Stevin H. Gehrke

Hierarchically Designed Agarose and Poly(Ethylene Glycol) Interpenetrating Network Hydrogels for Cartilage Tissue Engineering

A new method for encapsulating cells in interpenetrating network (IPN) hydrogels of superior mechanical integrity was developed. In this study, two biocompatible materials-agarose and poly(ethylene glycol) (PEG) diacrylate-were combined to create a new IPN hydrogel with greatly enhanced mechanical performance. Unconfined compression of hydrogel samples revealed that the IPN displayed a fourfold increase in shear modulus relative to a pure PEG-diacrylate network (39.9 vs. 9.9 kPa) and a 4.9-fold increase relative to a pure agarose network (8.2 kPa). PEG and IPN compressive failure strains were found to be 71% ± 17% and 74% ± 17%, respectively, while pure agarose gels failed around 15% strain. Similar mechanical property improvements were seen when IPNs-encapsulated chondrocytes, and LIVE/DEAD cell viability assays demonstrated that cells survived the IPN encapsulation process. The majority of IPN-encapsulated chondrocytes remained viable 1 week postencapsulation, and chondrocytes exhibited glycosaminoglycan synthesis comparable to that of agarose-encapsulated chondrocytes at 3 weeks postencapsulation. The introduction of a new method for encapsulating cells in a hydrogel with enhanced mechanical performance is a promising step toward cartilage defect repair. This method can be applied to fabricate a broad variety of cell-based IPNs by varying monomers and polymers in type and concentration and by adding functional groups such as degradable sequences or cell adhesion groups. Further, this technology may be applicable in other cell-based applications where mechanical integrity of cell-containing hydrogels is of great importance.

Molecular and Functional Analyses of Amino Acid Decarboxylases Involved in Cuticle Tanning in Tribolium castaneum

Aspartate 1-decarboxylase (ADC) and 3,4-dihydroxyphenylalanine decarboxylase (DDC) provide β-alanine and dopamine used in insect cuticle tanning. β-Alanine is conjugated with dopamine to yield N-β-alanyldopamine (NBAD), a substrate for the phenol oxidase laccase that catalyzes the synthesis of cuticle protein cross-linking agents and pigment precursors. We identified ADC and DDC genes in the red flour beetle, Tribolium castaneum (Tc), and investigated their functions. TcADC mRNA was most abundant prior to the pupal-adult molt. Injection of TcADC double-stranded (ds) RNA (dsTcADC) into mature larvae resulted in depletion of NBAD in pharate adults, accumulation of dopamine, and abnormally dark pigmentation of the adult cuticle. Injection of β-alanine, the expected product of ADC, into dsTcADC-treated pupae rescued the pigmentation phenotype, resulting in normal rust-red color. A similar pattern of catechol content consisting of elevated dopamine and depressed NBAD was observed in the genetic black mutants of Tribolium, in which levels of TcADC mRNA were drastically reduced. Furthermore, from the Tribolium black mutant and dsTcADC-injected insects both exhibited similar changes in material properties. Dynamic mechanical analysis of elytral cuticle from beetles with depleted TcADC transcripts revealed diminished cross-linking of cuticular components, further confirming the important role of oxidation products of NBAD as cross-linking agents during cuticle tanning. Injection of dsTcDDC into larvae produced a lethal pupal phenotype, and the resulting grayish pupal cuticle exhibited many small patches of black pigmentation. When dsTcDDC was injected into young pupae, the resulting adults had abnormally dark brown body color, but there was little mortality. Injection of dsTcDDC resulted in more than a 5-fold increase in levels of DOPA, indicating that lack of TcDDC led to accumulation of its substrate, DOPA.

Controlling the swelling characteristics of temperature-sensitive cellulose ether hydrogels

Abstract Novel temperature-sensitive hydrogels; have been synthesized by chemically crosslinking cellulose ethers; hydroxypropyl cellulose, methyl cellulose and hydroxypropylmethyl celluloses. These gels are highly swollen at low temperatures, gradually deswelling as temperature rises, usually sharply over a limited temperature range. Linear polymer type, crosslink density and ionic content have been varied in order to determine the effects of controllable network parameters on the important properties (degree of swelling, transition temperature and sharpness of transition) of the gels obtained. The transition temperature itself is about that of the observed linear polymer lower critical solution temperature (LCST). Producing gels from increasingly hydrophilic cellulose ethers raises the transition temperature and increases the swelling degree at comparable crosslink densities. However, decreasing crosslinking has little effect on swelling above the transition temperature. In contrast, increasing ionic content increases swelling at all temperatures. The transition temperature is not affected by the degree of crosslinking, but increases with increasing ionic content. The volume transition of the hydroxypropyl cellulose gel is significantly sharper than for the other gels. Crosslinking and ionic content have little effect on transition sharpness. The trends observed with cellulose ether gels are consistent with those observed in gels produced by conventional copolymerization/crosslinking synthesis methods, though some differences are noted.

Chemical aspects of gel extraction

Abstract Dilute aqueous solutions can be effectively concentrated with crosslinked partially hydrolysed polyacrylamide gels. Because the gels are near their critical point, they can be regenerated with small changes in pH. The selectivity of concentrating nonelectrolyte solutes can be adjusted by changes in gel crosslinking; the selectivity of separating electrolytes seems largely the result of Donnan equilibria. The regeneration of the gels is effected largely by altering the ionization of carboxylic acid groups within the gel, and less influenced by ion pairing. The regeneration is compromised at high salt concentrations which reduce the gels swelling.

