New 3D Printed Scaffolds Based on Walstromite Synthesized by Sol-Gel Method.

This study explores the potential utilization of walstromite (BaCa2Si3O9) as a foundational material for creating new bioceramics in the form of scaffolds through 3D printing technology. To achieve this objective, this study investigates the chemical-mineralogical, morphological, and structural characteristics, as well as the biological properties, of walstromite-based bioceramics. The precursor mixture for walstromite synthesis is prepared through the sol-gel method, utilizing pure reagents. The resulting dried gelatinous precipitate is analyzed through complex thermal analysis, leading to the determination of the optimal calcination temperature. Subsequently, the calcined powder is characterized via X-ray diffraction and scanning electron microscopy, indicating the presence of calcium and barium silicates, as well as monocalcium silicate. This powder is then employed in additive 3D printing, resulting in ceramic scaffolds. The specific ceramic properties of the scaffold, such as apparent density, absorption, open porosity, and compressive strength, are assessed and fall within practical use limits. X-ray diffraction analysis confirms the formation of walstromite as a single phase in the ceramic scaffold. In vitro studies involving immersion in simulated body fluid (SBF) for 7 and 14 days, as well as contact with osteoblast-like cells, reveal the scaffold's ability to form a phosphate layer on its surface and its biocompatibility. This study concludes that the walstromite-based ceramic scaffold exhibits promising characteristics for potential applications in bone regeneration and tissue engineering.

Hypotrochoidal scaffolds for cartilage regeneration.

The main function of articular cartilage is to provide a low friction surface and protect the underlying subchondral bone. The extracellular matrix composition of articular cartilage mainly consists of glycosaminoglycans and collagen type II. Specifically, collagen type II fibers have an arch-like organization that can be mimicked with segments of a hypotrochoidal curve. In this study, a script was developed that allowed the fabrication of scaffolds with a hypotrochoidal design. This design was investigated and compared to a regular 0-90 woodpile design. The mechanical analyses revealed that the hypotrochoidal design had a lower component Young's modulus while the toughness and strain at yield were higher compared to the woodpile design. Fatigue tests showed that the hypotrochoidal design lost more energy per cycle due to the damping effect of the unique microarchitecture. In addition, data from cell culture under dynamic stimulation demonstrated that the collagen type II deposition was improved and collagen type X reduced in the hypotrochoidal design. Finally, Alcian blue staining revealed that the areas where the stress was higher during the stimulation produced more glycosaminoglycans. Our results highlight a new and simple scaffold design based on hypotrochoidal curves that could be used for cartilage tissue engineering.

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.

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.

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.

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.

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.

Gelatin Meshes Enriched with Graphene Oxide and Magnetic Nanoparticles Support and Enhance the Proliferation and Neuronal Differentiation of Human Adipose-Derived Stem Cells.

The field of tissue engineering is constantly evolving due to the fabrication of novel platforms that promise to stimulate tissue regeneration in the scenario of accidents. Here, we describe the fabrication of fibrous nanostructured substrates based on fish gelatin (FG) and enriched with graphene oxide (GO) and magnetic nanoparticles (MNPs) and demonstrate its biological properties in terms of cell viability and proliferation, cell adhesion, and differentiation. For this purpose, electrospun fibers were fabricated using aqueous precursors containing either only GO and only MNP nanospecies, or both of them within a fish gelatin solution. The obtained materials were investigated in terms of morphology, aqueous media affinity, tensile elasticity, and structural characteristics. The biological evaluation was assessed against adipose-derived stem cells by MTT, LDH, Live/Dead assay, cytoskeleton investigation, and neuronal trans-differentiation. The results indicate an overall good interaction and show that these materials offer a biofriendly environment. A higher concentration of both nanospecies types induced some toxic effects, thus 0.5% GO, MNPs, and GO/MNPs turned out to be the most suitable option for biological testing. Moreover, a successful neuronal differentiation has been shown on these materials, where cells presented a typical neuronal phenotype. This study demonstrates the potential of this scaffold to be further used in tissue engineering 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.

