Human T-Cell Responses to Metallic Ion-Doped Bioactive Glasses.

Biomaterials are extensively used as replacements for damaged tissue with bioactive glasses standing out as bone substitutes for their intrinsic osteogenic properties. However, biomaterial implantation has the following risks: the development of implant-associated infections and adverse immune responses. Thus, incorporating metallic ions with known antimicrobial properties can prevent infection, but should also modulate the immune response. Therefore, we selected silver, copper and tellurium as doping for bioactive glasses and evaluated the immunophenotype and cytokine profile of human T-cells cultured on top of these discs. Results showed that silver significantly decreased cell viability, copper increased the T helper (Th)-1 cell percentage while decreasing that of Th17, while tellurium did not affect either cell viability or immune response, as evaluated via multiparametric flow cytometry. Multiplex cytokines assay showed that IL-5 levels were decreased in the copper-doped discs, compared with its undoped control, while IL-10 tended to be lower in the doped glass, compared with the control (plastic) while undoped condition showed lower expression of IL-13 and increased MCP-1 and MIP-1ß secretion. Overall, we hypothesized that the Th1/Th17 shift, and specific cytokine expression indicated that T-cells might cross-activate other cell types, potentially macrophages and eosinophils, in response to the scaffolds.

A unified in vitro evaluation for apatite-forming ability of bioactive glasses and their variants.

The aim of this study was to propose and validate a new unified method for testing dissolution rates of bioactive glasses and their variants, and the formation of calcium phosphate layer formation on their surface, which is an indicator of bioactivity. At present, comparison in the literature is difficult as many groups use different testing protocols. An ISO standard covers the use of simulated body fluid on standard shape materials but it does not take into account that bioactive glasses can have very different specific surface areas, as for glass powders. Validation of the proposed modified test was through round robin testing and comparison to the ISO standard where appropriate. The proposed test uses fixed mass per solution volume ratio and agitated solution. The round robin study showed differences in hydroxyapatite nucleation on glasses of different composition and between glasses of the same composition but different particle size. The results were reproducible between research facilities. Researchers should use this method when testing new glasses, or their variants, to enable comparison between the literature in the future.

Development of Super-Paramagnetic Iron Oxide Nanoparticle-Coated Melt Electrowritten Scaffolds for Biomedical Applications.

Polycaprolactone (PCL) is usually the material chosen for melt electrowriting (MEW) due to its biocompatibility, mechanical strength, and melt processability. This work first investigates the effect of different processing parameters to obtain optimum PCL-MEW scaffolds. Secondly, to increase PCL`s hydrophilicity and cell affinity, and to enable coating with superparamagnetic iron oxide nanoparticles (SPIONs) and silica-coated-SPIONs (Si-SPIONs), the scaffolds are modified with alkaline surface treatment. Finally, SPIONs and Si-SPIONs are successfully coated on MEW scaffolds. Results show that reproducible scaffolds are fabricated. Additionally, the alkaline treatment does not change the three-dimensional morphology of scaffolds while reducing fiber diameter. Furthermore, SEM images and ATR-FTIR results confirmed that SPIONs and Si-SPIONs-were coated on scaffolds. A cytocompatibility assay showed a non-toxic effect on MG-63 osteoblast-like cells in all scaffolds. Additionally, higher MC3T3-E1 pre-osteoblastic cell adhesion efficiency and proliferation are achieved for the alkaline-treated scaffolds and SPIONs/Si-SPIONs-coated scaffolds. All samples demonstrated the ability to generate heat, useful for hyperthermia-treatment, when subjected to an alternating magnetic field. Overall, the findings suggest that the strategy of coating PCL-MEW scaffolds with SPIONs/Si-SPIONs has the potential to improve scaffold performance for biomedical applications, especially for producing magnetically responsive MEW scaffolds.

The unexplored role of alkali and alkaline earth elements (ALAEs) on the structure, processing, and biological effects of bioactive glasses.

