Human Annulus Fibrosus Research Articles

A data-driven microstructure-based model for predicting circumferential behavior and failure in degenerated human annulus fibrosus

The degeneration of the intervertebral disc annulus fibrosus poses significant challenges in understanding and predicting its mechanical behavior. In this article, we present a novel approach, enriched with detailed insights into microstructure and degeneration progression, to accurately predict the mechanics of the degenerated human annulus. Central to this framework is a fully three-dimensional continuum-based model that integrates hydration state and multiscale structural features, including proteoglycan macromolecules and interpenetrating collagen fibrillar networks across various hierarchical levels within the multi-layered lamellar/inter-lamellar soft tissue, capable of sustaining deformation-induced damage. To ensure accurate and comprehensive predictions of the degenerated annulus mechanical behavior, we establish a data-driven correlation between disc degeneration grade and individual age, which influences the composition and mechanical integrity of annulus constituents while accounting for regional variations. The methodology includes a thorough identification of age- and grade-related evolutions of model inputs, followed by a detailed quantitative evaluation of the model predictive capabilities, with a focus on circumferential behavior and failure. The model successfully replicates experimental data, accurately capturing stiffness, transverse response (Poisson's ratio), and ultimate properties across different annulus regions, while also accommodating the modulation of the age/grade relationship. The reduction rates between normal and severe degeneration align reasonably well with experimental data, with the inner region exhibiting the largest decrease in stiffness (34.63%) and no significant change observed in the outer region. Failure stress drops considerably in both regions (49.86% in the inner and 45.33% in the outer), while failure strain decreases by 36.39% in the outer and 24.74% in the inner. Our findings demonstrate that the proposed framework significantly enhances the predictive accuracy of annulus mechanics across a spectrum of degeneration levels, from normal to severely degenerated states. This approach promises improved predictive accuracy, deeper insights into disc health and injury risk, and a robust foundation for further research on the impact of degeneration on disc integrity. Statement of SignificanceUnderstanding and predicting the mechanical behavior of degenerated human annulus fibrosus remains a significant challenge due to the complex interplay of structural, biochemical, and age-related factors. This study presents a microstructure-based approach to address this challenge by integrating hydration state, detailed structural features across hierarchical scales, and deformation-induced damage and failure, alongside age-related changes and degeneration grade factors. This approach enables accurate simulations of annulus mechanics across regions, with model results thoroughly compared to available data, reinforcing its applicability in capturing degeneration effects. By capturing the intricate interactions between microstructure and mechanical behavior in degenerated discs, the model lays a strong foundation for improving clinical assessments and guiding future treatment strategies for disc-related conditions.

Three-dimensional fiber patterning in the annulus fibrosus can be derived from vertebral endplate topography.

The annulus fibrosus (AF) of the Intervertebral disc (IVD) is composed of concentric lamellae of helically wound collagen fibers. Understanding the spatial variation of collagen fiber orientations in these lamellae, and the resulting material anisotropy, is crucial to predicting the mechanical behavior of the complete IVD. This study builds on a prior model predicated on path-independent displacement of fiber endpoints during vertebral body growth to predict a complete, three-dimensional annulus fibrosus fiber network from a small number of subject-independent input parameters and vertebral endplate topographies obtained from clinical imaging. To evaluate the model, it was first fit to mid-plane fiber orientations obtained using polarized light microscopy in a population of bovine caudal discs for which computed tomography images vertebral endplates were also available. Additionally, the model was used to predict the trajectories based on human lumbar disc geometries and results were compared to previously reported data. Finally, the model was employed to investigate potential disc-related variations in fiber angle distributions. The model was able to accurately predict experimentally measured fiber distributions in both bovine and human discs using only endplate topography and three input parameters. Critically, the model recapitulated previously observed asymmetry between the inclinations of right- and left-handed fibers in the posterolateral aspect of the human AF. Level to level variation of disc height and aspect ratio in the human lumbar spine was predicted to affect absolute values of fiber inclination, but not this asymmetry. Taken together these results suggest that patient-specific distributions of AF fiber orientation may be readily incorporated into computational models of the spine using only disc geometry and a small number of subject-independent parameters.

Composite Hydrogel Sealants for Annulus Fibrosus Repair.

