Miura Shigenori, Nie Minghao, Emoto Kazuo, Takeuchi Shoji
Department of Mechano-Informatics, Graduate School of Information Science and Technology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Department of Molecular Biology and Biochemistry, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
ACS Biomater Sci Eng. 2025 Jan 13;11(1):442-450. doi: 10.1021/acsbiomaterials.4c01097. Epub 2024 Dec 18.
Engineered skin models with sensory innervation are a growing and challenging field of research aimed at applications in regenerative medicine, biosensing, and drug screening. Researchers are attempting to fabricate innervated skin tissues using collagen sponges, cell culture inserts, and microfluidic devices to partially mimic the layered structure of the skin. However, innervation of the full-thickness skin model has not yet been achieved. Here, using the anchoring culture device we previously reported, which is a powerful tool to construct a full-thickness three-dimensional (3D) skin model with balanced tissue contraction forces, we drastically improved dermal layer innervation using a composite hydrogel of collagen and Matrigel (Coll:MG). To determine the preferable hydrogel matrix for neurite extension in the 3D skin construct, DRG neural spheroids were placed at the bottom of the dermal layer composed of various hydrogel scaffold, including type I collagen from different origins (dermis or tendon) and Coll:MG composite hydrogel with different compositions. We showed that the Coll:MG (2:1) composite hydrogel significantly increased vertical neurite extension in the dermal layer, concomitant with the reduced tissue shrinkage during the culture. In contrast, in the collagen-only hydrogel, neurite extension occurred mostly in the horizontal direction, and tissues sometimes detached from the anchors due to significant shrinkage, indicating that tissue shrinkage may affect the direction of neurite extension. To exemplify this idea, 3D skin constructed in the device was partially detached from the anchors to comply with the cell-induced tissue shrinkage and reduce the strain on the tissue. The data showed that the partial allowance of in-plane tissue strain remarkably increased vertical neurite extension compared to the control cultures. Collectively, our results strongly suggest that neurite extension angles can be modulated by adjusting the tissue strain during the culture. Our findings highlight the importance of controlling tissue strain for the advancement of an innervated skin model.
具有感觉神经支配的工程皮肤模型是一个不断发展且具有挑战性的研究领域，旨在用于再生医学、生物传感和药物筛选。研究人员正尝试使用胶原海绵、细胞培养插入物和微流控装置制造有神经支配的皮肤组织，以部分模拟皮肤的分层结构。然而，全层皮肤模型的神经支配尚未实现。在此，我们使用我们之前报道的锚定培养装置，这是构建具有平衡组织收缩力的全层三维（3D）皮肤模型的有力工具，通过使用胶原蛋白和基质胶（Coll:MG）的复合水凝胶，大幅改善了真皮层的神经支配。为了确定3D皮肤构建物中神经突延伸的最佳水凝胶基质，将背根神经节（DRG）神经球置于由各种水凝胶支架组成的真皮层底部，这些水凝胶支架包括不同来源（真皮或肌腱）的I型胶原以及不同组成的Coll:MG复合水凝胶。我们发现，Coll:MG（2:1）复合水凝胶显著增加了真皮层中神经突的垂直延伸，同时培养过程中组织收缩减少。相比之下，在仅含胶原蛋白的水凝胶中，神经突延伸大多发生在水平方向，并且由于显著收缩，组织有时会从锚定物上脱落，这表明组织收缩可能会影响神经突延伸的方向。为了验证这一想法，在该装置中构建的3D皮肤部分从锚定物上分离，以顺应细胞诱导的组织收缩并减少组织上的应变。数据表明，与对照培养相比，平面内组织应变的部分允许显著增加了神经突的垂直延伸。总体而言，我们的结果有力地表明，神经突延伸角度可通过在培养过程中调节组织应变来调控。我们的发现突出了控制组织应变量对于推进有神经支配皮肤模型的重要性。
