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Dissolution and gelation procedures have a great influence on gelation time, microstructure and mechanical properties of reconstituted collagen products. We have investigated the dissolution of atelocollagen in CO2/water solutions at low temperature (4 °C) at different CO2 pressures (0.3–0.9 MPa), as well as gelation kinetics and physico-chemical properties of the hydrogel obtained after CO2 removal. Compared to conventional methods, the CO2-assisted technique resulted in faster soluble collagen dissolution and faster gelation into transparent gels characterized by thin 10 nm fibrils. Electrophoresis and CD spectroscopy demonstrated that the process did not denature the soluble collagen. The possibility to obtain collagen dissolution and gelation without the use of chemical agent other than water and CO2 makes this process particularly appealing for biomedical applications.

The main limitation of drug-enhanced radiotherapy concerns the difficulty to evaluate the effectiveness of cancer targeting after drug administration hindering the standardization of therapies based on current radiosensitizing compounds. The challenge regards the development of systems able to combine imaging and radiotherapy enhancement in order to perform highly reliable cancer theragnosis. For these reasons, gold-magnetite hybrid nanoparticles (H-NPs) are proposed as innovative theranostic nanotools for imaging-guided radiosensitization in cancer treatment. In this work we propose a novel method for the synthesis of hydrophilic and superparamagnetic Tween20-stabilized gold-magnetite H-NPs. Morphology and chemical composition of nanoparticles were assessed by transmission electron microscopy, x-ray diffraction analysis and ion-coupled plasma optical emission spectroscopy. Colloidal stability and magnetic properties of nanoparticles were determined by dynamic light scattering and magnetometry. The potentialities of H-NPs for magnetic resonance imaging were studied using a human 4T-MRI scanner. Nanoparticles were proven to induce concentration-dependent contrast enhancement in T2*-weighted MR-images. The cytotoxicity, the cellular uptake and the radiosensitization activity of H-NPs were investigated in human osteosarcoma MG63 cell cultures and murine 3T3 fibroblasts, using specific bioassays and laser scanning confocal microscopy. H-NPs did not exhibit significant toxicity and were demonstrated to be internalized by cells. A significant x-ray enhancement at specific H-NPs exposure concentrations was evidenced on MG63 cell line. [ABSTRACT FROM AUTHOR]

Targeted Radionuclide Therapy (TRT) is a medical technique exploiting radionuclides to combat cancer growth and spread. TRT requires a supply of radionuclides that are currently produced by either cyclotrons or nuclear research reactors. In this context, the ISOLPHARM project investigates the production of innovative radionuclides for medical applications. This production will be based on the forthcoming SPES facility at the Legnaro National Laboratories (LNL) of the National Institute for Nuclear Physics (INFN), an ISOL facility where high-purity radioactive beams will be used to produce carrier-free radiopharmaceuticals. Previous studies demonstrated that a significant amount of 111 Ag, an innovative β/γ emitter suitable for TRT with theranostic applications, can be obtained at the SPES facility. The present work describes the first imaging study on phantoms with 111 Ag performed by the ISOLPHARM collaboration. This is a fundamental step to pave the way for the upcoming in vivo studies on the 111 Ag-based radiopharmaceutical currently being developed. The imaging potential of this radionuclide was investigated by acquiring phantom images with Cerenkov Luminescence Imaging (CLI) and digital autoradiography (ARG)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The current treatments for wounds often fail to induce adequate healing, leaving wounds vulnerable to persistent infections and development of drug-resistant microbial biofilms. New natural-derived nanoparticles were studied to impair bacteria colonization and hinder the formation of biofilms in wounds. The nanoparticles were fabricated through polyelectrolyte complexation of chitosan (CS, polycation) and hyaluronic acid (HA, polyanion). UV-induced photo-crosslinking was used to enhance the stability of the nanoparticles. To achieve this, HA was methacrylated (HAMA, degree of modification of 20 %). Photo-crosslinked nanoparticles obtained from HAMA and CS had a diameter of 478 nm and a more homogeneous size distribution than nanoparticles assembled solely through complexation (742 nm). The nanoparticles were loaded with the antimicrobial agent bacitracin (BC), resulting in nanoparticles with a diameter of 332 nm. The encapsulation of BC was highly efficient (97 %). The BC-loaded nanoparticles showed significant antibacterial activity against gram-positive bacteria Staphylococcus aureus, Methicillin-resistant S. aureus and S. epidermidis. Photo-crosslinked HAMA/CS nanoparticles loaded with BC demonstrated inhibition of biofilm formation and a positive effect on the proliferation of mammalian cells (L929). These crosslinked nanoparticles have potential for the long-term treatment of wounds and controlled antibiotic delivery at the location of a lesion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Molecularly imprinted polymers (MIPs) are synthetic receptors made by template-assisted synthesis. MIPs might be ideal receptors for sensing devices, given the possibility to custom-design selectivity and affinity toward a targeted analyte and their robustness and ability to withstand harsh conditions. However, the synthesis of MIP is an inherently random process that produces a statistical distribution of binding sites, characterized by a variety of affinities. This is verified both for bulk MIP materials and for MIP's thin layers. In the present work, we aimed at assessing the effects of inhomogeneous versus homogeneous imprinted binding sites on electrochemical sensing measurements, and the possible implications on the sensor's performance. In the example of an Electrochemical Impedance Spectroscopy (EIS) sensor for the 17β-estradiol (E2) hormone, the scenario of inhomogeneous binding sites was studied by modifying electrodes with an E2-MIP polyaniline (PANI) thin layer, called the "Imprinted PANI layer". In contrast, the condition of discrete and uniform binding sites was epitomized by electrodes modified with a thin PANI layer purposedly doped with E2-MIP nanoparticles (nanoMIPs), which were referred to as "nanoMIP-doped PANI". The behaviors of the two EIS sensors were compared. Interestingly, the sensitivity of the nanoMIP-doped PANI was almost twice with respect to that of the imprinted PANI layer, strongly suggesting that the homogeneity of the binding sites has a fundamental role in the sensor's development. The nanoMIP-doped PANI sensor, which showed a response for E2 in the range 36.7 pM-36.7 nM and had a limit of detection of 2.86 pg/mL, was used to determine E2 in wastewater.

