Characterization of aqueous interactions of copper-doped phosphate-based glasses by vapour sorption

Owing to their adjustable dissolution properties, phosphate-based glasses (PGs) are promising materials for the controlled release of bioinorganics, such as copper ions. This study describes a vapour sorption method that allowed for the investigation of the kinetics and mechanisms of aqueous interactions of PGs of the formulation 50P₂O₅–30CaO–(20–x)Na₂O–xCuO (x = 0, 1, 5 and 10 mol.%). Initial characterization was performed using ³¹P magic angle spinning nuclear magnetic resonance and attenuated total reflectance–Fourier transform infrared spectroscopy. Increasing CuO content resulted in chemical shifts of the predominant Q² NMR peak and of the (POP)_as and (PO⁻) Fourier transform infrared absorptions, owing to the higher strength of the POCu bond compared to PONa. Vapour sorption and desorption were gravimetrically measured in PG powders exposed to variable relative humidity (RH). Sorption was negligible below 70% RH and increased exponentially with RH from 70 to 90%, where it exhibited a negative correlation with CuO content. Vapour sorption in 0% and 1% CuO glasses resulted in phosphate chain hydration and hydrolysis, as evidenced by protonated Q⁰(1H) and Q¹(1H) species. Dissolution rates in deionized water showed a linear correlation (R² > 0.99) with vapour sorption. Furthermore, cation release rates could be predicted based on dissolution rates and PG composition. The release of orthophosphate and short polyphosphate species corroborates the action of hydrolysis and was correlated with pH changes. In conclusion, the agreement between vapour sorption and routine characterization techniques in water demonstrates the potential of this method for the study of PG aqueous reactions.     

Selectivity of binding of PEGs and PEG-like oligomers to anti-PEG antibodies induced by methoxyPEG-proteins

The use of methoxypoly(ethylene glycol) (mPEG) in PEG conjugates of proteins and non-protein therapeutic agents has led to the recognition that the polymer components of such conjugates can induce anti-PEG antibodies (anti-PEGs) that may accelerate the clearance and reduce the efficacy of the conjugates. Others have classified anti-PEGs as “methoxy-specific” or “backbone-specific”. The results of our previous research on anti-PEGs in the sera of rabbits immunized with mPEG or hydroxyPEG (HO-PEG) conjugates of three unrelated proteins were consistent with that classification (Sherman, M.R., et al., 2012. Bioconjug. Chem. 23, 485–499). Enzyme-linked immunosorbent assays (ELISAs) were performed on rabbit antisera and rabbit monoclonal anti-PEGs with competitors including 10 kDa mPEG, 10 kDa PEG diol and six linear or cyclic oligomers of oxyethylene (CH₂CH₂O), with molecular weights of ca. 150–264 Da. Our results demonstrate that (1) the binding affinities of anti-mPEGs depend more on the backbone lengths of the polymers and the hydrophobicities of their end-groups than on their resemblance to the methoxy terminus of the immunogenic polymer; (2) anti-PEGs raised against HO-PEG-proteins are not directed against the terminal hydroxy group, but against the backbone; (3) rabbit anti-PEGs bind to and distinguish among PEG-like oligomers with as few as three oxyethylene groups; and (4) none of the monoclonal or polyclonal anti-PEGs was absolutely “methoxy-specific” or “backbone-specific”, but displayed distinct relative selectivities. If these results are relevant to human immune responses, the clinical use of stable conjugates of HO-PEG with proteins and non-protein therapeutic agents would be expected to produce fewer and less intense immune responses than those induced by conjugates with mPEG or PEGs with larger alkoxy groups.     

