Decellularized and Crosslinked Porcine Peripheral Nerve Based Hydrogel for Neural Tissue Regeneration

Introduction:: Xenogeneic decellularized matrices or hydrogels derived from decellularized tissues or organs are highly attractive scaffolds for surgical applications due to their low immunogenicity, biocompatibility, tunability, and tissue specific extracellular matrix (ECM) components. Previously, porcine decellularized nerve matrix has shown potential in peripheral nerve regeneration¹. However, decellularized porcine peripheral nerve based hydrogels lacked sufficient mechanical strength to be used as an injectable vehicle. Hence, a need arises to develop decellularization technique that can efficiently retain extracellular matrix proteins integrity, that can provide sufficient mechanical strength. Here, we explore the decellularization of different branches of porcine peripheral nerves (peroneal, tibial, sural, and sciatic) using novel SDD method and assessed the decellularization efficiency, gelation kinetics, injectability, and mechanical properties of resulting hydrogels. We further crosslinked the decellularized matrix pre-gel solution, and analyzed the effect of crosslinking on gelation kinetics, mechanical strength, and in vitro biodegradation. Our goal is to generate a decellularized peripheral nerve-based hydrogel with ECM protein composition and mechanical properties that mimics native nerves, which can be used for neural regeneration after injury.

Materials and Methods:: Porcine peripheral nerves with different branches (peroneal, sural, tibial, and sciatic nerve) were isolated from mini pigs, and decellularized using a previously published protocol for rodent sciatic nerve ². Decellularization efficiency was assessed using immunofluorescence staining for removal of cellular components (DAPI- nuclear presence, S100- Schwann cells, MBP- myelin, and β-tubulin III- neurons) and preservation of ECM proteins (collagen-I, collagen–IV, and laminin). The residual lipids from the decellularized porcine peripheral nerves were removed using dichloromethane and ethanol solvent mixture treatment. The gelation kinetics of pre-gel solutions was assessed using a U.V. absorbance study. Also, the mechanical strength, time for gelation and complex viscosity w.r.t to increase to time and temperature was assessed using Anton Parr MCR 302 rheometer. Furthermore, the decellularized porcine pre-gel solution was crosslinked using genipin and 4s arm PEG-succinimidyl glutarate. The mechanical strength and gelation kinetics were assessed using rheometer. In vitro biodegradation of non/crosslinked will be assessed by measuring the change in mass of hydrogel at different time points. The impact of crosslinker on cellular proliferation on bone marrow derived mesenchymal stem cells (BM-MSCs) will be studied using alamar blue reagent, live dead staining. Immunocytochemistry staining for neuronal markers (β-tubulin III, NF-200) will be used for analysis of BM-MSCs neuronal differentiation within crosslinked decellularized porcine peripheral nerve-based hydrogels. The statistical difference was analyzed using One WAY ANOVA.

Results, Conclusions, and Discussions:: DAPI staining was used to assess nuclear presence after decellularization. Decellularization resulted in near complete removal of nuclei as evidenced by no visible DAPI staining compared to fresh nerve controls. Collagen-I, Collagen IV and laminin are key ECM components in peripheral nerve and should be preserved during decellularization. Staining for Collagen-I, Collagen IV and laminin demonstrated robust preservation of these ECM proteins. These results demonstrated that the decellularization method employed was effective at removing cellular components and retaining the native ECM. Gelation kinetics of the nerve-based hydrogels was conducted, and results demonstrated that digested, decellularized, porcine sciatic nerve branches can successfully form hydrogels. We also found that removing residual lipids from decellularized porcine peripheral nerves using dichloromethane and ethanol solvent treatment resulted in significant improvement on gelation kinetics and thermal gelled hydrogels had higher mechanical stiffness. Furthermore, crosslinking of decellularized porcine peripheral nerves was further able to tune mechanical strength of hydrogels, varying the crosslinker concentration and using different crosslinker (genipin, 4s arm PEG-succinimidyl glutarate). At present, we are studying the in vitro biodegradation and neuronal differentiation potential of non/crosslinked decellularized and delipided porcine peripheral nerves.This study explores the decellularization of different porcine sciatic nerve branches and subsequent fabrication into hydrogels to be used for neural regeneration after injury. We demonstrated the ability to robustly remove the cellular components while preserving the native ECM proteins. We also demonstrated the ability to fabricate injectable hydrogels from these decellularized porcine nerve branches. We found that the presence of lipids delays gelation kinetics and ultimately affects the mechanical strength of hydrogel. The crosslinking of decellularized porcine peripheral nerve matrix affects the gelation kinetics and mechanical stiffness of hydrogels. Overall, we studied the decellularized porcine peripheral nerve branches as a promising scaffold neural regeneration after injury.

Acknowledgements (Optional): : NIH-R21NS123
Pruitt Chair Funding

References (Optional): : 1.Lin et al, Acta Biomaterilia, 2018, 326-338.
2.McCrary et al, Tissue Engineering Part C: Methods, 2020, 23-36.