CN106390195A - 一种胶原膜的改性方法 - Google Patents
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Abstract
本发明公开了一种胶原膜的改性方法:以培养板为模板,加入胶原蛋白,冷冻过夜;将所得样品真空冷冻干燥24h,制备胶原膜;将表没食子儿茶素没食子酸酯进行溶液配制;将制备的胶原膜置于EGCG中预交联,浸泡于含EGCG的0.05mol/L醋酸溶液,于4℃交联24h,lXPBS缓冲液漂洗3次;将制备好的复合膜浸泡在1%的PEG20000中,浸泡过夜,再用PBS缓冲液冲洗三次,冷冻干燥过夜。本发明的有益效果在于:提供了一种拥有更好机械性能、抗炎性和生物相容性的胶原复合膜,提高了以前胶原膜的机械性能,降低了药物‑胶原复合膜的生物毒性,具有很好的应用前景,为引导骨再生术等相关骨增量手术提供了新选择。
Description
技术领域
本发明属于生物材料领域,具体涉及一种胶原膜的改性方法。
背景技术
牙科手术中常需要应用引导骨再生手术进行骨增量。胶原是细胞外基质的一种成分,已经广泛应用于引导骨再生术中。胶原膜有较好的生物相容性,并能促进成骨细胞黏附、增殖、迁移和分化,也能有阻挡成纤维细胞快速入侵骨组织生长位点、隔开软组织与硬组织的生长等屏障功能。
然而,作为一种外源性移植物,胶原仍然会引起一定的炎症反应,也因为它的易降解性而缺乏较好的机械性能。科学家们采用交联剂与胶原进行交联,从而提高它的机械性能。常用的交联剂是戊二醛,但它容易引发炎症和产生一定的细胞毒性。因此,胶原膜长期的保存和使用,不仅仅需要提高机械性能,更要抗炎和提高生物相容性。
表没食子儿茶素没食子酸酯(EGCG)是一种来自绿茶的多酚,它拥有众多生物学功能,近年来广受关注。科学家们发现,EGCG拥有抗癌、抗氧化、抗炎症、抗纤维化和促进成骨的作用。同时,它也可以作为一种胶原的交联剂,既可以增强胶原的机械性能,也不会破坏胶原的三股螺旋结构。因此,EGCG-胶原复合膜可以用于引导骨再生等手术中,并拥有良好的前景。
EGCG与胶原膜交联后,随着EGCG浓度的增加,胶原膜的机械性随之增加。然而,EGCG作为一种药物,较大浓度的EGCG仍然拥有一定的细胞毒性。Li等人的研究表明不同浓度的EGCG会有不同的作用,不同浓度的EGCG可以有抗癌作用也可能有促癌作用。因此,对EGCG的细胞毒性仍然需要进一步的解决。
因此,现有的胶原膜的机械性能、抗炎性和生物相容性都有待提高。
发明内容
本发明针对现有技术的不足,提供一种胶原膜的改性方法,
其中,所述改性胶原膜是通过加载一种聚合物在用药物-胶原复合膜上得到的。
其中,所述改性胶原膜所述加载的聚合物为聚乙二醇(PEG)。
其中,所述改性胶原膜所用药物为:表没食子儿茶素没食子酸酯(EGCG)。
本发明还提供了聚合物改性的药物-胶原复合膜的制备方法:先用药物交联胶原膜,再加载聚合物,以得到聚合物改性的药物-胶原复合膜。
其中,所述制备方法中改性胶原膜所述加载的聚合物为:聚乙二醇(PEG)。
其中,所述制备方法中改性胶原膜所用药物为:表没食子儿茶素没食子酸酯(EGCG)。
1、一种胶原膜的改性方法,其特征在于,包括以下步骤:
A.以培养板为模板,加入胶原蛋白2mL,在-26℃低温冰箱冷冻过夜;
B.在-53℃、1 Pa条件下,将步骤A所得样品在冻干机真空冷冻干燥24 h,制备胶原膜;
C.将表没食子儿茶素没食子酸酯按照0.64%,0.064%,0.0064%梯度浓度进行溶液配制;
D. 将步骤B中制备的胶原膜置于0.64%EGCG中预交联10 min后,浸泡于含0.64%EGCG的0.05 mol/L醋酸溶液,于4℃交联24 h,lXPBS缓冲液漂洗3次。同样的方法制备加载0.064%EGCG,0.0064%EGCG的的胶原复合膜;
