New strontium-based coatings show activity against pathogenic bacteria in spine infection.

Infections of implants and prostheses represent relevant complications associated with the implantation of biomedical devices in spine surgery. Indeed, due to the length of the surgical procedures and the need to implant invasive devices, infections have high incidence, interfere with osseointegration, and are becoming increasingly difficult to threat with common therapies due to the acquisition of antibiotic resistance genes by pathogenic bacteria. The application of metal-substituted tricalcium phosphate coatings onto the biomedical devices is a promising strategy to simultaneously prevent bacterial infections and promote osseointegration/osseoinduction. Strontium-substituted tricalcium phosphate (Sr-TCP) is known to be an encouraging formulation with osseoinductive properties, but its antimicrobial potential is still unexplored. To this end, novel Sr-TCP coatings were manufactured by Ionized Jet Deposition technology and characterized for their physiochemical and morphological properties, cytotoxicity, and bioactivity against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538P human pathogenic strains. The coatings are nanostructured, as they are composed by aggregates with diameters from 90 nm up to 1 µm, and their morphology depends significantly on the deposition time. The Sr-TCP coatings did not exhibit any cytotoxic effects on human cell lines and provided an inhibitory effect on the planktonic growth of E. coli and S. aureus strains after 8 h of incubation. Furthermore, bacterial adhesion (after 4 h of exposure) and biofilm formation (after 24 h of cell growth) were significantly reduced when the strains were cultured on Sr-TCP compared to tricalcium phosphate only coatings. On Sr-TCP coatings, E. coli and S. aureus cells lost their organization in a biofilm-like structure and showed morphological alterations due to the toxic effect of the metal. These results demonstrate the stability and anti-adhesion/antibiofilm properties of IJD-manufactured Sr-TCP coatings, which represent potential candidates for future applications to prevent prostheses infections and to promote osteointegration/osteoinduction.

Ionized jet deposition of silver nanostructured coatings: Assessment of chemico-physical and biological behavior for application in orthopedics.

Infection is one of the main issues connected to implantation of biomedical devices and represents a very difficult issue to tackle, for clinicians and for patients. This study aimed at tackling infection through antibacterial nanostructured silver coatings manufactured by Ionized Jet Deposition (IJD) for application as new and advanced coating systems for medical devices. Films composition and morphology depending on deposition parameters were investigated and their performances evaluated by correlating these properties with the antibacterial and antibiofilm efficacy of the coatings, against Escherichia coli and Staphylococcus aureus strains and with their cytotoxicity towards human cell line fibroblasts. The biocompatibility of the coatings, the nanotoxicity, and the safety of the proposed approach were evaluated, for the first time, in vitro and in vivo by rat subcutaneous implant models. Different deposition times, corresponding to different thicknesses, were selected and compared. All silver coatings exhibited a highly homogeneous surface composed of nanosized spherical aggregates. All coatings having a thickness of 50 nm and above showed high antibacterial efficacy, while none of the tested options caused cytotoxicity when tested in vitro. Indeed, silver films impacted on bacterial strains viability and capability to adhere to the substrate, in a thickness-dependent manner. The nanostructure obtained by IJD permitted to mitigate the toxicity of silver, conferring strong antibacterial and anti-adhesive features, without affecting the coatings biocompatibility. At the explant, the coatings were still present although they showed signs of progressive dissolution, compatible with the release of silver, but no cracking, delamination or in vivo toxicity was observed.

Advantages and limitations of using cell viability assays for 3D bioprinted constructs.

Bioprinting shows promise for bioengineered scaffolds and three-dimensional (3D) disease models, but assessing the viability of embedded cells is challenging. Conventional assays are limited by the technical problems that derive from using multi-layered bioink matrices dispersing cells in three dimensions. In this study, we tested bioprinted osteogenic bioinks as a model system. Alginate- or gelatin-based bioinks were loaded with/without ceramic microparticles and osteogenic cells (bone tumor cells, with or without normal bone cells). Despite demonstrating 80%-90% viability through manual counting and live/dead staining, this was time-consuming and operator-dependent. Moreover, for the alginate-bioprinted scaffold, cell spheroids could not be distinguished from single cells. The indirect assay (alamarBlue), was faster but less accurate than live/dead staining due to dependence on hydrogel permeability. Automated confocal microscope acquisition and cell counting of live/dead staining was more reproducible, reliable, faster, efficient, and avoided overestimates compared to manual cell counting by optical microscopy. Finally, for 1.2 mm thick 3D bioprints, dual-photon confocal scanning with vital staining greatly improved the precision of the evaluation of cell distribution and viability and cell-cell interactions through thez-axis. In summary, automated confocal microscopy and cell counting provided superior accuracy for the assessment of cell viability and interactions in 3D bioprinted models compared to most commonly and currently used techniques.

