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.

Graphene-Oxide Porous Biopolymer Hybrids Enhance In Vitro Osteogenic Differentiation and Promote Ectopic Osteogenesis In Vivo.

Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.

Nanocomposite particles with improved microstructure for 3D culture systems and bone regeneration.

Nano-apatite and gelatin-alginate hydrogel microparticles have been prepared by a one-step synthesis combined with electrostatic bead generation, for the reconstruction of bone defects. Based on the analysis of bone composition, architecture and embryonic intramembranous ossification, a bio-inspired fabrication has been developed. Accordingly, the mineral phase has been in situ synthesized, calcifying the hydrogel matrix while the latter was crosslinked, finally generating microparticles that can assemble into a bone defect to ensure interconnected pores. Although nano-apatite-biopolymer composites have been widely investigated, microstructural optimization to provide improved distribution and stability of the mineral is rarely achieved. The optimization of the developed method progressively resulted in two types of formulations (15P and 7.5P), with 15 and 7.5 (wt%) phosphate content in the initial precursor. The osteolytic potential was investigated using differentiated macrophages. A commercially available calcium phosphate bone graft substitute (Eurocer 400) was incorporated into the hydrogel, and the obtained composites were in vitro tested for comparison. The cytocompatibility of the microparticles was studied with mouse osteoblast-like cell line MC3T3-E1. Results indicated the best in vitro performance have been obtained for the sample loaded with 7.5P. Preliminary evaluation of biocompatibility into a critical size (3 mm) defect in rabbits showed that 7.5P nanocomposite is associated with newly formed bone in the proximity of the microparticles, after 28 days.

Fabrication of k-Carrageenan/Alginate/Carboxymethyl Cellulose basedScaffolds via 3D Printing for Potential Biomedical Applications.

Three-dimensional (3D) printing technology was able to generate great attention because of its unique methodology and for its major potential to manufacture detailed and customizable scaffolds in terms of size, shape and pore structure in fields like medicine, pharmaceutics and food. This study aims to fabricate an ink entirely composed of natural polymers, alginate, k-carrageenan and carboxymethyl cellulose (AkCMC). Extrusion-based 3D printing was used to obtain scaffolds based on a crosslinked interpenetrating polymer network from the alginate, k-carrageenan, carboxymethyl cellulose and glutaraldehide formulation using CaCl2, KCl and glutaraldehyde in various concentrations of acetic acid. The stabile bonding of the crosslinked scaffolds was assessed using infrared spectroscopy (FT-IR) as well as swelling, degradation and mechanical investigations. Moreover, morphology analysis (µCT and SEM) confirmed the 3D printed samples' porous structure. In the AkCMC-GA objects crosslinked with the biggest acetic acid concentration, the values of pores and walls are the highest, at 3.9 × 10-2 µm-1. Additionally, this research proves the encapsulation of vitamin B1 via FT-IR and UV-Vis spectroscopy. The highest encapsulation efficiency of vitamin B1 was registered for the AkCMC-GA samples crosslinked with the maximum acetic acid concentration. The kinetic release of the vitamin was evaluated by UV-Vis spectroscopy. Based on the results of these experiments, 3D printed constructs using AkCMC-GA ink could be used for soft tissue engineering applications and also for vitamin B1 encapsulation.

Three-Dimensional-Printed Sodium Alginate and k-Carrageenan-Based Scaffolds with Potential Biomedical Applications.

This work reports the development of a marine-derived polysaccharide formulation based on k-Carrageenan and sodium alginate in order to produce a novel scaffold for engineering applications. The viscoelastic properties of the bicomponent inks were assessed via rheological tests prior to 3D printing. Compositions with different weight ratios between the two polymers, without any crosslinker, were subjected to 3D printing for the first time, to the best of our knowledge, and the fabrication parameters were optimized to ensure a controlled architecture. Crosslinking of the 3D-printed scaffolds was performed in the presence of a chloride mixture (CaCl2:KCl = 1:1; v/v) of different concentrations. The efficiency of the crosslinking protocol was evaluated in terms of swelling behavior and mechanical properties. The swelling behavior indicated a decrease in the swelling degree when the concentration of the crosslinking agent was increased. These results are consistent with the nanoindentation measurements and the results of the macro-scale tests. Moreover, morphology analysis was also used to determine the pore size of the samples upon freeze-drying and the uniformity and micro-architectural characteristics of the scaffolds. Overall, the registered results indicated that the bicomponent ink, Alg/kCG = 1:1 may exhibit potential for tissue-engineering applications.

