CN114479119B - 一种应用于牙周炎治疗的负载ZIF-8的GelMA水凝胶的制备方法 - Google Patents
一种应用于牙周炎治疗的负载ZIF-8的GelMA水凝胶的制备方法 Download PDFInfo
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Abstract
本研究公开了一种应用于牙周炎治疗的负载2‑甲基咪唑锌盐(ZIF‑8)的甲基丙烯酰化明胶(GelMA)复合光敏水凝胶制品及其制备方法。利用物理方法将ZIF‑8与GelMA混合得到复合光敏水凝胶。本发明制备工艺简单,重复性好,具有很好的生物相容性,能够促进大鼠骨髓间充质干细胞的早期粘附与增殖,具有抗菌能力,并提高成骨相关活性。在的牙周炎大鼠模型中显著促进了破坏牙槽骨的修复。
Description
技术领域
本发明属于牙科技术领域,具体涉及一种应用于牙周炎治疗的载有ZIF-8的GelMA复合光敏水凝胶制品及其制备方法。
背景技术
牙周炎是由牙菌斑的微生物引起的,它可以逐渐破坏牙齿的支撑结构,即牙槽骨、牙周膜和牙龈,最终导致牙齿脱落。牙周炎是世界上最常见的流行病之一,全球有7.43亿人受其影响。它还与糖尿病、阿尔茨海默病等一些全身性疾病密切相关。目前牙周炎的主要治疗策略是清除刺激物和促、促进牙周组织修复,尤其是牙槽骨,目的是恢复支撑功能。前者的治疗方法包括刮治和根面平整。后者的治疗方法包括引导组织再生(GTR)。然而,目前牙槽骨再生的治疗能力仍然有限。不完全的牙槽骨修复影响牙周炎的治疗效果,降低患者的生活质量。因此,有必要寻找一种有效的策略来促进牙槽骨再生。
由于牙周袋的解剖结构不规则,注射剂型是牙周炎治疗中常用的给药方式。注射剂由载体和药物两个重要部分组成。载药载体被无创地注射到牙周袋中,并在局部持续释放。通过这种方法,不仅改善了患者的治疗舒适度,还提高了药物的生物利用度。值得注意的是,龈沟液持续的冲刷可以清除牙周袋中的异物。因此,理想的牙周炎治疗载体应具有良好的注射流动性和注射后的稳定性。
明胶甲基丙烯酰基(GelMA)是由甲基丙烯酸酐修饰的明胶,具有体温下呈液态和紫外光交联的特性。GelMA具有与天然细胞外基质(ECM)相似的特性,因为它含有精氨酸-甘氨酸-天冬氨酸(RGD)序列和基质金属蛋白酶(MMP)敏感基序,允许细胞粘附和重塑。GelMA具有独特的理化性质和良好的生物相容性,有望成为牙周炎领域的理想载体。然而,在一些生物医学应用中,单纯的GelMA很少能达到目标,因此需要添加一些物质来赋予它特定的功能。例如,生物活性玻璃赋予GelMA成骨能力,洗必泰赋予GelMA抗菌能力。因此,我们需要寻找一种物质对GelMA进行修饰,使其适合牙周炎的治疗。
分子筛咪唑骨架-8(ZIF-8)是金属有机骨架(MOF)中的重要成员,由咪唑配体(Im)桥联的锌离子(Zn2+)组成。ZIF-8可持续释放在抗菌和成骨过程中起关键作用的Zn2+,在许多生物领域具有广阔的应用前景。ShunYao等人建立了一种包裹ZIF-8的甲基丙烯酸化透明质酸(MEHA)微针,该微针具有抗菌性能和良好的生物相容性,可促进全层感染创面的愈合。YiyuanXue等人制备了原位负载ZIF-8的聚己内酯/胶原(PCL/Col)膜,通过释放Zn2+促进体内外成骨。值得注意的是,以往的研究表明,在牙周炎的治疗策略中,成骨和抗菌功能的协同作用有助于牙槽骨再生。因此,ZIF-8可能在牙周炎的治疗中脱颖而出。
发明内容
为了解决上述问题,本发明提供了一种应用于牙周炎治疗的负载ZIF-8的GelMA可注射光敏水凝胶,制备方法简单易行,具有促进大鼠骨髓间充质干细胞成骨分化和抗菌作用,能够促进大鼠牙槽骨再生。
本发明首先提供一种负载ZIF-8的GelMA复合水凝胶(GelMA-Z)的制备方法,该方法包括:
将冻干GelMA粉末在45℃避光条件下溶于含有酰基膦酸锂盐(LAP)去离子水中,制备GelMA溶液,然后将ZIF-8和GelMA溶液在45℃避光条件下均匀混合制得复合水凝胶前驱体,复合水凝胶前驱体在蓝光下可交联形成水凝胶。
优选的是,所述的GelMA的浓度为10%。
