Evaluation of water properties in HEA–HEMA hydrogels swollen in aqueous-PEG solutions using thermoanalytical techniques

Hydrogels are polymeric materials used in many pharmaceutical and biomedical applications due to their ability to form 3D hydrophilic polymeric networks, which can absorb large amounts of water. In the present work, polyethylene glycols (PEG) were introduced into the hydrogel liquid phase in order to improve the mechanical properties of hydrogels composed of 2-hydroxyethylacrylate and 2-hydroxyethylmethacrylate (HEA–HEMA) synthesized with different co-monomer compositions and equilibrated in water or in 20 % water–PEG 400 and 600 solutions. The thermoanalytical techniques [differential scanning calorimetry (DSC) and thermogravimetry (TG)] were used to evaluate the amount and properties of free and bound water in HEA–HEMA hydrogels. The internal structure and the mechanical properties of hydrogels were studied using scanning electron microscopy and friability assay. TG “loss-on-drying” experiments were applied to study the water-retention properties of hydrogels, whereas the combination of TG and DSC allowed estimating the total amount of freezable and non-freezing water in hydrogels. The results show that the addition of viscous co-solvent (PEG) to the liquid medium results in significant improvement of the mechanical properties of HEA–HEMA hydrogels and also slightly retards the water loss from the hydrogels. A redistribution of free and bound water in the hydrogels equilibrated in mixed solutions containing 20 vol% of PEGs takes place.

Lipopeptides: from self-assembly to bioactivity

This Feature Article discusses several classes of lipopeptide with important biomedical applications as antimicrobial and antifungal agents, in immune therapies and in personal care applications among others. Two main classes of lipopeptide are considered: (i) bacterially-expressed lipopeptides with a cyclic peptide headgroup and (ii) linear lipopeptides (with one or more lipid chains) based on bio-derived and bio-inspired amino acid sequences with current clinical applications. The applications are briefly summarized, and the biophysical characterization of the molecules is reviewed, with a particular focus on self-assembly. For several of these types of biomolecule, the formation of micelles above a critical micelle concentration has been observed while others form bilayer structures, depending on conditions of pH and temperature. As yet, there are few studies on the possible relationship between self-assembly into structures such as micelles and bioactivity of this class of molecule although this is likely to attract further attention.

Enzymatically-triggered, isothermally responsive polymers: re-programming poly(oligoethylene glycols) to respond to phosphatase

Polymers which can respond to externally applied stimuli have found much application in the biomedical field due to their (reversible) coil–globule transitions. Polymers displaying a lower critical solution temperature are the most commonly used, but for blood-borne (i.e., soluble) biomedical applications the application of heat is not always possible, nor practical. Here we report the design and synthesis of poly(oligoethylene glycol methacrylate)-based polymers whose cloud points are easily varied by alkaline phosphatase-mediated dephosphorylation. By fine-tuning the density of phosphate groups on the backbone, it was possible to induce an isothermal transition: A change in solubility triggered by removal of a small number of phosphate esters from the side chains activating the LCST-type response. As there was no temperature change involved, this serves as a model of a cell-instructed polymer response. Finally, it was found that both polymers were non cytotoxic against MCF-7 cells (at 1 mg·mL–1), which confirms promise for biomedical applications.

An adhesive elastomeric supramolecular polyurethane healable at body temperature

In this paper, we report the synthesis and healing ability of a non-cytotoxic supramolecular polyurethane network whose mechanical properties can be recovered efficiently (> 99%) at the temperature of the human body (37 ºC). Rheological analysis revealed an acceleration in the drop of the storage modulus above 37 ºC, on account of the dissociation of the supramolecular polyurethane network, and this decrease in viscosity enables the efficient recovery of the mechanical properties. Microscopic and mechanical characterisation has shown that this material is able to recover mechanical properties across a damage site with minimal contact required between the interfaces and also demonstrated that the mechanical properties improved when compared to other low temperature healing elastomers or gel-like materials. The supramolecular polyurethane was found to be non-toxic in a cytotoxicity assay carried out in human skin fibroblasts (cell viability > 94% and non-significantly different compared to the untreated control). This supramolecular network material also exhibited excellent adhesion to pig skin and could be healed completely in situ post damage indicating that biomedical applications could be targeted, such as artificial skin or wound dressings with supramolecular materials of this type.

