Introduction

Scientific knowledge on the biology of intervertebral disc (ID) degeneration has improved considerably over the past decades. Genetic inheritance, aging, nutritional compromise and repetitive loading have been found to promote the disorganization of normal extracellular matrix turnover along with the degradation of its major structural proteins [1, 18]. Consequently, the structural integrity and hydration levels of the disc are compromised, leading to loss of the disc’s functional properties.

Intervertebral disc herniation is the most prevalent clinical manifestation of this wear process. The type of herniation (protrusion, extrusion or sequestration) together with age and duration of symptoms have been correlated to different degrees of molecular changes within the disc cells and its extracellular matrix [16]. In addition, serial magnetic resonance imaging (MRI) studies have documented the spontaneous resorption of non-operatively treated herniated discs [17, 31]. Contact between the herniated fragment of the ID and the epidural space was thought to trigger an autoimmune response with inflammatory cell infiltration and in situ neovascularization, leading to the progressive resorption of the herniated fragment and regression of symptoms. Mounting evidence has shown that growth factors, matrix metalloproteinases (MMPs) and inflammatory mediators, such as cytokines, play a key role in ID homeostasis and pathology [9, 15]. However, the role of such molecules in the pathogenesis of ID herniation as well as in the spontaneous regression of herniated disc fragments remains only partially elucidated.

Matrix metalloproteinases are a family of proteolytic enzymes that are known to be implicated in both the normal extracellular matrix turnover and degradation [28]. Currently, 24 members of the MMP family have been identified with a wide array of potential substrates. The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) group of enzymes is a subfamily of metalloproteinases with the ability to degrade aggrecan acting at a different cleavage site than MMPs [25]. Among several members of the ADAMTS family, ADAMTS-4 plays a central role in cartilage degradation.

The involvement of several MMPs and ADAMTS in the pathogenesis and regression of ID herniation has been studied mainly by means of biochemistry and immunohistochemistry [26, 29]. However, few studies have investigated the mRNA transcription levels of the above enzymes, particularly in human ID herniation samples. A quantitative molecular analysis of the mRNA expression levels of the MMPs will provide insights into the molecular mechanism that regulates the expression of MMPs in disc herniation. In addition, the majority of the presented studies examined a limited number of MMPs in relatively small and heterogenic groups of ID specimens. Therefore, the present study aimed to concomitantly examine the transcript levels of a large number of MMPs (-1, -3, -8, -9, -13 and -14) and ADAMTS-4 in herniated and control disc specimens to elucidate their mRNA expression profile, as well as to investigate their correlation with the clinicopathological profile of patients suffering from symptomatic lumbar ID herniation.

Materials and methods

Tissue samples

Intervertebral disc tissue samples were collected from a total of 63 patients who underwent posterior open discectomy for lumbar ID herniation in our department during a recruitment period of 1 year (2008). The patients reported symptoms of radiculopathy prior to surgery. Patients suffering from spinal stenosis, spondylolisthesis, scoliosis or systemic inflammatory disorders were excluded from the study. After excision, an experienced pathologist, in addition to the clinicians present, examined the tissue samples and identified the pulposus sections to limit the percentage of end-plates or annulus. Samples were then immediately refrigerated at −80°C until RNA extraction. Ten cadaveric ID tissue samples were also obtained within 12 h of the patient succumbing to the disease. The samples were used as controls of normal MMP mRNA expression. Donors were aged between 20 and 46 years and were known to have a negative history of low back pain, spinal trauma or systemic inflammatory disease. All the patients or relatives signed an informed consent form approved by the university ethics committee to participate in the study. The study design was in accordance with the Declaration of Helsinki guidelines.

Prior to surgery, an MRI examination was routinely performed to be used for ID herniation classification. To determine the type of herniation, recommendations of the combined task force from the North American Spine Society, the American Society of Spine Radiology and the American Society of Neuroradiology based on the imaging characterization of Milette were used [5]. Preoperative pain intensity was measured subjectively using the visual analog scale score (VAS). Patient employment was defined as heavy, provided it included manual labor with repetitive loading of the spine. A summary of patient and control group data are shown in Table 1a, b. There was no significant difference in terms of age, gender, body mass index, employment and smoking habits between the control and ID herniation groups (min P > 0.24).

