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Three-dimensional printing has opened up new perspectives in bone substitution, facilitating the production of customized scaffolds. The advances of the last few years have placed this technology at the forefront of personalized medicine and virtual surgical planning. This article presents an overview of additive manufacturing of bioceramics for bone regeneration, covering both the additive manufacturing methods and the consolidation strategies that can be applied. We highlight the main progress made in recent years, with an insight into drug delivery applications and the advances in the translation of this technology to the biomedical industry and the clinics. Furthermore, the main challenges and future trends of this new medical technology are identified and discussed., Peer Reviewed, Postprint (author's final draft)

Antibiotic resistance is a leading cause of death worldwide. In this paper, we explore new alternatives in the treatment of infections. Noble metal nanoparticles could help to mitigate this problem. In this work, palladium nanoparticles were synthesized by laser ablation in order to explore their antimicrobial capacity. To obtain palladium nanoparticles, a palladium plate immersed in water, or methanol, was ablated, using two pulsed lasers that emit radiation with wavelengths of 532 nm and 1064 nm, respectively. Pure Pd-NPs with crystalline microstructure and rounded shape were obtained. The nanoparticles’ size is more homogeneous if the laser wavelength is 532 nm, and it decreases when methanol is used as solvent, reaching mean diameters smaller than 6 nm. With the objective of studying antimicrobial activity against Staphylococcus aureus, the Pd-NPs were immobilized on the surface of titanium discs. The release of palladium ions was recorded during the first seven days, and the cytotoxicity of the immobilized NPs was also tested with L929 mouse fibroblast cell line. Palladium nanoparticles synthesized by means of the infrared laser in methanol showed a strong inhibitory effect on S. aureus and good cytocompatibility, with no toxic effect on fibroblast cells., Peer Reviewed, Postprint (published version)

Endothelial coverage of an exposed cardiovascular stent surface leads to the occurrence of restenosis and late-stent thrombosis several months after implantation. To overcome this difficulty, modification of stent surfaces with topographical or biochemical features may be performed to increase endothelial cells’ (ECs) adhesion and/or migration. This work combines both strategies on cobalt-chromium (CoCr) alloy and studies the potential synergistic effect of linear patterned surfaces that are obtained by direct laser interference patterning (DLIP), coupled with the use of Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR) peptides. An extensive characterization of the modified surfaces was performed by using AFM, XPS, surface charge, electrochemical analysis and fluorescent methods. The biological response was studied in terms of EC adhesion, migration and proliferation assays. CoCr surfaces were successfully patterned with a periodicity of 10 µm and two different depths, D (˜79 and 762 nm). RGD and YIGSR were immobilized on the surfaces by CPTES silanization. Early EC adhesion was increased on the peptide-functionalized surfaces, especially for YIGSR compared to RGD. High-depth patterns generated 80% of ECs’ alignment within the topographical lines and enhanced EC migration. It is noteworthy that the combined use of the two strategies synergistically accelerated the ECs’ migration and proliferation, proving the potential of this strategy to enhance stent endothelialization., Peer Reviewed, Postprint (published version)

The project is aimed at investigating the effect of laser surface modification of dental-grade zirconia (3Y-TZP) to guide cellular behaviour. A nanosecond laser was employed to fabricate groove patterns on the surface of 3Y-TZP, yielding three different topographies according to the groove periodicity (30, 50 and 100µm). The resulting topography, microstructure, hardness and hydrothermal degradation resistance of the modified zirconia was assessed by means of advanced characterization techniques. A cellular study was conducted to evaluate the behaviour of human mesenchymal stem cells on the patterned samples in terms of adhesion and cell morphology. The results showed grooves of approximately 1.7µm heigh with pronounced pile-up due to the melting and re-solidification of the material. The phase analysis revealed that no phase transformation is induced during the laser modification; however, a non-homogeneous monoclinic phase distribution was observed on the patterned surfaces after hydrothermal degradation. Finally, all the patterned surfaces allowed cell attachment, increased cell area and favoured cell elongation and alignment in the direction of the laser patterns., Postprint (published version)

The version of record is available online at: http://dx.doi.org/10.1007/s10237-022-01577-2, Scaffolds are microporous biocompatible structures that serve as material support for cells to proliferate, differentiate and form functional tissue. In particular, in the field of bone regeneration, insertion of scaffolds in a proper physiological environment is known to favour bone formation by releasing calcium ions, among others, triggering differentiation of mesenchymal cells into osteoblasts. Computational simulation of molecular distributions through scaffolds is a potential tool to study the scaffolds’ performance or optimal designs, to analyse their impact on cell differentiation, and also to move towards reduction in animal experimentation. Unfortunately, the required numerical models are often highly complex and computationally too costly to develop parametric studies. In this context, we propose a computational parametric reduced-order model to obtain the distribution of calcium ions in the interstitial fluid flowing through scaffolds, depending on several physical parameters. We use the well-known Proper Orthogonal Decomposition (POD) with two different variations: local POD and POD with quadratic approximations. Computations are performed using two realistic geometries based on a foamed and a 3D-printed scaffolds. The location of regions with high concentration of calcium in the numerical simulations is in fair agreement with regions of bone formation shown in experimental observations reported in the literature. Besides, reduced-order solutions accurately approximate the reference finite element solutions, with a significant decrease in the number of degrees of freedom, thus avoiding computationally expensive simulations, especially when performing a parametric analysis. The proposed reduced-order model is a competitive tool to assist the design of scaffolds in osteoinduction research., The authors acknowledge the financial support from the Ministerio de Ciencia e Innovación (MCIN/ AEI/10.13039/501100011033) through the grants PID2020-113463RBC32, PID2020-113463RB-C33, CEX2018-000797-S and PID2019-103892RB-I00, and the Generalitat de Catalunya for the Serra Hunter Fellowship of ME and the ICREA Academia Award of MPG., Peer Reviewed, Postprint (published version)

The passivation of titanium dental implants is performed in order to clean the surface and obtain a thin layer of protective oxide (TiO2) on the surface of the material in order to improve its behavior against corrosion and prevent the release of ions into the physiological environment. The most common chemical agent for the passivation process is hydrochloric acid (HCl), and in this work we intend to determine the capacity of citric acid as a passivating and bactericidal agent. Discs of commercially pure titanium (c.p.Ti) grade 4 were used with different treatments: control (Ctr), passivated by HCl, passivated by citric acid at 20% at different immersion times (20, 30, and 40 min) and a higher concentration of citric acid (40%) for 20 min. Physical-chemical characterization of all of the treated surfaces has been carried out by scanning electronic microscopy (SEM), confocal microscopy, and the ‘Sessile Drop’ technique in order to obtain information about different parameters (topography, elemental composition, roughness, wettability, and surface energy) that are relevant to understand the biological response of the material. In order to evaluate the corrosion behavior of the different treatments under physiological conditions, open circuit potential and potentiodynamic tests have been carried out. Additionally, ion release tests were realized by means of ICP-MS. The antibacterial behavior has been evaluated by performing bacterial adhesion tests, in which two strains have been used: Pseudomonas aeruginosa (Gram–) and Streptococcus sanguinis (Gram+). After the adhesion test, a bacterial viability study has been carried out (‘Life and Death’) and the number of colony-forming units has been calculated with SEM images. The results obtained show that the passivation with citric acid improves the hydrophilic character, corrosion resistance, and presents a bactericide character in comparison with the HCl treatment. The increasing of citric acid concentration improves the, Peer Reviewed, Postprint (author's final draft)

