CN116672504A - 一种适用于糖尿病创面无瘢痕修复的3d打印生物材料及其制备方法 - Google Patents
一种适用于糖尿病创面无瘢痕修复的3d打印生物材料及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种适用于糖尿病创面无瘢痕修复的3D打印生物材料及其制备方法,该3D打印生物材料含有以下浓度的组分:10~30%(w/v)ECMMA、5×10‑5~40×10‑5%(w/v)EVs、5×10‑5~40×10‑5%(w/v)Cu‑EGCG、0.1~0.2%(w/v)光引发剂,制备时,按浓度配比,先将ECMMA溶于光引发剂中,混合均匀后形成溶液A,再将溶液A和Cu‑EGCG、EVs混合均匀,形成ECM/EVs/Cu‑EGCG水凝胶即可,是一种具有适当孔隙率、机械强度、吸水性能、拉伸及压缩性能、体外降解、低细胞毒性及较好的生物相容性的水凝胶材料,能够改善糖尿病溃疡创面炎症微环境,起到促进糖尿病创面微血管生长、抑制过度炎症反应、促进创面愈合、减少瘢痕挛缩等效果。
Description
技术领域
本发明属于生物组织工程技术领域,具体的说,是一种适用于糖尿病创面无瘢痕修复的3D打印生物材料及其制备方法。
背景技术
皮肤是人体最大的器官,有着屏障、吸收、分泌、感觉、体温调节等重要功能。目前,在我国,糖尿病发病率逐年升高,而在糖尿病患者中,因皮肤微血管功能障碍导致的糖尿病创面最为常见,且为付出代价最高的并发症之一。临床上针对糖尿病创面的保守治疗包括抗生素治疗、含银敷料换液、高压氧治疗、电刺激等,但是由于糖尿病创面多存在感染及炎症失衡情况,保守治疗常不能愈合,进而需要进行外科手术干预。
在临床的手术治疗方式(刃厚皮、筋膜瓣、肌皮瓣等)中,容易存活的刃厚皮片常被外科医生选择作为创面修复的手段之一。自体刃厚皮片厚度适中,移植后成活率高,可多次取皮,能解决皮源不足的难题。但糖尿病创面存在细胞外基质(ECM)生成、沉积及重塑异常等突出问题,上述方案治疗后的糖尿病创面局部瘢痕化程度较高,弹性差,导致愈合后的创面在摩擦、受压部位——尤其是足部容易再次破溃甚至并发严重感染,不仅为患者及社会带来沉重的经济负担,还导致截肢甚至死亡等严重后果(截肢后五年死亡率高达40-59%)。
糖尿病引起的皮肤结构与组分的改变会直接影响皮肤功能。目前的研究表明:糖尿病发生后皮肤的机械特性会发生改变,包括:抗拉强度、牵引力、形变程度等,此外皮肤的屏障功能也会受到影响。因此,寻找在有效解决糖尿病创面缺血、长期异常炎症状态等问题的同时补充ECM、抑制局部纤维化的治疗手段具有必要性。
现有技术中,公开号为CN114225096A的发明专利提供了一种促进伤口愈合的复合水凝胶及其制备方法和应用,该专利采用两种天然生物聚合物SFMA(甲基丙烯酰化丝素蛋白)和DEXMA(甲基丙烯酰化葡聚糖)为原料,通过在SFMA与DEXMA交联形成的水凝胶基体上负载富含血小板的血浆和脱细胞真皮基质形成复合水凝胶。两种具有不同物理化学性质的生物聚合物的协同结合使复合水凝胶的各种性质得以微调,包括力学性能、膨胀性等,另外,通过水凝胶中负载PRP(高浓度血小板血浆)来实现生长因子的缓慢释放,同时载入ADM(脱细胞真皮基质)赋予该材料的生物活性,对促进糖尿病伤口的愈合具有显著的潜力。该专利提供的水凝胶的主要成分为SFMA(甲基丙烯酰化丝素蛋白)和DEXMA(甲基丙烯酰化葡聚糖),都与皮肤组分相差甚远,而ADM仅作为添加物在水凝胶中使用,其提供的生物活性还有待研究。
发明内容
