CN116139337A - 重组人源胶原蛋白交联海绵及其制备方法 - Google Patents
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Abstract
本发明公开了一种重组人源胶原蛋白交联海绵及其制备方法。所述方法先将硅烷偶联剂在碱性条件下水解形成水解液,然后加入重组人源胶原蛋白,混合均匀后先冷冻干燥形成海绵,再加热使其交联固化,制得重组人源胶原蛋白交联海绵。本发明制备方法简单,制得的重组人源胶原蛋白交联海绵具有优异的力学性能、稳定的生物降解性以及无细胞毒性,作为人体修复材料具有广泛的应用前景。
Description
技术领域
本发明属于生物材料技术领域,涉及一种重组人源胶原蛋白交联海绵及其制备方法。
背景技术
重组人源胶原蛋白是根据人Ⅲ型胶原蛋白α1链胶原域甘氨酸-X-Y(Gly-X-Y)三肽的重复特征序列,经人工设计合成一段人源胶原基因单体,并通过一系列体位剪切酶链接而构建的具有同向六串人源胶原蛋白基因单体的表达载体,再转入到巴氏毕赤酵母菌经高密度发酵、分离和纯化而得到的(中国专利ZL201110327865.5)。
重组人源胶原蛋白作为一类蛋白质分子,其具有高效水溶性,导致其在水中缺乏机械强度和结构稳定性,极大地限制了其进一步应用。为了减缓重组人源胶原蛋白的生物降解并提高它们在水溶液中的机械稳定性,通常通过化学或物理方式对其进行交联。一般物理交联所需工序要求较高并且复杂,且交联效果有限。化学交联相对工艺简单,交联效果较好,但是现有广泛使用的化学交联剂(如戊二醛和六亚甲基异氰酸酯、1,4-丁二醇二缩水甘油醚等)被用于交联胶原蛋白时存在明显的缺点。戊二醛作为常用的交联剂,其残留易引发局部组织钙化反应和细胞毒性反应等。1,4-丁二醇二缩水甘油醚虽然表现出较低细胞毒性,但是经证实其具有导致癌症的风险,根据ISO 10993标准,其残留量被要求严格控制在2ppm以下。因此,需要寻找新型的化学交联剂,使其能够显著提高溶液稳定性和力学强度的同时,具有良好的生物相容性。目前还没有用无机相通过杂化的形式交联重组人源胶原蛋白和调控其性能的报道。
发明内容
针对现有交联重组人源胶原蛋白存在的机械强度和结构稳定性较差、化学交联后生物毒性大等问题,本发明提供一种力学性能优异、生物降解稳定和无细胞毒性的重组人源胶原蛋白交联海绵及其制备方法。
本发明的技术方案如下:
重组人源胶原蛋白交联海绵的制备方法,包括以下步骤:
步骤1,将硅烷偶联剂加入水中,调节pH=9~10,搅拌使其充分水解形成颗粒,得到水解液;
步骤2,将重组人源胶原蛋白加入水解液中,搅拌至混合均匀,将混合溶液倒入模具中,冷冻干燥形成重组人源胶原蛋白海绵,再将重组人源胶原蛋白海绵在40~80℃下交联固化2~10h,制得重组人源胶原蛋白交联海绵。
优选地,步骤1中,硅烷偶联剂选自γ-(2,3-环氧丙氧)丙基三甲氧基硅烷(GPTMS)和γ-缩水甘油醚氧丙基三乙氧基硅烷类含环氧基团的硅烷(GPTES)、四乙氧基硅烷(TEOS)、乙烯基三乙氧基硅烷(VTEO)、3-氨丙基三甲氧基硅烷(APTMS)和3-氨丙基三乙氧基硅烷(APTES)类含氨基的硅烷中的一种或多种,更优选为γ-(2,3-环氧丙氧)丙基三甲氧基硅烷或四乙氧基硅烷。
优选地,步骤1中,加入共溶剂促进硅烷偶联剂水解,例如以乙醇为共溶剂。
优选地,步骤1中,水解时间为30~60min。
优选地,步骤2中,重组人源胶原蛋白由保藏编号为CGMCC No.5021的巴斯德毕赤酵母Pichia pastoris发酵产生,已在中国专利ZL201110327865.5充分公开。
优选地,步骤2中,混合溶液中,重组人源胶原蛋白的浓度为10~15wt.%,硅烷偶联剂的浓度为5~15wt.%,更优选为5~10wt.%。
优选地,步骤2中,搅拌时间为1~10h。