优选的是,所述的ZIF-8的浓度为0.05%-0.2%。
优选的是,所述的LAP的浓度为0.2%
优选的是,蓝光波长为405nm。
优选的是,蓝光交联时间为15秒。
本发明还提供上述负载ZIF-8的GelMA复合水凝胶在牙周炎治疗领域的应用。
本发明的有益效果
GelMA-Z具有可注射性和光交联特性。
GelMA-Z具有很好的生物相容性。
GelMA-Z具有促进大鼠骨髓间充质干细胞成骨分化的能力。
GelMA-Z对大肠杆菌和金黄色葡萄球菌有出色的抗菌能力。
GelMA-Z在体外实验中具有优异的促进成骨细胞分化能力。
GelMA-Z在牙周炎动物模型中能够牙槽骨修复。
附图说明
图1为本发明在实施例1的条件下,制备的复合水凝胶的扫描电子显微镜图;
图2为本发明在实施例1的条件下,制备的复合水凝胶的X射线衍射图像;
图3为本发明在实施例1的条件下,制备的复合水凝胶释放Zn2+的浓度;
图4为本发明在实施例1的条件下,制备的复合水凝胶的茜素红染色图;
图5为本发明在实施例2的条件下,制备的复合水凝胶释放Zn2+的浓度;
图6为本发明在实施例2的条件下,制备的复合水凝胶的茜素红染色图;
图7为本发明在实施例3的条件下,制备的复合水凝胶释放Zn2+的浓度;
图8为本发明在实施例3的条件下,制备的复合水凝胶的茜素红染色图;
图9为本发明在实施例4的条件下,制备的复合水凝胶与大肠杆菌和金黄色葡萄球菌共培养的细菌生长曲线;
图10为本发明在实施例5的条件下,制备的复合水凝胶与大鼠骨髓间充质干细胞共培养的细胞增殖趋势图;
图11为本发明在实施例6的条件下,制备的复合水凝胶治疗大鼠牙周炎4周后,牙槽骨micro-CT图。
具体实施方式:
本发明首先提供一种负载ZIF-8的GelMA复合水凝胶的制备方法,该方法包括:
步骤一:将冻干GelMA在45℃避光条件下溶于含有酰基膦酸锂盐(LAP)去离子水中。
步骤二:将步骤一的GelMA溶液与ZIF-8混合,在45℃避光条件下搅拌均匀制备复合水凝胶前驱体。
步骤三;将步骤二得到的复合水凝胶前驱体使用蓝光交联得到复合水凝胶。
按照本发明,将冻干的GelMA粉末在45℃避光条件下溶于去离子水中。所述的GelMA溶液浓度优选为10%,所述的LAP浓度为0.2%。
按照本发明,将GelMA溶液与ZIF-8混合,在45℃避光条件下搅拌均匀制备复合水凝胶前驱体。所述的ZIF-8浓度优选为0.05%-0.2%。
按照本发明,复合水凝胶前驱体使用蓝光交联得到复合水凝胶。蓝光波长优选为405nm,交联时间优选为15秒。
本发明还提供上述制备方法得到的负载ZIF-8的GelMA复合水凝胶在牙周炎领域的应用。
实施例1
将冷冻干燥的GelMA粉末(0.1g)在45℃避光条件下溶解在含有酰基膦酸锂盐(LAP)(0.2%w/v)的去离子水(1.0mL)中。然后,通过0.22μm的膜过滤,制备无菌的GelMA溶液。随后,将无菌ZIF-8(0.05%w/v)分散到无菌的GelMA溶液中,在45℃避光条件下搅拌均匀制得复合水凝胶前驱液。将前驱液注入圆柱体模具中405nm波长蓝光交联15秒制得复合水凝胶。
将实施例1得到的复合水凝胶使用扫描电子显微镜对样品的形貌进行了研究,图1显示水凝胶具有多孔结构。
将实施例1得到的复合水凝胶冻干3天后,将样品粉末进行X射线衍射扫描。图2显示复合水凝胶具有ZIF-8的特征峰。
将实施例1得到的复合水凝胶使用原子吸收光谱法测定Zn2+的释放量。图3显示在特定时间点复合水凝胶释放的累计Zn2+浓度,7天时其浓度达到0.69μg/mL。
将实施例1得到的复合水凝胶与大鼠骨髓间充质干细胞共培养21天后茜素红染色。图4显示茜素红染色结果,复合水凝胶能促进细胞钙结节形成。
实施例2
将冷冻干燥的GelMA粉末(0.1g)在45℃避光条件下溶解在含有酰基膦酸锂盐(LAP)(0.2%w/v)的去离子水(1.0mL)中。然后,通过0.22μm的膜过滤,制备无菌的GelMA溶液。随后,将无菌ZIF-8(0.1%w/v)分散到无菌的GelMA溶液中,在45℃避光条件下搅拌均匀制得复合水凝胶前驱液。将前驱液注入圆柱体模具中405nm波长蓝光交联15秒制得复合水凝胶。
