Visualizing patterns of intervertebral disc damage with dual-energy computed tomography: assessment of diagnostic accuracy in an ex vivo spine biophantom.

BACKGROUND: Previously, dual-energy computed tomography (DECT) has been established for imaging spinal fractures as an alternative modality to magnetic resonance imaging (MRI). PURPOSE: To analyze the diagnostic accuracy of DECT in visualizing intervertebral disc (IVD) damage. MATERIAL AND METHODS: The lumbar spine of a Great Dane dog was used as an ex vivo biophantom. DECT was performed as sequential volume technique on a single-source CT scanner. IVDs were imaged before and after an injection of sodium chloride solution and after anterior discectomy in single-source sequential volume DECT technique using 80 and 135 kVp. Chondroitin/Collagen maps (cMaps) were reconstructed at 1 mm and compared with standard CT. Standardized regions of interest (ROI) were placed in the anterior anulus fibrosus, nucleus pulposus, and other sites. Three blinded readers classified all images as intact disc, nucleus lesion, or anulus lesion. Additionally, clinical examples from patients with IVD lesions were retrospectively identified from the radiological database. RESULTS: Interrater reliability was almost perfect with a Fleiss kappa of 0.833 (95% confidence interval [CI] 0.83-0.835) for DECT, compared with 0.780 (95% CI 0.778-0.782) for standard CT. For overall detection accuracy of IVD, DECT achieved 91.0% sensitivity (95% CI 83.6-95.8) and 92.0% specificity (95% CI 80.8-97.8). Standard CT showed 91.0% sensitivity (95% CI 83.6-95.8) and 78.0% specificity (95% CI 64.0-88.5). CONCLUSION: DECT reliably identified IVD damage in an ex vivo biophantom. Clinical examples of patients with different lesions illustrate the accurate depiction of IVD microstructure. These data emphasize the diagnostic potential of DECT cMaps.

Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades.

Intervertebral disc (IVD) herniation and degeneration is a major source of back pain. In order to regenerate a herniated and degenerated disc, closure of the anulus fibrosus (AF) is of crucial importance. For molecular characterization of AF, genome-wide Affymetrix HG-U133plus2.0 microarrays of native AF and cultured cells were investigated. To evaluate if cells derived from degenerated AF are able to initiate gene expression of a regenerative pattern of extracellular matrix (ECM) molecules, cultivated cells were stimulated with bone morphogenetic protein 2 (BMP2), transforming growth factor ß1 (TGFß1) or tumor necrosis factor-α (TNFα) for 24 h. Comparative microarray analysis of native AF tissues showed 788 genes with a significantly different gene expression with 213 genes more highly expressed in mild and 575 genes in severe degenerated AF tissue. Mild degenerated native AF tissues showed a higher gene expression of common cartilage ECM genes, whereas severe degenerated AF tissues expressed genes known from degenerative processes, including matrix metalloproteinases (MMP) and bone associated genes. During monolayer cultivation, only 164 differentially expressed genes were found. The cells dedifferentiated and altered their gene expression profile. RTD-PCR analyses of BMP2- and TGFß1-stimulated cells from mild and severe degenerated AF tissue after 24 h showed an increased expression of cartilage associated genes. TNFα stimulation increased MMP1, 3, and 13 expression. Cells derived from mild and severe degenerated tissues could be stimulated to a comparable extent. These results give hope that regeneration of mildly but also strongly degenerated disc tissue is possible.

Intervertebral disc needle puncture injury can be repaired using a gelatin-poly (γ-glutamic acid) hydrogel: an in vitro bovine biomechanical validation.

