CN115317669A - 一种具有微结构仿生人工皮肤及其制备方法与应用 - Google Patents
一种具有微结构仿生人工皮肤及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种具有微结构仿生人工皮肤的制备方法，在真皮层和表皮层之间通过静电纺丝和静电直写制备微结构层；所述微结构层的制备方法如下：(1)在真皮层上方表面将微结构层生物墨水进行高压电纺丝，得到多孔网格结构的薄膜；(2)在所述薄膜上将微结构层生物墨水制备得到静电直写纤维，即得到一种微结构层。本发明采用多工艺复合的方法制备具有微结构仿生人工皮肤，可以克服单一工艺带来的局限，并模拟天然皮肤的微结构、改善人工皮肤的生理功能，同时该复合工艺也可以提高人工皮肤的制造自动化水平和制造效率。
Description
技术领域
本发明属于生物制造技术领域，尤其涉及一种具有微结构仿生人工皮肤及其制备方法与应用。
背景技术
皮肤作为人体的第一道防线，易受到外源性有害物质、烧伤以及交通事故等的伤害，目前治疗皮肤损伤的策略主要包括自体移植、同种异体移植和异种移植，但是自体皮肤移植受到供体部位短缺和额外疼痛的限制，由于供体数量有限以及免疫反应问题，同种异体和异种皮肤移植也无法修复大面积皮肤损伤。因此，人工皮肤有望成为皮肤再生的替代品。
除了修复受损皮肤外，人工皮肤还被用于皮肤疾病研究、药物开发以及化妆品效果测试等领域，而目前这些主要借助于2D细胞培养和动物实验；然而，2D细胞培养可能无法模拟皮肤微环境的自然生理，动物实验既昂贵又费时，且随着动物保护意识的增强，以动物为对象的试验逐渐被废弃。
近年来，3D生物打印已被广泛用于构建良好的人体组织和器官，相比于传统的方法，该技术具有可以制造复杂组织结构、沉积不同类型细胞、构造细胞梯度等优点。为了克服传统皮肤组织工程方案的局限性，人们已尝试通过3D生物打印方法来创建皮肤模型，基于不同的打印原理，生物打印皮肤可分为三大类：基于喷墨的、基于挤出的以及激光辅助的生物打印。与其他生物打印皮肤相比，挤出生物打印皮肤具有成型材料和粘度范围广、制造效率高以及易于实现同时多种材料和细胞的打印等优势。
过去研究人员通过使用先进的生物材料、细胞和各种培养技术，致力于开发三维工程皮肤等效物，以创建能够概括天然皮肤独特的结构和相应的生物功能。在天然皮肤中，真皮层和表皮层的交界处呈波纹形状，并在表皮层中形成网状脊，真皮-表皮交界处的起伏形态对于皮肤的几个关键特征至关重要，首先提供对表皮层的机械支撑以及氧气和营养供应，并增强真皮层和表皮层的粘附，这对于皮肤的结构稳定性很重要，最重要的是交界处的起伏形态可以控制细胞微环境，以影响细胞的生理状态。然而包括挤出生物打印技术在内制造的人工皮肤在真皮-表皮交界处通常具有平面界面设计。虽然一些研究在皮肤模型中也创建了交界处起伏的微结构，但大多数研究都是通过光刻或者立体光固化技术等先制造模板，然后再翻模得到人工皮肤，这些技术涉及的工艺繁琐，生产周期长，也难于构建具有三维多孔结构的皮肤层。
为此，能够提供一种工艺简单、生产周期短的具有微结构仿生人工皮肤的制备方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种具有微结构仿生人工皮肤及其制备方法与应用，本发明采用多工艺复合的方法制备具有微结构仿生人工皮肤，可以克服单一工艺带来的局限，并模拟天然皮肤的微结构、改善人工皮肤的生理功能，同时该复合工艺也可以提高人工皮肤的制造自动化水平和制造效率。
为了实现上述目的，本发明采用如下技术方案：
一种具有微结构仿生人工皮肤的制备方法，在真皮层和表皮层之间通过静电纺丝和静电直写制备微结构层。
