CN114644739B - 一种高粘附性水凝胶、制备方法及应用 - Google Patents
一种高粘附性水凝胶、制备方法及应用 Download PDFInfo
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- CN114644739B CN114644739B CN202011501646.XA CN202011501646A CN114644739B CN 114644739 B CN114644739 B CN 114644739B CN 202011501646 A CN202011501646 A CN 202011501646A CN 114644739 B CN114644739 B CN 114644739B
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Abstract
本发明提供了一种高粘附性水凝胶、制备方法及应用。包括以下步骤:使用含有自由基聚合基团的材料改性明胶;使用含有自由基聚合基团的材料改性原弹性蛋白;硫醇化的肝素的制备;可光交联改性明胶、改性原弹性蛋白和硫醇化的肝素水凝胶的制备。本发明的高粘附性水凝胶的制备方法,具有结构稳定性、良好的生物相容性和降解可控等性能。且存在三维网络结构,利于细胞的粘附和生长。综合以上性能优势,本发明的高粘附性且具有抗凝血功能的水凝胶在心肌血管化和心肌补片等方面有着广泛的应用前景。
Description
技术领域
本发明涉及医用材料领域,具体涉及一种高粘附性水凝胶、制备方法及应用。
背景技术
在人体组织中,心肌的微血管密度极高,是代谢最为旺盛的组织之一。每年由于心肌梗死失去生命的患者高达60万,其中10%的急性心梗患者是死亡在发病现场或在去医院的路上。针对如此高危且频发的疾病,一旦发病,冠脉支架置入或者外科搭桥是目前最主要治疗手段,但是已死亡的心肌不可恢复,心功能必然进一步下降,且死亡心肌的进一步瘢痕化,不仅影响心动周期收缩的协调性,也影响心肌功能的恢复,因此植入厚层微血管化心肌组织可能成为未来梗死性心肌病及终末期心脏病的终极治疗手段。但所有模拟人体心肌组织结构和功能的植入材料都必须解决植入物易于血栓化这一难题。这种移植物的血管化包括两方面的含义:(1)构建移植物内在的,具有灌注功能的成熟血管网络;(2)实现移植后,移植物与宿主血管网络的快速吻合和长期整合。
在现阶段的研究中有学者采用以内皮细胞或内皮前体细胞为基础的细胞共培养策略。考虑体内新生血管形成的现实情况,采用其他细胞与ECs共培养以获得更好的组织血管化似乎是一个更明智的选择。通过调控活性生物支架及水凝胶,即调控天然或人工合成材料,使其有利于移植细胞生存或诱导宿主细胞,进而促进移植物血管化是一个较好的选择,因此受到越来越多的关注。水凝胶制备技术操作方便,极易于小型化、自动化生产,但其缺点是与固态基质相比,其生物力学性能差。因此,将有良好机械强度及生物相容性,且具有粘黏性的水凝胶材料和形成心肌细胞能力的种子细胞复合,是有望构建出可用于移植、修复或替代自体心肌的生物材料。
随着组织工程材料的发展,已有研究表明:水凝胶作为植入物有较少的免疫排斥,同时其结构与细胞外基质结构(ECM)类似,有利于构建组织损伤后再生微环境,促进组织结构的再建。在心肌组织方面,例如利用壳聚糖与脱细胞猪心脏基质混合,冻干制成三维支架,结果发现大鼠心室肌细胞的存活和基因表达都明显高于对照组。通过调节原材料比例发现,弹性模量在13.3±4kPa时,有益效果最佳。但其通过引入冰醋酸冻干成型,强度有限,不适合作为植入材料。另外,Sherrell与他的研究团队利用胶原蛋白制成高度生物相容性的水凝胶材料,该水凝胶可以被制成适合心脏补片的各种形状。通过复合碳纳米管,改善了支架材料的韧性,杨氏模量也提高了2-3倍。