Outer Annulus Fibrosus Research Articles

Single cell RNA sequencing reveals a shift in cell function and maturation of endogenous and infiltrating cell types in response to acute intervertebral disc injury.

Intervertebral disc (IVD) degeneration contributes to disabling back pain. Degeneration can be initiated by injury and progressively leads to irreversible cell loss and loss of IVD function. Attempts to restore IVD function through cell replacement therapies have had limited success due to knowledge gaps in critical cell populations and molecular crosstalk after injury. Here, we used single cell RNA sequencing to identify the transcriptional changes of endogenous and infiltrating IVD cell populations, as well as the potential of resident mesenchymal stem cells (MSCs) for tissue repair. Control and Injured (needle puncture) tail IVDs were extracted from 12 week old female C57BL/6 mice 7 days post injury and clustering analyses, gene ontology, and pseudotime trajectory analyses were used to determine transcriptomic divergences in the cells of the injured IVD, while immunofluorescence was utilized to determine mesenchymal stem cell (MSC) localization. Clustering analysis revealed 11 distinct cell populations that were IVD tissue specific, immune, or vascular cells. Differential gene expression analysis determined that Outer Annulus Fibrosus, Neutrophils, Saa2-High MSCs, Macrophages, and Krt18+ Nucleus Pulposus (NP) cells were the major drivers of transcriptomic differences between Control and Injured cells. Gene ontology of DEGs suggested that the most upregulated biological pathways were angiogenesis and T cell related while wound healing and ECM regulation categories were downregulated. Pseudotime trajectory analyses revealed that cells were driven towards increased cell differentiation due to IVD injury in all IVD tissue clusters except for Krt18+ NP which remained in a less mature cell state. Saa2-High and Grem1-High MSCs populations drifted towards more IVD differentiated cells profiles with injury and localized distinctly within the IVD. This study strengthens the understanding of heterogeneous IVD cell populations response to injury and identifies targetable MSC populations for future IVD repair studies.

Hydration State Throughout Porcine Lumbar Intervertebral Discs: Comparing Fresh and Frozen-Thawed Specimens.

Water content in intervertebral discs (IVDs) is essential for physiological and mechanical function. Freezing post-mortem tissue prior to biomechanical testing is a common practice to prevent tissue degradation, but this process has been theorized to alter hydration within IVDs. The hydration state throughout porcine lumbar IVDs, a common lumbar surrogate, is unknown as are the effects of freezing on porcine IVD hydration. Nineteen porcine lumbar spines were stored in one of the three conditions: frozen (- 20 °C) wrapped in saline-soaked gauze, frozen (- 20 °C) without saline, or fresh. Water content was measured in four disc regions within each of 89 discs: nucleus pulposus (NP), inner (AF-A), intermediate (AF-B), and outer (AF-C) annulus fibrosus. A three-factor, repeated measure analysis of variance was conducted for storage condition, spinal level, and repeated measure disc region. No significant differences were observed in spinal level or storage condition as a main effect. Mean hydration was significantly different in each disc region with mass percentage of water found to be 88.8 ± 1.7% in NP, 79.6 ± 3.8% in AF-A, 71.9 ± 3.7% in AF-B, and 62.3 ± 3.3% in AF-C. No significant differences were shown in NP and AF-C regions between storage conditions. Two significant differences in storage condition were observed in AF-A and AF-B regions, but there is likely no biological difference in these populations. Water content throughout porcine lumbar IVD was determined and results suggest one freeze-thaw cycle at - 20 °C does not alter the overall hydration within the porcine lumbar IVD.

Stimuli-Responsive Delivery Systems for Intervertebral Disc Degeneration.

