Development of Protein-Displaying Peptide Hydrogels for Tissue Engineering

摘要 组织工程用新型生物材料的开发是一个影响广泛的新兴研究领域 关于美国的公共卫生。该领域的进展有可能治疗无数的医学 皮肤损伤(即撕裂和烧伤)、心脏损伤、严重脊椎损伤和骨骼等情况 休息一下。目前，用于组织工程的大多数合成材料是结合生长的聚合物 将因子或多肽制成物质基质。结合方式的范围从共价(对于小肽) 到吸附(对于更大的感兴趣的蛋白质)。如果全长蛋白质，这一领域将显著进步。 可以结合并展示在组织工程平台中，具有高亲和力和特异性。 刺激反应性多肽水凝胶在材料科学和组织科学领域受到越来越多的关注。 最近的工程界。这些类型的材料组装成纳米级纤维，这些纤维被水合成 形成刚性凝胶材料。这些材料的优点包括，容易获得纯单体单元，非 毒性、可注射性，在某些情况下，还具有抗菌性能。然而，缺乏的是能够 通过高亲和力的非共价结合作用结合并展示功能蛋白。在 在拟议研究计划的指导阶段，噬菌体展示技术将使用病毒- 来源于噬菌体Q？的类颗粒(VLP)，这是一种主要由芬兰人利用的病毒纳米颗粒 多种生物技术应用的实验室。该提案旨在开发一个平台来展示 Q？VLP外表面的多肽文库，用于定向进化实验 与凝胶形成肽MAX8特异相互作用的变体。我们将在屏幕上显示选定的多肽 并评估结合亲和力和凝胶掺入之间的关系。材料 然后，将严格表征新合成材料的性能。 拟议研究的独立阶段将直接从指导阶段的结果发展起来。 在我的小组中发起的研究计划将使用获得的关于多肽亲和标签的使用的信息 用于将大分子结合到水凝胶中，以开发一种新的组织工程方法。这个 在指导阶段确定的多肽亲和标签将与骨形态发生蛋白2(BMP2)在 一个或两个末端，并作为凝胶原纤维形成的成核点。修改后的BMP2蛋白将是 集成到多肽水凝胶中，并对材料的相关性能进行评估，如蛋白质释放 和装载能力。这些新材料将作为组织培养实验的支架，以促进 骨祖细胞的生长和分化。这种特殊方法的一个优点是，材料 是由凝胶成分简单混合而成，从而使多种生长因子并入 轻而易举。这将在未来的化身中扩展为包括多个增长因素，以更好地模拟原生 细胞外基质。

Abstract 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.