A lack of primary stability and osteointegration in metallic implants may result in implant loosening and failure. Adding porosity to metallic implants reduces the stress shielding effect and improves implant performance, allowing the surrounding bone tissue to grow into the scaffold. However, a bioactive surface is needed to stimulate implant osteointegration and improve mechanical stability. In this study, porous titanium implants were produced via powder sintering to create different porous diameters and open interconnectivity. Two strategies were used to generate a bioactive surface on the metallic foams: (1) an inorganic alkali thermochemical treatment, (2) grafting a cell adhesive tripeptide (RGD). RGD peptides exhibit an affinity for integrins expressed by osteoblasts, and have been reported to improve osteoblast adhesion, whereas the thermochemical treatment is known to improve titanium implant osseointegration upon implantation. Bioactivated scaffolds and control samples were implanted into the tibiae of rabbits to analyze the effect of these two strategies in vivo regarding bone tissue regeneration through interconnected porosity. Histomorphometric evaluation was performed at 4 and 12 weeks after implantation. Bone-to-implant contact (BIC) and bone in-growth and on-growth were evaluated in different regions of interest (ROIs) inside and outside the implant. The results of this study show that after a long-term postoperative period, the RGD-coated samples presented higher quantification values of quantified newly formed bone tissue in the implant’s outer area. However, the total analyzed bone in-growth was observed to be slightly greater in the scaffolds treated with alkali thermochemical treatment. These results suggest that both strategies contribute to enhancing porous metallic implant stability and osteointegration, and a combination of both strategies might be worth pursuing., Peer Reviewed, Postprint (author's final draft)

We report a simple approach to fabricate free-standing perforated 2D nanomembranes hosting well-ordered 1D metallic nanostructures to obtain hybrid materials with nanostructured surfaces for fexible electronics. Nanomembranes are formed by alternatively depositing perforated poly(lactic acid) (PLA) and poly(3,4-ethylenedioxythiophene) layers. Copper metallic nanowires (NWs) were incorporated into the nanoperforations of the top PLA layer by electrodeposition and further coated with silver via a transmetallation reaction. The combination of 2D polymeric nanomembranes and aligned 1D metallic NWs allows merging the fexibility and conformability of the ultrathin soft polymeric nanomembranes with the good electrical properties of metals for biointegrated electronic devices. Thus, we were able to tailor the nanomembrane surface chemistry as it was corroborated by SEM, EDX, XPS, CV, EIS and contact angle. The obtained hybrid nanomembranes were fexible and conformable showing sensing capacity towards H2O2 with good linear concentration range (0.35–10 mM), sensitivity (120 µA cm-2 mM-1) and limit of detection (7 µm). Moreover, the membranes showed good stability, reproducibility and selectivity towards H2O2, Peer Reviewed, Postprint (published version)

Hydrogels have been recently proposed as suitable materials to generate reactive oxygen and nitrogen species (RONS) upon gas-plasma treatment, and postulated as promising alternatives to conventional cancer therapies. Acting as delivery vehicles that allow a controlled release of RONS to the diseased site, plasma-treated hydrogels can overcome some of the limitations presented by plasma-treated liquids in in vivo therapies. In this work, we optimized the composition of a methylcellulose (MC) hydrogel to confer it with the ability to form a gel at physiological temperatures while remaining in the liquid phase at room temperature to allow gas-plasma treatment with suitable formation of plasma-generated RONS. MC hydrogels demonstrated the capacity for generation, prolonged storage and release of RONS. This release induced cytotoxic effects on the osteosarcoma cancer cell line MG-63, reducing its cell viability in a dose-response manner. These promising results postulate plasma-treated thermosensitive hydrogels as good candidates to provide local anticancer therapies., Peer Reviewed, Postprint (published version)

BACKGROUND Orthopedic implant-related infection remains one of the most serious complications after orthopedic surgery. In recent years, there has been an increased scientific interest to improve prevention and treatment strategies. However, many of these strategies have focused on chemical measures. AIM To analyze the effect of alternating current electrical fields on bacterial adherence to titanium surfaces. METHODS Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were exposed to 6.5 V electrical currents at different frequencies: 0.5 Hz, 0.1 Hz, and 0.05 Hz. After exposure, a bacterial count was then performed and compared to the control model. Other variables registered included the presence of electrocoagulation of the medium, electrode oxidation and/or corrosion, and changes in pH of the medium. RESULTS The most effective electrical model for reducing S. aureus adhesion was 6.5 V alternating current at 0.05 Hz achieving a 90% adhesion reduction rate. For E. coli, the 0.05 Hz frequency model also showed the most effective results with a 53% adhesion reduction rate, although these were significantly lower than S. aureus. Notable adhesion reduction rates were observed for S. aureus and E.coli in the studied conditions. However, the presence of electrode oxidation makes us presume these conditions are not optimal for in vivo use. CONCLUSION Although our findings suggest electrical currents may be useful in preventing bacterial adhesion to metal surfaces, further research using other electrical conditions must be examined to consider their use for in vivo trials., Peer Reviewed, Postprint (published version)

High-dose systemic chemotherapy constitutes a main strategy in the management of bone metastases, employing drugs like doxorubicin (DOX), related with severe side effects. To solve this issue, Cold Atmospheric Plasmas (CAP) have been proposed as potential non-invasive anti-cancer agents capable of improving the efficacy of traditional drugs. Here, we investigate the cytotoxic effects of Plasma Conditioned Medium (PCM) in combination with DOX in prostate cancer cells from bone metastases (PC-3) as well as in non-malignant bone-cells. PCM was able to enhance the cytotoxic potential of DOX both in monolayer and in a 3D bioengineered model mimicking the bone matrix. The combined treatment of PCM + DOX resulted in a profound downregulation of the redox defenses (CAT1, SOD2, GPX1) and drug resistance genes (MRP1, MDR1, BCRP1), resulting in an enhanced uptake of DOX coupled to an overload of intracellular ROS. Besides, PCM improved the cytotoxic potential of DOX interfering on the migratory and clonogenic potential of PC-3 cells. Importantly, non-malignant bone cells were unaffected by the combination of PCM + DOX. Overall, these new findings may represent a new therapeutic approach for the management of bone metastatic prostate cancer in the future., Peer Reviewed, Postprint (published version)

Since the discovery that nanostructured surfaces were able to kill bacteria, many works have been published focusing on the design of nanopatterned surfaces with antimicrobial properties. Synthetic bone grafts, based on calcium phosphate (CaP) formulations, can greatly benefit from this discovery if adequate nanotopographies can be developed. However, CaP are reactive materials and experience ionic exchanges when placed into aqueous solutions which may in turn affect cell behaviour and complicate the interpretation of the bactericidal results. The present study explores the bactericidal potential of two nanopillared CaP prepared by hydrolysis of two different sizes of a-tricalcium phosphate (a-TCP) powders under biomimetic or hydrothermal conditions. A more lethal bactericidal response toward Pseudomonas aeruginosa (~75% killing efficiency of adhered bacteria) was obtained from the hydrothermally treated CaP which consisted in a more irregular topography in terms of pillar size (radius: 20–60 nm), interpillar distances (100–1500 nm) and pillar distribution (pillar groups forming bouquets) than the biomimetically treated one (radius: 20–40 nm and interpillar distances: 50–200 nm with a homogeneous pillar distribution). The material reactivity was greatly influenced by the type of medium (nutrient-rich versus nutrient-free) and the presence or not of bacteria. A lower reactivity and superior bacterial attachment were observed in the nutrient-free medium while a lower attachment was observed for the nutrient rich medium which was explained by a superior reactivity of the material paired with the lower tendency of planktonic bacteria to adhere on surfaces in the presence of nutrients. Importantly, the ionic exchanges produced by the presence of materials were not toxic to planktonic cells. Thus, we can conclude that topography was the main contributor to mortality in the bacterial adhesion tests., Peer Reviewed, Postprint (published version)

