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

[16626]范·威，伯纳德·j .软骨是骨骼末端的光滑组织，能承受巨大的负荷，并使关节活动自如。然而，当软骨因受伤或老化而受损时，就会出现骨关节炎。软骨的再生能力有限，到目前为止，还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，而且发病率呈上升趋势。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）的团队将使用一种新型生物反应器，该反应器安装在离心机内，提供压力脉冲来模拟膝盖在行走过程中承受的负荷，以培养组织。该项目为生物制药行业制造用于再生医学的新组织提供了支持技术。莫尔豪斯大学的非裔美国人的加入有望增加少数族裔本科生和博士生在新兴的生物技术领域攻读学位的机会。关节软骨（AC）是运动关节的结缔组织，具有高度有序的结构，可以承受巨大的负载转移，并且具有易于运动的无摩擦表面。然而，AC很容易受到损伤或老化的损害，导致骨关节炎，并且由于它是神经和无血管的，因此再生能力有限。为了提高健康AC再生的成功率，需要能够同时引入多种刺激的新型生物反应器，包括振荡静水压力，灌注含有软骨因子的新鲜培养基，水凝胶和三层结构培养，以及具有细胞类型和生长因子梯度的3D打印。NSF goal的中心假设是，当有策略地结合(1)MSCs和AChs以最佳比例组织共培养，(2)振荡静水压力模拟体内条件，通过在独特的离心生物反应器中培养完成，(3)使用TGF?Ò1刺激生长因子；(4)含有抗氧化营养成分的水凝胶通过减少活性氧来促进软骨形成；(5)通过基于流动的直接墨水书写的生物制造技术，在细胞类型、力学性能和生长因子上分层构建三层软骨组织。通过更好地理解共同培养和创造健康软骨所需的机械和生化刺激的复杂相互作用，以及允许研究所涉及的细胞机制的新方案，骨关节炎的软骨再生医学将得到显著进展。相关知识将通过使用原子力显微镜来确定组织和细胞弹性模量，并绘制细胞表面β -整合素分布，这些分布与产生健康的ECM有关。这些信息将与组织学和质谱相结合，以充分表征表型表达。重要的是，GOALI的工业合作伙伴Regeneron Pharmaceuticals， Inc.将发挥关键作用，通过qRT-PCR和下一代测序提供全面的伴随基因型信息，以作为培养条件的功能差异评估软骨和成骨mRNA标记物的存在。最后，将获得关于使用新型热敏水凝胶增材制造具有机械性能和材料含量梯度的复合生物结构的新知识。该奖项由CBET部门的生物技术和生化工程项目颁发，由人力资源开发部的历史黑人学院和大学本科项目（HBCU-UP）和工业创新与伙伴关系部门的GOALI项目共同资助。

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.