Rebecca Kuntz Willits

Effect of collagen gel stiffness on neurite extension

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.

Synthetic polymers alter the structure of cervical mucus

Mucosal sites have an innate defense system--which includes immune cells, antibodies, and mucus--to protect the body from opportunistic pathogens. Some sexually transmitted diseases (STDs), such as HIV, utilize host defense mechanisms to evade detection by infecting motile immune cells present at the site. The infected cells migrate through the mucus layer and penetrate the epithelium undetected. A new strategy for preventing STDs could involve inhibiting cell migration through the mucus. One method for inhibiting migration is to alter the barrier property of mucus by modifying its gel structure. Mucin, the structural component of mucus, is a high molecular weight anionic molecule, which forms an entangled fiber network through non-covalent interactions. The addition of nonionic or cationic polymers, such as poly(ethylene glycol) (PEG) or poly(vinyl pyridine) (PVP), altered the overall gel structure as revealed by scanning electron microscopy (SEM), while anionic poly(acrylic acid) had little effect on the structure. Acid residues on mucin associate with PEG through hydrogen bonds to form regions of coalesced fibers within the mucus. PVP, however, interacts with mucin via electrostatic bonds, forming a gel that had areas of aggregated fibers adjacent to regions with virtually no fibers. These results suggest that addition of small amounts of certain synthetic polymers will modify mucus structure; these changes should alter the barrier properties of mucus.

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

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.

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

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.

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

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.

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.

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.

Enhanced schwann cell attachment and alignment using one-pot "Dual Click" GRGDS and YIGSR derivatized nanofibers

Using metal-free click chemistry and oxime condensation methodologies, GRGDS and YIGSR peptides were coupled to random and aligned degradable nanofiber networks postelectrospinning in a one-pot reaction. The bound peptides are bioactive, as demonstrated by Schwann cell attachment and proliferation, and the inclusion of YIGSR with GRGDS alters the expression of the receptor for YIGSR. Additionally, aligned nanofibers act as a potential guidance cue by increasing the aspect ratio and aligning the actin filaments, which suggest that peptide-functionalized scaffolds would be useful to direct SCs for peripheral nerve regeneration.

Polyethlyene Glycol Microgels to Deliver Bioactive Nerve Growth Factor

Delivery of bioactive molecules is a critical step in fabricating materials for regenerative medicine, yet, this step is particularly challenging in hydrated scaffolds such as hydrogels. Although bulk photocrosslinked poly(ethylene glycol) (PEG) hydrogels have been used for a variety of tissue engineering applications, their capability as drug delivery scaffolds has been limited due to undesirable release profiles and reduction in bioactivity of molecules. To solve these problems, this article presents the fabrication of degradable PEG microgels, which are micron-sized spherical hydrogels, to deliver bioactive nerve growth factor (NGF). NGF release and activity was measured after encapsulation in microgels formed from either 3 kDa or 6 kDa PEG to determine the role of hydrogel mesh size on release. Microgels formed from 6 kDa PEG were statistically larger and had a higher swelling ratio than 3 kDa PEG. The 6 kDa PEG microgels provided a Fickian release with a reduced burst effect and 3 kDa microgels provided anomalous release over ≥20 days. Regardless of molecular weight of PEG, NGF bioactivity was not significantly reduced compared to unprocessed NGF. These results demonstrate that microgels provide easy mechanisms to control the release while retaining the activity of growth factors. As this microgel-based delivery system can be injected at the site of nerve injury to promote nerve repair, the potential to deliver active growth factors in a controlled manner may reduce healing time for neural tissue engineering applications.

Tuning the Mechanical Properties of Poly(Ethylene Glycol) Microgel‐Based Scaffolds to Increase 3D Schwann Cell Proliferation

2D in vitro studies have demonstrated that Schwann cells prefer scaffolds with mechanical modulus approximately 10× higher than the modulus preferred by nerves, limiting the ability of many scaffolds to promote both neuron extension and Schwann cell proliferation. Therefore, the goals of this work are to develop and characterize microgel-based scaffolds that are tuned over the stiffness range relevant to neural tissue engineering and investigate Schwann cell morphology, viability, and proliferation within 3D scaffolds. Using thiol-ene reaction, microgels with surface thiols are produced and crosslinked into hydrogels using a multiarm vinylsulfone (VS). By varying the concentration of VS, scaffold stiffness ranges from 0.13 to 0.76 kPa. Cell morphology in all groups demonstrates that cells are able to spread and interact with the scaffold through day 5. Although the viability in all groups is high, proliferation of Schwann cells within the scaffold of G* = 0.53 kPa is significantly higher than other groups. This result is ≈ 5× lower than previously reported optimal stiffnesses on 2D surfaces, demonstrating the need for correlation of 3D cell response to mechanical modulus. As proliferation is the first step in Schwann cell integration into peripheral nerve conduits, these scaffolds demonstrate that the stiffness is a critical parameter to optimizing the regenerative process.

Comparison of neurite growth in three dimensional natural and synthetic hydrogels

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.