Current methods for tattoo removal are long, costly and have drawbacks such as scarring among others. Looking for alternatives, here we assess the feasibility of non-thermal plasma (NTP) as a standalone method for the removal of tattoos. We report the results of atmospheric pressure plasma jet treatment of real tattoo inks suspensions in water and in gelatin (liquid or as a solid film), selected as a model of skin. Analysis of the residual color and the temperature effect in all the samples after plasma treatment reveals significant differences between water and the skin model. Kinetic considerations and the extension of our results to more realistic scenarios allow us to conclude that NTP cannot compete with the current laser technology in a real application., Peer Reviewed, Postprint (published version)

Concave surfaces have shown to promote bone regeneration in vivo. However, bone scaffolds obtained by direct ink writing, one of the most promising approaches for the fabrication of personalized bone grafts, consist mostly of convex surfaces, since they are obtained by microextrusion of cylindrical strands. By modifying the geometry of the nozzle, it is possible to print 3D structures composed of non-cylindrical strands and favor the presence of concave surfaces. In this work, we compare the in vivo performance of 3D-printed calcium phosphate scaffolds with either conventional cylindrical strands or star-shaped strands, in a rabbit femoral condyle model. Monocortical defects, drilled in contralateral positions, are randomly grafted with the two scaffold configurations, with identical composition. The samples are explanted eight weeks post-surgery and assessed by µ-CT and resin-embedded histological observations. The results reveal that the scaffolds containing star-shaped strands have better osteoconductive properties, guiding the newly formed bone faster towards the core of the scaffolds, and enhance bone regeneration, although the increase is not statistically significant (p > 0.05). This new approach represents a turning point towards the optimization of pore shape in 3D-printed bone grafts, further boosting the possibilities that direct ink writing technology offers for patient-specific applications., Peer Reviewed, Postprint (published version)

Passivation of titanium alloy dental meshes cleans their surface and forms a thin layer of protective oxide (TiO2) on the surface of the material to improve resistance to corrosion and prevent release of ions to the physiological environment. The most common chemical agent for the passivation process of titanium meshes is hydrochloric acid (HCl). In this work, we introduce the use of Piranha solution (H2SO4 and H2O2) as a passivating and bactericidal agent for metallic dental meshes. Meshes of grade 5 titanium alloy (Ti6Al4V) were tested after different treatments: as-received control (Ctr), passivated by HCl, and passivated by Piranha solution. Physical-chemical characterization of all treated surfaces was carried out by scanning electron microscopy (SEM), confocal microscopy and sessile drop goniometry to assess meshes’ topography, elemental composition, roughness, wettability and surface free energy, that is, relevant properties with potential effects for the biological response of the material. Moreover, open circuit potential and potentiodynamic tests were carried out to evaluate the corrosion behavior of the differently-treated meshes under physiological conditions. Ion release tests were conducted using Inductively Coupled Plasma mass spectrometry (ICP-MS). The antibacterial activity by prevention of bacterial adhesion tests on the meshes was performed for two different bacterial strains, Pseudomonas aeruginosa (Gram-) and Streptococcus sanguinis (Gram+). Additionally, a bacterial viability study was performed with the LIVE/DEAD test. We complemented the antibacterial study by counting cells attached to the surface of the meshes visualized by SEM. Our results showed that the passivation of titanium meshes with Piranha solution improved their hydrophilicity and conferred a notably higher bactericidal activity in comparison with the meshes passivated with HCl. This unique response can be attributed to differences in the obtained nanotextures of the TiO2 layer., Peer Reviewed, Postprint (published version)

Data associated with the study called "Selectively targeting tumorigenicity in osteosarcoma". Each file corresponds to the raw data of one of the figures of the paper, 1

Cold atmospheric plasma (CAP) is a potential anticancer therapy. CAP has cytotoxic effects when applied either directly to cancer cell cultures or indirectly through plasma-conditioned liquids. This protocol describes how to treat adherent cultures of human cancer cell lines with CAP or plasma-conditioned medium and determine cell viability following treatment. The protocol also includes details on how to quantify the reactive oxygen and nitrogen species present in medium following CAP treatment, using chemical probes using UV-visible or fluorescence spectroscopy. CAP treatment takes ~30 min, and 3 h are required to complete quantification of reactive oxygen and nitrogen species. By providing a standardized protocol for evaluation of the effects of CAP and plasma-conditioned medium, we hope to facilitate the comparison and interpretation of results seen across different laboratories., Peer Reviewed, Postprint (author's final draft)

The present experimental trial uses two types of dental implants, one made of titanium (Ti6Al4V) and the other one of zirconia (ZrO2), but both of identical design, to compare their stability and micro-movements values under load. One of each type of implant (n = 42) was placed into 21 cow ribs, recording the insertion torque and the resonance frequency using a specific transducer. Subsequently, a prosthetic crown made of PMMA was screwed onto each of the implants in the sample. They were then subjected to a static compression load on the vestibular cusp of the crown. The resulting micromovements were measured. The zirconia implants obtained a higher mean of both IT and RFA when compared with those of titanium, with statistically significant differences in both cases (p = 0.0483 and p = 0.0296). However, the micromovement values when load was applied were very similar for both types, with the differences between them (p = 0.3867) not found to be statistically significant. The results show that zirconia implants have higher implant stability values than titanium implants. However, the fact that there are no differences in micromobility values implies that caution should be exercised when applying clinical protocols for zirconia based on RFA, which only has evidence for titanium., Peer Reviewed, Postprint (published version)

The performance and properties of Titanium (Ti) and its alloys can be fully exploited for biomedical applications such as orthopedic and dental implants, when porous structures are used. Such Ti structures can matched the bone mechanical properties, providing a solution to the stress-shielding effect problem and facilitating implant biointegration. Powder metallurgy (PM) combined with space holder (SH) technology is an appealing process to produce porous implants. This article briefly discusses the suitability of the PM with SH technology to produce porous structures from the perspectives of both design and manufacture for the production of medical devices., Peer Reviewed, Postprint (published version)

One possibility to prevent prosthetic infections is to produce biomaterials resistant to bacterial colonization by anchoring membrane active antimicrobial peptides (AMPs) onto the implant surface. In this perspective, a deeper understanding of the mode of action of the immobilized peptides should improve the development of AMP-inspired infection-resistant biomaterials. The aim of the present study was to characterize the bactericidal mechanism against Staphylococcus epidermidis of the AMP BMAP27(1–18), immobilized on titanium disks and on a model resin support, by applying viability counts, Field Emission Scanning Electron Microscopy (FE-SEM), and a fluorescence microplate assay with a membrane potential-sensitive dye. The cytocompatibility to osteoblast-like MG-63 cells was investigated in monoculture and in co-culture with bacteria. The impact of peptide orientation was explored by using N- and C- anchored analogues. On titanium, the ~50 % drop in bacteria viability and dramatically affected morphology indicate a contact-killing action exerted by the N- and C-immobilized peptides to the same extent. As further shown by the fluorescence assay with the resin-anchored peptides, the bactericidal effect was mediated by rapid membrane perturbation, similar to free peptides. However, at peptide MBC resin equivalents the C-oriented analogue proved more effective with more than 99 % killing and maximum fluorescence increase, compared to half-maximum fluorescence with more than 90 % killing produced by the N-orientation. Confocal microscopy analyses revealed 4–5 times better MG-63 cell adhesion on peptide-functionalized titanium both in monoculture and in co-culture with bacteria, regardless of peptide orientation, thus stimulating further studies on the effects of the immobilized BMAP27(1–18) on osteoblast cells., The authors acknowledge the financial support of departmental research funds (Department of Medicine, University of Udine, Italy). The authors also gratefully thank the Generalitat de Catalunya for funding through project 2017SGR-1165 and the Ministry of Science and Innovation, Spain, for financial support through the RTI2018-098075-B-C21 project, co-funded by the EU through the European Regional Development Funds., Peer Reviewed, Postprint (author's final draft)

