Advanced Strategies for 3D Bioprinting of Tissue and Organ Analogs Using Alginate Hydrogel Bioinks

Alginate is a natural polysaccharide that typically originates from various species of algae. Due to its low cost, good biocompatibility, and rapid ionic gelation, the alginate hydrogel has become a good option of bioink source for 3D bioprinting. However, the lack of cell adhesive moieties, erratic biodegradability, and poor printability are the critical limitations of alginate hydrogel bioink. This review discusses the pivotal properties of alginate hydrogel as a bioink for 3D bioprinting technologies. Afterward, a variety of advanced material formulations and biofabrication strategies that have recently been developed to overcome the drawbacks of alginate hydrogel bioink will be focused on. In addition, the applications of these advanced solutions for 3D bioprinting of tissue/organ mimicries such as regenerative implants and in vitro tissue models using alginate-based bioink will be systematically summarized.     

Extraction and Characterization of Collagen from Elasmobranch Byproducts for Potential Biomaterial Use

With the worldwide increase of fisheries, fish wastes have had a similar increase, alternatively they can be seen as a source of novel substances for the improvement of society’s wellbeing. Elasmobranchs are a subclass fished in high amounts, with some species being mainly bycatch. They possess an endoskeleton composed mainly by cartilage, from which chondroitin sulfate is currently obtained. Their use as a viable source for extraction of type II collagen has been hypothesized with the envisaging of a biomedical application, namely in biomaterials production. In the present work, raw cartilage from shark (Prionace glauca) and ray (Zeachara chilensis and Bathyraja brachyurops) was obtained from a fish processing company and submitted to acidic and enzymatic extractions, to produce acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC). From all the extractions, P. glauca PSC had the highest yield (3.5%), followed by ray ASC (0.92%), ray PSC (0.50%), and P. glauca ASC (0.15%). All the extracts showed similar properties, with the SDS-PAGE profiles being compatible with the presence of both type I and type II collagens. Moreover, the collagen extracts exhibited the competence to maintain their conformation at human basal temperature, presenting a denaturation temperature higher than 37 °C. Hydrogels were produced using P. glauca PSC combined with shark chondroitin sulfate, with the objective of mimicking the human cartilage extracellular matrix. These hydrogels were cohesive and structurally-stable at 37 °C, with rheological measurements exhibiting a conformation of an elastic solid when submitted to shear strain with a frequency up to 4 Hz. This work revealed a sustainable strategy for the valorization of fisheries’ by-products, within the concept of a circular economy, consisting of the use of P. glauca, Z. chilensis, and B. brachyurops cartilage for the extraction of collagen, which would be further employed in the development of hydrogels as a proof of concept of its biotechnological potential, ultimately envisaging its use in marine biomaterials to regenerate damaged cartilaginous tissues.     

Development of Biocomposite Alginate-Cuttlebone-Gelatin 3D Printing Inks Designed for Scaffolds with Bone Regeneration Potential

Fabrication of three-dimensional (3D) scaffolds using natural biomaterials introduces valuable opportunities in bone tissue reconstruction and regeneration. The current study aimed at the development of paste-like 3D printing inks with an extracellular matrix-inspired formulation based on marine materials: sodium alginate (SA), cuttlebone (CB), and fish gelatin (FG). Macroporous scaffolds with microporous biocomposite filaments were obtained by 3D printing combined with post-printing crosslinking. CB fragments were used for their potential to stimulate biomineralization. Alginate enhanced CB embedding within the polymer matrix as confirmed by scanning electron microscopy (ESEM) and micro-computer tomography (micro-CT) and improved the deformation under controlled compression as revealed by micro-CT. SA addition resulted in a modulation of the bulk and surface mechanical behavior, and lead to more elongated cell morphology as imaged by confocal microscopy and ESEM after the adhesion of MC3T3-E1 preosteoblasts at 48 h. Formation of a new mineral phase was detected on the scaffold’s surface after cell cultures. All the results were correlated with the scaffolds’ compositions. Overall, the study reveals the potential of the marine materials-containing inks to deliver 3D scaffolds with potential for bone regeneration applications.     

A 3D-Printed Polycaprolactone/Marine Collagen Scaffold Reinforced with Carbonated Hydroxyapatite from Fish Bones for Bone Regeneration

In bone tissue regeneration, extracellular matrix (ECM) and bioceramics are important factors, because of their osteogenic potential and cell–matrix interactions. Surface modifications with hydrophilic material including proteins show significant potential in tissue engineering applications, because scaffolds are generally fabricated using synthetic polymers and bioceramics. In the present study, carbonated hydroxyapatite (CHA) and marine atelocollagen (MC) were extracted from the bones and skins, respectively, of Paralichthys olivaceus. The extracted CHA was characterized using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) analysis, while MC was characterized using FTIR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The scaffolds consisting of polycaprolactone (PCL), and different compositions of CHA (2.5%, 5%, and 10%) were fabricated using a three-axis plotting system and coated with 2% MC. Then, the MC3T3-E1 cells were seeded on the scaffolds to evaluate the osteogenic differentiation in vitro, and in vivo calvarial implantation of the scaffolds was performed to study bone tissue regeneration. The results of mineralization confirmed that the MC/PCL, 2.5% CHA/MC/PCL, 5% CHA/MC/PCL, and 10% CHA/MC/PCL scaffolds increased osteogenic differentiation by 302%, 858%, 970%, and 1044%, respectively, compared with pure PCL scaffolds. Consequently, these results suggest that CHA and MC obtained from byproducts of P. olivaceus are superior alternatives for land animal-derived substances.     

Synthesis and Characterization of 2-Decenoic Acid Modified Chitosan for Infection Prevention and Tissue Engineering

Chitosan nanofiber membranes are recognized as functional antimicrobial materials, as they can effectively provide a barrier that guides tissue growth and supports healing. Methods to stabilize nanofibers in aqueous solutions include acylation with fatty acids. Modification with fatty acids that also have antimicrobial and biofilm-resistant properties may be particularly beneficial in tissue regeneration applications. This study investigated the ability to customize the fatty acid attachment by acyl chlorides to include antimicrobial 2-decenoic acid. Synthesis of 2-decenoyl chloride was followed by acylation of electrospun chitosan membranes in pyridine. Physicochemical properties were characterized through scanning electron microscopy, FTIR, contact angle, and thermogravimetric analysis. The ability of membranes to resist biofilm formation by S. aureus and P. aeruginosa was evaluated by direct inoculation. Cytocompatibility was evaluated by adding membranes to cultures of NIH3T3 fibroblast cells. Acylation with chlorides stabilized nanofibers in aqueous media without significant swelling of fibers and increased hydrophobicity of the membranes. Acyl-modified membranes reduced both S. aureus and P. aeruginosa bacterial biofilm formation on membrane while also supporting fibroblast growth. Acylated chitosan membranes may be useful as wound dressings, guided regeneration scaffolds, local drug delivery, or filtration.