CN117298337A - 一种骨修复水凝胶支架及其制备方法 - Google Patents
一种骨修复水凝胶支架及其制备方法 Download PDFInfo
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- CN117298337A CN117298337A CN202311502182.8A CN202311502182A CN117298337A CN 117298337 A CN117298337 A CN 117298337A CN 202311502182 A CN202311502182 A CN 202311502182A CN 117298337 A CN117298337 A CN 117298337A
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- hydrogel
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- bone repair
- bone
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Abstract
本发明公开了一种骨修复水凝胶支架及其制备方法。其中所述骨修复水凝胶支架，其特征在于，包括以下浓度的组分：10wt％‑20wt％甲基丙烯酰化明胶、10wt％‑20％甲基丙烯酰化海藻酸钠、100‑200mM含钙交联剂及1～5wt％谷氨酰胺转氨酶。本发明通过三重交联提高水凝胶的强度，交联方式温和，有效提高了水凝胶支架的强度，能够促进骨髓间充质干细胞的成骨分化。材料的制备过程中不引入有机溶剂，制备的水凝胶材料生物相容性优异。且该材料可以通过光固化生物3D打印技术成型，具备定制化制备骨缺损填充材料的能力。本发明所制得的骨修复水凝胶支架可以完全降解，能够引导骨缺损部位的骨组织再生，替代原有的植入水凝胶材料。
Description
技术领域
本发明属于组织工程技术领域，具体地涉及一种骨修复水凝胶支架及其制备方法。
背景技术
骨缺损修复涉及到细胞成骨分化，可以分化为成骨细胞的细胞来源有骨髓间充质干细胞、骨祖细胞等。其中骨髓间充质干细胞的诱导分化涉及物理信号的刺激，例如培养介质的机械强度。研究表明培养介质的机械强度越高，骨髓间充质干细胞成骨分化的能力越强；反之则向脂肪或软骨分化。因此，构建具有高机械强度的细胞培养材料能够促进骨髓间充质干细胞向成骨分化。通常情况下，人工合成的、分子链较长的、具有高交联度的高分子材料构建的水凝胶具备更高的机械强度，因此一般选用人工合成的高分子作为骨缺损填充物。此外，还有一些生物相容性优异的金属材料，例如金属钛，也可通过3D打印的个性化定制手段构建骨缺损补片。
但人工合成高分子材料在制备过程中不可避免地会使用一些有毒有害的试剂，制备工艺复杂，且小分子溶剂残留易造成严重的危害，降低材料的生物相容性，甚至造成患者致敏、致畸等不良后果。金属材料在体内无法降解，始终只能作为骨替代物，无法促进自体骨再生。
因此，需要研制出一种材料制备简单、生物相容性优异、能完全降解的骨修复水凝胶支架及其制备方法。
发明内容
针对人工合成高分子材料制备复杂、金属材料无法降解等缺点，本发明提供了一种天然高分子材料构建的骨修复水凝胶支架及其制备方法。所述水凝胶支架利用天然高分子材料明胶和海藻酸钠构建水凝胶材料，用特定交联方式提高水凝胶的机械强度，以促进骨髓间充质干细胞的成骨分化。
本发明的技术目的是通过以下技术方案来实现的：
