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. 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. 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. 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.

IntroductionWhole-organ intervertebral disc (IVD) culture is widely used to study IVD degeneration and evaluate regenerative therapies. Spatial visualization of resident cell viability is essential, yet commonly used assays, such as Calcein AM/Ethidium Homodimer-1 (EthD-1), lactate dehydrogenase (LDH)/EthD-1, and MTT/DAPI, lack standardized protocols that facilitate implementation.MethodsIn this study, we optimized and compared protocols for all three staining methods in bovine caudal IVDs and evaluated their strengths and limitations using appropriate positive and negative controls.ResultsSelection of suitable controls was critical for reliable viability assessment. Calcein AM/EthD-1 provided a straightforward approach but required protocol modifications, including Collagenase P pre-treatment, to ensure adequate tissue penetration. This method also requires immediate processing and imaging after harvesting. In contrast, LDH/EthD-1 and MTT/DAPI allows for less time-sensitive workflows and enables repeated staining. However, LDH/EthD-1 is unsuitable for short-term experiments, as LDH signal persists for up to 36 h after cell death, except under extreme conditions such as repeated snap-freezing. MTT/DAPI proved more suitable for short-term applications.DiscussionThese findings provide practical guidance for selecting and implementing viability assays in IVD culture, facilitating method choice based on specific experimental objectives and time constraints. Moreover, the insights gained may also be applicable to other dense tissues, such as tendon and cartilage.

The physiological relevance of dorsal root ganglion cultures is limited by monolayer formats that disrupt native multicellular organization. We present an in vitro model in which sensory neurons and glial cells are rapidly assembled into defined geometries using a sound-driven hydrodynamic process that requires no scaffold. This initial physical organization enables spontaneous self-assembly that restores essential features of native tissue. Glial cells envelop neurons with reduced gliosis, neurons regain an upright soma morphology, and axons extend in three dimensions. Mechanosensitive signaling shifts, with decreased nuclear Yes-associated protein (YAP) in glia and increased levels in neurons. The assembled in vitro system exhibits elevated neuronal calcium activity, enhanced cross-neuron correlation, and cluster-dependent activation of extracellular signal-regulated kinase (ERK) and calcitonin gene-related peptide (CGRP). Proteomic analysis shows reduced inflammatory, adhesion, and migratory pathways. This hydrodynamically assembled in vitro platform reveals how multicellular architecture governs neuron-glia communication and provides a scalable tool for studying sensory neurobiology.

Acoustic fields enable contactless manipulation of cells and multicellular assemblies, offering unique opportunities for three-dimensional (3D) cell culture and mechanobiology. However, standardized and reproducible methodologies that combine sound-guided cell assembly with controlled acoustic stimulation and downstream molecular analysis are still lacking. We developed a reproducible workflow integrating sound-guided assembly and acoustic stimulation of human bone marrow–derived mesenchymal stromal cell (hBMSC) spheroids within 3D fibrin hydrogels. Using a sound-induced morphogenesis (SIM) platform, spheroids were rapidly organized into circular patterns through low-frequency acoustic. Pattern geometry and stability were quantitatively assessed by image-based radial profile analysis. To evaluate biological responsiveness to assembly and stimulation, spheroids were analyzed under three conditions (randomly dispersed, sound-assembled, and sound-assembled with daily acoustic stimulation) followed by transcriptomic profiling using RNA sequencing and validation by RT-qPCR. Sound-guided assembly consistently generated stable, high-density annular spheroid patterns that were maintained over culture duration and unaffected by subsequent acoustic stimulation. Transcriptomic analysis revealed reproducible changes in gene expression associated with cell–cell interactions, stress regulation, and mechanosensing in assembled spheroids compared with dispersed controls. Additional acoustic stimulation induced a distinct transcriptional signature involving genes related to matrix remodeling and mechanotransduction, confirming biological sensitivity to applied mechanical cues. This method provides a standardized, non-invasive approach to couple spatial cell organization with controlled mechanical stimulation in 3D culture systems. The workflow is compatible with conventional cell culture practices, adaptable to other cell types and hydrogels, and suitable for mechanistic studies investigating how physical cues regulate cellular gene expression. It offers a transferable methodological framework for mechanobiology research and engineered tissue model development.

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.

