Poly(ethylene Glycol) Methyl Ether Methacrylate-Based Injectable Hydrogels: Swelling, Rheological, and In Vitro Biocompatibility Properties with ATDC5 Chondrogenic Lineage.

Here, we present the synthesis of a series of chemical homopolymeric and copolymeric injectable hydrogels based on polyethylene glycol methyl ether methacrylate (PEGMEM) alone or with 2-dimethylamino ethyl methacrylate (DMAEM). The objective of this study was to investigate how the modification of hydrogel components influences the swelling, rheological attributes, and in vitro biocompatibility of the hydrogels. The hydrogels' networks were formed via free radical polymerization, as assured by 1H nuclear magnetic resonance spectroscopy (1H NMR). The swelling of the hydrogels directly correlated with the monomer and the catalyst amounts, in addition to the molecular weight of the monomer. Rheological analysis revealed that most of the synthesized hydrogels had viscoelastic and shear-thinning properties. The storage modulus and the viscosity increased by increasing the monomer and the crosslinker fraction but decreased by increasing the catalyst. MTT analysis showed no potential toxicity of the homopolymeric hydrogels, whereas the copolymeric hydrogels were toxic only at high DMEAM concentrations. The crosslinker polyethylene glycol dimethacrylate (PEGDMA) induced inflammation in ATDC5 cells, as detected by the significant increase in nitric oxide synthase type II activity. The results suggest a range of highly tunable homopolymeric and copolymeric hydrogels as candidates for cartilage regeneration.

Injectable hybrid hydrogels physically crosslinked based on carrageenan and green graphene for tissue repair.

Injectable and biocompatible novel hybrid hydrogels based on physically crosslinked natural biopolymers and green graphene for potential use in tissue engineering are reported. Kappa and iota carrageenan, locust bean gum and gelatin are used as biopolymeric matrix. The effect of green graphene content on the swelling behavior, mechanical properties and biocompatibility of the hybrid hydrogels is investigated. The hybrid hydrogels present a porous network with three-dimensionally interconnected microstructures, with lower pore size than that of the hydrogel without graphene. The addition of graphene into the biopolymeric network improves the stability and the mechanical properties of the hydrogels in phosphate buffer saline solution at 37 °C without noticeable change in the injectability. The mechanical properties of the hybrid hydrogels were enhanced by varying the dosage of graphene between 0.025 and 0.075 w/v%. In this range, the hybrid hydrogels preserve their integrity during mechanical test and recover the initial shape after removing the applied stress. Meanwhile, hybrid hydrogels with graphene content of up to 0.05 w/v% exhibit good biocompatibility for 3T3-L1 fibroblasts; the cells proliferate inside the gel structure and show higher spreading after 48 h. These injectable hybrid hydrogels with graphene have promising future as materials for tissue repair.

Effect of Different Plasticizers on Thermal, Crystalline, and Permeability Properties of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Films.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) films were prepared using a cast film technique. Dioxane was chosen over other polymer solvents as it resulted in homogenous films with better morphology. Several plasticizers with different molecular weights and concentrations were added to the biopolymer solution prior to casting. Thermal, crystalline, and permeability properties were analyzed by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and both water vapor and oxygen transmission rate analysis. In general, the addition of plasticizers decreased the glass transition temperature (Tg), cold crystallization temperatures (Tcc), melting temperatures, as well as crystallinity degrees and increased the crystallite sizes and water vapor and oxygen transmission rates. The use of isosorbide and low-molecular-weight poly(ethylene glycol) (PEG) lowered the Tg around 30 °C at the highest used concentration, also being the most effective in increasing the crystallite size. When considering isosorbide and low-molecular-weight poly(ethylene glycol) (PEG) as very good plasticizers for PHBH, the question of which plasticizer to use strongly relies on the desired PHBH application.

Preparation of Poly(vinyl Alcohol) Microparticles for Freeze Protection of Sensitive Fruit Crops.

