双抗的α-MEM培养基反复冲洗骨髓腔，并使用移液枪吹打均匀，得到均匀的骨髓细胞悬液，利用rBMSC贴壁生长的特性，接种于一次性聚苯乙烯细胞培养瓶(Corning康宁75cm2透气盖直角培养瓶)中，在温度为37℃、5％CO2/95％空气、饱和湿度的细胞培养箱内进行培养，隔天换液去除悬浮细胞，长满后得到原代rBMSC。
大鼠ASC提取：对4周龄、体重为80-100g的雄性SD大鼠实施三倍剂量1％戊巴比妥钠腹腔注射处死后，在75％乙醇中浸泡10分钟，在无菌条件下从其腹股沟皮下剥离脂肪组织，去除血管后将脂肪组织剪碎，浸泡0.1％I型胶原酶置于37℃水浴45分钟进行消化，然后加入等体积α-MEM培养基(含10％FBS和1％双抗)终止消化。以1800rpm离心10分钟×2次，去除上层漂浮的脂肪和上清液，将细胞重悬在α-MEM培基中并转移至细胞培养瓶，在温度为37℃、5％CO2/95％空气、饱和湿度的细胞培养箱内进行培养，隔天换液去除悬浮细胞，长满后得到原代rASC。
流式细胞鉴定：通过对文献中报道的定义非造血间充质干细胞(MSC)的三种细胞表面抗原(CD29、CD45、CD90)进行荧光染色和流式细胞计数的方法，来鉴定所提取细胞的纯度。具体操作步骤如下：细胞传代至P3～P5时，将其消化、离心并重悬于PBS中。各实验组取两份500μL细胞悬液分别用于阴性对照和荧光染色。将细胞悬液在300rcf(1787rpm)转速下离心，吸去400μL上清液，按照抗体说明书向剩余100μL细胞悬液中加入一定体积的免疫荧光抗体，避光、冰上孵育30分钟。离心并用PBS清洗3次，最后得到500μL细胞悬液，进行流式细胞测定，每组计数10000个细胞，平行测定3次。处理数据时，以未经染色的阴性对照作为阴性筛选标准，先选出CD29、CD90双阳性的细胞，然后在这些细胞中再选出CD45阴性的细胞，计算这些细胞占细胞总数的百分比。流式测定干细胞表面标志抗原(CD29+/CD45-/CD90+)鉴定提取的rASC和rBMSC的结果如图1中A所示。
(2)外泌体的提取
提取并培养rBMSCs/rASCs至P3～P5、融合达到90％后，PBS冲洗并将培基更换为包含2％无外泌体血清的成骨诱导培基(OI，含100nM地塞米松、10mMβ-甘油磷酸钠和0.05mM抗坏血酸)或普通培基(Ctrl)。培养48小时后，收集上清液培养基，使用0.22μm过滤器过滤上清液去除悬浮细胞，在4℃下10000×g离心30分钟去除细胞碎片，然后100000×g离心90分钟获得外泌体并重悬于50μL PBS中，储存于-80℃以备进一步研究。根据不同干细胞来源和培养基条件，将所得外泌体分为4组：BMSC-OI-exo、BMSC-ctrl-exo、ASC-OI-exo和ASC-ctrl-exo。
(3)外泌体的鉴定
将外泌体溶液直接滴在超薄碳膜支持铜网上风干，采用TEM(Hitachi，日本)观察其形态，结果如图1中B所示。通过纳米颗粒追踪分析(NTA；NanoSight NS300，Malvern，UK)测定溶液中外泌体的粒径分布和浓度，结果如图1中C所示。通过Western blot检测外泌体表面的CD63和CD9的特异性抗原，具体步骤如下：将外泌体样品加入上样缓冲液，95℃水浴处理5分钟，在120V电压下45分钟将样品加载至12％SDS-PAGE聚丙烯酰胺凝胶中，然后采用100mA电流1.5小时转膜至硝酸纤维素膜；在4℃下用一抗孵育过夜后，用TBST洗涤3次后，加入HRP标记的山羊抗兔IgG二抗37℃孵育1小时后再用TBST洗涤3次；最后采用增强化学发光法检测蛋白质，并用Image Quant LAS 4000微型生物分子成像仪(GE Healthcare，Uppsala，Sweden)成像；结果如图1中D所示。
实施例2外泌体骨诱导性能评价
通过ALP活性定量、ALP染色、矿化染色和成骨相关基因rtPCR评价4组外泌体的骨诱导性能
(1)体外ALP活性定量
