CN116474171A - 一种兼具促骨感觉神经重塑、血管再生和骨快速修复的复合支架材料及其制备方法 - Google Patents
一种兼具促骨感觉神经重塑、血管再生和骨快速修复的复合支架材料及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种兼具促骨感觉神经重塑、血管再生和骨快速修复的复合支架材料及其制备方法。其制备过程包括：①通过仿生矿化及原位致孔技术制备具有多孔核‑壳结构羟基磷灰石(HA)涂层的内核颗粒；②再将上述颗粒置于含第一种生长因子的缓冲液中通过超高比表面积在孔隙内静电吸附生长因子，进一步在其表面原位自聚合多巴胺(DA)对孔隙进行包覆，得到复合颗粒；③对动物软组织处理获得脱细胞基质，依次加入第二种生长因子、复合颗粒，通过低速离心脱泡及低温冷冻干燥技术制得复合支架材料。该复合支架材料具有优异的降解性能、生物相容性、因子控释性能，促神经化、血管化及骨诱导性能，可实现不同大小的难治性骨缺损的治疗。
Description
技术领域
本发明涉及生物医学技术领域，特别涉及一种兼具促骨感觉神经重塑、血管再生和骨快速修复的复合支架材料及其制备方法和应用。
背景技术
由创伤和其他疾病引起的临界大小骨缺损是一个具有挑战性的临床难题。这一挑战源于骨修复是一个复杂的多阶段过程，涉及炎症、神经血管网络重建、快速骨矿化和骨重塑等阶段。针对这一过程的一个或多个阶段的骨愈合策略已经被开发出来用以加速骨愈合的过程。例如，双VEGF/BMP-2递送体系被设计为先促进血管生成以加速骨的愈合，以实现促血管化成骨。然而神经系统在骨再生过程中的作用，在很大程度上被低估了。事实上，骨受到感觉神经和自主神经纤维的支配，分布于骨膜、骨髓、生长板和矿化小梁/皮质骨。越来越多的证据表明，神经系统通过直接或间接调节成骨细胞和破骨细胞的活性，在骨骼发育和代谢中发挥不可替代的作用，因此可以开发新的骨愈合策略，利用骨愈合过程中神经的调节功能。临床研究和动物研究表明，感觉神经的丢失容易导致骨丢失增加，以至于骨折愈合延迟。秦岭等人报道，感觉神经元分泌的降钙素基因相关肽(CGRP)对调节血管网络重建起到正向。此外，感觉神经分泌物质人血清P物质(SP)、CGRP等多肽可调节间充质干细胞MSCs的增殖和成骨分化。因此，感觉神经在调节骨形成方面发挥着重要的生物学功能。由于感觉神经在骨代谢和稳态中的多种作用，在骨缺损部位重建感觉神经网络被认为是调节新骨形成的可行策略。
新骨痂中感觉神经的萌发和分枝是骨修复的重要上游特征。目前的数据表明，NGF-TrkA信号对于感觉神经重塑至关重要。虽然NGF的原位直接释放可以增强感觉神经再生和随后的骨成骨和骨重塑，但这些方法受到这些因子容易失活和爆炸性释放的限制。天然ECM通过其固有的局部结合、控释可溶性生物活性因子的能力来调节组织再生，已被广泛应用于骨修复。脱细胞的细胞外基质具有细胞粘附和延展性，还能增强神经突的伸长和控制神经突生长取向的特性。考虑到上述优点，ECM可作为NGF释放的理想载体，实现快速的感觉神经神经支配。
综上，本发明针对现有骨修复材料的缺陷以及市场的需求，开发出一种骨修复材料及其制备方法。本发明的骨修复材料具有对生长因子在内的多种蛋白质独立控制释放，释放时间可在数天至数月间调控；优异的促神经化成骨性能、成血管性能以及骨重塑功能；并具有优秀的生物相容性和生物可降解性，与成骨过程相匹配的降解速率。
发明内容
针对现有技术的骨修复材料忽视了神经支配在骨修复材料中的作用，支架无生物活性等问题，本发明提供一种兼具促骨感觉神经重塑、血管再生和骨快速修复的复合支架材料及其制备方法。
本发明采取以下技术方案：
兼具促骨感觉神经重塑、血管再生和骨快速修复的复合支架材料的制备方法，包括如下步骤：
