Extracellular vesicles (EVs), defined as cell-secreted nanoscale vesicles that carry bioactive molecules, have emerged as a promising therapeutic strategy in tumor and tissue regeneration. Their potential in repairing intervertebral disc degeneration (IDD) through multidimensional regulatory mechanisms is a rapidly advancing field of research. This paper provided an overview of the mechanisms of EVs in IDD repair, thoroughly reviewed recent literature on EVs for IDD, domestically and internationally, and summarized their therapeutic mechanisms. In IDD repair, EVs could act through different mechanisms at the molecular, cellular, and tissue levels. At the molecular level, EVs could treat IDD by inhibiting inflammatory reactions, suppressing oxidative stress, and regulating the synthesis and decomposition of extracellular matrix. At the cellular level, EVs could treat IDD by inhibiting cellular pyroptosis, ferroptosis, and apoptosis and promoting cell proliferation and differentiation. At the tissue level, EVs could treat IDD by inhibiting neovascularization. EVs have a strong potential for clinical application in the treatment of IDD and deserve more profound study.
Extracellular Vesicles/physiology* ; Humans ; Intervertebral Disc Degeneration/therapy* ; Apoptosis ; Cell Proliferation ; Oxidative Stress ; Cell Differentiation ; Extracellular Matrix/metabolism* ; Animals ; Pyroptosis

The lumbar intervertebral disc exhibits a complex physiological structure with interactions between various segments, and its components are extremely complex. The material properties of different components in the lumbar intervertebral disc, especially the water content (undergoing dynamic change as influenced by age, degeneration, mechanical loading, and proteoglycan content) - critically determine its mechanical properties. When the lumbar intervertebral disc is under continuous pressure, water seeps out, and after the pressure is removed, water re-infiltrates. This dynamic fluid exchange process directly affects the mechanical properties of the lumbar intervertebral disc, while previous isotropic modeling methods have been unable to accurately reflect such solid-liquid phase behaviors. To explore the load-bearing mechanism of the lumbar intervertebral disc and establish a more realistic mechanical model of the lumbar intervertebral disc, this study developed a solid-liquid biphasic, fiber-reinforced finite element model. This model was used to simulate the four movements of the human lumbar spine in daily life, namely flexion, extension, axial rotation, and lateral bending. The fluid pressure, effective solid stress, and liquid pressure-bearing ratio of the annulus fibrosus and nucleus pulposus of different lumbar intervertebral discs were compared and analyzed under the movements. Under all the movements, the fluid pressure distribution was closer to the nucleus pulposus, while the effective solid stress distribution was more concentrated in the outer annulus fibrosus. In terms of fluid pressure, the maximum fluid pressure of the lumbar intervertebral disc during lateral bending was 1.95 MPa, significantly higher than the maximum fluid pressure under other movements. Meanwhile, the maximum effective solid stress of the lumbar intervertebral disc during flexion was 2.43 MPa, markedly higher than the maximum effective solid stress under other movements. Overall, the liquid pressure-bearing ratio under axial rotation was smaller than that under other movements. Based on the solid-liquid biphasic modeling method, this study more accurately revealed the dominant role of the liquid phase in the daily load-bearing process of the lumbar intervertebral disc and the solid-phase mechanical mechanism of the annulus fibrosus load-bearing, and more effectively predicted the solid-liquid phase co-load-bearing mechanism of the lumbar intervertebral disc in daily life.
Humans ; Finite Element Analysis ; Intervertebral Disc/physiology* ; Lumbar Vertebrae/physiology* ; Weight-Bearing/physiology* ; Biomechanical Phenomena ; Stress, Mechanical ; Computer Simulation ; Models, Biological

