Modulation of TRPV4 protects against degeneration induced by sustained loading and promotes matrix synthesis in the intervertebral disc.

While it is well known that mechanical signals can either promote or disrupt intervertebral disc (IVD) homeostasis, the molecular mechanisms for transducing mechanical stimuli are not fully understood. The transient receptor potential vanilloid 4 (TRPV4) ion channel activated in isolated IVD cells initiates extracellular matrix (ECM) gene expression, while TRPV4 ablation reduces cytokine production in response to circumferential stretching. However, the role of TRPV4 on ECM maintenance during tissue-level mechanical loading remains unknown. Using an organ culture model, we modulated TRPV4 function over both short- (hours) and long-term (days) and evaluated the IVDs' response. Activating TRPV4 with the agonist GSK101 resulted in a Ca2+ flux propagating across the cells within the IVD. Nuclear factor (NF)-κB signaling in the IVD peaked at 6 h following TRPV4 activation that subsequently resulted in higher interleukin (IL)-6 production at 7 days. These cellular responses were concomitant with the accumulation of glycosaminoglycans and increased hydration in the nucleus pulposus that culminated in higher stiffness of the IVD. Sustained compressive loading of the IVD resulted in elevated NF-κB activity, IL-6 and vascular endothelial growth factor A (VEGFA) production, and degenerative changes to the ECM. TRPV4 inhibition using GSK205 during loading mitigated the changes in inflammatory cytokines, protected against IVD degeneration, but could not prevent ECM disorganization due to mechanical damage in the annulus fibrosus. These results indicate TRPV4 plays an important role in both short- and long-term adaptations of the IVD to mechanical loading. The modulation of TRPV4 may be a possible therapeutic for preventing load-induced IVD degeneration.

Analysis of the clinical efficacy of visualization of percutaneous endoscopic lumbar discectomy combined with annulus fibrosus suture in lumbar disc herniation.

The objective of this study is to compare the clinical effectiveness of visualization of percutaneous endoscopic lumbar discectomy (VPELD) combined with annulus fibrosus suture technique and simple percutaneous endoscopic lumbar discectomy (PELD) technique in the treatment of lumbar disc herniation. A retrospective analysis was conducted on 106 cases of lumbar disc herniation treated with foraminoscopic technique at our hospital from January 2020 to February 2022. Among them, 33 cases were treated with VPELD combined with annulus fibrosus suture in group A, and 73 cases were treated with PELD in group B. The preoperative and postoperative visual analogue scale (VAS), functional index (Oswestry Disability Index, ODI), healing of the annulus fibrosus, intervertebral space height, and postoperative recurrence were recorded and compared between the two groups. All patients underwent preoperative and postoperative MRI examinations, and the average follow-up period was 12 ± 2 months. Both groups showed significant improvements in postoperative VAS and ODI scores compared to the preoperative scores (P < 0.05), with no statistically significant difference between the groups during the same period (P > 0.05). There was no significant decrease in intervertebral space between the two groups after surgery (P > 0.05). Group A showed significantly lower postoperative recurrence rate and better annulus fibrosus healing compared to group B (P < 0.05). The VPELD combined with annulus fibrosus suture technique is a safe, feasible, and effective procedure for the treatment of lumbar disc herniation. When the indications are strictly adhered to, this technique can effectively reduce the postoperative recurrence rate and reoperation rate. It offers satisfactory clinical efficacy and can be considered as an alternative treatment option for eligible patients.

An Approach to Quantify Anisotropic Multiaxial Failure of the Annulus Fibrosus.

