Abstract
Engineering 3D microstructures with predetermined properties is critical for stem cell niche studies. We have developed a multiphoton femtosecond laser-based 3D printing platform, which generates complex protein microstructures in minutes. Here, we used the platform to test a series of fabrication and reagent parameters in precisely controlling the mechanical properties of protein micropillars. Atomic force microscopy was utilized to measure the reduced elastic modulus of the micropillars, and transmission electron microscopy was used to visualize the porosity of the structures. The reduced elastic modulus of the micropillars associated positively and linearly with the scanning power. On the other hand, the porosity and pore size of the micropillars associated inversely and linearly with the scanning power and reagent concentrations. While keeping the elastic modulus constant, the stiffness of the micropillars was controlled by varying their height. Subsequently, 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. Our results showed that the traction forces of all groups showed positive relationship with stiffness, and that the chondrocytes and bNPCs generated the highest and lowest traction forces, respectively.
Highlights
Materials on a variety of surfaces with a resolution of ≤ 50 nm
When the frame size was changed from 512*512 to 2048*2048, this resulted in a dramatic increase in the energy density per pixel from 2.48 nJ/μ m2 to 39.68 nJ/μ m2, which resulted in a saturation of the reduced elastic modulus of ~50 kPa (Fig. 1E)
The protein micropillars showed inverse linear association with porosity, suggesting that the control of reduced elastic modulus is likely to be mediated by the change in porosity of the microstructures
Summary
Materials on a variety of surfaces with a resolution of ≤ 50 nm. despite the high resolution, this process is confined to two-dimensional applications. The processing conditions are free of any harsh organic solvent or cross-linker, which can deteriorate biocompatibility, and a resolution of ~100 nm can be accomplished due to the two-photon absorption capability This method can achieve the nano-sized features that are necessary to address the current tissue engineering challenges[3]. We recently described a multiphoton-based fabrication platform of user-defined protein microstructures and micropatterns with submicron features, controllable voxels, morphology, topology and porosity[15], and demonstrated that it can be used as a simple “write-and-seed” miniaturized culture platform for cell niche studies It has the major advantage of being a one-step simple and biocompatible system as it is mould-, coating- and label-free. Using micropillar arrays with a fixed modulus but different stiffness, we demonstrated the application of this platform in the measurement of single cell traction forces in multiple cell types
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