OBJECTIVE: To explore mechanisms of acid-sensing ion channel 1 (ASIC1) mediated lumbar nucleus pulposus cell apoptosis through extracellular-signalregulated protein kinase 5 (ERK5) signaling pathway and mitochondrial dysfunction pathway. METHODS: Totally 34 patients with degenerative lumbar disc herniation (LDH) admitted from January 2020 to December 2022 were collected as research objects, including 21 males and 13 females;aged from 29 to 52 years old with an average of (37.43±4.75) years old;22 patients with grade Ⅱ and 12 patients with grade Ⅳ, according to Pfirrmann grading criteria;15 patients with L_4,5 and 19 patients with L₅S₁. The expression of ASIC1 in nucleus pulposus of LDH patients was measured by immunohistochemical staining. Nucleus pulposus cells were cultured by primary culture method, identified by toluidine blue staining and immunohistochemical staining, and the expression of ASIC1 protein was located by immunofluorescence staining. According to the addition of siRNA-ASIC1, ASIC1 overexpression plasmid, and ERK5 inhibitors, the nucleus pulpocyte was divided into three groups, named as SIRNA-silenced group, overexpression group, and inhibitor group, with 3 patients in each group. Cells of each group were collected at 72 h after intervention, expression of ASIC1, ERK5, BCL-xL/BCL-2-associated Death promoter (Bad), B-cell lymphoma-2 associated X (Bax) and B-cell lymphoblast-2 gene (Bcl-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR);intracellular calcium ion levels were detected by calcium ion kit, mitochondrial membrane potential was detected by JC-1 kit, and apoptosis was observed by AV-PI kit. RESULTS: In LDH patients with grade Ⅳ, nucleus pulposus tissue removed during operation revealed poor elasticity, white color and poor ductility, and immunohistochemical results showed increased ASIC1 expression. There was no significant difference in mRNA relative expression of ASIC1 between siRNA silencing group (0.31±0.03) and inhibitor group (0.39±0.05) (P>0.05). The mRNA relative expression level of ERK5 in siRNA silencing group(0.32±0.05) was significantly higher than that in inhibitor group (0.15±0.04)(P<0.05), which suggested ERK5 was the downstream molecule of ASIC1. The mRNA relative expression levels of apoptosis promoting factor Bad and Bax in siRNA silencing group and inhibitor group were lower than those in overexpression group(P<0.05), the relative expression level of anti-apoptosis factor Bcl-2 mRNA was significantly increased (P<0.05). The calcium content in overexpression group was higher than that in siRNA silencing and inhibitor groups (P<0.05), the normal proportion of mitochondrial membrane potential in overexpression group was lower than that in siRNA silencing and inhibitor group (P<0.05), and the apoptosis rate in overexpression group was higher than that in siRNA silencing and inhibitor group (P<0.05). CONCLUSION After the activation of ASIC1 channel protein, calcium ions could enter the cells and act as a second messenger molecule to regulate apoptosis of nucleus pulposus cells by ERK5 signaling pathway and mitochondrial disorder pathway.
Humans ; Acid Sensing Ion Channels/physiology* ; Male ; Female ; Apoptosis ; Middle Aged ; Adult ; Signal Transduction ; Mitogen-Activated Protein Kinase 7/physiology* ; Mitochondrial Diseases/genetics* ; Nucleus Pulposus/metabolism* ; Intervertebral Disc Degeneration/metabolism* ; Mitochondria/metabolism* ; Intervertebral Disc Displacement/genetics*

OBJECTIVE: To investigate the effect of midazolam on pain in lumbar disc herniation model rats based on p38 MAPK signaling pathway. METHODS: Fifty SPF-grade Sprague-Dawley healthy rats, half male and half female, were selected and randomly divided into normal group, model group, and low-dose, medium-dose, high-dose groups. Model group and low-dose, medium-dose, high-dose groups were initially modeled for lumbar disc herniation. Intraperitoneal injection of saline was performed in rats of normal and model groups; and in the low-dose, medium-dose, and high-dose groups, intraperitoneal injection of midazolam was performed with doses of 30, 60, and 90 mg/kg, respectively. Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), 5-hydroxytryptamine (5-HT), β-endorphin (β-EP), substance P (SP), neuropeptide Y (NPY) were detected in the serum of rats by enzyme-linked immunoassay. The expression of p38 MAPK and matrix metalloproteinase-3(MMP-3) protein were detected by Western blot in the tissues of rats of each group. RESULTS: The levels of TNF-α, IL-1β and β-EP were higher and the level of 5-HT was lower in the model group than in the normal group(P<0.05);the levels of TNF-α, IL-1β and β-EP were lower and the level of 5-HT was higher in the low-dose, medium-dose and high-dose groups than in the model group(P<0.05). The levels of SP and NPY increased in the model group compared with the normal group (P<0.05) and the levels of SP and NPY decreased in the low-dose, medium-dose and high-dose groups compared with the model group (P<0.05). The expression of p38 MAPK and MMP-3 increased in the model group compared with the normal group (P<0.05); the expression of p38 MAPK and MMP-3 decreased in the low-dose, medium-dose and high-dose compared with the model group(P<0.05). CONCLUSION Midazolam may ameliorate the immune inflammatory response in rats with a model of lumbar disc herniation, possibly regulated through the p38MAPK signaling pathway.
Rats ; Male ; Female ; Animals ; Intervertebral Disc Displacement/pathology* ; Rats, Sprague-Dawley ; Matrix Metalloproteinase 3/metabolism* ; Midazolam ; Tumor Necrosis Factor-alpha/metabolism* ; Serotonin/metabolism* ; MAP Kinase Signaling System/physiology* ; Pain ; p38 Mitogen-Activated Protein Kinases/metabolism*