优选地,步骤2中,将混合溶液倒入模具中,先在-70℃快速冷冻,再进行冷冻干燥。
优选地,步骤2中,交联固化温度为50~60℃,交联固化时间为4~6h。
进一步地,本发明提供上述重组人源胶原蛋白交联海绵在制备骨缺损修复材料、口腔黏膜修复材料、皮肤组织修复材料等中的应用。
本发明利用硅烷在碱性条件下发生水解形成具备大量活性基团的类二氧化硅颗粒,并以无机颗粒为交联的位点,在一定条件下与重组人源胶原蛋白链段上存在的氨基、羧基发生共价交联反应形成稳定交联结构,并通过冷冻干燥实现大比表面积多孔重组人源胶原蛋白交联海绵。
与现有技术相比,本发明的有益效果是:
(1)本发明的重组人源胶原蛋白交联海绵以重组人源胶原蛋白作为原料,有效地避免用于人体时排异及动物病毒毒性问题;
(2)本发明以硅烷偶联剂水解后形成的无机颗粒作为交联位点,避免了有毒化学试剂的使用,同时赋予了交联后的重组人源胶原蛋白海绵结构稳定性好、降解速度减缓以及力学性能明显提升的优点,在人体修复材料领域具有潜在的应用前景。
(3)本发明制备方法工艺简单、批次稳定、易操作、可规模化生产。
附图说明
图1为γ-(2,3-环氧丙氧)丙基三甲氧基硅烷水解溶液在光学显微镜下的图,a)为水解溶液PH为9-10,b)为水解溶液PH为7-8;
图2为重组人源胶原蛋白交联海绵样品实物图与SEM图,a)为GPTMS交联的重组人源胶原蛋白交联海绵实物图,b)为TEOS交联的重组人源胶原蛋白交联海绵实物图,c)为GPTMS交联的重组人源胶原蛋白交联海绵的SEM图,d)为TEOS交联的重组人源胶原蛋白交联海绵的SEM图;
图3为不同含量GPTMS交联的重组人源胶原蛋白交联海绵的拉伸强度与断裂伸长图;
图4为重组人源胶原蛋白交联海绵样品溶胀度及降解稳定性结果图,a)不同含量GPTMS的重组人源胶原蛋白交联海绵溶胀率图,b)为不同含量GPTMS的重组人源胶原蛋白交联海绵3天降解率图;
图5不同含量(0wt%、1wt%、5wt%、15wt%)GPTMS重组人源胶原蛋白交联海绵(I-SCF)样品的体外细胞毒测试结果图。
具体实施方式
下面通过具体实施例和附图对本发明作进一步详细说明。下述实施例中,所使用材料、试剂等,如无特殊说明,均可从商业途径购买得到。重组人源化胶原蛋白购自江苏江山聚源生物技术有限公司。
对比例1
首先取8.5g去离子水中,并调节PH至9-10,搅拌30min。随后1.5g重组人源化胶原蛋白加入到溶液中,搅拌溶解2.5h。结束后,将混合溶液倒入模具中,放入-70℃冰箱中急冻5h,再经冷冻干燥24h后得到重组人源胶原蛋白海绵,随后在60℃条件下烘干6h得到重组人源胶原蛋白交联海绵。
实施例1
首先将0.5gγ-(2,3-环氧丙氧)丙基三甲氧基硅烷加入8.0g去离子水中,并调节PH至9-10,搅拌水解30min。随后1.5g重组人源化胶原蛋白加入到溶液中,搅拌溶解2.5h。结束后,将混合溶液倒入模具中,放入-70℃冰箱中急冻5h,再经冷冻干燥24h后得到重组人源胶原蛋白海绵,随后在60℃条件下后固化6h得到重组人源胶原蛋白交联海绵。
实施例2
首先将0.5g四乙氧基硅烷加入7.0g去离子水中,加入1g乙醇作为共溶剂,并调节PH至9-10,搅拌水解30min。随后1.5g重组人源化胶原蛋白加入到溶液中,搅拌溶解2.5h。结束后,将混合溶液倒入模具中,放入-70℃冰箱中急冻5h,再经冷冻干燥24h后得到重组人源胶原蛋白海绵,随后在60℃条件下后固化6h得到重组人源胶原蛋白交联海绵。
实施例3
首先将1.0gγ-(2,3-环氧丙氧)丙基三甲氧基硅烷加入7.5g去离子水中,并调节PH至9-10,搅拌水解30min。随后1.5g重组人源化胶原蛋白加入到溶液中,搅拌溶解2.5h。结束后,将混合溶液倒入模具中,放入-70℃冰箱中急冻5h,再经冷冻干燥24h后得到重组人源胶原蛋白海绵,随后在60℃条件下后固化6h得到重组人源胶原蛋白交联海绵。
