Michael-type addition reactions for the in situ formation of poly(vinyl alcohol)-based hydrogels

Michael-type addition reactions offer the possibility to obtain in situ formation of polymeric hydrogels in the absence of a radical mechanism for the networking process. We explored such a synthetic route for obtaining a poly(vinyl alcohol) (PVA)-based hydrogel as a potential biomaterial for applications in vitro-retinal replacement surgery. The presence of radicals in the reaction medium can represent a risk for in situ surgical treatment. To circumvent this problem we have applied nucleophilic addition to ad hoc modified PVA macromers. The gel formation has been studied with respect to the timing required in this surgery and in terms of the structural characteristics of the obtained network.     

Synthesis and physicochemical characterization of end-linked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition

The synthesis of novel hybrid hydrogels by stepwise copolymerization of multiarm vinyl sulfone-terminated poly(ethylene glycol) macromers and alpha-omega cysteine oligopeptides via Michael-type additions is described. Cross-linking kinetics, studied by in situ rheometry, can be controlled by pH and the presence of charged amino acid residues in close proximity to the Cys, which modulates the pK(a) of the thiol group. These end-linked networks were characterized by their equilibrium swelling in water, by their viscoelastic properties in the swollen state, and by their soluble fraction. It was demonstrated that structure and properties are very sensitive to the preparation state including stoichiometry and precursor concentration and less sensitive to the pH during cross-linking. For each network the concentration of elastically active chains (nu) was calculated from experimentally determined sol fractions using Miller-Macosko theory and compared to values obtained from swelling and rheometry studies and by calculation from Flory's classical network models. Hydrogels were also prepared with varying macromer structures, and their properties were shown to respond to both macromer functionality and molecular weight.     

Cross-linking and degradation of step-growth hydrogels formed by thiol-ene photoclick chemistry

Thiol-ene photoclick hydrogels have been used for a variety of tissue engineering and controlled release applications. In this step-growth photopolymerization scheme, four-arm poly(ethylene glycol) norbornene (PEG4NB) was cross-linked with dithiol containing cross-linkers to form chemically cross-linked hydrogels. While the mechanism of thiol-ene gelation was well described in the literature, its network ideality and degradation behaviors are not well-characterized. Here, we compared the network cross-linking of thiol-ene hydrogels to Michael-type addition hydrogels and found thiol-ene hydrogels formed with faster gel points and higher degree of cross-linking. However, thiol-ene hydrogels still contained significant network nonideality, demonstrated by a high dependency of hydrogel swelling on macromer contents. In addition, the presence of ester bonds within the PEG-norbornene macromer rendered thiol-ene hydrogels hydrolytically degradable. Through validating model predictions with experimental results, we found that the hydrolytic degradation of thiol-ene hydrogels was not only governed by ester bond hydrolysis, but also affected by the degree of network cross-linking. In an attempt to manipulate network cross-linking and degradation of thiol-ene hydrogels, we incorporated peptide cross-linkers with different sequences and characterized the hydrolytic degradation of these PEG-peptide hydrogels. In addition, we incorporated a chymotrypsin-sensitive peptide as part of the cross-linkers to tune the mode of gel degradation from bulk degradation to surface erosion.     

Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions

We present polymeric hydrogel biomaterials that are biomimetic both in their synthesis and degradation. The design of oligopeptide building blocks with dual enzymatic responsiveness allows us to create polymer networks that are formed and functionalized via enzymatic reactions and are degradable via other enzymatic reactions, both occurring under physiological conditions. The activated transglutaminase enzyme factor XIIIa was utilized for site-specific coupling of prototypical cell adhesion ligands and for simultaneous cross-linking of hydrogel networks from factor XIIIa substrate-modified multiarm poly(ethylene glycol) macromers. Ligand incorporation is nearly quantitative and thus controllable, and does not alter the network's macroscopic properties over a concentration range that elicits specific cell adhesion. Living mammalian cells can be encapsulated in the gels without any noticeable decrease in viability. The degradation of gels can be engineered to occur, for example, via cell-secreted matrix metalloproteinases, thus rendering these gels interesting for biomedical applications such as drug delivery systems or smart implants for in situ tissue engineering.     