A natural biogenic fluorapatite as a new biomaterial for orthopedics and dentistry: antibacterial activity of lingula seashell and its use for nanostructured biomimetic coatings.

Calcium phosphates are widely studied in orthopedics and dentistry, to obtain biomimetic and antibacterial implants. However, the multi-substituted composition of mineralized tissues is not fully reproducible from synthetic procedures. Here, for the first time, we investigate the possible use of a natural, fluorapatite-based material, i.e., Lingula anatina seashell, resembling the composition of bone and enamel, as a biomaterial source for orthopedics and dentistry. Indeed, thanks to its unique mineralization process and conditions, L. anatina seashell is among the few natural apatite-based shells, and naturally contains ions having possible antibacterial efficacy, i.e., fluorine and zinc. After characterization, we explore its deposition by ionized jet deposition (IJD), to obtain nanostructured coatings for implantable devices. For the first time, we demonstrate that L. anatina seashells have strong antibacterial properties. Indeed, they significantly inhibit planktonic growth and cell adhesion of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The two strains show different susceptibility to the mineral and organic parts of the seashells, the first being more susceptible to zinc and fluorine in the mineral part, and the second to the organic (chitin-based) component. Upon deposition by IJD, all films exhibit a nanostructured morphology and sub-micrometric thickness. The multi-doped, complex composition of the target is maintained in the coating, demonstrating the feasibility of deposition of coatings starting from biogenic precursors (seashells). In conclusion, Lingula seashell-based coatings are non-cytotoxic with strong antimicrobial capability, especially against Gram-positive strains, consistently with their higher susceptibility to fluorine and zinc. Importantly, these properties are improved compared to synthetic fluorapatite, showing that the films are promising for antimicrobial applications.

Bone on-a-chip: a 3D dendritic network in a screening platform for osteocyte-targeted drugs.

Age-related musculoskeletal disorders, including osteoporosis, are frequent and associated with long lasting morbidity, in turn significantly impacting on healthcare system sustainability. There is therefore a compelling need to develop reliable preclinical models of disease and drug screening to validate novel drugs possibly on a personalized basis, without the need ofin vivoassay. In the context of bone tissue, although the osteocyte (Oc) network is a well-recognized therapeutic target, currentin vitropreclinical models are unable to mimic its physiologically relevant and highly complex structure. To this purpose, several features are needed, including an osteomimetic extracellular matrix, dynamic perfusion, and mechanical cues (e.g. shear stress) combined with a three-dimensional (3D) culture of Oc. Here we describe, for the first time, a high throughput microfluidic platform based on 96-miniaturized chips for large-scale preclinical evaluation to predict drug efficacy. We bioengineered a commercial microfluidic device that allows real-time visualization and equipped with multi-chips by the development and injection of a highly stiff bone-like 3D matrix, made of a blend of collagen-enriched natural hydrogels loaded with hydroxyapatite nanocrystals. The microchannel, filled with the ostemimetic matrix and Oc, is subjected to passive perfusion and shear stress. We used scanning electron microscopy for preliminary material characterization. Confocal microscopy and fluorescent microbeads were used after material injection into the microchannels to detect volume changes and the distribution of cell-sized objects within the hydrogel. The formation of a 3D dendritic network of Oc was monitored by measuring cell viability, evaluating phenotyping markers (connexin43, integrin alpha V/CD51, sclerostin), quantification of dendrites, and responsiveness to an anabolic drug. The platform is expected to accelerate the development of new drug aimed at modulating the survival and function of osteocytes.

Incorporation/Enrichment of 3D Bioprinted Constructs by Biomimetic Nanoparticles: Tuning Printability and Cell Behavior in Bone Models.

