Towards Complex Tissues Replication: Multilayer Scaffold Integrating Biomimetic Nanohydroxyapatite/Chitosan Composites.

This study explores an approach to design and prepare a multilayer scaffold mimicking interstratified natural tissue. This multilayer construct, composed of chitosan matrices with graded nanohydroxyapatite concentrations, was achieved through an in situ biomineralization process applied to individual layers. Three distinct precursor concentrations were considered, resulting in 10, 20, and 30 wt% nanohydroxyapatite content in each layer. The resulting chitosan/nanohydroxyapatite (Cs/n-HAp) scaffolds, created via freeze-drying, exhibited nanohydroxyapatite nucleation, homogeneous distribution, improved mechanical properties, and good cytocompatibility. The cytocompatibility analysis revealed that the Cs/n-HAp layers presented cell proliferation similar to the control in pure Cs for the samples with 10% n-HAp, indicating good cytocompatibility at this concentration, while no induction of apoptotic death pathways was demonstrated up to a 20 wt% n-Hap concentration. Successful multilayer assembly of Cs and Cs/n-HAp layers highlighted that the proposed approach represents a promising strategy for mimicking multifaceted tissues, such as osteochondral ones.

Micro- and Nanostructured Fibrous Composites via Electro-Fluid Dynamics: Design and Applications for Brain.

The brain consists of an interconnected network of neurons tightly packed in the extracellular matrix (ECM) to form complex and heterogeneous composite tissue. According to recent biomimicry approaches that consider biological features as active components of biomaterials, designing a highly reproducible microenvironment for brain cells can represent a key tool for tissue repair and regeneration. Indeed, this is crucial to support cell growth, mitigate inflammation phenomena and provide adequate structural properties needed to support the damaged tissue, corroborating the activity of the vascular network and ultimately the functionality of neurons. In this context, electro-fluid dynamic techniques (EFDTs), i.e., electrospinning, electrospraying and related techniques, offer the opportunity to engineer a wide variety of composite substrates by integrating fibers, particles, and hydrogels at different scales-from several hundred microns down to tens of nanometers-for the generation of countless patterns of physical and biochemical cues suitable for influencing the in vitro response of coexistent brain cell populations mediated by the surrounding microenvironment. In this review, an overview of the different technological approaches-based on EFDTs-for engineering fibrous and/or particle-loaded composite substrates will be proposed. The second section of this review will primarily focus on describing current and future approaches to the use of composites for brain applications, ranging from therapeutic to diagnostic/theranostic use and from repair to regeneration, with the ultimate goal of providing insightful information to guide future research efforts toward the development of more efficient and reliable solutions.

Towards nanostructured red-ox active bio-interfaces: Bioinspired antibacterial hybrid melanin-CeO2 nanoparticles for radical homeostasis.

Redox-active nano-biointerfaces are gaining weight in the field of regenerative medicine since they can act as enzymes in regulating physiological processes and enabling cell homeostasis, as well as the defense against pathogen aggression. In particular, cerium oxide nanoparticles (CeO2 NPs) stand as intriguing enzyme-mimicking nanoplatforms, owing to the reversible Ce+3/Ce+4 surface oxidation state. Moreover, surface functionalization leads to higher catalytic activity and selectivity, as well as more tunable enzyme-mimicking performances. Conjugation with melanin is an adequate strategy to boost and enrich CeO2 NPs biological features, because of melanin redox properties accounting for intrinsic antioxidant, antimicrobial and anti-inflammatory power. Herein, hybrid Melanin/CeO2 nanostructures were designed by simply coating the metal-oxide nanoparticles with melanin chains, obtained in-situ through ligand-to-metal charge transfer mechanism, according to a bioinspired approach. Obtained hybrid nanostructures underwent detailed physico-chemical characterization. Morphological and textural features were investigated through TEM, XRD and N2 physisorption. The nature of nanoparticle-melanin interaction was analyzed through FTIR, UV-vis and EPR spectroscopy. Melanin-coated hybrid nanostructures exhibited a relevant antioxidant activity, confirmed by a powerful quenching effect for DPPH radical, reaching 81 % inhibition at 33 µg/mL. A promising anti-inflammatory efficacy of the melanin-coated hybrid nanostructures was validated through a significant inhibition of BSA denaturation after 3 h. Meanwhile, the enzyme-mimicking activity was corroborated by a prolonged peroxidase activity after 8 h at 100 µg/mL and a relevant catalase-like action, by halving the H2O2 level in 30 min at 50 µg/mL. Antimicrobial assays attested that conjugation with melanin dramatically boosted CeO2 biocide activity against both Gram (-) and Gram (+) strains. Cytocompatibility tests demonstrated that the melanin coating not only enhanced the CeO2 nanostructures biomimicry, resulting in improved cell viability for human dermal fibroblast cells (HDFs), but mostly they proved that Melanin-CeO2 NPs were able to control the oxidative stress, modulating the production of nitrite and reactive oxygen species (ROS) levels in HDFs, under physiological conditions. Such remarkable outcomes make hybrid melanin-CeO2 nanozymes, promising redox-active interfaces for regenerative medicine.