本发明提供一种骨修复水凝胶支架，其配方包括以下浓度的组分：10wt％-20wt％甲基丙烯酰化明胶、10wt％-20wt％甲基丙烯酰化海藻酸钠、100-200mM CaCl2及1～5wt％谷氨酰胺转氨酶。
进一步地，本发明所述甲基丙烯酰化明胶通过以下方法获得：在8wt％-12w％明胶溶液中加入6wt％-10wt％的甲基丙烯酸酐,透析MW:12000-14000Da，除去未反应的甲基丙烯酸酐后冻干收集即得。
进一步地，本发明所述甲基丙烯酰化海藻酸钠通过以下方法获得：在8wt％-12wt％海藻酸钠溶液中加入6wt％-10wt％的甲基丙烯酸酐反应，结束后透析MW:12000-14000Da，除去未反应的甲基丙烯酸酐后冻干收集即得。
进一步地，本发明所述的骨修复水凝胶支架还负载有一种或几种促进骨再生的药物。
进一步地，本发明所述促进骨再生的药物的配方为：80-120μg的甘草酸/表没食子儿茶素没食子酸酯脂质体、1-2mg/mL光引发剂及0.5-0.6mg/mL阻光剂。
进一步地，本发明所述甘草酸/表没食子儿茶素没食子酸酯脂质体通过以下方法制备：将卵磷脂、胆固醇溶于氯仿，溶解后加入甘草酸乙醇溶液，除去氯仿和乙醇，加入表没食子儿茶素没食子酸酯水溶液复溶薄膜，超声分散即得。
更进一步地，本发明所述卵磷脂、胆固醇、甘草酸、表没食子儿茶素没食子酸酯的质量比为90-100:15-20:10-20:0.05-0.15；优选地，氯仿与乙醇的体积比0.8-1.2：0.8-1.2。
优选地，本发明所述阻光剂为柠檬黄；优选地，所述光引发剂选自苯基(2,4,6-三甲基苯甲酰基)磷酸锂或Irgacure 2959；更优选地，所述光引发剂为苯基(2,4,6-三甲基苯甲酰基)磷酸锂。
本发明的另一技术目的是提供一种骨修复水凝胶支架的制备方法，包括以下步骤：将甲基丙烯酰化明胶溶液、甲基丙烯酰化海藻酸钠溶液混匀得生物墨水，将水凝胶支架打印成型，去除未固化的生物墨水，浸入含钙交联剂中交联，去除多余的含钙交联剂，浸入谷氨酰胺转氨酶溶液中继续交联即得；优选地，所述水凝胶支架打印采用EFL光固化打印机；优选地，所述EFL光固化打印机的条件为光强度：10～12mW/cm2、曝光时间：12～16s。
本发明的另一技术目的是提供一种骨修复水凝胶支架的制备方法，包括以下步骤：将甲基丙烯酰化明胶溶液、甲基丙烯酰化海藻酸钠溶液及甘草酸/表没食子儿茶素没食子酸酯脂质体混匀得生物墨水，将水凝胶支架打印成型，去除未固化的生物墨水，浸入含钙交联剂中交联，去除多余的含钙交联剂，浸入谷氨酰胺转氨酶溶液中继续交联即得；优选地，所述水凝胶支架打印采用EFL光固化打印机；优选地，所述EFL光固化打印机的条件为光强度：10～12mW/cm2、曝光时间：12～16s。
本发明相对于现有技术具有如下有益效果：
1)、制备过程不引入有机溶剂，材料的生物相容性好；
2)、通过三重交联提高水凝胶的强度，交联方式温和，水凝胶支架强度可有效提高，能够促进骨髓间充质干细胞的成骨分化；
3)、本发明采用3D打印技术，根据设计打印出适应于不同损伤的仿生骨组织结构支架，定制化程度更高。此外，所打印出的微孔结构支架，更加有利于细胞的粘附和迁移；
4)、本发明制备的水凝胶材料可完全降解，不会在体内残留，阻碍自体骨再生。
5)、本发明引入脂质体负载水不溶性药物甘草酸和EGCG，能够提高成骨分化效果。
附图说明
图1是本发明的AlgMA和Alg、GelMA和Gel的核磁氢谱图。
具体实施方式