本发明的目的是提供一种适用于糖尿病创面无瘢痕修复的3D打印生物材料,即ECM/EVs/Cu-EGCG水凝胶,在促进糖尿病血管化进而促进创面愈合中均具有显著作用,且效果优于目前市场上的真皮材料。其中ECMMA以dECM(脱细胞真皮基质)为主体材料,能够充分模拟损伤组织周围微环境,并具有紫外光成型和温敏特性,能够实现定制化微米级别打印孔隙结构;Cu-EGCG能够兼顾EGCG的治疗性抗炎和抗氧化活性以及铜离子的血管生成活性且无细胞毒性,同时具备可打印性;EVs则具有提高皮肤修复性能的优势。为此,本发明还提供了该3D打印生物材料的制备方法,可实现产业化规模生产,对于一次性解决糖尿病创面炎症具有良好的应用前景。
本发明通过下述技术方案实现:一种适用于糖尿病创面无瘢痕修复的3D打印生物材料,含有以下浓度的组分:10~30%(w/v)ECMMA、5×10-5~40×10-5%(w/v)EVs、5×10-5~40×10-5%(w/v)Cu-EGCG、0.1~0.2%(w/v)光引发剂。
所述ECMMA是以dECM为主体材料,采用甲基丙烯酸缩水甘油酯经酰胺化反应接枝而制得。
所述EVs是以小鼠骨髓干细胞为原料,取其外泌体经浓缩、纯化而制得。
所述Cu-EGCG是分别制备CaCl2溶液和Na2CO3溶液,在Na2CO3溶液中加入PSS溶液,将Na2CO3和PSS的混合溶液快速加入CaCl2溶液中,搅拌、离心后收集固体颗粒,再加入EGCG和CuCl2溶液,搅拌后加入MOPS缓冲液,洗涤后,得到包覆Cu离子和EGCG的CaCO3粒子,即得。
所述光引发剂为苯基-2,4,6-三甲基苯甲酰基亚磷酸锂。
一种制备上述3D打印生物材料的方法,按浓度配比,先将ECMMA溶于光引发剂中,混合均匀后形成溶液A,再将溶液A和Cu-EGCG、EVs混合均匀,形成ECM/EVs/Cu-EGCG水凝胶。
本发明中,ECM是指细胞外基质,dECM是指脱细胞基质,EVs是指细胞外囊泡,EGCG是指表没食子儿茶素没食子酸酯。
本发明与现有技术相比,具有以下优点及有益效果:
(1)本发明旨在解决目前糖尿病创面复合植皮技术中因组织工程真皮材料孔径小,血管化效果差,瘢痕形成明显的缺陷,而提出了一种新的可采用3D打印制备的天然来源的脱细胞真皮为支架的组织工程真皮,即:ECM/EVs/Cu-EGCG水凝胶,具有在紫外光照下成型的能力,是一种具有适当孔隙率、机械强度、吸水性能、拉伸及压缩性能、体外降解、低细胞毒性及较好的生物相容性的水凝胶材料。
(2)本发明涉及的ECMMA是以dECM为主体材料,dECM是一种天然的生物代谢产品,具有很好的细胞支撑作用,能够增加创面愈合后真皮的厚度增加,增加皮肤柔软度,减少瘢痕挛缩以及改善局部组织功能,在本发明中,dECM通过甲基丙烯酸酐化改性后,仍然能够保持其天然结构和功能,同时,还具有温敏及光敏性,能够在紫外光照下成型,能够通过调控温度,能实现快速“推出”,完成高精度3D生物打印——保持再生修复所需要的定制化微米级别打印孔隙。已知的,通过SFMA与DEXMA交联形成的水凝胶系统则并不存在100%联通的孔隙结构。
(3)本发明在制备ECMMA时,通过合理控制其pH值、胃蛋白酶降解时间、酶反应温度等参数条件,可以尽可能多的保留其活性成分(胶原蛋白、糖胺聚糖等),使其在作为水凝胶使用时,充分还原伤口床的基质微环境,促进组织修复细胞进行生物活动,尤其适用于糖尿病创面修复,可改善糖尿病溃疡创面炎症微环境、促进血管新生,减少瘢痕挛缩。
(4)大量研究表明,在糖尿病状况下,内皮细胞经历了显著的基因转录变化,导致稳态功能的受损以及促炎症和促纤维化等因子的基因表达上调;此外,表观遗传改变在糖尿病并发症的发生和发展中扮演着重要的角色,即糖尿病状态导致内皮细胞功能失调状态下的整体RNA-染色体相互作用。为此,本发明采用间充质干细胞(vs成纤维细胞)来源的EVs,来提升糖尿病创面皮肤的修复性能。首先,EVs含大量核酸等物质,对炎症和血管化有丰富的调控网络,可作为非病理性细胞外基质的核酸组分补充,其中,在本发明中,EVs还可与ECMMA的原位组织再生调控作用以及Cu-EGCG的促血管化、炎症调节作用产生协同作用,共同调控糖尿病创面异常微环境,最终使其无瘢痕愈合。
