Introduction: A key challenge in stem cell research is the ability to direct stem cell growth and differentiation towards specific tissue lineages. While complex interactions between soluble factors and extracellular matrix (ECM) have been investigated extensively, physical properties such as substrate elasticity and stiffness are only recently gaining attention as tools for influencing stem cell differentiation and proliferation. It has been demonstrated that substrate stiffness can be used to direct mesenchymal stem cell (MSC) differentiation into mature neuronal, muscle and bone tissues. In this study, we hypothesized that increasing substrate stiffness would improve MSC attachment and proliferation on chitosan fibers.
Materials and Methods: Chitosan fibers were produced by extrusion of chitosan solutions into aqueous ammonia, followed by air drying. Fiber stiffness was modified through various combinations of thermal annealing and chemical crosslinking. Fibers were either dried at room temperature, annealed at 195 °C, or crosslinked with heparin. Monotonic tensile testing was done to determine tensile strength, elastic modulus, and breaking strain of processed fibers. Ovine bone marrow-derived MSCs were isolated, characterized and cultured on fibers with stiffness values of 7, 20, 91 and 128 MPa for 9 days. MSC proliferation was assessed using Alamar Blue, viability was evaluated using Calcein AM and cytoskeletal organization was visualized using rhodamine-phalloidin.
Results and Discussion: MSCs maintained more than 95% cell viability on all fibers during the culture. Results also demonstrated that MSC attachment and proliferation rate increased in proportion to fiber stiffness. Fibers with the lowest mechanical properties, those dried at room temperature, exhibited a lower rate of cell proliferation as compared to cells seeded on fibers with the highest mechanical properties, i.e. annealed at 195ºC. Interestingly, the covalent incorporation of heparin onto fibers reduced fiber stiffness but improved MSC attachment and proliferation, probably through growth factor and ECM protein binding. Actin fiber density and alignment increased with chitosan fiber stiffness.
Conclusion: Results demonstrated that increasing chitosan fiber stiffness significantly improved MSC attachment, proliferation and cytoskeletal organization. Growing MSC on chitosan fibers with tunable stiffness may provide an alternate method for influencing MSC performance in tissue engineering scaffolds.
Acknowledgment: The authors would like to thank the Perri Foundation for partial funding of this project through the Department of Cardiovascular Surgery at Children’s Hospital of Michigan.