CN112980009B - 一种纳米复合多孔凝胶支架及其构建方法与应用 - Google Patents
一种纳米复合多孔凝胶支架及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一种纳米复合多孔凝胶支架及其构建方法与应用。本发明基于物理/化学双交联的方法,在水凝胶内部网络物理嵌合纳米材料,通过乳液模板法构建纳米复合多孔凝胶支架。并进一步利用核酸适体对纳米复合多孔凝胶支架进行功能化设计,从而构建可募集内源干细胞的纳米复合多孔凝胶支架,避免了目前干细胞递送治疗中涉及的细胞免疫源性、低存活率等问题;同时,结合黑磷纳米片并构建连通多孔结构,有效提高了水凝胶支架材料的骨诱导活性及骨整合性,预期可更好地满足骨缺损临床治疗需求。
Description
技术领域
本发明属于水凝胶支架领域,尤其涉及一种纳米复合多孔凝胶支架及其构建方法与应用。
背景技术
在单一水凝胶体系中添加第二种可交联的生物高分子组分,可构建双网络(DN)水凝胶。一般来说,DN水凝胶的其中一个交联网络作为刚性骨架,另一个网络作为韧性基质,两个相互均匀渗透的交联网络,可以综合两种不同凝胶材料的理化特性,从而改善或赋予单凝胶体系特定性能,包括机械性能、多孔性、降解性以及导电性等。水凝胶的贯通多孔结构对于骨再生修复中细胞的浸润、营养的渗透、废物的清除、组织的整合以及血管的长入是十分必要的。水凝胶的多孔结构可以为血管生成提供更充足的空间,改善细胞与凝胶体之间的相互作用,促进骨骼的向内生长,形成材料/骨的混合层,改善水凝胶与宿主骨之间的机械联锁,为这个关键界面提供了更大的机械稳定性。目前,制备多孔水凝胶支架的常用方法,包括冷冻干燥、颗粒浸出、相分离技术、乳液模板法等。利用冷冻干燥制备的多孔水凝胶,通常难以保持适当的结构稳定性和机械性能;颗粒浸出法所需使用的制孔剂通常难以完全去除,并且所得到的孔结构及孔隙连通性也较差;相分离技术过程通常较为繁琐,制备过程中所需使用有机溶剂难以完全去除,从而会存在生物安全性的问题。利用明胶制备新型的水包空气乳液模板以构建的多孔复合水凝胶体系,过程简单经济,无需加入任何有机溶剂,可以有效避免有机溶剂对细胞活性的影响,十分有利于水凝胶材料在组织工程中的应用。
二维黑磷纳米材料(BPNSs)作为新型的二维纳米活性材料,具有良好的生物矿化及骨诱导活性[Liu X,Miller A L,2nd,Park S,et al.Two-Dimensional BlackPhosphorus and Graphene Oxide Nanosheets Synergistically Enhance CellProliferation and Osteogenesis on 3D Printed Scaffolds[J].ACS Appl MaterInterfaces,2019,11(26):23558-23572.],但裸露的BPNSs易于降解且有一定的细胞毒性。将BPNSs与水凝胶材料的结合,有利于解决其各自在骨再生修复中所面临的问题[Wang Z,Zhao J,Tang W,et al.Multifunctional Nanoengineered Hydrogels Consisting ofBlack Phosphorus Nanosheets Upregulate Bone Formation[J].Small,2019,15(41):e1901560.]。