Reproducing in vitro a model of the bone microenvironment is a current need. Preclinical in vitro screening, drug discovery, as well as pathophysiology studies may benefit from in vitro three-dimensional (3D) bone models, which permit high-throughput screening, low costs, and high reproducibility, overcoming the limitations of the conventional two-dimensional cell cultures. In order to obtain these models, 3D bioprinting offers new perspectives by allowing a combination of advanced techniques and inks. In this context, we propose the use of hydroxyapatite nanoparticles, assimilated to the mineral component of bone, as a route to tune the printability and the characteristics of the scaffold and to guide cell behavior. To this aim, both stoichiometric and Sr-substituted hydroxyapatite nanocrystals are used, so as to obtain different particle shapes and solubility. Our findings show that the nanoparticles have the desired shape and composition and that they can be embedded in the inks without loss of cell viability. Both Sr-containing and stoichiometric hydroxyapatite crystals permit enhancing the printing fidelity of the scaffolds in a particle-dependent fashion and control the swelling behavior and ion release of the scaffolds. Once Saos-2 cells are encapsulated in the scaffolds, high cell viability is detected until late time points, with a good cellular distribution throughout the material. We also show that even minor modifications in the hydroxyapatite particle characteristics result in a significantly different behavior of the scaffolds. This indicates that the use of calcium phosphate nanocrystals and structural ion-substitution is a promising approach to tune the behavior of 3D bioprinted constructs.

Ionized Jet Deposition of Calcium Phosphates-Based Nanocoatings: Tuning Coating Properties and Cell Behavior by Target Composition and Substrate Heating.

Calcium phosphate-based coatings are widely studied in orthopedics and dentistry for their similarity to the mineral component of bone and their capability to promote osseointegration. Different calcium phosphates have tunable properties that result in different behaviors in vitro, but the majority of studies focus only on hydroxyapatite. Here, different calcium phosphate-based nanostructured coatings are obtained by ionized jet deposition, starting with hydroxyapatite, brushite and beta-tricalcium phosphate targets. The properties of the coatings obtained from different precursors are systematically compared by assessing their composition, morphology, physical and mechanical properties, dissolution, and in vitro behavior. In addition, for the first time, depositions at high temperature are investigated for the further tuning of the coatings mechanical properties and stability. Results show that different phosphates can be deposited with good composition fidelity even if not in a crystalline phase. All coatings are nanostructured and non-cytotoxic and display variable surface roughness and wettability. Upon heating, higher adhesion and hydrophilicity are obtained as well as higher stability, resulting in better cell viability. Interestingly, different phosphates show very different in vitro behavior, with brushite being the most suitable for promoting cell viability and beta-tricalcium phosphate having a higher impact on cell morphology at the early timepoints.

Customized biofilm device for antibiofilm and antibacterial screening of newly developed nanostructured silver and zinc coatings.

BACKGROUND: Bacterial colonisation on implantable device surfaces is estimated to cause more than half of healthcare-associated infections. The application of inorganic coatings onto implantable devices limits/prevents microbial contaminations. However, reliable and high-throughput deposition technologies and experimental trials of metal coatings for biomedical applications are missing. Here, we propose the combination of the Ionized Jet Deposition (IJD) technology for metal-coating application, with the Calgary Biofilm Device (CBD) for high-throughput antibacterial and antibiofilm screening, to develop and screen novel metal-based coatings. RESULTS: The films are composed of nanosized spherical aggregates of metallic silver or zinc oxide with a homogeneous and highly rough surface topography. The antibacterial and antibiofilm activity of the coatings is related with the Gram staining, being Ag and Zn coatings more effective against gram-negative and gram-positive bacteria, respectively. The antibacterial/antibiofilm effect is proportional to the amount of metal deposited that influences the amount of metal ions released. The roughness also impacts the activity, mostly for Zn coatings. Antibiofilm properties are stronger on biofilms developing on the coating than on biofilms formed on uncoated substrates. This suggests a higher antibiofilm effect arising from the direct contact bacteria-coating than that associated with the metal ions release. Proof-of-concept of application to titanium alloys, representative of orthopaedic prostheses, confirmed the antibiofilm results, validating the approach. In addition, MTT tests show that the coatings are non-cytotoxic and ICP demonstrates that they have suitable release duration (> 7 days), suggesting the applicability of these new generation metal-based coatings for the functionalization of biomedical devices. CONCLUSIONS: The combination of the Calgary Biofilm Device with the Ionized Jet Deposition technology proved to be an innovative and powerful tool that allows to monitor both the metal ions release and the surface topography of the films, which makes it suitable for the study of the antibacterial and antibiofilm activity of nanostructured materials. The results obtained with the CBD were validated with coatings on titanium alloys and extended by also considering the anti-adhesion properties and biocompatibility. In view of upcoming application in orthopaedics, these evaluations would be useful for the development of materials with pleiotropic antimicrobial mechanisms.

