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.

ISSLS PRIZE in Basic Science 2024: superiority of nucleus pulposus cell- versus mesenchymal stromal cell-derived extracellular vesicles in attenuating disc degeneration and alleviating pain.

PURPOSE: To investigate the therapeutic potential of extracellular vesicles (EVs) derived from human nucleus pulposus cells (NPCs), with a specific emphasis on Tie2-enhanced NPCs, compared to EVs derived from human bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a coccygeal intervertebral disc degeneration (IDD) rat model. METHODS: EVs were isolated from healthy human NPCs cultured under standard (NPCSTD-EVs) and Tie2-enhancing (NPCTie2+-EVs) conditions. EVs were characterized, and their potential was assessed in vitro on degenerative NPCs in terms of cell proliferation and senescence, with or without 10 ng/mL interleukin (IL)-1ß. Thereafter, 16 Sprague-Dawley rats underwent annular puncture of three contiguous coccygeal discs to develop IDD. Phosphate-buffered saline, NPCSTD-EVs, NPCTie2+-EVs, or BM-MSC-derived EVs were injected into injured discs, and animals were followed for 12 weeks until sacrifice. Behavioral tests, radiographic disc height index (DHI) measurements, evaluation of pain biomarkers, and histological analyses were performed to assess the outcomes of injected EVs. RESULTS: NPC-derived EVs exhibited the typical exosomal morphology and were efficiently internalized by degenerative NPCs, enhancing cell proliferation, and reducing senescence. In vivo, a single injection of NPC-derived EVs preserved DHI, attenuated degenerative changes, and notably reduced mechanical hypersensitivity. MSC-derived EVs showed marginal improvements over sham controls across all measured outcomes. CONCLUSION: Our results underscore the regenerative potential of young NPC-derived EVs, particularly NPCTie2+-EVs, surpassing MSC-derived counterparts. These findings raise questions about the validity of MSCs as both EV sources and cellular therapeutics against IDD. The study emphasizes the critical influence of cell type, source, and culture conditions in EV-based therapeutics.

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.

Elimination of Lumbar Plexus Injury by Changing the Entry Point and Traction Direction of the Psoas Major Muscle in Transpsoas Lateral Lumbar Spine Surgery.

Background and Objectives: The lateral approach is commonly used for anterior column reconstruction, indirect decompression, and fusion in patients with lumbar degenerative diseases and spinal deformities. However, intraoperative lumbar plexus injury may occur. This is a retrospective comparative study to investigate and compare neurological complications between the conventional lateral approach and a modified lateral approach at L4/5. Materials and Methods: Patients with a lumbar degenerative disease requiring single-level intervertebral fusion at L4/5 were included and categorized into group X and group A. Patients in group X underwent conventional extreme lateral interbody fusion, while those in group A underwent a modified surgical procedure that included splitting of the anterior third of the psoas muscle, which was dilated by the retractor on the anterior third of the intervertebral disc. The incidence of lumbar plexus injury, defined as a decrease of ≥1 grade on manual muscle testing of hip flexors and knee extensors and sensory impairment of the thigh for ≥3 weeks, on the approach side, was investigated. Results: Each group comprised 50 patients. No significant between-group differences in age, sex, body mass index, and approach side were observed. There was a significant between-group difference in intraoperative neuromonitoring stimulation value (13.1 ± 5.4 mA in group X vs. 18.5 ± 2.3 mA in group A, p < 0.001). The incidence of neurological complications was significantly higher in group X than in group A (10.0% vs. 0.0%, respectively, p < 0.05). Conclusions: In our modified procedure, the anterior third of the psoas muscle was entered and split, and the intervertebral disc could be reached without damaging the lumbar plexus. When performing lumbar surgery using the lateral approach, lumbar plexus injury can be avoided by following surgical indication criteria based on the location of the lumbar plexus with respect to the psoas muscle and changing the transpsoas approach to the intervertebral disc.

Homing of vertebral-delivered mesenchymal stromal cells for degenerative intervertebral discs repair - an in vivo proof-of-concept study.

