CN115120773A - 一种可诱导丝蛋白纤维麦克烯导电凝胶的制备方法和应用 - Google Patents
一种可诱导丝蛋白纤维麦克烯导电凝胶的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种可诱导丝蛋白纤维麦克烯导电凝胶的制备方法和应用。本发明的制备方法包括：制备再生丝蛋白原液；配制一定浓度的再生丝蛋白水溶液，在其中加入麦克烯、辣根过氧化物酶及过氧化氢溶液混合，得到水凝胶前驱液；待水凝胶前驱液成胶后于乙醇溶液中浸泡，然后取出凝胶烘干，从而获得可诱导丝蛋白纤维麦克烯导电凝胶。本发明所获得的丝蛋白纤维麦克烯导电凝胶兼具可导电、价廉、环保、生物相容性好、可降解、自带抗菌性、促成血管、促成骨、抗炎等优点；其不仅可直接促进间充质干细胞分化促进成骨，还可以促进血管内皮细胞血管生成，也可间接促进巨噬细胞M2极化改善成骨免疫环境；其在骨缺损修复中具有良好的应用前景。
Description
技术领域
发明涉及一种可诱导丝蛋白纤维麦克烯导电凝胶的制备方法和应用，属于生物医用材料技术领域。
背景技术
骨作为人体承重的重要器官，对于人的运动能力至关重要。因各种原因导致的骨缺损不但影响肢体功能，严重时还可导致瘫痪、残疾，严重降低患者生存质量。应用材料对骨缺损进行修复是治疗骨缺损的唯一方法。另外，研究表明，电刺激(ES)在生理环境下具有较强的促成骨、促成血管以及抗炎作用。根据文献报道，ES主要通过促进碱性磷酸酶(ALP)、胶原纤维I(COL-1)等成骨蛋白的表达产生成骨作用；通过上调细胞核中CD31+的表达以促进成血管；通过上调TGF-β、CD206、Arg-1、IL-10等抗炎因子、下调TGF-α、NF-kB p65、CCR7等炎症因子、促进巨噬细胞向M2趋化等方式促进局部抗炎、抗感染作用。然而，骨缺损部位的导电性欠佳，如果植入的修复材料具有良好导电性，则可使骨缺损的修复锦上添花。目前金属材料的骨修复材料仍然是骨修复领域的最常见的植入物。部分金属材料虽然具有较好的导电性，但需要考虑到导电会促进金属材料的腐蚀，减少植入材料的使用寿命。另外如果要满足骨修复这一目的，就需要修复材料本身疏松多孔，从而有利于成骨细胞的生长。同时金属材料的生物相容性相对较差，部分人体会产生排异反应，需要在骨愈合后二次手术取出。此外，金属支架的加工难度较大，需要较为复杂的生产工艺，且原材料也不能从自然界直接获得，需要对矿石等进行提炼，价格高昂。随着可降解高分子材料和生物陶瓷材料的长足发展，一些可降解的非金属材料支架开始出现并商品化。但这些非金属支架的力学强度普遍较差，在应用于承重骨骼的修复时常出现支架断裂的情况。其降解产物多为酸性，会导致支架周围组织炎症，继而诱发感染，增加二次手术风险。
天然桑蚕丝来源的丝蛋白水凝胶由于其良好的生物相容性、易于获得、溶胀性好、天然抗菌、疏松多孔的优点，是作为一种用于制备可降解高分子骨修复支架材料的研究热点。已有文献报道将丝蛋白水凝胶作为支架用于骨修复，但由于单纯的丝蛋白水凝胶导电性很差，需要向丝蛋白水凝胶中添加导电材料才可使水凝胶具有良好的导电性。
麦克烯(MXene)作为一种受到科研关注的过渡金属化合物，其在人体内可降解且具有较好的导电性、生物相容性，因此其拥有巨大的生物医学应用潜力。其本身在人体内可促进成骨蛋白的表达，如碱性磷酸酶(ALP)以及I型胶原纤维，进而促进骨缺损的修复。若将丝蛋白水凝胶与麦克烯结合，则可使该产物兼备二者优点。目前还未见相关报道。
发明内容
本发明的目的是合成丝蛋白纤维麦克烯导电凝胶，使其兼具可导电、可降解、生物相容性好、天然抗菌、促成骨、促成血管、抗炎的特性，增强骨缺损部位的导电能力，促进骨修复。
为了实现上述目的，本发明提供了一种可诱导丝蛋白纤维麦克烯导电凝胶的制备方法，包括如下步骤：
步骤1：制备再生丝蛋白原液；
步骤2：取步骤1所得的再生丝蛋白原液配制成一定浓度的再生丝蛋白水溶液，再加入麦克烯、辣根过氧化物酶及过氧化氢溶液混合，得到水凝胶前驱液；
