CN114569797A - 一种含槲皮素、氧化镁的人工骨膜、制备方法及应用 - Google Patents
一种含槲皮素、氧化镁的人工骨膜、制备方法及应用 Download PDFInfo
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- CN114569797A CN114569797A CN202210463030.0A CN202210463030A CN114569797A CN 114569797 A CN114569797 A CN 114569797A CN 202210463030 A CN202210463030 A CN 202210463030A CN 114569797 A CN114569797 A CN 114569797A
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- Prior art keywords
- magnesium oxide
- artificial periosteum
- quercetin
- periosteum
- artificial
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
本发明涉及生物医学材料技术领域，具体公开了一种含槲皮素、氧化镁的人工骨膜，所述人工骨膜为含有氧化镁纳米粒子及槲皮素的纳米纤维膜。还公开了该人工骨膜的制备方法包括如下步骤：1）将可降解的高分子材料、氧化镁纳米粒子及槲皮素溶解于有机溶液中，室温下搅拌溶解，得到静电纺丝液：2）将所述静电纺丝液通过静电纺丝法制备成人工骨膜。并且公开了该人工骨膜在制备骨膜组织修复材料上的应用。本发明制备得到的人工骨膜具备促细胞迁移、促成骨、促血管形成作用，能加速骨缺损区域骨再生，能诱导细胞粘附、增殖、分化和矿化以及利于局部新生血管形成，能更好的满足临床需要。
Description
技术领域
本发明涉及生物医学材料技术领域，具体涉及一种含槲皮素、氧化镁的人工骨膜、制备方法及应用。
背景技术
骨膜是覆盖在骨组织表面上的高度血管化的致密结缔组织膜，在骨发育和骨折愈合过程中至关重要，可为新骨形成提供细胞支持和营养支持，促进骨骼的快速再生。在节段性骨缺损的治疗中，及时且丰富的血管长入是取得良好预后效果的前提之一，骨不连或者愈合延迟作为节段性骨缺损最常见的后遗症，通常与骨膜的剥离或损伤相关，而保留骨膜或使用带骨膜移植物能显著改善骨移植物的骨整合性能及加快骨缺损重建进程。
在骨折修复过程中，成骨与血管形成耦合作用是骨折修复的重要保证，骨诱导作用有利于骨折区域内骨组织矿化，同时及时且丰富的新生血管网，可以为新生骨组织提供丰富的血运及营养保障，因此合格的人工骨膜应该同时具备骨诱导及促血管形成的作用。
现有技术中，不断改善人工骨膜的促成骨、成血管功能是研发的重点和难点。静电纺丝制备的纤维膜因其与细胞外基质结构、骨膜结构相似，具有网格状、三维立体结构，能诱导成骨细胞粘附、增殖、分化和矿化以及利于局部新生血管形成，被认为是良好的仿生人工骨膜。如申请号为2019110618338的中国专利公开了一种负载去铁胺的人工骨膜及制备方法，其由可降解的高分子为主要原材料，并加入具有成骨功能的DFO分子，通过静电纺丝的方法制备DFO-高分子材料共混人工骨膜，通过纤维共混、表面吸附，和(或)负载加入聚合物微球后再与可降解的高分子材料溶液混合，之后可选择利用化学键将带有氨基的DFO键合到DFO-高分子材料共混人工骨膜表面，达到缓慢持久释放的效果。其具有良好的成骨活性，然而在成血管上表现差强人意，同时制备过程复杂，制备成本高。因此，开发功能更好的人工骨膜产品具有极为重要的意义。
发明内容
本发明所解决的技术问题在于提供一种促成骨、成血管功能好的含槲皮素、氧化镁的人工骨膜，并提供了该人工骨膜的制备方法，以降低操作难度，降低制备成本。
本发明所解决的技术问题采用以下技术方案来实现：
