The modulation of cellular responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases macrophage collagenase production in vitro

We examined the regulation of collagenase production by the monocyte/macrophage THP-1 cell line when these cells were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies. Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response. Copolymer hydrogels containing 2-ethoxyethyl methacrylate (EMA) or methyl methacrylate (MMA) also induced a high response, while PHEMA hydrogels induced a low level response and the phosphorylated hydrogel induced no response. This pattern was altered when the morphology of the hydrogels was changed to that of a sponge. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels. Sponges containing EMA and MMA produced low level response relative to the TCP control. PHEMA and phosphorylated sponges produced little and no response respectively. The dramatically reduced enzyme response to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge.     

Amyloid fibres of Sup35 support a prion-like mechanism of inheritance in yeast

Hydrogels were prepared from poly(vinyl alcohol) and chitosan in various blend ratios. The water contents of the hydrogels were in the range of 65 to 75 wt %. The attachment and growth of fibroblast cells (L-929) on the hydrogels were studied with a cell culture method. On the hydrogels with more than 15 wt % chitosan content, the attached cells were able not only to remain viable but also to proliferate. The relative cell attachment after incubation for 30 h increased with increasing chitosan content in the hydrogels. Cell attachment and growth on the hydrogel with 40 wt % chitosan content exceeded those on collagen, a widely-used mammalian cell culture substrate. The morphology of the cells attached onto the hydrogels with a lower chitosan content was spherical, but in hydrogels with more than 15 wt % chitosan content, the number of spindle-shaped cells increased with increasing chitosan content.     

Effect of additives on gelation and tissue adhesion of gelatin-poly(L-glutamic acid) mixture

Gelation and tissue adhesion of mixtures of gelatin and poly (L-glutamic acid) (PLGA) aqueous solution were investigated in the presence of additives following the addition of a water-soluble carbodiimide (WSC) that induced chemical cross linking between gelatin and PLGA. To prevent spontaneous gelation of the mixed solution through physical cross linking between gelatin molecules at room temperature, additives were added to the mixed solution. Among the additives studied, starch and urea were effective in preventing the spontaneous physical gelation. The mixed gelatin and PLGA solution set to a cross-linked hydrogel within scores of second by WSC addition, irrespective of the presence of urea, whereas the viscosity of the solution with added starch was too high to measure the gelation time. The cross-linked gelatin-PLGA hydrogels with and without urea showed higher bonding strength to soft tissues than fibrin glue. This was in marked contrast to gelatin-PLGA hydrogels with soluble starch. Irrespective of the presence of urea, the gelatin-PLGA hydrogels gradually biodegraded in the back subcutis of mice over 3 months and no severe inflammatory response to the hydrogels was observed. These findings indicate that urea is promising as an additive to prevent spontaneous physical gelation of the mixed gelatin and PLGA aqueous solution without changing the characteristics of WSC-induced cross linking and tissue adhesion of the formed hydrogel.     

Novel hydrophilic cyclic monomers in hydrogel synthesis

A range of hydrogels was prepared by the copolymerisation of 2-hydroxyethyl methacrylate (HEMA) with various hydrophilic cyclic monomers (CM) derived from cis-1,2-dihydroxy-3,5-cyclohexadiene (DHCD). The specific systems investigated were the cis-1,2-bis(2,3-epoxybutanoyloxy)-, cis-1,2-bis(10,11-epoxyundecanoyloxy)-, cis-1,2-bis(trimethylsiloxy)- and cis-1,2-bis(tert-butyldimethylsiloxy)-3,5-cyclohexadienes. The EWC, tensile strength and initial modulus of the poly HEMA-CM hydrogels derived from the epoxy compounds showed lower EWCs but similar mechanical properties compared with polyHEMA itself. Treatment of the hydrogel derived from cis-1,2-bis(2,3-epoxybutanoyloxy)-3,5-cyclohexadiene with 20% sodium hydroxide increased the EWC value markedly but produced highly fragile materials.     

