Stages Of Intervertebral Disc Degeneration Research Articles

An injectable anti-vascularization functionalized hydrogel for degenerative nucleus pulposus repair

Neovascularization and inflammatory cell invasion within the nucleus pulposus (NP) constitute pivotal pathological changes during the acceleration stage of intervertebral disc degeneration (IDD). Mesenchymal stem cells (MSCs), renowned for their remarkable capacity in intervertebral disc (IVD) regeneration, also exhibit the capability to secrete pro-angiogenic factors, expediting IDD progression under hypoxic conditions. Consequently, we developed a hydrogel comprised of methacrylated hyaluronic acid (HAMA), rat tail collagen I (COL), and MSCs, incorporating the vascular endothelial growth factor receptor (VEGFR) inhibitor cabozantinib (Cabo@HAMA-COL/MSCs hydrogel). This innovative construct aimed to facilitate NP regeneration while mitigating vascularization and inflammation. Our findings revealed that the hydrogel aptly mimicked the mechanical characteristics of NP tissue, exhibiting injectability, low cytotoxicity, and the preservation of the cellular phenotype of NP cells. Co-culturing of MSCs and human umbilical vein endothelial cells (HUVECs) promoted migration, tube formation, and sprouting of HUVECs, which will be inhibited by cabozantinib. In vivo experiments demonstrated that Cabo@HAMA-COL/MSCs hydrogel maintained disc height, protected NP, and alleviated vascularization and inflammation in a puncture-induced rat caudal IDD model. Consequently, our results substantiate that Cabo@HAMA-COL/MSCs hydrogel can prevent IDD degeneration by ameliorating the vascularization-inflammation pathological microenvironment, offering a promising therapeutic strategy for IDD.

A novel in-vitro model of intervertebral disc degeneration using hyperphysiological loading

Intervertebral disc (IVD) degeneration includes changes in tissue biomechanics, physical attributes, biochemical composition, disc microstructure, and cellularity, which can all affect the normal function of the IVD, and ultimately may lead to pain. The purpose of this research was to develop an in-vitro model of degeneration that includes the evaluation of physical, biomechanical, and structural parameters, and that does so over several load/recovery periods. Hyperphysiological loading was used as the degenerative initiator with three experimental groups employed using bovine coccygeal IVD specimens: Control; Single-Overload; and Double-Overload. An equilibrium stage comprising a static load followed by two load/recovery periods was followed by six further load/recovery periods. In the Control group all load/recovery periods were the same, comprising physiological cyclic loading. The overload groups differed in that hyperphysiological loading was applied during the 4th loading period (Single-Overload), or the 4th and 5th loading period (Double-Overload). Overloading led to a significant reduction in disc height compared to the Control group, which was not recovered in subsequent physiological load/recovery periods. However, there were no significant changes in stiffness. Overloading also led to significantly more microstructural damage compared to the Control group. Taking all outcome measures into account, the overload groups were evaluated as replicating clinically relevant aspects of moderate IVD degeneration. Further research into a potential dose–effect, and how more severe degeneration can be replicated would provide a model with the potential to evaluate new treatments and interventions for different stages of IVD degeneration.

Role of Autophagy and Pyroptosis in Intervertebral Disc Degeneration.

Intervertebral disc degeneration is a chronic degenerative disease caused by the interaction of genetic and environmental factors, mainly manifested as lower back pain. At present, the diagnosis of intervertebral disc degeneration mainly relies on imaging. However, early intervertebral disc degeneration is usually insidious, and there is currently a lack of relevant clinical biomarkers that can reliably reflect early disease progression. Pyroptosis is a regulatory form of cell death triggered by the activation of inflammatory bodies and caspase, which can induce the formation of plasma membrane pores and cell swelling or lysis. Previous studies have shown that during the progression of intervertebral disc degeneration, sustained activation of inflammasomes leads to nuclear cell pyroptosis, which can occur in the early stages of intervertebral disc degeneration. Moreover, intervertebral disc nucleus pulposus cells adapt to the external environment through autophagy and maintain cellular homeostasis and studying the mechanism of autophagy in IDD and intervening in its pathological and physiological processes can provide new ideas for the clinical treatment of IDD. This review analyzes the effects of pyroptosis and autophagy on IDD by reviewing relevant literature in recent years, in order to explore the relationship between pyroptosis, autophagy and IDD.

