Poster BB1 - Dorsal Root Ganglia Density Influences the Health and Fasciculation of Stretch-Grown Axonal Tracts

Introduction: Peripheral nerve injuries (PNI) affect 13-23 people per 100,000 in the United States per year. PNI may result in severe consequences such as lost ability to move or feel a limb, or more minor symptoms such as temperature intolerance, muscle atrophy and weakness, and/or localized or radiating pain. For patients requiring surgical intervention, current strategies typically involve bridging a segmental defect with an autograft, acellular allograft, or nerve guidance tube, and may involve electrical stimulation and/or physical therapy. Unfortunately, even these state-of-the-art treatment strategies often fail to restore complete function and sensation, and often leave patients with residual deficits. Our previous work has developed Tissue Engineered Nerve Grafts (TENGs) comprised of long, living axonal tracts that are “stretch-grown” in custom mechano-bioreactors [1], [2]. TENGs have been shown to accelerate host axonal regeneration when used to bridge segmental nerve defects and may serve as a viable replacement for conventional autografts [4], [3].
We are interested in optimizing axonal “stretch-growth” parameters to improve TENG bio-fabrication, consistency, and potency for improved therapeutic outcomes. In this project, we investigated how DRG density affected TENG phenotype, fascicle density, fascicle size, overall TENG health, and percent yield.

Materials and

Methods: TENGs were fabricated by stretch growing axonal tracts projecting from 20, 40, or 80 DRG in custom mechano-bioreactors. Rows of DRG were plated in parallel on a towing membrane that is mechanically pulled and a base membrane that remains stationary. The TENGs were stretch grown for 8 days to a length of 1.5cm. They were then encapsulated in a collagen mixture and cultured for 40-50 days. TENGs were then fixed, blocked and stained with Hoechst and Phalloidin for fluorescent microscopy. Fascicle count and diameter were quantified at various locations along the axonal tracts. An established semi-quantitative TENG Health Score was also applied. Percent yield was quantified by counting the number of TENGs in each experimental group that survived until the end of the stretching phase.

Results, Conclusions, and Discussions: In the current study, there was a 43% yield of the 20 and 40 DRG, while the 80 DRG had a 57% yield. The group that experienced the highest degree of neuronal death prior to the beginning of stretch was the 20 DRG group. Interestingly, fascicle count in all groups was significantly higher on the base membrane side than at the mid-point or on the towing membrane side of the TENG, suggesting better outgrowth for the base membrane DRG, as seen in Figure 1. Comparatively, fascicle diameter was larger in the middle of the TENG than in either membrane side in all groups, suggesting a greater degree of fasciculation. Quantification showed that, on average, 40 DRG had higher fascicle count compared to the 20 DRG or 80 DRG groups.
Tissue Engineered Nerve Grafts have the potential to serve as an axon-based therapeutic for the surgical repair of PNI segmental defects in humans. Before any clinical determination can be made, the optimization of these TENGs is crucial for success. In this study, we investigated whether changing the DRG density would have an impact on neuronal health and/or axon density and degree of fasciculation of TENGs. Ongoing studies are further investigating these parameters in vitro, with the best performing TENGs designs being further evaluated in vivo in the rat sciatic nerve injury model.

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