CN116392638A - 载万古霉素多级孔人工骨支架模型的3d打印构建及制备方法 - Google Patents
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Abstract
本发明公开了一种载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，是通过3D打印聚己内酯（PCL）/β‑磷酸三钙（β‑TCP）制备外层多孔支架与冷冻干燥法制备的聚己内酯（PCL）/矿化牛源Ⅰ型胶原（MC）/盐酸万古霉素（VCM）内层多孔支架复合构建具有多级孔结构的人工骨支架用于感染性骨修复的一种制备方法（多级孔支架以下简称PT‑MCP‑V），属于生物医用材料领域。PCL/β‑TCP外层支架中β‑TCP的含量为60%，PCL/MC/VCM内层支架中MC和VCM含量分别为60%和5%；PCL/β‑TCP外层支架填充密度60%，实际填充走线距离400~500μm，大孔径有利于血管生成和骨组织长入，β‑TCP的加入提升了支架的成骨能力。本发明将3D打印方法和冷冻干燥方法结合构建复合PT‑MCP‑V人工骨支架，具有多级孔结构，力学强度高，可药物缓释等优点。
Description
技术领域
本发明涉及一种载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，具体涉及一种由3D打印聚己内酯（PCL）/β-磷酸三钙（β-TCP）外层多孔支架和冷冻干燥法制备的聚己内酯（PCL）/矿化牛源Ⅰ型胶原（MC）/万古霉素（VCM）内层多孔支架复合制备而成的多级孔支架人工骨模型的构建及制备方法，属于生物医用材料领域。
背景技术
骨缺损的修复和重建是骨科临床面临的问题之一。近年来，由于交通、工业的快速发展，同时先天性、感染、肿瘤等疾病的多发造成的骨损伤呈上升趋势，临床上常用的骨材料有自体骨、同种异体骨、异体骨，但仍存在各种各样的缺陷，如自体骨数量稀少且在手术过程中会造成较大的骨损伤；异体骨具有极大的排异性、移植难度大等问题。
为了克服移植带来的问题，人工骨材料一直备受关注，且人工骨材料的使用也呈逐年上升趋势，例如凝胶、钛合金、高聚物、硫酸钙、胶原基材料、生物活性玻璃等。然而，胶原基材料具有很好的应用前景，因为胶原是天然骨组织中关键的组成成分，能够促进早期矿化及支持新骨的生成，同时材料可被新骨替代。羟基磷灰石是天然骨组织的主要无机成分。在体外利用仿生原理，可以制备出与天然骨具有相似成分和分级结构的复合物。胶原在酸性溶液中溶解，通过调节pH，羟基磷灰石晶体在胶原分子表面沉积，生成矿化胶原。通过高分子材料与矿化胶原复合的方式来制备良好的骨修复支架，但是支架力学强度较低，孔结构可控性不易调节，可满足的骨修复部位力学强度要求范围有限（矿化胶原蛋白与生物陶瓷的复合物及其制造方法:CN201210040283.3[P]. 2012-08-15.）。
聚己内酯（PCL）具有无毒、良好的生物相容性、良好的载药性能和可降解性且降解产物可随人体代谢到外部而被美国FDA批准用于临床的高分子材料。PCL具有合适的力学强度，但表现出疏水性特性，将其与无机材料结合可有效改善疏水性能。此外，与矿化胶原结合，可有效改善单独矿化胶原的力学强度低、结构不可控的问题。为实现良好的抗感染性能，可负载VCM进行药物缓释，实现局部骨感染的治疗（Qiu X, Li S, Li X, et al.Experimental study of β-TCP scaffold loaded with VAN/PLGA microspheres in thetreatment of infectious bone defects[J]. Colloids and Surfaces B:Biointerfaces, 2022, 213: 112424.）。