Synthesis and characterization of lanthanum bonded agar-carbomer hydrogel:a promising tool for biomedical research

Agar-Carbomer(branched poly(acrylic acid)) hydrogel,an injectable bio-resorbable scaffold with a controlled nanostructure specifically designed for neural cell housing,was developed together with a new protocol for building three dimensional biohybrid cell/hydrogel systems.In order to overcome classic structural analysis inconveniences due to the high water amount,which affects instruments results and reliability,agar-Carbomer hydrogels were synthesized by microwave-assisted block copolymerization together with La3+ salts.Propylene glycol,glycerol and buffered saline solution were used as cross-linking agents and solvent,respectively.Biomaterial properties were not affected by the presence of lanthanum,and were checked via swelling and rheological analysis.Moreover,the presence of La3+ within the polymeric network was characterized by thermogravimetric analysis,environmental scanning electron microscopy and Fourier transformed infrared spectroscopy.The results showed that the rare earth presented uniform distribution in the hydrogel network due to the formation of chemical bonds after polymerization without being modified its luminescence emission spectrum that allowed hydrogel detection.These results made the obtained host-guest system a useful tool for analytical research studies concerning regenerative medical applications that could also be potentially taken up with in vivo experiments.     

Hemostatic capability of rapidly curable glues from gelatin, poly(L-glutamic acid), and carbodiimide

The hemostatic capability of rapidly curable glues composed of gelatin and poly(L-glutamic acid) (PLGA) was compared with that of the conventional fibrin glue. The hydrogels produced from mixed gelatin and PLGA aqueous solution within several seconds by addition of water-soluble carbodiimide (WSC) was applied to the dog spleen injured by needle pricking. The WSC-catalyzed gelatin-PLGA glues exhibited higher hemostatic capability than the fibrin glue. The total amount of bleeding from the injured spleen until hemostasis when the gelatin-PLGA hydrogel glues were applied was significantly smaller than that of the fibrin glue application. The gelatin-PLGA glue application enhanced the success rate of complete hemostasis to a significantly greater extent than the fibrin glue, while the frequency of glue applications until achieving complete hemostasis decreased. The gelatin PLGA hydrogels strongly adhered to the surface of dog spleen, whereas the fibrin hydrogel was easily detached from the spleen surface. It was concluded that this strong adhesion mechanically suppressed the bleeding, leading to enhanced hemostasis by the rapidly curable gelatin-PLGA glues.     

A new biological glue from gelatin and poly (L-glutamic acid)

This study describes the potentiality of hydrogels composed of gelatin and poly(L-glutamic acid) (PLGA) as a biological glue for soft tissues and compares its effectiveness with that of a conventional fibrin glue. Water-soluble carbodiimides (WSC) were used to crosslink the aqueous mixture of gelatin and PLGA. The mixed aqueous solution of gelatin and PLGA set to a hydrogel by use of WSC as rapidly as BOLHEAL fibrin glue. An addition of PLGA to gelatin aqueous solution reduced not only its gelation time but also the WSC concentration necessary for hydrogel formation. The cured hydrogel exhibited firm adhesion to the mouse skin and other soft tissues with a higher bonding strength than BOLHEAL fibrin glue. Cohesive failure in the hydrogel was observed when the gel-tissue bond was broken, in contrast to BOLHEAL fibrin glue. The bonding strength of the gelatin-PLGA hydrogel became higher with the increasing PLGA concentration. The inflammatory reaction around the gelatin-PLGA hydrogel subcutaneously implanted in mice was mild, and the hydrogel was gradually absorbed with time in vivo. A toxicity test demonstrated that the concentration of WSC necessary as a biological glue was low enough not to induce its toxicity.     

