Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

The University of Akron

The integrity of the vaginal mucosa is critical to protecting women from infection, particularly sexually transmitted diseases. For example, breakdown of the mucosa, including the cell lining and/or mucus layer, due to vaginal infections has been shown to increase the risk of HIV infection. There is considerable interest in the development of new topical microbicides for women; many of these topical agents contain polymers. One potential mechanism for altering the barrier properties of a three-dimensional fibrous gel, such as cervical mucus, to cell penetration is to alter the fiber structure. In previous studies, we have shown that addition of synthetic polymers, such as polyvinyl pyridine (PVP) and polyethylene glycol (PEG), modify the fiber structure and mechanical properties of human cervical mucus. Here we investigated the ability of peripheral blood monocytes to migrate through structurally altered mucus gels. Adding PVP to mucus increased both the average fiber spacing and the rate of random migration of monocytes; addition of PEG to mucus also caused an increased random migration rate, although changes in overall fiber spacing were not obvious. In both cases, the addition of small amounts of polymer to cervical mucus decreased the barrier property of mucus with respect to cell migration. This result raises questions about the safety of polymeric agents as ingredients in topical microbicides.

The effect of synthetic polymers on the migration of monocytes through human cervical mucus.

Although collagen is commonly used as components of tissue-engineered nerve-guidance channels, little is known about the effect of the mechanical properties of commonly used gel concentrations on the extension of neurites. This study focused on neurite extension of dissociated chick dorsal root ganglia in vitro over a range of collagen concentrations (0.4-2.0 mg/ml). Neurite length increased in all gels between day 1 and day 4, except at the highest collagen concentration, where a 9% decrease was noted at day 4. Although maximum neurite extension was seen in lower concentration gels (0.6-0.8 mg/ml), mechanical stiffness of each gel significantly increased with increasing concentration, from 2.2 Pa at 0.4 mg/ml to 17.0 Pa at 2.0 mg/ml. A previous model of mechanical stiffness versus neurite outgrowth did not fit this data well, likely because of interactions between the growth cone and the collagen fibers. Overall, these results provided insight regarding factors that influence neurite elongation and may be utilized to further optimize tissue-engineered scaffolds.

Effect of collagen gel stiffness on neurite extension.

This study aimed to reveal the influence of brief DC electric stimulation on neurite outgrowth and outgrowth rates after application. Chick embryo dorsal root ganglia neurite outgrowth, rates, and overall alignment to EF were measured before stimulation and at two time points after stimulation. The presence of a 25 V/m EF for 10 min increased overall neurite outgrowth over controls for up to 48 h after stimulation and all growth was symmetric. These results demonstrate that even 10 min of stimulation, which is approximately 80% shorter than previous studies, promotes enhanced nerve growth.

Short-duration, DC electrical stimulation increases chick embryo DRG neurite outgrowth.

The extension of neurites on 2D collagen-coated substrates and within 3D collagen gels is based on various chemical and mechanical environmental factors. However, extrapolating results from 2D studies to 3D environments are difficult, especially with regard to neural outgrowth. The aim of this study was to investigate the effects of inhibitory molecules on nerve growth in 3D environments as compared to 2D surfaces. E9 chick dorsal root ganglion cells were seeded within collagen gels as well as onto collagen-coated glass and were exposed, for 24 h, to one of three experimental peptide sequences; arginine-glycine-aspartic acid-threonine (RGDT), cyclo(RGD-D-Phe-Val) (cRGD), or aspartic acid-glycine-glutamic acid-alanine (DGEA). In 3D collagen gels, only the cRGD peptide sequence reduced neurite extension across a variety of gel concentrations. In contrast, on 2D surfaces, both RGD peptides reduced the number of cells expressing neurites, but cRGD still exhibited superior inhibition of neurite expression. Further evaluation of cRGD results revealed that the peptide altered neurite growth vs. stiffness to a more linear relationship that is more typical of non-adhesive environments. Overall, these results further demonstrate the importance of peptide confirmation and sequence when investigating cell behavior in 3D environments.

The effect of soluble peptide sequences on neurite extension on 2D collagen substrates and within 3D collagen gels.