Bioactive Composite Methacrylated Gellan Gum for 3D-Printed Bone Tissue-Engineered Scaffolds.

Gellan gum (GG) was chemically modified with methacrylic moieties to produce a photocrosslinkable biomaterial ink, hereinafter called methacrylated GG (GGMA), with improved physico-chemical properties, mechanical behavior and stability under physiological conditions. Afterwards, GGMA was functionalized by incorporating two different bioactive compounds, a naturally derived eumelanin extracted from the black soldier fly (BSF-Eumel), or hydroxyapatite nanoparticles (HAp), synthesized by the sol-gel method. Different ink formulations based on GGMA (2 and 4% (w/v)), BSF-Eumel, at a selected concentration (0.3125 mg/mL), or HAp (10 and 30% wHAp/wGGMA) were developed and processed by three-dimensional (3D) printing. All the functionalized GGMA-based ink formulations allowed obtaining 3D-printed GGMA-based scaffolds with a well-organized structure. For both bioactive signals, the scaffolds with the highest GGMA concentration (4% (w/v)) and the highest percentage of infill (45%) showed the best performances in terms of morphological and mechanical properties. Indeed, these scaffolds showed a good structural integrity over 28 days. Given the presence of negatively charged groups along the eumelanin backbone, scaffolds consisting of GGMA/BSF-Eumel demonstrated a higher stability. From a mechanical point of view, GGMA/BSF-Eumel scaffolds exhibited values of storage modulus similar to those of GGMA ones, while the inclusion of HAp at 30% (wHAp/wGGMA) led to a storage modulus of 32.5 kPa, 3.5-fold greater than neat GGMA. In vitro studies proved the capability of the bioactivated 3D-printed scaffolds to support 7F2 osteoblast cell growth and differentiation. BSF-Eumel and HAp triggered a different time-dependent physiological response in the osteoblasts. Specifically, while the ink with BSF-Eumel acted as a stimulus towards cell proliferation, reaching the highest value at 14 days, a higher expression of alkaline phosphatase activity was detected for scaffolds consisting of GGMA and HAp. The overall findings demonstrated the possible use of these biomaterial inks for 3D-printed bone tissue-engineered scaffolds.

Advances in the Physico-Chemical, Antimicrobial and Angiogenic Properties of Graphene-Oxide/Cellulose Nanocomposites for Wound Healing.

Graphene oxide (GO) and its reduced form (rGO) have recently attracted a fascinating interest due to their physico-chemical properties, which have opened up new and interesting opportunities in a wide range of biomedical applications, such as wound healing. It is worth noting that GO and rGO may offer a convenient access to its ready dispersion within various polymeric matrices (such as cellulose and its derivative forms), owing to their large surface area, based on a carbon skeleton with many functional groups (i.e., hydroxyl, carboxyl, epoxy bridge, and carbonyl moieties). This results in new synergic properties due to the presence of both components (GO or rGO and polymers), acting at different length-scales. Furthermore, they have shown efficient antimicrobial and angiogenic properties, mostly related to the intracellular formation of reactive oxygen species (ROS), which are advantageous in wound care management. For this reason, GO or rGO integration in cellulose-based matrixes have allowed for designing highly advanced multifunctional hybrid nanocomposites with tailored properties. The current review aims to discuss a potential relationship between structural and physico-chemical properties (i.e., size, edge density, surface chemistry, hydrophilicity) of the nanocomposites with antimicrobials and angiogenic mechanisms that synergically influence the wound healing phenomenon, by paying particular attention to recent findings of GO or rGO/cellulose nanocomposites. Accordingly, after providing a general overview of cellulose and its derivatives, the production methods used for GO and rGO synthesis, the mechanisms that guide antimicrobial and angiogenic processes of tissue repair, as well as the most recent and remarkable outcomes on GO/cellulose scaffolds in wound healing applications, will be presented.