Application of single and cooperative different delivery systems for the treatment of intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration (IDD) is the most universal pathogenesis of low back pain (LBP), a prevalent and costly medical problem across the world. Persistent low back pain can seriously affect a patient's quality of life and even lead to disability. Furthermore, the corresponding medical expenses create a serious economic burden to both individuals and society. Intervertebral disc degeneration is commonly thought to be related to age, injury, obesity, genetic susceptibility, and other risk factors. Nonetheless, its specific pathological process has not been completely elucidated; the current mainstream view considers that this condition arises from the interaction of multiple mechanisms. With the development of medical concepts and technology, clinicians and scientists tend to intervene in the early or middle stages of intervertebral disc degeneration to avoid further aggravation. However, with the aid of modern delivery systems, it is now possible to intervene in the process of intervertebral disc at the cellular and molecular levels. This review aims to provide an overview of the main mechanisms associated with intervertebral disc degeneration and the delivery systems that can help us to improve the efficacy of intervertebral disc degeneration treatment.

SIRT1 Inhibits Apoptosis by Promoting Autophagic Flux in Human Nucleus Pulposus Cells in the Key Stage of Degeneration via ERK Signal Pathway.

Background The application of biomolecular interventions in the early stage of intervertebral disc degeneration (IVDD) is considered an ideal method for the treatment of IVDD. However, the precise definition of the “early stage” of IVDD is unclear. Silent information regulation 2 homologue-1 (SIRT1) can protect human degenerative nucleus pulposus (NP) cells from apoptosis by activating autophagy. However, the mechanism of this effect is still unclear. This study tried to confirm the “early stage” of IVDD and the role of NP cell autophagy during IVDD as well as to determine the mechanism by which SIRT1 protects NP cells. Methods The characteristics of the NP in various stages of degeneration were assessed to confirm the “early stage” of IVDD. Then, autophagy and apoptosis were detected in NP cells after SIRT1 upregulation/downregulation. Finally, LY294002 and PD98059 were used to inhibit the AKT/ERK pathway to determine the mechanism by which SIRT1 regulates autophagy in NP cells. Results Our data showed that mildly degenerative (Pfirrmann grade III with normal height of intervertebral disc) NP cells may be the key target for biomolecular interventions in IVDD and that SIRT1 protects human mildly degenerative NP cells from apoptosis by activating autophagy via the ERK signalling pathway. Conclusion Our data showed that SIRT1 inhibits apoptosis by promoting the autophagic flux in NP cells via the ERK signalling pathway during the key stage of degeneration. These findings will assist in the development of novel therapeutic approaches for IVDD treatment.

Intervertebral disc degeneration associated with vertebral marrow fat, assessed using quantitative magnetic resonance imaging

ObjectiveTo investigate the potential clinical application of quantitative MRI in assessing the correlation between lumbar vertebrae bone marrow fat deposition and intervertebral disc degeneration.Materials and methodsA total of 104 chronic lower-back pain volunteers underwent 3.0-T MRI with T2-weighted imaging, T2 mapping, and iterative decomposition of water and fat with echo asymmetry and least squares estimation (IDEAL-IQ) between August 2018 and June 2019. Each disc was assessed with T2 value by T2 mapping, and the L1-S1 vertebral bone marrow fat fraction was assessed by IDEAL-IQ. The differences and relationship between T2 value and the adjacent vertebral bone marrow fat fraction values within the five Pfirrmann groups, five age groups, and five lumbar levels were statistically analyzed.ResultsThe vertebral bone marrow fat fraction had a significant negative correlation with T2 values of nucleus pulposus’ T2 values (p < 0.001). However, the significant negative correlation was only found between T2 values of nucleus pulposus and adjacent vertebral bone marrow fat in Pfirrmann II–III, L1/2-L5/S1 level, and 40–49 years’ age groups. Pfirrmann grades of the intervertebral disc were positively correlated with adjacent vertebrae bone marrow fat fraction (p < 0.05).ConclusionLumbar bone marrow fat deposition significantly increases during the early stages of intervertebral disc degeneration. Quantitative measurements of bone marrow fat deposition and water content of intervertebral discs have a predictive value and are an important supplement to the qualitative traditional classification strategies for the early stages of intervertebral disc degeneration.

