Peripheral Nerve Decellularization for In Vitro Extracellular Matrix Hydrogel Use: A Comparative Study

Abstract

The rise of tissue-engineered biomaterials has introduced more clinically translatable models of disease, including three-dimensional (3D) decellularized extracellular matrix (dECM) hydrogels. Specifically, decellularized nerve hydrogels have been utilized to model peripheral nerve injuries and disorders in vitro; however, there lacks standardization in decellularization methods. Here, rat sciatic nerves of varying preparations were decellularized using previously established methods: sodium deoxycholate (SD)-based, 3-((3-cholamidopropyl)­dimethylammonio)-1-propanesulfonate (CHAPS)-based, and apoptosis-mediated. These nerves were characterized for cellular debris removal, ECM retention, and low cytotoxicity with cultured Schwann cells. The best preparations of each decellularization method were digested into dECM hydrogels, and rheological characterization, gelation kinetics, and confocal reflectance imaging of collagen fibril assembly were performed. It was determined that the SD-based method with nerve epineurial removal best maintained the overall ECM composition and mechanical properties of physiological peripheral nerves while efficiently stripping the scaffolds of tissue-specific cells and debris. This method was then utilized as a culture platform for quiescent Schwann cells and cancer–nerve crosstalk. Hydrogel-embedded Schwann cells were found to have high viability and act in a more physiologically relevant manner than those cultured in monolayers, and the hydrogel platform allowed for the activation of Schwann cells following treatment with cancer secreted factors. These findings establish a standard for peripheral nerve decellularization for usage as a dECM hydrogel testbed for in vitro peripheral nerve disease modeling and may facilitate the development of treatments for peripheral nerve disease and injury.

Engineering cell-derived extracellular matrix for peripheral nerve regeneration

“…Once adequate cd-ECMs have been deposited, cellular components should be cleared up from ECMs through decellularization processing. Cellular components are the main source of allogeneic and xenogenic tissue immunogenicity, which may negatively influence or even terminate the regeneration process, possibly with graft rejection as a result [ 160 , 161 ]. Nevertheless, ECM molecules are highly conserved among species, and their immunogenicity is extremely low or even negligible [ 162 ].…”

Detergent-Based Decellularization for Anisotropic Cardiac-Specific Extracellular Matrix Scaffold Generation

“…Preserving structural ECM proteins and other biomacromolecules is also critical to maintain ECM function [16]. Decellularization ECM has been widely applied to tissues and organs reconstruction, including corneal [17][18][19], musculoskeletal [20][21][22], cardiac [23][24][25], and neural tissues [26][27][28]. Additionally, various decellularization methods have been employed to generate decellularized ECM from cell sheets [17,[29][30][31], tissues [32][33][34][35], and organs [36][37][38][39][40].…”

Pancreatic Tumor-Derived Extracellular Vesicles Stimulate Schwann Cell Phenotype Indicative of Perineural Invasion via IL-8 Signaling

“…Recent studies highlight cancer exosomes as contributors to tumor innervation, the relative opposite and common comorbidity of PNI 11 . While preliminary reports suggest pancreatic TEVs may activate SCs, the complete mechanism of this interaction and its role in PNI has yet to be explored 12 .…”

“…The role of the ECM in TEV function must not be overlooked. The ECM promotes biochemical and biomechanical signaling that allows for physiological cellular behavior 12 . To take advantage of this unique interaction, biomedical engineers have employed three-dimensional (3D) scaffolds created from natural or synthetic polymers to achieve mechanical and/or compositional similarity to the native tissues 11 .…”

“…Previous studies of PNI have implemented Matrigel to model the crosstalk between SCs and PDAC cells 4,5 ; however, Matrigel and comparable products often vary batch-to-batch, have limited applications, and are unable to completely capture the physiological activity of cells within the TME 16 . More recently, tissue engineering has given researchers the ability to fabricate customizable biomaterials from native tissues through the process of decellularization 12 . This allows for more biomimetic replications of the TME.…”