(598b) Size Tunable Fabrication of Architectural Micropatterns in Hydrogels Using Magnetic Alginate Microparticles (MAMs) As Porogens in Magnetic Templating

Hydrogels are highly hydrophobic, biocompatible polymeric materials that have immense potential in tissue engineering as tissue substitutes for both injury repair in vivo and as tissue models in vitro. Their ability to recapitulate the water content, bioactivity and mechanical nature of biological tissues has led to their widespread use in many biomedical applications and settings. However, bulk processing of hydrogels leads to a polymer network that is nanoporous in nature, in contrast with biological tissues that are characterized by possessing microporosity that mediates cell migration, nutrient transport, and metabolite clearance. Furthermore, tissues like peripheral nerve are characterized by possessing aligned hierarchical structure that relates to aligned porosity in the underlying tissue. Thus, formulation techniques that allow control over pore distribution, interconnectivity, and alignment are of interest.

To address this, our lab has developed a bottom-up technique we call magnetic templating in which magnetic alginate microparticles (MAMs) are dispersed in a pre-crosslinked polymer solution, aligned under a static magnetic field and the polymer is then photopolymerized around MAMs. MAMs are then subsequently cleared, leaving aligned micropores in place. MAMs are currently fabricated using a flow-focusing microfluidic device. Magnetic nanoparticle coating greatly affects the stability of nanoparticles in alginate solutions, with sterically stable nanoparticles being highly stable compared to electrostatically stable nanoparticles. Systematic studies of droplet formation reveal droplet diameter is controlled by the flow rates and viscosities of the carrier and droplet phase. Droplet size increases with increasing droplet phase flow rate and viscosity and decreases with increasing carrier phase flow rate or viscosity. MAMs between 30 – 90 µm in diameter were used for magnetic templating of bulk glycidyl methacrylate hyaluronic acid/collagen hydrogels and hydrogels were then characterized for their physical and mechanical character using confocal microscopy, nano-CT, and indentation. Confocal microscopy showed that areal density of MAM chains is dependent on MAM size, increasing with decreasing MAM size and that void size shows a 25% increase in diameter upon MAM removal (Figure 1, left). Indentation of templated gels showed that the mechanical stiffness of hydrogels, specifically their relaxation modulus, does not depend on the size of the MAMs when the indentation is parallel to chain orientation but is dependent on MAM size for indentation in the perpendicular direction in a range varying from 1.5-10 kPa. Furthermore, indentation also revealed that there is anisotropic mechanical stiffness, with stiffer mechanical properties in the orientation perpendicular to alignment. MAM chain and micropore structure were characterized based on image analysis (Imaris) of nano CT (MAMs) and confocal microscopy (micropores) to quantify alignment and connectivity. Imaris processing of NanoCT images shows chains are highly parallel to each other, with some chains interconnecting and producing channels of 1-5 mm long covering a 3D volume. The magnetic templating process is one amenable for the incorporation of size-controllable microchannels in polymer networks and as a tool for increasing porosity of the scaffold. The incorporation of micropatterns is useful in tuning mechanical properties and can be varied in a range of stiffnesses. Because pores can be tuned and the physical and mechanical aspects of scaffolds be studied as a function of the MAM diameter and magnetic content, structure-property relationships can be studied and explored for the fabrication of tissues including nerve, and muscle and for studying in vitro migration of fibroblasts and macrophages. Ongoing work will study the effect of structure-property relationships on Schwann cell migration as a model for peripheral nerve repair and use Imaris to correlate cell migration depth with average channel length and interconnectivity.

Figure 1: (Left) In red (rhodamine) are images of vertically aligned MAM chains and (center) in green (FITC) are images of horizontally aligned chains in magnetically templated hydrogels with a) 30, b) 40, c) 54, and d) 62 μm MAMs. Scale bar = 100 μm. (right) Processed nano computed tomography images of templated hydrogels using Imaris.