Dynamic compressive loading influences degradation behavior of PEG-PLA hydrogels

Biodegradable hydrogels are attractive 3D environments for cell and tissue growth. In cartilage tissue engineering, mechanical stimulation has been shown to be an important regulator in promoting cartilage development. However, the impact of mechanical loading on the gel degradation kinetics has not been studied. In this study, we examined hydrolytically labile gels synthesized from poly(lactic acid)-b-poly(ethylene glycol)-b-poly-(lactic acid) dimethacrylate macromers, which have been used for cartilage tissue engineering. The gels were subject to physiological loading conditions in order to examine the effects of loading on hydrogel degradation. Initially, hydrogels were formed with two different cross-linking densities and subject to a dynamic compressive strain of 15% at 0.3, 1, or 3 Hz. Degradation behavior was assessed by mass loss, equilibrium swelling and compressive modulus as a function of degradation time. From equilibrium swelling, the pseudo-first-order reaction rate constants were determined as an indication of degradation kinetics. The application of dynamic loading significantly enhanced the degradation time for the low cross-linked gels (P < 0.01) while frequency showed no statistical differences in degradation rates or bulk erosion profiles. In the higher cross-linked gels, a 3 Hz dynamic strain significantly increased the degradation kinetics resulting in an overall faster degradation time by 6 days compared to gels subject to the 0.3 and 1 Hz loads (P < 0.0001). The bioreactor set-up also influenced overall degradation behavior where the use of impermeable versus permeable platens resulted in significantly lower degradation rate constants for both cross-linked gels (P < 0.001). The compressive modulus exponentially decreased with degradation time under dynamic loading. Together, our findings indicate that both loading regime and the bioreactor setup influence degradation and should be considered when designing and tuning a biodegradable hydrogel where mechanical stimulation is employed.     

Rheological properties of ovalbumin hydrogels as affected by surfactants addition

The gel properties of ovalbumin mixtures with three different surfactants (sodium perfluorooctanoate, sodium octanoate and sodium dodecanoate) have been studied by rheological techniques. The gel elasticities were determined as a function of surfactant concentration and surfactant type. The fractal dimension of the formed structures was evaluated from plots of storage modulus against surfactant concentration. The role of electrostatic, hydrophobic and disulfide SS interactions in these systems has been demonstrated to be the predominant. The viscosity of these structures tends to increase with surfactant concentration, except for the fluorinated one. Unfolded ovalbumin molecules tend to form fibrillar structures that tend to increase with surfactant concentration, except for the fluorinated one. This fact has been related to the particular nature of this molecule.     

Synthesis and degradation test of hyaluronic acid hydrogels

Hyaluronic acid (HA) hydrogels prepared with three different crosslinking reagents were assessed by in vitro and in vivo degradation tests for various tissue engineering applications. Adipic acid dihydrazide grafted HA (HA-ADH) was synthesized and used for the preparation of methacrylated HA (HA-MA) with methacrylic anhydride and thiolated HA (HA-SH) with Traut's reagent (imminothiolane). (1)H NMR analysis showed that the degrees of HA-ADH, HA-MA, and HA-SH modification were 69, 29, and 56 mol%, respectively. HA-ADH hydrogel was prepared by the crosslinking with bis(sulfosuccinimidyl) suberate (BS(3)), HA-MA hydrogel with dithiothreitol (DTT) by Michael addition, and HA-SH hydrogel with sodium tetrathionate by disulfide bond formation. According to in vitro degradation tests, HA-SH hydrogel was degraded very fast, compared to HA-ADH and HA-MA hydrogels. HA-ADH hydrogel was degraded slightly faster than HA-MA hydrogel. Based on these results, HA-MA hydrogels and HA-SH hydrogels were implanted in the back of SD rats and their degradation was assessed according to the pre-determined time schedule. As expected from the in vitro degradation test results, HA-SH hydrogel was in vivo degraded completely only in 2 weeks, whereas HA-MA hydrogels were degraded only partially even in 29 days. The degradation rate of HA hydrogels were thought to be controlled by changing the crosslinking reagents and the functional group of HA derivatives. In addition, the state of HA hydrogel was another factor in controlling the degradation rate. Dried HA hydrogel at 37 degrees C for a day resulted in relatively slow degradation compared to the bulk HA hydrogel. There was no adverse effect during the in vivo tests.     