Bioactive glass has been employed in several medical applications since its inception in 1969. The compositions of these materials have been investigated extensively with emphasis on glass network formers, therapeutic transition metals, and glass network modifiers. Through these experiments, several commercial and experimental compositions have been developed with varying chemical durability, induced physiological responses, and hydroxyapatite forming abilities. In many of these studies, the concentrations of each alkali and alkaline earth element have been altered to monitor changes in structure and biological response. This review aims to discuss the impact of each alkali and alkaline earth element on the structure, processing, and biological effects of bioactive glass. We explore critical questions regarding these elements from both a glass science and biological perspective. Should elements with little biological impact be included? Are alkali free bioactive glasses more promising for greater biological responses? Does this mixed alkali effect show increased degradation rates and should it be employed for optimized dissolution? Each of these questions along with others are evaluated comprehensively and discussed in the final section where guidance for compositional design is provided.

3D bioprinting of multifunctional alginate dialdehyde (ADA)-gelatin (GEL) (ADA-GEL) hydrogels incorporating ferulic acid.

The present work explores the 3D extrusion printing of ferulic acid (FA)-containing alginate dialdehyde (ADA)-gelatin (GEL) scaffolds with a wide spectrum of biophysical and pharmacological properties. The tailored addition of FA (≤0.2 %) increases the crosslinking between FA and GEL in the presence of calcium chloride (CaCl2) and microbial transglutaminase, as confirmed using trinitrobenzenesulfonic acid (TNBS) assay. In agreement with an increase in crosslinking density, a higher viscosity of ADA-GEL with FA incorporation was achieved, leading to better printability. Importantly, FA release, enzymatic degradation and swelling were progressively reduced with an increase in FA loading to ADA-GEL, over 28 days. Similar positive impact on antibacterial properties with S. epidermidis strains as well as antioxidant properties were recorded. Intriguingly, FA incorporated ADA-GEL supported murine pre-osteoblast proliferation with reduced osteosarcoma cell proliferation over 7 days in culture, implicating potential anticancer property. Most importantly, FA-incorporated and cell-encapsulated ADA-GEL can be extrusion printed to shape fidelity-compliant multilayer scaffolds, which also support pre-osteoblast cells over 7 days in culture. Taken together, the present study has confirmed the significant potential of 3D bioprinting of ADA-GEL-FA ink to obtain structurally stable scaffolds with a broad spectrum of biophysical and therapeutically significant properties, for bone tissue engineering applications.

Physical, Mechanical, and Biological Properties of PMMA-Based Composite Bone Cement Containing Silver-Doped Bioactive and Antibacterial Glass Particles with Different Particles Sizes.

In the present work, antibacterial composite bone cement was designed by introducing a bioactive and antibacterial glass into a commercial formulation. The effect of glass particles' addition on the curing parameters of the polymeric matrix was evaluated; moreover, the influence of the glass particle size on the glass dispersion, compressive and bending strength, bioactivity, and antibacterial effect was estimated. The results evidence a delay in the polymerization kinetics of the composite cement, which nevertheless complies with the requirements of the ISO standard. Morphological characterization provides evidence of good dispersion of the glass in the polymeric matrix and its exposition on the cement surface. The different glass grain sizes do not affect the composites' bioactivity and compressive strength, while a slight reduction in bending strength was observed for samples containing glass powders with greater dimensions. The size of the glass particles also appears to have an effect on the antibacterial properties, since the composites containing larger glass particles do not produce an inhibition halo towards the S. aureus strain. The obtained results demonstrate that, by carefully tailoring the glass amount and size, a multifunctional device for artificial joint fixing, temporary prostheses, or spinal surgery can be obtained.

Poly(ε-caprolactone)/bioactive glass composite electrospun fibers for tissue engineering applications.

In this work, composite electrospun fibers containing innovative bioactive glass nanoparticles were produced and characterized. Poly(ε-caprolactone), benign solvents, and sol-gel B- and Cu-doped bioactive glass powders were used to fabricate fibrous scaffolds. The retention of bioactive glass nanoparticles in the polymer matrix, the electrospinnability of this novel solution and the obtained electrospun composites were extensively characterized. As a result, composite electrospun fibers characterized by biocompatibility, bioactivity, and exhibiting overall properties adequate for both hard and soft tissue engineering applications, have been produced. The addition of these bioactive glass nanoparticles was, indeed, able to impart bioactive properties to the fibers. Cell culture studies show promising results, demonstrating proliferation and growth of cells on the composite fibers. Wettability, degradation rate, and mechanical performance were also tested and are in line with previous results.