As a major cause of low back pain, intervertebral disc degeneration is an increasingly prevalent chronic disease worldwide that leads to huge annual financial losses. The intervertebral disc consists of the inner nucleus pulposus, outer annulus fibrosus, and sandwiched cartilage endplates. All these factors collectively participate in maintaining the structure and physiological functions of the disc. During the unavoidable degeneration stage, the degenerated discs are surrounded by a harsh microenvironment characterized by acidic, oxidative, inflammatory, and chaotic cytokine expression. Loss of stem cell markers, imbalance of the extracellular matrix, increase in inflammation, sensory hyperinnervation, and vascularization have been considered as the reasons for the progression of intervertebral disc degeneration. The current treatment approaches include conservative therapy and surgery, both of which have drawbacks. Novel stimuli-responsive delivery systems are more promising future therapeutic options than traditional treatments. By combining bioactive agents with specially designed hydrogels, scaffolds, microspheres, and nanoparticles, novel stimuli-responsive delivery systems can realize the targeted and sustained release of drugs, which can both reduce systematic adverse effects and maximize therapeutic efficacy. Trigger factors are categorized into internal (pH, reactive oxygen species, enzymes, etc.) and external stimuli (photo, ultrasound, magnetic, etc.) based on their intrinsic properties. This review systematically summarizes novel stimuli-responsive delivery systems for intervertebral disc degeneration, shedding new light on intervertebral disc therapy.

Cellular behavior and extracellular matrix turnover in bovine annulus fibrosus cells under hydrostatic pressure and deviatoric strain.

Intervertebral disc herniation is a common spinal disorder that is often treated with discectomy when conservative measures fail. To devise therapeutic strategies for tears in the annulus fibrosus (AF), the regenerative capability of AF cells under spinal loading needs to be addressed. We hypothesized that the compressive loading associated with deformation in AF cells reduces synthetic and degradative activities in extracellular matrix and cell proliferation. We evaluated expression of key matrix molecules and cell proliferation by RT-PCR and immunohistochemistry by inner and outer bovine AF cells incubated under hydrostatic pressure (HP), arc-bending strain (Strain), and combined HP and Strain (HP/Strain) mimicking spinal loading. Inner AF cells showed significantly increased levels of aggrecan core protein, chondroitin sulfate N-acetylgalactosaminyltransferase-1, and tissue inhibitor of metalloproteinases-2 by 6 days under HP (p < 0.05), with a tendency toward increased matrix metalloproteinase-13. Outer AF cells demonstrated a significant decline in collagen type-2 under Strain and HP/Strain (p < 0.05) and a tendency toward suppression of collagen type-1 and elastin expression compared to HP and unloaded control. On the other hand, proliferating cell nucleus antigen increased significantly under Strain and HP/Strain in inner AF and declined under unloaded and HP in outer AF (p < 0.05). Immunohistology findings supported reductions in gene expressions of matrix molecules. Thus, changes in HP/Strain in AF appear to diminish synthetic and degradative activities while increasing cell proliferation. To promote regeneration, continuous overloading should be avoided, as it converts the synthetic activity to a state in which tissue repair is limited.

Assessment of a Discogenic Pain Animal Model Induced by Applying Continuous Shear Force to Intervertebral Discs