Introduction: Cell transplantation shows promising results for intervertebral disc (IVD) repair, however, contemporary strategies present concerns regarding needle puncture damage, cell retention, and straining the limited nutrient availability. Mesenchymal stromal cell (MSC) homing is a natural mechanism of long-distance cellular migration to sites of damage and regeneration. Previous ex vivo studies have confirmed the potential of MSC to migrate over the endplate and enhance IVD-matrix production. In this study, we aimed to exploit this mechanism to engender IVD repair in a rat disc degeneration model. Methods: Female Sprague Dawley rats were subjected to coccygeal disc degeneration through nucleus pulposus (NP) aspiration. In part 1; MSC or saline was transplanted into the vertebrae neighboring healthy or degenerative IVD subjected to irradiation or left untouched, and the ability to maintain the IVD integrity for 2 and 4 weeks was assessed by disc height index (DHI) and histology. For part 2, ubiquitously GFP expressing MSC were transplanted either intradiscally or vertebrally, and regenerative outcomes were compared at days 1, 5, and 14 post-transplantation. Moreover, the homing potential from vertebrae to IVD of the GFP+ MSC was assessed through cryosection mediated immunohistochemistry. Results: Part 1 of the study revealed significantly improved maintenance of DHI for IVD vertebrally receiving MSC. Moreover, histological observations revealed a trend of IVD integrity maintenance. Part 2 of the study highlighted the enhanced DHI and matrix integrity for discs receiving MSC vertebrally compared with intradiscal injection. Moreover, GFP rates highlighted MSC migration and integration in the IVD at similar rates as the intradiscally treated cohort. Conclusion: Vertebrally transplanted MSC had a beneficial effect on the degenerative cascade in their neighboring IVD, and thus potentially present an alternative administration strategy. Further investigation will be needed to determine the long-term effects, elucidate the role of cellular homing versus paracrine signaling, and validate our observations on a large animal model.

Three-dimensional morphological analysis of the thoracic pedicle and related radiographic factors in adolescent idiopathic scoliosis.

BACKGROUND: This study aimed to investigate the laterality of the pedicle morphology at the apical vertebra (AV) level and identify the radiographic factors associated with the laterality ratio of the pedicle morphology at the AV level in patients with adolescent idiopathic scoliosis (AIS). METHODS: Overall, 684 pedicles in 57 AIS patients aged 10-20 years, who underwent preoperative computed tomography (CT) and had Lenke type 1 or 2 with right convex main thoracic curves (MTC), were evaluated. Pedicle diameters of the MTC were assessed. We defined and compared the region containing two vertebrae adjacent to the AV (APEX±1) and the region containing two vertebrae adjacent to the neutral vertebra. We analyzed the pedicle diameter and laterality ratio of APEX±1 and performed multiple linear regression analysis to identify the radiographic factors associated with the laterality of the pedicle diameter. RESULTS: On the concave side of APEX±1, the pedicles of 15 patients (26.3%) did not accept a 4-mm-diameter pedicle screw (PS), even with 25% cortical bone width expansion. Laterality ratio differences in the pedicle diameters of the cortical bone width in APEX±1 were large in patients with more proximal AV level (p < 0.001) and smaller apical vertebral rotation (AVR) (p = 0.029). CONCLUSIONS: Preoperative planning to accurately select and insert the PS in AIS should be based on the anatomical limitations in APEX±1, AV level, and AVR degree. In APEX±1, the correlation between AVR and the laterality ratio of the pedicle diameter may be useful for pathoetiological interpretation of the AIS deformity.

Trans-Sacral Epiduroscopic Ho:YAG Laser Ablation of the Ligamentum Flavum in a Live Pig.

Introduction: For the aging population, surgery for lumbar spinal canal stenosis (LSCS) requires minimally invasive procedures. Recently, trans-sacral epiduroscopic laser decompression for lumbar disc herniation has been reported with good results. In this study, we devised a new method to perform trans-sacral epiduroscopic laser ablation of the ligamentum flavum (LF), known to be the major cause of LSCS. Using a live pig, this study aims to evaluate the efficacy, safety, and drawbacks of this procedure. Methods: Using an epiduroscope, we observed intra-spinal canal structures and then examined the feasibility and problems of a decompression procedure to ablate the LF using holmium:YAG (Ho:YAG) laser. The pig was observed for behavioral changes and neurological deficits after the procedure. Histological analysis was performed to evaluate the amount of tissue ablation and damage to surrounding tissues. Results: Although it was possible to partially ablate the LF using the Ho:YAG laser under epiduroscopy, it was difficult to maintain a clear field of view, and freely decompressing the target lesion has been a challenge. After the first two experiments, the pig neither showed abnormal behavior nor any signs of pain or paresis. However, in the third experiment, the pig died during the operation. On autopsy, no thermal or mechanical injury was noted around the ablated site, including the dura mater and nerve root. Histological analysis showed that the LF and lamina were deeply ablated as the laser power increased, and no damage was noted on surrounding tissues beyond a depth of 500 µm. Conclusions: Although Ho:YAG laser could ablate the ligamentum and bone tissues without causing damage to surrounding tissues, it was difficult to completely decompress the LF under epiduroscopy. This method is a potentially highly invasive procedure that requires caution in its clinical application and needs further improvement in terms of the instruments and techniques used.

