CN108567992A - 一种用于脊椎损伤血管快速修复的3d打印生物墨水及其制备方法 - Google Patents

一种用于脊椎损伤血管快速修复的3d打印生物墨水及其制备方法 Download PDF

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CN108567992A
CN108567992A CN201810235786.3A CN201810235786A CN108567992A CN 108567992 A CN108567992 A CN 108567992A CN 201810235786 A CN201810235786 A CN 201810235786A CN 108567992 A CN108567992 A CN 108567992A
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薛巍
宋镕光
阮淼亮
戴箭
王永周
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Jinan University
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Abstract

本发明公开一种用于脊椎损伤血管快速修复的3D打印生物墨水及其制备方法，涉及生物3D打印领域。该方法包括壳材料墨水的制备、内层细胞层墨水的制备、3D打印制备等步骤。本发明的生物墨水通过使用同轴方法同时打印2～4种材料，外层材料为内层细胞提供有效保护，避免打印时细胞沉降的问题，同时实现细胞种类、密度和分布可控，加入生长因子等促进脊髓损伤血管的快速修复，外层提供良好的力学性能，内层则更加有利于细胞的生长繁殖。本发明的3D打印生物墨水弥补了现有生物墨水的不足，将生物3D打印更好地应用于脊椎损伤血管的修复。

