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Intervertebral disc degeneration (IDD) is a chronic skeletal muscle degenerative disease, which is considered the main cause of low back pain. It seriously affects the quality of life of patients and consequently brings a heavy economic burden to their families and the society. Although IDD is considered a natural process in degenerative lesions, it is mainly caused by aging, trauma, genetic susceptibility and other factors. It is closely related to changes in the tissue structure and function, including the progressive destruction of extracellular matrix, cell aging, cell death of the intervertebral disc (IVD), inflammation, and impairment of tissue biomechanical function. Currently, the treatment of IDD is aimed at alleviating symptoms rather than at targeting pathological changes in the IVD. Furthermore, the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway is closely related to various pathological processes in IDD, and the activation of the MAPK/ERK pathway promotes the degradation of the IVD extracellular matrix, cell aging, apoptosis, and inflammatory responses. It also induces autophagy and oxidative stress that accelerate the IVD process. In our current review, we summarize the latest developments in the negative regulation of IDD after activation of the MAPK/ERK signaling pathway and emphasize on its influence on IDD. Targeting this pathway may become an attractive treatment strategy for IDD in the near future., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Intervertebral disc (IVD) degeneration (IVDD) leads to structural and functional changes. Biomaterials for restoring IVD function and promoting regeneration are currently being investigated; however, such approaches require validation using animal models that recapitulate clinical, biochemical, and biomechanical hallmarks of the human pathology. Herein, we comprehensively characterized a sheep model of chondroitinase-ABC (ChABC) induced IVDD. Briefly, ChABC (1 U) was injected into the L 1/2 , L 2/3 , and L 3/4 IVDs. Degeneration was assessed via longitudinal magnetic resonance (MR) and radiographic imaging. Additionally, kinematic, biochemical, and histological analyses were performed on explanted functional spinal units (FSUs). At 17-weeks, ChABC treated IVDs demonstrated significant reductions in MR index (p = 0.030) and disc height (p = 0.009) compared with pre-operative values. Additionally, ChABC treated IVDs exhibited significantly increased creep displacement (p = 0.004) and axial range of motion (p = 0.007) concomitant with significant decreases in tensile (p = 0.034) and torsional (p = 0.021) stiffnesses and long-term viscoelastic properties (p = 0.016). ChABC treated IVDs also exhibited a significant decrease in NP glycosaminoglycan: hydroxyproline ratio (p = 0.002) and changes in microarchitecture, particularly in the NP and endplates, compared with uninjured IVDs. Taken together, this study demonstrated that intradiscal injection of ChABC induces significant degeneration in sheep lumbar IVDs and the potential for using this model in evaluating biomaterials for IVD repair, regeneration, or fusion., (© 2020 Wiley Periodicals LLC.)

Intervertebral disc degeneration (IDD) is a spinal degenerative disease and one of the most important causes of musculoskeletal disability. Matrix metalloproteinase (MMP)-mediated extracellular matrix degradation is the core process of IDD. The regulators of MMPs in the intervertebral disc are still not fully known. In this study, using quantitative reverse transcription PCR, luciferase reporter assay, Western blotting, immunofluorescence, flow cytometry, and Cell Counting Kit-8 assay, we found that the miR-874-3p expression level was significantly decreased in IDD patients. MiR-874-3p could target and repress MMP2 and MMP3 expression in nucleus pulposus cells. These results could improve the understanding of IDD and provide a possible diagnostic marker and treatment candidate for IDD. The miR-874-3p/MMP2/MMP3 axis might also provide direction for future cancer and inflammation investigations., (Copyright © 2021. Published by Elsevier B.V.)

Objective: To investigate if the modification of human adipose-derived mesenchymal stem cells (hADSCs) by the antioxidants superoxide dismutase 2 (Sod2) and catalase (Cat) can attenuate the pathological conditions of intervertebral disc degeneration (IVD)., Methods: In vitro, MTT assay and qRT-PCR was used to detect cell proliferation and gene expressions in hADSCs transduced with Ad-null (an adenovirus vector containing no transgene expression cassette), Ad-Sod2 (recombinant adenovirus Sod2) and Ad-Cat. IVD mouse models were generated by needle puncture and treated with hADSCs with/without Ad-null/Ad-Sod2/Ad-Cat. X-ray evaluation, magnetic resonance imaging (MRI) analysis, histological analysis, immunohistochemistry, Western blots, ELISAs and qRT-PCR were performed., Results: hADSCs transduced with Ad-Sod2 and Ad-Cat showed enhanced cell proliferation with the upregulation of SOX9, ACAN, and COL2. In vivo, IVD mice injected with hADSCs showed increased disc height index, MRI index and mean T2 intensities, as well as the attenuated histologic grading of the annulus fibrosus (AF) and NP accompanied by the upregulation of GAG and COL2, which were further improved in the Ad-Sod2 hADSC + IVD and Ad-Cat hADSC + IVD groups. Furthermore, the increased expression of IL-1β, IL-6 and TNF-α was reduced in IVD mice injected with hADSCs. Compared with the hADSC + IVD group, the Ad-Sod2 hADSC/Ad-Cat hADSC + IVD groups had lower expression of pro-inflammatory factors., Conclusion: Modification of hADSCs by the antioxidants Sod2 and Cat improved the pathological condition of intervertebral disc tissues with increased GAG and COL2 expression, as well as reduced inflammation, thereby demonstrating a therapeutic effect in IVD., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-β, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Mechanical overloading-induced nucleus pulposus (NP) cells senescence plays an important role in the pathogenesis of intervertebral disc degeneration (IVDD). The silent mating type information regulator 2 homolog-1 (SIRT1)-mediated pathway preserves the normal NP cell phenotype and mitochondrial homeostasis under multiple stresses. We aimed to investigate the role of SIRT1 in IVDD by assessing the effects of SIRT1 overexpression on high-magnitude compression-induced senescence in NP cells. High-magnitude compression induced cellular senescence and mitochondrial dysfunction in human NP cells. Moreover, SIRT1 overexpression tended to alleviate NP cell senescence and mitochondrial dysfunction under compressive stress. Given the mitophagy-inducing property of SIRT1, activity of mitophagy was evaluated in NP cells to further demonstrate the underlying mechanism. The results showed that SIRT1-overexpression attenuated senescence and mitochondrial injury in NP cells subjected to high-magnitude compression. However, depletion of PINK1, a key mitophagic regulator, impaired mitophagy and blocked the protective role of SIRT1 against compression induced senescence in NP cells. In summary, these results suggest that SIRT1 plays a protective role in alleviating NP cell senescence and mitochondrial dysfunction under high-magnitude compression, the mechanism of which is associated with the regulation of PINK1-dependent mitophagy. Our findings may provide a potential therapeutic approach for IVDD treatment.

Degradation of extracellular matrix (ECM) in intervertebral disks (IVDs) during IVD degeneration plays a vital role in low back pain (LBP). In healthy IVDs, synthesis and degradation of ECM are kept in balance by matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs. MMPs are enzymes responsible for ECM degradation, and their expression levels are known to increase in degenerated disks. However, the exact pathophysiological concentration of MMP-1 in the degenerated disks of patients with chronic LBP has not been reported previously. Factors secreted by human mesenchymal stem cells (hMSCs) have shown positive results in cell therapy of degenerated disks. The aim of this study was to investigate the pathophysiological MMP-1 concentration (in ng/mL) in degenerated disk tissue and to evaluate if conditioned media (CM) from hMSCs could mitigate the effects of MMP-1 at the detected levels in a 3D in vitro disk cell (DC) pellet model. Tissue levels of MMP-1 were quantified in disk tissue collected from 6 chronic LBP patients undergoing surgery. DC pellet cultures were performed to investigate the effects of MMP-1 alone and the effects of conditioned media (CM) in the presence of MMP-1. MMP-1 was introduced in the pellets on day 14 at concentrations of 5, 50, or 100 ng/mL. The pellets were harvested on day 28 and evaluated for cell viability, proliferation, and ECM production. The mean concentration of MMP-1 in disk tissue was 151 ng/mL. Results from pellet cultures demonstrated a higher number of viable cells, glycosaminoglycan production, and ECM accumulation in the CM group even in the presence of MMP-1 compared to the controls. However, the level decreased with increasing MMP-1 concentration. The results demonstrated that CM has the ability to mitigate matrix degradation property of MMP-1 up to 50 ng/mL suggesting that CM could potentially be used to treat early stages of disk degeneration., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

Approximately 40% of people under 30 and over 90% of people 55 or older suffer from moderate-to-severe levels of degenerative intervertebral disc (IVD) disease in their lumbar spines. Surgical treatments are sometimes effective; however, the treatment of back pain related to IVD degeneration is still a challenge; therefore, new treatments are necessary. Apoptosis may be important in IVD degeneration because suppressing cell apoptosis inside the IVD inhibits degeneration. Caspase-3, the primary effector of apoptosis, may be a key treatment target. We analyzed caspase-3's role in two different types of IVD degeneration using caspase-3 knockout (Casp-3 KO) mice. Casp-3 KO delayed IVD degeneration in the injury-induced model but accelerated it in the age-induced model. Our results suggest that this is due to different pathological mechanisms of these two types of IVD degeneration. Apoptosis was suppressed in the IVD cells of Casp-3 KO mice, but cellular senescence was enhanced. This would explain why the Casp-3 KO was effective against injury-induced, but not age-related, IVD degeneration. Our results suggest that short-term caspase-3 inhibition could be used to treat injury-induced IVD degeneration.