Electrospun fibers coated with nanostructured biomimetic hydroxyapatite: A new platform for regeneration at the bone interfaces.

Reconstruction of gradient organic/inorganic tissues is a challenging task in orthopaedics. Indeed, to mimic tissue characteristics and stimulate bone regeneration at the interface, it is necessary to reproduce both the mineral and organic components of the tissue ECM, as well as the micro/nano-fibrous morphology. To address this goal, we propose here novel biomimetic patches obtained by the combination of electrospinning and nanostructured bone apatite. In particular, we deposited apatite on the electrospun fibers by Ionized Jet Deposition, a plasma-assisted technique that allows conformal deposition and the preservation in the coating of the target's stoichiometry. The damage to the substrate and fibrous morphology is a polymer-dependent aspect, that can be avoided by properly selecting the substrate composition and deposition parameters. In fact, all the tested polymers (poly(l-lactide), poly(D,l-lactide-co-glycolide, poly(ε-caprolactone), collagen) were effectively coated, and the morphological and thermal characterization revealed that poly(ε-caprolactone) suffered the least damage. The coating of collagen fibers, on the other hand, destroyed the fiber morphology and it could only be performed when collagen is blended with a more resistant synthetic polymer in the nanofibers. Due to the biomimetic composition and multiscale morphology from micro to nano, the poly(ε-caprolactone)-collagen biomimetic patches coated with bone apatite supported MSCs adhesion, patch colonization and early differentiation, while allowing optimal viability. The biomimetic coating allowed better scaffold colonization, promoting cell spreading on the fibers.

Conservation of Archaeological Bones: Assessment of Innovative Phosphate Consolidants in Comparison with Paraloid B72.

Aqueous solutions of diammonium hydrogen phosphate (DAP) have been recently proposed for consolidation of archeological bones, as an alternative to traditional products. Here, we investigated several routes to improve the performance of the DAP-based treatment, namely increasing the DAP concentration, adding calcium ions and adding ethanol to the DAP solution. Archaeological bones dated to about 1-0.8 million years ago were used for the tests. After preliminary screening by FTIR microscopy and FEG-SEM among different formulations, confirming the formation of new hydroxyapatite phases, the most promising formulation was selected, namely a 3 M DAP solution. The strengthening ability of this formulation was systematically compared to that of the most widely used commercial consolidant, namely Paraloid B72. The performance of the two treatments was evaluated in terms of Knoop and Vickers microhardness, resistance to scratch and resistance to material loss by peeling off. The results of the study show that the DAP treatment was able to improve the bone surface properties and also the resistance to material loss by peeling off, which is more dependent on in-depth consolidation. Paraloid B72 led to the formation of a layer of acrylic resin on the bone surface, which influenced the mechanical tests. Nonetheless, Paraloid B72 was able to penetrate in depth and substantially decrease the material loss by peeling off, even more effectively than DAP. The results of this study indicate that the potential of the DAP treatment for bone consolidation is confirmed.

Foot Orthosis and Sensorized House Slipper by 3D Printing.