Bioinspired 3D scaffolds with antimicrobial, drug delivery, and osteogenic functions for bone regeneration.

A major clinical challenge today is the large number of bone defects caused by diseases or trauma. The development of three-dimensional (3D) scaffolds with adequate properties is crucial for successful bone repair. In this study, we prepared biomimetic mesoporous bioactive glass (MBG)-based scaffolds with and without ceria addition (up to 3 mol %) to explore the biological structure and chemical composition of the marine sponge Spongia Agaricina (SA) as a sacrificial template. Micro-CT examination revealed that all scaffolds exhibited a highly porous structure with pore diameters primarily ranging from 143.5 µm to 213.5 µm, facilitating bone ingrowth. Additionally, smaller pores (< 75 µm), which are known to enhance osteogenesis, were observed. The undoped scaffold displayed the highest open porosity value of 90.83%. Cytotoxicity assessments demonstrated that all scaffolds were noncytotoxic and nongenotoxic toward osteoblast cells. Moreover, scaffolds with higher CeO2 content promoted osteogenic differentiation of dental pulp stem cells, stimulating calcium and osteocalcin secretion. The scaffolds also exhibited antimicrobial and antibiofilm effects against Staphylococcus aureus (S. aureus) as well as drug delivery ability. Our research findings indicated that the combination of MBG, natural biological structure, and the addition of Ce exhibited a synergistic effect on the structure and biological properties of scaffolds for applications in bone tissue engineering.

Scaffolds and Surfaces with Biomedical Applications.

The engineering of scaffolds and surfaces with enhanced properties for biomedical applications represents an ever-expanding field of research that is continuously gaining momentum [...].

Double-Reinforced Fish Gelatin Composite Scaffolds for Osteochondral Substitutes.

Genipin crosslinked composite blends of fish gelatin/kappa-carrageenan (fG/κC) with different concentrations of graphene oxide (GO) for osteochondral substitutes were prepared by a simple solution-blending method. The resulting structures were examined by micro-computer tomography, swelling studies, enzymatic degradations, compressions tests, MTT, LDH, and LIVE/DEAD assays. The derived findings revealed that genipin crosslinked fG/κC blends reinforced with GO have a homogenous morphology with ideal pore dimensions of 200-500 µm for bones alternative. GO additivation with a concentration above 1.25% increased the blends' fluid absorption. The full degradation of the blends occurs in 10 days and the gel fraction stability increases with GO concentration. The blend compression modules decrease at first until fG/κC GO3, which has the least elastic behavior, then by raising the GO concentration the blends start to regain elasticity. The MC3T3-E1 cell viability reveals less viable cells with the increase of GO concentration. The LDH together with the LIVE/DEAD assays reports a high concentration of live and healthy cells in all types of composite blends and very few dead cells at the higher GO content.

3D double-reinforced graphene oxide - nanocellulose biomaterial inks for tissue engineered constructs.

