Formulation of organic and inorganic hydrogel matrices for immobilization of β‐glucosidase in microfluidic platform

The aim of this study was to formulate silica and alginate hydrogels for immobilization of β‐glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate–polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized β‐glucosidase was loaded into glass–silicon–glass microreactors and catalysis of 4‐nitrophenyl β‐d ‐glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.     

Photo-cross-linked PLA-PEO-PLA hydrogels from self-assembled physical networks: mechanical properties and influence of assumed constitutive relationships

Poly(lactide)-block-poly(ethylene oxide)-block-poly(lactide) (PLA-PEO-PLA) triblock copolymers are known to form physical hydrogels in water as a result of the polymer's amphiphilicity. Their mechanical properties, biocompatibility, and biodegradability have made them attractive for use as soft tissue scaffolds. However, the network junction points are not covalently cross-linked, and in a highly aqueous environment these hydrogels adsorb more water, transform from gel to sol, and lose the designed mechanical properties. In this article, a hydrogel was formed by the use of a novel two-step approach. In the first step, the end-functionalized PLA-PEO-PLA triblock was self-assembled into a physical hydrogel through hydrophobic micelle network junctions, and in the second step, this self-assembled physical network structure was locked into place by photo-cross-linking the terminal acrylate groups. In contrast with physical hydrogels, the photo-cross-linked gels remained intact in phosphate-buffered solution at body temperature. The swelling, degradation, and mechanical properties were characterized, and they demonstrated an extended degradation time (approximately 65 days), an exponential decrease in modulus with degradation time, and a tunable shear modulus (1.6-133 kPa). We also discuss the various constitutive relationships (Hookean, neo-Hookean, and Mooney-Rivlin) that can be used to describe the stress-strain behavior of these hydrogels. The chosen model and assumptions used for data fitting influenced the obtained modulus values by as much as a factor of 3.5, which demonstrates the importance of clearly stating one's data fitting parameters so that accurate comparisons can be made within the literature.     

Application of a dense gas technique for sterilizing soft biomaterials

Sterilization of soft biomaterials such as hydrogels is challenging because existing methods such as gamma irradiation, steam sterilization, or ethylene oxide sterilization, while effective at achieving high sterility assurance levels (SAL), may compromise their physicochemical properties and biocompatibility. New methods that effectively sterilize soft biomaterials without compromising their properties are therefore required. In this report, a dense-carbon dioxide (CO(2) )-based technique was used to sterilize soft polyethylene glycol (PEG)-based hydrogels while retaining their structure and physicochemical properties. Conventional sterilization methods such as gamma irradiation and steam sterilization severely compromised the structure of the hydrogels. PEG hydrogels with high water content and low elastic shear modulus (a measure of stiffness) were deliberately inoculated with bacteria and spores and then subjected to dense CO(2) . The dense CO(2) -based methods effectively sterilized the hydrogels achieving a SAL of 10(-7) without compromising the viscoelastic properties, pH, water-content, and structure of the gels. Furthermore, dense CO(2) -treated gels were biocompatible and non-toxic when implanted subcutaneously in ferrets. The application of novel dense CO(2) -based methods to sterilize soft biomaterials has implications in developing safe sterilization methods for soft biomedical implants such as dermal fillers and viscosupplements.     

Synthesis and characterizations of in situ cross-linkable gelatin and 4-arm-PPO-PEO hybrid hydrogels via enzymatic reaction for tissue regenerative medicine