将实施例2得到的复合水凝胶使用原子吸收光谱法测定Zn2+的释放量。图5显示在特定时间点复合水凝胶释放的累计Zn2+浓度,7天时其浓度达到0.91μg/mL。
将实施例2得到的复合水凝胶与大鼠骨髓间充质干细胞共培养21天后茜素红染色。图6显示茜素红染色结果,复合水凝胶能促进细胞钙结节形成。
实施例3
将冷冻干燥的GelMA粉末(0.1g)在45℃避光条件下溶解在含有酰基膦酸锂盐(LAP)(0.2%w/v)的去离子水(1.0mL)中。然后,通过0.22μm的膜过滤,制备无菌的GelMA溶液。随后,将无菌ZIF-8(0.2%w/v)分散到无菌的GelMA溶液中,在45℃避光条件下搅拌均匀制得复合水凝胶前驱液。将前驱液注入圆柱体模具中405nm波长蓝光交联15秒制得复合水凝胶。
将实施例3得到的复合水凝胶使用原子吸收光谱法测定Zn2+的释放量。图7显示在特定时间点复合水凝胶释放的累计Zn2+浓度,7天时其浓度达到0.91μg/mL。
将实施例3得到的复合水凝胶与大鼠骨髓间充质干细胞共培养21天后茜素红染色。图8显示茜素红染色结果,复合水凝胶能促进细胞钙结节形成。
实施例4
将冷冻干燥的GelMA粉末(0.1g)在45℃避光条件下溶解在含有酰基膦酸锂盐(LAP)(0.2%w/v)的去离子水(1.0mL)中。然后,通过0.22μm的膜过滤,制备无菌的GelMA溶液。随后,将无菌ZIF-8(0.2%w/v)分散到无菌的GelMA溶液中,在45℃避光条件下搅拌均匀制得复合水凝胶前驱液。将前驱液注入圆柱体模具中405nm波长蓝光交联15秒制得复合水凝胶。
将实施例4得到的复合水凝胶与大肠杆菌和金黄色葡萄球菌共培养验证抗菌能力。图9在共培养条件下复合水凝胶明显抑制大肠杆菌和金黄色葡萄球菌的生长。
实施例5
将冷冻干燥的GelMA粉末(0.1g)在45℃避光条件下溶解在含有酰基膦酸锂盐(LAP)(0.2%w/v)的去离子水(1.0mL)中。然后,通过0.22μm的膜过滤,制备无菌的GelMA溶液。随后,将无菌ZIF-8(0.2%w/v)分散到无菌的GelMA溶液中,在45℃避光条件下搅拌均匀制得复合水凝胶前驱液。将前驱液注入圆柱体模具中405nm波长蓝光交联15秒制得复合水凝胶。
将实施例5得到的复合水凝胶与大鼠间充质干细胞共培养1、3、5天,使用CCK-8试剂盒检测细胞增值情况。图10显示细胞增殖无明显抑制,细胞存活率达90%以上,根据DINEN ISO标准(10993/5),复合水凝胶具有良好的生物相容性。
实施例6
将冷冻干燥的GelMA粉末(0.1g)在45℃避光条件下溶解在含有酰基膦酸锂盐(LAP)(0.2%w/v)的去离子水(1.0mL)中。然后,通过0.22μm的膜过滤,制备无菌的GelMA溶液。随后,将无菌ZIF-8(0.2%w/v)分散到无菌的GelMA溶液中,在45℃避光条件下搅拌均匀制得复合水凝胶前驱液。将前驱液注入圆柱体模具中405nm波长蓝光交联15秒制得复合水凝胶。
将实施例5得到的复合水凝胶注射到大鼠牙周炎区域。图11显示复合水凝胶促进大鼠牙槽骨再生,与牙周炎组相比CEJ-ABC距离缩短了33.4%。
Claims (3)
1.一种应用于牙周炎治疗的负载ZIF-8的GelMA复合水凝胶的制备方法,其特征在于,该方法包括:
步骤一:将冻干的甲基丙烯酰化明胶GelMA在45°C避光条件下溶于0.2% w/v苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐LAP的去离子水中;
步骤二:将步骤一的质量浓度为10% GelMA溶液与0.05%-0.2% w/v ZIF-8混合,在45°C避光条件下搅拌均匀制备复合水凝胶前驱体;
步骤三;将步骤二得到的复合水凝胶前驱体使用蓝光交联得到复合水凝胶。
2.根据权利要求1所述的一种应用于牙周炎治疗的负载ZIF-8的GelMA复合水凝胶的制备方法,其特征在于,所述步骤三的蓝光波长为405nm,交联时间为15秒。
3.权利要求1-2任何一项所述的制备方法得到的负载ZIF-8的GelMA复合水凝胶。
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