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摘要:
皮肤作为人体最大的器官，有着屏障功能、免疫应答、防止水分流失和排泄废物等重要作用。大面积的严重皮肤损伤患者会由于缺乏充足的可移植皮肤而死亡。生物3D打印技术的发展为可移植皮肤的制造提供了解决办法。生物3D打印技术可以定制化制造功能性的皮肤替代品，有望解决移植皮肤短缺的困难。本文简述了皮肤创伤修复原理，比较了用于皮肤修复的生物材料墨水、细胞和主要的生物3D打印技术，分析了光电技术在3D打印皮肤上的应用潜力，并总结了生物3D打印技术在皮肤修复应用中所面临的挑战和未来的发展方向，提出了光电技术在生物3D打印中的应用需求。
Abstract:The skin is the largest organ of the human body, which plays an important role in barrier function, immune response, preventing water loss and excreting waste. Patients with large-scale severe skin injuries will die due to lack of adequate skin grafts. The development of 3D bioprinting technology provides a solution for the manufacture of transplantable skin. Firstly, the principles of skin wound repairing are described. Secondly, the bioinks, cells and main 3D bioprinting technologies used in skin wound repairing are compared. Then, the opto-electronic technologies involved are analyzed, and the challenges and future development of 3D bioprinting in the application of skin repair are summarized. Finally, the application requirements of opto-electronic technology in 3D bioprinting are proposed.
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Key words:
- 3D bioprinting /
- skin /
- bioink /
- cell /
- opto-electronic technology
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Overview: The skin is the first line of defense against external stimuli. Therefore, the skin is most vulnerable to injury, and serious skin injury may be life-threatening, so repairing damaged skin is of great significance. Because 3D bioprinting is able to accurately place a variety of different types of cells, even stem cells and appendages, and can repeatably create skin substitutes to replace the injured or damaged parts of the skin, making it similar to the skin appearance and function, 3D bioprinting makes up for the shortcomings of conventional skin wound repairing treatment, and is currently one of the most likely manufacturing methods to develop skin substitutes.
In order to improve the accuracy of printed skin, the degree of adaptation to the wound, and the effect of skin wound repairing, more and more opto-electronic technologies have been applied in 3D bioprinting. Piezoelectric and laser pulse technology can be used in the nozzle to obtain droplets with more uniform cell distribution and droplet diameters that are more suitable for inkjet printing. Digital mask projection technology uses digital micromirror to control the mask to print photosensitive materials to obtain high-resolution customized patterns. Laser-induced forward transfer technology adjusts the energy, spot size, and duration of the pulsed laser beam to cover the laser energy absorbing layer with bioink containing cells. Near-infrared fluorescence technology is used to monitor the printing process in real time, so as to adjust and plan the printing path in real time and obtain the skin with higher degree of compatibility with the defective skin.
In this article, firstly, the skin tissue structures and the principles of skin wound repairing are described. Secondly, the bioinks, cells and main 3D bioprinting technologies used in skin wound repairing are compared. Then, the opto-electronic technologies involved are analyzed. Finally, the application requirements of opto-electronic technology in 3D bioprinting are proposed.
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图 2 用于皮肤的生物3D打印技术。