Hydrothermal (H) processes accelerate the hydrolysis reaction of a-tricalcium phosphate (a-TCP) compared to the long-established biomimetic (B) treatments. They are of special interest for patient-specific 3D-printed bone graft substitutes, where the manufacturing time represents a critical constraint. Altering the reaction conditions has implications for the physicochemical properties of the reaction product. However, the impact of the changes produced by the hydrothermal reaction on the in vivo performance was hitherto unknown. The present study compares the bone regeneration potential of 3D-printed a-TCP scaffolds hardened using these two treatments in rabbit condyle monocortical defects. Although both consolidation processes resulted in biocompatible scaffolds with osseointegrative and osteoconductive properties, the amount of newly formed bone increased by one third in the hydrothermal vs the biomimetic samples. B and H scaffolds consisted mostly of high specific surface area calcium-deficient hydroxyapatite (38 and 27 m2 g-1, respectively), with H samples containing also 10 wt.% ß-tricalcium phosphate (ß-TCP). The shrinkage produced during the consolidation process was shown to be very small in both cases, below 3%, and smaller for H than for B samples. The differences in the in vivo performance were mainly attributed to the distinct crystallisation nanostructures, which proved to have a major impact on permeability and protein adsorption capacity, using BSA as a model protein, with B samples being highly impermeable. Given the crucial role that soluble proteins play in osteogenesis, this is proposed to be a relevant factor behind the distinct in vivo performances observed for the two materials. Statement of significance The possibility to accelerate the consolidation of self-setting calcium phosphate inks through hydrothermal treatments has aroused great interest due to the associated advantages for the development of 3D-printed personalised bone scaffolds. U, Peer Reviewed, Postprint (published version)

A dual approach employing peptidic biofunctionalization and laser micro-patterns on dental zirconia was explored, with the aim of providing a flexible tool to improve tissue integration of restorations. Direct laser interference patterning with a femtosecond Ti:Sapphire laser was employed, and two periodic grooved patterns were produced with a periodicity of 3 and 10 µm. A platform containing the cell-adhesive RGD and the osteo- genic DWIVA peptides was used to functionalize the grooved surfaces. Topography and surface damage were characterized by confocal laser scanning (CLSM), scanning electron and scanning transmission electron micro- scopy techniques. The surface patterns exhibited a high homogeneity and subsurface damage was found in the form of nano-cracks and nano-pores, at the bottom of the valleys. Accelerated tests in water steam were carried out to assess hydrothermal degradation resistance, which slightly decreased after the laser treatment. Interest- ingly, the detrimental effects of the laser modification were reverted by a post-laser thermal treatment. The attachment of the molecule was verified trough fluorescence CLSM and X-ray photoelectron spectroscopy. Finally, the biological properties of the surfaces were studied in human mesenchymal stem cells. Cell adhesion, morphology, migration and differentiation were investigated. Cells on grooved surfaces displayed an elongated morphology and aligned along the patterns. On these surfaces, migration was greatly enhanced along the grooves, but also highly restricted in the perpendicular direction as compared to flat specimens. After bio- functionalization, cell number and cell area increased and well-developed cell cytoskeletons were observed. However, no effects on cell migration were found for the peptidic platform. Although some osteogenic potential was found in specimens grooved with a periodicity of 10 µm, the largest effects were observed from the biomolecule, which favored upregulation of several ge, Peer Reviewed, Postprint (author's final draft)

Osteosarcoma is the most common primary bone tumor, and its first line of treatment presents a high failure rate. The 5-year survival for children and teenagers with osteosarcoma is 70% (if diagnosed before it has metastasized) or 20% (if spread at the time of diagnosis), stressing the need for novel therapies. Recently, cold atmospheric plasmas (ionized gases consisting of UV–Vis radiation, electromagnetic fields and a great variety of reactive species) and plasma-treated liquids have been shown to have the potential to selectively eliminate cancer cells in different tumors through an oxidative stress-dependent mechanism. In this work, we review the current state of the art in cold plasma therapy for osteosarcoma. Specifically, we emphasize the mechanisms unveiled thus far regarding the action of plasmas on osteosarcoma. Finally, we review current and potential future approaches, emphasizing the most critical challenges for the development of osteosarcoma therapies based on this emerging technique., Peer Reviewed, Postprint (published version)

One of the major problems faced by metallic implants is the high probability of bacterial infections, with significant consequences for the patient. In this work, a thermochemical treatment is proposed to obtain silver-doped calcium titanate coatings on the Ti surface to improve the bioactivity of porous 3D-printed Ti structures and simultaneously provide them with antibacterial properties. A complete characterization of the new coating, the study of the ion release and the analysis of its cytotoxicity were carried out together with evaluation of the natural apatite forming in simulated body fluid (SBF). Moreover, the antibacterial properties of the coatings were assessed against Pseudomona aeruginosa and Escherichia coli as gram-negative and Staphylococcus aureus and Staphylococcus epidermidis as gram-positive bacterial strains. Ag ions were integrated into the Ca titanate layer and Ag nanoparticles were formed within the entire 3D Ti surface. Ca and Ag ions were released from both porous and solid samples into the Hanks' solution for 48 h. The treated surfaces showed no cytotoxicity and an apatite layer precipitated on the entire porous surface when the samples were immersed in SBF. The release of Ag from the surface had a strong antibacterial effect and prevented bacterial adhesion and proliferation on the surface. Moreover, the nanostructured topography of the coating resulted also in a reduction of bacterial adhesion and proliferation, even in absence of Ag. In conclusion, the cost-effective approach here reported provided protection against the most predominant bacterial colonizers to the Ti porous implants, while maintaining their bioactivity., Postprint (published version)

Atmospheric pressure plasma jets have been shown to impact several cancer cell lines, both in vitro and in vivo. These effects are based on the biochemistry of the reactive oxygen and nitrogen species generated by plasmas in physiological liquids, referred to as plasma-conditioned liquids. Plasma-conditioned media are efficient in the generation of reactive species, inducing selective cancer cell death. However, the concentration of reactive species generated by plasma in the cell culture media of different cell types can be highly variable, complicating the ability to draw precise conclusions due to the differential sensitivity of different cells to reactive species. Here, we compared the effects of direct and indirect plasma treatment on non-malignant bone cells (hOBs and hMSCs) and bone cancer cells (SaOs-2s and MG63s) by treating the cells directly or exposing them to previously treated cell culture medium. Biological effects were correlated with the concentrations of reactive species generated in the liquid. A linear increase in reactive species in the cell culture medium was observed with increased plasma treatment time independent of the volume treated. Values up to 700 µM for H2O2 and 140 µM of NO2- were attained in 2 mL after 15 min of plasma treatment in AdvDMEM cell culture media. Selectivity towards bone cancer cells was observed after both direct and indirect plasma treatments, leading to a decrease in bone cancer cell viability at 72 h to 30% for the longest plasma treatment times while maintaining the survival of non-malignant cells. Therefore, plasma-conditioned media may represent the basis for a potentially novel non-invasive technique for bone cancer therapy., Peer Reviewed, Postprint (published version)