Imaging Evaluation of Intervertebral Disc Degeneration and Painful Discs-Advances and Challenges in Quantitative MRI.

In recent years, various quantitative and functional magnetic resonance imaging (MRI) sequences have been developed and used in clinical practice for the diagnosis of patients with low back pain (LBP). Until now, T2-weighted imaging (T2WI), a visual qualitative evaluation method, has been used to diagnose intervertebral disc (IVD) degeneration. However, this method has limitations in terms of reproducibility and inter-observer agreement. Moreover, T2WI observations do not directly relate with LBP. Therefore, new sequences such as T2 mapping, T1ρ mapping, and MR spectroscopy have been developed as alternative quantitative evaluation methods. These new quantitative MRIs can evaluate the anatomical and physiological changes of IVD degeneration in more detail than conventional T2WI. However, the values obtained from these quantitative MRIs still do not directly correlate with LBP, and there is a need for more widespread use of techniques that are more specific to clinical symptoms such as pain. In this paper, we review the state-of-the-art methodologies and future challenges of quantitative MRI as an imaging diagnostic tool for IVD degeneration and painful discs.

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.

Anatomy of the L5 nerve root in the pelvis for safe sacral screw placement: a cadaveric study.

OBJECTIVE: Previous reports have focused on the complications of L5 nerve root injury caused by anterolateral misplacement of the S1 pedicle screws. Anatomical knowledge of the L5 nerve root in the pelvis is essential for safe and effective placement of the sacral screw. This cadaveric study aimed to investigate the course of the L5 nerve root in the pelvis and to clarify a safe zone for inserting the sacral screw. METHODS: Fifty-four L5 nerve roots located bilaterally in 27 formalin-fixed cadavers were studied. The ventral rami of the L5 nerve roots were dissected along their courses from the intervertebral foramina to the lesser pelvis. The running angles of the L5 nerve roots from the centerline were measured in the coronal plane. In addition, the distances from the ala of the sacrum to the L5 nerve roots were measured in the sagittal plane. RESULTS: The authors found that the running angles of the L5 nerve roots changed at the most anterior surface of the ala of the sacrum. The angles of the bilateral L5 nerve roots from the right and left L5 intervertebral foramina to their inflection points were 13.77° ± 5.01° and 14.65° ± 4.71°, respectively. The angles of the bilateral L5 nerve roots from the right and left inflection points to the lesser pelvis were 19.66° ± 6.40° and 20.58° ± 5.78°, respectively. There were no significant differences between the angles measured in the right and left nerve roots. The majority of the L5 nerves coursed outward after changing their angles at the inflection point. The distances from the ala of the sacrum to the L5 nerve roots in the sagittal plane were less than 1 mm in all cases, which indicated that the L5 nerve roots were positioned close to the ala of the sacrum and had poor mobility. CONCLUSIONS: All of the L5 nerve roots coursed outward after exiting the intervertebral foramina and never inward. To prevent iatrogenic L5 nerve root injury, surgeons should insert the S1 pedicle screw medially with an angle > 0° toward the inside of the S1 anterior foramina and the sacral alar screw laterally with an angle > 30°.

L5 nerve root injury caused by anterolateral malpositioning of loosened S1 pedicle screws: illustrative cases.

BACKGROUND: Although malpositioning of pedicle screws into the spinal canal and intervertebral foramen can cause spinal nerve root injuries, there are few reports of L5 nerve root injuries when S1 pedicle screws have been inserted anterolaterally. The authors report two cases of L5 nerve root injury caused by anterolateral malpositioning of loosened S1 pedicle screws. OBSERVATIONS: In both patients, S1 pedicle screws were inserted toward the outside of the S1 anterior foramen, and the tip of the screws perforated the anterior sacral cortex. L5 nerve root impairment was not observed immediately after surgery. However, severe leg pain in the L5 area was observed after the S1 pedicle screws became loosened. In case 1, the symptoms could not be controlled with conservative treatment. Reoperation was performed 3 months after the initial surgery. In case 2, the symptoms gradually improved with conservative treatment because the area around the loosened S1 screw was surrounded by newly formed bone that stabilized the screws, as observed with computed tomography 1 year after surgery. LESSONS: Surgeons should recognize that anterolateral malpositioning of S1 pedicle screws can cause L5 nerve root injury. The screws should be inserted in the correct direction without loosening.