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Despite the use of therapeutic ultrasound in the treatment of soft tissue pathologies, there remains some controversy regarding its efficacy. In order to develop new treatment protocols, it is a common practice to carry out in vitro studies in cell cultures before conducting animal tests. The lack of reproducibility of the experimental results observed in the literature concerning in vitro experiments motivated us to establish a methodology for characterizing the acoustic field in culture plate wells. In this work, such acoustic fields are fully characterized in a real experimental configu-ration, with the transducer being placed in contact with the surface of a standard 12-well culture plate. To study the non-thermal effects of ultrasound on fibroblasts, two different treatment protocols are proposed: long pulse (200 cycles) signals, which give rise to a standing wave in the well with the presence of cavitation (ISPTP max = 19.25 W/cm), and a short pulse (five cycles) of high acoustic pressure, which produces a number of echoes in the cavity (ISPTP = 33.1 W/cm, with Pmax = 1.01 MPa). The influence of the acoustic intensity, the number of pulses, and the pulse repetition frequency was studied. We further analyzed the correlation of these acoustic parameters with cell via-bility, population, occupied surface, and cell morphology. Lytic effects when cavitation was present, as well as mechanotransduction reactions, were observed., This research was funded by Spanish Research Agency, grant number DPI2016-80254-R, and the CICYT, grant number MAT2014-51918-C2-1-R

The photophysical properties of naphthalimide-based fluorophores can be easily tuned by chemical manipulation of the substituents on that privileged scaffold. Replacement of a OMe group at position 6 in 2-(hydroxyl)ethyl-naphthalimide derivatives by diverse amines, including 2-(hydroxyl)ethylamine, trans-(4-acetamido)cyclohexylamine and azetidine increases the solvatochromic (ICT) character, while this replacement in 2-(dimethylamino)ethyl-naphthalimide analogues (PET fluorophores) decrease their solvent polarity sensitivity or even reversed them to solvatochromic fluorophores. These fluorophores resulted macrophage nucleus imaging probes, which bind DNA as intercalants and showed low cytotoxicity in human cancer cells, The work was supported by grants SAF2012-32209, FU2015-67284-R, CTQ2017-85658-R form the Spanish Ministerio de Economía y Competividad, Agencia Estatal de Investigación and the European Regional Development Fund; and the CSIC grant 201580E073

The article deals with a comparative analysis of the parameters of the polymerization in physiological conditions of three commercially available alkyl cyanoacrylates, n-butyl cyanoacrylate (GLUBRAN 2), n-hexyl cyanoacrylate (IFABOND), and n-octyl cyanoacrylate (EVOBOND), the cell behavior of the corresponding polymers and the application of these adhesives in the fixation of surgical polypropylene meshes for hernia repair in an animal model of rabbits. The results obtained demonstrate that the curing process depends on the nature of the alkyl residue of the ester group of cyanoacrylate molecules, being the heat of polymerization lower for the octyl derivative in comparison with the hexyl and butyl, and reaching a maximum temperature of 35 °C after a time of mixing with physiological fluids of 60-70 s. The cell behavior demonstrates that the three systems do not present toxicity for fibroblasts and low adhesion of cells, which is a positive result for application as tissue adhesives, especially for the fixation of abdominal polypropylene meshes for hernia repair. The animal experimentation indicates the excellent tolerance of the meshes fixed with the cyanoacrylic adhesives, during at least a period of 90 d, and guarantees a good adhesion for the application of hernia repair meshes.