Tannic-acid-mediated synthesis and characterization of magnetite-gold nanoplatforms for photothermal therapy.

Aim: The design of new hybrid nanoplatforms (HNPs) through the innovative and eco-friendly use of tannic acid (TA) for the synthesis and stabilization of the nanoplatforms. Materials & methods: The size, morphology, composition and magnetic and plasmonic properties of HNPs were investigated together with their ability to generate heat under laser irradiation and the hemotoxicity to explore their potential use for biomedical applications. Results & conclusion: The use of TA allowed the synthesis of the HNPs by adopting a simple and green method. The HNPs preserved the peculiar properties of both magnetic and plasmonic nanoparticles and did not show any hemotoxic effect.

The aim of this research was to prepare new nanoparticles (called nanoplatforms) made from two parts: a magnetic core and the addition of gold particles. These particles can be used for cancer treatment because, when stimulated by light, they are able to release heat, which can kill cancer cells. In particular, in this work, we investigated the preparation of these particles using green methods, without the use of toxic reagents. The obtained nanoparticles were studied to investigate their size, shape, composition, magnetic properties, ability to generate heat and possible toxic effect toward blood cells. The results show that these particles can be produced with green methods, release heat and are not toxic.

UV-Cured Bio-Based Acrylated Soybean Oil Scaffold Reinforced with Bioactive Glasses.

In this study, a bio-based acrylate resin derived from soybean oil was used in combination with a reactive diluent, isobornyl acrylate, to synthetize a composite scaffold reinforced with bioactive glass particles. The formulation contained acrylated epoxidized soybean oil (AESO), isobornyl acrylate (IBOA), a photo-initiator (Irgacure 819) and a bioactive glass particle. The resin showed high reactivity towards radical photopolymerisation, and the presence of the bioactive glass did not significantly affect the photocuring process. The 3D-printed samples showed different properties from the mould-polymerised samples. The glass transition temperature Tg showed an increase of 3D samples with increasing bioactive glass content, attributed to the layer-by-layer curing process that resulted in improved interaction between the bioactive glass and the polymer matrix. Scanning electron microscope analysis revealed an optimal distribution on bioactive glass within the samples. Compression tests indicated that the 3D-printed sample exhibited higher modulus compared to mould-synthetized samples, proving the enhanced mechanical behaviour of 3D-printed scaffolds. The cytocompatibility and biocompatibility of the samples were evaluated using human bone marrow mesenchymal stem cells (bMSCs). The metabolic activity and attachment of cells on the samples' surfaces were analysed, and the results demonstrated higher metabolic activity and increased cell attachment on the surfaces containing higher bioactive glass content. The viability of the cells was further confirmed through live/dead staining and reseeding experiments. Overall, this study presents a novel approach for fabricating bioactive glass reinforced scaffolds using 3D printing technology, offering potential applications in tissue engineering.

In situ reduction of Ag on magnetic nanoparticles with gallic acid: effect of the synthesis parameters on morphology.

Aim: Synthesis of Fe3O4-Ag composite nanoparticles (NPs) by a new in situ reduction of Ag NPs on the surface of Fe3O4 NPs using gallic acid as a reducing agent. Materials & methods: The influence of process parameters on NP morphology and functionalization was evaluated by means of field-emission scanning/scanning transmission electron microscopy and Fourier-transform IR spectroscopy. Results & conclusion: The synthesis conditions affected the morphology of the obtained NPs, evidence of the formation of polydispersed aggregates, nanoflower-like or nanodumbbell nanocomposites. In particular, well-defined nanodumbbells were obtained in aqueous media, with an NP/gallic acid ratio of 10:1, while the presence of a silica shell did not improve the morphology of Ag NPs nucleated on the Fe3O4 core.

Nanoparticles (NPs) have been extensively investigated in the biomedical field for their use in diagnosing and treating tumors. The aim of this work was to develop a simple and green method to produce NPs formed from iron oxides (which are magnetic) and silver. Magnetic NPs can be moved utilizing an external magnetic field, which can be used to localize NPs in a particular location, such as in tumors. Silver NPs have antibacterial properties and can be used to generate heat to kill tumor cells when exposed to light. This study showed that, by varying factors when synthesizing these NPs (e.g., amount of reagents, presence of coatings on the particles), it is possible to obtain multifunctional NPs with different shapes and sizes.