1)首先向去离子水中加入无机盐和造孔剂制备得到改良仿生矿化溶液，然后将可降解的内核微粒孵育在改良仿生矿化溶液，每天更换仿生矿化溶液，孵育7天以形成具有多孔结构的矿物质包被层，矿化颗粒用去离子水洗涤、干燥备用；
2)取步骤1)中得到的矿化颗粒分散于含第一种生长因子的0.2％ BSA溶液(pH＝7.4)中，37℃下旋转孵育4.0小时，负载第一种生长因子的矿化颗粒；
3)将步骤2)获得的矿化颗粒放置于含多巴胺DA的10mM Tris缓冲溶液(pH＝8.5)中，反应持续2-6小时，得到含第一种生长因子的复合颗粒；
4)对动物软组织进行处理获取脱细胞基质；
5)将步骤4)中制备的脱细胞基质用PBS缓冲液多次冲洗，并将脱细胞基质的pH值调整为7.20，加入第二种生长因子并充分混合，通过静电吸附作用获得含第二种生长因子的脱细胞基质；
6)向步骤5)获得的脱细胞基质中加入步骤3)获得的复合颗粒，搅拌静电吸附，分散均匀，低速离心脱泡得到半流体状复合材料的混合物，并将混合物转移到模具中，于-20℃至-80℃的温度下静置24小时得到冷冻的混合物；
7)将步骤6)制备的冷冻的混合物在0℃以下进行真空冷冻干燥获得复合支架材料。
上述技术方案中，进一步地，步骤1)中，改良仿生矿化溶液中的无机盐具体是通过依次向去离子水中加入以下试剂并形成对应浓度：141mM NaCl，4mM KCl，0.5mM MgSO4，1.0mM MgCl2，25mM NaHCO3，20.0mM HEPES，5mM CaCl2，和2mM KH2PO4；之后再加入造孔剂，仿生矿化溶液的pH值应调整为6.80。
进一步地，步骤1)中，仿生矿化工艺中使用的造孔剂包括聚甲基丙烯酸酯、甲基丙烯酸甲酯、聚乙二醇、聚乙烯醇和聚乙烯醇缩丁醛中的一种或多种，浓度为1-10mg/mL。
进一步地，所述可降解的内核微粒为β-TCP颗粒、磷酸镁颗粒、聚乳酸-羟基乙酸共聚物(PLGA)、聚乙二醇(PEG)、聚乳酸(PLA)和聚氨酯(PU)中的一种或多种，可降解的内核微粒与改良仿生矿化溶液的用量比例为25mg:50mL。
进一步地，步骤2)中的旋转孵育工序中，旋转速率等于100转/min，所处温度为37℃；所述的第一种生长因子为BMP-2、胰岛素样生长因子、或成纤维生长因子，分散于BSA溶液中浓度为1-10mg/mL。
进一步地，步骤3)中，DA的Tris溶液中DA浓度为2mg/ml，需用10mM Tris缓冲溶液在超声作用下溶解，溶液pH＝8.5，孵育过程需要全程避光。
进一步地，步骤4)中，所述脱细胞基质来源于包括猪、牛或者人的软组织；所述软组织包括皮肤、血管、韧带、肌腱、隔膜和小肠系膜中的一种或多种。
进一步地，步骤4)具体为，将动物软组织清洗后剪成所需规格尺寸的组织原材料，然后放入1.0％脱氧胆酸钠溶液中，并置于37℃，120rpm的摇床上脱细胞24小时后，将其置于40U/mLDNaseI与10mM MgCl的溶液浸泡90分钟，以完全去除细胞残留物，最后，用0.1％过氧乙酸/4％乙醇溶液灭菌处理2小时后，将其离心去除灭菌液后用0.9％生理盐水振荡浴清洗30min，循环3次后低温冷冻保存脱细胞基质。
进一步地，步骤5)中，所述的第二种生长因子为神经生长因子、转化生长因子β、碱性成纤维生长因子、胰岛素样生长因子、血管内皮细胞生长因子、成纤维细胞生长因子、酸性和碱性纤维生长因子中的一种或多种，浓度为1-10mg/mL。
进一步的，所述骨修复材料可以根据骨缺损大小裁剪成合适尺寸后使用，也可以通过影像学数据利用3D打印技术定制化摸具，构建符合骨缺损大小的骨修复材料。
相对于现有技术，本发明具有以下技术特点：