本发明通过三重交联提高水凝胶的强度，交联方式温和，有效提高了水凝胶支架的强度，能够促进骨髓间充质干细胞的成骨分化。具体的交联方式为：1.甲基丙烯酰化的明胶和海藻酸钠通过碳碳双键进行第一重交联；2.利用钙离子与海藻酸钠进行第二重交联；3.利用转谷氨酰胺酶(mTG酶)通过谷氨酰胺的γ-羰基和甲基丙烯酰化明胶(GelMA)赖氨酸残基的ε-氨基的酯化进行第三重交联。所利用的三重交联方式温和，不影响蛋白质、酶等活性物质的存活，且Ca2+可以促进骨修复。材料的制备过程中不引入有机溶剂，制备的水凝胶材料生物相容性优异。且该材料可以通过光固化生物3D打印技术成型，具备定制化制备骨缺损填充材料的能力。本发明所制得的水凝胶支架可以完全降解，能够引导骨缺损部位的骨组织再生，替代原有的植入水凝胶材料。
此外，水凝胶还能作为药物储存库，缓释一种或几种促进骨再生的药物，更好地诱导骨再生。本发明同时研究了三重交联3D打印水凝胶负载表没食子儿茶素没食子酸酯(EGCG)和甘草酸(GA)，用于抑制骨组织炎症累积和抑制破骨细胞分化。综合治疗效果优于不负载药物的三重交联3D打印水凝胶。
为了更好地说明本发明的技术目的、技术方案和优点，现结合附图与具体实施例对本发明做进一步说明。
一、骨修复水凝胶支架的制备
一)、前处理
1)、改性明胶(GelMA)的合成
称取20g的明胶溶于400mL的蒸馏水中，充分溶解后，缓慢滴加12mL甲基丙烯酸酐，反应5小时。反应结束后透析(MW:12000-14000Da)三天，充分除去未反应的GMA后冻干收集即得。
2)、改性海藻酸钠(AlgMA)的合成
称取20g的海藻酸钠溶于400mL的蒸馏水中，充分溶解后，缓慢滴加12mL甲基丙烯酸酐，反应5小时。反应结束后透析(MW:12000-14000Da)三天，充分除去未反应的甲基丙烯酸酐后冻干收集即得。
3)、甘草酸/表没食子儿茶素没食子酸酯脂质体的制备
利用薄膜法制备脂质体：称取90mg的卵磷脂、15mg的胆固醇溶于19mL的氯仿，超声分散5min充分溶解后，加入1mL甘草酸乙醇溶液(20mg/mL)，使用旋转蒸发仪除去氯仿和乙醇，得到白色的薄膜。加入21mL EGCG水溶液(5μg/mL)复溶薄膜，超声分散15min(30％，开5s，关5s)，得到浓度为5mg/ml LIP@GA@EGCG脂质体溶液。
二)、骨修复水凝胶支架的制备
实施例1
实施例1的3D打印骨修复水凝胶支架包括以下浓度的组分：10％ GelMA、10％AlgMA、100mM CaCl2、1％谷氨酰胺转氨酶(mTG酶)
本实施例所述伤口敷料，包括以下步骤：
1).称取前处理所得0.1g GelMA溶于1mL去离子水，得到10％ GelMA溶液；
2).称取前处理所得0.1g AlgMA溶于1mL去离子水，得到10％ AlgMA溶液；
3).称取0.11g CaCl2溶于10mL去离子水，得到100mM CaCl2溶液；
4).称取0.1g mTG酶溶于10mL去离子水，得到1％mTG酶溶液；
5).将上述GelMA溶液和AlgMA溶液按体积比1：1混合均匀得到生物墨水，在EFL光固化打印机中将水凝胶打印成型，用PBS溶液清洗除去未固化的生物墨水，浸入100mMCaCl2溶液中，交联3min，得到双重交联后的支架，用PBS洗去多余的CaCl2溶液，浸入1％mTG酶溶液中，继续交联3min，得到三重交联后的支架。
实施例2
实施例2的3D打印骨修复水凝胶支架包括以下浓度的组分：10％ GelMA、10％AlgMA、100mM CaCl2、3％谷氨酰胺转氨酶(mTG酶)
本实施例所述伤口敷料，包括以下步骤：
1).称取前处理所得0.1g GelMA溶于1mL去离子水，得到10％ GelMA溶液；
2).称取前处理所得0.1g AlgMA溶于1mL去离子水，得到10％ AlgMA溶液；
