A common variant rs2054564 in ADAMST17 is associated with susceptibility to lumbar spondylosis.

The molecular pathophysiology underlying lumbar spondylosis development remains unclear. To identify genetic factors associated with lumbar spondylosis, we conducted a genome-wide association study using 83 severe lumbar spondylosis cases and 182 healthy controls and identified 65 candidate disease-associated single nucleotide polymorphisms (SNPs). Replication analysis in 510 case and 911 control subjects from five independent Japanese cohorts identified rs2054564, located in intron 7 of ADAMTS17, as a disease-associated SNP with a genome-wide significance threshold (P = 1.17 × 10-11, odds ratio = 1.92). This association was significant even after adjustment of age, sex, and body mass index (P = 7.52 × 10-11). A replication study in a Korean cohort, including 123 case and 319 control subjects, also verified the significant association of this SNP with severe lumbar spondylosis. Immunohistochemistry revealed that fibrillin-1 (FBN1) and ADAMTS17 were co-expressed in the annulus fibrosus of intervertebral discs (IVDs). ADAMTS17 overexpression in MG63 cells promoted extracellular microfibrils biogenesis, suggesting the potential role of ADAMTS17 in IVD function through interaction with fibrillin fibers. Finally, we provided evidence of FBN1 involvement in IVD function by showing that lumbar IVDs in patients with Marfan syndrome, caused by heterozygous FBN1 gene mutation, were significantly more degenerated. We identified a common SNP variant, located in ADAMTS17, associated with susceptibility to lumbar spondylosis and demonstrated the potential role of the ADAMTS17-fibrillin network in IVDs in lumbar spondylosis development.

Multifunctional Nanofibrous Scaffolds with Angle-Ply Microstructure and Co-Delivery Capacity Promote Partial Repair and Total Replacement of Intervertebral Disc.

There is an urgent clinical need for the treatment of annulus fibrosus (AF) impairment caused by intervertebral disc (IVD) degeneration or surgical injury. Although repairing injured AF through tissue engineering is promising, the approach is limited by the complicated angle-ply microstructure, inflammatory microenvironment, poor self-repairing ability of AF cells and deficient matrix production. In this study, electrospinning technology is used to construct aligned core-shell nanofibrous scaffolds loaded with transforming growth factor-ß3 (TGFß3) and ibuprofen (IBU), respectively. The results confirm that the rapid IBU release improves the inflammatory microenvironment, while sustained TGFß3 release enhances nascent extracellular matrix (ECM) formation. Biomaterials for clinical applications must repair local AF defects during herniectomy and enable AF regeneration during disc replacement, so a box defect model and total IVD replacement model in rat tail are constructed. The dual-drug delivering electrospun scaffolds are assembled into angle-ply structure to form a highly biomimetic AF that is implanted into the box defect or used to replace the disc. In two animal models, it is found that biomimetic scaffolds with good anti-inflammatory ability enhance ECM formation and maintain the mechanical properties of IVD. Findings from this study demonstrate that the multifunctional nanofibrous scaffolds provide inspirations for IVD repair.

Delineation of Inner Annulus Fibrosus and Nucleus Pulposus on Routine T2-weighted MR Images.

STUDY DESIGN: Retrospective study of 150 IVDs. OBJECTIVE: Assessment of costume algorithm ability to delineate the IAF and NP on routine T2 images. SUMMARY OF BACKGROUND DATA: Central hyperintense region on T2-weighted MR images of normal lumbar IVDs represents a combination of IAF and NP. Ability to identify NP as distinct from IAF can help improve our understanding of IVD morphology in-vivo. METHODS: Sagittal T2-weighted TSE MR images of 150 lumbar IVDs from 25 patients were analyzed. MR images were processed using a custom algorithm that markedly increased the signal intensity of structures with inherent signal intensity within 2 defined intensity thresholds. Signal intensity and contrast-to- noise ratio between outer annulus fibrosus, IAF, and NP were assessed at baseline and after processing. To assess consistency of underlying T2 differences, similar analysis was done on 108 discs from 18 patients in whom additional sagittal T2-weighted STIR images were available. RESULTS: Following image processing, apparent IAF and NP were rendered visible in 86% and 84.3% IVDs on T2-weighted TSE and STIR images respectively. While signal intensity of these 2 regions was inherently different (P< 0.001) before processing on TSE and STIR images, their visualization was facilitated by a significant increase (P<0.001) in contrast-to-noise ratio after processing. Nonvisualization of NP was associated with disc degeneration (P<0.001). CONCLUSION: Inherent differences exist in signal intensities of normal NP and IAF on T2-weighted MR images. Accentuating these differences using image postprocessing techniques can render these 2 structures visible.

Intradiscal quantitative chemical exchange saturation transfer MRI signal correlates with discogenic pain in human patients.

Low back pain (LBP) is often a result of a degenerative process in the intervertebral disc. The precise origin of discogenic pain is diagnosed by the invasive procedure of provocative discography (PD). Previously, we developed quantitative chemical exchange saturation transfer (qCEST) magnetic resonance imaging (MRI) to detect pH as a biomarker for discogenic pain. Based on these findings we initiated a clinical study with the goal to evaluate the correlation between qCEST values and PD results in LBP patients. Twenty five volunteers with chronic low back pain were subjected to T2-weighted (T2w) and qCEST MRI scans followed by PD. A total of 72 discs were analyzed. The average qCEST signal value of painful discs was significantly higher than non-painful discs (p = 0.012). The ratio between qCEST and normalized T2w was found to be significantly higher in painful discs compared to non-painful discs (p = 0.0022). A receiver operating characteristics (ROC) analysis indicated that qCEST/T2w ratio could be used to differentiate between painful and non-painful discs with 78% sensitivity and 81% specificity. The results of the study suggest that qCEST could be used for the diagnosis of discogenic pain, in conjunction with the commonly used T2w scan.