优选的，所述微结构层的制备方法如下：
(1)在真皮层上方表面将微结构层生物墨水进行高压电纺丝，得到多孔网格结构的薄膜；
(2)在所述薄膜上将微结构层生物墨水制备得到静电直写纤维，即得到一种微结构层。
优选的，所述薄膜的厚度为100-300μm；
所述静电直写纤维的高度为50-350μm，纤维与纤维之间的间隔为50-200μm。
优选的，所述高压电纺丝的参数为：电压4-7kV，静电纺丝喷头与真皮层上方表面的垂直距离10-15cm，电纺速度0.3-1.5ml/h。
优选的，所述静电直写的电压为3-4kV。
优选的，所述真皮层是通过将真皮层生物墨水通过生物打印机制备得到三维多孔真皮层，所述真皮层的厚度为1.5-4mm。
优选的，所述生物打印机参数打印速度为3-8mm/s，挤出速度为0.5-1.5mm3/s。
优选的，所述真皮层生物墨水包括成纤维细胞。
优选的，所述表皮层是通过将表皮层生物墨水通过生物打印机在所述微结构层上方打印得到致密的表皮层，所述表皮层的厚度为0.2-0.5mm。
优选的，所述表皮层生物墨水包括角质形成细胞。
优选的，还包括人工皮肤的培养，培养方法为：浸没培养和气-液界面培养。
优选的，所述培养的具体步骤为：将制备的人工皮肤在37℃、5％CO2的环境下，完全浸没在培养基中3-7天，随后进行气-液界面培养14-21天。
培养基采用DMEM/Ham’s F12(3：1)，补充有10％胎牛血清、1％青霉素/链霉素、0.1-1μg/ml氢化可的松、1-8μg/ml胰岛素、0-2mmol/L Ca2+、0.1-0.3mmol/L腺嘌呤、1-8μg/ml转铁蛋白。
如上述所述制备方法得到的具有微结构仿生人工皮肤。
如上述所述制备方法得到的人工皮肤在制备组织修复产品中的应用。
经由上述的技术方案可知，与现有技术相比，本发明的有益效果如下：
(1)本发明制备方法采用多工艺复合制备含微结构仿生人工皮肤克服了单一工艺带来的局限；
(2)本发明采用挤出生物打印技术使得真皮层易于创建互连互通的宏观孔结构，同时墨水组分固化后具有微孔结构，这有利于营养物质的供应和代谢废物的排出，同时可供细胞附着生长，为细胞提供优越的微环境；
(3)本发明微结构层的静电纺丝膜的加入有利于微结构的制备以及后续表皮层的播种，同时静电直写具有的高精度有利于创建真皮-表皮交界处微结构的几何尺寸；
(4)本发明方法制备过程自动化程度高，制备效率高。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，本描述中的附图仅仅是本发明的实施例。
图1为本发明是实施例制备人工皮肤的工艺流程图；
其中，1-真皮层生物墨水，2-挤出喷头，3-微结构层生物墨水，4-静电纺丝喷头，5-微结构层生物墨水，6-静电直写喷头，7-表皮层生物墨水，8-喷墨喷头，9-真皮层，10-多孔网格结构的薄膜，11-静电直写纤维，12-表皮层。。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、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。
实施例1
如图1，一种具有微结构仿生人工皮肤的制备方法，具体包括以下步骤：
(1)制备真皮层：将真皮层生物墨水1装入挤出喷头2中，采用生物打印机制备人工皮肤的三维多孔真皮层9；真皮层生物墨水1包含2×106/ml成纤维细胞、7％甲基丙烯酸酰化明胶和5％明胶，三维多孔真皮层的平均厚度1.5mm；其中，生物打印机的打印速度为5mm/s，挤出速度为1mm3/s；
