Human Disc Degeneration Research Articles

Autophagy expression and its significance in chondrocytes from a degenerate model of human cervical endplate

To explore the autophagy expression and examine its significance in chondrocytes in a degenerate model of human cervical vertebrae endplate. Cartilage endplates were obtained from 48 hospitalized patients with cervical vertebral fracture or dislocation at our hospital between February 2012 to August 2012. They were divided into cervical spondylosis group with cervical spondylotic myelopathy (n = 31) and control group (n = 17).Endplate chondrocytes were isolated by enzyme digestion and cultured in vitro. The cells were stained with toluidine blue and hematoxylin and eosin; laser scanning confocal microscope and monodansylcadaverine (MDC) were used to observe autophagy in endplate chondrocytes; reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of typeIIcollagen and aggrecan and Western blot for the protein of LC3. A degenerative cell model of human cervical endplate chondrocytes was established successfully in vitro. Compared with the common group, the cellular morphologies of degenerative group showed spindle changes. Autophagic body was stained with MDC.Intracellular and perinuclear LC3 protein was detected by laser confocal microscopy. Compared with the control group, the mRNA expressions of aggrecan (0.715 ± 0.194) and typeII collagen (0.628 ± 0.254) markedly decreased (0.845 ± 0.186,0.913 ± 0.254, P < 0.05) and LC3-II/LC3-I declined in cervical spondylosis group. Autophagy plays an important pathogenic role in the process of human cervical disc degeneration. And regulating its expression may improve disc degeneration in endplate cartilage cells.

Characterization of miRNA expression in human degenerative lumbar discs

Characterization of miRNA expression in human degenerative lumbar discs

Ionizing radiation exposure and the development of intervertebral disc degeneration—no case to answer

In the following perspective article, Mellor and Breen provide a counterpoint to a previous perspective on the potential link between ionizing radiation exposure and intervertebral disc degeneration in humans [1]. The previous perspective asked, is this link a myth or reality? It suggested the potential for such a link. Mellor and Breen offer a drastically alternate view, in essence, that the question itself is flawed. To support their perspective, they explain the different units of radiation measurement and their conversion to risk in humans and how this impacts the previous perspective. They explain the variable sensitivity of different body tissues to radiation and highlight that neither human research nor any of the multiple international regulatory agencies have ever suggested that the intervertebral disc is sensitive to radiation. Finally, they claim that it is impossible to predict with any certainty the effects of low-level radiation on the intervertebral discs.

Increased periostin gene expression in degenerative intervertebral disc cells

Background contextDisc degeneration is a multifactorial disease that may cause clinical symptoms such as chronic back pain or radiculopathy in the extremities. Periostin, an extracellular matrix protein involved in the process of fibrosis, expressed in tissues subjected to mechanical stress such as intervertebral disc. However, the expression of periostin during disc degeneration has not yet been studied. PurposeThe aim of this study is to elucidate the difference in gene expression profiles between degenerative and nondegenerative intervertebral discs for a better understanding of disc degeneration. Study designDegenerative and nondegenerative nucleus pulposus cells were isolated from elderly patients with degenerative disc disease and younger patients with adolescent idiopathic scoliosis, respectively. MethodsAffymetrix GeneChip Human arrays were used to derive gene expression profiles for disc degeneration, and gene expressions of periostin and other degeneration-related markers were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blot analysis. Immunohistochemical analysis of periostin and Gomori trichrome stain was performed to show the relationship of periostin, fibrosis, and disc degeneration. The mechanical stress experiment was designed to demonstrate the relationship of periostin, stress, and disc degeneration. ResultsFourteen genes were identified to express at significantly different levels between degenerative and nondegenerative groups. An increase of periostin gene expression was observed in human degenerative nucleus pulposus cells for the messenger RNA and protein levels. Histological examination demonstrated an increased positive staining of periostin in degenerative discs from human tissues and rat needle-punctured tails and more fibrosis with architectural disorder and fragmentation in human degenerative disc as compared with nondegenerative discs. The expression of periostin was significantly induced by stress in human degenerative nucleus pulposus cells but not in nondegenerative cells. ConclusionsThis study demonstrates for the first time an upregulation of periostin in addition to the expression levels of Type I collagen and matrix metalloproteinase-2 in human disc degeneration. It suggests that periostin may be a candidate gene that shows promise as a new prognostic marker and a therapeutic target that is worth further study to expand our knowledge of its role in disc degeneration.