The cholecystokinin type 2 receptor (CCK2-R) represents an ideal target for cancer therapy since it is overexpressed in several tumors and is associated with poor prognosis. Nastorazepide (Z-360), a selective CCK2-R antagonist, has been widely investigated as a CCK2-R ligand for targeted therapy; however, its high hydrophobicity may represent a limit to cell selectivity and optimal in vivo biodistribution. Here, we present three new fluorescent Z-360 derivatives ( IP-002 G -Rho, IP-002 L -Rho, and IP-002 M -Rho ) in which nastorazepide was linked, through spacers bearing different saccharides (glucose (G), lactose (L), and maltotriose (M)), to sulforhodamine B. A fourth compound ( IP-002 H -Rho ) with no pendant sugar was also synthesized as a control. Through two-dimensional (2D) and three-dimensional (3D) in vitro studies, we evaluated the compound association with and selectivity for CCK2-R-overexpressing cells (A431-CCK2-R + ) vs CCK2-R-underexpressing cells (A431 WT). 2D in vitro studies highlighted a progressive increase of IP-002 x -Rho association with A431-CCK2-R + cells according to the linker hydrophilicity, that is, maltotriose > lactose > glucose > hydrogen, with IP-002 M -Rho showing a 2.4- and a 1.36-fold higher uptake than IP-002 G -Rho and IP-002 L -Rho , respectively. Unexpectedly, IP-002 H -Rho showed a similar cell association to that of IP-002 L -Rho but with no difference between the two tested cell lines. On the contrary, association with A431-CCK2-R + cells as compared to the A431 WT was found to be 1.08-, 1.14-, and 1.37-fold higher for IP-002 G -Rho , IP-002 L -Rho , and IP-002 M -Rho , respectively, proving IP-002 M -Rho to be the best-performing compound, as also confirmed by competition studies. Trafficking studies on A431-CCK2-R + cells incubated with IP-002 M -Rho suggested the coexistence of receptor-mediated endocytosis and simple diffusion. On the contrary, a high and selective uptake of IP-002 M -Rho by A431-CCK2-R + cells only was observed on 3D scaffolds embedded with cells, underlining the importance of 3D models in in vitro preliminary evaluation.

The treatment of bone defects is a clinical challenge. Bone tissue engineering is gaining interest as an alternative to current treatments, with the development of 3D porous structures (scaffolds) helpful in promoting bone regeneration by ensuring temporary functional support. In this work, methacrylated silk fibroin (SilMA) sponges were investigated as scaffolds for bone tissue engineering by exploiting the combination of physical (induced by NaCl salt during particulate leaching) and chemical crosslinking (induced by UV-light exposure) techniques. A biomimetic approach was adopted to better simulate the extracellular matrix of the bone by introducing either natural (mussel shell-derived) or synthetic-origin hydroxyapatite nanoparticles into the SilMA sponges. The obtained materials were characterized in terms of pore size, water absorption capability and mechanical properties to understand both the effect of the inclusion of the two different types of nanoparticles and the effect of the photocrosslinking. Moreover, the SilMA sponges were tested for their bioactivity and suitability for bone tissue engineering purposes by using osteosarcoma cells, studying their metabolism by an AlamarBlue assay and their morphology by scanning electron microscopy. Results indicate that photocrosslinking helps in obtaining more regular structures with bimodal pore size distributions and in enhancing the stability of the constructs in water. Moreover, the addition of naturally derived hydroxyapatite was observed to be more effective at activating osteosarcoma cell metabolism than synthetic hydroxyapatite, showing a statistically significant difference in the AlamarBlue measurement on day 7 after seeding. The methacrylated silk fibroin/hydroxyapatite nanocomposite sponges developed in this work were found to be promising tools for targeting bone regeneration with a sustainable approach.

Damages to the intervertebral disc (IVD) due to improper loading or degeneration result in back pain, which is a common disease affecting an increasing number of patients. Different strategies for IVD remediation have been developed, from surgical treatment to disc replacement, by using both metallic and non-metallic materials. Hydrogels are very attractive materials due to their ability to simulate the properties of many soft tissues; moreover, their chemical composition can be varied in order to assure performances similar to the natural disc. In particular, for the replacement of the IVD outer ring, namely, the anulus fibrosus , the shear properties are of paramount importance. In this work, we produced hydrogels through the photo-induced crosslinking of different mixtures composed of two hydrophilic monofunctional and difunctional polymers, namely, poly(ethyleneglycol) methyl ether methacrylate (PEGMEMA) and poly(ethyleneglycol) dimethacrylate (PEGDMA), together with a hydrophobic molecule, i.e., tert-butyl acrylate (tBA). By changing the ratio among the precursors, we demonstrated the tunability of both the shear properties and hydrophilicity. The structural properties of hydrogels were studied by solid-state nuclear magnetic resonance (NMR). These experiments provided insights on both the structure and molecular dynamics of polymeric networks and, together with information obtained by differential scanning calorimetry (DSC), allowed for correlating the physical properties of the hydrogels with their chemical composition.

Currently, optical sensors based on molecularly imprinted polymers (MIPs) have been attracting significant interest. MIP sensing relies on the combination of the MIP's selective capability, which is conveyed to the polymeric material by a template-assisted synthesis, with optical techniques that offer exquisite sensitivity. In this work, we devised an MIP nanoparticle optical sensor for the ultralow detection of serum albumin through time-resolved fluorescence spectroscopy. The Fluo-nanoMIPs (∅~120 nm) were synthetized using fluorescein-O-methacrylate (0.1×, 1×, 10× mol:mol versus template) as an organic fluorescent reporter. The ability of 0.1× and 1×Fluo-nanoMIPs to bind albumin (15 fM-150 nM) was confirmed by fluorescence intensity analyses and isothermal titration calorimetry. The apparent dissociation constant (K app ) was 30 pM. Conversely, the 10× fluorophore content did not enable monitoring binding. Then, the time-resolved fluorescence spectroscopy of the nanosensors was studied. The 1×Fluo-nanoMIPs showed a decrease in fluorescence lifetime upon binding to albumin (100 fM-150 nM), K app = 28 pM, linear dynamic range 3.0-83.5 pM, limit of detection (LOD) 1.26 pM. Selectivity was confirmed testing 1×Fluo-nanoMIPs against competitor proteins. Finally, as a proof of concept, the nanosensors demonstrated detection of the albumin (1.5 nM) spiked in wine samples, suggesting a possible scaling up of the method in monitoring allergens in wines.

The simultaneous interrogation of both lossy mode (LMR) and surface plasmon (SPR) resonances was herein exploited for the first time to devise a sensor in combination with soft molecularly imprinting of nanoparticles (nanoMIPs), specifically entailed of the selectivity towards the protein biomarker human serum transferrin (HTR). Two distinct metal-oxide bilayers, i.e. TiO 2 -ZrO 2 and ZrO 2 -TiO 2 , were used in the SPR-LMR sensing platforms. The responses to binding of the target protein HTR of both sensing configurations (TiO 2 -ZrO 2 -Au-nanoMIPs, ZrO 2 -TiO 2 -Au-nanoMIPs) showed femtomolar HTR detection, LODs of tens of fM and K Dapp  ~ 30 fM. Selectivity for HTR was demonstrated. The SPR interrogation was more efficient for the ZrO 2 -TiO 2 -Au-nanoMIPs configuration (sensitivity at low concentrations, S = 0.108 nm/fM) than for the TiO 2 -ZrO 2 -Au-nanoMIPs one (S = 0.061 nm/fM); while LMR was more efficient for TiO 2 -ZrO 2 -Au-nanoMIPs (S = 0.396 nm/fM) than for ZrO 2 -TiO 2 -Au-nanoMIPs (S = 0.177 nm/fM). The simultaneous resonance monitoring is advantageous for point of care determinations, both in terms of measurement's redundancy, that enables the cross-control of the measure and the optimization of the detection, by exploiting the individual characteristics of each resonance., (© 2023. The Author(s).)

