CN116425562A - 一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架及其制备方法和应用 - Google Patents
一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架及其制备方法和应用 Download PDFInfo
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- CN116425562A CN116425562A CN202310338461.9A CN202310338461A CN116425562A CN 116425562 A CN116425562 A CN 116425562A CN 202310338461 A CN202310338461 A CN 202310338461A CN 116425562 A CN116425562 A CN 116425562A
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- calcium phosphate
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- biphasic calcium
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 title claims abstract description 162
- 239000001506 calcium phosphate Substances 0.000 title claims abstract description 126
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- 229910000389 calcium phosphate Inorganic materials 0.000 title claims abstract description 125
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- 238000002360 preparation method Methods 0.000 title claims abstract description 24
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- C04B38/06—Porous mortars, concrete, artificial stone or ceramic ware; Preparation thereof by burning-out added substances by burning natural expanding materials or by sublimating or melting out added substances
- C04B38/063—Preparing or treating the raw materials individually or as batches
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Abstract
本发明公开了一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架及其制备方法和应用。本发明采用了化学共沉淀法并煅烧制备Zn离子掺杂双相磷酸钙，再分别按一定比例复合以同样方法制备的Si、Mg、Sr三种活性离子掺杂双相磷酸钙进行两种不同活性离子的化学组成改性，加入甲基纤维素和聚乙烯醇调和成打印浆料，利用3D打印技术3D打印构建具有三维连通大孔和微纳米级微孔复合的多级孔结构进行结构改性，协同改性的磷酸钙陶瓷支架起促成骨作用。具有制备工艺简单、离子成分分布均匀，力学和生物学性能良好的特点，对拓展双相磷酸钙陶瓷支架在非承重骨缺损修复的临床应用具有重要意义。
Description
技术领域
本发明属于骨缺损修复生物医用陶瓷支架领域，特别涉及一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架及其制备方法和应用。
背景技术
羟基磷灰石(Hydroxyapatite，HA)和β-磷酸三钙(Beta-tricalcium phosphate，β-TCP)是备受关注的骨修复材料之一，因为它们是天然骨的主要成分。羟基磷灰石因化学组成与人体骨骼的无机成分相似，具有良好的生物相容性及骨传导性能，力学性能好，然而其结构稳定不易降解。β-磷酸三钙具有良好的生物可降解吸收性，能被骨直接吸收，但是力学性能不佳，且相对人体骨生成速率而言，其降解速率较快。由羟基磷灰石(HA)和β-磷酸三钙(β-TCP)混合组成的双相磷酸钙(Biphasic calcium phosphate，BCP)材料，则同时兼具β-TCP可降解吸收和HA高生物活性的特性，同时获得适当的降解率，它们降解产物钙(Ca)、磷(P)可进入活体循环系统形成新骨，是理想的硬组织替代材料。Silva A等(Silva A,Rinco U,Jacob R,et al.The effectiveness of hydroxyapatite-beta tricalciumphosphate incorporated into stem cells from human exfoliated deciduous teethfor reconstruction of rat calvarial bone defects[J].Clinical OralInvestigations,2021:1-14.)采用直径为6mm的Wistar大鼠颅骨产生的双皮质缺损，将实验动物随机分为几组，有：阴性对照组、自体骨组、BCP组，每组各10只。4周和8周后，通过组织计量学分析评估新生骨和残留生物材料颗粒的存在，实验结果表明，阴性对照组没有自发修复，植入BCP修复大鼠颅骨缺损，虽不如自体移植明显，但亦有良好的效果。