(5)Cu-EGCG通过金属Cu离子与多酚EGCG通过金属配位形成共价键结合,结合了EGCG的治疗性抗炎和抗氧化活性以及铜离子的血管生成活性,可增加3D打印真皮支架的促血管化及抗炎性能,以期改善糖尿病溃疡创面炎症微环境、促进血管新生。
综上所述,本发明提出了一种可以一次性解决糖尿病创面炎症失衡、血管化障碍和基质形成异常三大问题的治疗手段,采用ECMMA提供创面修复的真皮组织支架,可提供细胞长入及营养物质交换的尺寸孔隙,在起到替代真皮的同时,能够对糖尿病创面进行EVs和Cu-EGCG的缓释,能够改善糖尿病溃疡创面炎症微环境,起到促进糖尿病创面微血管生长、抑制过度炎症反应、促进创面愈合、减少瘢痕挛缩等效果。
附图说明
图1为实施例2成胶前后示意图。
图2为实施例1、2、3的压力-应变曲线。
图3为细胞增殖实验统计图。
图4为体外血管生成实验结果。
具体实施方式
下面将本发明的发明目的、技术方案和有益效果作进一步详细的说明。
应该指出,以下详细说明都是示例性的,旨在对所要求的本发明提供进一步的说明,除非另有说明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
本发明的目的在于克服复合植皮技术中组织工程真皮材料孔径小,血管化效果差,瘢痕形成明显的不足之处,提出一种新的3D打印的天然来源的脱细胞真皮为支架的组织工程真皮。其中已有的dECM成分可以加速真皮组织的再生,为保证临床治疗的效果,真皮替代物的组分应与正常皮肤相似。而dECM作为创面修复的组织模板,使创面愈合后真皮的厚度增加,增加皮肤柔软度,减少瘢痕挛缩,改善局部组织功能。此外,作为基础材料的dECM在经过甲基丙烯酸酐改性后不仅保持了天然dECM的结构和功能,同时它还拥有在紫外光照下成型的能力。使其能够克服现有材料的不足之处,而提供一种具有适当孔隙率、机械强度、吸水性能、拉伸及压缩性能、体外降解、低细胞毒性及较好的生物相容性的水凝胶材料。
本发明通过甲基丙烯酸酐化的dECM在添加EVs和Cu-EGCG后,能够形成甲基丙烯酸酐化ECM/EVs/Cu-EGCG(EECE)凝胶。3D打印技术的发展使人工真皮有了更强的灵活性,可将上述水凝胶作为3D打印墨水,设计成拥有利于细胞长入及营养物质交换的尺寸孔隙的真皮支架,复合植皮后,在起到替代真皮的同时,能够对糖尿病创面进行EVs和Cu-EGCG的缓释,起到促进糖尿病创面微血管生长、抑制过度炎症反应的作用进而促进创面的愈合。因此,本发明在促进糖尿病血管化进而促进创面愈合中具有显著作用,且效果优于目前市场上的真皮材料。
进一步的,本发明的技术方案可概况如下:
ECMMA的制备:将新鲜猪皮去除表皮和皮下组织,切成1×1cm块状,相继使用0.25wt%胰蛋白酶混合1mM乙二胺四乙酸(EDTA)的溶液、1wt%的Triton-X-100混合25mMEDTA的1PBS溶液、含有10mM氯化镁(MgCl2)的30U/mL DNase溶液孵育处理,去除细胞成分及脂肪制成猪真皮dECM,冻干后溶于胃蛋白酶乙酸溶液。dECM溶液用1M NaOH滴定至pH中性(pH 7-8),加入甲基丙烯酸缩水甘油酯(Glycidyl Methacrylate,GMA)在37℃反应过夜。然后在37℃透析3天,以去除未交联GMA。将透析后溶液速冻后放入低温干燥机冻干成粉末。
EVs的制备:将小鼠骨髓干细胞培养上清离心去除细胞或细胞碎片,依次加入结合缓冲液混匀及结合树脂混匀,离心去除上清,得到EVs初步浓缩液;置入纯化柱静置后离心,去除废液,重复两次洗涤和离心后,将纯化柱放入低吸附蛋白的离心管中,加入洗脱液进行洗脱并离心,后再次将滤液加入纯化柱静置后离心,得到浓缩的EVs溶液;然后通过SuperEV(超纯尺寸排阻色谱柱)实现EVs的纯化;最后将纯化的EVs溶液再次进行结合、纯化等步骤,得到浓缩的纯净EVs溶液。