Fluorescence,aggregation properties and FT-IR microspectroscopy of elastin and collagen fibers

Histological and histochemical observations support the hypothesis that collagen fibers can link to elastic fibers. However, the resulting organization of elastin and collagen type complexes and differences between these materials in terms of macromolecular orientation and frequencies of their chemical vibrational groups have not yet been solved. This study aimed to investigate the macromolecular organization of pure elastin, collagen type I and elastin–collagen complexes using polarized light DIC-microscopy. Additionally, differences and similarities between pure elastin and collagen bundles (CB) were investigated by Fourier transform-infrared (FT-IR) microspectroscopy. Although elastin exhibited a faint birefringence, the elastin–collagen complex aggregates formed in solution exhibited a deep birefringence and formation of an ordered-supramolecular complex typical of collagen chiral structure. The FT-IR study revealed elastin and CB peptide NH groups involved in different types of H-bonding. More energy is absorbed in the vibrational transitions corresponding to CH, CH₂ and CH₃ groups (probably associated with the hydrophobicity demonstrated by 8-anilino-1-naphtalene sulfonic acid sodium salt [ANS] fluorescence), and to νCN, δNH and ωCH₂ groups of elastin compared to CB. It is assumed that the α-helix contribution to the pure elastin amide I profile is 46.8%, whereas that of the B-sheet is 20% and that unordered structures contribute to the remaining percentage. An FT-IR profile library reveals that the elastin signature within the 1360–1189 cm⁻¹ spectral range resembles that of Conex–Toray aramid fibers.     

In situ characterization of change in superficial organic components of thermoacidophilic archaeon Acidianus manzaensis YN-25

For the first time, synchrotron radiation (SR) -based carbon K-edge X-ray absorption near edge structure (XANES) spectroscopy in-situ characterization was conducted to evaluate the evolution of superficial (about 10 nm) organic components of extracellular polymeric substances (EPS) of thermoacidophilic archaeon Acidianus manzaensis YN-25 acclimated with different energy substrates (FeS₂, CuFeS₂, S⁰, FeSO₄). The atomic force microscopy (AFM) morphology scanning showed that the strain acclimated with different energy substrates varied a lot in EPS amount. XANES results showed clear associations between the energy substrates and the changes in organic composition in terms of typical function groups (CO, CO and CN). The chalcopyrite- and pyrite-acclimated cells contained higher proportion of proteins but less proportion of polysaccharides than the S⁰-acclimated cells. The FeSO₄-acclimated cells contained the highest proportion of proteins, while the S⁰-acclimated cells contained more lipids and polysaccharides. The results of linear-combination and peak fitting of the K-edge XANES for the extracellular superficial organic component C is consistent with the trend in comparison with the results of FTIR and spectrophotometric determination, but there are significant differences in the values. These differences are caused by the inconsistencies of measurement depth between XANES and the latter two characterization methods.     

Premature degradation of poly(α-hydroxyesters) during thermal processing of Bioglass®-containing composites

Bioactive, biodegradable composites are increasingly being explored as bone replacement materials and as scaffolds for tissue engineering. Their properties are not only dependent on the properties of the filler and matrix, but are also determined by their interaction. This study investigated the effect on poly(d,l-lactide) (PDLLA) matrix when processed at high-temperatures in the presence of Bioglass^® particulate filler. Composites with different filler contents were compounded at elevated temperatures by co-extrusion followed by compression moulding and compared with composites of similar composition prepared by thermally induced phase separation (TIPS), a low-temperature processing route. It was found that the inclusion of Bioglass^® in PDLLA under elevated temperatures resulted in the degradation of the matrix, leading to a reduction in the mechanical properties of the composites and in the molecular weight of the matrix. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy showed the presence of a peak at 1600 cm^?1 in the composite material, particularly when processed at elevated temperatures, whereas no peak at this wavelength was discernible for the pure PDLLA. Furthermore, time-based ATR-FTIR spectra taken at elevated temperatures on the TIPS-processed composites showed an increase in the intensity of the peak at 1600 cm^?1 and a concomitant reduction of the CO stretch peak at 1745 cm^?1 with time. This suggested the formation of a carboxylate salt by-products as a consequence of a reaction at the interface between the Bioglass^® filler and the PDLLA matrix. Therefore, the results confirmed that this degradation was not solely due to shear effects during the extrusion process. This work thereby supports the assertion that, in the presence of Bioglass^® filler particles, poly(α-hydroxyester)-based composites should not be processed at elevated temperatures.     