The effectiveness of the system for predicting the results of surgical treatment of patients with lumbar disc herniation

Objective . To evaluate the effectiveness of a multivariate logistic regression model for the predicting surgical treatment results in patients with lumbar disc herniation. Materials and Methods . Study design: monocentric retrospective study. The study included patients operated on for lumbar disc herniation at levels L4–L5, L5–S1, with a 3-year follow-up. Two groups were identified: Group I included 350 patients (their data served as a basis for creation of multivariate logistic regression predicting model), and Group II – 514 patients (in this group, the effectiveness of the model was evaluated). Group II was divided into two subgroups: Subgroup IIa (recurrence probability 50 %) – 17 (3.3 %) patients. Patients in Subgroup IIa underwent microdisectomy, and in Subgroup IIb – spinal fusion. In order to obtain homogeneous pre-operative indicators of both group parameters, the PSM method was used. Statistical calculations were performed in the RStudio program. Results . In Group II, significant differences in indicators in the subgroups were noted for the following parameters (p < 0.05): smoking, disc height index, segmental volume of movement, lumbar lordosis angle, type of intervertebral hernia (except for sequestration), Modic changes, and stage of intervertebral disc degeneration according to Pfirrmann. In Subgroup IIa, 8 (1.6 %) reoperations were performed, in Subgroup IIb – 2 (0.4 %). Using the PSM method, the data of groups I and II were flattened out for significantly different indicators. The sample size was 37 patients in each group. The number of reoperations in the groups differed statistically significantly: Group I – 35 % [22 %; 51 %]; Group II – 5 % [1 %; 18 %]. The risk of reoperation in Group II is 0.13 [0.03; 0.58] times lower than in Group I (p = 0.002). Conclusions . The proposed system for predicting the results of surgical treatment of patients with intervertebral disc hernia can be used as a tool to determine the surgical tactics aimed at reducing the frequency of reoperations.

Gene Expression Changes in an Early Stage of Intervertebral Disc Degeneration Induced by Passive Cigarette Smoking: Erratum.

Gene Expression Changes in an Early Stage of Intervertebral Disc Degeneration Induced by Passive Cigarette Smoking: Erratum.

Changes in essential and trace elements content in degenerating human intervertebral discs do not correspond to patients’ clinical status

Abstract: Aim: To date, there is no paper considering disc degeneration process in aspect of some elements content in degenerating discs tissue, clinical status of patients, and imaging analysis. Concentration of essential and trace elements in disc tissue may be a consequence of both environmental and genetic factors. Methods: The material of 19 intervertebral discs was harvested from 17 patients during lumbar discectomy. Control was 9 healthy discs obtained from organ donors. Atomic absorption spectrometry was used to asses levels of Cu, Fe, Mn, Pb, Zn, Na, Mg, K, Ca, and P in the tissue, as well as dry weight (d.w.) of the tissue. Results : All 10 essential and trace elements were detected in all samples. Significant increase of Ca, Mg, Fe, and P, and decrease of Cu and K in operated discs was found; remaining changes between sick and healthy discs were not significant. There were no age/elements, Pfirrmann grade /elements, or Modic Grade changes/ elements correlations. Significant positive correlation was found between Mg and Zn, K and Fe, Ca and Zn, Ca and Mg, P and Zn, P and Mg, and P and Ca. Negative correlation was indicated only between age and Na. The study is one to analyze and asses contents of essential and trace elements in intervertebral discs. Calcium levels were higher in degenerating disc group than in healthy one. Conclusion: Lack of correlation between calcium content and stage of intervertebral disc degeneration as well as age of patients in degenerating disc group is a surprising result.

Intervention of rAAV-hTERT-Transducted Nucleus Pulposus Cells in Early Stage of Intervertebral Disc Degeneration: A Study in Canine Model.