骨髓间充质干细胞(BMSCs)是一种骨源性细胞,具有自我更新能力和多潜能祖细胞的特点,可以分化为多种细胞类型。基于外源干细胞的植入体通常受到许多问题的限制,例如移植后存活率低、免疫排斥、耗时的体外细胞培养步骤以及可能的安全性和道德问题等。由于BMSCs细胞在软骨下骨、滑液和滑膜等其它关节组织中大量存在,利用BMSCs细胞向骨损伤部位迁移进行骨缺损修复,是一种有效的方式,因此通过原位招募内源性BMSCs细胞修复骨缺损受到了广泛关注[Chen W B,Sun Y Y,Gu X P,et al.Conditioned medium ofmesenchymal stem cells delays osteoarthritis progression in a rat model byprotecting subchondral bone,maintaining matrix homeostasis,and enhancingautophagy[J].Journal of Tissue Engineering and Regenerative Medicine,2019,13(9):1618-1628.]。核酸适体Apt19S可以代替生长因子用于干细胞的招募,其化学稳定性更好,不需要特定的结构构象就可以保持其生物活性[Hou Z,Meyer S,Propson N E,etal.Characterization and target identification of a DNAaptamer that labelspluripotent stem cells[J].Cell Res,2015,25(3):390-3.]。目前,将BPNSs联合Apt应用于双网络水凝胶的研究还未有报道。
发明内容
为解决凝胶支架孔隙率和力学性能不足问题,同时兼顾促成骨分化性能等生物学性能的问题,本发明基于物理/化学双交联的方法,在水凝胶内部网络物理嵌合纳米材料,通过乳液模板法构建纳米复合多孔凝胶支架。并进一步利用核酸适体对纳米复合多孔凝胶支架进行功能化设计,从而构建可募集内源干细胞的纳米复合多孔凝胶支架,所述物理交联是DNA聚合物碱基对之间的氢键所形成的高分子网络,所述化学交联是GelMA聚合物通过紫外光引发自由基聚合所形成的高分子网络。
本发明的目的通过下述方案实现。
一种纳米复合多孔凝胶支架的构建方法,包括以下步骤:
(1)将预溶液进行预聚合反应,得预聚物;所述预溶液中含有双键改性明胶(GelMA)、脱氧核苷酸(DNA)和光引发剂;
(2)将预聚物进行乳化反应,然后置于紫外光下交联,得到纳米复合多孔凝胶支架。
优选的,所述DNA在预溶液中的浓度为5-50mg/mL。
优选的,所述双键改性明胶在预溶液中的浓度为0.05-0.15g/mL。
优选的,所述乳化反应的搅拌速率为5000-20000rpm,搅拌时间30-60s。
进一步优选的,所述DNA在预溶液中的浓度为10-50mg/mL;所述乳化反应的搅拌速率为15000-20000rpm。
优选的,所述预溶液中含有双键改性明胶、脱氧核苷酸和光引发剂的PBS溶液。
优选的,所述光引发剂在预溶液中的浓度为0.005-0.015g/mL。
优选的,所述预聚合反应的温度为90℃-100℃,反应的时间为1-10min,搅拌速度为50-200rpm。
优选的,步骤(2)中,紫外光照的时间为1-5min,紫外光波长为365nm,强度为5-15mW。
优选的,所述光引发剂为光引发剂Irgacure 2959。
优选的,所述双键改性明胶的制备包括以下步骤:
(1)明胶在加热条件下,搅拌溶于PBS中;
(2)加入甲基丙烯酸酐,在加热条件下反应;
(3)将反应液转移到透析袋中,透析至反应物无小分子杂质;
(4)使用冻干机冻干透析液,制备干燥的GelMA;
所述透析的温度为35℃-45℃;所述双键改性明胶(GelMA)合成条件为明胶和甲基丙烯酸酐用量(g:mL)为1:0.5-1:4,反应温度45℃-65℃,透析膜截留分子量5000-10000,透析时间4-8d。
以上所述的构建方法制备的一种纳米复合多孔凝胶支架。
以上所述的一种纳米复合多孔凝胶支架在制备可募集内源干细胞的纳米复合多孔凝胶支架的应用,包括以下步骤:
(1)将双键改性明胶溶解于PBS溶液中;
(2)向步骤(1)所得溶液中依次加入DNA、光引发剂和纳米材料,搅拌至混合均匀得到预溶液;
(3)将预溶液进行90℃-100℃高温处理1-10min,搅拌速度为50-200rpm,高温处理后室温静置得到预聚物;
(4)将核酸适体加入预聚物中,搅拌至分散均匀;
(5)将预聚物转移至模具中,进行搅拌乳化,搅拌速率为5000-20000rpm,搅拌时间30~60s,紫外光引发自由基聚合,得到可募集内源干细胞的纳米复合多孔凝胶支架。
优选的,所述纳米材料为纳米黑磷材料(BPNSs),浓度为25-500ppm或锂藻土(Laponite),浓度0.5-3.0w/v%。
优选的,所述核酸适体为双键修饰的Apt19S。
优选的,所述可募集内源干细胞的纳米复合多孔凝胶支架制备条件:改性明胶GelMA浓度为5w/v%-15w/v%,DNA为5-50mg/mL,BPNSs浓度为50ppm-500ppm,Apt19S浓度为1μM-10μM,光引发剂Irgacure 2959浓度0.5w/v%-1.5w/v%,紫外光照1-5min,紫外光波长:365nm,强度:5-15mW。
本发明以含双键的改性明胶GelMA和DNA聚合物作为有机相,以BPNSs作为骨诱导活性组分,以Apt19S作为招募干细胞的活性分子,构建一种可募集内源干细胞的纳米复合多孔凝胶支架。
与现有技术相比,本发明具有如下优点:
(1)本发明基于物理/化学双交联,通过乳液模板法构建可募集内源干细胞的纳米复合多孔凝胶支架,不仅保证了骨修复在空间结构上所需的孔隙结构,同时赋予该多孔凝胶支架良好的干细胞募集性能,为核酸适体在骨再生医疗领域的研究应用提供了新的可能。
(2)本发明通过控制反应条件(DNA浓度和BPNSs浓度等),使所制备的可募集内源干细胞的纳米复合多孔凝胶支架在保持良好细胞相容性的同时具有良好的促细胞成骨分化的作用。
附图说明
图1为实施例1中GelMA凝胶体的SEM图片。
图2为实施例1中DNA/Gel多孔复合水凝胶支架的实物图。
图3为实施例2中DNA/Gel多孔复合水凝胶支架的SEM图片。
图4为实施例2中Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架的实物图。
图5为实施例3中所使用BPNSs的TEM图片。
图6为实施例4中DNA/Gel复合乳液及其交联后的凝胶支架照片。
图7为实施例4中Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架的SEM图片。
图8为实施例2中干细胞在DNA/Gel多孔复合水凝胶支架培养5天后的细胞活性效果图。
图9为实施例2中rBMSCs细胞在DNA/Gel多孔复合水凝胶支架和BP-DNA/Gel纳米复合多孔凝胶支架表面培养7天后OCN基因表达水平图。
图10为实施例2中利用Transwell实验表征Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架对rBMSCs细胞的趋化作用图。
具体实施方式
为了更好的理解本发明,下面结合实施例对本发明作进一步说明,但是本发明要求保护的范围并不局限于实施例所示的范围。
以下实施例采用Micro-CT扫描重建以分析水凝胶支架的孔隙率,采用动态热机械分析仪测试水凝胶支架的力学性能。
实施例1
步骤一:改性明胶(GelMA)合成
(1)将5g明胶加入100mL PBS溶液中,50℃下搅拌至溶解;
(2)按照投料比(明胶:甲基丙烯酸酐=1g:1.5mL)加入7.5mL甲基丙烯酸酐,50℃下反应2h;
(3)将反应液转移到截留分子量为5000的透析袋中,40℃透析7d;
(4)对预冻好的透析液进行冷冻干燥,即可得到所需的GelMA。
步骤二:脱氧核苷酸/改性明胶(DNA/Gel)多孔复合凝胶支架的制备
