Magnetically‐actuated microcages for cells entrapment, fabricated by laser direct writing via two photon polymerization

The manipulation of biological materials at cellular level constitutes a sine qua non and provocative research area regarding the development of micro/nano‐medicine. In this study, we report on 3D superparamagnetic microcage‐like structures that, in conjunction with an externally applied static magnetic field, were highly efficient in entrapping cells. The microcage‐like structures were fabricated using Laser Direct Writing via Two‐Photon Polymerization (LDW via TPP) of IP‐L780 biocompatible photopolymer/iron oxide superparamagnetic nanoparticles (MNPs) composite. The unique properties of LDW via TPP technique enabled the reproduction of the complex architecture of the 3D structures, with a very high accuracy i.e., about 90 nm lateral resolution. 3D hyperspectral microscopy was employed to investigate the structural and compositional characteristics of the microcage‐like structures. Scanning Electron Microscopy coupled with Energy Dispersive X‐Ray Spectroscopy was used to prove the unique features regarding the morphology and the functionality of the 3D structures seeded with MG‐63 osteoblast‐like cells. Comparative studies were made on microcage‐like structures made of IP‐L780 photopolymer alone (i.e., without superparamagnetic properties). We found that the cell‐seeded structures made by IP‐L780/MNPs composite actuated by static magnetic fields of 1.3 T were 13.66 ± 5.11 folds (p < 0.01) more efficient in terms of cells entrapment than the structures made by IP‐L780 photopolymer alone (i.e., that could not be actuated magnetically). The unique 3D architecture of the microcage‐like superparamagnetic structures and their actuation by external static magnetic fields acted in synergy for entrapping osteoblast‐like cells, showing a significant potential for bone tissue engineering applications

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

In this review, we present the most recent and relevant research that has been done regarding the fabrication of 3D micro/nanostructures for tissue engineering applications. First, we make an overview of 3D micro/nanostructures that act as backbone constructs where the seeded cells can attach, proliferate and differentiate towards the formation of new tissue. Then, we describe the fabrication of 3D micro/nanostructures that are able to control the cellular processes leading to faster tissue regeneration, by actuation using topographical, mechanical, chemical, electric or magnetic stimuli. An in-depth analysis of the actuation of the 3D micro/nanostructures using each of the above-mentioned stimuli for controlling the behavior of the seeded cells is provided. For each type of stimulus, a particular recent application is presented and discussed, such as controlling the cell proliferation and avoiding the formation of a necrotic core (topographic stimulation), controlling the cell adhesion (nanostructuring), supporting the cell differentiation via nuclei deformation (mechanical stimulation), improving the osteogenesis (chemical and magnetic stimulation), controlled drug-delivery systems (electric stimulation) and fastening tissue formation (magnetic stimulation). The existing techniques used for the fabrication of such stimuli-actuated 3D micro/nanostructures, are briefly summarized. Special attention is dedicated to structures’ fabrication using laser-assisted technologies. The performances of stimuli-actuated 3D micro/nanostructures fabricated by laser-direct writing via two-photon polymerization are particularly emphasized

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

The fabrication of complex, reproducible, and accurate micro-and nanostructured interfaces that impede the interaction between material’s surface and different cell types represents an important objective in the development of medical devices. This can be achieved by topographical means such as dual-scale structures, mainly represented by microstructures with surface nanopatterning. Fabrication via laser irradiation of materials seems promising. However, laser-assisted fabrication of dual-scale structures, i.e., ripples relies on stochastic processes deriving from laser–matter interaction, limiting the control over the structures’ topography. In this paper, we report on laser fabrication of cell-repellent dual-scale 3D structures with fully reproducible and high spatial accuracy topographies. Structures were designed as micrometric “mushrooms” decorated with fingerprint-like nanometric features with heights and periodicities close to those of the calamistrum, i.e., 200–300 nm. They were fabricated by Laser Direct Writing via Two-Photon Polymerization of IP-Dip photoresist. Design and laser writing parameters were optimized for conferring cell-repellent properties to the structures, even for high cellular densities in the culture medium. The structures were most efficient in repelling the cells when the fingerprint-like features had periodicities and heights of ≅200 nm, fairly close to the repellent surfaces of the calamistrum. Laser power was the most important parameter for the optimization protocol

3D Biomimetic Magnetic Structures for Static Magnetic Field Stimulation of Osteogenesis

We designed, fabricated and optimized 3D biomimetic magnetic structures that stimulate the osteogenesis in static magnetic fields. The structures were fabricated by direct laser writing via two-photon polymerization of IP-L780 photopolymer and were based on ellipsoidal, hexagonal units organized in a multilayered architecture. The magnetic activity of the structures was assured by coating with a thin layer of collagen-chitosan-hydroxyapatite-magnetic nanoparticles composite. In vitro experiments using MG-63 osteoblast-like cells for 3D structures with gradients of pore size helped us to find an optimum pore size between 20–40 µm. Starting from optimized 3D structures, we evaluated both qualitatively and quantitatively the effects of static magnetic fields of up to 250 mT on cell proliferation and differentiation, by ALP (alkaline phosphatase) production, Alizarin Red and osteocalcin secretion measurements. We demonstrated that the synergic effect of 3D structure optimization and static magnetic stimulation enhances the bone regeneration by a factor greater than 2 as compared with the same structure in the absence of a magnetic field