Bioactive Hydroxyapatite Aerogels with Piezoelectric Particles.

Open-cell foams based on hydroxyapatite (HAp) can mimic the extracellular matrix (ECM) to better replace damaged hard tissues and assist in their regeneration processes. Aerogels of HAp nanowires (NW) with barium titanate (BT) particles were produced and characterized regarding their physical and chemical properties, bioactivity, and in vitro cytotoxicity. Considering the role of piezoelectricity (mainly due to collagen) and surface charges in bone remodeling, all BT particles, of size 280 nm and 2 and 3 µm, contained BaTiO3 in their piezoelectric tetragonal phase. The synthesized nanowires were verified to be AB-type carbonated hydroxyapatite. The aerogels showed high porosity and relatively homogeneous distribution of the BT particles. Barium titanate proved to be non-cytotoxic while all the aerogels produced were cytotoxic for an extract concentration of 1 mg/mL but became non-cytotoxic at concentrations of 0.5 mg/mL and below. It is possible that these results were affected by the higher surface area and quicker dissolution rate of the aerogels. In the bioactivity assays, SEM/EDS, it was not easy to differentiate between the apatite deposition and the surface of the HAp wires. However, a quantitative EDS analysis shows a possible CaP deposition/dissolution cycle taking place.

Elevated fucose content enhances the cryoprotective performance of anionic polysaccharides.

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Here, a subset of 26 natural polysaccharides of various chemical composition was probed for their potential in enhancing the metabolic post-thaw viability (PTV) of cryopreserved Vero cells. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor™ CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18-35.7 mol%), uronic acids (UA) (13.5-26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. Principal component analysis (PCA) revealed that fucose enhances cell survival by a charge-independent, MW-scaling mechanism (PC1), drastically different from the charge-dominated ice growth disruption of UA (PC2). Its neutral nature and unique properties distinguishable from other neutral monomers suggest fucose may play a passive role in conformational adaptability of polysaccharide to ice growth inhibition, or an active role in cell membrane stabilization through binding. Ultimately, fucose-rich anionic polysaccharides may indulge in polymer-ice and polymer-cell interactions that actively disrupt ice and minimize lethal volumetric fluctuations due to a balanced hydrophobic-hydrophilic character. Our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection.

Polyvinylpyrrolidone Nanofibers Incorporating Mesoporous Bioactive Glass for Bone Tissue Engineering.

Composite biomaterials that combine osteoconductive and osteoinductive properties are a promising approach for bone tissue engineering (BTE) since they stimulate osteogenesis while mimicking extracellular matrix (ECM) morphology. In this context, the aim of the present research was to produce polyvinylpyrrolidone (PVP) nanofibers containing mesoporous bioactive glass (MBG) 80S15 nanoparticles. These composite materials were produced by the electrospinning technique. Design of experiments (DOE) was used to estimate the optimal electrospinning parameters to reduce average fiber diameter. The polymeric matrices were thermally crosslinked under different conditions, and the fibers' morphology was studied using scanning electron microscopy (SEM). Evaluation of the mechanical properties of nanofibrous mats revealed a dependence on thermal crosslinking parameters and on the presence of MBG 80S15 particles inside the polymeric fibers. Degradation tests indicated that the presence of MBG led to a faster degradation of nanofibrous mats and to a higher swelling capacity. The assessment of in vitro bioactivity in simulated body fluid (SBF) was performed using MBG pellets and PVP/MBG (1:1) composites to assess if the bioactive properties of MBG 80S15 were kept when it was incorporated into PVP nanofibers. FTIR and XRD analysis along with SEM-EDS results indicated that a hydroxy-carbonate apatite (HCA) layer formed on the surface of MBG pellets and nanofibrous webs after soaking in SBF over different time periods. In general, the materials revealed no cytotoxic effects on the Saos-2 cell line. The overall results for the materials produced show the potential of the composites to be used in BTE.

Phototoxic Potential of Different DNA Intercalators for Skin Cancer Therapy: In Vitro Screening.

