Injectable decellularized nucleus pulposus tissue exhibits neuroinhibitory properties

Abstract

Background Chronic low back pain (LBP) is a leading cause of disability, but treatments for LBP are limited. Degeneration of the intervertebral disc due to loss of neuroinhibitory sulfated glycosaminoglycans (sGAGs) allows nerves from dorsal root ganglia to grow into the core of the disc. Treatment with a decellularized tissue hydrogel that contains sGAGs may inhibit nerve growth and prevent disc‐associated LBP. Methods A protocol to decellularize porcine nucleus pulposus (NP) was adapted from previous methods. DNA, sGAG, α‐gal antigen, and collagen content were analyzed before and after decellularization. The decellularized tissue was then enzymatically modified to be injectable and form a gel at 37°C. Following this, the mechanical properties, microstructure, cytotoxicity, and neuroinhibitory properties were analyzed. Results The decellularization process removed 99% of DNA and maintained 74% of sGAGs and 154% of collagen compared to the controls NPs. Rheology demonstrated that regelled NP exhibited properties similar to but slightly lower than collagen‐matched controls. Culture of NP cells in the regelled NP demonstrated an increase in metabolic activity and DNA content over 7 days. The collagen content of the regelled NP stayed relatively constant over 7 days. Analysis of the neuroinhibitory properties demonstrated regelled NP significantly inhibited neuronal growth compared to collagen controls. Conclusions The decellularization process developed here for porcine NP tissue was able to remove the antigenic material while maintaining the sGAG and collagen. This decellularized tissue was then able to be modified into a thermally forming gel that maintained the viability of cells and demonstrated robust neuroinhibitory properties in vitro. This biomaterial holds promise as an NP supplement to prevent nerve growth into the native disc and NP in vivo.

Detergent-Free Decellularization of Notochordal Cell-Derived Matrix Yields a Regenerative, Injectable, and Swellable Biomaterial

“…Epitopes of Galα1-3-Galβ1-(3)­4GlcNAc-R (α-Gal) are an important point in tissue transplantation, as they may be recognized by antibodies within the body, leading to graft rejection, but also remodeling . However, recent studies suggest a lack of α-Gal within the porcine NP . Thus, in this study, we chose to first focus on the removal of DNA to prevent transmission of endogenous viruses.…”

“…To this end, we restricted the buffer volume to 0.01 mL/mg of tissue dry weight, effectively creating an incompletely swollen gel during the decellularization process. Similar to a recent previously published protocol, , with our protocol, overall GAG and collagen content were not of statistically significant difference with decellularization despite differences in median values. A high GAG/collagen ratio of ≈27 ± 5 is characteristic for NCM and was only slightly reduced in dNCM to ≈21.3.…”

“… 36 However, recent studies suggest a lack of α-Gal within the porcine NP. 37 Thus, in this study, we chose to first focus on the removal of DNA to prevent transmission of endogenous viruses.…”

Research progress in decellularized extracellular matrix hydrogels for intervertebral disc degeneration

“…Research has shown that cross-linking chondroitin sulfate or type I collagen in dECM hydrogels provides mechanical properties similar to those of normal NP tissue. 142,151 It is evident from numerous studies that to obtain dECM hydrogels with better mechanical properties, cross-linking polymers are required in addition to conventional gelling methods; the most common is the natural biological cross-linker genipin, although high concentrations of genipin may affect cellular activity. One of the most important aspects of future research is to find a cross-linking agent that has good mechanical properties and is nontoxic.…”

Development of 2-D and 3-D culture platforms derived from decellularized nucleus pulposus

“…To fabricate ECM-derived bioscaffolds, decellularization techniques have been developed to remove immunogenic cellular components from tissues, while striving to preserve the native ultrastructure and cell-instructive biochemical composition of the ECM ( Crapo et al, 2011 ). Various groups have reported NP decellularization protocols that can extract cellular content to obtain scaffolds enriched in structural ECM components ( Wachs et al, 2017 ; Piening et al, 2022 ; Illien-Jünger et al, 2016 ; Mercuri, Gill, and Simionescu 2011 ; Fernandez et al, 2016 ; Xu et al, 2019 ; Chan et al, 2013 ). Despite its low cellularity, decellularization of the NP is challenging because it contains a high concentration of GAGs that are more easily extracted during processing as compared to highly crosslinked collagens ( Tsuchiya et al, 2014 ; Peloso et al, 2016 ; Hanai et al, 2020 ).…”