Study Design: An in vivo and in vitro study of the correlation between Paraoxonase 1 (PON1) and intervertebral disc degeneration (IVDD)., Objective: The aim of this study is to clarify the expression and role of PON1 on the process of IVDD., Summary of Background Data: IVDD is responsible for most of the spinal degenerative diseases. Inflammation and oxidative stress can deteriorate the living environment of nucleus pulposus (NP) cells, leading to IVDD. PON1 is an enzyme reported to have anti-inflammatory and anti-oxidative effects. There is no study about the correlation of PON1 expression with IVDD., Methods: Immunohistochemical (IHC), hematoxylin and eosin (H&E) staining, and Western blot examined the expression of PON1 in 88 human disc samples (male: female 43: 45) and rat models (n = 5 each group). The level of PON1 is measured in the tumor necrosis factor (TNF)-α and oxidative stress (H2O2)-induced degenerative NP cell models using Western blot and reverse transcription-polymerase chain reaction (RT-qPCR). The TNF-α, interleukin (IL)-1β, Mito superoxide (SOX), aggrecan, and collagen II are detected in nucleus pulposus (NP) cells transfected with si-RNA of PON1 using Enzyme-Linked Immunosorbent Assay (ELISA), mitoSOX staining Western blot, and RT-qPCR., Results: The expression of PON1 is significantly suppressed in human and rat degenerative intervertebral discs. The level of PON1 is significantly decreased in TNF-α and oxidative stress (H2O2)-induced degenerative NP cell models. ELISA results show that the level of TNF-α and IL-1β obviously increased; Mito SOX staining indicates that the Mito SOX fluorescence significantly increased, and the expression of aggrecan and collagen reduced in NP cells transfected with si-RNA of PON1., Conclusion: Our study indicates that low PON1 expression is predictive of severe IVDD; PON1 plays an important role of keeping the homeostatic balance of intervertebral discs, and therapeutic approach regarding PON1 may be helpful to alleviate IVDD in the future., Level of Evidence: N/A.

The expression of heparan sulfate endosulfatases (Sulfs) was investigated in the intervertebral disc (IVD) to clarify their role in IVD homeostasis. Sulf-1 and -2 expression were elucidated in normal and degenerated human IVD. Age-related effects on Sulf expression, type II collagen levels, and structural changes were analyzed in IVDs of wild-type (WT) and Sulf-1 knockout (Sulf-1⁻/⁻) mice. The effect of recombinant Sulf-1 (100 ng/ml) and Sulf-1 knockdown on heparan sulfate proteoglycan and collagen expression in ATDC5 cells were examined. Finally, the effect of Sulf-1 on transforming growth factor (TGF) β1-induced signaling was evaluated. Results show that Sulf-1 and -2 levels were higher in degenerated human IVDs. In WT mice, Sulf-1 and -2 expression generally declined as the animals aged. In particular, Sulf-1 in the nucleus pulposus was higher compared with Sulf-2 at the age of 1 and 6 months and significantly declined with aging. Sulf-1⁻/⁻ mice showed more severe IVD pathology than WT mice, with lower type II collagen levels in nucleus pulposus, and degeneration with type I collagen in annulus fibrosus. In vitro, Sulf-1 induced type II collagen expression and significantly increased TGF-β1-induced Smad2/3 phosphorylation in ATDC5 cells. In conclusion, Sulf-1 might play a critical role from development to maintenance of IVD homeostasis by regulating collagen expression.

Study Design: Animal experiment: a rat model of lumbar disc herniation (LDH) induced painful radiculopathies., Objective: To investigate the role and mechanism of AMP-activated protein kinase (AMPK) in dorsal root ganglia (DRG) neurons in LDH-induced painful radiculopathies., Summary of Background Data: Overactivation of multiple pain signals in DRG neurons triggered by LDH is crucial to the development of radicular pain. AMPK is recognized as a cellular energy sensor, as well as a pain sensation modulator, but its function in LDH-induced pain hypersensitivity remains largely unknown., Methods: The LDH rat model was established by autologous nucleus pulposus transplantation into the right lumbar 5 (L5) nerve root. At different time points after AMPK agonist metformin (250 mg/kg/d) or mammalian target of rapamycin (mTOR) inhibitor rapamycin (5 mg/kg) intraperitoneal administration, thermal and mechanical sensitivity were evaluated by measuring paw withdrawal latency (PWL) and 50% paw withdrawal thresholds (PWT). The levels of AMPK, mTOR, and p70S6K phosphorylation were determined by Western blot. We also investigated the proportion of p-AMPK positive neurons in the right L5 DRG neurons using immunofluorescence., Results: LDH evoked persistent thermal hyperalgesia and mechanical allodynia on the ipsilateral paw, as indicated by the decreased PWL and 50% PWT. These pain hypersensitive behaviors were accompanied with significant inhibition of AMPK and activation of mTOR in the associated DRG neurons. Pharmacological activation of AMPK in the DRG neurons not only suppressed mTOR/p70S6K signaling, but also alleviated LDH-induced pain hypersensitive behaviors., Conclusion: We provide a molecular mechanism for the activation of pain signals based on AMPK-mTOR axis, as well as an intervention strategy by targeting AMPK-mTOR axis in LDH-induced painful radiculopathies., Level of Evidence: N/A.

Intervertebral disc (IVD) degeneration is the major contributor to low back pain, a highly prevalent musculoskeletal problem that represents the leading cause of disability. Proinflammatory M1 macrophages were identified in degenerated IVDs. However, their role in the pathogenesis of IVD degeneration and the underlying mechanism was largely unknown. In this study, we explored the combined effects of molecules secreted by M1 macrophages on nucleus pulposus cells, by treating rat nucleus pulposus cells (rNP) with the conditioned medium collected from M1-polarized RAW264.7 cells (MФCM). We found that MФCM caused molecular changes associated with IVD degeneration, including increased expression of key matrix catabolic genes (Adamts4, Adamts5, Mmp3, and Mmp13), reduced the expression of major matrix-associated anabolic genes ( Sox9, Acan, and Col2a1), and upregulated transcription of inflammation-related genes ( IL-1b, IL-6, Ccl2, and Ccl3), in rNP cells. Moreover, we found that MФCM activated both ERK and JNK pathways in these cells, and that inhibition of JNK pathway attenuated MФCM-induced expression of both catabolic and inflammatory genes, whereas ERK inhibition only suppressed induction of catabolic, but not inflammatory genes. Together, our data demonstrated that proinflammatory macrophages promoted the degenerative phenotypes in rNP cells in part through ERK and JNK signaling, and suggested that inhibition of these pathways may serve as a potential therapeutic approach for the treatment of IVD degeneration., (© 2018 Wiley Periodicals, Inc.)

