Photopatterned Dendrons for Tissue Engineering Applications

组织工程和再生医学领域的目标是替换或修复因损伤或疾病而受损的组织和器官。产生实验室培养的组织的理想方案是从合适的来源(理想情况下是患者)采集细胞，在培养中培养它们，直到有足够数量的细胞，然后将它们转移到三维(3D)聚合物支架上，使组织成形。在这里，它们被给予必要的化学和物理线索，使它们能够发展成功能结构，然后被植入患者体内。然而，要使这成为现实，特别是对于更复杂的组织，必须克服一些问题和技术障碍。其中一个问题涉及用于形成3D细胞支架的材料。为了让细胞附着在支架上并在其上生长，材料必须具有细胞识别的粘附点。组织工程中常用的许多聚合物具有良好的生物相容性和生物降解性，这两者都是支架的理想特性，但细胞粘附区有限。有许多策略可以改变这些材料的表面性质，以极大地提高细胞粘附性，但都存在诸如使用昂贵的专门设备或聚合物降解等缺点。然而，组织工程和再生医学策略中更基本的问题是细胞的空间排列。在自然组织中，多种细胞类型在确定的三维结构中的复杂排列对组织的功能和生存有很大的影响。目前，我们不能在实验室中重建这种组织，而是在很大程度上依赖于细胞的随机播种，如果我们想要将血管和神经整合到实验室培养的组织中，这是特别有问题的。这项研究提案将通过开发一种新的策略来解决这两个问题，即用显示多个细胞粘附肽拷贝的分子来形成聚合物材料的图案。这最终将允许多种细胞类型在特定的排列中顺序种植和生长，重建在真实组织中发现的组织。为了充分利用许多聚合物中的化学功能和有限数量的官能团，这些官能团可以在不发生降解的情况下以化学方式引入其他聚合物中，通过将树枝连接到支架表面，可用粘合剂位置的有效数量将显著增加。这些支化分子的末端含有多个基团，因此，通过将细胞粘附肽附着到每个端基上，聚合物的每个可用官能团将显示多种粘附性物种。虽然树枝状聚合物的改性可以作为一种独立的技术来增加细胞粘附性，但细胞图案化的问题也将通过用树枝状物以特定图案装饰聚合物表面来解决。该技术的基础是通过笼形连接物分子的共价连接对支架进行初始修饰，即被保护基团掩盖的反应性化学官能团。这些笼子可以通过暴露在紫外线(UV)下和由此产生的未掩蔽的化学基团来选择性地移除，然后用于附着细胞黏附的树突。使用该技术的图案化将通过图案化掩模的UV曝光来实现，从而产生相应的未老化官能团的图案。Dendrons将被附着到反应性的、暴露在紫外线下的区域，而其余的表面功能将被困在笼子里，细胞将生长在粘合剂图案上。然后将重复该过程以用于其他类型的后续图案化。最终，这项技术的实用性将通过肌肉和神经细胞的图案化和共培养来证明，以促进这两种细胞之间特定部位突触连接的形成。

The fields of tissue engineering and regenerative medicine aim to replace or repair tissues and organs compromised by injury or disease. The ideal scenario for generating a laboratory-grown tissue is to harvest cells from a suitable source (ideally the patient), grow them in culture until there are a sufficient number and then transfer them to a three-dimensional (3D) polymer scaffold to give shape to the tissue. Here they are given the necessary chemical and physical cues to enable them to develop into a functional construct which is then implanted into the patient.However, there are a number of problems and technical hurdles that must be overcome for this to become a reality, especially for more complex tissues. One problem concerns the materials used to form the 3D cell scaffolds. For cells to adhere to and grow on a scaffold, the material must possess adhesive sites for the cells to recognize. Many polymers commonly used in tissue engineering have excellent biocompatibility and biodegradability, both desirable properties of a scaffold, but have limited cell-adhesive areas. A number of strategies exist to alter the surface properties of these materials in order to greatly increase cell adhesion, but all suffer from drawbacks such as the use of expensive, specialized equipment or degradation of the polymer. A more fundamental problem in tissue engineering and regenerative medicine strategies, however, is the spatial arrangement of cells. In natural tissues, the complex arrangement of multiple cell types in defined 3D architectures has a great influence on the function and survival of the tissue. Currently, we can not recreate this organization in the laboratory, instead largely relying on random seeding of cells, and this is particularly problematical if we want to incorporate blood vessels and nerves into lab-grown tissues.This research proposal will address both of these problems by developing a novel strategy to pattern polymeric materials with molecules that display multiple copies of cell-adhesive peptides. This will ultimately allow sequential seeding and growth of multiple cell types in defined arrangements, recreating the organization found in real tissues. To fully exploit the chemical functionality found in many polymers and the limited number of functional groups that can be chemically introduced into others without degradation occurring, the effective number of available adhesive sites will be dramatically increased by coupling dendrons to scaffold surfaces. These branched molecules contain multiple groups at the tips of their branches so, by attaching cell-adhesive peptides to each end group, each of the polymer's available functional groups will be made to display multiple adhesive species.Although modification of polymers with dendrons could be used as a stand-alone technique for increasing cell adhesion, the problem of cell patterning will also be addressed by decorating polymer surfaces with the dendrons in specific patterns. The basis of this technique is the initial modification of the scaffolds by covalent attachment of caged linker molecules, i.e. reactive chemical functionalities which are masked by a protecting group. These cages can be selectively removed by exposure to ultraviolet (UV) light and the resulting unmasked chemical group then used to attach cell-adhesive dendrons. Patterning using this technique will be achieved by UV exposure through patterned masks, resulting in a corresponding pattern of uncaged functional groups. Dendrons will be attached to the reactive, UV-exposed areas leaving the rest of the surface functionalities caged, and cells will be grown on the adhesive patterns. The process will then be repeated for subsequent patterning of other cell types. Ultimately, the utility of this technique will be demonstrated by patterning and co-culture of muscle and nerve cells to promote the site-specific formation of synaptic connections between the two cell types.