To investigate the efficacy of recombinant adeno-associated virus (rAAV)-human telomerase reverse transcriptase (hTERT)-transducted nucleus pulposus cells (NPCs) in disc degeneration process in a canine disc degeneration model. The intervertebral disc degeneration of lumbar (L) 1-2, L3-4, and L5-6, from 12 female mongrels was prepared with the 20-gauge biopsy gun. Four weeks after animal model preparation, intervention experiment with rAAV-hTERT-transducted NPCs was conducted: group A, L1-2, serum-free medium with rAAV-hTERT modified NPCs; group B, L3-4, serum-free medium with NPCs; group C, L5-6, serum-free medium alone. Canines underwent digital radiography and magnetic resonance imaging 1 day before intervention, and 4, 8, and 12 weeks after intervention to evaluate the change of disc height and hydration status of interventional intervertebral discs. Twelve weeks after intervention, histological, biomechanical, and biochemical studies were carried out. The rAAV-hTERT-transducted NPCs were constructed successfully. The mRNA level of hTERT from rAAV-hTERT-transfected NPCs increased obviously. There was no significant change of disc height index observed between groups and within groups. The relative grayscale index (RGI) was maintained 8 weeks after the intervention in group A, whereas in group B and group C, the RGI decreased significantly (p<0.05). No significant differences of the angle of lateral bending and extension-flexion bending were observed in group A compared with other groups (p>0.05). The morphology of disc structure was preserved in group A. In group B, the structure of inner annulus was broken down and the jelly-like nucleus pulposus (NP) tissue transmitted into the fibrocartilaginous tissue. In group C, the jelly-like NP tissue was completely replaced by fibrocartilaginous tissue. In the NP, the content of proteoglycan (PG) and collagen II was higher in group A than in group C (p<0.05). The content of PG was 13, 8.9, and 15.6 times higher than the content of collagen II in group A, group B, and group C, respectively. In 12 weeks of observation, rAAV-hTERT-transducted NPCs could delay the degeneration process in the canine model which was superior than the capacity of NPCs in preserving structure integrity, content of extracellular matrix, and mechanical stability.

Annulus fibrosus cell characteristics are a potential source of intervertebral disc pathogenesis.

In the end stage of intervertebral disc degeneration, cartilage, bone, endothelial cells, and neurons appear in association with the worsening condition. The origin of the abnormal cells is not clear. This study investigated the properties of progenitor cells in the annulus fibrosus (AF) using one in vitro and two in vivo models. Cultivation of rabbit AF cells with chondrogenic media significantly increased expressions of collagen and aggrecan. Upon exposure to osteogenic conditions, the cultures showed increased mineralization and expression of osteopontin, runx2, and bmp2 genes. Two models were used in the in vivo subcutaneous implantation experiments: 1) rabbit AF tissue in a demineralized bone matrix (DBM) cylinder (DBM/AF), and, 2) rat intact and needle punctured lumbar discs. Bone formation in the AF tissue was detected and hypertrophic chondrocytes and osteoblasts were present 1 month after implantation of the DBM/AF to nude mice. In addition to collagen I and II, immunostaining shows collagen X and osteocalcin expression in DBM/AF specimens 4 months after implantation. Similar changes were detected in the injured discs. Almost the entire needle punctured disc had ossified at 6 months. The results suggest that AF cells have characteristics of progenitor cells and, under appropriate stimuli, are capable of differentiating into chondrocytes and osteoblasts in vitro as well as in vivo. Importantly, these cells may be a target for biological treatment of disc degeneration.

A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype

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.

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

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

Evaluation of platelet-rich plasma and hydrostatic pressure regarding cell differentiation in nucleus pulposus tissue engineering

Generation of a biological nucleus pulposus (NP) replacement by tissue engineering appears to be a promising approach for the therapy of early stages of intervertebral disc degeneration. Thereby, autologous mesenchymal stem cells (MSCs) represent an attractive cell source compared to cells of the NP that are already altered in their phenotype due to degenerative processes. This study compares the influence of 3D pellet culture and alginate beads, as well as that of different media compositions, by the addition of human platelet-rich plasma (PRP) or transforming growth factor (TGF-β1 ) in interaction with hydrostatic pressure on chondrogenic differentiation of human MSCs compared to NP cells. We found that gene expression of the chondrogenic markers aggrecan, collagen type 2 and collagen type 1 and Sox9 was considerably lower in cells cultivated with PRP compared to TGF-β1 . Immunohistology confirmed this result at protein level in pellet culture. Additionally, the pellet culture system was found to be more suitable than alginate beads. A positive influence of hydrostatic pressure could only be shown for individual donors. In summary, in comparison to TGF-β1 , human PRP did not induce adequate chondrogenic differentiation for both culture systems and cell types used. The mixture of growth factors in PRP promoted proliferation rather than chondrogenic differentiation. Based on these results, an application of PRP in human NP tissue-engineering approaches cannot be recommended.