Interleukin-1 receptor antagonist deficient mice provide insights into pathogenesis of human intervertebral disc degeneration

ObjectivesInterleukin 1 (IL-1) is potentially important in the pathogenesis of intervertebral disc (IVD) degeneration; increasing production of matrix degradation enzymes and inhibiting matrix synthesis. Although IL-1 polymorphisms have been linked...

The role of the ERK1/2 pathway as an alternative to the aging-diminished cyclic AMP pathway in calcitonin-mediated chondrogenesis in human nucleus pulposus

Human disc degeneration initiated by aging in the central nucleus pulposus (hNP) is an irreversible process and the recovery has become seriously emerging. In this study, the related mechanisms of calcitonin on the regeneration of hNP and the effects of calcitonin on the age-related alterations were examined. The harvested hNP population was designated as YhNP (from young donor, age <50) and OhNP (from old donor, age >50). Primary OhNP cells showed more hypertrophic phenotypes than YhNP. However, calcitonin (10−8–10−6 M) was able to induce the same chondrogenesis in both YhNP and OhNP by elevating chondrogenic specific-mRNA and protein expressions. Their cell viabilities were increased with calcitonin treatment. No significant differences of calcitonin receptor (CTR) were expressed between YhNP and OhNP cells. Interestingly, in calcitonin-induced pathways for chondrogenesis, highly increased cyclic AMP (cAMP) was detected in YhNP but was strongly diminished by aging in OhNP after calcitonin treatment. However, to maintain the chondrogenesis, calcitonin-induced an alterative phosphorylated ERK1/2 (p-ERK) in both cells. After inhibiting ERK1/2 by PD98059, calcitonin-induced chondrogenesis in OhNP was almost restrained while YhNP cells were not affected. Our results demonstrated that the regeneration of calcitonin on hNP was maintained with aging which was satisfied by an alternative signaling pathway. Therefore, calcitonin shows great potential for clinical therapy for disc regeneration without aging considerations.

Differential expression of galectin‐1 and its interactions with cells and laminins in the intervertebral disc

Galectin-1 (Gal-1), an endogenous β-galactoside-binding protein, binds to laminins, which are highly expressed in the nucleus pulposus (NP) of the intervertebral disc (IVD). The objective of this study is to evaluate the expression of Gal-1 protein in IVD tissues during aging and the effect of Gal-1 on IVD cell adhesion to laminins. Tissues from rat, porcine, and human (scoliosis or disc degeneration) IVDs were used to evaluate Gal-1 expression via immunostaining, RT-PCR, and Western blot analysis. Attachment of isolated IVD cells (porcine and human) on select laminin isoforms (LM-111 and LM-511) was compared with/without pre-incubation with exogenous Gal-1. A biotinylated Gal-1(B-Gal-1) was used to evaluate for binding to IVD cells and to select for IVD cells by magnetic activated cell sorting (MACS). NP cells expressed high levels of Gal-1 protein as compared to anulus fibrosus (AF) cells in immature tissues, while exogenous Gal-1 increased both NP and AF cell attachment to laminins and exhibited a similar binding to both cell types in vitro. With aging, Gal-1 levels in NP tissue appeared to decrease. In addition, incubation with B-Gal-1 was able to promote the retention of more than 50% of IVD cells via MACS. Our results provide new findings for the presence and functional role of Gal-1 within IVDs. Similar staining patterns for Gal-1 and LM-511 in IVD tissue suggest that Gal-1 may serve as an adhesion molecule to interact with both cells and laminins. This MACS protocol may be useful for selecting pure IVD cells from mixed cells of pathological tissue.

