bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the Correlation among the Expression of Inflammatory Cytokines, Degeneration of the Intervertebral Disc, and Prominent Symptoms in Degenerative Human Lumbar Intervertebral Discs
Authors: Sainoh T. , Orita S. , Yamauchi K. , Sakuma Y. , Kubota G. , Oikawa Y. , Inage K. , Sato J. , Fujimoto K. , Takahashi K. , Ohtori S.
Published: May 2014

Introduction

Low back pain (LBP) disease is reportedly associated with inflammatory cytokines such as tumor necrosis factor (TNF-α) and interleukin-6 (IL-6), as well as with nerve growth factor (NGF) that is expressed in increased amounts within degenerative intervertebral discs (IVDs). In the present study, we aimed to assess the correlation between the local expression of TNF-α, IL-6, and NGF within degenerative human lumbar IVDs and the degree of degeneration, as well as the correlation between the expression of these mediators and the prominent symptoms.

Materials and Methods

In total, 58 IVD samples of patients who underwent surgery for various lumbar disorders were examined. The expression level of these mediators in each sample was determined using enzyme-linked immunosorbent assay. The samples were grouped according to the degree of IVD degeneration using Pfirrmann grading on magnetic resonance imaging, and the grades were correlated with the individual expression levels. The chief complaints of the patients were then recorded and assigned to the LBP group and the leg pain group, and were compared with the individual expression levels.

Results

A gradual increase in TNF-α (R: 0.391) and IL-6 (R: 0.388) expression levels were observed with a progression in the degree of degeneration, but NGF (R: − 0.164) exhibited a minimal decrease in the expression level. With regard to the chief complaints, the LBP group only exhibited a significant increase in the TNF-α expression level.

Conclusion

We noted a trend of increasing expression levels of TNF-α and IL-6 with the progression of IVD degeneration, which indicates that these cytokines may have an important role in the degenerative process of IVD at any stage. Moreover, NGF may have an important effect during the early stage of the progression of degeneration. With regard to the symptoms, TNF-α expression was significantly greater in the LBP group. Thus, we propose that TNF-α may be significantly involved in LBP. Disclosure of Interest None declared
Disc Space Excessive Distraction is a Potent Risk Factor for Adjacent Segment Degeneration after Anterior Cervical Discectomy and Fusion
Authors: Huang X. , Li F. , Shen H. , Li M.
Published: May 2014

Introduction

We reported that excessive disc space distraction caused by insertion of large cages during anterior cervical discectomy and fusion (ACDF) induces adjacent segment degeneration (ASD). Previous reports indicated that over distraction of disc space was an independent risk factor for ASD after posterior lumbar interbody fusion. We therefore hypothesize that the incidence of ASD after ACDF with larger size of cages should be higher than that ACDF with normal cages due to this loss of disc space distraction.

Materials and Methods

Radiographic ASD is defined as development of spondylolisthesis >3 mm, decrease in disc height >3 mm, or intervertebral angle at flexion lesser than −5 degrees. Symptomatic ASD is defined by a decrease of ≥ 4 points in Japanese Orthopedic Association score. A 130 patients with cervical spondylotic radiculopathy were treated with either ACDF with larger size of cages ( n = 45) or ACDF with normal cages ( n = 85) and followed up for a minimum of 2 years (mean, 39.2 months).

Results

Disc space distraction in the normal cage group was significantly smaller (0.4 mm) as compared with the larger size group (1.8 mm). The incidence of clinical ASD in the normal cage group (2%, 1/45) was also significantly less than that in the larger size group (15%, 13/85). Multivariate analysis showed that disc space distraction was the most significant risk factor.

Conclusion

Among various risk factors for ASD after ACDF, the excessive distraction of disc space proved to be a potent risk factor for ASD. Disclosure of Interest None declared
Correlation of Intervertebral Disc Degeneration among Different Spinal Regions in the Entire Spine in a Population-Based Cohort: The Wakayama Spine Study
Authors: Teraguchi M. , Hashizume H. , Yoshimura N. , Yamada H. , Akune T. , Yoshida M.
Published: May 2014

Introduction

A thorough literature review about the correlation of disc degeneration (DD) among the cervical, thoracic, and lumbar region revealed no studies that focused on a population-based cohort analysis of DD in the entire spine in humans using MRI. To further elucidate the role of intraindividual factors in the development of DD, we should examine DD in the entire spine. The purpose of this study, was to examine the correlation of DD in the cervical, thoracic, and lumbar region, respectively.

Materials and Methods

This was a cross-sectional study of a cohort from western Japan, comprising 975 (men, 324; women, 641) of 1,011 participants (age, 21-97 years). The participants were subjected to the entire spine MRI and all intervertebral discs in the cervical (C2/3-7/Th1), thoracic (Th1/2-Th12/L1), and lumbar (L1/2-L5/S) region on sagittal T2-weighted images were evaluated according to the Pfirrmann classification system (1-3= nondegeneration; 4, 5=degeneration). The Spearman rank correlation analysis was used to determine the correlation among the number of DD at each region mutually after adjustment for age.

Results

The Spearman rank correlation coefficient was 0.347 (95%CI: 0.5-0.59) between the cervical region and thoracic region, 0.300 (95%CI: 0.42-0.52) between the lumbar region and thoracic region, and 0.246 (95%CI: 0.34-0.44) between the cervical region and lumbar region.