Targeted treatment of triple-negative-breast cancer through pH-triggered tumour associated macrophages using smart theranostic nanoformulations.

Triple-negative breast cancer (TNBC) represents 15-25 % of the new breast cancer cases diagnosed worldwide every year. TNBC is among the most aggressive and worst prognosis breast cancer, mainly because targeted therapies are not available. Herein, we developed a magnetic theranostic hybrid nanovehicle for targeted treatment of TNBC through pH-triggered tumour associated macrophages (TAMs) targeting. The lipid core of the nanovehicle was composed of a Carnaúba wax matrix that simultaneously incorporated iron oxide nanoparticles and doxorubicin (DOX) - a chemotherapeutic drug. These drug-loaded wax nanovehicles were modified with a combination of two functional and complementary molecules: (i) a mannose ligand (macrophage targeting) and (ii) an acid-sensitive sheddable polyethylene glycol (PEG) moiety (specificity). The TAMs targeting strategy relied on the mannose - mannose receptor recognition exclusively after acid-sensitive "shedding" of the PEG in the relatively low tumour microenvironment pH. The pH-induced targeting capability towards TAMs was confirmed in vitro in a J774A.1 macrophage cell line at different pH (7.4 and 6.5). Biocompatibility and efficacy of the final targeted formulations were demonstrated in vitro in the TNBC MDA-MB-231 cell line and in vivo in an M-Wnt tumour-bearing (TNBC) mouse model. A preferential accumulation of the DOX-loaded lipid nanovehicles in the tumours of M-Wnt-tumour bearing mice was observed, which resulted both on an efficient tumour growth inhibition and a significantly reduced off-target toxicity compared to free DOX. Additionally, the developed magnetic hybrid nanovehicles showed outstanding performances as T2-contrast agents in magnetic resonance imaging (r2 ≈ 400-600 mM-1·s-1) and as heat generating sources in magnetic hyperthermia (specific absorption rate, SAR ≈ 178 W·g-1Fe). These targeted magnetic hybrid nanovehicles emerge as a suitable theranostic option that responds to the urgent demand for more precise and personalized treatments, not only because they are able to offer localized imaging and therapeutic potential, but also because they allow to efficiently control the balance between safety and efficacy.

Insights into the Effect of Magnetic Confinement on the Performance of Magnetic Nanocomposites in Magnetic Hyperthermia and Magnetic Resonance Imaging.

The combination of superparamagnetic iron oxide nanoparticles (SPIONs) and lipid matrices enables the integration of imaging, drug delivery, and therapy functionalities into smart theranostic nanocomposites. SPION confinement creates new interactions primarily among the embedded SPIONs and then between the nanocomposites and the surroundings. Understanding the parameters that rule these interactions in real interacting (nano)systems still represents a challenge, making it difficult to predict or even explain the final (magnetic) behavior of such systems. Herein, a systematic study focused on the performance of a magnetic nanocomposite as a magnetic resonance imaging (MRI) contrast agent and magnetic hyperthermia (MH) effector is presented. The effect of stabilizing agents and magnetic loading on the final physicochemical and, more importantly, functional properties (i.e., blocking temperature, specific absorption rate, relaxivity) was studied in detail.

Eumelanin from the Black Soldier Fly as Sustainable Biomaterial: Characterisation and Functional Benefits in Tissue-Engineered Composite Scaffolds.

An optimized extraction protocol for eumelanins from black soldier flies (BSF-Eumel) allows an in-depth study of natural eumelanin pigments, which are a valuable tool for the design and fabrication of sustainable scaffolds. Here, water-soluble BSF-Eumel sub-micrometer colloidal particles were used as bioactive signals for developing a composite biomaterial ink for scaffold preparation. For this purpose, BSF-Eumel was characterized both chemically and morphologically; moreover, biological studies were carried out to investigate the dose-dependent cell viability and its influence on human mesenchymal stem cells (hMSCs), with the aim of validating suitable protocols and to find an optimal working concentration for eumelanin-based scaffold preparation. As proof of concept, 3D printed scaffolds based on methacrylated hyaluronic acid (MEHA) and BSF-Eumel were successfully produced. The scaffolds with and without BSF-Eumel were characterized in terms of their physico-chemical, mechanical and biological behaviours. The results showed that MEHA/BSF-Eumel scaffolds had similar storage modulus values to MEHA scaffolds. In terms of swelling ratio and stability, these scaffolds were able to retain their structure without significant changes over 21 days. Biological investigations demonstrated the ability of the bioactivated scaffolds to support the adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells.