BACKGROUND: In clinical practice, specific customization is needed to address foot pathology, which must be disease and patient-specific. To date, the traditional methods for manufacturing custom functional Foot Orthoses (FO) are based on plaster casting and manual manufacturing, hence orthotic therapy depends entirely on the skills and expertise of individual practitioners. This makes the procedures difficult to standardize and replicate, as well as expensive, time-consuming and material-wasting, as well as difficult to standardize and replicate. 3D printing offers new perspectives in the development of patient-specific orthoses, as it permits addressing all the limitations of currently available technologies, but has been so far scarcely explored for the podiatric field, so many aspects remain unmet, especially for what regards customization, which requires the definition of a protocol that entails all stages from patient scanning to manufacturing. METHODS: A feasibility study was carried out involving interdisciplinary cooperation between industrial engineers and podiatrists. To that end: (i) For patient-specific data acquisition, 3D scanning of the foot is compared to traditional casting. (ii) a modelling GD workflow is first created to design a process permitting easy creations of customized shapes, enabling the end user (the podiatrist) to interactively customize the orthoses. Then, (iii) a comparison is made between different printing materials, in order to reproduce the same mechanical behavior shown by standard orthoses. To do this, the mechanical properties of standard materials (Polycarbonate sheets), cut and hand-shaped, are compared with four groups of 3D printed samples: poly(ethylene glycol) (PETG), poly(acrylonitrile-butadiene.styrene) (ABS), polycarbonate (PC) and poly(lactic acid) (PLA) obtained by Fused Filament Fabrication (FFF). RESULTS: Differences found between the foot plaster model obtained with the plaster slipper cast in a neutral position and the model of the real foot obtained with 3D scanning in the same position can be ascribed to the non-stationarity of the patient during the acquisition process, and were limited by a locking system with which no substantial differences in the almost entire sole of the foot scan were observed. CONCLUSIONS: Using the designed GD workflow, podiatrists with limited CAD skills can easily design and interactively customize foot orthoses to adapt them to the patients' clinical needs. 3D printing enables the complex shape of the orthoses to be reproduced easily and quickly. Compared to Polycarbonate sheets (gold standard), all the printed materials were less deformable and reached lower yield stress for comparable deformation. No modifications in any of the materials as a result of printing process were observed.

FT-IR Spectral Signature of Sensitive and Multidrug-Resistant Osteosarcoma Cell-Derived Extracellular Nanovesicles.

Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, the outcome is unsatisfactory, and multidrug resistance (MDR) is a pivotal process in OS treatment failure. OS-derived extracellular vesicles (EVs) promote drug resistance to chemotherapy and target therapy through different mechanisms. The aim of this study was to identify subpopulations of osteosarcoma-EVs by Fourier transform infrared spectroscopy (FT-IR) to define a specific spectral signature for sensitive and multidrug-resistant OS-derived EVs. EVs were isolated from sensitive and MDR OS cells as well as from mesenchymal stem cells by differential centrifugation and ultracentrifugation. EVs size, morphology and protein expression were characterized. FT-IR/ATR of EVs spectra were acquired in the region of 400-4000 cm-1 (resolution 4 cm-1, 128 scans). The FT-IR spectra obtained were consistently different in the EVs compared to cells from which they originate. A specific spectral signature, characterized by a shift and a new band (1601 cm-1), permitted to clearly distinguish EVs isolated by sensitive and multidrug-resistant OS cells. Our data suggest that FT-IR spectroscopy allows to characterize and define a specific spectral signature for sensitive and MDR OS-derived EVs.

Antibacterial and Antibiofilm Activity of Nanostructured Copper Films Prepared by Ionized Jet Deposition.

Metal coatings represent good strategies to functionalize surfaces/devices and limit bacterial contamination/colonization thanks to their pleiotropic activity and their ability to prevent the biofilm formation. Here, we investigated the antibacterial and antibiofilm capacity of copper coatings deposited through the Ionized Jet Deposition (IJD) on the Calgary Biofilm Device (CBD) against the growth of two gram-negative and two gram-positive pathogenic strains. Three areas (i.e., (+)Cu, (++)Cu, and (+++)Cu based on the metal amount) on the CBD were obtained, presenting nanostructured coatings with high surface homogeneity and increasing dimensions of aggregates from the CBD periphery to the centre. The coatings in (++)Cu and (+++)Cu were efficient against the planktonic growth of the four pathogens. This antibacterial effect decreased in (+)Cu but was still significant for most of the pathogens. The antibiofilm efficacy was significant for all the strains and on both coated and uncoated surfaces in (+++)Cu, whereas in (++)Cu the only biofilms forming on the coated surfaces were inhibited, suggesting that the decrease of the metal on the coatings was associated to a reduced metal ion release. In conclusion, this work demonstrates that Cu coatings deposited by IJD have antibacterial and antibiofilm activity against a broad range of pathogens indicating their possible application to functionalize biomedical devices.

3D Printing and Bioprinting to Model Bone Cancer: The Role of Materials and Nanoscale Cues in Directing Cell Behavior.