Iron Oxide-Au Magneto-Plasmonic Heterostructures: Advances in Their Eco-Friendly Synthesis.

In recent years, nanotechnologies have attracted considerable interest, especially in the biomedical field. Among the most investigated particles, magnetic based on iron oxides and Au nanoparticles gained huge interest for their magnetic and plasmonic properties, respectively. These nanoparticles are usually produced starting from processes and reagents that can be the cause of potential human health and environmental concerns. For this reason, there is a need to develop simple, green, low-cost, and non-toxic synthesis methods and reagents. This review aims at providing an overview of the most recently developed processes to produce iron oxide magnetic nanoparticles, Au nanoparticles, and their magneto-plasmonic heterostructures using eco-friendly approaches, focusing the attention on the microorganisms and plant-assisted syntheses and showing the first results of the development of magneto-plasmonic heterostructures.

Superparamagnetic and highly bioactive SPIONS/bioactive glass nanocomposite and its potential application in magnetic hyperthermia.

Magnetic bioactive glass-ceramics are biomaterials applied for magnetic hyperthermia in bone cancer treatment, thereby treating the bone tumor besides regenerating the damaged bone. However, combining high bioactivity and high saturation magnetization remains a challenge since the thermal treatment step employed to grow magnetic phases is also related to loss of bioactivity. Here, we propose a new nanocomposite made of superparamagnetic iron oxide nanoparticles (SPIONs) dispersed in a sol-gel-derived bioactive glass matrix, which does not need any thermal treatment for crystallization of magnetic phases. The scanning and transmission electron microscopies, X-ray diffraction, and dynamic light scattering results confirm that the SPIONs are actually embedded in a nanosized glass matrix, thus forming a nanocomposite. Magnetic and calorimetric characterizations evidence their proper behavior for hyperthermia applications, besides evidencing inter-magnetic nanoparticle interactions within the nanocomposite. Bioactivity and in vitro characterizations show that such nanocomposites exhibit apatite-forming properties similar to the highly bioactive parent glass, besides being osteoinductive. This methodology is a new alternative to produce magnetic bioactive materials to which the magnetic properties only rely on the quality of the SPIONs used in the synthesis. Thereby, these nanocomposites can be recognized as a new class of bioactive materials for applications in bone cancer treatment by hyperthermia.

Tellurium: A new active element for innovative multifunctional bioactive glasses.

Bioactive glasses have been widely investigated for their ability to release ions with therapeutic effect. In this paper, a silica based bioactive glass was doped with a low amount of tellurium dioxide (1 and 5 mol%) to confer antibacterial and antioxidant properties. The obtained glasses were characterized in terms of morphology, composition, structure, characteristic temperatures and in vitro bioactivity. Moreover, comprehensive analyses were carried out to estimate the cytocompatibility, the antibacterial and antioxidant properties of Te-doped glasses. The performed characterizations demonstrated that the Te insertion did not interfere with the amorphous nature of the glass, the substitution of SiO2 with TeO2 led to a slight decrease in Tg and a TeO2 amount higher than 1 mol% can induce a change in the primary crystal field. In vitro bioactivity test demonstrated the Te-doped glass ability to induce the precipitation of hydroxyapatite. Finally, the biological characterization showed a strong antibacterial and antioxidant effects of Te-containing glasses in comparison with the control glass, demonstrating that Te is a promising element to enhance the biological response of biomaterials.

Shock waves induce activity of human osteoblast-like cells in bioactive scaffolds.

BACKGROUND: Bone replacement is frequently needed in periodontal, orthopedic, and maxillofacial diseases. To avoid complications with autografts and allografts, artificial grafts (scaffolds) are candidates for stimulating bone regeneration after colonization with osteoblasts. Moreover, osteoblast activity can be induced by biological or physical stimulation. In this research, extracorporeal shock waves were used to improve the ability of human osteoblasts to colonize scaffolds and to induce their osteogenic properties. METHODS: Osteoblasts, treated with shock waves, were seeded on glass-ceramic macroporous scaffolds. Cells in scaffolds were counted after detachment and examined for calcium nodule formation (Alizarin staining), for differentiation markers (real time polymerase chain reaction), and for scaffold colonization (scanning electron microscope). RESULTS: Shock waves initially increased both the number and the activity of osteoblasts in the scaffold, but subsequently increased only osteoblast activity. Moreover, shock waves favored scaffold colonization even in the deeper layers. CONCLUSIONS: The calcium deposits and differentiation markers studied have demonstrated that shock waves increase osteoblast migration and penetration into scaffolds. CLINICAL RELEVANCE: This study may provide an important starting point for the introduction of shock waves to boost bone formation through osteoblast stimulation in diseases characterized by bone defects.