Poly(vinyl alcohol) (PVA) displays ice recrystallization inhibition (IRI) properties as many antifreeze proteins found in cold tolerant organisms. The molecular architecture and composition (molecular weight and distribution of pendant OH and acetate groups) have been studied to improve the antifreezing properties of PVA, suggesting that the molecular architecture of PVA plays an important role in IRI activity. The present work deals with the preparation of PVA microparticles using an alkaline treatment. The effect of PVA molecular weight on the morphology and antifreezeing properties of PVA microparticles was investigated. The antifreezeing property of PVA microparticles on the susceptibility of flower bud tissues to freeze damage was also evaluated. The alkaline treatment of an aqueous PVA solution produced stable polymer chain aggregates with spherical shapes. The average size of the PVA microparticles increased significantly with the increasing molecular weight of the PVA macromolecule precursor. The PVA microparticles inhibited the growth of ice crystals and blocked ice growth at concentrations as low as 0.01 % w/v. The effect of impeding ice crystal growth by preventing the joining of adjacent ice crystals is attributed to the larger size of the PVA particles adsorbed on the ice surface compared to the aggregated PVA macromolecules in saline solution. The thermal hysteresis activity of PVA macromolecules and microparticles was not detected by differential scanning calorimetry analysis. The PVA microparticles reduced the incidence of freeze injuries in flower bud tissues by 55% and their application, considering the low toxicity of PVA, has a high potential for freeze protection in fruit crops.

Isosorbide plasticized corn starch filled with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) microparticles: Properties and behavior under environmental factors.

In this work, new green and fully biodegradable composites, based on corn starch, plasticized with two different amounts of isosorbide and filled by poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microparticles, were obtained by melt processing. The analysis of their morphologies, crystallinity, structural interactions and dynamomechanical properties as well as the evaluation of their moisture resistance and biodegradability in soil, were performed in function of the plasticizer and/or microparticle amount. The analysis of morphology, crystallinity and structural interactions showed that the plasticization process was completed under the melting processing conditions used. The microparticles were homogeneously dispersed in the thermoplastic starch matrix without suffering any deformation or breaking during the processing. Biocomposites with adequate storage modulus values were obtained, especially the TPS plasticized with 35% of isosorbide and filled with 5 wt% of PHBV microparticles. The incorporation of PHBV microparticles leads to biocomposites with higher moisture resistance. All the biocomposites were completely biodegraded in soil in a short period of time. The performed study demonstrated that these biocomposites could be used for applications in the packaging industry.

Improvement of endothelial function by Gunnera tinctoria extract with antioxidant properties.

BACKGROUND: Gunnera tinctoria has been collected by Mapuche-Pewenche people for food and medicinal purposes. The high polyphenol content of methanolic extract from G. tinctoria leaves with chemical constituents such as ellagic acid and quercetin derivatives suggests its application to prevent endothelial dysfunction and oxidative stress. The aim of this study was to provide evidence of the protective effect of this extract on endothelial function by reducing oxidative stress induced by high D-glucose and H2O2, as well as by stimulating nitric oxide (NO) levels in human umbilical vein endothelial cells (HUVECs). RESULTS: A methanolic extract with a high content of polyphenols (520 ± 30 mg gallic acid equivalents/g dry extract) was obtained from G. tinctoria leaves. Its main constituent was ellagic acid. The results of Ferric reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays of the extract confirmed its antioxidant activity by inhibition pathway of radical species. The incubation of HUVECs with the extract decreased the apoptosis and reactive oxygen species (ROS) synthesis induced by high extracellular concentration of D-glucose or hydrogen peroxide. The extract increased endothelial NO levels and reduced vasoconstriction in human placental vessels. CONCLUSIONS: This study provides evidence about the antioxidant and endothelial protective properties of methanolic G. tinctoria leaf extract. The extract improves the availability of NO in HUVECs, inhibiting the production of ROS and vasoconstriction.

Carrageenan-based physically crosslinked injectable hydrogel for wound healing and tissue repairing applications.

