High-Throughput Craniofacial tissue engineering

描述（由申请人提供）：高通量技术已经显著改变了基因组学、蛋白质组学和药物发现和制剂等领域。该技术同样可以彻底改变用于组织工程应用的生物材料的发展。该提案的一个基本组成部分是应用和推进我们的全自动化，高通量发现方法，以推动hESC组织工程更接近临床应用。现有的hESC和iPSC方法的两个关键的剩余限制是1）与干细胞分化成功能细胞类型如软骨细胞和成骨细胞相关的低效率和长时间，和2）用于组织工程的现有可降解材料的次优性能（例如机械性能、生物相容性）。因此，我们建议开发高通量策略以快速优化关键细胞群的同质群体的生产，以及提供改善的细胞性能和低炎症的可降解生物材料。因此，我们的具体目标是：目标1。开发高效、快速的方法，将人胚胎干细胞（hESC）和诱导多能干细胞（iPSC）分化为同质的颅面细胞群。我们将使用hESC和iPSC细胞的高通量方法来确定可溶性（生长因子和小分子）和不溶性因子（合成聚合物表面）的最佳组合，这些因子能够将ES细胞转化为颅面前体细胞和完全定向的成骨和软骨细胞。目标二：开发可生物降解、非炎症和机械适当的3D支架系统，可以有效地将颅面细胞输送到损伤部位。将合成透明质酸（软骨）和聚（2-氨基酯）（骨）聚合物的高通量文库，并分别评估其形成凝胶或固体多孔支架的能力。然后评价有利材料的软骨细胞或骨细胞相容性。目的3：评估在目的1和2中开发的组织工程构建体在体内产生软骨和骨的性能。小动物模型将用于测试颅骨临界尺寸缺损中的骨生成，而软骨生成将在皮下进行评价。

DESCRIPTION (provided by applicant): High throughout technologies has already significantly revolutionized fields such as genomics, proteomics, and drug discovery and formulation. This technology can similarly revolutionize the development of biomaterials for tissue engineering applications. A fundamental component of this proposal is to apply and advance our fully automated, high throughput discovery methods to push hESC tissue engineering closer towards clinical applications. Two key remaining limitations of existing hESC and iPSC methods are 1) the low efficiency and long time associated with stem cell differentiation into functional cell types such as chondrocytes and osteoblasts, and 2) suboptimal performance (e.g. mechanical properties, biocompatibility) of existing degradable materials used for tissue engineering. As such, we propose to develop both high throughput strategies to rapidly optimize the production of homogenous populations of key cell populations and degradable biomaterials that provide for improved cellular performance and low inflammation. Accordingly our specific aims are: Aim1. Develop efficient, rapid methods to differentiate human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into homogenous populations of craniofacial cells. We will use high-throughput approaches on hESC and iPSC cells to identify the optimal combinations of soluble (growth factor and small molecules), and insoluble factors (synthetic polymer surfaces) capable of committing ES cells to craniofacial precursor cells and fully committed osteogenic and chondrogenic cells. Aim 2: Develop biodegradable, non-inflammatory and mechanically appropriate 3D scaffold systems that can effectively deliver craniofacial cells to the site of injury. High-throughput libraries of hyaluronic acid (cartilage) and poly(2-amino ester) (bone) polymers will be synthesized and assessed for ability to form gels or solid porous scaffolds, respectively. Favorable materials will then be evaluated for either chondrocyte or bone cell compatibility. Aim 3: Assess performance of tissue engineered constructs developed in Aim 1 and 2 to generate cartilage and bone in vivo. Small animal model will be used to test osteogenesis in a cranial critical sized defect, while chondrogenesis will be evaluated subcutaneously.