The improvement of culturing techniques to model the environment and physiological conditions surrounding tumors has also been applied to the study of extracellular vesicles (EVs) in cancer research. EVs role is not only limited to cell-to-cell communication in tumor physiology, they are also a promising source of biomarkers, and a tool to deliver drugs and induce antitumoral activity. In the present review, we have addressed the improvements achieved by using 3D culture models to evaluate the role of EVs in tumor progression and the potential applications of EVs in diagnostics and therapeutics. The most employed assays are gel-based spheroids, often utilized to examine the cell invasion rate and angiogenesis markers upon EVs treatment. To study EVs as drug carriers, a more complex multicellular cultures and organoids from cancer stem cell populations have been developed. Such strategies provide a closer response to in vivo physiology observed responses. They are also the best models to understand the complex interactions between different populations of cells and the extracellular matrix, in which tumor-derived EVs modify epithelial or mesenchymal cells to become protumor agents. Finally, the growth of cells in 3D bioreactor-like systems is appointed as the best approach to industrial EVs production, a necessary step toward clinical translation of EVs-based therapy.

The decellularized extracellular matrix (ECM) obtained from human and porcine adipose tissue (AT) is currently used to prepare regenerative medicine bio-scaffolds. However, the influence of these natural biomaterials on host immune response is not yet deeply understood. Since macrophages play a key role in the inflammation/healing processes due to their high functional plasticity between M1 and M2 phenotypes, the evaluation of their response to decellularized ECM is mandatory. It is also necessary to analyze the immunocompetence of macrophages after contact with decellularized ECM materials to assess their functional role in a possible infection scenario. In this work, we studied the effect of four decellularized adipose matrices (DAMs) obtained from human and porcine AT by enzymatic or chemical methods on macrophage phenotypes and fungal phagocytosis. First, a thorough biochemical characterization of these biomaterials by quantification of remnant DNA, lipids, and proteins was performed, thus indicating the efficiency and reliability of both methods. The proteomic analysis evidenced that some proteins are differentially preserved depending on both the AT origin and the decellularization method employed. After exposure to the four DAMs, specific markers of M1 proinflammatory and M2 anti-inflammatory macrophages were analyzed. Porcine DAMs favor the M2 phenotype, independently of the decellularization method employed. Finally, a sensitive fungal phagocytosis assay allowed us to relate the macrophage phagocytosis capability with specific proteins differentially preserved in certain DAMs. The results obtained in this study highlight the close relationship between the ECM biochemical composition and the macrophage’s functional role.

In patients with comorbidities, a large number of wounds become chronic, representing an overwhelming economic burden for healthcare systems. Engineering the microenvironment is a paramount trend to activate cells and burst-healing mechanisms. The extrusion bioprinting of advanced dressings was performed with novel composite bioinks made by blending adipose decellularized extracellular matrix with plasma and human dermal fibroblasts. Rheological and microstructural assessments of the composite hydrogels supported post-printing cell viability and proliferation over time. Embedded fibroblasts expressed steady concentrations of extracellular matrix proteins, including type 1, 3 and 4 collagens and fibronectin. ELISA assessments, multiplex protein arrays and ensuing bioinformatic analyses revealed paracrine activities corresponding to wound-healing activation through the modulation of inflammation and angiogenesis. The two modalities of advanced dressings, differing in platelet number, showed differences in the release of inflammatory and angiogenic cytokines, including interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The conditioned media stimulated human-dermal-cell proliferation over time. Our findings open the door to engineering the microenvironment as a strategy to enhance healing.

Polymer scaffolds play an important role in tissue engineering applications. Poly(ethylene glycol) based hydrogels have received a lot of attention in this field because of their high biocompatibility and ease of processing. However, in many cases they do not exhibit proper tissue invasion and nutrient transport because of their dense structure. In the present work, several approaches were developed and compared to each other to produce interconnected macroporous poly(ethylene glycol) hydrogels by including different types of porogens in the photocrosslinking reaction. The swelling capacity of the resulting hydrogels was analyzed and compared to non-porous hydrogel samples. Moreover, the obtained materials were characterized by means of mechanical properties and porosity using rheometry, scanning electron microscopy, and mercury intrusion porosimetry. Results showed that interconnected and uniform pores were obtained when a porogen template was used during hydrogel fabrication by photocrosslinking. On the other side, when the porogen particles were dispersed into the macromer solution before matrix photocrosslinking the interconnexion was negligible. The templates must be dissolved before the hydrogel's cell-seeding in vitro, while the dispersed porogen can be used in situ in the in vitro seeding tests.

Despite the immense need for effective treatment of spinal cord injury (SCI), no successful repair strategy has yet been clinically implemented. Multifunctional biomaterials, based on porcine adipose tissue-derived extracellular matrix (adECM) and reduced graphene oxide (rGO), were recently shown to stimulate in vitro neural stem cell growth and differentiation. Nevertheless, their functional performance in clinically more relevant in vivo conditions remains largely unknown. Before clinical application of these adECM-rGO nanocomposites can be considered, a rigorous assessment of the cytotoxicity and biocompatibility of these biomaterials is required. For instance, xenogeneic adECM scaffolds could still harbour potential immunogenicity following decellularization. In addition, the toxicity of rGO has been studied before, yet often in experimental settings that do not bear relevance to regenerative medicine. Therefore, the present study aimed to assess both the in vitro as well as in vivo safety of adECM and adECM-rGO scaffolds. First, pulmonary, renal and hepato-cytotoxicity as well as macrophage polarization studies showed that scaffolds were benign in vitro. Then, a laminectomy was performed at the 10th thoracic vertebra, and scaffolds were implanted directly contacting the spinal cord. For a total duration of 6 weeks, animal welfare was not negatively affected. Histological analysis demonstrated the degradation of adECM scaffolds and subsequent tissue remodeling. Graphene-based scaffolds showed a very limited fibrous encapsulation, while rGO sheets were engulfed by foreign body giant cells. Furthermore, all scaffolds were infiltrated by macrophages, which were largely polarized towards a pro-regenerative phenotype. Lastly, organ-specific histopathology and biochemical analysis of blood did not reveal any adverse effects. In summary, both adECM and adECM-rGO implants were biocompatible upon laminectomy while establishing a pro-regenerative microenvironment, which justifies further research on their therapeutic potential for treatment of SCI.