Recent findings demonstrate that leptin plays a significant role in chondrocyte and osteoblast differentiation. However, the mechanisms by which leptin acts on cartilage endplate (CEP) cells to give rise to calcification are still unclear. The aim of this study was to evaluate the effects of leptin that induced mineralization of CEP cells in vitro and in vivo. We constructed a rat model of lumbar disc degeneration and determined that leptin was highly expressed in the presence of CEP calcification. Rat CEP cells treated with or without leptin were used for in vitro analysis using RT-PCR and Western blotting to examine the expression of osteocalcin (OCN) and runt-related transcription factor 2 (Runx2). Both OCN and Runx2 expression levels were significantly increased in a dose- and time-dependent manner. Leptin activated ERK1/2 and STAT3 phosphorylation in a time-dependent manner. Inhibition of phosphorylated ERK1/2 using targeted siRNA suppressed leptin-induced OCN and Runx2 expression and blocked the formation of mineralized nodules in CEP cells. We further demonstrated that exogenous leptin induced matrix mineralization of CEP cells in vivo. We suggest that leptin promotes the osteoblastic differentiation of CEP cells via the MAPK/ERK signal transduction pathway and may be used to investigate the mechanisms of disc degeneration., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Nucleus pulposus (NP) cell senescence is a typical pathological feature within the degenerative intervertebral disc. As a potential inducing and aggregating factor of disc degeneration, mechanical overloading affects disc biology in multiple ways. The present study was to investigate the NP cell senescence-associated phenotype under intermittent high compression in an ex vivo disc bioreactor culture, and the role of the p38-MAPK pathway in this regulatory process. Porcine discs were cultured in culture chambers of a self-developed mechanically active bioreactor and subjected to different magnitudes of dynamic compression (low-magnitude and high-magnitude: 0.1 and 1.3 MPa at a frequency of 1.0 Hz for 2 h per day respectively) for 7 days. Non-compressed discs were used as controls. The inhibitor SB203580 was used to study the role of the p38-MAPK pathway in this process. Results showed that intermittent high-magnitude compression clearly induced senescence-associated changes in NP cells, such as increasing β-galactosidase-positive NP cells, decreasing PCNA-positive NP cells, promoting the formation of senescence-associated heterochromatic foci (SAHF), up-regulating the expression of senescence markers (p16 and p53), and attenuating matrix production. However, inhibition of the p38-MAPK pathway partly attenuated the effects of intermittent high-magnitude (1.3 MPa) compression on those described NP cell senescence-associated parameters. In conclusion, intermittent high-magnitude compression can induce NP cell senescence-associated changes in an ex vivo disc bioreactor culture, and the p38-MAPK pathway is involved in this process., (© 2017 The Author(s).)

Degeneration of IVD is a progressive and irreversible process and can be evaluated with immunohistochemical examination or radiological grading. MMPs are a family of proteolytic enzymes and involved in the degradation of the matrix components of the IVD. We aimed to compare MMP-1, -2, -3, and -9 expressions with demographic features, visual analogue scale (VAS), Oswestry Disability Index (ODI) and radiological (MRI) grades. The study involved 60 participants. We recorded data about age, complaint, radiological imaging, expression levels of MMP-1, -2, -3, and -9, ODI and VAS for back pain retrospectively. Intervertebral disc degeneration was graded on a 0-5 scale according to the Pfirrmann classification. As a result of the study, the median age was 52.09±12.74years. There were statistical significances between age and MMP-1, and MMP-2. There was a close correlation between grade and MMP-9. We found correlation between the VAS and the MMP-9 expression. In addition, there was relationship between expression of MMP-2 and MMP-1, MMP-3, MMP-9. In conclusion, the expressions of MMP-1 and -2 are increased with aging. There was no relationship between radiological evaluation of IVDD and aging. Increased expression of MMPs affected IVDD positively. The relationship with MMPs is not explained. This study adds to our understanding of the interaction between MMPs and IVDD., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The molecular mechanisms of intervertebral disc degeneration (IDD) remain elusive. We found that miR-155 is down-regulated in degenerative nucleus pulposus (NP), and more severe degeneration is correlated with higher matrix metallopeptidase 16 (MMP-16) expression. MMP-16 also degraded matrix aggrecan. Here, we addressed the in vivo miR-155-mediated pathological impact on IDD using a classic puncture mouse model. Lentiviral upregulated-miR-155 or downregulated-miR-155 was transduced into the discs of C57 mice, which was validated by real-time polymerase chain reaction (real-time PCR) and in situ hybridization. Immunohistochemistry and western blotting revealed that up-regulation of miR-155 resulted in down-regulation of MMP-16 and an increase in aggrecan and collagen type II in mouse NP; whereas, down-regulation of miR-155 resulted in up-regulation of MMP-16 and a decrease in aggrecan in mouse NP. Radiographic and histological analysis showed that the up-regulation of miR-155 attenuated IDD, while down-regulation of miR-155 resulted in the deterioration of IDD. These findings indicate that decreased miR-155 contributed to the up-regulation of MMP-16 in vivo, and MMP-16 further degraded aggrecan and collagen type II, leading to the dehydration and degeneration of discs. Our findings revealed a therapeutic role for miR-155 in IDD. © 2017 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1323-1334, 2017., (© 2017 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society.)

BACKGROUND Degrading enzymes play an important role in the process of disc degeneration. The objective of this study was to investigate the correlation between the expression of high temperature requirement serine protease A1 (HtrA1) in the nucleus pulposus and the T2 value of the nucleus pulposus region in magnetic resonance imaging (MRI). MATERIAL AND METHODS Thirty-six patients who had undergone surgical excision of the nucleus pulposus were examined by MRI before surgery. Pfirrmann grading of the target intervertebral disc was performed according to the sagittal T2-weighted imaging, and the T2 value of the target nucleus pulposus was measured according to the median sagittal T2 mapping. The correlation between the Pfirrmann grade and the T2 value was analyzed. The expression of HtrA1 in the nucleus pulposus was analyzed by RT-PCR and Western blot. The correlation between the expression of HtrA1 and the T2 value was analyzed. RESULTS The T2 value of the nucleus pulposus region was 33.11-167.91 ms, with an average of 86.64±38.73 ms. According to Spearman correlation analysis, there was a rank correlation between T2 value and Pfirrmann grade (P<0.0001), and the correlation coefficient (rs)=-0.93617. There was a linear correlation between the mRNA level of HtrA1 and T2 value in nucleus pulposus tissues (a=3.88, b=-0.019, F=112.63, P<0.0001), normalized regression coefficient=-0.88. There was a linear correlation between the expression level of HtrA1 protein and the T2 value in the nucleus pulposus tissues (a=3.30, b=-0.016, F=93.15, P<0.0001) and normalized regression coefficient=-0.86. CONCLUSIONS The expression of HtrA1 was strongly related to the T2 value, suggesting that HtrA1 plays an important role in the pathological process of intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration is responsible for various spine pathologies and present clinical treatments are insufficient. Concurrently, the mechanisms behind IVD degeneration are still not completely understood, so as to allow development of efficient tissue engineering approaches. A model of rat IVD degeneration directly coupled to herniation is here proposed in a pilot study. Disc injury is induced by needle puncture, using two different needles gauges: a low caliber 25-G needle and a high caliber 21-G needle. Histological, biochemical, and radiographic degeneration was evaluated at 2 and 6 weeks post-injury. We show that the larger caliber needle results in a more extended histological and radiographic degeneration within the IVD, compared to the smaller one. TUNEL quantification indicates also increased cell death in the 21-G group. Analyses of collagen type I (Picrosirius red staining), collagen type II (immunofluorescence), and GAG content (Blyscan assay) indicate that degeneration features spontaneously recover from 2 to 6 weeks, for both needle types. Moreover, we show the occurrence of hernia proportional to the needle gauge. The number of CD68+ macrophages present, as well as cell apoptosis within the herniated tissue are both proportional to hernia volume. Moreover, hernias formed after lesion tend to spontaneously diminish in volume after 6 weeks. Finally, MMP3 is increased in the hernia in the 21-G group at 2 weeks. This model, by uniquely combining IVD degeneration and IVD herniation in the same animal, may help to understand mechanisms behind IVD pathophysiology, such as hernia formation and spontaneous regression. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:258-268, 2017., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Intervertebral disk degeneration is a progressive and debilitating disease with multifactorial causes. Nitric oxide (NO) might contribute to the cell death pathway. We evaluated the presence of the constitutive form of the neuronal NOS (nNOS) in both health and degenerated intervertebral disk through qPCR and immunohistochemistry. We also analyzed the potential role of nNOS modulation in the tail needle puncture model of intervertebral disk degeneration. Male Wistar rats were submitted to percutaneous disk puncture with a 21-gauge needle of coccygeal vertebras. The selective nNOS pharmacological inhibitor N (ω) -propyl-L-arginine (NPLA) or a nNOS-target siRNA (siRNAnNOShum&#95;4400) was injected immediately after the intervertebral disk puncture with a 30-gauge needle. Signs of disk degeneration were analyzed by in vivo magnetic resonance imaging and histological score. We found that intact intervertebral disks express low levels of nNOS mRNA. Disk injury caused a 4 fold increase in nNOS mRNA content at 5 h post disk lesion. However, NPLA or nNOS-target siRNA slight mitigate the intervertebral disk degenerative progress. Our data show evidence of the nNOS presence in the intervertebral disk and its upregulation during degeneration. Further studies would disclose the nNOS role and its potential therapeutical value in the intervertebral disk degeneration.