Surface modification of poly(ε-caprolactone) using a dielectric barrier discharge in atmospheric pressure glow discharge mode

The role of roughening and functionalization processes involved in modifying the wettability of poly(ε-caprolactone) (PCL) after treatment by an atmospheric pressure glow discharge plasma is discussed. The change in the ratio of CO/C–O bonds is a significant factor influencing the wettability of PCL. As the contact angle decreases, the level of CO bonds tends to rise. Surface roughness alterations are the driving force for lasting increases in wettability, while the surface functional species are shorter lived. We can approximate from ageing that the increase in wettability for PCL after plasma treatment is 55–60% due to roughening and 40–45% due to surface functionalization for the plasma device investigated.     

Rat calvaria osteoblast behavior and antibacterial properties of O2 and N2 plasma-implanted biodegradable poly(butylene succinate)

Poly(butylene succinate), a novel biodegradable aliphatic polyester with excellent processability and mechanical properties, was modified by O₂ or N₂ plasma immersion ion implantation (PIII). X-ray photoelectron spectroscopy and contact angle measurements were carried out to reveal the surface characteristics of the treated and control specimens. The in vitro effects of the materials on seeded osteoblasts were detected by cell viability assay, alkaline phosphatase activity test, and real-time polymerase chain reaction analysis. Plate counting was performed to investigate the antibacterial properties. Our results show that both PIII treatments significantly improve the hydrophilicity of PBSu, and CO and nitrogen groups (CNH and CNH₂) can be detected on the PBSu after O₂ and N₂ PIII, respectively. The modified samples exhibit similar compatibility to osteoblasts, which is better than that of the control, but O₂ PIII and N₂ PIII produce different effects according to the osteogenic gene expressions of seeded osteoblasts on the materials. Moreover, the N₂ plasma-modified PBSu exhibits anti-infection effects against Staphylococcus aureus and Escherichia coli but no such effects can be achieved after O₂ PIII.     

Radical scavenging abilities and hepatoprotective effect of [N,N′-Bis (salicylidene) ethane-1, 2-diaminato] oxovanadium (IV) complex in CCl4-treated rats

The antioxidant activity of [N, N′-Bis (salicylidene) ethane-1, 2-diaminato] oxovanadium (IV) complex (VO-salen complex) was evaluated using different in vitro evaluating systems including superoxide anion (O₂^?) and hydrogen peroxide (H₂O₂) scavenging activities. In addition, the inhibitory effects of this compound on protein oxidation and inhibition of Fe²⁺/ascorbate-induced lipid peroxidation were studied using rat liver homogenate. In vitro results revealed that the VO-salen complex has strong inhibitory effects on protein oxidation and lipid peroxidation of the liver homogenate along with a concentration-dependent quenching of H₂O₂ and O₂^? radicals. In an in vivo approach, hepatoprotective potential of the VO-salen complex against liver damages induced by CCl₄ treatment was also investigated. After intraperitoneal injection of CCl₄ to rats, various biochemical changes associated with liver injury and/or oxidative stress were measured. The results showed that the sera levels of ALT, AST, ALP and the content of hepatic thiobarbituric acid reactive substances (TBARS) were all increased and the glutathione (GSH) content and the hepatic superoxide dismutase (SOD) and catalase (CAT) activities were decreased in CCl₄-treated rats. However, simultaneous treatment of rats with VO-salen (0.6 mg/kg) and CCl₄ significantly attenuated the sera levels of ALT, AST, ALP and the hepatic TBARS content. In addition, by VO-salen therapy, the hepatic SOD and CAT activities and the GSH content were all restored back almost to their normal levels. The liver damages were also significantly ameliorated as compared to the CCl₄-treated rats. Based on these results, the VO-salen complex might be considered as an effective antioxidant and hepatoprotective agent suitable for further biological evaluation.     