Intervertebral disc (IVD) herniation is a leading cause of disability and lower back pain, causing enormous socioeconomic burdens. The standard of care for disc herniation is nucleotomy, which alleviates pain but does not repair the annulus fibrosus (AF) defect nor recover the biomechanical function of the disc. Existing bioadhesives for AF repair are limited by insufficient adhesion and significant mechanical and geometrical mismatch with the AF tissue, resulting in the recurrence of protrusion or detachment of bioadhesives. Here, we report a composite hydrogel sealant constructed from a composite of a three-dimensional (3D)-printed thermoplastic polyurethane (TPU) mesh and tough hydrogel. We tailored the fiber angle and volume fraction of the TPU mesh design to match the angle-ply structure and mechanical properties of native AF. Also, we proposed and tested three types of geometrical design of the composite hydrogel sealant to match the defect shape and size. Our results show that the sealant could mimic native AF in terms of the elastic modulus, flexural modulus, and fracture toughness and form strong adhesion with the human AF tissue. The bovine IVD tests show the effectiveness of the composite hydrogel sealant for AF repair and biomechanics recovery and for preventing herniation with its heightened stiffness and superior adhesion. By harnessing the combined capabilities of 3D printing and bioadhesives, these composite hydrogel sealants demonstrate promising potential for diverse applications in tissue repair and regeneration.

High glucose enhances fibrosis in human annulus fibrosus cells by activating mTOR, PKCδ, and NF-κB signaling pathways.

Low back pain stands as a significant factor in disability, largely resulting from intervertebral disc degeneration (IVDD). High glucose (HG) levels have been implicated in the pathogenesis of IVDD. However, the detailed mechanism of HG in IVDD is largely unknown. Our clinical results revealed that fibrosis markers such as CTGF, Col1a1, ATF4, and EIF2 are highly expressed in advanced-stage IVDD patients. Stimulation of human annulus fibrosus cells (HAFCs) with HG, but not mannitol, promotes fibrosis protein production. Ingenuity Pathway Analysis in the GSE database found that the mTOR, PKCδ, and NF-κB pathways were significantly changed during IVDD. The mTOR, PKCδ, and NF-κB inhibitors or siRNAs all abolished HG-induced fibrosis protein production. In addition, treatment of HAFCs with HG enhances the activation of mTOR, PKCδ, and NF-κB pathways. Thus, HG facilitates fibrosis in IVDD through mTOR, PKCδ, and NF-κB pathways. These results underscore the critical role of HG as a fibrotic factor in the progression of IVDD.

Polymeric Nanoparticles Enable mRNA Transfection and Its Translation in Intervertebral Disc and Human Joint Cells, Except for M1 Macrophages.

Chronic lower back pain caused by intervertebral disc degeneration and osteoarthritis (OA) are highly prevalent chronic diseases. Although pain management and surgery can alleviate symptoms, no disease-modifying treatments are available. mRNA delivery could halt inflammation and degeneration and induce regeneration by overexpressing anti-inflammatory cytokines or growth factors involved in cartilage regeneration. Here, we investigated poly(amidoamine)-based polymeric nanoparticles to deliver mRNA to human joint and intervertebral disc cells. Human OA chondrocytes, human nucleus pulposus (NP) cells, human annulus fibrosus (AF) cells, fibroblast-like synoviocytes (FLS) and M1-like macrophages were cultured and transfected with uncoated or PGA-PEG-coated nanoparticles loaded with EGFP-encoding mRNA. Cell viability and transfection efficiency were analyzed for all cell types. Nanoparticle internalization was investigated in FLS and M1-like macrophages. No significant decrease in cell viability was observed in most conditions. Only macrophages showed a dose-dependent reduction of viability. Transfection with either nanoparticle version resulted in EGFP expression in NP cells, AF cells, OA chondrocytes and FLS. Macrophages showed internalization of nanoparticles by particle-cell co-localization, but no detectable expression of EGFP. Taken together, our data show that poly (amidoamine)-based nanoparticles can be used for mRNA delivery into cells of the human joint and intervertebral disc, indicating its potential future use as an mRNA delivery system in OA and IVDD, except for macrophages.

Developmental morphogens direct human induced pluripotent stem cells toward an annulus fibrosus-like cell phenotype.