In vitro comparison between commercially and manually mixed antibiotic-loaded bone cements.

PURPOSE: The purpose of this study is the evaluation of the differences and, eventually, of the advantages or disadvantages of manual formulations with respect to industrial ones. METHODS: Medical-grade bone cements (Palacos R® and Palacos LV®), based on poly-methyl methacrylate (PMMA) and used clinically in several cemented prosthetic devices were manually enriched with gentamicin sulphate during preparation and then compared with a commercially-available, antibiotic-loaded cement (Palacos R+G®) by means of an in vitro antibacterial test (inhibition zone evaluation). The purpose of this study was to evaluate the differences and advantages or disadvantages, if any, of manual formulations compared to commercial ones. The use of a different antibiotic (vancomycin) alone or in addition to gentamicin-containing bone cements was also considered. RESULTS AND CONCLUSION: The commercial formulation produces an inhibition zone that is a bit larger and more regular than the manually mixed preparation. The vancomycin halo is smaller but clearer than the gentamicin halo. The addition of vancomycin to gentamicin-containing bone cements does not significantly increase the halo dimensions but could be an interesting strategy in the prevention of multiple and resistant infections.

Enhancing Mechanical Properties and Biological Performances of Injectable Bioactive Glass by Gelatin and Chitosan for Bone Small Defect Repair.

Bioactive glass (BG) represents a promising biomaterial for bone healing; here injectable BG pastes biological properties were improved by the addition of gelatin or chitosan, as well as mechanical resistance was enhanced by adding 10 or 20 wt% 3-Glycidyloxypropyl trimethoxysilane (GPTMS) cross-linker. Composite pastes exhibited bioactivity as apatite formation was observed by Scanning Electron Microscopy (SEM) and X-Ray Diffraction (XRD) after 14 days immersion in simulated body fluid (SBF); moreover, polymers did not enhance degradability as weight loss was >10% after 30 days in physiological conditions. BG-gelatin-20 wt% GPTMS composites demonstrated the highest compressive strength (4.8 ± 0.5 MPa) in comparison with the bulk control paste made of 100% BG in water (1.9 ± 0.1 MPa). Cytocompatibility was demonstrated towards human mesenchymal stem cells (hMSC), osteoblasts progenitors, and endothelial cells. The presence of 20 wt% GPTMS conferred antibacterial properties thus inhibiting the joint pathogens Staphylococcus aureus and Staphylococcus epidermidis infection. Finally, hMSC osteogenesis was successfully supported in a 3D model as demonstrated by alkaline phosphatase release and osteogenic genes expression.

Competitive Surface Colonization of Antibacterial and Bioactive Materials Doped with Strontium and/or Silver Ions.

Nowadays, there is a large amount of research aimed at improving the multifunctional behavior of the biomaterials for bone contact, including the concomitant ability to induce apatite formation (bioactivity), fast and effective osteoblasts colonization, and antibacterial activity. The aim of this study is to develop antibacterial and bioactive surfaces (Ti6Al4V alloy and a silica-based bioactive glass) by chemical doping with strontium and/or silver ions. The surfaces were characterized by Scanning Electron Microscopy equipped with Energy Dispersive X ray Spectroscopy (SEM-EDS), X-ray photoelectron spectroscopy (XPS), and Transmission Electron Microscopy (TEM). To better focus on the cells-bacteria competition for the implant surface, in addition to the standard assays for the evaluation of the bacteria adhesion (ISO22196) and for single-cell cultures or biofilm formation, an innovative set of co-cultures of cells and bacteria is here proposed to simulate a competitive surface colonization. The results suggest that all the bioactive tested materials were cytocompatible toward the bone progenitor cells representative for the self-healing process, and that the doped ones were effective in reducing the surface colonization from a pathogenic drug-resistant strain of Staphylococcus aureus. The co-cultures experiments demonstrated that the doped surfaces were able to protect the adhered osteoblasts from the bacteria colonization as well as prevent the infection prior to the surface colonization by the osteoblasts.