The impact of repeated in-hospital reprocessing on 100% cotton fabric continues to be debated. We analyzed the properties of surgical gowns and drapes over 15 months of clinical use. The amount of linting fibers and the water absorption rate increased significantly, but microbial and blood penetration was preserved.

Extrusion-based bioprinting is one of the most widespread technologies due to its affordability, wide range of processable materials, and ease of use. However, the formulation of new inks for this technique is based on time-consuming trial-and-error processes to establish the optimal ink composition and printing parameters. Here, a dynamic printability window was modeled for the assessment of the printability of polysaccharide blend inks of alginate and hyaluronic acid with the intent to build a versatile predictive tool to speed up the testing procedures. The model considers both the rheological properties of the blends (viscosity, shear thinning behavior, and viscoelasticity) and their printability (in terms of extrudability and the ability of forming a well-defined filament and detailed geometries). By imposing some conditions on the model equations, it was possible to define empirical bands in which the printability is ensured. The predictive capability of the built model was successfully verified on an untested blend of alginate and hyaluronic acid chosen to simultaneously optimize the printability index and minimize the size of the deposited filament.

Thermoplastic molded regenerated silk fibroin was proposed as a structural material in tissue engineering applications, mainly for application in bone. The protocol allows us to obtain a compact non-porous material with a compression modulus in the order of a Giga Pascal in dry conditions (and in the order of tens of MPa in wet conditions). This material is produced by compressing a lyophilized silk fibroin powder or sponge into a mold temperature higher than the glass transition temperature. The main purpose of the produced resin was the osteofixation and other structural applications in which the lack of porosity was not an issue. In this work, we introduced the use of citric acid in the thermoplastic molding protocol of silk fibroin to obtain porosity inside the structural material. The citric acid powder during the compression acted as a template for the pore formation. The mean pore diameter achieved by the addition of the higher amount of citric acid was around 5 μm. In addition, citric acid could effectively crosslink the silk fibroin chain, improving its mechanical strength. This effect was proved both by evaluating the compression modulus (the highest value recorded was 77 MPa in wet conditions) and by studying the spectra obtained by Fourier transform infrared spectroscopy. This protocol may be applied in the near future to the production of structural bone scaffolds.

The combination of non-specific deformable nanogels and plasmonic optical probes provides an innovative solution for specific sensing using a generalistic recognition layer. Soft polyacrylamide nanogels that lack specific selectivity but are characterized by responsive behavior, i.e., shrinking and swelling dependent on the surrounding environment, were grafted to a gold plasmonic D-shaped plastic optical fiber (POF) probe. The nanogel-POF cyclically challenged with water or alcoholic solutions optically reported the reversible solvent-to-phase transitions of the nanomaterial, embodying a primary optical switch. Additionally, the non-specific nanogel-POF interface exhibited more degrees of freedom through which specific sensing was enabled. The real-time monitoring of the refractive index variations due to the time-related volume-to-phase transition effects of the nanogels enabled us to determine the environment's characteristics and broadly classify solvents. Hence the nanogel-POF interface was a descriptor of mathematical functions for substance identification and classification processes. These results epitomize the concept of responsive non-specific nanomaterials to perform a multiparametric description of the environment, offering a specific set of features for the processing stage and particularly suitable for machine and deep learning. Thus, soft MathMaterial interfaces provide the ground to devise devices suitable for the next generation of smart intelligent sensing processes.

Methacrylated silk (Sil-MA) is a chemically modified silk fibroin specifically designed to be crosslinkable under UV light, which makes this material applicable in additive manufacturing techniques and allows the prototyping and development of patient-specific 2D or 3D constructs. In this study, we produced a thin grid structure based on crosslinked Sil-MA that can be withdrawn and ejected and that can recover its shape after rehydration. A complete chemical and physical characterization of Sil-MA was first conducted. Additionally, we tested Sil-MA biocompatibility according to the International Standard Organization protocols (ISO 10993) ensuring the possibility of using it in future trials. Sil-MA was also tested to verify its ability to support osteogenesis. Overall, Sil-MA was shown to be biocompatible and osteoconductive. Finally, two different additive manufacturing technologies, a Digital Light Processing (DLP) UV projector and a pneumatic extrusion technique, were used to develop a Sil-MA grid construct. A proof-of-concept of its shape-memory property was provided. Together, our data support the hypothesis that Sil-MA grid constructs can be injectable and applicable in bone regeneration applications.

A polymeric multimode waveguide, characterized by a pioneering spoon-shaped geometry, was herein proposed for the first time to devise Surface Plasmon Resonance (SPR) biochemical sensors. The plasmon excitation was enabled by layering a gold nanofilm of ∼60 nm onto the spoon-waveguide. As a consequence of the waveguide's extra-ordinary geometry, two distinct sensing regions were identified: a planar one, located on the spoon's neck, and a concave one on the bowl, with angled surfaces. The bulk sensitivity (S n ) is correlated both to the way the light was launched in/collected from the sensor (parallel or orthogonal to the main axis of the waveguide) and to the sensing area interrogated (planar-neck or angled-bowl), indicating that the sensor's performance can be conveniently tuned, depending on the chosen measuring configuration. The SPR sensor's characterization showed S n equal to 750 nm/RIU for the neck and to 950 nm/RIU for the bowl. To further inspect the peculiar sensing-features and assess the application niches, the spoon-shaped waveguide was functionalized with two kinds of receptors, both specific for human serum albumin (HSA): an antibody on the bowl region (high S n ); molecularly imprinted nanoparticles (nanoMIPs) on the neck region (low S n ). The experimental results showed a limit of detection (LOD) for the immune-sensor of 280 pM and an LOD for the nanoMIP-sensor of 4.16 fM. The overall response of the HSA multi-sensor encompassed eight orders of magnitude, suggesting that the spoon-shaped waveguide's provides multi-scale detection and holds potential to devise multi-analyte sensing platforms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Despite the tremendous technological advances that metal additive manufacturing (AM) has made in the last decades, there are still some major concerns guaranteeing its massive industrial application in the biomedical field. Indeed, some main limitations arise in dealing with their biological properties, specifically in terms of osseointegration. Morphological accuracy of sub-unital elements along with the printing resolution are major constraints in the design workspace of a lattice, hindering the possibility of manufacturing structures optimized for proper osteointegration. To overcome these issues, the authors developed a new hybrid multifunctional composite scaffold consisting of an AM Ti6Al4V lattice structure and a silk fibroin/gelatin foam. The composite was realized by combining laser powder bed fusion (L-PBF) of simple cubic lattice structures with foaming techniques. A combined process of foaming and electrodeposition has been also evaluated. The multifunctional scaffolds were characterized to evaluate their pore size, morphology, and distribution as well as their adhesion and behavior at the metal-polymer interface. Pull-out tests in dry and hydrated conditions were employed for the mechanical characterization. Additionally, a cytotoxicity assessment was performed to preliminarily evaluate their potential application in the biomedical field as load-bearing next-generation medical devices.