Osteoarthritis (OA) pain arises from dynamic crosstalk between degraded cartilage and sensitized sensory nerves. Cartilage-derived signals, including nerve growth factor (NGF), pro-inflammatory cytokines, and matrix-degrading enzymes, promote nerve sprouting and hyperexcitability. While sensory afferents release neuropeptides that further amplify inflammation and cartilage degeneration. Altered joint mechanics additionally activate mechanosensitive ion channels, linking biomechanical stress to nociceptive signaling. This review summarizes current knowledge on cartilage–sensory nerve interactions in OA and their contribution to pain progression. We discuss key molecular mediators, biomarkers, and therapeutic targets, and provide an overview of in vivo, in vitro, and ex vivo model platforms for studying cartilage–sensory nerve crosstalk, highlighting emerging predictive systems for mechanistic and translational research. Together, these insights support the development of mechanism-based pain phenotyping and personalized therapeutic strategies for OA. Core Take-Home Messages and Clinical Significance •Cartilage actively contributes to OA pain by engaging in bidirectional crosstalk with sensory nerves.•Inflammatory mediators, neuropeptides, and mechanosensitive ion channels form an integrated network driving OA pain.•Predictive in vitro and ex vivo models provide tractable platforms to study cartilage–sensory nerve crosstalk and support translational pain research.•Mechanism-linked neural, inflammatory and mechanosensitive biomarkers enable OA pain phenotyping beyond structural severity. The Translational Potential of this Article This review highlights cartilage–sensory nerve crosstalk as a key mechanism underlying osteoarthritis pain, moving beyond a structure-centric view of disease progression. Mechanistic insights into neuroinflammatory and mechanosensitive pathways support the development of biomarkers for pain phenotyping and patient stratification. These insights have direct implications for clinical trial design and interpretation, particularly in addressing discordance between structural and symptomatic outcomes.

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.

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.

Herein we show an accessible technique based on Faraday waves that assist the rapid assembly of osteoinductive β-Tricalcium phosphate (β-TCP) particles as well as human osteoblast pre-assembled in spheroids. The hydrodynamic forces originating at ‘seabed’ of the assembly chamber can be used to tightly aggregate inorganic and biological entities at packing densities that resemble those of native tissues. Additionally, following a layer-by-layer assembly procedure, centimeter scaled osteoinductive three-dimensional and cellularized constructs have been fabricated. We showed that the intimate connection between biological building blocks is essential in engineering living system able of localized mineral deposition. Our results demonstrate, for the first time, the possibility to obtain three-dimensional cellularized and acellularized anisotropic constructs using Faraday waves.

Purpose This study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization. Methods hAFCs from spinal trauma patients were stimulated with TNF-α or IL-1β. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 μM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model. Results IL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1β treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation. Conclusion Cxb can inhibit PGE-2 production in hAFCs in an IL-1β-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM.

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.

Chronic discogenic back pain is associated with increased inflammatory cytokine levels that can influence the proximal peripheral nervous system, namely the dorsal root ganglion (DRG). However, transition to chronic pain is widely thought to involve glial activation in the spinal cord. In this study, an in vitro model was used to evaluate the communication between DRG and spinal cord glia. Primary neonatal rat DRG cells were treated with/without inflammatory cytokines (TNF-α, IL-1β, and IL-6). The conditioned media were collected at two time points (12 and 24 h) and applied to spinal cord mixed glial culture (MGC) for 24 h. Adult bovine DRG and spinal cord cell cultures were also tested, as an alternative large animal model, and results were compared with the neonatal rat findings. Compared with untreated DRG-conditioned medium, the second cytokine-treated DRG-conditioned medium (following medium change, thus containing solely DRG-derived molecules) elevated CD11b expression and calcium signal in neonatal rat microglia and enhanced Iba1 expression in adult bovine microglia. Cytokine treatment induced a DRG-mediated microgliosis. The described in vitro model allows the use of cells from large species and may represent an alternative to animal pain models (3R principles).

Abstract It has been shown that painful intervertebral discs (IVDs) were associated with a deeper innervation. However, the effect of the disc's degenerative microenvironment on neuronal outgrowth remains largely unknown. The focus of this study was to determine the influence of hypoxia on dorsal root ganglion (DRG) neurite outgrowth. Toward this aim, the DRG‐derived cell line ND7/23 was either directly subjected to 2% or 20% oxygen conditions or exposed to conditioned medium (CM) collected from IVDs cultured under 2% or 20% oxygen. Viability and outgrowth analysis were performed following 3 days of exposure. Results obtained with the cell line were further validated on cultures of rabbit spinal DRG explants and dissociated DRG neurons. Results showed that hypoxia significantly increased neurite outgrowth length in ND7/23 cells, which was also validated in DRG explant and primary cell culture, although hypoxia conditioned IVD did not significantly increase ND7/23 neurite outgrowth. While hypoxia dramatically decreased the outgrowth frequency in explant cultures, it significantly increased collateral sprouting of dissociated neurons. Importantly, the hypoxia‐induced decrease of outgrowth frequency at the explant level was not due to inhibition of outgrowth branching but rather to neuronal necrosis. In summary, hypoxia in DRG promoted neurite sprouting, while neuronal necrosis may reduce the density of neuronal outgrowth at the tissue level. These findings may help to explain the deeper neo‐innervation found in the painful disc tissue. Highlights Hypoxia promoted elongation and branching of neurite outgrowth at single cell level, but reduced outgrowth density at tissue level, possibly due to hypoxia‐induced neuronal necrosis; these findings may help to explain the deeper neo‐innervation found in clinically painful tissues.