Tears in the annulus fibrosus (AF) of the intervertebral disk (IVD) occur due to multiaxial loading on the spine. However, most existing AF failure studies measure uniaxial stress, not the multiaxial stress at failure. Delamination theory, which requires advanced structural knowledge and knowledge about the interactions between the AF fibers and matrix, has historically been used to understand and predict AF failure. Alternatively, a simple method, the Tsai-Hill yield criteria, could describe multiaxial failure of the AF. This yield criteria uses the known tissue fiber orientation and an equation to establish the multiaxial failure stresses that cause failure. This paper presents a method to test the multiaxial failure stress of the AF experimentally and evaluate the potential for the Tsai-Hill model to predict these failure stresses. Porcine AF was cut into a dogbone shape at three distinct angles relative to the primary lamella direction (parallel, transverse, and oblique). Then, each dogbone was pulled to complete rupture. The Cauchy stress in the material's fiber coordinates was calculated. These multiaxial stress parameters were used to optimize the coefficients of the Tsai-Hill yield. The coefficients obtained for the Tsai-Hill model vary by an order of magnitude between the fiber and transverse directions, and these coefficients provide a good description of the AF multiaxial failure stress. These results establish both an experimental approach and the use of the Tsai-Hill model to explain the anisotropic failure behavior of the tissue.

The mechanical properties measurement could be affected by forceps: a technical note on sampling human annulus fibrosus.

PURPOSE: To compare the mechanical properties of human annulus fibrosus obtained by forceps versus bistoury and observe whether the measurement could be affected by forceps sampling method. METHODS: In this study, the mechanical properties of the the extracellular matrix (ECM) of human annulus fibrosus, including elastic modulus and stiffness, were investigated using atomic force microscope (AFM). Tissue was obtained from patients during operation using a bistoury or nucleus pulposus forceps. Tissues obtained with the nucleus pulposus forceps were considered as the forceps group and those obtained with a bistoury were considered as the bistoury group. RESULTS: There was no significant difference observed between the forceps and bistoury group according to histological staining. The elastic modulus of the forceps group was 0.41 ± 0.08 MPa, and that of bistoury group was 0.53 ± 0.13 MPa, and the difference between the two groups was statistically significant (p < 0.05). The stiffness of the forceps group was 0.024 ± 0.003 N/m, and that of the bistoury group was 0.037 ± 0.003 N/m, and the difference between the two groups was statistically significant (p < 0.05). CONCLUSION: The results indicate that the forceps sampling method has a substantial negative effect on the micromechanical properties of the ECM of the annulus fibrosus. Bistoury sampling method is recommended as the experimental subject for exploring the micromechanics mechanisms of cervical degenerative disease.

A novel repair strategy using knotless squeeze anchors for lumbar disc herniation with endplate junction lesions under biportal endoscopic spinal surgery.

BACKGROUND: Annulus fibrosus-endplate (AF-EP) junction lesions are important determinants for lumbar disc herniation (LDH). Utilizing biportal endoscopic spinal surgery (BESS), we introduce a novel repair method using bioabsorbable PushLock anchors with suture fibers to stretch disconnected AF tissues to the vertebral cortex. METHODS: The viewing and working portals are established to excise herniated disc materials causing radiculopathy. Through the working portal, a suture strand is passed through the intact AF tissue near the lesion and retrieved using the Suture Crossing Device. Then, the knotless suture limbs are secured into the cortical bone socket of the vertebral body with a PushLock anchor. CONCLUSION: The procedure is a simple, safe, and feasible knotless suturing technique for the treatment of LDH with AF-EP junction lesions.

Annulus Fibrosus Injury Induces Acute Neuroinflammation and Chronic Glial Response in Dorsal Root Ganglion and Spinal Cord-An In Vivo Rat Discogenic Pain Model.

