SOD2 orchestrates redox homeostasis in intervertebral discs: A novel insight into oxidative stress-mediated degeneration and therapeutic potential.

Low back pain (LBP) is a pervasive global health concern, primarily associated with intervertebral disc (IVD) degeneration. Although oxidative stress has been shown to contribute to IVD degeneration, the underlying mechanisms remain undetermined. This study aimed to unravel the role of superoxide dismutase 2 (SOD2) in IVD pathogenesis and target oxidative stress to limit IVD degeneration. SOD2 demonstrated a dynamic regulation in surgically excised human IVD tissues, with initial upregulation in moderate degeneration and downregulation in severely degenerated IVDs. Through a comprehensive set of in vitro and in vivo experiments, we found a suggestive association between excessive mitochondrial superoxide, cellular senescence, and matrix degradation in human and mouse IVD cells. We confirmed that aging and mechanical stress, established triggers for IVD degeneration, escalated mitochondrial superoxide levels in mouse models. Critically, chondrocyte-specific Sod2 deficiency accelerated age-related and mechanical stress-induced disc degeneration in mice, and could be attenuated by ß-nicotinamide mononucleotide treatment. These revelations underscore the central role of SOD2 in IVD redox balance and unveil potential therapeutic avenues, making SOD2 and mitochondrial superoxide promising targets for effective LBP interventions.

Recombinant Laminin-511 Fragment (iMatrix-511) Coating Supports Maintenance of Human Nucleus Pulposus Progenitor Cells In Vitro.

The angiopoietin-1 receptor (Tie2) marks specific nucleus pulposus (NP) progenitor cells, shows a rapid decline during aging and intervertebral disc degeneration, and has thus sparked interest in its utilization as a regenerative agent against disc degeneration. However, the challenge of maintaining and expanding these progenitor cells in vitro has been a significant hurdle. In this study, we investigated the potential of laminin-511 to sustain Tie2+ NP progenitor cells in vitro. We isolated cells from human NP tissue (n = 5) and cultured them for 6 days on either standard (Non-coat) or iMatrix-511 (laminin-511 product)-coated (Lami-coat) dishes. We assessed these cells for their proliferative capacity, activation of Erk1/2 and Akt pathways, as well as the expression of cell surface markers such as Tie2, GD2, and CD24. To gauge their regenerative potential, we examined their extracellular matrix (ECM) production capacity (intracellular type II collagen (Col2) and proteoglycans (PG)) and their ability to form spherical colonies within methylcellulose hydrogels. Lami-coat significantly enhanced cell proliferation rates and increased Tie2 expression, resulting in a 7.9-fold increase in Tie2-expressing cell yields. Moreover, the overall proportion of cells positive for Tie2 also increased 2.7-fold. Notably, the Col2 positivity rate was significantly higher on laminin-coated plates (Non-coat: 10.24% (±1.7%) versus Lami-coat: 26.2% (±7.5%), p = 0.010), and the ability to form spherical colonies also showed a significant improvement (Non-coat: 40.7 (±8.8)/1000 cells versus Lami-coat: 70.53 (±18.0)/1000 cells, p = 0.016). These findings demonstrate that Lami-coat enhances the potential of NP cells, as indicated by improved colony formation and proliferative characteristics. This highlights the potential of laminin-coating in maintaining the NP progenitor cell phenotype in culture, thereby supporting their translation into prospective clinical cell-transplantation products.

Investigation of the Mitigation of DMSO-Induced Cytotoxicity by Hyaluronic Acid following Cryopreservation of Human Nucleus Pulposus Cells.

To develop an off-the-shelf therapeutic product for intervertebral disc (IVD) repair using nucleus pulposus cells (NPCs), it is beneficial to mitigate dimethyl sulfoxide (DMSO)-induced cytotoxicity caused by intracellular reactive oxygen species (ROS). Hyaluronic acid (HA) has been shown to protect chondrocytes against ROS. Therefore, we examined the potential of HA on mitigating DMSO-induced cytotoxicity for the enhancement of NPC therapy. Human NPC cryopreserved in DMSO solutions were thawed, mixed with equal amounts of EDTA-PBS (Group E) or HA (Group H), and incubated for 3-5 h. After incubation, DMSO was removed, and the cells were cultured for 5 days. Thereafter, we examined cell viability, cell proliferation rates, Tie2 positivity (a marker of NP progenitor cells), and the estimated numbers of Tie2 positive cells. Fluorescence intensity of DHE and MitoSOX staining, as indicators for oxidative stress, were evaluated by flow cytometry. Group H showed higher rates of cell proliferation and Tie2 expressing cells with a trend toward suppression of oxidative stress compared to Group E. Thus, HA treatment appears to suppress ROS induced by DMSO. These results highlight the ability of HA to maintain NPC functionalities, suggesting that mixing HA at the time of transplantation may be useful in the development of off-the-shelf NPC products.

N-acetylcysteine attenuates oxidative stress-mediated cell viability loss induced by dimethyl sulfoxide in cryopreservation of human nucleus pulposus cells: A potential solution for mass production.