1)该复合支架材料中使用的具有长效缓释性能的矿化颗粒为自行设计的功能性填料，以如β-TCP颗粒的可降解内核颗粒、改良仿生矿化溶液、致孔剂、生长因子和多巴胺等组分作为原料，并结合多步矿化涂层的构建、功能性蛋白的原位负载、蛋白原位封装等组合工艺制得；将可降解的β-TCP等颗粒作为内核，使用自行设计的改良仿生矿化溶液在β-TCP表面所构建的多孔羟基磷灰石矿化涂层，致孔剂的引入在矿化涂层上形成大量的放射状孔隙结构，使其具有更大的比表面积，以实现生长因子的高效负载。同时，多巴胺在负载有BMP-2等功能性蛋白的矿化颗粒表面聚合形成一层聚多巴胺涂层实现对负载有生长因子的孔隙进行原位包封，得到复合颗粒如β-TCP/BMP-2/聚多巴胺复合颗粒，能进一步的提高蛋白质的保存和独立控释性能，释放时间可在数天至数月间调控；
2)该复合支架材料中使用的脱细胞材料，是通过一系列前期预处理动物组织步骤和多项改良配方后的生物化学溶液对原材料进行脱细胞处理所获得。通过上述改良后脱细胞工艺所制得的脱细胞基质保留了其天然纳米结构，从而保护表面含有的大量生长因子亲和位点，以实现各类生长因子的特异性吸附与控制释放；保护了脱细胞基质原有的三维支架结构，并有效除去了原材料中会引起人体免疫反应的抗原，具有一定的机械性能和优秀的生物相容性；保留了细胞外基质的主要成分，能够更好的促进细胞黏附、迁移和生长；
3)该复合支架材料通过将上述矿化颗粒和脱细胞基质通过模具塑型、低温冷冻干燥技术等组合工艺制得。通过控制不同组分调控多种蛋白的独立控制释放速率，以实现调控骨组织修复不同阶段的神经、血管和骨的生长速率。通过脱细胞材料缓释神经诱导相关蛋白促进早期的骨感觉神经再生，利用聚多巴胺封装的矿化颗粒长时间的释放骨诱导蛋白及骨修复所需的氨基酸和钙磷离子，以实现骨缺损的快速愈合，更精准的模拟正常骨修复过程，并调控骨感觉神经、血管再生，骨矿化和骨重塑过程。本发明制得的复合支架材料具有优异的促神经化成骨性能，通过促进缺损处组织感觉神经化从而释放神经肽提高骨组织生长速率；具有优秀的骨重塑功能，新骨中感觉神经通过神经支配优化成骨过程，避免异位成骨。
附图说明
图1实施例1中复合支架材料(简称为ECM@S/BMP-2)的制备示意图；
图2复合支架材料的代表性扫描电子显微镜图。
图3实施例2骨修复材料的因子释放速率图：材料BMP-2、NGF释放速率图；
图4实施例5骨修复材料体内自适应成骨性能图：A)每组术后4周或8周颅骨的Micro CT重建图像；B-E)不同各组的骨体积(BV)、骨体积分数(BV/TV)、骨小梁数(Tb.N)的定量数据。
图5实施例5骨修复材料体内促神经再生性能图：A)每组术后28天，免疫组织荧光染色感觉神经特异性靶标降钙素基因相关肽(Calcitonin Gene-Related Peptide，CGRP)。
图6实施例5骨修复材料体内血管再生性能图：A)每组术后28天，免疫组织荧光染色血管特异性靶标CD31。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例的技术方案进行清楚、完整的描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本发明的保护范围。
除非另作定义，本公开所使用的技术术语或者科学术语应当为本发明所属领域内有一般技能的人士所理解的通常意义。
本发明中的改良仿生矿化溶液(即改良模拟体液)的配方如下：依次向去离子水中加入以下试剂并形成对应浓度：141mM NaCl，4mM KCl，0.5mM MgSO4，1.0mM MgCl2，25mMNaHCO3，20.0mM HEPES，5mM CaCl2，和2mM KH2PO4；之后再加入1-10mg/mL造孔剂，仿生矿化溶液的pH值应调整为6.80。
所述的造孔剂包括聚甲基丙烯酸酯、甲基丙烯酸甲酯、聚乙二醇、聚乙烯醇和聚乙烯醇缩丁醛中的一种或多种。