Regulation of Neurotrophic Factor Expression in Nerve Cells and Intervertebral Disk Cells by Inflammatory Cytokines, Implications for Pain Pathways

IntroductionIntervertebral disk (IVD) degeneration and prolapse contribute significantly to low back pain and sciatica cases. Within the IVD, the nucleus pulposus cells are known to produce a number of catabolic cytokines which have been shown to elicit a wide range of effects including increased production of degradation enzymes and decreased production of matrix molecules. Here, we investigate the hypothesis that cytokines induce neurotrophic and angiogenic factors in nucleus pulposus cells and nerve cells and induce neurite outgrowth within nerve cells.Materials and MethodsHuman NP cells were isolated via collagenase digestion from human IVD obtained from surgical samples. Following expansion in monolayer culture, cells were transferred to alginate bead cultures at P2 and maintained for 2 weeks before stimulation with cytokines to enable re-differentiation to take place. Cells were then stimulated with IL-1 (0.001 to 100 ng/mL), IL-6 (1 to 100 ng/mL), or IL-8 (1 to 100 ng/mL) for 48 hours, RNA extracted and real time PCR performed to investigate expression of nucleus growth factor (NGF), BDNF, NTF3, and VEGF together with the housekeeping genes GAPDH and 18 s. In addition, SHSY-5Y nerve cell line were cultured in the presence of retinoic acid for 7 days before stimulation with IL-1, IL-6, IL-8, or TNF (1 to 100 ng/mL) to investigate the gene expression of NGF, BDNF, and NTF3 together with effects on neurite outgrowth.ResultsHuman NP cells showed a biphasic response to IL-1 in terms of NGF expression with expression decreased at low doses of IL-1 (0.001–0.01 ng/mL) but an increase in NGF expression at high IL-1 doses (1 ng/mL–100 ng/mL) (Fig. 1). A Small but nonsignificant increase in NTF3 was seen following IL-1 stimulation, whereas IL-6 treatment of NP cells showed a 10-fold increase in NTF 3 expression in some patient samples. Within SH-SY5Y cells interestingly, NGF and BDNF expression was not observed but NTF-3 was downregulated by IL-1 and IL-8 treatments and stimulated by IL-6 stimulation. TNF and IL-1 appeared to induce the greatest effects on neurite outgrowth and number of neurites per cell, both of which increased at doses above 1 ng/mL stimulation. VEGF was also upregulated in human NP cells following IL-1 stimulation even at low doses.ConclusionHere, we demonstrate that a number of cytokines which are upregulated during human disk degeneration and prolapse induce a number of neurotrophic factors and angiogenic factors within human NP cells in 3D cultures. A number of cytokines also induce neurotrophic factor expression and neurite outgrowth in a nerve cell line suggesting a role for these cytokines both within the nerve ingrowth seen in an intact disk and intact disks and following prolapse.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declared

Regulation of Neurotrophic Factor Expression in Nerve Cells and Intervertebral Disk Cells by Inflammatory Cytokines, Implications for Pain Pathways

