C. Allan Guymon

Biotinylated Biodegradable Nanotemplated Hydrogel Networks for Cell Interactive Applications

We describe the synthesis of a novel biotinylated nanotextured degradable hydrogel that can be rapidly surface engineered with a diverse range of biotinylated moieties. The hydrogel is synthesized by reacting methacrylated biotin-PEG with dimethacrylated P LA-b- PEG-b-P LA (LPLDMA, PEG = poly(ethylene glycol), PLA = poly(lactic acid)),or dimethacrylated PEG-b-P LA-b- PEG (PLPDMA). Methacrylated biotin-PEG is prepared by reacting biotin-PEG-OH with methacrylic anhydride. Biotin-PEG-OH is prepared by reacting alpha-hydroxy-omega-amine PEG with N-hydroxysuccinimide-biotin. Confirmation of the final product is determined using (1)H NMR and Fourier transform infrared spectroscopy (FTIR). The integrity and surface presentation of the biotin units is observed spectrophotometrically using the HABA/avidin assay. To produce nanostructured polymer topography, a self-assembling lyotropic liquid crystalline mesophase is used as a polymerization template, generating biotinylated hydrogels with highly organized lamellar matrix geometry. Traditionally processed isotropic hydrogels are used for comparison. Scanning electron microscopy shows that isotropic hydrogels have a smooth glassy appearance while lamellar templated hydrogels have defined surface topographical features that enhance preosteoblast human palatal mesenchymal cell (HEPM) attachment. Engineering the surfaces of the hydrogels with cell adhesive Arg-Gly-Asp (RGD) peptide sequences using the biotin-avidin interaction significantly enhances cell attachment. Surface engineering of cell adhesive peptides in conjunction with the lamellar template induced surface topography generates additive enhancements in cell attachment.

Photopolymerized microfeatures for directed spiral ganglion neurite and Schwann cell growth

Cochlear implants (CIs) provide auditory perception to individuals with severe hearing impairment. However, their ability to encode complex auditory stimuli is limited due, in part, to poor spatial resolution caused by electrical current spread in the inner ear. Directing nerve cell processes towards target electrodes may reduce the problematic current spread and improve stimulatory specificity. In this work, photopolymerization was used to fabricate micro- and nano-patterned methacrylate polymers to probe the extent of spiral ganglion neuron (SGN) neurite and Schwann cell (SGSC) contact guidance based on variations in substrate topographical cues. Micropatterned substrates are formed in a rapid, single-step reaction by selectively blocking light with photomasks which have parallel line-space gratings with periodicities of 10-100 μm. Channel amplitudes of 250 nm-10 μm are generated by modulating UV exposure time, light intensity, and photoinitiator concentration. Gradual transitions are observed between ridges and grooves using scanning electron and atomic force microscopy. The transitions stand in contrast to vertical features generated via etching lithographic techniques. Alignment of neural elements increases significantly with increasing feature amplitude and constant periodicity, as well as with decreasing periodicity and constant amplitude. SGN neurite alignment strongly correlates (r = 0.93) with maximum feature slope. Multiple neuronal and glial types orient to the patterns with varying degrees of alignment. This work presents a method to fabricate gradually-sloping micropatterns for cellular contact guidance studies and demonstrates spatial control of inner ear neural elements in response to micro- and nano-scale surface topography.

Contribution of monomer functionality and additives to polymerization kinetics and liquid crystal phase separation in acrylate‐based polymer‐dispersed liquid crystals (PDLCs)

Fundamental control of the polymerization behaviour of polymer‐dispersed liquid crystals (PDLCs) is critical to the formation of high‐performance devices by polymer‐induced phase separation (PIPS). Previous PDLC research has shown that monomer functionality and additives such as surfactants or reactive diluents can impart significant changes to the electro‐optical behaviour of a system, especially in acrylate‐based materials. The influence of monomer functionality and additives on the polymerization kinetics and LC phase separation were examined in the formation of acrylate‐based PDLCs. Real‐time infrared (RTIR) spectroscopy was utilized to simultaneously monitor polymerization rate, double bond conversion and LC phase separation. In the formation of PDLCs by PIPS, increasing acrylate monomer functionality reduces the polymerization rate, overall double bond conversion and the extent of LC phase separation. Interestingly, the additives octanoic acid and N‐vinylpyrrolidone (NVP) increase the polymerization rate but suppress LC phase separation. During PDLC formation, both octanoic acid and NVP enhance the solubility of the LC in the growing polymer matrix, reducing the rate of liquid–gel demixing and decreasing nematic fraction in PDLCs. As a non‐reactive component, octanoic acid increases the polymerization rate by plasticizing the crosslinked polymerization. NVP, a reactive diluent added to decrease viscosity, increases polymerization rate through favourable copolymerization with acrylate monomer.

