Regions Of Intervertebral Disc Research Articles

ADVANCED BIOREACTOR STUDIES OF REGION-SPECIFIC RESPONSE IN THE INTERVERTEBRAL DISC TO COMPRESSION, FLEXION/EXTENSION, AND TORSION

Intervertebral disc (IVD) degeneration is inadequately understood due to the lack of in vitro systems that fully mimic the mechanical and biological complexity of this organ. We have recently made an advancement by developing a bioreactor able to simulate physiological, multiaxial IVD loading and maintain the biological environment in ex vivo IVD models [1].To validate this new bioreactor system, we simulated natural spine movement by loading 12 bovine IVDs under a combination of static compression (0.1 MPa), cyclic flexion/extension (±3˚, ±6˚ or 0-6˚) and cyclic torsion (±2˚, ±4˚ or 0-4˚) for more than 10’000 (0.2 Hz) or 100’000 (1 Hz) cycles over 14 days. A higher number of cycles increased the release of glycosaminoglycans and nitric oxide, as an inflammation marker, whereas fewer cycles maintained these two factors at physiological levels. All applied protocols upregulated the expression of MMP13 in the outermost annulus fibrosus (AF), indicating a collagen degradation response. This was supported by fissures observed in the AF after a longer loading duration. Increasing loading cycles induced high cell death in the nucleus pulposus and inner AF, while with fewer cycles, high cell viability was maintained in all IVD regions, irrespective of the magnitude of rotation.Less frequent multiaxial loading maintains IVD homeostasis while more frequent loading initiates an IVD degenerative profile. Specifically, the morphological and molecular changes were localized in the AF, which can be associated with combined flexion/extension and torsion. More loading cycles induced region-specific cell death and a higher release of extracellular matrix molecules from the innermost IVD regions, likely associated with longer exposure to static compression. Altogether, we demonstrated the advantages of the multiaxial bioreactor to study region-specific response in the IVD, which will allow a more profound investigation of IVD degeneration under different combinations of motions.

Towards Image Guided Magnetic Resonance Elastography via Active Driver Positioning Robot.

This work demonstrates the initial prototype of an MRE driver positioning system, allowing capture of displacement wave patterns from various mechanical vibration loading angles under different vibration frequencies through MR imaging. Three different configurations of the MRE driver positioning robot are tested with an intervertebral disc (IVD) shaped gel phantom. Both the octahedral shear stress signal to noise ratio (OSS-SNR) and estimated stiffness show statistically significant dependence on driver configuration in each of the three phantom IVD regions. This dependence demonstrates that driver configuration is a critical factor in MRE, and that the developed robot is capable of producing a range of configurations. This work presents the first demonstration of an active, imaging guided MRE driver positioning system, with significance for the future application of MRE to a wider range of human tissues.

Toward the Next Generation of Spine Bioreactors: Validation of an Ex Vivo Intervertebral Disc Organ Model and Customized Specimen Holder for Multiaxial Loading

A new generation of bioreactors with integrated six degrees of freedom (6 DOF) aims to mimic more accurately the natural intervertebral disc (IVD) load. We developed and validated in a biological and mechanical study a specimen holder and corresponding ex vivo IVD organ model according to the bioreactor requirements for multiaxial loading and a long-term IVD culture. IVD height changes and cell viability were compared between the 6 DOF model and the standard 1 DOF model throughout the 3 weeks of cyclic compressive loading in the uniaxial bioreactor. Furthermore, the 6 DOF model and holder were loaded for 9 days in the multiaxial bioreactor under development using the same conditions, and the IVDs were evaluated for cell viability. The interface of the IVD model and specimen holder, enhanced with fixation screws onto the bone, was tested in compression, torsion, lateral bending, and tension. Additionally, critical motions such as tension and bending were assessed for a combination of side screws and top screws or side screws and adhesive. The 6 DOF model loaded in the uniaxial bioreactor maintained similar cell viability in the IVD regions as the 1 DOF model. The viability was high after 2 weeks throughout the whole IVD and reduced by more than 30% in the inner annulus fibrous after 3 weeks. Similarly, the IVDs remained highly viabile when cultured in the multiaxial bioreactor. In both models, IVD height changes after loading were in the range of typical physiological conditions. When differently directed motions were applied, the holder-IVD interface remained stable under hyper-physiological loading levels using a side screw approach in compression and torsion and the combination of side and top screws in tension and bending. We thus conclude that the developed holding system is mechanically reliable and biologically compatible for application in a new generation of multiaxial bioreactors.