One of the biggest challenges in the biocompatibility of implantable metals is the prevention of the stress shielding effect, which is related to the coupling of the bone-metal mechanical properties. This stress shielding phenomenon provokes bone resorption and the consequent adverse effects on prosthesis fixation. However, it can be inhibited by adapting the stiffness of the implant material. Since the use of titanium (Ti) porous structures is a great alternative not only to inhibit this effect but also to improve the osteointegration of orthopedic and dental implants, a brief description of the techniques used for their manufacturing and a review of the current commercialized implants produced from porous Ti assemblies are compiled in this work. As powder metallurgy (PM) with space holder (SH) is a powerful technology used to produce porous Ti structures, it is here discussed its potential for the fabrication of medical devices from the perspectives of both design and manufacture. The most important parameters of the technique such as the size and shape of the initial metallic particles, the SH and binder type of materials, the compaction pressure of the green form, and in the sintering stage, the temperature, atmosphere, and time are reviewed according to the bibliography reported. Furthermore, the importance of the porosity and its types together with the influence of the mentioned parameters in the final porosity and, consequently, in the ultimate mechanical properties of the structure are discussed. Finally, a few examples of the PM-SH application for the manufacturing of orthopedic implants are presented., Peer Reviewed, Postprint (author's final draft)

Improving cell-material interactions is a major goal in tissue engineering. In thisregard, functionalization of biomaterials with cell instructive molecules from theextracellular matrix stands out as a powerful strategy to enhance their bioactivity andachieve optimal tissue integration. However, current functionalization strategies, likethe use of native full-length proteins, are associated with drawbacks, thus urging theneed of developing new methodologies. In this regard, the use of synthetic peptidesencompassing specific bioactive regions of proteins represents a promising alterna-tive. In particular, the combination of peptide sequences with complementary or syn-ergistic effects makes it possible to address more than one biological target at thebiomaterial surface. In this review, an overview of the main strategies using peptidesto install multifunctionality on biomaterials is presented, mostly focusing on the com-bination of the RGD motif with other peptides sequences. The evolution of theseapproaches, starting from simple methods, like using peptide mixtures, to moreadvanced systems of peptide presentation, with very well defined chemical proper-ties, are explained. For each system of peptide's presentation, three main aspects ofmultifunctionality—improving receptor selectivity, mimicking the extracellular matrixand preventing bacterial colonization while improving cell adhesion—are highlighted., Peer Reviewed, Postprint (published version)

Surface topography and physical-chemical properties usually play a key-role in both osseointegration improvement and bacterial colonization reduction over the surface of dental implants. The aim of this study is to compare the chemical and bacteriological behavior of two different acid passivation surface treatments on titanium c.p. grade 3 used for dental implant manufacturing. Surface roughness was evaluated using White Light Interferometry (WLI) in order to determine different roughness parameters such as average roughness (Sa), the spacing parameter (Sm) and the hybrid parameter of surface index area (SIA). Contact angle (CA) and surface free energy (SFE) were evaluated in order to establish the surface wettability of the different groups of samples. Titanium ion-release from the different samples was also been analyzed in Hank’s solution medium at 37 °C by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) at different immersion times. Bacterial viability adhesion assays were done using S. sanguinis (CECT 480, Spain) as a bacterial strain model of primary colonizer in oral biofilm formation. The bacteria attachment and morphology on Ti surfaces were determined using a live/dead staining method after 4 h of incubation and further analyzed by scanning electron microscope (SEM). Acid passivation surface treatments produced a statistically-significant (p < 0.05) roughness increase in all the evaluated parameters (Sa, Sm, SAI). The treatment with citric acid decreased the static contact angle (CA) and caused an increase in surface free energy (SFE) with a high polarization and oxidizing character. These physical-chemical surface characteristics obtained by means of citric acid passivation caused the bactericidal behavior as it has been proved in bacterial studies., Peer Reviewed, Postprint (published version)

Conventional direct ink writing uses circular nozzles and, therefore, results in cylindrical strands. 3D printing with non-circular nozzles adds new degrees of freedom to this versatile technology, and allows obtaining structures with higher specific surface area or even introducing concave surfaces in the printed architecture. This is an enticing prospect for countless applications, including tissue engineering, chemical reaction catalysts, water evaporators and electrochemical energy storage devices. Despite this, it has been hardly explored by the 3D-printing community. Herein, we develop for the first time 3D printed structures with complex filament section morphologies using a custom-made modular nozzle and a self-setting ceramic ink. The fast elastic recovery of the ink allows obtaining good shape fidelity in the printed filaments, permitting the creation of intricate surfaces with up to 30% concavity and increasing up to 2.5 times the specific surface area compared to cylindrical strands. The use of non-circular nozzles introduces some specific constraints in the printing process. The geometry of the nozzle determines the stable printing directions, and nozzle orientation becomes a critical parameter to achieve a stable printing. Strand torsion, a phenomenon that remains unnoticed with circular nozzles, may result in relevant changes in the geometrical features of the printed structures., Peer Reviewed, Postprint (published version)

HO radicals are the most important reactive species generated during water treatment by non-thermal plasma devices. In this letter, we report the first quantification of the steady-state concentration and lifetime of plasma-produced hydroxyl radicals in water solutions at pH 3 and 7, and we discuss the differences based on their reactivity with other plasma-generated species. Finally, we show to what extent the use of chemical probes to quantify short-lived reactive species has an influence on the results and that it should be taken into account., Peer Reviewed, Postprint (published version)

Actual polymer bioresorbable stents (BRS) generate a risk of device thrombosis as a consequence of the incomplete endothelialization after stent implantation. The material-tissue interactions are not fully controlled and stent fabrication techniques do not allow personalized medical solutions. This work investigates the effect of different functionalization strategies onto solvent-cast poly(l-lactic acid) (PLLA) surfaces with the capacity to enhance surface endothelial adhesion and the fabrication of 3D printed BRS. PLLA films were obtained by solvent casting and treated thermally to increase mechanical properties. Surface functionalization was performed by oxygen plasma (OP), sodium hydroxide (SH) etching, or cutinase enzyme (ET) hydrolysis, generating hydroxyl and carboxyl groups. A higher amount of carboxyl and hydroxyl groups was determined on OP and ET compared to the SH surfaces, as determined by contact angle and X-ray photoelectron spectroscopy (XPS). Endothelial cells (ECs) adhesion and spreading was higher on OP and ET functionalized surfaces correlated with the increase of functional groups without affecting the degradation. To verify the feasibility of the approach proposed, 3D printed PLLA BRS stents were produced by the solvent-cast direct writing technique, Peer Reviewed, Postprint (published version)

