Whole transcriptomic analysis of mesenchymal stem cells cultured in Nichoid micro-scaffolds

Mesenchymal stem cells (MSCs) are known to be ideal candidates for clinical applications where not only regenerative potential but also immunomodulation ability is fundamental. Over the last years, increasing efforts have been put into the design and fabrication of 3D synthetic niches, conceived to emulate the native tissue microenvironment and aiming at efficiently controlling the MSC phenotype in vitro. In this panorama, our group patented an engineered microstructured scaffold, called Nichoid. It is fabricated through two-photon polymerization, a technique enabling the creation of 3D structures with control of scaffold geometry at the cell level and spatial resolution beyond the diffraction limit, down to 100 nm. The Nichoid’s capacity to maintain higher levels of stemness as compared to 2D substrates, with no need for adding exogenous soluble factors, has already been demonstrated in MSCs, neural precursors, and murine embryonic stem cells. In this work, we evaluated how three-dimensionality can influence the whole gene expression profile in rat MSCs. Our results show that at only 4 days from cell seeding, gene activation is affected in a significant way, since 654 genes appear to be differentially expressed (392 upregulated and 262 downregulated) between cells cultured in 3D Nichoids and in 2D controls. The functional enrichment analysis shows that differentially expressed genes are mainly enriched in pathways related to the actin cytoskeleton, extracellular matrix (ECM), and, in particular, cell adhesion molecules (CAMs), thus confirming the important role of cell morphology and adhesions in determining the MSC phenotype. In conclusion, our results suggest that the Nichoid, thanks to its exclusive architecture and 3D cell adhesion properties, is not only a useful tool for governing cell stemness but could also be a means for controlling immune-related MSC features specifically involved in cell migration

polymer nanostructuring by two photon absorption

Two-photon polymerization (2PP) is an innovative technology that in recent years showed a tremendous potential for three-dimensional structuring of photopolymers at the submicron scale. It is based on the nonlinear absorption of ultrashort laser pulses in transparent photosensitive materials. 2PP has been so far exploited in various fields, including photonics, microfluidics, regenerative medicine and MEMS prototyping. The versatility of this technology relies also on the photomaterials; indeed, polymers are easy to process, low cost and they allow the tailoring of their chemical and mechanical properties. 2PP nanotechnology is here exploited to produce micro and nanostructures that can be easily customized both in the geometry and in polymer functionalization. In particular, atomic force microscopy tips are fabricated on top of commercial cantilevers to demonstrate the technology feasibility and customizability. Moreover nanoporous membranes that can be fabricated by 2PP as a single custom product or as a mould for mass production through replica moulding are realized to evaluate the scalability of the fabrication process

Natural and Synthetic Polymers for Bone Scaffolds Optimization

Bone tissue is the structural component of the body, which allows locomotion, protects vital internal organs, and provides themaintenance ofmineral homeostasis. Several bone-related pathologies generate critical-size bone defects that our organism is not able to heal spontaneously and require a therapeutic action. Conventional therapies span frompharmacological to interventionalmethodologies, all of them characterized by several drawbacks. To circumvent these effects, tissue engineering and regenerative medicine are innovative and promising approaches that exploit the capability of bone progenitors, especially mesenchymal stem cells, to dierentiate into functional bone cells. So far, several materials have been tested in order to guarantee the specific requirements for bone tissue regeneration, ranging from the material biocompatibility to the ideal 3D bone-like architectural structure. In this review, we analyse the state-of-the-art of the most widespread polymeric scaold materials and their application in in vitro and in vivo models, in order to evaluate their usability in the field of bone tissue engineering. Here, we will present several adopted strategies in scaold production, from the dierent combination of materials, to chemical factor inclusion, embedding of cells, and manufacturing technology improvement