发现该支架支持心脏细胞的存活和分化。但其引入的纳米材料存在明显的团聚和渗出,在功能性和安全性方面都存在隐患。在神经组织领域,已有研究发现通过改变GelMA/MeTro的比值,材料表现出可调的力学性能,该水凝胶支架在外周神经损伤修复领域已展现出应用前景。肝素的应用领域,研究学者利用明胶复合肝素,形成冷冻凝胶。在体外实验中发现共混肝素凝胶较单一明胶凝胶更具有功能性,但共混方式和冷冻成型方式在应用方面存在明显的限制。已有相似的研究较多,但是总结可以发现,单一的原料制成的支架材料都存在某方面的短板。因此研究有必要朝着多元复合的方向发展,以建立优势互补的新材料,其中肝素化修饰的稳定明胶/弹性蛋白水凝胶在抗凝血领域具有应用前景。
发明内容
为了克服现有材料的不足,本发明所要解决的技术问题是提供一种高粘附性水凝胶、制备方法及应用。
为解决上述技术问题,本发明采用的技术方案如下:
提供一种高粘附性水凝胶,其为改性明胶/改性原弹性蛋白/硫醇化肝素光交联水凝胶,由可光交联改性明胶、改性原弹性蛋白和硫醇化的肝素经自由基聚合反应得到,该水凝胶具有稳定的三维网络多孔结构。
按上述方案,所述改性明胶为甲基丙烯酸酐(MA)改性明胶或甲基丙烯酸缩水甘油酯(GMA)改性明胶;
用于改性原弹性蛋白为甲基丙烯酸酐(MA)改性原弹性蛋白、甲基丙烯酸缩水甘油酯(GMA)原弹性蛋白。
按上述方案,所述的硫醇化的肝素为C=N双键还原为C-N单键的硫醇化的肝素。
提供一种高粘附性水凝胶的制备方法,包括以下步骤:
(1)使用含有自由基聚合基团的材料改性明胶;
(2)使用含有自由基聚合基团的材料改性原弹性蛋白;
(3)硫醇化的肝素的制备:提供还原末端为醛基的肝素衍生物;将带有醛基的肝素衍生物与可通过氨醛缩合反应引入巯基的材料经氨醛缩合引入巯基;对上述产物进行还原,将C=N双键还原为C-N单键,获得硫醇化的肝素;
(4)可光交联改性明胶、改性原弹性蛋白和硫醇化的肝素水凝胶的制备:
将上述可光交联改性明胶,改性原弹性蛋白和硫醇化的肝素在光敏性引发剂作用下,经自由基聚合反应光交联获得水凝胶。
本发明中,用于改性明胶的可产生自由基聚合基团的材料包括甲基丙烯酸酐(MA)、甲基丙烯酸缩水甘油酯(GMA)等。
本发明中,用于改性原弹性蛋白的可产生自由基聚合基团的材料包括甲基丙烯酸酐(MA)、甲基丙烯酸缩水甘油酯(GMA)等。
本发明中,用于可通过氨醛缩合引入巯基的材料包括半胱氨酸,对巯基苯胺等。
本发明中,用于还原碳氮双键的的材料为具有一定还原性的材料,如氰基硼氢化钠等。
本发明中,用于引发自由基聚合的光敏性引发剂为如2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮/光引发剂(2-hydroxy-4'-(2-hydroxyethoxy)-2-methyl-propiophe(I2959),苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐(Lithium phenyl-2,4,6-trimethylbenzoylphosphinate(LAP))等。
本发明中:步骤(4)中使用的溶剂为含有冰醋酸的二甲亚砜。
本发明中,所述的可光交联GelMA/MeTro/硫醇化肝素水凝胶的制备方法中,光引发剂浓度为0.5wt.%-1.0wt.%,紫外波长为320-390nm,强度为6.0-10mW/cm2,光照时间为50-100s。
按上述方案,体系中GelMA和MeTro的总浓度为5wt.%-20wt.%;GelMA:MeTro的质量比比为3:7-6:4;体系中硫醇化肝素的含量为10wt.%-20wt.%。
按上述方案,所述的步骤(1)具体过程为将含有自由基聚合基团的材料逐滴加入明胶溶液中,恒温搅拌反应,反应温度为50-70℃,反应时间为2-4h,将产物透析过滤,冻干后保存。