Electrochemistry, although an ancient field of knowledge, has become of paramount importance in the synthesis of materials at the nanoscale, with great interest not only for fundamental research but also for practical applications. One of the promising fields in which electrochemistry meets nanoscience and nanotechnology is micro/nanoscale motors. Micro/nano motors, which are devices able to perform complex tasks at the nanoscale, are commonly multifunctional nanostructures of different materials - metals, polymers, oxides- and shapes -spheres, wires, helices- with the ability to be propelled in fluids. Here, we first introduce the topic of micro/nanomotors and make a concise review of the field up to day. We have analyzed the field from different points of view (e.g. materials science and nanotechnology, physics, chemistry, engineering, biology or environmental science) to have a broader view of how the different disciplines have contributed to such exciting and impactful topic. After that, we focus our attention on describing what electrochemical technology is and how it can be successfully used to fabricate and characterize micro/nanostructures composed of different materials and showing complex shapes. Finally, we will review the micro and nanomotors fabricated using electrochemical techniques with applications in biomedicine and environmental remediation, the two main applications investigated so far in this field. Thus, different strategies have thus been shown capable of producing core-shell nanomaterials combining the properties of different materials, multisegmented nanostructures made of, for example, alternating metal and polymer segments to confer them with flexibility or helicoidal systems to favor propulsion. Moreover, further functionalization and interaction with other materials to form hybrid and more complex objects is also shown., Peer Reviewed, Postprint (published version)

Myocardial infarction is caused by an interruption of coronary blood flow, leading to one of the main death causes worldwide. Current therapeutic approaches are palliative and not able to solve the loss of cardiac tissue. Cardiosphere derived cells (CDCs) reduce scarring, and increase viable myocardium, with safety and adequate biodistribution, but show a low rate engraftment and survival after implantation. In order to solve the low retention, we propose the encapsulation of CDCs within three-dimensional alginate-poly-L-lysine-alginate matrix as therapy for cardiac regeneration. In this work, we demonstrate the encapsulation of CDCs in alginate matrix, with no decrease in viability over a month, and showing the preservation of CDCs phenotype, differentiation potential, gene expression profile and growth factor release after encapsulation, moving a step forward to clinical translation of CDCs therapy in regeneration in heart failure., Peer Reviewed, Postprint (published version)

One limitation of wearable electronics, and at the same time a challenge, is the lack of energy storage devices with multiple functionalities produced using clean and environmental-friendly strategies. Here, a multifunctional conductive hydrogel containing poly(3,4-ethylenedioxythiophene) (PEDOT) and alginate was fabricated, to be used as electrodes in supercapacitors, by applying water-mediated self-assembly and polymerization processes at room temperature. The interpenetration of both polymers allowed combining flexibility and self-healing properties within the same hydrogel together with the intrinsic biocompatibility and sustainability of such materials. Initially, PEDOT : polystyrene sulfonate and alginate aqueous solutions were mixed in two different proportions (1:1 and 1:3) and ionically cross-linked with CaCl2. Subsequently, re-interpenetration of poly(hydroxymethyl-3,4-ethylenedioxythiophene) (PHMeDOT) by anodic polymerization in CaCl2 aqueous solution was achieved. Re-interpenetrated 1:3 PEDOT/alginate hydrogels showed excellent capacitance values (35 mF/cm2) and good capacitance retention. On the other hand, the electrochemical properties were not significantly changed after many cutting/self-healing cycles as was observed by cyclic voltammetry. Therefore, this sustainably produced hydrogel shows promising properties as wearable energy storage devices., Postprint (author's final draft)

Dental implants can fail due to various factors, in which bad tissue integration is believed to have a significant role. Specific properties of the implant surface, such as its chemistry and roughness, are of paramount importance to address specific cell responses, such as the adsorption of proteins, as well as the adhesion and differentiation of cells, which are suitable for biomaterial and tissue engineering. In this study, an acrylate-containing coating was produced on titanium surfaces through the atmospheric pressure plasma treatment of a liquid precursor, 2-hydroxyethyl methacrylate. A hydrophilic coating was obtained, showing retention of the monomer chemistry as assessed by FTIR analysis and XPS. Enhanced fibroblast adhesion and decreased Staphylococcus aureus and Escherichia coli adhesion were recorded, showing that this is a suitable method to produce biocompatible coatings with a reduced bacterial adhesion., Peer Reviewed, Postprint (published version)

The use of oxidative stress generated by Cold Atmospheric Plasma (CAP) in oncology is being recently studied as a novel potential anti-cancer therapy. However, the beneficial effects of CAP for treating osteosarcoma have mostly been demonstrated in 2-dimensional cultures of cells, which do not mimic the complexity of the 3-dimensional (3D) bone microenvironment. In order to evaluate the effects of CAP in a relevant context of the human disease, we developed a 3D tissue-engineered model of osteosarcoma using a bone-like scaffold made of collagen type I and hydroxyapatite nanoparticles. Human osteosarcoma cells cultured within the scaffold showed a high capacity to infiltrate and proliferate and to exhibit osteomimicry in vitro. As expected, we observed significantly different functional behaviors between monolayer and 3D cultures when treated with Cold Plasma-Activated Ringer's Solution (PAR). Our data reveal that the 3D environment not only protects cells from PAR-induced lethality by scavenging and diminishing the amount of reactive oxygen and nitrogen species generated by CAP, but also favours the stemness phenotype of osteosarcoma cells. This is the first study that demonstrates the negative effect of PAR on cancer stem-like cell subpopulations in a 3D biomimetic model of cancer. These findings will allow to suitably re-focus research on plasma-based therapies in future., Peer Reviewed, Postprint (published version)

Implants for orthopedic applications need to be biocompatible and bioactive, with mechanical properties similar to those of surrounding natural bone. Given this scenario titanium (Ti) scaffolds obtained by Direct Ink Writing technique offer the opportunity to manufacture customized structures with controlled porosity and mechanical properties. Considering that 3D Ti scaffolds have a significant surface area, it is necessary to develop strategies against the initial bacterial adhesion in order to prevent infection in the early stages of the implantation, while promoting cell adhesion to the scaffold. The challenge is not only achieving a balance between antibacterial activity and osseointegration, it is also to develop a homogeneous coating on the inner and outer surface of the scaffold. The purpose of this work was the development of a single-step electrodeposition process in order to uniformly cover Ti scaffolds with a layer of calcium phosphate (CaP) loaded with chlorhexidine digluconate (CHX). Scaffold characterization was assessed by scanning electron microscopy, Energy dispersive X-ray spectroscopy, X-ray diffraction, micro-Raman microscopy and compressive strength tests. Results determined that the surface of scaffolds was covered by plate-like and whisker-like calcium phosphate crystals, which main phases were octacalcium phosphate and brushite. Biological tests showed that the as-coated scaffolds reduced bacteria adhesion (73 ± 3% for Staphylococcus aureus and 70 ± 2% for Escherichia coli). In vitro cell studies and confocal analysis revealed the adhesion and spreading of osteoblast-like SaOS-2 on coated surfaces. Therefore, the proposed strategy can be a potential candidate in bone replacing surgeries., Peer Reviewed, Postprint (author's final draft)

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (ß-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic ß-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of ß-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to ß-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significance Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered ß-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA, Peer Reviewed, Postprint (author's final draft)