Senescence is a crucial driver of intervertebral disc degeneration (IDD). Disc cells are exposed to high oxygen tension due to neovascularization in degenerative discs. However, the effect of oxygen tension on disc cell senescence was unknown. Herein, rat nucleus pulposus (NP) cells were cultured under 20% O 2 or 1% O 2 . Consequently, ROS induced by 20% O 2 caused DNA damage and then activated p53-p21-Rb and p16-Rb pathways via ERK signaling to induce NP cell senescence. It also induced catabolic and proinflammatory phenotype of NP cells via MAPK and NF- κ B pathways. Furthermore, 20% O 2 was found to upregulate Nox4 in NP cells. Small interfering RNA against Nox4 reduced ROS production induced by 20% O 2 and consequently suppressed premature senescence of NP cells. On the contrary, NP cells overexpressing Nox4 produced more ROS and rapidly developed senescent signs. In consistent with the in vitro studies, the expression of Nox4, p21, and Rb was upregulated in rat degenerative discs. This study, for the first time, demonstrates that Nox4 is an oxygen-sensing enzyme and a main ROS source in NP cells. Nox4-dependent ROS are genotoxic and a potent trigger of NP cell senescence. Nox4 is a potential therapeutic target for disc cell senescence and IDD.

Destruction of extracellular matrix (ECM) leads to degeneration of the intervertebral disk (IVD), which is a major contributor to many spine disorders. IVD degeneration is induced by pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), which are secreted by immune cells, including macrophages and neutrophils. The cytokines modulate ECM-modifying enzymes such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human annulus fibrosus (AF) cells. The resulting imbalance in catabolic and anabolic enzymes can cause generalized back, neck, and low back pain (LBP). Photobiomodulation (PBM) is known to regulate inflammatory responses and wound healing. The aim of this study was to mimic the degenerative IVD microenvironment, and to investigate the effect of a variety of PBM conditions (wavelength: 635, 525, and 470 nm; energy density: 16, 32, and 64 J/cm(2)) on the production of ECM-modifying-enzymes by AF cells under degenerative conditions induced by macrophage-conditioned medium (MCM), which contains pro-inflammatory cytokines such as TNF-α and IL-β secreted by macrophage during the development of intervertebral disk inflammation. We showed that the MCM-stimulated AF cells express imbalanced ratios of TIMPs (TIMP-1 and TIMP-2) and MMPs (MMP-1 and MMP-3). PBM selectively modulated the production of ECM-modifying enzymes in AF cells. These results suggest that PBM can be a therapeutic tool for degenerative IVD disorders.

Background: Mechanical loading plays an important role in the regulation of extracellular matrix (ECM) homeostasis as well as pathogenesis of intervertebral disc (IVD) degeneration. The human annulus fibrosus (hAF) in the IVD is subjected to contact shear stress during body motion. However, the effects of shear stress on hAF cells remain unclear. This aim of the study was to investigate the expression of the ECM (COLI, COLIII and aggrecan) and matrix metalloproteinase (MMP-1, MMP-3 and ADAMTS-4) genes in hAF cells following fluid-induced shear stress in a custom-fabricated bio-microfluidic device., Methods: hAF cells were harvested from degenerated disc tissues in routine spine surgery, staged by magnetic resonance imaging, expanded in monolayers and then seeded onto the bio-microfluidic device. The experimental groups were subjected to 1 and 10 dyne/cm(2) shear stress for 4 h, and no shear stress was applied to the control group. We used real time polymerase chain reaction for gene expression., Results: Shear stress of 1 dyne/cm(2) exerted an anabolic effect on COLI and COLIII genes and catabolic effects on the aggrecan gene, while 10 dyne/cm(2) had an anabolic effect on the COLI gene and a catabolic effect on COLIII and aggrecan genes. The COLI gene was upregulated in a stress-dependent manner. Expression of MMP-1 was significantly higher in the 10 dyne/cm(2) group compared to the control group (P < 0.05), but was similar in the control and 1 dyne/cm(2) groups. Expression of MMP-3 and ADAMTS-4 were similar in all three groups., Conclusion: Taken together, hAF cells responded to shear stress. The findings help us understand and clarify the effects of shear stress on IVD degeneration as well as the development of a new therapeutic strategy for IVD degeneration.

Lumbar intervertebral disc degeneration (IDD) is a common clinical pathology and has become a focus for research in recent years. Matrix metalloproteinases (MMPs) are enzymes responsible for the degradation of almost all extracellular matrix proteins (ECM). The over-expression of MMPs or tissue inhibitors of metalloproteinases (TIMPs) may disrupt the dynamic balance of the ECM. Therefore, in the current study, the expression levels of MMP-1 and TIMP-1 in lumbar IDD patients were evaluated in an attempt to elucidate their role in IDD pathogenesis and progression. In total, 60 IDD patients were recruited as the experimental group, along with 20 cases of lumbar vertebral injury without disc degeneration as the control group. Preoperative venous blood samples were collected, and intervertebral disc tissues were collected from the lesion during surgery. Serum and tissue levels of MMP-1 and TIMP-1 were quantified by enzyme-linked immunosorbent assay and immunohistochemical staining, respectively. Serum and tissue MMP-1 levels in IDD patients were significantly higher than those in the control group (P < 0.05). Additionally, sub-group analysis revealed that severe IDD patients had higher MMP-1 levels compared with mild or moderate IDD patients (P < 0.05). However, there were no significant differences in TIMP- 1 levels in either the serum or tissues of IDD patients compared to patients in the control group (P > 0.05). These results demonstrate that MMP-1 expression is increased in IDD, with higher expression observed in more severe cases, whereas TIMP-1 expression was similarly expressed in both normal and degenerated discs.

Objective: To observe the effect of acupotomy lysis at acupoints around the neck on expression of matrix metalloproteinase 1 (MMP-1), MMP-2 and tissue inhibitor of metalloproteinase 1 (TIMP-1) genes and ultrastructure of pulpiform nucleus in cervical intervertebral disc degeneration (IVDD) rats, so as to explore its mechanism underlying easing IVDD., Methods: SD rats were randomly allocated to control (n = 15), model (n = 14), Jiaji (EX-B 2, n = 13), cervico-acupoint (n = 14) and medication groups (n = 14). The cervical IVDD model was established by using static-dynamic imbalance method. For rats of the Jiaji (EX-B 2) and cervico-acupoint groups, EX-B 2-points of the cervical 2-7 segments, and peri-cervical acupoints: bilateral "Naokong" (GB 19) , "Naohu" (GV 17), "Dazhui" (GV 14), bilateral "Quyuan" (SI 13) and bilateral "Tianzong" (SI 11) were separately punctured with a needle-knife, once every 5 days for 3 times, and for rats of the medication group, Brufen Capsules (15 mg · kg(-1) · d(-1)) and Jingfukang Granule (0.5 mg · kg(-1) · d(-1)) were given by intragastric administration, once daily for 10 days. The expression levels of MMP-1, MMP-3 and TIMP-1 genes in the pulpiform nucleus of cervical intervertebral discs were detected by RT-PCR and changes of the ultrastructure of the pulpiform nucleus observed under transmission electron microscope., Results: Compared to the control group, the expression levels of MMP-1 mRNA and MMP-3 mRNA of the cervical intervertebral disc tissues were significantly up-regulated in the model group (P < 0.05), and that of TIMP-1 mRNA was obviously down-regulated in the model group (P < 0.05). After the treatment, the increased expression of MMP-1 mRNA and MMP-3 mRNA and the decreased expression of TIMP-1 mRNA were reversed by acupotomy lysis and medication (P < 0.05) except TIMP-1 mRNA in the medication group (P > 0.05). No significant differences were found between the Jiaji (EX-B 2) and cervico-acupoint groups in down-regulating MMP-1 mRNA and MMP-3 mRNA expression and up-regulating TIMP-1 mRNA expression (P > 0.05). Results of electron microscope examinations showed that the ultrastructural injury changes of cells of the pulpiform nucleus were relatively milder in the Jiaji (EX-B 2) and cervico-acupoint groups, followed by the medication group in comparison with those of the model group., Conclusion: Acupotomy lysis at acupoints around the neck can improve the ultrastructural changes of cells of the pulpiform nucleus of cervical intervertebral discs in IVDD rats, which is possibly by regulating the expression of MMP-1, MMP-3 and TIMP-1 genes.

