Lumbar disc (LD) herniation and aging are prevalent conditions that can result in substantial morbidity. This study aimed to clarify the mechanisms connecting the LD aging and herniation, particularly focusing on cellular senescence and molecular alterations in the nucleus pulposus (NP). We performed a detailed analysis of NP samples from a diverse cohort, including individuals of varying ages and those with diagnosed LD herniation. Our methodology combined histological assessments with single-nucleus RNA sequencing to identify phenotypic and molecular changes related to NP aging and herniation. We discovered that cellular senescence and a decrease in nucleus pulposus progenitor cells (NPPCs) are central to both processes. Additionally, we found an age-related increase in NFAT1 expression that promotes NPPC senescence and contributes to both aging and herniation of LD. This research offers fresh insights into LD aging and its associated pathologies, potentially guiding the development of new therapeutic strategies to target the root causes of LD herniation and aging.
Intervertebral Disc Displacement/metabolism* ; Humans ; Aging/pathology* ; Nucleus Pulposus/pathology* ; Male ; Female ; Transcriptome ; Middle Aged ; Lumbar Vertebrae/pathology* ; Adult ; Cellular Senescence ; Stem Cells/pathology* ; Aged ; Intervertebral Disc Degeneration/metabolism*

PURPOSE: To investigate the molecular responses of various genes and proteins related to disc degeneration upon treatment with cytokines that affect disc-cell proliferation and phenotype in living human intervertebral discs (IVDs). Responsiveness to these cytokines according to the degree of disc degeneration was also evaluated. MATERIALS AND METHODS: The disc specimens were classified into two groups: group 1 (6 patients) showed mild degeneration of IVDs and group 2 (6 patients) exhibited severe degeneration of IVDs. Gene expression was analyzed after treatment with four cytokines: recombinant human bone morphogenic protein (rhBMP-2), transforming growth factor-beta (TGF-beta), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). Molecular responses were assessed after exposure of cells from the IVD specimens to these cytokines via real-time polymerase chain reaction and immunofluorescence staining. RESULTS: mRNA gene expression was significantly greater for aggrecan, type I collagen, type II collagen, alkaline phosphatase, osteocalcin, and Sox9 in group 1 than mRNA gene expression in group 2, when the samples were not treated with cytokines. Analysis of mRNA levels for these molecules after morphogen treatment revealed significant increases in both groups, which were much higher in group 1 than in group 2. The average number of IVD cells that were immunofluorescence stained positive for alkaline phosphatase increased after treatment with rhBMP-2 and TGF-beta in group 1. CONCLUSION: The biologic responsiveness to treatment of rhBMP-2, TGF-beta, TNF-alpha, and IL-1beta in the degenerative living human IVD can be different according to the degree of degeneration of the IVD.
Adult ; Aggrecans/genetics/metabolism ; Alkaline Phosphatase/genetics/metabolism ; Biological Products/pharmacology/*therapeutic use ; Bone Morphogenetic Protein 2/pharmacology/therapeutic use ; Collagen Type I/genetics/metabolism ; Collagen Type II/genetics/metabolism ; Cytokines/*pharmacology/*therapeutic use ; Female ; Fluorescent Antibody Technique ; Gene Expression Regulation/drug effects ; Humans ; Interleukin-1/pharmacology/therapeutic use ; Intervertebral Disc/*drug effects/*pathology ; Intervertebral Disc Degeneration/*drug therapy/genetics/*pathology ; Male ; Middle Aged ; Osteocalcin/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Recombinant Proteins/pharmacology/therapeutic use ; SOX9 Transcription Factor/genetics/metabolism ; Transforming Growth Factor beta/pharmacology/therapeutic use ; Tumor Necrosis Factor-alpha/pharmacology

Intervertebral disc (IVD) degeneration is implicated as a major cause of low back pain. The alternated phenotypes, reduced cell survival, decreased metabolic activity, loss of matrix production and dystrophic mineralization of nucleus pulposus (NP) cells may be key contributors to progressive IVD degeneration. IVD is the largest avascular structure in the body, characterized by low oxygen tension in vivo. Hypoxia-inducible factor (HIF) is a master transcription factor that is induced upon hypoxia and directs coordinated cellular responses to hypoxic environments. This review summarizes relevant studies concerning the involvement of HIF in the regulation of biological behaviors of NP cells. We describe current data on the expression of HIF in NP cells and further discuss the various roles that HIF plays in the regulation of the phenotype, survival, metabolism, matrix production and dystrophic mineralization of NP cells. Here, we conclude that HIF may be a promising target for the prevention and treatment of IVD degeneration.
Animals ; Basic Helix-Loop-Helix Transcription Factors/genetics/*metabolism ; Cell Survival ; Extracellular Matrix/metabolism ; Humans ; Hypoxia-Inducible Factor 1/genetics/*metabolism ; Intervertebral Disc/*cytology/metabolism ; Intervertebral Disc Degeneration/*metabolism/*pathology