活性离子掺杂改性磷酸钙陶瓷支架材料，可以不同程度上改善材料的成骨和成血管性能。锌离子(Zn2+)具有维持骨骼的正常生长发育、参与蛋白质和核酸的形成与代谢，参与免疫过程和细胞间的信号传导、维持膜的稳定性、增强成骨细胞活性、抑制破骨细胞活性，促进成骨细胞骨钙素的分泌从而使得骨基质更加成熟等作用(Beattie J H,AvenellA.Trace element nutrition and bone metabolism[J].Nutrition research reviews,1992,5(1):167-188.)，而缺锌则会导致骨质疏松、骨密度降低、骨骼发育畸形等不良后果(Oteiza P I,Mackenzie GG.Zinc,oxidant-triggered cell signaling,and humanhealth[J].Molecular Aspects of Medicine,2005,26(4-5):245-255.)；硅(Si)是人体健康所必须的微量元素之一，存在于人的结缔组织和骨组织中，在骨骼的生长和修复中起重要作用。在骨矿化过程中，活性钙化位点处发现了大量硅的存在，表明了硅积极与了骨的形成。在动物实验模型中，缺乏硅会导致胶原形成的减少、骨骼的异常生长以及生长迟缓。镁(Mg)是人体中含量第四高的元素，在骨中的含量为0.6wt.％(Myers H M.Calciumphosphates in oral biology and medicine[J].Calcium Phosphate Biomaterials inPreventive and Restorative Dentistry.Farmington,CT:Karger,1991:154-71.)，镁离子(Mg2+)缺乏会使骨生长受损、导致骨质疏松。研究表明镁离子在骨代谢中具有较大作用，它会影响成骨细胞和破骨细胞的活性从而促进骨生长。Mg2+在生物磷灰石钙化前期含量较高，随着矿化进行，含量逐渐降低(Bigi A,Foresti E,Gregorini R,Ripamonti A,RoveriN,Shah JS.The role of magnesium on the structure of biologicalapatites.Calcif Tissue Int.1992,50:439–44.)；锶离子(Sr2+)能抑制骨吸收，在骨质疏松骨缺损修复领域受到关注。其生物学机制在于ERK/p38信号通路介导了Sr掺杂调控成骨和成血管，进而促进血管化骨组织的再生过程，在OVX症骨缺损修复领域有重要应用前景。此外，Sr掺杂还能调控巨噬细胞(M0)从促炎的M1极化向抗炎的M2极化转化，进而为软骨组织的修复创造更好的微环境(Chong Y W,Bi C,Wei W,et al.Strontium released bi-lineage scaffolds with immunomodulatory properties induce a pro-regenerativeenvironment for osteochondral regeneration[J].Materials Science andEngineering:C,2019,103(C):109833.)。而既往实验研究表明，在双相磷酸钙中替代多离子掺杂容易造成BCP出现杂相和结构的改变。且不同离子间组合通常具有协同效应，有望进一步增强骨修复效果。
大量的体外体内实验研究表明，物理化学性质和结构特性会影响钙磷陶瓷的成骨活性。骨诱导陶瓷独特的物相组成和多孔结构特征使其能够与宿主系统中的信号分子和细胞外基质相互作用，创造出有利于新骨形成的微环境。孔连通结构良好、孔径尺寸适宜、孔隙率高是实现材料引导细胞迁移增殖、新生骨组织和血管组织生长，并保障骨组织再生修复过程中营养物质输运和新陈代谢的关键。研究表明，与天然骨组织结构相似的骨修复材料，不仅可为成骨细胞提供迁移、黏附、增殖和成骨分化的空间，还有利于营养物质体内废物和输运和交换，从而促进骨组织的重建。此外，孔径在150-500μm且孔隙率高的多孔支架材料能形成矿化骨，在骨缺损修复和骨组织工程构建领域具有良好的应用效果。因此，调控生物陶瓷支架材料的三维孔道结构和尺寸至关重要。与缺损组织三维形态相似的多孔支架材料的构建对于实现个性化治疗和精准化组织缺损修复至关重要。目前添加造孔剂法、有机泡沫浸渍法、浇注成型等多种技术可以构建具有不同孔隙率和孔径尺寸的多孔陶瓷支架。但是以上方法不能做到孔结构的设计和调控，其相比之下，3D(three-dimensional)打印技术能做到个性化和精准化的调控，能够获得与病损组织三维形态几乎相符的精确几何结构，使其在三维尺寸、形态、不同成分的有序堆积和空间布局的精确调控等方面成为可能。然而，当前的生物陶瓷支架材料仍存在一些缺陷，例如诱导成骨活性差、降解速与宿主骨组织生成速率不匹配，成分和生物学功能单一、力学性能欠佳等。