The possibility to prepare molecularly imprinted nanoparticles from silk fibroin was recently demonstrated starting from methacrylated silk fibroin and choosing a protein as template. Here, we attempted the imprinting of fibroin-based molecularly imprinted polymers (MIPs), called bioMIPs, using as a template hepcidin that is a iron-metabolism regulator-peptide, possessing a hairpin structure. A homogeneous population (PDI < 0.2) of bioMIPs with size ~50 nm was produced. The bioMIPs were selective for the template; the estimated dissociation constant for hepcidin was K D = 3.6 ± 0.5 10 -7 M and the average number of binding sites per bioMIP was equal to 2. The bioMIPs used in a competitive assay for hepcidin in serum showed a detection range of 1.01 10 -7 - 6.82 10 -7 M and a limit of detection of 3.29 10 -8 M., (© 2022. The Author(s).)

Soft, deformable, molecularly imprinted nanoparticles (nanoMIPs) were combined to nano-plasmonic sensor chips realized on poly (methyl methacrylate) (PMMA) substrates to develop highly sensitive bio/chemical sensors. NanoMIPs (d mean < 50 nm), which are tailor-made nanoreceptors prepared by a template assisted synthesis, were made selective to bind Bovine Serum Albumin (BSA), and were herein used to functionalize gold optical nanostructures placed on a PMMA substrate, this latter acting as a slab waveguide. We compared nanoMIP-functionalized non-optimized gold nanogratings based on periodic nano-stripes to optimized nanogratings with a deposited ultra-thin MIP layer (<100 nm). The sensors performances were tested by the detection of BSA using the same setup, in which both chips were considered as slab waveguides, with the periodic nano-stripes allocated in a longitudinal orientation with respect to the direction of the input light. Result demonstrated the nanoMIP-non optimized nanogratings showed superior performance with respect to the ultra-thin MIP-optimized nanogratings. The peculiar deformable character of the nano-MIPs enabled to significantly enhance the limit of detection (LOD) of the plasmonic bio/sensor, allowing the detection of the low femtomolar concentration of analyte (LOD ∼ 3 fM), thus outpassing of four orders of magnitude the sensitivies achieved so far on optimized nano-patterned plasmonic platforms functionalized with ultra-thin MIP layers. Thus, deformable nanoMIPs onto non-optimized plasmonic probes permit to attain ultralow detections, down to the quasi-single molecule. As a general consideration, the combination of more plasmonic transducers to different kinds of MIP receptors is discussed as a mean to attain the detection range for the selected application field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cennamo, Bossi, Arcadio, Maniglio and Zeni.)

Surface Enhanced Raman Scattering (SERS) active gold nanostars represent an opportunity in the field of bioimaging and drug delivery. The combination of gold surface chemical versatility with the possibility to tune the optical properties changing the nanoparticles shape constitutes a multimodal approach for the investigation of the behavior of these carriers inside living cells. In this work, SERS active star-shaped nanoparticles were functionalized with doxorubicin molecules and covered with immuno-mimetic thiolated polyethylene glycol (PEG). Doxorubicin-conjugate gold nanoparticles show an intense Raman enhancement, a good stability in physiological conditions, and a low cytotoxicity. The strong adsorption of the anticancer drug doxorubicin in close contact with the gold nanostars surface enables their use as SERS tag imaging probes in vivo. Upon laser irradiation of the nanoparticles, a strong SERS signal is generated by the doxorubicin molecules close to the nanostars surface, enabling the localization of the nanoparticles inside the cells. After long time irradiation, the SERS signal drops, indicating the thermally driven delivery of the drug inside the cell. Therefore, the combination of SERS and laser scanning confocal microscopy is a powerful technique for the real-time analysis of drug release in living cells.

In the tissue-engineering field silk fibroin can be tailored to the target applications by modifying its secondary structure and molecular weight, and functionalizing the molecule with specific active groups linked to the amino acid side chains. To better tune the silk fibroin molecular weight and structural properties, we propose the creation of a lower molecular weight fibroin-derived material through a selective and tunable enzymatic attack on the fibroin chain. Cleavage at specific amino acid sites leads to precise silk fibroin fragmentation and, thus, lower molecular weight materials whose length and properties can be tuned with the enzyme concentration. The cleavage increased the presence of free amino groups, hence reactivity, and aqueous solutions of the resulting polymer remained stable for up to seven days. Films of fragmented fibroin were prepared and characterized, demonstrating that the fragmentation did not affect β-sheet formation after methanol treatment, but differences were detected after the water-vapor annealing process, confirmed by structural and thermal analyses. The adopted fragmentation method is fast, controllable and precise, allowing the creation of a silk-derived material class that is stable in water, with a tunable molecular weight and secondary structure rearrangements, and is thus a versatile tool for the further tunability and modulation of bioengineered constructs.

Nanosized biomimetics prepared by the strategy of molecular imprinting, that is, the stamping of recognition sites by means of a template-assisted synthesis, are demonstrating potential as plastic antibodies in medicine, proving effective for cell imaging and targeted therapies. Most molecularly imprinted nanoparticles (MIP-NPs) are currently made of soft matter, such as polyacrylamide and derivatives. Yet, MIP-NPs biocompatibility is crucial for their effective translation into clinical uses. Here, we propose the original idea to synthesize fully biocompatible molecularly imprinted nanoparticles starting from the natural polymer silk fibroin (MIP SF-NPs), which is nontoxic and highly biocompatible. The conditions to produce MIP SF-NPs of different sizes ( d mean ∼ 50 nm; d mean ∼ 100 nm) were set using the response surface method. The stamping of a single, high affinity ( K D = 57 × 10 -9 M), and selective recognition site per silk fibroin nanoparticle was demonstrated, together with the confirmation of nontoxicity. Additionally, MIP SF-NPs were used to decorate silk microfibers and silk nanofibers, providing a general means to add entailed biofunctionalities to materials.

