Glioblastoma (GBM) is a devastating cancer of the brain with an extremely poor prognosis. For this reason, besides clinical and preclinical studies, novel in vitro models for the assessment of cancer response to drugs and radiation are being developed. In such context, three-dimensional (3D)-engineered cellular microenvironments, compared to unrealistic two-dimensional (2D) monolayer cell culture, provide a model closer to the in vivo configuration. Concerning cancer treatment, while X-ray radiotherapy and chemotherapy remain the current standard, proton beam therapy is an appealing alternative as protons can be efficiently targeted to destroy cancer cells while sparing the surrounding healthy tissue. However, despite the treatment's compelling biological and medical rationale, little is known about the effects of protons on GBM at the cellular level. In this work, we designed novel 3D-engineered scaffolds inspired by the geometry of brain blood vessels, which cover a vital role in the colonization mechanisms of GBM cells. The architectures were fabricated by two-photon polymerization (2PP), cultured with U-251 GBM cells and integrated for the first time in the context of proton radiation experiments to assess their response to treatment. We employed Gamma H2A.X as a fluorescent biomarker to identify the DNA damage induced in the cells by proton beams. The results show a higher DNA double-strand breakage in 2D cell monolayers as compared to cells cultured in 3D. The discrepancy in terms of proton radiation response could indicate a difference in the radioresistance of the GBM cells or in the rate of repair kinetics between 2D cell monolayers and 3D cell networks. Thus, these biomimetic-engineered 3D scaffolds pave the way for the realization of a benchmark tool that can be used to routinely assess the effects of proton therapy on 3D GBM cell networks and other types of cancer cells.

Biomimicking biological niches of healthy tissues or tumors can be achieved by means of artificial microenvironments, where structural and mechanical properties are crucial parameters to promote tissue formation and recreate natural conditions. In this work, three-dimensional (3D) scaffolds based on woodpile structures were fabricated by two-photon polymerization (2PP) of different photosensitive polymers (IP-S and SZ2080) and hydrogels (PEGDA 700) using two different 2PP setups, a commercial one and a customized one. The structures' properties were tuned to study the effect of scaffold dimensions (gap size) and their mechanical properties on the adhesion and proliferation of bone marrow mesenchymal stem cells (BM-MSCs), which can serve as a model for leukemic diseases, among other hematological applications. The woodpile structures feature gap sizes of 25, 50, and 100 μm and a fixed beam diameter of 25 μm, to systematically study the optimal cell colonization that promotes healthy cell growth and potential tissue formation. The characterization of the scaffolds involved scanning electron microscopy and mechanical nanoindenting, while their suitability for supporting cell growth was evaluated with live/dead cell assays and multistaining 3D confocal imaging. In the mechanical assays of the hydrogel material, we observed two different stiffness ranges depending on the indentation depth. Larger gap woodpile structures coated with fibronectin were identified as the most promising scaffolds for 3D BM-MSC cellular models, showing higher proliferation rates. The results indicate that both the design and the employed materials are suitable for further assays, where retaining the BM-MSC stemness and original features is crucial, including studies focused on BM disorders such as leukemia and others. Moreover, the combination of 3D scaffold geometry and materials holds great potential for the investigation of cellular behaviors in a co-culture setting, for example, mesenchymal and hematopoietic stem cells, to be further applied in medical research and pharmacological studies.

A major obstacle in glioma research is the lack of in vitro models that can retain cellular features of glioma cells in vivo. To overcome this limitation, a 3D-engineered scaffold, fabricated by two-photon polymerization, is developed as a cell culture model system to study patient-derived glioma cells. Scanning electron microscopy, (live cell) confocal microscopy, and immunohistochemistry are employed to assess the 3D model with respect to scaffold colonization, cellular morphology, and epidermal growth factor receptor localization. Both glioma patient-derived cells and established cell lines successfully colonize the scaffolds. Compared to conventional 2D cell cultures, the 3D-engineered scaffolds more closely resemble in vivo glioma cellular features and allow better monitoring of individual cells, cellular protrusions, and intracellular trafficking. Furthermore, less random cell motility and increased stability of cellular networks is observed for cells cultured on the scaffolds. The 3D-engineered glioma scaffolds therefore represent a promising tool for studying brain cancer mechanobiology as well as for drug screening studies.

