Annulus Fibrosus Regions Research Articles

Effect of Link N on the Expression of Neurotrophins and Substance P Release by Human Intervertebral Disc Cells Stimulated with Proinflammatory Cytokines

IntroductionAlthough there are different causes of low back pain, intervertebral disc (IVD) degeneration is one of them. In healthy discs, nociceptive nerve fibers and mechanoreceptors penetrate up...

Disc degeneration reduces the delamination strength of the annulus fibrosus in the rabbit annular disc puncture model

Background contextDegenerative disc disease is a common pathologic disorder accompanied by both structural and biochemical changes. Changes in stress distribution across the disc can lead to annulus fibrosus (AF) damage that can affect the strength and integrity of the disc. Given that some present degeneration therapies incorporate biological regrowth of the nucleus pulposus (NP), it is crucial that the AF remains capable of containing this newly grown material. PurposeTo examine the resistance of AF to delamination using an adhesive peel test in experimentally degenerated rabbit discs. Study designExperimentally induced disc degeneration; excised AF tissue study. MethodsDisc degeneration was induced in eight New Zealand white rabbits by annular puncture; four additional rabbits served as controls. In experimental rabbits, an 18-gauge needle was inserted into the anterolateral AF region of levels L2–L3 and L4–L5, and disc height was monitored by X-ray. Animals were sacrificed at 4 and 12 weeks postsurgery and magnetic resonance images and X-rays were taken. Four discs were excised from the experimental animals; two punctured (L2–L3 and L4–L5) and two controls (L3–L4 and L6–L7). The same four discs were also excised from the age-matched control animals and served as nonpunctured control discs. To determine resistance to delamination, AF samples were dissected from each disc and subjected to a mechanical peel test at 0.5 mm/s. ResultsMagnetic resonance imaging and X-ray images confirmed dehydration of the NP and reduced disc height, similar to that found in clinical degeneration. Resistance to delamination was significantly lower in punctured/degenerated discs compared with both the nonpunctured discs from the same animal (27% lower) and the nonpunctured control discs (30% lower) (p=.024). ConclusionsThe findings of this study suggest that degeneration increases the potential for delamination between AF layers. Given this substantial change to the integrity of the AF after degeneration, clinical treatments should not only target rehydration or regrowth of the NP, but should also target repair and strengthening of the AF to confine the NP.

Quantitative analysis of annulus fibrosus and nucleus pulposus derived from T2 mapping, diffusion-weighted and diffusion tensor MR imaging

Quantitative information on annulus fibrosus (AF) and nucleus pulposus (NP) is of interest in early diagnosis of degenerated intervertebral disc (IVD) disease. In this present study, we developed a practical framework to analyse and visualise three feature maps of the IVD: the T2 relaxation time, the apparent diffusion coefficient and the anisotropy. They are derived from advanced Magnetic Resonance Imaging sequences such as T2 mapping, diffusion-weighted and diffusion tensor sequences. These features were quantified using regression analysis with least squares and signal intensity-based direction-oriented methods. Based on these maps, a threshold-based geometrical computing procedure was developed to compute the geometrical repartition maps. As a first case study, in vivo features of AF and NP regions of the L4/5 IVD were analysed and visualised. Practical relevance of our developed framework for a clinical purpose is discussed. In fact, our framework allows useful information of IVD to be quantified and used for early diagnosis as well as for further modelling purpose.

Energy metabolism of intervertebral disc under mechanical loading

Intervertebral disc (IVD) degeneration is closely associated with low back pain (LBP), which is a major health concern in the U.S. Cellular biosynthesis of extracellular matrix (ECM), which is important for maintaining tissue integrity and preventing tissue degeneration, is an energy demanding process. Due to impaired nutrient support in avascular IVD, adenosine triphosphate (ATP) supply could be a limiting factor for maintaining normal ECM synthesis. Therefore, the objective of this study was to investigate the energy metabolism in the annulus fibrosus (AF) and nucleus pulposus (NP) of porcine IVD under static and dynamic compressions. Under compression, pH decreased and the contents of lactate and ATP increased significantly in both AF and NP regions, suggesting that compression can promote ATP production via glycolysis and reduce pH by increasing lactate accumulation. A high level of extracellular ATP content was detected in the NP region and regulated by compressive loading. Since ATP can serve not only as an intra-cellular energy currency, but also as a regulator of a variety of cellular activities extracellularly through the purinergic signaling pathway, our findings suggest that compression-mediated ATP metabolism could be a novel mechanobiological pathway for regulating IVD metabolism.

