Design a hydrogel scaffold to guide axonal growth in 3-D

生物分子的空间排列对调控细胞的运动和功能起着至关重要的作用。在设计用于神经组织再生的合成模拟物时，我们认为必须考虑模拟物的三维特性。因此，我们首先尝试创建一种具有交替细胞许可/非许可区域的三维图案化基质来实现体外轴突引导。为了实现这一点，壳聚糖基水凝胶将首先被细胞不允许的聚乙二醇经光不稳定的硝基苄基修饰，从而使水凝胶基质细胞不允许。然后，细胞不允许的聚乙二醇会在选定的区域使用光化学被选择性地移除，并被细胞允许的多肽取代。将使用聚焦的双光子激光来激活光反应。由于光子的量子特性，为了激活相同的光反应，双光子激光器的波长是常规单光子激光器的两倍。这种波长的红移使该过程更适合于多肽的固定化。这项研究将进一步推进，以创造一种具有交替细胞许可/非许可区域的3D图案基质，并结合生物分子的浓度梯度，以提供更好的3D神经指导。长期以来，人们一直推测，结合触觉信号(交替的细胞允许区和非允许区)和趋化信号(生物分子的浓度梯度)的生物模拟装置将促进和引导脊髓损伤后的轴突生长；然而，在3-D中同时呈现这两种信号的方法一直难以实现。在这项研究中，我们旨在更好地描绘细胞和材料在体外的三维相互作用，试图为植入装置的设计奠定基础。这项拟议的项目是新颖的，因为这是第一次将触角线索和化学线索结合在一起，以引导3D水凝胶结构中的轴突生长。最终，我们的目标是克服脊髓损伤，并预计本文创造的基本知识将导致克服其他中枢神经系统疾病的策略，例如脑损伤，在这些疾病中再生至关重要。

The spatial arrangement of biomolecules plays a critical role in manipulating cell motility and function. In designing a synthetic analog for nerve tissue regeneration, we believe that the analog's 3-dimensional character must be considered. Therefore, we first attempt to create a 3-D patterned matrix with alternating cell permissive/non-permissive regions to achieve axonal guidance in vitro. In order to achieve this, chitosan based hydrogel will be initially modified by cell non-permissive polyethylene glycol (PEG) via photo-labile nitrobenzyl groups, to render the hydrogel matrix cell non-permissive. The cell non-permissive PEG will then be selectively removed in selected regions using photochemistry and replaced by cell permissive peptides. Focused two-photon laser will be used to activate the photoreaction. Due to the quantum characteristics of photons, the wavelength of the two-photon laser is double that of the conventional single photon laser to activate the same photo-reaction. This red-shift of wavelength makes the process more adaptable for peptide immobilization. The study will be further advanced to create a 3-D patterned matrix with alternating cell permissive/non-permissive regions in combination with a concentration gradient of biomolecules to provide better nerve guidance in 3-D. It has long been speculated that a bio-mimetic device incorporating both haptotactic cues (alternating cell permissive and non-permissive regions) and chemotactic cues (concentration gradient of biomolecules) will promote and guide axonal growth after spinal cord injuries; however the means to present both cues in 3-D has been elusive. In this study, we aim to better delineate the interaction of cells and materials in vitro in 3-D, in an attempt to lay the foundation for the design of a device for implantation. The proposed project is novel in that this is the first study to incorporate both haptotactic cues and chemotactic cues to guide axonal growth within a 3-D hydrogel construct. Ultimately, we aim to overcome spinal cord injuries and anticipate that the fundamental knowledge created herein will lead to strategies to overcome other central nervous system disorders, such as brain injuries, where regeneration is critical.