BACKGROUND: Chronic discogenic pain includes degeneration-driven changes under the mechanical macroenvironment of an internal disc, which leads to the progressive changes of biochemical microenvironment that induce abnormal ingrowth of the nociceptor. The propriety of the animal model reflecting the pathologic natural history has not been assessed. OBJECTIVES: This study investigated the biochemical evidence of chronic discogenic pain by employing a discogenic pain animal model induced by shear force. STUDY DESIGN: Animal study utilizing rats in vivo model of a shear force device. METHODS: Fifteen rats were divided into 3 groups (n = 5/group) according to the period for which sustained dorsoventral shear force was applied (1 week or 2 weeks); the control group received the spinous attachment unit, without a spring. Pain data were collected using von Frey hairs on the hind paws. Growth factor and cytokine abundance was analyzed in the dorsal root ganglion (DRG) and plasma. RESULTS: After the shear force devices were installed, the significant variables were found to markedly increase in the DRG tissues of the 2-week group; however, they were not altered in the 1-week group. Specifically, interleukin (IL)-6, neurogrowth factor (NGF), transforming growth factor (TGF)-alpha, platelet-derived growth factor (PDGF)-beta, and vascular endothelial growth factor (VEGF) were increased. Meanwhile, the plasma levels of tumor necrosis factor-alpha, IL-1beta, IL-5, IL-6, IL-12, and NGF were increased in the 1-week group; whereas, TGF-alpha, PDGF-beta, and VEGF were increased in the 2-week group. LIMITATIONS: The limitations include the general limitations of quadrupedal animals, the poor precision and flexural deformation of shear force devices, inaccuracies regarding the evaluation of histological denaturation, and short intervention and observational periods. CONCLUSIONS: This animal model effectively generated biochemical responses to shear loading with evidence of neurological changes induced without direct macrodamage to the outer annulus fibrosus. Chemical internals were induced by mechanical externals among the contributing factors of chronic discogenic pain. KEY WORDS: Discogenic pain, dorsal root ganglion, intervertebral discs, low back pain, shear force, interleukin, growth factor, animal model

Physiological and degenerative loading of bovine intervertebral disc in a bioreactor: A finite element study of complex motions

Intervertebral disc (IVD) degeneration and regenerative therapies are commonly studied in organ-culture experiments with uniaxial compressive loading. Recently, in our laboratory, we established a bioreactor system capable of applying loads in six degrees-of-freedom (DOF) to bovine IVDs, which replicates more closely the complex multi-axial loading of the IVD in vivo. However, the magnitudes of loading that are physiological (able to maintain cell viability) or mechanically degenerative are unknown for load cases combining several DOFs. This study aimed to establish physiological and degenerative levels of maximum principal strains and stresses in the bovine IVD tissue and to investigate how they are achieved under complex load cases related to common daily activities.The physiological and degenerative levels of maximum principal strains and stresses were determined via finite element (FE) analysis of bovine IVD subjected to experimentally established physiological and degenerative compressive loading protocols. Then, complex load cases, such as a combination of compression + flexion + torsion, were applied on the FE-model with increasing magnitudes of loading to discover when physiological and degenerative tissue strains and stresses were reached.When applying 0.1 MPa of compression and ±2–3° of flexion and ±1–2° of torsion the investigated mechanical parameters remained at physiological levels, but with ±6–8° of flexion in combination with ±2–4° of torsion, the stresses in the outer annulus fibrosus (OAF) exceeded degenerative levels.In the case of compression + flexion + torsion, the mechanical degeneration likely initiates at the OAF when loading magnitudes are high enough. The physiological and degenerative magnitudes can be used as guidelines for bioreactor experiments with bovine IVDs.

EX VIVO ORGAN MODEL AND SAMPLE HOLDER FOR INTERVERTEBRAL DISC STUDIES IN A NEW GENERATION OF MULTIAXIAL BIOREACTORS

A novel ex vivo intervertebral disc (IVD) organ model and corresponding sample holder were developed according to the requirements for six degrees of freedom loading and sterile culture in a new generation of multiaxial bioreactors. We tested if the model can be maintained in long-term IVD organ culture and validated the mechanical resistance of the IVD holder in compression, tension, torsion, and bending.An ex vivo bovine caudal IVD organ model was adapted by retaining 5-6 mm of vertebral bone to machine a central cross and a hole for nutrient access through the cartilaginous endplate. A counter cross was made on a customized, circular IVD holder. The new model was compared to a standard model with a minimum of bone for the cell viability and height changes after 3 weeks of cyclic compressive uniaxial loading (0.02-0.2 MPa, 0.2 Hz, 2h/ day; n= 3 for day 0, n= 2 for week 1, 2, and 3 endpoints). Mechanical tests were conducted on the assembly of IVD and holder enhanced with different combinations of side screws, top screws, and bone adhesive (n=3 for each test).The new model retained a high level of cell viability after three weeks of in vitro culture (outer annulus fibrosus 82%, inner annulus fibrosus 69%, nucleus pulposus 75%) and maintained the typical values of IVD height reduction after loading (≤ 10%). The holder-IVD interface reached the following highest average values in the tested configurations: 320.37 N in compression, 431.86 N in tension, 1.64 Nm in torsion, and 0.79 Nm in bending.The new IVD organ model can be maintained in long-term culture and when combined with the corresponding holder resists sufficient loads to study IVD degeneration and therapies in a new generation of multiaxial bioreactors.