Table 1 Patient clinical characteristics
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RNA extraction, reverse transcription and real-time PCR

Total RNA was isolated from fresh tissue and homogenized with a power homogenizer using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration and purity were determined on a UV spectrophotometer (Hitachi Instruments Inc., USA) by absorbance measurements (260-nm absorbance and 260/280-nm absorbance ratio). RNA integrity was examined by 1% agarose gel electrophoresis and ethidium bromide staining.

Reverse transcription reactions for the preparation of first-strand cDNA from 2 μg of total RNA were performed using the AffinityScriptTM Multi Temperature cDNA synthesis kit (Stratagene, La Jolla, CA, USA). Random hexamers were used as amplification primers. Real-time PCR reactions were performed using the Mx3000P real-time PCR system (Stratagene, La Jolla, CA, USA) with SYBR® Green I Master Mix (Stratagene). Data were collected and analyzed using the Mx3000P real-time PCR software version 2.00, Build 215 Schema 60 (Stratagene). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were used as internal controls and their geometric mean in each sample was employed to normalize the mRNA expression levels of the MMPs. The primer pair sequences used are shown in Table 2. PCR products were analyzed by electrophoresis on 2% agarose gels, stained with ethidium bromide and photographed on a UV light transilluminator. MMPs or ADAMTS-4 transcription levels were calculated using the formula: normalized sample or control = (1 + EMMP)−∆CtMMP/(1 + EGAPDH)−∆CtGAPDH. Procedures were repeated with a cDNA template synthesized three times from the same RNA. The mRNA levels of each sample for every gene tested represent the mean value of data acquired from three independent RT-PCR experiments. Reproducibility of the real-time PCR results for the same samples was 99%.

Table 2 Primer sequences used for quantitative real-time RT-PCR
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Statistical analysis

The one-sample Kolmogorov–Smirnov test was used to assess the normality of the distribution of the mRNA expression values for the genes studied. Accordingly, the mRNA expression of the MMPs/ADAMTS-4 in the control and herniated groups, as well as in groups of different clinicopathological and MRI features, were compared using non-parametric procedures (Kruskal–Wallis and Mann–Whitney tests). The Spearman rank correlation test (non-parametric) was employed to examine pairwise MMPs/ADAMTS-4 mRNA correlations in control and herniated discs. Probability values or differences less than 0.05 were considered to be significant. Statistical calculations were performed using SPSS software, version 11.

Results

MMPs and ADAMTS-4 mRNA levels in normal and herniated discs

Transcript levels of the MMPs/ADAMTS-4 examined in the present study did not differ significantly between normal and herniated IDs (Table 3). The mRNA of the MMPs/ADAMTS-4 examined was not detectable in all control or herniated ID samples. Specifically, no expression of MMP-1 mRNA was noted in either control or herniated samples. Expression of MMP-3, -13 and -14 mRNA ranged between 40 and 90% in the control and 63 and 66% in the herniated IDs. Finally, MMP-8 and ADAMTS-4 mRNA was expressed in 40% of the control group and only in 19 and 34% of the herniated discs, respectively (Table 4). Notably, 26% of the herniated ID specimens exhibited a complete absence of mRNA expression for the MMPs and ADAMTS-4 examined. However, all of these samples expressed both housekeeping genes, beta-actin and GAPDH, demonstrating that these results were indicative of the samples’ molecular profile and not due to low cell numbers or bad RNA quality.

Table 3 Quantitative MMP mRNA expression in normal and herniated lumbar discs
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Table 4 Detected mRNA expression of MMPs and ADAMTS-4 in normal and herniated lumbar discs
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MMPs and ADAMTS-4 mRNA co-expression analysis pairwise

In the group of control discs, only a limited number of correlations were observed among the genes studied. Specifically, MMP-9 transcript levels were found to be positively correlated with those of MMP-13 and -14 (P < 0.001 and P = 0.001, respectively, Pearson correlation). MMP-13 and -14 mRNA were also found to be co-expressed in the normal discs (P < 0.001, Pearson correlation). However, in the group of herniated discs, multiple positive correlations were observed among almost all of the MMP transcript levels studied (Spearman correlation; Table 5a, b).