Photodynamic therapy is a minimally invasive procedure used in the treatment of several diseases, including some types of cancer. It is based on photosensitizer molecules, which, in the presence of oxygen and light, lead to the formation of reactive oxygen species (ROS) and consequent cell death. The selection of the photosensitizer molecule is important for the therapy efficiency; therefore, many molecules such as dyes, natural products and metallic complexes have been investigated regarding their photosensitizing potential. In this work, the phototoxic potential of the DNA-intercalating molecules-the dyes methylene blue (MB), acridine orange (AO) and gentian violet (GV); the natural products curcumin (CUR), quercetin (QT) and epigallocatechin gallate (EGCG); and the chelating compounds neocuproine (NEO), 1,10-phenanthroline (PHE) and 2,2'-bipyridyl (BIPY)-were analyzed. The cytotoxicity of these chemicals was tested in vitro in non-cancer keratinocytes (HaCaT) and squamous cell carcinoma (MET1) cell lines. A phototoxicity assay and the detection of intracellular ROS were performed in MET1 cells. Results revealed that the IC50 values of the dyes and curcumin in MET1 cells were lower than 30 µM, while the values for the natural products QT and EGCG and the chelating agents BIPY and PHE were higher than 100 µM. The IC50 of MB and AO was greatly affected by irradiation when submitted to 640 nm and 457 nm light sources, respectively. ROS detection was more evident for cells treated with AO at low concentrations. In studies with the melanoma cell line WM983b, cells were more resistant to MB and AO and presented slightly higher IC50 values, in line with the results of the phototoxicity assays. This study reveals that many molecules can act as photosensitizers, but the effect depends on the cell line and the concentration of the chemical. Finally, significant photosensitizing activity of acridine orange at low concentrations and moderate light doses was demonstrated.

Cosmetic emulsion based on the fucose-rich polysaccharide FucoPol: Bioactive properties and sensorial evaluation.

In this study, the physicochemical characteristics, bioactive properties, and sensorial evaluation of a O/W cosmetic formulation containing FucoPol, a fucose-containing bacterial polysaccharide, were assessed. The stability of the FucoPol-based cream, named F-cream, was demonstrated over a period of 2 months at different temperatures (4, 20 and 30 °C), during which it maintained the organoleptic characteristics and pH (5.88-6.19), with minimal variations on the apparent viscosity. Furthermore, no breaking mechanisms occurred upon centrifuging the samples (accelerated stability test) kept at 4 °C and at 30 °C for 60 days. The F-cream presented a shear-thinning and solid-liquid behavior consistent with its envisaged use for topical applications, proving to be a suitable candidate for an anti-aging application due to its antioxidant capacity and effective photoprotection, maintaining cellular preservation. Moreover, the formulation was proven non-cytotoxic for HaCaT cells at concentrations between 0.78 and 12.5 mg/mL, promoting HFFF2 cell migration (46-70 % of wound closure) at a concentration of 2.5 mg/mL, and HaCaT cell migration at a concentration of 10 mg/mL (95-98 % of wound closure). Upon application over the skin, the F-cream provided a hydration and softness with desired spreadability with no residues after application. These findings show that FucoPol has good potential to be used as a functional and/or active ingredient in cosmetic formulations, forming an emulsified cream with appealing sensorial properties that can act as a moisturizer with photoprotection, antioxidant, and regeneration properties.

Bioactivity Enhancement of Plasma-Sprayed Hydroxyapatite Coatings through Non-Contact Corona Electrical Charging.

Atmospheric plasma spray (APS) remains the only certified industrial process to produce hydroxyapatite (Hap) coatings on orthopaedic and dental implants intended for commercialization. Despite the established clinical success of Hap-coated implants, such as hip and knee arthroplasties, a concern is being raised regarding the failure and revision rates in younger patients, which are increasing rapidly worldwide. The lifetime risk of replacement for patients in the 50-60 age interval is about 35%, which is significantly higher than 5% for patients aged 70 or older. Improved implants targeted at younger patients are a necessity that experts have been alerted to. One approach is to enhance their bioactivity. For this purpose, the method with the most outstanding biological results is the electrical polarization of Hap, which remarkably accelerates implant osteointegration. There is, however, the technical challenge of charging the coatings. Although this is straightforward on bulk samples with planar faces, it is not easy on coatings, and there are several problems regarding the application of electrodes. To the best of our knowledge, this study demonstrates, for the first time, the electrical charging of APS Hap coatings using a non-contact, electrode-free method: corona charging. Bioactivity enhancement is observed, establishing the promising potential of corona charging in orthopedics and dental implantology. It is found that the coatings can store charge at the surface and bulk levels up to high surface potentials (>1000 V). The biological in vitro results show higher Ca2+ and P5+ intakes in charged coatings compared to non-charged coatings. Moreover, a higher osteoblastic cellular proliferation is promoted in the charged coatings, indicating the promising potential of corona-charged coatings when applied in orthopedics and dental implantology.