Chronic painful intervertebral disc (IVD) degeneration (i.e., discogenic pain) is a major source of global disability needing improved knowledge on multiple-tissue interactions and how they progress in order improve treatment strategies. This study used an in vivo rat annulus fibrosus (AF) injury-driven discogenic pain model to investigate the acute and chronic changes in IVD degeneration and spinal inflammation, as well as sensitization, inflammation, and remodeling in dorsal root ganglion (DRG) and spinal cord (SC) dorsal horn. AF injury induced moderate IVD degeneration with acute and broad spinal inflammation that progressed to DRG to SC changes within days and weeks, respectively. Specifically, AF injury elevated macrophages in the spine (CD68) and DRGs (Iba1) that peaked at 3 days post-injury, and increased microglia (Iba1) in SC that peaked at 2 weeks post-injury. AF injury also triggered glial responses with elevated GFAP in DRGs and SC at least 8 weeks post-injury. Spinal CD68 and SC neuropeptide Substance P both remained elevated at 8 weeks, suggesting that slow and incomplete IVD healing provides a chronic source of inflammation with continued SC sensitization. We conclude that AF injury-driven IVD degeneration induces acute spinal, DRG, and SC inflammatory crosstalk with sustained glial responses in both DRGs and SC, leading to chronic SC sensitization and neural plasticity. The known association of these markers with neuropathic pain suggests that therapeutic strategies for discogenic pain need to target both spinal and nervous systems, with early strategies managing acute inflammatory processes, and late strategies targeting chronic IVD inflammation, SC sensitization, and remodeling.

Study on the clinical effect of percutaneous transforaminal endoscopic discectomy combined with annulus fibrosus repair in the treatment of single-segment lumbar disc herniation in young and middle-aged patients.

Objective: To explore the clinical effect of percutaneous transforaminal endoscopic discectomy (PTED) combined with annulus fibrosus repair in the treatment of single-segment lumber disc herniation (LDH) in young and middle-aged patients. Methods: Ninty-six patients with single-segment LDH admitted to Baoding First Central Hospital from March 2021 to November 2022 were selected in the retrospective study. The patients were divided into endoscopic group and combined group according to different surgical methods. The surgical conditions, VAS score and ODI score the two groups of patients were compared, as well as the postoperative review results. Results: There were 50 patients in the endoscopic group the average operation time was 43.68 ± 10.77 minutes, the average intraoperative blood loss was 35.38 ± 10.02 ml, there were seven cases of surgical segment recurrence and 10 cases of postoperative intervertebral instability at the surgical segment. There were 46 patients in the combined group, the average operation time was 52.26 ± 8.39 minutes, the average intraoperative blood loss was 39.23 ± 9.02ml, there was one case of surgical segment recurrence and two cases of surgical segment intervertebral instability. The operation time (t=-4.328, P<0.01), postoperative recurrence cases (χ2=4.386, P<0.05) and intervertebral instability cases (χ2=5.366, P<0.05) of the two groups of patients). The difference was statistically significant. There was no significant difference in intraoperative blood loss between the two groups (t=-1.965, P>0.05). For six months after surgery, the differences in VAS and ODI scores between the two groups were statistically significant. In addition, there were statistically significant differences in the VAS scores and ODI scores of the two groups of patients at each time point after surgery compared with those before surgery (P<0.05). Conclusion: The clinical efficacy of PTED combined with annulus fibrosus repair showed better clinical efficacy than PTED alone, and it can reduce the occurrence of surgical segment recurrence and intervertebral instability, suggesting that PTED combined with annulus fibrosus repair may be worthy of promotion in clinical practice.

Combined flexion and compression negatively impact the mechanical integrity of the annulus fibrosus.

PURPOSE: To observe the effect of static flexion, in combination with compression, on the intralamellar and interlamellar matrix properties of the annulus fibrosus. METHODS: C3/C4 cervical functional spinal units of porcine specimens were selected. Following preloading, all specimens were loaded under 1200 N axial compression in either a neutral or static end range flexion posture (15º) for 2 h. Following loading, six annulus samples were dissected from each disc: four single-layer and two multi-layer samples. The multi-layer samples underwent peel tests to quantify the mechanical properties of the interlamellar matrix while the single-layer samples underwent tensile tests to quantify the mechanical properties of the intralamellar matrix. Statistical comparisons between properties were performed to determine differences between postural condition, extraction location, and extraction depth. RESULTS: Flexion elicited a decrease in lamellar adhesive strength (p = 0.045) and in single-layer failure strain (p = 0.03) when compared to a neutral posture. Flexion also had extraction depth-specific effects namely increased intralamellar matrix stiffness in the inner annulus when compared to neutral (p = 0019). Flexion also resulted in a significant decrease in toe region strain for the inner region of the annulus (p = 0.035). The inner region of the annulus was shown to have a significant increase in stress at 30% strain when compared to the outer region after flexion (p = 0.041). CONCLUSION: The current findings suggest that the mechanical properties of the interlamellar and intralamellar matrices are sensitive to flexion, creating an environment that promotes an increased potential for damage to occur.