步骤3：待水凝胶前驱液成胶后于乙醇溶液中浸泡，然后取出凝胶烘干，从而获得可诱导丝蛋白纤维麦克烯导电凝胶。
优选地，所述步骤1中制备再生丝蛋白原液的方法包括：将天然桑茧或生桑蚕丝经脱胶、烘干后制成脱胶蚕丝，将脱胶蚕丝溶解，然后经过透析除去杂质并浓缩后获得再生丝蛋白原液。
优选地，所述脱胶是采用碳酸钠溶液进行脱胶；所述溶解是采用溴化锂溶液溶解；所述透析是采用截留分子量为14000的透析袋；所述浓缩是采用高分子聚合物进行透析浓缩。
优选地，所述高分子聚合物为分子量为6000～12000的聚乙二醇。
优选地，所述步骤2中再生丝蛋白水溶液的浓度为2～8wt％。
优选地，所述步骤2的水凝胶前驱液中麦克烯的浓度为1～10mg/mL，辣根过氧化物酶的浓度为800～1000U/mL，过氧化氢的浓度为0.4～0.6wt％。
优选地，所述步骤3中乙醇溶液的体积浓度为75％，所述浸泡的时间为24～36h。
本发明还提供了上述的可诱导丝蛋白纤维麦克烯导电凝胶的制备方法制备所得的可诱导丝蛋白纤维麦克烯导电凝胶在制备骨缺损修复的产品中的应用。
与现有技术相比，本发明的有益效果在于：
(1)本发明所获得的丝蛋白纤维麦克烯导电凝胶兼具可导电、价廉、环保、生物相容性好、可降解、自带抗菌性、促成血管、促成骨、抗炎等优点；其不仅可直接促进间充质干细胞分化促进成骨，也可以促进血管内皮细胞血管生成，还可间接促进巨噬细胞M2极化改善成骨免疫环境；其在骨缺损修复中具有良好的应用前景；
(2)本发明选用的原材料桑茧自然界存量巨大且可直接获得；且原材料加工后废料均可回收，对环境无害；此外，本发明的工艺流程简单，适于规模化制备。
附图说明
图1为本发明的可诱导丝蛋白纤维麦克烯导电凝胶的制备工艺流程图；
图2为透射电镜(TEM)观察麦克烯再生丝蛋白溶液的共孵育结果，SF-0h：单纯丝蛋白溶液；SF-72h：单纯丝蛋白溶液37℃水浴孵育72小时；MXene：4mg/mL单纯麦克烯水溶液，MXene/SF-72h：4mg/mL麦克烯丝蛋白溶液37℃水浴孵育72小时；
图3为本发明制备的可诱导丝蛋白纤维麦克烯导电凝胶的SEM图及对应的SEM-EDS图，SF：丝蛋白水凝胶，2M/SF：2mg/mL麦克烯丝蛋白水凝胶，4M/SF：4mg/mL麦克烯丝蛋白水凝胶，8M/SF：8mg/mL麦克烯丝蛋白水凝胶；
图4为本发明制备的可诱导丝蛋白纤维麦克烯导电凝胶的导电能力测试结果，SF：丝蛋白水凝胶，2M/SF：2mg/mL麦克烯丝蛋白水凝胶，4M/SF：4mg/mL麦克烯丝蛋白水凝胶，8M/SF：8mg/mL麦克烯丝蛋白水凝胶。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
以下实施例中，如无特殊说明，百分比代表的是质量百分比，所用到的试剂和材料为常规市售产品。
实施例1
本实施例提供了一种丝蛋白纤维麦克烯导电凝胶的制备方法，其工艺流程如图1所示，具体包括如下步骤：
1、丝蛋白的制备：
步骤1)：将天然桑茧浸入5％碳酸钠溶液中煮沸30min，重复两次进行脱胶，然后烘干制成脱胶蚕丝；
步骤2)：将步骤1)获得脱胶蚕丝于60℃水浴下用9.7mol/L溴化锂溶液按照质量比1：10溶解并加热1小时；
步骤3)：将步骤2)获得的再生丝蛋白液冷却至室温(25℃)后，装入截留分子量为14000的透析袋于去离子水中透析72小时，期间每8小时更换去离子水；
步骤4)：在步骤3)透析完毕后置于30％的分子量为9000-10000的聚乙二醇水溶液中透析24小时进行浓缩，将得到的再生丝蛋白原液配制为5％的再生丝蛋白(RSF)溶液。
2、丝蛋白纤维麦克烯导电凝胶的制备：
步骤1)：将5％的再生丝蛋白溶液与辣根过氧化物酶(HRP，900U/mL)、过氧化氢溶液(过氧化氢浓度0.5％)和麦克烯(2mg/mL)混合，得到前驱液；其中HRP、过氧化氢和麦克烯(吉林省一一科技有限公司，货号：22-3-6-2-Dp)的浓度均为终浓度，即在前驱液中的浓度；