In situ cross-linkable hybrid hydrogels composed of gelatin and 4-arm-polypropylene oxide-polyethylene oxide (Tetronic) was developed as an injectable scaffold for tissue regeneration. The gelatin was modified by hydroxyphenyl propionic acid (HPA) and the Tetronic was conjugated with tyramines (Tet-TA). The hydrogels were rapidly formed by mixing the polymer solutions containing horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). The gelation time and mechanical properties of the hydrogels could be controlled by varying the HRP and H(2)O(2) concentrations. In vitro degradation study of the hybrid hydrogels was carried out using collagenase and the prolonged proteolytic degradation was obtained due to the presence of the Tetronic. Human dermal fibroblast (hDFB) was cultured in the hydrogel matrices to evaluate the cyto-compatibility. The encapsulated cells were shown to be highly viable and spread over the gel matrices, suggesting that the hybrid hydrogels have an excellent cyto-compatibility. The hydrogels were also subcutaneously injected in the back of mice and the results demonstrated that the hydrogels were rapidly formed at the injected site. From these results, we demonstrate that the in situ cross-linkable hydrogels formed by hybridization of gelatin and Tetronic via enzyme-mediated reactions hold great promise for use as injectable matrices for tissue regenerative medicine due to their tunable physico-chemical properties and excellent bioactivity.     

组织工程用纤维增强天然多糖水凝胶的进展

天然多糖水凝胶具有良好的生物相容性,然而其力学性能调节幅度小,无法满足组织工程应用巨大的需求。通过纤维增强法,不仅可显著提高天然多糖水凝胶的力学性能,还能调节复合水凝胶的降解性能、促进细胞粘附、增殖与分化行为及其组织沉积。常用的天然多糖组织工程水凝胶的纤维增强方法有物理共混法、化学作用法、静电驱动法与自组装法等。本文综述了纤维增强水凝胶的结构与功能特点,讨论了纤维增强对组织工程水凝胶的意义,以期对纤维增强组织工程水凝胶的发展起到促进作用。     

Synthesis and characterization of some cellulose/chondroitin sulphate hydrogels and their evaluation as carriers for drug delivery

Mixed hydrogels based on natural, biodegradable and biocompatible polysaccharides, such as cellulose (C) and chondroitin sulphate (CS) in various mixing ratios were prepared by a crosslinking technique and characterized by swelling behaviour, FTIR spectroscopy, scanning electron microscopy, toxicity and biocompatibility tests.The mixed cellulose/chondroitin sulphate hydrogels have been loaded with 7-[2-nitroxiacetyl-oxy-3-(4-acetyl-amino-phenoxy)-propyl]-8-morpholino-1,3-dimethyl-xanthine, a novel nitric oxide donor compound with a lower toxicity and a higher anti-inflammatory activity than its parent molecules, paracetamol and theophylline. Swelling and release kinetics have been also studied. It has been established that an increase of CS content in hydrogels composition leads to a higher swelling ratio for all formulations and to a decreased released amount of nitric oxide donor compound. It has been found that the swelling occurs by an anomalous swelling mechanism, while the release of nitric oxide donor compound follows a diffusion controlled mechanism.     

Biodegradable thermoresponsive hydrogels for aqueous encapsulation and controlled release of hydrophilic model drugs

A series of hydrogels with both thermoresponsive and completely biodegradable properties was developed for aqueous encapsulation and controlled release of hydrophilic drugs in response to temperature change. The hydrogels were prepared in phosphate-buffered saline (pH 7.4) through free radical polymerization of N-isopropylacrylamide (NIPAAm) monomer and a dextran macromer containing multiple hydrolytically degradable oligolactate-2-hydroxyethyl methacrylate units (Dex-lactateHEMA). Swelling measurement results demonstrated that four gels with feeding weight ratios of NIPAAm:Dex-lactateHEMA = 7:2, 6:3, 5:4, and 4:5 (w/w) were thermoresponsive by showing a lower critical solution temperature at approximately 32 degrees C. The swelling and degradation of the hydrogels strongly depended on temperature and hydrogel composition. An empirical mathematical model was established to describe the fast water absorption at the early stage and deswelling at the late stage of the hydrogels at 37 degrees C. Two hydrophilic model drugs, methylene blue and bovine serum albumin, were loaded into the hydrogels during the synthesis process. The molecular size of the drugs, the hydrophilicity and degradation of the hydrogels, and temperature played important roles in controlling the drug release.     