Fabrication and Evaluation of a Novel Integrated Annulus Fibrosus-Nucleus Pulposus Hybrid Scaffold

The scope of this study was to fabricate and evaluate a novel integrated annulus fibrosus-nucleus pulposus scaffold composed of bone matrix gelatin (BMG) and cartilage extracellular matrix (ECM) respectively. Scaffolds were fabricated by a serial physicochemical and free-drying process. The physiochemical property and compatibility of the composite scaffold with intervertebral disc cells were evaluated. HE staining showed no residual cells in both annulus fibrosus and nucleus pulposus scaffolds. SEM observation revealed that the integration of annulus fibrosus region and nucleus pulposus region was well. The pore diameter of annulus fibrosus scaffold is about 401.4±13.1 μm and the nucleus pulposus scaffold 112.4±21.8 μm, which demonstrated that the composite scaffolds possess interconnected pores structure. The average compressive elastic modulus of the integrated scaffold was 49.06 ±15.57 kpa, which was lower than that of porcine coccyx intervertebral disc, 135.9±28.9 kPa. SEM observation and LIVE/DEAD staining showed good adhesion and high viability of intervertebral disc cells in the integrated scaffold. In conclusion, the integrated annulus fibrosus-nucleus pulposus scaffold can be used for intervertebral disc tissue engineering because of its non-immunogenicity, preservation of ECM composition, good physicochemical property and high biocompatibility with interbertebral disc cells.

Link-N Peptide: A Stepping Stone towards Biological Repair of Intervertebral Disk Degeneration

Introduction Back pain is a fairly common problem which affects a large portion of the population across all ages and has an impact on quality of life. Intervertebral disk (IVD) degeneration is the single most common implicated cause of back pain. Presently, there is no medical treatment or therapeutic agent to address this problem and surgery is the only offered option. Link-N peptide represents the 16 amino acid sequence from the N-terminus of the link protein that stabilizes the proteoglycan aggregates present in cartilage and disk. Link-N peptide is released from the link protein as a result of proteolysis, and has been suggested to play a role in matrix homeostasis by promoting new matrix synthesis. We evaluated its regenerative potential in intact human IVD. Materials and Methods Lumbar IVDs were obtained through organ donations via Transplant Quebec. Disks from seven individuals, five disks per spine, were harvested. Cells were isolated from nucleus pulposus (NP) and inner annulus fibrosus regions of the disks. Single cells were beaded in 1.2% alginate and cultured in DMEM containing 1 g/L glucose and 10% FBS. Alginate beads were exposed to 10–10000 ng/mL Link-N peptide for 48 hours. Intact disks were prepared for organ culture by parallel cuts through the adjacent vertebral bodies close to the end plates, and the remaining bone and the calcified part of the cartilage endplates were removed using a high-speed bone burr. Disks were maintained and cultured with no external load applied in DMEM containing 1 g/L glucose and supplemented with 1% FBS. Link-N was conjugated with 5-TAMRA dye then injected into the center of the disk. The distribution of Link-N in the medium and within the disk was studied to determine whether Link-N is retained in the disk. Disks from adjacent levels were matched for the degree of degeneration and were injected in their NP region with 50µCi 35SO4 along with 0.1 mg or 1 mg of Link-N in 100 µL of medium per disk and harvested after 48 hours. Sustained effect of Link-N was evaluated by injecting the disk with Link-N and injecting 35SO4 1 week later. Proteoglycan synthesis was evaluated by measuring 35SO4 incorporation. Results When human lumbar disk cells from NP and iAF regions beaded in alginate were exposed to Link-N peptide for 48 hours, proteoglycan synthesis was observed to increase in a dose-dependent manner with the maximal response at 1000 ng/mL Link-N. Fluorescently labelled Link-N peptide was injected into the disks to determine if Link-N is retained in the disks matrix or freely diffuses throughout the tissue and equilibrates with surrounding medium. Samples were taken continually from the surrounding medium and from the disk tissue at the termination of the experiment. Fluorescent-Link-N was detectable in the medium at 24 hours and reached equilibrium after 48 hours. The fluorescent peptide was found in the NP and NP/iAF junction but not in the remaining AF. Thus loss of Link-N appears to occur by diffusion through the endplates (Fig. 1). Cell viability was maintained in the NP, at >96%, after injection of 1 mg of Link-N/disk. Disks injected with Link-N showed increased proteoglycan synthesis in the NP and iAF compared to adjacent level control disks matched for grade of degeneration. To evaluate the duration of the effect, disks were injected with 35SO4 1 week after the injection of Link-N. Proteoglycan synthesis remained elevated in Link-N injected disks compared to adjacent level control disks suggesting a sustained effect. Conclusion Link-N peptide has previously been shown to promote matrix protein synthesis by bovine disk cells in monolayer and pellet cultures. In this work, we show that Link-N can promote proteoglycan synthesis not only in human disk cells cultured in 3D constructs, but also in intact adult human disks where the cells are in their native environment. Recently, an increase in disk height measured by MRI was shown in an in vivo rabbit model, where degenerated disks were injected with Link-N. If a similar restoration of disk function could be achieved in the human disks, then Link-N could be a promising candidate for biologically induced disk repair, and could provide an alternative to surgical intervention for early stage disk degeneration. Link-N has a significant cost advantage over growth factors, such as BMP7, TGFβ and, GDF5. Based on prior in vivo studies in the rabbit, Link-N is over 100 times less expensive than recombinant growth factors that have a similar repair response. Thus, Link-N peptide injection could be both effective and cost-efficient therapy for retarding the ongoing degenerative process in early stage disk disease and help relieve back pain. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Annulus fibrosus tissue engineering using lamellar silk scaffolds