EFFECT OF NUCLEOTOMY ON AXIAL AND RADIAL COMPRESSION STRAIN DISTRIBUTIONS OF INTERVERTEBRAL DISC

Surgery of nucleus pulposus removal is one of the methods used for the treatment of lumbar disc herniation in the clinic. In order to understand the strain states of the lumbar intervertebral disc (IVD) after nucleotomy, the axial compression experiments of porcine lumbar IVD were implemented using digital image correlation (DIC) technology. The normal IVDs are named as control group; the IVDs treated with trypsin by removing the nucleus pulposus are named as experimental group. The distributions and the change rules of both axial and radial strains in different regions of the IVD with load were studied, and the experimental results of two groups were compared. The results show that both axial and radial strains of different regions increased with stress in two groups, and the variation had non-uniformity. The axial strain in the posterior region of the annulus fibrosus (AF) was larger than that in the anterior region, and the inner AF was larger than that of the outer AF. The axial strain increase of the experimental group was larger than that of the control group obviously. In the posterior region, the radial strain increase was larger than that in the anterior region, and the outer AF was significantly larger than that of the inner AF. The radial strain direction of the experimental group in the inner AF was towards the center of the nucleus pulposus, which was opposite to that of the control group, and the radial strain increase was evidently smaller than that of the control group. In the outer AF, the radial strain direction of the experimental group was the same as that of the control group and the radial strain increase was significantly larger than that.

Spatiotemporal Characterization of Human Early Intervertebral Disc Formation at Single-Cell Resolution.

The intervertebral disc (IVD) acts as a fibrocartilaginous joint to anchor adjacent vertebrae. Although several studies have demonstrated the cellular heterogeneity of adult mature IVDs, a single-cell transcriptomic atlas mapping early IVD formation is still lacking. Here, the authors generate a spatiotemporal and single cell-based transcriptomic atlas of human IVD formation at the embryonic stage and a comparative mouse transcript landscape. They identifytwo novel human notochord (NC)/nucleus pulposus (NP) clusters, SRY-box transcription factor 10 (SOX10)+ and cathepsin K (CTSK)+ , that are distributed in the early and late stages of IVD formation and they are validated by lineage tracing experiments in mice. Matrisome NC/NP clusters, T-box transcription factor T(TBXT)+ and CTSK+ , are responsible for the extracellular matrix homeostasis. The IVD atlas suggests that a subcluster of the vertebral chondrocyte subcluster might give rise to an inner annulus fibrosus of chondrogenic origin, while the fibroblastic outer annulus fibrosus preferentially expresseds transgelin and fibromodulin . Through analyzing intercellular crosstalk, the authors further find that notochordal secreted phosphoprotein 1 (SPP1) is a novel cue in the IVD microenvironment, and it is associated with IVD development and degeneration. In conclusion, the single-cell transcriptomic atlas will be leveraged to develop preventative and regenerative strategies for IVD degeneration.

Ccn2a-FGFR1-SHH signaling is necessary for intervertebral disc homeostasis and regeneration in adult zebrafish.