Li等通过结合H2O2发泡法和微球烧结法的优点，制造出具有适当均匀的大孔和丰富的微孔的BCP生物陶瓷，研究发现，得到的BCP生物陶瓷可以很好地引发和调节体外生物反应，如降解、骨样磷灰石形成、蛋白质吸附、细胞扩散、血管生成和成骨分化。通过优化孔径分布和微纳米形貌，进一步提高了BCP生物陶瓷的骨诱导性，以满足再生医学的要求。(LiX,Wang Y,Chen F,et al.Design of macropore structure and micro-nano topographyto promote the early neovascularization and osteoinductivity of biphasiccalcium phosphate bioceramics[J].Materials&Design,2022,216:110581.)但研究仅对BCP生物陶瓷的结构进行改性，本发明在制备含三维连通大孔和微纳米形貌支架对结构改性的同时，对BCP支架通过离子掺杂对成分进行改性。Lu等探究了不同含量的锌离子掺入双相磷酸钙陶瓷的的理化性质、体外细胞学反应、体内骨诱导和骨缺损再生的影响。研究结果显示，当掺杂锌的含量等于或高于2.5mol.％时，BCP支架可以长期缓慢释放锌离子。掺入量为2.5mol.％时，BCP的骨诱导活性明显增强，具有最高的促骨生成和适当的破骨细胞生成活性，加速了新骨的形成。(Lu T,Yuan X,Zhang L,et al.Enhancing osteoinduction andbone regeneration of biphasic calcium phosphate scaffold thought modulatingthe balance between pro-osteogenesis and anti-osteoclastogenesis by zincdoping[J].Materials Today Chemistry,2023,29:101410.)但研究只进行了单离子掺杂改性BCP支架的理化性能和成骨性，而人体骨组织是含有多种活性离子共同作用的，本发明在锌最佳掺量的基础上复合了镁、锶、硅三种离子掺杂BCP，研究其对BCP支架在理化性能和体外细胞学反应的影响。
发明内容
为了克服现有技术存在的不足，本发明的目的是提供一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架及其制备方法和应用。
本发明的首要目的在于克服现有技术两种及以上离子掺杂进双相磷酸钙会导致材料晶体结构稳定性发生改变影响其物相组成，从而限制了双相磷酸钙的应用的缺点与不足，提供一种先掺杂再以不同比例进行复合的离子掺杂双相磷酸钙复合支架的制备方法。
本发明的另一目的在于提供上述具有宏观大孔和微纳米级小孔的离子掺杂双相磷酸钙复合陶瓷支架的制备及应用。
本发明的目的至少通过如下技术方案之一实现。
本发明采用了化学共沉淀的方法合成了一种将活性离子直接掺杂进入双相磷酸钙中复合另一种活性离子掺杂双相磷酸钙的双相磷酸钙粉体。
本发明提供的一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架的制备方法，包括如下步骤：
(1)将钙源、锌源、磷源与水搅拌混合均匀，得到反应溶液，调节所述反应溶液的pH值，陈化、多次离心洗涤得到沉淀，经冷冻干燥、低温煅烧，将煅烧得到的产物球磨、烘干、过筛，得到锌离子掺杂的双相磷酸钙粉体；
(2)将上述锌源替换成另一种活性离子源，按照步骤(1)相同操作，得到另一种活性离子掺杂的双相磷酸钙粉体；
(3)将步骤(1)得到的锌离子掺杂的双相磷酸钙粉体和步骤(2)得到的另一种离子掺杂的双相磷酸钙粉体复合，均匀混合，得到离子掺杂并复合的双相磷酸钙粉体；
(4)将步骤(3)中所得到的粉体与增稠剂均匀混合，滴加粘结剂得到粘稠状打印浆料；将所述粘稠状打印浆料进行3D打印，得到支架素坯；
(5)放入烘箱干燥，再进行高温烧结，得到所述一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架。
进一步地，所述钙源为硝酸钙，其溶液浓度为0.20-0.55mol/L。
进一步地，所述磷源为磷酸氢二铵、磷酸二氢铵、磷酸氢二钠、磷酸二氢钠中的至少一种。
进一步地，所述钙源与磷源的体积比为(1.3-2.0):1。
进一步地，所述锌源为硝酸锌。
进一步地，所述另一种活性离子源为硝酸锶、硝酸镁、正硅酸乙酯中的至少一种。
进一步地，锌、镁、锶离子中任一种离子源与钙源的摩尔比为(0.02-0.5):1，硅离子按硅酸根方式掺杂，硅酸根与磷源的摩尔比为(0.01-0.20):1。
进一步地，步骤(1)中，用与去离子水体积比为1:1的稀氨水溶液来调节反应溶液的pH值为8.00-10.35，搅拌速率为200-500r/min，搅拌时间为60-120min，陈化的时间为18-30h。
进一步地，步骤(1)中，溶液的离心速率为4000-5000r/min，洗涤液为去离子水，所述冷冻干燥的时间为48-72h，所述煅烧的温度为850-900℃，煅烧的时间为2-4h。
进一步地，步骤(3)中，按水和粉体物料的质量比为(1-3):1的比例湿法球磨，其中大球小球的质量比为3:2，300r/min的速率下球磨2-3h，放入60℃烘干12h；将干燥后的粉体过53μm以下(270目)筛，即可得到待打印粉末样品。
进一步地，步骤(3)中，锌离子掺杂的双相磷酸钙粉体与另一种活性离子掺杂的双相磷酸钙粉体的质量百分比为(0.05-2):1，优选为(0.25-1):1。
进一步地，步骤(4)中，所述增稠剂为甲基纤维素，所述粘结剂为聚乙烯醇。离子掺杂并复合的双相磷酸钙粉体、甲基纤维素、聚乙烯醇按照质量比为100:(3-5):(80-110)的比例均匀混合30min-60min，制成粘稠状打印浆体。
进一步地，步骤(4)所述3D打印的喷头温度为5-30℃，3D打印的平台温度为5-30℃，3D打印的喷头气压为0.3-0.6Mpa，3D打印的打印速率为5-20mm/s。
进一步地，步骤(4)所述3D打印的纤维直径为0.4-0.6mm，纤维间距为0.6-1.2mm，打印支架直径为5-14mm，打印高度为2-15mm。
进一步地，步骤(5)所述干燥包括：先将支架素坯在常温条件下干燥24-48h，然后在60℃的条件下干燥24-48h。
进一步地，步骤(5)所述烧结温度为1050-1150℃，分三阶段升温，升温速率为2-10℃/min，保温时间为2-4h，并以同样的升温速率降至室温。
本发明提供一种由上述制备方法制得的一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架，具体来说，是一种3D打印具有连通宏观大孔和微纳米孔活性离子掺杂双相磷酸钙复合陶瓷支架，该陶瓷支架能够应用在制备非承重骨缺损修复填充材料中。
与现有技术相比，本发明具有如下优点和有益效果：