PREPARATION OF POROUS NANOCOMPOSITE SCAFFOLDS WITH HONEYCOMB MONOLITH STRUCTURE BY ONE PHASE SOLUTION FREEZE-DRYING METHOD

Biodegradable porous nanocomposite scaffolds of poly(lactide-co-glycolide) (PLGA) and L-lactic acid (LAc)oligomer surface-grafted hydroxyapatite nanoparticles (op-HA) with a honeycomb monolith structure were fabricated with the single-phase solution freeze-drying method. The effects of different freezing temperatures on the properties of the scaffolds, such as microstructures, compressive strength, cell penetration and cell proliferation were studied. The highly porous and well interconnected scaffolds with a tunable pore structure were obtained. The effect of different freezing temperature (4℃, -20℃, -80℃ and -196℃) was investigated in relation to the scaffold morphology, the porosity varied from 91.2% to 83.0% and the average pore diameter varied from (167.2 ± 62.6) μm to (11.9± 4.2)μm while the σ10 increased significantly. The cell proliferation were decreased and associated with the above-mentioned properties. Uniform distribution of op-HA particles and homogeneous roughness of pore wall surfaces were found in the 4℃ frozen scaffold. The 4℃ frozen scaffold exhibited better cell penetration and increased cell proliferation because of its larger pore size, higher porosity and interconnection. The microstructures described here provide a new approach for the design and fabrication of op-HA/PLGA based scaffold materials with potentially broad applicability for replacement of bone defects.     

A potentiostatic study of oxygen transmissibility and permeability through hydrogel membranes

The permeability characteristics of membranes prepared from hydrogels of poly(2-hydroxyethylmethacrylate) (PHEMA) and poly(2-hydroxyethylmethacrylate-co-N,N-dimethylacrylamide) (PHNDA) are described. True values of the permeability and transmissibility coefficients of oxygen in the membranes are determined by using electrochemical procedures involving the measurement of the steady state current either in membranes with different thickness or in a single membrane in which its thickness is varied with layers of moistened paper. Comparison of the results obtained for the transport properties in these hydrogels with others obtained in other hydrogels permit to conclude that the degree of swelling rather than the chemical nature of the hydrogels affects the permeation properties. The chemical structure presumably only affects in high degree the chemical stability and flexibility of the hydrogel membranes.     

Characterization of protein release through glucose-sensitive hydrogel membranes

Glucose-sensitive phase-reversible hydrogels have been prepared based on the specific interaction between polymer-bound glucose and concanavalin A (Con-A). The main goal of this study was to characterize the release of model proteins (insulin and lysozyme) through the hydrogel membrane as the free glucose concentration in the environment was changed. The diffusion of the model proteins through the hydrogel membrane was examined using a diffusion cell. Porous poly(hydroxyethyl methacrylate) (PHEMA) membranes were used to sandwich the mixture of glucose-containing polymers and Con-A in between the donor and receptor chambers. The porous PHEMA membranes allowed diffusion of glucose, insulin and lysozyme, while preventing loss of glucose-containing polymers and Con-A in the sol state. The release rate of model proteins through the glucose-sensitive hydrogel membrane was dependent on the concentration of free glucose. The release rate of the proteins did not remain constant, however, due to the change in free glucose concentration resulting from diffusion of glucose from the receptor chamber to the donor chamber. This study demonstrated the possibility that the glucose-sensitive phase-reversible hydrogels can be used to regulate the insulin release as a function of the free glucose concentration in the environment.     

In vitro sorption and desorption of basic fibroblast growth factor from biodegradable hydrogels

In vitro interaction of basic fibroblast growth factor (bFGF) with biodegradable gelatin hydrogels was investigated, focusing on its sorption into the hydrogels and desorption from them. Basic bFGF was sorbed to the hydrogel of acidic gelatin with an isoelectric point (IEP) of 5.0 over time at 4 degrees C, in contrast to that of basic gelatin with an IEP of 9.0 and type I collagen. The bFGF sorption was almost independent of the sorption temperature except for 4 degrees C and the hydrogel water content. Fluorescent microscopic observation revealed that bFGF was sorbed into the interior of the acidic gelatin hydrogel. The binding molar ratio of bFGF to the acidic gelatin was around 1.0. The bFGF sorption to the acidic gelatin hydrogel increased when gelatin was further carboxylated. bFGF was sorbed into the acidic gelatin hydrogel more slowly than into the poly(acrylic acid) (PAAc) hydrogel, probably because of the lower density of negative charge of gelatin. The bFGF sorption decreased with an increase in solution ionic strength, indicating that an electrostatic interaction was the main driving force for bFGF sorption to the acidic gelatin hydrogel. However, even at higher ionic strengths of solution, the sorbed bFGF was not desorbed from the acidic gelatin hydrogel, in contrast to the PAAc hydrogel.     