Low back pain is the leading cause of disability worldwide and in many patients the source of pain can be attributed to pathological changes within the intervertebral disc (IVD). As present treatment options fail to address the underlying biological problem, novel therapies are currently subject to intense research. The physiologic IVD microenvironment features a highly complex interaction of biochemical and mechanical factors influencing cell metabolism and extracellular matrix turnover and is therefore difficult to simulate for research purposes on IVD pathology. The first whole organ culture models were not able to sufficiently replicate human in vivo conditions as mechanical loading, the predominant way of IVD nutrient supply and waste exchange, remained disregarded. To mimic the unique IVD niche more realistically, whole organ culture bioreactors have been developed, allowing for dynamic loading of IVDs and nutrient exchange. Recent advancements on bioreactor systems have facilitated whole organ culture of various IVDs for extended periods. IVD organ culture bioreactors have the potential to bridge the gap between in vitro and in vivo systems and thus may give valuable insights on IVD pathology and/or potential novel treatment approaches if the respective model is adjusted according to a well-defined research question. In this review, we outline the potential of currently utilized IVD bioreactor systems and present suggestions for further developments to more reliably investigate IVD biology and novel treatment approaches.

OBJECTIVE:Ischemia-related risk factors are consistently correlated with discogenic pain, but it remains unclear how the ischemia-associated hypoxia and acidosis influence the peripheral sensory nervous system, namely the dorsal root ganglion (DRG), either directly or indirectly via intervertebral disc (IVD) mediation.METHODS:Bovine tail IVD organ cultures were preconditioned in different hypoxic and/or acidic conditions for 3 days to collect the conditioned medium (CM). The DRG-derived ND7/23 cells were either treated by the IVD CM or directly stimulated by hypoxic and/or acidic conditions. Neuronal sensitization was evaluated using calcium imaging (Fluo-4) after 3 days.RESULTS:We found that direct exposure of DRG cell line to hypoxia and acidosis increased both spontaneous and bradykinin-stimulated calcium response compared to normoxia-neutral pH cultures. Hypoxia and low pH in combination showed stronger effect than either parameter on its own. Indirect exposure of DRG to hypoxia-acidosis-stressed IVD CM also increased spontaneous and bradykinin-stimulated response, but to a lower extent than direct exposure. The impact of direct hypoxia and acidosis on DRG was validated in a primary sheep DRG cell culture, showing the same trend.CONCLUSION:Our data suggest that targeting hypoxia and acidosis stresses both in IVD and DRG could be a relevant objective in discogenic pain treatment.

Soft tissue injury is very common and associated with pain, tissue swelling and even malformation if not treated on time. Treating methods include cryotherapy, electrical therapy, ultrasound therapy and anti-inflammatory drug, but none of them is completely satisfying. In this work, for a better therapeutic effect, drug therapy and pulsed electromagnetic field (PEMF) therapy were combined. We constructed a drug delivery system using the tetra-PEG/agar hydrogel (PA). By incorporating Fe3O4 NPs into the hydrogel network, a magnetism-responsive property was achieved in the system. The cytotoxicity and in vivo study showed a good biocompatibility of the PA/Fe3O4 hydrogel. A magnetism-controlled release was attained by the incorporation of Fe3O4. Finally, in vivo study showed a better performance of the DS-loaded PA/Fe3O4 compared with the commercially available DS ointment regarding the recovery of the injured soft tissue. Therefore, this magnetism-responsive hydrogel may represent a promising alternative to treat soft tissue injury.

Objective: This study aimed to characterize the mesenchymal stem cell (MSC) subpopulation migrating towards a degenerated intervertebral disc (IVD) and to assess its regenerative potential. Design: Based on initial screening for migration towards C-C motif chemokine ligand 5 (CCL5), the migration potential of CD146+ and CD146-mesenchymal stem cells (MSCs) was evaluated in vitro and in a degenerated organ culture model (degeneration by high-frequency loading in a bioreactor). Discogenic differentiation potential of CD146+ and CD146-MSCs was investigated by in vitro pellet culture assay with supplementation of growth and differentiation factor-6 (GDF6). Furthermore, trypsin degenerated IVDs were treated by either homing or injection of CD146+ or CD146-MSCs and glycosaminoglycan synthesis was evaluated by Sulphur 35 incorporation after 35 days of culture. Results: Surface expression of CD146 led to a higher number of migrated MSCs both in vitro and in organ culture. CD146+ and CD146- pellets responded with a similar up-regulation of anabolic markers. A higher production of sulfated glycosaminoglycans (sGAG)/DNA was observed for CD146+ pellets, while in organ cultures, sGAG synthesis rate was higher for IVDs treated with CD146-MSCs by either homing or injection. Conclusions: The CD146+ MSC subpopulation held greater migration potential towards degenerative IVDs, while the CD146- cells induced a stronger regenerative response in the resident IVD cells. These findings were independent of the application route (injection vs migration). From a translational point of view, our data suggests that CD146+ MSCs may be suitable for re-population, while CD146-MSCs may represent the primary choice for stimulation of endogenous IVD cells. (C) 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.