(1)本发明通过化学共沉淀法制备的掺杂两种活性离子改善双相磷酸钙的性能的方法，协同激活细胞和诱导干细胞成骨分化的活性，进而提高双相磷酸钙的促成骨性能。通过直接化学共沉淀法制备的一种离子掺杂双相磷酸钙(BCP)复合另一种离子掺杂双相磷酸钙的方式，很好地解决了直接由两种或两种以上离子掺杂双相磷酸钙导致双相磷酸钙结构不稳定、晶相发生改变从而导致材料的成骨性能下降的问题。相较于传统制备方法，不需要复杂的制备条件和设备，制备方法简单易行，离子分布更加均匀。能够促进骨髓间充质干细胞在陶瓷表面的增殖和成骨分化，提高了双相磷酸钙的成骨性能，作为骨缺损修复材料具有独特的优势和应用潜力。
(2)本发明制备出的硅离子掺杂双相磷酸钙在复合了锌掺杂双相磷酸钙后，使得在高温下烧结维持了无毒副作用、晶相生物活性更好的β-TCP物相，而没有生成更易降解α-TCP转变成结构更加稳定，从而进一步促进细胞的黏附、增殖和成骨性能。同样，镁离子掺杂双相磷酸钙在复合了锌掺杂双相磷酸钙、锶离子掺杂磷酸钙在复合了锌掺杂双相磷酸钙都在一定程度上改善了双相磷酸钙的理化性能和生物活性，扩展了双相磷酸钙在化学组成上的改性的思路和方法。
(3)本发明通过调整不同比例的离子掺杂双相磷酸钙的复合比，筛选出较优的物理化学性能(如高孔隙率、较大的抗压强度、较适宜的降解速率)和生物学性能(如促进骨细胞的黏附、增殖和分化)的复合比，提高骨组织再生速度等。
(4)本发明通过离子掺杂再复合的方式对双相磷酸钙在组成上进行改性，同时运用3D打印技术打印了互连接孔隙的复杂网络三维结构支架对双相磷酸钙在结构上进行改性，同时对双相磷酸钙在化学组成和结构上对其改性。具有足够的、相互连接孔隙的复杂网络三维结构支架不仅有利于新生血管的形成，还能促进干细胞的黏附以及增殖和成骨分化。
附图说明
图1为对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙粉体X射线衍射谱图。
图2为对比例1、2的未掺杂双相磷酸钙粉体和硅离子掺杂双相磷酸钙粉体扫描电子显微镜图。
图3为对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙在高温烧结后陶瓷支架X射线衍射谱图。
图4为对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙表面及断面的扫描电子显微镜(SEM)结构图。
图5为小鼠骨髓间充质干细胞(mBMSCs)在对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙陶瓷支架表面培养1天、3天后的细胞增殖情况结果。
图6小鼠骨髓间充质干细胞与对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙共培养7天和14天后的碱性磷酸酶(ALP)活性结果。
图7a、图7b、图7c分别为对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙陶瓷支架成骨分化相关基因表达情况结果。
图8为对比例1未掺杂双相磷酸钙陶瓷支架与实施例2镁离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙陶瓷支架的抗压强度图。
图9小鼠骨髓间充质干细胞与对比例1未掺杂和实施例3锶离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙共培养7天和14天后的碱性磷酸酶(ALP)活性结果。
具体实施方式
以下结合实例对本发明的具体实施作进一步说明，但本发明的实施和保护不限于此。需指出的是，以下若有未特别详细说明之过程，均是本领域技术人员可参照现有技术实现或理解的。所用试剂或仪器未注明生产厂商者，视为可以通过市售购买得到的常规产品。
对比例1
为与实施例制备得到的离子掺杂双相磷酸钙陶瓷支架进行对比，对比例1制备了未掺杂离子的双相磷酸钙陶瓷支架。
未掺杂离子的双相磷酸钙(BCP)粉体按以下方法制备：称取26.412g(0.20mol)磷酸氢二铵加入1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成磷酸氢二铵溶液A；称取73.207g(0.31mol)四水硝酸钙到1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成硝酸钙溶液B；将磷酸氢二铵溶液A滴加到硝酸钙溶液B中，滴加过程中350r/min持续搅拌，搅拌均匀，得到反应溶液，同时用氨水调节反应溶液的pH值为8.60(±0.2)；滴加结束后继续搅拌60min，搅拌速率为350r/min，搅拌均匀。随后陈化24h、离心取沉淀，离心速率为4000r/min，每次离心5min，离心洗涤三次。随后进行72h的冷冻干燥，900℃低温煅烧2h，升温速率为5℃/min，得到未掺杂离子的β-TCP/HA比例为7:3双相磷酸钙粉体。按水料比为1:1的比例湿法球磨，其中大球小球的质量比为3:2，300r/min的速率下球磨2h，放入60℃烘干12h，将干燥后粉体过53μm的筛，得到粒度小于53μm的打印粉体。
具有三维连通孔结构的未掺杂双相磷酸钙陶瓷支架的制备按如下方法制备：用搅拌器将5g的上述制备的未掺杂离子的BCP粉体，0.15g甲基纤维素混合均匀，再加入4.2g浓度为8％的聚乙烯醇(PVA)溶液(称取8g PVA溶于92mL去离子水中，用磁力搅拌子并置于95℃水浴锅中搅拌加热30min，使PVA完全溶解至透明澄清。所述聚乙烯醇为PVA1799)，充分搅拌得到均匀粘稠状浆料，将浆料转移到3D打印料筒中。导入STL格式的圆柱形模型文件，模型尺寸设为10×10×3mm，3D打印的喷头温度为25℃，平台温度为27℃，纤维直径为0.4mm，填充间距设为800μm、打印层厚设为320μm、打印速度设为6mm/s、压力设为0.36MPa。将打印得到的支架常温干燥24h，60℃条件干燥24h，最后经过1100℃烧结分三阶段升温，升温速率为2℃/min，保温时间为2h，并以同样的升温速率降至室温。得到所述具有三维连通孔结构未掺杂离子的双相磷酸钙陶瓷支架，标记为BCP。