β-磷酸三钙（β-tricalcium phosphate, β-TCP），化学式为β-Ca3(PO4)2，属于六角晶系，β-TCP具有优异的力学强度、生物相容、骨传导与骨诱导作用（Bohner M, Santoni B L G, Döbelin N. β-tricalcium phosphate for bonesubstitution: Synthesis and properties[J]. Acta biomaterialia, 2020, 113: 23-41.），是骨修复材料中比较理想的生物陶瓷材料，但由于其孔隙率过低、脆性大，所以在使用过程中常采用与高分子聚合物混合的方式改善性能。
多孔支架在含有较大的孔径的同时包含微孔，这样可以促进骨向内生长，目前研究表明：大孔通常认为大小在100~1000μm之间，可以促进血管生成从而促进骨向内生长。此外，在体外成熟过程中通过大孔供应营养物质和去除代谢废物提高了支架在体内植入后的成功率。大孔中存在微孔，通常认为在10~100μm可促进毛细管力的增加和细胞相互作用和蛋白质吸附的表面积增加，导致物理效应，两者协同促进骨修复（Karageorgiou V, KaplanD. Porosity of 3D biomaterial scaffolds and osteogenesis[J]. Biomaterials,2005, 26(27): 5474-5491.）。熔融3D打印技术（FDM）可完成多孔支架对大孔的需求，同时由于其可个性化的特点，作为应用于组织工程的一项技术，展现出巨大的发展潜力。冷冻干燥作为常用的制造微孔技术，常用于骨修复支架制备。本发明将两种技术复合构建多级孔人工骨支架，构建满足大孔中存在微孔需求的支架，两者共同促进骨修复，具有极大的临床应用潜力。
发明内容
本发明提供了一种载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，多级孔人工骨支架PT-MCP-V具有优良的力学强度、药物缓释性能，且内外层材料均可降解，骨材料表面及内部被吸收后，可促进骨组织再生，最终为自体骨所代替。
本发明提供了一种载万古霉素多级孔人工骨支架模型，首先，通过三维建模软件构建目标支架的数字模型；然后，采用3D打印方式将聚己内酯（PCL）/β-磷酸三钙（β-TCP）复合丝通过逐层打印的方式由数字模型转换为目标外层多孔支架，构成相互交连的大孔（400~500μm）支架；此外，通过3D打印机配套软件可实现机器完成启动→暂停→启动这一功能。通过这一操作，得以将冷冻干燥制备的PCL/MC/VCM微孔（10~30μm）支架与PCL/β-TCP支架结合，构成PT-MCP-V多级孔人工骨支架。外层多孔支架提高了单独内层多孔支架的力学强度，减缓了单独内层多孔支架的药物释放速度。内层多孔支架PCL/MC/VCM中含有的牛源Ⅰ型胶原、羟基磷灰石模拟人骨中的主要成分，具有良好的生物活性并可在植入后促进骨再生，诱导骨细胞增殖的成分。同时，VCM可在感染部位进行局部释放防止骨感染的发生。
本发明中，外层多孔支架由50~60%质量含量的β-TCP复合丝用3D打印方式制备，β-TCP的加入使支架在生物体内会随着降解逐渐溶出钙磷离子，为新生组织提供原料进行原位固化形成新骨。通过3D打印的方式可实现孔径、孔隙率可调控，实现个性化定制支架。内层多孔支架为40~80%重量百分比的MC均匀分散在高分子聚合物PCL溶液中通过冷冻干燥制得。同时，内层支架负载VCM，植入到目标部位，实现局部药物释放，降低感染的同时降低全身用药的毒性，通过外层3D打印支架的作用可使单独内层PCL/MC/VCM支架的药物释放速度得到降低，得以使药物缓释。内层支架中VCM含量占内层支架重量的5%。PCL/β-TCP外层支架填充密度为60%，填充走线距离为400~500μm。