In this study, a novel injectable hydrogel based on iota and kappa carrageenan, locust bean gum and gelatin was prepared for wound healing and tissue repairing applications. This injectable hydrogel was obtained via physical crosslinking. FTIR analysis confirmed the physical interaction between the biopolymeric components of the hydrogel. The prepared injectable hydrogel exhibited shear-thinning characteristics and could be injected for minimally invasive applications. Also, the hydrogel showed a porous structure, physiological and mechanical stability and biocompatibility. The in vitro cell culture studies showed that fibroblasts were able to grow, adhere and spread inside the hydrogel, indicating that hydrogel could support tissue repair. Moreover, hydrogel could be useful for the delivery of biomolecules. Vascular endothelial growth factor was encapsulated within the hydrogel and subsequently released, which accelerated the migration of human umbilical vein endothelial cells and facilitated in vitro wound healing. Overall, the results indicate that hydrogel can be a potential injectable delivery vehicle for wound healing and tissue repair.

Properties and behavior under environmental factors of isosorbide-plasticized starch reinforced with microcrystalline cellulose biocomposites.

Green biocomposites based on corn starch plasticized with isosorbide and glycerol and filled with microcrystalline cellulose (MCC) were processed. The structural interactions, dynamomechanical properties, water absorption, and soil biodegradability were investigated by different techniques considering the effect of the type and amount of plasticizer and the MCC addition. The changes along the time of the structural interactions and the dynamomechanical properties of these materials and the influence of the retrogradation phenomenon were also studied. The use of isosorbide as plasticizer instead of glycerol improved the stiffness, the water absorption and the biodegradation rate of thermoplastic starch (TPS). Moreover, no evidence of retrogradation for isosorbide was observed. An isosorbide content of 35% and the addition of MCC filler enhanced in a greater extent these properties with a complete biodegradability in 7 month. These green biocomposites can be an alternative for food packaging applications.

Overexpression of ZePrx in Nicotiana tabacum Affects Lignin Biosynthesis Without Altering Redox Homeostasis.

Class III plant peroxidases (Prxs) are involved in the oxidative polymerization of lignins. Zinnia elegans Jacq. Basic peroxidase (ZePrx) has been previously characterized as capable of catalyzing this reaction in vitro and the role in lignin biosynthesis of several of its Arabidopsis thaliana homologous has been previously confirmed. In the present work, ZePrx was overexpressed in Nicotiana tabacum to further characterize its function in planta with particular attention to its involvement in lignin biosynthesis. Since Prxs are known to alter ROS levels by using them as electron acceptor or producing them in their catalytic activity, the impact of this overexpression in redox homeostasis was studied by analyzing the metabolites and enzymes of the ascorbate-glutathione cycle. In relation to the modification induced by ZePrx overexpression in lignin composition and cellular metabolism, the carbohydrate composition of the cell wall as well as overall gene expression through RNA-Seq were analyzed. The obtained results indicate that the overexpression of ZePrx caused an increase in syringyl lignin in cell wall stems, suggesting that ZePrx is relevant for the oxidation of sinapyl alcohol during lignin biosynthesis, coherently with its S-peroxidase nature. The increase in the glucose content of the cell wall and the reduction of the expression of several genes involved in secondary cell wall biosynthesis suggests the occurrence of a possible compensatory response to maintain cell wall properties. The perturbation of cellular redox homeostasis occurring as a consequence of ZePrx overexpression was kept under control by an increase in APX activity and a reduction in ascorbate redox state. In conclusion, our results confirm the role of ZePrx in lignin biosynthesis and highlight that its activity alters cellular pathways putatively aimed at maintaining redox homeostasis.

A Novel Hydrocolloid Film Based on Pectin, Starch and Gunnera tinctoria and Ugni molinae Plant Extracts for Wound Dressing Applications.