Introduction Intervertebral disk (IVD) degeneration and prolapse contribute significantly to low back pain and sciatica cases. Within the IVD, the nucleus pulposus cells are known to produce a number of catabolic cytokines which have been shown to elicit a wide range of effects including increased production of degradation enzymes and decreased production of matrix molecules. Here, we investigate the hypothesis that cytokines induce neurotrophic and angiogenic factors in nucleus pulposus cells and nerve cells and induce neurite outgrowth within nerve cells. Materials and Methods Human NP cells were isolated via collagenase digestion from human IVD obtained from surgical samples. Following expansion in monolayer culture, cells were transferred to alginate bead cultures at P2 and maintained for 2 weeks before stimulation with cytokines to enable re-differentiation to take place. Cells were then stimulated with IL-1 (0.001 to 100 ng/mL), IL-6 (1 to 100 ng/mL), or IL-8 (1 to 100 ng/mL) for 48 hours, RNA extracted and real time PCR performed to investigate expression of nucleus growth factor (NGF), BDNF, NTF3, and VEGF together with the housekeeping genes GAPDH and 18 s. In addition, SHSY-5Y nerve cell line were cultured in the presence of retinoic acid for 7 days before stimulation with IL-1, IL-6, IL-8, or TNF (1 to 100 ng/mL) to investigate the gene expression of NGF, BDNF, and NTF3 together with effects on neurite outgrowth. Results Human NP cells showed a biphasic response to IL-1 in terms of NGF expression with expression decreased at low doses of IL-1 (0.001–0.01 ng/mL) but an increase in NGF expression at high IL-1 doses (1 ng/mL–100 ng/mL) (Fig. 1). A Small but nonsignificant increase in NTF3 was seen following IL-1 stimulation, whereas IL-6 treatment of NP cells showed a 10-fold increase in NTF 3 expression in some patient samples. Within SH-SY5Y cells interestingly, NGF and BDNF expression was not observed but NTF-3 was downregulated by IL-1 and IL-8 treatments and stimulated by IL-6 stimulation. TNF and IL-1 appeared to induce the greatest effects on neurite outgrowth and number of neurites per cell, both of which increased at doses above 1 ng/mL stimulation. VEGF was also upregulated in human NP cells following IL-1 stimulation even at low doses. Conclusion Here, we demonstrate that a number of cytokines which are upregulated during human disk degeneration and prolapse induce a number of neurotrophic factors and angiogenic factors within human NP cells in 3D cultures. A number of cytokines also induce neurotrophic factor expression and neurite outgrowth in a nerve cell line suggesting a role for these cytokines both within the nerve ingrowth seen in an intact disk and intact disks and following prolapse. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Mesenchymal Stem Cells Mediate Disk Regeneration by Suppression of Fibrosis in Nucleus Pulposus

Introduction Confluent fibrosis, manifested as over-accumulation of fibrous matrix during tissue repair or remodeling, leads to scar formation and often organ failure. Studies suggest that 68% of protruded intervertebral disks and 44% of prolapsed IVD exhibit signs of fibrosis in the nucleus pulposus (NP) and that disk scarring indicates advanced stages of degeneration. It is not clear how fibrosis contributes to the initiation or progression of disk degeneration, and conversely if the prevention of fibrotic events plays a role in IVD repair or regeneration. Mesenchymal stem cells (MSCs) may arrest IVD degeneration and cross-talk with IVD cells via cell-cell contact or long-range signaling, implying MSCs may modulate disk microenvironment and function indirectly. Lupine is a reliable model for IVD degeneration studies as their lumbar spine bears biological relevance to humans regarding mechanics and age-related changes. Studies showed that puncturing to the annulus could transform the NP into a fibrocartilaginous phenotype, recapitulating the fibrotic features of human disk degeneration. We here tested if MSCs can rectify IVD degeneration and its function through modulation of fibrotic events, and investigated the pathways of the action. Materials and Methods Total 6-month-old NZW rabbits with puncture-induced lumbar disk degeneration were intradiscally implanted with Chinchilla bone marrow MSCs, labeled by Qtracker and encapsulated in oligopeptide-hydrogel at 5 x 10E3 cells per disk ( n = 8). Saline or hydrogel alone was injected as control. Treated disk levels were monitored by 3T MRI and radiographs up to 1 year. At 4 to 12 weeks posttreatment, the mechanical integrity of NP and joint segment were investigated by confined compression and motion stiffness tests. The collagen fibril diameter ( n = 120) and the porosity of the fibril meshwork in the NP were analyzed in situ using scanning electron microscopy and morphometric analyzer. The elastic modulus of collagen fibrils ( n = 75) in the NP was measured by nanoindentation using atomic force microscopy. Collagen I and II content was assessed by immunofluorescence. Human bone marrow MSC-conditioned media (MSC-CM) was collected and used to culture alginate-encapsulated NP cells harvested from patients with Pfirrmann grade III/IV disk degeneration. After 7 days, expression of fibrotic genes was studied using real-time PCR ( n = 3). Results T2-weighted MRI and disk height analysis validated that MSCs arrested the degeneration. Motion segment stiffness was significantly attenuated in MSC-treated disks, indicating MSCs alleviated ankylosis caused by the degeneration and restored the spinal segment function. MSCs regenerated the swelling pressure and compressive strength of the NP, implying that MSCs regenerated joint function through preserving the biphasic tissue properties. Collagen molecules in the degenerated NP aggregated into gigantic fibrils, about 2-fold larger in diameter, resulting in fewer fibers and larger interfibrillar space. However, MSCs inhibited the deregulated higher-order assembly of fibrils. MSCs significantly reduced collagen I deposition and prevented the increase of fibril elastic modulus in the NP associated with degeneration. These suggest that the abnormal collagen fibrils are not solely resulted from fibril fusion but also a heterotypic change of collagen composition, and that the positive effects of MSCs are coupled to a regulation of fibrillogenesis in the NP matrix. MSC-CM repressed the expression of fibrosis-related genes MMP12 and HSP47 in degenerative human NP cells. Conclusion MSC transplantation can alleviate fibrotic diseases including pulmonary, renal, and cardiac fibrosis through pathways independent of MSC differentiation. Our findings imply that MSCs also rescue disk degeneration and recover disk function by suppressing fibrotic events. The action of MSCs is elicited through a regulation of fibrillogenesis in part via paracrine signaling to the degenerative NP cells. We propose a model in which MSCs reinforce the mechanical integrity of NP by normalizing the interplay between collagen meshwork and proteoglycan. Disk repair by augmenting the antifibrotic function of MSCs or use of other antifibrotic agents warrants further investigation. This study is supported by the Research Grant Council of Hong Kong and HKU Foundation. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration

IntroductionThe longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model.MethodsForty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental loaded and distal-unloaded control discs were harvested and analyzed by real-time reverse transcription-polymerase chain reaction (PCR) messenger RNA quantification for catabolic genes [matrix metalloproteinase (MMP)-1a, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5], anti-catabolic genes [tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3], ECM genes [aggrecan-1, collagen type 1-α1, and collagen type 2-α1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, and IL-6]. Immunohistochemistry for MMP-3, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 was performed to assess their protein expression level and distribution. The presence of MMP- and aggrecanase-cleaved aggrecan neoepitopes was similarly investigated to evaluate aggrecanolytic activity.ResultsQuantitative PCR demonstrated up-regulation of all MMPs and ADAMTS-4 but not ADAMTS-5. TIMP-1 and TIMP-2 were almost unchanged while TIMP-3 was down-regulated. Down-regulation of aggrecan-1 and collagen type 2-α1 and up-regulation of collagen type 1-α1 were observed. Despite TNF-α elevation, ILs developed little to no up-regulation. Immunohistochemistry showed, in the nucleus pulposus, the percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope increased from 7 through 56 days with increased MMP-3 and decreased TIMP-1 and TIMP-2 immunopositivity. The percentage of immunopositive cells of aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with decreased TIMP-3 immunopositivity. In the annulus fibrosus, MMP-cleaved aggrecan neoepitope presented much the same expression pattern. Aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with increased ADAMTS-4 and ADAMTS-5 immunopositivity.ConclusionsThis rat tail sustained static compression model mimics ECM metabolic imbalances of MMPs, aggrecanases, and TIMPs in human degenerative discs. A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 signifies an advanced stage of intervertebral disc degeneration.

Ionizing radiation exposure and the development of intervertebral disc degeneration in humans: myth or reality

Ionizing radiation exposure and the development of intervertebral disc degeneration in humans: myth or reality