Osteochondral (OC) regeneration faces several limitations in orthopedic surgery, owing to the complexity of the OC tissue that simultaneously entails the restoration of articular cartilage and subchondral bone diseases. In this study, novel biofunctional hierarchical scaffolds composed of a horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) cartilage-like layer (HRP-SF layer) fully integrated into a HRP-SF/ZnSr-doped β-tricalcium phosphate (β-TCP) subchondral bone-like layer (HRP-SF/dTCP layer) were proposed as a promising strategy for OC tissue regeneration. For comparative purposes, a similar bilayered structure produced with no ion incorporation (HRP-SF/TCP layer) was used. A homogeneous porosity distribution was achieved throughout the scaffolds, as shown by micro-computed tomography analysis. The ion-doped bilayered scaffolds presented a wet compressive modulus (226.56 ± 60.34 kPa) and dynamic mechanical properties (ranging from 403.56 ± 111.62 to 593.56 ± 206.90 kPa) superior to that of the control bilayered scaffolds (189.18 ± 90.80 kPa and ranging from 262.72 ± 59.92 to 347.68 ± 93.37 kPa, respectively). Apatite crystal formation, after immersion in simulated body fluid (SBF), was observed in the subchondral bone-like layers for the scaffolds incorporating TCP powders. Human osteoblasts (hOBs) and human articular chondrocytes (hACs) were co-cultured onto the bilayered structures and monocultured in the respective cartilage and subchondral bone half of the partitioned scaffolds. Both cell types showed good adhesion and proliferation in the scaffold compartments, as well as adequate integration of the interface regions. Osteoblasts produced a mineralized extracellular matrix (ECM) in the subchondral bone-like layers, and chondrocytes showed GAG deposition. The gene expression profile was different in the distinct zones of the bilayered constructs, and the intermediate regions showed pre-hypertrophic chondrocyte gene expression, especially on the BdTCP constructs. Immunofluorescence analysis supported these observations. This study showed that the proposed bilayered scaffolds allowed a specific stimulation of the chondrogenic and osteogenic cells in the co-culture system together with the formation of an osteochondral-like tissue interface. Hence, the structural adaptability, suitable mechanical properties, and biological performance of the hierarchical scaffolds make these constructs a desired strategy for OC defect regeneration., This study was funded by the Portuguese Foundation for Science and Technology (FCT) for the HierarchiTech project (M-ERA-NET/0001/2014). The project FROnTHERA (NORTE-01-0145-FEDER-000023), was supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The FCT distinctions attributed to JMO (IF/00423/2012 and IF/01285/ 2015), and ALO (IF/00411/2013). VPR (PD/BD/113806/ 2015) and JBC (PD/BD/113803/2015) were awarded PhD scholarships under the financial support from FCT/MCTES and FSE/POCH, PD/59/2013. I.F.C. was awarded an FCT Ph.D. scholarship (SFRH/BD/99555/2014).

Herein, multilayer polysaccharide films were proposed and characterized as biomaterials for the local and controlled release of an antitumoral drug. To that aim, multilayer films of alginate (Alg) and chitosan (Chi) were built up through spray assisted layer-by-layer (LbL) technique employing an automatic equipment. A specific drug against breast cancer, tamoxifen (TMX), was incorporated in different intermediate positions of the multilayer Alg/Chi films. Our findings highlight that Alg/Chi multilayer films can be employed for sustained and local TMX delivery and their therapeutic effect can be modulated and optimized by the number of bilayers deposited over the loaded tamoxifen, the quantity of tamoxifen loaded in several intermediate positions and the total area of the film., The authors would like to thank Rosa Ana Ramirez and Raquel de Roba for their assistance regarding cell culture experiments. Financial support from MINECO (MAT2017-83014-C2-1-P and MAT2017-84277- R) is gratefully acknowledged. Rebeca Hernández thanks MEC for a Ramón y Cajal contract and Miryam Criado thanks MEC for a FPU fellowship.

Chondroitin sulfate (CS) is a relevant family of polysaccharides that participates in a large variety of biological events that are related to neural processes by regulating various growth factors through the pattern and degree of sulfation of the polysaccharide. However, their own complexity makes their optimization for biomedical applications a difficult undertaking. Thus, a different perspective has to be taken. Herein, we show that the particular sulfate distribution within the disaccharide repeating-unit plays a key role in the binding of growth factors (GFs). In particular, this disposition modulates the surface charge of the helical structure that, interestingly, has a significant influence on the binding capacity of CSs with several GFs. This fact should be carefully considered in the design of new ligands with improved activity as GFs ligands., The authors gratefully acknowledge the financial support provided by the Spanish Ministerio de Economía y Competitividad (Grant MAT2015-65184-C2-2-R) and by CSIC project iCOOPB20237. The authors thank Prof. Jian Liu (School of Pharmacy, University of North Carolina) for providing a plasmid expression of the chondroitin, lyase ABC, and V. M. Moreno for cloning the 4-O-endo-sulfatase from Bacteroides thetaiotaomicron.

