Mammalian cell cryopreservation by using liquid marbles.

Liquid marbles (LMs) are nonsticky droplets covered by micro- or nanometrically scaled particles and obtained by simply rolling small amounts of a liquid in a very hydrophobic powder. Since pioneer work by Aussillous and Quéré, a wide palette of hydrophobic materials for the preparation of LMs, as well as potential applications, have been reported. Because of the bioinspired origin of this concept, the applicability of LMs in biomedicine is gaining increasing attention, with remarkable advances in their use as microbioreactors for blood typing, drug screening, and tumor growth, among others. Herein, we explore the novel use of LMs as a biotechnological tool for the cryopreservation of mammalian cells as an alternative to conventional methods, which typically require the use of cryopreservant agents that commonly associate with some degree of cell toxicity. Murine L929 fibroblasts, a reference cell line for cytotoxicity studies, and poly(tetrafluoroethylene), a hydrophobic polymer widely used in cardiovascular surgery, were selected for the preparation of the cell-containing LMs. Our results reveal that there is a safe range of droplet volumes and cell densities that can be successfully used to cryopreserve mammalian cell lines and recover them after thawing without significantly affecting major cellular parameters such as adhesion, morphology, viability, proliferation, and cell cycle. We envision that progress in the exploration of cell-containing LMs could also open their impact as microreactors for the miniaturization of cytotoxicity procedures of drugs and materials in which powerful tools for cell evaluation such as flow cytometry could be used because of the elevated amount of cells handled.

Different cell responses induced by exposure to maghemite nanoparticles.

Recent advances in nanotechnology have permitted the development of a wide repertoire of inorganic magnetic nanoparticles (NPs) with extensive promise for biomedical applications. Despite this remarkable potential, many questions still arise concerning the biocompatible nature of NPs when in contact with biological systems. Herein, we have investigated how controlled changes in the physicochemical properties of iron oxide NPs at their surface (i.e., surface charge and hydrodynamic size) affect, first, their interaction with cell media components and, subsequently, cell responses to NP exposure. For that purpose, we have prepared iron oxide NPs with three different coatings (i.e., dimercaptosuccinic acid - DMSA, (3-aminopropyl)triethoxysilane - APS and dextran) and explored the response of two different cell types, murine L929 fibroblasts and human Saos-2 osteoblasts, to their exposure. Interestingly, different cell responses were found depending on the NP concentration, surface charge and cell type. In this sense, neutral NPs, as those coated with dextran, induced negligible cell damage, as their cellular internalization was significantly reduced. In contrast, surface-charged NPs (i.e., those coated with DMSA and APS) caused significant cellular changes in viability, morphology and cell cycle under certain culture conditions, as a result of a more active cellular internalization. These results also revealed a particular cellular ability to detect and remember the original physicochemical properties of the NPs, despite the formation of a protein corona when incubated in culture media. Overall, conclusions from these studies are of crucial interest for future biomedical applications of iron oxide NPs.

Deep eutectic solvent-assisted synthesis of biodegradable polyesters with antibacterial properties.

Bacterial infection related to the implantation of medical devices represents a serious clinical complication, with dramatic consequences for many patients. In past decades, numerous attempts have been made to develop materials with antibacterial and/or antifouling properties by the incorporation of antibiotic and/or antiseptic compounds. In this context, deep eutectic solvents (DESs) are acquiring increasing interest not only as efficient carriers of active principle ingredients (APIs) but also as assistant platforms for the synthesis of a wide repertoire of polymer-related materials. Herein, we have successfully prepared biodegradable poly(octanediol-co-citrate) polyesters with acquired antibacterial properties by the DES-assisted incorporation of quaternary ammonium or phosphonium salts into the polymer network. In the resulting polymers, the presence of these salts (i.e., choline chloride, tetraethylammonium bromide, hexadecyltrimethylammonium bromide, and methyltriphenylphosphonium bromide) inhibits bacterial growth in the early postimplantation steps, as tested in cultures of Escherichia coli on solid agar plates. Later, positive polymer cytocompatibility is expected to support cell colonization, as anticipated from in vitro preliminary studies with L929 fibroblasts. Finally, the attractive elastic properties of these polyesters permit matching those of soft tissues such as skin. For all of these reasons, we envisage the utility of some of these antibacterial, biocompatible, and biodegradable polyesters as potential candidates for the preparation of antimicrobial wound dressings. These results further emphasize the enormous versatility of DES-assisted synthesis for the incorporation, in the synthesis step, of a wide palette of APIs into polymeric networks suitable for biomedical applications.

In situ precipitation of amorphous calcium phosphate and ciprofloxacin crystals during the formation of chitosan hydrogels and its application for drug delivery purposes.