按上述方案,所述的步骤(2)的具体过程为:将原弹性蛋白和MA于PBS溶液中,在0-4℃下充分充分反应12小时以上后终止反应;将产物溶液透析过滤,冻干后保存。
按上述方案,所述的步骤(3)中:还原末端为醛基的肝素衍生物的制备:将肝素钠溶解于亚硝酸中,充分混匀,冰浴反应,然后中和至终止反应,透析,冻干得到样品并保存。
进一步的,所述的步骤(1)的制备方法中:GelMA溶液透析袋截留分子量为12-14kDa。
进一步的,所述的步骤(2)的制备方法中,MeTro溶液透析袋截留分子量为3000-3500。
进一步的,所述的步骤(3)中所述的透析为:在NH4HCO3溶液中用截留分子量为3500的透析袋透析。
进一步地,所述的步骤(3)冰浴反应过程中,每5-10min振荡一次。
明胶具有一系列优良的性能,如亲水性、侧链反应活性高、良好的生物相容性等。最主要的是含有精氨酸-甘氨酸-天门冬氨酸(RGD)氨基酸序列,RGD是一种细胞黏附多肽,由精氨酸、甘氨酸和天冬氨酸组成,存在于多种细胞外基质中。它能和细胞膜上11种整合素产生特异性结,有利于细胞的粘附增殖和分化。同时明胶来源广泛、价格低廉、生物相容性好,含有可降解的位点。原弹性蛋白是一种高弹性蛋白,具有结构完整性,可调节人体组织中的细胞功能。
本发明先提供含有自由基聚合基团的材料改性的明胶如GelMA,在凝胶中可提供生理细胞结合基元和蛋白酶敏感降解位点,可通过浓度控制降解速率,和使用含有自由基聚合基团的材料改性原弹性蛋白如MeTro,改性后明胶具有光敏特性,可作为良好的拟细胞外基质材料,可为细胞增殖和分化提供良好的三维环境。改性原弹性蛋白(MeTro)也具有光敏性,它们与GelMA混合后在光照的条件下可形成贯穿的多孔网络结构,有利于细胞的增殖和分化;具有良好的韧性,可通过调整改性明胶和改性原弹性蛋白的比例,调整其弹性模量,使水凝胶伸缩性接近心肌组织。另外,生物材料在与血液接触时存在表面血栓形成的问题,这影响了生物材料在体内使用的安全性,将肝素被引入了凝胶系统,提供肝素化处理的生物材料,可以提高材料的抗凝血性能,降低血栓形成的可能,同时还增加生物材料的组织相容性。除了解决血栓问题外,肝素处理后的材料还可以通过抑制白细胞的激活来抑制炎症的发生,具有抗炎的功能。本发明通过提供硫醇化的肝素,基于硫醇化的肝素形成二硫键的结构,可加强肝素与凝胶结合,使凝胶体系更为稳定。然后在光引发剂存在的条件下,一定波长范围的紫外光刺激后,碳碳双键活化断裂形成自由基,当自由基发生接触后形成稳定的碳碳单键,从而形成网状结构。另一方面:紫外辐照下巯基与双键结合成稳定的键合。具体过程如下:引发剂在UV光的激发下产生活性自由基,第一步该自由基夺取巯基(-SH)上的一个H原子,产生巯基自由基;第二步攻击C=C,打开其不饱和的π键,产生烷基自由基。最后烷基自由基夺取巯基(-SH)化合物上的氢原子,再次产生巯基自由基,使得引发链增长或烷基自由基碰到巯基自由基引起双基终止反应。过程见图4。
本发明最终提供的改性明胶/改性原弹性蛋白/硫醇化肝素水凝胶具有三维多孔结构,在SEM下可观察到水凝胶内部的多孔结构孔径,且可根据原料的浓度调节其孔径结构,使得水凝胶材料三维环境适合细胞生长;可通过调节GelMA/MeTro体系配比,实现力学性能如模量的调节,可作为心肌补片用于心肌梗死支架;通过巯基引入的肝素,均匀稳定的分布于凝胶中,使该水凝胶具有良好的抗凝血性和抗炎性,应用于心肌补片具有优势;同时其具有生物可降解性,在心肌修复中作为一次植入材料具有潜在优势。本申请首次通过巯基引入肝素,提高GelMA/MeTro凝胶体系综合性能,扩展了其应用领域,可应用在心肌血管化中,作为厚层微血管化心肌组织的植入材料。目前尚未有研究报道GelMA/MeTro体系通过巯基方式引入肝素制备GelMA/MeTro/硫醇化肝素水凝胶。