Therapeutic interventions for intervertebral disc herniation remain scarce due to the inability of endogenous annulus fibrosus (AF) cells to respond to injury and drive tissue regeneration. Unlike other orthopedic tissues, such as cartilage, delivery of exogenous cells to the site of annular injury remains underdeveloped, largely due to a lack of an ideal cell source and the invasive nature of cell isolation. Human induced pluripotent stem cells (iPSCs) can be differentiated to specific cell fates using biochemical factors and are, therefore, an invaluable tool for cell therapy approaches. While differentiation protocols have been developed for cartilage and fibrous connective tissues (e.g., tendon), the signals that regulate the induction and differentiation of human iPSCs toward the AF fate remain unknown. iPSC-derived sclerotome cells were treated with various combinations of developmental signals including transforming growth factor beta 3 (TGF-β3), connective tissue growth factor (CTGF), platelet derived growth factor BB (PDGF-BB), insulin-like growth factor 1 (IGF-1), or the Hedgehog pathway activator, Purmorphamine, and gene expression changes in major AF-associated ECM genes were assessed. The top performing combination treatments were further validated by using three distinct iPSC lines and by assessing the production of upregulated ECM proteins of interest. To conduct a broader analysis of the transcriptomic shifts elicited by each factor combination, and to compare genetic profiles of treated cells to mature human AF cells, a 96.96 Fluidigm gene expression array was applied, and principal component analysis was employed to identify the transcriptional signatures of each cell population and treatment group in comparison to native AF cells. TGF-β3, in combination with PDGF-BB, CTGF, or IGF-1, induced an upregulation of key AF ECM genes in iPSC-derived sclerotome cells. In particular, treatment with a combination of TGF-β3 with PDGF-BB for 14 days significantly increased gene expression of collagen II and aggrecan and increased protein deposition of collagen I and elastin compared to other treatment groups. Assessment of genes uniquely highly expressed by AF cells or SCL cells, respectively, revealed a shift toward the genetic profile of AF cells with the addition of TGF-β3 and PDGF-BB for 14 days. These findings represent an initial approach to guide human induced pluripotent stem cells toward an AF-like fate for cellular delivery strategies.

FUNCTIONAL ACTIVITY OF MONOMERIC C-REACTIVE PROTEIN IN INTERVERTEBRAL DISC

Monomeric C reactive protein (mCRP) presents important proinflammatory effects in endothelial cells, leukocytes, or chondrocytes. However, CRP in its pentameric form exhibits weak anti-inflammatory activity. It is used as a biomarker to follow severity and progression in infectious or inflammatory diseases, such as intervertebral disc degeneration (IVDD). This work assesses for the first time the mCRP effects in human intervertebral disc cells, trying to verify the pathophysiological relevance and mechanism of action of mCRP in the etiology and progression of IVD degeneration.We demonstrated that mCRP induces the expression of multiple proinflammatory and catabolic factors, like nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and lipocalin 2 (LCN2), in human annulus fibrosus (AF) and nucleus pulposus (NP) cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signaling of mCRP.Our results indicate that the effect of mCRP is persistent and sustained, regardless of the proinflammatory environment, as it was similar in healthy and degenerative human primary AF cells. This is the first article that demonstrates the localization of mCRP in intravertebral disc cells of the AF and NP and that provides evidence for the functional activity of mCRP in healthy and degenerative human AF and NP disc cells.

INVESTIGATION OF INTERVERTEBRAL DISC HERNIATION THROUGH A HUMAN ANULUS FIBROSUS PRO-INFLAMMATORY MODEL

Intervertebral disc (IVD) herniation involves a chronic inflammatory process and failure of the annulus fibrosus (AF), in a process still under investigated. Inflammation can lead to disorganization of extracellular matrix (ECM) and fibrotic alterations, among others. Most of the ongoing research relies on in vivo or ex vivo models that do not truly mimic the human microenvironment. Here, we developed a 3D in vitro model aimed at recapitulating inflammation progression in human AF (hAF) to improve the understanding of AF failure.hAF of herniated samples from low back pain patients were collected and digested as previously established. hAF cells were cultured in 2D and 3D, embedded in collagen-type 1 hydrogels. hAF cells in 2D and 3D were then stimulated with IL-1β (10ng/mL), in acute (48h) and chronic (7days) conditions. After 7 days, cell morphology together with extracellular matrix (ECM) (collagen I and fibronectin) were assessed by immunofluorescence. Inflammatory cytokine secretion were analyzed by a protein array.2D experiments showed alteration of cell morphology, with cell area reduction upon inflammatory stimulation. The profile of inflammatory cytokines secreted showed that: i) IL-1α increases with time in culture, while TNF-α decreases; and ii) IL-1α and MIG/CXCL9 are increased in chronic vs acute inflammation. ECM analysis showed no differences. In 3D culture, AF cell morphologic alterations with inflammation were also observed. Concerning ECM production, collagen I production was reduced with chronic inflammation, while fibronectin remained unaltered. Collagen II and aggrecan appear to increase with chronic inflammation. Cytokine production from 3D culture is currently being assessed. ECM alterations are being complemented by characterization at biomechanical (nanoindentation) level.Our data show that chronic inflammation is crucial to understand AF failure mechanisms and improve the resemblance of human IVD herniation.

