3D Differentiation of Mesenchymal Stem Cells in Peptide Amphiphile Matrices

描述(由申请人提供)：能够与成体干细胞相互作用，引导其向特定终末细胞类型分化的新材料将在新兴的再生医学领域发挥重要作用。这项拟议的工作详细介绍了一种在三维支架内诱导人间充质干细胞(HMSCs)分化为成骨细胞系的策略，并将其应用于骨再生。拟议的支架将由多肽两亲性(PA)组成，这是完全由常见生物成分(多肽和脂肪酸)制成的合成材料。众所周知，这里描述的这一家族的PA在水溶液中形成高纵横比的纳米纤维，产生在纳米结构上类似于细胞外基质的纤维凝胶。PA将配备地塞米松(Dex)，地塞米松(Dex)通过可水解肼键合在PA上。地塞米松在二维培养中诱导hMSCs成骨是众所周知的，但一种可注射的三维地塞米松释放凝胶能够在体内分化hMSCs并支持其增殖的报道尚未见报道。一旦地塞米松释放的PA被合成，地塞米松的释放将在没有细胞的情况下从PA凝胶中测量到缓冲液中。酰肼键的零级水解有望以缓慢、持续的方式释放地塞米松。释放动力学确定后，hMSCs将在所建议的地塞米松释放凝胶内进行三维培养，该凝胶将与稀释剂PA构建以提供结构支撑。凝胶的表征将使用电子显微镜、小角X射线散射、流变学和圆二色谱来完成。染色、基因表达和蛋白质表达的组合将被用来量化成骨和矿物质产生的程度。在地塞米松释放PA的同时，还将开发一种展示模拟生长因子骨形态发生蛋白2(BMP-2)的肽序列的PA。建议的BMP-2模拟PA可能与地塞米松释放的PA协同作用，最终产生比单独使用其中任何一种成分更快的骨生成和愈合。将进行几个体外实验来比较这两种成骨方法，这两种方法通过不同的细胞途径发挥作用。如果成功，本文开发和研究的地塞米松释放和BMP-2模拟PA支架可能作为注射材料与hMSCs一起用于人类骨发育。这种治疗可以显著改善因骨缺损、癌骨切除或骨质疏松性骨折而导致骨再生能力严重受损的患者的生活质量。 公共卫生相关性：在人口老龄化的背景下，癌症、创伤或骨质疏松症引起的骨缺陷是一个日益严重的问题。这项拟议的研究将探索一种可注射材料，当与成人间充质干细胞混合时，当人体的自然再生能力受到影响时，这种材料可能能够促进骨再生。这项工作的结果将为未来旨在改善严重骨缺损症患者生活质量的研究提供参考。

DESCRIPTION (provided by applicant): New materials that are capable of interacting with adult stem cells to direct their differentiation toward a certain terminal cell type will play an important role in the emerging field of regenerative medicine. The proposed work details a strategy for inducing the differentiation of human mesenchymal stem cells (hMSCs) into osteoblastic lineage inside a three dimensional scaffold, with applications in bone regeneration. The proposed scaffold will be comprised of peptide amphiphiles (PAs), which are synthetic materials made entirely from common biological components (peptides and fatty acids). PAs of the family described here are well-known to form high aspect ratio nanofibers in aqueous solution, yielding a fibrous gel that resembles the extracellular matrix in nanostructure. PAs will be outfitted with dexamethasone (Dex) bound to the PA via a hydrolysable hydrazone linkage. Dex is well-known to induce osteogenesis of hMSCs in 2-dimensional culture, but an injectable, 3-dimensional, Dex-releasing gel capable of differentiating hMSCs and supporting their proliferation in vivo has never been reported. Once a Dex-releasing PA has been synthesized, Dex release will be measured from the PA gels into buffer in the absence of cells. Zero-order hydrolysis of the hydrazone bond is expected to release Dex in a slow, sustained manner. After release kinetics have been determined, hMSCs will be cultured in three dimensions inside the proposed Dex-releasing gel, which will be constructed with a diluent PA to provide structural support. Characterization of the gel will be accomplished using electron microscopy, small-angle X-ray scattering, rheology, and circular dichroism. A combination of staining, gene expression, and protein expression will be used to quantify the extent of osteogenesis and mineral production. In parallel with the Dex-releasing PA, a PA that displays a peptide sequence mimicking the growth factor bone morphogenic protein 2 (BMP-2) will also be developed. The proposed BMP-2-mimetic PA may function in synergy with the Dex-releasing PA, eventually yielding faster bone production and healing than either component alone. Several in vitro experiments will be performed to compare the two methods of osteogenesis, which function through different cellular pathways. If successful, the Dex-releasing and BMP-2-mimetic PA scaffolds developed and studied here may be used as injectable materials in conjunction with hMSCs for bone development in humans. Such a treatment may drastically improve the quality of life of patients with severely compromised bone regeneration capacities due to bone defects, removal of cancerous bone, or osteoporotic fractures. PUBLIC HEALTH RELEVANCE: Bone defects due to cancer, trauma, or osteoporosis are an increasing problem in the context of an aging population. The proposed research will investigate an injectable material that when mixed with adult mesenchymal stem cells may be capable of promoting bone regeneration when the body's natural regeneration capacity is compromised. The results of this work will inform future studies aimed at improving the quality of life of patients with severe bone defects.