Background: Rats are a widely accepted preclinical model for evaluating intervertebral disc (IVD) degeneration and regeneration. IVD morphology is commonly assessed using histology, which forms the foundation for quantifying the state of IVD degeneration. IVD degeneration severity is evaluated using different grading systems that focus on distinct degenerative features. A standard grading system would facilitate more accurate comparison across laboratories and more robust comparisons of different models and interventions.Aims: This study aimed to develop a histology grading system to quantify IVD degeneration for different rat models.Materials & Methods: This study involved a literature review, a survey of experts in the field, and a validation study using 25 slides that were scored by 15 graders from different international institutes to determine inter‐ and intra‐rater reliability.Results: A new IVD degeneration grading system was established and it consists of eight significant degenerative features, including nucleus pulposus (NP) shape, NP area, NP cell number, NP cell morphology, annulus fibrosus (AF) lamellar organization, AF tears/fissures/disruptions, NP‐AF border appearance, as well as endplate disruptions/microfractures and osteophyte/ossification. The validation study indicated this system was easily adopted, and able to discern different severities of degenerative changes from different rat IVD degeneration models with high reproducibility for both experienced and inexperienced graders. In addition, a widely‐accepted protocol for histological preparation of rat IVD samples based on the survey findings include paraffin embedding, sagittal orientation, section thickness < 10 μm, and staining using H&E and/or SO/FG to facilitate comparison across laboratories.Conclusion: The proposed histological preparation protocol and grading system provide a platform for more precise comparisons and more robust evaluation of rat IVD degeneration models and interventions across laboratories.

Low back pain is the main cause of disability and is associated with intervertebral disc degeneration. Contemporary treatments are limited to palliative therapeutics or aggressive surgical interventions; however, current advancements in cell therapy offer to fill this breach. Clinical data suggest that cell transplantation can accomplish pain relief without any observed adverse effects. Despite a large variety of preclinical studies and preliminary clinical investigations, controversy remains on the optimal cell type and transplantation strategies. The translational potential of this article lies in the aim to update on the current state of intervertebral disc cell therapy and to identify current obstacles.

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab‐to‐lab variability jeopardizes the much‐needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources.Methods: The most commonly applied methods for NP cell extraction, expansion, and re‐differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re‐differentiation media and techniques were also investigated.Results: Recommended protocols are provided for extraction, expansion, and re‐differentiation of NP cells from common species utilized for NP cell culture.Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species‐specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross‐lab comparisons on NP cells worldwide.

Background: Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin‐1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2⁺ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.Purpose: Here, we present a comprehensive protocol for sorting of Tie2⁺ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence‐activated cell sorting‐based methodology to sort and analyze Tie2⁺ NPCs.Methods: We present flow cytometry protocols to isolate the Tie2⁺ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2⁺ NPCs from the IVD cell population during the isolation process. A cross‐species phylogenetic analysis of Tie2 across species is presented.Results: Our protocols are efficient towards labeling and isolation of Tie2⁺ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species.Conclusions: Current identification of Tie2⁺ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2⁺ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2⁺ fraction is the subject of future research.

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 β-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.