PURPOSE: The subtle impairments of the disc due to anular punctures may have an immediate effect on the functional integrity due to the altered intradiscal pressure, hence the subsequent catabolic degradation. This study evaluates functional restoration of needle puncture injured intervertebral discs with a newly developed injectable hydrogel using the quantitative discomanometry (QD) test. The proposed hydrogel is composed of gelatin and poly (γ-glutamic acid) (γ-PGA) and crosslinked with 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC). METHODS: Thirty-six bovine motion segments were distributed into six groups. Needle puncture injured discs were created in all discs except for those in the first group (intact). The second group included injured discs that received no treatment (injury). The remaining four groups included injured discs repaired with injected hydrogels fabricated with different polymer solutions and EDC concentrations including: gelatin/γ-PGA solution crosslinked with the EDC solution at a 10:1 and 40:1 ratio to form the GP/E(10:1) and GP/E(40:1) groups, respectively, and gelatin and γ-PGA solution crosslinked with the EDC solution at a 10:1 ratio to form the G/E(10:1) or P/E(10:1) groups. The QD tests were performed to evaluate disc integrity of all six groups. RESULTS: Among all hydrogel repair groups, the GP/E(10:1) group was found to have the highest leakage and saturate pressure and was the only group comparable to the intact one. CONCLUSIONS: Restoration of disc integrity secondary to needle puncture injury can be achieved via the repair with the newly developed gelatin hydrogel incorporated with γ-PGA and EDC. These slides can be retrieved under Electronic Supplementary Material.

Pro-Inflammatory Stimuli Influence Expression of Intercellular Adhesion Molecule 1 in Human Anulus Fibrosus Cells through FAK/ERK/GSK3 and PKCδ Signaling Pathways.

OBJECTIVE: Intervertebral disc (IVD) degeneration and disc herniation are major causes of lower back pain, which involve the presence of inflammatory mediators and tissue invasion by immune cells. Intercellular adhesion molecule 1 (ICAM1, also termed CD54) is an adhesion molecule that mediates cell-cell interactions, particularly between immune cells and target tissue. The aim of this study was to examine the intracellular signaling pathways involved in inflammatory stimuli-induced ICAM1 expression in human anulus fibrosus (AF) cells. METHODS: Quantitative reverse transcription-polymerase chain reaction (qPCR), western blotting, and flow cytometry were performed to dissect the roles of different signaling pathways in inflammatory stimuli-mediated ICAM1 expression. RESULTS: Using qPCR and western blot analyses, a significant increase in ICAM1 expression was observed in AF cells after stimulation of lipopolysaccharide (LPS) plus interferon-gamma (IFNγ) in a time-dependent manner. Flow cytometry revealed ICAM1 upregulation on the surface of AF cells. Importantly, LPS plus IFNγ treatment also significantly promoted Chemokine ligand (CCL)2 expression, but not CCL3. The enhanced ICAM1 expression was abolished after incubation with antibody against CCL2. In AF cells, treatment with LPS plus IFNγ activated the FAK/ERK/GSK3 signaling pathways, promoted a time-dependent increase in PKCδ phosphorylation, and promoted PKCδ translocation to the nucleus. Treatment with the pharmacological PKCδ inhibitor; rottlerin, effectively blocked the enhanced productions of ICAM1 and CCL2. CONCLUSIONS: Inflammatory stimuli in AF cells are part of a specific pathophysiology in IVD degeneration and disc herniation that modulates CCL2/ICAM1 activation through the FAK/ERK/GSK3 and PKCδ signaling pathways in AF cells.

Chemokine CCL25 Induces Migration and Extracellular Matrix Production of Anulus Fibrosus-Derived Cells.

Intervertebral disc degeneration is a major source of back pain. For intervertebral disc regeneration after herniation a fast closure of anulus fibrosus (AF) defects is crucial. Here, the use of the C-C motif chemokine ligand 25 (CCL)25 in comparison to differentiation factors such as transforming growth factor (TGF)ß3, bone morphogenetic protein (BMP)2, BMP7, BMP12, and BMP14 (all in concentrations of 10, 50 and 100 ng/mL) was tested in an in vitro micro mass pellet model with isolated and cultivated human AF-cells (n = 3) to induce and enhance AF-matrix formation. The pellets were differentiated (serum-free) with supplementation of the factors. After 28 days all used factors induced proteoglycan production (safranin O staining) and collagen type I production (immunohistochemical staining) in at least one of the tested concentrations. Histomorphometric scoring revealed that TGFß3 delivered the strongest induction of proteoglycan production in all three concentrations. Furthermore, it was the only factor able to facilitate collagen type II production, even higher than in native tissue samples. CCL25 was also able to induce proteoglycan and collagen type I production comparable to several BMPs. CCL25 could additionally induce migration of AF-cells in a chemotaxis assay and therefore possibly aid in regeneration processes after disc herniation by recruiting AF-cells.