Conclusion

Our population-based study using the whole spine MRI revealed a highly significant intraindividual correlation between DD in the cervical region and DD in the thoracic or lumbar region. Needless to say, the biomechanical environments among the three regions are fundamentally different. This distinct individual correlation for DD could be attributed to underlying genetic factor or endogenous factors, and so on. To further elucidate the role of intraindividual factors to DD, investigations on the extent of degenerative changes using the whole spine MRI could provide useful fundamental data concerning degenerative spinal change. Disclosure of Interest None declared
Body Mass Index and its Association with Lumbar Disc Herniation and Sciatica: A Large-Scale, Population-Based Study
Authors: Samartzis D. , Karppinen J. , Luk K. DK , Cheung K. MC
Published: May 2014

Introduction

Elevated body mass index (BMI) or overweight and obesity are pandemics. Lumbar disc herniation and sciatica occur in every population and present severe socioeconomic consequences. However, little is known regarding the role of BMI with lumbar disc herniation and sciatica. As such, the following large-scale study addressed the association of BMI, in particular overweight and obesity, with disc herniation, its global lumbar involvement and its implications with the development of sciatica.

Materials and Methods

A cross-sectional study of 2,596 Southern Chinese (mean age: 42 years; 60% females) was conducted assessing T2-weighted MRI, environmental and lifestyle factors, as well as clinical profiles of sciatica. On imaging, the presence of disc bulge/extrusion (DBE) and other spinal phenotypes from L1-S1 were assessed. Disc degeneration was assessed and graded based on the Schneiderman et al criteria and summated of all levels to produce a degenerative disc disease (DDD) score (potential score range: 0-15). A total DBE (TDBE) score of L1-S1 was calculated (potential score range: 0-10). Asian-modified BMI values and categories of underweight, normal weight, overweight, and obese were obtained for each subject.

Results

DBE was noted in 46.3% of the subjects, mainly occurring at L4-S1. The mean DDD and TDBE scores were 0.7 and 2.83, respectively. Historical prevalence of sciatica was 44.6%, with 17.9% reporting sciatica at the time of assessment. The mean BMI was 22.9 kg/m2 (7.2% underweight, 47.9% normal weight, 36.1% overweight, and 8.9% obese). Elevated DDD score was significantly correlated with an elevated TDBE score (r=0.610; p< 0.001). TDBE score significantly increased with elevated BMI categories ( p< 0.001). Multivariate analyses adjusting for covariates noted that elevated BMI was significantly associated with DBE (normal weight [Ref]; underweight OR: 0.71[95% CI: 0.49-1.03]; overweight OR: 1.26[95% CI: 1.04-1.52]; obese OR: 1.78[95% CI: 1.30-2.44]). TDBE score (OR: 1.36; 95% CI: 1.15-1.60) and obesity (OR: 1.68; 95% CI: 1.25-2.24) were significantly related with sciatica. Worse functional and disability scores were associated with sciatica ( p< 0.05).

Conclusion

Based on the largest population-based study to assess the role of BMI and its association with disc herniation, this study definitely noted that overweight and obesity significantly increased the likelihood of having lumbar disc herniation, its global severity, and the risk of developing sciatica. Disclosure of Interest None declared
Regeneration of the Intervertebral Disc with Nucleus Pulposus Cell-Seeded Collagen II/Hyaluronan/Chondroitin-6-Sulfate Tri-Copolymer Constructs in a Rabbit Disc Degeneration Model
Authors: Huang B. , Zhuang Y. , Li C. , Liu L. , Zhou Y.
Published: May 2014

Introduction

Advancement in tissue engineering provides a promising approach to recover the functionality of the degenerated intervertebral disc. In our study, a nucleus pulposus (NP) cell-seeded collagen II/hyaluronan/chondroitin-6-sulfate (CII/HyA/CS) tri-copolymer construct was implanted into the disc space directly after nucleotomy in a rabbit model. The aim of this study was to investigate whether the NP cell-seeded CII/HyA/CS tri-copolymer constructs could regenerate the degenerated disc in vivo after implantation into the rabbit nucleotomy model.

Materials and Methods

NP cells isolated from thoracic and lumbar spines of New Zealand white rabbits of about 3 weeks of age and 1 kg in weight were labeled with a 5- (and-6)-carboxyflurescein diacetate succinimidyl ester (CFDA-SE) fluorescent dye and seeded within the CII/HyA/CS scaffold by a centrifugation method. After in vitro culture for 1 week, NP cell-seeded CII/HyA/CS tri-copolymer constructs were allografted into the disc defects of recipient rabbit immediately after nucleotomy of the lumbar spine. The Bradner disc index and the T2-weighted signal intensity index were determined using lateral plane radiographs and magnetic resonance imaging at 4, 12, and 24 weeks after the operation. Finally, the operated discs were explanted for gross morphological observation, histological evaluation, and cell viability assessment. Animals with only nucleotomy and cell-free CII/HyA/CS scaffold implantation served as controls.

Results

In our study, we could demonstrate that the T2-weighted signal intensity index of the operated discs decreased in all three groups 1 month after surgery and the index of the cell-containing scaffold insertion group was significantly higher than that of the other two groups. After 24 weeks, the index of the cell-containing scaffold insertion group increased significantly. However, further decline was observed in both the noninsertion group and the scaffold insertion group. In radiographic analysis, the narrowing of the intervertebral disc space was significantly retarded by the cell-scaffold hybrids implantation up to 24 postoperative weeks. Moreover, the gross morphology and histological evaluation indicated that the allografted NP cells were viable and showed extracellular matrix production.

