CN116688224A - 促进骨软骨再生的复合水凝胶支架及其制备方法和应用 - Google Patents
促进骨软骨再生的复合水凝胶支架及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了促进骨软骨再生的复合水凝胶支架，包括MAA‑CM‑10和KGN。本发明将CM‑10经甲基丙烯酸改性，一方面较容易地将其锚定在可光交联的水凝胶或组织工程支架如甲基丙烯酰化明胶中，另一方面，由于CM‑10的牢牢铆合，可以减少其突释并能长期发挥促骨软骨再生作用。本发明通过脂质体包载Kartogenin后与MAA‑CM‑10的联合使用也具有类似作用，可以通过RUNX家族信号通路协同促进骨软骨再生。
Description
技术领域
本发明涉及促进骨软骨再生的复合水凝胶支架及其制备方法和应用。
背景技术
目前骨软骨损伤修复技术方案包括微骨折术、细胞移植、软骨移植、基因治疗及组织工程技术等。其中，组织工程技术是研究热点，也是骨软骨再生最有潜力的技术。TGF-β1是目前在骨软骨再生中常用的生长因子，但存在免疫反应、半衰期短、价格昂贵、容易变性等缺点，因此寻找新的促骨软骨生长因子一直是骨软骨组织工程的研究热点。近年来，生物活性肽由于其具有保质期长、稳定性好、成本及免疫原性低和药物传递容易等优点，在组织工程中的应用愈加广泛。细胞调节蛋白(Cytomudulin，CM)是最近发现的类TGF-β的短肽家族，研究表明其可以通过与TGF-β类似的作用机制促进细胞基质的表达，也可诱导MSCs的软骨分化。KGN是一种小分子化合物，合成简单且无抗原性，是目前研究热门的软骨诱导刺激因子。有文献显示TGF-β1联合Kartogenin可协同促进骨软骨再生，协同作用机制可能与RUNX家族有关。
微骨折术、细胞移植、软骨移植等技术虽可用于软骨再生，但存在修复效果差、免疫反应、供体源有限等缺点。基因治疗虽已成功用于软骨损伤修复，但由于其仍存在安全性和治疗效果的不确定性，还需大量试验去进一步评估。组织工程是目前骨软骨损伤修复的重要技术。TGF-β1是目前常用的促骨软骨再生的生长因子，但存在免疫反应、半衰期短、价格昂贵、容易变性等缺点。此外，由于其与支架材料之间大多是依靠单纯的物理结合，无法被牢牢锚定在组织工程支架中而导致生长因子短时间的快速释放，副作用大，且很难维持长效的作用。CM-10是细胞调节蛋白的家族成员之一，具有稳定性好、易于保存、性价比高、免疫反应性低等优点。有研究显示其在促骨软骨再生方面有类似TGF-β1的作用，但其在水溶性条件下作用不明显。
发明内容
针对现有技术存在的不足，本专利将CM-10经甲基丙烯酸(Methacrylic acid，MAA)改性(MAA-CM-10)，一方面较容易地将其锚定在可光交联的水凝胶或组织工程支架如甲基丙烯酰化明胶(Gelatin Methacryloyl，GelMA)中，另一方面由于CM-10的牢牢铆合，可以减少其突释并能长期发挥促骨软骨再生作用。有文献显示TGF-β1联合Kartogenin可协同促进骨软骨再生，本申请通过脂质体包载Kartogenin后与MAA-CM-10的联合使用也具有类似作用，可以通过RUNX家族信号通路协同促进骨软骨再生。
为实现上述目的，本发明提供了如下技术方案：促进骨软骨再生的复合水凝胶支架，包括MAA-CM-10和KGN。
作为优选，MAA-CM-10和KGN的质量比为20:1～5:1。
作为优选，还包括水凝胶或组织工程支架。
作为优选，MAA-CM-10、水凝胶或组织工程支架的质量比为1:40～1:50。