本发明提供了上述载万古霉素多级孔人工骨支架的3D打印构建及制备方法，其特征是由SolidWorks构建多级孔支架模型，3D打印PCL/β-TCP外层多孔支架，冷冻干燥制备PCL/MC/VCM内层多孔支架。具体地，本发明提供的3D打印构建方法为：
在构建PT-MCP-V多级孔人工骨支架模型的过程中使用3D打印方式构建外层结构并预留出内层支架的空间，步骤如下：
（1）采用SolidWorks三维建模软件，构建一个空心圆柱模型；
（2）使用3D打印切片软件对空心圆柱进行切片，设置打印参数：层高为0.3mm，打印速度为30 mm/s，打印温度为90℃，填充密度为60%，理论线间距0.66mm；
（3）将制得的PCL/β-TCP复合丝放入3D打印机中打印外层支架；
（4）当3D打印机按指令打印至指定位置时，暂停打印并置入制备的内层PCL/MC/VCM多孔支架，继续打印，得到PT-MCP-V多级孔人工骨复合支架。
进一步地，所述的内层PCL/MC/VCM支架占PT-MCP-V多级孔支架的体积百分比是12.4%。
进一步地，上述的3D打印构建方法具体描述如下：
（1）外层3D打印PCL/β-TCP支架填充密度60%，理论填充走线距离660μm，打印速度30mm/s，实际填充走线距离400~500μm；
（2）内层冷冻干燥制备PCL/MC/VCM支架孔径10~30μm，孔隙率为（57.94±0.50）%；PCL/MC/VCM（φ 10mm × H 10mm）支架压缩强度为（1.16±0.08）MPa，压缩模量为（1.27±0.06）MPa，20天累计药物释放百分比为（72.19±1.75）%；
（3）将上述内、外层支架复合构建PT-MCP-V多级孔人工骨支架，即通过SolidWorks构建的多级孔支架模型，将PCL/MC/VCM支架置入3D打印PCL/β-TCP支架，复合构建PT-MCP-V多级孔支架，即内层为孔径10~30μm的PCL/MC/VCM支架，外层为线间距400~500μm的PCL/β-TCP支架。PT-MCP-V（φ 20mm × H 20mm）多级孔支架孔隙率为（51.19±0.34）%，与单独内层支架相差7%，保持在50%以上；压缩强度（10.82±0.12）MPa，压缩模量（12.35±0.42）MPa，相较于单独的内层支架PCL/MC/VCM（φ 10mm × H 10mm），压缩强度提升了927%，压缩模量提升966%；20天累计药物释放百分比为（70.14±1.94）%。
进一步地，采用SolidWorks建模软件构建一个外层尺寸为：直径（φ）为2x mm、高（H）为 2x mm，内层尺寸为：直径（φ）为 x mm 、高（H）为 x mm的空心圆柱。为了保证3D打印支架的精度，上述尺寸中5≤x≤10。
当3D打印机按指令打印至指定位置时（即打印高度为1.5x mm处）暂停打印将直径为x mm、高为 x mm的内层PCL/MC/VCM多孔支架放入预留孔，随后进行续打，得到PT-MCP-V多级孔人工骨支架。将制备好的支架进行灭菌（15kGy的钴-60）后保存待用。
本发明提供了上述PCL/β-TCP复合丝的制备方法，具体包括以下步骤：
（1）沉淀法制备β-TCP：按照摩尔比Ca/P=0.43向去离子水中添加磷酸和碳酸钙混合均匀（保持磷酸的浓度为0.3M，碳酸钙浓度为0.13M），经过充分反应90min后，得到磷酸/碳酸钙溶液；使用分液漏斗将上述磷酸/碳酸钙溶液缓慢滴入0.13M的氢氧化钙溶液，所得混合溶液中摩尔比Ca/P=0.86，充分反应4h后，向溶液中添加碳酸钙粉末，使所得溶液中摩尔比Ca/P=1.5，过夜反应后静置5h，然后将沉淀进行离心15min后放入球磨机中球磨2h，取球磨后产物继续离心15min，放入80℃的真空干燥箱中充分干燥，将干燥后的产物经过研磨后放入马弗炉于1200℃烧结，取冷却后产物研磨并过200目筛子（保持粒径在74μm以下），得到粒径均匀的β-TCP；