Table 5 Spearman correlation tables demonstrating the co-expression profile of MMPs in the groups of A. normal and B. herniated lumbar discs
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The above-mentioned analysis was performed taking into consideration the mRNA expression status of the MMPs and ADAMTS-4 (mRNA expression or not) in the groups of control and herniated ID specimens. Our results showed that the MMP-8 expression status was significantly correlated to MMP-13 in the control group (P = 0.035, Pearson). Multiple positive correlations were found in the herniated group concerning the mRNA expression status of almost all of the MMPs and ADAMTS-4 (Table 6a, b).

Table 6 Correlation tables demonstrating the pairwise co-expression profile of MMPs based on their expression status (expression or not) in the groups of C. normal and D. herniated lumbar discs
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Correlation of transcript levels with clinicopathological features

Age and gender

Patients >60 years were found to have significantly higher MMP-8 transcript levels than those between 40 and 60 years old (P = 0.005, Mann–Whitney test). Gender did not significantly affect the transcript levels of any of the MMPs or ADAMTS-4 investigated.

Body mass index

Our data did not show any significant correlation between BMI and mRNA levels of the MMPs/ADAMTS-4 included in the present study.

Duration of symptoms

The duration of patient symptoms appeared to significantly affect the transcript levels of MMP-9 and -13 (P = 0.017 and P = 0.037, respectively; Kruskal–Wallis test). In detail, patients suffering from radicular pain for 3–12 months exhibited higher MMP-9 and -13 mRNA levels, compared to those in pain for <3 months (P = 0.008 and P = 0.020, respectively, Mann–Whitney test; Fig. 1). MMP-9 transcript levels were also significantly elevated in patients with chronic pain (>12 months) compared to those in pain for <3 months (P = 0.016, Mann–Whitney test).

Fig. 1
figure 1

Normalized transcript levels of a MMP-9 and b MMP-13 in herniated disc specimens with respect to the duration of patient symptoms (P = 0.017 and P = 0.037, respectively; Kruskal–Wallis test)

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Pain intensity

High pain intensity (VAS between 8 and 10) significantly correlated with elevated MMP-9 mRNA levels, while a trend was observed in the same patient group for higher MMP-8 and -13 mRNA levels compared to discs excised from patients with low pain (VAS < 5) (P = 0.024, P = 0.061 and P = 0.063, respectively; Mann–Whitney test).

Level and type of ID herniation

The level or type of disc herniation (protrusion, extrusion or sequestration) as assessed by MRI did not significantly affect the transcript levels of the MMPs examined in the present study, with the exception of MMP-9. Specifically, sequestered discs exhibited significantly lower MMP-9 mRNA levels compared to protruding discs (P = 0.035; Mann–Whitney test). The ADAMTS-4 mRNA expression status was significantly associated with the ID herniation type (P = 0.032).

Employment

Patient employment, either light (n = 27) or heavy physical work (n = 36), did not correlate with MMPs/ADAMTS-4 transcript levels.

Smoking

Tobacco use was associated with lower MMP-3 mRNA levels in herniated discs (P = 0.048; Mann–Whitney test). The frequency of MMP-3 mRNA expression was significantly higher in non-smoking patients (P = 0.048; x 2 test). Furthermore, the amount of cigarette smoking was found to affect MMP-3 and -13 transcript levels. Specifically, discs removed from patients smoking more than 20 cigarettes per day exhibited significantly lower MMP-3 and -13 mRNA levels than non-smoking patients (P = 0.034 and P = 0.041, respectively, Mann–Whitney test; Fig. 2).

Fig. 2
figure 2

Normalized transcript levels of a MMP-3 and b MMP-13 in smoking and non-smoking patients. Significantly lower MMP-3 (c) and MMP-13 (d) mRNA levels were observed in discs removed from patients smoking more than 20 cigarettes per day compared to non-smoking patients (P = 0.034 and P = 0.041, respectively; Mann–Whitney test)

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Our data also revealed a significant association between smoking habits, duration of patient pain and pain intensity. Specifically, smoking was found to be negatively correlated to the duration of symptoms and positively correlated to the intensity of pain (P = 0.001 and P = 0.01, respectively; Spearman correlation). Furthermore, the duration of symptoms was negatively associated with pain intensity (P = 0.01; Spearman correlation).

