Introduction: Bone fractures caused by injuries due to aging and stress require reconstruction to restore tissue function. As we age, current grafting-based treatments are limited by supply and donor match rates. Additionally, there are limited options for bone tissue regeneration for bone diseases such as osteoporosis, leaving these individuals with high risks of fractures and health decline. Most bone homeostasis declines due to aging as the tissue undergoes changes in its extracellular matrix (ECM) composition, impacting the bone cells that contribute to bone mineralization and strength. To overcome such limitations in treatments and intervention methods, we will incorporate calcium phosphate and vitamin C to 3D-bioprintable bone ECM for bone regeneration.
Materials and
Methods: For a deeper understanding of the role of aging in bone ECM, we will develop a 3D microphysicological system recapitulating the 3D composition/structure of bone ECM. Specifically, to engineer the 3D bone matrix, 1-5% w/v hydroxyapatite (HA) was dissolved in 4-25% w/v Ascorbic Acid (AA). The HA/AA solution was mixed with collagen at final concentration of 3mg/ml and fibronectin at concentration of 5-25μg/ml. Human primary osteoblasts (HOBs) at density 0.1 million/ml are embedded in the ECM and human umbilical endothelial cells (HUVECs) are used to mimic the bone microvasculature. The structure roughness, porosity, composition, viscoelasticity, and other mechanical properties of the ECM are measured by SEM, FTIR, AFM, and rheology. Finally, the biocompatibility of the ECM and bioprinting on HOBs is measured by apoptosis staining for caspase 3/6 in vitro and by implanting 3D-bioprinted scaffolds into C57Bl/6 mice. Mechanical properties were evaluated in young (passage < 4 cells) and aged (passage >4 cells), as well as in secondary osteoporotic models as established by treatment of devices or mice using glucocorticoids.
Results, Conclusions, and Discussions: Our results from SEM, FTIR, and rheology demonstrated differences in 3D ECM porosity, roughness, stiffness, and viscoelastic behavior. Specifically, AA in the ECM supported the formation of COL bundles and improved matrix mechanical properties for cellular health and regenerative abilities of 3D bioprinted bone scaffolds. Overall our 3D micro physiological systems permit us to fine-tune the microenvironment that cells are sensing and measure the dynamic changes of bone regeneration in vitro, avoiding animal studies. Finally, these platforms will enable us to identify new molecular mechanisms in aging bone diseases and develop new bioengineered tools for bone tissue regeneration, such as implantable bone scaffolds.
Acknowledgements (Optional): This work was supported by Georgetown Funding (91252), the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (R21AR076497), and the Georgetown University Aging and Alzheimer's Research Training NIH T32 Program (GR414768).