Synthetic biomimetic hydrogels incorporated with ephrin-A1 for therapeutic angiogenesis

Eph receptors and ephrin ligands are essential for vascular development and angiogenic remodeling. In this work, we developed biomimetic poly(ethylene glycol)-diacrylate hydrogels incorporated with ephrin-A1 and examined their angiogenic properties. Ephrin-A1 was covalently immobilized on the surface of hydrogels by chemical modification and photopolymerization. Ephrin-A1 immobilized on hydrogels was found to retain its capacity to stimulate endothelial cell adhesion in a dose-dependent manner as similar findings were observed on polystyrene culture wells pre-adsorbed with ephrin-A1. Cell adhesion stimulated by ephrin-A1 was abolished by treatment with soluble RGDS and anti-alpha(v)beta3 integrin but not anti-alpha(v)beta5 integrin antibodies, suggesting that ephrin-A1 activates cell adhesion through alpha(v)beta3 integrins. Also, surface immobilized ephrin-A1 was found to induce endothelial tubule formation with luminal diameters ranging 5-30 microm on hydrogels. The results of these studies demonstrate that pro-angiogenic properties of ephrin-A1 are preserved in hydrogels and suggest potential applications of this hydrogel system in regenerative medicine and tissue engineering.     

Fine surface structure of enterotoxemic Escherichia coli O139:K12 strains associated with swine edema disease

The fine structure of the cell surface of seven enterotoxemic Escherichia coli (ETEEC) O139:K12 strains isolated from piglets with edema disease were examined electron microscopically using both the negative-staining method and the freeze-substitution fixation method. Densely packed, fine fibers were observed; they consisted of a capsule layer approximately 25 nm thick around the cell surfaces of strains 107/86, IW-2, ED-3, ED-43, and ED-61, all of which have a capacity to adhere strongly to HEp-2 cells. In contrast, no such structure was observed on the surface of strains RK-O139 or ED-1, both of which adhere only weakly to HEp-2 cells. These results suggest that the capsule structure might be associated with the ability to adhere to HEp-2 cells and, as a result, also potentially play some role in ETEEC infection. Received: 29 April 1996 / Accepted: 13 August 1996     

Preparation and characterization of polyvinyl alcohol-gelatin hydrogel membranes for biomedical applications

The purpose of this research was to design and develop hydrogels by esterification of polyvinyl alcohol (PVA) with gelatin. The membranes were characterized by Fourier Transform Infrared (FTIR) spectroscopy, x-ray diffraction (XRD), and differential scanning calorimetry. The viscosity of the esterified product (as solution) was compared with the mixture of PVA and gelatin of the same composition. The mechanical properties of the hydrogels were characterized by tensile tests. Swelling behavior and hemocompatibility of the membrane were also evaluated. The diffusion coefficient of salicylic acid (SA), when the receptor compartment contained Ringer's solution, through the membrane was determined. SA was used as a model drug. FTIR spectra of the membranes indicated complete esterification of the free carboxylic groups of gelatin. XRD studies indicated that the crystallinity of the membranes was mainly due to gelatin. The comparison of viscosity indicated an increase in segment density within the molecular coil. The membrane had sufficient strength and water-holding capacity. Hemocompatibility suggested that the hydrogel could be tried as wound dressing and as an implantable drug delivery system. The diffusion coefficient of SA through the membrane was found to be 1.32×10⁻⁵ cm²/s. The experimental results indicated that the hydrogel could be tried for various biomedical applications. Published: March 16, 2007 Formerly College of Pharmacy, University of Delhi, Pushp Vihar, New Delhi-110017 India     

Antifouling properties of tough gels against barnacles in a long-term marine environment experiment

In the marine environment, the antifouling (AF) properties of various kinds of hydrogels against sessile marine organisms (algae, sea squirts, barnacles) were tested in a long-term experiment. The results demonstrate that most hydrogels can endure at least 2 months in the marine environment. In particular, mechanically tough PAMPS/PAAm DN and PVA gels exhibited AF activity against marine sessile organisms, especially barnacles, for as long as 330 days. The AF ability of hydrogels toward barnacles is explained in terms of an ‘easy-release’ mechanism in which the high water content and the elastic modulus of the gel are two important parameters.     