(2)制备微结构层：将微结构层生物墨水3加入到静电纺丝喷头4中，通过在真皮层上方表面进行高压电纺丝，高压电纺丝的参数为：电压5kV，静电纺丝喷头与真皮层上方表面的垂直距离10cm，电纺速度1ml/h，从而形成多孔网格结构的薄膜10，在形成200μm的薄膜后，通过调整静电纺丝喷头4与薄膜表面的距离为2mm后，并结合生物打印机的运动及电压使静电纺丝喷头4变成静电直写喷头6，在薄膜上方表面采用相同的微结构层生物墨水5按照提前设计好的几何尺寸进行微结构的制备静电直写纤维11，静电纺丝纤维高度200μm，纤维与纤维之间的间隔100μm；微结构层生物墨水3/5包含8％聚己内酯和2％明胶；其中，生物打印机运动参数为：打印速度30mm/s，挤出速度0.5mm3/s，电压为3.4kV；
(3)制备表皮层：将表皮层生物墨水7装入喷墨喷头8中，采用生物打印机在微结构上方打印致密的表皮层12；表皮层生物墨水7包含4×106/ml角质形成细胞、10％胎牛血清和1％青霉素与链霉素混合液的DMEM培养基，表皮层的平均厚度0.2-0.5mm；
(4)培养人工皮肤：按照常规组织工程皮肤的培养方式，经过浸没培养和气-液界面培养，将上述构建的人工皮肤进行培养后得到功能性的具有微结构仿生人工皮；其中，培养的具体步骤为：将制备的人工皮肤在37℃、5％CO2的环境下，完全浸没在培养基中5天，随后进行气-液界面培养18天，培养基为DMEM/Ham’s F12(3：1)，补充有10％胎牛血清、1％青霉素/链霉素、0.4μg/ml氢化可的松、5μg/ml胰岛素、1.8mmol/L Ca2+、0.18mmol/L腺嘌呤、5μg/ml转铁蛋白。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (10)
1.一种具有微结构仿生人工皮肤的制备方法，其特征在于，在真皮层和表皮层之间通过静电纺丝和静电直写制备微结构层。
2.根据权利要求1所述的一种具有微结构仿生人工皮肤，其特征在于，所述微结构层的制备方法如下：
(1)在真皮层上方表面将微结构层生物墨水进行高压电纺丝，得到多孔网格结构的薄膜；
(2)在所述薄膜上将微结构层生物墨水制备得到静电直写纤维，即得到一种微结构层。
3.根据权利要求2所述的一种具有微结构仿生人工皮肤的制备方法，其特征在于，所述薄膜的厚度为100-300μm；
所述静电直写纤维的高度为50-350μm，纤维与纤维之间的间隔为50-200μm。
4.根据权利要求1所述的一种具有微结构仿生人工皮肤的制备方法，其特征在于，所述真皮层是通过将真皮层生物墨水通过生物打印机制备得到三维多孔真皮层，所述真皮层的厚度为1.5-4mm。
5.根据权利要求4所述的一种具有微结构仿生人工皮肤的制备方法，其特征在于，所述真皮层生物墨水包括成纤维细胞。
6.根据权利要求1所述的一种具有微结构仿生人工皮肤的制备方法，其特征在于，所述表皮层是通过将表皮层生物墨水通过生物打印机在所述微结构层上方打印得到致密的表皮层，所述表皮层的厚度为0.2-0.5mm。
7.根据权利要求6所述的一种具有微结构仿生人工皮肤的制备方法，其特征在于，所述表皮层生物墨水包括角质形成细胞。
8.根据权利要求1所述的一种具有微结构仿生人工皮肤的制备方法，其特征在于，还包括人工皮肤的培养，培养方法为：浸没培养和气-液界面培养。
9.如权利要求1-8任一项所述制备方法得到的具有微结构仿生人工皮肤。
10.如权利要求1-8任一项所述制备方法得到的人工皮肤在制备组织修复产品中的应用。
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