CN110917409B - 二甲双胍缓释tHA/PCL引导组织再生膜及其制备方法 - Google Patents

二甲双胍缓释tHA/PCL引导组织再生膜及其制备方法 Download PDF

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CN110917409B
CN110917409B CN201911242734.XA CN201911242734A CN110917409B CN 110917409 B CN110917409 B CN 110917409B CN 201911242734 A CN201911242734 A CN 201911242734A CN 110917409 B CN110917409 B CN 110917409B
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高翔
宋锦璘
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Stomatological Hospital of Chongqing Medical University
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Abstract

本发明公开了一种二甲双胍缓释tHA/PCL引导组织再生膜及其制备方法，二甲双胍缓释tHA/PCL引导组织再生膜包括tHA/PCL复合纳米纤维膜和含有二甲双胍的CMC/PLL涂层，其制备方法：1)制备tHA；2)通过静电纺丝技术制备出tHA/PCL复合纳米纤维膜；3)利用静电吸附层层自组装技术，在tHA/PCL复合纳米纤维膜的表面制备含有二甲双胍的CMC/PLL涂层；4)采用生物交联剂京尼平对CMC/PLL涂层进行化学交联。本发明利用二甲双胍对纳米颗粒tHA的细胞毒性作用的调控作用，改善了tHA/PCL复合纤维材料的生物相容性和成骨活性，能促进牙周组织的修复再生。

Description

二甲双胍缓释tHA/PCL引导组织再生膜及其制备方法
技术领域
本发明涉及医疗材料技术领域，特别涉及一种引导组织再生膜及其制备方法。
背景技术
牙周炎是牙周组织的一种慢性感染性疾病,在世界范围内均有较高的患病率，其主要症状和临床病理包括牙龈出血和炎症、牙周袋的形成、牙槽骨的吸收。其中，牙槽骨丧失将引起牙周支持组织的不可逆性破坏，最终导致牙齿的脱落。因此，牙周治疗的最终目标是重建因牙周炎而丧失的牙周支持组织，而修复再生新的牙槽骨组织是实现这一目标的关键。目前，对于轻度吸收破坏的牙槽骨，可利用组织的自愈能力，通过消除局部菌斑，控制炎症的方法实现牙槽骨组织的自我修复。对于重度破坏吸收的牙周组织，常常需要借助引导组织再生术(Guided tissue regeneration,GTR)以获得正常牙周支持组织的修复重建。GTR技术的理论基础是通过将一个物理性屏障膜(引导组织再生膜)置于牙槽骨缺损表面，阻止龈上皮及结缔组织与根面接触，同时诱导周围组织中牙周膜干细胞(Periodontalligament stem cells,PDLSCs)向成骨细胞分化，参与牙槽骨组织修复再生，最终重建被破坏的牙周支持组织。牙槽骨组织正常修复大约需要3个月左右的时间。在口腔环境中，由于口腔卫生差、吸烟、牙龈厚度不足等原因，GTR术后常常出现牙龈退缩，导致屏障膜早期暴露进而影响牙周组织修复效果。因此，为提高GTR术后疗效，缩短牙周组织修复重建时间，研发一种具有高诱导活性的引导组织再生膜是目前口腔生物材料领域的研究热点。
发明内容
有鉴于此，本发明的目的是提供一种二甲双胍缓释tHA/PCL引导组织再生膜及其制备方法，以解决骨组织再生及修复的技术问题。