Fabrication, Structural and Biological Characterization of Zinc-Containing Bioactive Glasses and Their Use in Membranes for Guided Bone Regeneration.

Polymeric membranes are widely used in guided bone regeneration (GBR), particularly in dentistry. In addition, bioactive glasses can be added to the polymers in order to develop a matrix that is osteoconductive and osteoinductive, increasing cell adhesion and proliferation. The bioactive glasses allow the insertion into its network of therapeutic ions in order to add specific biological properties. The addition of zinc into bioactive glasses can promote antibacterial activity and induce the differentiation and proliferation of the bone cells. In this study, bioactive glasses containing zinc (0.25, 0.5, 1 and 2 mol%) were developed and structurally and biologically characterized. The biological results show that the Zn-containing bioactive glasses do not present significant antibacterial activity, but the addition of zinc at the highest concentration does not compromise the bioactivity and promotes the viability of Saos-2 cells. The cell culture assays in the membranes (PCL, PCL:BG and PCL:BGZn2) showed that zinc addition promotes cell viability and an increase in alkaline phosphatase (ALP) production.

Biocompatibility, Bioactivity, and Antibacterial Behaviour of Cerium-Containing Bioglass®.

The main reason for the increased use of dental implants in clinical practice is associated with aesthetic parameters. Implants are also presented as the only technique that conserves and stimulates natural bone. However, there are several problems associated with infections, such as peri-implantitis. This disease reveals a progressive inflammatory action that affects the hard and soft tissues surrounding the implant, leading to implant loss. To prevent the onset of this disease, coating the implant with bioactive glasses has been suggested. In addition to its intrinsic function of promoting bone regeneration, it is also possible to insert therapeutic ions, such as cerium. Cerium has several advantages when the aim is to improve osseointegration and prevent infectious problems with dental implant placement. It promotes increased growth and the differentiation of osteoblasts, improves the mechanical properties of bone, and prevents bacterial adhesion and proliferation that may occur on the implant surface. This antibacterial effect is due to its ability to disrupt the cell wall and membrane of bacteria, thus interfering with vital metabolic functions such as respiration. In addition, its antioxidant effect reverses oxidative stress after implantation in bone. In this work, Bioglass 45S5 with CeO2 with different percentages (0.25, 0.5, 1, and 2 mol%) was developed by the melt-quenching method. The materials were analyzed in terms of morphological, structural, and biological (cytotoxicity, bioactivity, and antibacterial activity) properties. The addition of cerium did not promote structural changes to the bioactive glass, which shows no cytotoxicity for the Saos-2 cell line up to 25 mg/mL of extract concentration for all cerium contents. For the maximum cerium concentration (2 mol%) the bioactive glass shows an evident inhibitory effect for Escherichia coli and Streptococcus mutans bacteria. Furthermore, all samples showed the beginning of the deposition of a CaP-rich layer on the surface of the material after 24 h.

Clay-Based Nanocomposite Hydrogels for Biomedical Applications: A Review.

In recent decades, new and improved materials have been developed with a significant interest in three-dimensional (3D) scaffolds that can cope with the diverse needs of the expanding biomedical field and promote the required biological response in multiple applications. Due to their biocompatibility, ability to encapsulate and deliver drugs, and capacity to mimic the extracellular matrix (ECM), typical hydrogels have been extensively investigated in the biomedical and biotechnological fields. The major limitations of hydrogels include poor mechanical integrity and limited cell interaction, restricting their broad applicability. To overcome these limitations, an emerging approach, aimed at the generation of hybrid materials with synergistic effects, is focused on incorporating nanoparticles (NPs) within polymeric gels to achieve nanocomposites with tailored functionality and improved properties. This review focuses on the unique contributions of clay nanoparticles, regarding the recent developments of clay-based nanocomposite hydrogels, with an emphasis on biomedical applications.

Liposomes encapsulating methylene blue and acridine orange: An approach for phototherapy of skin cancer.

