Multiphoton photochemical crosslinking-based fabrication of protein microstructures : studies on mechanical properties, adhesion maturation and traction force

Abstract

Extracellular matrix is the microenvironment which interacts with cells to regulate their activities. Understanding the complexity and the function of cell niche is crucial for future development of cell therapy and regenerative medicines. The extracellular matrix contains a lot of signals, such as the biological and physical signals. Besides them, a great deal of recent evidence shows that the mechanical signal plays a very important role in cell behaviors. Recently we have developed a multiphoton femtosecond laser-based 3D printing platform which fabricates complex structures in minutes. Here, we first used the platform to test a series of fabrication and reagent parameters, such as scanning power, scanning speed, and substrate and photosensitizer concentrations, in precisely controlling the mechanical properties of protein microstructures. Atomic force microscopy was used to measure the reduced elastic modulus of the microstructures. The reduced elastic modulus of the microstructures associated positively and linearly with the scanning power, and nonlinearly with scanning speed, scanning cycle, number and energy density. Some studies modulated stiffness by changing substrate to crosslinker ratio and demonstrated that cells respond to stiffness through generating traction force by cytoskeleton. However in most of these studies both stiffness and elastic modulus were changed simultaneously and the separated roles of stiffness and elastic modulus on cell activities were not fully investigated yet. Here we provide evidence to distinguish the differential roles of stiffness and elastic modulus in cell adhesion maturation. Based on the precise control of elastic modulus by multiphoton femtosecond laser-based 3D printing platform, we successfully decoupled stiffness from elastic modulus to investigate their respective influences on cell adhesion maturation. Results demonstrated that expression of adhesion proteins, particularly integrin alpha v, pFAK and paxillin, responded positively to stiffness, while elastic modulus made no difference on adhesion formation characterized by integrin alpha v, pFAK and paxillin. Finally, the single cell traction forces of rabbit chondrocytes, human dermal fibroblasts, human mesenchymal stem cells, and bovine nucleus pulposus cells (bNPCs) were successfully measured by culturing the cells on micropillar arrays of different stiffness. Results showed that the traction forces of all groups showed positive relationship with stiffness, and that the rabbit chondrocytes and bNPCs generated the highest and lowest traction forces, respectively.