Low back pain is a highly prevalent, chronic, and costly medical condition predominantly triggered by intervertebral disc degeneration (IDD). IDD is often caused by structural and biochemical changes in intervertebral discs (IVD) that prompt a pathologic shift from an anabolic to catabolic state, affecting extracellular matrix (ECM) production, enzyme generation, cytokine and chemokine production, neurotrophic and angiogenic factor production. The IVD is an immune-privileged organ. However, during degeneration immune cells and inflammatory factors can infiltrate through defects in the cartilage endplate and annulus fibrosus fissures, further accelerating the catabolic environment. Remarkably, though, catabolic ECM disruption also occurs in the absence of immune cell infiltration, largely due to native disc cell production of catabolic enzymes and cytokines. An unbalanced metabolism could be induced by many different factors, including a harsh microenvironment, biomechanical cues, genetics, and infection. The complex, multifactorial nature of IDD brings the challenge of identifying key factors which initiate the degenerative cascade, eventually leading to back pain. These factors are often investigated through methods including animal models, 3D cell culture, bioreactors, and computational models. However, the crosstalk between the IVD, immune system, and shifted metabolism is frequently misconstrued, often with the assumption that the presence of cytokines and chemokines is synonymous to inflammation or an immune response, which is not true for the intact disc. Therefore, this review will tackle immunomodulatory and IVD cell roles in IDD, clarifying the differences between cellular involvements and implications for therapeutic development and assessing models used to explore inflammatory or catabolic IVD environments.

The cartilaginous endplates (CEP) are key components of the intervertebral disc (IVD) necessary for sustaining the nutrition of the disc while distributing mechanical loads and preventing the disc from bulging into the adjacent vertebral body. The size, shape, and composition of the CEP are essential in maintaining its function, and degeneration of the CEP is considered a contributor to early IVD degeneration. In addition, the CEP is implicated in Modic changes, which are often associated with low back pain. This review aims to tackle the current knowledge of the CEP regarding its structure, composition, permeability, and mechanical role in a healthy disc, how they change with degeneration, and how they connect to IVD degeneration and low back pain. Additionally, the authors suggest a standardized naming convention regarding the CEP and bony endplate and suggest avoiding the term vertebral endplate. Currently, there is limited data on the CEP itself as reported data is often a combination of CEP and bony endplate, or the CEP is considered as articular cartilage. However, it is clear the CEP is a unique tissue type that differs from articular cartilage, bony endplate, and other IVD tissues. Thus, future research should investigate the CEP separately to fully understand its role in healthy and degenerated IVDs. Further, most IVD regeneration therapies in development failed to address, or even considered the CEP, despite its key role in nutrition and mechanical stability within the IVD. Thus, the CEP should be considered and potentially targeted for future sustainable treatments.

Background: This study investigates the native presence and potential anabolic effects of interleukin (IL)‐4 and IL‐10 in the human intervertebral disc (IVD).Methods: Human nucleus pulposus (NP) cells cultured in 3D from trauma and degenerate IVDs and NP explants were stimulated with 10 ng/mL IL‐4, IL‐10, or each in combination with 1 ng/mL IL‐1β stimulation. The role of IL‐4 and IL‐10 in the IVD was evaluated using immunohistochemistry, gene expression, and Luminex multiplex immunoassay proteomics (73 secreted) and phosphoproteomics (21 phosphorylated proteins).Results: IL‐4, IL‐4R, and IL‐10R expression and localization in human cartilage endplate tissue were demonstrated for the first time. No significant gene expression changes were noted under IL‐4 or IL‐10 stimulation. However, IL‐1β stimulation significantly increased MMP3, COX2, TIMP1, and TRPV4 expression in NP cells from trauma IVDs. Combined IL‐4 and IL‐1β treatment induced a significant increase in protein secretion of IL‐1α, IL‐7, IL‐16, IL‐17F, IL‐18, IFNγ, TNF, ST2, PROK1, bFGF2, and stem cell factor exclusively in NP cells from degenerated IVDs. Conversely, the secretome profile of explants revealed an IL‐4–mediated decrease in CXCL13 following treatment with IL‐1β. Combined IL‐10 and IL‐1β treatment increased neurotrophic growth factor secretion compared with IL‐10 baseline.Conclusions: The NP cell phenotype affects the pleiotropic role of IL‐4, which can induce a pro‐inflammatory response in the presence of catabolic stimuli and enhance the effects of IL‐1β in degenerated IVDs. Environmental factors, including 3D culture and hypoxia, may alter IL‐4's role. Finally, IL‐10's potential neurotrophic effects under catabolic stimuli warrant further investigation to clarify its role in IVD degeneration.

