AF cell derived exosomes regulate endothelial cell migration and inflammation: Implications for vascularization in intervertebral disc degeneration.

AIMS: The intervertebral disc is the largest avascular organ of the body. Vascularization of the disc has been typically regarded as a pathological feature of intervertebral disc degeneration (IDD). However, the underlying mechanism of vascularization in IDD is still unclear. The current study aimed to investigate the role of AF cell derived exosome (AF-exo) in the interaction with human umbilical vein endothelial cells (HUVECs) and its potential role in the regulation of vascularization in IDD. MAIN METHODS: Human AF tissues were obtained from patients with IDD and idiopathic scoliosis. The AF-exo were isolated and identified by transmission electron microscopy (TEM), nanoparticle trafficking analysis (NTA) and Western blotting. Then, the AF-exo were used for HUVECs cultures. The migration of HUVECs was observed in 2D and 3D cultures. The inflammatory phenotype of HUVECs was examined by Real-time PCR and enzyme-linked immunosorbent assay (ELISA). Additionally, apoptosis of HUVECs were analyzed by flow cytometry. KEY FINDINGS: Here, we for the first time found that AF cells could secrete AF-exo and that the AF-exo could be phagocytosed by HUVECs. Additionally, we found that degenerated AF-exo exerted pro-vascularization effect on HUVECs by promoting cell migration (in 2D and 3D cultures) and inflammatory factor expression including IL-6, TNF-α, MMP-3, MMP-13 and VEGF, whereas the application of non-degenerated AF-exo demonstrated inverse effects. SIGNIFICANCE: These results showed that AF-exo is an essential regulator mediating intercellular communication between AF cells and HUVECs, suggesting its important role in vascularization in the intervertebral disc.

Constant compression decreases vascular bud and VEGFA expression in a rabbit vertebral endplate ex vivo culture model.

SUMMARY OF BACKGROUND DATA: The vascular buds in the vertebral endplate (VEP) are the structural foundation of nutrient exchange in the intervertebral disc (IVD). VEGF is closely related to angiogenesis in the endplate and intervertebral disc degeneration (IDD). OBJECTIVE: To investigate the effects of static load on vascular buds and VEGF expression in the VEP and to further clarify the relation between IDD and VEGF. METHODS: IVD motion segments were harvested from rabbit lumbar spines and cultured under no-loading conditions (controls) or in custom-made apparatuses under a constant compressive load (0.5 MPa) for up to 14 days. Tissue integrity and the number of vascular buds were determined, and the concentrations and expression of Aggrecan, COL2a1, and VEGFA in the VEPs were assessed after 3, 7, and 14 days of culturing and then compared with those of fresh tissues. RESULTS: Under the constant compression, the morphological integrity of the VEPs was gradually disrupted, and immunohistochemistry results showed a significant decrease in the levels of Agg and COL2a1. During the static load, the number of vascular buds in the VEPs was gradually reduced from the early stage of culture, and ELISA showed that the constant compressive load caused a significant decrease in the VEGFA and VEGFR2 protein concentrations, which were consistent with the immunohistochemistry results. Western blot and RT-PCR results also showed that the loading state caused a significant decrease in VEGFA expression compared with that of fresh and control samples. CONCLUSIONS: Constant compression caused degeneration of the VEP as well as a decreased number of vascular buds, thereby accelerating disc degeneration. VEGFA is involved in this process. We anticipate that regulating the expression of VEGFA may improve the condition of the lesions to the vascular buds in the endplates, thus enhancing the nutritional supply function in IVD and providing new therapeutic targets and strategies for the effective prevention and treatment of IDD.

Morphometric Analysis of Lumbar Arteries and Relationship with Intervertebral Discs: A study of Surgical Anatomy on Human Fresh Cadavers.

AIM: To examine the morphological structure of anterior lumbar spinal region. MATERIAL AND METHODS: Fifteen fresh human cadavers were studied in our forensic medicine institution. Within the first 24 hours after death, cadavers were subject to a routine autopsy, by which the intestines were removed and the structures adjacent to the lumbar arteries (LAs) were examined. The following characteristics of the LA were examined and recorded: diameter, number of LA, intervertebral disc, and vertebral corpus relationship, and LA anatomical variations. RESULTS: In 14 of the 15 cadavers (93.3%), the first four LAs originated from the abdominal aorta. In one (6.7%) cadaver, the fourth LA originated from the right aortic trunk together with the median sacral artery (MSA), and this was observed only in the right side. In one cadaver, the first LAs emerged as a common trunk. Twelve cadavers did not have a fifth LA. In one cadaver (6.7%), the fifth LAs emerged as in a pair, distally from the origin of the MSA. Two cadavers (13.3%) had fifth LAs that emerged from the common trunk with the MSA and this occurred only on the right side. LAs which emerged from the right side of the abdominal aorta passed under the inferior vena cava. Of the first four LA, the third LA had the largest vessel diameter, and the first LA had the narrowest vessel diameter. Fifth LAs had one of the narrowest vessel diameters of all LAs. CONCLUSION: We consider that anatomical study of LAs using fresh cadavers can be useful to ensure safer anterior lumbar spinal surgeries.

