GOALI: Enhancing Cartilage Tissue Engineering through Synergistic Influence of Co-Culture, Mechano-Chemical Factors, and 3D Printed Scaffolds in a Novel Centrifugal Bioreactor

1606226货车Wie，Bernard J. Carnival，骨骼末端的光滑组织，可以承受巨大的负荷，并提供关节活动的便利。然而，当软骨因损伤或老化而受损时，就会发生骨关节炎。软骨再生能力有限，迄今为止，没有能够恢复其功能的治疗方法。骨关节炎影响着许多美国人，并且呈上升趋势。在这个项目中，来自华盛顿州立大学、Regeneron制药公司、和莫尔豪斯学院，一所历史悠久的黑人学院，将使用一种新型的生物反应器，该生物反应器安装在离心机内，离心机提供压力脉冲，以模拟膝盖在行走时所承受的负荷，以生长组织。 该项目为生物制药行业提供了制造再生医学新组织的技术。参与非裔美国人从莫尔豪斯预计将增加机会，少数民族本科生和博士生寻求学位的新兴领域的生物技术。关节软骨（AC），结缔组织内衬运动关节，具有高度有序的结构，以承受巨大的负荷转移和无摩擦表面，以方便运动。然而，AC易受损伤或老化引起的病变的影响，导致骨关节炎，并且由于其是无神经和无血管的，因此再生能力有限。为了提高再生健康AC的成功率，需要能够同时引入多种刺激的新生物反应器，包括振荡静水压力，用含有软骨形成因子的新鲜培养基灌注，以及在水凝胶和三层结构中培养，用细胞类型和生长因子的梯度3D打印。在这个NSF目标的中心假设是，最佳的软骨形成将发生时，战略性地结合（1）组织共培养的MSC和乙酰胆碱在最佳比例，（2）振荡静水压力，以模拟体内条件下，通过培养在一个独特的离心生物反应器，（3）生长因子刺激，通过使用TGF？1;（4）显示出通过减少活性氧来增强软骨形成的负载抗氧化剂营养品的水凝胶，和（5）通过用基于流动的直接墨水书写的在细胞类型、机械性质和生长因子方面渐变的3-D构建体的生物制造来模拟三层软骨组织。骨关节炎的软骨再生医学将通过改善对创造健康软骨所需的共培养、机械和生化刺激的复杂相互作用的理解，以及通过允许研究所涉及的细胞机制的新协议，来显著推进。相关的知识将通过使用原子力显微镜来确定组织和细胞的弹性模量，并映射细胞表面β 1-整合素的分布，涉及生产健康的ECM。该信息将与组织学和质谱法相结合，以充分表征表型表达。重要的是，GOALI的工业合作伙伴Regeneron Pharmaceuticals，Inc.将通过qRT-PCR和下一代测序提供全面的伴随基因型信息，以区别评估软骨形成与成骨mRNA标记物作为培养条件的函数的存在，从而发挥关键作用。最后，该奖项由CBET分部生物技术和生物化学工程项目颁发，由历史上的黑人学院和大学本科生项目（HBCU-UP）共同资助。人力资源开发司和工业创新和伙伴关系司的GOALI方案提供了支助。

1606226 Van Wie, Bernard J.Cartilage, the smooth tissue at the end of bones, can withstand enormous loads and provides ease of motion in articulating joints. However, when cartilage is damaged due to injury or aging, osteoarthritis develops. Cartilage has a limited ability to regenerate and to date, no treatment capable of restoring its function exists. Osteoarthritis affects many Americans and is on the rise. In this project, a team from Washington State University, Regeneron Pharmaceuticals, Inc., and Morehouse College, a Historically Black College, will use a novel bioreactor housed within a centrifuge that supplies pulses of pressure to simulate loads experienced by knees during walking for growing the tissue. The project provides enabling technology for the biopharmaceutical industry for manufacturing new tissues for regenerative medicine. Involving African Americans from Morehouse is expected to enhance opportunities for minority undergraduate and PhD students seeking degrees in the burgeoning field of biotechnology.Articular cartilage (AC), a connective tissue lining moving joints, has a highly ordered structure for withstanding enormous load transfers and a frictionless surface for ease of motion. However, AC is vulnerable to lesions due to injury or aging, leading to osteoarthritis, and has a limited ability to regenerate because it is aneural and avascular. To improve success in regenerating healthy AC, new bioreactors capable of introducing multiple stimuli simultaneously, including oscillating hydrostatic pressures, perfusing with fresh medium containing chondrogenic factors, and culturing in hydrogels and tri-layered constructs, 3D printed with a gradient of cell types and growth factors, are needed. The central hypothesis in this NSF GOALI is that optimal chondrogenesis will occur when strategically combining (1) tissue co-culture of MSCs and AChs at optimum ratios, (2) oscillating hydrostatic pressures to mimic in vivo conditions, accomplished by cultivating in a unique centrifugal bioreactor, (3) growth factor stimulation through use of TGF?Ò1; (4) antioxidant nutraceutical laden hydrogels shown to enhance chondrogenesis by reducing reactive oxygen species, and (5) mimicry of the tri-layered cartilage tissue through biomanufacturing with flow-based direct-ink-writing of 3-D constructs gradated in cell type, mechanical properties and growth factor. Cartilage regenerative medicine for osteoarthritis will be advanced significantly by improved understanding of the complex interplay of co-culture and mechanical and biochemical stimuli needed to create healthy cartilage, and by new protocols that will allow study of the cellular mechanisms involved. Relevant knowledge will be gained by using atomic force microscopy to determine tissue and cellular elastic moduli, and to map cell surface beta1-integrin distributions that are implicated in producing healthy ECM. This information will be combined with histology and mass spectrometry to fully characterize phenotypic expression. Importantly, the GOALI industrial partner, Regeneron Pharmaceuticals, Inc. will play a key role by providing comprehensive companion genotypic information via qRT-PCR and next generation sequencing to differentially assess the presence of chondrogenic versus osteogenic mRNA markers as a function of culture conditions. Finally, new knowledge will be gained about additive manufacturing of composite bio-constructs with mechanical property and material content gradients using novel thermosensitive hydrogels.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Historically Black Colleges and Universities Undergraduate Program (HBCU-UP)of the Division of Human Resource Development and by the GOALI Program of the Division of Industrial Innovation and Partnerships.