3).称取0.11g CaCl2溶于10mL去离子水，得到100mM CaCl2溶液；
4).称取0.3g mTG酶溶于10mL去离子水，得到3％mTG酶溶液；
5).将上述GelMA溶液和AlgMA溶液按体积比1：1混合均匀得到生物墨水，在EFL光固化打印机中将水凝胶打印成型，用PBS溶液清洗除去未固化的生物墨水，浸入100mMCaCl2溶液中，交联3min，得到双重交联后的支架，用PBS洗去多余的CaCl2溶液，浸入3％mTG酶溶液中，继续交联3min，得到三重交联后的支架。
实施例3
实施例3的3D打印骨修复水凝胶支架包括以下浓度的组分：10％ GelMA、10％AlgMA、100mM CaCl2、5％谷氨酰胺转氨酶(mTG酶)
本实施例所述伤口敷料，包括以下步骤：
1).称取前处理所得0.1g GelMA溶于1mL去离子水，得到10％ GelMA溶液；
2).称取前处理所得0.1g AlgMA溶于1mL去离子水，得到10％ AlgMA溶液；
3).称取0.11g CaCl2溶于10mL去离子水，得到100mM CaCl2溶液；
4).称取0.5g mTG酶溶于10mL去离子水，得到5％mTG酶溶液；
5).将上述GelMA溶液和AlgMA溶液按体积比1：1混合均匀得到生物墨水，在EFL光固化打印机中将水凝胶打印成型，用PBS溶液清洗除去未固化的生物墨水，浸入100mMCaCl2溶液中，交联3min，得到双重交联后的支架，用PBS洗去多余的CaCl2溶液，浸入5％mTG酶溶液中，继续交联3min，得到三重交联后的支架。
实施例4
实施例4的3D打印骨修复水凝胶支架包括以下浓度的组分：10％ GelMA、10％AlgMA、100μg LIP@GA@EGCG脂质体、100mM CaCl2、3％谷氨酰胺转氨酶(mTG酶)
本实施例所述伤口敷料，包括以下步骤：
1).称取(前处理所得)0.1g GelMA溶于1mL去离子水，得到10％ GelMA溶液；
2).称取(前处理所得)0.1g AlgMA溶于1mL去离子水，得到10％ AlgMA溶液；
3).称取0.11g CaCl2溶于10mL去离子水，得到100mM CaCl2溶液；
4).称取0.3g mTG酶溶于10mL去离子水，得到3％mTG酶溶液；
5).将上述GelMA溶液、AlgMA溶液按体积比1：1混合后加入前处理所得20μLLIP@GA@EGCG脂质体(浓度为100μg/mL),混合均匀得到生物墨水，在EFL光固化打印机中将水凝胶打印成型，用PBS溶液清洗除去未固化的生物墨水，浸入100mM CaCl2溶液中，交联3min，得到双重交联后的支架，用PBS洗去多余的CaCl2溶液，浸入3％mTG酶溶液中，继续交联3min，得到三重交联后的支架。
实施例5
实施例5的3D打印骨修复水凝胶支架包括以下浓度的组分：10％ GelMA、10％AlgMA、100μg LIP@GA@EGCG脂质体、200mM CaCl2、3％谷氨酰胺转氨酶(mTG酶)
本实施例所述伤口敷料，包括以下步骤：
1).称取(前处理所得)0.1g GelMA溶于1mL去离子水，得到10％ GelMA溶液；
2).称取(前处理所得)0.1g AlgMA溶于1mL去离子水，得到10％ AlgMA溶液；
3).称取0.22g CaCl2溶于10mL去离子水，得到200mM CaCl2溶液；
4).称取0.3g mTG酶溶于10mL去离子水，得到3％mTG酶溶液；