The goal of this work was to investigate methods to retain bioactivity of nerve growth factor (NGF) after encapsulation in poly(lactic-co-glycolic acid) (PLGA) discs for controlled release. Poly(ethylene glycol) (PEG) was chosen as a porogen not only to control the release rate of NGF but also because it has been used to help maintain bioactivity of molecules in organic solvents. NGF and PEG were encapsulated in PLGA via standard dissolution-evaporation techniques with methylene chloride as the solvent. Morita et al. (Pharm Res 2000; 17:1367-1373) indicated that colyophilization of PEG and bioactive factors before exposure to organic solvents increased the retention of bioactivity. Therefore, various PEG:NGF mass ratios were colyophilized before encapsulation within PLGA to examine retained activity of NGF. When PEG was not colyophilized before encapsulation, NGF activity was lost during the fabrication process. In contrast, colyophilization of PEG and NGF supported retention of NGF activity during the entire fabrication process. The amount of PEG encapsulated was the dominating factor in the rate of NGF release regardless of the fabrication method. These results demonstrate the usefulness of PEG in both acting as a porogen to modulate release and aiding in the retention of activity of NGF. This process may be extended to other methods to enhance activity of growth factors after exposure to organic solvents.

Maintaining bioactivity of NGF for controlled release from PLGA using PEG.

Electrical therapies have been found to aid repair of nerve injuries and have been shown to increase and direct neurite outgrowth during stimulation. This enhanced neural growth existed even after the electric field (EF) or stimulation was removed, but the factors that may influence the enhanced growth, such as stimulation media or surface coating, have not been fully investigated. This study characterized neurite outgrowth and branching under various conditions: EF magnitude and application time, ECM surface coating, medium during EF application and growth supplements. A uniform, low-magnitude EF (24 or 44 V m(-1)) was applied to dissociated chick embryo dorsal root ganglia seeded on collagen or laminin-coated surfaces. During the growth period, cells were either exposed to NGF or N2, and during stimulation cells were exposed to either unsupplemented media (Ca(2+)) or PBS (no Ca(2+)). Parallel controls for each experiment included cells exposed to the chamber with no stimulation and cells remaining outside the chamber. After brief electrical stimulation (10 min), neurite length significantly increased 24 h after application for all conditions studied. Of particular interest, increased stimulation time (10-100 min) further enhanced neurite length on laminin but not on collagen surfaces. Neurite branching was not affected by stimulation on any surface, and no preferential growth of neurites was noted after stimulation. Overall, the results of this report suggest that short-duration electric stimulation is sufficient to enhance neurite length under a variety of conditions. While further data are needed to fully elucidate a mechanism for this increased growth, these data suggest that one focus of those investigations should be the interaction between the growth cone and the substrata.

Applied electric field enhances DRG neurite growth: influence of stimulation media, surface coating and growth supplements.

Modular, bioactive, macroporous scaffolds were formed by crosslinking poly(ethylene glycol) (PEG) microspheres around living cells. Hydrogel microspheres were produced from reactive PEG derivatives in aqueous sodium sulfate solutions without the use of surfactants or copolymers. Microspheres were formed following thermally induced phase separation if the gel point was reached prior to extensive coarsening of the PEG-rich domains. Three types of PEG microspheres with different functionalities were used to form scaffolds: one type provided mechanical support, the second type provided controlled delivery of the angiogenesis-promoting molecule, sphingosine 1-phosphate (S1P) and the third type served as a slowly dissolving non-cytotoxic porogen. Scaffolds were formed by centrifuging microspheres in the presence of HepG2 hepatoma cells, resulting in a homogenous distribution of cells. During overnight incubation at 37 degrees C, the microspheres reacted with serum proteins in cell culture medium to stabilize the scaffolds. Within 2 days in culture, macropores formed due to the dissolution of the porogenic PEG microspheres, without affecting cell viability. Gradients in porosity were produced by varying the buoyancy of the porogenic microspheres. Conjugated RGD cell adhesion peptides and the delivery of S1P promoted endothelial cell infiltration through macropores in the scaffolds. The scaffolds presented here differ from previous hydrogel scaffolds in that: (i) cells are not encapsulated in hydrogel; (ii) macropores form in the presence of cells; and (iii) scaffold properties are controlled by the modular assembly of different microspheres that perform distinct functions.