Bone cancer, both primary and metastatic, is characterized by a low survival rate. Currently, available models lack in mimicking the complexity of bone, of cancer, and of their microenvironment, leading to poor predictivity. Three-dimensional technologies can help address this need, by developing predictive models that can recapitulate the conditions for cancer development and progression. Among the existing tools to obtain suitable 3D models of bone cancer, 3D printing and bioprinting appear very promising, as they enable combining cells, biomolecules, and biomaterials into organized and complex structures that can reproduce the main characteristic of bone. The challenge is to recapitulate a bone-like microenvironment for analysis of stromal-cancer cell interactions and biological mechanics leading to tumor progression. In this review, existing approaches to obtain in vitro 3D-printed and -bioprinted bone models are discussed, with a focus on the role of biomaterials selection in determining the behavior of the models and its degree of customization. To obtain a reliable 3D bone model, the evaluation of different polymeric matrices and the inclusion of ceramic fillers is of paramount importance, as they help reproduce the behavior of both normal and cancer cells in the bone microenvironment. Open challenges and future perspectives are discussed to solve existing shortcomings and to pave the way for potential development strategies.

Ionized jet deposition of antimicrobial and stem cell friendly silver-substituted tricalcium phosphate nanocoatings on titanium alloy.

Orthopedic infections pose severe societal and economic burden and interfere with the capability of the implanted devices to integrate in the host bone, thus significantly increasing implants failure rate. To address infection and promote integration, here nanostructured antibacterial and bioactive thin films are proposed, obtained, for the first time, by Ionized Jet Deposition (IJD) of silver-substituted tricalcium phosphate (Ag-TCP) targets on titanium. Coatings morphology, composition and mechanical properties are characterized and proof-of-concept of biocompatibility is shown. Antimicrobial efficacy is investigated against four Gram positive and Gram negative bacterial strains and against C. albicans fungus, by investigating the modifications in planktonic bacterial growth in the absence and presence of silver. Then, for all bacterial strains, the capability of the film to inhibit bacterial adhesion is also tested. Results indicate that IJD permits a fine control over films composition and morphology and deposition of films with suitable mechanical properties. Biological studies show a good efficacy against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and against fungus Candida albicans, with evidences of efficacy against planktonic growth and significant reduction of bacterial cell adhesion. No cytotoxic effects are evidenced for equine adipose tissue derived mesenchymal stem cells (ADMSCs), as no reductions are caused to cells viability and no interference is assessed in cells differentiation towards osteogenic lineage, in the presence of silver. Instead, thanks to nanostructuration and biomimetic composition, tricalcium phosphate (TCP) coatings favor cells viability, also when silver-substituted. These findings show that silver-substituted nanostructured coatings are promising for orthopedic implant applications.

Nanostructure and biomimetics orchestrate mesenchymal stromal cell differentiation: An in vitro bioactivity study on new coatings for orthopedic applications.

The choice of the appropriate material having suitable compositional and morphological surface characteristics, is a crucial step in the development of orthopedic implants. The purpose of this paper is to elucidate, on this regard, the influence of two important hits, i.e., biogenic apatite with bone-like composition and nanostructured morphology, providing the evidence of the efficacy of nanostructured biogenic apatite coatings in favoring adhesion, growth, proliferation, and in vitro osteogenic differentiation of human mesenchymal stromal cells (hMSCs) isolated from the bone marrow. The specific features of this coating in terms of topographical and biochemical cues, obtained by Ionized Jet Deposition, are perceived by hMSCs, as suggested by changes in different morphologic parameters as Aspect Ratio or Elongation index, suggesting the impact exerted by the nanostructure on early adhesion events, cytoskeleton organization, and cells fate. In addition, the nanostructured CaP coating sustained the metabolic activity of the cells and facilitated the osteogenic differentiation of MSC by supporting the osteogenesis-related gene expression. These findings support the use of a combined approach between technological advancement and instructive surfaces, both from the topographical and the biochemical point of view, in order to manufacture smart biomaterials able to respond to different needs of the orthopedic practice.

Perfused Platforms to Mimic Bone Microenvironment at the Macro/Milli/Microscale: Pros and Cons.