Load-induced changes in the diffusion tensor of ovine anulus fibrosus: A pilot MRI study.

PURPOSE: To assess the feasibility of diffusion tensor imaging (DTI) for evaluating changes in anulus fibrosus (AF) microstructure following uniaxial compression. MATERIALS AND METHODS: Six axially aligned samples of AF were obtained from a merino sheep disc; two each from the anterior, lateral, and posterior regions. The samples were mechanically loaded in axial compression during five cycles at a rate and maximum compressive strain that reflected physiological conditions. DTI was conducted at 7T for each sample before and after mechanical testing. RESULTS: The mechanical response of all samples in unconfined compression was nonlinear. A stiffer response during the first loading cycle, compared to the remaining cycles, was observed. Change in diffusion parameters appeared to be region-dependent. The mean fractional anisotropy increased following mechanical testing. This was smallest in the lateral (2% and 9%) and largest in the anterior and posterior samples (17-25%). The mean average diffusivity remained relatively constant (<2%) after mechanical testing in the lateral and posterior samples, but increased (by 5%) in the anterior samples. The mean angle made by the principal eigenvector with the spine axis in the lateral samples was 73° and remained relatively constant (<2%) following mechanical testing. This angle was smaller in the anterior (55°) and posterior (47°) regions and increased by 6-16° following mechanical testing. CONCLUSION: These preliminary results suggest that axial compression reorients the collagen fibers, such that they become more consistently aligned parallel to the plane of the endplates. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. MAGN. RESON. IMAGING 2017;45:1723-1735.

Advances and Prospects in Biomaterials for Intervertebral Disk Regeneration.

Low-back and neck-shoulder pains caused by intervertebral disk degeneration are highly prevalent among middle-aged and elderly people globally. The main therapy method for intervertebral disk degeneration is surgical intervention, including interbody fusion, disk replacement, and diskectomy. However, the stress changes caused by traditional fusion surgery are prone to degeneration of adjacent segments, while non-fusion surgery has problems, such as ossification of artificial intervertebral disks. To overcome these drawbacks, biomaterials that could endogenously regenerate the intervertebral disk and restore the biomechanical function of the intervertebral disk is imperative. Intervertebral disk is a fibrocartilaginous tissue, primarily comprising nucleus pulposus and annulus fibrosus. Nucleus pulposus (NP) contains high water and proteoglycan, and its main function is absorbing compressive forces and dispersing loads from physical activities to other body parts. Annulus fibrosus (AF) is a multilamellar structure that encloses the NP, comprises water and collagen, and supports compressive and shear stress during complex motion. Therefore, different biomaterials and tissue engineering strategies are required for the functional recovery of NP and AF based on their structures and function. Recently, great progress has been achieved on biomaterials for NP and AF made of functional polymers, such as chitosan, collagen, polylactic acid, and polycaprolactone. However, scaffolds regenerating intervertebral disk remain unexplored. Hence, several tissue engineering strategies based on cell transplantation and growth factors have been extensively researched. In this review, we summarized the functional polymers and tissue engineering strategies of NP and AF to endogenously regenerate degenerative intervertebral disk. The perspective and challenges of tissue engineering strategies using functional polymers, cell transplantation, and growth factor for generating degenerative intervertebral disks were also discussed.

Simultaneous quantification of cefuroxime and clindamycin in human lumbar anulus fibrosus, nucleus pulposus and serum via UPLC-MS/MS.