Icariin regulates stem cell migration for endogenous repair of intervertebral disc degeneration by increasing the expression of chemotactic cytokines

BackgroundIcariin (ICA) can promote the migration and bone formation of bone marrow mesenchymal stem cells. This study explored a potential role of ICA in recruiting stem cell niches (SCNs) within the intervertebral disc region (ISN)-derived stem cells (ISN-SCs) to treat intervertebral disc degeneration (IVDD).Materials and methodsEdU staining, transwell, and wound healing tests were used to analyze the function of ICA on ISN-SCs proliferation and migration ability. Simultaneously, the IVDD rat model was constructed by the acupuncture and divided into Sham, Sham + ICA, IVDD, and IVDD + ICA groups. H&E and PAS staining were performed to detect the pathological changes of IVDD tissues. Immunofluorescence was performed to discover relevant marker expression on the surface of stem cells in the IVDD tissues. Western blot and qPCR were executed to find the protein and mRNA expression of related cytokines in the IVDD tissues.ResultsISN-SCs treated with 1 μM ICA obtained the better ability of proliferation and migration. H&E staining showed that the annulus fibrosus in the IVDD group was obviously hyperplasia with cavities and fissures; the nucleus pulposus was reduced. PAS staining showed that the content of polysaccharides was significantly reduced in the nucleus pulposus of IVDD group. However, the ICA treatment alleviated the pathological trends of the IVDD tissues. Simultaneously, ICA treatment increased significantly the expression of stem cells and IGF-1, TGF-β, SDF-1, CCL-5, Collagen I, Collagen II, Aggrecan, and SOX9 in IVDD tissues.ConclusionsICA treatment promoted the migration of stem cell in IVDD by increasing the expression of chemotactic cytokines, including IGF-1, TGF-β, SDF-1, and CCL-5.

20. Biomechanical effect of facet joint violation on adjacent segment motion and disc strain following single level lumbar fusion with transpedicular screw and rod instrumentation

<h3>BACKGROUND CONTEXT</h3> Facet joint violation (FV) is a complication occurring while placing pedicle screws at the upper-most level using an entry point and trajectory too close to the superior facet joint. The incidence of FV has been reported up to be as high as 50%. However, little is known about how FV affects superior adjacent level spinal stability and especially intervertebral disc (IVD) strain after lumbar fusion. <h3>PURPOSE</h3> To investigate the biomechanical effects of FV in terms of mobility and disc surface strains at the upper level adjacent to L4-5 pedicle screw-rod fixation (PSR) in cadaveric specimens. The disc strain was studied with a new optical technique of soft tissue strain analysis (3D digital image correlation [DIC] technique). <h3>STUDY DESIGN/SETTING</h3> Biomechanic testing in human cadaveric spine specimens. <h3>PATIENT SAMPLE</h3> A total of 14 human cadaveric L3-S1 specimens. <h3>OUTCOME MEASURES</h3> 1. Upper adjacent segment range of motion. 2. Disc surface strain/stress. <h3>METHODS</h3> A total of 14 human cadaveric L3-S1 specimens were divided into two equal groups. L4-5 pedicle-rod fixation was performed in a different fashion in each group ie, facet joint preservation (FP) and facet joint violation (FV). The violation of the facet joint was defined by denuding the facet joint capsule and removing of the external cortical surface of the superior articular process with a 3 mm Leksell-rongeur. The entry point of these pedicle screws was located at the junction between the inferolateral aspect of the facet joint. All specimens were tested multi-directionally under pure moment loading (7.5Nm). DIC was performed with cameras positioned laterally (left side) to capture the change in principal disc strains (Pmax and Pmin) at the upper adjacent level (L3-4) under peak load. Optical motion tracking was simultaneously performed on the right side. The IVD region was divided into four similar sized quarters and included upper and lower endplates. Analysis of variance of upper adjacent level range of motion (ROM) and disc strain, normalized to intact, was performed between the groups. Statistical significant was set at P≤0.05. <h3>RESULTS</h3> Normalized ROM was significantly greater with FV than FP at upper adjacent level (L3-4) during right axial rotation (1.22 vs 0.99, 23% difference, p=0.047). Normalized IVD Pmax strains at the upper adjacent level (L3-4) were significantly greater with FV than FP in the anterior quadrant during right axial rotation (1.80 vs 0.83 (ratio to intact), 97% difference, p=0.003). However, there were no significant differences in ROM and disc Pmax or Pmin strains during flexion, extension and lateral bending. <h3>CONCLUSIONS</h3> Facet violations at the uppermost level of fusion construct during pedicle screw-rod fixation were associated with adjacent level torsional instability and the increase of IVD strain. This study suggested that the preservation of uppermost facet joint of fusion segment was helpful for alleviating adjacent segment mobility and stress concentrations of the IVD above the fusion segment. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Development of a standardized histopathology scoring system for human intervertebral disc degeneration: an Orthopaedic Research Society Spine Section Initiative.