Human Disc Nucleus Properties and Vertebral Endplate Permeability

Experimental quantification of relationships between vertebral endplate morphology, permeability, disc cell density, glycosaminoglycan (GAG) content, and degeneration in samples harvested from human cadaveric spines. To test the hypothesis that variation in endplate permeability and porosity contributes to changes in intervertebral disc cell density and overall degeneration. Cells within the intervertebral disc are dependent on diffusive exchange with capillaries in the adjacent vertebral bone. Previous findings suggest that blocked routes of transport negatively affect disc quality, yet there are no quantitative relationships between human vertebral endplate permeability, porosity, cell density, and disc degeneration. Such relationships would be valuable for clarifying degeneration risk factors and patient features that may impede efforts at disc tissue engineering. Fifty-one motion segments were harvested from 13 frozen cadaveric human lumbar spines (32-85 years) and classified for degeneration using the magnetic resonance imaging-based Pfirrmann scale. A cylindrical core was harvested from the center of each motion segment that included vertebral bony and cartilage endplates along with adjacent nucleus tissue. The endplate mobility, a type of permeability, was measured directly using a custom-made permeameter before and after the cartilage endplate was removed. Cell density within the nucleus tissue was estimated using the picogreen method, while the nuclear GAG content was quantified using the dimethylmethylene blue technique. Specimens were imaged at 8 μm resolution using microCT; bony porosity was calculated. Analysis of variance, linear regression, and multiple comparison tests were used to analyze the data. RESULTS.: Nucleus cell density increased as the disc height decreased (R² = 0.13; P = 0.01) but was not related to subchondral bone porosity (P > 0.5), total mobility (P > 0.4), or age (P > 0.2). When controlling for disc height, however, a significant, negative effect of age on cell density was observed (P = 0.03). In addition to this, GAG content decreased with age nonlinearly (R² = 0.83, P < 0.0001) and a cell function measurement, GAGs/cell, decreased with degeneration (R² = 0.24; P < 0.0001). Total mobility (R² = 0.14; P < 0.01) and porosity (R² = 0.1, P < 0.01) had a positive correlation with age. Although cell density increased with degeneration, cell function indicated that GAGs/cell decreased. Because permeability and porosity increase with age and degeneration, this implies that cell dysfunction, rather than physical barriers to transport, accelerates disc disease.

Beneficial effects of hyperbaric oxygen on human degenerated intervertebral disk cells via suppression of IL‐1β and p38 MAPK signal

Nucleus pulposus cells (NPCs) from degenerating disks produce catabolic and inflammatory factors, including interleukin (IL)-1, nitric oxide (NO), prostaglandin E2 (PGE-2), and matrix metalloproteinaes (MMPs). An imbalance between MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) has been proposed to exist in the degenerating disk. This study evaluates the effects of hyperbaric oxygen (HBO) on the human degenerated NPCs. NPCs were maintained in alginate bead culture. All hyperoxic cells were exposed to 100% O(2) at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber. p38 MAPK phosphorylation of the NPCs was detected using the phosphor-kinase array kit. RNA was isolated for real-time quantitative polymerase chain reaction (Q-PCR) analysis of aggrecan and type II collagen gene expression. The amounts of IL-1β, NO, PGE-2, MMP-3, and TIMP-1 in the conditioned media were quantified by enzyme-linked immunosorbent assay (ELISA). Our data showed that HBO treatment decreased expression of IL-1β, increased the gene expression of aggrecan and type II collagen, suppressed the phosphorylation of p38 MAPK, decreased NO, PGE-2, and MMP-3, and increased TIMP-1 expression in NPCs as compared with the atmospheric treatment. These results support the hypothesis that IL-1β and the p38 MAPK signal may be responsible for many of the inflammatory and catabolic changes seen in the human disk degeneration, and support our proposal that HBO treatment-induced increase of the anabolic factor (TIMP-1)/catabolic factor (MMP-3) ratio may provide a therapeutic approach to slow the course of intervertebral disk degeneration.

Human intervertebral disc internal strain in compression: The effect of disc region, loading position, and degeneration

The primary function of the disc is mechanical; therefore, degenerative changes in disc mechanics and the interactions between the annulus fibrosus (AF) and nucleus pulposus (NP) in nondegenerate and degenerate discs are important to functional evaluation. The disc experiences complex loading conditions, including mechanical interactions between the pressurized NP and the surrounding fiber-reinforced AF. Our objective was to noninvasively evaluate the internal deformations of nondegenerate and degenerate human discs under axial compression with flexion, neutral, and extension positions using magnetic resonance imaging and image correlation. The side of applied bending (e.g., anterior AF in flexion) had higher tensile radial and compressive axial strains, and the opposite side of bending exhibited tensile axial strains even though the disc was loaded under axial compression. Degenerated discs exhibited higher compressive axial and tensile radial strains, which suggest that load distribution through the disc subcomponents are altered with degeneration, likely due to the depressurized NP placing more of the applied load directly on the AF. The posterior AF exhibited higher compressive axial and higher tensile radial strains than the other AF regions, and the strains were not correlated with degeneration, suggesting this region undergoes high strains throughout life, which may predispose it to failure and tears. In addition to understanding internal disc mechanics, this study provides important new data into the changes in internal strain with degeneration, data for validation of finite element models, and provides a technique and baseline data for evaluating surgical treatments.