Anatomical Study of the Lumbar Segmental Arteries in Relation to the Oblique Lateral Interbody Fusion Approach.

OBJECTIVE: To use computed tomography angiography to evaluate the regional anatomy of the lumbar segmental arteries (LAs) associated with the surgical field in oblique lateral interbody fusion (OLIF). METHODS: Computed tomography angiography images from 50 patients were reviewed. In the sagittal plane, distances from the LA to the upper and inferior edges of the vertebral body were measured in the anterior quarter of the anterior and median lines of the intervertebral disc (IVD). LAs were classified as types I-IV based on the zone in which they passed through the vertebral body. RESULTS: The LA branch angles were acute (<90°) at L1-L3 and blunt (>90°) at L4-L5. The average distances from the LA to the upper and inferior edges of the vertebral body in the anterior quarter position revealed that La1.2 > Lb1.2 and Lb3.4.5 > La3.4.5. For the IVD of L1-L2, Lb1 < La2; IVD of L2-L3, Lb2 < La3; IVD of L3-L4, Lb3 > La4; IVD of L4-L5, Lb4 > La5. In zone I, the most frequent LA type was type IV at L1 (n = 41; 85.4%) and L2 (n = 42; 84.0%), type III at L3 (n = 20; 40.0%), and type II at L4 (n = 36; 80.0%) and L5 (n = 5; 83.3%). In zone II, the most frequent LA type was type III at L1 (n = 38; 79.2%), L2 (n = 39; 78.0%), L3 (n = 43; 86.0%), and L4 (n = 28; 62.2%), whereas type II was the most frequent LA type at L5 (n = 5; 83.3%). In zone III, type III was the most frequent LA type at L1-L4. In zone IV, type IV was the most frequent LA type at L3 (n = 44; 88.0%), L4 (n = 42; 93.3%), and L5 (n = 6; 100%). CONCLUSIONS: The risk of LA injury during OLIF is the least when the cage is placed in zones II and III. Care is required during OLIF in zone IV of L3-L5. The fixation pin should be fixed on the upper edge of the lower vertebral body at L1-L2 and L2-L3, and on the lower edge of the upper vertebral body at L3-L4 and L4-L5.

Effect of diminished flow in rabbit lumbar arteries on intervertebral disc matrix changes using MRI T2-mapping and histology.

BACKGROUND: Impaired lumbar artery flow has been reported in clinical and epidemiological studies to be associated with low back pain and lumbar disc degeneration. However, it has not been experimentally demonstrated that impaired lumbar artery flow directly induces intervertebral disc (IVD) degeneration by affecting IVD matrix metabolism. The purpose of this study was to evaluate whether ligation of the lumbar artery can affect degenerative changes in the rabbit IVD. METHODS: New Zealand White rabbits (n = 20) were used in this study. Under general anesthesia, the third and fourth lumbar arteries were double-ligated using vascular clips. The blood flow to the L3/L4 disc (cranial disc) was reduced by ligation of the third lumbar artery and that of the L5/L6 disc (caudal disc) by ligation of the fourth lumbar artery. The blood flow to the L4/L5 disc (bilateral disc) was decreased by ligation of both the third and fourth lumbar arteries. The L2/L3 disc was used as the control. Disc height was radiographically monitored biweekly until 12 weeks after surgery. The rabbits were sacrificed at 4, 8, and 12 weeks after surgery and magnetic resonance imaging (MRI) T2-mapping, histology and immunohistochemistry were assessed. RESULTS: Lumbar artery ligation did not induce significant changes in disc height between control and ischemic discs (cranial, bilateral and caudal discs) during the 12-week experimental period. T2-values of ischemic discs had no significant trend to be lower than those of the control L2/L3 discs. Histologically, Safranin-O staining changed following ligation of corresponding IVD lumbar arteries. Histological grading scores for disc degeneration, which correlated significantly with MRI T2-values, had significant changes after the surgery. Immunohistochemical analysis showed that the ligation of lumbar arteries significantly affected a change in the percentage of HIF-1α immunoreactive cells of ischemia discs compared to that of control discs four weeks after the surgery (p < 0.05). CONCLUSIONS: The MRI and histology results suggest that diminished flow in lumbar arteries induce mild changes in the extracellular matrix metabolism of rabbit IVDs. These matrix changes, however, were not progressive and differed from the degenerative disc changes seen in the process of human IVD degeneration.

