CN112826635A - 一种促进血管化乳房支架的制作方法 - Google Patents

一种促进血管化乳房支架的制作方法 Download PDF

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CN112826635A
CN112826635A CN202110103742.7A CN202110103742A CN112826635A CN 112826635 A CN112826635 A CN 112826635A CN 202110103742 A CN202110103742 A CN 202110103742A CN 112826635 A CN112826635 A CN 112826635A
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layer
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姜文彬
孙家明
汪振星
陈雳风
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Tongji Medical College of Huazhong University of Science and Technology
Union Hospital Tongji Medical College Huazhong University of Science and Technology
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Abstract

本发明提供一种促进血管化乳房支架的制作方法，包括如下步骤，通过影像学CT、MRI扫描、DSA血管造影等方式得到乳房的影像学资料，然后利用CAD软件进行建模，输出为所需打印乳房的片层STL格式文件，以生物可降解材料和活细胞为打印原料，最后利用3D打印机逐层叠加的原理将材料堆积成型。本发明通过将活细胞直接用作打印原料的生物3D打印技术可以实现组织支架与细胞的一体化构建，大大缩短了传统组织工程构建策略中所需的体外操作时间。

Description

一种促进血管化乳房支架的制作方法
技术领域
本发明涉及组织工程乳房再造领域，具体是一种促进血管化乳房支架的制作方法。
背景技术
乳腺癌是全球女性发病率最高的恶性肿瘤，乳腺癌改良根治切除术(全乳切除)，使女性失去乳房正常形态，乳房重建是解决该问题的唯一手段，由于现有的乳房重建方式存在诸多不足，所以我国实际接受该手术的比例不足5％。
目前乳房填充材料主要包括医用材料、自体移植物及组织工程产品三大类。医用材料又包括硅胶、聚丙烯酰胺凝胶、三酰甘油(甘油三酯)、透明质酸等植入型假体和液体石蜡、凡士林等注射用混合物。这些材料易出现假体破裂或渗漏、炎症反应、肉芽肿、包膜挛缩、假体移位、感染、蒙道尔病等并发症。
3D打印技术是一种以数字模型文件为基础，运用粉末状金属或塑料等可粘合材料，通过逐层打印的方式来构造物体的快速成型技术。目前3D打印技术所用的材料主要有硅胶、树脂、石膏、金属粉末等。有些3D打印材料不适用于制作乳房代用品，如金属、树脂等；而有些则技术难度大，成本高，时间长。
发明内容
针对上述现有技术的不足，本发明提供一种促进血管化乳房支架的制作方法，采用生物相容性好的水凝胶材料，这一类材料可通过成分的调控达到接近机体软组织的弹性模量，将其与活细胞结合直接用作打印原料，可以实现组织支架与细胞的一体化构建，大大缩短了传统组织工程构建策略中所需的体外操作时间。
本发明提供的技术方案：一种促进血管化乳房支架的制作方法，其特征在于包括如下步骤：
(1)通过影像学CT、MRI扫描或DSA血管造影方式得到患者的乳腺影像学数据，然后利用CAD软件进行三维建模，得到片层STL格式文件；
(2)基于以上数据分析与模型制作，设计出乳房支架的多孔结构；
(3)将聚乙二醇二丙烯酸酯(PEGDA)以10％-15％w/v、光引发剂LAP以2.0％-3.0％w/v、光吸收剂以0.05％-0.1％w/v的浓度溶于磷酸盐缓冲溶液，得到可吸收液态水凝胶材料；
(4)将步骤(3)所得的可吸收液态水凝胶材料进行无菌处理，加入离心管，移液枪反复吹打混匀，按照100万-200万个细胞/毫升在液态水凝胶内混入取自于患者自身的脂肪干细胞或骨髓间充质干细胞；
(5)对步骤(2)所得的模型进行分层切片处理，将步骤(4)中所得的打印原料一同放入光固化3D打印机，逐层打印成固态且具有生物活性的乳房支架。
进一步的，所述步骤(5)中3D打印的参数为：层高20μm，光强14mW/cm2，基层层数20，基层曝光时间32s，片层曝光时间32s，剥离距离6mm，剥离速度18mm/min，剥离回复速度100mm/min，光源波长为405nm。
进一步的，所述步骤(3)中所述的光吸收剂为柠檬黄。
进一步的，所述步骤(4)中的脂肪干细胞或骨髓间充质干细胞在术中无菌条件下进行抽取，然后显微镜下计数板计数后转入离心管1000转/分离心，去除上层液体后备用。抽取细胞分泌的细胞因子或细胞外囊泡，可促进血管新生与颗粒脂肪存活，有利于大体积乳房支架中血管的长入，进一步加速了脂肪组织的再生。