Plasma Derived Products for Polypropylene Mesh Integration in Abdominal Wall Defects: Procedure Description and Partial Results.

Introduction: Abdominal wall surgery for parietal defects is done by implanting a type of mesh in the surrounding tissue above or beneath the fascia layer of the abdominal wall. The most common type of mesh used is polypropylene which sometimes takes a lot of time to be covered by the fibrous tissue. In an attempt to accelerate the cellular binding on the mesh and so to increase the recovery rate, we developed a protocol with plasma derived products to accelerate the mesh integration. Platelet rich fibrin (PRF) and platelet rich plasma (PRP) were evaluated in promoting the collagen synthesis and cell proliferation on the mesh surface. Material and Methods: We evaluated 32 patients with different types of abdominal wall defects which required polypropylene mesh implants in open surgery with the mesh implanted above the aponeurosis layer. We divided the patients into 3 groups: standard procedure, mesh augmented with PRF only, mesh augmented with PRP only. Results: Even though the number of patients involved in the study has a very small impact for a statistical analysis, the pattern observed in our prospective study reveals from the beginning that augmenting the standard procedure with plasma derived products improve the outcome (mesh integration) up to 65% faster integration. Conclusion: The technique that we used to augment the standard implant is cost-effective and simple to use in the surgical theatre.

Glial contribution to cyclodextrin-mediated reversal of cholesterol accumulation in murine NPC1-deficient neurons in vivo.

Niemann-Pick type C disease is a rare and fatal lysosomal storage disorder presenting severe neurovisceral symptoms. Disease-causing mutations in genes encoding either NPC1 or NPC2 protein provoke accumulation of cholesterol and other lipids in specific structures of the endosomal-lysosomal system and degeneration of specific cells, notably neurons in the central nervous system (CNS). 2-hydroxypropyl-beta-cyclodextrin (CD) emerged as potential therapeutic approach based on animal studies and clinical data, but the mechanism of action in neurons has remained unclear. To address this topic in vivo, we took advantage of the retina as highly accessible part of the CNS and intravitreal injections as mode of drug administration. Coupling CD to gold nanoparticles allowed us to trace its intracellular location. We report that CD enters the endosomal-lysosomal system of neurons in vivo and enables the release of lipid-laden lamellar inclusions, which are then removed from the extracellular space by specific types of glial cells. Our data suggest that CD induces a concerted action of neurons and glial cells to restore lipid homeostasis in the central nervous system.

Double-Cross-Linked Networks Based on Methacryloyl Mucin.

Mucin is a glycoprotein with proven potential in the biomaterials field, but its use is still underexploited for such applications. The present work aims to produce a synthesis of methacryloyl mucin single-network (SN) hydrogels and their double-cross-linked-network (DCN) counterparts. Following the synthesis of the mucin methacryloyl derivative, various SN hydrogels are prepared through the photopolymerization of methacrylate bonds, using reaction media with different pH values. The SN hydrogels are converted into DCN systems via supplementary cross-linking in tannic acid aqueous solution. The chemical modification of mucin is described, and the obtained product is characterized; the structural modification of mucin is assessed through FTIR spectroscopy, and the circular dichroism and the isoelectric point of methacryloyl mucin is evaluated. The affinity for aqueous media of both SN and DCN hydrogels is estimated, and the mechanical properties of the systems are assessed, both at macroscale through uniaxial compression and rheology tests and also at microscale through nanoindentation tests.

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.

Electrospinning Fabrication and Cytocompatibility Investigation of Nanodiamond Particles-Gelatin Fibrous Tubular Scaffolds for Nerve Regeneration.