Improved stimuli-response and mechanical properties of nanostructured poly(N-isopropylacrylamide-co-dimethylsiloxane) hydrogels generated through photopolymerization in lyotropic liquid crystal templates

Temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM) hydrogels are widely studied stimuli-responsive systems due to their significant volume changes at biologically relevant temperatures and a potential wide range of applications including drug delivery, cell cultures, chemical sensors, and desalination. The successful performance of PNIPAM gels often requires a rapid response rate with a significant degree of deswelling when heated above the lower critical solution temperature. However, it is often difficult to design PNIPAM hydrogels with appropriate mechanical strength for the gels to remain functional in a working environment. Herein, lyotropic liquid crystals (LLCs) are utilized to generate a hexagonal nanostructure in PNIPAM hydrogels in order to improve material properties and transport characteristics. Cross-linked methacrylated poly(dimethylsiloxane) (PDMS) was incorporated into PNIPAM gels at varying concentrations through photopolymerization in the hexagonal LLC phase in order to modulate mechanical properties. The hexagonal LLC nanostructure dramatically increases the hydrogel deswelling rate compared to traditional isotropic PNIPAM–PDMS hydrogels of the same chemical composition. Additionally, the ordered LLC structure allows for considerable incorporation of PDMS into the hydrogel without significantly decreasing the water content of the gels. Interestingly, the hexagonal nanostructured hydrogels exhibit similar compressive moduli compared to isotropic hydrogel controls despite having considerably higher water content. These results may be utilized to generate stimuli-sensitive hydrogels with an appropriate rate and degree of deswelling while maintaining necessary mechanical integrity of the gel for use in numerous biomedical and industrial applications.

Fast deswelling kinetics of nanostructured poly(N-isopropylacrylamide) photopolymerized in a lyotropic liquid crystal template.

The thermal-response and mechanical properties of poly(N-isopropylacrylamide) (PNIPAm) are improved by controlling the polymer nanostructure through photopolymerization in a bicontinuous cubic lyotropic liquid crystal (LLC) template. The bicontinuous cubic nanostructure increases the rate and amount of water expelled from PNIPAm for heating above the lower critical solution temperature (LCST) relative to an isotropic PNIPAm hydrogel while maintaining the mechanical integrity of the polymer. These results could allow development of PNIPAm hydrogels with proper water uptake, deswelling kinetics, volume transition, and mechanical properties required for successful performance in a growing number of advanced biological and industrial applications.

Neural pathfinding on uni- and multidirectional photopolymerized micropatterns.

Overcoming signal resolution barriers of neural prostheses, such as the commercially available cochlear impant (CI) or the developing retinal implant, will likely require spatial control of regenerative neural elements. To rationally design materials that direct nerve growth, it is first necessary to determine pathfinding behavior of de novo neurite growth from prosthesis-relevant cells such as spiral ganglion neurons (SGNs) in the inner ear. Accordingly, in this work, repeating 90° turns were fabricated as multidirectional micropatterns to determine SGN neurite turning capability and pathfinding. Unidirectional micropatterns and unpatterned substrates are used as comparisons. Spiral ganglion Schwann cell alignment (SGSC) is also examined on each surface type. Micropatterns are fabricated using the spatial reaction control inherent to photopolymerization with photomasks that have either parallel line spacing gratings for unidirectional patterns or repeating 90° angle steps for multidirectional patterns. Feature depth is controlled by modulating UV exposure time by shuttering the light source at given time increments. Substrate topography is characterized by white light interferometry and scanning electron microscopy (SEM). Both pattern types exhibit features that are 25 μm in width and 7.4 ± 0.7 μm in depth. SGN neurites orient randomly on unpatterned photopolymer controls, align and consistently track unidirectional patterns, and are substantially influenced by, but do not consistently track, multidirectional turning cues. Neurite lengths are 20% shorter on multidirectional substrates compared to unidirectional patterns while neurite branching and microfeature crossing events are significantly higher. For both pattern types, the majority of the neurite length is located in depressed surface features. Developing methods to understand neural pathfinding and to guide de novo neurite growth to specific stimulatory elements will enable design of innovative biomaterials that improve functional outcomes of devices that interface with the nervous system.

Microtopographical features generated by photopolymerization recruit RhoA/ROCK through TRPV1 to direct cell and neurite growth.