BackgroundHistopathological analysis of intervertebral disc (IVD) tissues is a critical domain of back pain research. Identification, description, and classification of attributes that distinguish abnormal tissues form a basis for probing disease mechanisms and conceiving novel therapies. Unfortunately, lack of standardized methods and nomenclature can limit comparisons of results across studies and prevent organizing information into a clear representation of the hierarchical, spatial, and temporal patterns of IVD degeneration. Thus, the following Orthopaedic Research Society (ORS) Spine Section Initiative aimed to develop a standardized histopathology scoring scheme for human IVD degeneration.MethodsGuided by a working group of experts, this prospective process entailed a series of stages that consisted of reviewing and assessing past grading schemes, surveying IVD researchers globally on current practice and recommendations for a new grading system, utilizing expert opinion a taxonomy of histological grading was developed, and validation performed.ResultsA standardized taxonomy was developed, which showed excellent intra‐rater reliability for scoring nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous end plate (CEP) regions (interclass correlation [ICC] > .89). The ability to reliably detect subtle changes varied by IVD region, being poorest in the NP (ICC: .89‐.95) where changes at the cellular level were important, vs the AF (ICC: .93‐.98), CEP (ICC: .97‐.98), and boney end plate (ICC: .96‐.99) where matrix and structural changes varied more dramatically with degeneration.ConclusionsThe proposed grading system incorporates more comprehensive descriptions of degenerative features for all the IVD sub‐tissues than prior criteria. While there was excellent reliability, our results reinforce the need for improved training, particularly for novice raters. Future evaluation of the proposed system in real‐world settings (eg, at the microscope) will be needed to further refine criteria and more fully evaluate utility. This improved taxonomy could aid in the understanding of IVD degeneration phenotypes and their association with back pain.

Region- and degeneration dependent stiffness distribution in intervertebral discs derived by shear wave elastography

Information on the local stiffness characteristics of the intervertebral disc (IVD) is crucial for the understanding of its structure-function properties in health and disease and may improve numerical modeling. Previous studies have attempted to map local tissue stiffness by sectioning the disc and performing mechanical testing on these discrete tissue units, which is technically challenging and may bias the results. Shear wave elastography (SWE) represents a nondestructive alternative that can provide spatially continuous elasticity estimates. We investigated the feasibility of SWE for human intervertebral disc elasticity mapping in a laboratory setting.To this end, global spinal segment mechanical behavior was determined in 6 loading directions and served as ground truth data for the validation of the approach. Subsequently, the cranial spinal vertebra was removed and shear wave elastographic scans of the IVD were acquired. SWE-measurements were reconstructed into three-dimensional elastographic maps, discretized into distinct IVD regions and correlated with global segment mechanical parameters. SWE-derived Young’s modulus estimates were compared among different regions and as a function of their state of degeneration.We found annulus shear wave speed to be moderately correlated with segment mechanical behavior irrespective of the loading direction whereas shear wave speed in the nucleus pulposus showed a very weak association (mean (SD) absolute Pearson correlation coefficients: 0.51 (0.14) and 0.17 (0.12), respectively). Young’s modulus mapping of the intervertebral disc revealed stiffness to be highest in the ventral annulus with a stiffness decrease both circumferentially towards the dorsal aspect as well as towards the center of the disc. SWE hence provides a valid alternative to disc sectioning and piecewise mechanical testing.