On‐chip magnetophoretic capture in a model of malaria‐infected red blood cells

The search for new rapid diagnostic tests for malaria is a priority for developing anefficient strategy to fight this endemic disease, which affects more than 3 billionpeople worldwide. In this study, we characterize systematically an easy‐to‐operatelab‐on‐chip, designed for the magnetophoretic capture of malaria‐infected red bloodcells (RBCs). The method relies on the positive magnetic susceptibility of infectedRBCs with respect to blood plasma. A matrix of nickel posts fabricated in a siliconchip placed face down is aimed at attracting infected cells, while healthy cells se-diment on a glass slide under the action of gravity. Using a model of infected RBCs,that is, erythrocytes with methemoglobin, we obtained a capture efficiency of about70% after 10 min in static conditions. By proper agitation, the capture efficiencyreached 85% after just 5 min. Sample preparation requires only a 1:10 volume di-lution of whole blood, previously treated with heparin, in a phosphate‐bufferedsolution. Nonspecific attraction of untreated RBCs was not observed in the sametime interval

Synthetic niche substrates engineered via two-photon laser polymerization for the expansion of human mesenchymal stromal cells

The present study reports on the development of an innovative culture substrate, micro-fabricated by two-photon laser polymerization (2PP) in a hybrid organic-inorganic photoresin. It was previously demonstrated that this substrate is able to guide spontaneous homing and colonization of mesenchymal stromal cells by the presence of synthetic microniches. Here, the number of niches covering the culture substrate was increased up to 10% of the total surface. Human bone marrow-derived mesenchymal stromal cells were expanded for 3 weeks and then their proliferation, clonogenic capacity and bilineage differentiation potential towards the osteogenic and adipogenic lineage were evaluated, both by colorimetric assays and by real-time polymerase chain reaction. Compared with cells cultured on glass substrates, cells expanded on 2PP substrates showed a greater colony diameter, which is an index of clonogenic potential. Following medium conditioning on 2PP-cultured cells, the expression of RUNX2 and BSP genes, as well as PPAR-gamma, was significantly greater than that measured on glass controls. Thus, human cells expanded on the synthetic niche substrate maintained their proliferative potential, clonogenic capacity and bilineage differentiation potential more effectively than cells expanded on glass substrates and in some aspects were comparable to non-expanded cells. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd

The effect of cell morphology on the permeability of the nuclear envelope to diffusive factors

A recent advance in understanding stem cell differentiation is that the cell is able to translate its morphology, i.e., roundish or spread, into a fate decision. We hypothesize that strain states in the nuclear envelope (NE) cause changes in the structure of the nuclear pore complexes. This induces significant changes in the NE's permeability to the traffic of the transcription factors involved in stem cell differentiation which are imported into the nucleus by passive diffusion. To demonstrate this, we set up a numerical model of the transport of diffusive molecules through the nuclear pore complex (NPC), on the basis of the NPC deformation. We then compared the prediction of the model for two different cell configurations with roundish and spread nuclear topologies with those measured on cells cultured in both configurations. To measure the geometrical features of the NPC, using electron tomography we reconstructed three-dimensional portions of the envelope of cells cultured in both configurations. We found non-significant differences in both the shape and size of the transmembrane ring of single pores with envelope deformation. In the numerical model, we thus assumed that the changes in pore complex permeability, caused by the envelope strains, are due to variations in the opening configuration of the nuclear basket, which in turn modifies the porosity of the pore complex mainly on its nuclear side. To validate the model, we cultured cells on a substrate shaped as a spatial micro-grid, called the "nichoid," which is nanoengineered by two-photon laser polymerization, and induces a roundish nuclear configuration in cells adhering to the nichoid grid, and a spread configuration in cells adhering to the flat substrate surrounding the grid. We then measured the diffusion through the nuclear envelope of an inert green-fluorescent protein, by fluorescence recovery after photobleaching (FRAP). Finally, we compared the diffusion times predicted by the numerical model for roundish vs. spread cells, with the measured times. Our data show that cell stretching modulates the characteristic time needed for the nuclear import of a small inert molecule, GFP, and the model predicts a faster import of diffusive molecules in the spread compared to roundish cells