将rBMSC细胞悬液以5000细胞/孔的密度接种于96孔板内，培养24小时后，将培养基更换为包含外泌体的α-MEM培养基(含2％无外泌体血清、1％双抗)，置于培养箱中继续培养4天，取出检测ALP活性。检测ALP活性的实验步骤如下：配置ALP缓冲液(0.1mol/L甘氨酸，1mmol/L MgCl2·6H2O)和ALP工作液(1mg/mL PNPP-Na/ALP缓冲液)。吸去细胞培养基并用PBS清洗2次，每孔加入1mL的1％NP-40裂解液并37℃恒温振荡90分钟使细胞裂解，BCA法测定并计算细胞裂解液的总蛋白量。每孔取50μL细胞裂解液转移至96孔板，加入200μLALP工作液孵育15分钟显色后，测定405nm吸光度(OD值)。计算各组的405nm OD值/(总蛋白量×孵育时间)，单位为405nm OD value/min/mg protein，将所得数据对空白对照组数据进行标准化处理，得到各组的相对ALP活性数据。
(2)ALP染色和矿化染色
将rBMSC细胞悬液以2×104细胞/孔的密度接种于24孔板内，培养24小时后，将培养基更换为包含最佳浓度外泌体的α-MEM培养基(含2％无外泌体血清、1％双抗)，置于培养箱中继续培养7天后，使用BCIP/NBT碱性磷酸酶显色试剂盒(碧云天，中国江苏)进行ALP染色。细胞培养21天后，吸去培养基，每孔加入1mg/mL、pH为4.2的茜素红溶液，37℃孵育过夜。染色完成后用PBS小心反复冲洗孔板底部至洗液呈无色透明，加入少量PBS保持湿润，用倒置显微镜(LeicaDMI6000B，德国)观察拍摄ALP染色和矿化沉积情况。
(3)成骨相关基因表达检测(rtPCR)
将rBMSC细胞悬液以3×104细胞/孔的密度接种于24孔板内，培养24小时后，将培养基更换为包含最佳浓度外泌体的α-MEM培养基(含2％无外泌体血清、1％双抗)，置于培养箱中继续培养7天后，分别使用Trizol(Takara)和PrimeScript RT试剂盒(Takara)从细胞中提取RNA并反转录为互补DNA(cDNA)。稀释后的cDNA与TB GreenTM Premix Ex TaqTM(Takara)、引物混合进行RT-qPCR，以β-actin为内参对成骨分化相关基因进行定量。本研究中使用的引物序列见下表。
图2中显示了4组外泌体ALP活性定量、ALP染色和矿化染色以及成骨相关基因rtPCR表达的情况。根据图2中结果可知不同条件/细胞来源的外泌体具有不同程度的成骨诱导性能，其中BMSC-OI-exo具有最佳成骨诱导性能。
实施例3MBG支架和exo-MBG支架制备及材料表征
(1)MBG支架和exo-MBG支架制备
40℃水浴下，依次加入300g无水乙醇，6mL 1mol/L HCl和24gF127，剧烈搅拌至完全溶解、溶液呈澄清状态；依次加入4.56g四水硝酸钙和1.29mL磷酸三乙酯，滴加31.2g正硅酸乙酯，保持40℃水浴并剧烈搅拌反应24小时，将产物60℃旋蒸得到粘稠的MBG溶胶。将PU海绵剪成直径5mm的圆片作为支架大孔模板，将6gMBG溶胶和2.5g高分子微球均匀混合、灌注于PU海绵并反复挤压至均匀分布于海绵上。在烘箱中60℃烘干72小时，然后转入马弗炉550℃煅烧6小时，得到具有大孔/微孔/介孔多级结构的MBG支架。
将MBG支架进行高压灭菌器灭菌后，将50uL BMSC-OI-exo浓缩液滴加于MBG支架，在4℃静置4小时使外泌体完全吸附到支架的表面微孔中。然后，将支架冻干过夜得到负载外泌体的MBG支架(exo-MBG)。
(2)材料表征
采用场发射扫描电子显微镜镜(FE-SEM,Hitachi S-4800)观察MBG支架及复合支架的大孔结构和表面微孔形貌；采用高分辨透射电子显微镜(HRTEM,JEM-2010,JEOL,Japan)观察MBG的介孔形貌；采用EDS表征支架的元素组成；采用X射线衍射(XRD,Rigaku D/max 2550VB/PC,Japan)对MBG(介孔生物玻璃)进行物相分析；采用氮气吸附脱附(ASAP2010N,Micrometrics Instrument Corp.,USA)对MBG的介孔进行分析，根据Barrett-Joyner-Helen(BJH)公式计算平均孔径；采用材料万能性能试验机(AG-2000A,Shimadzu,Japan)测量其抗压强度(载荷速度1mm/min)。