The effect of DMSA-functionalized magnetic nanoparticles on transendothelial migration of monocytes in the murine lung via a beta(2) integrin-dependent pathway

Magnetic nanoparticles surface-functionalized with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute an innovative and promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. Transendothelial migration of leukocytes in the lung is a side effect of endovenous administration of MNPs-DMSA. Using cytologic and phenotypic analysis of murine bronchoalveolar lavage cells, we identified monocytes/macrophages as the main subpopulation of leukocytes involved in this process. Moreover, ultrastructural analysis revealed the presence of nanoparticles inside of numerous macrophages from bronchoalveolar lavage. MNPs-DMSA at concentrations as high as 1 X 10(15) nanoparticles/mL had no toxic effects on macrophages, as evidenced by 3-(4, 5-dimethylthiazolyi-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Notably, MNPs-DMSA up-regulated the mRNA expression of E, L- and P-selectin and macrophage-1 antigen in the murine lung. Upregulation of these cell adhesion molecules was associated with an increased concentration of tumor necrosis factor-alpha in lung. Finally, the critical relevance of the beta(2) integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our results characterize mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung, and identify beta(2) integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. (C) 2009 Elsevier Ltd. All rights reserved.

Tissue response around silicon nitride implants in rabbits

The chemical and dimensional stability associated with suitable fracture toughness and propitious tribological characteristics make silicon nitride-based ceramics potential candidates for biomedical applications, mainly as orthopedic implants. Considering this combination of properties, silicon nitride components were investigated in relation to their biocompatibility. For this study, two cylindrical implants were installed in each tibia of five rabbits and were kept in the animals for 8 weeks. During the healing time, tissue tracers were administrated in the animals so as to evaluate the bone growth around the implants. Eight weeks after the surgery, the animals were euthanized and histological analyses were performed. No adverse reactions were observed close to the implant. The osteogenesis process occurred during the entire period defined by the tracers. However, this process occurred more intensely 4 weeks after the surgery. In addition, the histological analyses showed that bone growth occurred preferentially in the cortical areas. Different kinds of tissue were identified on the implant surface, characterized by lamellar bone tissue containing osteocytes and osteons, by a noncalcified matrix containing osteoblasts, or by the presence of collagen III, which may change to collagen I or remain as a fibrous tissue. The results demonstrated that silicon nitride obtained according to the procedure proposed in this research is a biocompatible material. (c) 2007 Wiley Periodicals, Inc.

Synthesis and characterization of glass-ceramic microspheres for thermotherapy

Glass microspheres containing radionuclides are used to treat liver cancer. A promising alternative therapy is being developed based on the magnetic hyperthermia which is related to the heat supplied by a magnetic material under an alternating current magnetic field. The advantage of this option is that most of killed cells are cancer cells which are more susceptible to the temperature raise. In the present work aluminum iron silicate glasses containing minor glass modifiers and nucleating agents were synthesized as irregular shape particles which were further transformed in microspheres by using a petrol liquefied gas-oxygen torch. The optimized processing parameters which lead to microspheres that give a response to the magnetic field were determined. The dissolution rate in water at 90 degrees C was determined to be 3 x 10(-8) g cm(-2) min(-1). The microsphere size distribution was determined by laser scattering. The crystalline phase responsible for the ferromagnetic response was identified as magnetite. Since this phase has a high saturation magnetization and high Curie temperature, it is potentially useful for biomedical applications. The hysteresis magnetic loop was measured for materials produced in different conditions, and some of them showed to be appropriated for thermotherapy. The ratio Fe(3+)/Fe(total) was determined by Mossbauer spectroscopy. (C) 2010 Elsevier B.V. All rights reserved.

Two-Photon Polymerization for Fabricating Structures Containing the Biopolymer Chitosan

Two-photon polymerization is a powerful tool for fabricating three-dimensional micro/nano structures for applications ranging from nanophotonics to biology. To tailor such structure for specific purposes it is often important to dope them. In this paper we report on the fabrication of structures, with nanometric surface features (resolution of approximately 700 nm), using two-photon polymerization of an acrylic resin doped with the biocompatible polymer chitosan using a guest-host scheme. The fluorescence background in the Raman spectrum indicates the presence of chitosan throughout the structure. Mechanical characterization reveals that chitosan does not affect the mechanical properties of the host acrylic resin and, consequently, the structures exhibit excellent integrity. The approach presented in this work can be used in the fabrication of micro- and nanostructures containing biopolymers for biomedical applications.