一种含槲皮素、氧化镁的人工骨膜，所述人工骨膜为含有氧化镁纳米粒子及槲皮素的纳米纤维膜。
进一步地，所述人工骨膜为将可降解的高分子材料、氧化镁纳米粒子及槲皮素溶解于有机溶液中得到静电纺丝液，然后通过静电纺丝法制备而成的纳米纤维膜。
进一步地，所述可降解的高分子材料为可降解聚酯类合成高分子材料，可降解聚酯类合成高分子材料为聚乳酸、聚己内酯、PLGA、聚乳酸-己内酯共聚物和聚乳酸-羟基乙酸-己内酯共聚物中的一种或几种；所述有机溶剂为六氟异丙醇、二甲基甲酰胺、三氟乙醇、乙酸和甲酸中的一种。
进一步地，所述可降解的高分子材料为PLGA，所述有机溶剂由六氟异丙醇、二甲基甲酰胺按1:1的体积比混合而成。
进一步地，人工骨膜中，所述氧化镁纳米粒子的质量占比为15~25%，所述槲皮素的质量占比为0~1%。
优选的，所述氧化镁纳米粒子的粒径为50~100nm，槲皮素纯度大于95%。
进一步地，人工骨膜中，所述氧化镁纳米粒子的质量占比为20%，所述槲皮素的质量占比为0.01~0.1%。
一种含槲皮素、氧化镁的人工骨膜的制备方法，包括如下步骤：
1）将可降解的高分子材料、氧化镁纳米粒子及槲皮素溶解于有机溶液中，室温下搅拌溶解，得到静电纺丝液；
2）将所述静电纺丝液通过静电纺丝法制备成人工骨膜。
进一步地，步骤1）中，搅拌过程中进行超声处理，超声频率为20~30 kHZ，超声处理时间大于0.5h。超声处理使原料溶解更为充分，在溶剂中分布均匀。
进一步地，步骤2）中，静电纺丝过程中，电压参数为正电压10~15KV，负电压为1~3KV；以滚筒为纺丝膜收集装置，滚筒的转动速度为50~200 rpm，静电纺丝液的流动速度为0.2~0.4ml/h，收集距离为12~18cm，得到的纺丝膜即为人工骨膜，人工骨膜厚度为25~75um。条件及参数控制较为重要，控制不当会导致纺丝丝径要么太细不方便连续纺织，要么太粗导致丝径超过纳米级别，进而导致纺丝膜空隙率下降，进而进一步影响其性能。
本发明还提出了如上所述的含槲皮素、氧化镁的人工骨膜在制备骨膜组织修复材料上的应用。
有益效果：本发明所述的含槲皮素、氧化镁的人工骨膜，其中采用的氧化镁纳米粒子可以在降解过程中释放镁离子，而槲皮素是从中草药及水果中提取的类黄酮物质，分子式为C15H10O7，中文别名3,3',4',5,7-五羟基黄酮，具有多种生物活性和神经保护、抗过敏、抗氧化、抗炎、免疫调节、抗微生物和抗肿瘤作用，本发明的申请人在研究中发现适当剂量的槲皮素与氧化镁纳米粒子结合后具有很好的协同作用，比单纯的槲皮素或单纯的氧化镁纳米粒子结合后具有更佳的骨诱导、促成血管作用，并且能够通过调控多个信号通路参与骨骼稳态的维持。
本发明制备得到的人工骨膜具备促细胞迁移、促成骨、促血管形成作用，能加速骨缺损区域骨再生，实现更快速修复颅骨缺损，适宜浓度的槲皮素可增强氧化镁纳米粒子成骨成血管的能力。
本发明采用静电纺丝法制备的人工骨膜具有与细胞外基质结构、骨膜结构相似，具有网状、高度多孔的表面及微观结构，能诱导细胞粘附、增殖、分化和矿化以及利于局部新生血管形成，能更好的满足临床需要。
本发明所述的含槲皮素、氧化镁的人工骨膜的制备方法操作简单，易于控制，产品稳定，且原材料价格便宜，易于转化，适合大量生产应用。
本发明所述的含槲皮素、氧化镁的人工骨膜能进一步应用为制备骨膜组织修复材料，具有很好的临床开发应用价值。
附图说明
图1为实施例1的扫描电镜图。
图2为实施例2的扫描电镜图。
图3为对照实施例1的扫描电镜图。
图4为对照实施例2的扫描电镜图。
图5为对照实施例3的扫描电镜图。
图6为红外光谱结果图。
图7为纺丝膜的细胞生物相容性分析结果示意图。
图8为纺丝膜对细胞迁移的影响示意图。
图9为纺丝膜对大鼠BMSC成骨分化的影响示意图。
图10为纺丝膜对促血管形成的作用的影响示意图。
图11为大鼠皮下植入人工骨膜评估生物相容性及促血管形成的影响。
图12为纺丝膜促大鼠颅骨缺损骨修复试验结果示意图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例进一步阐述本发明。
实施例1