对比例2
为与实施例制备得到的复合锌离子掺杂双相磷酸钙陶瓷支架进行对比，对比例2制备了硅离子掺杂双相磷酸钙陶瓷支架。
未复合的硅离子掺杂的双相磷酸钙粉体按以下方法制备：称取25.356g(0.20mol)磷酸氢二铵和1.792mL正硅酸乙酯(溶于无水乙醇中)加入到1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成混合水溶液A；称取73.207g四水硝酸钙1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成硝酸钙溶液B；将混合水溶液A滴加到硝酸钙溶液B中，滴加过程中350r/min持续搅拌，搅拌均匀，得到反应溶液，同时用氨水调节反应溶液的pH值为8.68(±0.2)；滴加结束后继续搅拌60min，搅拌速率为350r/min，搅拌均匀。随后陈化24h、离心取沉淀，离心速率为4000r/min，每次离心5min，离心洗涤三次。随后进行72h的冷冻干燥，900℃低温煅烧2h，升温速率为5℃/min，得到未复合4mol.％硅离子掺杂β-磷酸三钙/羟基磷灰石比例为7:3双相磷酸钙粉体。按水料比为1:1的比例湿法球磨，其中大球小球的质量比为3:2，300r/min的速率下球磨2h，放入60℃烘干12h，将干燥后粉体过53μm的筛，得到粒度小于53μm的打印粉体。
具有三维连通孔结构的未复合的硅离子掺杂双相磷酸钙陶瓷支架的制备按如下方法制备：用搅拌器将5g的上述制备的未复合的硅离子掺杂BCP粉体，0.15g甲基纤维素混合均匀，再加入4.2g浓度为8％的聚乙烯醇溶液(称取8g PVA溶于92mL去离子水中，用磁力搅拌子并置于95℃水浴锅中搅拌加热30min，使PVA完全溶解至透明澄清。所述聚乙烯醇为PVA1799)，充分搅拌得到均匀粘稠状浆料，将浆料转移到3D打印料筒中。导入STL格式的圆柱形模型文件，模型尺寸设为10×10×3mm，3D打印的喷头温度为25℃，平台温度为27℃，纤维直径为0.4mm，纤维间距设为800μm、打印层厚设为320μm、打印速度设为6mm/s、压力设为0.36MPa。将打印得到的支架常温干燥24h，60℃条件干燥24h，最后经过1150℃烧结分三阶段升温，升温速率为2℃/min，保温时间为2h，并以同样的升温速率降至室温。得到所述具有三维连通孔结构未复合的4mol.％硅离子掺杂的双相磷酸钙陶瓷支架，标记为Si-BCP。
实施例1
硅离子掺杂的双相磷酸钙粉体按以下方法制备：称取25.356g磷酸氢二铵和1.792mL正硅酸乙酯(溶于无水乙醇中)(P+Si为0.2mol)加入到1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成混合水溶液A；称取73.207g四水硝酸钙1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成硝酸钙溶液B；将混合水溶液A滴加到硝酸钙溶液B中，滴加过程中350r/min持续搅拌，搅拌均匀，得到反应溶液，同时用氨水调节反应溶液的pH值为8.68(±0.2)；滴加结束后继续搅拌60min，搅拌速率为350r/min，搅拌均匀。随后陈化24h、离心取沉淀，离心速率为4000r/min，每次离心5min，离心洗涤三次。随后进行72h的冷冻干燥，900℃低温煅烧2h，升温速率为5℃/min，得到4mol.％硅离子的β-磷酸三钙/羟基磷灰石比例为7:3双相磷酸钙粉体。按水料比为1:1的比例湿法球磨，其中大球小球的质量比为3:2，300r/min的速率下球磨2h，放入60℃烘干12h，将干燥后粉体过53μm的筛，得到粒度小于53μm的打印粉体。
锌离子掺杂的双相磷酸钙粉体按以下方法制备：称取26.412g(0.20mol)磷酸氢二铵加入1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成磷酸氢二铵溶液A；称取73.207g四水硝酸钙和2.305g六水硝酸锌混合溶解于1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成混合水溶液B；将磷酸氢二铵溶液A滴加到混合水溶液B中，滴加过程中350r/min持续搅拌，搅拌均匀，得到反应溶液，同时用氨水调节反应溶液的pH值为8.70(±0.2)；滴加结束后继续搅拌60min，搅拌速率为350r/min，搅拌均匀。随后陈化24h、离心取沉淀，离心速率为4000r/min，每次离心5min，离心洗涤三次。随后进行72h的冷冻干燥，900℃低温煅烧2h，升温速率为5℃/min，得到2.5mol.％锌离子的β-磷酸三钙/羟基磷灰石比例为7:3双相磷酸钙粉体。按水料比为1:1的比例湿法球磨，其中大球小球的质量比为3:2，300r/min的速率下球磨2h，放入60℃烘干12h，将干燥后粉体过53μm的筛，得到粒度小于53μm的打印粉体。
将所述得到的4mol.％硅离子掺杂的双相磷酸钙粉体与得到的2.5mol.％锌离子掺杂的双相磷酸钙粉体按质量百分比为1:1的比例进行复合，混合均匀。
具有三维连通孔结构的硅离子掺杂的双相磷酸钙复合锌掺杂离子的双相磷酸钙陶瓷支架的制备按如下方法制备：用搅拌器将5g的上述制备的硅离子掺杂并复合锌掺杂离子的BCP粉体，0.15g甲基纤维素混合均匀，再加入4.2g浓度为8％的聚乙烯醇溶液(称取8gPVA溶于92mL去离子水中，用磁力搅拌子并置于95℃水浴锅中搅拌加热30min，使PVA完全溶解至透明澄清。所述聚乙烯醇为PVA1799)，充分搅拌得到均匀粘稠状浆料，将浆料转移到3D打印料筒中。导入STL格式的圆柱形模型文件，模型尺寸设为10×10×3mm，3D打印的喷头温度为24℃、平台温度为27℃、纤维直径为0.4mm、填充间距设为800μm、打印层厚设为320μm、打印速度设为6mm/s、压力设为0.36MPa。将打印得到的支架常温干燥24h，60℃条件干燥24h，最后经过1150℃烧结分三阶段升温，升温速率为2℃/min，保温时间为2h，并以同样的升温速率降至室温。得到所述具有三维连通孔结构的硅离子掺杂的双相磷酸钙复合锌掺杂离子的双相磷酸钙陶瓷支架，标记为Si/Zn-BCP。