Electrically polarized HAp-coated Ti: In vitro bone cell–material interactions

Electrically polarized bulk sintered hydroxyapatite (HAp) compacts have been shown to accelerate mineralization and bone tissue ingrowth in vivo. In this work, a comprehensive study has been carried out to investigate the influence of surface charge and polarity on in vitro bone cell adhesion, proliferation and differentiation on electrically polarized HAp-coated Ti. Uniform and crack free sol–gel derived HAp coatings of 20 ± 1.38 μm thickness were polarized by application of an external d.c. field of 2.0 kV cm^?1 at 400 °C for 1h. In vitro bioactivity of polarized HAp coatings was evaluated by soaking in simulated body fluid, and bone cell–material interactions were studied by culturing with human fetal osteoblast cells (hFOB) for a maximum period of 11 days. Scanning electron microscopic observation showed that accelerated mineralization on negatively charged surfaces favored rapid cell attachment and faster tissue ingrowth over non-polarized HAp coating surfaces, while positive charge on HAp coating surfaces restricted apatite nucleation with limited cellular response. Immunochemistry and confocal microscopy confirmed that the cell adhesion and early stage differentiation were more pronounced on negatively charged coating surfaces as hFOB cells expressed higher vinculin and alkaline phosphatase proteins on negatively charged surface compared to cells grown on all other surfaces. Our results in this study are process independent and potentially applicable to any other commercially available coating techniques.     

Polarization of hydroxyapatite: Influence on osteoblast cell proliferation

Hydroxyapatite (HA) has been used clinically to treat bone defects. However, modifications of the surface properties of HA could improve and control bone matrix deposition and localized host tissue integration. The aim of this study was to investigate the effect of developing a surface charge on HA discs with respect to osteoblast activity in vitro. HA discs (12 mm × 2 mm) were sintered in either air or water vapour. The HA discs were then electrically polarized (positive and negative surfaces) or non-polarized (controls) and seeded with MC3T3-E1 cells. Polarized HA sintered in water vapour was shown to retain six times more charge than polarized HA sintered in air. Picogreen analysis demonstrated that at 4 h cell number was significantly higher on the negatively and positively charged HA surface (water sintered) in comparison to the non-charged water and air-sintered HA controls. At 7 days there was a significant increase in cell number on the negatively charged HA (air sintered) sample in comparison to the negatively charged water vapour sintered HA sample and the non-charged water vapour sintered control sample. Also at 7 days, the picogreen data showed a significant increase in cell number on the positively charged water-treated HA sample in comparison to both the air- and water-treated HA non-charged control HA samples. An alamarBlue assay at 7 days demonstrated significant cell metabolic activity on the charged surfaces (both positive and negative) in comparison to the non-charged HA and the tissue culture plastic controls. This study demonstrated that all of the HA discs tested supported cell viability/attachment. However, cell attachment/proliferation/metabolic activity was significantly increased as a result of developing a charge on the HA surface.     

Poly(vinyl alcohol) microspheres with pH- and thermosensitive properties as temperature-controlled drug delivery

One of the most important inconveniences of the pH- and temperature-sensitive hydrogels is the loss of thermosensitivity when relatively large amounts of a pH-sensitive monomer are co-polymerized with N-isopropylacrylamide (NIPAAm). In order to overcome this drawback, we propose here a method to prepare thermosensitive poly(vinyl alcohol) (PVA) microspheres with a higher content of carboxylic groups that preserve thermosensitive properties. Moreover, PVA possesses excellent mechanical properties, biocompatibility and non-toxicity. PVA microspheres were obtained by suspension cross-linking of an acidified aqueous solution of the polymer with glutaraldehyde. Poly(N-isopropylacrylamide-co-N-hydroxymethyl acrylamide) (poly(NIPAAm-co-HMAAm)), designed to have a lower critical solution temperature (LCST) corresponding to that of the human body, was grafted onto PVA microspheres in order to confer them with thermosensitivity. Then, the pH-sensitive functional groups (COOH) were introduced by reaction between the un-grafted OH groups of PVA and succinic anhydride. The pH- and temperature-sensitive PVA microspheres display a sharp volume transition under physiological conditions around the LCST of the linear polymer. The microspheres possess good drug-loading capacity without losing their thermosensitive properties. Under simulated physiological conditions, the release of drugs is controlled by temperature.     