（2）PCL/β-TCP复合丝的制备：将PCL在1，4-二氧六环中于常温下制备并充分搅拌均匀，保持浓度为10%（w/v）；加入β-TCP后继续搅拌，得到混合均匀的PCL/β-TCP溶液；然后在室温下将该溶液在大量水中沉淀以去除溶剂。将沉淀物置于-30℃~-20℃冰箱预冻2h后，放置在-96℃冷冻干燥机中进一步去除有机残留和水分。将冻干后的PCL/β-TCP复合材料切割成直径（5±0.5）mm的颗粒，将切割后的PCL/β-TCP颗粒在螺杆挤出机中加热，加热温度为70℃，挤出直径为（1.75±0.05）mm的PCL/β-TCP复合丝。
本发明提供了上述内层PCL/MC/VCM多孔支架的制备方法，具体包括以下步骤：
（1）矿化牛源Ⅰ型胶原的制备：牛源Ⅰ型胶原凝胶（Ⅰ型牛胶原固含量为1.36%，分子量270~300KDa）溶解在0.5M的乙酸溶液中，配置成0.001g/mL的胶原溶液，按0.12mol钙离子/克胶原凝胶的量向胶原溶液中加入含钙离子水溶液，得到钙/胶原复合溶液，以摩尔比Ca/P=1.66向所得钙/胶原溶液中滴加0.5M的磷酸溶液，得到含钙/磷/胶原复合溶液，搅拌2h后，将钙/磷/胶原溶液调节pH值保持8，继续搅拌12h后取出静置24h，倒去上清液，获得沉淀，将沉淀物加5-10mL的去离子水重复离心5~7次，直到上清液pH=7；将MC在-30℃~-20℃下预冻2h，在-96℃的冷冻干燥机中完全干燥，将冷冻干燥后的MC用研钵研磨并过100目筛子（保持粒径在150μm以下），得到粒径均匀的MC粉末，置于4℃冰箱中冷藏备用。
（2）内层PCL/MC/VCM多孔支架的制备：使用溶剂1，4-二氧六环溶解PCL，配置溶液浓度为0.1g/ml的PCL溶液，常温下进行搅拌溶解，向完全溶解后的PCL溶液中添加MC和VCM，得到PCL/MC/VCM混合溶液，保持溶液整体浓度为25%（w/v），所述的MC和VCM在PCL/MC/VCM中的质量百分比分别为60%和5%，在常温下充分搅拌。然后，在常温下将PCL/MC/VCM混合溶液倒入提前预冻的聚四氟乙烯模具中并在-30℃~-20℃进行预冻2h后，将预冻后的PCL/MC/VCM支架于-96℃冷冻干燥机中进行冻干去除有机溶剂，得到内层PCL/MC/VCM多孔支架。
本发明的有益效果：
（1）采用SolidWorks构建多级孔人工骨支架模型，通过将3D打印PCL/β-TCP外层支架与冷冻干燥PCL/MC/VCM内层支架复合制备，形成内外层不同孔径的多级孔支架，协同促进骨组织再生。
（2）PT-MCP-V（φ 20mm × H 20mm）多级孔支架相对于单独PCL/MC/VCM（φ 10mm× H 10mm）内层支架压缩强度从1.16MPa提高到10.82MPa，使得多级孔支架符合更广泛的人松质骨力学范畴；
（3）多级孔支架的孔隙率保持在50%以上，外层支架的加入使得PT-MCP-V支架孔隙率降低，一定程度上减缓了药物的释放速度，外层支架具有400~500μm的大孔径，内层具有10~30μm的微孔，符合骨修复对孔隙率和孔径的需求；
（4）内、外层支架均具有促进MC3T3-E1(小鼠胚胎成骨细胞前体细胞)增殖的效果，具有良好的生物相容性。
附图说明
图1为本发明复合构建PT-MCP-V多级孔人工骨支架示意图。
图2为实施例1中PCL/β-TCP复合丝（a, d）、PCL/β-TCP支架（b, e）及PCL/MC/VCM（c, f）支架微观形貌。a，b，c为低倍数，d，e，f为高倍数。
图3为实施例1中PCL/MC/VCM支架抗菌实验示意图，未载药（a）与载药（b）。
图4为产品的释放曲线；其中a为VCM标准释放曲线，b为实施例1中PT-MCP-V多级孔支架药物释放曲线，c为实施例2中PT-MCP-V多级孔支架药物释放曲线。