Discussion

The present study investigated the mRNA expression profile of MMPs in a homogeneous, yet comparatively large, group of surgically resected lumbar-herniated IDs and in a control group of cadaveric specimens. Specifically, the transcript levels of several MMPs and ADAMTS-4 were examined by means of quantitative real-time PCR and correlated with the clinicopathological profile of the patients. Members of the major subgroups of metalloproteinases were included in the analysis, e.g., the collagenases (-1, -8, -13), the gelatinases (-9), the stromelysins (-3) and the membrane-type MMPs (-14) as well as ADAMTS-4, one of the most commonly studied members of the ADAMTS family of enzymes.

Numerous studies in literature have examined the protein expression levels of MMPs and ADAMTS in human herniated discs [10, 19, 20, 26, 29]. Using immunohistochemical techniques, Weiler et al. [29] found a significant correlation between MMP protein activity and histological signs of degeneration in human IDs. Similarly, Roberts et al. [26] found increased MMP activity in ID herniation compared to other disc disorders and control specimens. Nevertheless, limited evidence exists regarding the molecular profile of MMPs in ID herniation. In a recent study, Bachmeier et al. [3] examined the transcript levels of several MMPs in a group of lumbar-degenerated and -herniated discs. The authors found a substantial up-regulation of MMP-3 and -8 mRNA levels. However, a limited number of controls were used in the study (two samples), which impeded the use of standard statistical analysis. Our analysis did not show any significant differences between herniated and control discs in the mRNA expression levels of any of the MMPs examined. Given the increased MMP activity that has been observed in ID herniation compared to control specimens, [26] our results suggest that post-transcriptional modifications occur in these settings.

The findings of this study demonstrated multiple positive correlations between MMPs and ADAMTS-4 transcript levels in herniated discs. Fewer correlations, however, were observed in the control group. MMP production in ID tissue is mediated by cytokines, growth factors and inflammatory mediators. The role of interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) has been documented in vitro [15]. Growth factors are known to affect the process of ID degeneration by inducing neovascularization and regulating MMP expression levels [23]. However, MMPs also play a regulatory role in the interactions between macrophages and chondrocytes. In recent studies, Haro et al. demonstrated a role for MMP-3 and -7 in the release of soluble bioactive factors, thus affecting macrophage infiltration in ID tissue [7, 8]. MMPs are also capable of activating other MMPs. Therefore, it appears that a cascade of events can be triggered by the initial activation of even only one enzyme. This process explains the transcriptional co-expression of several MMPs observed and indicates a possible synergistic effect of multiple MMPs in promoting the resorption process of herniated discs.

Several studies have documented the involvement of ADAMTS-4 in both articular cartilage and ID degradation [21, 22]. Recently, Pockert et al. [24] examined the expression of several members of the ADAMTS family, including ADAMTS-4, in ID samples of patients suffering from degenerative disc disease. Patients with radicular pain were excluded from their study. These authors found a significantly increased mRNA expression in degenerated compared with non-degenerated IDs, which, however, also expressed ADAMTS mRNA. In a study of herniated ID samples without any control group, Hatano et al. [11] demonstrated the expression of ADAMTS-4 mRNA and protein, particularly in cases of transligamentous extrusion and sequestration. Our analysis showed that ADAMTS-4 mRNA was equally expressed in herniated and control ID samples. However, it was less frequently expressed in the two groups compared with MMPs, with the exception of MMP-8. Our findings are in accordance with Roberts et al. [26] who also found decreased aggrecanase activity, at the protein level, compared with MMPs in herniated discs. Imaging studies have suggested that aggrecan degradation occurs relatively early in the process of ID degeneration preceding the structural breakdown of the collagen network. The frequency of ADAMTS-4 mRNA expression in this study was higher in early compared to late stages of disc herniation, reinforcing the above hypothesis. We can only speculate that ADAMTS-4 plays a minor role in disc herniation. However, further in-depth studies are necessary to elucidate the exact role of ADAMTS-4 in the herniation process.