Background: This randomized controlled trial was carried out to (1) evaluate the effect of nucleoplasty with coblation on the PLA2 activity in the degenerative intervertebral disks of an animal model and (2) explore the possible therapeutic mechanism of coblation in addition to the current theory, which focuses on decreasing the intradiskal pressure in the treatment of intervertebral disk degeneration., Methods: Thirty-six animal models of intervertebral disk degeneration were successfully established and then randomly divided into two groups: the coblation group (n = 18) and coblation control group (n = 18). Nucleoplasty using coblation was performed in the coblation group. L4-5 and L5-6 intervertebral disk samples were harvested and analyzed for PLA2 activity in different groups at different time points., Results: The PLA2 activity in the coblation control group was significantly higher than that in the control group (194.86 ± 11.80 and 80.68 ± 5.56, respectively; P < 0.01). There was a significant decrease in the PLA2 activity 1 week after coblation than at the real time after coblation (154.39 ± 7.99 and 184.98 ± 9.43, respectively; P < 0.001). The PLA2 activity at 1 month after coblation remained at a lower level than those at 1 week and at the real time after coblation (142.63 ± 10.72, 154.39 ± 7.99, and 184.98 ± 9.43, respectively), but there was no significant decrease in the PLA2 activity between 1 week and 1 month after coblation., Conclusions: Coblation appeared to effectively degrade the PLA2 activity in the degenerative intervertebral disks of this animal model. This represents a potential mechanism for the clinical use of coblation in the treatment of low back pain.

Sulfatase 1 (SULF1) plays a key role in cell signaling involving in cell growth, differentiation, proliferation, and migration. Abnormal SULF1 expression has been implicated in the development of various cancers and diseases of the skeletal and nervous systems. The present study aims to examine the difference in SULF1 expression between degenerative and non-degenerative intervertebral discs (IVDs) to provide an enhanced understanding of disc degeneration. Degenerative and non-degenerative disc tissues were surgically harvested from patients and experimental rats. Disc degeneration-specific genes were identified by microarray analysis. The gene expression of SULF1 was measured by sulfatase assay, reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blotting. Also, the presence of SULF1 in human and rat discs was confirmed by immunohistochemistry. More specifically in human cells, an increase of SULF1 gene expression was observed in degenerative cells at both mRNA and protein levels, as well as in time- and dose-dependent manner in response to TNF-α treatment. Increased staining of SULF1 was detected in degenerative discs compared to non-degenerative discs for humans and rats. These findings show an upregulation of SULF1 in degenerative discs for the first time, and suggest that there is a link between SULF1 and disc degeneration., (© 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Background/aims: The pathogenesis of lumbar disc degeneration (LDD) involved activation of matrix metalloproteinase 13 (MMP13) by differential expression of fibroblast growth factor receptor 1 (FGFR1) and FGFR3. Nevertheless, the molecular regulation of FGFR1 and FGFR3 in the lumber disc cells remains elusive., Methods: We examined the FGFR1 and FGFR3 levels and microRNAs (miRNAs) levels in the resected LDD discs, compared to the traumatized, non-LDD discs. We analyzed the binding of miR-100 to the 3'UTR of FGFR3 mRNA and its effects on FGFR3 translation by bioinformatics analysis and by luciferase-reporter assay, respectively. We modified miR-100 levels in a human nucleus pulposus SV40 cell line (HNPSV), and examined the effects on the expression of FGFR3 and MMP13, by RT-qPCR, Western blot and ELISA., Results: The levels of FGFR1 and miR-100 were significantly higher, while the levels of FGFR3 were significantly lower, in LDD discs, compared to the control non-LDD discs. The levels of FGFR3, but not the levels of FGFR1, inversely correlated with the levels of miR-100. Moreover, miR-100 was found to bind to the 3'UTR of FGFR3 mRNA to prevent its translation. In miR-100-modified HNPSV cells, we found that miR-100 decreased FGFR3 levels, and increased MMP13 levels., Conclusion: miR-100 may activate MMP13 through 3'UTR-suppressoin of FGFR3 mRNA to facilitate development of LDD., (© 2015 S. Karger AG, Basel.)

SIRT1 could protect degenerative human NP cells against apoptosis, and there were extensive and intimate connection between apoptosis and autophagy. Up to now, the role of autophagy in the process of human IVD degeneration is unclear. We sought to explore the relationship between autophagy and human IVD degeneration and to understand whether autophagy is involved in the protective effect of SIRT1 against apoptosis in NP cells. Our results showed that the autophagosomes number, the mRNA level of LC3 and Beclin-1, the protein expression of LC3-II/I and Beclin-1, decreased in NP from DDD. Resveratrol could increase the protein expression of LC3-II/I and Beclin-1, and reduce apoptosis in degenerative NP cells. In contrast, the protein levels of LC3-II/I and Beclin-1 were down-regulated and apoptosis level was significantly up-regulated in treatment with nicotinamide or SIRT1-siRNA transfection. Further analysis identified that the expression of cleaved Caspase3 and apoptosis incidence significantly increased with the pretreatment of bafilomycin A, whether resveratrol was added or not. These suggested that autophagy may play an important role in IVD degeneration, and SIRT1 protected degenerative human NP cells against apoptosis via promoting autophagy. These findings would aid in the development of novel therapeutic approaches for degenerative disc disease treatment.

Matrix metalloproteinases (MMPs) have been known to play a pivotal role in the age- and/or disease-related degradation of intervertebral discs. We aimed to explore as to whether the expression of these enzymes is correlated to disc degeneration caused by increasing age and severity of herniation in the East Asian population. Thus, we studied the expressions of MMP-1 (collagenase), MMP-2 (gelatinase) and MMP-14 (membrane-type protease) in 65 patients diagnosed with lumbar disc herniation. The patients were divided into 3 groups according to their age, and the severity of herniation was graded on the basis of magnetic resonance imaging (MRI). Immunohistochemistry analysis was conducted to determine the expression of different MMPs in the post-surgery disc specimens. The results showed that expressions of these three enzymes were directly and positively related to the degree of disc degradation. Whereas, the MMP-1 expression was found to be elevated with the increasing age, the MMP-2 and MMP-14 remained unchanged in groups of different ages. A direct correlation between the expressions of MMP-2 and MMP-14 suggested a role of MMP-14 in the modulation of MMP-2 expression.

Objective: To observe the effect of electroacupuncture(EA) stimulation of "Jiaji" (EX-B 2) on the expression of matrix metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1) proteins in the lumbar disc in rabbits with lumbar intervertebral disc degeneration so as to explore its mechanism in relieving intervertebral disc degeneration., Methods: A total of 36 New Zealand rabbits were randomly divided into normal, sham operation (sham), model, and EA groups, with 9 rabbits in each group. The lumbar intervertebral disc degeneration model was established by using a custom-made external loading device to axially compress the lumbar discs (L4, L5) for 28 days in reference to Kroeber and colleagues' methods. After modeling, EA stimulation (2 Hz/15 Hz, 1-2 mA) was applied to bilateral "Jiaji" (EX-B 2) areas for 20 min, once daily for 28 days. The expression levels of MMP-13 and TIMP-1 proteins of the lumbar intervertebral disc (L4-L5) tissues were assayed by Western blot and immunoflorescence methods, separately., Results: Compared to the normal and sham groups, MMP-13 expression levels at the time-points of day 28 in the model group, on day 28 (pre-EA) in the EA group were significantly up-regulated (P < 0.01); and TIMP-1 expression levels on day 28 in both model and EA groups were significantly decreased (P < 0.01). Following EA treatment, the expression level of MMP-13 was notably lower in the EA group than in the model group, and that of TIMP-1 was remarkably higher in the EA group than in the model group (P < 0.01). No significant differences were found in the expression levels of MMP-13 and TIMP-1 from day 1 to day 56 in the same one group of both normal and sham groups, and between these two groups (P > 0.05)., Conclusion: Electroacupuncture at "Jiaji" (EX-B 2) can effectively suppress intervertebral disc degeneration induced up-regulation of MMP-13 protein and down-regulation of TIMP-1 protein in the disc tissue in rabbits, which may contribute to its effect in bettering lumbago in clinic.