Porous metal lattice structures have a very high potential in biomedical applications, setting as innovative new generation prosthetic devices. Laser powder bed fusion (L-PBF) is one of the most widely used additive manufacturing (AM) techniques involved in the production of Ti6Al4V lattice structures. The mechanical and failure behavior of lattice structures is strongly affected by geometrical imperfections and defects occurring during L-PBF process. Due to the influence of multiple process parameters and to their combined effect, the mechanical properties of these structures are not yet properly understood. Despite the major commitment to characterize and better comprehend lattice structures, little attention has been paid to the impact that single struts have on the overall lattice properties. In this work, the authors have investigated the tensile strength and fatigue behavior of thin L-PBF Ti6Al4V lattice struts at different building orientations (0°, 15°, 45°, and 90°). This investigation has been focused on the effect that microstructural defects (particularly porosity) and actual surface geometry (including surface texture and geometrical errors such as varying cross-section shape and size) have on the mechanical performances of the struts in relation to their building direction. The results have shown that there is a tendency, particularly for low printing angles, of fatigue life to decrease with decreasing of the building angle. This is mainly due to the surge in surface texture and loss in cross-sectional regularity. On the other hand, the monotonic tensile test results have shown a low sensitivity to these factors. The strut failure behavior has been examined employing dynamic digital image correlation (DIC) of tensile tests and scanning electron imaging (SEM) of the fracture surfaces., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The request of new materials, matching strict requirements to be applied in precision and patient-specific medicine, is pushing for the synthesis of more and more complex block copolymers. Amphiphilic block copolymers are emerging in the biomedical field due to their great potential in terms of stimuli responsiveness, drug loading capabilities and reversible thermal gelation. Amphiphilicity guarantees self-assembly and thermoreversibility, while grafting polymers offers the possibility of combining blocks with various properties in one single material. These features make amphiphilic block copolymers excellent candidates for fine tuning drug delivery, gene therapy and for designing injectable hydrogels for tissue engineering. This manuscript revises the main techniques developed in the last decade for the synthesis of amphiphilic block copolymers for biomedical application. Strategies for fine tuning the properties of these novel materials during synthesis are discussed. A deep knowledge of the synthesis techniques and their effect on the performance and the biocompatibility of these polymers is the first step to move them from the lab to the bench. Current results predict a bright future for these materials in paving the way towards a smarter, less invasive, while more effective, medicine., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Peri-implant mucositis and peri-implantitis are biofilm-related diseases causing major concern in oral implantology, requiring complex anti-infective procedures or implant removal. Microbial biosurfactants emerged as new anti-biofilm agents for coating implantable devices preserving biocompatibility. This study aimed to assess the efficacy of rhamnolipid biosurfactant R89 (R89BS) to reduce Staphylococcus aureus and Staphylococcus epidermidis biofilm formation on titanium., Methods: R89BS was physically adsorbed on titanium discs (TDs). Cytotoxicity of coated TDs was evaluated on normal lung fibroblasts (MRC5) using a lactate dehydrogenase assay. The ability of coated TDs to inhibit biofilm formation was evaluated by quantifying biofilm biomass and cell metabolic activity, at different time-points, with respect to uncoated controls. A qualitative analysis of sessile bacteria was also performed by scanning electron microscopy., Results: R89BS-coated discs showed no cytotoxic effects. TDs coated with 4 mg/mL R89BS inhibited the biofilm biomass of S. aureus by 99%, 47% and 7% and of S. epidermidis by 54%, 29%, and 10% at 24, 48 and 72 h respectively. A significant reduction of the biofilm metabolic activity was also documented. The same coating applied on three commercial implant surfaces resulted in a biomass inhibition higher than 90% for S. aureus, and up to 78% for S. epidermidis at 24 h., Conclusions: R89BS-coating was effective in reducing Staphylococcus biofilm formation at the titanium implant surface. The anti-biofilm action can be obtained on several different commercially available implant surfaces, independently of their surface morphology.

The first wave of the COVID-19 pandemic brought about a broader use of masks by both professionals and the general population. This resulted in a severe worldwide shortage of devices and the need to increase import and activate production of safe and effective surgical masks at the national level. In order to support the demand for testing surgical masks in the Italian context, Universities provided their contribution by setting up laboratories for testing mask performance before releasing products into the national market. This paper reports the effort of seven Italian university laboratories who set up facilities for testing face masks during the emergency period of the COVID-19 pandemic. Measurement set-ups were built, adapting the methods specified in the EN 14683:2019+AC. Data on differential pressure (DP) and bacterial filtration efficiency (BFE) of 120 masks, including different materials and designs, were collected over three months. More than 60% of the masks satisfied requirements for DP and BFE set by the standard. Masks made of nonwoven polypropylene with at least three layers (spunbonded-meltblown-spunbonded) showed the best results, ensuring both good breathability and high filtration efficiency. The majority of the masks created with alternative materials and designs did not comply with both standard requirements, resulting in suitability only as community masks. The effective partnering between universities and industries to meet a public need in an emergency context represented a fruitful example of the so-called university "third-mission".

Microbial biofilms strongly resist host immune responses and antimicrobial treatments and are frequently responsible for chronic infections in peri-implant tissues. Biosurfactants (BSs) have recently gained prominence as a new generation of anti-adhesive and antimicrobial agents with great biocompatibility and were recently suggested for coating implantable materials in order to improve their anti-biofilm properties. In this study, the anti-biofilm activity of lipopeptide AC7BS, rhamnolipid R89BS, and sophorolipid SL18 was evaluated against clinically relevant fungal/bacterial dual-species biofilms ( Candida albicans , Staphylococcus aureus , Staphylococcus epidermidis ) through quantitative and qualitative in vitro tests. C. albicans - S. aureus and C. albicans - S. epidermidis cultures were able to produce a dense biofilm on the surface of the polystyrene plates and on medical-grade silicone discs. All tested BSs demonstrated an effective inhibitory activity against dual-species biofilms formation in terms of total biomass, cell metabolic activity, microstructural architecture, and cell viability, up to 72 h on both these surfaces. In co-incubation conditions, in which BSs were tested in soluble form, rhamnolipid R89BS (0.05 mg/ml) was the most effective among the tested BSs against the formation of both dual-species biofilms, reducing on average 94 and 95% of biofilm biomass and metabolic activity at 72 h of incubation, respectively. Similarly, rhamnolipid R89BS silicone surface coating proved to be the most effective in inhibiting the formation of both dual-species biofilms, with average reductions of 93 and 90%, respectively. Scanning electron microscopy observations showed areas of treated surfaces that were free of microbial cells or in which thinner and less structured biofilms were present, compared to controls. The obtained results endorse the idea that coating of implant surfaces with BSs may be a promising strategy for the prevention of C. albicans - Staphylococcus spp. colonization on medical devices, and can potentially contribute to the reduction of the high economic efforts undertaken by healthcare systems for the treatment of these complex fungal-bacterial infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ceresa, Rinaldi, Tessarolo, Maniglio, Fedeli, Tambone, Caciagli, Banat, Diaz De Rienzo and Fracchia.)