步骤2)：将步骤1)形成的前驱液在室温(25℃)下静置30分钟，前驱液初步成胶；
步骤3)：将步骤2)获得产物于75％(体积分数)乙醇溶液中浸泡24～36小时；
步骤4)：将步骤3)产物烘干，获得丝蛋白纤维麦克烯导电凝胶。
实施例2
本实施例提供了一种丝蛋白纤维麦克烯导电凝胶的制备方法，步骤同实施例1，与实施例1不同的是，麦克烯在前驱液中的浓度为4mg/mL。
实施例3
本实施例提供了一种丝蛋白纤维麦克烯导电凝胶的制备方法，步骤同实施例1，与实施例1不同的是，麦克烯在前驱液中的浓度为8mg/mL。
对比例
丝蛋白水凝胶的制备：制备方法同实施例1，与实施例1不同的是，未加入麦克烯。
使用透射电镜(TEM)对麦克烯的再生丝蛋白溶液进行时间梯度观察，其中，单纯的丝蛋白溶液和单纯的麦克烯水溶液作为对照组，可见到麦克烯与丝蛋白共孵育后可促进丝蛋白纤维的生长，如图2所示；
使用日立S-4800系统对上述实施例1～3制备的丝蛋白纤维麦克烯导电凝胶的微观结构进行观察，其中，对比例制备的丝蛋白水凝胶作为对照，可见凝胶本身为疏松多孔结构，SEM-EDS分析可见麦克烯呈现点状均匀分布于丝蛋白凝胶中，如图3所示；
电化学分析测试了上述实施例1～3制备的丝蛋白纤维麦克烯导电凝胶的电导率，其中，对比例制备的丝蛋白水凝胶作为对照，发现随着麦克烯的加入，凝胶的导电能力明显提高，且加入的麦克烯浓度越高，导电能力越好，如图4所示。
上述制备的丝蛋白纤维麦克烯导电凝胶在骨缺损修复中的应用：
根据骨缺损的形状、大小、深度对丝蛋白纤维麦克烯导电凝胶进行塑型，经过75％乙醇浸泡以及紫外灯照射杀菌后植入骨缺损部位，在骨缺损两侧植入皮下电极后，消毒、缝皮。手术后1周内每日观察伤口有无红肿化脓、换药。术后12周内每日对骨缺损部位使用100mV/mm电压予以电刺激30分钟，约12周后骨缺损得到明显修复。
Claims (8)
1.一种可诱导丝蛋白纤维麦克烯导电凝胶的制备方法，其特征在于，包括如下步骤：
步骤1：制备再生丝蛋白原液；
步骤2：取步骤1所得的再生丝蛋白原液配制成一定浓度的再生丝蛋白水溶液，再加入麦克烯、辣根过氧化物酶及过氧化氢溶液混合，得到水凝胶前驱液；
步骤3：待水凝胶前驱液成胶后于乙醇溶液中浸泡，然后取出凝胶烘干，从而获得可诱导丝蛋白纤维麦克烯导电凝胶。
2.如权利要求1所述的可诱导丝蛋白纤维麦克烯导电凝胶的制备方法，其特征在于，所述步骤1中制备再生丝蛋白原液的方法包括：将天然桑茧或桑蚕丝经脱胶、烘干后制成脱胶蚕丝，将脱胶蚕丝溶解，然后经过透析除去杂质并浓缩后获得再生丝蛋白原液。
3.如权利要求2所述的可诱导丝蛋白纤维麦克烯导电凝胶的制备方法，其特征在于，所述脱胶是采用碳酸钠溶液进行脱胶；所述溶解是采用溴化锂溶液溶解；所述透析是采用截留分子量为14000的透析袋；所述浓缩是采用高分子聚合物进行透析浓缩。
4.如权利要求3所述的可诱导丝蛋白纤维麦克烯导电凝胶的制备方法，其特征在于，所述高分子聚合物为分子量为6000～12000的聚乙二醇。
5.如权利要求2所述的可诱导丝蛋白纤维麦克烯导电凝胶的制备方法，其特征在于，所述步骤2中再生丝蛋白水溶液的浓度为2～8wt％。
6.如权利要求1所述的可诱导丝蛋白纤维麦克烯导电凝胶的制备方法，其特征在于，所述步骤2的水凝胶前驱液中麦克烯的浓度为1～10mg/mL，辣根过氧化物酶的浓度为800～1000U/mL，过氧化氢的浓度为0.4～0.6wt％。
7.如权利要求1所述的可诱导丝蛋白纤维麦克烯导电凝胶的制备方法，其特征在于，所述步骤3中乙醇溶液的体积浓度为75％，所述浸泡的时间为24～36h。
8.权利要求1～7中任意一项所述的可诱导丝蛋白纤维麦克烯导电凝胶的制备方法制备所得的可诱导丝蛋白纤维麦克烯导电凝胶在制备骨缺损修复的产品中的应用。
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