In silico feasibility assessment of extracorporeal delivery of low-intensity pulsed ultrasound to intervertebral discs within the lumbar spine

Low intensity pulsed ultrasound (LIPUS) may have utility for non-invasive treatment of discogenic lower back pain through stimulating, remodeling and accelerating healing of injured or degenerated intervertebral disc (IVD) tissues. This study investigates the feasibility of delivering LIPUS to lumbar IVDs between L2 and S1 spine vertebra using a planar extracorporeal phased array (8 × 8 cm, 1024 elements, 500 kHz). Three 3D anatomical models with heterogenous tissues were generated from patient CT image sets and used in the simulation-based analysis. Time-reversal acoustic modeling techniques were applied to optimize posterior-lateral placement of the array with respect to the body to facilitate energy deposition in discrete target regions spanning the annulus fibrosus and central nucleus of each IVD. Forward acoustic and biothermal simulations were performed with time-reversal optimized array placements and driving amplitude/phase settings to predict LIPUS intensity distributions at target sites and to investigate off-target energy deposition and heating potential. Simulation results demonstrate focal intensity gain of 5–168 across all IVD targets and anatomical models, with greater average intensity gain (>50) and energy localization in posterior, posterolateral, and lateral target sites of IVDs. Localized LIPUS delivery was enhanced in thinner patient anatomies and in the high lumbar levels (L2-L3 and L3-L4). Multiple amplitude/phasing illumination patterns could be sequenced at a fixed array position for larger regional energy coverage in the IVD. Biothermal simulations demonstrated that LIPUS-appropriate exposures of 100 mW cm−2 ISPTA to the target disc region would result in <1 °C global peak temperature elevation for all cases. Hence, simulations suggest that spatially-precise extracorporeal delivery of therapeutically relevant LIPUS doses to discrete regions of lumbar IVDs is feasible and may be useful in clinical management of discogenic back pain.

MRI During Spinal Loading Reveals Intervertebral Disc Behavior Corresponding to Discogram Findings of Annular Fissures and Pain Provocation.

Retrospective analysis of prospectively collected data. The aim of this study was to investigate whether spinal loading, depicted with magnetic resonance imaging (MRI), induces regional intervertebral disc (IVD) differences associated with presence and width of annular fissure and induced pain at discography. Annular fissures play a role in low back pain (LBP) but cannot be accurately characterized with conventional MRI. Recently, annular fissures were suggested to influence different load-induced IVD behavior during MRI when comparing LBP-patients and controls. Thus, the loading effect could characterize behavior related to annular fissures noninvasively with MRI. Lumbar spines of 30 LBP-patients were investigated with MRI with and without loading, discography and CT. Five IVD regions were outlined on sagittal MRI images. Difference in normalized signal intensity (SI) with and without loading was calculated for each region. Eighty-three CT-discograms were graded regarding presence and width of fissures. Discograms were classified as pain-positive if a concordant pain response was obtained at a pressure <50 psi. Comparing IVDs with outer fissures with IVDs without fissures, loading induced different behavior in the two ventral regions and in the posterior region. Higher SI increase in the central region was induced in IVDs with narrower fissures compared to IVDs with wider fissures. In the group of pain-negative discograms, a SI decrease was induced in the dorsal region whereas lack of such in the pain-positive group. The spinal loading-effect, depicted with MRI, reveals different regional behaviors between IVDs with outer fissures compared to those without, and between IVDs with narrow and broad fissures, as well as within posterior annulus between pain-positive and pain-negative discograms. Findings are of importance for future attempts to uncover phenotypes of painful IVDs. 2.

Effects of photobiomodulation on annulus fibrosus cells derived from degenerative disc disease patients exposed to microvascular endothelial cells conditioned medium

Intervertebral disc (IVD) degeneration with chronic low back pain is associated with neo-vascularisation into the deeper IVD regions. During this process, endothelial cells (ECs), which are primarily responsible for angiogenesis, interact with the adjacent annulus fibrosus (AF) cells, which are the first line of defence against the invasion of vascular structures into deeper IVD regions. However, the accumulation of inflammatory and catabolic enzymes that results from this interaction promotes matrix degradation and an inflammatory response. Thus, regulating the production of these mediators and catabolic enzymes could ameliorate IVD degeneration. Photobiomodulation (PBM) therapy is a non-invasive stimulation known to have biologically beneficial effects on wound healing, tissue repair, and inflammation. Here, we examined the effects of PBM, administered at various wavelengths (645, 525, and 465 nm) and doses (16, 32, and 64 J/cm2), on EC-stimulated human AF cells. Our results show that PBM selectively inhibited the EC-mediated production of inflammatory mediators, catabolic enzymes, and neurotrophins by human AF cells in a dose- and wavelength-dependent manner. These results suggest that PBM could be a superior and advanced treatment strategy for IVD degeneration.

