MiR-874-3p plays a protective role in intervertebral disc degeneration by suppressing MMP2 and MMP3.

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.

Sod2 and catalase improve pathological conditions of intervertebral disc degeneration by modifying human adipose-derived mesenchymal stem cells.

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-1ß, 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.

Human Levels of MMP-1 in Degenerated Disks Can Be Mitigated by Signaling Peptides from Mesenchymal Stem Cells.

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.

Inflammatory cytokine and catabolic enzyme expression in a goat model of intervertebral disc degeneration.

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-1ß [IL-1ß], 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-ß, 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.

Caspase-3 knockout inhibits intervertebral disc degeneration related to injury but accelerates degeneration related to aging.

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.

Paraoxonase 1 Was Negatively Associated With Intervertebral Disc 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)-1ß, 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-1ß 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.

AMP-Activated Protein Kinase Activation in Dorsal Root Ganglion Suppresses mTOR/p70S6K Signaling and Alleviates Painful Radiculopathies in Lumbar Disc Herniation Rat Model.

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.

Proinflammatory macrophages promote degenerative phenotypes in rat nucleus pulpous cells partly through ERK and JNK signaling.

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.

Leptin regulates disc cartilage endplate degeneration and ossification through activation of the MAPK-ERK signalling pathway in vivo and in vitro.

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.

Correlation Between Expression of High Temperature Requirement Serine Protease A1 (HtrA1) in Nucleus Pulposus and T2 Value of Magnetic Resonance Imaging.

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.

The Presence of the Neuronal Nitric Oxide Synthase Isoform in the Intervertebral Disk.

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_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.

Joint analysis of IVD herniation and degeneration by rat caudal needle puncture model.

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.

Photobiomodulation on human annulus fibrosus cells during the intervertebral disk degeneration: extracellular matrix-modifying enzymes.

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-1ß), 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-ß 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.

Fluid-induced, shear stress-regulated extracellular matrix and matrix metalloproteinase genes expression on human annulus fibrosus cells.

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.

Effect of percutaneous nucleoplasty with coblation on phospholipase A2 activity in the intervertebral disks of an animal model of intervertebral disk degeneration: a randomized controlled trial.

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.

Increased sulfatase 1 gene expression in degenerative intervertebral disc cells.

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.

Expression profiles of MMP-1 and TIMP-1 in lumbar intervertebral disc 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.

Expression of matrix metalloproteinases is positively related to the severity of disc degeneration and growing age in the East Asian lumbar disc herniation patients.

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.

Correlation of matrix metalloproteinase-3 expression with patient age, magnetic resonance imaging and histopathological grade in lumbar disc degeneration.

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.

Rheological and dynamic integrity of simulated degenerated disc and consequences after cross-linker augmentation.

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.