5).将上述GelMA溶液、AlgMA溶液按体积比1：1混合后加入前处理所得20μLLIP@GA@EGCG脂质体(浓度为100μg/mL),混合均匀得到生物墨水，在EFL光固化打印机中将水凝胶打印成型，用PBS溶液清洗除去未固化的生物墨水，浸入200mM CaCl2溶液中，交联3min，得到双重交联后的支架，用PBS洗去多余的CaCl2溶液，浸入3％mTG酶溶液中，继续交联3min，得到三重交联后的支架。
实施例6
实施例6的3D打印骨修复水凝胶支架包括以下浓度的组分：10％ GelMA、10％AlgMA、100μg LIP@GA@EGCG脂质体、150mM CaCl2、3％谷氨酰胺转氨酶(mTG酶)
本实施例所述伤口敷料，包括以下步骤：
1).称取(前处理所得)0.1g GelMA溶于1mL去离子水，得到10％ GelMA溶液；
2).称取(前处理所得)0.1g AlgMA溶于1mL去离子水，得到10％ AlgMA溶液；
3).称取0.165g CaCl2溶于10mL去离子水，得到150mM CaCl2溶液；
4).称取0.3g mTG酶溶于10mL去离子水，得到3％mTG酶溶液；
5).将上述GelMA溶液、AlgMA溶液按体积比1：1混合后加入前处理所得20μLLIP@GA@EGCG脂质体(浓度为100μg/mL),混合均匀得到生物墨水，在EFL光固化打印机中将水凝胶打印成型，用PBS溶液清洗除去未固化的生物墨水，浸入150mM CaCl2溶液中，交联3min，得到双重交联后的支架，用PBS洗去多余的CaCl2溶液，浸入3％mTG酶溶液中，继续交联3min，得到三重交联后的支架。
对比例1
对比例1的3D打印骨修复水凝胶支架包括以下浓度的组分：10％ GelMA、10％AlgMA。
本实施例所述伤口敷料，包括以下步骤：
1).称取0.1g GelMA溶于1mL水，得到10％ GelMA溶液；
2).称取0.1g AlgMA溶于1mL水，得到10％ AlgMA溶液；
3).将上述GelMA溶液和AlgMA溶液按体积比1：1混合均匀得到生物墨水，在EFL光固化打印机中将水凝胶打印成型。
对比例2
对比例2的3D打印骨修复水凝胶支架包括以下浓度的组分：10％ GelMA、10％AlgMA、100mM CaCl2。
本实施例所述伤口敷料，包括以下步骤：
1).称取0.1g GelMA溶于1mL去离子水，得到10％ GelMA溶液；
2).称取0.1g AlgMA溶于1mL去离子水，得到10％ AlgMA溶液；
3).称取0.11g CaCl2溶于10mL去离子水，得到100mM CaCl2溶液；
4).将上述GelMA溶液和AlgMA溶液按体积比1：1混合均匀得到生物墨水，在EFL光固化打印机中将水凝胶打印成型，用PBS溶液清洗除去未固化的生物墨水，浸入100mMCaCl2溶液中，交联3min，得到双重固化的支架。
二、效果测试
1.核磁检测
如图1所示：NMR结果分析：4.98ppm处的氨基亚甲基峰(-CH2NH2)GelMA、AlgMA氢谱图的峰值均下降，由于氨基亚甲基峰是甲基丙烯酰基改性的结合位点，表明氨基亚甲基峰发生反应，甲基丙烯酰化增加，且在5.6和6.2ppm左右的反应中形成新的丙烯酸质子峰(＝CH2)，表明前处理的GelMA、AlgMA皆合成成功。
2.水凝胶的储能模量
表1流变学测得的水凝胶支架的储能模量(Pa)
在实施例1-6及对比例1-2的水凝胶支架完全交联后，用旋转流变仪对水凝胶支架的储能模量进行检测。检测结果如表1所示：实施例1-3的模量逐渐增强，代表mTG酶能够增强水凝胶的交联程度。实施例4的模量与实施例3相差不大，证明脂质体的添加不会影响水凝胶的机械性能。实施例5、6在增加氯化钙含量后，交联程度增大，储能模量进一步升高。对比例1-2表明钙离子交联也对水凝胶强度的提升有一定的作用。