BACKGROUND: The biodegradable and biocompatible nature of pectin-based films is of particular interest in wound dressing applications, due to its non-toxicity, pH-sensitivity and gelling activity. An approach to improve the mechanical properties, the release profile of bioactive compounds as well as the performance in wet environments of pectin-based films is mixing with other biopolymers. OBJECTIVE: To prepare hydrocolloid films based on crosslinked pectin / starch blend loaded with bioactive extracts from leaves of G. tinctoria and U. molinae with controlled release of bioactive compounds and healing property. METHODS: The hydrocolloid films were characterized by FTIR, SEM, and TGA-FTIR techniques and their tensile properties, water uptake, and polyphenolic release profile in aqueous media were evaluated. The dermal anti inflammatory activity of the hydrocolloid films was assessed by the mouse ear inflammation test. The wound healing property of the loaded hydrocolloid films was explored in a rat model and in a clinical trial (sacrum pressure ulcer). RESULTS: The films showed an adequate water-uptake capacity between 100-160%. The release of active compounds from the hydrocolloid films followed the Korsmeyer-Peppas equation. The mechanical properties of hydrocolloid films were not affected by the plant extracts within the concentration range used. The incorporation of the bioactive extracts in the polysaccharide films inhibited the topical edematous response by about 50%. The topical application of the loaded hydrocolloid film on the pressure ulcer is completely closed after 17 days without showing any adverse reaction. CONCLUSION: A novel hydrocolloid matrix was produced from crosslinked starch-pectin, which exhibited suitable chemical-physical properties to be used as a carrier of plant extracts with wound healing properties.

Poly(hydroxybutyrate-co-hydroxyvalerate) microparticles embedded in κ-carrageenan/locust bean gum hydrogel as a dual drug delivery carrier.

A novel composite hydrogel was prepared as a dual drug delivery carrier. Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) microparticles were prepared to encapsulate simultaneously ketoprofen and mupirocin, as hydrophobic drug models. These microparticles were embedded in a physically crosslinked hydrogel of κ-carrageenan/locust bean gum. This composite hydrogel showed for both drugs a slower release than the obtained release from microparticles and hydrogel separately. The release of both drugs was observed during a period of 7 days at 37 °C. Different kinetic models were analyzed and the results indicated the best fitting to a Higuchi model suggesting that the release was mostly controlled by diffusion. Also, the drug loaded microparticles were spherical with average mean particle size of 1.0 µm, mesoporous, and distributed homogeneously in the hydrogel. The composite hydrogel showed a thermosensitive swelling behavior reaching 183% of swelling ratio at 37 °C. The composite hydrogel showed the elastic component to be higher than the viscous component, indicating characteristics of a strong hydrogel. The biocompatibility was evaluated with in vitro cytotoxicity assays and the results indicated that this composite hydrogel could be considered as a potential biomaterial for dual drug delivery, mainly for wound healing applications.

Improvement of endothelial function by Gunnera tinctoria extract with antioxidant properties

BACKGROUND: Gunnera tinctoria has been collected by Mapuche-Pewenche people for food and medicinal purposes. The high polyphenol content of methanolic extract from G. tinctoria leaves with chemical constituents such as ellagic acid and quercetin derivatives suggests its application to prevent endothelial dysfunction and oxidative stress. The aim of this study was to provide evidence of the protective effect of this extract on endothelial function by reducing oxidative stress induced by high D-glucose and H2O2, as well as by stimulating nitric oxide (NO) levels in human umbilical vein endothelial cells (HUVECs). RESULTS: A methanolic extract with a high content of polyphenols (520 ± 30 mg gallic acid equivalents/g dry extract) was obtained from G. tinctoria leaves. Its main constituent was ellagic acid. The results of Ferric reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays of the extract confirmed its antioxidant activity by inhibition pathway of radical species. The incubation of HUVECs with the extract decreased the apoptosis and reactive oxygen species (ROS) synthesis induced by high extracellular concentration of D-glucose or hydrogen peroxide. The extract increased endothelial NO levels and reduced vasoconstriction in human placental vessels. CONCLUSIONS: This study provides evidence about the antioxidant and endothelial protective properties of methanolic G. tinctoria leaf extract. The extract improves the availability of NO in HUVECs, inhibiting the production of ROS and vasoconstriction.

Hydrocortisone loaded poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) nanoparticles for topical ophthalmic administration: Preparation, characterization and evaluation of ophthalmic toxicity.

Poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) nanoparticles (PHBV-NPs) to encapsulate hydrocortisone (HC) for topical ophthalmic administration were prepared and characterized. The technique used to prepare the nanoparticles (NPs) was emulsification/solvent evaporation. The obtained size was 237.3 ±â€¯2.7 nm, suitable for topical ocular administration. The obtained results for the entrapment efficiency were between 1 and 2.5% and for the drug loading were around 0.5%. The release behaviour of HC from the PHBV-NPs was also analyzed, adjusting this to a Higuchi kinetic model. For the new drug delivery system developed the ocular toxicity profile was determined by viability studies carried out on bovine keratocytes, by a Hen's Egg Test - Chorioallantoic Membrane (HET-CAM) and by a Bovine Corneal Opacity and Permeability assay (BCOP). The obtained results concluded that the new system is no cytotoxic on bovine keratocytes and is neither irritating nor produces any alteration in the transparency and in the permeability of the cornea. Confocal studies were also performed and confirmed that PHBV-NPs are able to penetrate efficiently into the corneal tissue. This novel PHBV-based drug delivery system could be a good option for topical ophthalmic administration of drugs.

Entrapment of chitosan, pectin or κ-carrageenan within methacrylate based hydrogels: Effect on swelling and mechanical properties.

Composite hydrogels were obtained by the entrapment of chitosan, pectin or κ-carrageenan within methacrylate-based hydrogels to improve their swelling and the mechanical properties. The results indicated that the water uptake (WU) of κ-carrageenan and chitosan hydrogels were until 3.5 and 2.2 times higher than the WU of the synthetic hydrogel, respectively. The surface morphologies of the hydrogels showed that the pectin and κ-carrageenan favors the formation of larger and more defined pores. The mechanical properties indicated that the pectin increased slightly the mechanical properties and the κ-carrageenan improves the mechanical properties of the synthetic hydrogel reaching up 400 N of compression load. Therefore, the entrapment of κ-carrageenan within synthetic hydrogels improved both the swelling and the mechanical properties. The biocompatibility of the hydrogels was evaluated with in vitro cytotoxicity assays and the results indicated that they could be considered as candidates for biomedical use.

Corn starch plasticized with isosorbide and filled with microcrystalline cellulose: Processing and characterization.

Innovative, green and fully biodegradable biocomposites, based on plasticized corn starch, were prepared by melt processing and filled with microcrystalline cellulose (MCC). Isosorbide and glycerol were used as plasticizers. The effect of the type and content of the plasticizer and the filler addition on processing, surface morphologies, crystallinity and thermal stability were investigated. Aged materials were analyzed too to study the retrogradation phenomena along the time. The processing using isosorbide required lower temperatures but greater shear and processing times leading to more homogeneous biocomposites. With isosorbide, higher crystallinity and no retrogradation signals were observed as clearly occurred with glycerol. The thermal stability of biocomposites was high too. These properties enhanced in a greater extent when the isosorbide content was 35% and with the MCC incorporation, the last due to a good adhesion of MCC to the matrix. These biocomposites could be a good option for packaging applications.

Starch films loaded with donut-shaped starch-quercetin microparticles: Characterization and release kinetics.

Starch films loaded with donut-shaped starch-quercetin microparticles were prepared from two different botanical origins. The quercetin release kinetics through the films were studied. The donut-shaped starch-quercetin microparticles were prepared by thermal aqueous-alcoholic treatment. The quercetin loading percentage and therefore the antioxidant activity were higher for the microparticles from legume than those of cereal origins. The starch-quercetin microparticles also showed higher thermal stability than the starch granules. The starch films were produced using the solution casting method. The films with more microparticles content showed higher thermal stability. In-vitro release studies of the quercetin through the films were performed in aqueous-ethanolic medium. The quercetin released reached the equilibrium in 1 to 4 days for the films of cereal starch and in more than a week for the films of legume origin. The release data were fitted to Peppas-Sahlin model that suggests the release kinetics were controlled mainly by fickian diffusion. The produced biofilms can be utilized mainly for active food packaging applications.