Hydrogels of poly(ethylene glycol): mechanical characterization and release of a model drug

Thermosensitive polymer networks were synthesized from poly(ethylene glycol), hexamethylene diisocyanate and 1,2,6-hexanetriol in stoichiometric proportions. By varying the amount of 1,2,6-hexanetriol and the molar mass of the poly(ethylene glycol), a wide range of networks with different crosslinking densities was prepared. The networks obtained were characterized by the temperature dependence of their degree of equilibrium swelling in water and by their Young's moduli. For each network, the molecular weight between crosslinks was estimated. The structure of the hydrogels was analysed with respect to scaling laws, and it was found that the results obtained with PEG 1500 and PEG 6000 hydrogels are in agreement with theoretical predictions, whereas those obtained with PEG 400 hydrogels are in disagreement. The release properties of PEG hydrogels were studied by the determination of the diffusion coefficient for acebutolol chlorhydrate and by an analysis of the effect of temperature on these coefficients. Finally, these release properties were correlated with the swelling and structural properties of the hydrogels.     

Effect of charged groups on the adsorption and penetration of proteins onto and into carboxymethylated poly(HEMA) hydrogels

A range of carboxymethylated poly(hydroxyethyl methacrylate) (CM-PHEMA) hydrogels with varying degrees of carboxymethylation was synthesized for a systematic study of the effects of ionized groups ('charge') on the uptake by hydrogel matrices of the proteins, lysozyme and human serum albumin (HSA). Using a radiolabel-tracer technique, X-ray photoelectron spectroscopy, and laser scanning confocal microscopy, we attempted to differentiate between protein molecules that were irreversibly adsorbed onto the hydrogel surface and those that penetrated into the hydrogel matrix. The effective pore size of the CM-PHEMA hydrogels was modelled and compared with the known molecular dimensions of the two proteins. The effects of the presence of varying amounts of ionized groups in the hydrogel matrix differed for the two proteins. For lysozyme, increased uptake was observed at higher carboxymethylation; this is interpreted as resulting from a combination of electrostatic attraction and increasing ease of penetration of the protein into the more porous hydrogel matrix. For HSA, on the other hand, the uptake was primarily by surface adsorption, with little diffusive penetration into the matrix.     

Observations on the β→ζ m massive transformation in two-phase (β+ζ) alloys of the Cu−Ga−Ge system

Optical metallography, transmission electron microscopy, and thermal arrest measurements, have been used to study the transformation, on quenching, of an initial two-phase (β+ζ) microstructure, in the Cu?Ga?Ge system. The two phases bcc (β) and hcp (ζ) coexist in a narrow temperature range near 635°C in a ternary Cu-21.0Ga-1.5Ge alloy. The microstructure at high temperature typically consists of β regions outlining ζ grains. Upon rapid quenching of such a structure, the hexagonal ζ grains remain stable, but each β region transforms via a massive transformation to an hcp ζ _m structure without change of composition. Growth of such ζ _m grains takes place only from certain original β/ζ boundaries, but not from others. A ξ _m grain adopts the same crystallographic orientation as the adjacent ζ grain at which it initiated. However, the coherent ζ/ζ _m “boundary” which develops exhibits a lattice parameter discontinuity across it, due to the difference in composition. The process amounts to massive growth without conventional nucleation. The significance of these findings, particularly in terms of the desirability of the presence or absence of certain initial crystallographic relationships between the β and ζ grains is discussed.     