根据本发明的一种具体实例，所述的可降解的内核微粒可以采用β-TCP颗粒，平均直径约为10μm；此外，也可以为磷酸镁颗粒、聚乳酸-羟基乙酸共聚物(PLGA)、聚乙二醇(PEG)、聚乳酸(PLA)和聚氨酯(PU)中的任一种。实施例1
过筛后的β-TCP放入改良后的模拟体液中进行仿生矿化，矿化并负载BMP-2后，将其浸泡在含有DA的Tris缓冲液中于表面形成DA涂层，后用去离子水清洗并冻干后获得β-TCP/BMP-2/聚多巴胺复合颗粒。将获得的新鲜猪皮进行清洗、裁切、匀浆后，使用脱细胞液和过氧乙酸进行脱细胞和除菌，并加入NGF亲和负载，获得含NGF的脱细胞基质。按矿化后复合颗粒质量和脱细胞基质干重比例为1:2称取两者，称取ECM 10g，干重为6.8g，称取复合颗粒3.4g，将两者放入匀浆器内，并逐步加入等量生理盐水搅拌混合均匀，将获得半流体状复合材料置于容器内冷冻冻干后获得复合支架材料。
复合支架材料制备流程图参见图1。制得的复合支架材料如图2电镜图所示，脱细胞基质的三维空间结构保持完整，并且孔径大小均一，具有良好的多孔结构。
实施例2
将实施例1中的材料裁切为等质量小块，放入EP管中，加入4mL SBF模拟体液，放入37℃，100rpm气浴摇床内模拟体外释放4周，在特定时间点取出所有释放液，冷冻避光保存，同时补充等量SBF到释放体系中。通过NGF和BMP-2对应ELISA试剂盒，检测释放液中各个时间点各个因子的浓度，并计因子的累积释放量。
骨修复材料因子释放速率参见图3。从图3中可以看出，该材料对NGF和BMP-2局均有缓释功能，并且均能持续释放30天以上。而且与ECM结合的NGF在前15天的释放速率相对较快，在20天左右达到峰值，能够更快的促进周围组织感觉神经化，从而促成骨。
实施例3
8周大鼠腹腔注射4％戊巴比妥钠(40mg kg-1)麻醉后，将大鼠从矢状中线处切开，暴露颅骨，用取骨钻沿颅骨中心线左右两侧各钻一个直径为6mm的圆形缺损，将实施例1中的支架材料裁切成半径为3mm，厚度约为0.2mm的圆形薄片，填入骨缺损处。空白组经相同处理，不植入任何材料。6周后人道处死大鼠，将大鼠颅骨取出，剔除大量软组织，并在10％的福尔马林溶液中固定48h，进行Micro CT扫描，对缺损部位进行分析。
骨修复材料的自适应调节BMP-2介导的骨形成能力参见图4。如图4所示，相较于空白组，实验组在4w观察到大片增生性新骨，并在8w基本完全覆盖缺损部位，显示出优异成骨性能。
实施例4
将实施例3获得的组织标本用10％ EDTA溶液脱钙处理20天。然后将制备的组织脱水，并用石蜡包埋。组织切片后，将片子用梯度酒精脱蜡和脱水，并用胰蛋白酶法修复表面抗原，PBS清洗3次，每次5min。洗净后的片子用0.5％(v/v)Triton X-100渗透20min，并用1％(w/v)山羊血清白蛋白在37℃下封闭1小时。然后用抗TUBB3、CD31的抗体在4℃孵育过夜，并与二抗在37℃下孵育1小时。最后，用0.1g/mL 4',6-二脒基-2-苯基吲哚(DAPI)染色细胞核5min，用徕卡荧光显微镜对样品进行成像和观察。
骨修复材料的神经重塑功能和血管再生性能参见图5、6。如图5所示，相较于空白组，实验组免疫荧光显示其有大量的CGRP阳性染色，有大量感觉神经重塑；图6显示实验组新骨生成处有较多环状的CD31染色，有大量的毛细血管再生。
以上实施例的说明只是用于帮助理解本发明方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求保护范围内。
Claims (10)
1.一种兼具促骨感觉神经重塑、血管再生和骨快速修复的复合支架材料的制备方法，其特征在于，包括如下步骤：
1)首先向去离子水中加入无机盐和造孔剂制备得到改良仿生矿化溶液，然后将可降解的内核微粒孵育在改良仿生矿化溶液，每天更换仿生矿化溶液，孵育7天以形成具有多孔结构的矿物质包被层，矿化颗粒用去离子水洗涤、干燥备用；