图1为对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙粉体X射线衍射谱图，可以看出，对比例1-2和实施例1粉体均为β-TCP(JCPDF NO.090169)和HA(JCPDF NO.090432)物相比约为7:3的双相磷酸钙粉体，无其他杂相。
图2为对比例1、2的未掺杂双相磷酸钙粉体和硅离子掺杂双相磷酸钙粉体扫描电子显微镜图。由SEM图可以看出制备的粉体含有两种不同形貌和大小不同的晶粒，粒度更大的呈棱形的是β-TCP晶粒、粒度小的呈六面体形貌的是HA晶粒，与XRD结果一致。
图3为对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙在高温烧结后陶瓷支架X射线衍射谱图。X射线衍射谱图，在高温烧结时，对比例2的Si-BCP组主物相为α-TCP，而未掺杂组及Si/Zn-BCP组，均为β-TCP和HA组成的双相磷酸钙陶瓷，说明4％mol的Si的掺杂在高温烧结下会导致磷酸钙晶格失稳，结构发生变化，向α-TCP转变；而复合了Zn的Si/Zn-BCP组，均为纯的β-TCP和HA组成的双相磷酸钙陶瓷，说明Zn2+的替代可以稳定晶格，促成α-TCP晶相的消失，转化成更稳定的无毒副作用的β-TCP。图5细胞增殖和图6的ALP活性的结果也表明，α-TCP的存在抑制小鼠骨髓间充质干细胞的增殖和ALP蛋白的活性，而复合了Zn的Si/Zn-BCP极大地改善了这一效果，甚至相较于对比例1，有显著的促进作用。
图4为对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙表面及断面的扫描电子显微镜结构图。图4中，从表面宏观图(a)可知，对比例1、2和实施例1的支架均有连通的近似350μm×350μm方形的宏观大孔结构，其孔范围在150-500μm之间，具有形成矿化骨的潜在条件；从表面微观图(b)可知，对比例1、2和实施例1的表面均存在不同程度的微孔，相较于对比例1，对比例2硅离子掺杂双相磷酸钙表面存在更多的微孔，微孔的存在有利于宿主内环境中生长因子的吸附，有利于成骨，但过多的孔隙同时也会造成支架机械性能的下降，因而适宜的孔隙结构是骨修复材料的关键。而复合了锌离子掺杂双相磷酸钙支架表面的孔数量有所减少，说明锌离子掺杂有利于双相磷酸钙陶瓷的烧结，可在一定程度上提高支架的力学强度；断面微观图(b)有类似的结果；从断面宏观图(c)可知，对比例1、2和实施例1的支架均有连通的近似400μm×150μm长方形的宏观大孔结构。
图5为小鼠骨髓间充质干细胞在对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙陶瓷支架表面培养1天、3天后的细胞增殖情况结果。对比例1、2和实施例1陶瓷支架经过超声、干燥后放入高温高压灭菌锅灭菌后(每组设置6个平行)，烘干后放入48孔板用基础培养基浸润24h后，支架表面按2×10 4cells/孔的密度接种细胞，分别于1、3天后用cck-8试剂盒检测细胞增殖情况。从图中可以看出，随着时间的增长，三组细胞数量均有明显的增长趋势，说明细胞呈正常生长态势，且具有连通的宏观大孔为细胞生长提供空间，有利于细胞的增殖。细胞培养3d后，对比例2组相较于对比例1组存在抑制效果，这可能是因为α-TCP的存在，使得细胞活性降低，实施例1的细胞增殖效果显著优于对比例1、对比例2，说明硅掺杂复合锌掺杂能很好地促进小鼠骨髓间充质干细胞的增殖。
图6小鼠骨髓间充质干细胞与对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙共培养7天和14天后的ALP活性结果。同上步骤灭菌、烘干、浸润后，支架表面按3×10 4cells/孔的密度接种细胞，隔天更换成骨诱导液，培养7d和14d后进行ALP活性检测和成骨分化相关基因表达情况的检测。图6显示，3组均有较好的ALP活性，而ALP活性和基质矿化紧密相关，微纳米孔结构的存在可以很好地吸附蛋白，为骨的矿化提供条件，说明微纳米孔结构能很好促进ALP活性。实施例1的7天、14天的ALP活性均显著优于对比例1、对比例2，说明硅掺杂复合锌掺杂能很好地促进小鼠骨髓间充质干细胞的ALP活性。
图7a、图7b、图7c分别为对比例1、2和实施例1未掺杂、未复合、及硅离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙陶瓷支架成骨分化相关基因表达情况结果(图7a、图7b、图7c分别是Col-I(Collagen-I)基因、Runx2(Runt-related transcription factor 2)基因、OCN(Osteocalcin)基因的结果图)。在成骨分化相关基因表达检测中，在与双相磷酸钙陶瓷支架共培养时，实施例1与对比例1、对比例2相比，细胞的Col-I、Runx2和OCN三种成骨分化相关基因的表达量都有一定幅度的上调，说明具有三维连通孔结构的硅离子掺杂的双相磷酸钙复合锌掺杂离子的双相磷酸钙陶瓷支架的促成骨性能得到了显著提高。
实施例2
