Poster P5 - Decellularized Human Peripheral Nerve Base Injectable Hydrogel for Injured Spinal Cord Regeneration

Introduction: Spinal cord injury (SCI) can result in permanent loss of sensory and motor functions. According to the World Health Organization, it is estimated that 15.4 million people were living with SCI globally in 2021. Currently, there are no FDA-approved treatments for locomotor recovery after SCI. Decellularized human peripheral nerves have shown the potential to promote axonal regeneration in peripheral nerve diseases. However, developing an injectable hydrogel using decellularized human nerves is still a challenge due to the presence of lipids. In this study, we developed an injectable hydrogel using decellularized human sciatic nerve (iHPN). Developing an injectable hydrogel allows us to develop a minimum invasiveness hydrogel solution that can be directly administered at the site of injury. Furthermore, the presence of pro-regenerative cues like Collagen I, laminin, and various growth factors can promote axonal regeneration in the injured spinal cord. In the current study, human nerves were decellularized modifying the Hudson protocol previously published by our lab, and delipidated using various organic solvents. The nerves were then digested and neutralized to p.H. 7.4 and crosslinked with a natural crosslinker, genipin, to match the CNS native mechanical strength. The resultant hydrogels exhibited injectability and mechanical strength that can support neuronal cell growth. Overall, iHPN has the potential to promote axonal regeneration in the injured spinal cord and can also be used clinically as a delivery vehicle for SCI regeneration.

Materials and

Methods: Human nerves were obtained from the National Disease Research Institute (NDRI). Human sciatic nerves (from 5 different donors) were decellularized modifying our lab's previously published Hudson protocol. Briefly, Triton X-100 detergent was used instead of Triton X-200 followed by the DNAase incubation step. The nerves were then delipidated using either dichloromethane: ethanol (2:1 v: v) or hexane: isopropanol (3:2 v: v) to compare their effectiveness. In some instances, the nerves were delipidated twice for better digestion. To evaluate decellularization and delipidation efficiency, an immunohistochemistry analysis was done for the absence of nuclei content (using DAPI stain), ECM retention (Collagen I, and laminin), and lipid content (using Oil Red O staining). The decellularized and delipidated nerves (at different concentrations of 10 mg/mL,15 mg/mL, and 20 mg/mL) were digested using pepsin (1 mg/mL), neutralized using 1 N NaOH and 10x PBS to p.H. 7.4, and a pre-gel solution was obtained. 5 mM genipin was added as a crosslinker and the hydrogels underwent gelation at 37°C. The rheological assessment like time sweep (for gelation time), frequency sweep (for mechanical stiffness), and shear thinning (for injectability analysis) was performed using 8 mm sandblasted parallel plate geometry at 37°C.Uncrosslinked hydrogels (neutralized pre-gel solution kept at 37°C without genipin) served as a control for rheological assessment. Native fresh nerves are referred to as FHSN, decell only as dHSN, decellularized and delipidated (once) as idHSN, and decellularized and delipidated (twice) as iidHSN.

Results, Conclusions, and Discussions: DAPI staining revealed the absence of nuclei in dHSN and idHSN, while cells were present in fresh native nerves. Immunostaining also revealed that both decellularized only and decellularized and delipidated nerves retained ECM (Collagen I and laminin) (Figures 1 and 2) This indicates that the nerves were devoid of any cells while they retained ECM. Oil Red O stain revealed a decreased amount of lipids across idHSN & iidHSN in comparison to the FHSN and dHSN samples (Figure 3). We did not observe any significant difference in lipid content among the nerves delipidated with either dichloromethane: ethanol or hexane: isopropanol; however, the nerves delipidated using hexane: isopropanol did not digest as well as the dichloromethane: ethanol nerves. Hence, the nerves delipidated using dichloromethane: ethanol was used for further studies. During digestion, several concentrations (10mg/mL, 15mg/mL, and 20mg/mL) of mg nerve tissue to mL of pepsin were tested to find an optimum concentration. 15mg/mL was found to provide better digestion of nerves and optimum viscosity while 10mg/mL concentration was too liquid and not forming hydrogel while 20mg/mL was too viscous and difficult to handle. The rheological assessment using time sweep shows that both uncrosslinked and 5 mM genipin crosslinked hydrogels started gelating within 4 minutes and underwent complete gelation within 14 minutes at 37°C as evidenced by an increase in storage modulus (G’) (Figure 4). Furthermore, frequency sweep shows that uncrosslinked hydrogels have mechanical stiffness (G’) of around 10-12 Pa while 5 mM genipin crosslinked hydrogels have G’ of 400-500 Pa (Figure 5). The increase in mechanical stiffness is attributed to genipin crosslinking the amino group present on ECM like Collagen I. Additionally, previously biomaterials with mechanical stiffness 500-2000 Pa have been shown to promote axonal regeneration. Both uncrosslinked and 5mM genipin crosslinked hydrogels exhibited shear thinning properties demonstrating the injectability of the hydrogels. We are currently studying biocompatibility (using neuronal and glial cells) and biodegradation of hydrogels. Overall, iHPN is a clinically relevant injectable hydrogel formulation that can be used to promote axonal regeneration and a cell delivery vehicle for SCI treatment.

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