CN113476655A - 一种3d打印骨修复复合支架及制备方法 - Google Patents

一种3d打印骨修复复合支架及制备方法 Download PDF

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CN113476655A
CN113476655A CN202110886672.7A CN202110886672A CN113476655A CN 113476655 A CN113476655 A CN 113476655A CN 202110886672 A CN202110886672 A CN 202110886672A CN 113476655 A CN113476655 A CN 113476655A
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printing
gelatin
bone repair
polycaprolactone
hydroxyapatite
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邹琴
王晨鑫
李玉宝
刘杰
林明玥
张瑞
李雨帆
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Sichuan University
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Abstract

本发明公开了一种3D打印骨修复复合支架及制备方法，该支架包括明胶、聚己内酯和纳米羟基磷灰石，所述明胶、聚己内酯和纳米羟基磷灰石的质量比为1:1:2。通过冰乙酸溶解得到明胶‑聚己内酯‑纳米羟基磷灰石浆料，采用挤出沉积式3D打印骨修复复合支架。本发明骨修复支架材料同时发挥明胶和PCL的双重优良性质，具有良好的生物相容性，良好的生物降解性。

Description

一种3D打印骨修复复合支架及制备方法
技术领域
本发明属于骨修复复合材料技术领域，具体涉及一种3D打印骨修复复合支架及制备方法。
背景技术
3D打印技术是一项新型数字化快速成型技术，也称为增材制造技术，它是计算机辅助下，以物体的计算机辅助设计(CAD)模型或CT扫描等数据为基础，利用金属粉末，陶瓷，高分子等原材料，通过“分层制造、逐层叠加”的方式快速构造任意复杂结构的物理模型。骨科较常用的3D打印技术主要包括光固化立体打印、选择性激光烧结、熔融沉积成型、金属直接熔融、喷墨打印等。注射式(挤出式)3D打印直接采用压缩空气或通过压缩空气直线电动机推动的活塞将注射筒中的材料连续挤出，对于黏度较大的“生物墨水”的打印优势更为明显。3D打印挤出沉积成形技术作为一种新型3D打印成形技术，是基于连续式流态直写技术的成形方法。其工作原理：通过压缩容器内空气或电机推动螺杆产生的压力挤出容料腔或注射器中的流态材料(墨水或浆料)，并根据导入的模型以连续丝的状态在基板上沉积成形。因具有材料范围广、设备成本低、成形能力强等优点，挤出沉积技术被广泛应用于生物3D打印领域。
传统的骨组织工程支架使用聚己内酯为材料制备的组织工程支架，聚己内酯(PCL)作为一种高分子聚酯，材料来源广泛，具有良好的生物相容性及生物降解性，然而PCL难溶于水，常需氯仿等有机溶剂作为黏结剂，但氯仿难以完全去除易导致毒性反应，并且人工合成聚合材料降解产生酸性产物，易引起炎症反应，所以成骨性能和生物相容性较差。而且传统的骨组织工程支架使用明胶为材料制备的组织工程支架，明胶做为一种天然的生物材料，已成为骨组织再生领域中的研究热点。其具有仿生特性，生物学性能好、骨传导性和骨结合能力强，在骨修复方面具有明显的优势。但往往单一的明胶支架力学机械性能较差，在骨修复过程中无法起到支撑作用，且降解速度过快，所以骨修复能力和支撑能力较差。但实际应用中支架需要具有一定的力学强度且需要较高的成骨性能和生物相容性。