实施例4
首先将1.5gγ-(2,3-环氧丙氧)丙基三甲氧基硅烷加入7.0g去离子水中,并调节PH至9-10,搅拌水解30min。随后1.5g重组人源化胶原蛋白加入到溶液中,搅拌溶解2.5h。结束后,将混合溶液倒入模具中,放入-70℃冰箱中急冻5h,再经冷冻干燥24h后得到重组人源胶原蛋白海绵,随后在60℃条件下后固化6h得到重组人源胶原蛋白交联海绵。
对比例2
首先将0.5gγ-(2,3-环氧丙氧)丙基三甲氧基硅烷加入8.0g去离子水中,并调节PH至7-8,搅拌水解30min。随后1.5g重组人源化胶原蛋白加入到溶液中,搅拌溶解2.5h。结束后,将混合溶液倒入模具中,放入-70℃冰箱中急冻5h,再经冷冻干燥24h后得到重组人源胶原蛋白海绵,随后在60℃条件下后固化6h得到重组人源胶原蛋白交联海绵。
表征例
1.拉伸测试
按照医药行业标准YY/T 0471.4-2004的测试方法分别测定各重组人源胶原蛋白交联海绵的机械性能。具体步骤如下:将重组人源胶原蛋白交联海绵样品裁剪为2cm×8cm大小,然后固定在质构仪上,样品的夹持距离为5cm。在温度为25℃、相对湿度为50%的恒温恒湿条件下进行单轴拉伸实验,测试重组人源胶原蛋白交联海绵的拉伸强度。
2.溶胀度测试
将重组人源胶原蛋白交联海绵精确称量,其重量为Wd,然后加到PBS中,常温下放置24小时,把充分膨胀的重组人源胶原蛋白交联海绵取出,用吸水纸去除重组人源胶原蛋白交联海绵表面的游离水后,称量重组人源胶原蛋白交联海绵重量为Wg,计算膨胀度(%)=(Wg-Wd/Wd)×100%。
用按上述方法测得的溶胀度可以间接判断重组人源胶原蛋白交联海绵交联情况。溶胀度越低表明待测交联海绵的交联程度越高,结构越稳定。
3.降解率测试
首先重组人源胶原蛋白交联海绵精确称量,并记录数据Wo;
将备用的海绵分布放入过量PBS水溶液中,并放置于37℃恒温培养箱中,分别浸泡3天;取出重组人源胶原蛋白交联海绵并用蒸馏水冲洗三次,然后在冷冻室冷冻过夜,冷冻干燥,并进行称重计为Wt。按以下公式计算水中降解率(Wo-Wt)/Wo×100%。
按上述方法测得的降解率可判断重组人源胶原蛋白交联海绵降解稳定性情况。降解程度越低,说明表面结构越稳定。
4.体外细胞毒性测试
体外细胞毒性测试根据EN ISO 10993-5:2009《医疗器械的生物学评价--第5部分:体外细胞毒性试验》标准,检测细胞增值率,进行体外细胞毒性测试。
①先将重组人源胶原蛋白交联海绵与RPMI1640培养液按0.001g/ml混合,置于37℃,5%二氧化碳孵箱中浸提24小时,用0.22μm微孔滤膜过滤除菌,得到浸提液;
②将1×105个/mL的L929细胞悬浮液接种于96孔细胞培养板,置于37℃二氧化碳培养箱中培养24小时,待细胞贴壁生长后,去除上清液,分成对照组和实验组两组;
③对照组加入RPMI1640培养液;实验组加入含50%上述浸提液的RPMI1640培养液;将对照组和实验组分别置于37℃二氧化碳培养箱中继续培养,于2天后取出,培养板每孔加入MTT溶液(5mg/ml)20μL,于37℃继续培养4小时,终止培养;
④小心吸弃孔内培养上清液,每孔加入200μL DMSO,振荡10分钟混匀后,用酶联免疫检测仪在630nm下分别测定其吸光度值;
⑤根据下面的公式计算细胞相对增值率(RCR),RCR(%)=(实验组平均吸光度值/对照组平均吸光度值)×100%;
⑥细胞相对增值率RCR与细胞毒性分级关系如下:
RCR不小于100%,细胞毒性分级为0级;
RCR为75~99%,细胞毒性分级为1级;
RCR为50~74%,细胞毒性分级为2级;
RCR为25~49%,细胞毒性分级为3级;
RCR为1~24%,细胞毒性分级为4级;
RCR为0%,细胞毒性分级为5级。
用按上述上方法测得的细胞相对增值率判断重组人源胶原蛋白交联海绵的生物相容性。RCR越高,表明待测重组人源胶原蛋白交联海绵的生物相容性越好。