The advent of improved fabrication technologies, particularly 3D printing, has enabled the engineering of bone tissue for patient-specific healing and the fabrication of in vitro tissue models for ex vivo testing. However, inks made from natural polymers often fall short in terms of mechanical strength, stability, and the induction of osteogenesis. Our research focused on developing novel printable formulations using a gelatin/pectin polymeric matrix that integrate synergistic reinforcement components i.e. graphene oxide (GO) and oxidized nanocellulose fibers (CNF). Using 3D printing technology and the aforementioned biomaterial composite inks, bone-like scaffolds were created. To simulate critical-sized flaws and demonstrate scaffold fidelity, 3D scaffolds were successfully printed using formulations with varied GO concentrations (0.25, 0.5, and 1% wt with respect to polymer content). The addition of GO to hydrogel inks enhanced not only the compressive modulus but also the printability and scaffold fidelity compared to the pure colloid-gelatin/pectin system. Due to its strong potential for 3D bioprinting, the sample containing 0.5% GO is shown to have the greatest perspectives for bone tissue models and tissue engineering applications.

Novel Green Crosslinked Salecan Hydrogels and Preliminary Investigation of Their Use in 3D Printing.

Salecan, a kind of polysaccharide, is produced by the Agrobacterium ZX09 salt tolerant strain. In this study, green crosslinked citric acid-salecan hydrogels are explored as novel materials with a high potential for use in regenerative medicine. The impact of salecan and citric acid on the final crosslinked hydrogels was intensively studied and estimated in terms of the whole physicochemical properties and antimicrobial activity. FTIR spectra demonstrated the successful green crosslinking of salecan through its esterification with citric acid where the formation of strong covalent bonds collaboratively helped to stabilize the entire hydrogel systems in a wet state. Hydrogels presented a microporous morphology, good swelling capacity, pH responsiveness, great mechanical stability under stress conditions and good antibacterial activity, all related to the concentration of the biopolymers used in the synthesis step. Additionally, salecan hydrogels were preliminary investigated as printing inks. Thanks to their excellent rheological behavior, we optimized the citrate-salecan hydrogel inks and printing parameters to render 3D constructs with great printing fidelity and integrity. The novel synthesized salecan green crosslinked hydrogels enriches the family of salecan-derived hydrogels. Moreover, this work not only expands the application of salecan hydrogels in various fields, but also provides a new potential option of designing salecan-based 3D printed scaffolds for customized regenerative medicine.

Manufacturing of scaffolds with interconnected internal open porosity and surface roughness.

Manufacturing of three-dimensional scaffolds with multiple levels of porosity are an advantage in tissue regeneration approaches to influence cell behavior. Three-dimensional scaffolds with surface roughness and intra-filament open porosity were successfully fabricated by additive manufacturing combined with chemical foaming and porogen leaching without the need of toxic solvents. The decomposition of sodium citrate, a chemical blowing agent, generated pores within the scaffold filaments, which were interconnected and opened to the external environment by leaching of a water-soluble sacrificial phase, as confirmed by micro-CT and buoyancy measurements. The additional porosity did not result in lower elastic modulus, but in higher strain at maximum load, i.e. scaffold ductility. Human mesenchymal stromal cells cultured for 24 h adhered in greater numbers on these scaffolds when compared to plain additive-manufactured ones, irrespectively of the scaffold pre-treatment method. Additionally, they showed a more spread and random morphology, which is known to influence cell fate. Cells cultured for a longer period exhibited enhanced metabolic activity while secreting higher osteogenic markers after 7 days in culture. STATEMENT OF SIGNIFICANCE: Inspired by the function of hierarchical cellular structures in natural materials, this work elucidates the development of scaffolds with multiscale porosity by combining in-situ foaming and additive manufacturing, and successive porogen leaching. The resulting scaffolds displayed enhanced mechanical toughness and multiscale pore network interconnectivity, combined with early differentiation of adult mesenchymal stromal cells into the osteogenic lineage.

Salicin and Hederacoside C-Based Extracts and UV-Absorbers Co-Loaded into Bioactive Lipid Nanocarriers with Promoted Skin Antiaging and Hydrating Efficacy.