BACKGROUND: This study aimed to develop a sensitive, accurate method for simultaneously quantifying cefuroxime and clindamycin in human serum, lumbar anulus fibrosus and nucleus pulposus. METHODS: Cefuroxime and clindamycin were quantified using ultra high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple-reaction-monitoring mode on a triple-quadrupole AB Qtrap 5500 system in positive ion mode. Internal standards were D3-cefuroxime and D3,13C-clindamycin. Samples were pretreated by precipitating total protein. RESULTS: The method showed high sensitivity and good linearity over broad calibration ranges from 100 to 100 000 ng/mL for cefuroxime and 10 to 10 000 ng/mL for clindamycin in serum, and from 10 to 10 000 ng/mL for cefuroxime and 1 to 1 000 ng/mL for clindamycin in lumbar nucleus pulposus. In all sample types, correlation coefficients were greater than 0.99, intra- and inter-day precision (relative standard deviation) was less than 15%, and accuracy (relative error) was within 14% for both analytes. This method was effective at quantifying penetration of cefuroxime and clindamycin in patients undergoing oblique lumbar interbody fusion surgery. CONCLUSIONS: A very sensitive, specific method for simultaneous detection of cefuroxime and clindamycin has been developed for human lumbar anulus fibrosus, nucleus pulposus and serum samples.

Vascularization of the human intervertebral disc: A scoping review.

Intervertebral discs (IVDs) are often referred to as the largest avascular structures of the human body, yet a collective resource characterizing the vascularization of the IVD does not exist. To address this gap, the objective of this study was to conduct a comprehensive search of the literature to review and summarize current knowledge of the prevalence and localization of blood supply in human IVDs, with a scoping review. A comprehensive search of peer-reviewed publications on the topic of IVD vascularization in humans was conducted across six electronic databases: PubMed, EMBASE, MEDLINE, Scopus, Web of Science, and BIOSIS Previews. Studies of humans were included regardless of age, sex, ethnicity, and health status, with the exception of IVD herniation. Two independent reviewers screened titles and abstracts and full-texts according to eligibility criteria. The review was conducted and reported according to Preferred Reporting Items for Systematic Reviews Extension for Scoping Reviews guidelines. Our search yielded 3122 articles, with 22 articles meeting the inclusion criteria. The study samples ranged in age from fetal to >90 years and included both sexes, various health statuses, and used different methodologies (eg, histology, medical imaging, and gross dissection) to assess vasculature. Overall, consistent observations were that (a) the nucleus pulposus of the IVD is avascular throughout life, (b) both the cartilage endplates and annulus fibrosus receive considerable blood supply early in life that diminishes over the lifespan, and (c) vascular ingrowth into the cartilage endplates and inner layers of the annulus fibrosus is commonly associated with damaged or disrupted tissue, irrespective of age. Histology and immunohistochemistry are often used to report vascularization of the IVD. The body of the current literature suggests that the IVD should not be generalized as an avascular tissue. Instead, vascularization of the IVD differs based on the constituent tissues, their age, and state of degeneration or damage.

Galectins-1 and -3 in Human Intervertebral Disc Degeneration: Non-Uniform Distribution Profiles and Activation of Disease Markers Involving NF-κB by Galectin-1.

Degeneration of the human intervertebral disc (IVD) is assumed to underlie severe clinical symptoms, in particular chronic back pain. Since adhesion/growth-regulatory galectins are linked to arthritis/osteoarthritis pathogenesis by activating a pro-degradative/-inflammatory gene expression signature, we hypothesized a similar functional involvement of galectins in IVD degeneration. Immunohistochemical evidence for the presence of galectins-1 and -3 in IVD is provided comparatively for specimens of spondylochondrosis, spondylolisthesis, and spinal deformity. Immunopositivity was detected in sections of fixed IVD specimens in each cellular compartment with age-, disease-, and galectin-type-related differences. Of note, presence of both galectins correlated with IVD degeneration, whereas correlation with age was seen only for galectin-3. In addition, staining profiles for these two galectins showed different distribution patterns in serial sections, an indication for non-redundant functionalities. In vitro, both galectins bound to IVD cells in a glycan-dependent manner. However, exclusively galectin-1 binding triggered a significant induction of functional disease markers (i.e., IL6, CXCL8, and MMP1/3/13) with involvement of the nuclear factor-kB pathway. This study thus gives direction to further network analyses and functional studies on galectins in IVD degeneration. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2204-2216, 2019.