本发明二甲双胍缓释tHA/PCL引导组织再生膜，其包括tHA/PCL复合纳米纤维膜和形成在tHA/PCL复合纳米纤维膜上的含有二甲双胍的CMC/PLL涂层，所述含有二甲双胍的CMC/PLL涂层由交错叠加的含有二甲双胍的CMC涂层和含有二甲双胍的PLL涂层组成。
本发明二甲双胍缓释tHA/PCL引导组织再生膜的制备方法，其特征在于：包括以下步骤：
1)制备tHA，其包括：
在室温下，将Ca(NO3)2加入浓度为2mg/ml的多巴胺水溶液中配成混合溶液，待搅拌反应30min后，逐滴加入预先配置好的Na2HPO4水溶液直至溶液中的钙磷比达到1.67；滴加完成后，使该混合溶液在60℃、PH＝8.5条件下继续搅拌反应12小时，而后陈化24小时；反应结束后，将混合溶液通过离心沉淀、洗涤、烘干处理，获得以聚多巴胺为模板生长形成的仿生纳米羟基磷灰石；
2)用六氟异丙醇作为溶剂配制聚己内酯溶液，随后将仿生纳米羟基磷灰石加入聚己内酯溶液中，通过静电纺丝技术制备出tHA/PCL复合纳米纤维膜；
3)将tHA/PCL复合纳米纤维膜放入多聚赖氨酸溶液中，并在37℃摇床中反应24小时，然后取出tHA/PCL复合纳米纤维膜，随后将tHA/PCL复合纳米纤维膜交替放入含二甲双胍的羧甲基壳聚糖溶液和含二甲双胍的多聚赖氨酸溶液中，利用静电吸附层层自组装技术，在tHA/PCL复合纳米纤维膜的表面制备含有二甲双胍的CMC/PLL涂层；
4)采用生物交联剂京尼平对含有二甲双胍的CMC/PLL涂层进行化学交联。
优选地，步骤2)中所述的聚己内酯溶液的浓度为8％；步骤3)中羧甲基壳聚糖溶液中二甲双胍的浓度为100μM，多聚赖氨酸溶液中二甲双胍的浓度为100μM。
优选的，在步骤3)中，tHA/PCL复合纳米纤维膜每次在含二甲双胍的羧甲基壳聚糖溶液和含二甲双胍的多聚赖氨酸溶液中的浸泡时间为5min。
本发明的有益效果：
1、本发明二甲双胍缓释tHA/PCL引导组织再生膜，其CMC/PLL涂层中含有的二甲双胍对纳米颗粒tHA的细胞毒性作用具有潜在的调控作用；通过在tHA/PCL复合纳米纤维表面构建缓释涂层实现二甲双胍在牙周局部微环境中的长期缓慢释放，能提高牙周组织细胞抵抗tHA诱发的细胞ROS氧化应激损伤的能力，改善了tHA/PCL复合纤维材料的生物相容性和成骨活性，能促进牙周组织的修复再生。
2、本发明二甲双胍缓释tHA/PCL引导组织再生膜及其制备方法，利用羧甲基壳聚糖(CMC)和多聚赖氨酸(PLL)作为聚电解质材料，通过聚多巴胺共价接枝技术、层层自组装技术和京尼平交联技术的有机结合，构建药物缓释涂层。相比传统的单纯利用层层自组装静电吸附建立的药物涂层，本发明构建的CMC/PLL涂层不仅降解缓慢，能够实现所加载生物活性小分子的缓慢释放，而且由于其基底层与聚多巴胺层的共价结合，不易从材料表面剥脱，提高了缓释涂层在体内环境中应用的稳定性。
附图说明
图1为二甲双胍缓释tHA/PCL引导组织再生膜的制备流程示意图。
图2为利用PDA为模板合成的仿生纳米羟基磷灰石(tHA)在电子显微镜下的形貌观察图。
图3为通过扫描电子显微镜获得的通过静电纺丝技术制得的tHA含量不同的tHA/PCL复合纳米纤维膜。
图4为干细胞在不同纳米纤维膜上的增殖活性数据对比图，从图中可以看出。在相同磷灰石含量(10％)下，干细胞在tHA/PCL复合纳米纤维膜表面上的增殖活性高于在传统HA/PCL复合纳米纤维膜表面的增殖活性；
图5为在电子显微镜下观察随着tHA含量的增加，纤维表面干细胞内ROS水平表现出上升趋势的观察图；
图6为随着tHA含量的增加，纤维表面干细胞内ROS水平的上升趋势图；
图7为在tHA刺激环境下，二甲双胍对人牙周膜干细胞(Human periodontalligament stem cells,hPDLSCs)细胞内ROS及自噬信号分子LC-3基因表达的影响。