(1)将0.1g GelMA在50℃下溶解于1.0mL去离子水中得到GelMA溶液;
(2)依次加入10mg DNA(来源于鲑鱼睾丸的脱氧核糖核酸钠盐,Sigma-Aldrich,分子量约为~1.3×106g/mol),0.005g光引发剂Irgacure 2959,45℃下搅拌至混合均匀;
(3)对预溶液进行90℃高温处理5min,室温静置得到预聚物;
(4)将已物理交联的预聚物注射于模具中进行高速搅拌乳化(搅拌速率见表1),并置于紫外光下(365nm,5mW)交联5min,即可得到DNA/Gel多孔复合水凝胶支架。图1所示为GelMA凝胶体经冷冻干燥后的SEM照片,该孔隙结构是由于冷冻干燥、冰晶挥发所导致的。图2所示为DNA/Gel多孔复合水凝胶支架的实物图。
表1
由表1可以看出乳化速率在5000-20000rpm之间,所得多孔水凝胶具有较高的孔隙率,多孔水凝胶支架的压缩模量主要受材料组分的影响,乳化速率的影响不明显。未经乳化(0rpm)复合预聚物是无法制备具有多孔结构的水凝胶支架,乳化速度过低(2000rpm),复合预聚物的乳化效果不佳,水凝胶支架的孔隙率较低。
步骤三:核酸适体功能化BP-DNA/Gel纳米复合多孔凝胶支架的构建
(1)将0.15g GelMA在50℃下溶解于1.0mL去离子水中得到GelMA溶液;
(2)依次加入10mg DNA(来源于鲑鱼睾丸的脱氧核糖核酸钠盐,Sigma-Aldrich,分子量约为~1.3×106g/mol),500μL 600ppm BPNSs、0.005g光引发剂Irgacure 2959,45℃下搅拌至混合均匀。
(3)对预溶液进行90℃高温处理5min,室温静置得到预聚物;
(4)将2μM Apt19S加入预聚物中充分搅拌至均匀分散。之后注射于模具中进行高速搅拌乳化,并置于紫外光下(365nm,5mW)交联10min固化得到Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架。
实施例2
步骤一:改性明胶(GelMA)合成
(1)将5g明胶加入100mL PBS溶液中,50℃下搅拌至溶解;
(2)按照投料比(明胶:甲基丙烯酸酐=1g:2mL)加入10mL甲基丙烯酸酐,50℃下反应1h;
(3)将反应液转移到截留分子量为7000的透析袋中,40℃透析7d;
(4)对预冻好的透析液进行冷冻干燥,即可得到所需的GelMA。
步骤二:脱氧核苷酸/改性明胶(DNA/Gel)多孔复合凝胶支架的制备
(1)将0.1g GelMA在50℃下溶解于1.0mL去离子水中得到GelMA溶液;
(2)依次加入10mg DNA(来源于鲑鱼睾丸的脱氧核糖核酸钠盐,Sigma-Aldrich,分子量约为~1.3×106g/mol),0.005g光引发剂Irgacure 2959,45℃下搅拌至混合均匀;
(3)对预溶液进行95℃高温处理5min,室温静置物理交联;
(4)将已物理交联的预聚物注射于模具中进行高速搅拌乳化,并置于紫外光下(365nm,5mW)交联5min,即可得到DNA/Gel多孔复合水凝胶支架。图3所示为DNA/Gel多孔复合水凝胶支架的SEM照片,可以观察到明显的孔洞及孔窗结构。
步骤三:核酸适体功能化BP-DNA/Gel纳米复合多孔凝胶支架的构建
(1)将0.15g GelMA在50℃下溶解于1.0mL去离子水中得到GelMA溶液;
(2)依次加入30mg DNA(来源于鲑鱼睾丸的脱氧核糖核酸钠盐,Sigma-Aldrich,分子量约为~1.3×106g/mol),500μL 600ppm BPNSs、0.0075g光引发剂Irgacure 2959,45℃下搅拌至混合均匀;
(3)对预溶液进行95℃高温处理5min,室温静置得到预聚物;
