A major limitation of existing 3D implantable structures for bone tissue engineering is that most of the cells rapidly attach on the outer edges of the structure, restricting the cells penetration into the inner parts and causing the formation of a necrotic core. Furthermore, these structures generally possess a random spatial arrangement and do not preserve the isotropy on the whole volume. Here, we report on the fabrication and testing of an innovative 3D hierarchical, honeycomb-like structure (HS), with reproducible and isotropic arhitecture, that allows in ‘volume’ migration of osteoblasts. In particular, we demonstrate the possibility to control the 3D spatial cells growth inside these complex architectures by adjusting the free spaces inside the structures. The structures were made of vertical microtubes arranged in a mulitlayered configuration, fabricated via laser direct writing by two photons polymerization of the IP-L780 photopolymer. In vitro tests performed in MG-63 osteoblast-like cells demonstrated that the cells migration inside the 3D structures is conducted by the separation space between the microtubes layers. Specifically, for layers separation between 2 and 10 μ m, the cells gradually penetrated between the microtubes. Furthermore, these structures induced the strongest cells osteogenic differentiation and mineralization, with ALP activity 1.5 times stronger, amount of calcified minerals 1.3 times higher and osteocalcin secretion increased by 2.3 times compared to the other structures. On the opposite, for layers separation less than 2 μ m and above 10 μ m, the cells were not able to make interconnections and exhibited poor mineralization ability.

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.

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.

We reported on three-dimensional (3D) superparamagnetic scaffolds that enhanced the mineralization of magnetic nanoparticle-free osteoblast cells. The scaffolds were fabricated with submicronic resolution by laser direct writing via two photons polymerization of Ormocore/magnetic nanoparticles (MNPs) composites and possessed complex and reproducible architectures. MNPs with a diameter of 4.9 ± 1.5 nm and saturation magnetization of 30 emu/g were added to Ormocore, in concentrations of 0, 2 and 4 mg/mL. The homogenous distribution and the concentration of the MNPs from the unpolymerized Ormocore/MNPs composite were preserved after the photopolymerization process. The MNPs in the scaffolds retained their superparamagnetic behavior. The specific magnetizations of the scaffolds with 2 and 4 mg/mL MNPs concentrations were of 14 emu/g and 17 emu/g, respectively. The MNPs reduced the shrinkage of the structures from 80.2 ± 5.3% for scaffolds without MNPs to 20.7 ± 4.7% for scaffolds with 4 mg/mL MNPs. Osteoblast cells seeded on scaffolds exposed to static magnetic field of 1.3 T deformed the regular architecture of the scaffolds and evoked faster mineralization in comparison to unstimulated samples. Scaffolds deformation and extracellular matrix mineralization under static magnetic field (SMF) exposure increased with increasing MNPs concentration. The results are discussed in the frame of gradient magnetic fields of ~3 × 10⁻⁴ T/m generated by MNPs over the cells bodies.

Glass is an alternative solution to polymer for the fabrication of three-dimensional (3D) microfluidic biochips. Femtosecond (fs) lasers are nowadays the most promising tools for transparent glass processing. Specifically, the multiphoton process induced by fs pulses enables fabrication of embedded 3D channels with high precision. The subtractive fabrication process creating 3D hollow structures in glass, known as fs laser-assisted etching (FLAE), is based on selective removal of the laser-modified regions by successive chemical etching in diluted hydrofluoric acid solutions. In this work we demonstrate the possibility to generate embedded hollow channels in photosensitive Foturan glass volume by high repetition rate picosecond (ps) laser-assisted etching (PLAE). In particular, the influence of the critical irradiation doses and etching rates are discussed in comparison of two different wavelengths of ultraviolet (355 nm) and visible (532 nm) ranges. Fast and controlled fabrication of a basic structure composed of an embedded micro-channel connected with two open reservoirs, commonly used in the biochip design, are achieved inside glass. Distinct advantages such as good aspect-ratio, reduced processing time for large areas, and lower fabrication cost are evidenced.

The fabrication of 3D microstructures is under continuous development for engineering bone substitutes. Collagen/chitosan (Col/CT) blends emerge as biomaterials that meet the mechanical and biological requirements associated with bone tissue. In this work, we optimize the osteogenic effect of 3D microstructures by their functionalization with Col/CT blends with different blending ratios. The structures were fabricated by laser direct writing via two-photons polymerization of IP-L780 photopolymer. They comprised of hexagonal and ellipsoidal units 80 µm in length, 40 µm in width and 14 µm height, separated by 20 µm pillars. Structures’ functionalization was achieved via dip coating in Col/CT blends with specific blending ratios. The osteogenic role of Col/CT functionalization of the 3D structures was confirmed by biological assays concerning the expression of alkaline phosphatase (ALP) and osteocalcin secretion as osteogenic markers and Alizarin Red (AR) as dye for mineral deposits in osteoblast-like cells seeded on the structures. The structures having ellipsoidal units showed the best results, but the trends were similar for both ellipsoidal and hexagonal units. The strongest osteogenic effect was obtained for Col/CT blending ratio of 20/80, as demonstrated by the highest ALP activity, osteocalcin secretion and AR staining intensity in the seeded cells compared to all the other samples.