Stabilized magnetic nanoparticles are the subject of intense research for targeting applications and this work deals with the design, preparation and application of specific core-shell nanoparticles based on ionic crosslinked chitosan. The nanometric size of the materials was demonstrated by dynamic light scattering (DLS) and field emission scanning electron microscopy (FESEM) that also proved an increase of the size of chitosan nanoparticles (NPs) with the magnetite content. Steady oscillatory rheology measurements revealed a gel-like behavior of aqueous dispersions of chitosan NPs with concentrations ranging from 0.5% to 2.0% (w/v). The cytotoxicity of all the materials synthesized was analyzed in human fibroblasts cultures using the Alamar Blue and lactate dehydrogenase (LDH) assays. The measured specific power absorption under alternating magnetic fields (f = 580 kHz, H = 24 kA/m) indicated that magnetic core-shell chitosan NPs can be useful as remotely driven heaters for magnetic hyperthermia. © 2013 Elsevier Ltd.

Nanocomposites were prepared with segmented polyurethanes and Cloisite 30B by either solution mixing or in situ polymerization and characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis and X-ray diffraction. Cytotoxicity and genotoxicity were assessed with lymphocytes while cell viability was measured by the methyl tetrazolium assay using fibroblasts. It was found that in situ polymerization rendered exfoliated nanocomposites with higher glass transition temperature, tensile modulus and thermal stability compared to nanocomposites obtained by solution mixing. The mitotic index of lymphocytes was significantly reduced at high clay concentrations (6 wt% and 10 wt%), while fibroblast viability improved in the presence of extract obtained after days 2 and 7.

This article reports the fabrication and characterization of NPs based on the self-assembling of polymeric drugs with amphiphilic character synthetized from oleyl 2-acetamido-2-deoxy-α-D-glucopyranoside methacrylate and vinyl pyrrolidone (OAGMA-VP). NPs were spherical, with an apparent hydrodynamic diameter between 91 and 226 nm and with zeta potential values that ensure stability. Atomic concentrations of C, O, and N, determined by X-ray photoelectron spectroscopy (XPS) of NPs, compared well with the corresponding theoretical values. High resolution XPS C1s spectra suggest that the carbons bound to heteroatoms or carbonyl groups are preferentially situated on the surface of the NP samples. ToF-SIMS spectra analyzed by principal component analysis (PCA) indicated that ions coming from acetyl and oleyl groups of OAGMA play important roles in the outer structure of NPs. Water contact angle and surface tension values of NPs were characteristic of hydrophilic surfaces, confirming the location of VP sequences on the surface. Cell culture assays showed that copolymeric NPs did not compromise biocompatibility of human fibroblasts according to ISO standard, but they were cytotoxic on a human glioblastoma cell line (A-172), This work was supported by Ministerio de Economiá y Competitividad, (MAT2010-18155). M.F.-G. also thanks to the JAE-doc program of CSIC. The assistance of Dr. Lara Gamble and Winston Ciridon in the SIMS and XPS assays and the support of NESAC/BIO (NIH EB-002027) is appreciated

The preparation, characterization, and analysis of physicochemical and biological properties of a new bioactive polymer system, based on a copolymer of an acrylic derivative of triflusal (a molecule with chemical structure related to aspirin with antiaggregating activity for platelets) is described and evaluated as thin bioactive coating for vascular grafts and coronary stents. The acrylic monomer derived from triflusal (THEMA) provides random copolymers when it is polymerized with butyl acrylate (BA), according to their reactivity ratios, r(THEMA) = 1.05 and r(BA) = 0.33. The copolymer THBA70, containing a molar composition f(THEMA) = 0.45 and f(BA) = 0.55 presents the optimal properties of stability, flexibility, and adhesion, with a T(g) = 21 ± 2 °C, to be applied as bioactive and biostable coatings for vascular grafts and coronary stents. Thin films of this copolymer system present an excellent biocompatibility and a good inherent antiaggregant activity for platelets.