One of the essential requirements to create nanoparticle (NP)-based applications and functions is the ability to control their deposition in specific locations. Many methods have been proposed, with wet direct writing (DW) techniques such as inkjet printing being the most employed. These methods generally depend on off-line and solvent-based NP synthesis leading to contamination and impurity in the final NP film as well as inhomogeneity in the deposition caused by solution-substrate interactions. This paper introduces a dry aerosol direct writing (dADW) method, which combines spark ablation-based and solvent-free NP synthesis with spatially selective deposition using aerodynamic focusing in a vacuum chamber. The challenge is to print high-resolution lines and spots of nanoparticles with a diameter < 100 nm. We study two aerodynamic nozzle concepts, a converging nozzle (CN) and a sheath gas nozzle (SGN), and investigate numerically how their design, as well as operating parameters, relate to the deposition process performance. This is quantified by three criteria: contraction factor, focusing ratio, and collection efficiency. We also compared our numerical results to experimental assays by manufacturing two SGNs and three CNs and evaluating the performance of each nozzle in terms of resolution, sharpness and thickness of the line. Using one of the SGN designs with an outlet diameter of 248 µm and an aerosol to total flow rate ratio of 0.17, we achieved a high-resolution line with a width of 67 µm, i.e., equal to 27% of the nozzle diameter, when printing < 100 nm Au NPs. The presented additive manufacturing method enables, therefore, the creation of high-resolution and sharp patterns of metallic nanoparticles, which can be employed in a wide range of applications, ranging from interconnects to optical and gas sensors.

The design of advanced functional devices often requires the use of intrinsically curved geometries that belong to the realm of non-Euclidean geometry and remain a challenge for traditional engineering approaches. Here, it is shown how the simple deflection of thick meta-plates based on hexagonal cellular mesostructures can be used to achieve a wide range of intrinsic (i.e., Gaussian) curvatures, including dome-like and saddle-like shapes. Depending on the unit cell structure, non-auxetic (i.e., positive Poisson ratio) or auxetic (i.e., negative Poisson ratio) plates can be obtained, leading to a negative or positive value of the Gaussian curvature upon bending, respectively. It is found that bending such meta-plates along their longitudinal direction induces a curvature along their transverse direction. Experimentally and numerically, it is shown how the amplitude of this induced curvature is related to the longitudinal bending and the geometry of the meta-plate. The approach proposed here constitutes a general route for the rational design of advanced functional devices with intrinsically curved geometries. To demonstrate the merits of this approach, a scaling relationship is presented, and its validity is demonstrated by applying it to 3D-printed microscale meta-plates. Several applications for adaptive optical devices with adjustable focal length and soft wearable robotics are presented.

The ability to tune the localised surface plasmon resonance (LSPR) behaviour of metal nanostructures has great importance for many optical sensor applications such as metal (plasmon) enhanced fluorescence spectroscopy and surface-enhanced Raman scattering (SERS). In this paper, we used Aerosol Direct Writing (ADW) to selectively deposit fine gold nanoparticles (AuNPs) patterns. A low-temperature thermal post-treatment (below 200 °C) provides enough energy to merge and transform AuNPs into larger features significantly different from non-thermally treated samples. The optical behaviour of non-treated and thermally treated AuNP films was investigated by photoluminescence (PL) spectroscopy. The PL measurements showed a red-shift, compared to bulk gold, using 488 nm and 514 nm laser excitation, and a blue-shift using 633 nm laser excitation. The thermal post-treatment leads to a further blue-shift compared to non-treated samples in the presence of both 514 and 633 nm laser. Finally, the AuNPs patterns were employed as a SERS-active substrate to detect low-concentrated (10⁻⁸M) rhodamine B. This method's ability to selectively deposit 3D gold nanostructures and tune their optical behaviour through a low-temperature thermal treatment allows optimisation of the optical response and enhancement of the Raman signal for specific bio-analytes.

