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.

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.

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.

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.

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.

Porosity imaged by a vector projection algorithm correlates with fractal dimension measured on 3D models obtained by microCT.

Porosity is an important factor to consider in a large variety of materials. Porosity can be visualized in bone or 3D synthetic biomaterials by microcomputed tomography (microCT). Blocks of porous poly(2-hydroxyethyl methacrylate) were prepared with polystyrene beads of different diameter (500, 850, 1160 and 1560 µm) and analysed by microCT. On each 2D binarized microCT section, pixels of the pores which belong to the same image column received the same pseudo-colour according to a look up table. The same colour was applied on the same column of a frontal plane image which was constructed line by line from all images of the microCT stack. The fractal dimension Df of the frontal plane image was measured as well as the descriptors of the 3D models (porosity, 3D fractal dimension D3D, thickness, density and separation of material walls. Porosity, thickness Df and D3D increased with the size of the porogen beads. A linear correlation was observed between Df and D3D. This method provides quantitative and qualitative analysis of porosity on a single frontal plane image of a porous object.

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.

Osteoblast responsive biosilica-enriched gelatin microfibrillar microenvironments.

Engineering of scaffolds for bone regeneration is often inspired by the native extracellular matrix mimicking its composite fibrous structure. In the present study, we used low loadings of diatomite earth (DE) biosilica to improve the bone regeneration potential of gelatin electrospun fibrillar microenvironments. We explored the effect of increasing the DE content from 1 % to 3 % and 5 %, respectively, on the physico-chemical properties of the fibrous scaffolds denoted FG_DE1, FG_DE3, FG_DE5, regarding the aqueous media affinity, stability under simulated physiological conditions, morphology characteristics, and local mechanical properties at the surface. The presence of biosilica generated composite structures with lower swelling degrees and higher stiffness when compared to gelatin fibers. Increasing DE content led to higher Young modulus, while the stability of the protein matrix in PBS, at 37 °C, over 21 was significantly decreased by the presence of diatomite loadings. The best preosteoblast response was obtained for FG_DE3, with enhanced mineralization during the osteogenic differentiation when compared to the control sample without diatomite. 5 % DE in FG_DE5 proved to negatively influence cells' metabolic activity and morphology. Hence, the obtained composite microfibrillar scaffolds might find application as osteoblast-responsive materials for bone 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.

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.

Porous calcium alginate-gelatin interpenetrated matrix and its biomineralization potential.

Artificial bone composites exhibit distinctive features by comparison to natural tissues, due to a lack of self-organization and intimate interaction apatite-matrix. This explains the need of "bio-inspired materials", in which hydroxyapatite grows in contact with self-assembling natural polymers. The present work investigates the function of a rational design in the hydroxyapatite-forming potential of a common biopolymer. Gelatin modified through intrinsic interactions with calcium alginate led through freeze-drying to porous hydrogels, whose architecture, constitutive features and chemistry were investigated with respect to their role on biomineralization. The apatite-forming ability was enhanced by the porosity of the materials, while the presence of alginate-reinforced Gel elastic chains, definitely favored this phenomenon. Depending on the concentration, polysaccharide chains act as "ionic pumps" enhancing the biomineralization. The mineralization-promoting effect of the peptide-polysaccharide network strictly depends on the hydrogels structural, compositional and morphological features derived from the interaction between the above mentioned two components.

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.

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 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.

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.

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.

Poly(2-hydroxyethyl methacrylate-co-dodecyl methacrylate-co-acrylic acid): synthesis, physico-chemical characterisation and nafcillin carrier.

In the present study polymeric microbeads of poly(2-hydroxyethyl methacrylate-co-dodecyl methacrylate-co-acrylic acid) or p(HEMA-co-dDMA-co-AA) were synthesised and characterized through FT-IR and scanning electron microscopy (SEM); their swelling behavior against saline solution was explored and their in vitro cytotoxicity was evaluated. Further, in order to elucidate kinetic aspects regarding the ternary system p(HEMA-co-dDMA-co-AA), a mathematical model of the reactivity ratios of the comonomers in the terpolymer has been conceived and analyzed. An intensified tendency of AA units accumulation in the copolymer has been noticed, in spite of HEMA units, while dDMA conserves in the copolymer the fraction from the feed. Three compositions have been selected for nafcillin-loading and their in vitro release capacity was evaluated. The compositions of 80:10:10 and 75:10:15 M ratios appear suitable for further in vivo testing, in order to be used as drug delivery systems in the treatment of different osseous diseases.