Junxuan Ma

Intervertebral disc (IVD) degeneration (IVDD) is a major cause of low back pain, yet treatment options remain limited. Robust IVDD models are essential for discovering and validating new regenerative treatments. Ex vivo whole organ bioreactor cultures using bovine IVDs are a well‐established approach, with various degeneration models developed on this platform. However, most existing models replicate only isolated aspects of IVDD, failing to reflect its complex nature. There is a critical need for in vitro models that more accurately simulate the full spectrum of degeneration phenotypes observed in patients. Combining multiple well‐established degeneration models offers a promising strategy. In this study, we investigated the combined effects of enzyme (papain) and cytokine (tumor necrosis factor alpha [TNFα]) based degeneration inducers on bioreactor loaded bovine IVDs. While papain injection led to a 5.5‐fold higher glycosaminoglycan loss and tissue void formation, TNFα induced inflammatory and catabolic changes relevant to IVDD, including significant aggrecanase‐1 (ADAMTS4) upregulation and a 2.65‐fold increase in interleukin 6 release. Both effects were evident when combined, enabling the manifestation of multiple aspects of IVDD in one model. To also explore implications on nociception, primary bovine dorsal root ganglion neurons were cultured and treated with conditioned medium from the induced degenerative IVDs. Nociceptors treated with degenerative medium showed a 1.51‐fold higher proportion of neurons with a response compared to treatment with control IVD medium. By expanding the range of degenerative changes and bridging them to pain‐associated features, this model provides a valuable platform for testing novel regenerative therapies.

Biomarkers are becoming pivotal in understanding the complex pathophysiology of disc herniation and guiding novel therapeutic strategies. Recent research highlights the value of molecular and cellular biomarkers in delineating disease progression, treatment monitoring and patient stratification. This review summarizes current advances in the identification and validation of emerging biomarkers across genomic, transcriptomic, proteomic, and metabolomic domains, emphasizing their potential to bridge basic mechanistic insights with clinical translation. Particular attention is given to the interplay between inflammatory mediators, extracellular matrix turnover, and immune cell activity as indicators of lumbar disc herniation diagnosis and prognosis. Despite encouraging progress, standardization of biomarker validation protocols, inter-study comparability, and large-scale clinical implementation remain major challenges. Future directions include the integration of multi-omics technologies and bioinformatic tools to identify predictive biomarker panels with diagnostic and prognostic utility towards personalized medicine.

Purpose: Intervertebral disc degeneration (IVDD) is a major contributor to low back pain (LBP), with mechanical overloading recognized as a driver of disc pathology. This study investigated early responses of bovine caudal intervertebral discs (IVDs) to repetitive multiaxial loading and assessed effects on nociceptive sensitization. Methods: Whole IVDs were cultured ex vivo for 3 or 7 days either free-swelling or under multiaxial loading (0.2 MPa compression, 0–6° flexion, 0–4° torsion, 0.2 Hz, 4 h/day). Disc height, biomechanics, gene expression, biochemical markers, collagen integrity, and cell viability were evaluated. Conditioned medium (CM) from IVDs was applied to primary dorsal root ganglion (DRG) neurons to assess calcium responses and neurite outgrowth. Results: Multiaxial loading induced rapid, region-specific molecular changes, particularly in the outer annulus fibrosus, with upregulation of inflammatory (IL6) and catabolic (MMP13) genes and mild increases in glycosaminoglycan and nitric oxide release. DRG neurons exposed to CM from loaded IVDs exhibited enhanced capsaicin-evoked calcium responses and increased neurite branching, especially in CGRP-positive nociceptors, indicating early discogenic sensitization. Collagen denaturation showed a trend toward higher levels in loaded IVDs, while cell viability, tissue structure, and biomechanical properties remained preserved. Conclusion: Multiaxial loading at the selected magnitude induced early molecular signs of degeneration and neuronal sensitization without detectable alterations in cell viability, tissue structure, or biomechanics, contributing to our understanding of mechanically induced impact on the IVD. Ex vivo multiaxial loading in bioreactors provides a physiologically relevant platform to study the initiation and progression of IVDD and associated discogenic pain mechanisms.

