High-Throughput Craniofacial tissue engineering

描述(由申请人提供)：高通量技术已经使基因组学、蛋白质组学以及药物发现和配方等领域发生了重大变革。这项技术同样可以革命性地开发用于组织工程应用的生物材料。这项建议的一个基本组成部分是应用和推进我们的全自动、高通量的发现方法，以推动hESC组织工程更接近临床应用。现有的hESC和IPSC方法的两个主要局限性是：1)干细胞分化为软骨细胞和成骨细胞等功能细胞类型的效率低且时间长；2)用于组织工程的现有可降解材料的性能(如机械性能、生物相容性)不佳。因此，我们建议开发高通量策略，以快速优化关键细胞群体的同质群体的生产，并开发可降解生物材料，以提供更好的细胞性能和低炎症。因此，我们的具体目标是：Aim1。开发高效、快速的方法将人类胚胎干细胞(HESCs)和诱导的多能干细胞(IPSCs)分化为同源的颅面细胞群。我们将在hESC和IPSC细胞上使用高通量方法来确定能够将ES细胞定向到颅面部前体细胞和完全定向成骨和软骨细胞的最佳组合。目的2：研制可生物降解、非炎症性、机械适宜性的三维支架系统，有效地将颅面细胞输送至损伤部位。将合成高通量的透明质酸(软骨)和聚(2-氨基酯)(骨)聚合物文库，并分别评估其形成凝胶或固体多孔支架的能力。然后将对有利的材料进行软骨细胞或骨细胞相容性评估。目的3：评估目标1和目标2中开发的组织工程化构建物在体内生成软骨和骨的性能。小动物模型将被用来测试颅骨关键大小的缺损中的成骨，而软骨生成将被评估在皮下。

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.