综合以上功能,这种高粘附性且具有抗凝血功能的水凝胶在心肌补片和心肌血管化等应用方面存在巨大的潜力,通过性能调整有望成为新型的修复心肌梗死支架材料。
本发明的有益效果:
1.本发明提供的改性明胶/改性原弹性蛋白/硫醇化肝素水凝胶具有三维多孔结构,具有结构稳定性、良好的生物相容性和降解可控等性能。可通过调节GelMA/MeTro体系配比,实现力学性能如模量的调节,可作为心肌补片用于心肌梗死支架;通过巯基引入的肝素,均匀稳定的分布于凝胶中,使该水凝胶具有良好的抗凝血性和抗炎性,应用于心肌补片具有优势;同时其具有生物可降解性,在心肌修复中作为一次植入材料具有潜在优势。与现有支架材料相比,拥有稳定且可控结构的同时,通过天然材料的复合改善了支架的力学性能。制备的支架不涉及小分子交联剂是引入,作为植入材料,更加安全可靠。
2.本发明首次提供了一种通过巯基方式引入肝素制备GelMA/MeTro/硫醇化肝素水凝胶的方法,制备过程实验条件温和且简易,且通过光固化的方式成型可实现多形状、多尺寸的凝胶个性化定制。
附图说明
图1硫醇化的肝素的制备过程图。
图2GelMA和MeTro通过氨基与甲基丙烯酸酐接枝C=C的示意图。
图3可光交联GelMA、MeTro和硫醇化的肝素水凝胶的制备示意图。
图4巯基-C=C双键反应机理。
图515%GelMa/MeTro(40:60)水凝胶材料扫描电镜表面形态。
图6为不同配比水凝胶的力学强度。
图7为15%肝素修饰的15%GelMa/MeTro(40:60)水凝胶的力学强度。
图8为各组分水凝胶的细胞粘附性对照。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细阐述。
实例1:普通GelMA/MeTro的制备
(1)GelMA的制备
称取10g明胶加入100mL PBS溶液中,于50℃温浴下搅拌1h至完全溶解,制备10%(w/v)Gel溶液;将8ml的MA以0.5mL/min的速度逐步滴入10%(w/v)Gel溶液。搅拌反应2h,得到GelMA溶液;将GelMA溶液置于透析袋(截留分子量为12-14kDa)中透析,再用0.22μm滤膜过滤后冻干保存。
(2)MeTro的制备
称取10g原弹性蛋白溶解于100mL PBS溶液中,于4℃条件下完全溶解;将8%(v/v)的MA加入溶液中,在4℃下充分反应12小时后加入3倍稀释的冷(4℃)DPBS终止反应;得到MeTro溶液,在4℃的蒸馏水中透析过滤后冻干常温保存。
(3)可光交联GelMA、MeTro水凝胶的制备
将上述得到一定量的GelMA溶解于光引发剂I2959溶液(0.5wt.%)中超声分散10min,制得5%(w/v)、7.5%(w/v)、10%(w/v)、15%(w/v)、20%(w/v)溶液。在40℃水浴环境下搅拌至完全溶解。置于6.9mW/cm2紫外灯下光照50秒。得到不同浓度的GelMA水凝胶。
将上述得到一定量的MeTro溶解于光引发剂I2959溶液(0.5wt.%)中超声分散10min,制得5%(w/v)、7.5%(w/v)、10%(w/v)、15%(w/v)、20%(w/v)溶液。在40℃水浴环境下搅拌至完全溶解。置于6.9mW/cm2紫外灯下光照50秒。得到不同浓度的MeTro水凝胶。
将上述得到一定量的GelMA和MeTro按质量比的比例为40:60溶解光引发剂I2959溶液(0.5wt.%)中超声分散10min,制得5%(w/v)、7.5%(w/v)、10%(w/v)、15%(w/v)溶液。在40℃水浴环境下搅拌至完全溶解。置于6.9mW/cm2紫外灯下光照50秒。得到不同质量分数的GelMA/MeTro水凝胶。
实施例2:高粘附性且具有抗凝血功能的水凝胶的制备