Optimization of photopolymerized bioerodible hydrogel properties for adhesion prevention

The aim of this study was to optimize the properties of a lubricious bioerodible hydrogel barrier for the prevention of postoperative adhesions. Water-soluble macromers based on block copolymers of poly(ethylene glycol) (PEG) and poly(lactic acid) or poly(glycolic acid) with terminal acrylate groups were used, and these macromers were gelled in vivo by exposure to long wavelength ultraviolet light. The precursor was photopolymerized from buffered saline solution while in contact with the tissues. This resulted in the conformal coating of the tissue with an adherent hydrogel film, while forming a nonadhesive barrier at the free surface, on the treated wound site. The hydrogels were evaluated in two animal models of postsurgical adhesions, first in a rat cecum abrasion model and then in a rabbit uterine horn ischemia model. In the rat cecum model, six of seven animals treated with a hydrogel, with glycolide in the precursor as the comonomer, showed no adhesions; untreated animals and animals treated with precursor, but not gelled with light, showed consistent dense adhesions. In the rabbit uterine horn ischemia model, using hydrogels with lactide in the precursor as the comonomer, and PEG of molecular weight from 6,000 to 18,500 Da, adhesions were dramatically reduced, with occurrence in none of seven animals treated with a gel containing PEG 10,000. By contrast, the seven animals in the control group demonstrated a mean of 35% involvement of the horn length in dense, fibrous adhesions. These materials, photopolymerized in vivo in direct contact with the tissues, appear to form an adherent hydrogel barrier that is highly effective in reducing postoperative adhesions in the models used.(ABSTRACT TRUNCATED AT 250 WORDS)     

A liposomal hydrogel for the prevention of bacterial adhesion to catheters

The adhesion of bacteria to medical implants and the subsequent development of a biofilm frequently results in the infection of surrounding tissue and may require removal of the device. We have developed a liposomal hydrogel system that significantly reduces bacterial adhesion to silicone catheter material. The system consists of a poly (ethylene glycol)-gelatin hydrogel in which liposomes containing the antibiotic ciprofloxacin are sequestered. A poly (ethylene glycol)-gelatin-liposome mixture was applied to a silicone surface that had been pre-treated with phenylazido-modified gelatin. Hydrogel cross-linking and attachment to surface-immobilized gelatin was accomplished through the formation of urethane bonds between gelatin and nitrophenyl carbonate-activated poly (ethylene glycol). Liposomal hydrogel-coated catheters were shown to have an initial ciprofloxacin content of 185+/-16 microg cm(-2). Ciprofloxacin was released over seven days with an average release rate of 1.9+/-0.2 microg cm(-2) h(-1) for the first 94 h. In vitro assays using a clinical isolate of Pseudomonas aeruginosa established the antimicrobial efficacy of the liposomal hydrogel. A modified Kirby-Bauer assay produced growth-inhibition zone diameters of 39+/-1 mm, while bacterial adhesion was completely inhibited on catheter surfaces throughout a seven-day in vitro adhesion assay. This new antimicrobial coating shows promise as a prophylactic and/or treatment for catheter-related infection.     

Massive and martensitic transformations in the Ag−Al system

The Ag?Al system in the composition range of stability of the high temperature β phase exhibits two-phase field characteristics which make it an ideal system for examining massive phase transformations. Experiments have established that the β phase can be retained to room temperature following cooling rates of the order of 10⁵ deg per sec. Subsequently, the M_s temperatures have been determined for the composition range Ag-24.4 to 25.0 at. pct Al. Since the ζ phase can also be retained, it has been possible to examine the β→ζ _m , β→μ _m and ζ→μ _m 3composition-invariant, nonmartensitic phase transformation at a single composition. In addition, the two high-temperature phases, β and ζ, are associated with a congruent point at Ag-24.5 at. pct Al, the width of the two-phase field increasing at higher and lower aluminum concentrations. This has permitted an investigation of the morphology of the ζ _m -product for a range of compositions and cooling rates, the latter determining the extent of undercooling at which the reaction occurs. In particular, that morphology representative of a given degree of supercooling has been compared for several compositions and the interdependence of cooling rate and parent/product coherency (as revealed by the shape of the resulting ζ _m -phase) has been examined.     