OBJECTIVE: To analyze dynamic electromyography characteristics and related factors of lumbar back muscle activity in patients with lumbar disc herniation, and to clarify the clinical significance of dynamic electromyography in the diagnosis and treatment of patients with lumbar disc herniation(LDH). METHODS: From September 2014 to March 2021, 40 patients with lumbar disc herniation(LDH group) were detected by surface electromyography telemeter. There were 14 males and 26 females, aged from 20 to 61 years old, with an average of(40.68±10.56) years old, the course of illness was from 1 to 120 months, with an average of (17.75±27.56) months. In addition, 12 normal people were recruited as the control group. There were 2 males and 10 females. The age ranged from 24 to 53 years old, with an average of(36.50±10.30) years old. All subjects were subjected to dynamic electromyographic tests of the subthoracic erector spinae, lumbar erector spinae, and multifidus muscles during static standing and trunk flexion and extension. Compare the EMG activity data (average EMG amplitude, median frequency, original EMG graph) of the tested muscles between patients with lumbar disc herniation and normal people, and analyze the correlation between the general data of patients with lumbar disc herniation and the tested muscle EMG data. RESULTS: When standing still, the average electromyographic amplitude of the erector spinal muscle of the right and left thoracic segments of the subjects in the LDH group increased compared with the control group, and the difference was significant(P<0.05). In the trunk flexion and extension, the average electromyographic amplitude of the right and left proximal thoracic erector spinae, the right left lumbar erector spinae, and the right left multifidus muscle of the subjects in the LDH group are all larger than the control group, and the difference was significant(P<0.05). In the trunk flexion and extension, the median frequencies of the right left proximal thoracic erector spinae、the right left lumbar erector spinae, and the right left multifidus muscle of the subjects in the LDH group were all larger than the normal control group, and the difference was significant (P<0.05). During trunk flexion and extension, the original electromyographic patterns of subjects in the LDH group were significantly different from those in the control group. During the maintenance of the maximum trunk flexion of the subjects in the LDH group, there was a high level of electromyographic activity of the lower back muscles, and the electromyographic static signals that should appear regularly in the original signal could not be distinguished. When the trunk was flexed and extended, had gender, age, weight and height of subjects in the LDH group were not significantly correlated with the average EMG amplitude and median frequency of bilateral proximal thoracic, lumbar erector spinae and bilateral multifidus muscles respectively(P>0.05). CONCLUSION Patients with lumbar disc herniation have characteristic surface EMG changes in the back muscles that are different from those of normal people. These features can more objectively reflect the patient's muscle condition and can be an effective indicator for the diagnosis and treatment effect evaluation of patients with lumbar disc herniation. It can be seen that surface electromyography is not only a detection method, it can be considered in the routine diagnosis and treatment plan of LDH to guide clinical work.
Male ; Female ; Humans ; Young Adult ; Adult ; Middle Aged ; Electromyography ; Intervertebral Disc Displacement/therapy* ; Lumbar Vertebrae ; Paraspinal Muscles ; Range of Motion, Articular/physiology* ; Muscle, Skeletal

