D13 - Injectable Hydrogel Derived from Decellularized Human Peripheral Nerve to Ameliorate Neuroinflammation at the Injured Spinal Cord Site

Introduction: : Spinal cord injury (SCI) is a debilitating condition that often results in permanent loss of motor function. One of the major challenges in treating SCI is the limited intrinsic regenerative capacity of the central nervous system, compounded by a strong and persistent neuroinflammatory response at the injury site. In this study, we developed an injectable hydrogel (iHPN) derived from decellularized human peripheral nerves, engineered to mimic the mechanical properties of native spinal cord tissue (Fig.1A). iHPN is a biodegradable platform that supports the growth of human SH-SY5Y neuroblastoma cells and primary human brain neurons. Furthermore, iHPN modulates the inflammatory response by attenuating the reactivity of human astrocytes, human microglia, and mouse RAW 264.7 macrophages in vitro. When implanted into a rat contusion model of SCI, iHPN reduced lesion volume, enhanced myelination, and mitigated neuroinflammation mediated by astrocyte and microglial cells (Fig.1A). However, no significant improvement in locomotor recovery was observed in treated animals. Collectively, these results demonstrate that iHPN holds promise as a minimally invasive biomaterial platform for SCI therapy, particularly for applications involving drug or cell delivery and axonal support.

Materials and

Methods: : Deidentified human sciatic nerves from six cadavers were decellularized using a modified Hudson protocol to remove cellular content while preserving extracellular matrix (ECM) proteins (Collagen I/IV, laminin). Two delipidation methods were tested: (i) n-hexane:isopropanol (3:1 v:v) and (ii) dichloromethane:ethanol (2:1 v:v). Decellularization and delipidation efficiency were assessed using dsDNA quantification, Oil Red O staining, and sulfo-vanillin assays (n=3). Delipidated nerves were digested with pepsin and crosslinked with genipin to form the injectable hydrogel (iHPN). Rheological properties were evaluated for gelation kinetics and stiffness (n=3), and injectability was tested through 25G, 27G, and 30G needles. The protein content in digested decellularized and delipidated nerve solution was quantified using LC MS/MS. Human astrocytes and RAW 264.7 macrophages were cultured in iHPN with or without lipopolysaccharide (LPS) to model inflammation (n=3). Astrocyte reactivity was assessed via GFAP immunostaining and IL-1β ELISA. Macrophage polarization was examined on days 3 and 7 using CD206/CD86 immunostaining and ELISA for IL-4, IL-10, and TNF-α. Human SH-SY5Y cells and brain neurons were cultured in iHPN to assess viability (LIVE/DEAD) and neuronal differentiation (β-tubulin III). In vivo, female Sprague Dawley rats (n=6) received a T9–T11 contusion injury and iHPN injection within 15 minutes. Behavioral recovery was evaluated using Basso, Beattie, Bresnahan and horizontal ladder tests. After 4 weeks, spinal cords were analyzed by immunostaining (GFAP, Iba-1, CD86, CD206, TMEM119, β-tubulin III, MAP-2, GAP43), H&E for lesion volume, and Luxol Fast Blue for remyelination. Animals injected with saline were used as control. Statistical analysis was performed using one-way ANOVA.

Results, Conclusions, and Discussions:: The modified Hudson protocol effectively decellularized human sciatic nerves, removing cellular debris while preserving key extracellular matrix (ECM) proteins such as Collagen I, Collagen IV, and Laminin. LC MS/MS based proteomics analysis revealed the retention of various ECM and growth factors in the decellularized and delipidated nerves. The additional delipidation step using dichloromethane-ethanol significantly reduced lipid content, as confirmed by Oil Red O staining and sulfo-vanillin assays. Nerves treated with dichloromethane-ethanol also demonstrated better digestion compared to those treated with n-hexane: isopropanol. Rheological analysis showed that the decellularized and delipidated nerves could be digested with pepsin to form a shear-thinning injectable hydrogel and were able to pass through 25 G and 27 G needles (Fig. 1B). When crosslinked with 5 mM genipin, the hydrogel had a mechanical stiffness of 400 Pascals and gelled completely within 10-12 minutes at 37°C, mimicking CNS tissue properties (Fig. 1C). The biodegradability of the hydrogel was demonstrated, with approximately 60% degradation over seven days in 1x PBS, indicating a suitable degradation rate for CNS applications (Fig. 1D). The genipin-crosslinked iHPN hydrogel supported human SH-SY5Y cell viability and enhanced neuronal differentiation compared to traditional tissue culture plastic (TCP). Astrocytes cultured within the iHPN maintained a quiescent state even under inflammatory conditions induced by LPS, as shown by lower GFAP expression (Fig. 2A) and IL-1β secretion, suggesting the hydrogel’s anti-inflammatory properties. Moreover, macrophages cultured in iHPN display an anti-inflammatory (M2) phenotype, with higher CD206 expression, lower CD86 expression (Fig. 2A), increased IL-4 (Fig. 2B), and reduced TNF-α secretion (Fig. 2C), further supporting the immunomodulatory potential of iHPN. Furthermore, iHPN when injected into rat spinal cord model, was seen to decrease astrocytes reactivity (decreased GFAP expression) and promote axonal regeneration (higher neurofilament, NF-200) (Fig. 2D and E). This eventually decreases lesion volume (Fig. 3A and B) and promote remyelination, promoting tissue repair (Fig.3C).However, there was no significant improvement in BBB and horizontal ladder score among iHPN and saline treated animals. These results suggest that ECM and growth factors retained in iHPN provide pro-regenerative and anti-inflammatory cues at the SCI site.

Acknowledgements and/or References (Optional):: We acknowledge the use of tissues procured by the National Disease Research Interchange (NDRI) with support from NIH grant U42OD11158. The authors would like to acknowledge the funding support received by NIH (P0225954) and Pruitt chair funding awarded to PI Christine E Schmidt. The authors also acknowledge Dr. Benjamin Keselowsky at the University of Florida for providing RAW 264.7 cells used in this study. The authors acknowledge Samantha Shumard for helping in the decellularization of human peripheral nerves.