E. 将制备好的EGCG-胶原复合膜浸泡在1%的PEG20000中,于25℃浸泡过夜,再用PBS缓冲液冲洗三次,于-20℃条件下冷冻干燥过夜。
其中,所述步骤A中培养板的直径为3.5cm/孔,所述步骤E还包括胶原复合膜的生物相容性试验。
本发明的有益效果在于:创造性提供了一种拥有更好机械性能、抗炎性和生物相容性的胶原复合膜,提高了以前胶原膜的机械性能,克服了容易引发炎症等长期困扰本领域的技术问题,降低了药物-胶原复合膜的生物毒性,具有很好的应用前景,为引导骨再生术等相关骨增量手术提供了新选择。
附图说明
图1为本发明改性胶原膜的制备示意图。
图2为PEG改性EGCG-胶原复合膜材料表面结构图。
图3为MG63细胞在PEG改性EGCG-胶原复合膜上的形态特征图。
图4为MG63细胞在PEG改性EGCG-胶原复合膜上的活细胞数量特征图。
图5为MG63细胞在PEG改性EGCG-胶原复合膜上的CCK-8细胞活性特征图。
具体实施方式
实施例1:一种胶原膜的改性方法,包括以下步骤:
一、PEG改性的EGCG-胶原复合膜的制备
其中PEG改性的EGCG-胶原复合膜的制备示意图如图1所示。
制备流程一:胶原膜与EGCG复合,制得EGCG-胶原复合膜,该复合膜抑制细胞活力。
制备流程一:胶原膜与EGCG复合,制得EGCG-胶原复合膜,再与PEG改性,该复合膜促进细胞活力。
1、胶原膜的制备
以直径为3.5cm/孔的培养板为模具,加入胶原蛋白2 mL。-26℃低温冰箱冷冻过夜,在一53℃、1 Pa条件下,冻干机真空冷冻干燥24 h,制备胶原膜。
2、EGCG(0.64%,0.064%,0.0064%)梯度浓度溶液配制。
3、EGCG交联胶原膜
将制备的胶原膜置于0.64%EGCG中预交联10 min后,浸泡于含0.64%EGCG的0.05 mol/L醋酸溶液,于4℃交联24 h,l X PBS缓冲液漂洗3次。同样的方法制备加载0.064%EGCG,0.0064%EGCG的的胶原复合膜。
4、PEG化
将制备好的EGCG-胶原复合膜浸泡在1%的PEG20000中,于25℃浸泡过夜,再用PBS缓冲液冲洗三次,于-20℃条件下冷冻干燥过夜。
二、PEG改性的EGCG-胶原复合膜的生物相容性试验
1、细胞培养
MG63细胞株常规养于高糖DMEM培养基中(内含10%胎牛血清,100kU/L青霉素,100kU/L链霉菌)在5%CO2、37℃条件下培养,当融合度达到80%时进行处理。
2、加载EGCG、PEG的胶原膜对成骨细胞处理及观测
分多组进行:每个培养板作一组,编号A1—A15组。将各组细胞置于事先制备好的PEG改性的EGCG-胶原复合膜上进行培养,做空白组,每个培养板继续在前述环境下培养,换液时同时重新配置培养液梯度。
3、MG63细胞形态的免疫荧光染色。
用含2%多聚甲醛的PBS固定第3天的细胞,固定5分钟(pH 7.4),将样品在PBS中洗涤3次(每次5分钟),用含有1%牛血清白蛋白和0.1%Triton X-100的PBS预处理1小时,然后在1%Tween20中保温20分钟。之后用PBS洗涤5分钟后,培养在用PBS1:100稀释的Rhodamine-phalloidin (Life technologies, Thermo, USA)中15分钟。然后用PBS冲洗3次,每次5分钟。添加300nM的DAPI染色溶液(Life technologies, Thermo, USA),覆盖细胞和培养基1-5分钟,避光。然后,用PBS冲洗细胞2-3次以去除染色液,采取图像(图2)。结果显示,PEG改性后的EGCG-胶原复合膜, 明显促进了细胞的铺展, 细胞有更好的型态。
4、CCK-8法(测量细胞增殖活性)