(4)将5μM Apt19S加入预聚物中充分搅拌至均匀分散。之后注射于模具中进行高速搅拌乳化,并置于紫外光下(365nm,5mW)交联5min固化得到Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架。图4所示为Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架的实物图。
实施例3
步骤一:改性明胶(GelMA)合成
(1)将5g明胶加入100mL PBS溶液中,50℃下搅拌至溶解;
(2)按照投料比(明胶:甲基丙烯酸酐=1g:1mL)加入5mL甲基丙烯酸酐,50℃下反应3h;
(3)将反应液转移到截留分子量为10000的透析袋中,40℃透析7d;
(4)对预冻好的透析液进行冷冻干燥,即可得到所需的GelMA。
步骤二:脱氧核苷酸/改性明胶(DNA/Gel)多孔复合凝胶支架的制备
(1)将0.08g GelMA在50℃下溶解于1.0mL去离子水中得到GelMA溶液;
(2)依次加入如表2中用量的DNA(来源于鲑鱼睾丸的脱氧核糖核酸钠盐,Sigma-Aldrich,分子量约为~1.3×106g/mol),0.015g光引发剂Irgacure 2959,45℃下搅拌至混合均匀;
(3)对预溶液进行95℃高温处理1min,室温静置得到预聚物;
(4)将预聚物注射于模具中进行高速搅拌乳化,并置于紫外光下(365nm,5mW)交联1min,即可得到DNA/Gel多孔复合凝胶支架。
表2
由表2可以看出DNA浓度在5-50mg/mL范围内,水凝胶支架具有良好的力学性能及较高的孔隙分布。相比于不含有DNA的水凝胶支架,DNA的引入显著增强了水凝胶支架的力学强度。DNA浓度过高(60mg/mL)会导致体系粘度过大,而无法形成均匀多孔结构,孔隙率及力学强度有所下降。
步骤三:核酸适体功能化BP-DNA/Gel纳米复合多孔凝胶支架的构建
(1)将0.16g GelMA在50℃下溶解于1.0mL去离子水中得到GelMA溶液;
(2)依次加入80mg DNA(来源于鲑鱼睾丸的脱氧核糖核酸钠盐,Sigma-Aldrich,分子量约为~1.3×106g/mol),1.0mL 200ppm BPNSs、0.03g光引发剂Irgacure 2959,45℃下搅拌至混合均匀。图5所示为所使用BPNSs的TEM图片;
(3)对预溶液进行95℃高温处理1min,室温静置得到预聚物;
(4)将1μM Apt19S加入预聚物中充分搅拌至均匀分散。之后注射于模具中进行高速搅拌乳化,并置于紫外光下(365nm,5mW)交联2min固化得到Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架。
实施例4
步骤一:改性明胶(GelMA)合成
(1)将5g明胶加入100mL PBS溶液中,50℃下搅拌至溶解;
(2)按照投料比(明胶:甲基丙烯酸酐=1g:2mL)加入10mL甲基丙烯酸酐,50℃下反应1h;
(3)将反应液转移到截留分子量为7000的透析袋中,40℃透析7d;
(4)对预冻好的透析液进行冷冻干燥,即可得到所需的GelMA。
步骤二:脱氧核苷酸/改性明胶(DNA/Gel)多孔复合凝胶支架的制备
(1)将0.1g GelMA在50℃下溶解于2.0mL去离子水中得到GelMA溶液;
(2)依次加入5mg DNA(来源于鲑鱼睾丸的脱氧核糖核酸钠盐,Sigma-Aldrich,分子量约为~1.3×106g/mol),0.01g光引发剂Irgacure 2959,45℃下搅拌至混合均匀;
(3)对预溶液进行95℃高温处理5min,室温静置物理交联;
(4)将已物理交联的预聚物注射于模具中进行高速搅拌乳化,并置于紫外光下(365nm,5mW)交联5min,即可得到DNA/Gel多孔复合水凝胶支架。图6所示为DNA/Gel复合乳液照片及其交联后的多孔凝胶支架。