含槲皮素、氧化镁的人工骨膜的制备方法包括如下步骤：
1）将0.96 g PLGA、0.24 g 氧化镁纳米粒子、1.2mg槲皮素溶解在4 ml二甲基甲酰胺和4 ml六氟异丙醇中，室温下连续搅拌24h，并通过超声处理，制备成静电纺丝液；
2）将步骤1）得到的静电纺丝液装入注射器中，应用静电纺丝装置，电压参数为正电压12KV，负电压为2KV，保持恒定速度0.3ml/h速度进行推注，在离针头15cm处用滚筒收集纺丝膜，滚筒转速为100rpm，得到厚度为25-75μm的纺丝膜即为人工骨膜。
本实施例得到的人工骨膜含0.1%槲皮素、20%氧化镁纳米粒子。
实施例2
含槲皮素、氧化镁的人工骨膜的制备方法包括如下步骤：
1）将0.96 g PLGA、0.24 g 氧化镁纳米粒子、0.12mg槲皮素溶解在4 ml二甲基甲酰胺和4 ml六氟异丙醇中，室温下连续搅拌24h，并通过超声处理，制备成静电纺丝液；
2）将步骤1）得到的静电纺丝液装入注射器中，应用静电纺丝装置，电压参数为正电压12KV，负电压为2KV，保持恒定速度0.3ml/h速度进行推注，在离针头15cm处用滚筒收集纺丝膜，滚筒转速为100rpm，得到厚度为25-75μm的纺丝膜即为人工骨膜。
本实施例得到的人工骨膜含0.01%槲皮素、20%氧化镁纳米粒子。
对照实施例1
用0.96 g PLGA、0.24 g 氧化镁纳米粒子、12mg槲皮素溶解在4 ml二甲基甲酰胺和4 ml六氟异丙醇中，室温下连续搅拌24h，并通过超声处理，制备成静电纺丝液；将静电纺丝液进行纺丝得到纺丝膜，纺丝膜为含1%槲皮素、20%氧化镁纳米粒子的人工骨膜。
对照实施例2
用0.96 g PLGA、0.24 g 氧化镁纳米粒子溶解在4 ml二甲基甲酰胺和4 ml六氟异丙醇中，室温下连续搅拌24h，并通过超声处理，制备成静电纺丝液；将静电纺丝液进行纺丝得到含有20% 氧化镁纳米粒子及PLGA的纺丝膜。
对照实施例3
用0.96 g PLGA溶解在4 ml二甲基甲酰胺和4 ml六氟异丙醇中，室温下连续搅拌24h，并通过超声处理，制备成静电纺丝液；将静电纺丝液进行纺丝得到仅含PLGA的纺丝膜。
对照实施例4
用1.2mg槲皮素溶解在4 ml二甲基甲酰胺和4 ml六氟异丙醇中，室温下连续搅拌24h，并通过超声处理，制备成静电纺丝液；将静电纺丝液进行纺丝得到仅含纯槲皮素的纺丝膜。
各实施例中，采用的氧化镁纳米粒子的粒径为50~100nm，槲皮素纯度大于95%。
将各实施例得到的纺丝膜进一步进行性能检测和实验。
其中Que表示对照实施例4的含纯槲皮素的纺丝膜，PLGA表示对照实施例3的含纯PLGA的纺丝膜，PLGA/氧化镁纳米粒子表示对照实施例2含20wt%氧化镁纳米粒子与PLGA的纺丝膜，0.01%Que、0.1%Que、1%Que分别表示实施例2、实施例1、对照实施例1得到的纺丝膜。
（一）表面形态检测。
将实施例1、实施例2、对照实施例1、对照实施例2、对照实施例3得到的人工骨膜进行表面形态的检测，电镜扫描图结果如图1~图5所示，从扫描电镜的结果来看，可见PLGA基材的纳米纺丝纤维膜由纳米纤维不规则交联在一起，纺丝之间存在多孔样结构，形成了具有高度多孔的表面及微观结构，且实施例1~实施例3中的纺丝膜由于负载氧化镁纳米粒子及槲皮素，纺丝上存在结点状的凸起，这种结点凸起加高度多孔的表面结构利于细胞粘附、迁移及微血管形成。
（二）负载结果检测。
将实施例1、实施例2、对照实施例1、对照实施例2、对照实施例3及对照实施例4得到的纺丝膜进行红外光谱分析，结果如图6所示，结果表明，人工骨膜成功负载氧化镁纳米粒子及槲皮素。
（三）、人工骨膜的细胞生物相容性分析。
将实施例1、实施例2、对照实施例1、对照实施例2、对照实施例3制备成的纺丝膜对大鼠骨髓间充质干细胞的活死细胞染色，结果如图7的A部分和B部分所示。通过live/deadcell染色，通过荧光显示各组纺丝膜间的死细胞数量无明显差别，且与空白组死细胞数量无明显差别，提示五种仿生骨膜均无明显细胞毒性，生物安全度高。