图5为实施例1中PCL/MC/VCM支架和实施例1和2中PT-MCP-V支架孔隙率。
图6为实施例1中PCL/MC/VCM支架和实施例1和2中PT-MCP-V支架应力应变（a）、压缩强度（b）及压缩模量（c）示意图。
图7为实施例1和2中CCK-8检测PCL/MC/VCM支架和PCL/β-TCP支架细胞增殖。
实施方式
下面通过实施例来进一步说明本发明，但不局限于以下实施例。
实施例
沉淀法制备β-TCP：将29.98g磷酸溶入1000ml纯水中，称取13.13g碳酸钙均匀加入磷酸溶液中，充分反应90min，得到磷酸/碳酸钙溶液，称取10.13g氢氧化钙融入1000ml纯水中，用分液漏斗将磷酸/碳酸钙溶液缓慢滴加入氢氧化钙溶液中，4h后向溶液中加入13.13g的碳酸钙粉末，过夜反应后静置5h，将沉淀物进行离心15min后放入球磨机中球磨2h，取球磨后产物继续离心15min，放入80℃真空干燥箱干燥24h，研磨干燥前驱体放入马弗炉于1200℃烧结5.5h，取冷却后产物研磨过200目筛子（保持粒径在74μm以下），得到β-TCP粉末。
PCL/β-TCP复合丝制备：将PCL溶解在1，4-二氧六环中，浓度为10%（w/v），常温下搅拌2h。加入β-TCP后继续搅拌2h，得到混合均匀的PCL/β-TCP溶液。然后在室温下将该溶液在大量水中沉淀以去除溶剂。将沉淀物置于-30℃~-20℃冰箱预冻2h后，放置在-96℃冷冻干燥机中进一步去除有机残留和水分。将冻干后的PCL/β-TCP复合材料切割成直径约5 mm的颗粒，将切割后的PCL/β-TCP颗粒在螺杆挤出机中加热，加热温度为70℃，挤出直径为（1.75±0.05）mm 的复合丝。
矿化牛源Ⅰ型胶原材料制备：将5.51g牛源Ⅰ型胶原凝胶（Ⅰ型牛胶原固含量为1.36%，分子量270~300KDa）溶解在75ml的0.0005mol/mL的乙酸溶液中，配置成0.001g/mL的胶原溶液，按0.12摩尔钙离子/克胶原凝胶的量向胶原溶液中加入含钙离子水溶液，得到钙/胶原复合溶液，以Ga/P=1.66向所得钙/胶原溶液中滴加含有磷酸根溶液，得到含钙/磷/胶原复合溶液，搅拌2h后，将钙/磷/胶原溶液调节pH值保持8，继续搅拌12h后取出静置24h，倒去上清液，获得沉淀，将沉淀物加纯化水重复离心7次，直到上清液pH=7。将矿化胶原在-20℃~-30℃下预冻2h, 在-96℃下进行冷冻干燥24h，将冷冻干燥后的MC用研钵研磨并过100目筛子（保持粒径在150μm以下），得到MC粉末并置于4℃冰箱中冷藏备用。
PCL/MC/VCM多孔支架制备：使用溶剂1，4-二氧六环溶解PCL，配置溶液浓度为0.1g/ml的PCL溶液，常温下搅拌2h，向完全溶解后的PCL溶液中添加MC和VCM，得到PCL/MC/VCM混合溶液，所述的MC和VCM含量分别为溶质总重量的60%和5%，在常温下搅拌1h。在常温下将PCL/MC/VCM混合溶液倒入提前预冻的聚四氟乙烯模具中并在-30℃~-20℃进行预冻2h，将预冻后的PCL/MC/VCM支架于-96℃冷冻干燥机中进行冻干去除有机溶剂，24h后取出得到内层PCL/MC/VCM支架。
3D打印PT-MCP-V多级孔支架：采用Solid Works建模软件构建一个外层尺寸为φ20mm × H 20mm，内层尺寸为φ 10mm × H 10mm的空心圆柱。 如图1所示，外层支架是空心的，空心部分用于放置内层支架，通过3D打印将数字模型转化成实物。
将建立的模型导入3D打印软件进行切片，3D打印机按切片指令打印至指定位置时（即打印高度15mm处），暂停打印，置入尺寸为φ 10mm × H 10mm的PCL/MC/VCM多孔支架，，随后进行续打，将制备完成的PT-MCP-V（φ 20mm × H 20mm）多级孔支架进行灭菌（15kGy的钴-60）后待用。