Intervertebral Disc Segmentation and Diagnostic Application Based on Wavelet Denoising and AAM Model in Human Spine Image.

To solve the problem of location and segmentation of intervertebral discs in spinal MRI images, a method of intervertebral disc segmentation and degeneration classification diagnosis based on wavelet image denoising and independent component analysis-active appearance model (ICA-AAM) was proposed. Firstly, the spinal MRI image is decomposed by wavelet transform, and the noise is filtered by soft threshold method. Then, aiming at the inadequacy of PCA method in AAM in describing data details, ICA is used instead of PCA to model shape and texture models, and an improved AAM segmentation model is formed. Finally, the intervertebral discs in MRI images are segmented by AAM model, and the degeneration classification of intervertebral discs is diagnosed according to the gray level characteristics of the segmented region. The experimental results show that the method can accurately locate and segment the intervertebral disc region and make classification diagnosis.

Engineering a biomimetic integrated scaffold for intervertebral disc replacement

Tissue engineering technology provides a promising alternative to restore physiological functionality of damaged intervertebral disc (IVD). Advanced tissue engineering strategies for IVD have increasingly focused on engineering IVD regions combined the inner nucleus pulposus (NP) and surrounding annulus fibrosus (AF) tissue. However, simulating the cellular and matrix structures and function of the complex structure of IVD is still a critical challenge. Toward this goal, this study engineered a biomimetic AF-NP composite with circumferentially oriented poly(ε-caprolactone) microfibers seeded with AF cells, with an alginate hydrogel encapsulating NP cells as a core. Fluorescent imaging and histological analysis showed that AF cells spread along the circumferentially oriented PCL microfibers and NP cells colonized in the alginate hydrogel similar to native IVD, without obvious migration and mixing between the AF and NP region. Engineered IVD implants showed progressive tissue formation over time after subcutaneous implantation in nude mice, which were indicated by deposition and organization of extracellular matrix and enhanced mechanical properties. In terms of form and function of IVD-like tissue, our engineered biomimetic AF-NP composites have potential application for IVD replacement.

A method for characterising human intervertebral disc glycosaminoglycan disaccharides using liquid chromatography-mass spectrometry with multiple reaction monitoring.

Intervertebral disc (IVD) degeneration results in the depletion of proteoglycans and glycosaminoglycans (GAGs), which can lead to structural and mechanical loss of IVD function, ingrowth of nociceptive nerve fbres and eventually discogenic pain. Specifc GAG types as well as their disaccharide paterns can be predictive of disease and degeneration in several tissues but have not been comprehensively studied within the IVD. A highly sensitive mass spectrometry based technique with multiple reaction monitoring (MRM) was used to provide characterisation of chondroitin sulphate (CS), hyaluronic acid (HA), heparan sulphate (HS) and their disaccharide sulphation paterns across diferent anatomical regions of human IVDs. Principal component analysis further distinguished important regional variations and proposed potential ageing variations in GAG profles. CS was the GAG in greatest abundance in the IVD followed by HA and HS. Principal component analysis identifIed clear separation of GAG profiles between nucleus pulposus and annulus fibrosus in young and old specimens. Distinct patterns of predominantly expressed disaccharides of CS and HS between young and old IVD samples, provided preliminary evidence that important alterations in disaccharides occur within IVDs during ageing. This technique offered a novel approach to identify and quantify specific GAG disaccharides in human IVDs and the data presented were the first to offer insight into the spatial distribution as well as association with ageing of GAGs and GAG disaccharide sulphation patterns across the human IVD.