Low- and high-resolution nuclear magnetic resonance (NMR) characterisation of hyaluronan-based native and sulfated hydrogels

Hyaluronan-based hydrogels were synthesised using different crosslinking agents, such as 1,3-diaminopropane (1,3-DAP) and 1,6-diaminohexane (1,6-DAE). The hydrogels were sulfated to provide materials (Hyal-1,3-DAP, Hyal-1,6-DAE, HyalS-1,3-DAP and HyalS-1,6-DAE) that were characterised by both high- and low-resolution nuclear magnetic resonance (NMR) spectroscopy. The (13)C NMR spectra of the materials were analysed to identify, characterise and study the crosslinking degree of the hydrogels. The crosslinking degree was also determined by potentiometric titration and the effectiveness of the two techniques was compared. Measurements of longitudinal relaxation times (spin-lattice) and of NOE enhancement were used to study the mobility of the hydrogels. Low-resolution NMR studies allowed the determination of the water transport properties in the hydrogels. In addition, the swelling degree for the various hydrogels was calculated as a function of the longitudinal and transversal relaxation times of the water molecules. Lastly, the self-diffusion coefficients of the water in interaction with the four polysaccharides were measured by the pulsed field gradient spin echo (PFGSE) sequence.     

Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures

We report a four-step procedure that optimizes the methodology for isolation of highly purified rat Kupffer cells (KC). We combined the previously reported techniques of enzymatic tissue treatment, density gradient centrifugation, centrifugal elutriation and selective adherence. ED-2 immunophenotyping and non-specific esterase histochemistry were used for cell identification. This combination resulted in a satisfactorily high yield of 80-100 x 10(6)KCs per liver, over 95% positive for ED-2 and 98% viable cells. Cultures of isolated KCs were functionally intact and exhibited a concentration and time-dependent LPS-induced TNF-alpha and nitric oxide production.     

Combined influence of substrate stiffness and surface topography on the antiadhesive properties of Acr-sP(EO-stat-PO) hydrogels

Biomaterials that prevent nonspecific protein adsorption and cell adhesion are of high relevance for diverse applications in tissue engineering and diagnostics. One of the most widely applied materials for this purpose is Poly(ethylene glycol) (PEG). We have investigated how micrometer line topography and substrate elasticity act upon the antiadhesive properties of PEG-based hydrogels. In our studies we apply bulk hydrogel cross-linked from star-shaped poly(ethylene oxide-stat-propylene oxide) macromonomers. Substrate surfaces were topographically patterned via replica molding. Additionally, the mechanical properties were altered by variations in the cross-linking density. Surface patterns with dimensions in the range of the cells' own size, namely 10 μm wide grooves, induced significant cell adhesion and spreading on the Acr-sP(EO-stat-PO) hydrogels. In contrast, there was only little adhesion to smaller and larger pattern sizes and no adhesion at all on the smooth substrates, regardless the rigidity of the gel. The effect of varied substrate stiffness on cell behavior was only manifest in combination with topography. Softer substrates with line patterns lead to significantly higher cell adhesion and spreading than stiff substrates. We conclude that the physical and mechanical surface characteristics can eliminate the nonadhesive properties of PEG-based hydrogels to a large extent. This has to be taken into account when designing surfaces for biomedical application such as scaffolds for tissue engineering which rely on the inertness of PEG.     