Figure 2. Main process methods of 3D bioprinting for skin application.
表 1 天然聚合物类生物材料的特点及应用
Table 1. The characteristics and applications of natural polymer biomaterials
生物材料 特点 应用 纤维蛋白 天然存在于血液中，在凝血酶的作用下变成水凝胶状，在早期愈合中模拟了临时的ECM，所以可以促进血液凝结和伤口愈合[19]。 使用稀释的血浆来源的纤维蛋白在transwell系统中共培养角质形成细胞和成纤维细胞可增加胶原蛋白的表达，同时减少其迁移[20]；创建了一个由成纤维细胞和含有纤维蛋白原的人血浆组成的真皮隔室，制造出了双层皮肤替代品[21]。 胶原蛋白 具有螺旋结构的三条多肽链组成，在37 ℃下具有一定的结构稳定性，而且其含有精氨酸-甘氨酸-天冬氨酸(RGD)序列的原纤维结构为细胞提供了结构和生物学支持，可促进细胞附着和增殖[22]。 制备了含有成纤维细胞和角质形成细胞的胶原蛋白-糖胺聚糖支架，用于烧伤创面治疗[23]；采用了生物相容性交联方法或设计良好的胶原蛋白基质内的聚合物网格，通过pH值或温度控制或两者共同引发的其凝胶化[24-25]。 明胶 高分子量多肽，通常是从胶原蛋白的水解中获得的不可逆变形式，与胶原蛋白相似，明胶结构中RGD残基的存在可促进细胞粘附，增殖和迁移，表现出较低的抗原性[26]。 明胶与藻酸盐混合可通过成分比例和温度的控制来提高可打印性(两种聚合物均具有热响应性)，并有助于打印后的交联[27]。 藻酸盐 一种生物相容性良好的带负电荷的多糖，具有高剪切稀化和可快速胶凝后打印的性能[28]。 通过增加藻酸盐的粘度或使用如含Ca2+的化学交联剂提高可打印性[27]；缺乏能使细胞粘附的RGD结构域，因此通常将天然蛋白(包括纤维蛋白，壳聚糖，胶原蛋白，HA和最常见的明胶)与藻酸盐混合[29]。 壳聚糖 一种在无脊椎动物和真菌的外骨骼中大量发现的多糖，止痛和止血的聚合物，也可用作抗菌、消炎药和伤口愈合剂[30]。 双挤压平台逐层沉积多糖壳聚糖和聚(γ-谷氨酸)，壳聚糖的氨基和γ-谷氨酸的羧基之间形成的静电相互作用为打印结构提供了稳定性，使细胞具有良好的活力[31]；非离子热敏交联，即将壳聚糖转化为羟丙基几丁质(HPCH)，有利于细胞的存活和增殖[32]。 dECM 除去组织或器官的非细胞部分，为细胞的信息交流提供可调节的环境并支持细胞迁移。而且，ECM可以通过使用不同的方法获得，并重新用作组织再生的支架[33]。 使猪皮肤组织脱细胞形成了可打印的dECM生物墨水，可促进皮肤的稳定性，增强表皮组织，促进体内的新血管形成和再上皮形成以及伤口闭合[34]；采用脱细胞工艺制备了猪皮粉，并将猪皮粉与具有良好可打印性的海藻酸盐混合，开发了新型生物墨水，打印的细胞显示出更高的代谢活性[35]。 GelMA GelMA可以方便地调节支架力学性能、表面硬度、孔隙率、降解性等，具有亲水性，可吸收渗出液，保持创面润洁，具有光敏特性和温敏特性[36]。 在光交联的明胶中培养永生化的人角质形成细胞制造了具有一定屏障功能的表皮[37]；在不同结构的GelMA打印物培养了人脐静脉内皮细胞、小鼠成肌细胞和成纤维细胞，使细胞可以按照GelMA光固化形成的图案生长和增殖[38]。 
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表 2 用于皮肤修复的生物3D打印的主要细胞类型及应用
Table 2. The main cell types and applications of 3D bioprinting for skin repairing
细胞类型 简介 应用 角质形成细胞 表皮中的主要细胞类型。 Lee等人[45]利用胶原和角质形成细胞作为生物墨水，打印了伤口特异性组织工程皮肤产品；Kim等人[46]通过挤出打印和喷墨打印使用角质形成细胞直接打印了皮肤模型。 成纤维细胞 存在于皮肤的真皮层中，负责细胞外基质和非纤维成分的产生[47]。 Won等人[48]将基于皮肤脱细胞的细胞外基质(dECM)粉末和成纤维细胞作为生物墨水，发现细胞中的基因表达与皮肤形态生物学相似；Shi等人[49]通过挤压成型用藻酸钠/明胶复合材料和成纤维细胞制造了一种新型的真皮替代支架，与人体皮肤组织具有相似的理化特性。 内皮细胞 内皮细胞的存在可以增加血管化形成，有利于各种生长因子的分泌和信号交流。 Baltaza等人[50]在真皮层中合并了内皮细胞自组装的血管床，用于促进皮肤移植物的血管生成和灌注；Huyan等人[51]制造了由成纤维细胞、角质形成细胞和微血管内皮细胞组成的双层结构，具有显着的血管生成现象。 黑色素细胞 黑色素细胞，其分泌的黑色素可以作为调节皮肤颜色和抵御紫外线的应用。 Ng等人[52]应用3种不同类型的皮肤细胞(角质形成细胞，黑素细胞和成纤维细胞)制造了含色素的人体皮肤结构，并显示了与皮肤供体相似的浅色色素沉着；Min等人[53]将黑色素细胞和角质形成细胞打印在真皮层的顶部，并在真皮-表皮交界处观察到雀斑状色素沉着现象。 脂肪充间质干细胞 从脂肪组织中提取的具有多向分化潜能的干细胞。 显示出免疫调节炎症现象，促进新血管形成以及在再生过程中刺激增殖，还能够自我更新并分化为各种谱系，以替换受损组织[54-55]。 骨髓充间质干细胞 从骨髓穿刺物中分离出来的干细胞。 移植同种异体骨髓间充质干细胞后，几乎没有观察到排斥反应，可分化为角质形成细胞[56]；骨髓充间质干细胞表达更高数量的胶原蛋白，可加速愈合过程，增加血管生成，以及直接分化成表达角质形成细胞特异性标记的上皮细胞来改善皮肤愈合[57-59]。 胎盘干细胞 从胎盘分离的干细胞。 胎盘获得的干细胞对供体没有风险，并且含有大量的再生细胞，具有免疫调节和免疫抑制特性[60]。
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表 3 用于皮肤修复的主要生物3D打印技术及其用于皮肤修复的特点
Table 3. The main 3D bioprinting technology for skin repair and its characteristics
3D打印技术 所应用的主要光电技术 特点 喷墨打印 在无载体的情况下，精确沉积感光细胞，以预定的排列方式在细胞层上沉积了成熟的和分化的感光体[84]；首先，通过调节施加到压电打印头的电压波形的参数来产生均匀的微滴。然后微滴被固化，从而形成微颗粒[85]。 打印精度高，可打印复杂的微米级结构；仅适用于低粘度生物材料墨水，交联困难，且细胞密度较低，否则会出现喷嘴堵塞。 光固化打印 数字微镜阵列设备(DMD)具有大约200万个微镜阵列，可以分别控制它们以控制在制造阶段投射到单体溶液的光学图案，并同时移动载物台，紫外光用于诱导光敏生物材料的光聚合[86-90]。 高分辨率，无喷嘴堵塞；材料需要与光引发剂混合，但是光引发剂会影响细胞活性。 激光辅助打印 利用激光能量将充满细胞的生物墨水，通过无接触无喷嘴的打印方式，将液滴推进到接收基质上[88, 91-92]。 无喷嘴，高细胞密度的精确沉积；高能量的激光束影响细胞活性。 挤出打印 利用激光扫描等技术实时监测挤出微丝的直径，便于调整工艺参数[49, 80, 93]。 生物材料墨水的可挤出粘度范围大，可混合的细胞密度高；挤出微丝的分辨率低，且挤出力会大大降低细胞存活率。
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表 4 用于皮肤修复的主要生物3D打印技术及其用于皮肤修复的特点
Table 4. The main 3D bioprinting technology for skin repair and its characteristics
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