Cu-EGCG的制备:首先制备20mM CaCl2和Na2CO3原液,PSS以1mg/mL的浓度分别溶于两种溶液中,将10mL的Na2CO3和PSS混合物快速加入10mL的CaCl2和PSS混合物中,将得到的混合物连续搅拌30s,然后离心(10000rpm 5min)收集固体颗粒,用去离子水洗涤3次。加入EGCG(0.5mL,11mg/mL,24mM)和CuCl2(0.5mL,3.2mg/mL,24mM)水溶液,搅拌10s。加入5mLMOPS缓冲液(100mM,20.9g/L,pH8.0),通过洗涤去除多余的EGCG和CuCl2,得到包覆一层Cu-EGCG的CaCO3粒子。重复上述包覆工艺2次,得到了包覆3层Cu-EGCG的CaCO3颗粒。
实施例1:ECM/EVs/Cu-EGCG凝胶
本实施例的ECM/EVs/Cu-EGCG凝胶包括以下浓度的组分:10%(w/v)ECMMA、50μg/mL的EVs、50μg/mL Cu-EGCG、0.1%(w/v)的LAP。
其制备步骤如下:
(1)按上述方式分别制备ECMMA冻干粉末、EVs浓缩液和Cu-EGCG;
(2)配置浓度为0.1%(w/v)的LAP溶液;
(3)将ECMMA冻干粉末溶于0.1%(w/v)的LAP溶液中,混合均匀并配置浓度为10%(w/v)的ECMMA生物墨水,记为溶液A;
(4)配置浓度为100μg/mL的EVs溶液,记为溶液B;
(5)配置浓度为100μg/mL的Cu-EGCG水溶液,记为溶液C;
(6)将溶液A、B、C按体积比为2∶1∶1混合均匀,形成稳定的ECM/EVs/Cu-EGCG凝胶。
实施例2:ECM/EVs/Cu-EGCG凝胶
本实施例的ECM/EVs/Cu-EGCG凝胶包括以下浓度的组分:15%(w/v)ECMMA、50μg/mL的EVs、50μg/mL Cu-EGCG、0.1%(w/v)的LAP。
其制备步骤如下:
(1)按上述方式分别制备ECMMA冻干粉末、EVs浓缩液和Cu-EGCG;
(2)配置浓度为0.1%(w/v)的LAP溶液;
(3)将ECMMA冻干粉末溶于0.1%(w/v)的LAP溶液中,混合均匀并配置浓度为15%(w/v)的ECMMA生物墨水,记为溶液A;
(4)配置浓度为100μg/mL的EVs溶液,记为溶液B;
(5)配置浓度为100μg/mL的Cu-EGCG水溶液,记为溶液C;
(6)将溶液A、B、C按体积比为2∶1∶1混合均匀,形成稳定的ECM/EVs/Cu-EGCG凝胶。
实施例3:ECM/EVs/Cu-EGCG凝胶
本实施例的ECM/EVs/Cu-EGCG凝胶包括以下浓度的组分:20%(w/v)ECMMA、50μg/mL的EVs、50μg/mL Cu-EGCG、0.1%(w/v)的LAP。
其制备步骤如下:
(1)按上述方式分别制备ECMMA冻干粉末、EVs浓缩液和Cu-EGCG;
(2)配置浓度为0.1%(w/v)的LAP溶液;
(3)将ECMMA冻干粉末溶于0.1%(w/v)的LAP溶液中,混合均匀并配置浓度为20%(w/v)的ECMMA生物墨水,记为溶液A;
(4)配置浓度为100μg/mL的EVs溶液,记为溶液B;
(5)配置浓度为100μg/mL的Cu-EGCG水溶液,记为溶液C;
(6)将溶液A、B、C按体积比为2∶1∶1混合均匀,形成稳定的ECM/EVs/Cu-EGCG凝胶。
实施例4:ECM/EVs/Cu-EGCG凝胶
本实施例的ECM/EVs/Cu-EGCG凝胶包括以下浓度的组分:20%(w/v)ECMMA、100μg/mL的EVs、100μg/mL Cu-EGCG、0.1%(w/v)的LAP。
其制备步骤如下:
(1)按上述方式分别制备ECMMA冻干粉末、EVs浓缩液和Cu-EGCG;
(2)配置浓度为0.1%(w/v)的LAP溶液;
(3)将ECMMA冻干粉末溶于0.1%(w/v)的LAP溶液中,混合均匀并配置浓度为20%(w/v)的ECMMA生物墨水,记为溶液A;