Bioresorbable stents (BRS) potential in treating coronary heart disease is still to be further developed. Current trends include research with new polymeric materials, the need for thinner struts combined with appropriate mechanical properties, radiopacity and optimized local drug delivery. This work presents a novel solvent-cast direct-write (SC-DW) printing system to manufacture BRS onto a rotating cylinder with poly-l-lactic acid (PLLA) and poly(l-lactic-co- ¿ -caprolactone) (PLCL) inks. Printed stents were characterized in terms of mechanical, thermal and biological properties with human umbilical vein endothelial cells (HUVECs). Expansion assays showed that stents withstood pressures of at least 16 atm and the indirect cytotoxicity test indicated that stents were biocompatible. Polymeric inks were further modified with the addition of 3 radiopaque agents, namely iodine, triiodobenzoic acid (TIBA) and barium sulfate (BaSO) to render stents radiopaque. Subsequent characterization showed a general increase in strut thickness with respect to control PLLA or PLCL stents, which in turn resulted in higher resistance to compression. Microcomputed tomography was used to assess stents’ radiopacity, showing that TIBA and BaSO-containing stents presented high X-ray attenuation values and maintained their radiopacity after 3 months incubation time., Peer Reviewed, Postprint (published version)

A conducting nanocomposite hydrogel is developed for the detection of L-lactate. The hydrogel is based on a mixture of alginate (Alg) and poly(3,4-ethylenedioxythiophene) (PEDOT), which is loaded with gold nanoparticles (GNP). In this novel hydrogel, Alg provides 3D structural support and flexibility, PEDOT confers conductivity and sensing capacity, and GNP provides signal amplification with respect to simple voltammetric and chronoamperometric response. The synergistic combination of the properties provided by each component results in a new flexible nanocomposite with outstanding capacity to detect hydrogen peroxide, which has been used to detect the oxidation of L-lactate. The hydrogel detects hydrogen peroxide with linear response and limits of detection of 0.91 µM and 0.02 µM by cyclic voltammetry and chronoamperometry, respectively. The hydrogel is functionalized with lactate oxidase, which catalyzes the oxidation of L-lactate to pyruvate, forming hydrogen peroxide. For L-lactate detection, the functionalized biosensor works in two linear regimes, one for concentrations lower than 5 mM with a limit of detection of 0.4 mM, and the other for concentrations up to 100 mM with a limit of detection of 3.5 mM. Because of its linear range interval, the developed biosensor could be suitable for a wide number of biological fluids., Peer Reviewed, Postprint (author's final draft)

Diamond-like carbon (DLC) coatings have been extensively studied over the last two decades in the field of materials engineering. The addition of effective bactericidal agents like silver suggests the potential application of silver-doped DLC coatings in the biomedical sector. In this paper, DLC coatings containing about 2 at. % Ag were grown on CoCrMo substrates by pulsed filtered cathodic arc co-deposition using pure graphite and silver targets. Morphology, structure and chemical composition of the coatings were characterized by SEM, XPS, XRD, Raman and HRTEM. Wettability and surface free energy were also measured. Mechanical properties were analyzed by means of nanoindentation and Rockwell-C adhesion tests. Corrosion resistance of coated and uncoated samples was determined by potentio-dynamic polarization tests in Hank's solution. Cytotoxicity and antibacterial activity against S. aureus and P. aeruginosa were also studied. For comparative purposes, nitrogen-doped DLC coatings obtained by RF magnetron sputtering were also prepared and characterized. Well-adhered and corrosion resistant Ag-DLC nanostructured coatings of up to 1.5 µm thick and 21 GPa in hardness were obtained. The doping concentration of silver studied in this work seems to be optimal for the preparation of non-cytotoxic coatings with a significant bactericidal activity., This work has been financed by the Ministry of the Economy, Industry and Competitiveness of Spain under the project BIOPLASMA (MAT2015-67103-C4)., Peer Reviewed, Preprint

In the present study, amorphous tricalcium phosphate (TCP) has been synthetized by a wet route to obtain low temperature a-TCP at 650 °C (LT-aTCP) and compare its structural, physical–chemical and thermal properties with those of a-TCP obtained by the conventional solid-state reaction method at 1400 °C (HT-aTCP). Even if no significant differences were observed concerning the values of lattice parameters measured by Rietveld refinement, LT-aTCP presented lower crystallinity and higher crystal strains than HT-aTCP. The reactivity in water of the a-TCP obtained by the two different routes was assessed. Both raw samples appeared relatively inert in solution and did not favour the nucleation of calcium deficient apatite (CDA); the LT-aTCP and HT-aTCP were converted into apatite only after milling. The mechanical process leads to a decrease in crystallinity and the formation of an amorphous phase, which is supported in this work by Raman spectroscopy. The faster rate of conversion of milled LT-aTCP compared to HT-aTCP can be assigned to its higher specific surface area, lower crystallinity and higher residual crystal strain; these favour the dissolution of the a-TCP phase. Finally, the setting properties of a-TCP-based bone cements were compared regarding their synthesis route. Although the synthesis route does not significantly affect the setting times, the kinetic of conversion into CDA was faster for LT-aTCP than for HT-aTCP. Thus, the modulation of the dissolution rate of a-TCP-based cement determined by the preparation route and the grinding process allows control of the overall setting reaction., Peer Reviewed, Postprint (author's final draft)

Porosity plays a key role on the osteogenic performance of bone scaffolds. Direct Ink Writing (DIW) allows the design of customized synthetic bone grafts with patient-specific architecture and controlled macroporosity. Being an extrusion-based technique, the scaffolds obtained are formed by arrays of cylindrical filaments, and therefore have convex surfaces. This may represent a serious limitation, as the role of surface curvature and more specifically the stimulating role of concave surfaces in osteoinduction and bone growth has been recently highlighted. Hence the need to design strategies that allow the introduction of concave pores in DIW scaffolds. In the current study, we propose to add gelatin microspheres as a sacrificial material in a self-setting calcium phosphate ink. Neither the phase transformation responsible for the hardening of the scaffold nor the formation of characteristic network of needle-like hydroxyapatite crystals was affected by the addition of gelatin microspheres. The partial dissolution of the gelatin resulted in the creation of spherical pores throughout the filaments and exposed on the surface, increasing filament porosity from 0.2 % to 67.9 %. Moreover, the presence of retained gelatin proved to have a significant effect on the mechanical properties, reducing the strength but simultaneously giving the scaffolds an elastic behavior, despite the high content of ceramic as a continuous phase. Notwithstanding the inherent difficulty of in vitro cultures with this highly reactive material an enhancement of MG-63 cell proliferation, as well as better spreading of hMSCs was recorded on the developed scaffolds., Peer Reviewed, Postprint (published version)

In the recent decades, zinc (Zn) and its alloys have been drawing attention as promising candidates for bioresorbable cardiovascular stents due to its degradation rate more suitable than magnesium (Mg) and iron (Fe) alloys. However, its mechanical properties need to be improved in order to meet the criteria for vascular stents. This work investigates the mechanical properties, biodegradability and biocompatibility of Zn-Mg and Zn-Cu alloys in order to determine a proper alloy composition for optimal stent performance. Nanoindentation measurements are performed to characterize the mechanical properties at the nanoscale as a function of the Zn microstructure variations induced by alloying. The biodegradation mechanisms are discussed and correlated to microstructure, mechanical performance and bacterial/cell response. Addition of Mg or Cu alloying elements refined the microstructure of Zn and enhanced yield strength (YS) and ultimate tensile strength (UTS) proportional to the volume fraction of secondary phases. Zn-1Mg showed the higher YS and UTS and better performance in terms of degradation stability in Hanks’ solution. Zn-Cu alloys presented an antibacterial effect for S. aureus controlled by diffusion mechanisms and by contact. Biocompatibility was dependent on the degradation rate and the nature of the corrosion products., Peer Reviewed, Objectius de Desenvolupament Sostenible::3 - Salut i Benestar, Postprint (published version)