Do early stages of lumbar intervertebral disc degeneration really cause instability? Evaluation of an in vitro database

Early stages of intervertebral disc degeneration are postulated to cause instability. In the literature, however, some authors report the opposite. These contradictory positions are probably supported by the mostly small number of segments which are investigated. The aim of this project therefore was to investigate the influence of intervertebral disc degeneration on lumbar spine rotational stability using a large data set. The flexibility data from all spine specimens tested in our institute so far were collected in a large in vitro database. From this database, all lumbar spine specimens were selected, which had been tested for flexibility under pure moment loads of ±7.5 N m and for which radiographs were accessible. 203 segments met these criteria. Their radiographic degree of disc degeneration was determined on a scale from 0 (no degeneration) to 3 (severe degeneration) and their influence on the respective range of motion and neutral zone was examined. The different lumbar levels differ in flexibility, which increases the variability of the data if pooled together. To minimise this effect a statistical model was fitted. The model-based mean estimates showed a decrease of the range of motion from grade 0 to 3 in flexion/extension (by 3.1°, p < 0.05) and lateral bending (by 3.4°, p < 0.05). In contrast, in axial rotation the range of motion tended to increase; however, not only from grade 0 to 1 but also towards grade 3 (by 0.2°) (p > 0.05). The neutral zone was affected in a similar way but to a smaller degree (p > 0.05). In conclusion, the results indicated that early stages of intervertebral disc degeneration do not necessarily cause rotational instability. In contrast, stability increased in flexion/extension and lateral bending. Only in axial rotation stability tended to decrease.

Micro-CT quantification of subchondral endplate changes in intervertebral disc degeneration

The intervertebral disc (IVD) is dependent on nutrient provision through a cartilage layer with underlying subchondral bone, analogous to joint cartilage. In the joint, subchondral bone remodeling has been associated with osteoarthritis (OA) progression due to compromised nutrient and gas diffusion and reduced structural support of the overlaying cartilage. However, subchondral bone changes in IVD degeneration have never been quantified before. The aim of this study is to determine the subchondral bone changes at different stages of IVD degeneration by micro-CT. Twenty-seven IVDs including the adjacent vertebral endplates were obtained at autopsy. Midsagittal slices, graded according the Thompson score, were scanned. Per scan 12 standardized cylindrical volumes of interest (VOI) were selected. Six VOIs contained the bony endplate and trabeculae (endplate VOIs) and six accompanying VOIs only contained trabecular bone (vertebral VOIs). Bone volume as percentage of the total volume (BV/TV) of the VOI, trabecular thickness (TrTh) and connectivity density (CD) were determined. An increase in BV/TV and TrTh was found in endplate VOIs of IVDs with higher Thompson score whereas these values remained stable or decreased in the vertebral VOIs. The increase in bone volume combined with the increase in TrTh in endplate VOIs strongly suggest that the subchondral endplate condenses to a more dense structure in degenerated IVDs. This may negatively influence the diffusion and nutrition of the IVD. The endplate differences between intact and mild degenerative IVDs (grade II) indicate an early association of subchondral endplate changes with IVD degeneration.

