Craniofacial tissue engineering with human embryonic an*

描述（由申请人提供）：我们建议使用我们在生物材料开发方面建立的和有据可查的历史，作为组织替代的支架，作为颅面组织再生中干细胞方法的平台。这项工作的主要假设包括：i）胚胎来源的人干细胞可以分化成颅面组织产生细胞，例如成骨细胞和软骨细胞，ii）高通量技术可以用于筛选大量的培养基和聚合物候选物，用于组织工程支架的潜在用途，和iii）干细胞与三维聚合物支架的适当组合可用于生产复杂的颅面组织。为了验证这些假设，我们提出以下几点：目的1：研究人胚胎干细胞在体外向颅面组织前体细胞的分化。我们以前的工作调查在体外分化成各种细胞类型和组织的人胚胎干细胞将继续评估分化成颅面组织形成所需的细胞类型的必要条件。 目的2：利用高通量筛选技术研究人类干细胞与聚合物表面的相互作用。最近，我们已经开发出了使用纳升级微阵列的各种单体和聚合物的细胞/聚合物相互作用的快速筛选技术。将通过细胞增殖和颅面组织的组织特异性标志物的存在来评估成功。目标3：评估分化和未分化的人胚胎干细胞接种聚合物支架结合各种基因和生长因子的体外组织生产。支持人类干细胞分化为颅面组织的聚合物材料将被制成三维支架，其中包含各种基因和生长因子，并通过各种标记物的组织学染色进行分析。目标4：来自先前目标的最佳候选物将用分化和未分化的细胞接种，并皮下植入或植入大鼠的临界尺寸缺损模型中。无胸腺大鼠的皮下模型将用于评估体内环境中干细胞接种支架的组织产生，包括细胞分化为多种表型的支架。无胸腺大鼠的颅骨缺损模型是临床相关模型，通常用作骨再生的测量。

DESCRIPTION (provided by applicant): We propose to use our established and well documented history in the development of biomaterials as scaffolding for tissue replacement as a platform for stem cell approaches in craniofacial tissue regeneration. The governing hypotheses of this work include: i) that human stem cells of embryonic origin can be differentiated into craniofacial tissue producing cells such as osteoblasts and chondrocytes, ii) that highthroughput techniques can be used for the screening of large numbers of media and polymer candidates for potential use with tissue engineering scaffolds, and iii) that the appropriate combination of stem cells with 3-dimensional polymer scaffolds can be used for the production of complex craniofacial tissues. To test these hypotheses, we propose the following: Aim 1: Examine the in vitro differentiation of human embryonic stem cells into precursors of craniofacial tissues. Our previous work investigating the in vitro differentiation of human embryonic stem cells into various cell types and tissues will be continued to assess conditions necessary for differentiation into cell types necessary for craniofacial tissue formation. Aim 2: Investigate the interaction of human stem cells with polymer surfaces using high-throughput screening technology. Recently, we have developed techniques for the rapid screening of cell/polymer interactions using nanoliter-scale microarrays of various monomers and polymers. Success will be assessed through cell proliferation and the presence of tissue specific markers for craniofacial tissues. Aim 3: Assess in vitro tissue production by differentiated and undifferentiated human embryonic stem cell seeded polymeric scaffolds incorporating various genes and growth factors. Polymeric materials that support the differentiation of human stem cells into craniofacial tissues will be fabricated into 3-dimensional scaffolds incorporating various genes and growth factors and analyzed by histological staining of various markers. Aim 4: Optimal candidates from the previous aims will be seeded with differentiated and undifferentiated cells and implanted either subcutaneously or in a critical sized defect model in rats. The subcutaneous model in athymic rats will be used to assess tissue production by stem cell seeded scaffolds in an in vivo environment, including scaffolds with cells differentiated into multiple phenotypes. The cranial defect model in athymic rats is a clinically relevant model that is commonly used as a measure of bone regeneration.