(4)配置浓度为200μg/mL的EVs溶液,记为溶液B;
(5)配置浓度为200μg/mL的Cu-EGCG水溶液,记为溶液C;
(6)将溶液A、B、C按体积比为2∶1∶1混合均匀,形成稳定的ECM/EVs/Cu-EGCG凝胶。
实施例5:ECM/EVs/Cu-EGCG凝胶
本实施例的ECM/EVs/Cu-EGCG凝胶包括以下浓度的组分:30%(w/v)ECMMA、100μg/mL的EVs、100μg/mL Cu-EGCG、0.2%(w/v)的LAP。
其制备步骤如下:
(1)按上述方式分别制备ECMMA冻干粉末、EVs浓缩液和Cu-EGCG;
(2)配置浓度为0.2%(w/v)的LAP溶液;
(3)将ECMMA冻干粉末溶于0.2%(w/v)的LAP溶液中,混合均匀并配置浓度为30%(w/v)的ECMMA生物墨水,记为溶液A;
(4)配置浓度为200μg/mL的EVs溶液,记为溶液B;
(5)配置浓度为200μg/mL的Cu-EGCG水溶液,记为溶液C;
(6)将溶液A、B、C按体积比为2∶1∶1混合均匀,形成稳定的ECM/EVs/Cu-EGCG凝胶。
实施例6:ECM/EVs/Cu-EGCG凝胶
本实施例的ECM/EVs/Cu-EGCG凝胶包括以下浓度的组分:30%(w/v)ECMMA、200μg/mL的EVs、200μg/mL Cu-EGCG、0.2%(w/v)的LAP。
其制备步骤如下:
(1)按上述方式分别制备ECMMA冻干粉末、EVs浓缩液和Cu-EGCG;
(2)配置浓度为0.2%(w/v)的LAP溶液;
(3)将ECMMA冻干粉末溶于0.2%(w/v)的LAP溶液中,混合均匀并配置浓度为30%(w/v)的ECMMA生物墨水,记为溶液A;
(4)配置浓度为400μg/mL的EVs溶液,记为溶液B;
(5)配置浓度为400μg/mL的Cu-EGCG水溶液,记为溶液C;
(6)将溶液A、B、C按体积比为2∶1∶1混合均匀,形成稳定的ECM/EVs/Cu-EGCG凝胶。
实施例7:ECM/EVs/Cu-EGCG凝胶
本实施例的ECM/EVs/Cu-EGCG凝胶包括以下浓度的组分:30%(w/v)ECMMA、400μg/mL的EVs、400μg/mL Cu-EGCG、0.2%(w/v)的LAP。
其制备步骤如下:
(1)按上述方式分别制备ECMMA冻干粉末、EVs浓缩液和Cu-EGCG;
(2)配置浓度为0.2%(w/v)的LAP溶液;
(3)将ECMMA冻干粉末溶于0.2%(w/v)的LAP溶液中,混合均匀并配置浓度为30%(w/v)的ECMMA生物墨水,记为溶液A;
(4)配置浓度为800μg/mL的EVs溶液,记为溶液B;
(5)配置浓度为800μg/mL的Cu-EGCG水溶液,记为溶液C;
(6)将溶液A、B、C按体积比为2∶1∶1混合均匀,形成稳定的ECM/EVs/Cu-EGCG凝胶。
实施例8:3D打印的ECM/EVs/Cu-EGCG皮肤组织
采用3D打印机对实施例7的凝胶进行打印,打印过程中,控制环境温度为18℃,环境湿度为50%-55%,打印平台温度为4℃,喷嘴内径为100μm,喷嘴的移动速度为10mm/s,喷嘴初始高度距平台约为0.8mm,气泵压力为0.1-0.3MPa,单层规格20mm×20mm,打印层数4层,紫外光照交联时间20秒,即得ECM/EVs/Cu-EGCG皮肤组织。
实验部分:
(一)水凝胶压缩实验
采用压缩实验测量水凝胶最佳的成胶浓度,将溶液A、溶液B和溶液C混合均匀,通过TA流变仪(AR 1500Ex,TAinstrument,USA)测量得到的应力-应变曲线,提取每个水凝胶的压缩模量。在测试中,水凝胶被放置在两个压缩板之间,用平板探针以0.05mm/s到60%的速度进行压缩。
