Five agarose types (D1LE, D2LE, LM, MS8 and D5) were evaluated in tissue engineering and compared for the first time using an array of analysis methods. Acellular and cellular constructs were generated from 0.3–3%, and their biomechanical properties, in vivo biocompatibility (as determined by LIVE/DEAD, WST-1 and DNA release, with n = 6 per sample) and in vivo biocompatibility (by hematological and biochemical analyses and histology, with n = 4 animals per agarose type) were analyzed. Results revealed that the biomechanical properties of each hydrogel were related to the agarose concentration (p < 0.001). Regarding the agarose type, the highest (p < 0.001) Young modulus, stress at fracture and break load were D1LE, D2LE and D5, whereas the strain at fracture was higher in D5 and MS8 at 3% (p < 0.05). All agaroses showed high biocompatibility on human skin cells, especially in indirect contact, with a correlation with agarose concentration (p = 0.0074 for LIVE/DEAD and p = 0.0014 for WST-1) and type, although cell function tended to decrease in direct contact with highly concentrated agaroses. All agaroses were safe in vivo, with no systemic effects as determined by hematological and biochemical analysis and histology of major organs. Locally, implants were partially encapsulated and a pro-regenerative response with abundant M2-type macrophages was found. In summary, we may state that all these agarose types can be safely used in tissue engineering and that the biomechanical properties and biocompatibility were strongly associated to the agarose concentration in the hydrogel and partially associated to the agarose type. These results open the door to the generation of specific agarose-based hydrogels for definite clinical applications such as the human skin, cornea or oral mucosa.

Development of an ideal biomaterial for clinical use is one of the main objectives of current research in tissue engineering. Marine-origin polysaccharides, in particular agaroses, have been widely explored as scaffolds for tissue engineering. We previously developed a biomaterial based on a combination of agarose with fibrin, that was successfully translated to clinical practice. However, in search of novel biomaterials with improved physical and biological properties, we have now generated new fibrin-agarose (FA) biomaterials using 5 different types of agaroses at 4 different concentrations. First, we evaluated the cytotoxic effects and the biomechanical properties of these biomaterials. Then, each bioartificial tissue was grafted in vivo and histological, histochemical and immunohistochemical analyses were performed after 30 days. Ex vivo evaluation showed high biocompatibility and differences in their biomechanical properties. In vivo, FA tissues were biocompatible at the systemic and local levels, and histological analyses showed that biointegration was associated to a pro-regenerative process with M2-type CD206-positive macrophages. These results confirm the biocompatibility of FA biomaterials and support their clinical use for the generation of human tissues by tissue engineering, with the possibility of selecting specific agarose types and concentrations for applications requiring precise biomechanical properties and in vivo reabsorption times.

Because cartilage has limited regenerative capability, a fully efficient advanced therapy medicinal product is needed to treat severe cartilage damage. We evaluated a novel biomaterial obtained by decellularizing sturgeon chondral endoskeleton tissue for use in cartilage tissue engineering. In silico analysis suggested high homology between human and sturgeon collagen proteins, and ultra-performance liquid chromatography confirmed that both types of cartilage consisted mainly of the same amino acids. Decellularized sturgeon cartilage was recellularized with human chondrocytes and four types of human mesenchymal stem cells (MSC) and their suitability for generating a cartilage substitute was assessed ex vivo and in vivo. The results supported the biocompatibility of the novel scaffold, as well as its ability to sustain cell adhesion, proliferation and differentiation. In vivo assays showed that the MSC cells in grafted cartilage disks were biosynthetically active and able to remodel the extracellular matrix of cartilage substitutes, with the production of type II collagen and other relevant components, especially when adipose tissue MSC were used. In addition, these cartilage substitutes triggered a pro-regenerative reaction mediated by CD206-positive M2 macrophages. These preliminary results warrant further research to characterize in greater detail the potential clinical translation of these novel cartilage substitutes.

In this work, we propose a surface functionalization of magnetic nanoparticles (MNPs) with hydroxyl and/or amino groups by the use of poly(ethylene glycol) (PEG) and its mixture with other functionalization agents, bearing amine groups, namely, amine-terminated PEG (NH₂-PEG) and aminopropyltriethoxysilane (APTES). The proposed functionalization method enabled the preparation of MNPs that were nontoxic to human skin cells and could accelerate or delay human blood coagulation, which may be useful for designing materials with controlled hemostatic properties. Subsequently, these nanoparticles were used to obtain supramolecular magnetic hydrogels with the use of a short peptide hydrogelator: fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF). The hydrogel matrices were obtained which, along with MNPs themselves, were thoroughly studied using a wide range of physicochemical, mechanical, and biological methods. It was demonstrated that surface functionalizations of the obtained MNPs remarkably affected the mechanical properties of the final magnetic hydrogels, despite the relatively low content of these nanoparticles within the total hydrogel mass. This ability to fine-tune the mechanical properties of the hydrogels, combined with their biocompatibility and the versatile potential for functionalizing such nanoparticles, makes them excellent candidates as biomaterials for a wide range of biomedical applications.

Background: Tissue engineered urethral substitutes could offer alternative approaches for urethral regeneration. In this work, we generated and evaluated in vivo a multilayered urethral substitute generated with nanostructured fibrin-agarose biomaterials. Methods: A three-layered urethral substitute was generated and grafted to repair surgically induced penile urethra defects in rabbits (acellular multilayered urethral substitute graft (UREG)). Native urethras served as positive controls (positive control (P-CTR)), unrepaired defects as negative controls (negative control (N-CTR)), and oral mucosa autograft (AUTO), as the gold standard treatment. Results were analyzed after 3 months for surgical feasibility, complications, urine stain patterns and histological, histochemical and immunohistochemical analyses. Results: All procedures were feasible, with no severe complications. Both in N-CTR and AUTO groups, 2/5 animals developed urethral fistulas, with none in UREG. Altered urine stains appeared in 3/5 of N-CTR and AUTO animals, and in 1/5 of UREG. Histologically, the UREG implants resembled native urethra, with high expression of pancytokeratin, cytokeratins 7, 8 and 19, involucrin and uroplakin, and stromal expression of versican, biglycan and decorin, with similar collagen and glycosaminoglycan contents. However, UREG grafts showed lower alcian blue staining intensity, higher number of blood vessels and a discontinuous muscular layer at the grafting site. In contrast, N-CTR and AUTO groups failed to reproduce the structure and molecular composition of the native urethra. Conclusions: A multilayered fibrin-agarose urethral substitute generated by tissue engineering could support urethral regeneration in an animal model of urethral repair. These findings support its potential as a promising alternative for urethral reconstruction.