3.溶胀性能测试
表2水凝胶支架的平衡溶胀率
水凝胶支架在进入体液环境时会发生不同程度的溶胀，合适的溶胀率对水凝胶结构的维持至关重要。从表2中可以看出溶胀率随交联程度的增加而降低，这是由于交联程度的增加所导致的支架网络结构的更加紧密。明显可以看出，对比例1的溶胀率高达70.61±4.37％，对比例2为光固化和钙离子交联双重交联有所降低至56.39±6.59％，而实施例1-3为三重交联的溶胀率随着mTG酶的浓度增加交联度也随之增加。实施例5、6因为钙离子的浓度增加，凝胶网络交联密度增加，导致溶胀率更低。
4.降解性能测试
表3水凝胶支架在体外降解28天后质量残留率(％)
表3为施例1-6及对比例1-2的水凝胶支架在有酶和无酶的PBS溶液条件下28天的降解结果。表3显示降解至28天时，有酶条件下的水凝胶基本完全降解，而无酶的水凝胶降解至90％以上,该数据说明该水凝胶支架有着良好的生物可降解性。
5.生物相容性
测试方法：依照《GB/T 16886.5-2017医疗器械生物学评价第5部分：体外细胞毒性试验》进行。具体操作如下：
将实施例1-6及对比例1-2所得水凝胶支架依照《GB/T 16886.5-2017医疗器械生物学评价第5部分：体外细胞毒性试验》规定的浸提方法对水凝胶浸提24小时，收集浸提液。以小鼠成纤维细胞系L929为试验对象，将其接种于96孔板，贴壁后换用浸提液培养72小时，随后利用CCK-8法测量吸光度，计算细胞存活率。
表4 L929细胞与三重交联高强度3D打印水凝胶培养后的细胞存活率
为确定该水凝胶支架的细胞毒性，用CCK8法对3T3细胞进行了定量评价。如表4所示，在培养72小时后，所有水凝胶支架联合培养组对3T3细胞的细胞毒性都很低。共培养后，所有组分的细胞存活率达85％以上，说明该水凝胶支架具有良好的生物相容性和促进细胞增殖的能力。
6.体外成骨分化能力检测
将实施例1-6及对比例1-2所得水凝胶支架依照《GB/T 16886.5-2017医疗器械生物学评价第5部分：体外细胞毒性试验》规定的浸提方法对水凝胶浸提24小时，收集浸提液。将骨髓间充质干细胞接种在48孔板上，用水凝胶浸提液连续培养14天。培养完成后用4％多聚甲醛对细胞进行固定、PBS清洗，最后用茜素红染色液进行染色。
表5骨髓间充质干细胞与水凝胶支架培养14天后的茜素红染色吸光度
如表5所示，茜素红染色结果表明，支架模量越高，茜素红染色的吸光度越高，表明越能促进BMSCs的成骨分化。
将实施例1-6及对比例1-2所得水凝胶支架依照《GB/T 16886.5-2017医疗器械生物学评价第5部分：体外细胞毒性试验》规定的浸提方法对水凝胶浸提24小时，收集浸提液。将骨髓间充质干细胞接种在48孔板上，连续培养14天。培养完成后收集细胞，用定量实时PCR对OCN和OPN的表达量进行检测。
表6骨髓间充质干细胞与水凝胶支架培养7天后的OCN和OPN表达量
为评估支架的成骨分化潜力，用支架的条件培养基培养BMSCs，加入1μmβ受体激动剂7天，然后通过定量实时PCR(qRT-PCR)检测成骨相关基因的mRNA表达水平。表6显示了成骨基因的表达水平，包括骨桥蛋白(OPN)和骨钙素(OCN)。培养7天后，实施例1-6与对比例1-2相比，实施例1-6与成骨分化相关的OPN和OCN的mRNA表达明显升高，但增量不同，从qRT-PCR分析结果来看，实施例4的成骨相关指标水平在所有组中最高。证明除了机械强度增强会影响成骨分化能力外，甘草酸和EGCG联合使用也会提高BMSCs在水凝胶上的成骨分化能力。