In order to study the mechanical behavior of degeneration and nucleotomy of lumbar intervertebral disc, compression experiments with porcine lumbar intervertebral discs were carried out. The lumbar intervertebral discs with trypsin-treated and nucleus nucleotomy served as the experimental group and the normal discs as the control group. Considering the effects of load magnitude and loading rate, the relationship between stress and strain, instantaneous elastic modulus and creep property of intervertebral disc were obtained. The creep constitutive model was established. The results show that the strain and creep strain of the experimental group increase significantly with the increase of compression load and loading rate, whereas the instantaneous elastic modulus decreases obviously, compared with the control group. It indicates that the effect of load magnitude and loading rate on load-bearing capacity of intervertebral disc after nucleotomy is larger obviously than that of normal disc. The creep behavior of the experimental group can be still predicted by the Kelvin three-parameter solid model. The results will provide theoretical foundation for clinical treatment and postoperative rehabilitation of intervertebral disc disease.
Animals ; Biomechanical Phenomena ; Intervertebral Disc ; physiology ; surgery ; Lumbar Vertebrae ; Stress, Mechanical ; Swine ; Weight-Bearing

BACKGROUNDThe development of mechanically active culture systems helps increase the understanding of the role of mechanical stress in intervertebral disc (IVD) degeneration. Motion segment cultures allow for preservation of the native IVD structure, and adjacent vertebral bodies facilitate the application and control of mechanical loads. The purpose of this study was to establish loading and organ culture methods for rabbit IVD motion segments to study the effect of static load on the whole disc organ.

METHODSIVD motion segments were harvested from rabbit lumbar spines and cultured in no-loading 6-well plates (control conditions) or custom-made apparatuses under a constant, compressive load (3 kg, 0.5 MPa) for up to 14 days. Tissue integrity, matrix synthesis, and the matrix gene expression profile were assessed after 3, 7, and 14 days of culturing and compared with those of fresh tissues.

RESULTSThe results showed that ex vivo culturing of motion segments preserved tissue integrity under no-loading conditions for 14 days whereas the static load gradually destroyed the morphology after 3 days. Proteoglycan contents were decreased under both conditions, with a more obvious decrease under static load, and proteoglycan gene expression was also downregulated. However, under static load, immunohistochemical staining intensity and collagen Type II alpha 1 (COL2A1) gene expression were significantly enhanced (61.54 ± 5.91, P = 0.035) and upregulated (1.195 ± 0.040, P = 0.000), respectively, compared with those in the controls (P < 0.05). In contrast, under constant compression, these trends were reversed. Our initial results indicated that short-term static load stimulated the synthesis of collagen Type II alpha 1; however, sustained constant compression led to progressive degeneration and specifically to a decreased proteoglycan content.

CONCLUSIONSA loading and organ culture system for ex vivo rabbit IVD motion segments was developed. Using this system, we were able to study the effects of mechanical stimulation on the biology of IVDs, as well as the pathomechanics of IVD degeneration.

Animals ; Gene Expression Regulation ; Immunohistochemistry ; Intervertebral Disc ; metabolism ; physiology ; Intervertebral Disc Degeneration ; metabolism ; physiopathology ; Male ; Nucleus Pulposus ; metabolism ; physiology ; Organ Culture Techniques ; methods ; Rabbits ; Stress, Mechanical

BACKGROUNDThymosin beta-4 (TB-4) is considered key roles in tissue development, maintenance and pathological processes. The study aimed to prove TB-4 positive biological function on nucleus pulposus (NP) cell apoptosis and slowing the process of cell aging while increasing the cell proliferation.

METHODSTB-4 recombinant adeno-associated virus (AAV) was constructed and induced to human NP cells. Cell of same group were cultured without gene modification as controlled group. Proliferation capacity and cell apoptosis were observed during 6 passages of the cells. Morphology and expression of the TB-4 gene were documented as parameter of cell activity during cell passage.

RESULTSNP cells with TB-4 transfection has normal TB-4 expression and exocytosis. NP cells with TB-4 transfection performed significantly higher cell activity than that at the control group in each generation. TB-4 recombinant AAV-transfected human NP cells also show slower cell aging, lower cell apoptosis and higher cell proliferation than control group.

CONCLUSIONSTB-4 can prevent NP cell apoptosis, slow NP cell aging and promote NP cell proliferation. AAV transfection technique was able to highly and stably express TB-4 in human NP cells, which may provide a new pathway for innovation in the treatment of intervertebral disc degenerative diseases.