具体实施方式
下面结合附图和实施例对本发明作进一步描述。
本实施例中二甲双胍缓释tHA/PCL引导组织再生膜，其包括tHA/PCL复合纳米纤维膜和形成在tHA/PCL复合纳米纤维膜上的含有二甲双胍的CMC/PLL涂层，所述含有二甲双胍的CMC/PLL涂层由交错叠加的含有二甲双胍的CMC涂层和含有二甲双胍的PLL涂层组成。
本实施例中二甲双胍缓释tHA/PCL引导组织再生膜的制备方法，其包括以下步骤：
1)制备tHA，其包括：
在室温下，将Ca(NO3)2加入浓度为2mg/ml的多巴胺水溶液中配成混合溶液，待搅拌反应30min后，逐滴加入预先配置好的Na2HPO4水溶液直至溶液中的钙磷比达到1.67；滴加完成后，使该混合溶液在60℃、PH＝8.5条件下继续搅拌反应12小时，而后陈化24小时；反应结束后，将混合溶液通过离心沉淀、洗涤、烘干处理，获得以聚多巴胺(Polydopamine，PDA)为模板生长形成的仿生纳米羟基磷灰石(Polydopamine-templated hydroxyapatite，tHA)。
本步骤制得的仿生纳米羟基磷灰石(tHA)与水热法、化学沉淀法等传统方法合成的纳米羟基磷灰石(Nano-hydroxyapatite,nano-HA)相比，其tHA结构中富含聚多巴胺成分，且具有与天然骨组织磷灰石晶体类似的钙磷比和结晶度。
2)用六氟异丙醇作为溶剂配制聚己内酯(Polycaprolactone，PCL)溶液，随后将仿生纳米羟基磷灰石加入聚己内酯溶液中，通过静电纺丝技术制备出tHA/PCL复合纳米纤维膜。
由于制得的tHA/PCL复合纳米纤维膜富含聚多巴胺成分，使得其表现出比传统的nano-HA/PCL复合纳米纤维具有更优的细胞相容性和成骨活性。
3)将tHA/PCL复合纳米纤维膜放入多聚赖氨酸溶液中，并在37℃摇床中反应24小时，然后取出tHA/PCL复合纳米纤维膜，随后将tHA/PCL复合纳米纤维膜交替放入含二甲双胍(Metformin，Met)的羧甲基壳聚糖溶液和含二甲双胍的多聚赖氨酸溶液中，利用静电吸附层层自组装技术，在tHA/PCL复合纳米纤维膜的表面制备含有二甲双胍的CMC/PLL涂层。羧甲基壳聚糖(Carboxymethyl chitosan,CMC)，其带负电；多聚赖氨酸(Polylysine,PLL)，其带正电。在自组装过程中，由于tHA/PCL复合纳米纤维膜的仿生纳米羟基磷灰石(tHA)材料结构中存在大量的聚多巴胺膜成分，能利用聚多巴胺膜对-NH2和-SH基团的反应活性，方便、快捷的达到CMC涂层与基底tHA/PCL复合纳米纤维膜共价结合的目的，且结合力强。
4)采用生物交联剂京尼平对含有二甲双胍的CMC/PLL涂层进行化学交联。与传统的交联剂如戊二醛相比，京尼平的交联能力强于戊二醛，且不易被快速降解，有利于保持材料的稳定性，更适于药物缓释系统的构建；同时京尼平的生物相容性好，适于制备生物材料，且对细胞毒性极低。
通过化学交联提高了含有二甲双胍的CMC/PLL涂层的机械强度，减小了涂层的溶胀性，能避免CMC/PLL涂层吸水溶胀而破碎，造成加载药物的突释的问题。
本实施例中，步骤2)中所述的聚己内酯溶液的浓度为8％；步骤3)中羧甲基壳聚糖溶液中二甲双胍的浓度为100μM，多聚赖氨酸溶液中二甲双胍的浓度为100μM。在步骤3)中，tHA/PCL复合纳米纤维膜每次在含二甲双胍的羧甲基壳聚糖溶液和含二甲双胍的多聚赖氨酸溶液中的浸泡时间为5min，tHA/PCL复合纳米纤维膜在含二甲双胍的羧甲基壳聚糖溶液和含二甲双胍的多聚赖氨酸溶液中交替循环的次数为100次。