Degeneration of the intervertebral disc (IVD) represents a significant muscular skeletal disease. Recently, scaffolds composed of synthetic, natural and hybrid biomaterials have been investigated as options to restore the IVD; however, they lack the hallmark lamellar morphological features of annulus fibrosus (AF) tissue. The goal of regenerating the disc is to achieve anatomical morphology as well as restoration of mechanical and biological function. In this study, two types of scaffold morphology formed from silk fibroin were investigated towards the goal of AF tissue restoration. The first design mimics the lamellar features of the IVD that are associated with the AF region. The second is a porous spongy scaffold that serves as a control. Toroidal scaffolds were formed from the lamellar and porous silk material systems to generate structures with an outer diameter of 8 mm, inner diameter of 3.5 mm and a height of 3 mm. The inter-lamellar spacing in the lamellar scaffold was 150-250 µm and the average pore sizes in the porous scaffolds were 100-250 µm. The scaffolds were seeded with porcine AF cells and, after growth over defined time frames in vitro, histology, biochemical assays, mechanical testing and gene expression indicated that the lamellar scaffold generated results that were more favourable in terms of ECM expression and tissue function than the porous scaffold for AF tissue. Further, the seeded porcine AF cells supported the native shape of AF tissue in the lamellar silk scaffolds. The lamellar silk scaffolds were effective in the formation of AF-like tissue in vitro.

Nucleus pulposus cell-matrix interactions with laminins

The cells of the nucleus pulposus (NP) region of the intervertebral disc play a critical role in this tissue's generation and maintenance, and alterations in NP cell viability, metabolism, and phenotype with aging may be key contributors to progressive disc degeneration. Relatively little is understood about the phenotype of NP cells, including their cell-matrix interactions which may modulate phenotype and survival. Our previous work has identified strong and region-specific expression of laminins and laminin cell-surface receptors in immature NP tissues, suggesting laminin cell-matrix interactions are uniquely important to the biology of NP cells. Whether these observed tissue-level laminin expression patterns reflect functional adhesion behaviors for these cells is not known. In this study, we examined NP cell-matrix interactions with specific matrix ligands, including various laminin isoforms, using quantitative assays of cell attachment, spreading, and adhesion strength. NP cells were found to attach in higher numbers and exhibited rapid cell spreading and higher resistance to detachment force on two laminin isoforms (LM-511,LM-332) identified to be uniquely expressed in the NP region, as compared to another laminin isoform (LM-111) and several other matrix ligands (collagen, fibronectin). Additionally, NP cells were found to attach in higher numbers to laminins as compared to cells isolated from the disc's annulus fibrosus region. These findings confirm that laminin and laminin receptor expression documented in NP tissues translates into unique functional NP cell adhesion behaviors that may be useful tools for in vitro cell culture and biomaterials that support NP cells.