Photodynamic therapy uses photosensitizer molecules for the photo-mediated treatment of several diseases such as cancer and skin disorders. However, most of the photosensitizer molecules present problems such as aggregation and low solubility in physiological environments which hinders the treatment efficacy. To overcome these problems, the development of stable liposomes loading photosensitizing molecules as delivery systems can be explored as promising alternatives to enhance cellular uptake and the therapy's efficacy. In this work, liposomes composed by different lipids with or without surfactants were characterized for the encapsulation of photosensitizer molecules such as Methylene Blue (MB) and Acridine Orange (AO). Liposomes were produced by the thin-film hydration method followed by extrusion to reduce particle size and were characterized by Dynamic Light Scattering and Atomic Force Microscopy. Encapsulation efficiency was evaluated as well as the release profile of these molecules from the liposome systems. Cytotoxicity and phototoxicity studies were performed on keratinocytes with and without carcinoma. Results showed that liposome's stability depends on the composition of lipids regardless of the presence of surfactants. Most stable liposomes were those with cholesterol plus the surfactants Span® 80 or sodium cholate that were able to provide higher stability for the liposomes considering the MB and AO encapsulation. Encapsulation efficiency (EE) studies revealed that AO had greater affinity for the vesicles presenting high EE (>98%) while for MB the encapsulation was, in general, moderate (between 63% and 86%). Greater phototoxicity was observed for MET1 squamous cell carcinoma (SCC) cells treated with AO liposomes, achieving similar half-maximal inhibition concentration (IC50) as for the free drug. Finally, two different possible approaches were found, namely, MB-liposomes with potential as a cytotoxic agent for cancer cells; and AO liposomes with a great phototoxicity potential at very low concentrations.

Photoprotective effect of the fucose-containing polysaccharide FucoPol.

This study reports the performance of FucoPol, a fucose-containing bacterial polysaccharide, as a photostable agent with high absorption yield at concentrations as low as 0.02 % (w/v). FucoPol is non-cytotoxic, efficiently protects from UVA and UVB at concentrations of 0.02-2 % (w/v) and 0.2-2 % (w/v), respectively, has over 94 % overall photostability up to 1.5 h of irradiation time. The lowest concentration studied (0.02 %, w/v) shows a sun protection factor (SPF) of 2.61 ± 0.08. The SPF-to-concentration unit ratio showed that FucoPol is about 60-fold more photoprotective than combinations of common organic and inorganic UV filters. In vitro radiation exposure experiments of adhered Vero epithelial and PM1 keratinocytic cells in the presence of 0.25 % (w/v) FucoPol further showed that cell viability was preserved, and delayed radiation-induced cell death was prevented. Overall, FucoPol outperforms common cosmetic biopolymers like xanthan and fucogel. These results are very promising for the development of bio-based sunscreen formulations.

Development of a Cryoprotective Formula Based on the Fucose-Containing Polysaccharide FucoPol.

This study reports the performance of the polysaccharide FucoPol as an enhancer of cryoprotective formulations. FucoPol at a concentration of 0.25% (w/v) was added to several normothermic Dulbecco-derived solutions and hypothermic Euro-Collins, Custodiol-HTK, and Unisol-CV media, substituting some constituents in the latter class to develop FucoPol-based formulations that were tested for their ability to cryopreserve Vero cells. Supplementation yielded post-thaw cell recovery enhancements of at least 70% and averaged at 82%. The FucoPol-supplemented formulations Dulbecco(+)+FP and Unisol-CVS3 achieved cell viabilities capable of competing with the commercial cryogenic formula CryoStor CS5. Particularly in Unisol-CVS3, mannitol, glucose, gluconate, and dextran were all substituted by 0.25% FucoPol, and still, a similar viability was achieved. Multiparametric correlation clustering showed that FucoPol cryoprotection synergizes best with K+, Ca2+, and Cl- in its microenvironment. Component substitution analysis demonstrated that FucoPol acts consistently as a cryoprotectant, an impermeant, and a colloidal stabilizer, providing a combined alternative to glucose, mannitol, gluconate, and dextran, thus highlighting its potential in the development of high-performing cryoprotective formulas.

Demonstration of the cryoprotective properties of the fucose-containing polysaccharide FucoPol.

We report the cryoprotective potential of FucoPol, a fucose-containing bacterial exopolysaccharide produced by Enterobacter A47. In vitro cryopreservation assays of Vero, Saos-2, HFFF2 and C2C12 cell lines exposed to a validated non-cytotoxic 2.5 mg/mL FucoPol concentration demonstrated a consistent post-thaw metabolic viability increase. Calorimetric analysis showed a non-colligative, FucoPol concentration-dependent increase of the freezing point (Tf), with minimal change in melting point (Tm). Freezing point variation was corroborated by Polarized Optical Microscopy studies, also showing a reduction of ice crystal dimensions. Its proven shear-thinning behaviour and polyanionicity favour interactivity between the polysaccharide and the water-ice interface, resulting in ice growth inhibition. These findings demonstrate FucoPol's high promise as a bio-based, biodegradable approach to be implemented into cryopreservation formulations.

