Complex scaffolds composed of micro- and nano-structures are a key target in tissue engineering and the combination of sequential 3D printing and electrospinning enables the fabrication of these multi-scale structures. In this work, dual 3D printed and electrospun polycaprolactone (PCL) scaffolds with multiple mesh layers were successfully prepared. The scaffold macro- and micro-porosity were assessed by optical and scanning electron microscopy, showing that electrospun fibers formed aligned meshes within the pores of the scaffold. Consequently, the hydrophilicity of the scaffold increased with time, enhancing cell adhesion and growth. Additionally, compression tests in back and forth cycles demonstrated a good shape recovery behavior of the scaffolds. Biological results indicated that hybrid PCL scaffolds are biocompatible and enable a correct cell culture over time. Moreover, MC3T3-E1 preosteoblast culture on the scaffolds promoted the mineralization, increased the alkaline phosphatase (ALP) activity and upregulated the expression of early and late osteogenic markers, namely ALP and osteopontin (OPN), respectively. These results demonstrate that the sequential combination of 3D printing and electrospinning provides a facile method of incorporating fibers within a 3D printed scaffold, becoming a promising approach towards multi-scale hierarchical scaffolds capable of guiding the osteogenic differentiation.

Cellulose-containing residue from agar production was incorporated as a filler into soy protein-based hydrogels and revalorized without further purification. Rheological assessment of these hydrogels was carried out in order to confirm their shear-thinning behavior and their suitability for 3D printing. It was observed that all hydrogels behaved as weak gels, which are suitable for 3D printing and have good printability and shape fidelity. The addition of cellulose did not cause chemical crosslinking but physical interactions, which led to morphological changes, thereby promoting hardness and shape recovery of the 3D-printed products. The hydrogel with the highest residue content (8 wt %) showed the highest value (78%) in shape recovery. Furthermore, the physicochemical characterization of these 3D-printed products revealed that although they have high swelling capacity, they preserve their integrity in wet conditions. These results suggested the potential of the 3D-printed products developed using residues without further purification to promote circular economy, increasing the efficiency in resources utilization.

Sterilization is a crucial step in the process of developing bioinks for tissue engineering applications. In this work, alginate/gelatin inks were subjected to three sterilization methods: ultraviolet (UV) radiation, filtration (FILT), and autoclaving (AUTO). In addition, to simulate the sterilization effect in a real environment, inks were formulated in two different media, specifically, Dulbecco’s Modified Eagle’s Medium (DMEM) and phosphate-buffered saline (PBS). First, rheological tests were performed to evaluate the flow properties of the inks, and we observed that UV samples showed shear thinning behavior, which was favorable for threedimensional (3D) printing. Furthermore, the 3D-printed constructs developed with UV inks showed better shape and size fidelity than those obtained with FILT and AUTO. In order to relate this behavior to the material structure, Fourier transform infrared (FTIR) analysis was carried out and the predominant conformation in protein was determined by deconvolution of the amide I band, which confirmed that the prevalence of a-helix structure was greater for UV samples. This work highlights the relevance of sterilization processes, which are essential for biomedical applications, in the research field of bioinks.

Inks based on soybean protein isolate (SPI) were developed and their formulations were optimized as a function of the ink heat treatment and the content of other biopolymers to assess the effects of protein–polysaccharides and protein–protein interactions. First, the rheological behavior of the inks was analyzed in relation to the polyvinyl alcohol (PVA) concentration employed (20, 25, and 30 wt%) and, as a result of the analysis, the ink with 25 wt% PVA was selected. Additionally, sodium alginate (SA) and gelatin (GEL) were added to the formulations to improve the viscoelastic properties of the inks and the effect of the SA or GEL concentrations (1, 2, and 3 wt%) was studied. All inks showed shear thinning behavior and self-supporting abilities. Among all the 3D printed scaffolds, those with higher SA (3 wt%) or GEL (2 and 3 wt%) content showed higher shape fidelity and were selected for further characterization. Texture profile analysis demonstrated that the scaffolds prepared with previously heat-treated inks containing 3 wt% GEL showed the highest strength. Additionally, these scaffolds showed a higher water-uptake capacity profile.

In this study, novel scaffolds based on natural polymers were developed by combining 3D printing (3DP) and electrospinning (ES) techniques. ES ink was prepared with gelatin and poly(vinyl alcohol) (PVA), while 3DP ink was prepared with gelatin and chitin. Different biopolymers were used to confer unique properties to each ink and obtain a multilayered scaffold suitable for tissue regeneration. First, gelatin is able to exhibit the characteristics needed for both inks since gelatin chains contain arginineglycine-aspartic (RGD) motifs, an important sequence in the promotion of cell adhesion, which gives gelatin an improved biological behavior in comparison to other polymers. Additionally, PVA was selected for ES ink to facilitate gelatin spinnability, and chitin was incorporated into 3DP ink as reinforcement to provide mechanical support and protection to the overall design. In this work, chitin was extracted from fruit fly pupae. The high extraction yield and purity of the chitin obtained from the fruit fly pupae confirmed that this pupa is an alternative source to produce chitin. Once the chitin was characterized, both inks were prepared and rheological analysis was carried out in order to confirm the shear thinning behavior required for additive manufacturing processes. The combination of 3DP and ES processes resulted in porous scaffolds, which were proven biocompatible, highlighting their potential for biomedical applications.