Polymeric drugs based on random copolymers with antimitotic activity were obtained by free radical copolymerization of oleyl 2-acetamido-2-deoxy-R-D-glucopyranoside methacrylate (OAGMA) and 2-ethyl-(2- pyrrolidone) methacrylate (EPM) at low and high conversion and analyzed in terms of microstructure, physicochemical, and biological properties. Reactivity ratios of monomers were found to be rOAGMA ) 1.34 and rEPM ) 0.98, indicating the obtaining of statistical copolymers with random sequence distribution of the comonomeric units in the macromolecular chains. The glass transition temperature of the copolymers presents a negative deviation from the predicted values according to the Fox equation, suggesting a higher flexibility of the alternating diad. Copolymeric systems with OAGMA contents between 10-50 mol % presented thermosensitive behavior in a heating process showing cloud point temperatures (CPT) in the range 45-28 °C with increasing OAGMA content and hysteresis in one heating-and-cooling cycle. In vitro glycolipid release studies revealed the stability of the ester group in culture medium. The polymeric drugs with 30 and 50 mol % OAGMA presented antimitotic activity on a human glioblastoma line, but they were less toxic on normal human fibroblast cultures., Financial support from the Comisio´n Interministerial de Ciencia y Tecnología, CICYT, (MAT2007- 63355) and NoE EXPERTISSUES (UE Contract No. 500283- 2) is gratefully acknowledged

¿-Tocopheryl succinate (¿-TOS) is a well-known mitochondrially targeted anticancer compound, however, it is highly hydrophobic and toxic. In order to improve its activity and reduce its toxicity, new surfactant-free biologically active nanoparticles (NP) were synthesized. A methacrylic derivative of ¿-TOS (MTOS) was prepared and incorporated in amphiphilic pseudoblock copolymers when copolymerized with N-vinylpyrrolidone (VP) by free radical polymerization (poly(VP-co-MTOS)). The selected poly(VP-co-MTOS) copolymers formed surfactant-free NP by nanoprecipitation with sizes between 96 and 220 nm and narrow size distribution, and the in vitro biological activity was tested. In order to understand the structure-activity relationship three other methacrylic monomers were synthesized and characterized: MVE did not have the succinate group, SPHY did not have the chromanol ring, and MPHY did not have both the succinate group and the chromanol ring. The corresponding families of copolymers (poly(VP-co-MVE), poly(VP-co-SPHY), and poly(VP-co-MPHY)) were synthesized and characterized, and their biological activity was compared to poly(VP-co-MTOS). Both poly(VP-co-MTOS) and poly(VP-co-MVE) presented triple action: reduced cell viability of cancer cells with little or no harm to normal cells (anticancer), reduced viability of proliferating endothelial cells with little or no harm to quiescent endothelial cells (antiangiogenic), and efficiently encapsulated hydrophobic molecules (nanocarrier). The anticancer and antiangiogenic activity of the synthesized copolymers is demonstrated as the active compound (vitamin E or ¿-tocopheryl succinate) do not need to be cleaved to trigger the biological action targeting ubiquinone binding sites of complex II. Poly(VP-co-SPHY) and poly(VP-co-MPHY) also formed surfactant-free NP that were also endocyted by the assayed cells; however, these NP did not selectively reduce cell viability of cancer cells. Therefore, the chromanol ring of the vitamin E analogues has an important role in the biological activity of the copolymers. Moreover, when succinate moiety is substituted and vitamin E is directly linked to the macromolecular chain through an ester bond, the biological activity is maintained., Authors acknowledge Dr. Gema Rodriǵ uez, Lautaro Biancotto, David Goḿ ez, and Rosa Ana Ramiŕ ez for their help in AFM, SEM/TEM, and cell culture experiments. Authors also thank Dr. M.P. Murphy for providing MitoQ for the experiments. This work was funded by the Spanish Ministry of Economy and Competitiveness (MAT2010−18155), Eugenio Rodriǵ uez Pascual Foundation, CIBER BBN-ECO Foundation project, and the European University of Madrid.

Hydrogel networks that combine suitable physical and biomechanical characteristics for tissue engineering scaffolds are in demand. The aim of this work was the development of hydrogel networks based on agarose and chitosan using oxidized dextrins as low cytotoxicity crosslinking agents, paying special attention to the study of the influence of the polysaccharide composition and oxidation degree of the dextrins in the final characteristics of the network. The results show that the formation of an interpenetrating or a semi-interpenetrating polymer network was mainly dependent on a minimum agarose content and degree of oxidation of dextrin. Spectroscopic, thermal and swelling analysis revealed good compatibility with an absence of phase separation of polysaccharides at agarose:chitosan proportions of 50:50 and 25:75. The analysis of atomic force microscopy images showed the formation of a fibrillar microstructure whose distribution within the crosslinked chitosan depended mainly on the crosslinker. All materials exhibited the viscoelastic behaviour typical of gels, with a constant storage modulus independent of frequency for all compositions. The stiffness was strongly influenced by the degree of oxidation of the crosslinker. Cellular response to the hydrogels was studied with cells of different strains, and cell adhesion and proliferation was correlated with the homogeneity of the samples and their elastic properties. Some hydrogel formulations seemed to be candidates for tissue engineering applications such as wound healing or soft tissue regeneration. © 2013 Acta Materialia Inc.