Relationship Between Apoptosis of Endplate Microvasculature and Degeneration of the Intervertebral Disk.

OBJECTIVE: To explore the relationship between intervertebral disk degeneration and endplate microvasculature, and to determine the role of apoptosis in the pathophysiology underlying end plate microvasculature. METHODS: Twelve 6-month-old rabbits were randomly divided into group A (control group where animals underwent a sham operation, in which the loading device was implanted but without loading) and group B (degeneration group, where a calibrated spring within the loading device would immediately create static shear force of 50 N to the disk of L4-5). Paraffin-embedded midsagittal sections of the L4-5 disk were obtained 4 weeks after surgery in the both groups. Sections were stained with cluster of differentiation (CD) 31 immunohistochemistry to measure the blood vessel density in the endplate, with CD31 immunofluorescence and terminal dUTP nick-end labeling (TUNEL) to detect the apoptosis of vascular endothelial cells in the endplate. RESULTS: After 4 weeks, the microvasculature density was 91 ± 8 vessels/mm2 in group A and 47 ± 2 vessels/mm2 (P < 0.001) in group B, demonstrating that vessels were reduced in the endplate of intervertebral disk degeneration. CD31 immunofluorescence and TUNEL showed that apoptosis of vascular endothelial cells exists in the endplate of intervertebral disk degeneration. CONCLUSIONS: The results of this study suggest that apoptosis of vascular endothelial cells results in a decrease in endplate microvasculature density, further affecting the pathologic process of intervertebral disk degeneration.

Improved display of cervical intervertebral discs on water (iodine) images: incidental findings from single-source dual-energy CT angiography of head and neck arteries.

OBJECTIVE: To (a) assess the diagnostic performance of material decomposition (MD) water (iodine) images for the evaluation of cervical intervertebral discs (IVDs) in patients who underwent dual-energy head and neck CT angiography (HNCTA) compared with 70-keV images and (b) to explore the correlation of water concentration with the T2 relaxation time of IVDs. MATERIALS AND METHODS: Twenty-four consecutive patients who underwent dual-energy HNCTA and cervical spine MRI were studied. The diagnostic performance of water (iodine), 70-keV and MR images for IVD bulge and herniation was assessed. A subjective image score for each image set was recorded. The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of IVDs to the cervical spinal cord were compared between water (iodine) and 70-keV images. Disc water concentration as measured on water (iodine) images was correlated with T2 relaxation time. RESULTS: IVD evaluations for bulge and herniation did not differ significantly among the three image sets (pairwise comparisons; all p > 0.05). SNR and CNR were significantly improved on water (iodine) images compared with those on 70-keV images (p < 0.001). Although water (iodine) images showed higher image quality scores when evaluating IVDs compared with 70-keV images, the difference is not significant (all adjusted p > 0.05). IVD water concentration exhibited no correlation with relative T2 relaxation time (all p > 0.05). CONCLUSION: Water (iodine) images facilitated analysis of cervical IVDs by providing higher SNR and CNR compared with 70-keV images. The disc water concentration measured on water (iodine) images exhibited no correlation with relative T2 relaxation time. KEY POINTS: • There was no significant difference in cervical IVD evaluations for bulge and herniation among water (iodine) images, 70-keV images and MR images. • Water (iodine) images provided higher objective and subjective image quality than 70-keV images, though the difference of subjective evaluation was not statistically significant. • The disc water concentration exhibited no correlation with relative T2 relaxation time, which reflects the inferiority of the water (iodine) images in evaluating disc water content compared with T2 maps.

Nerves and blood vessels in degenerated intervertebral discs are confined to physically disrupted tissue.