Increased MMP‐2 activity during intervertebral disc degeneration is correlated to MMP‐14 levels

Intervertebral disc (IVD) degeneration is associated with the increased expression of several matrix metalloproteinases (MMPs), in particular MMP-2. However, little is known about the actual activity of MMP-2 in healthy and degenerated discs, or what mechanisms are involved in its activation. A major activation pathway involves complex formation with MMP-14 and a tissue inhibitor of metalloproteinases-2 (TIMP-2). In a series of 56 human IVDs, obtained at autopsy and graded according to the Thompson score (I-V), we analysed whether MMP-2 activity was increased in different stages of IVD degeneration and to what extent activation was related to the production of MMP-14 and TIMP-2. MMP-2 activation and production were quantified by gelatin zymography. Immunohistochemical staining of MMP-14 and TIMP-2 was quantified with a video overlay-based system. A positive correlation was observed between the amount of active MMP-2 and pro-MMP-2 and degeneration grade (p < 0.001, correlation coefficient (CC) 0.557; and p < 0.001, CC 0.556, respectively). MMP-2 activity correlated positively with MMP-14 and less strongly with TIMP-2 (p = 0.001, CC 0.436; and p = 0.03, CC 0.288, respectively). Moreover, immunopositivity for MMP-14 correlated to degeneration grade (p = 0.002, CC 0.398). IVD degeneration was associated with the activity of MMP-2 and the correlation of its activation with MMP-14 production suggests MMP-14 activates MMP-2 during degeneration. As MMP-14 is capable of activating several other enzymes that are also thought to be involved in IVD degeneration, it may be a key mediator of the degenerative process.

Gene Expression Changes in an Early Stage of Intervertebral Disc Degeneration Induced by Passive Cigarette Smoking

This study attempts to determine the molecular changes in intervertebral disc degeneration of rats induced by passive cigarette smoking. To quantitate and compare the gene expression levels in intervertebral discs from passively cigarette smoking rats and nonsmoking rats. The molecular mechanism of intervertebral disc degeneration has been investigated mainly in vitro but little in vivo, and gene expression analysis has been performed in a few studies. The cigarette smoking is a risk factor of low back pain. We developed a smoking box to create a rat model of intervertebral disc degeneration induced by passive cigarette smoking. Total RNA was extracted from intervertebral discs of rats that were raised in a cigarette-smoking box for 2 to 7 weeks. After synthesis of cDNA, the quantitative analysis of gene expression was performed by the real-time PCR. The remaining spines were subjected to the histologic examination. Histologic changes of the nucleus pulposus and the anulus fibrosus were detected after 2 weeks of smoking and were frequently found after 7 weeks. Collagen genes were downregulated remarkably after 7 weeks of smoking. No significant increase was observed in the expressions of matrix metalloproteinase-3, but the expression of tissue inhibitor of metalloproteinases-1 started to increase at 4 weeks of smoking. Aggrecan also started to be up-regulated at 4 weeks. Changes in gene expression by passive cigarette smoking precede the histologic changes in the intervertebral discs. Reactions to suppress the destruction of tissue matrix and to regenerate the intervertebral discs are occurring at the same time as the degenerative histologic changes.

Characterization of intervertebral disc degeneration by high‐resolution magic angle spinning (HR‐MAS) spectroscopy

The goal of this study was to determine the ability of high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to distinguish different stages of intervertebral disc degeneration (IVDD). 17 discs were removed from human cadavers and analyzed them using 1D and 2D (total correlation spectroscopy (TOCSY)) (1)H HR-MAS spectroscopy, and T(1) and T(2) relaxation time measurements to determine the chemical composition and changes in chemical environment of discs with increasing levels of degeneration (Thompson grade). Among the significant findings were that spectra were very similar for samples taken from annular and nuclear regions of discs, and that visually apparent changes were observed in the spectra of the annular and nuclear samples from discs with increasing Thompson grade. Area ratios of the N-acetyl to choline (Cho) regions, and Cho to carbohydrate (Carb) regions of the spectra allowed us to discriminate between discs of increasing Thompson grade with minimal overlap of individual ratios. Changes in T(1) and T(2) relaxation times of the chemical constituents of disc spectra were not significantly correlated to the degree of degeneration. The results of this study support the feasibility of using in vivo spectroscopy for detecting chemical changes associated with disc degeneration.

P104. Gene expression changes in an early stage of intervertebral disc degeneration induced by passive cigarette smoking

BACKGROUND CONTEXT: Many epidemiological studies have demonstrated that cigarette smoking is a risk factor for low back pain. They proposed an idea that vasospasms or arteriosclerotic changes caused by cigarette smoking may have a negative effect on the blood supply to vertebral bodies and other structures surrounding the discs, subsequently affecting disc nutrition.