Effects of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) microparticles on morphological, mechanical, thermal, and barrier properties in thermoplastic potato starch films.

Biocomposites of potato starch/poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microparticles were prepared through the solvent casting method. Glycerol was used as a plasticizer. The effects of concentrations of PHBV microparticles as filler and glycerol on crystallinity behavior, surface morphology, dynamic mechanical properties, and thermal stability were studied. Humidity absorption and the water vapor transmission rate (WVTR) were investigated as well. Wide angle X-ray scattering (WAXS) patterns revealed that the plasticizing process occurred successfully. Scanning electron microscopy (SEM) micrographs exhibited good homogeneity of the surfaces for the biocomposites with a lower glycerol concentration. Dynamic mechanical analysis (DMA) results confirmed the reinforcing effect of PHBV microparticles inside the matrix. Thermogravimetric analysis (TGA) indicated that the presence of PHBV microparticles increased the thermal stability of the starch. Results of humidity absorption tests showed that the high hydrophilicity of the starch was reduced once the PHBV microparticles had been incorporated. Also, increasing PHBV microparticles reduced the water vapor transmission rate. However, samples with reduced glycerol content absorbed less humidity and showed a lower water vapor transmission rate.

Preparation of starch nanoparticles loaded with quercetin using nanoprecipitation technique.

Nanoparticles of starches from different botanical origin were prepared by nanoprecipitation using 0.1M hydrochloric acid as non-solvent. The morphology and the particle size were analyzed using field emission scanning electron microscopy and dynamic light scattering. The nanoparticles were spherical and their sizes vary depending on the origin and the concentration of the starch solution. Starch nanoparticles loaded with quercetin were prepared. In-vitro release studies of the quercetin from the starch nanoparticles were performed in 35% ethanol as a release medium. The starch origin affects the quercetin loading percentage, the release kinetics and the antioxidant activity of the produced nanoparticles. The starch-quercetin nanoparticles from cereal origin showed the lowest loading percentage and the lowest fraction released of quercetin in comparison with nanoparticles from tuber and legume origin. The release kinetics seem to be controlled mainly by Fickian diffusion which have been revealed fitting the release data to the Peppas-Sahlin model.

Preparation of donut-shaped starch microparticles by aqueous-alcoholic treatment.

A simple method for producing donut-shaped starch microparticles by adding ethanol to a heated aqueous slurry of corn starch is presented. The obtained microparticles were analysed by SEM, XRD and DSC. The average size of microparticles was 14.1 ±â€¯0.3 µm with holes of an average size of 4.6 ±â€¯0.2 µm. The crystalline arrangement of the microparticles was of a V-type single helix. The change in crystallinity from A-type of the starch granules to a more open structure, where water molecules could penetrate easier within the microparticles, substantially increased their solubility and swelling power. The microparticles exhibited a higher gelatinization temperature and a lower gelatinization enthalpy than did the starch granules. The donut-shaped microparticles were stable for more than 18 months and can be used as a carrier of an active compound or as a filler in bioplastics.

Effect of nanocellulose as a filler on biodegradable thermoplastic starch films from tuber, cereal and legume.

Starches from different vegetal sources (tuber, cereal and legume) were plasticized with an invariant glycerol content and reinforced with cellulose nanocrystals by solution casting method. The influence of both, starch nature and filler amount, in the crystallinity and the extension of plasticization have been analyzed by X-ray diffraction. Thermoplastic starches (TPS) morphologies were obtained by scanning electron microscopy. Mechanical properties and thermal stability were analyzed by dynamomechanical and thermogravimetric analysis. Water absorption evolution was studied as well. A major extension in plasticization (high amylopectin starches) led to matrices with large starch-rich domains, a good thermal stability and resistance to water absorption but low stiffness. The incorporation of cellulose nanoparticles favoured plasticization and increased the rigidity in TPS films, as well as the thermal stability and moisture resistance. The aim of this work was to obtain bio-based thermoplastic starch films for replacing petroleum-derived ones in packaging industry, especially for short-life applications.