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

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-1ß. 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-1ß 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-1ß-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.

Potential molecular targets and drugs for basement membranes-related intervertebral disk degeneration through bioinformatics analysis and molecular docking.

BACKGROUND: Through bioinformatics analysis to identify the hub genes of Intervertebral disc degeneration (IVDD) associated with basement membranes (BMs) and find out the potential molecular targets and drugs for BMs-related annulus fibrosus (AF) degeneration based on bioinformatic analysis and molecular approach. METHODS: Intervertebral disc degeneration (IVDD) related targets were obtained from GeneCards, DisGenet and OMIM databases. BMs related genes were obtained from Basement membraneBASE database. The intersection targets were identified and subjected to protein-to-protein interaction (PPI) construction via STRING. Hub genes were identified and conducted Gene ontology (GO) and pathway enrichment analysis through MCODE and Clue GO in Cytospace respectively. DSigDB database was retrieved to predict therapeutic drugs and molecular docking was performed through PyMOL, AutoDock 1.5.6 to verify the binding energy between the drug and the different expressed hub genes. Finally, GSE70362 from GEO database was obtained to verify the different expression and correlation of each hub gene for AF degeneration. RESULTS: We identified 41 intersection genes between 3 disease targets databases and Basement membraneBASE database. PPI network revealed 25 hub genes and they were mainly enriched in GO terms relating to glycosaminoglycan catabolic process, the TGF-ß signaling pathway. 4 core targets were found to be significant via comparison of microarray samples and they showed strong correlation. The molecular docking results showed that the core targets have strong binding energy with predicting drugs including chitosamine and retinoic acid. CONCLUSIONS: In this study, we identified hub genes, pathways, potential targets, and drugs for treatment in BMs-related AF degeneration and IVDD.

Leukoreduced PRP enhanced proliferation and ECM production yet inhibited senescence, inflammation, and multi-differentiation potential of AFSCs by downregulating HMGB1.

BACKGROUND: This study assessed the role and potential mechanism of platelet-rich plasma (PRP) in the progression of intervertebral disk degeneration (IVDD). METHODS: Annulus fibrosus (AF)-derived stem cells (AFSCs) from New Zealand white rabbits received the transfection with high mobility group box 1 (HMGB1) plasmids and the subsequent treatment with bleomycin, 10% leukoreduced PRP or leukoconcentrated PRP. Dying cells were indicated by immunocytochemistry analysis for senescence-associated ß-galactosidase (SA-ß-gal) staining. The proliferation of these cells was evaluated based on the population doubling time (PDT). The expressions of HMGB1, pro-aging and anti-aging molecules, extracellular matrix (ECM)-related catabolic/anabolic factors, and inflammatory genes at the molecular or transcriptional levels were quantified via Western blot or reverse transcription-quantitative PCR (RT-qPCR). Besides, the adipocytes, osteocytes, and chondrocytes were separately dyed by Oil Red O, Alizarin Red S, and Safranin O staining. RESULTS: Bleomycin enhanced the senescent morphological changes and increased the PDT and the expressions of SA-ß-gal, pro-aging molecules, ECM-related catabolic factors, inflammatory genes, and HMGB1 while suppressing the expressions of anti-aging and anabolic molecules. Leukoreduced PRP reversed the effects of bleomycin and inhibited the differentiation of AFSCs into adipocytes, osteocytes, and chondrocytes. Besides, HMGB1 overexpression offset the roles of leukoreduced PRP in AFSCs. CONCLUSION: Leukoreduced PRP promotes cell proliferation and ECM production of AFSCs, while inhibiting their senescence, inflammation, and multi-differentiation potentials via downregulating HMGB1 expression.