作为优选，所述组织工程支架包括GelMA、甲基丙烯酰化透明质酸、甲基丙烯酰化壳聚糖或甲基丙烯酰化葡聚糖。
上述所述的促进骨软骨再生的复合水凝胶支架的制备方法，包括如下步骤：
1)、将卵磷脂、胆固醇和KGN溶入有机溶液中，使三者充分混合溶解，去除有机溶剂后得到固含物；
2)、用PBS对步骤1)所述的固含物进行水化再超声，然后通过聚碳酸酯膜进行过滤，得到负载KGN的脂质体Lipo@KGN；
3)、将MAA-CM-10、Lipo@KGN和BMSCs混入GelMA溶液中，获得水凝胶混合原液，通过光交联形成负载BMSCs的复合水凝胶支架GelMA@CM-10@Lipo@KGN；
4)、将步骤3)所述的复合水凝胶支架GelMA@CM-10@Lipo@KGN通过不完全软骨诱导培养基进行培养，BMSCs被诱导成软骨小球。
作为优选，所述有机溶剂包括三氯甲烷或甲醇。
上述所述的制备方法所制备的复合水凝胶支架。
上述所述的复合水凝胶支架在促进骨软骨再生方面的应用。
综上所述，本发明具有以下有益效果：
1、将负载BMSCs的复合水凝胶支架通过不完全软骨培养基培养21天，可成功将BMSCs诱导成软骨小球，通过不同组别(GelMA、GelMA@CM-10、GelMA+TGF-β1、GelMA@CM-10@Lipo@KGN、GelMA@Lipo@KGN+TGF-β1)之间的比较，结果显示，CM-10能在骨软骨再生方面与TGF-β1有类似作用，且加入KGN能协同促骨软骨再生；
2、CM-10作为一种TGF-β1模拟肽，它相比TGF-β1，存在稳定性好、易于保存、性价比高、免疫原性较低等优点，且有TGF-β1类似的骨软骨再生作用；将它联合KGN，结果显示，也能协同促骨软骨再生；
3、传统的3D培养，大部分以细胞球培养为主，该专利技术采用多孔性水凝胶支架进行BMSCs的3D培养，且进行软骨诱导，结果显示，软骨诱导效果良好；
4、通过多孔性水凝胶支架进行培养，相比传统的细胞球3D培养技术，能诱导出更大的软骨小球，且可用于动物模型任何形状及大小的缺损部位的填充及修复；
5、将该水凝胶混合原液注射到大鼠股骨髁间骨软骨缺损模型中，光交联30s形成一完全填充缺损的骨软骨再生水凝胶支架，通过6周和12周大体观观察、Micro-CT扫描和切片染色等评估骨软骨损伤修复效果，结果显示，CM-10能明显促进骨软骨再生，加入KGN后可协同促骨软骨再生；
6、此外，CM-10与GelMA通过C键结合铆合在支架中可达到一个长效稳定的促骨软骨再生作用，相比TGF-β1，在体内产生免疫反应的可能性明显降低，且在体内更稳定，不容易被快速释放分解掉；
7、Lipo@KGN通过非共价作用锚定在水凝胶支架中，使KGN达到缓释的作用，从而获得一个长效稳定的协同促骨软骨再生作用；
8、该复合水凝胶支架体内可塑性强，可用于动物模型任何形状及大小的缺损部位的填充及修复；同时循环压缩实验显示，反复的压缩后，力学强度无明显变化，这说明在反复力学强度刺激下，水凝胶支架内部结构不受破坏，因此在体内某些存在反复力学强度刺激的特殊部位，同样可以应用。
附图说明
图1为本发明专利应用于骨软骨再生研究的实验流程图；
图2为不同组别通过3D细胞培养形成软骨小球情况；
图3为不同组别3D细胞培养后提取RNA通过PCR技术检测的Aggrecan表达情况；
图4为不同组别3D细胞培养后提取RNA通过PCR技术检测Col2a1的表达情况；
图5为动物实验大鼠股骨髁间骨软骨缺损不同组别之间的骨软骨修复情况；
图6为体外3D细胞培养诱导成软骨小球，冰冻切片行番红染色结果；
图7为动物实验大鼠股骨髁间骨软骨缺损不同组别之间的软骨下骨修复情况；
图8为本发明的软骨小球的示意图。
