CN111588910A - 一种高强度细胞外基质海绵及其制备方法 - Google Patents

一种高强度细胞外基质海绵及其制备方法 Download PDF

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CN111588910A
CN111588910A CN202010456570.7A CN202010456570A CN111588910A CN 111588910 A CN111588910 A CN 111588910A CN 202010456570 A CN202010456570 A CN 202010456570A CN 111588910 A CN111588910 A CN 111588910A
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高秀岩
郑红霞
姜红
任孝敏
王京燕
董平格
高英娇
曲梁静
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Shandong Junxiu Biotechnology Co ltd
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Abstract

本发明公开了一种高强度细胞外基质海绵及其制备方法，这种高强度细胞外基质海绵呈白色蜂窝状结构，其制备方法包括以下步骤：取哺乳动物腹膜组织，机械方式联合碳酸氢钠溶液浸泡去除脂肪，EDTA‑NaOH溶液去除细胞核等免疫原性物质，采用不同浓度梯度的NaOH溶液洗脱，强酸处理，纯化水洗涤至pH值4‑7，冻干，辐照灭菌形成一种高强度细胞外基质海绵。

Description

一种高强度细胞外基质海绵及其制备方法
技术领域
本发明涉及医学材料制备，尤其涉及一种高强度细胞外基质海绵及其制备新方法，属于医用生物材料领域。
背景技术
细胞外基质是由细胞合成并分泌到细胞外间质中的大分子物质，主要由三类成分组成：1.结构蛋白：如胶原、弹性蛋白和基膜粘连蛋白等；2.多糖：如糖胺聚糖、蛋白聚糖、透明质酸和硫酸软骨素等；3.粘连蛋白：如纤粘连蛋白和层粘连蛋白等，此外，还有一些脂质和生长因子等。将同种或异种组织进行脱细胞等处理后可获得细胞外基质，将细胞外基质应用于组织修复，为组织细胞提供再生环境，可有效促进组织修复。
胶原蛋白作为细胞外基质的主要组成成分，具有低抗原性、可降解性、良好的生物相容性及促进细胞增殖等作用，常用来加工成胶原蛋白海绵，然而，其机械强度不足问题在一定程度上限制了其使用。
申请号为201110022008.4的专利介绍了一种胶原海绵及其制备方法，通过对胶原水溶液进行辐射交联，然后冷冻干燥制成，在交联的同时进行了灭菌，避免了制备过程引起的生物安全问题。申请号为200910059363.1的专利公开了一种制备软骨源性胶原海绵支架的方法以及由该方法得到的胶原海绵支架。发明方法包括：粉碎、脱细胞、去端肽、溶解、交联，得到的胶原海绵支架具有良好得生物相容性，抗原性极低，可由来源于同种动物多个个体的软骨制备出均一的海绵支架，其孔径及孔隙率均可控，可为软骨细胞、骨髓间充质干细胞提供适宜的生长增殖环境，特别对软骨缺损部位具有良好的修复效果，更利于标准化生产和临床使用。申请号为200710040444.8的专利公开了多孔胶原海绵的制备方法，将胶原蛋白溶于乙酸溶液中，将壳聚糖溶于乙酸溶液，二者混合后，采用冷冻干燥的方式获得海绵状材料。本发明通过对多孔胶原海绵制备过程中冷冻干燥工艺的参数进行精确控制，得到了孔径均匀的多孔胶原海绵。现有的胶原海绵制备技术仍然没有解决海绵机械强度差的问题。
发明内容
本发明针对现有胶原海绵力学强度差的问题，提供了一种高强度细胞外基质海绵及其制备方法。取材哺乳动物腹膜组织，进行去杂质和去免疫原物质处理后，经过强酸溶胀，冻干获得高强度细胞外基质海绵。通过这种方式制备的海绵，保留了细胞外基质中对组织再生有益的多种成分，对基质的纤维结构破坏小从而使其强度得到保持。其蓬松的三维结构为损伤组织细胞再生提供了很好的支架结构。尤其是，该海绵在吸收液体后，其强度不降反而升高，适合使用时进行缝合处理。
本发明制备一种可诱导骨再生的生物膜的策略为：取哺乳动物腹膜组织，机械方式联合碳酸氢钠溶液浸泡去除脂肪，EDTA-NaOH溶液去除细胞核等免疫原性物质，采用不同浓度梯度的NaOH溶液洗脱，强酸处理，纯化水洗涤至pH值4-7，冻干，辐照灭菌形成一种高强度细胞外基质海绵。
进一步的，本发明制备高强度细胞外基质海绵采用如下技术方案：
一方面，本发明提供一种高强度细胞外基质海绵的制备方法，包括：
1）取新鲜哺乳动物腹膜组织，机械去除表面大块脂肪后，使用1-3%NaHCO3浸泡6-12h，流水冲洗24h，去除剩余脂肪；
2）将步骤1）中组织转入0.5mol/LEDTA-2Na-2.5%NaOH溶液中，室温振摇15h后，改用2%NaOH溶液室温振摇2h，重复上述操作1次；
3）将步骤2）中的组织转入0.5%NaOH溶液中，室温振摇30min，重复3次；
4）将步骤3）中的组织转入pH值＜1的盐酸溶液中；
5）将步骤4）中的组织转入纯化水中洗涤，至pH值4-7；
6）低温冷冻干燥：-80℃冷冻24~48h，冷冻真空抽干；
7）灭菌。
可选的，所述步骤1）中哺乳动物包括猪、牛、狗、羊、兔和鼠，优选小牛。
可选的，所述步骤4）中，盐酸溶液的浓度为0.1mol/L-0.5mol/L，处理时间2-3h，优选1-5h。
另一方面，本发明提供一种高强度细胞外基质海绵，采用如上所述的制备方法制备获得。
可选的，所述的高强度细胞外基质海绵呈白色蜂窝状结构，耐缝合。
本发明的有益效果如下：
1.本发明提供的制备方法获得的一种高强度细胞外基质海绵强度高，可耐受临床缝线的牵拉，湿态下缝合强度可达10N；
2.本发明提供的一种高强度细胞外基质海绵有效去除了材料的免疫原性，保留了原组织的纤维结构，确保海绵力学性能的稳定性；
3.本发明提供的一种高强度细胞外基质海绵的制备方法简单有效，使用的试剂环境影响风险小，易去除，适合制备医学材料。
附图说明
图1为高强度细胞外基质海绵样品图片。
具体实施方式
下面结合具体实施例对本发明作进一步详细说明。需要说明的是，以下所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
实施例
取猪新鲜腹膜组织，机械去除表面大块脂肪后，使用3%NaHCO3浸泡12h，流水冲洗24h，去除剩余脂肪，将组织转入0.5mol/LEDTA-2Na-2.5%NaOH溶液中，室温振摇15h后，改用2%NaOH溶液室温振摇2h，重复上述操作1次。将组织转入0.5%NaOH溶液中，室温振摇30min，重复3次，将组织转入0.3mol/L的盐酸溶液中溶胀2h，纯化水洗涤至pH值6.5，-80℃冷冻24h，冷冻真空干燥，辐照灭菌后备用。

