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.

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.

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.

Chitosan-Based Materials Featuring Multiscale Anisotropy for Wider Tissue Engineering Applications.

We designed graphene oxide composites with increased morphological and structural variability using fatty acid-coupled polysaccharide co-polymer as the continuous phase. The matrix was synthesized by N, O-acylation of chitosan with palmitic and lauric acid. The obtained co-polymer was crosslinked with genipin and composited with graphene oxide. FTIR spectra highlighted the modification and multi-components interaction. DLS, SEM, and contact angle tests demonstrated that the conjugation of hydrophobic molecules to chitosan increased surface roughness and hydrophilicity, since it triggered a core-shell macromolecular structuration. Nanoindentation revealed a notable durotaxis gradient due to chitosan/fatty acid self-organization and graphene sheet embedment. The composited building blocks with graphene oxide were more stable during in vitro enzymatic degradation tests and swelled less. In vitro viability, cytotoxicity, and inflammatory response tests yielded promising results, and the protein adsorption test demonstrated potential antifouling efficacy. The robust and stable substrates with heterogeneous architecture we developed show promise in biomedical applications.

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.

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.

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.

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.