This study was done to evaluate whether injections of resveratrol, a natural compound found in the skin of grapes, had anabolic effects on degenerated intervertebral discs in a rabbit model. Two non-continuous lumbar discs were punctured in rabbits to induce disc degeneration. Four weeks and 6 weeks after puncture, the rabbits were treated by injections with dimethylsulfoxide (DMSO) or resveratrol. At 4, 8, and 16 weeks after initial injection, rabbits were sacrificed and the spine was extracted for magnetic resonance image (MRI), mRNA expression, and histological staining. Resveratrol treatment resulted in stronger signal intensity in T2-weighted images. MRI grade showed significantly lower in the resveratrol group than the DMSO group (P = 0.039). In the resveratrol group, aggrecan gene expression was significantly increased than that in the DMSO group at 16 weeks after injection (P = 0.027). MMP-13 mRNA levels in the resveratrol group were significantly decreased than those in the DMSO group at 8 and 16 weeks (P = 0.006 and P = 0.048, respectively). In hematoxylin and eosin stain, resveratrol-treated discs showed the features of regeneration. Histologic grade revealed improvement in resveratrol-treated discs, compared with DMSO-treated discs (P = 0.024). These anabolic effects on degenerated discs indicate that resveratrol is a promising candidate for treatment of degenerative disc disease.
Aggrecans/genetics/metabolism ; Anabolic Agents/*administration & dosage ; Animals ; Disease Models, Animal ; Drug Administration Schedule ; Intervertebral Disc Degeneration/*drug therapy/pathology ; Magnetic Resonance Imaging ; Matrix Metalloproteinase 13/genetics/metabolism ; RNA, Messenger/metabolism ; Rabbits ; Spine/radiography ; Stilbenes/*administration & dosage

OBJECTIVETo explore the role of transforming growth factor-β1 (TGF-β1) in intervertebral disc degeneration and its association with the pathological grading of disc degeneration.

METHODSNormal and degenerative intervertebral disc tissues were collected were classified into 5 grades of increasing degenerative changes. HE staining, immunohistochemistry, TUNEL staining and RT-PCR were used to detect the expression of TGF-β1 in the disc tissues.

RESULTSImmunohistochemistry and RT-PCR showed positive expressions of TGF-β1 and Bcl-2 in normal disc tissues, where Bax was expressed at have a trace level. In the degenerative disc tissues, TGF-β1 expression increased with the pathological grades; the expression levels of TGF-β1 showed significant differences between degenerative and normal tissues and between grade IV and grade I disc tissues (P<0.01).

CONCLUSIONTGF-β1 is an important factor participating in the disc degeneration and its expression level is closely related to the pathological grade of degenerative discs.

Adult ; Aged ; Female ; Humans ; Intervertebral Disc ; pathology ; Intervertebral Disc Degeneration ; classification ; metabolism ; pathology ; Male ; Middle Aged ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the role of platelet-derived growth factor-BB (PDGF-BB) in the degeneration of the hypertrophied ligamentum flavum (LF) in the lumbar spine.

METHODSSurgical specimens of degenerative hypertrophied LF were obtained from 11 patients with lumbar spinal stenosis (LSS, mean age 57.8 years), with those from 10 age-matched patients with lumbar disc herniation (LDH, mean age 53.5 years) as the normal controls. The thickness of the LF was measured preoperatively by axial T1-weighted magnetic resonance imaging (MRI) at the facet joint level. Immunohistochemistry was employed to determine the expression of PDGF β and PDGF-BB in the LF. The mRNA and protein expressions of PDGF-BB in the LF were detected using real-time PCR and Western blotting, respectively.

RESULTSThe thickness of the LF was 5.30±1.12 mm in the degenerative group and 2.80±1.53 mm in the control group, showing a significant difference between the two groups (P<0.001). PDGF-β and PDGF-BB were positive in the fibroblasts in hypertrophied LF. The mRNA and protein expressions of PDGF-BB were significantly higher in the degenerative group than in the control group (P=0.013 and 0.023, respectively).

CONCLUSIONThe high expression of PDGF-BB in the hypertrophied LF suggests its important role in the development of hypertrophy of LF in lumbar spinal canal stenosis.

Adult ; Aged ; Female ; Humans ; Hypertrophy ; metabolism ; Intervertebral Disc Degeneration ; metabolism ; pathology ; Ligamentum Flavum ; metabolism ; pathology ; Lumbar Vertebrae ; Male ; Middle Aged ; Proto-Oncogene Proteins c-sis ; metabolism ; Spinal Stenosis ; metabolism ; pathology

OBJECTIVETo observe the morphology and phenotypes of cells extracted from the endplate in the intervertebral discs and identify the factors affecting their biological characteristic.