图1为实施例2中水凝胶的成胶示意图。图2为各实施例成胶压缩实验的测试结果,从结果中可看到,随着浓度的增加,凝胶在同一压力下的形变随之减少,实施例1的压缩模量最小,在较小压力下已经超过80%形变;实施例3的压缩模量最高,为258kPa,但其形变超过40.6%后即破碎;实施例2的压缩模量为201kPa,可承受74%的形变仍能保持形状,接近人体皮肤的真皮层。
(二)生物相容性
使用人真皮来源的人微血管内皮细胞Human microvascular endothelial cells(HMEC)-1(Meisen,CTCC-001-0219)进行细胞增殖实验。按2000cells/孔接种到96孔板中,接种完毕将培养板放回37℃培养箱进行培养,待细胞贴壁后将培养基更换为浸提液,培养24小时、48小时、72小时后分别在培基中加入每孔10ul CCK8溶液,2小时后测量450nm处吸光度值。每组实验至少重复3次。
图3为细胞增殖实验统计图,如图所示随着实施例2、4、5、6、7中的Cu-EGCG浓度的增加,细胞活性呈逐步下降的趋势。但在实施例2和实施例4、实施例5组浸提液中培养3天后的HEMC-1细胞活性均大于90%,分别为100%±0.8%、96.3%±1.6%、93.9%±1.9,实例6和实例7浸提液中培养3天后的HMEC-1细胞活性分别为82.3%±3.3%、61.1%±3.8%。由此可以证明,实施例2和实施例4、实施例5较其他实施例呈现出良好的细胞相容性。
(三)体外血管生成实验
提前将96孔板和枪头预冷,于每孔中加入100μL基质胶,避免产生气泡。在37℃培养箱中放置45min凝固成胶。将HMEC-1细胞消化并用各组浸提液制成1.5*105/ml细胞悬液,在铺好基质胶的孔加入100μL重悬液(15000个细胞/孔),每组重复3孔。37℃孵育4小时后在显微镜下观察拍照,使用imageJ软件处理图片,统计成管结点数量(Number of Junctions)和成管总长度(Total Length)。
图4为体外血管生成实验结果,如图所示,对比其他实例,实施例5的浓度有明显的促进体外血管生成的作用,为最终的材料浓度。
以上所述,仅是本发明的较佳实施例,并非对本发明做任何形式上的限制,凡是依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化,均落入本发明的保护范围之内。
Claims (6)
1.一种适用于糖尿病创面无瘢痕修复的3D打印生物材料,其特征在于:含有以下浓度的组分:10~30%(w/v)ECMMA、5×10-5~40×10-5%(w/v)EVs、5×10-5~40×10-5%(w/v)Cu-EGCG、0.1~0.2%(w/v)光引发剂。
2.根据权利要求1所述的3D打印生物材料,其特征在于:所述ECMMA是以dECM为主体材料,采用甲基丙烯酸缩水甘油酯经酰胺化反应接枝而制得。
3.根据权利要求1所述的3D打印生物材料,其特征在于:所述EVs是以小鼠骨髓干细胞为原料,取其外泌体经浓缩、纯化而制得。
4.根据权利要求1所述的3D打印生物材料,其特征在于:所述Cu-EGCG是分别制备CaCl2溶液和Na2CO3溶液,在Na2CO3溶液中加入PSS溶液,将Na2CO3和PSS的混合溶液快速加入CaCl2溶液中,搅拌、离心后收集固体颗粒,再加入EGCG和CuCl2溶液,搅拌后加入MOPS缓冲液,洗涤后,得到包覆Cu离子和EGCG的CaCO3粒子,即得。
5.根据权利要求1所述的3D打印生物材料,其特征在于:所述光引发剂为苯基-2,4,6-三甲基苯甲酰基亚磷酸锂。
6.一种制备权利要求1~5任一项所述3D打印生物材料的方法,其特征在于:按浓度配比,先将ECMMA溶于光引发剂中,混合均匀后形成溶液A,再将溶液A和Cu-EGCG、EVs混合均匀,形成ECM/EVs/Cu-EGCG水凝胶。
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