CN110903510A - 一种可见光共聚交联多孔网状GelMA-dHAMMA水凝胶的制备方法 - Google Patents
一种可见光共聚交联多孔网状GelMA-dHAMMA水凝胶的制备方法 Download PDFInfo
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Abstract
本发明涉及医用生物敷料领域,具体地说,是一种可见光共聚交联多孔网状GelMA‑dHAMMA水凝胶的制备方法。具体的制作方法为:明胶通过甲基丙烯酸酐(MA)接枝改性形成甲基丙烯酰胺基明胶(GelMA);人脱细胞羊膜(dHAM)通过甲基丙烯酸酐(MA)接枝改性形成dHAMMA,将接枝改性后的人脱细胞羊膜dHAMMA研磨成粉状;将形成的粉状GelMA与dHAMMA按质量比2:1混合,加入光引发剂L0290行光交联;将光交联后的复合水凝胶冻干,于γ辐照灭菌,于无菌条件下封装。本发明具有良好的抗炎、抗感染及生物相容性,能够促进口腔黏膜创口快速愈合。
Description
技术领域
本发明涉及医用生物敷料领域,具体地说,是一种可见光共聚交联多孔网状GelMA-dHAMMA水凝胶的制备方法。
背景技术
口腔黏膜位置相对特殊,它覆盖口腔表面,前方与唇部皮肤相连,后方与咽喉黏膜相连,通过抵抗机械性刺激、限制毒性物质、微生物入侵及唾液的分泌等发挥口腔保护作用。严重粘膜病变、肿瘤手术等对口腔粘膜创伤严重,常引起较大的口腔粘膜缺损,以往采用打碘仿油纱包覆盖创面,邻近瓣转移修复如颊脂垫瓣、腭瓣、颈阔肌瓣等。不仅增加手术难度及创伤,而且引起供区不同程度的功能障碍及并发症。打碘仿油纱包覆盖创面,导致术后疤痕畸形,引起张口受限、舌活动受限等。理想的口腔黏膜缺损替代材料应具有以下特点:1)造价低;2)保存期长;3)使用方便,手术操作简单,易于固定;4)无抗原性;5)具有持久性;6)具有柔韧性;7)对细菌具有屏障作用;8)对不规则创面顺应性好;9)不会过度增生。目前尚无满足以上所有条件的理想替代物。
发明内容
为了解决上述技术问题,本发明旨在发明一种既有良好保护效果如抗炎、抗感染及促进愈合等,又具有良好的透气性、保水性及韧性的生物活性敷料,能够达到刺激缺损区细胞生长及血管形成,促进口腔黏膜缺损的快速修复,缺损区黏膜组织再生后,原敷料可生物降解或被新生组织爬行替代目的的复合水凝胶敷料的制备方法。
为了实现上述技术目的,本发明采用的具体技术方案如下:
一种可见光共聚交联多孔网状GelMA-dHAMMA水凝胶的制备方法,其特征在于,主要包括以下步骤:
步骤1:脱细胞羊膜的制备;
步骤2:dHAM通过甲基丙烯酸酐(MA)接枝改性形成dHAMMA;
步骤3:GelMA水凝胶电纺液的制备;
步骤4:多孔网状复合水凝胶的制备。
本发明进一步改进,所述步骤1包括以下流程:
采取样本前征得产妇知情同意,并实验经医院伦理委员会批准;
选择肝炎病毒抗原、梅毒抗体及人免疫缺陷病毒抗体均为阴性的剖腹产的产妇,无菌操作下取胎膜,钝性分离去除绒毛膜组织,保留羊膜层;
将羊膜转移到补充有抗生素及抗菌剂的无菌PBS(PH 7.4)中反复冲洗去除血凝块,然后用0.2%EDTA在37℃下处理30分钟;
接着于0.5mol/L氢氧化钠溶液中浸泡30秒;
此后将其转移到5%氯化铵中剧烈摇晃;
通过剧烈晃动和刮擦从羊膜中去除细胞,最后用PBS洗涤三次。
本发明进一步改进,所述步骤2包括以下流程:
脱细胞基质主要由胶原纤维组成,胶原纤维上多余的氨基,能够与酰氯或酸酐反应生成酰胺键,从而将甲基丙烯酰胺接枝到胶原纤维分子链上;
将甲基丙烯酰胺接枝到dHAM胶原纤维分子链上;
基于上述原理,将上述脱细胞羊膜浸泡于一定浓度的MA溶液中(MA溶于PBS中,浓度控制在4%V/V),避光条件下进行甲基丙烯酸酰酯化,反应温度控制在4℃,反应时间为12小时,形成dHAMMA
然后取出dHAMMA,用PBS溶液充分反复冲洗,去除未反应的MA;
将dHAMMA冻干,研磨成粉状备用。
本发明进一步改进,所述步骤3包括以下流程:
将20g明胶与200mlPBS混合放入预热的60℃水浴中搅拌溶解至透明黄色液体;
用泵将16ml甲基丙烯酸酐以4ml/min的速度在避光条件下滴入上述液体并充分搅拌2小时;
加入预热好的800mlPBS搅拌反应15min注入14-16KD透析袋中用去离子水透析一周(温度不宜过高,期间予以更换去离子水);
一周后将取出透析袋中溶液,选用0.45um滤纸过滤后放入-20℃冰箱预冻一夜,之后冻干备用。
本发明进一步改进,所述步骤4包括以下流程:
将GelMA400mg溶解于PBS中,得到浓度为10%(w/v)的GelMA溶液;
加入200mgdHAMMA均匀搅拌,之后加入0.1%(w/v)的苯基磷酸锂盐(L0290,TokyoChemical Industry, Japan)作为光引发剂;
加入L0290后,将混合溶液置于可见光光固化设备(395-480 nm,10.5 mm固化尖端)中20秒,形成0.2 cm厚的膜;
将合成的GeLMA-dHAMMA复合水凝胶用PBS冲液洗涤,于γ辐照灭菌,于无菌条件下封装,4℃保存。
本发明的有益效果:1、脱细胞羊膜通过接枝改性及光交联技术与GelMA共价结合,形成三维网络多孔结构复合水凝胶敷料,高度模拟天然细胞外基质结构,利于缺损区组织细胞的生长。
2、该双层敷料既具有良好的机械强度和顺应性,同时具有脱细胞羊膜的生物活性,不仅能够促进缺损区组织的再生愈合,同时能够为细胞生长、血管形成提供稳定的细胞外环境。
3、如果在敷料使用时加入抗菌药、止血药或止痛药,该敷料所具备的空间网络能够将药物缓慢持续的释放到创面起到持续治疗作用。
附图说明
图1为本发明的脱细胞羊膜的制作示意图;
图2为本发明的d-HAM通过甲基丙烯酸酐(MA)接枝改性形成dHAMMA制作示意图;
图3为本发明的GelMA水凝胶合成示意图;
图4为本发明的GelMA-dHAMMA复合水凝胶制备示意图。
具体实施方式
为了加深对本发明的理解,下面将结合实施例对本发明做进一步详细描述,该实施例仅用于解释本发明,并不对本发明的保护范围构成限定。
实施例,如图1-4所示:一、脱细胞羊膜的制备:采取样本前征得产妇知情同意,并实验经医院伦理委员会批准。选择肝炎病毒抗原、梅毒抗体及人免疫缺陷病毒抗体均为阴性的剖腹产的产妇,无菌操作下取胎膜,钝性分离去除绒毛膜组织,保留羊膜层。将羊膜转移到补充有抗生素及抗菌剂的无菌PBS(PH 7.4)中反复冲洗去除血凝块,然后用0.2%EDTA在37℃下处理30分钟;接着于0.5mol/L氢氧化钠溶液中浸泡30秒;此后将其转移到5%氯化铵中剧烈摇晃;通过剧烈晃动和刮擦从羊膜中去除细胞,最后用PBS洗涤三次。
二、dHAM通过甲基丙烯酸酐(MA)接枝改性:脱细胞基质主要由胶原纤维组成,胶原纤维上多余的氨基,能够与酰氯或酸酐反应生成酰胺键,从而将甲基丙烯酰胺接枝到胶原纤维分子链上。将甲基丙烯酰胺接枝到dHAM胶原纤维分子链上。基于上述原理,将上述脱细胞羊膜浸泡于一定浓度的MA溶液中(MA溶于PBS中,浓度控制在4%V/V),避光条件下进行甲基丙烯酸酰酯化,反应温度控制在4℃,反应时间为12小时,形成dHAMMA。然后取出dHAMMA,用PBS溶液充分反复冲洗,去除未反应的MA。将dHAMMA冻干,研磨成粉状备用。
三、GelMA水凝胶电纺液的制备:将20g明胶与200mlPBS混合放入预热的60℃水浴中搅拌溶解至透明黄色液体;用泵将16ml甲基丙烯酸酐以4ml/min的速度在避光条件下滴入上述液体并充分搅拌2小时;加入预热好的800mlPBS搅拌反应15min注入14-16KD透析袋中用去离子水透析一周(温度不宜过高,期间予以更换去离子水);一周后将取出透析袋中溶液,选用0.45um滤纸过滤后放入-20℃冰箱预冻一夜,之后冻干备用。