To the best of our knowledge, this is the first study in which the transcript MMP levels in human-herniated IDs are directly associated with smoking habits. Smoking was previously associated with the degree of disc degeneration and the occurrence of low back pain [6, 14]. It was suggested that cigarette smoking reduces blood circulation around the ID, thus limiting its nutrition and metabolite exchange [12]. Recently, Uei et al. [27] identified marked histological and molecular changes in the nucleus pulposus and annulus fibrosus of rat IDs after 7 weeks of passive cigarette smoking. A detrimental dose- and time-dependent effect of nicotine on cell proliferation and extracellular matrix synthesis has also been documented in vitro [2]. Our analysis showed that the amount of cigarette smoking significantly down-regulates the expression of MMP-3 and -13. Recent reports have shown MMP-3 to affect the resorption process of herniated ID tissue, directly through the proteolysis of extracellular matrix components and indirectly by inducing neoangiogenesis in the periphery of IDs and by promoting macrophage infiltration [7]. Similarly, MMP-13 is more potent than other MMPs in cleaving the helical region of collagen types I, III and particularly II which is found predominantly in the nucleus pulposus. Therefore, our data suggest a possible direct association of smoking with a reduced collagen degradation and resorption rate in herniated discs. Notably, the amount of cigarette smoking was significantly correlated with both the intensity and duration of patient symptoms (positive and negative correlations, respectively). Therefore, it appears that smoking is associated with intense, rather than chronic, pain. However, the latter patient characteristics may be under the influence of several diverse factors, such as individual, psychosocial and occupational. Psychosocial factors, in particular, have been found to independently affect the occurrence of low back pain, recovery rates, as well as the transition from acute to chronic pain. Therefore, whether smoking alone has a clinically significant effect on the resorption rate of herniated disc fragments and, consequently, on pain regression itself should be further investigated.

Our analysis demonstrated that the duration of patient symptoms was associated with increased transcript levels of MMP-13 and -9 indicating that the MMP mRNA expression is associated with chronic pain. Moreover, MMP-9 and -13 were found to be elevated in patients with intense pain. The two enzymes are known to readily degrade collagen and denatured collagens, e.g., gelatins. The duration of disc herniation is believed to affect significantly the extent of neovascularization around herniated ID fragments. Newly formed blood vessels were found to play an important role as a passage for infiltrating macrophages, resulting in the secretion of proteolytic enzymes such as MMPs [16]. Of note, up-regulation of MMP-9 mRNA expression was established in vitro in the process of neurogenesis and vascularization during embryonic tissue development [4]. Furthermore, inflammatory cytokines, such as tumor necrosis factor-a (TNF-α) and interleukin-1 (IL-1), are produced by infiltrating mononuclear cells immediately after the onset of disc herniation [30] and, in turn, these cytokines induce the expression of MMPs. TNF-α in particular also contributes to the clinical manifestations of disc herniation by inducing endoneuronal edema, nerve fiber demyelination and eventually pain, in association with phospholipase A2 (PLA2) and nitric oxide (NO) [13]. Therefore, we assume that in the setting of chronic inflammation, the increased expression of inflammation-related molecules accounts for the up-regulated expression of MMP-9 and -13 in addition to the effect of extended neovascularization around herniated ID fragments.

Conclusions

Our study provides evidence on the molecular profile of MMPs and ADAMTS-4 in lumbar ID herniation. Transcript levels of the MMPs studied and ADAMTS-4 did not differ significantly between herniated and control ID samples as opposed to the increased MMP protein levels and activity described earlier, leading to the speculation that post-transcriptional modifications occur. Multiple positive correlations were identified in MMP transcript levels that imply a synergistic effect on the activity of these levels in ID herniation. Smoking habits were found to have a negative dose-dependent effect on the transcript levels of specific MMPs and a positive correlation with pain intensity, suggesting an unfavorable role for smoking in the regression process of herniated disc fragments. Pain intensity and duration of patient symptoms significantly affected the expression levels of specific MMPs, possibly as a consequence of increased neovascularization and chronic inflammation.