(426f) Magnetically Templated Hydrogels for Peripheral Nerve Injury Repair

Peripheral nerve injuries (PNI) are a significant health problem and economic burden, costing $150 million dollars annually. There are various options currently on market for peripheral nerve injury (PNI) repair. Nerve wraps, conduits, and connects can be used for nerve regeneration for short distances between transected nerve stumps,¹ however, as the gap size increases, the only viable options for PNI repair are autografts and decellularized nerve allografts – cadaveric tissue that has been stripped of cellular content using detergents. Nevertheless, autografts require multiple surgeries and can cause functional defects at the donor site if multiple, large grafts are required, and decellularized nerve tissue is limited by donor availability and can only be used in PNI gaps less than 5 cm. Thus, there is a need for engineered solutions for PNIs with gaps greater than 2 cm. The success of allografts and autografts have been attributed to the microtubular, channel-like structure of the natural peripheral nerve extracellular matrix (ECM) that is maintained in both cases.²Our hypothesis is that a hydrogel-based scaffold templated with porous microchannels could be viable for PNI repair of large (> 2 cm) nerve gaps by mimicking the tubular structure of the peripheral nerve ECM and guiding axon growth in a unidirectional fashion.

We introduce a novel method of patterning three-dimensional, anisotropic porosity: magnetic templating. This process involves suspending magnetic alginate microparticles (MAMs) in a hydrogel precursor solution, aligning the MAMs in the presence of a magnetic field, crosslinking the hydrogel, and finally degrading the MAMs, leaving behind a tubular porous architecture in the templated hydrogel. Our hydrogels are composed of collagen to encourage cell adhesion and glycidyl methacrylate hyaluronic acid (GMHA), which we have chosen due to the presence of hyaluronan in the natural peripheral nerve ECM, and due to its biodegradability, tunable mechanical properties, and capability for chemical modifications. MAMs are comprised of calcium-crosslinked alginate and encapsulate magnetic iron oxide nanoparticles that lend the MAMs their magnetic properties. Previous work in this project has been conducted using MAMs made via an emulsion method. While good preliminary results were obtained using emulsion MAMs (eMAMs), this platform is limited due to issues with poor control over MAM size and magnetic content and batch-to-batch variability. We have developed a protocol for fabricating MAMs using microfluidic droplet generation to produce MAMs with uniform size and magnetic content. This method was chosen for improved reproducibility and tunability of the ultimate microarchitecture of the magnetically templated hydrogels.

This microfluidic MAM (µMAM) fabrication platform has allowed us to better tune MAM composition and examine corresponding effects on alignment and degradation. Using nano-computed tomography (nanoCT) to create 3D re-constructions of magnetically templated hydrogels, utilizing the magnetic iron oxide nanoparticles as the contrast agents in the scan, we can measure MAM chain length, alignment, and areal density within the MAM-templated hydrogel. To examine channel morphology, we used confocal microscopy of templated hydrogels after MAM degradation and subsequent filling of the channels with dextran-FITC for visualization. An orthophenanthroline absorbance assay and electron paramagnetic resonance (EPR) were both used as iron quantification methods to track clearance of iron oxide from the templated hydrogels. A ten-fold decrease in iron oxide content was measured after MAM dissolution. Using finite element analysis simulations in COMSOL to simulate arrays of permanent NdFeB magnets, we designed a magnetic templating setup that produces long, uniform magnetically-templated hydrogels (3 cm long currently, but longer fields are possible in theory) to demonstrate scalability for clinical translation.

Finally, we have conducted a preliminary in vivo study by inducing a 10 mm gap in a rat sciatic nerve to test three types of implants: a magnetically templated hydrogel, a non-templated hydrogel, and a fresh nerve isograft (which would represent the clinical treatment). For this pilot study, eMAMs were used to template the hydrogel implants. As this was a pilot study, a short four-week time point was chosen – although this timespan was not long enough for complete nerve reconnection in a rat model, we still observed promising results. The magnetically templated hydrogel implants achieved substantial cell infiltration and re-modeling while the non-templated hydrogel implants did not. Using immunofluorescent staining for neurofilament, epifluorescence microscopy, and image analysis, the areal density of axons was measured in cross-sections taken throughout the implants. With n = 2, the study showed that after four weeks, the magnetically templated hydrogel had an areal density of axons comparable to that of the isograft near the proximal end of the implant. Our results show promise for a microstructured biomaterial that could aid in PNI repair and has other potential tissue engineering applications.

1. Kehoe S, Zhang XF, Boyd D. FDA approved guidance conduits and wraps for peripheral nerve injury: A review of materials and efficacy. Injury. 2012;43(5):553-572. doi:10.1016/j.injury.2010.12.030.

2. Spivey EC, Khaing ZZ, Shear JB, Schmidt CE. The fundamental role of subcellular topography in peripheral nerve repair therapies. Biomaterials. 2012;33(17):4264-4276. doi:10.1016/j.biomaterials.2012.02.043.