High-Throughput Craniofacial tissue engineering

描述（由申请人提供）：高科技已经给基因组学、蛋白质组学和药物发现和配方等领域带来了重大变革。这项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，高通量发现方法，推动hESC组织工程更接近临床应用。现有hESC和iPSC方法的两个主要限制是：1)干细胞分化为功能细胞类型（如软骨细胞和成骨细胞）的效率低且时间长；2)用于组织工程的现有可降解材料的性能不理想（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和可降解生物材料的生产，从而提高细胞性能和降低炎症。因此，我们的具体目标是：开发高效、快速的方法将人类胚胎干细胞（hESCs）和诱导多能干细胞（iPSCs）分化为同质的颅面细胞群体。我们将对hESC和iPSC细胞使用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，这些因子能够将胚胎干细胞转化为颅面前体细胞和完全转化为成骨细胞和软骨细胞。目标2：开发可生物降解、非炎症性和机械适宜的3D支架系统，可以有效地将颅面细胞运送到损伤部位。将分别合成高通量透明质酸（软骨）和聚（2-氨基酯）（骨）聚合物文库，并评估其形成凝胶或固体多孔支架的能力。然后评估有利材料的软骨细胞或骨细胞相容性。目的3：评估目的1和2中开发的组织工程构建物在体内生成软骨和骨的性能。小动物模型将用于测试颅骨临界尺寸缺陷的成骨，而软骨形成将进行皮下评估。

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. PUBLIC HEALTH RELEVANCE: We believe that 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 and iPSC tissue engineering closer towards craniofacial clinical applications.