Tree gum-based renewable materials: Sustainable applications in nanotechnology,biomedical and environmental fields

The prospective uses of tree gum polysaccharides and their nanostructures in various aspects of food, water, energy, biotechnology, environment and medicine industries, have garnered a great deal of attention recently. In addition to extensive applications of tree gums in food, there are substantial non-food applications of these commercial gums, which have gained widespread attention due to their availability, structural diversity and remarkable properties as ‘green’ bio-based renewable materials. Tree gums are obtainable as natural polysaccharides from various tree genera possessing exceptional properties, including their renewable, biocompatible, biodegradable, and non-toxic nature and their ability to undergo easy chemical modifications. This review focuses on non-food applications of several important commercially available gums (arabic, karaya, tragacanth, ghatti and kondagogu) for the greener synthesis and stabilization of metal/metal oxide NPs, production of electrospun fibers, environmental bioremediation, bio-catalysis, biosensors, coordination complexes of metal–hydrogels, and for antimicrobial and biomedical applications. Furthermore, polysaccharides acquired from botanical, seaweed, animal, and microbial origins are briefly compared with the characteristics of tree gum exudates.     

Characterization of photo-cross-linked oligo[poly(ethylene glycol) fumarate] hydrogels for cartilage tissue engineering

Photo-cross-linkable oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels have been developed for use in tissue engineering applications. We demonstrated that compressive modulus of these hydrogels increased with increasing polymer concentration, and hydrogels with different mechanical properties were formed by altering the ratio of cross-linker/polymer in precursor solution. Conversely, swelling of hydrogels decreased with increasing polymer concentration and cross-linker/polymer ratio. These hydrogels are degradable and degradation rates vary with the change in cross-linking level. Chondrocyte attachment was quantified as a method for evaluating adhesion of cells to the hydrogels. These data revealed that cross-linking density affects cell behavior on the hydrogel surfaces. Cell attachment was greater on the samples with increased cross-linking density. Chondrocytes on these samples exhibited spread morphology with distinct actin stress fibers, whereas they maintained their rounded morphology on the samples with lower cross-linking density. Moreover, chondrocytes were photoencapsulated within various hydrogel networks. Our results revealed that cells encapsulated within 2-mm thick OPF hydrogel disks remained viable throughout the 3-week culture period, with no difference in viability across the thickness of hydrogels. Photoencapsulated chondrocytes expressed the mRNA of type II collagen and produced cartilaginous matrix within the hydrogel constructs after three weeks. These findings suggest that photo-cross-linkable OPF hydrogels may be useful for cartilage tissue engineering and cell delivery applications.     

Enhanced tissue adhesiveness of injectable gelatin hydrogels through dual catalytic activity of horseradish peroxidase

Development of bioadhesives with tunable mechanical strength, high adhesiveness, biocompatibility, and injectability is greatly desirable in all surgeries to replace or complement the sutures and staples. Herein, the dual catalytic activity of horseradish peroxidase is exploited to in situ form the hydroxyphenyl propionic acid‐gelatin/thiolated gelatin (GH/GS) adhesive hydrogels including two alternative crosslinks (phenol‐phenol and disulfide bonds) with fast gelation (few seconds – several minutes) and improved physicochemical properties. Their elastic moduli increase from 6.7 to 10.3 kPa by adding GS polymer that leads to the better stability of GH/GS hydrogels than GH ones. GH/GS adhesive strength is respectively 6.5‐fold and 15.8‐fold higher than GH‐only and fibrin glue that is due to additional disulfide linkages between hydrogels and tissues. Moreover, in vitro cell study with human dermal fibroblast showed the cell‐compatibility of GH/GS hydrogels. Taken together, GH/GS hydrogels can be considered as promising potential adhesive materials for various biomedical applications.     