Microbial degradation of phenanthrene by addition of a sophorolipid mixture

The influence of sophorolipids on microbial degradation of poorly soluble phenanthrene in liquid and soil suspension culture was evaluated in the work presented. Experiments were carried out in two parts. In the first part, important basic physico-chemical characteristics of the biosurfactant and the pollutant used were determined. The critical micelle concentration (CMC) and the solubilization ratio of the biosurfactant were found to be in a good range compared with synthetic surfactants. Also, a reduction to 71% of the detectable amount of phenanthrene was measured within 4 d in soil suspension without any biotic influence. In the second part, culture experiments were done with Sphingomonas yanoikuyae, the bacterium used throughout the work presented here with the aim to assess the toxicity of the sophorolipids on these bacteria and the effect of the surfactant on biodegradation. In exponential growth tests, no toxicity up to 1 g l(-1) sophorolipids could be detected, whereas in an agar plate test, slight growth hindrance was measured at a lower concentration of 250 mg l(-1). The above mentioned data were important for planning further experiments. In the following cultivations with liquid and soil suspension media, enhancements of the biodegradation with surfactant addition were measurable. Fluorescence measurements showed that this effect was not due to an increasing biomass, but to an augmentation of bioavailability of the phenanthrene through increasing the apparent dissolved pollutant. Surfactant addition had the consequence of decreasing the residual detectable pollutant concentration (after 36 h 0.5 compared with 2.3 mg l(-1) soil suspension) and increasing the maximal degradation rate (127 instead of 80 mg l(-1) soil suspension x 10 h). Therefore, the two main problems of biological soil remediation techniques, longer process time and residual pollutants, may be solved by the use of surfactants.     

Cellulose degradation and cellulase formation by Phialophora malorum

The formation of cellulases and -glucosidase and their location in the fungus Phialophora malorum was studied on some different carbon sources. The cellulases were found to be partly cell-free and partly cell-bound during growth on cellulose and carboxymethyl-cellulose. Glucose and cellobiose repressed the cellulase formation but a low carboxymethylcellulase activity was measurable on the glucose-grown mycelium. The unicellular stage did not appear to grow on carboxymethyl-cellulose or cellulose, but mycelium was formed on these carbon sources.     

Gel formation by fibrin oligomers without addition of monomers

Soluble fibrin oligomers were formed by reacting fibrinogen with thrombin under fine clotting conditions where the action of thrombin is the rate-determining step for polymerization, and by inhibiting the reaction shortly before gelation. Oligomeric fibrin was separated from unreacted fibrinogen and small oligomers by gel permeation chromatography. Electron microscopy revealed that the largest soluble fibrin oligomers resemble the protofibrils present in fine clots, but are somewhat shorter and entirely lack the twisted, trifunctional junctions that contribute to the elastic properties of fine clots. When thrombin was added to the soluble fibrin oligomers, polymerization resumed and clots were formed at a more rapid rate than from fibrinogen at the same concentration and resulted in a less-opaque clot under coarse clotting conditions. The results confirm a prediction of a theory for the polymerization of fibrin and provide additional evidence that the final state of a coarse fibrin clot depends on the mobility of protofibrils during its formation.     

Exogenously triggered, enzymatic degradation of photopolymerized hydrogels with polycaprolactone subunits: experimental observation and modeling of mass loss behavior

Degradation plays an important role in the evolution of the extracellular matrix secreted by chondrocytes encapsulated in PEG-based hydrogels. For this study, macromonomers were synthesized by methacrylating both ends of polycaprolactone-b-poly(ethylene glycol)-b-polycaprolactone (PEG-CAP) tri-block copolymers. These divinyl molecules were photopolymerized to form hydrogels with PEG-CAP crosslinks that were subsequently degraded upon exogenous addition of a lipase enzyme. The rate of degradation and subsequent mass loss depends on both the length of the polycaprolactone units and the concentration of enzyme. Control gels that did not receive lipase did not significantly degrade on the time scale of these experiments. A model was developed to predict mass loss using enzyme kinetics and a previously described statistical treatment of bulk network degradation. The model was used to predict mass loss profiles at the specific conditions used, and also to demonstrate the importance of potential changes in reaction rate and enzyme stability on temporal mass loss.     

Triphasic mixture model of cell-mediated enzymatic degradation of hydrogels

One critical component of engineering living tissue equivalents is the design scaffolds (often made of hydrogels) whose degradation kinetics can match that of matrix production by cells. However, cell-mediated enzymatic degradation of a hydrogel is a highly complex and nonlinear process that is challenging to comprehend based solely on experimental observations. To address this issue, this study presents a triphasic mixture model of the enzyme–hydrogel system, which consists of a solid polymer network, water and enzyme. On the basis mixture theory, the rubber elasticity theory and the Michaelis–Menton kinetics for degradation, the model naturally incorporates a strong coupling between gel mechanical properties, the kinetics of degradation and the transport of enzyme through the gel. The model is then used to investigate the particular problem of a single spherical enzyme-producing cell, embedded in a spherical hydrogel domain, for which the governing equations can be cast within the cento-symmetric assumptions. The governing equations are subsequently solved using an implicit nonlinear finite element procedure to obtain the evolution of enzyme concentration and gel degradation through time and space. The model shows that two regimes of degradation behaviour exist, whereby degradation is dominated either by diffusion or dominated by reaction kinetics. Depending on the enzyme properties and the initial hydrogel design, the temporal and spatial changes in gel cross-linking are dramatically impacted, a feature that is likely to strongly affect new tissue development.     