Surface properties play an important role in the functioning of a biomaterial in the biological environment. This work describes the influence of the changes that occurred on diamond-like carbon (DLC) and polymeric substrata by different nitrogen and ammonia plasmas treatments and its effects on the cell proliferation on these materials. All substrata were additionally subjected to the effect of neutral beams of nitrogen atoms and NH species for comparison purposes. Results about the proliferation, viability, and morphology of fibroblasts were correlated with surface chemical composition, surface tension, and topography. It was found that the presence of amine groups on the surface and the surface tension are beneficial factors for the cell growth. Surface roughness in DLC also plays a positive role in favoring cell adhesion and proliferation, but it can be detrimental for some of the treated polymers because of the accumulation of low molecular weight fragments formed as a result of the plasma treatments. Analysis of the overall results for each type of material allowed to define a unique parameter called 'factor of merit' accounting for the influence of the different surface characteristics on the cell deployment, which can be used to predict qualitatively the efficiency for cell growth. © The Author(s) 2011.

This paper reports a biological evaluation of a nonresorbable acrylic cement loaded with alendronate for the treatment of osteoporotic vertebral compression fractures. The cement formulation was based on polymethyl methacrylate and acrylic monomers; one of these had covalently linked vitamin E residues. The same cement in the absence of alendronate was used as a control. The setting of the charged cement presented a maximum polymerization temperature of 44ºC, a setting time of 24 min, a residual monomer content lower than 3 wt.%, a compressive strength of 99±10 MPa and an elastic modulus of 1.2±0.2 GPa. Cytotoxicity studies using human osteoblast cultures revealed that the leachable substances of the alendronate loaded cement collected between 1 and 7 days decreased cell viability to values lower than 80%. However, morphological changes and cellular damage in cells produced by the extracts decreased with the leak time. Cell adhesion and growth on charged cement was significantly lower than on the control. Implantation of the cement paste in the intra-femoral cavity of rabbits showed that initially the osteogenic activity was evident for the cement charged with alendronate, and the osteosynthesis process took place mainly in the trabeculae and was manifested by the presence of a non-mineralised osseous spicule. The interface between material and adjacent bone tissue was initially characterized by a variable fibrous response that in many cases it appeared reduced to thin connective tissue after a 24-week-period, Financial support from the Comisión Interministerial de Ciencia y Tecnología, CICYT, (MAT2007-63355) is gratefully acknowledged.

This paper describes a study on the preparation and characterisation of partially biodegradable microparticles of poly(e-caprolactone)/poly(ethyl methacrylate) (PCL/PEMA) as carriers of synthetic glycolipids with antimitotic activity against brain tumour cells. Microparticles prepared by suspension polymerisation of methacrylate in the presence of already polymerised PCL showed a predominantly spherical but complex morphology, with segregation of PCL micro/nano-domains towards the surface. Small diameter discs were prepared by compression moulding of blends of microparticles and the active principle under mild conditions. The in vitro behaviour of the discs and release of the glycolipid were studied in different simulated fluid models. Ingress of fluids increased with increasing hydrophobicity of the medium. Release of the glycolipid was sustained in all fluids, the most prolonged profile being in human synovial fluid and phosphate-buffered saline modified with 20 vol.% dioxane. Slow disintegration of the discs and partial degradation of the microparticles was evident in accelerated studies. The antimitotic activity of glycolipid released from the discs was proved against a human glioblastoma line. This activity, along with selectivity against human fibroblasts, could be controlled by the amount of drug charged in the disc., This research was supported by the Comisión Interministerial de Ciencia y Tecnología (MAT2007-63355), NoE EXPERTISSUES (UE Contract No. 500283-2), CSIC (PIF 200580F0061-62-63) and a Predoctoral Fellowship from the Spanish Ministry of Science and Education (AP2005-0246). M. Fernández was supported by the Ramón y Cajal programme (MEC-Spain).