Conventional and herbal active principles can be combined in a beneficial harmony using their best features and compensating for the certain weaknesses of each. The study will answer the question, "how can willow bark extract (Wbe) or ivy leaf extract (Ile) influence the photoprotective, skin permeation and hydration properties of Bioactive Lipid Nanocarriers (BLN) loaded with UV-filters and selected herbals?". BLN-Wbe/Ile-UV-filters were characterized for particle size, zeta potential, thermal behavior, entrapment efficiency and drug loading. The formulated BLN-hydrogels (HG) were subjected to in vitro release and permeation experiments. The in vitro determination of sun protection factors, as well as comparative in vitro photostability tests, rheology behavior and in vivo hydration status have been also considered for hydrogels containing BLN-Ile/Wbe-UV-filters. Photoprotection of BLN-HG against UVA rays was more pronounced as compared with the UVB (UVA-PF reached values of 30, while the maximum SPF value was 13). The in vitro irradiation study demonstrated the photostability of BLN-HG under UV exposure. A noteworthy cosmetic efficacy was detected by in vivo skin test (hydration effect reached 97% for the BLN-Wbe-UV-filters prepared with pomegranate oil). The research novelty, represented by the first-time co-optation of the active herbal extracts (Wbe and Ile) together with two synthetic filters in the same nanostructured delivery system, will provide appropriate scientific support for the cosmetic industry to design novel marketed formulations with improved quality and health benefices.

Electrospun Nanofibrous Membranes Based on Citric Acid-Functionalized Chitosan Containing rGO-TEPA with Potential Application in Wound Dressings.

The present research work is focused on the design and investigation of electrospun composite membranes based on citric acid-functionalized chitosan (CsA) containing reduced graphene oxide-tetraethylene pentamine (CsA/rGO-TEPA) as materials with opportune bio-properties for applications in wound dressings. The covalent functionalization of chitosan (CS) with citric acid (CA) was achieved through the EDC/NHS coupling system and was checked by 1H-NMR spectroscopy and FTIR spectrometry. The mixtures to be electrospun were formulated by adding three concentrations of rGO-TEPA into the 1/1 (w/w) CsA/poly (ethylene oxide) (PEO) solution. The effect of rGO-TEPA concentration on the morphology, wettability, thermal stability, cytocompatibility, cytotoxicity, and anti-biofilm activity of the nanofibrous membranes was extensively investigated. FTIR and Raman results confirmed the covalent and non-covalent interactions that appeared between the system's compounds, and the exfoliation of rGO-TEPA sheets within the CsA in the presence of PEO (CsA/P) polymer matrix, respectively. SEM analysis emphasized the nanofibrous architecture of membranes and the presence of rGO-TEPA sheets entrapped into the CsA nanofiber structure. The MTT cellular viability assay showed a good cytocompatibility with the highest level of cell development and proliferation registered for the CsA/P composite nanofibrous membrane with 0.250 wt.% rGO-TEPA. The designed nanofibrous membranes could have potential applications in wound dressings, given that they showed a good anti-biofilm activity against Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacterial strains.

Nanostructured Polyacrylamide Hydrogels with Improved Mechanical Properties and Antimicrobial Behavior.

This work proposes a simple method to obtain nanostructured hydrogels with improved mechanical characteristics and relevant antibacterial behavior for applications in articular cartilage regeneration and repair. Low amounts of silver-decorated carbon-nanotubes (Ag@CNTs) were used as reinforcing agents of the semi-interpenetrating polymer network, consisting of linear polyacrylamide (PAAm) embedded in a PAAm-methylene-bis-acrylamide (MBA) hydrogel. The rational design of the materials considered a specific purpose for each employed species: (1) the classical PAAm-MBA network provides the backbone of the materials; (2) the linear PAAm (i) aids the dispersion of the nanospecies, ensuring the systems' homogeneity and (ii) enhances the mechanical properties of the materials with regard to resilience at repeated compressions and ultimate compression stress, as shown by the specific mechanical tests; and (3) the Ag@CNTs (i) reinforce the materials, making them more robust, and (ii) imprint antimicrobial characteristics on the obtained scaffolds. The tests also showed that the obtained materials are stable, exhibiting little degradation after 4 weeks of incubation in phosphate-buffered saline. Furthermore, as revealed by micro-computed tomography, the morphometric features of the scaffolds are adequate for applications in the field of articular tissue regeneration and repair.