Nerves and blood vessels are found in the peripheral annulus and endplates of healthy adult intervertebral discs. Degenerative changes can allow these vessels to grow inwards and become associated with discogenic pain, but it is not yet clear how far, and why, they grow in. Previously we have shown that physical disruption of the disc matrix, which is a defining feature of disc degeneration, creates free surfaces which lose proteoglycans and water, and so become physically and chemically conducive to cell migration. We now hypothesise that blood vessels and nerves in degenerated discs are confined to such disrupted tissue. Whole lumbar discs were obtained from 40 patients (aged 37-75 years) undergoing surgery for disc herniation, disc degeneration with spondylolisthesis or adolescent scoliosis ('non-degenerated' controls). Thin (5-µm) sections were stained with H&E and toluidine blue for semi-quantitative assessment of blood vessels, fissures and proteoglycan loss. Ten thick (30-µm) frozen sections from each disc were immunostained for CD31 (an endothelial cell marker), PGP 9.5 and Substance P (general and nociceptive nerve markers, respectively) and examined by confocal microscopy. Volocity image analysis software was used to calculate the cross-sectional area of each labelled structure, and its distance from the nearest free surface (disc periphery or internal fissure). Results showed that nerves and blood vessels were confined to proteoglycan-depleted regions of disrupted annulus. The maximum distance of any blood vessel or nerve from the nearest free surface was 888 and 247 µm, respectively. Blood vessels were greater in number, grew deeper, and occupied more area than nerves. The density of labelled blood vessels and nerves increased significantly with Pfirrmann grade of disc degeneration and with local proteoglycan loss. Analysing multiple thick sections with fluorescent markers on a confocal microscope allows reliable detection of thin filamentous structures, even within a dense matrix. We conclude that, in degenerated and herniated discs, blood vessels and nerves are confined to proteoglycan-depleted regions of disrupted tissue, especially within annulus fissures.

Awareness of middle sacral artery pathway: A cadaveric study of the presacral area.

PURPOSE: To assess the anatomic path of the middle sacral artery (MSA) at the presacral area and its relationship to the spinal midline during an axial lumbar interbody fusion (AxiaLif) approach. METHODS: Fifty human cadavers (25 males, 25 females) were used in this study. A transabdominal approach was used to expose the anterior aspect of the L5/S1 intervertebral disc and the presacral space. We measured the size and distance from the spinal midline at the following positions: (a) middle of the L5/S1 disc level, (b) 1 cm below the sacral promontory (SP), and (c) 2 cm below the SP. Each parameter was measured three times by two observers, and the mean value analyzed. RESULTS: The MSA was present and originated from the left common iliac artery in all cadavers with a mean width of 2.14 mm. The position of the MSA in relation to the midline was most commonly on the left side (LS, 56%) followed by the right side (RS, 34%) and midline (ML, 10%). In the LS group, the distance from the midline is relatively constant in the three measured positions with a mean value of (a) 1.78 mm (range, 0-8.17 mm), (b) 2.08 mm (range, 0-7.10 mm), and (c) 2.06 mm (range, 0-9.76 mm). In the RS group, the distance from the midline increased from cephalad to caudad, with a mean value of (a) 1.44 mm (range, 0-9.64 mm), (b) 2.19 mm (range, 0-9.95 mm), and (c) 2.92 mm (range, 0-10.03 mm). CONCLUSIONS: Our study found the presacral anatomic path of the MSA was most commonly at the left of midline. In addition, the right-sided MSA variant had increasing distance from the midline along its anatomic path from cephalad to caudad. Our findings suggest an AxiaLif approach at the left of midline may place the MSA at greatest risk.

Atherosclerotic Disease and its Relationship to Lumbar Degenerative Disk Disease, Facet Arthritis, and Stenosis With Computed Tomography Angiography.

BACKGROUND: The intervertebral disk is the largest avascular structure in the body. It relies on passive diffusion from arteries at the periphery of the disk for nutrition. Previous studies have suggested a correlation between vascular disease and lumbar degenerative disk disease (DDD), but the association with facet arthritis and stenosis has not been evaluated. OBJECTIVE: To evaluate the degree of lumbar artery stenosis, aortic atherosclerosis on computed tomography angiography, and its relationship to lumbar DDD, facet arthritis, and spinal canal stenosis. DESIGN: Retrospective case review. SETTING: Academic tertiary care hospital. PARTICIPANTS: Not applicable. METHODS: A total of 300 lumbar arteries (150 lumbar artery pairs of the first to fifth lumbar arteries) were evaluated on consecutive computed tomography angiography scans. Severity of vascular disease of lumbar arteries was documented as normal, mild, moderate, severe, or occluded. Aortic vascular disease was documented along the posterior wall where the lumbar arteries originate. MAIN OUTCOME MEASUREMENTS: The relationship between vascular disease with DDD, facet arthritis, and spinal canal stenosis was examined and further evaluated controlling for age. RESULTS: Lumbar artery and aortic atherosclerosis had a positive relationship with DDD, facet arthritis, and spinal stenosis that was statistically significant (P < .05) even after controlling for age. The correlation coefficient was greatest in the younger age group when looking at lumbar artery vascular disease with DDD (0.73, confidence interval 0.50-0.96, P < .0001) and aortic vascular disease with DDD (0.72, confidence interval 0.49-0.94, P < .0001). The correlation of vascular disease with facet arthritis and stenosis was not strong in the older age group. CONCLUSION: Atherosclerotic disease of the lumbar arteries and aorta correlated with lumbar DDD, facet arthritis, and spinal canal stenosis after we adjusted for age, although the correlation with facet arthritis and spinal canal stenosis was not as strong in the older age group. LEVEL OF EVIDENCE: IV.