PMMA-Based Bone Cements and the Problem of Joint Arthroplasty Infections: Status and New Perspectives.

Polymethyl methacrylate (PMMA)-based bone cement is a biomaterial that has been used over the last 50 years to stabilize hip and knee implants or as a bone filler. Although PMMA-based bone cement is widely used and allows a fast-primary fixation to the bone, it does not guarantee a mechanically and biologically stable interface with bone, and most of all it is prone to bacteria adhesion and infection development. In the 1970s, antibiotic-loaded bone cements were introduced to reduce the infection rate in arthroplasty; however, the efficiency of antibiotic-containing bone cement is still a debated issue. For these reasons, in recent years, the scientific community has investigated new approaches to impart antibacterial properties to PMMA bone cement. The aim of this review is to summarize the current status regarding antibiotic-loaded PMMA-based bone cements, fill the gap regarding the lack of data on antibacterial bone cement, and explore the progress of antibacterial bone cement formulations, focusing attention on the new perspectives. In particular, this review highlights the innovative study of composite bone cements containing inorganic antibacterial and bioactive phases, which are a fascinating alternative that can impart both osteointegration and antibacterial properties to PMMA-based bone cement.

Electrospun Filaments Embedding Bioactive Glass Particles with Ion Release and Enhanced Mineralization.

Efforts in tissue engineering aim at creating scaffolds that mimic the physiological environment with its structural, topographical and mechanical properties for restoring the function of damaged tissue. In this study we introduce composite fibres made by a biodegradable poly(lactic acid) (PLLA) matrix embedding bioactive silica-based glass particles (SBA2). Electrospinning is performed to achieve porous PLLA filaments with uniform dispersion of bioactive glass powder. The obtained composite fibres show in aligned arrays significantly increased elastic modulus compared with that of neat polymer fibres during uniaxial tensile stress. Additionally, the SBA2 bioactivity is preserved upon encapsulation as highlighted by the promoted deposition of hydroxycarbonate apatite (HCA) upon immersion in simulated body fluid solutions. HCA formation is sequential to earlier processes of polymer erosion and ion release leading to acidification of the surrounding solution environment. These findings suggest PLLA-SBA2 fibres as a composite, multifunctional system which might be appealing for both bone and soft tissue engineering applications.

Tumor Targeting by Monoclonal Antibody Functionalized Magnetic Nanoparticles.

Tumor-targeted drug-loaded nanocarriers represent innovative and attractive tools for cancer therapy. Several magnetic nanoparticles (MNPs) were analyzed as potential tumor-targeted drug-loaded nanocarriers after functionalization with anti-Met oncogene (anti-Met/HGFR) monoclonal antibody (mAb) and doxorubicin (DOXO). Their cytocompatibility, stability, immunocompetence (immunoprecipitation), and their interactions with cancer cells in vitro (Perl's staining, confocal microscopy, cytotoxic assays: MTT, real time toxicity) and with tumors in vivo (Perl's staining) were evaluated. The simplest silica- and calcium-free mAb-loaded MNPs were the most cytocompatible, the most stable, and showed the best immunocompetence and specificity. These mAb-functionalized MNPs specifically interacted with the surface of Met/HGFR-positive cells, and not with Met/HGFR-negative cells; they were not internalized, but they discharged in the targeted cells DOXO, which reached the nucleus, exerting cytotoxicity. The presence of mAbs on DOXO-MNPs significantly increased their cytotoxicity on Met/HGFR-positive cells, while no such effect was detectable on Met/HGFR-negative cells. Bare MNPs were biocompatible in vivo; mAb presence on MNPs induced a better dispersion within the tumor mass when injected in situ in Met/HGFR-positive xenotumors in NOD/SCID-γnull mice. These MNPs may represent a new and promising carrier for in vivo targeted drug delivery, in which applied gradient and alternating magnetic fields can enhance targeting and induce hyperthermia respectively.