Disc Cell Senescence in Vivo: Increased Cell Senescence is Associated with Decreased Cell Proliferation in the Human Annulus

BACKGROUND CONTEXT: During disc degeneration, there is a well-recognized loss of cells, which puts the remaining cell population at risk for any diminution in cell number or cell function. Cell senescence is recognized as a potential factor playing a role in human disc degeneration. Senescent cells are viable but have lost the ability to divide. Senescent cells, however, continue to be metabolically active, and persist and accumulate over time.

Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs

Background context The population of senescent disc cells has been shown to increase in degenerated or herniated discs. However, the mechanism and signaling pathway involved in the senescence of nucleus pulposus (NP) chondrocytes are unknown. Purpose To demonstrate the mechanisms involved in the senescence of NP chondrocytes. Study design/setting Senescence-related markers were assessed in the surgically obtained human NP specimens. Patient sample NP specimens remaining in the central region of the intervertebral disc were obtained from 25 patients (mean: 49 years, range: 20–75 years) undergoing discectomy. Based on the preoperative magnetic resonance images, there were 3 patients with Grade II degeneration, 17 patients with Grade III degeneration, and 5 patients with Grade IV degeneration. Outcome measures We examined cell senescence markers (senescence-associated β-galactosidase [SA-β-gal], telomere length, telomerase activity, p53, p21, pRB, and p16) and the hydrogen peroxide (H 2O 2) content as a marker for an oxidative stress in the human NP specimens. Methods SA-β-gal expression, telomere length, telomerase activity, and H 2O 2 content as well as their relationships with age and degeneration grades were analyzed. For the mechanism involved in the senescence of NP chondrocytes, expressions of p53, p21, pRB, and p16 in these cells were assessed with immunohistochemistry and Western blotting. Results The percentages of SA-β-gal-positive NP chondrocytes increased with age (r=.82, p<.001), whereas the telomere length and telomerase activity declined (r=−.41, p=.045; r=−.52, p=.008, respectively) However, there was no significant correlation between age and H 2O 2 contents (p=.18). The NP specimens with Grade III or Grade IV degeneration showed significantly higher percentages of SA-β-gal-positive NP chondrocytes than those with Grade II degeneration (p=.01 and p=.025, respectively). Immunohistochemistry showed that the senescent NP chondrocytes in all the specimens expressed p53, p21, and pRB, but a few NP chondrocytes in only two specimens expressed p16. Western blotting showed that the expressions of p53, p21, and pRB displayed a corresponding pattern, that is, a strong p53 expression led to strong p21 and pRB expressions and vice versa. Conclusions Our in vivo study demonstrated that senescent NP chondrocytes increased or accumulated in the NP with increasing age and advancing disc degeneration. The NP chondrocytes in the aging discs exhibited characteristic senescent features such as an increased SA-β-gal expression, shortened telomeres, and decreased telomerase activity. We further demonstrated that the telomere-based p53-p21-pRB pathway, rather than the stress-based p16-pRB pathway, plays a more important role in the senescence of NP chondrocytes in an in vivo condition. Our results suggest that prevention or reversal of the senescence of NP chondrocytes can be a novel therapeutic target for human disc degeneration.