Nonviral overexpression of Scleraxis or Mohawk drives reprogramming of degenerate human annulus fibrosus cells from a diseased to a healthy phenotype.

Intervertebral disc (IVD) degeneration is a major contributor to low back pain (LBP), yet there are no clinical therapies targeting the underlying pathology. The annulus fibrosus (AF) plays a critical role in maintaining IVD structure/function and undergoes degenerative changes such as matrix catabolism and inflammation. Thus, therapies targeting the AF are crucial to fully restore IVD function. Previously, we have shown nonviral delivery of transcription factors to push diseased nucleus pulposus cells to a healthy phenotype. As a next step in a proof-of-concept study, we report the use of Scleraxis (SCX) and Mohawk (MKX), which are critical for the development, maintenance, and regeneration of the AF and may have therapeutic potential to induce a healthy, pro-anabolic phenotype in diseased AF cells. MKX and SCX plasmids were delivered via electroporation into diseased human AF cells from autopsy specimens and patients undergoing surgery for LBP. Transfected cells were cultured over 14 days and assessed for cell morphology, viability, density, gene expression of key phenotypic, inflammatory, matrix, pain markers, and collagen accumulation. AF cells demonstrated a fibroblastic phenotype posttreatment. Moreover, transfection of SCX and MKX resulted in significant upregulation of the respective genes, as well as SOX9. Transfected autopsy cells demonstrated upregulation of core extracellular matrix markers; however, this was observed to a lesser effect in surgical cells. Matrix-degrading enzymes and inflammatory cytokines were downregulated, suggesting a push toward a pro-anabolic, anti-inflammatory phenotype. Similarly, pain markers were downregulated over time in autopsy cells. At the protein level, collagen content was increased in both MKX and SCX transfected cells compared to controls. This exploratory study demonstrates the potential of MKX or SCX to drive reprogramming in mild to moderately degenerate AF cells from autopsy and severely degenerate AF cells from surgical patients toward a healthy phenotype and may be a potential nonviral gene therapy for LBP.

Celecoxib alleviates nociceptor sensitization mediated by interleukin-1beta-primed annulus fibrosus cells

PurposeThis 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.MethodshAFCs 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.ResultsIL-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.ConclusionCxb 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.

Terminal Complement Activation Is Induced by Factors Released from Endplate Tissue of Disc Degeneration Patients and Stimulates Expression of Catabolic Enzymes in Annulus Fibrosus Cells.

Terminal complement complex (TCC) deposition was identified in human degenerated discs. To clarify the role of terminal complement activation in disc degeneration (DD), we investigated respective activating mechanisms and cellular effects in annulus fibrosus (AF) cells. Isolated cells from human AF, nucleus pulposus (NP), and endplate (EP) were stimulated with human serum alone or with zymosan and treated with either the C3 inhibitor Cp40 or the C5 antibody eculizumab. Complement activation was determined via anaphylatoxin generation and TCC deposition detection. Thereby, induced catabolic effects were evaluated in cultured AF cells. Moreover, C5 cleavage under degenerative conditions in the presence of AF cells was assessed. Zymosan-induced anaphylatoxin generation and TCC deposition was significantly suppressed by both complement inhibitors. Zymosan induced gene expression of ADAMTS4, MMP1, and COX2. Whereas the C3 blockade attenuated the expression of ADAMTS4, the C5 blockade reduced the expression of ADAMTS4, MMP1, and COX2. Direct C5 cleavage was significantly enhanced by EP conditioned medium from DD patients and CTSD. These results indicate that terminal complement activation might be functionally involved in the progression of DD. Moreover, we found evidence that soluble factors secreted by degenerated EP tissue can mediate direct C5 cleavage, thereby contributing to complement activation in degenerated discs.