镁离子掺杂的双相磷酸钙粉体按以下方法制备：称取26.412g磷酸氢二铵加入1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成磷酸氢二铵溶液A；称取73.207g硝酸钙和4.611g六水硝酸镁混合溶解于1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成混合水溶液B；将磷酸氢二铵溶液A滴加到混合水溶液B中，滴加过程中350r/min持续搅拌，搅拌均匀，得到反应溶液，同时用氨水调节反应溶液的pH值为8.68(±0.2)；滴加结束后继续搅拌60min，搅拌速率为350r/min，搅拌均匀。随后陈化24h、离心取沉淀，离心速率为4000r/min，每次离心5min，离心洗涤三次。随后进行72h的冷冻干燥，900℃低温煅烧2h，升温速率为5℃/min，得到5mol.％镁离子掺杂的β-磷酸三钙/羟基磷灰石比例为7:3双相磷酸钙粉体。按水料比为1：1的比例湿法球磨，其中大球小球的质量比为3：2，300r/min的速率下球磨2h，放入60℃烘干12h，将干燥后粉体过53μm的筛，得到粒度小于53μm的打印粉体。
锌离子掺杂的双相磷酸钙粉体按以下方法制备：称取26.412g(0.20mol)磷酸氢二铵加入1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成磷酸氢二铵溶液A；称取73.207g四水硝酸钙和2.305g六水硝酸锌溶解于1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成混合水溶液B；将磷酸氢二铵溶液A滴加到混合水溶液B中，滴加过程中350r/min持续搅拌，搅拌均匀，得到反应溶液，同时用氨水调节反应溶液的pH值为8.70(±0.2)；滴加结束后继续搅拌60min，搅拌速率为350r/min，搅拌均匀。随后陈化24h、离心取沉淀，离心速率为4000r/min，每次离心5min，离心洗涤三次。随后进行72h的冷冻干燥，900℃低温煅烧2h，升温速率为5℃/min，得到2.5mol.％锌离子的β-磷酸三钙/羟基磷灰石比例为7:3双相磷酸钙粉体。按水料比为1:1的比例湿法球磨，其中大球小球的质量比为3:2，300r/min的速率下球磨2h，放入60℃烘干12h，将干燥后粉体过53μm的筛，得到粒度小于53μm的打印粉体。
将所述得到的5mol.％镁离子掺杂的双相磷酸钙粉体与得到的2.5mol.％锌离子掺杂的双相磷酸钙粉体按质量百分比为4:1的比例进行复合，混合均匀。
具有三维连通孔结构的镁离子掺杂的双相磷酸钙复合锌掺杂离子的双相磷酸钙陶瓷支架的制备按如下方法制备：用搅拌器将5g的上述制备的镁离子掺杂并复合锌掺杂离子的BCP粉体，0.15g甲基纤维素混合均匀，再加入4.2g浓度为8％的聚乙烯醇溶液(称取8gPVA溶于92mL去离子水中，用磁力搅拌子并置于95℃水浴锅中搅拌加热30min，使PVA完全溶解至透明澄清。所述聚乙烯醇为PVA1799)，充分搅拌得到均匀粘稠状浆料，将浆料转移到3D打印料筒中。导入STL格式的圆柱形模型文件，模型尺寸设为10×10×3mm，3D打印的喷头温度为25℃、平台温度为27℃、纤维直径为0.4mm、填充间距设为800μm、打印层厚设为320μm、打印速度设为6mm/s、压力设为0.36MPa。将打印得到的支架常温干燥24h，60℃条件干燥24h，最后经过1100℃烧结分三阶段升温，升温速率为2℃/min，保温时间为2h，并以同样的升温速率降至室温。具有三维连通孔结构的镁离子掺杂的双相磷酸钙复合锌掺杂离子的双相磷酸钙陶瓷支架的制备，标记为Mg/Zn-BCP。
图8为对比例1未掺杂双相磷酸钙陶瓷支架与实施例2镁离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙陶瓷支架的抗压强度图。相较于对比例1双相磷酸钙陶瓷支架的抗压强度17.65MPa，实施例2镁离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙的抗压强度23.50MPa，提高了33％，说明镁离子掺杂复合锌离子能显著提高双相磷酸钙支架的抗压强度。
实施例3
锶离子掺杂的磷酸钙粉体按以下方法制备：称取26.412g磷酸氢二铵加入1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成磷酸氢二铵溶液A；称取45.924g硝酸钙和19.682g六水硝酸锶混合溶解于1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成混合水溶液B；将磷酸氢二铵溶液A滴加到混合水溶液B中，滴加过程中350r/min持续搅拌，搅拌均匀，得到反应溶液，同时用氨水调节反应溶液的pH值为8.68(±0.2)；滴加结束后继续搅拌60min，搅拌速率为350r/min，搅拌均匀。随后陈化24h、离心取沉淀，离心速率为4000r/min，每次离心5min，离心洗涤三次。随后进行72h的冷冻干燥，900℃低温煅烧2h，升温速率为5℃/min，得到30mol.％锶离子掺杂磷酸钙粉体。按水料比为1:1的比例湿法球磨，其中大球小球的质量比为3:2，300r/min的速率下球磨2h，放入60℃烘干12h，将干燥后粉体过53μm的筛，得到粒度小于53μm的打印粉体。
锌离子掺杂的双相磷酸钙粉体按以下方法制备：称取26.412g(0.20mol)磷酸氢二铵加入1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成磷酸氢二铵溶液A；称取73.207g四水硝酸钙和2.305g六水硝酸锌溶解于1000mL的去离子水中，用磁力搅拌器搅拌30min使其充分溶解混合均匀，转速为400r/min，配制成混合水溶液B；将磷酸氢二铵溶液A滴加到混合水溶液B中，滴加过程中350r/min持续搅拌，搅拌均匀，得到反应溶液，同时用氨水调节反应溶液的pH值为8.70(±0.2)；滴加结束后继续搅拌60min，搅拌速率为350r/min，搅拌均匀。随后陈化24h、离心取沉淀，离心速率为4000r/min，每次离心5min，离心洗涤三次。随后进行72h的冷冻干燥，900℃低温煅烧2h，升温速率为5℃/min，得到2.5mol.％锌离子的β-磷酸三钙/羟基磷灰石比例为7:3双相磷酸钙粉体。按水料比为1:1的比例湿法球磨，其中大球小球的质量比为3:2，300r/min的速率下球磨2h，放入60℃烘干12h，将干燥后粉体过53μm的筛，得到粒度小于53μm的打印粉体。