Tensile behaviour of individual fibre bundles in the human lumbar anulus fibrosus.

Disc degeneration is a common medical affliction whose origins are not fully understood. An improved understanding of its underlying mechanisms could lead to the development of more effective treatments. The aim of this paper was to investigate the effect of (1) degeneration, (2) circumferential region and (3) strain rate on the microscale mechanical properties (toe region modulus, linear modulus, extensibility, phase angle) of individual fibre bundles in the anulus fibrosus lamellae of the human intervertebral disc. Healthy and degenerate fibre bundles excised from different circumferential regions in the outer anulus (posterolateral, lateral, anterolateral, anterior) were tensile tested at slow (0.1%/s), medium (1%/s) and fast (10%/s) strain rates using a micromechanical testing system. Our preliminary results showed that neither degeneration nor circumferential region significantly affected the fibre bundles' mechanical behaviour. However, when the fibre bundles were tested at higher strain rates, this resulted in significantly higher linear moduli and lower phase angles. These findings, compared with data from other studies investigating single and multiple lamellae sections, suggest that degeneration has minimal effect on outer anulus mechanics irrespective of structural level, and the inter- and intra-lamellar arrangement and continuity of the fibre bundles may influence the lamellae's regional behaviour and viscoelasticity.

Biomechanics of the human intervertebral disc: A review of testing techniques and results.

Many experimental testing techniques have been adopted in order to provide an understanding of the biomechanics of the human intervertebral disc (IVD). The aim of this review article is to amalgamate results from these studies to provide readers with an overview of the studies conducted and their contribution to our current understanding of the biomechanics and function of the IVD. The overview is presented in a way that should prove useful to experimentalists and computational modellers. Mechanical properties of whole IVDs can be assessed conveniently by testing 'motion segments' comprising two vertebrae and the intervening IVD and ligaments. Neural arches should be removed if load-sharing between them and the disc is of no interest, and specimens containing more than two vertebrae are required to study 'adjacent level' effects. Mechanisms of injury (including endplate fracture and disc herniation) have been studied by applying complex loading at physiologically-relevant loading rates, whereas mechanical evaluations of surgical prostheses require slower application of standardised loading protocols. Results can be strongly influenced by the testing environment, preconditioning, loading rate, specimen age and degeneration, and spinal level. Component tissues of the disc (anulus fibrosus, nucleus pulposus, and cartilage endplates) have been studied to determine their material properties, but only the anulus has been thoroughly evaluated. Animal discs can be used as a model of human discs where uniform non-degenerate specimens are required, although differences in scale, age, and anatomy can lead to problems in interpretation.

Biomaterials for intervertebral disc regeneration and repair.

The intervertebral disc contributes to motion, weight bearing, and flexibility of the spine, but is susceptible to damage and morphological changes that contribute to pathology with age and injury. Engineering strategies that rely upon synthetic materials or composite implants that do not interface with the biological components of the disc have not met with widespread use or desirable outcomes in the treatment of intervertebral disc pathology. Here we review bioengineering advances to treat disc disorders, using cell-supplemented materials, or acellular, biologically based materials, that provide opportunity for cell-material interactions and remodeling in the treatment of intervertebral disc disorders. While a field still in early development, bioengineering-based strategies employing novel biomaterials are emerging as promising alternatives for clinical treatment of intervertebral disc disorders.