Intervertebral disc (IVD) degeneration is the primary cause of back pain in humans. However, the cellular and molecular pathogenesis of IVD degeneration is poorly understood. This study shows that zebrafish IVDs possess distinct and non-overlapping zones of cell proliferation and cell death. We find that, in zebrafish, cellular communication network factor 2a (ccn2a) is expressed in notochord and IVDs. Although IVD development appears normal in ccn2a mutants, the adult mutant IVDs exhibit decreased cell proliferation and increased cell death leading to IVD degeneration. Moreover, Ccn2a overexpression promotes regeneration through accelerating cell proliferation and suppressing cell death in wild-type aged IVDs. Mechanistically, Ccn2a maintains IVD homeostasis and promotes IVD regeneration by enhancing outer annulus fibrosus cell proliferation and suppressing nucleus pulposus cell death through augmenting FGFR1-SHH signaling. These findings reveal that Ccn2a plays a central role in IVD homeostasis and regeneration, which could be exploited for therapeutic intervention in degenerated human discs.

Discogenic Low Back Pain: Anatomy, Pathophysiology and Treatments of Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration is a major contributing factor for discogenic low back pain (LBP), causing a significant global disability. The IVD consists of an inner core proteoglycan-rich nucleus pulposus (NP) and outer lamellae collagen-rich annulus fibrosus (AF) and is confined by a cartilage end plate (CEP), providing structural support and shock absorption against mechanical loads. Changes to degenerative cascades in the IVD cause dysfunction and instability in the lumbar spine. Various treatments include pharmacological, rehabilitation or surgical interventions that aim to relieve pain; however, these modalities do not halt the pathologic events of disc degeneration or promote tissue regeneration. Loss of stem and progenitor markers, imbalance of the extracellular matrix (ECM), increase of inflammation, sensory hyperinnervation and vascularization, and associated signaling pathways have been identified as the onset and progression of disc degeneration. To better understand the pain originating from IVD, our review focuses on the anatomy of IVD and the pathophysiology of disc degeneration that contribute to the development of discogenic pain. We highlight the key mechanisms and associated signaling pathways underlying disc degeneration causing discogenic back pain, current clinical treatments, clinical perspective and directions of future therapies. Our review comprehensively provides a better understanding of healthy IVD and degenerative events of the IVD associated with discogenic pain, which helps to model painful disc degeneration as a therapeutic platform and to identify signaling pathways as therapeutic targets for the future treatment of discogenic pain.

Glis1 and oxaloacetate in nucleus pulposus stromal cell somatic reprogramming and survival.

Regenerative medicine aims to repair degenerate tissue through cell refurbishment with minimally invasive procedures. Adipose tissue (FAT)-derived stem or stromal cells are a convenient autologous choice for many regenerative cell therapy approaches. The intervertebral disc (IVD) is a suitable target. Comprised of an inner nucleus pulposus (NP) and an outer annulus fibrosus (AF), the degeneration of the IVD through trauma or aging presents a substantial socio-economic burden worldwide. The avascular nature of the mature NP forces cells to reside in a unique environment with increased lactate levels, conditions that pose a challenge to cell-based therapies. We assessed adipose and IVD tissue-derived stromal cells through in vitro transcriptome analysis in 2D and 3D culture and suggested that the transcription factor Glis1 and metabolite oxaloacetic acid (OAA) could provide NP cells with survival tools for the harsh niche conditions in the IVD.