Genipin-crosslinked collagen scaffolds inducing chondrogenesis: a mechanical and biological characterization.

Articular cartilage degeneration is still an unsolved issue owing to its weak repairing capabilities, which usually result in fibrocartilage tissue formation. This fibrous tissue lacks of structural and bio-mechanical properties, degrading over time. Currently, arthroscopic techniques and autologous transplantation are the most used clinical procedures. However, rather than restoring cartilage integrity, these methods only postpone further cartilage deterioration. Therefore, tissue engineering strategies aimed at selecting scaffolds that remarkably support the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) could represent a promising solution, but they are still challenging for researchers. In this study, the influence of two different genipin (Gp) crosslinking routes on collagen (Coll)-based scaffolds in terms of hMSCs chondrogenic differentiation and biomechanical performances was investigated. Three-dimensional (3D) porous Coll scaffolds were fabricated by freeze-drying techniques and were crosslinked with Gp following a "two-step" and an in "bulk" procedure, in order to increase the physico-mechanical stability of the structure. Chondrogenic differentiation efficacy of hMSCs and biomechanical behavior under compression forces through unconfined stress-strain tests were assessed. Coll/Gp scaffolds revealed an isotropic and highly homogeneous pore distribution along with an increase in the stiffness, also supported by the increase in the Coll denaturation temperature (Td  = 57-63°C) and a significant amount of Coll and GAG deposition during the 3 weeks of chondrogenic culture. In particular, the presence of Gp in "bulk" led to a more uniform and homogenous chondral-like matrix deposition by hMSCs if compared to the results obtained from the Gp "two-step" functionalization procedure.

Trends in Managing Cardiac and Orthopaedic Device-Associated Infections by Using Therapeutic Biomaterials.

Over the years, there has been an increasing number of cardiac and orthopaedic implanted medical devices, which has caused an increased incidence of device-associated infections. The surfaces of these indwelling devices are preferred sites for the development of biofilms that are potentially lethal for patients. Device-related infections form a large proportion of hospital-acquired infections and have a bearing on both morbidity and mortality. Treatment of these infections is limited to the use of systemic antibiotics with invasive revision surgeries, which had implications on healthcare burdens. The purpose of this review is to describe the main causes that lead to the onset of infection, highlighting both the biological and clinical pathophysiology. Both passive and active surface treatments have been used in the field of biomaterials to reduce the impact of these infections. This includes the use of antimicrobial peptides and ionic liquids in the preventive treatment of antibiotic-resistant biofilms. Thus far, multiple in vivo studies have shown efficacious effects against the antibiotic-resistant biofilm. However, this has yet to materialize in clinical medicine.

Evidence of Modular Responsiveness of Osteoblast-Like Cells Exposed to Hydroxyapatite-Containing Magnetic Nanostructures.

The development of nanocomposites with tailored physical-chemical properties, such as nanoparticles containing magnetic iron oxides for manipulating cellular events at distance, implies exciting prospects in biomedical applications for bone tissue regeneration. In this context, this study aims to emphasize the occurrence of differential responsiveness in osteoblast-like cells to different nanocomposites with diverse features: dextran-grafted iron oxide (DM) nanoparticles and their hybrid nano-hydroxyapatite (DM/n-HA) counterpart. Here, responsiveness of cells in the presence of DMs or DM/n-HAs was evaluated in terms of cytoskeletal features. We observed that effects triggered by the DM are no more retained when DM is embedded onto the DM/n-HA nanocomposites. Also, analysis of mRNA level variations of the focal adhesion kinase (FAK), P53 and SLC11A2/DMT1 human genes showed that the DM/n-HA-treated cells retain tracts of physiological responsiveness compared to the DM-treated cells. Overall, a shielding effect by the n-HA component can be assumed, masking the DM's cytotoxic potential, also hinting a modular biomimicry of the nanocomposites respect to the physiological responses of osteoblast-like cells. In this view, the biocompatibility of n-HA together with the magnetic responsiveness of DMs represent an optimized combination of structural with functional features of the DM/n-HA nano-tools for bone tissue engineering, for finely acting within physiological ranges.