Conclusion

In our study, we had constructed rabbit NP cell-seeded CII/HyA/CS tri-copolymer implants in vitro. Immediately after nucleotomy of the recipient rabbit, we allografted the precultured cell-scaffold hybrids into the lacuna of the disc. Results documented survival of the allografted NP cells and extracellular matrix deposition, which finally resulted in maintenance of disc height and restoration of T2-weighted signal intensity on magnetic resonance imaging. Disclosure of Interest None declared
Pathogenesis of Ossification of Ligamentum Flavum
Authors: N. B. J.  , S. L. J.  , N. G.
Published: May 2014

Introduction

Ossification of ligamentum flavum (OLF) is a rare disorder. The present study aims at histopathological analysis of OLF with special emphasis on the alterations in the elastin fibers, collagen, matrix, cellularity, chodrocyte proliferation, and osteoid metaplasia.

Materials and Methods

The study involves histopathologic examination of surgically harvested ligamentum flavum (LF) specimens from 10 OLF, 5 with degenerative spinal stenosis and 5 normal LF as controls. The specimens were studied with hematoxylin and eosin, Verhoff van Gieson, toluidine blue, Masons trichrome, and Alcian blue. All specimens were analyzed qualitatively for (1) elastic fiber orientation, integrity, and loss, (2) collagen bundles,(3) fibrosis,(4) chondroid metaplasia,(5) chondrocyte proliferation, and (6) Osteoid metaplasia. The quantitative analysis was done using SCION image analysis software.

Results

All the 10 OLF specimens showed extensive loss of elastic fibers associated with cartilage matrix formation. There were many areas of irregularly oriented elastic fibers, areas of ossification and in between there was a transitional area of cartilage matrix with chondrocytes progressing toward ossification. Elastic fiber occupying ratio was 60.96, 40.25, and 22.66%; number of chondrocytes per mm2 was 37.56, 93.35, and 117.68; area of chondroid matrix (%) was 11.34, 32.37, and 73.56, respectively, and the area of osteoid matrix (%) was 0, 9.37, and 33.43 respectively for normal controls, degenerative group, and OLF group. The areas of cartilage matrix were more on the dorsal side compared with the dural side of LF. In contrast, cartilage matrix osteogenesis was more in dural than dorsal surface of LF.

Conclusion

In OLF, loss of elastic fibers was associated with proliferation of collagen fibers, fibrosis, chondrogenesis, and osteogenesis. The pathological process initiates in the dorsal surface with chondroid metaplasia gradually elevating the mature cartilage matrix toward the dural surface, where the initial ossification occurs. Disclosure of Interest None declared References Jolly SS. Fluoride balance studies in endemic fluorosis. Fluoride 1976;8:138–147 Jolly SS, Singh BM, Mathur OC, Malhotra KC. Epidemiological, clinical, and biochemical study of endemic dental and skeletal fluorosis in punjab. BMJ 1968;4(5628):427–429 Jolly SS, Singh ID, Prasad S, Sharma R, Singh BM, Mathur OC. An epidemiological study of endemic fluorosis in Punjab. Indian J Med Res 1969;57(7):1333–1346 Jolly SS, Prasad S, Sharma R, Rai B. Human fluoride intoxication in the Punjab. Fluoride 1971;4:64–79 Jolly SS, Prasad S, Sharma R, Chander R. Endemic fluorosis in Punjab-skeletal aspects. Fluoride 1973;6:4–18 Naidu MRC, Reddy DR, Reddy PK, Sastry Kolluri VR. [Fluorotic cervical spinal cord disease]. Rev Chir Orthop Repar Appar Mot 1989;75(4):228–231 Siddiqui AH. Fluorosis in Nalgonda District, Hyderabad-Deccan. BMJ 1955;2(4953):1408–1413
Collagen-Derived Peptide N-Acetylated Proline-Glycine-Proline in Intervetebral Disc Recruits Cartilage Endplate Stem Cells by Binding with CXC Receptor 2
Authors: Feng C.
Published: May 2014

Introduction

Degenerative disc disease (DDD) is a prevalent musculoskeletal disorder and is the major cause of low back pain. But our comprehension about development of the intervertebral disc (IVD) and pathogenesis of disc degeneration is unclear, which is an obstacle for appropriate therapies. Recently, some reports identified stem/progenitor cells within particular IVD niches and cartilage endplate. These cells together with mesenchymal stem cells (MSC) are recruited to nucleus pulposus(NP) and annulus fibrosus(AF) to generate regenerative functions in a healthy IVD. The possibility of stimulating these stem cells holds promise for future therapy for DDD. But factors which recruit stem cells to NP and AF are unknown. There are increasing evidences that N-acetylated proline-glycine-proline (N-AC-PGP), which is a degradation fragment of collagen, plays an important role in inflammatory cell recruitment. But whether it presents in IVD and whether it is a chemokine of stem cell have remained elusive.

Materials and Methods

In this study, we detected the presence of N-AC-PGP by liquid chromatography-mass spectrum in IVD with different degeneration grade and the enzymes responsible for generating N-AC-PGP, matrix metalloproteases 8 and 9 and prolyl endopeptidase (PE), are also quantified by ELISA in these IVD samples. To found out the role of N-AC-PGP in IVD, cartilage endplate stem cells (CESC) chemotaxis assay was performed by Tanswell assay.

Results

We demonstrate the correlation between N-AC-PGP content in IVD and content of MMP-8and9 and PE.N-AC-PGP increase with increasing degeneration grade of IVD. Then, we described the generation pathway of N-AC-PGP in IVD. Finally, we demonstrated that N-AC-PGP can recruit CESC by binding with CXC receptors 2 on CESC.