A HYBRID SCAFFOLD OF POLY(LACTIDE-CO-GLYCOLIDE) SPONGE FILLED WITH FIBRIN GEL FOR CARTILAGE TISSUE ENGINEERING

The poly(lactide-co-glyeolide) (PLGA) sponge fabricated by a gelatin porogen leaching method was filled with fibrin gel to obtain a hybrid scaffold for chondrocytes culture in vitro. The fibrin gel evenly distributed in the hybrid scaffold with visible fibrinogen fibers after drying. In vitro culture it was found that in the hybrid scaffold the chondrocytes distributed more evenly and kept a round morphology as that in the normal cartilage. Although the ehondrocytes seeded in the control PLGA sponges showed similar proliferation behavior with that in the hybrid scaffolds, they were remarkably elongated, forming a fibroblast-like morphology. Moreover, a larger amount of glycosaminoglycans was secreted in the hybrid scaffolds than that in the PLGA sponges after in vitro culture of chondrocytes for 4 weeks. The results suggest that the fibrin/PLGA hybrid scaffold 2 be favorably applied for cartilage tissue engineering.     

Structural and material mechanical properties of human vertebral cancellous bone

The structural Young's modulus (i.e. that of the cancellous framework) was determined by non-destructive compressive mechanical testing in the three orthogonal axes of 48 vertebral bone cubes. In addition, the material Young's modulus (i.e. of the trabeculae themselves) was estimated using an ultrasonic technique. Apparent and true density were determined by direct physical measurements. Significant mechanical anisotropy was observed: mean structural Young's modulus varied from 165 MPa in the supero-inferior direction to 43 MPa in the lateral direction. Structural Young's modulus correlated with apparent density, with power-law regression models giving the best correlations (r2 = 0.52-0.88). Mechanical anisotropy increased as a function of decreasing apparent density (p < 0.001). Material Young's modulus was 10.0 +/- 1.3 GPa, and was negatively correlated with apparent density (p < 0.001). In multiple regression models, material Young's modulus was a significant independent predictor of structural Young's modulus only in the supero-inferior direction. The data suggest the presence of two effects in vertebral bone associated with decreasing apparent density and, by implication, bone loss in general: (a) increased mechanical anisotropy, such that there is relative conservation of stiffness in the axial direction compared with the transverse directions; and (b) increased stiffness of the trabeculae themselves.     

Ibuprofen inhibits pyrogen-dependent expression of VCAM-1 and ICAM-1 on human endothelial cells

Leukocyte adhesion and transmigration through the endothelial cell (EC) layer plays a crucial role in inflammation. IL-1 alpha and TNF alpha increase EC-adhesiveness for leukocytes by stimulating surface expression of ICAM-1 (intercellular adhesion molecule 1, CD54), VCAM-1 (vascular cell adhesion molecule 1, CD106) and E-selectin (CD62E). In this study, the effects of ibuprofen on IL-1 alpha and TNF alpha-induced expression of ICAM-1, VCAM-1 and E-selectin on cultured human umbilical vein EC (HUVEC) were analyzed. Exposure to IL-1 alpha or TNF alpha resulted in an increased expression of VCAM-1, ICAM-1, and E-selectin. Ibuprofen was identified as a potent inhibitor of IL-1 alpha and TNF alpha-induced surface expression of VCAM-1 and a less potent inhibitor of pyrogen-induced expression of ICAM-1, whereas no effect on E-selectin was found. The effects of ibuprofen on VCAM-1 expression were dose-dependent (IC50 [IL-1 alpha]: 0.5 mM; IC50 [TNF alpha]: 0.5 mM) and time-dependent with maximum responses observed after 18 h. Moreover, ibuprofen abrogated pyrogen-dependent adhesion of leukocytes to HUVEC. Ibuprofen also inhibited VCAM-1 mRNA expression in pyrogen activated EC. VCAM-1-downregulation on EC by ibuprofen may contribute to the anti-inflammatory actions of the drug.