进一步分别试验实施例1、实施例2、对照实施例1、对照实施例2、对照实施例3制备成的纺丝膜对纺丝膜对大鼠骨髓间充质干细胞（BMSC）的及大鼠血管内皮祖细胞（EPC）细胞增殖的影响，结果如图7的C部分和D部分所示，通过CCK-8试剂盒检测结果提示实施例1、实施例2得到的纺丝膜相较于对照实施例2的纺丝膜而言具有明显促进细胞增殖作用，而实施例3得到的纺丝膜对细胞增殖具有抑制作用，进一步说明质量分数为0.01%~0.1%槲皮素的人工骨膜具有良好的生物相容性和促进细胞增殖的作用，可以用于动物体内试验。
（四）人工骨膜对细胞迁移的影响。
利用Transwell小室评估检测实施例1、实施例2、对照实施例1、对照实施例2、对照实施例3制备成的纺丝膜对BMSC、EPC细胞迁移的作用，结果如图8所示，图8的A部分和B部分为各实施例的纺丝膜对BMSC、EPC细胞迁移作用的显微镜照片，图8的C部分和D部分为BMSC、EPC细胞迁移数量的统计分析示意图。结合显微镜图及细胞迁移数量的统计，说明负载有0.01%~0.1%槲皮素、20%氧化镁的人工骨膜比仅负载20%氧化镁人工骨膜组具有更强的促进细胞迁移的作用，但高浓度（≥1%）槲皮素的人工骨膜反而会抑制细胞迁移。PLGA/氧化镁纳米粒子组比PLGA组更能促进更多的细胞迁移，而对于BMSCs，0.01%Que（p = 0.0015）、0.1%Que（p < 0.0001）、1%Que（p = 0.0010）均比PLGA/氧化镁纳米粒子更能促进BMSCs迁移；对于EPC细胞，0.01%Que与PLGA/氧化镁纳米粒子迁移细胞数无明显差异（p = 0.1940），0.1%Que比PLGA/氧化镁纳米粒子组更能促进EPC细胞迁移（p = 0.0002），而1%Que比PLGA/氧化镁纳米粒子会抑制EPC细胞迁移（p = 0.0002）。1%Que能促进BMSCs迁移，却抑制EPC细胞迁移。综上说明，0.01%、0.1%Que组能明显促进BMSCs及EPC细胞迁移，预示着该人工骨膜可促进局部干细胞及血管内皮细胞募集至骨折部位发挥修复作用。
（五）人工骨膜对大鼠BMSC成骨分化的影响。
分别验证实施例1、实施例2、对照实施例1、对照实施例2、对照实施例3制备成的纺丝膜对大鼠BMSC成骨相关基因的影响，方法为将各组含纺丝膜浸提液的成骨诱导培养基培养BMSCs，诱导12天后提取RNA，应用QPCR检测成骨相关基因的表达情况。
结果如图9A部分所示，显示含0.01%~0.1%槲皮素、20%氧化镁的人工骨膜比单纯含20%氧化镁人工骨膜组具有更强的促进成骨相关基因（RUNX2、ALP、BMP2、COL1A1、OPN）的表达，0.1%Que组比0.01%Que组更能促进成骨相关基因的表达，但高浓度（≥1%）槲皮素的人工骨膜反而会抑制成骨相关基因的表达。
分别验证实施例1、实施例2、对照实施例1、对照实施例2、对照实施例3制备成的纺丝膜对大鼠BMSC成骨相关蛋白的影响，结果如图9B部分所示，含0.01%~0.1%槲皮素、20%氧化镁的人工骨膜比单纯含20%氧化镁人工骨膜组具有更强的促进成骨相关蛋白（RUNX2、COL1）的表达，但高浓度（≥1%）槲皮素的人工骨膜反而会成骨相关蛋白的表达。
分别验证实施例1、实施例2、对照实施例1、对照实施例2、对照实施例3制备成的纺丝膜对大鼠BMSC成骨分化进行茜素红染色及ALP染色，结果如图9的C部分所示，结果表明实施例1和实施例2的人工骨膜具有最强的促进钙化灶沉积及分泌ALP的作用。
从成骨相关指标来看，说明0.1%Que能明显促进骨缺损部位干细胞成骨分化，且适宜浓度槲皮素能与Mg2+协同发挥更强的成骨作用，高浓度的Que对成骨有一定的抑制作用。
（六）人工骨膜对促血管形成的作用的影响。