The intervertebral disc nucleus pulposus (NP) has two phenotypically distinct cell types-notochordal cells (NCs) and non-notochordal chondrocyte-like cells. In human discs, NCs are lost during adolescence, which is also when discs begin to show degenerative signs. However, little evidence exists regarding the link between NC disappearance and the pathogenesis of disc degeneration. To clarify this, a rat tail disc degeneration model induced by static compression at 1.3 MPa for 0, 1, or 7 days was designed and assessed for up to 56 postoperative days. Radiography, MRI, and histomorphology showed degenerative disc findings in response to the compression period. Immunofluorescence displayed that the number of DAPI-positive NP cells decreased with compression; particularly, the decrease was notable in larger, vacuolated, cytokeratin-8- and galectin-3-co-positive cells, identified as NCs. The proportion of TUNEL-positive cells, which predominantly comprised non-NCs, increased with compression. Quantitative PCR demonstrated isolated mRNA up-regulation of ADAMTS-5 in the 1-day loaded group and MMP-3 in the 7-day loaded group. Aggrecan-1 and collagen type 2α-1 mRNA levels were down-regulated in both groups. This rat tail temporary static compression model, which exhibits decreased NC phenotype, increased apoptotic cell death, and imbalanced catabolic and anabolic gene expression, reproduces different stages of intervertebral disc degeneration., (© 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Cytokeratin 8 (CK8) is a member of the cytokeratins family with multiple functions on the basis of its unique structural hallmark. The aberrant expression of CK8 and its phosphorylation are pertinent with various diseases. We have previously shown that CK8 exists in normal human nucleus pulposus (NP) cells and decreases as the intervertebral disc degenerates. However, the underlying molecular regulatory machinery of CK8 in intervertebral disc degeneration (IDD) has not been clarified. Here, we collected NP samples from patients with idiopathic scoliosis as control and IDD as degenerate groups. We found that CK8 expression decreased in IDD with an increased phosphorylation in degenerate NP cells. Moreover, NP cells were cultured under different compressive load schemes for diverse time duration. We found that compressive loads resulted in phosphorylation and disassembly of CK8 in a time-dependent and degree-dependent manner in vitro. The activation of protein kinase C was a significant molecular factor contributing to this phenomenon. Taken together, this study is the first to address the molecular mechanisms of CK8 downregulation in NP cells. Importantly, our findings provide clues regarding a molecular link between compressive loads and CK8 alterations, which shed a novel light on the etiology of IDD.

Study Design: An in situ study using whole-organ culture system., Objective: To study the effect of disc degeneration at different stages on its rheological and dynamic properties and to investigate the efficacy of exogenous cross-linking therapy., Summary of Background Data: Disc degeneration can involve protein denaturation or microdefects to the disc's collagen fiber network. A disc degeneration model using whole-organ culture technique can be effectively used for the screening of treatments of degenerated discs. Exogenous cross-linking therapy has been shown to enhance the mechanical properties of the disc by cross-linking collagen. However, the efficacy of this therapy on the degenerated disc is unclear., Methods: A total of 40 porcine thoracic discs were assigned to 5 groups: intact discs, moderately degenerated discs, moderately degenerated discs with cross-linker augmentation, severely degenerated discs, and severely degenerated discs with cross-linker augmentation. The disc degeneration was simulated by trypsin digestion and mechanical fatigue loading. Rheological properties, dynamic properties, water content, and histological analysis were conducted after a 7-day incubation., Results: The mechanical properties of moderate degenerated discs significantly decrease both in rheological and dynamic properties, and laminate structure disorganization was observed. Mechanical defects of severely degenerated discs resulted in disc height loss, an increase in the aggregate modulus and stiffness modulus, and a decrease in the damping coefficient, hydraulic permeability, and water content. Cross-linker augmentation significantly recovered mechanical properties of moderately degenerated discs and restored the water content compared with the intact disc. However, the augmentation did not fully repair the severely degenerated discs., Conclusion: Trypsin-induced extracellular matrix damage resulted in a change of the disc's biomechanics. Cross-linker augmentation recovers the rheological and dynamic properties of moderately degenerated discs but not of the severely degenerated discs. The genipin cross-linker may be able to improve the proteoglycan depletion effect in the nucleus pulposus but may not be effective to restore the structural damage in the collagen molecule of the anulus fibrosus.

Lumbar disc degeneration (LDD) is associated with both genetic and environmental factors and affects many people worldwide. A hallmark of LDD is loss of proteoglycan and water content in the nucleus pulposus of intervertebral discs. While some genetic determinants have been reported, the etiology of LDD is largely unknown. Here we report the findings from linkage and association studies on a total of 32,642 subjects consisting of 4,043 LDD cases and 28,599 control subjects. We identified carbohydrate sulfotransferase 3 (CHST3), an enzyme that catalyzes proteoglycan sulfation, as a susceptibility gene for LDD. The strongest genome-wide linkage peak encompassed CHST3 from a Southern Chinese family–based data set, while a genome-wide association was observed at rs4148941 in the gene in a meta-analysis using multiethnic population cohorts. rs4148941 lies within a potential microRNA-513a-5p (miR-513a-5p) binding site. Interaction between miR-513a-5p and mRNA transcribed from the susceptibility allele (A allele) of rs4148941 was enhanced in vitro compared with transcripts from other alleles. Additionally, expression of CHST3 mRNA was significantly reduced in the intervertebral disc cells of human subjects carrying the A allele of rs4148941. Together, our data provide new insights into the etiology of LDD, implicating an interplay between genetic risk factors and miRNA.

Chondroadherin, a member of the leucine-rich repeat family, has previously been demonstrated to be fragmented in some juveniles with idiopathic scoliosis. This observation led us to investigate adults with disc degeneration. Immunoblotting analysis demonstrated that non-degenerate discs from three different age groups show no chondroadherin fragmentation. Furthermore, the chondroadherin fragments in adult degenerate disc and the juvenile scoliotic disc were compared via immunoblot analysis and appeared to have a similar size. We then investigated whether or not chondroadherin fragmentation increases with the severity of disc degeneration. Three different samples with different severities were chosen from the same disc, and chondroadherin fragmentation was found to be more abundant with increasing severity of degeneration. This observation led us to the creation of a neoepitope antibody to the cleavage site observed. We then observed that the cleavage site in adult degenerate discs and juvenile scoliotic discs was identical as confirmed by the neoepitope antibody. Consequently, investigation of the protease capable of cleaving chondroadherin at this site was necessary. In vitro digests of disc tissue demonstrated that ADAMTS-4 and -5; cathepsins K, B, and L; and MMP-3, -7, -12, and -13 were incapable of cleavage of chondroadherin at this site and that HTRA1 was indeed the only protease capable. Furthermore, increased protein levels of the processed form of HTRA1 were demonstrated in degenerate disc tissues via immunoblotting. The results suggest that chondroadherin fragmentation can be used as a biomarker to distinguish the processes of disc degeneration from normal aging.