Mechanically induced histochemical and structural damage in the annulus fibrosus and cartilaginous endplate: a multi-colour immunofluorescence analysis.

The annulus fibrosus (AF) and endplate (EP) are collagenous spine tissues that are frequently injured due to gradual mechanical overload. Macroscopic injuries to these tissues are typically a by-product of microdamage accumulation. Many existing histochemistry and biochemistry techniques are used to examine microdamage in the AF and EP; however, there are several limitations when used in isolation. Immunofluorescence may be sensitive to histochemical and structural damage and permits the simultaneous evaluation of multiple proteins-collagen I (COL I) and collagen II (COL II). This investigation characterized the histochemical and structural damage in initially healthy porcine spinal joints that were either unloaded (control) or loaded via biofidelic compression loading. The mean fluorescence area and mean fluorescence intensity of COL II significantly decreased (- 54.9 and - 44.8%, respectively) in the loaded AF (p ≤ 0.002), with no changes in COL I (p ≥ 0.471). In contrast, the EP displayed similar decreases in COL I and COL II fluorescence area (- 35.6 and - 37.7%, respectively) under loading conditions (p ≤ 0.027). A significant reduction (-31.1%) in mean fluorescence intensity was only observed for COL II (p = 0.043). The normalized area of pores was not altered on the endplate surface (p = 0.338), but a significant increase (+ 7.0%) in the void area was observed on the EP-subchondral bone interface (p = 0.002). Colocalization of COL I and COL II was minimal in all tissues (R < 0.34). In conclusion, the immunofluorescence analysis captured histochemical and structural damage in collagenous spine tissues, namely, the AF and EP.

Single-cell RNA-sequencing atlas of bovine caudal intervertebral discs: Discovery of heterogeneous cell populations with distinct roles in homeostasis.

Back and neck pain are significant healthcare burdens that are commonly associated with pathologies of the intervertebral disc (IVD). The poor understanding of the cellular heterogeneity within the IVD makes it difficult to develop regenerative IVD therapies. To address this gap, we developed an atlas of bovine (Bos taurus) caudal IVDs using single-cell RNA-sequencing (scRNA-seq). Unsupervised clustering resolved 15 unique clusters, which we grouped into the following annotated partitions: nucleus pulposus (NP), outer annulus fibrosus (oAF), inner AF (iAF), notochord, muscle, endothelial, and immune cells. Analyzing the pooled gene expression profiles of the NP, oAF, and iAF partitions allowed us to identify novel markers for NP (CP, S100B, H2AC18, SNORC, CRELD2, PDIA4, DNAJC3, CHCHD7, and RCN2), oAF (IGFBP6, CTSK, LGALS1, and CCN3), and iAF (MGP, COMP, SPP1, GSN, SOD2, DCN, FN1, TIMP3, WDR73, and GAL) cells. Network analysis on subpopulations of NP and oAF cells determined that clusters NP1, NP2, NP4, and oAF1 displayed gene expression profiles consistent with cell survival, suggesting these clusters may uniquely support viability under the physiological stresses of the IVD. Clusters NP3, NP5, oAF2, and oAF3 expressed various extracellular matrix (ECM)-associated genes, suggesting their role in maintaining IVD structure. Lastly, transcriptional entropy and pseudotime analyses found that clusters NP3 and NP1 had the most stem-like gene expression signatures of the NP partition, implying these clusters may contain IVD progenitor cells. Overall, results highlight cell type diversity within the IVD, and these novel cell phenotypes may enhance our understanding of IVD development, homeostasis, degeneration, and regeneration.