Chronic low back pain (LBP) represents the leading cause of disability. There are limited non-surgical options in treating LBP. Developing high-throughput in vitro models is important to screen for novel targets. LBP is associated with a complex cell-to-cell crosstalk. Particularly, LBP is initiated by the interaction between the intervertebral disc (IVD) and nociceptive neurons in the dorsal root ganglion (DRG). Furthermore, pain is influenced by the functional crosstalk among DRG neurons. Cells correctly crosstalk only when they are spatially arranged close. We demonstrate that the in vitro spatial organization of cells is essential to study their communication. Bovine DRG and tail IVD were collected from a local abattoir. The enzymatically dissociated DRG micro-tissue was fast assembled to recapitulate the densely packed multicellular architecture of DRG naïve tissue. (Figure1A∼B) This was achieved by the hydrodynamic forces generated from a mild vertical vibration (60 Hz and 0.5 g). Using this approach, DRG neurons could be assembled around a specimen of annulus fibrosus (AF) in a collagen-matrix-based hydrogel. AF primed with IL-1β and TNF-a (both 10 ng/mL) was compared with AF not treated in affecting the sprouting of nociceptors' axons. The nociceptors were labeled using immunofluorescence of calcitonin gene-related peptide (CGRP). In the assembled multicellular system, the YAP nuclei translocation in glia was reduced by a fold change of 3.4 compared to monolayer culture, indicating their distinct mechanobiological response. (Figure1C) The cellular calcium signal was highly correlated when they were assembled into a cluster. (Figure1D) In the AF∼DRG multi-organotypic system, when the neurons were close to the AF, the CGRP(+) axons' length was increased (∼50%) in the cytokine-primed-AF group compared to non-primed control. When the neurons were far from the AF, no such difference could be detected between groups. (Figure2B∼D) Notably, the CGRP(+) axon length was negatively correlated with the AF∼neuron distance in the cytokine-primed-AF group (Rho = −0.55, p < 0.001, Figure 2E). The hydrodynamic bio-assembly achieved an enrichment of neurons that were close to the AF. The spatial distance among organotypic compartments, and their accurate structural organization, impact cell-to-cell crosstalk and should be controlled to assure the robustness of in vitro models. Apart from the crosstalk between IVD and DRG neurons, other types of cells, e.g. , vessel cells, glia and inflammatory cells, need to be investigated as they are also key players in LBP. The hydrodynamic approaches empower a precise control of proper cellular spatial organization.

Discogenic pain is associated with deep nerve ingrowth in annulus fibrosus tissue (AF) of intervertebral disc (IVD). To model AF nerve ingrowth, primary bovine dorsal root ganglion (DRG) micro‐scale tissue units are spatially organised around an AF explant by mild hydrodynamic forces within a collagen matrix. This results in a densely packed multicellular system mimicking the native DRG tissue morphology and a controlled AF‐neuron distance. Such a multicellular organisation is essential to evolve populational‐level cellular functions and in vivo‐like morphologies. Pro‐inflammatory cytokine‐primed AF demonstrates its neurotrophic and neurotropic effects on nociceptor axons. Both effects are dependent on the AF‐neuron distance underpinning the role of recapitulating inter‐tissue/organ anatomical proximity when investigating their crosstalk. This is the first in vitro model studying AF nerve ingrowth by engineering mature and large animal tissues in a morphologically and physiologically relevant environment. The new approach can be used to biofabricate multi‐tissue/organ models for untangling pathophysiological conditions and develop novel therapies.

Introduction: Mechanical overloading can trigger a degenerative‐like cascade in an organ culture of intervertebral disc (IVD). Whether the overloaded IVD can influence the activation of nociceptors (i.e., the damage sensing neurons) remains unknown. The study aims to investigate the influence of overloaded IVD conditioned medium (CM) on the activation of nociceptors.Methods: In the static loading regime, force‐controlled loading of 0.2 MPa for 20 h/day representing “long‐term sitting and standing” was compared with a displacement‐controlled loading maintaining original IVD height. In the dynamic loading regime, high‐frequency‐intensity loading representing degenerative “wear and tear” was compared with a lower‐frequency‐intensity loading. CM of differently loaded IVDs were collected to stimulate the primary bovine dorsal root ganglion (DRG) cultures. Calcium imaging (Fluo‐4) and calcitonin gene‐related peptide (CGRP) immunofluorescent labeling were jointly used to record the calcium flickering in CGRP(+) nociceptors.Results: Force‐controlled loading led to a higher IVD cell death compared to displacement‐controlled loading. Both static and dynamic overloading (force‐controlled and high‐frequency‐intensity loadings) elevated the frequency of calcium flickering in the subsurface space of CGRP(+) nociceptors compared to their mild loading counterparts.Conclusion: In the organ culture system, IVD overloading mediated an altered IVD‐nociceptor communication suggesting a biological mechanism associated with discogenic pain.