Mussel Shell-Derived Macroporous 3D Scaffold: Characterization and Optimization Study of a Bioceramic from the Circular Economy.

Fish industry by-products constitute an interesting platform for the extraction and recovery of valuable compounds in a circular economy approach. Among them, mussel shells could provide a calcium-rich source for the synthesis of hydroxyapatite (HA) bioceramics. In this work, HA nanoparticles have been successfully synthesized starting from mussel shells (Mytilus edulis) with a two steps process based on thermal treatment to convert CaCO3 in CaO and subsequent wet precipitation with a phosphorus source. Several parameters were studied, such as the temperature and gaseous atmosphere of the thermal treatment as well as the use of two different phosphorus-containing reagents in the wet precipitation. Data have revealed that the characteristics of the powders can be tailored, changing the conditions of the process. In particular, the use of (NH4)2HPO4 as the phosphorus source led to HA nanoparticles with a high crystallinity degree, while smaller nanoparticles with a higher surface area were obtained when H3PO4 was employed. Further, a selected HA sample was synthesized at the pilot scale; then, it was employed to fabricate porous 3D scaffolds using the direct foaming method. A highly porous scaffold with open and interconnected porosity associated with good mechanical properties (i.e., porosity in the range 87-89%, pore size in the range 50-300 µm, and a compressive strength σ = 0.51 ± 0.14 MPa) suitable for bone replacement was achieved. These results suggest that mussel shell by-products are effectively usable for the development of compounds of high added value in the biomedical field.

Bioactive chitosan-based scaffolds with improved properties induced by dextran-grafted nano-maghemite and l-arginine amino acid.

Over the past years, fundamentals of magnetism opened a wide research area of interest, in the field of tissue engineering and regenerative medicine. The integration of magnetic nanoarchitectures into synthetic/natural scaffold formulations allowed obtaining "on demand" responsive structures able to guide the regeneration process. The aim of this work was the design and characterization of three-dimensional (3D) chitosan-based scaffolds containing dextran-grafted maghemite nanoarchitectures (DM) and functionalized with l-arginine (l-Arg) amino acid as bioactive agent. A homogeneous pore distribution and a high degree of interconnection were obtained for all the structures with DMs, which resulted well distributed inside the polymer matrix. All the results suggest that the simultaneous presence of DMs and l-Arg conferred interesting mechano-structural and bioactive properties toward osteoblast-like and human mesenchymal stem cells, differentially stimulating their proliferation both in the absence and in the presence of a time-dependent magnetic field. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1244-1252, 2019.

Simplified preparation and characterization of magnetic hydroxyapatite-based nanocomposites.

Authors aimed to provide a magnetic responsiveness to bone-mimicking nano-hydroxyapatite (n-HA). For this purpose, dextran-grafted iron oxide nanoarchitectures (DM) were synthesized by a green-friendly and scalable alkaline co-precipitation method at room temperature and used to functionalize n-HA crystals. Different amounts of DM hybrid structures were added into the nanocomposites (DM/n-HA 1:1, 2:1 and 3:1weight ratio) which were investigated through extensive physicochemical (XRD, ICP, TGA and Zeta-potential), microstructural (TEM and DLS), magnetic (VSM) and biological analyses (MTT proliferation assay). X-ray diffraction patterns have confirmed the n-HA formation in the presence of DM as a co-reagent. Furthermore, the addition of DM during the synthesis does not affect the primary crystallite domains of DM/n-HA nanocomposites. DM/n-HAs have shown a rising of the magnetic moment values by increasing DM content up to 2:1 ratio. However, the magnetic moment value recorded in the DM/n-HA 3:1 do not further increase showing a saturation behaviour. The cytocompatibility of the DM/n-HA was evaluated with respect to the MG63 osteoblast-like cell line. Proliferation assays revealed that viability, carried out in the absence of external magnetic field, was not affected by the amount of DM employed. Interestingly, assays also suggested that the DM/n-HA nanocomposites exhibit a possible shielding effect with respect to the anti-proliferative activity induced by the DM particles alone.