The role of SLRPs and large aggregating proteoglycans in collagen fibrillogenesis, extracellular matrix assembly, and mechanical function of fibrocartilage.

PURPOSE: Proteoglycans, especially small leucine rich proteoglycans (SLRPs), play major roles in facilitating the development and regulation of collagen fibers and other extracellular matrix components. However, their roles in fibrocartilage have not been widely reviewed. Here, we discuss both SLRP and large aggregating proteoglycan's roles in collagen fibrillogenesis and extracellular matrix assembly in fibrocartilage tissues such as the meniscus, annulus fibrosus (AF), and TMJ disc. We also discuss their expression levels throughout development, aging and degeneration, as well as repair. METHODS: A review of literature discussing proteoglycans and collagen fibrillogenesis in fibrocartilage was conducted and data from these manuscripts were analyzed and grouped to discuss trends throughout the tissue's architectural zones and developmental stage. RESULTS: The spatial collagen architecture of these fibrocartilaginous tissues is reflected in the distribution of proteoglycans expressed, suggesting that each proteoglycan plays an important role in the type of architecture presented and associated mechanical function. CONCLUSION: The unique structure-function relationship of fibrocartilage makes the varied architectures throughout the tissues imperative for their success and understanding the functions of these proteoglycans in developing and maintaining the fiber structure could inform future work in fibrocartilage replacement using tissue engineered constructs.

A Novel Testing Method to Quantify Mechanical Properties of the Intact Annulus Fibrosus Ring From Rat-Tail Intervertebral Discs.

The annulus fibrosus is the ring-like exterior of the intervertebral disc, which is composed of concentrically organized layers of collagen fiber bundles. The mechanical properties of the annulus have been studied extensively; however, tests are typically performed on extracted fragments or multilayered samples of the annulus and not on the annulus as a whole. The purpose of this study was twofold: (1) to develop a novel testing technique to measure the mechanical properties of the intact, isolated annulus; and (2) to perform a preliminary analysis of the rate-dependency of these mechanical properties. Twenty-nine whole annulus ring samples were dissected from 11 skeletally mature Sprague Dawley rat tails and underwent a tensile failure test at either 2%/s (n = 16) or 20%/s (n = 13). Force and displacement were sampled at 100 Hz and were subsequently normalized to stress and strain. Various mechanical properties were derived from the stress-strain curves and statistically compared between the rates. All mechanical variables, with the exception of initial failure stress, were found to be unaffected by rate. Interestingly, initial failure stress was higher for samples tested at the slower rate compared to the higher rate which is atypical for viscoelastic tissues. Although in general rate did not appear to impact the annulus ring response to tensile loading, this novel, intact annular ring testing technique provides an alternative way to quantify mechanical properties of the annulus.

Impact of Variations in Water Concentration on the Nanomechanical Behavior of Type I Collagen Microfibrils in Annulus Fibrosus.

Radial variation in water concentration from outer to inner lamellae is one of the characteristic features of annulus fibrosus (AF). In addition, water concentration changes are also associated with intervertebral disc (IVD) degeneration. Such changes alter the chemo-mechanical interactions among the biomolecular constituents at molecular level, affecting the load-bearing nature of IVD. This study investigates mechanistic impacts of water concentration on the collagen type I microfibrils in AF using molecular dynamics simulations. Results show, in axial tension, that increase in water concentration (WC) from 0% to 50% increases the elastic modulus from 2.7 GPa to 3.9 GPa. This is attributed to combination of shift in deformation from backbone straightening to combined backbone stretching- intermolecular sliding and subsequent strengthening of tropocollagen-water (TC-water-TC) interfaces through water bridges and intermolecular electrostatic attractions. Further increase in WC to 75% reduces the modulus to 1.8 GPa due to shift in deformation to polypeptide straightening and weakening of TC-water-TC interface due to reduced electrostatic attraction and increase in the number of water molecules in a water bridge. During axial compression, increase in WC to 50% results in increase in modulus from 0.8 GPa to 4.5 GPa. This is attributed to the combination of the development of hydrostatic pressure and strengthening of the TC-water-TC interface. Further increase in WC to 75% shifts load-bearing characteristic from collagen to water, resulting in a decrease in elastic modulus to 2.8 GPa. Such water-mediated alteration in load-bearing properties acts as foundations toward AF mechanics and provides insights toward understanding degeneration-mediated altered spinal stiffness.