Modular scaffolds assembled around living cells using poly(ethylene glycol) microspheres with macroporation via a non-cytotoxic porogen.

Over the past decade, it has been increasingly recognized that both chemical and mechanical properties of scaffolds influence neural cell behavior, ranging from growth to differentiation to migration. However, mechanical properties are difficult to control for in the design of scaffolds for nerve regeneration, as properties change over time for most biologically derived scaffolds. The focus of this project was to examine how the mechanical properties of a nondegradable scaffold, poly(ethylene glycol) (PEG) gels, influenced nerve cell behavior. Low concentration PEG gels, of 3, 4, or 5% PEG, with added collagen to alter chemical properties were examined for both their mechanical properties and their ability to support nerve expression and extension. Stiffness (G*) significantly increased with increased PEG concentration. The addition of chemically conjugated collagen significantly decreased the stiffness compared to plain gels. This phenomenon was confirmed to be an effect of the conjugate, and not the protein itself, as G* of gels containing conjugate, but no protein, was not significantly different than G* of gels with conjugated protein. PC12 cell neurite expression increased with decreasing PEG and increasing collagen concentration. At its best, the expression approached the value on collagen-coated tissue culture plastic, which is a substantial improvement over previous studies on PEG. Neurite extension of dorsal root ganglia was also improved on these same gels over gels with either higher PEG concentration or lower collagen amount. Overall, these results suggest that exploration of lower stiffness materials is necessary to improve neurite growth and extension in three-dimensional synthetic scaffolds.

Characterization of poly(ethylene glycol) gels with added collagen for neural tissue engineering.

Methods were developed to perform precipitation photopolymerization of PEG-diacrylate. Previously, comonomers have been added to PEG when precipitation polymerization was desired. In the present method, the LCST of the PEG itself was lowered by the addition of the kosmotropic salt sodium sulfate to an aqueous solution. Typical of a precipitation polymerization, small microparticles or microspheres (1-5 μm) resulted with relatively low polydispersity. However, aggregate formation was often severe, presumably because of a lack of stabilization of the phase-separated colloids. Microparticles were also produced by copoymerization of PEG-diacrylate with acrylic acid or aminoethylmethacrylate. The comonomers affected the zeta potential of the formed microparticles but not the size. The carboxyl groups of acrylic-acid-containing PEG microparticles were activated, and scaffolds were formed by mixing with amine-containing PEG microparticles. Although the scaffolds were relatively weak, human hepatoma cells showed excellent viability when present during microparticle cross-linking.

Poly(ethylene glycol) microparticles produced by precipitation polymerization in aqueous solution.

Within a 3D environment, the chemical and mechanical properties of a scaffold can significantly influence nerve behavior. How these properties influence with nerve cells is important for optimizing neurite extension within a scaffold. The purpose of this study was to investigate the effect of low concentration poly(ethylene glycol) (PEG) with added laminin on 3D growth of dissociated dorsal root ganglia. Because of its high affinity for neurite adhesion and ability to promote extension, laminin was conjugated to the PEG chain, as well as mixed in the gel, at various concentrations to provide chemical cues. Gel stiffness, as determined by G*, significantly decreased with decreasing PEG concentration and with increasing laminin conjugate. Extension within the gels increased as the concentration of laminin increased with no difference between how laminin was presented (mixed or conjugated) to the cells. For example, in 3% PEG, extension increased from 92.29 ± 5.27 μm to 146.35 ± 13.12 μm as laminin conjugate concentration increased from plain to 100 μg/ml. Results indicated that the chemical properties of the scaffold influenced neurite growth more than the mechanical properties as laminin concentration had a greater impact on growth than the stiffness of the gel over the range studied. Neurite length as a function of scaffold stiffness and adhesion properties was also characterized and demonstrated a positive linear relationship between rate of neurite extension and laminin concentration. This study further demonstrates the importance of characterizing interactions between cell behavior and the chemical and mechanical environment.