With axial loading during MRI diurnal T2-value changes in lumbar discs are neglectable: a cross sectional study

BackgroundAxial loading during MRI (alMRI) combined with T2 mapping recently was shown as a promising method to reveal biomechanical intervertebral disc (IVD) characteristics. This feasibility study aims to investigate whether there is a diurnal variation in the IVD T2-value when using alMRI. This is of importance for the planning of when to perform alMRI investigations and for interpretations of alMRI findings in relation to clinical symptoms.MethodsSix healthy volunteers (30 lumbar discs), were examined with alMRI at three different sessions during 1 day. To be representative for a low back pain cohort in terms of age and IVD degeneration the included participants had a wide age range (27-63y) and all Pfirrmann grades represented. The T2-values were measured in five IVD regions of interest (ROI). The ROIs were equally large in sagittal plane with ROI1 representing anterior parts of the IVD, ROI5 posterior IVD parts and ROI2–4 the parts in between.ResultsT2-values of the entire IVD varied between 38 and 138 ms at 7 am, 33-143 ms at 11.30 am, and 31-147 ms at 4 pm with large regional IVD variations at all time points. No significant alterations of the T2-values over the day were found, neither for the entire IVD (p = 0.4) nor for the various ROIs (p = 0.2–1.0). Neither when correlated to Pfirrmann grade, any significant diurnal T2-value changes were found.ConclusionsWith alMRI, only minor diurnal T2-value changes were found in the lumbar discs. Nonsignificant and neglectable diurnal changes are advantageous both for research purposes, as well as in the clinical setting, giving comparable and robust data regardless of at what time-point the alMRI is performed.

Transplantation of Hypoxic-Preconditioned Bone Mesenchymal Stem Cells Retards Intervertebral Disc Degeneration via Enhancing Implanted Cell Survival and Migration in Rats.

Objective Special hypoxic and hypertonic microenvironment in intervertebral discs (IVDs) decreases the treatment effect of cell transplantation. We investigated the hypothesis that hypoxic preconditioning (HP) could improve the therapeutic effect of bone mesenchymal stem cells (BMSCs) to IVD degeneration. Methods BMSCs from green fluorescent protein-transgenic rats were pretreated with cobalt chloride (CoCl2, 100 μM, 24 h) for hypoxic conditions in vitro. Apoptosis (related pathways of caspase-3 and bcl-2) and migration (related pathways of HIF-1α and CXCR4) were detected in BMSCs. In vivo, BMSCs and HP BMSCs (H-BMSCs) were injected into the rat model of IVD degeneration. The IVD height, survival, migration, and differentiation of transplanted BMSCs and matrix protein expression (collagen II, aggrecan, and MMP-13) were tested. Results H-BMSCs could extensively decrease IVD degeneration by increasing IVD height and collagen II and aggrecan expressions when compared with BMSCs. Significantly, more GFP-positive BMSCs were observed in the nucleus pulposus and annulus fibrosus regions of IVD. HP could significantly decrease BMSC apoptosis (activating bcl-2 and inhibiting caspase-3) and improve BMSC migration (increasing HIF-1α and CXCR4) in vitro. Conclusion HP could significantly enhance the capacity of BMSCs to repair DDD by increasing the survival and migration of implanted cells and increasing matrix protein expression.

A computational model for investigating the effects of changes in bioavailability of insulin-like growth factor-1 on the homeostasis of the intervertebral disc

Insulin-like growth factor-1 (IGF-1) is well-known for upregulating cell proliferation and biosynthesis of the extracellular matrix in the intervertebral disc (IVD). Pathological conditions, such as obesity or chronic kidney disease cause IGF-1 deficiency in plasma. How this deficiency impacts disc homeostasis remains unknown. Pro-anabolic approaches for the treatment of disc degeneration based on enhancing IGF-1 bioavailability to tissue-cells are considered, but knowledge of their effectiveness in enhancing cellular anabolism of a degenerated disc is limited.In this study, we developed a computational model for disc homeostasis specifically addressing the role of IGF-1 in modulating both extracellular matrix biosynthesis and cellularity in the IVD. This model was applied to investigate how changes in IGF-1 bioavailability, namely deficiency or enhancement of growth factor, affect disc health.In this study, it was found that IGF-1 deficiency mainly affects the biosynthesis of ECM components, especially in the most external regions of the IVD such as the cartilage endplates and the outer portion of annulus fibrosus. Also, a total of three approaches for increasing IGF-1 bioavailability as a therapy for degenerated IVDs were investigated. It was found that all these strategies are only beneficial to those disc regions receiving sufficient nutritional supply (i.e., the outmost IVD regions), while they exacerbate tissue degradation in malnourished regions (i.e., inner portion of the disc). This suggests that pro-anabolic growth factor-based therapies are limited in that their success strongly depends on an adequate nutritional supply to the IVD tissue, which is not guaranteed in degenerated discs.