图9为水凝胶支架是由前体溶液经紫外照射成胶，可以在不同的模具中形成不同形状的水凝胶支架，可塑性强；
图10为GelMA@CM-10@Lipo@KGN(循环压缩实验)。
具体实施方式
参照附图对本发明做进一步说明。
本实施例公开了促进骨软骨再生的复合水凝胶支架，其基于类TGF-β1模拟肽联合Kartogenin协同促进骨软骨再生的复合水凝胶支架，包括MAA-CM-10和KGN，且两者的质量比为20:1～5:1。
在一些技术方案中，复合水凝胶支架还包括水凝胶或组织工程支架，且MAA-CM-10、水凝胶或组织工程支架的质量比为1:40～1:50。其中，组织工程支架包括GelMA、甲基丙烯酰化透明质酸(HAMA)、甲基丙烯酰化壳聚糖(CSMA)、甲基丙烯酰化葡聚糖(DexMA)。
上述所述的促进骨软骨再生的复合水凝胶支架的制备方法，包括如下步骤：
1)、将卵磷脂、胆固醇和KGN溶入有机溶液中，使三者充分混合溶解，去除有机溶剂后得到固含物；其中，有机溶剂包括三氯甲烷或甲醇；
2)、用PBS对步骤1)所述的固含物进行水化再超声，然后通过聚碳酸酯膜进行过滤，得到负载KGN的脂质体Lipo@KGN；
3)、将MAA-CM-10、Lipo@KGN和BMSCs混入GelMA溶液中，获得水凝胶混合原液，通过光交联形成负载BMSCs的复合水凝胶支架GelMA@CM-10@Lipo@KGN；
4)、将步骤3)所述的复合水凝胶支架GelMA@CM-10@Lipo@KGN通过不完全软骨诱导培养基进行培养，BMSCs被诱导成规则的软骨小球。
上述所述的制备方法所制备的复合水凝胶支架，其在促进骨软骨再生方面的应用。
具体实施例：
实施例1：
1.将BMSCs混入5％GelMA溶液中，通过光交联30s，形成负载BMSCs的GelMA水凝胶支架(BMSCs(107/ml)、GelMA(5％))。
2.将该水凝胶支架通过不完全软骨诱导培养基(不含TGF-β1)培养21天，BMSCs在支架中不能被诱导成软骨小球。
3.将该水凝胶混合原液注射到大鼠股骨髁间骨软骨缺损模型中，光交联30s，可形成一完全填充缺损的骨软骨再生水凝胶支架，通过6周和12周观察骨软骨修复效果。
结果：通过3D细胞培养形成软骨小球情况如图2所示；3D细胞培养后提取RNA通过PCR技术检测Aggrecan和Col2a1的表达情况如图3和图4所示；动物实验大鼠股骨髁间骨软骨缺损的骨软骨修复情况如图5所示。
实施例2：
1.将CM-10甲基丙烯酸化为MAA-CM-10(MAA-CM-10购买于强耀生物公司，Purity:98.15％)；
2.将MAA-CM-10(12.5mg/ml)和BMSCs混入GelMA溶液中，通过光交联30s，形成负载BMSCs的复合水凝胶支架(GelMA@CM-10,最终浓度：MAA-CM-10(1.25mg/ml)、BMSCs(107/ml)、GelMA(5％))。
3.将该水凝胶支架通过不完全软骨诱导培养基(不含TGF-β1)培养21天，BMSCs在支架中可被诱导成不规则软骨小球。
4.将该水凝胶混合原液注射到大鼠股骨髁间骨软骨缺损模型中，光交联30s，可形成一完全填充缺损的骨软骨再生水凝胶支架，通过6周和12周观察骨软骨修复效果。
结果：通过3D细胞培养形成软骨小球情况如图2所示；3D细胞培养后提取RNA通过PCR技术检测Aggrecan和Col2a1的表达情况如图3和图4所示；动物实验大鼠股骨髁间骨软骨缺损的骨软骨修复情况如图5所示。
实施例3：
1.将BMSCs混入5％GelMA溶液中，通过光交联30s，形成负载BMSCs的GelMA水凝胶支架(BMSCs(107/ml)、GelMA(5％))。
