Development of Protein-Displaying Peptide Hydrogels for Tissue Engineering

摘要 新型组织工程生物材料的开发是一个新兴的研究领域，具有广泛的影响 在美国的公共卫生。该领域的进展有可能治疗无数的医学疾病， 皮肤损伤（即撕裂伤和烧伤）、心脏创伤、严重脊柱损伤和骨损伤等疾病 休息.目前，大多数用于组织工程的合成材料是聚合物， 因子或肽到材料基质中。掺入模式范围从共价（对于小肽） 吸附性（对于较大的感兴趣的蛋白质）。如果能在蛋白质中发现全长蛋白质， 可以以高亲和力和特异性掺入并展示在组织工程平台内。 刺激响应性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 最近的工程社区。这些类型的材料组装成纳米级纤维，这些纤维被水合， 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， 毒性、可注射性以及在某些情况下的抗菌性能。然而，缺乏的是能力， 通过高亲和性非共价结合相互作用掺入和展示功能蛋白。在 在拟议的研究计划的指导阶段，噬菌体展示技术将利用病毒- 类病毒颗粒（VLP）来源于噬菌体Q？，这是一种主要由芬兰人利用的病毒纳米颗粒。 实验室的无数生物技术应用。该建议旨在发展一个平台， 在定向进化实验中使用的Q？VLP外表面上的肽库， 与凝胶形成肽MAX 8特异性相互作用的变体。我们将在 QVLP的表面，并评估结合亲和力和凝胶掺入如何相关。材料 然后将严格表征新合成材料的性质。 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肽亲和标签使用的信息 用于将大分子掺入水凝胶中，以开发新型组织工程方法。的 在指导阶段鉴定的肽亲和标记物将与骨形态发生蛋白2（BMP 2）融合， 一个或两个末端，并作为凝胶原纤维形成的成核位点。修饰的BMP 2蛋白将被 并评估材料的相关性质， 和负载能力。这些新材料将作为组织培养实验中的支架，以促进 骨祖细胞的生长和分化。这种特殊方法的优点是， 通过简单混合胶凝组分配制，从而掺入多种生长因子 很容易这将在未来的化身中得到扩展，包括多种生长因子，以更好地模仿原生 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.