Solute Transport in Intervertebral Disc

Loss of nutrient supply to the human intervertebral disc (IVD) cells is thought to be a major cause of disc degeneration in humans. To address this issue, transport of molecules of different size have been analyzed by a combination of experimental and modeling studies. Solute transport has been compared for steady-state and transient diffusion of several different solutes with molecular masses in the range 3-70 kDa, injected into parts of the disc where degeneration is thought most likely to occur first and into the blood supply to the disc. Diffusion coefficients of fluorescently tagged dextran molecules of different molecular weights have been measured in vitro using the concentration gradient technique in thin specimens of disc outer annulus and nucleus pulposus. Diffusion coefficients were found to decrease with molecular weight following a nonlinear relationship. Diffusion coefficients changed more rapidly for solutes with molecular masses less than 10 kDa. Although unrealistic or painful, solutes injected directly into the disc achieve the largest disc coverage with concentrations that would be high enough to be of practical use. Although more practical, solutes injected into the blood supply do not penetrate to the central regions of the disc and their concentrations dissipate more rapidly. Injection into the disc would be the best method to get drugs or growth factors to regions of degeneration in IVDs quickly; else concentrations of solute must be kept at a high value for several hours in the blood supply to the discs.

Asporin, a susceptibility gene in osteoarthritis, is expressed at higher levels in the more degenerate human intervertebral disc

IntroductionAsporin, also known as periodontal ligament-associated protein 1 (PLAP1), is a member of the family of small leucine-rich proteoglycan (SLRP) family. It is present within the cartilage extracellular matrix (ECM), and is reported to have a genetic association with osteoarthritis. Its D14 allele has recently been found to be associated with lumbar disc degeneration in Asian subjects. There have been no studies, however, of this gene's normal immunohistochemical localization within the human intervertebral disc, or of expression levels in Caucasian individuals with disc degeneration.MethodsStudies were approved by our human subjects Institutional Review Board. Methods included immunohistochemical localization of asporin in the disc of humans and the sand rat (a small rodent with spontaneous age-related disc degeneration), and Affymetrix microarray analysis of asporin gene expression in vivo and in vitro.ResultsImmunohistochemical studies of human discs revealed that some, but not all, cells of the outer annulus expressed asporin. Fewer cells in the inner annulus contained asporin, and it was rarely present in cells in the nucleus pulposus. Similar patterns were found for the presence of asporin in lumbar discs of sand rats. Substantial relative gene expression levels were seen for asporin in both disc tissue and in annulus cells grown in three-dimensional culture. More degenerate human discs (Thompson grade 4) showed higher expression levels of asporin than did less degenerate (grade 1, 2 and 3) discs, P = 0.004.ConclusionsIn the discs of Caucasian subjects studied here, and in the sand rat, greater immunolocalization levels were found in the outer compared to inner annulus. Localization was rare in the nucleus. Gene expression studies showed greatest expression of asporin in the more degenerate human discs in vivo.

Injury‐induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse

Intervertebral disc degeneration has been widely studied in different animal models. To test the hypothesis that needle puncture could induce progressive biochemical and molecular changes in murine discs, we established a mouse tail model to investigate the pathogenesis and molecular mechanism of puncture-induced disc degeneration. Caudal discs in mouse tails were punctured using a 31G gauge needle at controlled depth under microscopic guidance. The progress of the disc degeneration was evaluated by radiographic analysis of disc height, histological grading and glycosaminoglycan (GAG) quantification pre-operation and 1, 2, 6 and 12 weeks post-puncture. Gene and protein expression of the extracellular matrix (ECM) was analysed by RT-PCR, in situ hybridization and immunohistochemistry. Histological study and disc height analysis revealed progressive degenerative changes in the punctured discs. Compared with the pre-operation control group, total GAG content decreased 40% (p < 0.05) and aggrecan (Acan), decorin (Dcn) and versican (Vcan; Cspg2) expression was down-regulated at 12 weeks post-puncture. A transient increase of Col2a1-expressing cells and elevation of collagen II protein in the nucleus pulposus (NP) was detected. Fibronectin (Fn1) expression was up-regulated 50% and deposition of collagen I in NP was observed at 12 weeks post-puncture. This study is the first to use an injury-induced model to study disc degeneration in mouse. The disc degeneration involves a transient transformation of NP from notochordal to chondrogenic and eventually into fibrocartilaginous phenotype. The degenerative changes have some similarity to human disc degeneration, suggesting that this model may potentially be used in future to study the molecular mechanism and dissect the pathways of disc degeneration.