如图2（a）为PCL/β-TCP复合丝的电镜图，采用image j软件进行测量得到复合丝直径为1.73mm，符合（1.75±0.05）mm的误差范围，图2（d）为复合丝的放大图，红色箭头所指为PCL中包裹的β-TCP颗粒；图2（b）（c）所示，采用image j软件进行测量得到外层PCL/β-TCP支架孔径为433μm，内层PCL/MC/VCM支架孔径为10μm~30μm，图2（e, f）为高倍数下的PCL/β-TCP支架和PCL/MC/VCM支架表明形貌；
图3为对PCL/MC/VCM（φ 10mm × H 10mm）进行抗菌实验，可以看出添加的VCM的内层支架出现了明显的抑菌圈，依据对葡萄球菌的抑菌圈直径解释标准中VCM的CLSI标准，支架的抑菌圈直径大于15mm，属于极度敏感的范围。
实施例
β-TCP、PCL/β-TCP、MC、PCL/MC/VCM制备步骤同例1。
3D打印PT-MCP-V多级孔支架：采用SolidWorks建模软件构建一个外层尺寸为φ10mm × H 10mm，内层尺寸为φ 5mm × H 5mm的空心圆柱。
将建立的模型导入3D打印软件进行切片，3D打印机按切片指令打印至指定位置时（即打印高度7.5mm处），暂停打印，置入尺寸为φ 5mm × H 5mm的PCL/MC/VCM多孔支架，随后进行续打，将制备完成的PT-MCP-V（φ 10mm × H 10mm）多级孔人工骨支架进行灭菌（15kGy的钴-60）后待用。
支架的药物释放曲线见图4，如图4（a）所示为在计算药物释放百分比前，通过Origin软件进行拟合绘制VCM的标准释放曲线，得到拟合公式为y=-0.01674+5.00357x，R2=0.99458。通过实验紫外分光光度计测量20天的药物释放实验评估PCL/MC/VCM支架和PT-MCP-V支架的药物释放性能。
图4（b）是将实施例1中的PT-MCP-V（φ 20mm × H 20mm）和PCL/MC/VCM（φ 10mm× H 10mm）支架置于PBS中20天累计药物释放百分比数据图，PT-MCP-V在20天内累计药物释放百分比为(70.1±1.94)%，而单独的PCL/MC/VCM 支架达到了(72.2±1.75)%。在药物释放到第6天时，两组支架累计药物释放百分比差值达到最大值10%；
如图4（c）所示，将PT-MCP-V支架置于PBS中8天药物释放百分比为(77.2±1.83)%。而单独的PCL/MC/VCM（φ 5mm × H 5mm）在第8天药物释放百分比为(91.9±2.47)%。在药物释放到第2天时，两组支架累计药物释放百分比差值达到最大值38%，极大的减缓了目标部位的药物浓度；
支架的孔隙率见图5，基于阿基米德原理，利用液体置换法测得实施例1中PT-MCP-V（φ 20mm × H 20mm）多级孔支架孔隙率为（51.2±0.34）%，符合人骨修复对孔隙率的要求；实施例2中PT-MCP-V（φ 10mm × H 10mm）的孔隙率为（50.64±0.61）%，符合人骨修复对孔隙率的要求；
力学性能见图6所示，图6（a）中压缩曲线表明，实施例1和例2中PT-MCP-V（φ 20mm× H 20mm）和PT-MCP-V（φ 10mm × H 10mm）支架的抗压强度相较于PCL/MC/VCM（φ 10mm× H 10mm）支架的抗压强度得到了明显的提升，而PCL/MC/VCM（φ 10mm × H 10mm）支架在应变为0.5时结构便发生了破坏而无法继续被压缩，得到的抗压强度无法满足人骨修复对力学的需求。
图6（b）（c）反映了支架的力学性能，通过测试：实施例1中PT-MCP-V（φ 20mm × H20mm）抗压强度可达到（10.8±0.12）MPa，压缩模量（12.35±0.42）MPa，相较于单独的PCL/MC/VCM（φ 10mm × H 10mm），压缩强度提升了927%，压缩模量提升966%；实施例2中PT-MCP-V（φ 10mm × H 10mm）的抗压强度可达到（9.0±0.33）MPa，压缩模量达（7.7±0.40）MPa，相较于PCL/MC/VCM（φ 10mm × H 10mm ）支架压缩强度提升了774%，压缩模量提升608%；