In situ forming collagen–hyaluronic acid membrane structures: Mechanism of self-assembly and applications in regenerative medicine

Bioactive, in situ forming materials have the potential to complement minimally invasive surgical procedures and enhance tissue healing. For such biomaterials to be adopted in the clinic, they must be cost-effective, easily handled by the surgeon and have a history of biocompatibility. To this end, we report a novel and facile self-assembling strategy to create membranes and encapsulating structures using collagen and hyaluronic acid (HA). Unlike membranes built by layer-by-layer deposition of oppositely charged biomolecules, the collagen–HA membranes described here form a diffusion barrier upon electrostatic interaction of the oppositely charged biomolecules, which is further driven by osmotic pressure imbalances. The resulting membranes have a nanofibrous architecture, a thicknesses of 130 μm and a tensile modulus (0.59 ± 0.06 MPa) that can increase 7-fold using carbodiimide chemistry (4.42 ± 1.46 MPa). Collagen–HA membranes support mesenchymal stem cell proliferation and have a slow and steady protein release profile (7% at day 28), offering opportunities for targeted tissue regeneration. We demonstrate the capacity to encapsulate cells by injecting HA into the collagen solution, and enhance allograft and implant biocompatibility through a coating technique. This study describes a novel mechanism of collagen–HA membrane formation and provides the groundwork to apply these membranes in a variety of tissue engineering applications.     

In vitro differentiation of chondrogenic ATDC5 cells is enhanced by culturing on synthetic hydrogels with various charge densities

We investigated the behavior of chondrogenic ATDC5 cells on synthetic polymer gels with various charge densities: negatively charged poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) gel, neutral poly(dimethylacrylamide) (PDMAAm) gel, and copolymer gels of 2-acrylamido-2-methyl-1-propanesulfonic acid and dimethylacrylamide P(AMPS-co-DMAAm) with different compositions (molar fractions of AMPS, F = 0.25, 0.5, 0.75). In insulin-free maintenance medium, the ATDC5 cells cultured on the highly negatively charged gels – PAMPS gel and the P(AMPS-co-DMAAm) copolymer gels (F = 0.75) – spread and became confluent at day 7, and interestingly formed nodules at day 14, expressing type II collagen and proteoglycan. This result demonstrates that the highly negatively charged gels can induce chondrogenic differentiation of ATDC5 cells even in insulin-free maintenance medium, in which the ATDC5 cells cultured on the standard polystyrene dish cannot differentiate into chondrocytes. In insulin-supplemented differentiation medium, ATDC5 cells cultured on the PDMAAm gel made focal adhesions, rapidly aggregated and formed large nodules within 7 days, expressing significantly greater levels of type II collagen and proteoglycan than cells cultured on the polystyrene dish and the negatively charged gels. These results showed that the neutral gel accelerated chondrogenic differentiation of ATDC5 cells cultured in the differentiation medium. We suggest that the highly negatively charged PAMPS gel and the neutral PDMAAm gel are interesting biomaterials for cartilage tissue engineering as a scaffold with the potential to induce chondrogenic differentiation.     

Thermally responsive nanoparticle-encapsulated curcumin and its combination with mild hyperthermia for enhanced cancer cell destruction

In this study, thermally responsive polymeric nanoparticle-encapsulated curcumin (nCCM) was prepared and characterized. The nCCM is ∼22 and 300 nm in diameter at 37 and 22 °C, respectively. The smaller size of the nCCM at 37 °C was found to significantly facilitate its uptake in vitro by human prostate adenocarcinoma PC-3 cancer cells. However, the intracellular nCCM decreases rapidly (rather than plateaus) after reaching its peak at ∼1.5 h during a 3-day incubation of the PC-3 cells with nCCM. Moreover, a mild hyperthermia (with negligible cytotoxicity alone) at 43 °C applied between 1 and 1.5 h during the 3-day incubation not only increases the peak uptake but also alters intracellular distribution of nCCM (facilitating its delivery into cell nuclei), which helps to retain a significantly much higher level of intracellular curcumin. These effects of mild hyperthermia could be due in part to the thermal responsiveness of the nCCM: they are more positively charged at 43 °C and can be more easily attracted to the negatively charged nuclear membrane to enter nuclei as a result of electrostatic interaction. Ultimately, a combination of the thermally responsive nCCM and mild hyperthermia significantly enhances the anticancer capability of nCCM, resulting in a more than 7-fold decrease in its inhibitory concentration to reduce cell viability to 50% (IC₅₀). Further mechanistic studies suggest injury pathways associated with heat shock proteins 27 and 70 should contribute to the enhanced cancer cell destruction by inducing cell apoptosis and necrosis. Overall, this study demonstrates the potential of combining mild hyperthermia and thermally responsive nanodrugs such as nCCM for augmented cancer therapy.     