Conclusion

These findings reveal a possible regeneration mechanism in IVD, which depends on the chemotaxis of N-AC-PGP for CESC and indicate a new therapy by using N-AC-PGP to stimulate stem cells in IVD. Disclosure of Interest None declared References Snelgrove RJ, Jackson PL, Hardison MT, et al. A critical role for LTA4H in limiting chronic pulmonary neutrophilic inflammation. Science 2010;330(6000):90–94 Gaggar A, Jackson PL, Noerager BD, et al. A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation. J Immunol 2008;180(8):5662–5669 Weathington NM, van Houwelingen AH, Noerager BD, et al. A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation. Nat Med 2006;12(3):317–323
Role of Semaphorins in Angiogenesis and Innervation in Human Intervertebral Disc Degeneration
Authors: Binch A. LA. , Phillips K. L , Chiverton N. , Cole A. , Michael A. R. , Breakwell L. , Cross A. K. , Le Maitre C. L.
Published: May 2014

Introduction

Although the intervertebral disc (IVD) is considered the largest aneural structure within the human body, during degeneration nociceptive nerves are seen to accompany blood vessels into the IVD yet it remains unclear how this process occurs. Semaphorins are axonal guidance molecules known to repulse nerves by altering the actin cytoskeleton of growth cones. Semaphorin 3A (sema3A) has recently been localized in healthy IVDs, suggesting an inhibitory role toward neural and vascular ingrowth. This study aimed to first identify the presence of the class 3 semaphorins and sema4D shown to be involved in promoting angiogenesis and their receptors within nucleus pulposus(NP) cells and an endothelial cell line and determine whether their expression could be altered by cytokine treatment.

Materials and Methods

Real-time PCR (RT-PCR) was performed to investigate gene expression of class 3 semaphorins; Sema3A-3F, sema4D, their receptors plexin A1-A4 and plexin D1 and neuropilin-1 (NRP-1) and NRP-2 in directly extracted RNA from human NP cells and NP cells cultured in alginate for 2weeks before treatment for 48hours with interleukin (IL)-1, IL-6, or tumor necrosis factor α(TNFα) at 0 to 100ng/mL. Similarly, HDMEC-1 endothelial cells were treated with IL-1, IL-6, or TNFα at 0 and 10ng/mL for 48hours, after which mRNA expression was investigated by RT-PCR.

Results

Expression of semaphorins and their receptors were present in normal and degenerate disc samples. In human NP cells, IL-6 inhibited semaphorin expression, whereas IL-1 and TNFα significantly increased sema3D over 10-fold ( p≤0.05). IL-1 upregulated all plexins, conversely TNFα significantly decreased plexin A2. NRP-2 showed a significant increase in response to IL-1. While NRP-1 was significantly decreased in response to IL-1. In HDMEC-1 cells IL-1 significantly increased sema3C and sema3D while TNF significantly increased sema3C. TNF significantly increased NRP-2 expression, however, NRP-1 was decreased by all cytokines. Plexin receptor expression was increased upon IL-6 and TNF treatment whereas IL-1 caused significant decrease in plexin A3 expression.

Conclusion

Here, we have demonstrated that several cytokines known to be upregulated during disc degeneration and disc prolapse, regulate expression of certain members of class 3 semaphorins, as well as their receptors in human NP cells and HDMEC-1 cells. This data suggests that the presence of these factors within the degenerate disc may be responsible for modulating the ingrowth of blood vessels and promote nerve ingrowth leading to discogenic pain. Disclosure of Interest None declared
Disc Degeneration after Temporary Fixation of the Motion Segment in Unstable Thoracolumbar A and B Fractures
Authors: Hoppe S. , Keel M. , Aghayev E. , Benneker L.
Published: May 2014

Introduction

Anterior stabilization with cages/iliac bone graft and corpectomy in addition with posterior spondylodesis is the gold standard for the treatment of anterior unstable Magerl types A and B fractures of the thoracolumbar junction.1,2 The effect of temporary fixation on the intervertebral disc as well as on progression of kyphosis, especially after dorsal implant removal to unblock the caudal nonfused segment, is unclear.

Materials and Methods

Retrospective case series of all patients treated with posterior stabilization in combination with vertebroplasty/kyphoplasty between 2000 and 2010 with secondary implant removal 8 to 12 months after the index operation. All cases were assessed for loss of reduction, Cobb angle, and disc height after implant removal by anteroposterior and lateral radiographs.

Results

Overall 20 (m: 13; f: 7) (age: 40.9 ± 14.4 years) out of 59 patients treated with bridging dorsal instrumentation with transpedicular vertebroplasty underwent implant removal. Fractures were classified as A 3.21 in 15, A 3.1 in 3, and B 2.3 in 2 cases. The fracture location was Th12 in 5, L1 in 11, L2 and L3 in 2 cases. The implant removal was performed 9.6 ± 3.2 month after initial surgery. In all but one case increase of kyphosis was found 6 month later. The mean increase of kyphosis was 8 ± 6 degrees (max: 12 degrees; min: 0 degrees), mainly in the upper motion segment. Significant loss of disc height was found in the upper motion segment compared with before implant removal, whereas vertebral height remains stable. Age highly correlated to loss of reduction and disc height (r = 0.51). No differences between the fracture location and types were found.