Development of New Hybrid Casein-Loaded PHEMA-PEGDA Hydrogels with Enhanced Mineralisation Potential.

Casein is a micellar protein rich in glutamic and aspartic acids as well as in phosphoserine. Considering its native affinity for calcium and the connection of sub-micelles through calcium phosphate nanoclusters, this protein holds promise for stimulating biomimetic mineralisation phenomena and direct binding with the mineral phase of hard tissues. In this work we prepared new hybrids based on casein embedded in a poly(2-hydroxyethyl methacrylate)-polyethyleneglycol diacrylate (PHEMA-PEGDA) hydrogel. The resulting materials were investigated structurally by Fourier transform infrared (FT-IR). Casein modified the water affinity and the rheological properties of the hybrids. The microstructure was explored by scanning electron microscopy (SEM) and the distribution of the protein was established by combined SEM micrographs and elemental mapping considering the casein-specific elements (P, N and S) not contained by the synthetic hydrogel matrix. The effect of casein on the mineralisation potential and stability of the mineral phase was investigated by FT-IR and SEM when alternating incubation in Ca/P solutions is performed. Increasing casein content in the hybrids leads to improved mineralisation, with localised formation of nanoapatite phase on the protein areas in the richest sample in protein. This behaviour was proved microstructurally by SEM and through overlapping elemental distribution of Ca and P from the newly formed mineral and P, S and N from the protein. This study indicates that nanoapatite-casein-PHEMA-PEGDA nanocomposites may be developed for potential use in bone repair and regeneration.

Newly crosslinked chitosan- and chitosan-pectin-based hydrogels with high antioxidant and potential anticancer activity.

Monoaldehydes, due to natural origin and therapeutic activity, have attracted great attention for their ability to crosslink chitosan hydrogels for biomedical applications. However, most studies have focused on single-component hydrogels. In this work, chitosan-based hydrogels, crosslinked for the first time with 2,3,4-trihydroxybenzaldehyde (THBA), were modified with pectin (PC), bioactive glass (BG), and rosmarinic acid (RA). All of these were not only involved in the crosslinking, but also modulated properties or imparted completely new ones. THBA functioned as a crosslinker, resulting in improved mechanical properties, high swelling capacity and delayed degradation and also imparted high antioxidant activity and antiproliferative effect on cancer cells without cytotoxicity for normal cells. Hydrogels containing PC showed enhanced mechanical strength, while the combination with BG gave improved stability in PBS. All hydrogels modified with BG exhibited the ability to mineralise in SBF. The addition of RA enhanced antioxidant and anticancer activities and promoting the mineralisation process.

3D Printed Composite Scaffolds of GelMA and Hydroxyapatite Nanopowders Doped with Mg/Zn Ions to Evaluate the Expression of Genes and Proteins of Osteogenic Markers.

As bone diseases and defects are constantly increasing, the improvement of bone regeneration techniques is constantly evolving. The main purpose of this scientific study was to obtain and investigate biomaterials that can be used in tissue engineering. In this respect, nanocomposite inks of GelMA modified with hydroxyapatite (HA) substituted with Mg and Zn were developed. Using a 3D bioprinting technique, scaffolds with varying shapes and dimensions were obtained. The following analyses were used in order to study the nanocomposite materials and scaffolds obtained by the 3D printing technique: Fourier transform infrared spectrometry and X-ray diffraction (XRD), scanning electron microscopy (SEM), and micro-computed tomography (Micro-CT). The swelling and dissolvability of each scaffold were also studied. Biological studies, osteopontin (OPN), and osterix (OSX) gene expression evaluations were confirmed at the protein levels, using immunofluorescence coupled with confocal microscopy. These findings suggest the positive effect of magnesium and zinc on the osteogenic differentiation process. OSX fluorescent staining also confirmed the capacity of GelMA-HM5 and GelMA-HZ5 to support osteogenesis, especially of the magnesium enriched scaffold.