(3)外泌体缓释速率测定
将exo-MBG支架置于离心管中，以1:10质量比加入PBS并置于37℃30rpm的恒温振荡箱内，每隔一段时间取出上清液并补充等量新鲜PBS，取出的缓释液无菌密封保存于4℃冰箱内待所有时间点取样完成后测试。通过NTA测定各时间点缓释液中的外泌体浓度，绘制释放曲线。
MBG支架的理化性能表征如图3所示，exo-MBG支架的外泌体形貌、外泌体释放曲线、粒径分析和ALP活性定量结果如图4所示。图4中结果表明支架微孔结构对外泌体形态和活性起保护作用，证明了冻干递送外泌体的多级微纳结构骨修复支架的具有显著提高的体内骨缺损修复效果。
实施例4支架应用于体内骨缺损修复的治疗效果。
将SD大鼠(8周龄，雌性，体重150–180g)按40mg/kg戊巴比妥钠的剂量腹腔注射进行麻醉，头顶手术部位采用盐酸利多卡因进行局部麻醉。将大鼠头部剃毛并碘伏消毒后，头顶中央纵向切开皮肤约8mm，用直径为5mm的环钻颅骨上创建两个圆形全层骨缺损，但保持硬脑膜完整，钻孔过程中使用无菌生理盐水冲洗钻头以免局部温度过高。植入支架材料后缝合。手术后，按分组分笼饲养，允许动物自由获得食物和水，每日监测潜在的并发症或异常行为。在术后2周/5周/8周时，每组取1只实验动物肌肉注射四环素(TE，25mg/kg)/茜素红(AR，30mg/kg)/钙黄绿素(CA，20mg/kg)，连续注射2天。术后12周，腹腔注射5倍剂量的麻醉剂处死，取出颅骨样品进行后续表征。
micro-CT扫描(PerkinElmer Quantum GX，美国)：扫描电压和电流分别设置为90kV和88μA，视野范围(FOV)25mm，立体像素50μm、高分辨率模式。扫描后使用Analyze 12.0(PerkinElmer)软件对数据进行重建和分析，设置阈值范围3000/8500(min/max)将骨组织从软组织中分离出来。选择缺损部位作为感兴趣区(ROI)用于测定骨体积/总体积(BV/TV)和骨小梁厚度(Tb.Th.)。
硬组织切片：样品浸泡4％多聚甲醛固定7天后，采用梯度乙醇溶液逐级脱水。将脱水样品嵌入
7200 VLC树脂中，并使用金刚石带锯(EXAKT，德国)切割成约150μm厚的薄片，然后研磨并抛光至最终厚度～40μm。
顺序荧光染色观察：采用激光共聚焦显微镜观察各个实验组硬组织切片的顺序荧光染色。三种染料所对应的激发/发射波长分别为：四环素405/580nm，茜素红543/617nm，钙黄绿色488/517nm。所得荧光图像用ImageJ软件进行定量分析，测定缺损区域不同染料荧光标记的面积，以及不同染料荧光条带之间的距离，分别代表对应时间点的新骨矿化沉积量，以及两个时间点之间的新骨矿化沉积速率。
VG染色：使用Van Gieson苦味酸品红溶液(1％的酸性品红水溶液和苦味酸饱和溶液按照1:9的比例混合)对切片进行染色2分钟，乙醇清洗2次。染色完成后拍摄倒置显微镜观察骨组织再生情况。
石蜡包埋与切片：样品浸泡4％中性多聚甲醛溶液(29.01g/L Na2HPO4，2.96g/LNaH2PO4·2H2O，40g/L多聚甲醛，pH＝7.4)，置于4℃冰箱内固定1周后，浸泡10％EDTA使样品脱钙至样品完全软化。采用梯度乙醇浸泡的方法进行脱水，依次在50％、70％乙醇中浸泡90分钟，85％、95％乙醇中浸泡60分钟，100％乙醇中浸泡60分钟×2次，二甲苯浸泡30分钟×3次。将脱水后的样品放在模具中，60℃石蜡浸泡样品3小时后，冷却并包埋得到蜡块，将蜡块切成4.5μm厚度的切片。
HE/Masson三色染色：依次浸泡二甲苯30分钟×2次、无水乙醇2分钟×2次，90％和80％乙醇各2分钟，纯水5分钟。依次用苏木素、Biebrich Scarlet染料、1％磷钼酸、AnilineBlue(苯胺蓝)进行Masson三色染色；或依次用苏木素、伊红进行HE染色。依次浸泡无水乙醇2分钟×2次，二甲苯2分钟×2次，然后用盖玻片和树脂进行封片，用倒置显微镜进行观察和拍摄。