The immobilization of more than one single substance within the structure of a biocompatible polymer provides multifunctional biomaterials with attractive and enhanced properties. In the context of bone tissue engineering, it could be of great interest to synthesize a biomaterial that simultaneously contains amorphous calcium phosphate (ACP), to favor calcium and phosphate precipitation and promote osteogenesis, and an antibiotic such as ciprofloxacin (CFX) that can, eventually, avoid infections resulting after surgical scaffold implantation. However, the co-immobilization of multiple substances is by no means a trivial issue because of the enhanced number of interactions that can take place. One of the main issues is controlling not only the diverse solid forms that individual substances can eventually adopt, but also the forces responsible for the self-organization of the individual components. The latter determines whether phase-separated structures or conjugated architectures are obtained and, consequently, may dramatically affect their functionality. Herein, we have observed-by SEM, TEM, and solid-state NMR-that enzymatically-assisted coprecipitation of ACP and CFX resulted in phase-separated structures. Thus, CFX crystals showed identical morphology to that obtained in the absence of ACP, but the size was smaller. Neither the size nor the morphology of ACP exhibited significant differences whether precipitated with or without CFX, but, in the former case, ACP was stabilized over a wider range of pH and temperature. Finally, by using this methodology and the ice segregation induced self-assembly process (ISISA), we have successfully co-immobilized ACP and CFX in chitosan-based scaffolds. Interestingly, the presence of ACP exerted significant control on the CFX release from these materials.

Synthesis of novel lidocaine-releasing poly(diol-co-citrate) elastomers by using deep eutectic solvents.

Poly(octanediol-co-citrate) elastomers containing high loading of lidocaine were synthesized at temperatures below 100 °C by means of using deep eutectic mixtures of 1,8-octanediol and lidocaine. The preservation of lidocaine integrity resulted in high-capacity drug-eluting elastomers.

Poly(diol-co-citrate)s as novel elastomeric perivascular wraps for the reduction of neointimal hyperplasia.

The synthesis of poly(diol-co-citrate) elastomers that are biocompatible with vascular cells and can modulate the kinetics of the NO release based on the diol of selection is reported. NO-mediated cytostatic or cytotoxic effects can be controlled depending on the NO dose and the exposure time. When implanted in vivo in a rat carotid artery injury model, these materials demonstrate a significant reduction of neointimal hyperplasia. This is the first report of a NO-releasing polymer fabricated in the form of an elastomeric perivascular wrap for the treatment of neointimal hyperplasia. These elastomers also show promise for other cardiovascular pathologies where NO-based therapies could be beneficial.

Biodegradable nitric oxide-releasing poly(diol citrate) elastomers.

We have developed novel poly(diol citrate) elastomers, which are capable of providing localized and sustained release of nitric oxide (NO). The elastomer prepolymer was obtained by condensation of citric acid, 1,8-octanediol, and N,N'-bis(2-hydroxyethyl)ethylenediamine at 130 degrees C for 40 min. Films were prepared by solvent casting followed by crosslinking at 80 degrees C for 4 days. Mechanical properties were tested. NO-releasing expanded poly(tetrafluoroethylene) (ePTFE) vascular grafts were fabricated by coating the graft's lumen with the prepolymer and crosslinking it at 80 degrees C for 4 days prior to diazeniumdiolation. Samples were diazeniumdiolated via exposure to pressurized NO. Cell compatibility was assessed by monitoring the proliferation of porcine aortic smooth muscle cells (PASMC) on the elastomers. Degradation in phosphate buffer saline (PBS) (pH = 7) at 37 degrees C was evaluated for up to 6 weeks. The secondary amine-containing poly(diol citrate) films had a Young's modulus that ranged from 5.91 to 32.64 MPa, an ultimate tensile stress that ranged from 1.47 to 10.71 MPa, and an elongation at break from 200 to 260%, depending on the content of secondary amine in the feed monomer. These elastomers were degradable and compatible with PASMC. Furthermore, degradation rate was found to be independent of the content of secondary amines in the prepolymer. The NO release from diazeniumdiolated films and ePTFE grafts was sustained for two days. In conclusion, these novel diazeniumdiolated polyester elastomers may be useful in medical devices that require blood contact or control of cell proliferation.

L929 fibroblast and Saos-2 osteoblast response to hydroxyapatite-betaTCP/agarose biomaterial.