发明内容
针对现有技术存在的技术问题，本发明提供了一种3D打印骨修复复合支架及制备方法，目的是解决单一聚己PCL支架难降解，且细胞不易在其上生长；单一明胶3D打印支架难以成型，降解速度快；聚己内酯(PCL)与明胶(Gel)难以共混打印等问题。
为了实现上述技术目的，本发明3D打印骨修复复合支架具体采用的技术方案如下：
一种3D打印骨修复复合支架，所述的复合支架包括明胶、聚己内酯和纳米羟基磷灰石，所述明胶、聚己内酯和纳米羟基磷灰石的质量比为1:1:2。
所述的明胶、聚己内酯和纳米羟基磷灰石通过分散剂冰乙酸混合搅拌形成打印浆料。
所述的明胶为A型明胶颗粒，所述的聚己内酯为分子量8万的颗粒，所述的羟基磷灰石粒径为35～40微米。
在本发明的另一方面，提供一种挤出沉积式3D打印骨修复复合支架的方法，包括以下步骤：
1)将明胶、聚己内酯溶于冰乙酸中充分溶解，加入羟基磷灰石粉末，搅拌直至粘度为20MPa.s～30MPa.s；
2)设定3D打印参数，包括料筒温度、接收板温度、打印气压调、打印速率，打印得到骨修复支架毛坯；
3)将骨修复支架毛坯浸没在交联剂中进行交联即可。
进一步的，所述料筒温度设定为10～18℃，接收板温度设定为5℃，打印气压调至0.4MPa，打印速率为12～14mm/s。
进一步的，所述骨修复支架毛坯为0°/45°/90°/135°米字形结构。
进一步的，所述交联剂为香草醛-无水乙醇溶液，所述香草醛质量比为10％。
本发明的有益效果为：
本发明所述骨修复支架的3D打印浆料组分为：明胶粉末，PCL颗粒，纳米羟基磷灰石粉末以及冰乙酸。纳米羟基磷灰石是人体骨的主要无机成分，同时在此3D打印浆料中起到了增稠剂的作用，解决了Gel难以成型的问题。本发明3D打印浆料配方成功的实现明胶与PCL的共混，解决了PCL难降解以及Gel机械性能差的问题，并且在羟基磷灰石作为增稠剂的作用下得到了粘度合适、适合连续挤出式的3D打印浆料。本发明还提供了基于该3D打印浆料的构建骨修复支架的方法和打印后经过交联的支架。运用此浆料进行3D打印，打印出0°/45°/90°/135°米字形结构支架，再经过质量比10％的香草醛-无水乙醇溶液交联12小时制得本发明产品。本发明生产出来的骨修复支架材料同时发挥明胶和PCL的双重优良性质，具有良好的生物相容性，良好的生物降解性，经过香草醛-无水乙醇溶液交联后具有良好的力学性能，是良好的细胞生长支持材料支架。
附图说明
图1是本发明的聚己内酯-明胶-纳米羟基磷灰石多孔支架支架在扫描电镜下的微观形貌图；
图2是本发明聚己内酯-明胶-纳米羟基磷灰石多孔支架支架与MG63细胞共培养后扫描电镜图；
图3是本发明的支架植入新西兰大白兔股骨髁部位4W、8W、12W后的micro-CT二维成像图。
具体实施方式
下面将结合本发明具体的实施例，对本发明技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供了一种3D打印骨修复复合支架，该复合支架体系为：聚己内酯(PCL)-明胶(Gel)-纳米羟基磷灰石，并采用冰乙酸对其体系溶解制备浆料。
本发明在PCL中加入生物性能优异的可降解物质-明胶，并采用了一种可以同时溶解PCL与明胶的溶剂-冰乙酸。将PCL与明胶在37℃条件下分别溶解于冰乙酸中，再将两者进行共混形成PCL-明胶的复合材料，解决了聚己内酯(PCL)与明胶(Gel)难以共混打印的问题。