Monomeric CRP regulates inflammatory responses in human intervertebral disc cells.

CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration. We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry. We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues. Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κβ signalling may be implicated in the mediation of this mCRP-induced state.

Toward a mechanically biocompatible intervertebral disc: Engineering of combined biomimetic annulus fibrosus and nucleus pulposus analogs.

Intervertebral disc (IVD) degeneration and accompanying lower back pain impose global medical and societal challenges, affecting over 600 million people worldwide. The IVD complex fibrocartilaginous structure is responsible for the spine biomechanical function. The nucleus pulposus (NP), composed of swellable glycosaminoglycan (GAG), transfers compressive loads to the surrounding fiber-reinforced annulus fibrosus (AF) lamellae, which stretches under tension. Together, these substructures allow the IVD to withstand extremely high and complex loads. Key to mimic the complete disc must consider the properties of its substructures. This study presents three novel substructures-a biomimetic silk-reinforced composite lamella for the AF, a GAG analog for the NP, and a novel biomimetic combined AF-NP construct. The biomimetic AF demonstrates nonlinear, hyperelastic, and anisotropic behavior similar to the native human AF, while the NP analog demonstrates mechanical behavior similar to the human NP. The synergized biomimetic AF-NP demonstrates similar behavior to the unconfined NP, with significantly increased deformations indicating improved performance. Validation of the AF-NP construct mechanics using a finite element model yields results compatible with native human IVD under various physiological loadings. The ability of our AF-NP construct to mimic the native IVD offers a revolutionary concept for the potential development of a fully functional IVD.

Single-cell RNA sequencing reveals resident progenitor and vascularization-associated cell subpopulations in rat annulus fibrosus.

One of the main causes of low back pain is intervertebral disc degeneration (IDD). Annulus fibrosus (AF) is important for the integrity and functions of the intervertebral disc (IVD). However, the resident functional cell components such as progenitors and vascularization-associated cells in AF are yet to be fully identified. Identification of functional AF cell subpopulations including resident progenitors and vascularization-associated cells. In this study, the single-cell RNA sequencing data of rat IVDs from a public database were analyzed using Seurat for cell clustering, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional analysis, StemID for stem cell identification, Monocle and RNA velocity for pseudotime differentiation trajectory validation, single-cell regulatory network inference and clustering (SCENIC) for gene regulatory network (GRN) analysis, and CellChat for cell-cell interaction analysis. Immunostaining on normal and degenerated rat IVDs, as well as human AF, was used for validations. From the data analysis, seven AF cell clusters were identified, including two newly discovered functional clusters, the Grem1 + subpopulation and the Lum +​subpopulation. The Grem1 + subpopulation had progenitor characteristics, while the Lum +​subpopulation was associated with vascularization during IDD. The GRN analysis showed that Sox9 and Id1 were among the key regulators in the Grem1 + subpopulation, and Nr2f2 and Creb5 could be responsible for the vascularization function in the Lum +​subpopulation. Cell-cell interaction analysis revealed highly regulated cellular communications between these cells, and multiple signaling networks including PDGF and MIF signaling pathways were involved in the interactions. Our results revealed two new functional AF cell subpopulations, with stemness and vascularization induction potential, respectively. These findings complement our knowledge about IVDs, especially the AF, and in return provide potential cell source and regulation targets for IDD treatment and tissue repair. The existence of the cell subpopulations was also validated in human AF, which strengthen the clinical relevance of the findings.

USP14 promotes pyroptosis of human annulus fibrosus cells derived from patients with intervertebral disc degeneration through deubiquitination of NLRP3.