Biphasic calcium phosphate, a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), has been successfully used as an excellent bone graft substitute because of the HA capacity for direct interaction with bone and the beta-TCP resorption properties. Agarose has been recently mixtured with ceramics as natural biodegradable binder to increase the biomaterial flexibility facilitating its placement into the bone defect. In this study, the behavior of L929 fibroblasts and Saos-2 osteoblasts cultured on hydroxyapatite-betaTCP/agarose disks has been evaluated. Both cell types adhere and proliferate on the biomaterial surface maintaining their characteristic morphology. Transitory changes on cell cycle, size, and complexity are observed. The biomaterial induces apoptosis in Saos-2 osteoblasts but not in fibroblasts. A transitory stimulation of fibroblast mitochondrial activity is observed. This effect remains in osteoblasts after 9 days of culture showing a higher sensitivity of this cell type. However, the intracellular reactive oxygen species content and the lactate dehydrogenase release of Saos-2 osteoblasts indicate that hydroxyapatite-betaTCP/agarose does not induce oxidative stress in this cell type and confirm the integrity of the osteoblast plasma membrane. These results underline the good biocompatibility of hydroxyapatite-betaTCP/agarose disks and its potential utility for bone substitution and repair.

In vitro positive biocompatibility evaluation of glass-glass ceramic thermoseeds for hyperthermic treatment of bone tumors.

A new kind of magnetic thermoseed for bone tissue engineering has been synthesized. The materials used are specially designed to restore bone tissue after tumor extirpation, because they exhibit bioactive behavior and the ability to act as thermoseeds for cancer treatment using hyperthermia. The L929 cell line of mouse fibroblasts has been used in a wide biocompatibility study concerning cell proliferation and morphology studies, mitochondrial function determination, lactate dehydrogenase measurement, and flow cytometry studies, including cell cycle analysis, cell size and complexity, and intracellular reactive oxygen species content. The results presented in this work indicate that these bioactive magnetic materials are highly biocompatible and show greater cell response for thermoseeds with a higher magnetic phase content. There were no significant alterations detected in the cell cycle, and the interaction between fibroblasts and the different mixtures did not induce significant apoptosis.

Endothelial cells derived from circulating progenitors as an effective source to functional endothelialization of NaOH-treated poly(epsilon-caprolactone) films.

Biomaterials have been widely used to prepare synthetic vascular grafts over the past thirty years, but the inherent thrombogenicity of their surface can lead to graft failure. Endothelial progenitor cells (EPC) are circulating premature cells able to differentiate in either myocardial or endothelial cells (EC). The therapeutic potential of these cells and its easy obtaining technique are important reasons why these cells could be used to improve the performance of vascular grafts. In this study, two different stages of differentiation of EC derived from EPC (EC(1) and EC(2)) were characterized and cultured on poly(epsilon-caprolactone) (PCL) films treated with NaOH (PCL-NaOH). We investigated by immunolabeling the expression of CD31, von Willebrand factor (vWF), and endothelial nitric oxide synthase (eNOS) in these cells during the differentiation process. The proliferation, cell cycle, and mitochondrial function of EC(2) cultured on PCL-NaOH were evaluated at different times. The effect of this biomaterial on the nitric oxide (NO) content was also measured. The mature EC obtained from circulating progenitor cells (EC(2)) showed an appropriate growth and functionality on NaOH-treated films. They conserved their capacity to define vessel-like structures in culture and increased their basal NO production. These results underline the potential usefulness of these EC(2) to get a functional endothelialization of polymers with applications in vascular tissue engineering.

Mitochondrial membrane potential and reactive oxygen species content of endothelial and smooth muscle cells cultured on poly(epsilon-caprolactone) films.

A transitory but significant stimulation of mitochondrial activity, increase of reactive oxygen species (ROS) and oxidative stress were previously observed in L929 fibroblasts cultured on poly(epsilon-caprolactone) (PCL) films. ROS, mainly formed in mitochondria, play a physiological role but an excessive production can promote endothelial dysfunction, cause oxidative injury to vascular cells, oxidize lipoproteins and accelerate atherothrombogenesis. On the other hand, mitochondria have a crucial position in programmed cell death control and are responsible for ATP synthesis through the coupling of oxidative phosphorylation to respiration. This coupling requires the existence of a mitochondrial membrane potential (Deltapsi(m)). The aim of the present study was to evaluate by flow cytometry the ROS content and Deltapsi(m) of both endothelial (EC) and smooth muscle cells (SMC) cultured on PCL films as a potential substrate for vascular graft development. Cell size, internal complexity and cell cycle were also analyzed to detect the possible appearance of the subG(1) cell fraction, characteristic of apoptotic cells. The effect of treating PCL films with NaOH before culture was also studied. PCL decreases the ROS content of EC during the culture but produces an increase of these levels in SMC after 7 days. PCL also induces variations of Deltapsi(m) which show a significant parallelism with the changes observed in ROS levels proving the importance and sensitivity of these measurements as indicators of the mitochondrial function. The treatment of PCL with NaOH decreases these effects demonstrating the benefits of increasing the surface hydrophilicity before cell culture which improves cell adhesion and proliferation and reduces oxidative stress. Since no important changes have been detected in subG(1) fraction of EC and SMC cultured on either PCL or PCL-NaOH, the changes of Deltapsi(m) observed in the present study cannot be related to apoptosis. These results confirm the potential utility of PCL as a suitable scaffold in Vascular Tissue Engineering.