Sponges from Plasma Treated Cellulose Nanofibers Grafted with Poly(ethylene glycol)methyl Ether Methacrylate.

In this work, cellulose nanofibers (CNF) were surface treated by plasma and grafted with poly(ethylene glycol)methyl ether methacrylate (PEGMMA) for increasing mechanical strength and hydrophobicity. The surface characteristics of the sponges were studied by scanning electron microscopy, micro-computed tomography, and Fourier transform infrared spectroscopy, which demonstrated successful surface modification. Plasma treatment applied to CNF suspension led to advanced defibrillation, and the resulting sponges (CNFpl) exhibited smaller wall thickness than CNF. The grafting of PEGMMA led to an increase in the wall thickness of the sponges and the number of larger pores when compared with the non-grafted counterparts. Sponges with increased hydrophobicity demonstrated by an almost 4 times increase in the water contact angle and better mechanical strength proved by 2.5 times increase in specific compression strength were obtained after PEGMMA grafting of plasma treated CNF. Cells cultivated on both neat and PEGMMA-grafted CNF sponges showed high viability (>99%). Remarkably, CNF grafted with PEGMMA showed better cell viability as compared with the untreated CNF sample; this difference is statistically significant (p < 0.05). In addition, the obtained sponges do not trigger an inflammatory response in macrophages, with TNF-α secretion by cells in contact with CNFpl, CNF-PEGMMA, and CNFpl-PEGMMA samples being lower than that observed for the CNF sample. All these results support the great potential of cellulose nanofibers surface treated by plasma and grafted with PEGMMA for biomedical applications.

Computed Tomography as a Characterization Tool for Engineered Scaffolds with Biomedical Applications.

The ever-growing field of materials with applications in the biomedical field holds great promise regarding the design and fabrication of devices with specific characteristics, especially scaffolds with personalized geometry and architecture. The continuous technological development pushes the limits of innovation in obtaining adequate scaffolds and establishing their characteristics and performance. To this end, computed tomography (CT) proved to be a reliable, nondestructive, high-performance machine, enabling visualization and structure analysis at submicronic resolutions. CT allows both qualitative and quantitative data of the 3D model, offering an overall image of its specific architectural features and reliable numerical data for rigorous analyses. The precise engineering of scaffolds consists in the fabrication of objects with well-defined morphometric parameters (e.g., shape, porosity, wall thickness) and in their performance validation through thorough control over their behavior (in situ visualization, degradation, new tissue formation, wear, etc.). This review is focused on the use of CT in biomaterial science with the aim of qualitatively and quantitatively assessing the scaffolds' features and monitoring their behavior following in vivo or in vitro experiments. Furthermore, the paper presents the benefits and limitations regarding the employment of this technique when engineering materials with applications in the biomedical field.

Development of thick paste-like inks based on superconcentrated gelatin/alginate for 3D printing of scaffolds with shape fidelity and stability.

Shape fidelity and integrity are serious challenges in the 3D printing of hydrogel precursors, as they can influence the overall performance of 3D scaffolds. This work reports the development of superconcentrated inks based on sodium alginate and fish gelatin as an appealing strategy to satisfy such challenges and dictate the quality of the printed scaffolds, without using crosslinking strategies during 3D printing. SEM micrographs and micro-CT images indicate the homogeneous distribution of the polysaccharide in the gelatin-based matrix, suggesting its potential to act as a reinforcing additive. The high concentration of gelatin aqueous solution (50 wt%) and substantial incorporation of alginate have facilitated the highly accurate printability and influence the in vitro stability and mechanical properties of the printed scaffolds. An improvement of the stiffness is dictated by the increase of alginate concentration from 20 wt% to 25 wt%, and an increase of Young modulus with about 46% is reached, confirming the reinforcing effect of polysaccharide. This study highlights the potential of paste-type inks to provide high resolution 3D printed structures with appealing structural and dimensional stability, in vitro degradability and mechanical properties for biomedical applications.