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

1606226 Van Wie，Bernard J.Cartilage，骨骼末端的光滑组织，可以承受巨大的载荷，并在关节连接时提供轻松的运动。然而，当软骨因损伤或老化而受损时，就会发生骨关节炎。软骨再生的能力有限，到目前为止，还没有能够恢复其功能的治疗方法。骨性关节炎影响着许多美国人，而且还在上升。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(一所历史上的黑人学院)的团队将使用一种安装在离心机内的新型生物反应器，离心机提供压力脉冲来模拟膝盖在行走过程中感受到的负荷，以生长组织。该项目为生物制药行业制造再生医学的新组织提供了使能技术。来自莫尔豪斯的非裔美国人的参与预计将增加少数族裔本科生和博士生在新兴生物技术领域寻求学位的机会。关节软骨(AC)是一种运动关节的结缔组织衬里，具有高度有序的结构，可以承受巨大的载荷转移，表面无摩擦，便于运动。然而，AC很容易因损伤或老化而受损，导致骨关节炎，而且由于它是无神经和无血管的，再生能力有限。为了提高健康AC的再生成功率，需要能够同时引入多种刺激的新的生物反应器，包括振荡的静水压力，灌流含有软骨形成因子的新鲜培养液，以及在水凝胶和三层结构中培养，三维打印细胞类型和生长因子的梯度。这一NSF目标的中心假设是，当战略性地结合(1)以最佳比例进行MSCs和ACHS的组织共培养，(2)通过在独特的离心式生物反应器中培养来实现振荡静水压力以模拟体内条件，(3)通过使用转化生长因子1刺激生长因子，(4)抗氧化性营养负载水凝胶被证明通过减少活性氧物种来促进软骨生成，以及(5)通过基于流动的构建物直接墨水写入细胞类型、机械性能和生长因子的3-D来模拟三层软骨组织，将会发生最佳的软骨生成。通过更好地了解共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，以及通过新的方案来研究相关的细胞机制，骨关节炎的软骨再生医学将得到显著的进步。相关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数，并绘制与产生健康的细胞外基质有关的细胞表面β1整合素分布图。这些信息将与组织学和质谱学相结合，以充分表征表型表达。重要的是，Goali的工业合作伙伴Regeneron制药公司将发挥关键作用，通过qRT-PCR和下一代测序提供全面的配套基因信息，以区别评估作为培养条件功能的软骨和成骨mRNA标记的存在。最后，将获得关于使用新型温敏水凝胶添加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部门生物技术和生物化学工程项目共同资助，由人力资源开发部门历史悠久的黑人学院和大学本科项目(HBCU-UP)和产业创新与合作部门的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.