Claims (5)

1.一种高强度细胞外基质海绵的制备方法，其特征在于，包括如下步骤：
1）取新鲜哺乳动物腹膜组织，机械去除表面大块脂肪后，使用1-3%NaHCO3浸泡6-12h，流水冲洗24h，去除剩余脂肪；
2）将步骤1）中组织转入0.5mol/L的EDTA-2Na-2.5%NaOH溶液中，室温振摇15h后，改用2%NaOH溶液室温振摇2h，重复上述操作1次；
3）将步骤2）中的组织转入0.5%NaOH溶液中，室温振摇30min，重复3次；
4）将步骤3）中的组织转入pH值＜1的盐酸溶液中；
5）将步骤4）中的组织转入纯化水中洗涤，至pH值4-7；
6）低温冷冻干燥：-80℃冷冻24h-48h，冷冻真空抽干；
7）灭菌。
2.根据权利要求1所述的制备方法，其特征在于，所述步骤1）中哺乳动物包括猪、牛、狗、羊、兔和鼠，优选小牛。
3.根据权利要求1所述的制备方法，其特征在于，所述步骤4）中，盐酸溶液的浓度为0.1mol/L-0.5mol/L，处理时间2-3h，优选1-5h。
4.一种高强度细胞外基质海绵，其特征在于，采用权利要求1-3任一项所述的制备方法获得。
5.根据权利要求4所述的一种高强度细胞外基质海绵，其特征在于，所述的高强度细胞外基质海绵呈白色蜂窝状结构，厚度为1mm-3mm。
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