Laser Direct Writing via Two-Photon Polymerization of 3D Hierarchical Structures with Cells-Antiadhesive Properties

Abstract

We report the design and fabrication by laser direct writing via two photons polymerization of innovative hierarchical structures with cell-repellency capability. The structures were designed in the shape of “mushrooms”, consisting of an underside (mushroom’s leg) acting as a support structure and a top side (mushroom’s hat) decorated with micro- and nanostructures. A ripple-like pattern was created on top of the mushrooms, over length scales ranging from several µm (microstructured mushroom-like pillars, MMP) to tens of nm (nanostructured mushroom-like pillars, NMP). The MMP and NMP structures were hydrophobic, with contact angles of (127 ± 2)° and (128 ± 4)°, respectively, whereas flat polymer surfaces were hydrophilic, with a contact angle of (43 ± 1)°. The cell attachment on NMP structures was reduced by 55% as compared to the controls, whereas for the MMP, a reduction of only 21% was observed. Moreover, the MMP structures preserved the native spindle-like with phyllopodia cellular shape, whereas the cells from NMP structures showed a round shape and absence of phyllopodia. Overall, the NMP structures were more effective in impeding the cellular attachment and affected the cell shape to a greater extent than the MMP structures. The influence of the wettability on cell adhesion and shape was less important, the cellular behavior being mainly governed by structures’ topography.

Laser Direct Writing of Dual-Scale 3D Structures for Cell Repelling at High Cellular Density

“…In our previous work reported in [ 37 ], the in vitro tests were carried out using a relatively low cell density, i.e., 5000 cells/sample, which led to about a 75% reduction in the adherence of the cells to the NMP structures, as compared to flat glass surfaces. In the present study, we used a cell density five times higher, i.e., 25,000 cells/sample and we checked if the optimized NMP structures preserved their cell-repellent potential.…”

“…In the last years, LDW via TPP has been successfully used for obtaining 3D architectures, in both micrometric and submicrometric ranges, capable of controlling the attachment of cells [ 37 , 38 , 39 ]. Recently, we have reported hierarchically mushroom-like constructs decorated with micrometric and submicrometric ripple-like features, that were able to impede the cellular attachment, when working at standard (i.e., relatively low) cellular densities in the culture medium, i.e., 5000 cells/sample [ 37 ].…”

“…In the last years, LDW via TPP has been successfully used for obtaining 3D architectures, in both micrometric and submicrometric ranges, capable of controlling the attachment of cells [ 37 , 38 , 39 ]. Recently, we have reported hierarchically mushroom-like constructs decorated with micrometric and submicrometric ripple-like features, that were able to impede the cellular attachment, when working at standard (i.e., relatively low) cellular densities in the culture medium, i.e., 5000 cells/sample [ 37 ]. That study addressed a broader application/concept, that was to obtain biomimetic dual-scale 3D structures with anti-adhesive properties, inspired by the size and geometry of the calamistrum of cribellate spiders [ 33 ], with a deviation in periodicity and height of less than 30%.…”

A Review on Stimuli-Actuated 3D Micro/Nanostructures for Tissue Engineering and the Potential of Laser-Direct Writing via Two-Photon Polymerization for Structure Fabrication

“…LDW via TPP allows the fabrication of highly complex free-form 3D micro/nanostructures, of high interest for scaffolding in tissue engineering and regenerative medicine [21,29,85]. Such structures known as scaffolds can be used for growing artificial living tissue which can be then implanted and integrated into a host natural tissue.…”

“…Such structures known as scaffolds can be used for growing artificial living tissue which can be then implanted and integrated into a host natural tissue. By selecting the appropriate biomaterials for structures' fabrication, LDW via TPP can be used for high-resolution and high-precision fabrication of 3D scaffolds, which allows for high reproducibility of extracellular matrices encountered in the living tissues [21,29,84,85]. Moreover, the near-IR incident wavelengths used in LDW via TPP do not induce cytotoxicity and can, therefore, be used for cells containing and manipulation.…”