Description

一种用于脊椎损伤血管快速修复的3D打印生物墨水及其制备 方法
技术领域
本发明涉及生物3D打印领域，尤其是一种用于脊椎损伤血管快速修复的3D打印生物墨水及其制备方法。
背景技术
生物3D打印技术在解决医学难题方面做出突出贡献，在细胞组织培养、组织工程领域的探究正在不断深入，然而生物墨水是打印的基础，其开发需求迫在眉睫。Armstrong,J.P.等通过在4℃下将泊洛沙姆F127、海藻酸盐、DMEM混合均匀，随后进行细胞封装，开发出可用于软骨打印的生物墨水。Armstrong,J.P.,et al."3D Bioprinting Using aTemplated Porous Bioink."Advanced Healthcare Materials.14,5,2016.专利CN201610269756.5通过生物大分子猪、牛或羊真皮源性细胞外基质纳米纤维微粉加入交联剂、促凝剂从而制备出可用于软组织支架的生物墨水。
脊椎损伤经常伴随着血管损伤，血管的修复对脊椎损伤的修复尤为重要，上述生物墨水在3D打印快速血管修复方面不能很好的解决这一问题。
发明内容
为了克服现有技术的缺点与不足，本发明的首要目的在于提供一种用于脊椎损伤血管快速修复的3D打印生物墨水的制备方法。
现有的生物打印喷头只能打印一种材料，难以兼顾力学强度和细胞生长环境等因素，同时还会产生因沉降导致的细胞密度以及种类空间不可控，在脊椎损伤血管修复上不能很好的应用。
本发明的另一目的在于提供通过上述制备方法制备得到的用于脊椎损伤血管快速修复的3D打印生物墨水。
本发明的目的通过下述技术方案实现：
一种用于脊椎损伤血管快速修复的3D打印生物墨水的制备方法，包括如下步骤：
(1)壳材料墨水的制备
以去离子水为溶剂，将明胶2％～6％与海藻酸钠1％～5％磁力搅拌均匀；加入生长因子VEGF 50～200ng/mL与抗菌消炎药物50～100μg/mL磁力搅拌均匀，紫外灭菌12～24h待用；
(2)内层细胞层墨水的制备
37±1℃温度下，将骨髓间充质干细胞(BMSC)1～3×106/mL、血管内皮细胞1～3×106/mL、平滑肌成纤维细胞1～3×106/mL、血管内皮生长因子(VEGF)20～200ng/mL、血小板生长因子(PDGF)20～200ng/mL与嗜碱性成纤维细胞生长因子(bFGF)20～200ng/mL加入至完全培养基DMEM中混合均匀；
(3)3D打印制备
采用同轴喷头，实现壳材料墨水和内层细胞层墨水的同步打印；打印机采用挤出打印设备，打印机壳材料墨水温度设定为37±1℃、打印速度为15mm/s～25mm/s；内层设置为37±1℃、打印速度5mm/s～10mm/s。
优选的，步骤(1)中所述的海藻酸钠的浓度为2％～5％。
优选的，步骤(1)中所述的生长因子VEGF的用量为50～100ng/mL。
优选的，步骤(1)中所述的抗菌消炎药物为链霉素、青霉素等。
优选的，步骤(2)中所述的血管内皮生长因子(VEGF)的用量为30～120ng/mL。
优选的，步骤(2)中所述的血小板生长因子(PDGF)的用量为30～120ng/mL。
优选的，步骤(2)中所述的嗜碱性成纤维细胞生长因子(bFGF)的用量为30～120ng/mL。
一种用于脊椎损伤血管快速修复的3D打印生物墨水，通过上述制备方法制备得到。
本发明的机理是：
生物3D打印技术在解决临床医学难题方面做出的贡献越来越大，在细胞培养、组织工程领域的探究不断深入，然而生物墨水是打印的基础，其开发需求迫在眉睫。脊椎损伤经常伴随着血管损伤，血管的修复对脊椎损伤的修复尤为重要，目前的生物墨水在3D打印快速血管修复方面不能很好的解决这一问题。针对现有技术的不足，本发明致力于一种用于脊椎损伤血管快速修复的3D打印生物墨水，弥补现有生物墨水的不足，将生物3D打印更好地应用于脊椎损伤血管的修复。
本发明相对于现有技术具有如下的优点及效果：
相比于传统的生物墨水，本发明的生物墨水通过使用同轴方法同时打印2～4种材料，外层材料为内层细胞提供有效保护，避免打印时细胞沉降的问题，同时实现细胞种类、密度和分布可控，加入生长因子等促进脊髓损伤血管的快速修复，外层提供良好的力学性能，内层则更加有利于细胞的生长繁殖。
附图说明
图1是采用本发明的3D打印生物墨水进行3D打印的同轴喷头示意图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
采用本发明的3D打印生物墨水进行3D打印的同轴喷头示意图，如图1所示。
实施例1
(1)壳材料墨水的制备
以去离子水为溶剂，将明胶2％与海藻酸钠5％磁力搅拌均匀；加入生长因子VEGF50ng/mL与抗菌消炎药物链霉素100μg/mL磁力搅拌均匀，紫外灭菌12h待用。
(2)内层细胞层墨水的制备
37℃温度下，将骨髓间充质干细胞(BMSC)1×106/mL、血管内皮细胞1×106/mL、平滑肌成纤维细胞1×106/mL、血管内皮生长因子(VEGF)30ng/mL、血小板生长因子(PDGF)30ng/mL与嗜碱性成纤维细胞生长因子(bFGF)30ng/mL加入至完全培养基DMEM中，混合均匀。
(3)打印制备
采用同轴喷头实现壳层材料和内层细胞的同步打印。打印机采用挤出打印设备，打印机壳层材料温度设定为37℃、打印速度为15mm/s；内层设置为37℃、打印速度5mm/s。
实施例2
(1)壳材料墨水的制备
以去离子水为溶剂，将6％明胶与2％海藻酸钠磁力搅拌均匀；加入生长因子VEGF100ng/mL与抗菌消炎药物链霉素50μg/mL磁力搅拌均匀，紫外灭菌16h待用。
(2)内层细胞层墨水的制备
37℃温度下，将骨髓间充质干细胞(BMSC)3×106/mL、血管内皮细胞3×106/mL、平滑肌成纤维细胞3×106/mL、血管内皮生长因子(VEGF)50ng/mL、血小板生长因子(PDGF)50ng/mL与嗜碱性成纤维细胞生长因子(bFGF)50ng/mL加入至完全培养基DMEM中，混合均匀。
(3)打印制备
采用同轴喷头，实现壳层材料和内层细胞的同步打印。打印机采用挤出打印设备，打印机壳层材料温度设定为37℃、打印速度为20mm/s；内层设置为37℃、打印速度10mm/s。
实施例3
(1)壳材料墨水的制备
以去离子水为溶剂，将5％明胶与2％海藻酸钠磁力搅拌均匀；加入生长因子VEGF60ng/mL与抗菌消炎药物链霉素70μg/mL磁力搅拌均匀，紫外灭菌18h待用。
(2)内层细胞层墨水的制备
37℃温度下，将骨髓间充质干细胞(BMSC)2×106/mL、血管内皮细胞2×106/mL、平滑肌成纤维细胞2×106/mL、血管内皮生长因子(VEGF)100ng/mL、血小板生长因子(PDGF)100ng/mL与嗜碱性成纤维细胞生长因子(bFGF)100ng/mL加入至完全培养基DMEM中，混合均匀。
(3)打印制备
采用同轴喷头，实现壳层材料和内层细胞的同步打印。打印机采用挤出打印设备，打印机壳层材料温度设定为37℃、打印速度为25mm/s；内层设置为37℃、打印速度10mm/s。
实施例4
(1)壳材料墨水的制备
以去离子水为溶剂，将3％明胶与3％海藻酸钠磁力搅拌均匀；加入生长因子VEGF70ng/mL与抗菌消炎药物链霉素80μg/mL磁力搅拌均匀，紫外灭菌20h待用。
(2)内层细胞层墨水的制备
37℃温度下，将骨髓间充质干细胞(BMSC)1×106/mL、血管内皮细胞2×106/mL、平滑肌成纤维细胞3×106/mL、血管内皮生长因子(VEGF)40ng/mL、血小板生长因子(PDGF)40ng/mL与嗜碱性成纤维细胞生长因子(bFGF)40ng/mL加入至完全培养基DMEM中，混合均匀。
(3)打印制备
采用同轴喷头，实现壳层材料和内层细胞的同步打印。打印机采用挤出打印设备，打印机壳层材料温度设定为37℃、打印速度为17mm/s；内层设置为37℃、打印速度8mm/s。
实施例5
(1)壳材料墨水的制备
以去离子水为溶剂，将3％明胶与2％海藻酸钠磁力搅拌均匀；加入生长因子VEGF浓度为80ng/mL与抗菌消炎药物链霉素90μg/mL磁力搅拌均匀，紫外灭菌24h待用。
(2)内层细胞层墨水的制备
37℃温度下，将骨髓间充质干细胞(BMSC)3×106/mL、血管内皮细胞2×106/mL、平滑肌成纤维细胞1×106/mL、血管内皮生长因子(VEGF)70ng/mL、血小板生长因子(PDGF)70ng/mL与嗜碱性成纤维细胞生长因子(bFGF)70ng/mL加入至完全培养基DMEM中，混合均匀。
(3)打印制备
采用同轴喷头，实现壳层材料和内层细胞的同步打印。打印机采用挤出打印设备，打印机壳层材料温度设定为37℃、打印速度为19mm/s；内层设置为37℃、打印速度9mm/s。
实施例6
(1)壳材料墨水的制备
以去离子水为溶剂，将6％明胶与4％海藻酸钠混合通过磁力搅拌均匀；加入生长因子VEGF，浓度为85ng/mL与抗菌消炎药物链霉素65μg/mL磁力搅拌均匀，紫外灭菌18h待用。
(2)内层细胞层墨水的制备
37℃温度下，将骨髓间充质干细胞(BMSC)2×106/mL、血管内皮细胞3×106/mL、平滑肌成纤维细胞2×106/mL、血管内皮生长因子(VEGF)120ng/mL、血小板生长因子(PDGF)120ng/mL与嗜碱性成纤维细胞生长因子(bFGF)120ng/mL加入至完全培养基DMEM中，混合均匀。
(3)打印制备
采用同轴喷头，实现壳层材料和内层细胞的同步打印。打印机采用挤出打印设备，打印机壳层材料温度设定为37℃、打印速度为16mm/s；内层设置为37℃、打印速度6mm/s。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (8)