In nature, cells exist in three-dimensional (3D) microenvironments with topography, stiffness, surface chemistry, and biological factors that strongly dictate their phenotype and behavior. The cellular microenvironment is an organized structure or scaffold that, together with the cells that live within it, make up living tissue. To mimic these systems and understand how the different properties of a scaffold, such as adhesion, proliferation, or function, influence cell behavior, we need to be able to fabricate cellular microenvironments with tunable properties. In this work, the nanotopography and functionality of scaffolds for cell culture were modified by coating 3D printed materials (DS3000 and poly(ethylene glycol)diacrylate, PEG-DA) with cellulose nanocrystals (CNCs). This general approach was demonstrated on a variety of structures designed to incorporate macro- and microscale features fabricated using photopolymerization and 3D printing techniques. Atomic force microscopy was used to characterize the CNC coatings and showed the ability to tune their density and in turn the surface nanoroughness from isolated nanoparticles to dense surface coverage. The ability to tune the density of CNCs on 3D printed structures could be leveraged to control the attachment and morphology of prostate cancer cells. It was also possible to introduce functionalization onto the surface of these scaffolds, either by directly coating them with CNCs grafted with the functionality of interest or with a more general approach of functionalizing the CNCs after coating using biotin-streptavidin coupling. The ability to carefully tune the nanostructure and functionalization of different 3D-printable materials is a step forward to creating in vitro scaffolds that mimic the nanoscale features of natural microenvironments, which are key to understanding their impact on cells and developing artificial tissues.

Surface-enhanced Raman scattering (SERS) substrates are of great interest for detecting low-concentrated analytes. However, issues such as multistep processing, cost, and possible presence of hazardous substances in the fabrication still represent a significant drawback. In this paper, an innovative direct writing method is introduced for solvent-free and spatially selective deposition of fine metal copper nanoparticles (CuNPs), with size distribution below 20 nm, generated in-line through a spark ablation method (SAM). The deposited CuNPs' morphology and composition were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), and energy-dispersive X-ray spectroscopy (EDS). The resulting CuNP patterns feature porous 3D microdomains with nanometric structures serving as hot spots for Raman signal enhancement. Low-temperature post-treatment (below 200 °C) of the deposited CuNPs significantly evolves its morphology and leads to sintering of NPs into a semicrystalline structure with sharp geometric features, which resulted in a more than 10-fold increase of the enhancement factor (up to 2.1 × 105) compared to non-heat-treated samples. The proposed method allows creating SERS substrates constituted by sharp 3D metallic nanopatterns selectively deposited onto specific regions, which paves the way for new printed, highly sensitive SERS-based sensors.

The development of cellular microenvironments suitable for neural tissue engineering purposes involves a plethora of research fields ranging from cell biology to biochemistry, neurosciences, physics, nanotechnology, mechanobiology. In the last two decades, this multi-disciplinary activity has led to the emergence of numerous strategies to create architectures capable of reproducing the topological, biochemical and mechanical properties of the extracellular matrix present in the central (CNS) and peripheral nervous system (PNS). Some of these approaches have succeeded in inducing the functional recovery of damaged areas in the CNS and the PNS to address the current lack of effective medical treatments for this type of injury. In this review, we analyze recent developments in the realization of two-dimensional and three-dimensional neuronal scaffolds following either top-down or bottom-up approaches. After providing an overview of the different fabrication techniques employed for tailoring the biomaterials, we draw on specific examples to describe the major features of the developed approaches. We then conclude with prospective proof of concept studies on guiding scaffolds and regenerative models on macro-scale brain implants targeting neural regeneration.

The development of advanced techniques of fabrication of three-dimensional (3D) microenvironments for the study of cell growth and proliferation has become one of the major motivations of material scientists and bioengineers in the past decade. Here, we present a novel residueless 3D structuration technique of poly(dimethylsiloxane) (PDMS) by water-in-PDMS emulsion casting and subsequent curing process in temperature-/pressure-controlled environment. Scanning electron microscopy and X-ray microcomputed tomography allowed us to investigate the impact of those parameters on the microarchitecture of the porous structure. We demonstrated that the optimized emulsion casting process gives rise to large-scale and highly interconnected network with pore size ranging from 500 μm to 1.5 mm that turned out to be nicely adapted to 3D cell culture. Experimental cell culture validations were performed using SaOS-2 (osteosarcoma) cell lines. Epifluorescence and deep penetration imaging techniques as two-photon confocal microscopy unveiled information about cell morphology and confirmed a homogeneous cell proliferation and spatial distribution in the 3D porous structure within an available volume larger than 1 cm³. These results open alternative scenarios for the fabrication and integration of porous scaffolds for the development of 3D cell culture platforms.