Student award winner in the undergraduate's degree category for the Society for Biomaterials 35th Annual Meeting, Orlando, Florida, April 13-16, 2011. Neurite growth in PEG gels: effect of mechanical stiffness and laminin concentration.

This research focused on developing a modular poly(ethylene glycol) (PEG) scaffold, assembled from PEG microgels and collagen I, to provide an environment to decouple the chemical and mechanical cues within a three-dimensional scaffold. We first characterized the microgel fabrication process, examining the size, polydispersity, swelling ratio, mesh size and storage modulus of the polymer particles. The resulting microgels had a low polydispersity index, PDI=1.08, and a diameter of ~1.6 μm. The mesh size of the microgels, calculated from the swelling ratio, was 47.53 Å. Modular hydrogels (modugels) were then formed by compacting N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimidyl group-activated microgels with PEG-4arm-amine and 0, 1, 10, or 100 μg ml(-1) collagen. The stiffness (G(∗)) of the modugels was not significantly altered with the addition of collagen, allowing for modification of the chemical environment independent from the mechanical properties of the scaffold. PC12 cell aggregation increased in modugels as collagen concentrations increased and cell viability in modugels was improved over bulk PEG hydrogels. Overall, these results indicate that further exploration of modular scaffolds formed from microgels could allow for a better understanding of the relationship between the chemical and mechanical properties and cellular behavior.

Modular poly(ethylene glycol) scaffolds provide the ability to decouple the effects of stiffness and protein concentration on PC12 cells.

The primary goal of this research was to characterize the effect of laminin on three-dimensional (3D) neurite growth. Gels were formed using type I collagen at concentrations of 0.4-2.0 mg mL(-1) supplemented with laminin at concentrations of 0, 1, 10, or 100 µg mL(-1). When imaged with confocal microscopy, laminin was shown to follow the collagen fibers; however, the addition of laminin had minimal effect on the stiffness of the scaffolds at any concentration of collagen. Individual neurons dissociated from E9 chick dorsal root ganglia were cultured in the gels for 24 h, and neurite lengths were measured. For collagen gels without laminin, a typical bimodal response of neurite outgrowth was observed, with increased growth at lower concentrations of collagen gel. However, alteration of the chemical nature of the collagen gel by the laminin additive shifted, or completely mitigated, the bimodal neurite growth response seen in gels without laminin. Expression of integrin subunits, α1, α3, α6 and β1, were confirmed by PCR and immunolabeling in the 3D scaffolds. These results provide insight into the interplay between mechanical and chemical environment to support neurite outgrowth in 3D. Understanding the relative impact of environmental factors on 3D nerve growth may improve biomaterial design for nerve cell regeneration.

The impact of laminin on 3D neurite extension in collagen gels.

Extracellular matrix incorporated within a scaffold plays an important role in assisting cell behavior in neural tissue engineering. In this study, we investigated how the concentration of fibronectin (FN) affected neurite growth when incorporated within a synthetic polymer gel made of poly(ethylene glycol) (PEG) or a natural polymer gel of collagen I. Mechanical and chemical properties of the scaffold were varied by using a range of concentrations of gels and FN. Rheology was used to determine the mechanical stiffness of hydrogels and neurite length and viability were measured to evaluate cell response. In both types of gels, increasing the concentration of the base scaffold (PEG or collagen) increased the mechanical stiffness as denoted by G∗. Neurite lengths in PEG gels increased with increasing FN concentration and decreased with increasing G∗. In collagen gels, FN reduced neurite extension for the lowest concentrations of collagen (0.4-0.6 mg/mL) while FN increased neurite extension for mid and high collagen concentrations (1.0-2.0 mg/mL). The results from these two different scaffolds indicate that both stiffness and FN concentration impact the growth of the neurite and that the addition of small amounts of FN (100 μg/ml) permits PEG gels to perform on par with similar stiffness collagen gels.

Rebecca Kuntz Willits

Department of Biomedical Engineering

The University of Akron

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

Rebecca Kuntz Willits

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering

The University of Akron

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

Department of Biomedical Engineering