Block Copolymers Containing (R)-3-Hydroxybutyrate and Isosorbide Succinate or (S)-Lactic Acid Mers

A new aliphatic block copolyester was synthesized in bulk from transesterification techniques between poly((R)-3-hydroxybutyrate) (PHB) and poly(isosorbide succinate) (PIS). Additionally, other two block copolyesters were synthesized in bulk either from transesterification reactions involving PHB and poly(l-lactide) (PLLA) or from ring-opening copolymerization of l-lactide and hydroxyl-terminated PHB, as result of a previous transesterification reactions with isosorbide. Two-component blends of PHB and PIS or PLLA were also prepared as comparative systems. SEC, MALDI-TOF mass spectrometry (MALDI-TOFMS), (1)H and (13)C NMR spectroscopy, WAXD, solubility tests, and TG thermal analysis were used for characterization. The block copolymer structures of the products were evidenced by MALDI-TOFMS, (13)C NMR, and WAXD data. The block copolymers and the corresponding binary blends presented different solubility properties, as revealed by solubility tests. Although the incorporation of PIS sequences into PHB main backbone did not enhance the thermal stability of the product, it reduced its crystallinity, which could be advantageous for faster biodegradation rate. These products, composed of PHB and PIS or PLLA sequences, are an interesting alternative in biomedical applications.

Bacterial cellulose-laponite clay nanocomposites

A novel material comprised of bacterial cellulose (BC) and Laponite clay with different inorganic organic ratios (m/m) was prepared by the contact of never-dried membranes of BC with a previous dispersion of clay particles in water. Field emission scanning electron microscopy (FE-SEM) data of composite materials revealed an effective adhesion of clay over the surface of BC membrane; inorganic particles also penetrate into the polymer bulk, with a significant change of the surface topography even at 5% of clay loading. As a consequence, the mechanical properties are deeply affected by the presence of clay, increasing the values of the Young modulus and the tensile strength. However the maximum strain is decreased when the clay content is increased in the composite in comparison to pristine BC. The main weight loss step of the composites is shifted towards higher temperatures compared to BC, indicating that the clay particles slightly protect the polymer from thermal and oxidative decomposition. (C) 2010 Elsevier Ltd. All rights reserved.

Effects of alloying elements on the cytotoxic response of titanium alloys

Titanium alloys, alloys, especially beta-type alloys containing beta-stabilizing elements, constitute a highly versatile category of metallic materials that have been under constant development for application in orthopedics and dentistry. This type of alloy generally presents a high mechanical strength-to-weight ratio, excellent corrosion resistance and low elastic modulus. The purpose of this study is to evaluate the cytotoxicity and adhesion of fibroblast cells on titanium alloy substrates containing Nb, Ta, Zr, Cu, Sn and Mo alloying elements. Cells cultured on polystyrene were used as controls. In vitro results with Vero cells demonstrated that the tested materials, except Cu-based alloy, presented high viability in short-term testing. Adhesion of cells cultured on disks showed no differences between the materials and reference except for the Ti-Cu alloy, which showed reduced adhesion attributed to poor metabolic activity. Titanium alloys with the addition of Nb, Ta, Zr, Sn and Mo elements show a promising potential for biomedical applications. (C) 2011 Elsevier B.V. All rights reserved.

Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

Encapsulation of single hMSCs in polyelectrolyte shells

The main objective of this Thesis was to encapsulate single viable cells within polyelectrolyte films using the Layer-by-Layer (LbL) technique. Most of the experiments used human mesenchymal stem cells (MSCs) whose characteristics (capacity of selfrenewal and potential to differentiate into several types of cells) make them particularly interesting to be used in biomedical applications. Also, most of the experiments used alginate (ALG) as the anionic polyelectrolyte and chitosan (CHI) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Hyaluronic acid (HA) was also tested as an anionic polyelectrolyte. At the beginning of the work, the experimental conditions necessary to obtain the encapsulation of individual cells were studied and established. Through fluorescence microscopy visualization by staining the cell nucleus and using polyelectrolytes conjugated to fluorescent dyes, it was possible to prove the obtainment of capsules containing one single cell inside. Capsules aggregation was an observed problem which, despite the efforts to design an experimental process to avoid this situation (namely, by playing with cell concentration and different means of re-suspending and stirring the cells), was not completely overcome. In a second part of the project, single cells were encapsulated within polyelectrolyte layers made of CHI/ALG, PAH/ALG and PAH/HA and their viability was evaluated through the resazurin reduction assay and the Live/Dead assay. In these experiments, during the LbL process, polyelectrolyte solutions were used at a concentration of 1mg/mL based on literature. In general, the viability of the encapsulated cells was shown to be very low/absent. Then, as a consequence of the lack of viability of cells encapsulated within polyelectrolyte layers, the LbL technique was applied in cells growing adherent to the surface of cell culture plates. The cells were cultured like in a sandwich, between the surface of the cell culture dish and the polyelectrolyte layers. Also here, the polyelectrolyte solutions were used at a concentration of 1mg/mL during the LbL process. Surprisingly, cell viability was also absent in these systems. A systematic study (dose-effect study) was performed to evaluate the effect of the concentration of the individual polyelectrolytes (ALG, CHI and PAH were studied) in cell viability. Experiments were performed using cells growing adherent to the surface of cell culture plates. The results pointed out that a very high (cytotoxic) concentration of polyelectrolytes had been in use. Also, in general, PAH was much more cytotoxic than CHI, whereas ALG was the less cytotoxic polyelectrolyte. Finally, using alginate and chitosan solutions with adequate concentrations (low concentrations: 50ng/mL and 1μg/mL), the encapsulation of single viable cells was again attempted. Once again, the encapsulated cells were not shown to be viable. In conclusion, the viability of the encapsulated cells is not only dependent on the cytotoxic characteristics (or combined cytotoxic characteristics) of the polyelectrolytes but it seems that, when detached from the culture plates, the cells become too fragile and lose their viability very easily.

New bacterial cellulose nanocomposites prepared by in situ radical polymerization of methacrylate monomers

Bacterial cellulose/polymethacrylate nanocomposites have received attention in numerous areas of study and in a variety of applications. The attractive properties of methacrylate polymers and bacterial cellulose, BC, allow the synthesis of new nanocomposites with distinct characteristics. In this study, BC/poly(glycidylmethacrylate) (BC/PGMA) and BC/poly(ethyleneglycol)methacrylate (BC/PPEGMA) nanocomposites were prepared through in situ free radical polymerization of GMA and PEGMA, respectively. Ammonium persulphate (APS) was used as an initiator and N,N’methylenebisacrilamide (MBA) was used as a crosslinker in BC/PGMA. Chemical composition, morphology, thermal stability, water absorption, mechanic and surface properties were determined through specific characterization techniques. The optimal polymerization was obtained at (1:2) for BC/PGMA, (1:2:0.2) ratio for BC/GMA/MBA and (1:20) for BC/PPEGMA, with 0.5% of initiator at 60 ºC during 6 h. A maximum of 67% and 87% of incorporation percentage was obtained, respectively, for the nanocomposites BC/PGMA/MBA and BC/PPEGMA. BC/PGMA nanocomposites exhibited an increase of roughness and compactation of the three-dimensional structure, an improvement in the thermal and mechanical properties, and a decrease in their swelling ability and crystallinity. On the other hand, BC/PPEGMA showed a decrease of stiffness of three-dimensional structure, improvement in thermal and mechanical properties, an increase in their swelling ability and a decrease the crystallinity. Both BC/polymethacrylate nanocomposites exhibited a basic surface character. The acid treatment showed to be a suitable strategy to modifiy BC/PGMA nanocomposites through epoxide ring-opening reaction mechanism. Nanocomposites became more compact, smooth and with more water retention ability. A decrease in the thermal and mechanical proprieties was observed. The new nanocomposites acquired properties useful to biomedical applications or/and removal of heavy metals due to the presence of functional groups.

Efeito do tratamento térmico do titânio sobre a proliferação de células pré-osteoblásticas

Titanium is a biomaterial widely employed in biomedical applications (implants, prostheses, valves, stents). Several heat treatments are usually used in order to obtain physical properties required to different applications. This work studied the influence of the heat treatment on microstructure of commercial pure titanium, and their consequences in growth and proliferation of MC3T3-E1 cells. Discs of titanium were treated in different temperatures, and characterized by optical microscopy, image analysis, wettabillity, roughness, hardness and X-ray diffraction. After the heat treatment, significant modifications in these properties were observed. Pattern images of titanium, before and after the cell culture, were compared by overlapping to analyze the influence of microstructure in microstructure and preferences guidance cells. However, in general, titanium discs that showed a higher residual strength also presented an increase of cells numbers on surface