Lumbar intervertebral disc allograft transplantation: the revascularisation pattern.

PURPOSE: Fresh frozen intervertebral disc allograft transplantation has been reported to be a viable treatment option for advanced degenerative disc diseases, but rapid degeneration of the postoperative allograft was found. Loss of nutrient supply is believed to be the most likely inducer because the disc allografts have to endure in an ischaemic environment until the nutrient pathway is re-established. The aim of this study was to focus on the revascularisation of the disc allograft after transplantation in goats. METHODS: Twenty male goats were used in this study. Intervertebral disc allograft transplantation was performed at L4/L5. Groups of five goats were killed at 1.5, 6 and 12 m postoperatively, respectively. The transplanted segments were harvested, fixed, sagittally cut and decalcified for H&E staining and immunochemistry to observe the blood vessel formation at the endplates, anterior outer annulus, posterior outer annulus, inner annulus and the nucleus. The blood vessel density and the sectional vessel area were measured. RESULTS: Blood vessels were first found in the marrow space of the bony endplate and the outer annulus at 1.5 month postoperatively. Then, they were able to penetrate to reach the cartilaginous endplate and the inner annulus after 6 months. Interestingly, the endplate area possessed the most abundant blood vessels, with the highest level of vessel density and area at the final follow-up. None of these newly formed vessels invaded the nucleus during the observation period. CONCLUSIONS: Revascularisation of the postoperative disc allograft has been determined, but its pattern was different from that in adult normal discs, suggesting that the typical nutrient diffusion pattern may be affected after transplantation.

Model of Disc Degeneration in Rat Tail Induced Through a Vascular Isolation of Vertebral Endplates.

Back pain is a major health problem. The degenerative cascade of the spine begins in the intervertebral disc, due to an impairment in the blood supply through the vertebral endplates. Our objective was to develop a novel disc degeneration model based on these premises, akin to the process in humans, in contrast to other proposed models (puncture, enzyme injection, aberrant loads,…) Material and methods: 37 Sprague-Dawley rats, 2 arms: (a) histological (n = 17, one died), en- bloc sections, Van Gieson staining, (Nisimura-Mochida criteria) and also collagen VI staining (tissue oxidative stress), four animals were euthanized every 2 weeks (2-8); and (b) imaging (n = 20, six wound sloughs), MRI 9.4 Tesla protocol, sequential disc volumetric analysis (24 h-8 weeks) in all animals. Disc degeneration was induced by means of vascular isolation of tail discs endplates either from one side or both. RESULTS: Isolation from both sides caused a progressive degeneration of the disc (p < 0.001 vs. controls), bigger than isolation from one side (p < 0.01 vs. both sides and p < 0.05 vs. controls), as rated by volumetric reduction; furthermore, tissue structural changes (Nisimura-Mochida) and collagen VI deposition confirmed these results. CONCLUSION: the model here described represents a novel and translational tool that reproduces the intervertebral disc degeneration in a similar way to that taking place in human beings.

Autoantibodies to full body vascular cell junctions colocalize with MYZAP, ARVCF, desmoplakins I and II and p0071 in endemic pemphigus in Colombia, South America.