Mass transfer in pH-sensitive hydrogels

Abstract This paper describes measurements of solute and solvent transport in swollen, swelling, and collapsing hydrogels. Solute diffusion in swollen gels follows Ficks law, with diffusion coefficients varying only slightly with the degree of swelling. Solvent diffusion into swelling gels is affected both by Ficks law and by the rates of ion exchange. Solvent diffusion in collapsing gels is faster than that in swelling gels, and is effectively described by Ficks law.

Factors Determining Hydrogel Permeability

Developing hydrogel membranes and coatings of appropriate permeability characteristics is key to the success of a number bioartificial organ technologies. Key principles relevant to the design and application of hydrogels for such applications were reviewed. The first key point is that permeability is a function of both transport and thermodynamic properties, the diffusion coefficient and partition coefficient, respectively, and that these parameters can be evaluated separately. Although the aspect of partitioning often emphasized is size exclusion, this review points out that many other relevant interactions come into play, especially hydrophobic and electrostatic interactions, and that these phenomena can dominate size exclusion. Similarly, while the diffusion coefficient also is strongly dependent upon size, other interactions can also cause diffusivity to deviate from theories which consider only solute size and gel swelling. For example, the heterogeneity of hydrogel networks can result in permeabilities that fail to decline as much as might be anticipated if networks were uniform.

Enhanced loading and activity retention of bioactive proteins in hydrogel delivery systems

A simple, general and effective technique is developed for increasing the loading of bioactive macromolecules into hydrogels using the principles of aqueous two-phase extraction. Model proteins, ovalbumin and alpha-amylase, were loaded into hydrated gels by soaking the gels in a buffered solution of protein containing 12 wt% PEG-10000 and 0.22 M salt (KCl, KBr or KI). The PEG and salts were expected to enhance protein sorption according to aqueous two-phase extraction heuristics. In the absence of the solution additives, the gels absorbed little protein. But protein loading up to 270 mg ovalbumin/g polymer and 67 mg alpha-amylase/g polymer was obtained when the PEG and salt were added; loading was not significantly dependent upon salt type. Ovalbumin release from hydrated gels was diffusion-controlled. The diffusion coefficient was 1.10 (-7) cm2/s, consistent with protein absorption into the gel rather than adsorption onto the surface. Release kinetics of both proteins from dried, glassy gels matched conventional behavior for release of absorbed drugs from glassy polymers. Finally, alpha-amylase activity was retained even after drying the loaded gel at 65 degreesC, conditions which denatured the enzyme when not absorbed in the gel.

Volume change kinetics of temperature-sensitive poly(vinyl methyl ether) gel

Abstract Thermally responsive gels of poly(vinyl methyl ether) (PVME) were formed by the γ-irradiation of solutions of linear PVME. Under precisely defined synthesis conditions, the shrinking rates of the gels could be increased to values well above the range observed for gels formed by copolymerization/crosslinking reactions. This enhancement in rate appears related to the development of a microporous structure which allows the convective expulsion of solvent from the network; this occurs more quickly than the diffusive motion of the network. The overall rate of a cyclical process using responsive gels can also be enhanced if one stage of the process, either swelling or shrinking, is reversed before equilibrium is achieved.

Permeability of responsive poly (N-isopropylacrylamide) gel to solutes

The diffusion (D) and partition (K) coefficients of dissolved solutes within a thermally responsive hydrogel, poly(N-isopropylacrylamide) (NIPAAm), were determined by independent measurement to give the permeability, defined as the product of D and K. For acetaminophen, the Yasuda et al. free volume theory successfully predicted the decrease in D as the swelling of the gel decreased with increasing temperature. In the collapsed gel, however, this theory overestimated D by 35 times. Ideal size exclusion theory failed to correlate observed partition coefficients (ratio of solute concentration in the gel to that in solution) for dilute solutions of acetaminophen, norethindrone and methyl orange. Examination of K as a function of solute character and the presence of structure breaking and structure forming salts demonstrates that hydrophobic interactions are often dominant in these systems. In the presence of ammonium sulfate, partition coefficients of nearly 200 were determined for methyl orange in collapsed gels, despite their relatively low water content (40%). As a result, permeability does not necessarily decrease with a decline in gel swelling or drop sharply upon gel collapse.

Tuning mechanical performance of poly(ethylene glycol) and agarose interpenetrating network hydrogels for cartilage tissue engineering

Hydrogels are attractive for tissue engineering applications due to their incredible versatility, but they can be limited in cartilage tissue engineering applications due to inadequate mechanical performance. In an effort to address this limitation, our team previously reported the drastic improvement in the mechanical performance of interpenetrating networks (IPNs) of poly(ethylene glycol) diacrylate (PEG-DA) and agarose relative to pure PEG-DA and agarose networks. The goal of the current study was specifically to determine the relative importance of PEG-DA concentration, agarose concentration, and PEG-DA molecular weight in controlling mechanical performance, swelling characteristics, and network parameters. IPNs consistently had compressive and shear moduli greater than the additive sum of either single network when compared to pure PEG-DA gels with a similar PEG-DA content. IPNs withstood a maximum stress of up to 4.0 MPa in unconfined compression, with increased PEG-DA molecular weight being the greatest contributing factor to improved failure properties. However, aside from failure properties, PEG-DA concentration was the most influential factor for the large majority of properties. Increasing the agarose and PEG-DA concentrations as well as the PEG-DA molecular weight of agarose/PEG-DA IPNs and pure PEG-DA gels improved moduli and maximum stresses by as much as an order of magnitude or greater compared to pure PEG-DA gels in our previous studies. Although the viability of encapsulated chondrocytes was not significantly affected by IPN formulation, glycosaminoglycan (GAG) content was significantly influenced, with a 12-fold increase over a three-week period in gels with a lower PEG-DA concentration. These results suggest that mechanical performance of IPNs may be tuned with partial but not complete independence from biological performance of encapsulated cells.