Conclusion

After temporary fixation of the intervertebral disc by internal fixation an increase of kyphosis in the segment above the augmented/fractured vertebra is to be expected, especially because of degeneration of the disc above the affected vertebra. This effect is age dependent. In how far this has clinical relevance is topic of ongoing investigations. Disclosure of Interest None declared References Harris MB. The role of anterior stabilization with instrumentation in the treatment of thoracolumbar burst fractures. Orthopedics 1992;15(3):347–350 Mariotti AJ, Diwan AD. Current concepts in anterior surgery for thoracolumbar trauma. Orthop Clin North Am 2002;33(2):403–412
Simulation of Degenerative and Proinflammatory Conditions for Testing of Cell Therapy Approaches in a Disc Organ Culture System
Authors: Teixeira G. Quelhas , Gonçalves R. , Boldt A. , Wilke H.-J. , Ignatius A. , Barbosa M. , Neidlinger-Wilke C.
Published: May 2014

Introduction

A successful cell therapy of disc degeneration requires long-term survival and metabolic activity of injected cells in the degenerative disc environment that is affected by low nutrient supply, decreased osmolarity, and proinflammatory conditions. A bovine disc organ culture system with simulation of degenerative and proinflammatory conditions was evaluated with regard to its suitability for testing the fate of injected cells.

Materials and Methods

Bovine caudal disc punches (up to six discs from each specimen) were exposed to needle puncture and proinflammatory factors (LPS, interleukin [IL]1β) for 2 days after a 6-day culture without stimulus, while untreated samples were used as controls. The induction of a proinflammatory response was evaluated by quantification of prostaglandin E2 (PGE2) in culture supernatants and gene expression analysis of proinflammatory cytokines, matrix metalloproteases (MMPs), collagen type II, and aggrecan was measured in isolated disc cells. The metabolic profile of disc punches was traced by quantification of glucose consumption and lactic acid production. To simulate normal versus degenerative conditions, cultures were maintained at 5 mM glucose and 400 mOsm or exposed to low glucose (0.05 mM) and reduced osmolarity (300 mOsm). PKH-labeled cells were injected within an albumin-based hydrogel scaffold. Discs were analyzed by histochemistry and by determination of glycosaminoglycan (GAG) content after 2 weeks.

Results

PGE2 increased for LPS 10 µg/mL, IL1β 10 ng/mL or 100 ng/mL treatments of 21.7 ± 0.8-fold, 5.5 ± 0.9-fold, and 9.6 ± 0.8-fold, respectively, when compared with control (Fig. 1A). Preliminary evaluations showed that LPS- and IL1β-treated discs increased expression of IL-6 and 8, MMP1 and MMP3, and decreased collagens type II and aggrecan expression (Fig. 1B). GAG content of the disc explants decreased (62.4 ± 9%) within 2 weeks. However, treated samples and controls showed similar glucose consumption and lactic acid production, with the concentrations never reaching levels of starvation or toxic environment. Fluorescent cells could be detected at each sampling time point with only little differences between culture conditions.

Conclusion

Simulation of degenerative or inflammatory conditions increased PGE2 release and expression of MMPs and ILs, and decreased matrix protein expression by disc cells. Monitoring of injected fluorescence-labeled cells allows characterization of cell reactions in a simulated degenerative environment. This approach is suitable for in vitro testing of regenerative or anti-inflammatory treatment strategies of disc degeneration. Acknowledgments The research described was financially supported by PEST founds, FP7-EU project GENODISC, Luso-German Integrated Actions 2012: CRUP/DAAD and by the German Spine Foundation. Disclosure of Interest None declared
Isolation of Stem Cells of the Degenerated Lumbar Intervertebral Disc Using a Low Cost and Very Effective Technique
Authors: Falavigna A. , Peletti-Figueiró M. , Aguiar I. Silvera , Roesch-Ely M. , Machado D. Cantarelli , Pêgas Henriques J. A.
Published: May 2014

Introduction

The limitations of regenerative therapy of the tissue using stem cells are, among others, the length of time until a satisfactory amount of viable cells is obtained for the therapy and the high financial cost. The purpose of this study, was to report a low cost and very effective technique to obtain large amounts of adult mesenchymal stem cells from the degenerated intervertebral disc in a short time.

Materials and Methods

The methodology of the isolation in this study gave rise to an intellectual property claim deposited at INPI under number BR 10 2013 031330 0. The intervertebral disc in this study was obtained from patients with lumbar disc degenerative disease refractory to conservative treatment, who required surgery as therapy. Isolation consisted of incubating the degenerated intervertebral disc without going through enzymatic or mechanical dissociation in DMEM medium supplemented with 10% SFB and 1% P/S in a humidified oven at 37°C for 3 days. To prove that these cells were adult mesenchymal stem cells, the supernatant cells were collected with a Pasteur pipette and cultured conventionally in culture containers adding growth factors (0.02 ng/µL of EGF and FGF) for another 3 days. For this purpose, cellular adhesion tests, with fibroblastic characteristics, of the presence/absence of specific surface antigens through flow cytometry (CD 105+, CD73+, CD90+, CD45-, CD34-, CD14-, CD11b-, CD19-, HLA-DR-) and of cell-differentiation for adipogenic and osteogenic tissue were performed.

Results

Using this process, a confluence of ∼100% of adult mesenchymal stem cells was found in the culture containers 3 days after the patient's intervertebral disc was removed. The isolated cells presented percentages of antigens with specific surfaces in flow cytometry that were positive for mesenchymal stem cells, could differentiate into adipogenic and osteogenic tissue, and had an adhesive characteristic with fibroblastic morphology. The greater the degeneration, the smaller was the percentage of growth and isolation of cells, however its differentiation was satisfactory for adipocytes and osteocytes.