As life expectancy increases, the population experiences progressive ageing. Ageing, in turn, is connected to an increase in bone-related diseases (i.e., osteoporosis and increased risk of fractures). Hence, the search for new approaches to study the occurrence of bone-related diseases and to develop new drugs for their prevention and treatment becomes more pressing. However, to date, a reliable in vitro model that can fully recapitulate the characteristics of bone tissue, either in physiological or altered conditions, is not available. Indeed, current methods for modelling normal and pathological bone are poor predictors of treatment outcomes in humans, as they fail to mimic the in vivo cellular microenvironment and tissue complexity. Bone, in fact, is a dynamic network including differently specialized cells and the extracellular matrix, constantly subjected to external and internal stimuli. To this regard, perfused vascularized models are a novel field of investigation that can offer a new technological approach to overcome the limitations of traditional cell culture methods. It allows the combination of perfusion, mechanical and biochemical stimuli, biological cues, biomaterials (mimicking the extracellular matrix of bone), and multiple cell types. This review will discuss macro, milli, and microscale perfused devices designed to model bone structure and microenvironment, focusing on the role of perfusion and encompassing different degrees of complexity. These devices are a very first, though promising, step for the development of 3D in vitro platforms for preclinical screening of novel anabolic or anti-catabolic therapeutic approaches to improve bone health.

Unravelling the Effect of Citrate on the Features and Biocompatibility of Magnesium Phosphate-Based Bone Cements.

In the framework of new materials for orthopedic applications, Magnesium Phosphate-based Cements (MPCs) are currently the focus of active research in biomedicine, given their promising features; in this field, the loading of MPCs with active molecules to be released in the proximity of newly forming bone could represent an innovative approach to enhance the in vivo performances of the biomaterial. In this work, we describe the preparation and characterization of MPCs containing citrate, an ion naturally present in bone which presents beneficial effects when released in the proximity of newly forming bone tissue. The cements were characterized in terms of handling properties, setting time, mechanical properties, crystallinity, and microstructure, so as to unravel the effect of citrate concentration on the features of the material. Upon incubation in aqueous media, we demonstrated that citrate could be successfully released from the cements, while contributing to the alkalinization of the surroundings. The cytotoxicity of the materials toward human fibroblasts was also tested, revealing the importance of a fine modulation of released citrate to guarantee the biocompatibility of the material.

Citrate Supplementation Restores the Impaired Mineralisation Resulting from the Acidic Microenvironment: An In Vitro Study.

Chronic metabolic acidosis leads to bone-remodelling disorders based on excessive mineral matrix resorption and inhibition of bone formation, but also affects the homeostasis of citrate, which is an essential player in maintaining the acid-base balance and in driving the mineralisation process. This study aimed to investigate the impact of acidosis on the osteogenic properties of bone-forming cells and the effects of citrate supplementation in restoring the osteogenic features impaired by the acidic milieu. For this purpose, human mesenchymal stromal cells were cultured in an osteogenic medium and the extracellular matrix mineralisation was analysed at the micro- and nano-level, both in neutral and acidic conditions and after treatment with calcium citrate and potassium citrate. The acidic milieu significantly decreased the citrate release and hindered the organisation of the extracellular matrix, but the citrate supplementation increased collagen production and, particularly calcium citrate, promoted the mineralisation process. Moreover, the positive effect of citrate supplementation was observed also in the physiological microenvironment. This in vitro study proves that the mineral matrix organisation is influenced by citrate availability in the microenvironment surrounding bone-forming cells, thus providing a biological basis for using citrate-based supplements in the management of bone-remodelling disorders related to chronic low-grade acidosis.

Nanodecoration of electrospun polymeric fibers with nanostructured silver coatings by ionized jet deposition for antibacterial tissues.

Silver-based nanomaterials are used as antibacterial agents in a number of applications, including wound dressing, where electrospun materials can effectively promote wound healing and tissue regeneration thanks to their biomimicry, flexibility and breathability. Incorporation of such nanomaterials in electrospun nonwovens is highly challenging if aiming at maximizing stability and antibacterial efficacy and minimizing silver detachment, without neglecting process straightforwardness and scalability. In this work nanostructured silver coatings were deposited by Ionized Jet Deposition (IJD) on Polylactic acid, a medical grade polyester-urethane and Polyamide 6,6 nanofibers. The resulting materials were thoroughly characterized to gain an in-depth view of coating morphology and substrate resistance to the low-temperature deposition process used. Morphology of silver coatings with well-cohesive grains having dimensions from a few tens to a few hundreds of nanometers was analyzed by SEM, TEM and AFM. TGA, DSC, FTIR and GPC showed that the polymers well withstand the deposition process with negligible effects on their properties, the only exception being the polylactic acid that resulted more susceptible to degradation. Finally, the efficacy against S. aureus and E. coli bacterial strains was demonstrated, indicating that electrospun fibers decorated with nanostructured silver by IJD represent a breakthrough solution in the field of antibacterial devices.