Fabrication and evaluation of biomimetic biodegradable tissue-engineered annulus fibrosus scaffold / 中国组织工程研究

BACKGROUND: It is still difficult to construct tissue-engineered anulus fibrosus scaffolds which have bionic structure, suitable biodegradability and good biocompatibility. OBJECTIVE: To fabricate bionic biodegradable scaffolds with polycaprolactone (PCL) and polydioxanone (PDS) and evaluate the feasibility as a tissue-engineered annulus fibrosus scaffold. METHODS: Five groups of scaffolds at different PCL/PDS proportions were prepared by melt spinning technique: PCL, PCL/PDS70/30, PCL/PDS50/50, PCL/PDS30/70, and PDS groups. Scanning electron microscopy was used to observe the structure and measure the fiber diameter and pore size of these prepared scaffolds. The mechanical properties and contact angle of the scaffolds were measured. The in vitro and in vivo biodegradation of the scaffolds were observation by in vitro simulation and subcutaneous implantation. The expression of inflammatory factors interleukin-1β and tumor necrosis factor-α in the biodegraded tissues was detected. Human Wharton’s jelly mesenchymal stem cells were cultured for 7 days. Cell viability and proliferation was determined by live/dead cell staining. This study was approved by the Medical Ethics Committee of Tianjin Hospital, China on March 2, 2016. RESULTS AND CONCLUSION: Scanning electron microscopy results showed that the thickness of the scaffold fibers was uniform and the angle between fibers was 60°. The mechanical properties analysis showed that the tensile and compressive modulus of the PDS group was the lowest, which did not meet the mechanical requirements of the anulus fibrosus; the tensile and compressive modulus in the PCL group was the highest, and those in the PCL/PDS70/30 and PCL/PDS50/50 group were moderate. Hydrophilicity test showed that higher PDS proportion led to better hydrophilicity. Biodegradation test showed that the biodegradation of pure PDS and PCL/PDS30/70 was too fast, that of PCL was too slow, and that of PCL/PDS70/30 and PCL/PDS50/50 was appropriate. Analysis of inflammatory response around the biodegraded tissue showed that higher proportion of PCL in the scaffold resulted in more severe inflammatory response. CCK-8 and live/dead cell staining showed that human Wharton’s jelly mesenchymal stem cells had good proliferative activity and high survival rate in the PCL/PDS70/30, PCL/PDS50/50, and PCL/PDS30/70 groups. These results suggest that scaffolds in the PCL/PDS70/30 and PCL/PDS50/50 groups can simulate the structure of natural annulus fibrosus, have appropriate biodegradability, excellent mechanical properties and good biocompatibility, which make it a suitable candidate for tissue-engineered annulus fibrosus scaffold.

Mesenchymal progenitor cells combined with pentosan polysulfate mediating disc regeneration at the time of microdiscectomy: a preliminary study in an ovine model.

OBJECT: Following microdiscectomy, discs generally fail to undergo spontaneous regeneration and patients may experience chronic low-back pain and recurrent disc prolapse. In published studies, formulations of mesenchymal progenitor cells combined with pentosan polysulfate (MPCs+PPS) have been shown to regenerate disc tissue in animal models, suggesting that this approach may provide a useful adjunct to microdiscectomy. The goal of this preclinical laboratory study was to determine if the transplantation of MPCs+PPS, embedded in a gelatin/fibrin scaffold (SCAF), and transplanted into a defect created by microdiscectomy, could promote disc regeneration. METHODS: A standardized microdiscectomy procedure was performed in 18 ovine lumbar discs. The subsequent disc defects were randomized to receive either no treatment (NIL), SCAF only, or the MPC+PPS formulation added to SCAF (MPCs+PPS+SCAF). Necropsies were undertaken 6 months postoperatively and the spines analyzed radiologically (radiography and MRI), biochemically, and histologically. RESULTS: No adverse events occurred throughout the duration of the study. The MPC+PPS+SCAF group had significantly less reduction in disc height compared with SCAF-only and NIL groups (p < 0.05 and p < 0.01, respectively). Magnetic resonance imaging Pfirrmann scores in the MPC+PPS+SCAF group were significantly lower than those in the SCAF group (p = 0.0213). The chaotropic solvent extractability of proteoglycans from the nucleus pulposus of MPC+PPS+SCAF-treated discs was significantly higher than that from the SCAF-only discs (p = 0.0312), and using gel exclusion chromatography, extracts from MPC+PPS+SCAF-treated discs also contained a higher percentage of proteoglycan aggregates than the extracts from both other groups. Analysis of the histological sections showed that 66% (p > 0.05) of the MPC+PPS+SCAF-treated discs exhibited less degeneration than the NIL or SCAF discs. CONCLUSIONS: These findings demonstrate the capacity of MPCs in combination with PPS, when embedded in a gelatin sponge and sealed with fibrin glue in a microdiscectomy defect, to restore disc height, disc morphology, and nucleus pulposus proteoglycan content.