Apoptosis ; genetics ; physiology ; Cell Line ; Cell Proliferation ; genetics ; physiology ; Cells, Cultured ; Cellular Senescence ; genetics ; physiology ; Dependovirus ; genetics ; Humans ; Immunohistochemistry ; Intervertebral Disc ; metabolism ; pathology ; Male ; Thymosin ; genetics ; metabolism

OBJECTIVETo investigate the effect of exercise load on apparent diffusion coefficient (ADC) and fractional anisotropy (FA) of normal lumbar intervertebral discs in magnetic resonance (MR) diffusion tensor imaging (DTI).

METHODSThirty healthy volunteers (24 males and 6 females, aged 19 to 25 years) underwent examinations with MR T2WI and DTI of the lumbar intervertebral discs before and after exercise load. Pfirrmann grading was evaluated with T2WI, and the B0 map, ADC map and FA map were reconstructed based on the DTI data to investigate the changes in ADC and FA after exercise.

RESULTSOf the 30 volunteers (150 intervertebral discs) receiving the examination, 27 with discs of Pfirrminn grade II were included for analysis. In these 27 volunteers, the average ADC and FA before exercise were (1.99 ± 0.18)×10⁻³ mm²/s and 0.155∓0.059, respectively. After exercise, ADC was lowered significantly to (1.93 ± 0.17)×10⁻³ mm²/s (P<0.05) and FA increased slightly to 0.1623 ± 0.017 (P>0.05).

CONCLUSIONDTI allows quantitatively analysis of the changes in water molecular diffusion and anisotropy of the lumbar intervertebral discs after exercise load, which can cause a decreased ADC and a increased FA value, and the change of ADC is more sensitive to exercise load.

Adult ; Anisotropy ; Diffusion Magnetic Resonance Imaging ; Diffusion Tensor Imaging ; Exercise ; Female ; Humans ; Intervertebral Disc ; physiology ; Lumbosacral Region ; physiology ; Male ; Reference Values ; Young Adult

BACKGROUNDRecent studies have indicated that human nucleus pulposus contain mesenchymal stem cells (NP-MSCs). However, the immunophenotypic variation of NP-MSCs in vitro was unclear. The present study was conducted to address the immunophenotypic variation of mesenchymal stem cells in nucleus pulposus under continuous proliferation in vitro and show the difference between mesenchymal stem cells and nucleus pulposus cell.

METHODSTissue samples were obtained from thoracolumbar burst fracture patients and degenerative disc disease patients who underwent discectomy and fusion procedures. Flow cytometric and laser scanning confocal microscope (LSCM) were used to detect the variation of mesenchymal stem cells in nucleus pulposus which were expressing CD105 and CD24 in condition with or without transforming growth factor β1 (TGF-β1).

RESULTSMore than 90% of the analyzed primary cells of mesenchymal stem cells in nucleus pulposus fulfilled the general immunophenotyping criteria for MSCs, such as CD44, CD105 and CD29, but the marker of mature NP cells characterized as CD24 was negative. In continuous cultures, the proportion of mesenchymal stem cells which were expressing CD44, CD105 and CD29 in nucleus pulposus gradually decreased. The mesenchymal stem cells in nucleus pulposus cells were positive for CD105 and CD29, with slight positivity for CD44. The CD24 expression gradually increased in proliferation. Biparametric flow cytometry and laser scanning confocal microscopy confirmed the presence of cells which were expressing CD105 and CD24 independently, and only a small part of cells expressed both CD105 and CD24 simultaneously. TGF-β1 could stimulate mesenchymal stem cells in nucleus pulposus to express CD24.

CONCLUSIONSNon-degenerative and degenerative NP contains mesechymal stem cells. The variation of CD24 can be used as a marker to identify the NP-MSCs differentiation into NP-like cells.

Adult ; Antigens, CD ; metabolism ; CD24 Antigen ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Endoglin ; Female ; Flow Cytometry ; Humans ; Hyaluronan Receptors ; metabolism ; Integrin beta1 ; metabolism ; Intervertebral Disc ; cytology ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Receptors, Cell Surface ; metabolism ; Young Adult