(1)GelMA的制备
称取10g明胶加入100mL PBS溶液中,于50℃温浴下搅拌1h至完全溶解,制备10%(w/v)Gel溶液;将8ml的MA以0.5mL/min的速度逐步滴入10%(w/v)Gel溶液。搅拌反应2h,得到GelMA溶液;将GelMA溶液置于透析袋(截留分子量为12-14kDa)中透析,再用0.22μm滤膜过滤后(-20℃)冻干保存。
(2)MeTro的制备
称取10g原弹性蛋白溶解于100mL PBS溶液中,于4℃条件下完全溶解;将8%(v/v)的MA加入溶液中,在4℃下充分反应12小时后加入3倍稀释的冷(4℃)DPBS终止反应;得到MeTro溶液,在4℃的蒸馏水中透析过滤后(-80℃)冻干常温保存。
(3)硫醇化的肝素的制备
制备过程分为以下步骤:a还原末端为醛基的肝素衍生物:称取1g肝素钠迅速溶解于亚硝酸(0.01mol/l,pH=2.65)中,充分混合,在0℃反应0.5h,每5-10min振荡一次。然后用体积比为Na2CO3/H2O/NaHCO3=3:5:5的混合溶液中和至p H=7终止反应,将得到的澄清溶液在NH4HCO3溶液中用截留分子量为3500的透析袋透析,冻干得到样品。b巯基化的肝素衍生物:将0.5g上述产物溶解于4mL二甲亚砜(含纯冰醋酸)中,加入过量半胱氨酸反应3h。用5倍体积的乙醇洗涤沉淀3次,沉淀用蒸馏水溶解后透析并冻干保存。c将上述产物溶解于二甲亚砜(含纯冰醋酸)中,加入氰基硼氢化钠还原成C-N单键。制得稳定的硫醇化肝素。
含有亚硝酸的弱酸性体系(pH在2.5-4.0)中,亚硝酸能将N位未取代的肝素降解为末端带醛基的低相对分子质量肝素,如图1所示。
(4)可光交联GelMA/MeTro/硫醇化肝素水凝胶的制备
将上述得到一定量的GelMA和MeTro按比例40:60溶解在光引发剂I2959溶液(0.5wt.%)中超声分散10min,制得5%(w/v)、7.5%(w/v)、10%(w/v)、15%(w/v)溶液。将硫醇化的肝素按溶液总量的10%(w/v)、15%(w/v)和20%(w/v)加入上述溶液。在40℃水浴环境下搅拌至完全溶解。置于6.9mW/cm2紫外灯下光照50秒。得到不同质量分数的GelMA/MeTro/硫醇化肝素水凝胶。
结合实例1和实例2:在不同配比的GelMA/MeTro凝胶基础上,制备了不同含量肝素修饰的不同配比的GelMA/MeTro水凝胶。将上述制备的10%(w/v)GelMA水凝胶、10%(w/v)MeTro水凝胶,10%和15%(w/v)GelMA/MeTro水凝胶和15%(w/v)肝素修饰的GelMA/MeTro/硫醇化肝素水凝胶,进行结构和性能对比,包括多孔结构、力学性能、细胞粘附性和抗凝血性能。结果表明:纯5wt.%-20wt%的GelMA和MeTro均能成胶,混合后不影响成胶性能,且复合改性肝素所制备的水凝胶具有三维多孔结构(见图5;其两组分不同配比及不同浓度可调节力学强度(见图6);GelMA/MeTro/硫醇化肝素水凝胶的力学强度、细胞粘附性较好(见图7-8),另外基于肝素的引物还可使其具有优异的抗凝血功能较好。
细胞粘附性实验:将小鼠成纤维细胞L929在水凝胶表面培养1天后,用PBS冲洗后进行Calcein-AM染色。通过剩余细胞的数量评价各成分水凝胶的粘附性。A)在15%的MeTro表面培养,B)在15%GelMA/MeTro(40:60)表面培养,C)在肝素修饰的GelMA/MeTro(40:60)表面培养。由图可看出,GelMA/MeTro/硫醇化肝素水凝胶具有优异的细胞粘附性。
Claims (9)
1.一种高粘附性水凝胶,其特征在于:为改性明胶/改性原弹性蛋白/硫醇化肝素光交联水凝胶,由可光交联改性明胶、改性原弹性蛋白和硫醇化的肝素经自由基聚合反应得到,该水凝胶具有稳定的三维网络多孔结构,所述改性明胶为甲基丙烯酸酐(MA)改性明胶或甲基丙烯酸缩水甘油酯(GMA)改性明胶;