Conclusion

The benefits of the technique described are that it allows: (1) obtaining quickly and (2) a large amount of adult mesenchymal cells, using a methodology (3) with a low financial cost. Disclosure of Interest A. Falavigna: Conflict with CNPq, FAPERGS, AOSpine Latin America M. Peletti-Figueiró: None declared I. Aguiar: None declared M. Roesch-Ely: None declared D. Machado: None declared J. A. Henriques: None declared
SDC2 and SDC4 Working Together to Keep the Homeostasis of IVD? A Preliminary Study
Authors: Zheng Z. , Wang J. , Liu H.
Published: May 2014

Introduction

Our previous work have proved that Syndecan 4 (SDC4) plays an important role in TNFa and IL1b induced disc degeneration by promoting extracellular matrix degradation, Syndecan 2 (SDC2) and SDC4 both are members of SDC family, however, expression and role of SDC2 in intervertebral disc is still unclear. The role of this study is to investigate the expression pattern and regulation of SDC2 and its and role in intervertebral disc degeneration.

Materials and Methods

Nucleus pulposus (NP) cells and annulus fibrosis (AF) cells were cultured separately. mRNA were extracted and expression of SDC2 in NP and AF cells were examined by q-PCR method. Expression of SDC2 was examined by q-PCR in 4, 8, 24hs after NP cells were treated by TNFa (20mg/L), IL1-b(10mg/L) and TGF-b (20mg) retrospectively. NP cells were pretreated with p38, JNK and NFkB signaling pathway inhibitors before treatment with TNFa, then expression of SDC2 was examined by qPCR.

Results

SDC2 was expressed in both NP and AF cells. NP cells have a higher expression level than AF cells of SDC2 in mRNA level. TNFa and IL1b could inhibit SDC2 expression in NP cells robustly. What's more, SDC2 expression was induced after TGFb treatment in NP cells. Inhibition of SDC2 expression by TNFa could be reduced by p38 and NFkB pathway inhibitor. JNK inhibitor has no role in Inhibition of SDC2 expression by TNFa.

Conclusion

Inflammatory cytokines could inhibit SDC2 expression and induce SDC4 expression in NP cells, p38 and NFkB pathway participant in this process. The different regulation pattern of SDC2 and SDC4 might play important role in keeping intervertebral disc homeostasis. Disclosure of Interest None declared
Adjacent Segment Disease Developing Dependence on Preoperative Adjacent Disc Degenerative Changes
Authors: Masevnin S. , Ptashnikov D. , Michailov D. , Smekalenkov O. , Hao M. , Zaborovskii N.
Published: May 2014

Introduction

The causes of adjacent segment disease (ASD) after lumbar spinal fusion are controversial today. Although the role of initial degenerative changes in the adjacent discs is not fully studied many authors have noted the special importance of them. Object To determine the importance of the preoperative degenerative changes of adjacent segment in the development of symptomatic ASD after short lumbar fusion.

Materials and Methods

Retrospective study of 78 patients who underwent short 360° fusion lumbar surgery from 2006 to 2010 for the treatment of degenerative lumbar spine with follow-up at least 3 years. All patients had no preoperative radiographic instability signs of the adjacent levels with normal height of the adjacent discs. We compare 2 groups: first group include 38 patients with the pre existing initial degenerative changes of adjacent discs according MRI and scored by Pfirrmann modified classification with 2-5 grade. The second group include 40 patients with no pre existing changes of the adjacent discs according MRI. There were 67% females. Mean age of 58 years (range 23-78). Mean follow-up of 4 years (3-7 years). MRI evaluation of adjacent segment condition and bend lateral radiographs were performed on the preoperative and follow-up visits. Adjacent segment condition was evaluated by MRI and scored with Pfirrmann modified classification.

Results

In the I group symptomatic ASD was found in 3 cases (7,9%) during 1 year follow-up. There were no cases with increasing the stage of degenerative changes by Pfirrmann according MRI. 3 years f/up in I group showed increasing symptomatic ASD to the 17 cases (44,7%). 76% of them required revision surgery. 11 patients had increasing the degenerative changes to 6-7 stage by Pfirrmann with no clinical manifestation. In the II group at 1 year f/up 2 patients (5%) had symptomatic ASD. At 3 years f/up the number of patients with symptomatic ASD was increased to 6 (15%). 50% of them required revision surgery.

Conclusion

Our study showed that the great role in the development of the symptomatic ASD after short lumbar fusion is belong to pre existing initial degenerative changes of adjacent discs. Patients with preoperative disc degenerative changes have statistically significant increasing risks of developing symptomatic ASD. Disclosure of Interest None declared References Hilibrand AS, Robbins M. Adjacent segment degeneration and adjacent segment disease: the consequences of spinal fusion? Spine J 2004;4(6, Suppl)190S-194S Park P, Garton HJ, Gala VC, Hoff JT, McGillicuddy JE. Adjacent segment disease after lumbar or lumbosacral fusion: review of the literature. Spine 2004;29(17):1938–1944
TGFβ Suppresses Galectin-3 Expression through Smad3 Signaling by NP Cells and its Role in Degenerative Disc Disease
Authors: Tian Y. , Cao P. , Yuan W. , Li J. , Shapiro I. M , Risbud M.
Published: May 2014

Introduction

Objective

To investigate TGFβ regulation of galectin-3 expression in nucleus pulposus (NP) cells and its role in degenerative disc disease.

Materials and Methods

qRT-PCR and Western blot were used to measure galectin-3 expression in NP. Transfections were used to determine role of Smad3 on TGFβ-mediated galectin-3 promoter activity. Lentiviral knockdown of Smad3 was performed to assess the role of Smad3 in galectin-3 expression.