The introduction of nanoparticles (NPs) into the breath-figure-templated self-assembly (BFTSA) process is an increasingly common method to selectively decorate a surface porous structure. In the field of prosthetic devices, besides controlling the morphology and roughness of the structure, NPs can enhance the osteointegration mechanism because of their specific ion release. Among the most widely used NPs, there are silica and hydroxyapatite (HAp). In this work, we propose a novel one-stage method to fabricate NP-decorated surface porous structures that are suitable for prosthetic coating applications. This technique combines the classical direct BFTSA process with the cavitation effect induced by an ultrasonic atomizer that generates a mist of water droplets with embedded NPs. Coatings were successfully obtained by combining a UV cross-linkable polymer precursor, alkoxy silicone, with synthesized HAp NPs, on Ti6Al4V alloy discs. The cross-linked polymeric surface porous structures at selected concentrations were then pyrolyzed in an ammonia atmosphere to obtain a silicon oxynitride (SiON) ceramic coating. Herein, we report the chemical and morphological analyses of both the polymeric and ceramic coatings as well as the effect of NPs at the interface.

Research in the field of radiopharmaceuticals is increasingly promoted by the widespread and growing interest in applying nuclear medicine procedures in both disease diagnosis and treatment. The production of radionuclides of medical interest is however a challenging issue. Along with the conventional techniques other innovative approaches are being investigated and, among those, the ISOLPHARM project is being developed at INFN-LNL (Istituto Nazionale di Fisica Nucleare - Laboratori Nazionali di Legnaro). Such technique foresees the employment of the SPES ISOL facility to produce isobarically pure Radioactive Ion Beams (RIBs), obtained thanks to electromagnetic mass separation and collected on appropriate substrates. The latter are successively recovered and dissolved, allowing thus the chemical separation and harvesting of the nuclides of interest, free from any isotopic contaminant. Although ISOLPHARM can be potentially employed for most of the routinely used medical radioisotopes, its innovation potential is better expressed considering its capability to provide carrier free unconventional nuclides, difficult to produce with state-of-art techniques, such as 111 Ag, a β - emitter potentially interesting for therapeutic applications. Thus, in the framework of ISOLPHARM, INFN supported a two-years experiment, called ISOLPHARM&#95;Ag, aimed at evaluating the feasibility of the production of a 111 Ag labelled radiopharmaceutical. The ISOL production yields are estimated by computing intensive Monte Carlo codes, that require an appropriate custom Information Technology infrastructure. The presented work is focused on the first part of the production chain including the capability to extract, ionize, and collect stable Ag beams with SPES technologies. MC calculations were used to estimate the expected 111 Ag in-target yields, whereas experiments with stable Ag were performed to test the ionization, transport and collection of Ag beams., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Soft molecularly imprinted nanogels (nanoMIPs), selective for human transferrin (HTR), were prepared via a template assisted synthesis. Owing to their soft matter, the nanoMIPs were observed to deform at binding to HTR: while no relevant changes were observed in the hydrodynamic sizes of HTR-free compared to HTR-loaded nanoMIPs, the HTR binding resulted in a significant increment of the nanoMIP stiffness, with the mean Young's modulus measured by AFM passing from 17 ± 6 kPa to 56 ± 18 kPa. When coupled to a plastic optical fibre (POF) plasmonic platform, the analyte-induced nanoMIP-deformations amplified the resonance shift, enabling to attain ultra-low sensitivities (LOD = 1.2 fM; linear dynamic range of concentrations from 1.2 fM to 1.8 pM). Therefore, soft molecularly imprinted nanogels that obey to analyte-induced deformation stand as a novel class of sensitivity-gain structures for plasmonic sensing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Silks, in particular silkworm silks, have been studied for decades as possible candidate materials for biomedical applications. Recently, great attentions have been paid to spider silks, mainly due to their unique and remarkable mechanical properties. Both materials express singular interactions with cells through specific biorecognition moieties on the core proteins making up the two silks. In this work, the silk from a Colombian spider, Linothele megatheloides (LM), which produces a single type of silk in a relatively large amount, was studied in comparison with silk from Bombyx mori silkworm, before and after degumming, with the evaluation of their chemical, mechanical and biological properties. Unexpected biological features in cell culture tests were found for the LM silk already at very early stage, so suggesting further investigation to explore its use for tailored biomedical applications., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

A wide variety of hydrogels have been proposed for tissue engineering applications, cell encapsulation, and bioinks for bioprinting applications. Cell-laden hydrogel constructs rely on natural hydrogels such as alginate, agarose, chitosan, collagen, gelatin, fibroin, and hyaluronic acid (HA), as well as on synthetic hydrogels such as poloxamers (Pluronics ® ) and polyethylene glycol (PEG). Alginate has become more and more important in the last years, thanks to the possibility to prepare alginate hydrogels suitable for cell encapsulation mainly because of the mild and reversible cross-linking conditions. In this paper alginate will be described in detail with respect to its chemistry, cross-linking behavior, biocompatibility, manufacturing capacity, and possible modifications.

Silk fibroin sponges have been widely studied and reported in literature for tissue engineering applications. Several fabrication methods have been proposed during the years to cover most of the demands in terms of properties, which should be adapted to the considered tissue. Most of these procedures are based on the secondary structure transition of the protein to the stable β crystalline form. This transition, known as physical cross-linking, makes the sponge resistant to dissolution in water, and, in general, increases the sponge stiffness. In our work, we propose an alternative method to ensure the stability of the sponge based on chemical cross-linking of a methacrylated version of silk fibroin (Sil-MA) obtained via chemical modification. The Sil-MA water solution with the addition of a photoinitiator (LAP) allows the opening, under UV radiation, of a double carbon-carbon bond and radical polymerization. The incorporation of air bubbles (that serves as a template for the pores) was accomplished by a mixer; then, the foam was stabilized under UV light and the excess water was removed by freeze-drying. Because of the cytotoxicity of the photoinitiator (found when used at a high concentration), an additional washing step in water has been introduced to eliminate the residues and improve the cells' viability. Fourier transform infrared (FTIR) analysis confirmed the functionalization of the protein. To evaluate the effect of the composition on the sponge properties, a 2 3 full factorial design of the experiment has been adopted. FTIR analysis revealed that the sponge composition did not affect the protein's secondary structure. The analysis of images obtained by SEM allowed some statistical measures of the porosity curves to be studied and modeled. The same modeling procedure was applied to the dissolution test in a simulated body fluid, to the water absorption, and to the cell viability (tested by the MTT and LDH assays). An empirical model for each property was built, showing how by changing the composition it is possible to tune the sponge properties.

