Microengineered Platforms for High-throughput Characterization of Cellular Microenvironments

骨关节炎是最常见的关节炎形式，在加拿大每10个人中就有1人受其影响。每年治疗关节炎的总费用估计为330亿美元。同样，加拿大每年有数百万患者接受骨畸形矫正手术。组织工程试图提供一种使用干细胞的替代治疗方法。来源于骨髓的间充质干细胞保留了向脂肪细胞、心肌细胞、骨细胞和软骨细胞分化的多系潜能。局部微环境的物理性质对间充质干细胞的分化起着重要的作用。许多研究旨在将干细胞与生物材料结合起来进行软骨和骨再生，但迄今为止，还没有研究系统地研究了在三维微环境中控制干细胞分化的物理特性的组合因素。在三维细胞微环境中，细胞通过处理各种化学和物理信号与周围环境相互作用。为了控制干细胞的细胞微环境，水凝胶因其高含水量、生物相容性和类似天然组织的机械特性而引起了人们的极大兴趣。然而，水凝胶的各种参数与控制干细胞的命运有关；参数的优化是必不可少的。因此，在一次实验中测试多个参数的高通量筛选技术，将有助于对水凝胶生成的细胞微环境进行系统的检测和优化。**高通量筛选技术是一种使用机器人液体处理设备快速制造化学、遗传和药理学材料微阵列并以高通量方式进行测试的方法。在本研究项目中，我们采用该技术开发了一种水凝胶微阵列，在标准显微镜载玻片上打印了数百微米大小的功能水凝胶点（直径：~500µm，厚度：~100µm）。我们将使用海藻酸盐水凝胶，它可以在紫外光下聚合。我们将测试27种海藻酸盐水凝胶的组合，每种组合有三种不同的参数和三种不同的条件来控制每种参数的物理性质。我们将使用先进的表征技术，如原子力显微镜和扫描电子显微镜来表征物理性质，如微观结构，刚度和附着力。开发的海藻酸盐微阵列将用于优化物理特性对间充质干细胞分化的影响。**虽然与生物反应相关的分子机制尚未明确，但该技术可能广泛应用于细胞微环境研究。此外，在这个程序中确定的物理性质可以用作设计参数工程新的生物材料和组织再生的微环境。在这个项目中，我们建议设计和应用高通量筛选技术来鉴定细胞-微环境相互作用，增加干细胞向骨和软骨分化。这项研究的结果将有很大的潜力对每年接受骨和软骨修复手术的大量患者产生积极的影响。因此，加拿大的骨科社区将受益于由成骨和软骨生物材料组成的新治疗技术，这些生物材料不仅支持，而且还诱导组织形成。开发的平台也将适用于许多组织再生应用，如心血管和神经组织，并有利于加拿大的生物材料和组织工程研究。

Osteoarthritis is the most common form of arthritis, affecting 1 out of every 10 people in Canada. The total cost of arthritis is estimated at $33 billion dollars per year. Similarly, millions of patients in Canada undergo procedures to correct bone deformities each year. Tissue engineering seeks to provide an alternate therapeutic approach using stem cells. Mesenchymal stem cells derived from bone marrow retain a multi-lineage potential to differentiate to fat cells, heart muscle cells, bone cells, and cartilage cells. The physical properties of local microenvironments will play an important role in the differentiation of mesenchymal stem cell. A number of studies have been aimed at combining stem cells with biomaterials for cartilage and bone regeneration, but to date, no study has systematically examined the combinatorial factors to control the physical properties for stem cell differentiation in a 3-D microenvironment. In the 3-D cellular microenvironment, cells interact with their surroundings by processing various chemical and physical signals. To control cellular microenvironments for stem cells, hydrogels have attracted great interests due to high water content, biocompatibility, and mechanical properties resembling natural tissues. However, various parameters of hydrogels are associated with controlling stem cell fate; the optimization of the parameters is essential. Therefore, a high-throughput screening technology to test many parameters in one experiment will facilitate the systematic examination and the optimization of cellular microenvironments generated by hydrogels.**High-throughput screening technology is a method of using robotic liquid handling devices to quickly fabricate microarrays of chemical, genetic, and pharmacological materials and conduct tests in a high-throughput manner. In this research program, we adopt this technology to develop a hydrogel microarray with hundreds of micrometer-sized functional hydrogel spots (diameter: ~500µm; thickness: ~100µm) printed on a standard microscope slide. We will use alginate hydrogels that can be polymerized by ultra violet light. We will test 27 combinations of alginate hydrogels with three different parameters and three different conditions of each parameter that control physical properties. We will characterize the physical properties, such as microstructure, stiffness, and adhesion, using advanced characterization techniques such as atomic force microscopy and scanning electron microscopy. The developed alginate microarray will be used to optimize the effects of physical properties on mesenchymal stem cell differentiation.**Although the molecular mechanisms associated with the biological responses have yet to be clarified, such technology may be widely applicable in cell-microenvironment research. Also, the physical properties identified in this program could be used as design parameters for engineering new biomaterials and microenvironments for tissue regeneration. In this program, we propose to engineer and apply high-throughput screening technology for the identification of cell-microenvironment interactions that increase stem cell differentiation into the bone and cartilage. The results of this research will have high potential to positively affect the large number of patients that undergo procedures to repair bone and cartilage each year. Thus, the orthopedic community in Canada would benefit from the new treatment techniques composed of osteogenic and chondrogenic biomaterials that not only support, but also induce tissue formation. The developed platforms will also be applicable to numerous tissue regeneration applications, such as cardiovascular and nerve tissues and beneficial for biomaterials and tissue engineering research in Canada.