Studying neural stem cell (NSC) behavior in 3D presents a pathway for understanding neurodegeneration caused by aging or disease. Our lab has developed a granular scaffold formed from microgels to study the impact of mechanobiology on aging in 3D model systems, however, temporal changes to this system are difficult to evaluate only via immunocytochemistry. NSCs typically require multiple markers to thoroughly identify their status via flow cytometry. Therefore, this project seeks to develop a degradable-on-demand system to release NSCs from the scaffolds at specific times and characterize them via flow cytometry. The incorporation of matrix metalloproteinase (MMP)-sensitive peptides could provide a more adaptable platform for
in vitro studies.
1 While NSCs clearly express some MMPs, particularly MMP-2 and MMP-9, there is not clear evidence that NSCs express MMP-1.
2 In this study, we hypothesize that MMP-1 sensitive granular scaffolds will enable degradation-on-demand via external MMP-1 addition, facilitating ease of study of NSC responses to the microenvironment without early reorganization by the cells. Polyethylene glycol granular scaffolds were fabricated to mimic mechanical properties of the brain, using an MMP-1 sensitive peptide in the crosslinker.
1, 3, 4 Microgels were characterized for swelling, size, and polydispersity to ensure uniformity. Granular scaffolds are assessed through rheology to determine shear modulus, and degradation is analyzed by measuring mass loss and cell collection in response to added MMP-1. The NSCs were checked for MMP-1 expression and then embedded within the scaffolds to evaluate material property effects on multipotency and mechanotransduction. As controls, we have previously found that nondegradable granular scaffolds, with a shear modulus of 0.25 kPa, supported NSC potency. The degradable scaffold will target a shear modulus in the range of 0.033-0.33 kPa to match that of standard brain tissue shear moduli.
5, 6 Cell number collected, and potency of the cells will be assessed at timepoints of 1, 3, 7, and 14 days. Ultimately, the development of this scaffold will allow for temporal assessment of cell response to 3D environments providing detailed results of the cellular properties over time.
(1) Lutolf, M. P.; Lauer-Fields, J. L.; Schmoekel, H. G.; Metters, A. T.; Weber, F. E.; Fields, G. B.; Hubbell, J. A. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics. Proc Natl Acad Sci U S A 2003, 100 (9), 5413-5418. DOI: 10.1073/pnas.0737381100 From NLM.
(2) Fujioka, H.; Dairyo, Y.; Yasunaga, K.; Emoto, K. Neural functions of matrix metalloproteinases: plasticity, neurogenesis, and disease. Biochem Res Int 2012, 2012, 789083. DOI: 10.1155/2012/789083 From NLM.
(3) Patterson, J.; Hubbell, J. A. Enhanced proteolytic degradation of molecularly engineered PEG hydrogels in response to MMP-1 and MMP-2. Biomaterials 2010, 31 (30), 7836-7845. DOI: https://doi.org/10.1016/j.biomaterials.2010.06.061.
(4) Leight, J. L.; Tokuda, E. Y.; Jones, C. E.; Lin, A. J.; Anseth, K. S. Multifunctional bioscaffolds for 3D culture of melanoma cells reveal increased MMP activity and migration with BRAF kinase inhibition. Proceedings of the National Academy of Sciences 2015, 112 (17), 5366-5371. DOI: doi:10.1073/pnas.1505662112.
(5) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 2006, 126 (4), 677-689. DOI: 10.1016/j.cell.2006.06.044 From NLM Medline.
(6) Qiao, Y.; Gong, J.; Jin, Z.; Tu, Y.; Yang, X. An optimized method of culturing neurons based on polyacrylamide gel. Biophys Rep 2024, 10 (1), 41-47. DOI: 10.52601/bpr.2023.230033 From NLM.