BACKGROUND: We previously described a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF). METHODS: Here we aimed to investigate disease autoreactivity to vessels in all body organs/systems. We compared 57 patients and 57 controls from the endemic area, matched by demographics, age, sex, and work activity. We performed immunofluorescence, immunohistochemistry, confocal microscopy, immunoblotting, indirect immune electron microscopy studies, and autometallographic studies. We performed ultrasonography on large patient arteries, investigating for vascular anomalies. In addition, we reviewed autopsies on seven patients who died affected by El Bagre-EPF. We immunoadsorbed any positive vessel immunofluorescence with desmoglein (Dsg1), investigating for new autoantigens. RESULTS: Overall, 57/57 patients affected by El Bagre-EPF displayed autoantibodies to vessels in all the organs/systems of the body via all methods (P < 0.01). The autoreactivity was polyclonal, and the patient's antibodies colocalized with commercial antibodies to desmoplakins I and II, p0071, ARVCF, and MYZAP (all from Progen Biotechnik, Germany; P < 0.01; all present at cell junctions). Immunoadsorption with Dsg1 on positive vessel immunofluorescence showed that the immune response against the vessels was directed against non-Dsg1 antigen(s). Autometallographic studies showed deposits of metals and metalloids in vessel cell junctions and in erythrocytes of 85% of patients (P < 0.01). CONCLUSIONS: Immune response to these vascular antigens is likely altering endothelial cells and vessel shapes, thus disturbing hemodynamic flow. The flow alterations likely lead to inflammation and may play a role in the atherogenesis often seen in these patients.

Influence of rabbit notochordal cells on symptomatic intervertebral disc degeneration: anti-angiogenic capacity on human endothelial cell proliferation under hypoxia.

OBJECTIVES: Symptomatic degenerative disc disease (DDD) is associated with neovascularization and nerve ingrowth into intervertebral discs (IVDs). Notochordal cells (NCs) are key cells that may lead to regeneration of IVDs. However, their activities under conditions of hypoxia, the real environment of IVD, are not well known. We hypothesized that NCs may inhibit neovascularization by interacting with endothelial cells (ECs) under hypoxia. DESIGN: Human IVDs were isolated and cultured to produce nucleus pulposus (NP) cell conditioned medium (NPCM). Immortalized human microvascular ECs were cultured in NPCM with notochordal cell-rich rabbit nucleus pulposus cells (rNC) under hypoxia. Vascular endothelial growth factor (VEGF), vascular cell adhesion molecule (VCAM), and interleukin-8 (IL-8) were analyzed by ELISA. Focal adhesion kinase (FAK), filamentous actin (F-actin), and platelet-derived growth factor (PDGF) were evaluated to investigate EC activity. Wound-healing migration assays were performed to examine EC migration. RESULTS: The VEGF level of EC cells cultured in NPCM was significantly higher under hypoxia compared to normoxia. VEGF expression was significantly decreased, and FAK, F-actin, PDGF expression were inhibited when ECs were cocultured with rNCs under hypoxia. ECs cocultured with rNC in NPCM showed significantly decreased migratory activity compared to those without rNC under hypoxia. CONCLUSIONS: The angiogenic capacity of ECs was significantly inhibited by NCs under hypoxia via a VEGF-related pathway. Our results suggest that NCs may play a key role in the development of IVDs by inhibiting vascular growth within the disc, and this may be a promising novel therapeutic strategy for targeting vascular ingrowth in symptomatic DDD.

[Biology and mechanobiology of the intervertebral disc]. / Biología y mecanobiología del disco intervertebral.

The intervertebral disc (IVD) is noted for its low cell content, and being the largest avascular structure of human body. The low amount of cells in the disc have to adapt to an anaerobic metabolism with low oxygen pressure and acidic pH. Apart from surviving in an adverse microenvironment, they are exposed to a high level of mechanical stress. The biological adaptation of cells to acidosis and hyperosmolarity conditions are regulated by mechanoproteins, which are responsible for converting a mechanical signal into a cellular response, thus modifying its gene expression. Mechanobiology helps us to better understand the pathophysiology of IVD and its potential biological repair.

A novel micro-CT-based method to monitor the morphology of blood vessels in the rabbit endplate.

PURPOSE: The aim of this study was to develop a novel method for observing the morphology of the blood vessels in the rabbit endplate. METHODS: Twenty 6-month-old rabbits were used in this study. The blood vessels in the L5 endplate in Group A were injected with iohexol and Group B with barium sulfate. Group C was the control group with saline. To optimize the study, Group B was divided into two subgroups: Group B-1 was injected with 100% (w/v) barium sulfate and Group B-2 with 50% (w/v). After injection, the L4-L5 vertebral body was excised and the cranial endplate of L5 was scanned using a micro-CT scanner. Models of the vertebral endplate and vessels were reconstructed using the 3D reconstruction software (Mimics 16.0) by calculating a bone threshold value, and then merged these two models to create a superimposed model. RESULTS: The 3D vessel models could not be created in Groups A and C, but they were clearly visualized in Group B. In the 3D model, the blood vessels extended from the subchondral bone to the endplate, and the density of the blood vessels in the area of the nucleus pulposus (NP) was higher than in the annulus fibrosus. CONCLUSIONS: The results of this study suggest that the blood vessels in the rabbit endplate can be clearly observed by the method described using barium sulfate [the 50% (w/v) gave better results compared with the 100% (w/v)]. The information from the 3D vessel structure could provide essential data to help us understand the nutrient pathways within the vertebral endplate.