2.将该水凝胶支架通过完全软骨诱导培养基(含TGF-β1)培养21天，BMSCs在支架中能被诱导成不规则软骨小球。
结果：通过3D细胞培养形成软骨小球情况如图2所示；3D细胞培养后提取RNA通过PCR技术检测Aggrecan和Col2a1的表达情况如图3和图4所示；动物实验大鼠股骨髁间骨软骨缺损的骨软骨修复情况如图5所示。
实施例4：
1.将CM-10甲基丙烯酸化为MAA-CM-10(Purity:98.15％)；
2.采用薄膜分散法，将卵磷脂(80mg)、胆固醇(20mg)和KGN(3mg)溶入三氯甲烷溶液中，充分混合溶解后，在旋转蒸发仪中，以100r/min和35℃的速度蒸发，去除有机溶剂后，在瓶底形成一层薄膜，然后用PBS进行水化再超声，最后依次通过孔径为0.45μm和0.22μm的聚碳酸酯膜过滤，即可得到孔径均匀的负载KGN的脂质体(Lipo@KGN)，KGN包封率为88.43％，最终浓度为0.34mg/ml。
3.将MAA-CM-10(12.5mg/ml)和Lipo@KGN(KGN：0.256mg/ml)和BMSCs混入GelMA溶液中，通过光交联30s，形成负载BMSCs的复合水凝胶支架(GelMA@CM-10@Lipo@KGN,最终浓度：MAA-CM-10(1.25mg/ml)、Lipo@KGN(KGN：0.102mg/ml)、BMSCs(107/ml)、GelMA(5％))。
4.将该水凝胶支架通过不完全软骨诱导培养基(不含TGF-β1)培养21天，BMSCs在支架中可被诱导成规则的软骨小球。
5.将该水凝胶混合原液注射到大鼠股骨髁间骨软骨缺损模型中，光交联30s，可形成一完全填充缺损的骨软骨再生水凝胶支架，通过6周和12周观察骨软骨修复效果。
结果：通过3D细胞培养形成软骨小球情况如图2所示；3D细胞培养后提取RNA通过PCR技术检测Aggrecan和Col2a1的表达情况如图3和图4所示；动物实验大鼠股骨髁间骨软骨缺损的骨软骨修复情况如图5所示。
实施例5：
1.采用薄膜分散法，将卵磷脂(80mg)、胆固醇(20mg)和KGN(3mg)溶入三氯甲烷溶液中，充分混合溶解后，在旋转蒸发仪中，以100r/min和35℃的速度蒸发，去除有机溶剂后，在瓶底形成一层薄膜，然后用PBS进行水化再超声，最后依次通过孔径为0.45μm和0.22μm的聚碳酸酯膜过滤，即可得到孔径均匀的负载KGN的脂质体(Lipo@KGN)，KGN包封率为88.43％，最终浓度为0.34mg/ml。
2.将Lipo@KGN(KGN：0.256mg/ml)和BMSCs混入GelMA溶液中，通过光交联30s，形成负载BMSCs的复合水凝胶支架(GelMA@Lipo@KGN,最终浓度：Lipo@KGN(KGN：0.102mg/ml)、BMSCs(107/ml)、GelMA(5％))。
4.将该水凝胶支架通过完全软骨诱导培养基(含TGF-β1)培养21天，BMSCs在支架中可被诱导成规则的软骨小球。
结果：通过3D细胞培养形成软骨小球情况如图2所示；3D细胞培养后提取RNA通过PCR技术检测Aggrecan和Col2a1的表达情况如图3和图4所示；动物实验大鼠股骨髁间骨软骨缺损的骨软骨修复情况如图5所示。
1、本申请成功制备了一种基于类TGF-β1模拟肽联合Kartogenin协同促进骨软骨再生的复合水凝胶支架；
2、本申请通过体内外实验，表明该支架中不仅CM-10有TGF-β1类似的促骨软骨再生作用，而且CM-10与KGN可协同促骨软骨再生；体外3D细胞培养诱导成软骨小球，冰冻切片行番红染色结果如图6所示，动物实验大鼠股骨髁间骨软骨缺损不同组别之间的软骨下骨修复情况如图7所示；