We investigated TNF-α and IL-1β regulation of ADAMTS-4 expression in nucleus pulposus (NP) cells and its role in aggrecan degradation. Real-time quantitative RT-PCR, Western blotting, and transient transfections with rat NP cells and lentiviral silencing with human NP cells were performed to determine the roles of MAPK and NF-κB in cytokine-mediated ADAMTS-4 expression and function. ADAMTS4 expression and promoter activity increased in NP cells after TNF-α and IL-1β treatment. Treatment of cells with MAPK and NF-κB inhibitors abolished the inductive effect of the cytokines on ADAMTS4 mRNA and protein expression. Although ERK1, p38α, p38β2, and p38γ were involved in induction, ERK2 and p38δ played no role in TNF-α-dependent promoter activity. The inductive effect of p65 on ADAMTS4 promoter was confirmed through gain and loss-of-function studies. Cotransfection of p50 completely blocked p65-mediated induction. Lentiviral transduction with shRNA plasmids shp65, shp52, shIKK-α, and shIKK-β significantly decreased TNF-α-dependent increase in ADAMTS-4 and -5 levels and aggrecan degradation. Silencing of either ADAMTS-4 or -5 resulted in reduction in TNF-α-dependent aggrecan degradation in NP cells. By controlling activation of MAPK and NF-κB signaling, TNF-α and IL-1β modulate expression of ADAMTS-4 in NP cells. To our knowledge, this is the first study to show nonredundant contribution of both ADAMTS-4 and ADAMTS-5 to aggrecan degradation in human NP cells in vitro., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Study Design: This is a quantitative study of heparanase isoforms expression in degenerative and nondegenerative intervertebral discs (IVDs)., Objective: To quantify the expression of both heparanase isoforms (HPSE1 and HPSE2) in IVD tissues as classified by different degeneration grades using the Pfirrmann grading system, and to correlate the expression with the loss of extracellular matrix molecules observed in patients with the disease., Summary of Background Data: The loss of proteoglycans as observed in IVD degeneration may occur due to the enhanced expression of matrix degrading enzymes, such as heparanase. However, the heparanase function in IVD degeneration remains unclear., Methods: This study comprised 53 surgical samples of degenerative discs obtained from patients with lumbar disc degeneration and 12 control samples collected from healthy individuals without any degenerative lumbar disc alterations who had accidental spine fractures.All patients underwent magnetic resonance imaging based on the Pfirrmann grading system for disc degeneration. Only the specimens that were classified according to magnetic resonance imaging evaluations as Pfirrmann grades I, II, III, and IV were analyzed.The tissue sections of the disc samples were subject to immunohistochemical staining with antibodies against the heparanase isoforms and to quantitative real time PCR to amplify heparanase isoforms cDNA. Protein and mRNA expressions were quantified. Analysis of variance and Student t test were used to compare the means of the study populations., Results: The data demonstrated a gradual increase in both the heparanase isoform protein expression and disc degeneration progression. Besides, mRNA expression of both heparanase isoforms were significantly higher in degenerative than nondegenerative IVDs., Conclusion: The overexpression of HPSE1 and HPSE2 in the intervertebral degenerated discs suggests a role for these factors in mediating extracellular matrix remodeling in degenerative discs during disease development.

High-temperature requirement serine protease A1 (HTRA1) is one of four known proteases belonging to the broadly conserved family of HTRA proteins. Although it was originally considered as representing an important modulator of tumorigenesis, an increasing number of reports have suggested that its influence on human disease may extend beyond cancer. HTRA1 has the capacity to degrade numerous extracellular matrix proteins, and as such, its potential involvement in diseases of the musculoskeletal system has been gaining increased attention. Musculoskeletal disease constitutes a wide variety of degenerative conditions that can manifest themselves in different ways such as joint and back pain, as well as deficiencies in skeletal bone quality, and ultimately result in significant suffering and reduced quality of life. Convincing data now exist to support a detrimental role for HTRA1 in the pathogenesis of joint and intervertebral disk degeneration. However, the function of HTRA1 in other closely related musculoskeletal diseases affecting bone and muscle remains unclear and largely unexplored. To help set the stage for future research, we discuss here some of the recent advances in our understanding of the role played by HTRA1 in musculoskeletal pathology., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Object: Disc-related disorders such as herniation and chronic degenerative disc disease (DDD) are often accompanied by acute or chronic pain. Different mediators have been identified in the development of radicular pain and DDD. Previous studies have not analyzed individual cytokine profiles discriminating between acute sciatic and chronic painful conditions, nor have they distinguished between different anatomical locations within the disc. The aim of this study was to elucidate the protein biochemical mechanisms in DDD., Methods: The authors determined expression levels of matrix metalloproteinase-3, transforming growth factor-β (TGF-β), tumor necrosis factor-α, interleukin-1α, and pro-substance P using enzyme-linked immunosorbent assay and Western blot analyses in patients suffering from DDD (n = 7), acute back pain due to herniated discs with radiculopathy (n = 7), and a control group (n = 7). Disc tissue samples from the anulus fibrosus (AF) and nucleus pulposus (NP) were analyzed. Statistical analysis was performed using nonparametric tests., Results: A distinct distribution of cytokines was found in different anatomical regions of intervertebral discs in patients with DDD and herniated NP. Increased TGF-β levels were predominantly found in DDD. Matrix metalloproteinase-3 was increased in acute herniated disc material. Increased levels of substance P were found in patients suffering from DDD but not in patients with disc herniation. The data showed significantly higher levels of proinflammatory cytokines in the AF and NP of patients with DDD, and the expression levels in the AF were even higher than in the NP, suggesting that the inflammatory response initiates from the AF., Conclusions: These results highlight the complex mechanisms involved during disc degeneration and the need to distinguish between acute and chronic processes as well as different anatomical regions, namely the AF and NP. They also highlight potential problems in disc nucleus replacement therapies because the results suggest a biochemical link between AF and NP cytokine expression.

Introduction: Degeneration of the intervertebral disc (IVD) is characterized by marked degradation and restructuring of the annulus fibrosus (AF). Although several matrix metalloproteinases (MMPs) have been found to be more prevalent in degenerate discs, their coordination and function within the context of the disease process are still not well understood. In this study, we sought to determine whether MMP-2 is associated with degenerative changes in the AF and to identify the manner by which AF cells use MMP-2., Methods: Two established animal models of disc degeneration, static compression and transannular needle puncture of rodent caudal discs, were examined for MMP-2 immunopositivity. With lentiviral transduction of an shRNA expression cassette, we screened and identified an effective shRNA sequence for generating stable RNA interference to silence MMP-2 expression in primary rat AF cells. Gelatin films were used to compare gelatinase activity and spatial patterns of degradation between transduced cells, and both noninfected and nonsense shRNA controls. The functional significance of MMP-2 was determined by assessing the ability for cells to remodel collagen gels., Results: Both static compression and 18-g annular puncture of rodent caudal discs stimulated an increase in MMP-2 activity with concurrent lamellar disorganization in the AF, whereas 22-g and 26-g needle injuries did not. To investigate the functional role of MMP-2, we established lentivirus-mediated RNAi to induce stable knockdown of transcript levels by as much as 88%, and protein levels by as much as 95% over a 10-day period. Culturing transduced cells on gelatin films confirmed that MMP-2 is the primary functional gelatinase in AF cells, and that MMP-2 is used locally in regions immediately around AF cells. In collagen gels, transduced cells demonstrated an inability to remodel collagen matrices., Conclusions: Our study indicates that increases in MMP-2 observed in human degenerate discs are mirrored in experimentally induced degenerative changes in rodent animal models. AF cells appear to use MMP-2 in a very directed fashion for local matrix degradation and collagen remodeling. This suggests that MMP-2 may have a functionally significant role in the etiology of degenerative disc disease and could be a potential therapeutic target.

Aim: The purpose of the present study is to analyze the expression of matrix metalloproteinase-3 (MMP-3), magnetic resonance imaging (MRI) grading and histopathological alterations of the intervertebral disc (IVD) for correlations with each other and with the age, gender and low back pain duration of the patients who had undergone operations for lumbar disc herniation (LDH)., Material and Methods: Forty-two patients were admitted to our clinic with signs of LDH and underwent surgery for LDH at 48 IVD levels. In all cases, specimens for histological and immunohistochemical analyses were removed from the IVD space. Lumbar IVD degeneration on MRI of the 48 IVDs from which surgical specimens had been obtained was classified into five grades using the Pfirrmann classification., Results: In the degenerated IVD, the expression of MMP-3, MRI grading and histopathological alterations of the IVD displayed significant correlation. Increased age is closely related with aforementioned alterations. There was no correlation between MMP-3 expression and age, gender and duration of the pain., Conclusion: For evaluating and treating IVD degeneration, MRI is a good and non-invasive diagnostic tool to determine the severity of degeneration. MMP-3 may be a therapeutic target of the degenerated IVD.

We examined the effects of ulinastatin on the expression of inducible nitric oxide synthase (iNOS), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-3 (MMP-3) in degenerated nucleus pulposus (NP) cells of rabbits induced by interleukin (IL)-1β in vitro. An in vitro NP cell culture model was set up with enzyme digestion. NP cells from adult white rabbits were divided into six groups: the normal control group, the ulinastatin control group (320 U/mL ulinastatin), the induced group (10 ng/mL IL-1β), and three inhibition groups (IL-1β followed by 160, 320, or 640 U/mL ulinastatin). After a 2-day culture, the NP cells were collected for immunohistochemical staining for MMP-2 and MMP-3 and spectrophotometric analysis of the amount of iNOS. Immunohistochemical staining showed that the expression of MMP-2 and MMP-3 proteins in NP cells decreased in the inhibition groups compared with the induced group, which was in inverse proportion to the ulinastatin concentration. Spectrophotometric results showed that, compared with the induced group, the iNOS content in each inhibition group decreased, most significantly in the 320 U/mL group. Ulinastatin effectively inhibited the increased expression of MMP-2, MMP-3, and iNOS in degenerated NP cells induced by IL-1β in vitro. It suggests that ulinastatin may potentially be useful for clinical therapy of intervertebral disc degeneration.