Electrospun composite cellulose acetate/iron oxide nanoparticles non-woven membranes for magnetic hyperthermia applications.

In the present work composite membranes were produced by combining magnetic nanoparticles (NPs) with cellulose acetate (CA) membranes for magnetic hyperthermia applications. The non-woven CA membranes were produced by electrospinning technique, and magnetic NPs were incorporated by adsorption at fibers surface or by addition to the electrospinning solution. Therefore, different designs of composite membranes were obtained. Superparamagnetic NPs synthesized by chemical precipitation were stabilized either with oleic acid (OA) or dimercaptosuccinic acid (DMSA) to obtain stable suspensions at physiological pH. The incorporation of magnetic NP into CA matrix was confirmed by scanning and transmission electron microscopy. The results showed that adsorption of magnetic NPs at fibers' surface originates composite membranes with higher heating ability than those produced by incorporation of magnetic NPs inside the fibers. However, adsorption of magnetic NPs at fibers' surface can cause cytotoxicity depending on the NPs concentration. Tensile tests demonstrated a reinforcement effect caused by the incorporation of magnetic NPs in the non-woven membrane.

Bio-inspired production of chitosan/chitin films from liquid crystalline suspensions.

Inspired by chitin based hierarchical structures observed in arthropods exoskeleton, this work reports the capturing of chitin nanowhiskers' chiral nematic order into a chitosan matrix. For this purpose, highly crystalline chitin nanowhiskers (CTNW) with spindle-like morphology and average aspect ratio of 24.9 were produced by acid hydrolysis of chitin. CTNW were uniformly dispersed at different concentrations in aqueous suspensions. The suspensions liquid crystalline phase domain was determined by rheological measurements and polarized optical microscopy (POM). Chitosan (CS) was added to the CTNW isotropic, biphasic and anisotropic suspensions and the solvent was evaporated to allow films formation. The Films' morphologies as well as the mechanical properties were explored. A correlation between experimental results and a theoretical model, for layered matrix' structures with fibers acting as a reinforcement agent, was established. The results evidence the existence of two different layered structures, one formed by chitosan layers induced by the presence of chitin and another formed by chitin nanowhiskers layers. By playing on the ratio chitin/chitosan one layered structure or the other can be obtained allowing the tunning of materials' mechanical properties.

Enhanced osteogenesis on biofunctionalized poly(ɛ-caprolactone)/poly(m-anthranilic acid) nanofibers.

Biofunctionalized nanofibers with a desired biological function can be used as a tissue engineering scaffold due to their small fiber diameters and porous structure. In the present study, poly(ɛ-caprolactone)/poly(m-anthranilic acid) nanofibers were biofunctionalized with covalent immobilization of bone morphogenetic protein-2 (BMP-2) through 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide activation. Fourier transform infrared analysis of the nanofiber surfaces confirmed the successful immobilization. The amount of immobilized BMP-2 was determined with bicinchoninic acid protein assay. The nanofibers before and after BMP-2 immobilization were non-cytotoxic and enhanced the attachment and proliferation of Saos-2 cells. Biofunctionalization of nanofibers with BMP-2 promoted in vitro osteogenic activity. The alkaline phosphatase activity and calcium mineralizatio of cells after 14 days of in vitro culture were enhanced on nanofibers with immobilized BMP-2.

Development of a new chitosan hydrogel for wound dressing.

Wound healing is a complex process involving an integrated response by many different cell types and growth factors in order to achieve rapid restoration of skin architecture and function. The present study evaluated the applicability of a chitosan hydrogel (CH) as a wound dressing. Scanning electron microscopy analysis was used to characterize CH morphology. Fibroblast cells isolated from rat skin were used to assess the cytotoxicity of the hydrogel. CH was able to promote cell adhesion and proliferation. Cell viability studies showed that the hydrogel and its degradation by-products are noncytotoxic. The evaluation of the applicability of CH in the treatment of dermal burns in Wistar rats was performed by induction of full-thickness transcutaneous dermal wounds. Wound healing was monitored through macroscopic and histological analysis. From macroscopic analysis, the wound beds of the animals treated with CH were considerably smaller than those of the controls. Histological analysis revealed lack of a reactive or a granulomatous inflammatory reaction in skin lesions with CH and the absence of pathological abnormalities in the organs obtained by necropsy, which supported the local and systemic histocompatibility of the biomaterial. The present results suggest that this biomaterial may aid the re-establishment of skin architecture.