Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham Women's Hospital, Cambridge, MA 02139, United States of America.
Future Vehicle Research Department, Toyota Research Institute North America, Toyota Motor North America Inc., 1555 Woodridge Ave, Ann Arbor, MI 48105, United States of America.
Biofabrication. 2021 Jun 1;13(3). doi: 10.1088/1758-5090/abff11.
Engineering three-dimensional (3D) sensible tissue constructs, along with the complex microarchitecture wiring of the sensory nervous system, has been an ongoing challenge in the tissue engineering field. By combining 3D bioprinting and human pluripotent stem cell (hPSC) technologies, sensible tissue constructs could be engineered in a rapid, precise, and controllable manner to replicate 3D microarchitectures and mechanosensory functionalities of the native sensory tissue (e.g. response to external stimuli). Here, we introduce a biofabrication approach to create complex 3D microarchitecture wirings. We develop an hPSC-sensory neuron (SN) laden bioink using highly purified and functional SN populations to 3D bioprint microarchitecture wirings that demonstrate responsiveness to warm/cold sense-inducing chemicals and mechanical stress. Specifically, we tailor a conventional differentiation strategy to our purification method by utilizing p75 cell surface marker and DAPT treatment along with neuronal growth factors in order to selectively differentiate neural crest cells into SNs. To create spatial resolution in 3D architectures and grow SNs in custom patterns and directions, an induced pluripotent stem cell (iPSC)-SN-laden gelatin bioink was printed on laminin-coated substrates using extrusion-based bioprinting technique. Then the printed constructs were covered with a collagen matrix that guided SNs growing in the printed micropattern. Using a sacrificial bioprinting technique, the iPSC-SNs were seeded into the hollow microchannels created by sacrificial gelatin ink printed in the gelatin methacryloyl supporting bath, thereby demonstrating controllability over axon guidance in curved lines up to several tens of centimeters in length on 2D substrates and in straight microchannels in 3D matrices. Therefore, this biofabrication approach could be amenable to incorporate sensible SN networks into the engineered skin equivalents, regenerative skin implants, and augmented somatosensory neuro-prosthetics that have the potential to regenerate sensible functions by connecting host neuron systems in injured areas.
工程三维（3D）敏感组织构建物，以及感觉神经系统的复杂微观结构布线，一直是组织工程领域的一个持续挑战。通过结合 3D 生物打印和人类多能干细胞（hPSC）技术，可以快速、精确和可控地工程化敏感组织构建物，以复制天然感觉组织的 3D 微观结构和机械感觉功能（例如对外界刺激的反应）。在这里，我们介绍了一种生物制造方法来创建复杂的 3D 微观结构布线。我们使用高度纯化和功能化的 SN 群体开发了一种 hPSC-感觉神经元（SN）负载生物墨水，用于 3D 生物打印微结构布线，该布线对热/冷感觉诱导化学物质和机械应激有反应。具体来说，我们通过利用 p75 细胞表面标志物和 DAPT 处理以及神经元生长因子来调整传统分化策略，以从神经嵴细胞中选择性地分化为 SN。为了在 3D 结构中创建空间分辨率并以定制的图案和方向生长 SN，使用挤出式生物打印技术将 iPSC-SN 负载明胶生物墨水打印在层粘连蛋白涂覆的基底上。然后，将打印的构建物覆盖胶原基质，引导 SN 在打印的微图案中生长。使用牺牲性生物打印技术，将 iPSC-SN 接种到牺牲性明胶墨水在明胶甲基丙烯酰化支撑浴中打印形成的中空微通道中，从而证明了在 2D 基底上长达几十厘米的弯曲线上和在 3D 基质中的直微通道中对轴突导向的可控性。因此，这种生物制造方法可以将敏感的 SN 网络整合到工程化的皮肤等效物、再生皮肤植入物和增强的体感神经假肢中，通过连接受伤区域中的宿主神经元系统，有可能再生敏感功能。