Staphylococcus aureus and Staphylococcus epidermidis are considered two of the most important pathogens, and their biofilms frequently cause device-associated infections. Microbial biosurfactants recently emerged as a new generation of anti-adhesive and anti-biofilm agents for coating implantable devices to preserve biocompatibility. In this study, R89 biosurfactant (R89BS) was evaluated as an anti-biofilm coating on medical-grade silicone. R89BS is composed of homologues of the mono- (75%) and di-rhamnolipid (25%) families, as evidenced by mass spectrometry analysis. The antimicrobial activity against Staphylococcus spp. planktonic and sessile cells was evaluated by microdilution and metabolic activity assays. R89BS inhibited S. aureus and S. epidermidis growth with minimal inhibitory concentrations (MIC 99 ) of 0.06 and 0.12 mg/mL, respectively and dispersed their pre-formed biofilms up to 93%. Silicone elastomeric discs (SEDs) coated by R89BS simple adsorption significantly counteracted Staphylococcus spp. biofilm formation, in terms of both built-up biomass (up to 60% inhibition at 72 h) and cell metabolic activity (up to 68% inhibition at 72 h). SEM analysis revealed significant inhibition of the amount of biofilm-covered surface. No cytotoxic effect on eukaryotic cells was detected at concentrations up to 0.2 mg/mL. R89BS-coated SEDs satisfy biocompatibility requirements for leaching products. Results indicate that rhamnolipid coatings are effective anti-biofilm treatments and represent a promising strategy for the prevention of infection associated with implantable devices.

Tissue regeneration requires scaffolds that exhibit mechanical properties similar to the tissues to be replaced while allowing cell infiltration and extracellular matrix production. Ideally, the scaffolds' porous architecture and physico-chemical properties can be precisely defined to address regenerative needs. We thus developed techniques to produce hybrid fibers coaxially structured with a polycaprolactone core and a 4-arm, polyethylene glycol thiol-norbornene sheath. We assessed the respective effects of crosslink density and sheath polymer size on the scaffold architecture, physical and mechanical properties, as well as cell-scaffold interactions in vitro and in vivo. All scaffolds displayed high elasticity, swelling and strength, mimicking soft tissue properties. Importantly, the thiol-ene hydrogel sheath enabled tunable softness and peptide tethering for cellular activities. With increased photopolymerization, stiffening and reduced swelling of scaffolds were found due to intra- and inter-fiber crosslinking. More polymerized scaffolds also enhanced the cell-scaffold interaction in vitro and induced spontaneous, deep cell infiltration to produce collagen and elastin for tissue regeneration in vivo. The molecular weight of sheath polymer provides an additional mechanism to alter the physical properties and biological activities of scaffolds. Overall, these robust scaffolds with tunable elasticity and regenerative cues offered a versatile and effective platform for tissue regeneration.

Aim: several strategies have been tested in recent years to prevent bacterial colonization of dental implants. Sericin, one of the two main silk proteins, possesses relevant biological activities and also literature reports about its potential antibacterial properties, but results are discordant and not yet definitive. The aim of this study was to evaluate the effectiveness of different experimental protocols in order to obtain a sericin-based coating on medical grade titanium (Ti) able to reduce microbial adhesion to the dental implant surface., Materials and Methods: different strategies for covalent bonding of sericin to Ti were pursued throughout a multi-step procedure on Ti-6Al-4V disks. The surface of grade 5 Ti was initially immersed in NaOH solution to obtain the exposure of functional -OH groups. Two different silanization strategies were then tested using aminopropyltriethoxysilane (APTES). Eventually, the bonding between silanized Ti-6Al-4V and sericin was obtained with two different crosslinking processes: glutaraldehyde (GLU) or carbodiimide/N-Hydroxy-succinimide (EDC/NHS). Micro-morphological and compositional analyses were performed on the samples at each intermediate step to assess the most effective coating strategy able to optimize the silanization and bioconjugation processes. Microbiological tests on the coated Ti-6Al-4V disks were conducted in vitro using a standard biofilm producer strain of Staphylococcus aureus (ATCC 6538) to quantify the inhibition of microbial biofilm formation (anti-biofilm efficacy) at 24 hours., Results: both silanization techniques resulted in a significant increase of silicon (Si) on the Ti-6Al-4V surfaces etched with NaOH. Differences were found between GLU and EDC/NHS bioconjugation strategies in terms of composition, surface micro-morphology and anti-biofilm efficacy. Ti-6Al-4V samples coated with GLU-bound sericin after silanization obtained via vapor phase deposition proved that this technique is the most convenient and effective coating strategy, resulting in a bacterial inhibition of about 53% in respect to the uncoated Ti-6Al-4V disks., Conclusions: The coating with glutaraldehyde-bound sericin after silanization in the vapor phase showed promising bacterial inhibition values with a significant reduction of S. aureus biofilm. Further studies including higher number of replicates and more peri-implant-relevant microorganisms are needed to evaluate the applicability of this experimental protocol to dental implants.

Bioactive coatings are usually applied to bone and dental prostheses to enhance the integration and their stability in the bone. Recently, silicon (Si) oxynitride ceramics have been demonstrated to possess osteoconductive properties due to the release of Si ions, particularly important in the early stage of bone formation. In addition, the pattern of the bone contacting surface has been reported to affect cells' differentiation and metabolic activity. In this work, we propose the Breath Figure (BF) process combined with a pyrolysis step, starting from a photo-crosslinkable alkoxy silicone precursor, as a method to realize bioactive patterned coating on metal bone and dental prostheses. Four different surface patterned coatings were applied to Ti4Al6V disks starting from solutions with different precursor concentrations. Morphology, chemical composition, and Si ions' release were evaluated and compared. Moreover, all samples underwent to biological in vitro testing with human mesenchymal stem cells (hMSCs) in comparison with the uncoated titanium alloy. The results indicated that the Si released from the coatings determined an increase in the cellular activity with the BF pattern influencing the hMSCs' initial adhesion and proliferation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1284-1294, 2019., (© 2018 Wiley Periodicals, Inc.)

The main limitation of drug-enhanced radiotherapy concerns the difficulty to evaluate the effectiveness of cancer targeting after drug administration hindering the standardization of therapies based on current radiosensitizing compounds. The challenge regards the development of systems able to combine imaging and radiotherapy enhancement in order to perform highly reliable cancer theragnosis. For these reasons, gold-magnetite hybrid nanoparticles (H-NPs) are proposed as innovative theranostic nanotools for imaging-guided radiosensitization in cancer treatment. In this work we propose a novel method for the synthesis of hydrophilic and superparamagnetic Tween20-stabilized gold-magnetite H-NPs. Morphology and chemical composition of nanoparticles were assessed by transmission electron microscopy, x-ray diffraction analysis and ion-coupled plasma optical emission spectroscopy. Colloidal stability and magnetic properties of nanoparticles were determined by dynamic light scattering and magnetometry. The potentialities of H-NPs for magnetic resonance imaging were studied using a human 4T-MRI scanner. Nanoparticles were proven to induce concentration-dependent contrast enhancement in T2*-weighted MR-images. The cytotoxicity, the cellular uptake and the radiosensitization activity of H-NPs were investigated in human osteosarcoma MG63 cell cultures and murine 3T3 fibroblasts, using specific bioassays and laser scanning confocal microscopy. H-NPs did not exhibit significant toxicity and were demonstrated to be internalized by cells. A significant x-ray enhancement at specific H-NPs exposure concentrations was evidenced on MG63 cell line.