3、本申请通过该多孔性水凝胶支架可实现对BMSCs进行3D培养，能诱导出软骨小球；软骨小球如图8所示；
4、本申请的该复合水凝胶支架在体内可塑性强，且循环压缩力学强度没有明显下降，还能产生长效缓慢的促骨软骨再生作用。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.促进骨软骨再生的复合水凝胶支架，其特征是，包括MAA-CM-10和KGN。
2.根据权利要求1所述的促进骨软骨再生的复合水凝胶支架，其特征是，MAA-CM-10和KGN的质量比为20:1～5:1。
3.根据权利要求1或2所述的促进骨软骨再生的复合水凝胶支架，其特征是，还包括水凝胶或组织工程支架。
4.根据权利要求3所述的促进骨软骨再生的复合水凝胶支架，其特征是，MAA-CM-10、水凝胶或组织工程支架的质量比为1:40～1:50。
5.根据权利要求4所述的促进骨软骨再生的复合水凝胶支架，其特征是，所述组织工程支架包括GelMA、甲基丙烯酰化透明质酸、甲基丙烯酰化壳聚糖或甲基丙烯酰化葡聚糖。
6.权利要求5所述的促进骨软骨再生的复合水凝胶支架的制备方法，其特征是，包括如下步骤：
1)、将卵磷脂、胆固醇和KGN溶入有机溶液中，使三者充分混合溶解，去除有机溶剂后得到固含物；
2)、用PBS对步骤1)所述的固含物进行水化再超声，然后通过聚碳酸酯膜进行过滤，得到负载KGN的脂质体Lipo@KGN；
3)、将MAA-CM-10、Lipo@KGN和BMSCs混入GelMA溶液中，获得水凝胶混合原液，通过光交联形成负载BMSCs的复合水凝胶支架GelMA@CM-10@Lipo@KGN；
4)、将步骤3)所述的复合水凝胶支架GelMA@CM-10@Lipo@KGN通过不完全软骨诱导培养基进行培养，BMSCs被诱导成软骨小球。
7.根据权利要求6所述的制备方法，其特征是，所述有机溶剂包括三氯甲烷或甲醇。
8.权利要求6或7所述的制备方法所制备的复合水凝胶支架。
9.权利要求8所述的复合水凝胶支架在促进骨软骨再生方面的应用。
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| CN117138058A (zh) * | 2023-10-31 | 2023-12-01 | 吉林农业科技学院 | 用于修复骨缺损的脂质体、水凝胶及制备方法与应用 |
| CN117618665A (zh) * | 2023-12-01 | 2024-03-01 | 新乡医学院 | 一种双微环境三层仿生水凝胶支架及其制备方法和应用 |
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| CN117138058A (zh) * | 2023-10-31 | 2023-12-01 | 吉林农业科技学院 | 用于修复骨缺损的脂质体、水凝胶及制备方法与应用 |
| CN117138058B (zh) * | 2023-10-31 | 2024-02-06 | 吉林农业科技学院 | 用于修复骨缺损的脂质体、水凝胶及制备方法与应用 |
| CN117618665A (zh) * | 2023-12-01 | 2024-03-01 | 新乡医学院 | 一种双微环境三层仿生水凝胶支架及其制备方法和应用 |
| CN117618665B (zh) * | 2023-12-01 | 2024-05-31 | 新乡医学院 | 一种双微环境三层仿生水凝胶支架及其制备方法和应用 |
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