本发明利用干细胞结合可降解生物支架，植入体内原位再生乳房组织的构建技术，由于其具有损伤小、排异少等优点，拥有较广泛的应用前景。组织工程乳房植入后，随着种子细胞的增殖分化与生物材料的降解，自身组织将完全替代外源性植入物。这种方法即避免了大面积供区损伤(皮瓣移植)，又规避了不可降解材料植入带来的风险(假体植入)。打印出的多孔带细胞水凝胶支架具有生物活性，可持续释放细胞因子，促进周围血管生成与组织长入，同时水凝胶采用PEGDA此类可吸收材料，可以逐渐被人体吸收，此期间其释放的细胞因子可以促进组织与血管的生长，完成乳房的原位再生。
本发明相比于现有技术，具有以下有益效果：
(1)具有生物活性的支架，可以持续释放细胞因子，促进机体血管和组织再生。
(2)通过直接打印活细胞的方式，解决了种子细胞在大体积复杂结构支架中无法均匀分布的问题；
(3)可在术中提取出种子细胞后，即刻进行组织工程移植物的构建，避免了冗长的体外细胞培养过程及潜在的污染风险；
(4)细胞可在接近于天然细胞外基质的环境中生长，利于保留活性与功能。除此之外，目前用于生物3D打印的墨水通常为生物相容性好的水凝胶材料，这一类材料可通过成分的调控达到接近机体软组织的弹性模量。
附图说明
图1是本发明的方法流程图；
图2是本发明的乳房模型分层切片图；
图3是本发明的乳房模型图；
图4是打印的乳房支架的示意图；
图5是组织工程乳房裸鼠皮下移植示意图；
图6是术后4周与12周乳房支架移植物的照片；
图7是乳房支架进行巴马香猪原位乳房缺损修复的对比照片；
图8是打印的乳房支架进行活细胞染色的实验结果图；
图9是打印的乳房支架进行压力性能测试的实验结果图；
具体实施方式
下面结合具体实施例和附图对本发明做进一步的说明。
实施例1
一种促进血管化乳房支架的制作方法，包括如下步骤：
(1)通过影像学CT、MRI扫描或DSA血管造影方式得到患者的乳腺影像学数据，然后利用CAD软件进行三维建模，得到片层STL格式文件，如图2所示，使支架大小符合乳房缺损患者的个体化需求；
(2)基于以上数据分析与模型制作，设计出乳房支架的多孔结构，如图3所示，方便血管的长入与生物支架中血管因子的释放；
((3)将聚乙二醇二丙烯酸酯(PEGDA)以12％w/v、光引发剂LAP以2.5％w/v、柠檬黄以0.05％w/v的浓度溶于磷酸盐缓冲溶液，得到可吸收液态水凝胶材料；
(4)将步骤(3)所得的可吸收液态水凝胶材料进行无菌处理，加入离心管，移液枪反复吹打混匀，按照100万个细胞/毫升在液态水凝胶内混入取自于患者自身的脂肪干细胞或骨髓间充质干细胞；
(5)对步骤(2)所得的模型进行分层切片处理，将步骤(4)中所得的打印原料一同放入光固化3D打印机，逐层打印成固态且具有生物活性的乳房支架。
所述步骤(5)中3D打印的参数为：层高20μm，光强14mW/cm2，基层层数20，基层曝光时间32s，片层曝光时间32s，剥离距离6mm，剥离速度18mm/min，剥离回复速度100mm/min，光源波长为405nm。光源波长为405nm，在此波长下，既能对生物墨水进行光固化打印，同时也不会影响细胞的活性与功能。
所述步骤(4)中的脂肪干细胞或骨髓间充质干细胞在术中无菌条件下进行抽取，然后显微镜下计数板计数后转入离心管1000转/分离心，去除上层液体后备用。抽取细胞分泌的细胞因子或细胞外囊泡，可促进血管新生与颗粒脂肪存活，有利于大体积乳房支架中血管的长入，进一步加速了脂肪组织的再生。
实施例2
一种促进血管化乳房支架的制作方法，包括如下步骤：
(1)通过影像学CT、MRI扫描或DSA血管造影方式得到患者的乳腺影像学数据，然后利用CAD软件进行三维建模，得到片层STL格式文件，如图2所示，使支架大小符合乳房缺损患者的个体化需求；
(2)基于以上数据分析与模型制作，设计出乳房支架的多孔结构，如图3所示，方便血管的长入与生物支架中血管因子的释放；
((3)将聚乙二醇二丙烯酸酯(PEGDA)以14％w/v、光引发剂LAP(苯基-2,4,6-三甲基苯甲酰基膦酸锂)以2％w/v、柠檬黄以0.07％w/v的浓度溶于磷酸盐缓冲溶液，得到可吸收液态水凝胶材料；
(4)将步骤(3)所得的可吸收液态水凝胶材料进行无菌处理，加入离心管，移液枪反复吹打混匀，按照150万个细胞/毫升在液态水凝胶内混入取自于患者自身的脂肪干细胞或骨髓间充质干细胞；
(5)对步骤(2)所得的模型进行分层切片处理，将步骤(4)中所得的打印原料一同放入光固化3D打印机，逐层打印成固态且具有生物活性的乳房支架。
所述步骤(5)中3D打印的参数为：层高20μm，光强14mW/cm2，基层层数20，基层曝光时间32s，片层曝光时间32s，剥离距离6mm，剥离速度18mm/min，剥离回复速度100mm/min，光源波长为405nm。光源波长为405nm，在此波长下，既能对生物墨水进行光固化打印，同时也不会影响细胞的活性与功能。
采用光固化3D打印技术打印出具有生物活性的，可持续刺激血管生成的水凝胶支架。所述支架结合医学影像数据和CAD建模，按照患者自身需求构建相应大小的生物支架，该支架内均匀分布有患者自身提取的细胞，可持续分泌细胞因子促进组织血管生成，随着时间的推移，机体组织逐渐再生，该支架也逐渐降解，完成缺损乳房的原位再生。
支架经过一系列性能测试，包括活死细胞染色和压缩性能测试，活死细胞染色的结果如图8所示，压缩性能测试的结果如图9所示、此外，进行细胞毒性测试，将所得支架植入裸小鼠体内三个月后小鼠正常存活，也可见细胞活死染色，以裸鼠作为动物模型植入生物活性支架，并与第一、三个月取材，可发现支架中有血管长入，并且有部分周围组织爬入多孔结构内，经切片染色发现为脂肪组织。
以上所述仅为本发明的具体实施方案的详细描述，并不以此限制本发明，凡在本发明的设计思路上所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (4)