METHODSThe intervertebral disc endplate were digested enzymatically, and the morphology of the obtained cells was examined under light microscope. Immunhistochemical analysis of collagen II and real-time PCR was carried out, and the morphologies, viability, cell growth, apoptosis and chondrocyte matrix production were compared between the cells isolated from the degenerative and normal vertebral endplates.

RESULTSThe cells in primary culture presented with spherical and oval morphology, and the cytoplasm was stained blue with toluidine blue. The morphologies of the cartilage endplate cells and the articular cells were almost identical. All the freshly isolated cells expressed collagen II. The degenerative vertebral endplate cells showed decreased expression of collagen II with increased apoptotic cells as compared with normal vertebral endplate cells.

CONCLUSIONThe intervertebral disc endplate cells, like articular cartilage cells, express cartilage-specific matrix proteins. Degenerative vertebral endplate cells show decreased cell vitality with increases cell apoptosis.

Adult ; Apoptosis ; physiology ; Cartilage ; metabolism ; pathology ; Cells, Cultured ; Chondrocytes ; metabolism ; pathology ; Collagen ; metabolism ; Female ; Growth Plate ; metabolism ; pathology ; Humans ; Intervertebral Disc ; metabolism ; pathology ; Intervertebral Disc Degeneration ; metabolism ; pathology ; Lumbar Vertebrae ; metabolism ; pathology ; Male ; Young Adult

OBJECTIVETo investigate the role of transforming growth factor (TGF)-beta1 in degeneration of the ligamentum flavum in the lumbar spine.

METHODSThe degenerative ligamentum flavum was obtained during surgery from 8 patients with lumbar spinal stenosis (mean age 58.6 years), and 8 young patients (mean age 24.2 years) with acute lumbar disc herniation were included as normal controls. The thickness of the ligamentum flavum was measured on preoperative magnetic resonance images, and the mRNA expressions of type-I collagen and TGF-beta1 in the ligamentum flavum were detected using reverse transcriptase-polymerase chain reaction. The protein expression and localization of TGF-beta1 were investigated by Western blotting and immunohistochemical staining, respectively.

RESULTSThe thickness of the ligamentum flavum were 4.70-/+0.40 mm in the degenerative group and 2.50-/+0.36 mm in the control group, showing significant difference between the two groups (P<0.001). The type-I collagen mRNA expression in the degenerative group was significantly higher than that in the control group (P=0.007). The mRNA and protein expressions of TGF-beta1 were significantly higher in the degenerative group than in the control group (P=0.008 and 0.004, respectively). Immunohistochemistry showed that TGF protein was localized in the fibroblasts within the ligamentum flavum.

CONCLUSIONDegenerative ligamentum flavum shows hypertrophy and fibrosis, and TGF-beta1 overexpression may be associated with in the development and progression of ligamentum flavum degeneration in the lumbar spine.

Adult ; Aged ; Female ; Humans ; Hypertrophy ; pathology ; Intervertebral Disc Degeneration ; metabolism ; pathology ; Ligamentum Flavum ; metabolism ; pathology ; Lumbar Vertebrae ; Magnetic Resonance Imaging ; Male ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Spinal Stenosis ; metabolism ; pathology ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Young Adult

OBJECTIVETo observe degenerative intervertebral disc and to examine innervation of degenerative discs in the rabbit anular-injury model.

METHODSTwo different magnitudes of anular injury at 5 mm depth were performed by 11 blade or 16 gauge needle at the L3-L4 or L5-L6 discs in New Zealand white rabbits (n=48, 2.5-3.0 kg). Disc degeneration was evaluated by radiographic, MRI and histological examination at different time points after surgery. To identify nerve ingrowth into disc, two general markers PGP 9.5 and GAP 43, for nerve fibers were examined by immunohistochemistry.

RESULTSignificant decreases in disc height and signal intensity in magnetic resonance imaging were observed in 11 blade group and 16 G puncture group (P<0.01). 16 G puncture group induced slower and more progressive disc degeneration companed with the stab group and control group. At the 12-week time point, nucleus pulposus tissues were extruded and scar tissues formed outside the disc. In stab discs, nerve ingrowth was scattered on the surface of injury site and in the deeper part of the scar tissues, more than 1 mm from the surface. However, in punctured discs, PGP 9.5 and GAP 43-immunoreative fibers were only observed in the outmost part of the scar tissues and superficial area. More nerve fibers were observed in stab group.

CONCLUSIONInnervation may act as a source of discogenic pain which is associated with intervertebral disc degeneration caused by disc anular injury.

Animals ; GAP-43 Protein ; metabolism ; Intervertebral Disc ; injuries ; innervation ; pathology ; Intervertebral Disc Degeneration ; diagnosis ; diagnostic imaging ; etiology ; Low Back Pain ; etiology ; Lumbar Vertebrae ; Male ; Nerve Fibers ; pathology ; Rabbits ; Radiography ; Random Allocation ; Ubiquitin Thiolesterase ; metabolism