2)取步骤1)中得到的矿化颗粒分散于含第一种生长因子的0.2％BSA溶液(pH＝7.4)中，37℃下旋转孵育4.0小时，负载第一种生长因子的矿化颗粒；
3)将步骤2)获得的矿化颗粒放置于含多巴胺DA的10mM Tris缓冲溶液(pH＝8.5)中，反应持续2-6小时，得到负载第一种生长因子的复合颗粒；
4)对动物软组织进行处理获取脱细胞基质；
5)将步骤4)中制备的脱细胞基质用PBS缓冲液多次冲洗，并将脱细胞基质的pH值调整为7.20，加入第二种生长因子并充分混合，通过静电吸附作用获得含第二种生长因子的脱细胞基质；
6)向步骤5)获得的脱细胞基质中加入步骤3)获得的复合颗粒，搅拌静电吸附，分散均匀，低速离心脱泡得到半流体状复合材料的混合物，并将混合物转移到模具中，于-20℃至-80℃的温度下静置24小时得到冷冻的混合物；
7)将步骤6)制备的冷冻的混合物在0℃以下进行真空冷冻干燥获得复合支架材料。
2.根据权利要求1所述的制备方法，其特征在于，步骤1)中，改良仿生矿化溶液中的无机盐具体是通过依次向去离子水中加入以下试剂并形成对应浓度：141mM NaCl，4mM KCl，0.5mM MgSO4，1.0mM MgCl2，25mM NaHCO3，20.0mM HEPES，5mM CaCl2，和2mM KH2PO4；之后再加入造孔剂，仿生矿化溶液的pH值应调整为6.80。
3.根据权利要求1所述的制备方法，其特征在于，步骤1)中，仿生矿化工艺中使用的造孔剂包括聚甲基丙烯酸酯、甲基丙烯酸甲酯、聚乙二醇、聚乙烯醇和聚乙烯醇缩丁醛中的一种或多种，浓度为1-10mg/mL。
4.根据权利要求1所述的制备方法，其特征在于，所述可降解的内核微粒为β-TCP颗粒、磷酸镁颗粒、聚乳酸-羟基乙酸共聚物(PLGA)、聚乙二醇(PEG)、聚乳酸(PLA)和聚氨酯(PU)中的一种或多种，可降解的内核微粒与改良仿生矿化溶液的用量比例为25mg:50mL。
5.根据权利要求1所述的制备方法，其特征在于，步骤2)中的旋转孵育工序中，旋转速率等于100转/min，所处温度为37℃；所述的第一种生长因子为BMP-2、胰岛素样生长因子、或成纤维生长因子，分散于BSA溶液中浓度为1-10mg/mL。
6.根据权利要求1所述的制备方法，其特征在于，步骤3)中，DA的Tris溶液中DA浓度为2mg/ml，需用10mM Tris缓冲溶液在超声作用下溶解，溶液pH＝8.5，孵育过程需要全程避光。
7.根据权利要求1所述的制备方法，其特征在于，步骤4)中，所述脱细胞基质来源于包括猪、牛或者人的软组织；所述软组织包括皮肤、血管、韧带、肌腱、隔膜和小肠系膜中的一种或多种。
8.根据权利要求1所述的制备方法，其特征在于，步骤4)具体为，将动物软组织清洗后剪成所需规格尺寸的组织原材料，然后放入1.0％脱氧胆酸钠溶液中，并置于37℃，120rpm的摇床上脱细胞24小时后，将其置于40U/mLDNaseI与10mM MgCl的溶液浸泡90分钟，以完全去除细胞残留物；最后，用0.1％过氧乙酸/4％乙醇溶液灭菌处理2小时后，将其离心去除灭菌液后用0.9％生理盐水振荡浴清洗30min，循环3次后低温冷冻保存脱细胞基质。
9.根据权利要求1所述的制备方法，其特征在于，步骤5)中，所述的第二种生长因子为神经生长因子、转化生长因子β、碱性成纤维生长因子、胰岛素样生长因子、血管内皮细胞生长因子、成纤维细胞生长因子、酸性和碱性纤维生长因子中的一种或多种，浓度为1-10mg/mL。
10.一种兼具促骨感觉神经重塑、血管再生和骨快速修复的复合支架材料，其特征在于，采用如权利要求1-9任一项所述的方法制得。
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