1.一种促进血管化乳房支架的制作方法，其特征在于包括如下步骤：
(1)通过影像学CT、MRI扫描或DSA血管造影方式得到患者的乳腺影像学数据，然后利用CAD软件进行三维建模，得到片层STL格式文件；
(2)基于以上数据分析与模型制作，设计出乳房支架的多孔结构；
(3)将聚乙二醇二丙烯酸酯以10％-15％w/v、光引发剂LAP以2.0％-3.0％w/v、光吸收剂以0.05％-0.1％w/v的浓度溶于磷酸盐缓冲溶液，得到可吸收液态水凝胶材料；
(4)将步骤(3)所得的可吸收液态水凝胶材料进行无菌处理，加入离心管，移液枪反复吹打混匀，按照100-200万个细胞/毫升在液态水凝胶内混入取自于患者自身的脂肪干细胞或骨髓间充质干细胞；
(5)对步骤(2)所得的模型进行分层切片处理，将步骤(4)中所得的打印原料一同放入光固化3D打印机，逐层打印成固态且具有生物活性的乳房支架。
2.根据权利要求1所述的促进血管化乳房支架的制作方法，其特征在于：所述步骤(5)中3D打印的参数为：层高20μm，光强14mW/cm2，基层层数20，基层曝光时间32s，片层曝光时间32s，剥离距离6mm，剥离速度18mm/min，剥离回复速度100mm/min，光源波长为405nm。
3.根据权利要求1所述的促进血管化乳房支架的制作方法，其特征在于：所述步骤(3)中所述的光吸收剂为柠檬黄。
4.根据权利要求1所述的促进血管化乳房支架的制作方法，其特征在于：所述步骤(4)中的脂肪干细胞或骨髓间充质干细胞在术中无菌条件下进行抽取，然后显微镜下计数板计数后转入离心管1000转/分离心，去除上层液体后备用，抽取的干细胞分泌细胞因子或细胞外囊泡，促进血管新生与颗粒脂肪存活，有利于大体积乳房支架中血管的长入，进一步加速了脂肪组织的再生。
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