This paper reports the electrospinning fabrication of flexible nanostructured tubular scaffolds, based on fish gelatin (FG) and nanodiamond nanoparticles (NDs), and their cytocompatibility with murine neural stem cells. The effects of both nanofiller and protein concentration on the scaffold morphology, aqueous affinity, size modification at rehydration, and degradation are assessed. Our findings indicate that nanostructuring with low amounts of NDs may modify the fiber properties, including a certain regional parallel orientation of fiber segments. NE-4C cells form dense clusters that strongly adhere to the surface of FG50-based scaffolds, while also increasing FG concentration and adding NDs favor cellular infiltration into the flexible fibrous FG70_NDs nanocomposite. This research illustrates the potential of nanostructured NDs-FG fibers as scaffolds for nerve repair and regeneration. We also emphasize the importance of further understanding the effect of the nanofiller-protein interphase on the microstructure and properties of electrospun fibers and on cell-interactivity.

Controllable four axis extrusion-based additive manufacturing system for the fabrication of tubular scaffolds with tailorable mechanical properties.

Many tubular tissues such as blood vessels and trachea can suffer long-segmental defects through trauma and disease. With current limitations in the use of autologous grafts, the need for a synthetic substitute is of continuous interest as possible alternatives. Fabrication of these tubular organs is commonly done with techniques such as electrospinning and melt electrowriting using a rotational collector. Current additive manufacturing (AM) systems do not commonly implement the use of a rotational axis, which limits their application for the fabrication of tubular scaffolds. In this study, a four axis extrusion-based AM system similar to fused deposition modeling (FDM) has been developed to create tubular hollow scaffolds. A rectangular and a diamond pore design were further investigated for mechanical characterization, as a standard and a biomimicry pore geometry respectively. We demonstrated that in the radial compression mode the diamond pore design had a higher Young's modulus (19,8 ± 0,7 MPa compared to 2,8 ± 0,5 MPa), while in the longitudinal tensile mode the rectangular pore design had a higher Young's modulus (5,8 ± 0,2 MPa compared to 0,1 ± 0,01 MPa). Three-point bending analyses revealed that the diamond pore design is more resistant to luminal collapse compared to the rectangular design. This data showed that by changing the scaffold pore design, a wide range of mechanical properties could be obtained. Furthermore, a full control over scaffold design and geometry can be achieved with the developed 4-axis extrusion-based system, which has not been reported with other techniques. This flexibility allow the manufacturing of scaffolds for diverse tubular tissue regeneration applications by designing suitable deposition patterns to match their mechanical pre-requisites.

Development of 3D Bioactive Scaffolds through 3D Printing Using Wollastonite-Gelatin Inks.

The bioactivity of scaffolds represents a key property to facilitate the bone repair after orthopedic trauma. This study reports the development of biomimetic paste-type inks based on wollastonite (CS) and fish gelatin (FG) in a mass ratio similar to natural bone, as an appealing strategy to promote the mineralization during scaffold incubation in simulated body fluid (SBF). High-resolution 3D scaffolds were fabricated through 3D printing, and the homogeneous distribution of CS in the protein matrix was revealed by scanning electron microscopy/energy-dispersive X-ray diffraction analysis (SEM/EDX) micrographs. The bioactivity of the scaffold was suggested by an outstanding mineralization capacity revealed by the apatite layers deposited on the scaffold surface after immersion in SBF. The biocompatibility was demonstrated by cell proliferation established by MTT assay and fluorescence microscopy images and confirmed by SEM micrographs illustrating cell spreading. This work highlights the potential of the bicomponent inks to fabricate 3D bioactive scaffolds and predicts the osteogenic properties for bone regeneration applications.

Bioinspired Hydrogel Coating Based on Methacryloyl Gelatin Bioactivates Polypropylene Meshes for Abdominal Wall Repair.