CCK-8检测结果见图7，随着培养时间的延长支架的吸光度在不断增大，表明支架可促进MC3T3-E1(小鼠胚胎成骨细胞前体细胞)增殖，具有良好的生物活性。
本发明通过3D打印和冷冻干燥方式结合构建的多级孔人工骨支架符合人松质骨力学范畴；多级孔人工骨支架的孔隙率大于50%，满足骨修复对孔隙率要求；多级孔人工骨支架的构建有效减缓了内层支架的药物释放速度；细胞在内外层支架上均产生了明显增殖，具有良好的生物相容性。在个性化定制骨修复阶段可以通过调整3D打印的填充率来调节多级孔支架的孔隙率、打印孔径以及力学强度，从而制备符合临床需求的人工骨支架，具有广阔的临床应用前景。
Claims (10)
1.一种载万古霉素多级孔人工骨支架模型，其特征在于：采用SolidWorks构建多级孔人工骨支架模型，通过将3D打印的PCL/β-TCP外层支架与冷冻干燥的PCL/MC/VCM内层支架复合制备，形成内外层不同孔径的多级孔支架，
外层支架中含有50~60%质量含量的β-TCP复合丝，β-TCP的加入使支架在生物体内会随着降解逐渐溶出钙磷离子，为新生组织提供原料进行原位固化形成新骨；内层支架为40~80%重量百分比的MC均匀分散在高分子聚合物PCL溶液中通过冷冻干燥制得，且内层支架负载VCM，植入到目标部位，内层支架中VCM含量占内层支架重量的5%。
2.根据权利要求1所述的载万古霉素多级孔人工骨支架模型，其特征在于：PCL/β-TCP外层支架填充密度为60%，实际填充走线距离为400~500μm；
外层支架尺寸为：直径为2x mm、高为 2x mm，内层支架尺寸为：直径为 x mm 、高为 xmm的空心圆柱，其中5≤x≤10。
3.一种权利要求1或2所述的载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，其特征在于：在构建PT-MCP-V多级孔人工骨支架模型的过程中使用3D打印方式构建外层结构并预留出内层支架的空间，步骤如下：
（1）采用SolidWorks三维建模软件，构建一个空心圆柱模型；
（2）使用3D打印切片软件对空心圆柱进行切片；
（3）将制得的PCL/β-TCP复合丝放入3D打印机中打印外层支架；
（4）当3D打印机按指令打印至指定位置时，暂停打印并置入制备的内层PCL/MC/VCM多孔支架，继续打印，得到PT-MCP-V多级孔人工骨复合支架。
4.根据权利要求3所述的载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，其特征在于：
外层3D打印PCL/β-TCP支架设置打印参数为：层高为0.3mm，打印速度为30 mm/s，打印温度为90℃，填充密度为60%，实际填充走线距离400~500μm。
5.根据权利要求3所述的载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，其特征在于：
内层冷冻干燥制备PCL/MC/VCM支架孔径10~30μm，孔隙率为（57.94±0.50）%；PCL/MC/VCM支架压缩强度为（1.16±0.08）MPa，压缩模量为（1.27±0.06）MPa，20天累计药物释放百分比为（72.19±1.75）%。
6.根据权利要求3所述的载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，其特征在于：
所得PT-MCP-V多级孔支架孔隙率为（51.19±0.34）%，保持在50%以上；压缩强度为（10.82±0.12）MPa，压缩模量为（12.35±0.42）MPa；20天累计药物释放百分比为（70.14±1.94）%。
