Development of Protein-Displaying Peptide Hydrogels for Tissue Engineering

描述（由申请人提供）：用于组织工程的新型生物材料的开发是一个新兴的研究领域，对美国的公共卫生具有广泛的影响。该领域的进展有可能治疗无数的医疗条件，如皮肤损伤（即撕裂伤和烧伤），心脏创伤，严重的脊柱损伤和骨折。目前，用于组织工程的大多数合成材料是将生长因子或肽掺入材料基质中的聚合物。掺入模式的范围从共价（对于小肽）到吸附（对于较大的目标蛋白质）。如果全长蛋白质能够以高亲和力和特异性整合并展示在组织工程平台内，该领域将显着进步。刺激响应性肽水凝胶近年来在材料科学和组织工程领域受到越来越多的关注。这些类型的材料组装成纳米级纤维，这些纤维被水合以形成刚性凝胶材料。这些材料的优点包括易于获得纯单体单元、无毒性、可注射性以及在某些情况下的抗菌性能。然而，缺乏的是通过高亲和力非共价结合相互作用掺入和展示功能蛋白的能力。在建议的研究计划的指导阶段，噬菌体展示技术将使用来自噬菌体Q？的病毒样颗粒（VLP）进行开发，一种病毒纳米颗粒，主要被芬兰实验室用于无数生物技术应用。该提案旨在开发一个在Q？外表面展示肽库的平台。用于定向进化实验以鉴定与凝胶形成肽MAX8特异性相互作用的变体的VLP。我们将展示选定的肽的表面上的Q？VLP和评估结合亲和力和凝胶掺入如何相关。然后将严格表征新合成材料的材料特性。拟议研究的独立阶段将直接从指导阶段的结果中发展。在我的小组中发起的研究计划将使用有关使用肽亲和标签将大分子掺入水凝胶中的信息来开发一种新的组织工程方法。在指导阶段鉴定的肽亲和标签将在一端或两端与骨形态发生蛋白2（BMP 2）融合，并作为凝胶原纤维形成的成核位点。修饰的BMP 2蛋白将被整合到肽水凝胶中，并评估材料的相关性质，如蛋白质释放和负载能力。这些新材料将作为组织培养实验的支架材料，促进骨祖细胞的生长和分化。这种特定方法的优点是通过简单混合胶凝组分来配制材料，从而使多种生长因子的掺入变得容易。这将在未来的化身扩展到包括多种生长因子，以更好地模拟天然细胞外基质。 公共卫生相关性：在美国，用于组织工程的生物材料的开发是一个新兴的研究领域，对公共卫生产生了广泛的影响。该领域处于起步阶段，但有可能影响各种疾病的治疗，这些疾病需要损伤组织的再生长，如脊髓损伤或烧伤。该提案描述了一种新的方法来呈现负责指导特定组织生长的蛋白质，用于潜在的治疗用途。

DESCRIPTION (provided by applicant): The development of novel bio-materials for tissue engineering is a burgeoning research field with broad impact on public health in the United States. Advances in the field have the potential to treat myriad medical conditions such as dermal injuries (i.e. lacerations and burns), cardiac trauma, severe spinal injuries, and bone breaks. Currently, most synthetic materials used for tissue engineering are polymers that incorporate growth factors or peptides into a material matrix. The mode of incorporation ranges from covalent (for small peptides) to adsorptive (for larger proteins of interest). The field would be significantly advanced if full-length proteins could be incorporated and displayed within tissue engineering platforms both with high affinity and specificity. Stimulus-responsive peptide hydrogels have received increasing attention in the materials science and tissue engineering communities of late. These types of materials assemble into nano-scale fibers that are hydrated to form rigid gel materials. The advantages of these materials include, facile access to pure monomer units, non- toxicity, injectability, and in certain cases, anti-bacterial properties. What lacks, however, is the ability to incorporate and display functional proteins through high-affinity non-covalent binding interactions. In the mentored phase of the proposed research program, phage display technology will be developed using virus- like particles (VLPs) derived from bacteriophage Q?, a viral nanoparticle principally utilized by the Finn laboratory for myriad bio-technological applications. The proposal aims to develop a platform for the display of peptide libraries on the exterior surfaces of Q? VLPs for use in directed evolution experiments to identify variants that interact specifically with a gel-forming peptide, MAX8. We will display the selected peptides on the surface of Q? VLPs and evaluate how binding affinity and gel-incorporation are correlated. The material properties of the newly synthesized materials will then be rigorously characterized. The independent phase of the proposed research will grow directly from the results of the mentored phase. The research program initiated in my group will use information garnered about the use of peptide affinity tags for incorporation of macromolecules into hydrogels to develop a novel tissue engineering approach. The peptide affinity tags identified in the mentored phase will be fused to bone morphogenetic protein 2 (BMP2) at one or both termini, and act as nucleation sites for gel fibril formation. The modified BMP2 proteins will be integrated into peptide hydrogels and the materials evaluated for relevant properties such as protein release and loading capacity. These new materials will serve as scaffolds in tissue culture experiments to promote the growth and differentiation of osteo-progenitor cells. An advantage of this particular approach is that materials are formulated by simple mixing of gelling components, thus making incorporation of multiple growth factors facile. This will be extended in future incarnations to include multiple growth factors to better mimic the native extracellular matrix. PUBLIC HEALTH RELEVANCE: The development of bio-materials for tissue engineering is a burgeoning research field with broad impact on public health in the United States. The field is in its infancy, but has the potential to affect the treatment of a variety of ailments that require the re-growth of injured tissue such as spinal cord injury or burn trauma. This proposal describes a novel way to present proteins responsible for directing the growth of specific tissues, within injectable gels for potential therapeutic use.