Poly(l-glutamic acid)/chitosan polyelectrolyte complex porous microspheres as cell microcarriers for cartilage regeneration

In this study a novel kind of porous poly(l-glutamic acid) (PLGA)/chitosan polyelectrolyte complex (PEC) microsphere was developed through electrostatic interaction between PLGA and chitosan. By adjusting the formula parameters chitosan microspheres with an average pore size of 47.5 ± 5.4 μm were first developed at a concentration of 2 wt.% and freeze temperature of −20 °C. For self-assembly of the PEC microspheres porous chitosan microspheres were then incubated in PLGA solution at 37 °C. Due to electrostatic interaction a large amount of PLGA (110.3 μg mg⁻¹) was homogeneously absorbed within the chitosan microspheres. The developed PEC microspheres retained their original size, pore diameters and interconnected porous structure. Fourier transform infrared spectroscopy, thermal gravimetric analysis and zeta potential analysis revealed that the PEC microspheres were successfully prepared through electrostatic interaction. Compared with microspheres fabricated from chitosan, the porous PEC microspheres were shown to efficiently promote chondrocyte attachment and proliferation. After injection subcutaneously for 8 weeks PEC microspheres loaded with chondrocytes were found to produce significant more cartilaginous matrix than chitosan microspheres. These results indicate that these novel fabricated porous PLGA/chitosan PEC microspheres could be used as injectable cell carriers for cartilage tissue engineering.     

One-year outcome of an interactive internet-based physical activity intervention among university students

ObjectiveThe purpose of the present study was to evaluate whether improvement in physical activity of students following a 4-month intervention of a university course was maintained 8 months later.MethodsData on 77 students who responded to our scheduled inquiries completely through 1 year were analyzed. Participants of the intervention group (n = 49) using the internet-based physical activity program exhibited significant increases in energy expenditures measured by IPAQ compared with the no-treatment control group (n = 28) through 1 year.ResultsParticipants who did not engage in regular university sports activities (baseline: 450 ± 351 kcal day⁻¹; post: 587 ± 320 kcal day⁻¹; 8-month follow-up: 580 ± 394 kcal day⁻¹) only exhibited significant increases in energy expenditures compared with those of the control group (baseline: 498 ± 341 kcal day⁻¹; post: 414 ± 242 kcal day⁻¹; 8-month follow-up: 347 ± 275 kcal day⁻¹).ConclusionThese results suggested that an internet-based interactive intervention could become a helpful tool in promoting and maintaining physical activity in the long term.     

Cardiopulmonary responses to robotic end-effector-based walking and stair climbing

BackgroundA recently developed robotic end-effector device (G-EO system, Reha Technology AG) can simulate walking and stair climbing. This approach has the potential to promote cardiovascular exercise training during rehabilitation. The aim of this study was to characterise cardiopulmonary responses of end-effector-based exercise in able-bodied subjects and to evaluate the feasibility of intensity-guided exercise testing.MethodsFive healthy subjects aged 33.7 ± 8.8 years (mean ± SD) performed a constant load test and an intensity-guided incremental exercise test. The outcome measures were steady-state and peak cardiopulmonary performance parameters including oxygen uptake (VO₂) and heart rate (HR).ResultsPassive end-effector-based stair climbing (VO₂ = 13.6 ± 4.5 mL/min/kg, HR = 95 ± 23 beats/min) showed considerably lower cardiopulmonary responses compared to reference data (VO₂ = 33.5 ± 4.8 mL/min/kg, HR = 159 ± 15 beats/min). Peak performance parameters during intensity-guided incremental exercise testing were: VO₂ = 35.8 ± 5.1 mL/min/kg and HR = 161 ± 27 beats/min, corresponding to a relative VO₂ = 76.0 ± 18.7% of predicted aerobic capacity and a relative HR = 87.3 ± 14.5% of age-predicted HR maximum.ConclusionEnd-effector-based exercise is a promising method for the implementation of cardiovascular exercise. Although end-effector-based stair climbing evoked lower cardiopulmonary responses than conventional stair climbing, active contribution during exercise elicited substantial cardiopulmonary responses within recommended ranges for aerobic training.