7.根据权利要求3所述的载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，其特征在于：所述的内层PCL/MC/VCM支架占PT-MCP-V多级孔支架的体积百分比是12.4%。
8.根据权利要求3所述的载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，其特征在于：PCL/β-TCP复合丝的制备方法，包括以下步骤：
（1）沉淀法制备β-TCP：按照摩尔比Ca/P=0.43向去离子水中添加磷酸和碳酸钙混合均匀，溶液中磷酸的浓度为0.3M，碳酸钙浓度为0.13M，经过充分反应90min后，得到磷酸/碳酸钙溶液；使用分液漏斗将上述磷酸/碳酸钙溶液缓慢滴入0.13M的氢氧化钙溶液，所得混合溶液中摩尔比Ca/P=0.86，充分反应4h后，向溶液中添加碳酸钙粉末，使所得溶液中摩尔比Ca/P=1.5，过夜反应后静置5h，然后将沉淀进行离心15min后放入球磨机中球磨2h，取球磨后产物继续离心15min，放入80℃的真空干燥箱中充分干燥，将干燥后的产物经过研磨后放入马弗炉于1200℃烧结，取冷却后产物研磨并过200目筛子，得到粒径均匀的β-TCP；
（2）PCL/β-TCP复合丝的制备：将PCL在1，4-二氧六环中于常温下制备并充分搅拌均匀，保持质量体积浓度为10%；加入β-TCP后继续搅拌，得到混合均匀的PCL/β-TCP溶液；然后在室温下将该溶液在大量水中沉淀以去除溶剂；将沉淀物置于-30℃~-20℃冰箱预冻2h后，放置在-96℃冷冻干燥机中进一步去除有机残留和水分；将冻干后的PCL/β-TCP复合材料切割成直径（5±0.5）mm的颗粒，将切割后的PCL/β-TCP颗粒在螺杆挤出机中加热，加热温度为70℃，挤出直径为（1.75±0.05）mm的PCL/β-TCP复合丝。
9.根据权利要求3所述的载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，其特征在于：内层PCL/MC/VCM多孔支架的制备方法，包括以下步骤：
（1）矿化牛源Ⅰ型胶原的制备：牛源Ⅰ型胶原凝胶溶解在0.5M的乙酸溶液中，配置成0.001g/mL的胶原溶液，按0.12mol钙离子/克胶原凝胶的量向胶原溶液中加入含钙离子水溶液，得到钙/胶原复合溶液，以摩尔比Ca/P=1.66向所得钙/胶原溶液中滴加0.5M的磷酸溶液，得到含钙/磷/胶原复合溶液，搅拌2h后，将钙/磷/胶原溶液调节pH值保持8，继续搅拌12h后取出静置24h，倒去上清液，获得沉淀，将沉淀物加5-10mL的去离子水重复离心5~7次，直到上清液pH=7；将MC在-30℃~-20℃下预冻2h，在-96℃的冷冻干燥机中完全干燥，将冷冻干燥后的MC用研钵研磨并过100目筛子，得到粒径均匀的MC粉末，置于4℃冰箱中冷藏备用；
（2）内层PCL/MC/VCM多孔支架的制备：使用溶剂1，4-二氧六环溶解PCL，配置溶液浓度为0.1g/ml的PCL溶液，常温下进行搅拌溶解，向完全溶解后的PCL溶液中添加MC和VCM，得到PCL/MC/VCM混合溶液，保持溶液质量体积浓度为25%，在常温下充分搅拌；然后，在常温下将PCL/MC/VCM混合溶液倒入提前预冻的聚四氟乙烯模具中并在-30℃~-20℃进行预冻2h，将预冻后的PCL/MC/VCM支架于-96℃冷冻干燥机中进行冻干去除有机溶剂，得到内层PCL/MC/VCM多孔支架。
10.根据权利要求9所述的载万古霉素多级孔人工骨支架模型的3D打印构建及制备方法，其特征在于：牛源Ⅰ型胶原凝胶中Ⅰ型牛胶原固含量为1.36%，分子量为270~300KDa。
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