将所述得到的30mol.％锶掺杂磷酸钙粉体与得到的2.5mol.％锌离子掺杂的双相磷酸钙粉体按质量百分比为7:3的比例进行复合，混合均匀。
具有三维连通孔结构的锶离子掺杂磷酸钙复合锌离子掺杂的双相磷酸钙陶瓷支架的制备按如下方法制备：用搅拌器将5g的上述制备的锶离子掺杂并复合锌掺杂离子的BCP粉体，0.15g甲基纤维素混合均匀，再加入4.2g浓度为8％的聚乙烯醇溶液(称取8g PVA溶于92mL去离子水中，用磁力搅拌子并置于95℃水浴锅中搅拌加热30min，使PVA完全溶解至透明澄清。所述聚乙烯醇为PVA1799)，充分搅拌得到均匀粘稠状浆料，将浆料转移到3D打印料筒中。导入STL格式的圆柱形模型文件，模型尺寸设为10×10×3mm，3D打印的喷头温度为25℃、平台温度为27℃、纤维直径为0.4mm、填充间距设为800μm、打印层厚设为320μm、打印速度设为6mm/s、压力设为0.36MPa。将打印得到的支架常温干燥24h，60℃条件干燥24h，最后经过1100℃烧结分三阶段升温，升温速率为2℃/min，保温时间为2h，并以同样的升温速率降至室温。具有三维连通孔结构的锶离子掺杂磷酸钙复合锌离子掺杂的双相磷酸钙陶瓷支架，标记为Sr/Zn-BCP,。
图9为小鼠骨髓间充质干细胞与对比例1未掺杂和实施例3锶离子掺杂双相磷酸钙复合锌离子掺杂双相磷酸钙共培养7天和14天后的碱性磷酸酶(ALP)活性结果。图9表明，2组均有较好的ALP活性，说明微纳米孔结构能很好地促进ALP活性。实施例3的7天、14天的ALP活性均显著优于对比例1，说明锶掺杂复合锌掺杂能很好的促进小鼠骨髓间充质干细胞的ALP活性。
以上实施例仅为本发明较优的实施方式，仅用于解释本发明，而非限制本发明，本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。
Claims (10)
1.一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架的制备方法，其特征在于，包括以下步骤：
(1)将钙源、锌源、磷源与水搅拌混合均匀，得到反应溶液，调节所述反应溶液的pH值，陈化、多次离心洗涤得到沉淀，经冷冻干燥、低温煅烧，将煅烧得到的产物球磨、烘干、过筛，得到锌离子掺杂的双相磷酸钙粉体；
(2)将上述锌源替换成另一种活性离子源，按照步骤(1)相同操作，得到另一种活性离子掺杂的双相磷酸钙粉体；
(3)将步骤(1)得到的锌离子掺杂的双相磷酸钙粉体和步骤(2)得到的另一种离子掺杂的双相磷酸钙粉体复合，均匀混合，得到离子掺杂并复合的双相磷酸钙粉体；
(4)将步骤(3)所得到的粉体与增稠剂均匀混合，滴加粘结剂得到粘稠状打印浆料；将所述粘稠状打印浆料进行3D打印，得到支架素坯；
(5)放入烘箱干燥，再进行高温烧结，得到所述一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架。
2.根据权利要求1所述的制备方法，其特征在于，步骤(1)中，所述钙源为硝酸钙，其溶液浓度为0.20-0.55mol/L；所述磷源为磷酸氢二铵、磷酸二氢铵、磷酸氢二钠、磷酸二氢钠中的至少一种；所述钙源与磷源的摩尔体积比为(1.3-2.0):1；所述锌源为硝酸锌；步骤(2)中，所述另一种活性离子源为硝酸锶、硝酸镁、正硅酸乙酯中的至少一种；锌、镁、锶离子中任一种离子源与钙源的摩尔比为(0.02-0.5):1，硅离子按硅酸根方式掺杂，硅酸根与磷源的摩尔比为(0.01-0.20):1。
3.根据权利要求1所述的制备方法，其特征在于，步骤(1)中，用稀氨水溶液调节反应溶液的pH值为8.00-10.35，搅拌速率为200-500r/min，搅拌时间为60-120min，陈化的时间为18-30h，溶液的离心速率为4000-5000r/min，洗涤液为去离子水，所述冷冻干燥的时间为48-72h，所述煅烧的温度为850-900℃，煅烧的时间为2-4h。
4.根据权利要求1所述的制备方法，其特征在于，步骤(3)中，按水和粉体物料的质量比为(1-3):1的比例湿法球磨，其中大球小球的质量比为3:2，300r/min的速率下球磨2-3h，放入60℃烘干12h；将干燥后的粉体过53μm以下筛，即可得到待打印粉末样品。
5.根据权利要求1所述的制备方法，其特征在于，步骤(3)中，锌离子掺杂的双相磷酸钙粉体与另一种活性离子掺杂的双相磷酸钙粉体的质量百分比为(0.05-2):1。
6.根据权利要求1所述的制备方法，其特征在于，步骤(4)中，所述增稠剂为甲基纤维素，所述粘结剂为聚乙烯醇；离子掺杂并复合的双相磷酸钙粉体、甲基纤维素、聚乙烯醇按照质量比为100:(3-5):(80-110)的比例均匀混合30min-60min，制成粘稠状打印浆料。
7.根据权利要求1所述的制备方法，其特征在于，步骤(4)中，3D打印的纤维直径为0.4-0.6mm，纤维间距为0.6-1.2mm，打印支架直径为5-14mm，打印高度为6-15mm，打印喷头温度为5-30℃，3D打印的平台温度为5-30℃，3D打印的喷头气压为0.3-0.6MPa，3D打印的打印速率为5-20mm/s。
8.根据权利要求1所述的制备方法，其特征在于，步骤(5)所述干燥包括：先将支架素坯在常温条件下干燥24-48h，然后在60℃的条件下干燥24-48h；步骤(5)所述烧结温度为1050-1150℃，分三阶段升温，升温速率为2-10℃/min，保温时间为2-4h，并以同样的降温速率降至室温。
9.由权利要求1-8任一项所述的制备方法制得的一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架。
10.权利要求9所述的一种具有三维连通多级孔结构的离子掺杂双相磷酸钙复合陶瓷支架在制备非承重骨缺损修复填充材料中的应用。
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