Results

A decrease in galectin-3 expression and promoter activity was observed in NP cells after TGFβ treatment. The same trend was observed in degenerative disc samples. CA-ALK5 treatment resulted in a decrease in basal galectin-3 promoter activity. While DN-ALK5 treatment can block the abolished the suppressive effect of TGFβ on galectin-3 promoter activity. The suppressive effect of Smad3 on galectin-3 promoter was confirmed through gain and loss of function studies. When cells are treated with DN-Smad3 (dominant negative Smad3) or Smad7, cytokine-mediated suppression in galectin-3 promoter activity is completely abolished. To further validate the role of Smad3 and to determine if there is cell type specificity, we measured galectin-3 promoter activity in Smad3 null and wild type mouse fibroblasts. Only in wild type cells is the promoter activity cytokine suppressible. Lentiviral transduction with sh-Smad3 significantly blocked TGFβ dependent decrease in galectin-3 expression.

Conclusion

By controlling the activation of Smad3 signaling, TGFβ modulates the expression of galectin-3 in NP cells. Galectin-3 may play an important role in degenerative disc diseases. To our knowledge, this is first study that shows that in NP cells TGFβ control galectin-3 transcription in Smad3-dependent fashion. Disclosure of Interest None declared
Role of BMI in Matrix Delamination during Intervertebral Disc Degeneration
Authors: Friedmann A. , Schwan S. , Heilmann A. , Goehre F. , Meisel H. J.
Published: May 2014

Introduction

Intervertebral disc (IVD) degeneration is a natural process during aging, contributed by mechanical, genetic, and biochemical parameters. Interactions between these parameters cause an instance of matrix degeneration in IVD tissue on the macroscopic and microscopic scale. The process of disc degeneration by means of matrix destruction should be promoted by an increased body mass index (BMI) representing increased mechanical loadings. The objective is to determine and correlate tissue changes of IVD degeneration in different states under consideration of patients’ BMI value.

Materials and Methods

As part of a clinical study, human IVD tissues from 60 patients removed during standard sequestrectomy as neurosurgical treatment after disc herniations are investigated regarding their microstructure using scanning electron microscopy. Specimens’ microstructure is investigated with focus on the delamination of the tissue matrix, as well as cellular changes. With digital image analyzing software, degeneration characteristics are evaluated and correlated with clinical relevant parameters.

Results

Matrix delamination is identified by digital image analysis as microstructural destruction in disc degeneration closely related to patients BMI on the one hand but on the other hand not related to patients’ age or gender. Cellular variations are proved as decrease in cell density and increase in cell size while creeping reduction of the thickness of the extracellular matrix.

Conclusion

Due to the future development of regenerative therapies for IVD degeneration it is essential to understand the mechanism of degenerative processes in disc tissue. This study demonstrates that structural variations in the microscale are characteristically for disc degeneration and may be the link or trigger for prolapse of degenerated disc tissue. Disclosure of Interest None declared
FasL on Human Nucleus Pulposus Cells Prevents Angiogenesis in the Disc by Inducing Fas-Mediated Apoptosis of Vascular Endothelial Cells
Authors: Sun Z. , Luo Z.
Published: May 2014

Introduction

The intervertebral disc is the largest avascular organ in the human body. However, with the progress of intervertebral disc degeneration (IDD), the disc tends to be vascularized increasingly via angiogenesis. It is well established that both human nucleus pulposus (NP) cells and vascular endothelial cells express FasL and Fas. However, the issue remains open as to whether there are certain active mechanisms preventing angiogenesis in the disc via the FasL-Fas machinery.

Materials and Methods

The study groups consisted of NP from cadavers and patients as below: Normal cadaveric donors as control ( n = 8; average age 41.8 ± 3.6 [range 37-47]) and patients with IDD undergoing discectomy as degenerative NP samples ( n = 20; average age 43.4 ± 5.4 [range 35-52]). Here, we established a coculture system of human NP cells and vascular endothelial (HMEC-1) cells. In adition, upregulated FasL of NP cells were cocultured with HMEC-1 cells.

Results

We found that normal NP cells were more capable of inducing apoptosis in HMEC-1 cells (14.2 ± 3.4%) than degenerate NP cells (6.7 ± 1.9%), p < 0.05 By upregulating the FasL expression in degenerate NP cells, we found that FasL played an essential role in the mediation of HMEC-1 cell apoptosis with the activation of downstream FADD and caspase-3. Moreover, we found an increased Fas expression in HMEC-1 cells following cocultured with NP cells, which might be closely linked with FasL produced by NP cells and enhance their interaction.

Conclusion

This is the first study showing FasL-Fas network might plays an important role in the molecular mechanisms of angiogenesis avoidance of human disc. Consequently, our findings might shed light on the pathogenesis in human IDD and provide a novel target for the treatment strategies for IDD. Disclosure of Interest None declared
Experimental Research on Repair of the Degenerated Intervertebral Disc by Bone Marrow Stromal Stem Cell
Authors: Wang J. , Cui X. , Xiang Q. , Zhou Y.
Published: May 2014

Introduction

Objective

To observe the effects of halting, reversing degeneration of the intervertebral disc repair or repairing the degenerated intervertebral disc after bone marrow, stromal stem cells were transplanted to the degenerated rat intervertebral disc.