Triphasic mixture model of cell-mediated enzymatic degradation of hydrogels

One critical component of engineering living tissue equivalents is the design scaffolds (often made of hydrogels) whose degradation kinetics can match that of matrix production by cells. However, cell-mediated enzymatic degradation of a hydrogel is a highly complex and nonlinear process that is challenging to comprehend based solely on experimental observations. To address this issue, this study presents a triphasic mixture model of the enzyme-hydrogel system, which consists of a solid polymer network, water and enzyme. On the basis mixture theory, the rubber elasticity theory and the Michaelis-Menton kinetics for degradation, the model naturally incorporates a strong coupling between gel mechanical properties, the kinetics of degradation and the transport of enzyme through the gel. The model is then used to investigate the particular problem of a single spherical enzyme-producing cell, embedded in a spherical hydrogel domain, for which the governing equations can be cast within the cento-symmetric assumptions. The governing equations are subsequently solved using an implicit nonlinear finite element procedure to obtain the evolution of enzyme concentration and gel degradation through time and space. The model shows that two regimes of degradation behaviour exist, whereby degradation is dominated either by diffusion or dominated by reaction kinetics. Depending on the enzyme properties and the initial hydrogel design, the temporal and spatial changes in gel cross-linking are dramatically impacted, a feature that is likely to strongly affect new tissue development.     

Tailoring the degradation of hydrogels formed from multivinyl poly(ethylene glycol) and poly(vinyl alcohol) macromers for cartilage tissue engineering

Tuning the degradation profiles of polymer cell carriers to match cell and tissue growth is an important design parameter for (cartilage) tissue engineering. In this study, degradable hydrogels were fabricated from divinyl, tetrafunctional poly(ethylene glycol) (PEG) and multivinyl, multifunctional poly(vinyl alcohol) (PVA) macromers to form homopolymer and copolymer gels. These gels were characterized by their volumetric swelling ratio and mass loss profiles as a function of degradation time. By variation of the macromer chemistry and functionality, the degradation time changed from less than 1 day for homopolymer PVA gels to 34 days for pure PEG gels. Furthermore, the degrading medium influenced mass loss, and a marked decrease in degradation time, from 34 to 12 days, was observed with the PEG gels when a chondrocyte-specific medium containing fetal bovine serum was employed. Interestingly, when copolymer gels of PEG and PVA were formed, PVA was released throughout the degradation (as determined by gel permeation chromatography) suggesting that covalent cross-linking of the PVA in the network was facilitated by copolymerizing with the PEG macromer. To assess these novel gels for cartilage tissue engineering applications, chondrocytes were photoencapsulated in the copolymer networks and cultured in vitro for up to 6 weeks. DNA, glycosaminoglycan (GAG), and total collagen contents increased with culture time, and the resulting neocartilaginous tissue at 6 weeks was homogeneously distributed as seen histologically. Biochemical analysis revealed that the constructs were comprised of 0.66 +/- 0.04 microg of DNA/mg wet weight (ww), 1.0 +/- 0.05% GAG/ww, and 0.29 +/- 0.07% total collagen/ww at 6 weeks. Furthermore, the compressive modulus increased during culture from 7 to 97 kPa as the neocartilaginous tissue evolved and the gel degraded. In summary, fabricating hydrogels through the copolymerization of PEG and PVA macromers is an effective tool for encapsulating chondrocytes, controlling gel degradation profiles, and generating cartilaginous tissue.     

Determination of glyceraldehyde formed in glucose degradation and glycation

Glyceraldehyde (GLA) was determined in glucose degradation and glycation. GLA was detected as a decahydroacridine-1,8-dione derivative on reversed phase HPLC using cyclohexane-1,3-dione derivatizing reagent. The glucose-derived GLA level was higher than the glycation-derived GLA level, because GLA was converted to intermediates and advanced glycation end products (AGE) in glycation. GLA was also generated from 3-deoxyglucosone and glucosone as intermediates of glucose degradation and glycation. This study suggests that glyceraldehyde is generated by hyperglycemia in diabetes, and that it is also formed in medicines such as peritoneal dialysis solution.     