以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，除此之外，本发明还可以其它方式实现，尽管参照上述实施例对本发明进行了详细的说明，所属领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，但这些修改或替换均在本发明的保护范围之内。
Claims (10)
1.一种骨修复水凝胶支架，其特征在于，包括以下浓度的组分：10wt％-20wt％甲基丙烯酰化明胶、10wt％-20wt％甲基丙烯酰化海藻酸钠、100-200mM含钙交联剂及1～5wt％谷氨酰胺转氨酶；优选地所述含钙交联剂为CaCl2。
2.根据权利要求1所述的骨修复水凝胶支架，其特征在于，所述甲基丙烯酰化明胶通过以下方法获得：在8wt％-12w％明胶溶液中加入6wt％-10wt％的甲基丙烯酸酐,透析MW:12000-14000Da，除去未反应的甲基丙烯酸酐后冻干收集即得。
3.根据权利要求1所述的骨修复水凝胶支架，其特征在于，所述甲基丙烯酰化海藻酸钠通过以下方法获得：在8wt％-12wt％海藻酸钠溶液中加入6wt％-10wt％的甲基丙烯酸酐反应，结束后透析MW:12000-14000Da，除去未反应的甲基丙烯酸酐后冻干收集即得。
4.一种如权利要求1～3中任意一项所述的骨修复水凝胶支架，其特征在于，还负载有一种或几种促进骨再生的药物。
5.根据权利要求4所述的骨修复水凝胶支架，其特征在于，所述促进骨再生的药物的配方为：80-120μg的甘草酸/表没食子儿茶素没食子酸酯脂质体、1-2mg/mL光引发剂及0.5-0.6mg/mL阻光剂。
6.根据权利要求5所述的骨修复水凝胶支架，其特征在于，所述甘草酸/表没食子儿茶素没食子酸酯脂质体通过以下方法制备：将卵磷脂、胆固醇溶于氯仿，溶解后加入甘草酸乙醇溶液，除去氯仿和乙醇，加入表没食子儿茶素没食子酸酯水溶液复溶薄膜，超声分散即得。
7.根据权利要求6所述的骨修复水凝胶支架，其特征在于，所述卵磷脂、胆固醇、甘草酸、表没食子儿茶素没食子酸酯的质量比为90-100:15-20:10-20:0.05-0.15；优选地，氯仿与乙醇体积比0.8-1.2：0.8-1.2。
8.根据权利要求5所述的骨修复水凝胶支架，其特征在于，所述阻光剂为柠檬黄；优选地，所述光引发剂选自苯基(2,4,6-三甲基苯甲酰基)磷酸锂或Irgacure 2959；更优选地，所述光引发剂为苯基(2,4,6-三甲基苯甲酰基)磷酸锂。
9.一种如权利要求1～3中任意一项所述的骨修复水凝胶支架的制备方法，其特征在于，包括以下步骤：将甲基丙烯酰化明胶溶液、甲基丙烯酰化海藻酸钠溶液混匀得生物墨水，将水凝胶支架打印成型，去除未固化的生物墨水，浸入含钙交联剂中交联，去除多余的含钙交联剂，浸入谷氨酰胺转氨酶溶液中继续交联即得；优选地，所述水凝胶支架打印采用EFL光固化打印机；优选地，所述EFL光固化打印机的条件为光强度：10～12mW/cm2、曝光时间：12～16s。
10.一种如权利要求1～8中任意一项所述的骨修复水凝胶支架的制备方法，其特征在于，包括以下步骤：将甲基丙烯酰化明胶溶液、甲基丙烯酰化海藻酸钠溶液及甘草酸/表没食子儿茶素没食子酸酯脂质体混匀得生物墨水，将水凝胶支架打印成型，去除未固化的生物墨水，浸入含钙交联剂中交联，去除多余的含钙交联剂，浸入谷氨酰胺转氨酶溶液中继续交联即得；优选地，所述水凝胶支架打印采用EFL光固化打印机；；优选地，所述EFL光固化打印机的条件为光强度：10～12mW/cm2、曝光时间：12～16s。
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