在96孔板中, 注入成骨细胞达到7000个/孔. 贴壁后,换成含2%胎牛血清的高糖DMEM配制的各种浓度的EGCG溶液,每孔200μl。继续培养24、48h,每孔换成新鲜培养液100μl,再加入10μl CCK-8溶液,细胞培养箱中孵育1h后在酶标仪上450nm波长处读取各孔吸亮度(A)值,计算细胞增殖率,公式为=(处理组A值-空白A值)/(对照组A值-空白A值)*100%。结果见图3。结果发现PEG改性后的EGCG-胶原复合膜上的细胞活性显著高于E-Col组。
5、活死细胞染色(CCK-8)
将实验孔样品经PBS液漂洗15s后,转移至盖玻片上,用含10μg/ml Calcein-AM的PBS液孵育30min,在共聚焦显微镜下检测成像。Calcein-AM显示绿色标示活细胞。计数活细胞数量(N活)。结果发现PEG改性后的EGCG-胶原复合膜上的活细胞数量显著高于E-Col组。
Claims (4)
1.一种胶原膜的改性方法,其特征在于,包括以下步骤:
A、以培养板为模板,加入胶原蛋白2mL,在-26℃低温冰箱冷冻过夜;
B、在-53℃、1Pa条件下,将步骤A所得样品在冻干机真空冷冻干燥24h,制备胶原膜;
C、将表没食子儿茶素没食子酸酯按照梯度浓度进行溶液配制;
D、将步骤B中制备的胶原膜置于EGCG中预交联10 min后,浸泡于含EGCG的0.05 mol/L醋酸溶液,于4℃交联24 h,lXPBS缓冲液漂洗3次;
E、将制备好的EGCG-胶原复合膜浸泡在1%的PEG20000中,于25℃浸泡过夜,再用PBS缓冲液冲洗三次,于-20℃条件下冷冻干燥过夜。
2.根据权利要求1所述的一种胶原膜的改性方法,其特征在于:所述步骤A中,培养板的直径为3.5cm/孔。
3.根据权利要求1所述的一种胶原膜的改性方法,其特征在于:所述步骤C中,梯度浓度为0.64%,0.064%,0.0064%。
4.根据权利要求1所述的一种胶原膜的改性方法,其特征在于:所述步骤E中,还包括胶原复合膜的生物相容性试验。
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CN107583099A (zh) * | 2017-07-24 | 2018-01-16 | 青岛金典生化器材有限公司 | 一种医用胶原蛋白膜敷料及其制备方法 |
CN108567994A (zh) * | 2018-06-29 | 2018-09-25 | 四川大学 | 一种改性胶原膜及其应用 |
CN108785744A (zh) * | 2018-06-29 | 2018-11-13 | 四川大学 | 一种改性胶原膜及其应用 |
CN108822318A (zh) * | 2018-06-29 | 2018-11-16 | 四川大学 | 一种聚乙二醇改性胶原膜及其应用 |
CN109096520A (zh) * | 2018-06-29 | 2018-12-28 | 四川大学 | 一种改性胶原及其制备方法 |
CN109331232A (zh) * | 2018-10-25 | 2019-02-15 | 四川大学华西医院 | 一种表没食子儿茶素没食子酸酯交联的小肠黏膜下层引导骨再生膜的制备方法 |
CN111888531A (zh) * | 2020-08-14 | 2020-11-06 | 广州润虹医药科技股份有限公司 | 一种引导性组织再生膜及其制备方法 |
CN113559323A (zh) * | 2021-07-31 | 2021-10-29 | 福建省博特生物科技有限公司 | 一种负载没食子酸胶原蛋白可注射水凝胶的制备方法及水凝胶的应用 |
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