图1为γ-(2,3-环氧丙氧)丙基三甲氧基硅烷硅烷水解溶液在光学显微镜下的图。其中a)为实施例1中水解溶液PH为9-10,可以看到均匀分布的颗粒。b)为对比例2中水解溶液PH为7-8,无明显颗粒生成。说明溶液PH影响硅烷偶联剂的水解程度。
图2为重组人源胶原蛋白交联海绵样品实物图与SEM图,a)为GPTMS交联的重组人源胶原蛋白交联海绵实物图,b)为TEOS交联的重组人源胶原蛋白交联海绵实物图,c)为GPTMS交联的重组人源胶原蛋白交联海绵的SEM图,d)为TEOS交联的重组人源胶原蛋白交联海绵的SEM图。可以看出,交联海绵均具有大比表面积和多孔的微观结构。
图3为不同含量GPTMS交联的重组人源胶原蛋白交联海绵样品拉伸强度与断裂伸长率图。可以看出,重组人源胶原蛋白交联海绵随GPTMS含量增加其拉伸强度逐渐升高,说明GPTMS加入明显增强了力学强度,断裂伸长率先增加后减小说明过高含量的无机粒子对断裂伸长率不利。
图4为重组人源胶原蛋白交联海绵样品溶胀度及降解稳定性图,a)不同含量GPTMS的重组人源胶原蛋白交联海绵溶胀率图,随GPTMS含量增加溶胀率越低,这表明GPTMS与重组胶原蛋白发生了共价交联且交联密度随其含量提高而提高;b)为不同含量GPTMS的重组人源胶原蛋白交联海绵3天降解率,降解度随GPTMS含量增加而越低,这表明GPTMS通过共价交联的有效地提高了胶原海绵的降解稳定性。
图5不同含量(0wt%、1wt%、5wt%、15wt%)GPTMS重组人源胶原蛋白交联海绵(I-SCF)样品体外细胞毒测试结果图,根据EN ISO 10993-5:2009《医疗器械的生物学评价--第5部分:体外细胞毒性试验》,随着GPTMS含量提高,细胞增殖率越高,表明重组人源胶原蛋白交联海绵生物相容性越好。
Claims (10)
1.重组人源胶原蛋白交联海绵的制备方法,其特征在于,包括以下步骤:
步骤1,将硅烷偶联剂加入水中,调节pH=9~10,搅拌使其充分水解形成颗粒,得到水解液;
步骤2,将重组人源胶原蛋白加入水解液中,搅拌至混合均匀,将混合溶液倒入模具中,冷冻干燥形成重组人源胶原蛋白海绵,再将重组人源胶原蛋白海绵在40~80℃下交联固化2~10h,制得重组人源胶原蛋白交联海绵。
2.根据权利要求1所述的制备方法,其特征在于,步骤1中,硅烷偶联剂选自γ-(2,3-环氧丙氧)丙基三甲氧基硅烷、γ-缩水甘油醚氧丙基三乙氧基硅烷、四乙氧基硅烷、乙烯基三乙氧基硅烷、3-氨丙基三甲氧基硅烷或3-氨丙基三乙氧基硅烷。
3.根据权利要求1所述的制备方法,其特征在于,步骤1中,加入共溶剂乙醇促进硅烷偶联剂水解。
4.根据权利要求1所述的制备方法,其特征在于,步骤1中,水解时间为30~60min;步骤2中,重组人源胶原蛋白由保藏编号为CGMCC No.5021的巴斯德毕赤酵母Pichia pastoris发酵产生。
5.根据权利要求1所述的制备方法,其特征在于,步骤2中,混合溶液中,重组人源胶原蛋白的浓度为10~15wt.%,硅烷偶联剂的浓度为5~15wt.%。
6.根据权利要求1所述的制备方法,其特征在于,步骤2中,混合溶液中,硅烷偶联剂的浓度为5~10wt.%。
7.根据权利要求1所述的制备方法,其特征在于,步骤2中,搅拌时间为1~10h;将混合溶液倒入模具中,先在-70℃快速冷冻,再进行冷冻干燥。
8.根据权利要求1所述的制备方法,其特征在于,步骤2中,交联固化温度为50~60℃,交联固化时间为4~6h。
9.根据权利要求1~8任一所述的制备方法制得的重组人源胶原蛋白交联海绵。
10.根据权利要求9所述的重组人源胶原蛋白交联海绵在制备骨缺损修复材料、口腔黏膜修复材料或皮肤组织修复材料中的应用。
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