How annulus defects can act as initiation sites for herniation.

PURPOSE: Both posture and loading rate are key factors in the herniation process and can determine the mechanism of disc failure. The aim of this study was to test the hypothesis that disruption visible with HR-MRI post-testing corresponds with microstructural features and further elucidate the mechanism by which this disruption weakens the disc. This will enable us to gain new insights into the herniation process. METHODS: Thirty ovine lumbar spinal segments were subjected to combinations of four loading conditions (0-12° flexion, 0-9° lateral bending, 0-4° axial rotation, 0-1500 N axial compression) for 1000 loading cycles at 2 Hz in a dynamic disc loading simulator. The discs were scanned in an ultra-high field MRI (11.7 T) then examined using brightfield microscopy to examine their microstructure. RESULTS: Four discs herniated and seven discs suffered nucleus displacement. These discs contained pre-existing defects in the central posterior annulus. Generally, following testing discs contained more posterior annulus disruption, Microstructural investigation revealed there was clear correspondence between HR-MRI and microstructural observations, and that the mid-outer annular-endplate junction had failed in all discs examined in this study. CONCLUSIONS: While all discs suffered outer annulus damage, only the discs containing pre-existing defects herniated. These pre-existing defects weakened the inner and mid annulus, allowing herniation to occur once the mid and outer annular wall was compromised. We propose this can occur during the degenerative cascade.

Spheroid-Based Tissue Engineering Strategies for Regeneration of the Intervertebral Disc.

Degenerative disc disease, a painful pathology of the intervertebral disc (IVD), often causes disability and reduces quality of life. Although regenerative cell-based strategies have shown promise in clinical trials, none have been widely adopted clinically. Recent developments demonstrated that spheroid-based approaches might help overcome challenges associated with cell-based IVD therapies. Spheroids are three-dimensional multicellular aggregates with architecture that enables the cells to differentiate and synthesize endogenous ECM, promotes cell-ECM interactions, enhances adhesion, and protects cells from harsh conditions. Spheroids could be applied in the IVD both in scaffold-free and scaffold-based configurations, possibly providing advantages over cell suspensions. This review highlights areas of future research in spheroid-based regeneration of nucleus pulposus (NP) and annulus fibrosus (AF). We also discuss cell sources and methods for spheroid fabrication and characterization, mechanisms related to spheroid fusion, as well as enhancement of spheroid performance in the context of the IVD microenvironment.

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-1ß) 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-1ß expression in human AF cells, and may further accelerate IVD degeneration.

FoxO1a mediated cadmium-induced annulus fibrosus cells apoptosis contributes to intervertebral disc degeneration in smoking.

Cadmium (Cd), a type of heavy metal that accumulates in the body because of smoking, mediates the toxic effect of smoking in many diseases, such as cardiovascular disease, osteoarthritis, and osteoporosis. However, the toxic effect of Cd on intervertebral disc tissues have not been reported. In the current study, we demonstrated that Cd induced the apoptosis of annulus fibrosus (AF) cells, which contributed to intervertebral disc degeneration (IVDD). Specifically, Cd induced the nuclear translocation of FoxO1a, which drives AF cells apoptosis through mitochondrial-related pathway. Phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signal pathway is also involved in this process. The combined use of LY29002, an inhibitor of PI3K, and small interfering RNA-targeting FoxO1a confirmed the relationship between the PI3K/AKT signal pathway and FoxO1a. In summary, present research explores the mechanism behind the contribution of smoking to IVDD and finds a new feasible target for preventing IVDD in smoking.