The presence of stem cells in potential stem cell niches of the intervertebral disc region: an in vitro study on rats.

The potential of stem cell niches (SCNs) in the intervertebral disc (IVD) region, which may be of great significance in the regeneration process, was recently proposed. To the best of our knowledge, no previous in vitro study has examined the characteristics of stem cells derived from the potential SCN of IVD (ISN). Therefore, increasing knowledge on ISN-derived stem cells (ISN-SCs) may provide a greater understanding of IVD degeneration and regeneration processes. We aimed to demonstrate the existence of ISN-SCs and to compare their characteristics with bone marrow mesenchymal stem cells (BMSCs) in vitro. Sprague-Dawley rats (male, 10-week-old) were used in this study. ISN tissues were separated by ophthalmic surgical instruments under a dissecting microscope according to the anatomical areas. BMSCs and cells isolated from the ISN tissues were cultured and expanded in vitro. Passage 4 populations were used for further analysis with respect to colony-forming ability, cellular immunophenotype, cell cycle, stem cell-related gene expression, and proliferation and multipotential differentiation capacities. In general, both of ISN-SCs and mesenchymal stromal cells (MSCs) met the minimal criteria for the definition of multipotent mesenchymal stromal cells, including adherence to plastic, specific surface antigen expression, and multipotent differentiation potential. Especially, ISN-SCs even showed greater potential of osteogenesis and chondrogenesis. The ISN-SCs also expressed stem cell-related genes that were comparable to those of BMSCs, and had colony-forming and self-renewal abilities. To the best of our knowledge, this is the first in vitro study aimed towards determining the existence and characteristics of ISN-SCs, which belong to the MSC family and with greater osteogenic and chondrogenic abilities than BMSCs according to our data. This finding may be of great significance for additional studies that investigate the migration of ISN-SCs into the IVD, and may provide a new perspective on different biological approaches for IVD self-regeneration.

Comparison of intervertebral disc displacements measured under applied loading with MRI at 3.0 T and 9.4 T

The purpose of this study was to compare displacement behavior of cyclically loaded cadaveric human intervertebral discs as measured noninvasively on a clinical 3.0T and a research 9.4T MRI system. Intervertebral discs were cyclically compressed at physiologically relevant levels with the same MRI-compatible loading device in the clinical and research systems. Displacement-encoded imaging was synchronized to cyclic loading to measure displacements under applied loading with MRI (dualMRI). Displacements from the two systems were compared individually using linear regression and, across all specimens, using Bland–Altman analysis. In-plane displacement patterns measured at 3.0T and 9.4T were qualitatively comparable and well correlated. Bland–Altman analyses showed that over 90% of displacement values within the intervertebral disc regions of interest lay within the limits of agreement. Measurement of displacement using dualMRI using a 3.0T clinical system is comparable to that of a 9.4T research system. Additional refinements of software, technique implementation, and image processing have potential to improve agreement between different MRI systems. Despite differences in MRI systems in this initial implementation, this work demonstrates that dualMRI can be reliably implemented at multiple magnetic field strengths, permitting translation of dualMRI for a variety of applications in the study of tissue and biomaterial biomechanics.

Modeling the role of IGF-1 on extracellular matrix biosynthesis and cellularity in intervertebral disc

The insulin-like growth factor-1 (IGF-1) is a well-known anabolic agent for intervertebral disc (IVD), promoting both proteoglycan (PG) biosynthesis and cell proliferation. Accordingly, it is believed that IGF-1 may play a central role in IVD homeostasis. Furthermore, the exogenous administration of IGF-1 has been proposed as a possible therapeutic strategy for disc degeneration. The objectives of this study were to develop a new computational framework for describing the mechanisms regulating IGF-mediated homeostasis in IVD, and to apply this numerical tool for investigating the effectiveness of exogenous administration of IGF-1 for curing disc degeneration. A diffusive–reactive model was developed for describing competitive binding of IGF-1 to its binding proteins and cell surface receptors, with the latter reaction initiating the intracellular signaling mechanism leading to PG production and cell proliferation. Because PG production increases cell metabolic rate, and cell proliferation increases nutritional demand, nutrients transport and metabolism were also included into the model, and co-regulated, together with IGF-1, IVD cellularity. The sustainability and the effectiveness of IGF-mediated anabolism were investigated for conditions of pathologically insufficient nutrient supply, and for the case of exogenous administration of IGF-1 to degenerated IVD. Results showed that pathological nutrients deprivation, by decreasing cellularity, caused a reduction of PG biosynthesis. Also, exogenous administration of IGF-1 was only beneficial in well-nourished regions of IVD, and exacerbated cell mortality in malnourished regions. These findings remark the central role of nutrition in IVD health, and suggest that adequate nutritional supply is paramount for achieving a successful IGF-based therapy for disc degeneration.