用于改性原弹性蛋白为甲基丙烯酸酐(MA)改性原弹性蛋白或甲基丙烯酸缩水甘油酯(GMA)原弹性蛋白;
所述的硫醇化的肝素为提供还原末端为醛基的肝素衍生物;将带有醛基的肝素衍生物与可通过氨醛缩合反应引入巯基的材料经氨醛缩合引入巯基;对上述产物进行还原,将C=N双键还原为C-N单键获得的硫醇化的肝素。
2.权利要求1所述的高粘附性水凝胶的制备方法,其特征在于:包括以下步骤:
(1)使用含有自由基聚合基团的材料改性明胶,获得可光交联改性明胶;
(2)使用含有自由基聚合基团的材料改性原弹性蛋白,获得改性原弹性蛋白;
(3)硫醇化的肝素的制备:提供还原末端为醛基的肝素衍生物;将带有醛基的肝素衍生物与可通过氨醛缩合反应引入巯基的材料经氨醛缩合引入巯基;对上述产物进行还原,将C=N双键还原为C-N单键,获得硫醇化的肝素;
(4)可光交联改性明胶、改性原弹性蛋白和硫醇化的肝素水凝胶的制备:将上述可光交联改性明胶,改性原弹性蛋白和硫醇化的肝素在光敏性引发剂作用下,经自由基聚合反应光交联获得水凝胶。
3.根据权利要求2所述的高粘附性水凝胶的制备方法,其特征在于:用于改性明胶的可产生自由基聚合基团的材料包括甲基丙烯酸酐(MA)、甲基丙烯酸缩水甘油酯(GMA);用于改性原弹性蛋白的可产生自由基聚合基团的材料包括甲基丙烯酸酐(MA)、甲基丙烯酸缩水甘油酯(GMA)。
4.根据权利要求2所述的高粘附性水凝胶的制备方法,其特征在于:用于可通过氨醛缩合引入巯基的材料包括半胱氨酸,对巯基苯胺。
5.根据权利要求2所述的高粘附性水凝胶的制备方法,其特征在于:用于引发自由基聚合的光敏性引发剂为2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮/光引发剂或苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐;
光引发剂浓度为0.5wt.%-1.0 wt.%,紫外波长为320-390 nm,强度为6.0-10 mW/cm2,光照时间为50-100 s。
6. 根据权利要求2所述的高粘附性水凝胶的制备方法,其特征在于:体系中可光交联改性明胶(GelMA )和改性原弹性蛋白(MeTro)的总浓度为5 wt.%-20 wt.%;GelMA:MeTro的质量比为3:7-6:4;体系中硫醇化的肝素的含量为10 wt.%-20 wt.%。
7. 根据权利要求2所述的高粘附性水凝胶的制备方法,其特征在于:所述的步骤(1)具体过程为将含有自由基聚合基团的材料逐滴加入明胶溶液中,恒温搅拌反应,反应温度为50-70℃,反应时间为 2-4h,将产物透析过滤,冻干后保存;
所述的步骤(2)的具体过程为:将原弹性蛋白和可产生自由基聚合基团的材料于PBS溶液中,在0-4℃下充分反应12小时以上后终止反应; 将产物溶液透析过滤,冻干后保存;
所述的步骤(3)中:还原末端为醛基的肝素衍生物的制备:将肝素钠溶解于亚硝酸中,充分混匀,冰浴反应,然后中和至终止反应,透析,冻干得到样品并保存。
8.根据权利要求2所述的高粘附性水凝胶的制备方法,其特征在于:所述的步骤(1)的制备方法中:GelMA溶液透析袋截留分子量为12-14kDa;所述的步骤(2)的制备方法中,MeTro溶液透析袋截留分子量为3000-3500;所述的步骤(3)中的透析为:在NH4HCO3溶液中用截留分子量为3500的透析袋透析;所述的步骤(3)冰浴反应过程中,每5-10min振荡一次。
9.权利要求1所述的高粘附性水凝胶应用于心肌补片或作为厚层微血管化心肌组织的植入材料的应用。
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