Controllable 2- and 3D Assembly of Mechanically Robust Skin Tissue Via Long Term Expression of DNA on Cell Membranes

通过细胞膜上 DNA 的长期表达实现机械鲁棒性皮肤组织的可控 2 和 3D 组装

• 批准号: 10328551
• 负责人: Jennifer N Cha
• 金额: $ 18.93万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-13 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10328551
• 关键词: 3-Dimensional; Adhesions; Affinity; Area; Autologous Transplantation; Binding; Biochemical; Biotin; Burn injury; Bypass; Cadherins; Cell Adhesion Molecules; Cell membrane; Cell surface; Cells; Cessation of life; Chimeric Proteins; Cicatrix; Collagen; Complementary DNA; Complex; Coupled; DNA; DNA Binding; DNA Sequence; DNA annealing; Dermis; Development; Dimensions; Engineering; Epidermal Growth Factor Receptor; Epithelial; Failure; Fibroblasts; Glass; Glues; Goals; Health; Immune response; Lead; Length; Linker DNA; Lipids; Location; Mechanics; Mediating; Methods; Movement; Nanostructures; Pathway interactions; Patients; Pattern; Polymers; Process; Property; Proteins; Publishing; Receptor Cell; Research; Resistance; Scheme; Shapes; Signal Transduction; Site; Skin; Skin Tissue; Skin Transplantation; Streptavidin; Structure; Surface; Tensile Strength; Thick; Time; Tissue Engineering; Tissues; Transplantation; Work; cell assembly; density; extracellular; flexibility; healing; improved; innovation; keratinocyte; mechanical properties; migration; preservation; programs; scaffold; trafficking; wound; wound bed

The proposed research plan will develop innovative bioconjugation and DNA-mediated cell assembly strategies for rapid creation of self-assembled multicellular scaffolds with programmable shapes, sizes, and dimensions. Over the past several decades, enormous strides have been made in developing cultured epithelial autografts (CEA) from patient-derived keratinocytes and fibroblasts (i.e. autografts) because they have the smallest chance of immune response and host rejection. However, the new skin tissue must be grown and formed into layers in a lengthy process and the weak mechanical properties of the transplanted skin may result in poor integration with the underlying wound area. In addition, the cells natural adhesion molecules that promote 2D structure are poorly optimized for flexibility and compliance, making it difficult to manipulate onto an underlying substrate or a wound. Patient movement is often inevitable for wounds and burns at certain locations and/or for patients with high burn percentages, which in turn can lead to skin transplant delamination and failure. As a result, the wound sites can become infected and form scar tissue, and in more extreme cases they may lead to intense suffering and even death. The proposed research will develop a DNA mediated bottom-up approach to rapidly generate large-area, close-packed skin cell arrays with predetermined final cell sheet thickness and controllable cell-cell spacing, joined together by reversible, programmable bonds. These cell sheets will boast significantly improved mechanical properties over current state-of-the-art, including robustness, compliance, resistance to tearing, and even self-healing. By combining DNA bound to the cells with complementary DNA freely mobile on the surface, the complementary DNA will act as ‘linker’ strands to bridge neighboring cells and drive interactions in both 2- and 3D to form close packed cell arrays. Having DNA linkers act as a ‘glue’ between cells should increase the mechanical stability of the formed tissues and also allow for self-healing. The DNA expressed on the cell membranes can also be used to engineer cell sheets with tunable adhesion forces to an underlying substrate to improve the overall mechanical strength of the final engineered tissue. To conjugate DNA to cell membranes while retaining long-term expression, the PIs have developed a new Affinity-Mediated Covalent Photoconjugation (AMCP) cell functionalization method where the PIs discovered that photocrosslinking protein tags to epidermal growth factor receptor (EGFR) allowed the attached proteins to bypass typical proteolytic pathways and return to the cell membrane. In the proposed research, the PIs will take advantage of the abundance of EGFR on skin cells to attach photocrosslinkable affibody-streptavidin fusion proteins, which in turn will be coupled with biotin-DNA, using the strong biotin-streptavidin interactions to increase ultimate tensile strength of the formed cell sheets. This method will allow tuning of both the number of fusion protein tags per cell and DNA strand density to preserve healthy intracellular signaling and proliferation.

拟议的研究计划将开发创新的生物结合和DNA介导的细胞组装 快速产生具有可编程形状，尺寸， 和尺寸。在过去的几十年里，在发展文化方面取得了巨大的进步。 来自患者来源的角质形成细胞和成纤维细胞的上皮自体移植物（CEA）（即自体移植物），因为它们具有 免疫反应和宿主排斥的可能性最小然而，新的皮肤组织必须生长， 并且可能导致移植皮肤的弱机械性能 与下方的伤口区域结合不良。此外，细胞的天然粘附分子， 2D结构在灵活性和顺应性方面优化得很差，使得难以在 底层基质或伤口。对于某些部位的伤口和烧伤，患者移动往往是不可避免的 和/或对于具有高烧伤百分比的患者，这又会导致皮肤移植分层和失败。 因此，伤口部位可能会受到感染并形成疤痕组织，在更极端的情况下， 导致巨大的痛苦甚至死亡 拟议的研究将开发一种DNA介导的自下而上的方法， 具有预定的最终细胞片厚度和可控的细胞-细胞间距的紧密堆积的皮肤细胞阵列， 通过可逆的可编程键连接在一起。这些细胞片将显著改善 机械性能优于当前最新技术水平，包括坚固性、顺应性、抗撕裂性，以及 甚至可以自我修复通过将结合到细胞的DNA与在表面上自由移动的互补DNA结合， 互补的DNA将作为“接头”链来桥接相邻的细胞，并驱动2- 和3D以形成紧密堆积的细胞阵列。让DNA连接体充当细胞之间的“胶水”， 所形成的组织的机械稳定性，并且还允许自愈。细胞上表达的DNA 膜也可用于设计具有可调粘附力的细胞片层 提高最终工程化组织的总体机械强度。 为了将DNA结合到细胞膜上，同时保持长期表达，PI开发了一种新的 亲和介导的共价光缀合（AMCP）细胞功能化方法，其中PI 发现表皮生长因子受体（EGFR）的光交联蛋白质标签允许附着在 蛋白质绕过典型的蛋白水解途径并返回细胞膜。在拟议的研究中， PI将利用表皮生长因子受体在皮肤细胞上的丰富性， 融合蛋白，其又将与生物素-DNA偶联，使用强的生物素-链霉亲和素相互作用， 增加所形成的电池片的极限拉伸强度。此方法将允许调整 每个细胞的融合蛋白标签和DNA链密度，以保持健康的细胞内信号传导和增殖。