Magnetic particle templating of hydrogels: engineering naturally derived hydrogel scaffolds with 3D aligned microarchitecture for nerve repair.

OBJECTIVE: Hydrogel scaffolds hold promise for a myriad of tissue engineering applications, but often lack tissue-mimetic architecture. Therefore, in this work, we sought to develop a new technology for the incorporation of aligned tubular architecture within hydrogel scaffolds engineered from the bottom-up. APPROACH: We report a platform fabrication technology-magnetic templating-distinct from other approaches in that it uses dissolvable magnetic alginate microparticles (MAMs) to form aligned columnar structures under an applied magnetic field. Removal of the MAMs yields scaffolds with aligned tubular microarchitecture that can promote cell remodeling for a variety of applications. This approach affords control of microstructure diameter and biological modification for advanced applications. Here, we sought to replicate the microarchitecture of the native nerve basal lamina using magnetic templating of hydrogels composed of glycidyl methacrylate hyaluronic acid and collagen I. MAIN RESULTS: Magnetically templated hydrogels were characterized for particle alignment and micro-porosity. Overall MAM removal efficacy was verified by 96.8% removal of iron oxide nanoparticles. Compressive mechanical properties were well-matched to peripheral nerve tissue at 0.93 kPa and 1.29 kPa, respectively. In vitro, templated hydrogels exhibited approximately 36% faster degradation over 12 h, and were found to guide axon extension from dorsal root ganglia. Finally, in a pilot in vivo study utilizing a 10 mm rat sciatic nerve defect model, magnetically templated hydrogels demonstrated promising results with qualitatively increased remodeling and axon regeneration compared to non-templated controls. SIGNIFICANCE: This simple and scalable technology has the flexibility to control tubular microstructure over long length scales, and thus the potential to meet the need for engineered scaffolds for tissue regeneration, including nerve guidance scaffolds.

Processing-Size Correlations in the Preparation of Magnetic Alginate Microspheres Through Emulsification and Ionic Crosslinking.

Magnetic alginate microspheres are biocompatible due to their alginate matrix, and motion-controllable by applied magnetic fields due to their magnetic character. Therefore, they have the potential of being used as vessels to a broad variety of materials, including drugs and therapeutic agents, facilitating entry to biological systems in a relatively non-invasive manner. Here, magnetic alginate microspheres were prepared through an emulsification and ionic cross-linking process, where a mixture of alginate and iron oxide magnetic nanoparticles was initially dispersed in a continuous phase, followed by gelation of this dispersed phase into microspheres by cross-linking the dispersion with calcium ions. The resulting magnetic alginate microspheres were found to be superparamagnetic and to respond to applied magnetic fields by chain formation. The effect of shear rate, alginate concentration, and magnetic nanoparticle concentration on microsphere size was investigated with the aim to control the size of microspheres with respect to process and formulation parameters. Two of these parameters, shear rate and alginate concentration, were used to correlate experimental results with a theoretical model for the case where the dispersed phase is more viscous than the continuous phase.

Human insulin fibril-assisted synthesis of fluorescent gold nanoclusters in alkaline media under physiological temperature.

Fluorescent insulin fibrils gold nanoclusters (Au NCs) have been synthesized through the reduction of gold by human insulin in fibrillated form. Likewise, nanocluster formation has been regulated by insulin, working as a protein-based template. Environment- and surface-controlled experiments have shown the optimized synthesis conditions is comprised of a pure aqueous alkaline solvent for insulin under constant heat at physiological temperature (37°C) prior to addition of the Au precursor (HAuCl4), followed by subsequent heating (37°C) and vigorous stirring after the addition of HAuCl4 until the completion of the synthetic approach. Microscopy experiments detected the presence of primordial fibril structures in samples of heated human insulin in the alkaline medium prior to addition of HAuCl4, while encountering more developed insulin fibrils in the terminal production of Au NCs. This investigation provides insight to the development of a novel synthesis of Au NCs in the alkaline medium, while providing a graphical description of the environmental and surface-dependent effects that were presented in the synthesis of human insulin nanoclusters. The study provides pertinent information for future synthetic procedures, as the protein state of several protein-nanoparticle systems may reflect on the results that were obtained herein.

Study of the aggregation of human insulin Langmuir monolayer.

The human insulin (HI) Langmuir monolayer at the air-water interface was systematically investigated in the presence and absence of Zn(II) ions in the subphase. HI samples were dissolved in acidic (pH 2) and basic (pH 9) aqueous solutions and then spread at the air-water interface. Spectroscopic data of aqueous solutions of HI show a difference in HI conformation at different pH values. Moreover, the dynamics of the insulin protein showed a dependence on the concentration of Zn(II) ions. In the absence of Zn(II) ions in the subphase, the acidic and basic solutions showed similar behavior at the air-water interface. In the presence of Zn(II) ions in the subphase, the surface pressure-area and surface potential-area isotherms suggest that HI may aggregate at the air-water interface. It was observed that increasing the concentration of Zn(II) ions in the acidic (pH 2) aqueous solution of HI led to an increase of the area at a specific surface pressure. It was also seen that the conformation of HI in the basic (pH 9) medium had a reverse effect (decrease in the surface area) with the increase of the concentration of Zn(II) ions in solution. From the compression-decompression cycles we can conclude that the aggregated HI film at air-water interface is not stable and tends to restore a monolayer of monomers. These results were confirmed from UV-vis and fluorescence spectroscopy analysis. Infrared reflection-absorption and circular dichroism spectroscopy techniques were used to determine the secondary structure and orientation changes of HI by zinc ions. Generally, the aggregation process leads to a conformation change from α-helix to ß-strand and ß-turn, and at the air-water interface, the aggregation process was likewise seen to induce specific orientations for HI in the acidic and basic media. A proposed surface orientation model is presented here as an explanation to the experimental data, shedding light for further research on the behavior of insulin as a Langmuir monolayer.