The surface modification by the formation of apatitic compounds, such as hydroxyapatite, improves biological fixation implants at an early stage after implantation. The structure, which is identical to mineral content of human bone, has the potential to be osteoinductive and/or osteoconductive materials. These calcium phosphates provoke the action of the cell signals that interact with the surface after implantation in order to quickly regenerate bone in contact with dental implants with mineral coating. A new generation of calcium phosphate coatings applied on the titanium surfaces of dental implants using laser, plasma-sprayed, laser-ablation, or electrochemical deposition processes produces that response. However, these modifications produce failures and bad responses in long-term behavior. Calcium phosphates films result in heterogeneous degradation due to the lack of crystallinity of the phosphates with a fast dissolution; conversely, the film presents cracks, which produce fractures in the coating. New thermochemical treatments have been developed to obtain biomimetic surfaces with calcium phosphate compounds that overcome the aforementioned problems. Among them, the chemical modification using biomineralization treatments has been extended to other materials, including composites, bioceramics, biopolymers, peptides, organic molecules, and other metallic materials, showing the potential for growing a calcium phosphate layer under biomimetic conditions., Peer Reviewed, Postprint (published version)

Sarcomas comprise a group of aggressive malignancies with very little treatment options beyond standard chemotherapy. Reposition of approved drugs represents an attractive approach to identify effective therapeutic compounds. One example is mithramycin (MTM), a natural antibiotic which has demonstrated a strong antitumour activity in several tumour types, including sarcomas. However, its widespread use in the clinic was limited by its poor toxicity profile. Results In order to improve the therapeutic index of MTM, we have loaded MTM into newly developed nanocarrier formulations. First, polylactide (PLA) polymeric nanoparticles (NPs) were generated by nanoprecipitation. Also, liposomes (LIP) were prepared by ethanol injection and evaporation solvent method. Finally, MTM-loaded hydrogels (HG) were obtained by passive loading using a urea derivative non-peptidic hydrogelator. MTM-loaded NPs and LIP display optimal hydrodynamic radii between 80 and 105 nm with a very low polydispersity index (PdI) and encapsulation efficiencies (EE) of 92 and 30%, respectively. All formulations show a high stability and different release rates ranging from a fast release in HG (100% after 30 min) to more sustained release from NPs (100% after 24 h) and LIP (40% after 48 h). In vitro assays confirmed that all assayed MTM formulations retain the cytotoxic, anti-invasive and anti-stemness potential of free MTM in models of myxoid liposarcoma, undifferentiated pleomorphic sarcoma and chondrosarcoma. In addition, whole genome transcriptomic analysis evidenced the ability of MTM, both free and encapsulated, to act as a multi-repressor of several tumour-promoting pathways at once. Importantly, the treatment of mice bearing sarcoma xenografts showed that encapsulated MTM exhibited enhanced therapeutic effects and was better tolerated than free MTM. Conclusions Overall, these novel formulations may represent an efficient and safer MTM-delivering alternative for sarcoma treatment., Peer Reviewed, Postprint (published version)

Multifunctional hydrogels are a class of materials offering new opportunities for interfacing living organisms with machines due to their mechanical compliance, biocompatibility, and capacity to be triggered by external stimuli. Here, we report a dual magnetic- and electric-stimuli-responsive hydrogel with the capacity to be disassembled and reassembled up to three times through reversible cross-links. This allows its use as an electronic device (e.g., temperature sensor) in the cross-linked state and spatiotemporal control through narrow channels in the disassembled state via the application of magnetic fields, followed by reassembly. The hydrogel consists of an interpenetrated polymer network of alginate (Alg) and poly(3,4-ethylenedioxythiophene) (PEDOT), which imparts mechanical and electrical properties, respectively. In addition, the incorporation of magnetite nanoparticles (Fe3O4 NPs) endows the hydrogel with magnetic properties. After structural, (electro)chemical, and physical characterization, we successfully performed dynamic and continuous transport of the hydrogel through disassembly, transporting the polymer–Fe3O4 NP aggregates toward a target using magnetic fields and its final reassembly to recover the multifunctional hydrogel in the cross-linked state. We also successfully tested the PEDOT/Alg/Fe3O4 NP hydrogel for temperature sensing and magnetic hyperthermia after various disassembly/re-cross-linking cycles. The present methodology can pave the way to a new generation of soft electronic devices with the capacity to be remotely transported., Peer Reviewed, Postprint (author's final draft)

In order to achieve the required properties of titanium implants, more resources and research are needed to turn into reality the dream of developing the perfect implant material. The objective of this study was to evaluate the viability of the Laser Directed Energy Deposition to produce biomedical Ti-Nb and Ti-Zr-Nb alloys from elemental powders (Ti, Nb and Zr). The Laser Directed Energy Deposition is an additive manufacturing process used to build a component by delivering energy and material simultaneously. The material is supplied in the form of particles or wire and a laser beam is employed to melt material that is selectively deposited on a specified surface, where it solidifies. Samples with different compositions are characterized to analyze their morphology, microstructure, constituent phases, mechanical properties, corrosion resistance and cytocompatibility. Laser-deposited Ti-Nb and Ti-Zr-Nb alloys show no relevant defects, such as pores or cracks. Titanium alloys with lower elastic modulus and a significantly higher hardness than Ti grade 2 were generated, therefore a better wear resistance could be expected from them. Moreover, their corrosion resistance is excellent due to the formation of a stable passive protective oxide film on the surface of the material; in addition, they also possess outstanding cytocompatibility., Peer Reviewed, Postprint (published version)

Coronene and corannulene are popular structures that have provided the basis for the investigation of extended carbon-based structures, like graphene, fullerene and their derivatives. Here we address the Jahn-Teller (JT) effects in electron paramagnetic resonance (EPR) spectroscopy of such molecules in ionic form. By density func- tional theory (DFT) and a Monte Carlo based sampling of the potential energy surface, we obtain three and five symmetry-related JT equivalent conformers in coronene and in corannulene ions, respectively. The structure and the interconversion between the JT distorted forms are discussed for their implication in EPR spectroscopy. The calculation of the g -tensor by DFT methods has also been benchmarked to serve as methodological approach for the investigation of more extended graphene molecules., Peer Reviewed, Postprint (published version)

The administration of cardiosphere-derived cells (CDCs) after acute myocardial infarction (AMI) is very promising. CDC encapsulation in alginate-poly-l-lysine-alginate (APA) could increase cell survival and adherence. The intrapericardial (IP) approach potentially achieves high concentrations of the therapeutic agent in the infarcted area. We aimed to evaluate IP therapy using a saline vehicle as a control (CON), a dose of 30 × 106 CDCs (CDCs) or APA microcapsules containing 30 × 106 CDCs (APA-CDCs) at 72 h in a porcine AMI model. Magnetic resonance imaging (MRI) was used to determine the left ventricular ejection fraction (LVEF), infarct size (IS), and indexed end diastolic and systolic volumes (EDVi; ESVi) pre- and 10 weeks post-injection. Programmed electrical stimulation (PES) was performed to test arrhythmia inducibility before euthanasia. Histopathological analysis was carried out afterwards. The IP infusion was successful in all animals. At 10 weeks, MRI revealed significantly higher LVEF in the APA-CDC group compared with CON. No significant differences were observed among groups in IS, EDVi, ESVi, PES and histopathological analyses. In conclusion, the IP injection of CDCs (microencapsulated or not) was feasible and safe 72 h post-AMI in the porcine model. Moreover, CDCs APA encapsulation could have a beneficial effect on cardiac function, reflected by a higher LVEF at 10 weeks., Peer Reviewed, Postprint (published version)