Background: Cell therapy is considered a promising strategy for intervertebral disc (IVD) regeneration. However, cell products often require long-term cryopreservation, which compromises cell viability and potency, thus potentially hindering commercialization and off-the-shelf availability. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant, however, DMSO is associated with cytotoxicity and cell viability loss. This study aimed to investigate the effects of DMSO on human nucleus pulposus cells (NPC) and the role of oxidative stress in DMSO-induced cytotoxicity. Furthermore, we examined the potential of antioxidant N-acetylcysteine (NAC) supplementation to mitigate the negative effects of DMSO. Methods: NPC were exposed to various concentrations of DMSO with or without a freezing cycle. Cell viability, cell apoptosis and necrosis rates, intracellular reactive oxygen species (ROS) levels, and gene expression of major antioxidant enzymes were evaluated. In addition, NAC was added to cryopreservation medium containing 10% DMSO and its effects on ROS levels and cell viability were assessed. Results: DMSO concentrations ≤1% for 24 h did not significantly affect the NPC viability, whereas exposure to 5 and 10% DMSO (most commonly used concentration) caused cell viability loss (loss of 57% and 68% respectively after 24 h) and cell death in a dose- and time-dependent manner. DMSO increased intracellular and mitochondrial ROS (1.9-fold and 3.6-fold respectively after 12 h exposure to 10% DMSO) and downregulated gene expression levels of antioxidant enzymes in a dose-dependent manner. Tempering ROS through NAC treatment significantly attenuated DMSO-induced oxidative stress and supported maintenance of cell viability. Conclusions: This study demonstrated dose- and time-dependent cytotoxic effects of DMSO on human NPC. The addition of NAC to the cryopreservation medium ameliorated cell viability loss by reducing DMSO-induced oxidative stress in the freeze-thawing cycle. These findings may be useful for future clinical applications of whole cells and cellular products.

Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells.

Previous work showed a link between Tie2+ nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% (p = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% (p = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 (p = 0.01). However, the addition of 1 µM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression (p = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics.

Association of NAT2 genetic polymorphism with the efficacy of Neurotropin® for the enhancement of aggrecan gene expression in nucleus pulposus cells: a pilot study.

BACKGROUND: Intervertebral disc degeneration, one of the major causes of low-back pain, results from altered biosynthesis/turnover of extracellular matrix in the disc. Previously, we reported that the analgesic drug Neurotropin® (NTP) had an anabolic effect on glycosaminoglycan synthesis in cultured nucleus pulposus (NP) cells via the stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1. However, its effect on the aggrecan core protein was not significantly detected, because of the data variance. A microarray analysis suggested that the effect of NTP on aggrecan was correlated with N-acetyltransferase 2 (NAT2), a drug-metabolizing enzyme. Specific NAT2 alleles are known to correlate with rapid, intermediate, and slow acetylation activities and side effects of various drugs. We investigated the association between the efficacy of NTP on aggrecan expression and the NAT2 genotype in cell donors. METHODS: NP cells were isolated from intervertebral disc tissues donated by 31 Japanese patients (28-68 years) who underwent discectomy. NTP was added to the primary cell cultures and its effect on the aggrecan mRNA was analyzed using real-time quantitative PCR. To assess acetylator status, genotyping was performed based on the inferred NAT2 haplotypes of five common single-nucleotide polymorphisms using allele-specific PCR. RESULTS: The phenotype frequencies of NAT2 in the patients were 0%, 42.0%, and 58.0% for slow, intermediate, and rapid acetylators, respectively. The proportions of responders to NTP treatment (aggrecan upregulation, ≥ 1.1-fold) in the intermediate and rapid acetylators were 76.9% and 38.9%, respectively. The odds ratio of the comparison of the intermediate acetylator status between responders and nonresponders was 5.2 (95% CI 1.06-26.0, P = 0.036), and regarding the 19 male patients, this was 14.0 (95% CI 1.54-127.2, P = 0.012). In the 12 females, the effect was not correlated with NAT2 phenotype but seemed to become weaker along with aging. CONCLUSIONS: An intermediate acetylator status significantly favored the efficacy of NTP treatment to enhance aggrecan production in NP cells. In males, this tendency was detected with higher significance. This study provides suggestive data of the association between NAT2 variants and the efficacy of NTP treatment. Given the small sample size, results should be further confirmed.

Effects of interleukin-17A in nucleus pulposus cells and its small-molecule inhibitors for intervertebral disc disease.

Intervertebral discs (IVD) degeneration, which is caused by ageing or mechanical stress, leads to IVD disease, including back pain and sciatica. The cytokine interleukin (IL)-17A is elevated in NP cells during IVD disease. Here we explored the pharmacotherapeutic potential of IL-17A for the treatment of IVD disease using small-molecule inhibitors that block binding of IL-17A to the IL-17A receptor (IL-17RA). Treatment of NP cells with IL-17A increased expression of cyclooxygenase-2 (COX-2), IL-6, matrix metalloproteinase (MMP)-3 and MMP-13. These increases were suppressed by an IL-17A-neutralizing antibody, and small molecules that were identified as inhibitors by binding to the IL-17A-binding region of IL-17RA. IL-17A signalling also altered sulphated glycosaminoglycan deposition and spheroid colony formation, while treatment with small-molecule inhibitors of IL-17A attenuated this response. Furthermore, mitogen-activated protein kinase pathways were activated by IL-17A stimulation and induced IL-6 and COX-2 expression, while small-molecule inhibitors of IL-17A suppressed their expression. Taken together, these results show that IL-17A is a valid target for IVD disease therapy and that small-molecule inhibitors that inhibit the IL-17A-IL-17RA interaction may be useful for pharmacotherapy of IVD disease.