New aspects of the formation of physical hydrogels of chitosan in a hydroalcoholic medium

New aspects concerning the mechanism of formation of chitosan physical hydrogels without any cross-linking agent were studied. The gelation took place during the evaporation of a hydroalcoholic solution of chitosan. We first demonstrated that it was possible to form a physical hydrogel from a hydrochloride form of chitosan. Chromatographic methods showed that during the gel formation, when the initial concentration is over C, the critical concentration of chain entanglement, the water and acid used for the solubilization of the polymer were both eliminated. This particular situation contributed to decrease the dielectric constant of the medium and the apparent charge density of chitosan chains, thus inducing the formation of a three-dimensional network through hydrophobic interactions and hydrogen bonding. In the gelation process, this step was kinetically determining. The speed of evaporation of water and acid were determined and different initial conditions were compared. Thus, we investigated the influence of: the initial polymer concentration, the nature of the counterion and the alcohol, the temperature and the geometry of the reactor. Our results allowed us to confirm the existence of a second critical initial concentration C, from which the evaporation of water became more difficult. We suggested that C corresponded to a reorganization of the solution involving the presence of gel precursors. Then, a mechanism of formation of physical hydrogels of chitosan in a hydroalcoholic medium could be proposed. For the first time, we demonstrated that it was possible to generate physical hydrogels in the presence of various diols, which size of the carbonated chain appeared as a limiting factor for the gelation process. These physical hydrogels of chitosan are currently used in our laboratory for tissue engineering in the treatment of third degree burns with the possibility to adapt their mechanical properties from the choice of both the acid or the alcohol used.     

Eldecalcitol induces apoptosis and autophagy in human osteosarcoma MG-63 cells by accumulating ROS to suppress the PI3K/Akt/mTOR signaling pathway

Eldecalcitol (ED-71) is a new type of vitamin D analog, and vitamin D has been reported to have therapeutic effects in infectious disease, autoimmune disease, and cancer. However, the anti-cancer effect of ED-71 remains unclear. The objective of this study was to explore the anti-cancer effect of ED-71 in human osteosarcoma cells and to identify the related mechanism. The CCK8 assay results showed that ED-71 inhibited MG-63 cell viability in dose and time dependent manners. Cloning and Transwell invasion assays showed that ED-71 inhibited clonal and invasion ability of MG-63 cells. Flow cytometry results showed ED-71 the G2/M cycle arrest rate, apoptosis, and intracellular ROS. Western blot was used to detect cleaved-caspase-3, Bax, Bcl-2, LC3-II/LC3-I, and P62 levels and the mTOR pathway. The increase of LC3-II and P62 indicated that ED-71 induced the formation of autophagosomes and inhibited autophagy flux. Furthermore, ED-71-induced apoptosis was weakened after adding 3-methyladenine and ED-71-induced early autophagy was weakened by caspase-3 inhibitor (Z-VAD-FMK), which indicated the two processes active each other in the presence of ED-71. Furthermore, N-acetylcysteine (NAC) pretreatment reversed the ED-71-treatment outcomes, including increased apoptosis and autophagy and inhibition of the PI3K/Akt/mTOR pathway. In conclusion, our results reveal that ED-71 induced G2/M arrest, apoptosis and autophagy in MG-63 cells by accumulating ROS to suppress the PI3K/Akt/mTOR signaling pathway     

DNA水凝胶的制备及应用

脱氧核糖核酸(Deoxyribonucleic acid,DNA)不仅可作为生物遗传的物质基础,又以其可编程性、功能多样性、生物相容性和生物可降解性等优点,在生物材料的构建方面表现出巨大的潜力。DNA水凝胶是一种主要由DNA参与形成的三维网状聚合物材料,同时因其保留的DNA生物性能与自身骨架的机械性能的完美融合使得它成为近年来最受关注的新兴功能高分子材料之一。目前,基于各种功能核酸序列或通过结合不同的功能材料制备的单组分或多组分DNA水凝胶,已广泛用于生物医学、分子检测及环境保护的研究或应用领域中。文中主要总结了近十几年来DNA水凝胶制备方法上的研究进展,探讨了DNA水凝胶的分类策略,并进一步综述了DNA水凝胶在药物运输、生物传感、细胞培养等方面的应用研究。最后对DNA水凝胶未来的发展方向以及可能面临的挑战进行了展望。