Periprosthetic UHMWPE Wear Debris Induces Inflammation, Vascularization, and Innervation After Total Disc Replacement in the Lumbar Spine.

BACKGROUND: The pathophysiology and mechanisms driving the generation of unintended pain after total disc replacement (TDR) remain unexplored. Ultrahigh-molecular-weight polyethylene (UHMWPE) wear debris from TDRs is known to induce inflammation, which may result in pain. QUESTIONS/PURPOSES: The purpose of this study was to determine whether (1) periprosthetic UHMWPE wear debris induces immune responses that lead to the production of tumor necrosis factor-α (TNFα) and interleukin (IL)-1ß, the vascularization factors, vascular endothelial growth factor (VEGF) and platelet-derived growth factor-bb (PDGFbb), and the innervation/pain factors, nerve growth factor (NGF) and substance P; (2) the number of macrophages is associated with the production of the aforementioned factors; (3) the wear debris-induced inflammatory pathogenesis involves an increase in vascularization and associated innervation. METHODS: Periprosthetic tissues from our collection of 11 patients with contemporary TDRs were evaluated using polarized light microscopy to quantify UHMWPE wear particles. The major reason for revision (mean implantation time of 3 years [range, 1-6 years]) was pain. For control subjects, biopsy samples from four patients with degenerative disc disease with severe pain and autopsy samples from three normal patients with no history of back pain were also investigated. Immunohistochemistry and histology were used to identify secretory factors, macrophages, and blood vessels. Immunostained serial sections were imaged at ×200 magnification and using MATLAB and NIH ImageJ, a threshold was determined for each factor and used to quantify positive staining normalized to tissue sectional area. The Mann-Whitney U test was used to compare results from different patient groups, whereas the Spearman Rho test was used to determine correlations. Significance was based on p < 0.05. RESULTS: The mean percent area of all six inflammatory, vascularization, and innervation factors was higher in TDR tissues when compared with normal disc tissues. Based on nonparametric data analysis, those factors showing the most significant increase included TNFα (5.17 ± 1.76 versus 0.05 ± 0.03, p = 0.02), VEGF (3.02 ± 1.01 versus 0.02 ± 0.002, p = 0.02), and substance P (4.15 ± 1.01 versus 0.08 ± 0.04, p = 0.02). The mean percent area for IL-1ß (2.41 ± 0.66 versus 0.13 ± 0.13, p = 0.01), VEGF (3.02 ± 1.01 versus 0.34 ± 0.29, p = 0.04), and substance P (4.15 ± 1.01 versus 1.05 ± 0.46, p = 0.01) was also higher in TDR tissues when compared with disc tissues from patients with painful degenerative disc disease. Five of the factors, TNFα, IL-1ß, VEGF, NGF, and substance P, strongly correlated with the number of wear particles, macrophages, and blood vessels. The most notable correlations included TNFα with wear particles (p < 0.001, ρ = 0.63), VEGF with macrophages (p = 0.001, ρ = 0.71), and NGF with blood vessels (p < 0.001, ρ = 0.70). Of particular significance, the expression of PDGFbb, NGF, and substance P was predominantly localized to blood vessels/nerve fibers. CONCLUSIONS: These findings indicate wear debris-induced inflammatory reactions can be linked to enhanced vascularization and associated innervation/pain factor production at periprosthetic sites around TDRs. Elucidating the pathogenesis of inflammatory particle disease will provide information needed to identify potential therapeutic targets and treatment strategies to mitigate pain and potentially avoid revision surgery. LEVEL OF EVIDENCE: Level III, therapeutic study.

Lumbar artery branches coursing vertically over the intervertebral discs of the lower lumbar spine: an anatomic study.

