José Pérez-Rigueiro

Perfused brain-on-chip platforms preserve tissue viability, structure and electrophysiological activity. No validated long-term adult or human brain OoCs exist, highlighting a need for advanced functional readouts and translational applications.

The European Pathfinder THOR project envisages the creation of a vascularized fragment of tissue that can be implanted in a patient using regenerated silk fibers as its building blocks. The selection of regenerated silk as the main building block of the scaffold relies heavily on its outstanding biocompatibility in comparison with either other artificial polymeric fibers or even natural silk fibers. Additionally, regenerated fibers produced through the Dynamic Dope Destabilization Spinning (D3S^TM) process are shown to exhibit high mechanical performance as reflected in values of strain at breaking and work to fracture comparable to those of the natural material. It is further shown that these fibers are endowed with the unique property of self-adhesion whereby hydrated fibers attach to one another and may sustain detachment forces of up to a few tens of MPa, a property that facilitates the generation of the scaffold with the fibers as its basic building block. Lastly, regenerated silk fibers are shown to be efficiently decorated with either peptides or small proteins, such as the vascular endothelial growth factor (VEGF), or with antibodies. The performance of both non-functionalized and decorated silk fibers is assessed in two different in vitro biological systems: (1) endothelial cell cultures, and (2) organotypic brain slice cultures. Together, these results support the use of regenerated silk fibers as versatile building blocks for biofunctional tissue scaffolds and provide experimental validation of the tissue engineering strategy established by the THOR project.

Cardiovascular diseases (CVDs) remain a leading source of morbidity and mortality worldwide. Although heart transplantation is an established treatment for selected patients with end-stage heart failure, the difficulty in obtaining enough donors and the problems associated with compatibility force the search for new strategies to mitigate these effects. Cardiac patches, a combination of therapeutic elements, such as cells and drugs deposited on a biomimetic scaffold, are particularly promising for repairing the long-term cardiac damage from a perspective based on tissue engineering. However, building these patches entails the compliance with extensive and exhaustive conditions and regulations. Key issues identified in the building process of cardiac patches are the cell–scaffold interaction, including the intensity and duration of this interaction. The present study is intended to develop a robust procedure that allows quantifying the adhesion between decellularized matrices and cells through single-cell force spectroscopy (SCFS) measurements. Determining this adhesion force is essential to ensure cell retention and therapeutic action in the damaged area and, therefore, to be able to design an advanced therapy medicinal product. The whole procedure is validated by measuring the adhesion of allogeneic adipose-derived mesenchymal stromal cells to a decellularized porcine myocardium patch.

Direct cellular reprogramming, the conversion of one somatic cell type into another, represents a remarkable advancement in regenerative medicine. Its potential to transform fibrotic tissue into functional parenchyma underscores its therapeutic promise. However, several critical challenges remain unresolved, including limited reprogramming efficiency, the long-term functional stability of converted cells, their integration within pre-existing cellular circuits, and safety concerns related to transgene integration and immunological responses to reprogramming-based viral vectors. Approaches based on the exogenous administration of recombinant proteins and miRNAs have also emerged, though these rely on factors that are naturally prone to exhaustion and degradation, potentially restricting their efficacy. This review is divided into three main sections. The first part addresses direct cellular reprogramming in the context of other cell-based applications, outlining its main applications and current biological limitations. The second part examines how different biomaterials, ranging from hydrogel scaffolds to nanoparticles, can modulate direct cellular reprogramming by providing mechanical and topographical cues and by enabling tighter control over the concentration and spatiotemporal dynamics of reprogramming factors and viral vectors. The third part discusses key findings in biomaterial-assisted reprogramming strategies, highlighting emerging opportunities for clinically translatable approaches. The convergence of regenerative biology and biomaterials science may ultimately generate advanced gel-based and hybrid cellular reprogramming platforms for in vitro testing and, in situ applications, for promoting cell fate stabilization and facilitating the regeneration of damaged tissues and organs.