乙酸，也叫醋酸、冰醋酸，是一种有机一元酸，为食醋主要成分。乙酸中的乙酰基，是生物化学中所有生命的基础。当它与辅酶A结合后，就成为了碳水化合物和脂肪新陈代谢的中心。通过分析发现，聚己内酯(PCL)具有在芳香化合物、酮类和极性溶剂中很好地溶解的性质；明胶具有溶于热水、甘油、丙二醇、乙酸、水杨酸等的性质。因此选择乙酸作为PCL与明胶的共同溶剂，同时乙酸具有一定的挥发性，满足挤出沉积式3D打印材料黏度可调可控、溶胶态到凝胶态的相变转换速度快的特点。
人体骨骼表现出的生物和机械性能是由于本身含有的有机蛋白质相(胶原蛋白和其他骨蛋白质)与无机矿物相(磷灰石)结合后，共同作用而产生的复合性能。目前骨修复材料主要包含高分子材料、无机陶瓷材料及其相互复合的衍生材料，其中复合材料可结合两种或两种以上生物材料的优点。同时，在浆料配置过程中发现，PCL/明胶/乙酸的复合材料由于粘稠度不足的问题，挤出沉积3D打印出的支架无法较好的成型，所以加入纳米羟基磷灰石粉末作为增稠剂，增强浆料粘稠度，使打印出的支架可以成型。
优选的，所述的明胶为A型明胶颗粒，所述的聚己内酯为分子量8万的颗粒，所述的羟基磷灰石粒径为35～40微米。
在本发明的另一方面，提供了一种挤出沉积式3D打印骨修复复合支架的方法，包括以下步骤：
1)将明胶、聚己内酯按一定比例混合均匀，所用溶剂冰乙酸易挥发且对生物体无毒，然后加入纳米级羟基磷灰石，混合后形成明胶-聚己内酯-羟基磷灰石复合浆料；
a.配置明胶-冰乙酸溶液，PCL-冰乙酸溶液：称取A型明胶和PCL各分别放入两个烧杯中，分别各加入分析纯冰乙酸，用保鲜膜将烧杯口封好，减少乙酸的挥发量。将两个烧杯放入37℃的恒温摇床中进行恒温震荡，10小时后，PCL与明胶充分溶解于冰乙酸中，得到透明无色的PCL-冰乙酸溶液和透明黄色的明胶-冰乙酸溶液。
b.将明胶-冰乙酸溶液倒入PCL-冰乙酸溶液中并充分进行搅拌，称取6g的羟基磷灰石粉末(过400目筛)加入到经过充分搅拌后的明胶-PCL-冰乙酸的混合溶液，再充分搅拌直至粘度合适。
2)设定3D打印参数，打印时的料筒温度设定在10～18℃之间，同时接收板温度设定在5℃左右，3D打印气压调至0.4MPa，打印速率为11mm/s，以确保浆料能够顺利挤出的同时在接收板上能够快速成型。采用米字型结构进行打印，其打印角度为0°/45°/90°/135°。打印后得到骨修复支架毛坯。
3)得到骨修复支架毛坯后，将其浸没香草醛-无水乙醇溶液中进行交联，交联12小时。
其中，香草醛-无水乙醇溶液(交联剂)中的质量比为10％。使用3D打印机打印所述组织工程支架时控制温度在14～18℃。
实施例1
一种新型挤出沉积式3D打印聚己内酯-明胶-纳米羟基磷灰石多孔支架的制备方法：
1)挤出沉积式3D打印聚己内酯-明胶-纳米羟基磷灰石浆料的制备
称取3g聚己内酯和3g明胶分别放入2个烧杯当中，再向两个烧杯中各加入10ml冰乙酸，用保鲜膜密封。将两个烧杯放置于37℃恒温摇床中轻微摇晃12小时，制得质量体积比均为30％的聚己内酯/乙酸与明胶/乙酸溶液。
12小时后，将两个烧杯同时取出，揭下保鲜膜，将明胶/乙酸溶液快速倒入进聚己内酯/乙酸溶液中，并充分搅拌0.5～1h。
将纳米羟基磷灰石研磨过筛，使用400目筛进行过筛。称量6g已过筛的纳米羟基磷灰石加入进聚己内酯/明胶混合溶液中，充分搅拌1～1.5h后直至粘稠，从而制得挤出沉积式3D打印聚己内酯-明胶-纳米羟基磷灰石浆料。
2)挤出沉积式3D打印聚己内酯-明胶-纳米羟基磷灰石多孔支架的制备
使用气压式挤出沉积3D打印机，对浆料进行3D打印。将装有打印浆料的3D打印料筒放置在打印机的料筒放置区内，调节设定参数，料筒温度为14～16℃，打印气压为0.3MPa，打印速率为12～14mm/s。打印出的支架参数：长20mm，宽20mm，高6.5mm，调试打印参数，并对支架形状、结构和尺寸进行设置，以保证多孔支架的表面结构、孔隙率和贯通性能。制得出支架后，使用扫描电子显微镜观察其微观结构，如图1所示。支架孔隙率大于60％，孔径大小为800～1200μm，使其有利于细胞黏附生长，组织与血管的长入。