Considering the potential of hydrogels to mimic the cellular microenvironment, methacryloyl gelatin (GelMA) and methacryloyl mucin (MuMA) were selected and compared as bioinspired coatings for commercially available polypropylene (PP) meshes for ventral hernia repair. Thin, elastic hydrated hydrogel layers were obtained through network-forming photo-polymerization, after immobilization of derivatives on the surface of the PP fibers. Fourier transform infrared spectroscopy (FTIR) proved the successful coating while the surface morphology and homogeneity were investigated by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). The stability of the hydrogel layers was evaluated through biodynamic tests performed on the coated meshes for seven days, followed by inspection of surface morphology through SEM and micro-CT. Taking into account that platelet-rich plasma (PRP) may improve healing due to its high concentration of growth factors, this extract was used as pre-treatment for the hydrogel coating to additionally stimulate cell interactions. The performed advanced characterization proved that GelMA and MuMA coatings can modulate fibroblasts response on PP meshes, either as such or supplemented with PRP extract as a blood-derived bioactivator. GelMA supported the best cellular response. These findings may extend the applicative potential of functionalized gelatin opening a new path on the research and engineering of a new generation of bioactive meshes.

Electrospun Fibre Webs Templated Synthesis of Mineral Scaffolds Based on Calcium Phosphates and Barium Titanate.

The current work focuses on the development of mineral scaffolds with complex composition and controlled morphology by using a polymeric template in the form of nonwoven fibre webs fabricated through electrospinning. By a cross-linking process, gelatine fibres stable in aqueous solutions were achieved, these being further subjected to a loading step with two types of mineral phases: calcium phosphates deposited by chemical reaction and barium titanate nanoparticles as decoration on the previously achieved structures. Thus, hybrid materials were obtained and subsequently processed in terms of freeze-drying and heat treating with the purpose of burning the template and consolidating the mineral part as potential bone implants with improved biological response by external stimulation. The results confirmed the tunable morphology, as well as the considerable applicability of both as-prepared and final samples for the development of medical devices, which encourages the continuation of research in the direction of assessing the synergistic contribution of barium titanate domains polarisation/magnetisation by external applied fields.

Fabrication and Biocompatibility Evaluation of Nanodiamonds-Gelatin Electrospun Materials Designed for Prospective Tissue Regeneration Applications.

Due to the reduced ability of most harmed tissues to self-regenerate, new strategies are being developed in order to promote self-repair assisted or not by biomaterials, among these tissue engineering (TE). Human adipose-derived mesenchymal stem cells (hASCs) currently represent a promising tool for tissue reconstruction, due to their low immunogenicity, high differentiation potential to multiple cell types and easy harvesting. Gelatin is a natural biocompatible polymer used for regenerative applications, while nanodiamond particles (NDs) are used as reinforcing nanomaterial that might modulate cell behavior, namely cell adhesion, viability, and proliferation. The development of electrospun microfibers loaded with NDs is expected to allow nanomechanical sensing due to local modifications of both nanostructure and stiffness. Two aqueous suspensions with 0.5 and 1% w/v NDs in gelatin from cold water fish skin (FG) were used to generate electrospun meshes. Advanced morpho- and micro-structural characterization revealed homogeneous microfibers. Nanoindentation tests confirmed the reinforcing effect of NDs. Biocompatibility assays showed an increased viability and proliferation profile of hASCs in contact with FG_NDs, correlated with very low cytotoxic effects of the materials. Moreover, hASCs developed an elongated cytoskeleton, suggesting that NDs addition to FG materials encouraged cell adhesion. This study showed the FG_NDs fibrous scaffolds potential for advanced TE applications.

Bioinspired 3D printable pectin-nanocellulose ink formulations.

The assessment of several ink formulations for 3D printing based on two natural macromolecular compounds is presented. In the current research we have exploited the fast crosslinking potential of pectin and the remarkable shear-thinning properties of carboxylated cellulose nanofibrils, which is known to induce a desired viscoelastic behavior. Prior to 3D printing, the viscoelastic properties of the polysaccharide inks were evaluated by rheological measurements and injectability tests. The reliance of the printing parameters on the ink composition was established through one-dimensional lines printing, the base units of 3D-structures. The performance of the 3D-printed structures after ionic cross-linking was evaluated in terms of mechanical properties and rehydration behavior. MicroCT was also used to evaluate the morphology of the 3D-printed objects regarding the effect of pectin/nanocellulose ratio on the geometrical features of scaffolds. The proportionality between the two polymers proved to be the determining factor for the firmness and strength of the printed objects.