Cell processes, including growth cones, respond to biophysical cues in their microenvironment to establish functional tissue architecture and intercellular networks. The mechanisms by which cells sense and translate biophysical cues into directed growth are unknown. We used photopolymerization to fabricate methacrylate platforms with patterned microtopographical features that precisely guide neurite growth and Schwann cell alignment. Pharmacologic inhibition of the transient receptor potential cation channel subfamily V member 1 (TRPV1) or reduced expression of TRPV1 by RNAi significantly disrupts neurite guidance by these microtopographical features. Exogenous expression of TRPV1 induces alignment of NIH3T3 fibroblasts that fail to align in the absence of TRPV1, further implicating TRPV1 channels as critical mediators of cellular responses to biophysical cues. Microtopographic features increase RhoA activity in growth cones and in TRPV1-expressing NIH3T3 cells. Further, Rho-associated kinase (ROCK) phosphorylation is elevated in growth cones and neurites on micropatterned surfaces. Inhibition of RhoA/ROCK by pharmacological compounds or reduced expression of either ROCKI or ROCKII isoforms by RNAi abolishes neurite and cell alignment, confirming that RhoA/ROCK signaling mediates neurite and cell alignment to microtopographic features. These studies demonstrate that microtopographical cues recruit TRPV1 channels and downstream signaling pathways, including RhoA and ROCK, to direct neurite and cell growth.

Material Stiffness Effects on Neurite Alignment to Photopolymerized Micropatterns

The ability to direct neurite growth into a close proximity of stimulating elements of a neural prosthesis, such as a retinal or cochlear implant (CI), may enhance device performance and overcome current spatial signal resolution barriers. In this work, spiral ganglion neurons (SGNs), which are the target neurons to be stimulated by CIs, were cultured on photopolymerized micropatterns with varied matrix stiffnesses to determine the effect of rigidity on neurite alignment to physical cues. Micropatterns were generated on methacrylate thin film surfaces in a simple, rapid photopolymerization step by photomasking the prepolymer formulation with parallel line–space gratings. Two methacrylate series, a nonpolar HMA-co-HDDMA series and a polar PEGDMA-co-EGDMA series, with significantly different surface wetting properties were evaluated. Equivalent pattern periodicity was maintained across each methacrylate series based on photomask band spacing, and the feature amplitude was tuned to a depth of 2 μm amplitude for all compositions using the temporal control afforded by the UV curing methodology. The surface morphology was characterized by scanning electron microscopy and white light interferometry. All micropatterned films adsorb similar amounts of laminin from solution, and no significant difference in SGN survival was observed when the substrate compositions were compared. SGN neurite alignment significantly increases with increasing material modulus for both methacrylate series. Interestingly, SGN neurites respond to material stiffness cues that are orders of magnitude higher (GPa) than what is typically ascribed to neural environments (kPa). The ability to understand neurite response to engineered physical cues and mechanical properties such as matrix stiffness will allow the development of advanced biomaterials that direct de novo neurite growth to address the spatial signal resolution limitations of current neural prosthetics.

Neuronal Differentiation of Induced Pluripotent Stem Cells on Surfactant Templated Chitosan Hydrogels

The development of effective tissue engineering materials requires careful consideration of several properties beyond biocompatibility, including permeability and mechanical stiffness. While surfactant templating has been used for over a decade to control the physical properties of photopolymer materials, the potential benefit of this technique with regard to biomaterials has yet to be fully explored. Herein we demonstrate that surfactant templating can be used to tune the water uptake and compressive modulus of photo-cross-linked chitosan hydrogels. Interestingly, templating with quaternary ammonium surfactants also hedges against property fluctuations that occur with changing pH. Further, we demonstrate that, after adequate surfactant removal, these materials are nontoxic, support the attachment of induced pluripotent stem cells and facilitate stem cell differentiation to neuronal phenotypes. These results demonstrate the utility of surfactant templating for optimizing the properties of biomaterials intended for a variety of applications, including retinal regeneration.

Polymerization of polymer/ferroelectric liquid crystal composites formed with branched liquid crystalline bismethacrylates

Polymerization and phase behaviour of a branched liquid crystalline bismethacrylate in a ferroelectric liquid crystal (FLC) were characterized. Addition of the monomer increases the temperature range of the smectic A phase, and, at relatively low concentrations of monomer, the temperature range increases to more than 10 times that observed in the neat FLC. Other phases such as the smectic C* phase are no longer exhibited as the monomer interferes with the inherent tilt of the FLC molecules. After polymerization, the polymer network phase separates and the phase transition temperatures return to values close to those of the FLC. The monomer also shows a high degree of orientational order before polymerization, which is retained to a large extent after polymerization. The order in the polymer network results in considerable birefringence at temperatures well above the clearing point of the pure FLC. This behaviour is induced by the order of the polymer network and interactions of the FLC molecules with the ...