Single Cell RNA-Sequence Analyses Reveal Uniquely Expressed Genes and Heterogeneous Immune Cell Involvement in the Rat Model of Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration is characterized by a loss of cellularity, and changes in cell-mediated activity that drives anatomic changes to IVD structure. In this study, we used single-cell RNA-sequencing analysis of degenerating tissues of the rat IVD following lumbar disc puncture. Two control, uninjured IVDs (L2-3, L3-4) and two degenerated, injured IVDs (L4-5, L5-6) from each animal were examined either at the two- or eight-week post-operative time points. The cells from these IVDs were extracted and transcriptionally profiled at the single-cell resolution. Unsupervised cluster analysis revealed the presence of four known cell types in both non-degenerative and degenerated IVDs based on previously established gene markers: IVD cells, endothelial cells, myeloid cells, and lymphoid cells. As a majority of cells were associated with the IVD cell cluster, sub-clustering was used to further identify the cell populations of the nucleus pulposus, inner and outer annulus fibrosus. The most notable difference between control and degenerated IVDs was the increase of myeloid and lymphoid cells in degenerated samples at two- and eight-weeks post-surgery. Differential gene expression analysis revealed multiple distinct cell types from the myeloid and lymphoid lineages, most notably macrophages and B lymphocytes, and demonstrated a high degree of immune specificity during degeneration. In addition to the heterogenous infiltrating immune cell populations in the degenerating IVD, the increased number of cells in the AF sub-cluster expressing Ngf and Ngfr, encoding for p75NTR, suggest that NGF signaling may be one of the key mediators of the IVD crosstalk between immune and neuronal cell populations. These findings provide the basis for future work to understand the involvement of select subsets of non-resident cells in IVD degeneration.

Peptide location fingerprinting identifies species- and tissue-conserved structural remodelling of proteins as a consequence of ageing and disease

Extracellular matrices (ECMs) in the intervertebral disc (IVD), lung and artery are thought to undergo age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins. Here we applied PLF to publicly available ageing human IVD (outer annulus fibrosus), ageing mouse lung and human arterial atherosclerosis datasets and bioinformatically identified novel target proteins alongside common age-associated differences within protein structures which were conserved between three ECM-rich organs, two species, three IVD tissue regions, sexes and in an age-related disease. We identify peptide yield differences across protein structures which coincide with biological regions, potentially reflecting the functional consequences of ageing or atherosclerosis for macromolecular assemblies (collagen VI), enzyme/inhibitor activity (alpha-2 macroglobulin), activation states (complement C3) and interaction states (laminins, perlecan, fibronectin, filamin-A, collagen XIV and apolipoprotein-B). Furthermore, we show that alpha-2 macroglobulin and collagen XIV exhibit possible shared structural consequences in IVD ageing and arterial atherosclerosis, providing novel links between an age-related disease and intrinsic ageing. Crucially, we also demonstrate that fibronectin, laminin beta chains and filamin-A all exhibit conserved age-associated structural differences between mouse lung and human IVD, providing evidence that ECM, and their associating proteins, may be subjected to potentially similar mechanisms or consequences of ageing across both species, irrespective of differences in lifespan and tissue function.

In vivo fluid transport in human intervertebral discs varies by spinal level and disc region.

BackgroundThe lumbar discs are large, dense tissues that are primarily avascular, and cells residing in the central region of the disc are up to 6–8 mm from the nearest blood vessel in adults. To maintain homeostasis, disc cells rely on nutrient transport between the discs and adjacent vertebrae. Thus, diminished transport has been proposed as a factor in age‐related disc degeneration.MethodsIn this study, we used magnetic resonance imaging (MRI) to quantify diurnal changes in T2 relaxation time, an MRI biomarker related to disc hydration, to generate 3D models of disc fluid distribution and determine how diurnal changes in fluid varied by spinal level. We recruited 10 participants (five males/five females; age: 21–30 years; BMI: 19.1–29.0 kg/m2) and evaluated the T2 relaxation time of each disc at 8:00 AM and 7:00 PM, as well as degeneration grade (Pfirrmann). We also measured disc height, volume, and perimeter in a subset of individuals as a preliminary comparison of geometry and transport properties.ResultsWe found that the baseline (AM) T2 relaxation time and the diurnal change in T2 relaxation time were greatest in the cranial lumbar discs, decreasing along the lumbar spine from cranial to caudal. In cranial discs, T2 relaxation times decreased in each disc region (nucleus pulposus [NP], inner annulus fibrosus [IAF], and outer annulus fibrosus [OAF]), whereas in caudal discs, T2 relaxation times decreased in the NP but increased in the AF.ConclusionsFluid transport varied by spinal level, where transport was greatest in the most cranial lumbar discs and decreased from cranial to caudal along the lumbar spine. Future work should evaluate what level‐dependent factors affect transport.

