(C-104) Injectable Decellularized Porcine Peripheral Nerve Based Hydrogel for Schwann Cells Delivery in Injured Spinal Cord

Introduction::
Porcine peripheral nerve-based acellular grafts and hydrogels comprised of extracellular matrix (ECM) proteins that are biocompatible, low immunogenic, and have neuronal restorative ability1. However, to date, there are challenges in the fabrication of injectable hydrogels using decellularized porcine peripheral nerves due to the weaker mechanical strength of hydrogel and the presence of lipid remnants in decellularized scaffolds. In this study, we aim to fabricate novel injectable hydrogel using decellularized porcine peripheral nerves for cellular delivery in neural tissue regeneration. Schwann cells (SCs) are currently studied in clinical trials for spinal cord injury treatment, however, the major lacunae of using direct transplantation are their lower retention (only ~5-34%) and viability at the site of injury2. Here, we will fabricate injectable hydrogel using different branches of decellularized porcine peripheral nerves (sciatic, and peroneal) and evaluate its potential for SCs delivery. To biomimic the injectable hydrogel mechanical strength with that of the naïve spinal cord, the effect of genipin-mediated crosslinking on tuning mechanical strength and biodegradation is studied. The success of this project will result in the development of a novel vehicle for cell delivery for spinal tissue regeneration.



Materials and Methods::
Porcine peripheral nerves will be isolated from mini pigs supplied by UF ACS and were decellularized using an established protocol for rodent sciatic nerves3. The decellularized nerves were then delipidized using a solution of ethanol and dichloromethane in a 1:2 ratio for 24 hours. To form the pre-gel solution, the decellularized and delipided nerves were digested in pepsin/acid solution for 48 hours at room temperature, then neutralized to a pH 7.6, and concentration was adjusted to 10mg/mL using 1X Phosphate Buffer Saline (PBS). Genipin was added to the neutralized pre-gel solution and the gelation kinetics of the hydrogels was measured using frequency sweep rheology. Uncrosslinked hydrogels were used as the control for this study. Currently, we are studying the potential of developed injectable hydrogels as cell delivery vehicles using rat SCs. For this, initially, the viability of cells in uncrosslinked and genipin crosslinked hydrogels will be evaluated using Alamar Blue reagent and Live Dead staining. In vitro biodegradation of genipin crosslinked and uncrosslinked injected hydrogels incubated in 1x PBS will be evaluated by measuring the difference in mass of hydrogel at different time points.



Results, Conclusions, and Discussions::
As shown in Figure 1, the mechanical strength of genipin crosslinked hydrogel in decellularized peroneal and sciatic nerves was significantly higher than uncrosslinked hydrogel. The increase in mechanical strength can be attributed to genipin-mediated crosslinking, the nucleophilic attack by the amino groups in the ECM onto the olefinic carbon of genipin4. Also, with the addition of genipin, the gelation of hydrogel was significantly faster than uncrosslinked hydrogel. The faster gelation kinetics can help in maintaining cellular retention and prevent cell leakage from the hydrogel in situ. Preliminary results show that the uncrosslinked hydrogels can support the rat SCs viability for 1 week time as estimated by the Alamar Blue reagent and live dead staining (Figure 2), and we will be studying the effect of genipin crosslinking on rat SCs viability and retention within the hydrogel. Prolonging SCs viability and retention at the site of injury would help in increasing the therapeutic effect of SCs for SCI regeneration. The effect of genipin-mediated crosslinking on the biodegradation of hydrogel will be studied. This study explores the gelation kinetics of porcine peripheral nerve-based hydrogels with the addition of crosslinkers for cell delivery treatments for injured spinal cord regeneration. We demonstrated hydrogel with and without genipin crosslinking had a storage modulus and viscosity that would be suitable for injection into an injury site and neuronal regeneration. We anticipate that genipin crosslinking would also result in prolonging biodegradation of hydrogel sufficient for neuronal regeneration and able to retain SCs at the site of injury. The outcomes of this study will result in novel cellular transplantation for injured spinal cord regeneration.



Acknowledgements (Optional): : The authors would like to acknowledge Pruitt Chair Funding.

References (Optional): :
1: Meder T, Prest T, Skillen C, Marchal L, Yupanqui VT, Soletti L, Gardner P, Cheetham J, Brown BN. Nerve-specific extracellular matrix hydrogel promotes functional regeneration following nerve gap injury. NPJ Regen Med. 2021 Oct 25;6(1):69. doi: 10.1038/s41536-021-00174-8. PMID: 34697304; PMCID: PMC8546053.

2: Cerqueira, Susana R et al. “Decellularized peripheral nerve supports Schwann cell transplants and axon growth following spinal cord injury.” Biomaterials vol. 177 (2018): 176-185. doi:10.1016/j.biomaterials.2018.05.049.

3 : Bousalis, Deanna et al. “Decellularized peripheral nerve as an injectable delivery vehicle for neural applications.” Journal of biomedical materials research. Part A vol. 110,3 (2022): 595-611. doi:10.1002/jbm.a.37312.
4: I. Dmour and M. O. Taha, “Chapter 2 - Natural and semisynthetic polymers in pharmaceutical nanotechnology,” in Organic Materials as Smart Nanocarriers for Drug Delivery, A. M. Grumezescu, Ed., William Andrew Publishing, 2018, pp. 35–100. doi: 10.1016/B978-0-12-813663-8.00002-6.