Objective: To investigate whether the presence of bacteria within non‐herniated intervertebral discs (IVDs) represents bacterial antigen signals within disc cell boundaries, consistent with in vivo presence and to assess the effects of bacterial exposure on human nucleus pulposus (NP) cells, focusing on immune response pathways and catabolic factor expression. Methods: Non‐herniated IVD tissue was analyzed using immunohistochemistry ( n = 79 discs) to detect bacterial presence and its correlation with catabolic factors. Bacterial survival was tested under IVD‐like conditions to simulate intradiscal growth potential. Human NP cells were treated with bacterial cell membrane components in both monolayer ( n = 3) and 3D cultures ( n = 3), with secretome analyzed via Luminex profiling. Co‐culture studies investigated bacterial internalization, with NP cells exposed to peptidoglycans or co‐cultured with S. aureus or C. acnes at physiologically relevant multiplicities of infection (MOI 0.01, n = 3) to assess intracellular signaling activation. Results: Immunohistochemical analysis revealed significant correlations between C. acnes intracellular staining and expression of catabolic markers: MMP‐3 ( p = 3.39 × 10 ⁻⁴ ); GSDMD ( p = 0.019); and the intracellular receptor: NOD2 ( p = 9.6 × 10 ⁻⁵ ), implicating these factors in immune surveillance by NP cells. The presence of NOD2 suggests activation of intracellular pathways that contribute to bacterial detection and trigger inflammatory responses. Stimulation of NP cells with peptidoglycan induced a strong catabolic secretome in both 2D and 3D cultures, whilst LPS showed limited effects. Low infectivity of NP cells with C. acnes and S. aureus suppressed VEGF, CXCL10 and CCL5. Effects of peptidoglycan and bacterial co‐culture were altered by TLR2/NOD2 inhibition, suggesting receptor‐specific but incomplete pathway dependence. Conclusion: This study identifies key bacterial receptors and signaling pathways in the IVD in response to bacteria, highlighting potential targets for therapeutic intervention in disc‐related inflammatory conditions. Our findings support the concept of an active immune role of NP cells in response to bacterial presence, challenging the notion of the disc as a sterile environment.

Introduction: The vertebral cartilage endplate (CEP), crucial for intervertebral disc health, is prone to degeneration linked to chronic low back pain, disc degeneration, and Modic changes (MC). While it is known that disc cells express toll-like receptors (TLRs) that recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), it is unclear if CEP cells (CEPCs) share this trait. The CEP has a higher cell density than the disc, making CEPCs an important contributor. This study aimed to identify TLRs on CEPCs and their role in pro-inflammatory and catabolic gene expression. Methods: Gene expression of TLR1–10 was measured in human CEPs and expanded CEPCs using quantitative polymerase chain reaction. Additionally, surface TLR expression was measured in CEPs grouped into non-MC and MC. CEPCs were stimulated with tumor necrosis factor alpha, interleukin 1 beta, small-molecule TLR agonists, or the 30 kDa N-terminal fibronectin fragment. TLR2 signaling was inhibited with TL2-C29, and TLR2 protein expression was measured with flow cytometry. Results: Ex vivo analysis found all 10 TLRs expressed, while cultured CEPCs lost TLR8 and TLR9 expression. TLR2 expression was significantly increased in MC1 CEPCs, and its expression increased significantly after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer upregulated TLR2 protein expression. The TLR2/1 and TLR2/6 ligands upregulated pro-inflammatory genes and matrix metalloproteases (MMP1, MMP3, and MMP13), and TLR2 inhibition inhibited their upregulation. Endplate resorptive capacity of TLR2 activation was confirmed in a CEP explant model. Conclusions: The expression of TLR1–10 in CEPCs suggests that the CEP is susceptible to PAMP and DAMP stimulation. Enhanced TLR2 expression in MC1, and generally in CEPCs under inflammatory conditions, has pro-inflammatory and pro-catabolic effects, suggesting a potential role in disc degeneration and MC.

Background: Immunohistochemistry (IHC) is a widely used method for localizing and semi‐quantifying proteins in tissue samples. Traditional IHC analysis often relies on manually counting 200 cells within a designated area, a time‐intensive and subjective process that can compromise reproducibility and accuracy. Advances in digital scanning and bioimage analysis tools, such as the open‐source software QuPath, enable semi‐automated cell counting, reducing subjectivity and increasing efficiency.Aims: This project developed a QuPath‐based script and detailed guide for semi‐automatic cell counting, specifically for tissues with low cellularity, such as intervertebral discs and cartilage.Methods and Results: The methodology was validated by demonstrating no significant differences between the manual counting and the semi‐automatic quantification (p = 0.783, p = 0.386) while showing a strong correlation between methods for both collagen type II staining (r = 0.9602, p < 0.0001) and N‐cadherin staining (r = 0.9044, p = 0.0001). Furthermore, a strong correlation (intraclass correlation coefficient (ICC) single raters = 0.853) between 3 individual raters with varying academic ranks and experiences in IHC analysis was shown using the semi‐automatic quantification method.Discusssion: The approach ensures high reproducibility and accuracy, with reduced variability between raters and laboratories. This semi‐automated method is particularly suited for tissues with a high extracellular matrix to cell ratio and low cellularity. By minimizing subjectivity and evaluation time, it provides a robust alternative to manual counting, making it ideal for applications where reproducibility and standardization are critical. While the methodology was effective in low‐cellularity tissues, its application in other tissue types warrants further exploration.Conclusions: These findings underscore the potential of QuPath to streamline IHC analysis and enhance inter‐laboratory comparability.