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?Ã’1; (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.