实施例2细胞与支架共培养试验
选取了MG63细胞(骨肉瘤细胞)来进行材料与细胞的共同培养。从液氮罐中取出冻存的MG63细胞株，快速放入提前37℃预热好的水浴锅中1-2min。放入离心机中，3000r/min离心3min。倒去冻存液，加入1ml培养基并吹打均匀后吸出加入至细胞培养瓶。换液，传代后将细胞与支架共同放置在24孔板中共同培养。7天后对其进行固定脱水。然后进行了扫描电镜对其进行分析。
如图2所示，可以看出，细胞在支架表面大量增殖并伸出尾足黏附在打印支架材料上。由此可以证明，实施例1制备的挤出沉积式3D打印支架具有优异的生物相容性以及优良的细胞黏附与细胞生长的特性。
实施例3生物学效应试验
将3只饲养观察2周后的新西兰大白兔称重后固定于实验手术台，自耳缘静脉注射3％戊巴比妥钠注射液(每公斤注射1mL)，在股骨髁手术区域附近备皮，常规消毒铺巾。于股骨髁顶部切开长约1cm的切口，制造φ5mm深6～7mm的缺损，于左右两条腿分别植入实施例1支架，植入支架后用骨蜡封闭，逐层缝合后使用聚维酮碘消毒。术后一天，注射青霉素钠溶液防止因感染导致动物死亡，并允许实验动物自由活动，所有动物单笼饲养。
分别在4W、8W、12W各处死一只实验兔。分离股骨与胫骨连接处软组织，暴露股骨髁，在离股骨髁下端1cm处截取样本，用4％多聚甲醛浸泡保存，进行大体观察与micro-CT扫描检测。
样本micro-CT二维分析如图3所示，从左至右依次为4周，8周，12周。从大体观察照片与CT二维图片可以看到，随着时间的推移，新骨逐渐向支架内生长，缺损区域有大量新骨生成，并且伴随着新骨的长入，支架在逐步进行降解，但仍能看出支架其形貌结构。这可能是由于明胶本身具有的优异的骨传导性和骨诱导能力，同时明胶与聚己内酯的混合改善了聚己内酯支架难以降解以及明胶支架极易降解，力学性能差的问题。
综上所述，本发明制备的挤出沉积式聚己内酯-明胶-纳米羟基磷灰石多孔支架具有良好的力学支撑性能，高贯通性，高孔隙率，具有良好的骨传导性以及骨诱导性能。是个性化的骨修复材料复合多孔支架，能够通过调节材料的孔隙率或者贯通性等方法提高材料的骨传导性，增加自体骨向骨修复材料内部生长，实现骨修复材料与自体骨的融合而起到固定和支撑的作用，动物实验表明，该支架能有效修复骨缺损，并能在体内进行降解。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (6)

1.一种3D打印骨修复复合支架，其特征在于，所述的复合支架包括明胶、聚己内酯和纳米羟基磷灰石，所述明胶、聚己内酯和纳米羟基磷灰石的质量比为1:1:2。
2.根据权利要求1所述的一种3D打印骨修复复合支架，其特征在于，所述的明胶、聚己内酯和纳米羟基磷灰石通过分散剂冰乙酸混合搅拌形成打印浆料。
3.根据权利要求1所述的一种3D打印骨修复复合支架，其特征在于，所述的明胶为A型明胶颗粒，所述的聚己内酯为分子量8万的颗粒，所述的羟基磷灰石粒径为35～40微米。
4.一种挤出沉积式3D打印骨修复复合支架的方法，其特征在于，包括以下步骤：
1)将明胶、聚己内酯溶于冰乙酸中充分溶解，加入羟基磷灰石粉末，搅拌直至粘度为20MPa.s～30MPa.s；
2)设定3D打印参数，包括料筒温度、接收板温度、打印气压调、打印速率，打印得到骨修复支架毛坯；
3)将骨修复支架毛坯浸没在交联剂中进行交联即可。
5.根据权利要求4所述的方法，其特征在于，所述料筒温度设定为10～18℃，接收板温度设定为5℃，打印气压调至0.4MPa，打印速率为12～14mm/s。
6.根据权利要求4所述的方法，其特征在于，所述交联剂为香草醛-无水乙醇溶液，所述香草醛质量比为10％。
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