Silk fibroin has acquired increasing interest in the last years for application in medicine and namely in tissue engineering. Several methods have been developed to process fibroin and for the fabrication of nets, sponges, films and gels. This paper deals with the fabrication and characterization of fibroin hydrogels obtained by using sodium oleate as gelation agent. Gels have been prepared by mixing Silk fibroin (SF) and Sodium oleate (SO) water solutions in different concentrations, and a quite wide frame of compositions have been explored. Rheological tests have been performed to determine the gelation times, scanning electron microscopies have been made to evaluate morphologies, FTIR analysis has been done to determine the conformation of the starting materials and of the resulting gels, water content has been measured and cytotoxicity tests have been performed to validate the potential biomedical use of the hydrogels. Depending on the SF and SO different gelation times have been obtained thanks to the formation of intermolecular bonds between the fibroin chains. The obtained fastest gelation of about 80 s could make this specific formulation compatible with in situ gelation. By changing composition, gels with different morphologies, rheological properties and water contents have been prepared.

Silk fibroin has acquired increasing interest for biomedical applications, and namely for the fabrication of scaffolds for tissue engineering, because of its highly positive biological interaction and the possibility to adapt the material to several application requirements by adopting different fabrication methods, in order to make films, sponges, fibers, nets or gels with predictable degradation times. For tissue engineering, in most cases porous scaffolds are required, in some cases possibly in situ forming and therefore fabricated in mild body-compatible conditions. In this work, we present a novel one-step method for the preparation of silk fibroin foams starting from water solutions and using low-pressure nitrous oxide gas as foaming agent. This foaming technique allows preparing fibroin porous scaffolds with easily tunable porosity, in mild processing conditions with the use of a relatively inert foaming agent saturating a fibroin water solution, that could be occasionally injected through a thin needle in the implantation site where expansion and foaming would occur. Optimal foaming processing conditions have been investigated, and the prepared foams have been characterized with Fourier Transform Infrared Spectroscopy (FTIR) compressive mechanical and rheological properties measurements, and by scanning electron microscopy and microCT.

Cell encapsulation in hydrogels is a technique that offers a variety of applications, ranging from drug delivery to biofabrication of three-dimensional scaffolds. The assembly of cell-laden hydrogel building blocks aims to generate complex biological constructs by manipulating microscale units. An important issue for the clinical implementation of this technique is the long-term storage of a large stock of cell/hydrogel building blocks. In this work, the impact of cryopreservation on the viability and functionality of cells encapsulated in alginate matrices is presented comparing different cryoprotective agents (CPAs). Human osteosarcoma MG63 cells were encapsulated in sodium alginate fiber constructs with wetspinning method and exposed to different formulations of cryopreservation media, containing dimethyl sulfoxide (DMSO), glycerol, and trehalose. The cell-laden fibers were subsequently slow-cooled down to -80°C and stored in liquid nitrogen. After thawing, viability and death pathway of encapsulated cells were investigated, and metabolic activity and proliferative capacity of cells released from the alginate matrix were evaluated. The viability of MG63 cells encapsulated in alginate matrix ranged from 71% ± 4% to 85% ± 2%, depending on the cryoprotective media formulation with no protracted harmful effects from the CPAs. On the other side, cells cryopreserved in encapsulated conditions and released from the hydrogel showed larger metabolic activity and proliferative capacity in tissue culture plate compared to cells cryopreserved in suspension, in particular when DMSO and glycerol were used as CPAs. Results have been correlated with the viscoelastic properties and water content changes of the alginate constructs loaded with the different CPAs.

The hydrolysis-condensation of trialkoxysilanes under strictly controlled conditions allows the production of silsesquioxanes (SSQs) with tunable size and architecture ranging from ladder to cage-like structures. These nano-objects can serve as building blocks for the preparation of hybrid organic/inorganic materials with selected properties. The SSQs growth can be tuned by simply controlling the reaction duration in the in situ water production route (ISWP), where the kinetics of the esterification reaction between carboxylic acids and alcohols rules out the extent of organosilane hydrolysis-condensation. Tunable SSQs with thiol functionalities (SH-NBBs) are suitable for further modification by exploiting the simple thiol-ene click reaction, thus allowing for modifying the wettability properties of derived coatings. In this paper, coatings were prepared from SH-NBBs with different architecture onto cotton fabrics and paper, and further functionalized with long alkyl chains by means of initiator-free UV-induced thiol-ene coupling with 1-decene (C10) and 1-tetradecene (C14). The coatings appeared to homogeneously cover the natural fibers and imparted a multi-scale roughness that was not affected by the click functionalization step. The two-step functionalization of cotton and paper warrants a stable highly hydrophobic character to the surface of natural materials that, in perspective, suggests a possible application in filtration devices for oil-water separation. Furthermore, the purification of SH-NBBs from ISWP by-products was possible during the coating process, and this step allowed for the fast, initiator-free, click-coupling of purified NBBs with C10 and C14 in solution with a nearly quantitative yield. Therefore, this approach is an alternative route to get sol-gel-derived, ladder-like, and cage-like SSQs functionalized with long alkyl chains., Competing Interests: The authors declare no conflict of interest.

Background: This study investigated the preparation of ordered patterned surfaces and/or microspheres from a natural-based polymer, using the breath figure and reverse breath figure methods., Methods: Poly(D,L-lactic acid) and starch poly(lactic acid) solutions were precipitated in different conditions - namely, polymer concentration, vapor atmosphere temperature and substrate - to evaluate the effect of these conditions on the morphology of the precipitates obtained., Results: The possibility of fine-tuning the properties of the final patterns simply by changing the vapor atmosphere was also demonstrated here using a range of compositions of the vapor phase. Porous films or discrete particles are formed when the differences in surface tension determine the ability of polymer solution to surround water droplets or methanol to surround polymer droplets, respectively. In vitro cytotoxicity was assessed applying a simple standard protocol to evaluate the possibility to use these materials in biomedical applications. Moreover, fluorescent microscopy images showed a good interaction of cells with the material, which were able to adhere on the patterned surfaces after 24 hours in culture., Conclusions: The development of patterned surfaces using the breath figure method was tested in this work for the preparation of both poly(lactic acid) and a blend containing starch and poly(lactic acid). The potential of these films to be used in the biomedical area was confirmed by a preliminary cytotoxicity test and by morphological observation of cell adhesion.