Development of a rat model with lumbar vertebral endplate lesion.

Vertebral endplate lesion (EPL) caused by severe disc degeneration is associated with low back pain. However, there is no suitable animal model to elucidate the pathophysiology of EPL. This study aimed to develop a rat model of EPL and evaluate rat behavior and imaging and histological findings. The L4-5 intervertebral discs of Sprague-Dawley rats were transperitoneally removed, except for the outer annulus fibrosus and cartilage endplate, in the EPL group. The L4-5 discs were not removed and simply exposed in the sham group. Changes around the vertebral endplate on magnetic resonance imaging (MRI) and computed tomography (CT) were evaluated. Additionally, pain-related behavioral and histological assessments were performed. In the EPL group, a low-signal area around the vertebral endplate was observed on T1-weighted and T2-weighted fat-saturated MRI at 8weeks or later after surgery. In the same group, CT showed osteosclerosis around the vertebral endplate at 12weeks after surgery. The sham group did not show abnormal imaging features on the MRI and CT. Behavioral evaluation showed that the EPL group had a significantly longer grooming time than the sham group. Conversely, the 12-week postoperative locomotion time and the 1- and 12-week postoperative standing times were significantly shorter in the EPL group than in the sham group. Histological evaluation showed a high degree of vertebral endplate degeneration and an increased number of osteoclasts and proportion of nerve fibers expressing calcitonin gene-related peptide in the EPL group compared to those in the sham group. Our rat EPL model showed pain-related behavioral patterns and an increased expression of pain-related neuropeptide. This model could contribute to the study of the pathophysiology of EPL and will help in the treatment of low back pain in the future.

Establishing a through-puncture model for assessing post-injection leakage in the intervertebral disc.

Needle injection through the outer annulus fibrosus of the intervertebral disc (IVD) is the most practical approach for delivery of therapeutic agents, which have been shown to potentially leak following needle retraction. The goal of this work was to establish a protocol for quantifying post-injection leakage and test its sensitivity to factors believed to affect needle track geometry. A through-puncture defect procedure, followed by controlled injection, was performed on bovine caudal IVDs. Sensitivity to needle size was tested by injection of saline into unconstrained discs with either a 30G, 26G, or 21G hypodermic needle. Sensitivity to axial load was tested by repeated injection via a 26G needle with either no constraint, fixed height, or 10% axial compressive strain. Sensitivity to flexion was tested by applying combined 0.2MPa compression and 15° of flexion following injection of 5% of disc volume. Needle diameter significantly affected maximum volume prior to leakage, ranging from 34.6 ± 31.9µL when using 21G to 115.6 ± 23.6µL when using 30G. While all unloaded discs leaked, axial compression decreased the incidence of leakage events by 50-100% depending on load history. Forward flexion resulted in a 22% incidence of leakage. Fluid injected into IVDs is at significant risk of leakage following needle retraction. This risk depends on factors which alter the geometry of the needle track, including needle size, pinching due to axial compression, and stretching as a result of forward flexion.

Histological Changes of Cervical Disc Tissue in Patients with Degenerative Ossification.