Alkaline-treated poly(epsilon-caprolactone) films: degradation in the presence or absence of fibroblasts.

In the first stage, we observed the study of the degradation behavior of alkaline-treated poly(epsilon-caprolactone) (PCL) in two biologically-related media: phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) for 18 months, finding a much accelerated degradation in the last one. As expected, the degradation in the presence of cells is much pronounced even considering that the study is limited to 6 months. The characterization of the degraded substrates by chemiluminescence (CL) allows to explain the modifications of the substrate and their relations with transitory oxidative stress phenomena described in the fibroblasts seeded onto the PCL membranes.

Transitory oxidative stress in L929 fibroblasts cultured on poly(epsilon-caprolactone) films.

Poly(epsilon-caprolactone) (PCL) is considered as a potential substrate for wide medical applications. In previous studies we carried out the in vitro biocompatibility assessment of PCL films using L929 mouse fibroblasts, obtaining good cell behaviour but a transitory stimulation of mitochondrial activity and cell retraction. Reactive oxygen species (ROS), mainly formed in mitochondria, can impair the function of several cellular components and produce cell oxidative stress by changing the normal red-ox status of the major cell antioxidants as glutathione. The aim of this study was to measure intracellular ROS production and glutathione content of L929 fibroblasts cultured on PCL films. Cell size, internal complexity, cell cycle and lactate dehydrogenase release were also evaluated. The films were treated with NaOH before culture to improve the cell-polymer interaction. PCL induces a transitory but significant oxidative stress in L929 fibroblasts. The treatment of PCL films with NaOH reduces this effect. PCL also induces transitory changes on cell size and complexity. Nevertheless, after 7 days in culture, cells reach control levels for all the studied parameters. Neither cell cycle nor membrane integrity appears affected by this oxidative stress respect to control cells at any culture time. These results underline the cytocompatibility of PCL films and, therefore, its potential utility as a suitable scaffold in tissue engineering.

Anisakis simplex only provokes allergic symptoms when the worm parasitises the gastrointestinal tract.

We analysed patients with allergic or digestive symptoms after seafood ingestion in order to assess a correct diet in Anisakis simplex sensitised individuals. A total of 120 patients who suffered allergic and/or digestive symptoms after marine food ingestion were studied. We performed skin prick tests for A. simplex and seafood, total serum and specific serum immunoglobulin E to A. simplex in the acute stage and 1 month later. A gastroscopy was carried out to find larvae in those patients with persistent abdominal pain. A challenge with non-infective larvae was performed to assess a correct diet. Some 96 patients were sensitised to A. simplex. Gastroscopy was performed in 47 and we detected larvae in 24. We compared symptoms, skin tests, total and specific IgE and the latency of appearance of symptoms in patients positive for Anisakis larvae, patients without larvae at gastroscopy and patients without digestive symptoms. There was no difference among the groups. We challenged 22 patients with frozen A. simplex larvae. After allowing deep-frozen seafood in the diet for more than 2 years, no patient suffered a reaction. At this time, we allowed all our patients well-frozen seafood without any allergic reaction occurring. Allergic symptoms are the most frequent manifestation of A. simplex parasitism. We could not find any patient allergic to the thermostable proteins of parasite.

Polyclonal autoantibodies against C1 inhibitor in a case of acquired angioedema.

BACKGROUND: Angioedema attributable to acquired C1 inhibitor (C1-INH) deficiency is a rare disease related to lymphoproliferative disorders or autoantibodies to Cl inhibitor. We describe a patient with angioedema and autoantibodies to C1 inhibitor. OBJECTIVE: To study the characteristics of autoantibodies to C1-INH in a patient with acquired angioedema. METHODS: Autoantibodies to Cl-INH were measured by enzyme-linked immunoadsorbent assay. Immunoglobulin (Ig)G autoantibody was purified by affinity chromatography on a protein G agarose column. We developed an enzyme-linked immunoadsorbent assay to determine whether the autoantibodies were directed against the C1-INH active center. RESULTS: IgM and mainly C1-INH IgG autoantibodies were detected; both had kappa and lambda chains. No monoclonal component was detected. The autoantibodies were directed against the Cl-INH active center. After various treatment strategies were attempted, an effective clinical response was attained with antifibrinolytic therapy. CONCLUSION: A case of acquired angioedema because of C1-INH deficiency was found to be attributable to the presence of polyclonal autoantibodies to C1-INH.