The response of cyclic tensile strain on the BMSCs co-cultured human degenerative anulus fibrosus cells / 天津医药

Objective To investigate the effects of different cyclic tensile strains on the proliferation and expression of bone marrow stromal cells (BMSCs)-cocultured human degenerated anulus fibrosus (AF) cells. Methods AF cells were isolated from a patient with degenerated intervertebral disc degeneration (IVD), which were co-cultured with BMSCs. The solely cultured AF cells were used as control group. The two groups of cells were expanded in monolayer, and cyclically strained for 3 hours, which were applied 0, 5%, 10%, 15%and 20%strains at a frequency of 0.25 Hz using BioDynamic test instrument. A flow cytometry method was used to examine the AF cell proliferation at 24 hours followed the application of cyclic tensile strains. After the total RNA was extracted, real-time PCR technology was used to detect the gene expression of collagenⅠand aggrecan. Results Under the same appropriate stress, the proliferative index (PI), the proportion of cells in the period of DNA synthesis, the expression of collagenⅠand aggrecan were significantly higher in the co-cultured group than those of control group (P<0.05). However, the best mechanical stimulation was different in the two groups. For the AF cells, the peaks of PI, the proportion of cells in the period of S period, the expression of collagenⅠand aggrecan were found in the 10% strain group, while for the co-cultured cells, they were found in the 15% strain group. Conclusion Co-culturing with BMSCs has a positive effect on the proliferation and expression of human degenerative fibrous ring cells, which can protect AF cells from bad stress stimulation.

Notochordal cells influence gene expression of inflammatory mediators of annulus fibrosus cells in proinflammatory cytokines stimulation.

OBJECTIVE: Notochordal cells in the intervertebral disc interact with nucleus pulposus (NP) cells and support the maintenance of disc homeostasis by regulation of matrix production. However, the influence of notochordal cells has not been evaluated in the annulus fibrosus (AF), which is the primary pain generator in the disc. We hypothesized that the notochordal cell has the capacity to modulate inflammatory mediators secreted by AF cells secondary to stimulation. METHODS: Notochordal and AF cells were isolated from adult New Zealand white rabbits. AF pellets were cultured with notochordal cell clusters or in notochordal cell-conditioned media (NCCM) for 24 or 48 hours with proinflammatory cytokines at varying concentrations. Gene expression in AF pellets were assayed for nitric oxide synthase (iNOS), cyclo-oxygenase (COX)-2, and interleukin (IL)-6 by real time reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: AF pellet in NCCM significantly decreased the iNOS and COX-2 messenger ribonucleic acid (mRNA) levels compared to AF pellets alone and AF pellets with notochordal cells (p < 0.05). AF pellet resulted in dose-dependent iNOS and COX-2 expression in response to IL-1beta, stimulation, demonstrating that 1 ng/ml for 24 hours yielded a maximal response. AF pellet in NCCM significantly decreased the expression of iNOS and COX-2 in response to 1ng/ml IL-1beta, stimulation at 24 hours (p < 0.05). There was no difference in IL-6 expression compared to AF pellets alone or AF pellets with notochordal cell clusters. CONCLUSION: We conclude that soluble factors from notochordal cells mitigate the gene expression of inflammatory mediators in stimulated AF, as expected after annular injury, suggesting that notochordal cells could serve as a novel therapeutic approach in symptomatic disc development.