ObjectiveTo explore the histological feature of the cervical disc degeneration in patients with degenerative ossification (DO) and its potential mechanisms. MethodsA total of 96 surgical segments, from cervical disc degenerative disease patients with surgical treatment, were divided into ossification group (group O, n=46) and non-ossification group (group NO, n=50) based on preoperative radiological exams. Samples of disc tissues and osteophytes were harvested during the decompression operation. The hematoxylin-eosin staining, Masson trichrome staining and Safranin O-fast green staining were used to compare the histological differences between the two groups. And the distribution and content of transforming growth factor (TGF)-β1, p-Smad2 and p-Smad3 between the two groups were compared by a semi-quantitative immunohistochemistry (IHC) method. ResultsFor all the disc tissues, the content of disc cells and collagen fibers decreased gradually from the outer annulus fibrosus (OAF) to the central nucleus pulposus (NP). Compared with group NO, the number of disc cells in group O increased significantly. But for proteoglycan in the inner annulus fibrosus (IAF) and NP, the content in group O decreased significantly. IHC analysis showed that TGF-β1, p-Smad2, and p-Smad3 were detected in all tissues. For group O, the content of TGF-β1 in the OAF and NP was significantly higher than that in group NO. For p-Smad2 in IAF and p-Smad3 in OAF, the content in group O were significantly higher than group NO. ConclusionHistologically, cervical disc degeneration in patients with DO is more severe than that without DO. Local higher content of TGF-β1, p-Smad2, and p-Smad3 are involved in the disc degeneration with DO. Further studies with multi-approach analyses are needed to better understand the role of TGF-β/Smads signaling pathway in the disc degeneration with DO.

Current Knowledge and Future Therapeutic Prospects in Symptomatic Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration is the main source of intractable lower back pain, and symptomatic IVD degeneration could be due to different degeneration mechanisms. In this article, we describe the molecular basis of symptomatic IVD degenerative disc diseases (DDDs), emphasizing the role of degeneration, inflammation, angiogenesis, and extracellular matrix (ECM) regulation during this process. In symptomatic DDD, pro-inflammatory mediators modulate catabolic reactions, resulting in changes in ECM homeostasis and, finally, neural/vascular ingrowth-related chronic intractable discogenic pain. In ECM homeostasis, anabolic protein-regulating genes show reduced expression and changes in ECM production, while matrix metalloproteinase gene expression increases and results in aggressive ECM degradation. The resultant loss of normal IVD viscoelasticity and a concomitant change in ECM composition are key mechanisms in DDDs. During inflammation, a macrophage-related cascade is represented by the secretion of high levels of pro-inflammatory cytokines, which induce inflammation. Aberrant angiogenesis is considered a key initiative pathologic step in symptomatic DDD. In reflection of angiogenesis, vascular endothelial growth factor expression is regulated by hypoxia-inducible factor-1 in the hypoxic conditions of IVDs. Furthermore, IVD cells undergoing degeneration potentially enhance neovascularization by secreting large amounts of angiogenic cytokines, which penetrate the IVD from the outer annulus fibrosus, extending deep into the outer part of the nucleus pulposus. Based on current knowledge, a multi-disciplinary approach is needed in all aspects of spinal research, starting from basic research to clinical applications, as this will provide information regarding treatments for DDDs and discogenic pain.

Development of a Perfusion Reactor for Intervertebral Disk Regeneration.

A tissue-engineered biological disk replacement has been proposed as a promising approach for the treatment of degenerative disk disease. A perfusion bioreactor would be a logical consideration to facilitate this scale-up as such reactors have been shown to improve nutrient delivery and provide beneficial mechanical forces that support the cultivation of large three-dimensional constructs. It was hypothesized that perfusion culture of tissue-engineered intervertebral disk (IVD) tissues would be capable of generating outer annulus fibrosus (oAF) and nucleus pulposus (NP) tissues comparable with established spinner reactor or static cultures, respectively, without compromising cellular viability, nutrient delivery, and tissue formation. In this study, the perfusion grown oAF and NP tissues did not show a significant difference in extracellular matrix (ECM) quantity or cellular phenotype when compared with their control conditions. In addition, they maintained cellular viability at the center core of the tissues and received enhanced diffusion of medium throughout the tissue when compared with static conditions. This study lays the groundwork for future studies to grow an entire IVD tissue to a physiologically relevant size.