Hif-αs Modulation of Sox9-Dependent Extracellular Matrix Production: Insight into Intervertebral Disc Cell Activity under Hypoxia

Introduction Intervertebral disc (IVD) degeneration leads to loss of mechanical function of motion segment and is attributed to deregulated extracellular matrix components in the nucleus pulposus (NP). The NP is rich in type II collagen (encoded by COL2A1) and aggrecan (encoded by AGC1), which are known to be regulated by the master chondrogenic transcription factor, Sox9.1 In situ studies of human degenerated NP illustrated a reduction of COL2A1 and absence of AGC1 expression, whereas SOX9 expression level remained high.2 This indicates that there may be other factors in the IVD that can regulate the Sox9-dependent matrix expression. Recent studies in aged rabbit IVD suggest that the abnormal NP cells may be contributed by a migration of the Sox9-expressing prechondrocytic cells from the annulus fibrosus (AF) adjacent to the endplate (EP).3, 4 Among the hypoxia inducible factor-α subunits (Hif-α), Hif-1α and Hif-2α (EPAS1) have been illustrated as important transcription factors in maintaining disc cell and matrix homeostasis, particularly in the hypoxic NP region.5-7 We hypothesized that Hif-αs may regulate the Sox9-dependent transcription of the extracellular matrix genes in response to IVD degeneration. First, we aimed at identification of Sox9 and Hif-αs co-expression in cells of non-degenerated and degenerated IVD. Second, we tested the modulatory effects of Hif-1α/Hif-2α on the expression of Col2a1 and Agc1 using Sox9-expressing mouse prechondrocytic cells as a model. Materials and Methods IVD were harvested from wild-type C57BL/6N mice at 3 and 6 month old. Disc degeneration was induced by an insertion of 25G needle into contralateral AF of 4-month old Lewis rat and discs were harvested post 2 to 4 weeks of operation. Lumbar IVD of scoliosis patients and degenerative disc disease patient were collected. All animal and human works were approved by local ethical committee. Comparative immunohistochemistry staining of Hif-1α, Hif-2α and Sox9 were studied in parrafilm sections of IVD. Luciferase-based promoter assays based on specific Col2a1 or Agc1 cis-acting elements via the overexpression for each of the HIF-α subunits and/or for Sox9 in ATDC5 cells were performed. Results Relative co-expression patterns of Hif-1α, Hif-2α and Sox9 were detected in AF, NP and EP of the mouse, rat and human IVD. Intracellular expression of Hif-1α, Hif-2α, and Sox9 were confirmed by immunofluorescence, indicating a possible role for Hif-αs-Sox9 tonic activity in the IVD. By luciferase-based promoter assay, Sox9 is demonstrated as a dominant transcription factor in the activation of transcription of Col2a1 and Agc1 in mouse chondroprogenitor cells. Overt additive transcriptional upregulation of Col2a1 and Agc1 was observed with a co-expression of HIF-1α and Sox9. Strikingly, Hif-2α inhibited the Sox9-dependent transcriptional upregulation of Col2a1 and Agc1. Conclusion Our findings indicate the presence of Hif-αs/Sox9 expressing cells in IVD. In addition, our data suggest that Hif-αs can modulate Sox9-mediated transcriptional regulation of Col2a1 and Agc1 in prechondrocytic cells, implicating a possible role of dynamic Hif-αs-Sox9 interaction in the IVD degeneration process. Further investigations of the orchestration mechanisms of Hif-αs/Sox9 in the IVD may provide insights in potential strategies to alleviate disc degeneration. Disclosure of Interest None declared References Richardson SM, Hoyland JA, Mobasheri R, Csaki C, Shakibaei M, Mobasheri A. 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