四、多孔网状复合水凝胶的制备:将GelMA400mg溶解于PBS中,得到浓度为10%(w/v)的GelMA溶液,加入200mgdHAMMA均匀搅拌,之后加入0.1%(w/v)的苯基磷酸锂盐(L0290,Tokyo Chemical Industry, Japan)作为光引发剂。加入L0290后,将混合溶液置于可见光光固化设备(395-480 nm,10.5 mm固化尖端)中20秒,形成0.2 cm厚的膜。将合成的GelMA-dHAMMA复合水凝胶用PBS冲液洗涤,于γ辐照灭菌,于无菌条件下封装,4℃保存。
临床使用时,可根据创面的需要必要时配置好含抗菌药、止血药或止痛药的药液,将敷料使用前于上述药液浸泡,要其肿胀吸附药物后覆盖于口腔黏膜缺损区,然后予以固定,待其自然吸收或手工取出。
以上显示和描述了本发明的基本原理、主要特征及优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定的权利要求书及其等效物界定。
Claims (5)
1.一种可见光共聚交联多孔网状GelMA-dHAMMA水凝胶的制备方法,其特征在于,主要包括以下步骤:
步骤1:脱细胞羊膜的制备;
步骤2:d-HAM通过甲基丙烯酸酐(MA)接枝改性形成d-HAMMA;
步骤3:GelMA水凝胶电纺液的制备;
步骤4:多孔网状复合水凝胶的制备。
2.根据权利要求1所述的一种可见光共聚交联多孔网状GelMA-dHAMMA水凝胶的制备方法,其特征在于,所述步骤1包括以下流程:
采取样本前征得产妇知情同意,并实验经医院伦理委员会批准;
选择肝炎病毒抗原、梅毒抗体及人免疫缺陷病毒抗体均为阴性的剖腹产的产妇,无菌操作下取胎膜,钝性分离去除绒毛膜组织,保留羊膜层;
将羊膜转移到补充有抗生素及抗菌剂的无菌PBS(PH 7.4)中反复冲洗去除血凝块,然后用0.2%EDTA在37℃下处理30分钟;
接着于0.5mol/L氢氧化钠溶液中浸泡30秒;
此后将其转移到5%氯化铵中剧烈摇晃;
通过剧烈晃动和刮擦从羊膜中去除细胞,最后用PBS洗涤三次。
3.根据权利要求1所述的一种可见光共聚交联多孔网状GelMA-dHAMMA水凝胶的制备方法,其特征在于,所述步骤2包括以下流程:
脱细胞基质主要由胶原纤维组成,胶原纤维上多余的氨基,能够与酰氯或酸酐反应生成酰胺键,从而将甲基丙烯酰胺接枝到胶原纤维分子链上;
将甲基丙烯酰胺接枝到dHAM胶原纤维分子链上;
基于上述原理,将上述脱细胞羊膜浸泡于一定浓度的MA溶液中(MA溶于PBS中,浓度控制在4%V/V),避光条件下进行甲基丙烯酸酰酯化,反应温度控制在4℃,反应时间为12小时,形成dHAMMA
然后取出dHAMMA,用PBS溶液充分反复冲洗,去除未反应的MA;
将dHAMMA冻干,研磨成粉状备用。
4.根据权利要求1所述的一种可见光共聚交联多孔网状GelMA-dHAMMA水凝胶的制备方法,其特征在于,所述步骤3包括以下流程:
将20g明胶与200mlPBS混合放入预热的60℃水浴中搅拌溶解至透明黄色液体;
用泵将16ml甲基丙烯酸酐以4ml/min的速度在避光条件下滴入上述液体并充分搅拌2小时;
加入预热好的800mlPBS搅拌反应15min注入14-16KD透析袋中用去离子水透析一周(温度不宜过高,期间予以更换去离子水);
一周后将取出透析袋中溶液,选用0.45um滤纸过滤后放入-20℃冰箱预冻一夜,之后冻干备用。
5.根据权利要求1所述的一种可见光共聚交联多孔网状GelMA-dHAMMA水凝胶的制备方法,其特征在于,所述步骤4包括以下流程:
将GeLMA400mg溶解于PBS中,得到浓度为10%(w/v)的GelMA溶液;
加入200mgdHAMMA均匀搅拌,之后加入0.1%(w/v)的苯基磷酸锂盐(L0290,TokyoChemical Industry, Japan)作为光引发剂;
加入L0290后,将混合溶液置于可见光光固化设备(395-480 nm,10.5 mm固化尖端)中20秒,形成0.2 cm厚的膜;
将合成的GeLMA-dHAMMA复合水凝胶用PBS冲液洗涤,于γ辐照灭菌,于无菌条件下封装,4℃保存。
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