Biomimetic method for combining the nucleus pulposus and annulus fibrosus for intervertebral disc tissue engineering

Tissue engineering strategies for the intervertebral disc (IVD) have traditionally focused either on the annulus fibrosus (AF) or the nucleus pulposus (NP) in isolation, or have simply compared AF cells and NP cells in identical culture conditions. Recently, others in the field have become aware of the advantage of combining the AF and NP into a more comprehensive strategy to address IVD tissue engineering, and have introduced biomimetic approaches to either AF or NP tissue engineering. Here, we introduced a new method for developing a biomimetic, cell-seeded IVD by electrospinning circumferentially-orientated polycaprolactone fibres (AF analogue), seeding them with cells (porcine chondrocytes) and then gelling a cell-agarose solution in the centre (NP analogue). Scanning electron microscopy images demonstrated a high degree of fibre alignment and, along with fluorescent actin staining, confirmed a preferred orientation of cells in the direction of the fibres. Viability assays and histology collectively demonstrated that cells were viable and well-distributed around the interface between the NP and AF regions. In addition, mechanical testing confirmed that the composite IVD scaffolds had higher moduli than the agarose hydrogels alone. As we enter the new decade and the fields of AF and NP tissue engineering begin to merge into a new interfacial and functional IVD tissue-engineering field, approaches such as the method presented here will serve as the foundation for continuously advancing technology that we ultimately endeavour to bring to the clinic for the treatment of patients severely afflicted by degenerative disc disease.

Human intervertebral disc internal strain in compression: The effect of disc region, loading position, and degeneration

The primary function of the disc is mechanical; therefore, degenerative changes in disc mechanics and the interactions between the annulus fibrosus (AF) and nucleus pulposus (NP) in nondegenerate and degenerate discs are important to functional evaluation. The disc experiences complex loading conditions, including mechanical interactions between the pressurized NP and the surrounding fiber-reinforced AF. Our objective was to noninvasively evaluate the internal deformations of nondegenerate and degenerate human discs under axial compression with flexion, neutral, and extension positions using magnetic resonance imaging and image correlation. The side of applied bending (e.g., anterior AF in flexion) had higher tensile radial and compressive axial strains, and the opposite side of bending exhibited tensile axial strains even though the disc was loaded under axial compression. Degenerated discs exhibited higher compressive axial and tensile radial strains, which suggest that load distribution through the disc subcomponents are altered with degeneration, likely due to the depressurized NP placing more of the applied load directly on the AF. The posterior AF exhibited higher compressive axial and higher tensile radial strains than the other AF regions, and the strains were not correlated with degeneration, suggesting this region undergoes high strains throughout life, which may predispose it to failure and tears. In addition to understanding internal disc mechanics, this study provides important new data into the changes in internal strain with degeneration, data for validation of finite element models, and provides a technique and baseline data for evaluating surgical treatments.

Relationship between solute transport properties and tissue morphology in human annulus fibrosus

Poor nutritional supply to the intervertebral disc is believed to be an important factor leading to disc degeneration. However, little is known regarding nutritional transport in human annulus fibrosus (AF) and its relation to tissue morphology. We hypothesized that solute diffusivity in human AF is anisotropic and inhomogeneous, and that transport behaviors are associated with tissue composition and structure. To test these hypotheses, we measured the direction-dependent diffusivity of a fluorescent molecule (fluorescein, 332 Da) in three regions of AF using a fluorescence recovery after photobleaching (FRAP) technique, and associated transport results to the regional variation in water content and collagen architecture in the tissue. Diffusivity in AF was anisotropic, with higher values in the axial direction than in the radial direction for all regions investigated. The values of the diffusion coefficient ranged from 0.38 +/- 0.25 x 10(-6) cm(2)/s (radial diffusivity in outer AF) to 2.68 +/- 0.84 x 10(-6) cm(2)/s (axial diffusivity in inner AF). In both directions, diffusivity decreased moving from inner to outer AF. Tissue structure was investigated using both scanning electron microscopy and environmental scanning electron microscopy. A unique arrangement of microtubes was found in human AF. Furthermore, we also found that the density of these microtubes varied moving from inner to outer AF. A similar trend of regional variation was found for water content, with the highest value also measured in inner AF. Therefore, we concluded that a relationship exists among the anisotropic and inhomogeneous diffusion in human AF and the structure and composition of the tissue.