1.一种用于脊椎损伤血管快速修复的3D打印生物墨水的制备方法，其特征在于包括如下步骤：
(1)壳材料墨水的制备
以去离子水为溶剂，将明胶2％～6％与海藻酸钠1％～5％磁力搅拌均匀；加入生长因子VEGF 50～200ng/mL与抗菌消炎药物50～100μg/mL磁力搅拌均匀，紫外灭菌12～24h待用；
(2)内层细胞层墨水的制备
37±1℃温度下，将骨髓间充质干细胞1～3×106/mL、血管内皮细胞1～3×106/mL、平滑肌成纤维细胞1～3×106/mL、血管内皮生长因子20～200ng/mL、血小板生长因子20～200ng/mL与嗜碱性成纤维细胞生长因子20～200ng/mL加入至完全培养基DMEM中，混合均匀；
(3)3D打印制备
采用同轴喷头，实现壳材料墨水和内层细胞层墨水的同步打印；打印机采用挤出打印设备，打印机壳材料墨水温度设定为37±1℃、打印速度为15mm/s～25mm/s；内层设置为37±1℃、打印速度5mm/s～10mm/s。
2.根据权利要求1所述的用于脊椎损伤血管快速修复的3D打印生物墨水的制备方法，其特征在于：
步骤(1)中所述的海藻酸钠的浓度为2％～5％。
3.根据权利要求1所述的用于脊椎损伤血管快速修复的3D打印生物墨水的制备方法，其特征在于：
步骤(1)中所述的生长因子VEGF的用量为50～100ng/mL。
4.根据权利要求1所述的用于脊椎损伤血管快速修复的3D打印生物墨水的制备方法，其特征在于：
步骤(1)中所述的抗菌消炎药物为链霉素、青霉素。
5.根据权利要求1所述的用于脊椎损伤血管快速修复的3D打印生物墨水的制备方法，其特征在于：
步骤(2)中所述的血管内皮生长因子的用量为30～120ng/mL。
6.根据权利要求1所述的用于脊椎损伤血管快速修复的3D打印生物墨水的制备方法，其特征在于：
步骤(2)中所述的血小板生长因子的用量为30～120ng/mL。
7.根据权利要求1所述的用于脊椎损伤血管快速修复的3D打印生物墨水的制备方法，其特征在于：
步骤(2)中所述的嗜碱性成纤维细胞生长因子的用量为30～120ng/mL。
8.一种用于脊椎损伤血管快速修复的3D打印生物墨水，其特征在于通过权利要求1～7任一项所述的制备方法制备得到。
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