X11 - 3D Printed Nerve Guidance Conduit For Biologics-Free Nerve Regeneration And Vascular Integration

Introduction: : There is a clinical need for an effective nerve guidance conduit to treat peripheral nerve injuries. Many studies have explored different materials and active cues to guide neural regeneration, with some success. However, none have demonstrated a comparable or better functional recovery than the clinical standard autograft. Autografts are often insufficient for reconstruction of an injury to long nerves such as the sciatic or brachial plexus. Synthetic nerve guidance conduits (NGCs) have been investigated for these injuries to guide axonal regeneration and lead to functional recovery. We have designed a biologics-free hydrogel-based multi-channel conduit with defined microscale features to guide axonal outgrowth. To investigate extraneural vascular infiltration and its effects on functional recovery, we also designed a multi-microchannel conduit with defined regularly spaced micropores, orthogonal to the axon guidance channels. Using our custom-built Rapid Projection, Image-guided, Dynamic (RaPID) bioprinting system, we are able to fabricate each hydrogel conduit within minutes from a milliliter-volume prepolymer vat. With our state-of-the-art printing platform, we have achieved NGCs with a consistent channel wall width of 10 microns. We implanted the NGCs for 17 weeks in a murine sciatic nerve transection injury model. We assessed the functional recovery by dynamic gait analysis throughout the recovery period and by compound muscle action potential (CMAP) electrophysiology before NGC harvesting. Both the non-porous and micro-porous conduit groups led to functional nerve regeneration on par with the autograft group. Further, both conduit groups resulted in restoration of bulk motor function to pre-injury performance.

Materials and

Methods: : Using our custom-built Rapid Projection, Image-guided, Dynamic (RaPID) bioprinting system, we are able to fabricate each hydrogel conduit within minutes from a milliliter-volume prepolymer vat. We have achieved NGCs with a consistent channel-wall width of 10 microns. We implanted the NGCs for 17 weeks in a murine sciatic nerve transection injury model. We assessed the functional recovery by dynamic gait analysis and compound muscle action potential (CMAP) electrophysiology.
Statistical analysis was performed using GraphPad Prism and G*Power v3.1.9.6. An a priori power analysis test was performed based on preliminary mouse behavioral testing data (effect size f was 0.948) to determine the minimum group size needed to achieve a power of 0.8 and an 𝛼 error probability of 0.05. The result of which was a group size of n = 5, which resulted in an actual power of 0.896. Descriptive statistics and their representation in figures were presented as mean ± standard deviation. Comparisons between two independent groups were conducted using Welch’s unpaired t-test. Outliers, when identified amongst repeated measurements, were removed using the ROUT method where Q = 1%. For comparing multiple groups, an ordinary one-way ANOVA was performed. For comparing multiple groups over time, either a repeated measures two-way ANOVA or mixed-effects model with the Geisser-Greenhouse correction was performed. The two-way ANOVA analysis cannot handle missing values, so a mixed-effects model was used in its stead when missing values were present. All ad hoc multiple comparisons testing was performed using Šídák’s with a 95% confidence interval.

Results, Conclusions, and Discussions:: We observed that the micro-porous conduit led to robust angiogenesis from distal external blood vessels, similar in density and morphology to the angiogenesis observed in the autograft. Both conduit groups resulted in similar functional nerve regeneration and full recovery of bulk motor function, via CMAP electrophysiology and dynamic gait analysis, respectively. Notably, both NGC groups performed not statistically different than that of the autograft group, which is the current clinical standard of surgical nerve reconstruction.
We have developed a synthetic therapeutic that can be implanted to aid nerve regeneration after a debilitating injury that performs similarly to the current clinical standard treatment. One major hurdle to clinically translating an implantable therapeutic is needing to include biologics such as cells or active biomolecules, which increases the regulatory burden. With this in mind, we systematically developed a biologics-free synthetic hydrogel nerve guidance conduit, composed of polymer backbones that have been used in many FDA-approved applications, capable of restoring motor function.
Overall, this study has demonstrated that in a mouse complete transection sciatic nerve injury model a RaPID 3D-printed PEGDA-GelMA composite hydrogel with 300-μm-wide hexagonal microchannels with 10-μm channel thickness leads to functional axonal regeneration after a 4-month recovery time period that is quantitatively similar in functional nerve regeneration performance to that of the clinically used reverse autograft surgical therapy and results in restoration of bulk motor function to pre-injury performance. This finding is remarkable in that the therapeutic conduit contained no xenogeneic, bioactive, or cellular components.

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