Intervertebral disc degeneration (IVDD) is a general disorder that results in low back pain and disability among many affected individuals. However, the current treatments for IVDD are limited to relieving the symptoms but do not solve the fundamental issue. In this study, the role of USP14 in mediating the activation of the NLRP3 inflammasome and the pyroptosis of AF cells from IVDD patients is determined in vitro, and gain- and loss-of-function assays of USP14 and the NLRP3 inflammasome are conducted. Pyroptosis of AF cells is detected by flow cytometry. The inflammatory cytokines (IL-1β and IL-18) and protein levels of NLRP3, active Caspase-1, Aggrecan, MMP3 and ADAMTS-5 are determined by ELISA and western blot analysis, respectively. The correlation between USP14 and NLRP3 is measured by coimmunoprecipitation and ubiquitination analysis. Upregulation of USP14 is accompanied by increased level of the NLRP3 inflammasome in AF cells from IVDD patients; furthermore, a positive correlation between them is observed. USP14 knockdown inhibits pyroptosis in AF cells by inducing ubiquitination of NLRP3, while overexpression of USP14 has the opposite effect, which is inhibited by the NLRP3 inflammasome inhibitor INF39. USP14 exerts its positive regulatory effect on AF cell pyroptosis by modulating the NLRP3/Caspase-1/IL-1β and IL-18 signaling axes.

Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways.

Degenerative disc disease (DDD) is an important cause of low back pain. Repetitive tensile stress from the daily motion of the spine predisposes it to injury of the annulus fibrosus (AF) which causes IVD degeneration. This study aims to determine the causal relationship between mechanical stretch and osteogenesis in the AF cells of IVD as affected by bone morphogenic proteins (BMPs), specifically BMP-2/6 heterodimers. Our results found that 15% tensile stress (high cyclic stretching, HCS) may induce the expression of osteogenesis-related markers (Runx2, osterix) by upregulating BMP-2/6 heterodimeric ligands and their receptors on the human AF cell line. HCS also induced transient phosphorylation of p38 mitogen-activated protein (MAP) kinase and SMAD1/5/8. Neutralizing antibodies to the BMP-2/6 receptor (ALK3) blocked the expression of Runx2 and osterix, as well as the phosphorylation of p38 and SMAD1/5/8. In addition, treatment with a p38 MAPK inhibitor (SB203580) or siRNA to neutralize the effects of SMAD1/5/8 suppressed tensile stress-induced Runx2 and osterix expression. Mechanical stretching induces activation of p38 MAP kinase and SMAD1/5/8 signaling pathways, followed by the upregulation of BMP-2/6 heterodimer expression, thereby stimulating osteogenic Runx2 and osterix expression on AF cells. HCS may accelerate the progression of IVD degeneration by promoting an osteogenic response.

Mechanical Stretch-Induced NLRP3 Inflammasome Expression on Human Annulus Fibrosus Cells Modulated by Endoplasmic Reticulum Stress.

Excessive mechanical loading is a major cause of spinal degeneration, typically originating from a tear in the annulus fibrosus (AF). Endoplasmic reticulum (ER) stress and NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome have been implicated in the pathogenesis of intervertebral disc (IVD) degeneration. However, the causal relationship between the mechanical stretching of AF cells and the NLRP3 inflammasome response associated with ER stress remains scarce. To elucidate the pathogenesis and regulatory mechanisms of mechanical stretch-induced IVD degeneration, human AF cell lines were subjected to different degrees of cyclic stretching to simulate daily spinal movements. Our results indicated that 15% high cyclic stretch (HCS) induced the expression of NLRP3 and interleukin-1 beta (IL-1β) and was also responsible for the increased expression of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2) and reactive oxygen species (ROS) in human AF cells. In addition, HCS increased the expression of glucose-regulated protein 78 (GRP78), an ER stress chaperone, which was neutralized with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. In addition, HCS was found to induce thioredoxin-interacting protein (TXNIP) expression and NLRP3 inflammasome activation, which can be suppressed by si-NOX2 or the NOX2 inhibitor GSK2795039. Consequently, HCS upregulated ER stress and ROS production, leading to increased NLRP3 and IL-1β expression in human AF cells, and may further accelerate IVD degeneration.