Photopolymerized thermosensitive hydrogels: synthesis, degradation, and cytocompatibility

In situ forming hydrogels based on thermosensitive polymers have attractive properties for tissue engineering. However, the physical interactions in these hydrogels are not strong enough to yield gels with sufficient stability for many of the proposed applications. In this study, additional covalent cross-links were introduced by photopolymerization to improve the mechanical properties and the stability of thermosensitive hydrogels. Methacrylate groups were coupled to the side chains of triblock copolymers (ABA) with thermosensitive poly( N-(2-hydroxypropyl) methacrylamide lactate) A blocks and a hydrophilic poly(ethylene glycol) B block. These polymers exhibit lower critical solution temperature (LCST) behavior in aqueous solution and the cloud point decreased with increasing amounts of methacrylate groups. These methacrylate groups were photopolymerized above the LCST to render covalent cross-links within the hydrophobic domains. The mechanical properties of photopolymerized hydrogels were substantially improved and their stability was prolonged significantly compared to nonphotopolymerized hydrogels. Whereas non-UV-cured gels disintegrated within 2 days at physiological pH and temperature, the photopolymerized gels degraded in 10 to 25 days depending on the degree of cross-linking. To assess biocompatibility, goat mesenchymal stem cells were seeded on the hydrogel surface or encapsulated within the gel and they remained viable as demonstrated by a LIVE/DEAD cell viability/cytotoxicity assay. Expression of alkaline phosphatase and production of collagen I demonstrated the functionality of the mesenchymal stem cells and their ability to differentiate upon encapsulation. Due to the improved mechanical properties, stability, and adequate cytocompatibility, the photopolymerized thermosensitive hydrogels can be regarded as highly potential materials for applications in tissue engineering.     

Hydrogels formed by multiple peptide ligation reactions to fasten corneal transplants

A stable cross-linked hydrogel was formed under mild aqueous conditions using pseudoproline peptide ligation chemistry. A cysteine-terminated lysine dendron containing four cysteines and a PEG macromolecule modified with terminal ester aldehydes were prepared. Upon mixing, the two macromers gave a stable hydrogel. This hydrogel along with sutures was used to successfully secure a corneal transplant in vitro.     

Ferulate–coniferyl alcohol cross-coupled products formed by radical coupling reactions

Radical coupling reactions between ethyl ferulate (Et-FA), a simple model for feruloyl polysaccharides in planta, and coniferyl alcohol (CA), a monolignol, were studied in order to better understand the polymer cross-coupling interactions among polysaccharides and monolignols or lignin, mediated by ferulate (FA), in plant cell walls. Cross-coupled FA/CA dimers produced in an aqueous buffer (pH 5.0) containing peroxidase/hydrogen peroxide were isolated and characterized by NMR. The total coupling products were characterized by 2D ¹³C–¹H correlation (HSQC) NMR spectroscopy and GC–MS. Results from this study showed that ferulate readily cross-couples with coniferyl alcohol through free radical coupling mechanisms producing a series of cross-coupled FA/CA dimers with β-O-4-, β-5-/8-5-, and 8-β-linkages; the syntheses and isolation of β-5- and 8-5-cross-coupled dimers are reported here. The transformation from 8-β-coupled FA/CA hydroxyl esters into lactones through intramolecular transesterification is demonstrated for the first time and mechanisms behind these transformations are discussed. The finding of both β-5- and 8-5-cross-coupled dimers in this study suggests that analogs of both may be present in plant cell walls. Finally it is suggested that ferulates in plants indeed react with monolignols through free radical mechanisms producing a more diverse array of cross-coupled dimers than previously reported.     

Glyoxal formation by Fenton-induced degradation of carbohydrates and related compounds

In this paper, we provide a systematic analysis of glyoxal (1) formation from a range of monosaccharides and related compounds, to determine their potential role as sources of this alpha-oxoaldehyde in vivo. Substrates were reacted with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) and the mixtures were analyzed by HPLC using the 6-hydroxy-2,4,5-triaminopyrimidine fluorimetric assay. The rank order of hexoses and their derivatives as glyoxal sources was found to be fructose > glucose = mannose = galactose > glucose-6-phosphate > mannitol. Within the pentose group, arabinose and ribose gave the higher yields of 1 followed by deoxyribose and its adenine N-glycosides and ribulose. Among the tested substrates, three-carbon compounds, that is, trioses and glycerol, but not glyceraldehyde-3-phosphate, were by far the most effective sources of 1. The effects of H(2)O(2) and Fe(2+)/EDTA concentrations as well as of other metal ions were also investigated.