步骤三:核酸适体功能化BP-DNA/Gel纳米复合多孔凝胶支架的构建
(1)将0.15g GelMA在50℃下溶解于2.0mL去离子水中得到GelMA溶液;
(2)依次加入15mg DNA(来源于鲑鱼睾丸的脱氧核糖核酸钠盐,Sigma-Aldrich,分子量约为~1.3×106g/mol),1mL 600ppm BPNSs、0.015g光引发剂Irgacure 2959,45℃下搅拌至混合均匀;
(3)对预溶液进行95℃高温处理5min,室温静置得到预聚物;
(4)将5μM Apt19S加入预聚物中充分搅拌至均匀分散。之后注射于模具中进行高速搅拌乳化,并置于紫外光下(365nm,5mW)交联5min固化得到Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架。
本发明首次将纳米黑磷(BPNSs)及分子适体(Apt)联合应用于多孔复合水凝胶支架中。相比于密实无孔水凝胶材料,Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架可以为细胞增殖提供了更充足的空间,Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架的SEM图如图7所示。Apt对纳米复合多孔凝胶支架的功能化设计为支架高效募集内源干细胞提供了可能,BPNSs通过物理嵌合于该多孔复合水凝胶支架中显著增强了支架的骨诱导活性。如图8所示为rBMSCs细胞在DNA/Gel多孔复合水凝胶支架上培养5天后细胞活死染色结果,多孔复合水凝胶支架表面无明显的死细胞,细胞均匀分布在多孔复合水凝胶支架表面及内部。图9所示为rBMSCs细胞在DNA/Gel多孔复合水凝胶支架(Control)和BP-DNA/Gel纳米复合多孔凝胶支架上培养7天后,OCN基因的mRNA表达水平。图10所示为Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架对rBMSCs细胞趋化作用的定量结果。以上研究结果表明Apt功能化BP-DNA/Gel纳米复合多孔凝胶支架能够有效刺激干细胞的迁移及定向成骨分化。
Claims (5)
1.一种纳米复合多孔凝胶支架在制备可募集内源干细胞的纳米复合多孔凝胶支架的应用,其特征在于,包括以下步骤:
(1)将双键改性明胶溶解于PBS溶液中;
(2)向步骤(1)所得溶液中依次加入DNA、光引发剂和纳米材料,搅拌至混合均匀得到预溶液;所述纳米材料为纳米黑磷材料;
(3)将预溶液进行90℃-100℃高温处理1-10 min,搅拌速度为50-200 rpm,高温处理后室温静置得到预聚物;
(4)将核酸适体加入预聚物中,搅拌至分散均匀;所述核酸适体为双键修饰的Apt19S;
(5)将预聚物转移至模具中,进行搅拌乳化,搅拌速率为5000-20000rpm,搅拌时间30~60s,再紫外光引发自由基聚合,得到可募集内源干细胞的纳米复合多孔凝胶支架;
所述DNA在预溶液中的浓度为5-50 mg/mL。
2.根据权利要求1所述的应用,其特征在于,所述双键改性明胶在预溶液中的浓度为0.05-0.15 g/mL。
3.根据权利要求1-2任一项所述的应用,其特征在于,所述DNA在预溶液中的浓度为10-50 mg/mL;所述乳化的搅拌速率为15000-20000rpm。
4.根据权利要求3所述的应用,其特征在于,步骤(5)中,紫外光照的时间为1-5 min,紫外光波长为365 nm,强度为5-15 mW。
5.根据权利要求4所述的应用,其特征在于,所述光引发剂为光引发剂Irgacure 2959;所述光引发剂在预溶液中的浓度为0.005-0.015 g/mL。
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