Adipokine human Resistin promotes obesity-associated inflammatory intervertebral disc degeneration via pro-inflammatory cytokine cascade activation

Adipokine human Resistin (hResistin), is known to be associated with insulin resistance and secrete low-grade pro-inflammatory cytokines in obesity. Although studies on low-grade inflammation of adipokine hResistin are known, studies on the effects and mechanisms of intervertebral disc degeneration (IVDD) are still lacking. Thus, we investigated the adipokine hResistin with or without pro-inflammatory cytokine IL-1β in intervertebral disc (IVD) cells such as human annulus fibrosus (hAF) and nucleus pulposus (hNP). The protein expression changes in IL-1β, IL-6, IL-8, MMP-1, MMP-3, and MMP-13, induced by the combined-hResistin and IL-1β stimulation on hAF cells, was significantly greater than that of the same induced by mono-IL-1β stimulation. Similarly, in the case of the protein expression change of inflammatory mediators induced by the combined-hResistin and IL-1β stimulation on hNP cells was also significantly greater than that of the same induced by mono-IL-1β stimulation. These results improve understanding of hResistin on inflammatory IVDD but also with other obesity-related inflammatory diseases.

Interleukin-1β More Than Mechanical Loading Induces a Degenerative Phenotype in Human Annulus Fibrosus Cells, Partially Impaired by Anti-Proteolytic Activity of Mesenchymal Stem Cell Secretome.

Mesenchymal stem/stromal cell (MSC)–based therapies for low back pain and intervertebral disc (IVD) degeneration have been emerging, despite the poor knowledge of their full mechanism of action. As failure of the annulus fibrosus (AF) is often associated with IVD herniation and inflammation, the objective of the present study was to investigate the impact of the MSC secretome on human AF cells exposed to mechanical loading and a pro-inflammatory environment. Human AF cells isolated from IVD biopsies from patients with adolescent idiopathic scoliosis (AIS) or disc degeneration (DD) were exposed to physiological cyclic tensile strain (CTS) for 72 h in a custom-made device, with or without interleukin (IL)-1β medium supplementation. AF cells stimulated with CTS + IL-1β were then treated with secretome from IL-1β–preconditioned MSCs for 48 h. AF cell metabolic activity, gene expression, protein secretion, matrix metalloproteinase (MMP) activity, and tissue inhibitor of MMPs (TIMP) concentration were evaluated. Expanded AF cells from AIS and DD patients revealed similar metabolic activity and gene expression profiles. CTS stimulation upregulated collagen type I (COL1A1) expression, while IL-1β significantly stimulated IL-6, IL-8, MMP-1, and MMP-3 gene expression and prostaglandin E2 production by AF cells but downregulated COL1A1. The combination of CTS + IL-1β had a similar outcome as IL-1β alone, accompanied by a significant upregulation of elastin. The MSC secretome did not show any immunomodulatory effect on CTS + IL-1β–stimulated AF cells but significantly decreased MMP-1, MMP-2, MMP-3, and MMP-9, while increasing the production of TIMP-1. The obtained results demonstrate a stronger impact of the inflammatory milieu on human AF cells than upper physiologic mechanical stress. In addition, a new MSC mechanism of action in degenerated IVD consisting of the modulation of AF MMP activity was also evidenced, contributing to the advancement of knowledge in AF tissue metabolism.

Fibrotic alterations in human annulus fibrosus correlate with progression of intervertebral disc herniation

BackgroundIntervertebral disc (IVD) herniation is characterized by annulus fibrosus failure (AF) in containing the nucleus pulposus (NP). IVD herniation involves cellular and extracellular matrix (ECM) alterations that have been associated with tissue fibrosis, although still poorly investigated.MethodsHere, fibrotic alterations in human AF were evaluated, by characterizing the herniated ECM. Human AF samples (herniated lumbar IVD (n = 39, age 24–83) and scoliosis controls (n = 6, age 15–21)) were processed for transmission electron microscopy and histological/immunohistochemical analysis of fibrotic markers. Correlations between the fibrotic markers in AF ECM and the degree of NP containment (protused, contained and uncontained) and patients’ age were conducted.ResultsOur results demonstrate that with herniation progression, i.e. loss of NP containment, human AF presents less stained area of sulphated glycosaminoglycans and collagen I, being collagen I fibres thinner and disorganized. On the other hand, fibronectin stained area and percentage of α-smooth muscle actin+ cells increase in human AF, while matrix metalloproteinase-12 (MMP12) production and percentage of macrophages (CD68+ cells) remain constant. These structural and biochemical fibrotic alterations observed in human AF with herniation progression occur independently of the age.ConclusionsThe characterization of human AF here conducted evidence the presence of fibrosis in degenerated IVD, while highlighting the importance of considering the herniation progression stage, despite the patients’ age, for a better understanding of the mechanisms behind AF failure and IVD herniation.