Intracellular Flow Cytometric Measurement of Extracellular Matrix Components in Porcine Intervertebral Disc Cells

The objective of this study was to develop and demonstrate the utility of a novel method of evaluating intracellular levels of extracellular matrix (ECM) components in intervertebral disc (IVD) cells using flow cytometry. By using this method, this study discriminated between cell populations in porcine IVD and examined the response of IVD cells to monolayer cultures, a traditional method of cell expansion, by measuring phenotypic attributes of ECM component production. It was found that monolayer cultures affected collagen production of IVD cells while there were differences in collagen type II production between the cells isolated from the annulus fibrosus (AF) and nucleus pulposus (NP) regions of IVD. Size distributions of fresh and cultured cells were also presented while the relationships between cell size and intracellular collagen level revealed heterogeneous cell populations in AF and NP regions. Furthermore, this study showed that the intracellular collagen signals of IVD cells were significantly enhanced by the treatments of Brefeldin-A and ascorbic acid. This suggests that Brefeldin-A and ascorbic acid could be used to increase the sensitivity of flow cytometric analysis on intracellular collagen levels by maximizing collagen accumulation inside cells. Since a unique feature of the flow cytometric screening tool is the ability to discriminate between various cell populations in a single sample, the flow cytometric method developed in this study may have the potential to identify specific collagen-producing cell populations from tissues or cell cultures.

Anisotropic Diffusive Transport in Annulus Fibrosus: Experimental Determination of the Diffusion Tensor by FRAP Technique

The annulus fibrosus (AF) of the intervertebral disc (IVD) exhibits a fiber-organized structure which is responsible for anisotropic and inhomogeneous mechanical and transport properties. Due to its particular morphology, nutrient transport within AF is regulated by complex transport kinetics. This work investigates the diffusive transport of a small solute in the posterior and anterior regions of AF since diffusion is the major transport mechanism for low molecular weight nutrients (e.g., oxygen and glucose) in IVD. Diffusion coefficient (D) of fluorescein (332 Da) in bovine coccygeal AF was measured in the three major (axial, circumferential, and radial) directions of the IVD by means of fluorescence recovery after photobleaching (FRAP) technique. It was found that the diffusion coefficient was anisotropic and inhomogeneous. In both anterior and posterior regions, the diffusion coefficient in the radial direction was found to be the lowest. Circumferential and axial diffusion coefficients were not significantly different in both posterior and anterior regions and their values were about 130% and 150% the value of the radial diffusion coefficient, respectively. The values of diffusion coefficients in the anterior region were in general higher than those of corresponding diffusion coefficients in the posterior region. This study represents the first quantitative analysis of anisotropic diffusion transport in AF by means of FRAP technique and provides additional knowledge on understanding the pathways of nutritional supply into IVD.

92. Internal strains in the lumbar intervertebral disc—weakness of the posterolateral regions?

HYPOTHESIS: Morphological and clinical signs of intervertebral disc (IVD) disruption are seen frequently in the posterior and posterolateral inner annulus fibrosus regions. The objectives of this study were to: (1) identify intradiscal circumferential and radial strain peaks during sub-maximal loads; (2) assess how bending loads modulate intradiscal strain patterns and if so, how can they explain mechanical IVD failure.

The composition and protein metabolism in the immature rabbit intervertebral disc.

The nucleus pulposus (NP) and annulus fibrosus (AF) of immature rabbit intervertebral discs (IVD) have been subjected to the dissociative extraction procedure of Sajdera and Hascall (1969). The soluble, insoluble and unextracted fractions so obtained were analysed for total nitrogen, collagen, tyrosine, uronic acid, hexosamine and sialic acid content. A high proportion of non-collagenous protein, hexose and sialic acid in the NP insoluble fraction suggests the presence of glycopeptides associated with collagen and/or proteoglycans. The levels of proteoglycan in the soluble NP and AF fraction are similar. Immature (soluble) collagen, however, resides largely in the AF region. The metabolism of rabbit IVD protein components was also investigated both chemically and by autoradiography. L-Tyrosine-3,5-H3 was administered intraperitoneally (3 mc/kg) to 4 week-old rabbits. Animals were sacrificed at various time intervals and the harvested tissues extracted as before and lumbar discs collected. The levels of L-Tyrosine-3,5-H3 in the NP and AF insoluble and soluble fractions were determined using a tritium scintillation counting procedure and localisation by autoradiography. Pronounced extracellular activity of proteoglycan and glycoprotein is not evident before 24 hours. Soluble collagen, however, is synthesized and dispersed within 4 hours of isotope administration.