PURPOSE: Bleeding from the lumbar artery is a potential complication during the transpsoas approach to the lower lumbar intervertebral discs. In this anatomic study, the morphological relationships between the branches of the lumbar artery and the lower intervertebral disc were investigated to assess the risk of injury to the branches of the lumbar segmental arteries. METHODS: We studied 88 sites (86 lumbar arteries) at the third and fourth lumbar vertebrae bilaterally in 22 formalin-fixed cadavers. The branches of the lumbar artery coursing along the lateral sides of the lower intervertebral disc [muscular branch, anastomotic branch, and branch supplying the spinal nerve and plexus (BSNP)] and the iliolumbar artery running upward over the L4-5 disc were identified. Branches crossing the intervertebral discs vertically were evaluated. RESULTS: Muscular branches with a lumen structure longer than 2 cm coursed vertically over the middle third of the intervertebral disc in 3 of 88 sites (3.4 %). Anastomotic branches ran downward in 13 of 88 (14.8 %), and iliolumbar arteries ran upward on the posterior third of the lateral sides of the disc in 2 of 88 (2.3 %). BSNPs ran downward through the posterior third of the disc at 18 of 88 sites (20.5 %). Overall, the arterial branches coursed vertically over the posterior third of the lateral sides of the intervertebral discs in approximately 30 % of subjects. CONCLUSIONS: Lumbar artery branches coursed vertically over the middle third and the posterior third of the lateral sides of the intervertebral discs in approximately 3 and 30 % of subjects, respectively.

The occurrence of vascular displacement into intervertebral disc space following the compensated sagittal imbalance of the spine: a case report and review of literature.

BACKGROUND: It is known that sagittal compensating mechanisms are created for counteracting sagittal imbalance problems; however, they can sometimes be associated with incidents which affect the plan of management. PURPOSE: The purpose of this study was to report a case of the occurrence of common iliac vessel displacement into the intervertebral disc space following one of the spinal compensatory mechanisms. MATERIAL AND METHODS: The authors demonstrated this case by showing the patient history, physical examination, imaging studies, and treatment strategy as well as by reviewing some related literature. RESULTS: An 81-year-old woman presented with a long history of low back pain with claudication. An upright plain radiograph and flexion-extension study demonstrated a progressive local thoracolumbar kyphosis and losing of lumbar lordosis with significant widening of the intervertebral disc space of L4-L5. An MRI scan and 3D volume rendering spiral computed tomography (3D-CT) revealed an abnormal content which was depicted as common iliac vessels inside the disc space of L4-L5. Consequently, a rare case of the occurrence of common iliac vessel displacement into the intervertebral disc space following one of the spinal compensatory mechanisms was reported. CONCLUSION: The occurrence of vascular displacement into the intervertebral disc space related to lumbar hyperextension, as a compensating mechanism, is a rare incident but can occur. Consequently, when this mechanism presents with abnormal widening of the intervertebral disc space, especially at the low lumbar level, it should raise surgeon's concern about the probability of vascular injury when performing a disc procedure. Thorough investigation with imaging studies and selecting the optimum surgical treatment are warranted.

Nerves are more abundant than blood vessels in the degenerate human intervertebral disc.

BACKGROUND: Chronic low back pain (LBP) is the most common cause of disability worldwide. New ideas surrounding LBP are emerging that are based on interactions between mechanical, biological and chemical influences on the human IVD. The degenerate IVD is proposed to be innervated by sensory nerve fibres and vascularised by blood vessels, and it is speculated to contribute to pain sensation. However, the incidence of nerve and blood vessel ingrowth, as well as whether these features are always associated, is unknown. We investigated the presence of nerves and blood vessels in the nucleus pulposus (NP) of the IVD in a large population of human discs. METHODS: Immunohistochemistry was performed with 61 human IVD samples, to identify and localise nerves (neurofilament 200 [NF200]/protein gene product 9.5) and blood vessels (CD31) within different regions of the IVD. RESULTS: Immunopositivity for NF200 was identified within all regions of the IVD within post-mortem tissues. Nerves were seen to protrude across lamellar ridges and through matrix towards NP cells. Nerves were identified deep within the NP and were in many cases, but not always, seen in close proximity to fissures or in areas where decreased matrix was seen. Fifteen percent of samples were degenerate and negative for nerves and blood vessels, whilst 16 % of all samples were degenerate with nerves and blood vessels. We identified 52% of samples that were degenerate with nerves but no blood vessels. Interestingly, only 4% of all samples were degenerate with no nerves but positive for blood vessels. Of the 85 samples investigated, only 6 % of samples were non-degenerate without nerves and blood vessels and 7% had nerves but no blood vessels. CONCLUSIONS: This study addresses the controversial topic of nerve and blood vessel ingrowth into the IVD in a large number of human samples. Our findings demonstrate that nerves are present within a large proportion of NP samples from degenerate IVDs. This study shows a possible link between nerve ingrowth and degeneration of the IVD and suggests that nerves can migrate in the absence of blood vessels.