分别将实施例1、实施例2、对照实施例1、对照实施例2、对照实施例3制备成的纺丝膜进行EPC细胞于基质胶上体外成管试验，并试验人工骨膜对血管形成相关基因（VEGFA、VWF）表达情况，用western-blot检测血管形成相关蛋白eNOS蛋白水平。结果如图10所示，图10的A部分为EPC细胞于基质胶上体外成管试验，图10的B部分为对A图结果进行totalnodes、total branches及branching length的统计分析，图10的C部分为各组人工骨膜对血管形成相关基因（VEGFA、VWF）表达情况分析结果，图10的D部分为western-blot检测血管形成相关蛋白eNOS蛋白水平检测表。总体结果表明实施例1和实施例2的人工骨膜比对照实施例3中仅负载有氧化镁纳米粒子的人工骨膜具有更强促血管形成作用，而高浓度（≥1%）槲皮素的人工骨膜反而会抑制血管形成。
将实施例1、对照实施例2和对照实施例3制备成的纺丝膜通过大鼠皮下植入人工骨膜试验进一步评估生物相容性及促血管形成的影响，结果如图11所示，观察样品的大体照、H&E染色及Masson染色、CD31免疫组化染色，结果提示负载有实施例1的人工骨膜比仅负载20%氧化镁的人工骨膜或不负载槲皮素或氧化镁的人工骨膜具有良好的生物相容性和更强促血管形成作用。
（七）人工骨膜对大鼠颅骨缺损骨修复的影响。
将实施例1、对照实施例2、对照实施例3制备而成的纺丝膜进行大鼠颅骨缺损骨修复的试验，结果如图12所示，说明不负载有槲皮素或氧化镁纳米粒子的人工骨膜基本没有修复效果，单纯负载20%氧化镁的人工骨膜有一定的修复效果，而负载有0.1%槲皮素、20%氧化镁纳米粒子的人工骨膜比单纯负载20%氧化镁的人工骨膜有更强促骨缺损修复的作用。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (10)
1.一种含槲皮素、氧化镁的人工骨膜，其特征在于，所述人工骨膜为含有氧化镁纳米粒子及槲皮素的纳米纤维膜。
2.根据权利要求1所述的含槲皮素、氧化镁的人工骨膜，其特征在于，所述人工骨膜为将可降解的高分子材料、氧化镁纳米粒子及槲皮素溶解于有机溶液中得到静电纺丝液，然后通过静电纺丝法制备而成的纳米纤维膜。
3.根据权利要求2所述的含槲皮素、氧化镁的人工骨膜，其特征在于，
所述可降解的高分子材料为可降解聚酯类合成高分子材料，可降解聚酯类合成高分子材料为聚乳酸、聚己内酯、PLGA、聚乳酸-己内酯共聚物和聚乳酸-羟基乙酸-己内酯共聚物中的一种或几种；所述有机溶剂为六氟异丙醇、二甲基甲酰胺、三氟乙醇、乙酸和甲酸中的一种。
4.根据权利要求3所述的含槲皮素、氧化镁的人工骨膜，其特征在于，所述可降解的高分子材料为PLGA，所述有机溶剂由六氟异丙醇、二甲基甲酰胺按1:1的体积比混合而成。
5.根据权利要求2所述的含槲皮素、氧化镁的人工骨膜，其特征在于，人工骨膜中，所述氧化镁纳米粒子的粒径为50~100nm，槲皮素纯度大于95%；
所述氧化镁纳米粒子的质量占比为15~25%，所述槲皮素的质量占比为0~1%。
6.根据权利要求5所述的含槲皮素、氧化镁的人工骨膜，其特征在于，人工骨膜中，所述氧化镁纳米粒子的质量占比为20%，所述槲皮素的质量占比为0.01~0.1%。
7.一种含槲皮素、氧化镁的人工骨膜的制备方法，其特征在于，包括如下步骤：
1）将可降解的高分子材料、氧化镁纳米粒子及槲皮素溶解于有机溶液中，室温下搅拌溶解，得到静电纺丝液；
2）将所述静电纺丝液通过静电纺丝法制备成人工骨膜。
8.根据权利要求7所述的含槲皮素、氧化镁的人工骨膜的制备方法，其特征在于，步骤1）中，搅拌过程中进行超声处理，超声频率为20~30 kHZ，超声处理时间大于0.5h。
9.根据权利要求7所述的含槲皮素、氧化镁的人工骨膜的制备方法，其特征在于，步骤2）中，静电纺丝过程中，电压参数为正电压10~15KV，负电压为1~3KV；以滚筒为纺丝膜收集装置，滚筒的转动速度为50~200 rpm，静电纺丝液的流动速度为0.2~0.4ml/h，收集距离为12~18cm，得到的纺丝膜即为人工骨膜，人工骨膜厚度为25~75um。
10.如权利要求1~6任一所述的含槲皮素、氧化镁的人工骨膜在制备骨膜组织修复材料上的应用。
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