Response of cyclic tensile strain on the human degenerative anulus fibrosus cells / 国际生物医学工程杂志

Objective To investigate the effects of different cyclic tensile strain on the proliferation of human anulus fibrosus cells from degenerated discs.Methods Anulus fibrosus(AF) cells were isolated from a degenerated human IVD,expanded in monolayer,and cyclically strained for 3 hours,applying 0,5％,10％,15％ and 20％ strains at a frequency of 0.25 Hz with the use of the DioDynamic test instrument.The flow cytometry method was used to examine the Af cells proliferation at 24 hours following application of the cyclic tensile strains.Results The proliferative index (PI) increased with the magnitude value of cyclic tensile strain except 20％ group.The most significant increase of proliferation index were found in 15％ group.Conclusion There might be some corelationships between magnitude of cyclic tensile strain and the proliferation of the degenerative AF cells.

Notochordal Cells Influence Gene Expression of Inflammatory Mediators of Annulus Fibrosus Cells in Proinflammatory Cytokines Stimulation

OBJECTIVE: Notochordal cells in the intervertebral disc interact with nucleus pulposus (NP) cells and support the maintenance of disc homeostasis by regulation of matrix production. However, the influence of notochordal cells has not been evaluated in the annulus fibrosus (AF), which is the primary pain generator in the disc. We hypothesized that the notochordal cell has the capacity to modulate inflammatory mediators secreted by AF cells secondary to stimulation. METHODS: Notochordal and AF cells were isolated from adult New Zealand white rabbits. AF pellets were cultured with notochordal cell clusters or in notochordal cell-conditioned media (NCCM) for 24 or 48 hours with proinflammatory cytokines at varying concentrations. Gene expression in AF pellets were assayed for nitric oxide synthase (iNOS), cyclo-oxygenase (COX)-2, and interleukin (IL)-6 by real time reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: AF pellet in NCCM significantly decreased the iNOS and COX-2 messenger ribonucleic acid (mRNA) levels compared to AF pellets alone and AF pellets with notochordal cells (p < 0.05). AF pellet resulted in dose-dependent iNOS and COX-2 expression in response to IL-1beta, stimulation, demonstrating that 1 ng/ml for 24 hours yielded a maximal response. AF pellet in NCCM significantly decreased the expression of iNOS and COX-2 in response to 1ng/ml IL-1beta, stimulation at 24 hours (p < 0.05). There was no difference in IL-6 expression compared to AF pellets alone or AF pellets with notochordal cell clusters. CONCLUSION: We conclude that soluble factors from notochordal cells mitigate the gene expression of inflammatory mediators in stimulated AF, as expected after annular injury, suggesting that notochordal cells could serve as a novel therapeutic approach in symptomatic disc development.

Histological Composition of the Extruded Intervertebral Disc in Lumbar Intervertebral Disc Herniation / 체질인류학회지

The aim of this study was to investigate histological composition (ratio of anulus fibrosus, cartilage endplate, and nucleus pulposus) of the herniated lumbar intervertebral disc fragments. The relative percentage of anulus fibrosus, cartilage endplate, and nucleus pulposus was examined in the herniated interverterbral disc fragments. The herniated intervertebral discs took out from the 31 patients were stained with H-E. The stained tissues were photographed, and the histological composition of the herniated intervertebral discs was analyzed after marking 3 components on the photograph with microscopic observation. The composition of the herniated lumbar intervertebral disc is 50.7% of nucleus pulposus, 41.3% of anulus fibrosus, and 8.0% of cartilage endplate. The proportion of the endplate in the herniated intervertebral disc in male was tend to be higher than that in female, and the proportion of the nucleus pulposus was tend to be decreased with aging, however, fail to reach statistical significance. This result may provide fundamental information for accessing clinical symptoms including pain in lumbar vertebral disc herniation.