Biomimetic Ligand Display in Proteolipobead Hybrid Matrices to Direct Stem Cell Chondrogenesis

ID：MPS/dmr/bmat(7623)1207480 PI：Gilchrist，Lane ORG：CUNY City College标题：在蛋白质微珠杂交基质中显示仿生配体以指导干细胞软骨生成内在优点：模拟干细胞微环境的元素以诱导再生控制是生物材料研究中的主要挑战之一。在主要的环境信号中，参与细胞-细胞相互作用的膜相关配体的存在(例如钙粘附素、Noch)和可溶性因子(例如转化生长因子-β)在细胞表面的隔离尚未在3D培养系统中得到很好的确定。由于3D环境更适合干细胞生态位的生物仿生，我们的长期目标是将微球支持的生物膜系统集成到3D基质中。这将使用一种复合的3D水凝胶支架架构来实现，该架构集成了位于微球支持的生物膜系统中的横向可移动的配体/结合因子，称为蛋白质脂珠。这将第一次允许各种配体/膜结合因子通过具有可控表面密度的新颖分子工程界面以3D形式向干细胞横向移动呈现。因此，这项提议的具体重点是建立一个平台，将横向可移动的N-钙粘连蛋白片段和转化生长因子-β3受体转化生长因子-β-R3受体(即β-葡聚糖)整合到悬浮在3D基质中的蛋白脂球中，以呈现给人骨髓间充质干细胞(HMSCs)。所提出的N-钙粘蛋白和转化生长因子-β-R3显示模式将用于研究N-钙粘蛋白介导的细胞-细胞通讯事件和转化生长因子-β3隔离在体外对MSC软骨形成的影响，并作为这一转型技术的概念验证。这项研究背后的具体假设是，将蛋白脂珠组件引入3D支架是一种可行的仿生手段，可以将配体/结合因子呈现给干细胞，并在干细胞利基中模拟细胞通讯。这些目的是为了检查是否可以实现这种配体呈递模式，并评估对干细胞活性和分化的影响。目的1将N-钙粘蛋白/转化生长因子-β-R3蛋白微球包埋于羧甲基纤维素3D载体中，探讨N-钙粘附素的展示和转化生长因子-β3生长因子的滞留。目的2通过监测骨髓间充质干细胞的活性和软骨分化情况，研究N-钙粘蛋白/转化生长因子-β-R3生物膜微球与人骨髓间充质干细胞的相互作用。BROADER影响：建议的活动对社会的好处是，该研究的贡献可以帮助跨越干细胞生物工程的主要技术障碍，以便人类最终能够指导干细胞再生过程。基本上，干细胞配体/结合因子呈现的整个维度还没有实现，所提出的支架设计可能导致在3D培养中控制干细胞的新的分子方法。此外，这项工作可能会导致软骨修复的创新疗法，这是一个重大的临床问题。为了超越研究成果的影响，我们将实施一种在促进培训和学习的同时推进发现的新机制。这项计划将专门为市中心的初中生和高中生量身定做，旨在激发人们对颜色分子基础的兴趣。这一教育推广活动整合了对CCNY(哈莱姆)社区受制裁和叛变的街头涂鸦艺术的UV-VIS颜料识别实地研究。将创建一个同时运行的在线染料和颜料数据库，该数据库将使用计算化学程序包进一步增强分子结构和与吸收相关的分子轨道可视化。在拟议的研究中，完全相同的颜料、染料和荧光团在涂料中具有双重用途，并作为分子报告基团。因此，参与的实验室处于有利地位，可以为对颜色的分子基础感兴趣的当地学生提供沿着这条学习途径的初步研究环境。干细胞生物材料工程的这项研究成果将通过会议上的口头陈述和CCNY的定期学生研究海报研讨会广泛传播。

ID: MPS/DMR/BMAT(7623) 1207480 PI: Gilchrist, Lane ORG: CUNY City CollegeTitle: Biomimetic Ligand Display in Proteolipobead Hybrid Matrices to Direct Stem Cell ChondrogenesisINTELLECTUAL MERIT: Mimicking elements of stem cell microenvironments to elicit regenerative control is one of the major challenges in biomaterials research. Of the major environmental cues, the presentation of membrane associated ligands involved in cell-cell interactions (e.g. cadherin, notch) and the sequestering of soluble factors (e.g. transforming growth factor-beta) at the cell surface have not yet been well established in 3D culture systems. As the 3D context is more appropriate for biomimicry of the stem cell niche, our long-term goal is to integrate microsphere-supported biomembrane systems into 3D matrices. This will be accomplished using a composite 3D hydrogel scaffold architecture that integrates laterally mobile ligands/bound factors positioned in microsphere-supported biomembrane systems known as proteolipobeads. For the first time, this will allow for the laterally mobile presentation of various ligands/membrane-bound factors to stem cells within a 3D format via novel, molecularly engineered interfaces with controlled surface densities. Thus, the specific focus of this proposal is to build a platform that incorporates laterally mobile N-Cadherin fragments and the TGF-beta3 receptor, TGF-beta-R3 (i.e., beta-glycan), in proteolipobeads suspended in a 3D matrix for presentation to human mesenchymal stem cells (hMSCs). The proposed mode of N-cadherin and TGF-beta-R3 display will be used to investigate the effects of N-cadherin-mediated cell-cell communication events and TGF-beta3 sequestering on MSC chondrogenesis in vitro and serve as the proof-of-concept for this transformative technology. The specific hypothesis behind the proposed research is that the introduction of proteolipobead assemblies into 3D scaffolds is a viable biomimetic means to present ligands/bound factors to stem cells and mimic cellular communication in the stem cell niche. The objectives are designed to examine if this mode of ligand presentation can be achieved and to assess the effects on stem cell viability and differentiation. Objective 1 is to embed N-cadherin/TGF-beta-R3 proteolipobeads in carboxymethylcellulose 3D constructs and probe N-Cadherin display and TGF-beta3 growth factor sequestration. Objective 2 is to investigate the interaction between N-cadherin/TGF-beta-R3 biomembrane-microspheres and hMSCs by monitoring MSC viability and chondrogenic differentiation.BROADER IMPACTS: The benefits to society of the proposed activity are that contributions from the research could help cross major technological barriers in stem cell bioengineering, so that humanity can ultimately guide stem cell regeneration processes. Essentially a whole dimension in stem cell ligand/bound factor presentation has not yet been achieved, and the proposed scaffold design may lead to new molecular methods for control of stem cells in 3D culture. In addition, this work may lead to innovative therapies for cartilage repair, which is a significant clinical problem. To go beyond the impact of the research outcomes, we will implement a new mechanism for advancing discovery while promoting training and learning. This program will be specifically tailored to inner-city middle and high school students and aimed at stimulating a fascination with the molecular basis for color. This educational outreach integrates UV-VIS pigment identification field studies of sanctioned and renegade street graffiti art in the CCNY (Harlem) neighborhood. A concurrently running online dye and pigment database will be created, that will be further augmented with molecular structure and absorption-relevant molecular orbital visualizations using computational chemistry packages. The very same pigments, dyes, and fluorophores have dual uses in paints and as molecular reporter groups in the proposed studies. Thus, the participating labs are well positioned to provide the initial research setting along this learning pathway for local students who take an interest in the molecular basis of color. The results of this research in stem cell biomaterial engineering will be broadly disseminated via oral presentations at conferences, and in regular student research poster symposia at CCNY.