Although intervertebral disc (IVD) degeneration is one of most common causes of morbidity, its etiology remains unclear. In healthy discs, the rates of synthesis and breakdown of the extracelluar matrix (ECM) are in equilibrium because of intricate regulation by growth factors and catabolic cytokines. Important among these physiologic growth factors are transforming growth factor-β (TGF-β1) and bone morphogenetic protein-2 (BMP-2). Disc degeneration is thought to be associated with a loss of this homeostasis between proteoglycan (PG) synthesis and cytokine-induced degradation leading to up-regulation of matrix metalloproteinases (MMP) families and down-regulation of extracelluar matrix production. Several strategies using biological agents have been attempted to manage IVD degeneration, improving the function and anabolic capabilities of IVD cells and inhibiting matrix degradation. The purpose of this study is to compare the effects of the anabolic cytokines BMP-2 and TGF-β1 with those of the catabolic cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) on porcine annulus fibrosus (AF). The results of this study show that the application of pro-inflammatory cytokines like tumor necrosis factor-α and interleukin-1β to normal annulus fibrosus cells leads to a significant increase in tissue levels of the degradative protease MMP-1. Treatment with a combination of minimum doses of both BMP-2 and TGF-β1 caused a greater decrease in MMP-1 and increase in aggrecan than either cytokine alone, suggesting a synergistic effect of the combined cytokines.

Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration. HTRA1 mRNA expression was significantly elevated in degenerated disc tissue and was associated with increased protein levels. However, these increases did not correlate with the appearance of rs11200638 single nucleotide polymorphism in the promoter region of the HTRA1 gene, as has previously been suggested. Recombinant HTRA1 induced MMP production in IVD cell cultures through a mechanism critically dependent on MEK but independent of IL-1β signaling. The use of a catalytically inactive mutant confirmed these effects to be primarily due to HTRA1 serine protease activity. HTRA1-induced fibronectin proteolysis resulted in the generation of various sized fragments, which when added to IVD cells in culture, caused a significant increase in MMP expression. Furthermore, one of these fragments was identified as being the amino-terminal fibrin- and heparin-binding domain and was also found to be increased within HTRA1-treated IVD cell cultures as well as in disc tissue from patients with IVD degeneration. Our results therefore support a scenario in which HTRA1 promotes IVD degeneration through the proteolytic cleavage of fibronectin and subsequent activation of resident disc cells.

Objective: Protein kinase Cδ (PKCδ) activation has been shown to be a principal rate-limiting step in matrix-degrading enzyme production in human articular chondrocytes. The aim of this study was to assess the role of the PKC pathways, specifically PKCδ, in intervertebral disc tissue homeostasis., Methods: Using in vitro, ex vivo, and in vivo techniques, we evaluated the pathophysiologic role of the PKCδ pathway by examining 1) proteoglycan deposition, 2) matrix-degrading enzyme production and activity, 3) downstream signaling pathways regulated by PKCδ, and 4) the effect on in vivo models of disc degeneration in genetically engineered PKCδ-knockout mice., Results: Studies of pathway-specific inhibitors revealed a vital role of the PKCδ/MAPK (ERK, p38, JNK) axis and NF-κB in disc homeostasis. Accordingly, in an in vivo model of disc injury, PKCδ-knockout mice were markedly resistant to disc degeneration., Conclusion: Suppression of the PKCδ pathway may be beneficial in the prevention and/or treatment of disc degeneration. The results of this study provide evidence for a potential therapeutic role of pathway-specific inhibitors of the PKCδ cascade in the future., (Copyright © 2012 by the American College of Rheumatology.)

Introduction: The 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., Methods: Forty-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., Results: Quantitative 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., Conclusions: This 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.

Matrix metalloproteinase (MMP) regulation and expression is important in the aging/degenerating human intervertebral disc. MMP-26 (also known as matrilysin-2 or endometase) is a newly discovered MMP which degrades type IV collagen, fibronectin, fibrinogen, vitronectin, denatured collagen types I-IV, insulin-like growth factor binding protein 1, and activated pro-MMP-9. Our objective here was to determine if it is present in human disc tissue and cultured disc cells. Immunohistochemistry and microarray gene expression analyses were used to evaluate the presence of MMP-26 in human disc tissue from healthy and degenerated discs. Immunohistochemistry was also applied to human annulus cells cultured in a collagen sponge. Cellular and matrix localization of MMP-26 was identified in the outer and inner annulus and in the nucleus pulposus. Fewer cells showed localization in the inner vs. outer annulus, and localization was sparse in the nucleus. During in vitro culture of annulus cells, MMP-26 was also expressed. Molecular analyses showed significant downregulation of expression of MMP-26 (p=0.03), and significant 9.8-fold upregulation of TGF-beta (p=0.01) in more degenerated discs vs. healthier discs. Findings document the first identification of MMP-26 in the disc at the molecular and protein levels. Results point to the potentially important role of MMP-26 in matrix modulation during disc health and degeneration., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The present study investigates the histological alterations and expression of matrix metalloproteinase 9 (MMP-9) in disc specimens of 43 patients who underwent surgery for lumbar disc herniation. The immunostaining for MMP-9 was evaluated semi-quantitatively. Histologic degeneration was scored between 0 and 12 depending on the degree of chondrocyte proliferation and presence of tears and clefts, granular changes and mucous degeneration. Herniation was graded as grade 1 (protrusion), grade 2 (extrusion) or grade 3 (sequestration) on MRI. Although there was no significant statistical difference between the histologic degeneration score and age, degenerative changes were more pronounced in higher grade of herniation (p<0.0001). MMP-9 expression was related to histologic degenerative score in all age groups (p=0.0065). MMP-9 was also related to herniation grade in patients younger than 30 years of age (p=0.0037). No significant association was found between MMP-9 expression and herniation grade in patients who were 30-60 years or over 60 years of age., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: To assess the expression of calpains and calpain-induced aggrecan fragmentation in early and advanced stages of degeneration of human intervertebral discs (IVDs)., Design: Disc tissue samples of 55 patients (mean age, 51.2 ± 22.3 years) who underwent intervertebral fusion were divided into groups with early and advanced degeneration based on the Thompson magnetic resonance imaging (MRI) scale. In advanced degeneration group, five patients (mean age, 35.5 ± 11.4 years) of lumbar disc herniation (LDH) were included. Protein levels of m- and μ-calpains and their inhibitor calpastatin were assayed, and immunohistochemical techniques were used to localize and quantify the production of the enzymes. To investigate calpain activity, we assayed purified aggrecan fragmentation in disc tissue by Western blotting and immunohistochemistry with VPGVA antibody, which recognizes the m-calpain generated neo-epitope GVA., Results: Discs at early stages of degeneration expressed low levels of m- and μ-calpains and calpastatin, and few cells expressed degenerative enzymes. At more advanced stages of degeneration, the expression and number of cells immunopositive for m-calpain, μ-calpain and calpastatin were significantly higher. Further finding showed that anti-GVA-reactive aggrecan fragments were significantly higher in discs at advanced compared with early stages of degeneration. Herniated disc samples showed stronger expression and more cells immunopositive for calpains, calpastatin and GVA in the nucleus pulposus than in the annulus fibrous., Conclusions: The expression of calpains, together with m-calpain-induced degradation products of extracellular matrix, was correlated with the degree of disc degeneration in human IVD tissue. These findings suggest that calpains may be involved in IVD degeneration via proteoglycan (PG) cleavage., (Copyright © 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: The complexity of the clinical, biochemical, hystochemical and immunologic aspects of the intervertebral disk, along with its molecular biology, justifies the object of our study on the extracellular matrix modifications in lumbar disk hernias and their impact on patient quality of life., Material and Method: The research lot was composed of 50 patients, aged between 18 and 73, who have undergone lumbar disk hernia surgery. MMP-9 (metalloproteinase-9) and TIMP-1 (tissue inhibitor of matrix metalloprotease 1) have been dosed in order to study the modifications on extracellular disk matrix, and quality of life assessment was carried out both in pre-operatory and post-operatory periods., Conclusions: Patients may prevent the appearance of degenerative processes of the intervertebral disk with care and responsibility by controlling their weight, avoiding intense physical activities and ceasing to smoke.