MBG和exo-MBG支架应用于体内骨缺损修复的治疗效果如图5所示。图5中结果表明冻干递送外泌体的多级微纳结构骨修复支架的具有显著提高的体内骨缺损修复效果。
Claims (10)
1.一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于包括以下步骤：
a.通过细胞培养上清液超离得到外泌体浓缩液；
b.将外泌体浓缩液吸附至具有大孔/微孔/介孔多级结构的MBG支架上并冷冻干燥。
2.根据权利要求1所述的一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于，步骤b为将具有大孔/微孔/介孔多级结构的介孔生物玻璃(MBG)支架灭菌，将外泌体浓缩液滴加至MBG支架上，静置使其在支架内部均匀分散并吸附入表面微孔内，冻干，得到冻干递送外泌体的多级微纳结构骨修复支架。
3.根据权利要求2所述的一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于，外泌体浓缩液以50-500μL/cm3的比例滴加至MBG支架上。
4.根据权利要求1所述的一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于，具有大孔/微孔/介孔多级结构的介孔生物玻璃(MBG)支架是通过溶剂挥发诱导自组装的溶胶凝胶法结合多模板法制备所得。
5.根据权利要求1所述的一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于，具有大孔/微孔/介孔多级结构的介孔生物玻璃(MBG)支架的制备方法为将6重量份MBG溶胶和1-3重量份的高分子微球(微孔模板剂)均匀混合后，将混合物以0.4-1g/cm3的质量体积比浇注在聚氨酯海绵(大孔模板)上，60℃烘干、550℃煅烧，得到具有大孔/微孔/介孔多级结构的MBG支架。
6.根据权利要求5所述的一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于，MBG溶胶的制备方法为以正硅酸乙酯、四水硝酸钙、磷酸三乙酯为原料，F127为介孔模板剂，将介孔模板剂8重量份加入80重量份的乙醇中，盐酸调节pH小于2，待介孔模板剂充分溶解后，加入四水硝酸钙1.52重量份、磷酸三乙酯0.46重量份和正硅酸乙酯10.4重量份，40±1℃下搅拌反应24h后蒸发浓缩得到粘稠的MBG溶胶。
7.根据权利要求1所述的一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于，外泌体浓缩液通过细胞培养上清液过滤和超速离心所得。
8.根据权利要求7所述的一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于，外泌体浓缩液的制备方法为提取并培养细胞，当细胞融合度达到80-90％时，将细胞培养基替换为含2-10％无外泌体血清的条件培养基，继续培养24-48小时后，收集上清液，用0.22μm的滤器去除悬浮细胞和细胞碎片，然后依次以10000×g速率离心30分钟、100,000×g离心90分钟，去除上清液并将外泌体沉淀重悬于50μL PBS中。
9.根据权利要求8所述的一种冻干递送外泌体的多级微纳结构骨修复支架的制备方法，其特征在于，所述的细胞选自不同种属的间充质干细胞(骨髓来源、脂肪来源等)、巨噬细胞、中性粒细胞、内皮细胞等贴壁培养细胞；条件培养基选自成骨分化诱导条件培养基、成脂分化诱导条件培养基、包含M-CSF或其他生长因子的条件培养基等。
10.一种冻干递送外泌体的多级微纳结构骨修复支架，其特征在于，由权利要求1-9任一项所述的方法制备得到。
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