The efficient design of biohybrid materials requires controlling the interaction between the cell and the material for a wide range of possible combinations. Single cell force spectroscopy (SCFS), an atomic force microscopy (AFM) experimental procedure based on the binding of an individual cell to an AFM cantilever and the assessment of the adhesion force between the cell and a target substrate, represents one of the most promising alternatives to characterize the interaction between cell and material. However, SCFS relies on the efficient binding of the cell to the AFM in order to avoid drawbacks, such as the detachment of the cell. In this work, three different versatile and robust procedures are presented that allow for the binding of either non-adherent (CD4⁺ T-lymphocytes) or adherent (mesenchymal stem cells, MSC) cells to the AFM probe. The three crosslinking strategies comprise (1) the streptavidin/biotin system, (2) sulfhydryl group-based crosslinkers, and (3) “click” (bioorthogonal) chemistry. Additionally, three decoration schemes of the functionalized AFM probes are explored: a specific antibody, concanavalin A, and direct binding of the cell through azide-derivatized membrane proteins. Differences are observed between these alternatives and it is found that the strength of the interaction (in decreasing order) is as follows: specific antibody, concanavalin A, and binding through azide-derivatized proteins.

Under benign conditions, bone tissue can regenerate itself without external intervention. However, this regenerative capacity can be compromised by various factors, most importantly related with the extent of the injury. Critical-sized defects, exceeding the body’s natural healing ability, demand the use of temporary or permanent devices like artificial joints or bone substitutes. While titanium is a widely used material for bone replacement, its integration into the body remains limited. This often leads to the progressive loosening of the implant and the need for revision surgeries, which are technically challenging, are commonly associated with high complication rates, and impose a significant economic burden. To enhance implant osseointegration, numerous studies have focused on the development of surface functionalization techniques to improve the response of the body to the implant. Yet, the challenge of achieving reliable and long-lasting prostheses persists. In this work, we address this challenge by applying a robust and versatile biofunctionalization process followed by the decoration of the material with oligopeptides. We immobilize four different peptides (RGD, CS-1, IKVAV, PHSRN) on R-THAB^® functionalized surfaces and find them to be highly stable in the long term. We also find that RGD is the best-performing peptide in in vitro cell cultures, enhancing adhesion, proliferation, and osteogenic differentiation of mesenchymal stem cells. To assess the in vivo effect of RGD-decorated Ti-6Al-4V implants, we develop a calvarial model in murine hosts. We find that the RGD-decoration remains stable for 1 week after the surgical procedure and reduces post-implantation macrophage-related inflammation. These results highlight the potential of peptide decoration on R-THAB^® functionalized surfaces to expedite the development of novel metallic biomaterials with enhanced biocompatibility properties, thereby advancing the field of regenerative medicine.

A versatile and robust procedure is developed that allows the identification of individual target molecules using antibodies bound to a DeepTip^TM functionalized atomic-force microscopy probe. The model system used for the validation of this process consists of a biotinylated anti-lactate dehydrogenase antibody immobilized on a streptavidin-decorated AFM probe. Lactate dehydrogenase (LDH) is employed as target molecule and covalently immobilized on functionalized MicroDeck^TM substrates. The interaction between sensor and target molecules is explored by recording force–displacement (F–z) curves with an atomic-force microscope. F–z curves that correspond to the genuine sensor–target molecule interaction are identified based on the following three criteria: (i) number of peaks, (ii) value of the adhesion force, and (iii) presence or absence of the elastomeric trait. The application of these criteria leads to establishing seven groups, ranging from no interaction to multiple sensor–target molecule interactions, for which force–displacement curves are classified. The possibility of recording consistently single-molecule interaction events between an antibody and its specific antigen, in combination with the high proportion of successful interaction events obtained, increases remarkably the possibilities offered by affinity atomic-force microscopy for the characterization of biological and biomimetic systems from the molecular to the tissue scales.

The interaction between the plant lipid transfer protein Pru p 3 and phytosphingosine was assessed using an atomic force microscope. Phytosphingosine was covalently immobilized on DeepTip^TM probes and Pru p 3 on MicroDeck^TM functionalized substrates. Single-molecular interaction events between both molecules were retrieved and classified and the distribution for each one of the identified types was calculated. A success rate of over 70% was found by comparing the number of specific Pru p 3-phytosphingosine interaction events with the total number of recorded curves. The analysis of the distribution established among the various types of curves was further pursued to distinguish between those curves that can mainly be used for assessing the recognition between phytosphingosine (sensor molecule) and Pru p 3 (target molecule) in the context of affinity atomic force microscopy, and those that entail details of the interaction and might be employed in the context of force spectroscopy. The successful application of these functionalized probes and substrates to the characterization of the low-intensity hydrophobic interaction characteristic of this system is a clear indication of the potential of exploiting this approach with an extremely wide range of different biological molecules of interest. The possibility of characterizing molecular assembly events with single-molecule resolution offers an advantageous procedure to plough into the field of molecular biomimetics.