Materials and Methods

Rats aged 2 weeks were selected and sacrificed by breaking the neck. The bone marrow stroma stem cells of the rats were separated, cultured, detected for their adherence rate, growth, and diving. The Wistar rats aged 8 weeks were divided into four groups at random with 15 rats for each group. Each group was further divided into three subgroups with five rats for each subgroup. Experimental animal models were established. The three subgroups of animals were sacrificed at appropriate time, respectively, observed for morphological changes of the intervertebral disc tissue under a light microscope, and immunohistochemically detected for expression of type I collagen in the fibrous rings.

Results

Primary culture and continuous culture indicate that the bone marrow stroma stem cells of the rats have an active proliferation and multiplication capacity. Hematoxylin and eosin staining indicates that group A is the normal group and the intervertebral disc shape is regular during various periods of time; in group B, the intervertebral space becomes narrow and the fibrous rings are arranged irregularly; in group C, the intervertebral space of the specimens is becoming narrow and the fibrous rings are arranged irregularly in an early stage; the histological changes of various periods of time are consistent with that of group B. An immunohistochemical qualitative analysis on the fibrous ring type I collagen indicates that the expression of fibrous ring type I collagen is more in group A and the expression of the fibrous ring type I collagen in group B is decreasing. In group C, the intervertebral disc structure is damaged and the expression of type I collagen in the fibrous rings decreases. In group D, the morphology of the fibrous rings and expression of type I collagen during various periods of time are similar to that of group B indicating that the alginate serves as a carrier for the stem cells and does not play a role in repairing the intervertebral disc.

Conclusion

The degeneration of the intervertebral disc may be delayed or the degenerated intervertebral disc repaired after the bone marrow stromal stem cells are transplanted to the degenerated rat intervertebral disc. Disclosure of Interest None declared
Collagen-Derived Peptide N-Acetylated Proline-Glycine-Proline in Intervetebral Disc Recruits Cartilage Endplate Stem Cells by Binding with CXC Receptor 2
Authors: Feng C. , Huang B. , Li H. , Zhou Y.
Published: May 2014

Introduction

Degenerative disc disease (DDD) is a prevalent musculoskeletal disorder and is the major cause of low back pain. But our comprehension about development of the intervertebral disc (IVD) and pathogenesis of disc degeneration is unclear, which is an obstacle for appropriate therapies.

Materials and Methods

Recently, some reports identified stem/progenitor cells within particular IVD niches and cartilage endplate. These cells together with mesenchymal stem cells are recruited to nucleus pulposus (NP) and annulus fibrosus (AF) to generate regenerative functions in a healthy IVD. The possibility of stimulating these stem cells holds promise for future therapy for DDD. But factors which recruit stem cells to NP and AF are unknown. There are increasing evidences that N-acetylated proline-glycine-proline (N-AC-PGP), which is a degradation fragment of collagen, plays an important role in inflammatory cell recruitment. But whether it presents in IVD and whether it is a chemokine of stem cell have remained elusive.

Results

In this study, we demonstrated the presence of N-AC-PGP in IVD and found the correlation between its content in IVD and degeneration grade of IVD. Then we described the generation pathway of N-AC-PGP in IVD. At last, we demonstrated that N-AC-PGP can recruit cartilage endplate stem cells (CESC) by binding with CXC receptors 2 on CESC.

Conclusion

These findings reveal a possible regeneration mechanism in IVD, which depends on the chemotaxis of N-AC-PGP for CESC and indicate a new therapy by using N-AC-PGP to stimulate stem cells in IVD. Disclosure of Interest None declared
Electrospun Scaffold Containing TGF-β1 Promotes Human Mesenchymal Stem Cell Differentiation toward a Nucleus Pulposus-Like Phenotype under Hypoxia
Authors: Zhou Y. , Cui X. , Wang J.
Published: May 2014

Introduction

Intervertebral disc degeneration is considered to have a close relation with nucleus pulposus calcification. Electrospunning provides promising strategies for fabricating nanofibrous scaffolds that resemble the extracellular matrix. Mesenchymal stem cells (MSCs) have great potential ability of proliferation and differentiation, which are suitable stem cell source for nucleus pulposus regeneration.

Materials and Methods

In this study, we fabricated two kinds of nanofibrous scaffolds. The first kind of scaffold was fabricated by blending TGF-β1, polyvinyl alcohol (PVA), and poly (lactic-co-glycolic acid) (PLGA) nanofibers and electrospinning (group I). The other kind of scaffold was fabricated using coaxial electrospinning, in which TGF-β1 and PVA formed the central core and PLGA formed the outer layer (group II). Human MSCs were seeded on both kinds of scaffolds and cultured in a hypoxia chamber (2% O2). Scaffold characterizations were examined using scanning electron microscopy, transmission electron microscopy, scaffold characterization, attenuated total reflectance-Fourier transform infrared spectroscopy. Cell proliferation and differentiation were evaluated after 3 weeks of cell culture.

Results

Results showed that both kinds of scaffolds shared similar diameter distributions and protein release. However, group I scaffolds were more hydrophilic than that of group II. Both kinds of scaffolds induced the MSCs to differentiate toward the nucleus pulposus-type phenotype in vitro. In addition, the expression of nucleus pulposus-associated genes (aggrecan, type II collagen, and Sox-9) in group I increased more than that in group II.

Conclusion

Our results indicated that electrospinning nanofibrous scaffolds containing TGF-β1 support the differentiation of MSCs toward the pulposus-type phenotype in a hypoxia chamber. Electrospinning scaffolds by blending TGF-β1, PVA, and PLGA nanofibers would be a more appropriate choice for nucleus pulposus regeneration. Disclosure of Interest None declared