当然在不同实施例中，聚己内酯溶液的浓度、羧甲基壳聚糖溶液和多聚赖氨酸溶液中二甲双胍的浓度均可根据需要做调整；并且tHA/PCL复合纳米纤维膜每次在含二甲双胍的羧甲基壳聚糖溶液和含二甲双胍的多聚赖氨酸溶液中的浸泡时间，以及tHA/PCL复合纳米纤维膜在含二甲双胍的羧甲基壳聚糖溶液和含二甲双胍的多聚赖氨酸溶液中交替循环的次数也可根据需要进行调整。
本实施例中二甲双胍缓释tHA/PCL引导组织再生膜，其tHA/PCL复合纳米纤维膜的生物学活性与tHA的含量为正相关关系，但与此同时，随着纤维中tHA含量的增加，纤维表面干细胞内活性氧(Reactive oxygen species,ROS)水平也表现出明显的上升趋势，当ROS蓄积超过一定浓度以后，材料出现明显的细胞毒性作用，表现为纤维表面细胞LDH释放率和凋亡率增加，细胞增殖和成骨分化效率受到抑制。
ROS是细胞有氧代谢过程中的一个副产品，包括超氧阴离子(O2 -)、过氧化物(H2O2)和含氧自由基等。正常情况下，生物体内的ROS处于一个较低水平，其在细胞信号转导、免疫反应以及基因的表达调控过程中发挥着重要作用，而过量的ROS则会对细胞内DNA、RNA、蛋白质和脂质等生物大分子造成氧化应激损伤，从而影响细胞的正常生理功能(增殖和分化)。
为了清除细胞内tHA介导所产生的过量ROS以及降低ROS对细胞造成的氧化应激损伤，本实施例在tHA/PCL复合纳米纤维膜上组装了含有二甲双胍的CMC/PLL涂层。CMC/PLL涂层中含有的二甲双胍对纳米颗粒tHA的细胞毒性作用具有潜在的调控作用，通过在tHA/PCL复合纳米纤维表面构建缓释涂层实现二甲双胍在牙周局部微环境中的长期缓慢释放，能提高牙周组织细胞抵抗tHA诱发的细胞ROS氧化应激损伤的能力，改善了tHA/PCL复合纤维材料的生物相容性和成骨活性，能促进牙周组织的修复再生。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (1)

1.一种二甲双胍缓释tHA/PCL引导组织再生膜，其特征在于：包括tHA/PCL复合纳米纤维膜和形成在tHA/PCL复合纳米纤维膜上的含有二甲双胍的CMC/PLL涂层，所述含有二甲双胍的CMC/PLL涂层由交错叠加的含有二甲双胍的CMC涂层和含有二甲双胍的PLL涂层组成；
所述二甲双胍缓释tHA/PCL引导组织再生膜的制备方法：包括以下步骤：
1)制备tHA，其包括：
在室温下，将Ca(NO3)2加入浓度为2mg/mL 的多巴胺水溶液中配成混合溶液，待搅拌反应30min后，逐滴加入预先配置好的Na2HPO4水溶液直至溶液中的钙磷比达到1.67；滴加完成后，使该混合溶液在60℃、pH ＝8.5条件下继续搅拌反应12小时，而后陈化24小时；反应结束后，将混合溶液通过离心沉淀、洗涤、烘干处理，获得以聚多巴胺为模板生长形成的仿生纳米羟基磷灰石；
2)用六氟异丙醇作为溶剂配制聚己内酯溶液，随后将仿生纳米羟基磷灰石加入聚己内酯溶液中，通过静电纺丝技术制备出tHA/PCL复合纳米纤维膜；
3)将tHA/PCL复合纳米纤维膜放入多聚赖氨酸溶液中，并在37℃摇床中反应24小时，然后取出tHA/PCL复合纳米纤维膜，随后将tHA/PCL复合纳米纤维膜交替放入含二甲双胍的羧甲基壳聚糖溶液和含二甲双胍的多聚赖氨酸溶液中，利用静电吸附层层自组装技术，在tHA/PCL复合纳米纤维膜的表面制备含有二甲双胍的CMC/PLL涂层；
4)采用生物交联剂京尼平对含有二甲双胍的CMC/PLL涂层进行化学交联；
步骤2)中所述的聚己内酯溶液的浓度为8％；步骤3)中羧甲基壳聚糖溶液中二甲双胍的浓度为100μM，多聚赖氨酸溶液中二甲双胍的浓度为100μM；
步骤3)中，tHA/PCL复合纳米纤维膜每次在含二甲双胍的羧甲基壳聚糖溶液和含二甲双胍的多聚赖氨酸溶液中的浸泡时间为5min。
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