Overall Structure Construction of an Intervertebral Disk Based on Highly Anisotropic Wood Hydrogel Composite Materials with Mechanical Matching and Buckling Buffering.

Natural intervertebral disks (IVDs) exhibit distinctive anisotropic mechanical support and dissipation performances due to their well-developed special microstructures. As the intact IVD structure degrades, the absence of function will lead to severe backache. However, the complete simulation for the characteristic structure and function of native IVD is unattainable using current methods. In this work, by overall construction of the two-phase structure of native IVD (extraction of the naturally aligned cellulose framework and in situ polymerization of the nanocomposite hydrogel), a complete wood framework IVD (WF-IVD) is manufactured containing elastic nanocomposite hydrogel-based nucleus pulposus (NP) and anisotropic wood cellulose hydrogel-based annulus fibrosus (AF). In addition to the imitation and construction of the natural structure, WF-IVD also achieves favorable mechanical matching and good biocompatibility and possesses unique mechanical buckling buffer characteristics owing to the aligned fiber bundles. This study offers a promising strategy for the mimicking and construction of complex native tissues.

Simultaneous detection of multiple mRNAs and proteins in bovine IVD cells and tissue with single cell resolution.

OBJECTIVES: Interactions of cells with their neighbors and influences by the surrounding extracellular matrix (ECM) is reflected in a cells transcriptome and proteome. In tissues comprised of heterogeneous cell populations or cells depending on ECM signalling cues such as those of the intervertebral disc (IVD), this information is obscured or lost when cells are pooled for the commonly used transcript analysis by quantitative PCR or RNA sequencing. Instead, these cells require means to analyse RNA transcript and protein distribution at a single cell or subcellular level to identify different cell types and functions, without removing them from their surrounding signalling cues. RESULTS: We developed a simple, sequential protocol combining RNA is situ hybridisation (RISH) and immunohistochemistry (IHC) for the simultaneous analysis of multiple transcripts alongside proteins. This allows one to characterize heterogeneous cell populations at the single cell level in the natural cell environment and signalling context, both in vivo and in vitro. This protocol is demonstrated on cells of the bovine IVD, for transcripts and proteins involved in mechanotransduction, stemness and cell proliferation. CONCLUSIONS: A simple, sequential protocol combining RISH and IHC is presented that allows for simultaneous information on RNA transcripts and proteins to characterize cells within a heterogeneous cell population and complex signalling environments such as those of the IVD.

A possible injectable tissue engineered nucleus pulposus constructed with platelet-rich plasma and ADSCs in vitro.

BACKGROUND: Injectable tissue engineered nucleus pulposus is a new idea for minimally invasive repair of degenerative intervertebral disc. The platelet-rich plasma (PRP) and adipose-derived stromal cells (ADSCs) could be harvested from autologous tissue easily. PRP contains numerous autologous growth factors and has reticulate fibrous structure which may have the potential to make ADSCs differentiate into nucleus pulposus-like cells. The goal of this study was to explore the feasibility of constructing a possible injectable tissue engineered nucleus pulposus with PRP gel scaffold and ADSCs. METHODS: After identification with flow cytometry, the rabbit ADSCs were seeded into PRP gel and cultured in vitro. At the 2nd, 4th, and 8th week, the PRP gel/ADSCs complex was observed by macroscopy, histological staining, BrdU immunofluorescence, and scanning electron microscopy. The glycosaminoglycans (GAG) in the PRP gel/ADSCs complex were measured by safranin O staining with spectrophotometry. In PRP gel/ADSCs complex, gene expression of HIF-1α, aggrecan, type II collagen were tested by RT-PCR. The injectability of this complex was evaluated. RESULTS: Macroscopically, the complex was solidified into gel with smooth surface and good elasticity. The safranin O dye was almost no positive staining at 2nd week; however, the positive staining of extracellular matrix was enhanced obviously at 4th and 8th week. The HE staining and SEM demonstrated that the cells were well-distributed in the reticulate scaffold. BrdU immunofluorescence showed that ADSCs can survive and proliferate in PRP gel at each time points. The level of GAG at 4th week was higher than those at 2nd week (P < 0.05), and significant difference was also noted between 4th and 8th week (P < 0.05). HIF-1α, aggrecan, type II collagen gene expression at 4th week were much more than those at 2nd week (P < 0.05), and significant differences were also noted between 4th and 8th week (P < 0.05). The flow rate of complex was 0.287 mL/min when passed through the 19-gauge needle with the 100 mmHg injection pressure. CONCLUSIONS: Our preliminary findings suggest that the PRP gel make it possible for rabbit ADSCs differentiated into nucleus pulposus-like cells after coculture in vitro. According to the results, it is a better feasible method for construction of autologous injectable tissue engineered nucleus pulposus.

Identification of significant gene biomarkers of low back pain caused by changes in the osmotic pressure of nucleus pulposus cells.

The incidence of intervertebral disc (IVD) degeneration disease, caused by changes in the osmotic pressure of nucleus pulposus (NP) cells, increases with age. In general, low back pain is associated with IVD degeneration. However, the mechanism and molecular target of low back pain have not been elucidated, and there are no data suggesting specific biomarkers of low back pain. Therefore, the research aims to identify and verify the significant gene biomarkers of low back pain. The differentially expressed genes (DEGs) were screened in the Gene Expression Omnibus (GEO) database, and the identification and analysis of significant gene biomarkers were also performed with various bioinformatics programs. A total of 120 patients with low back pain were recruited. Before surgery, the degree of pain was measured by the numeric rating scale (NRS), which enables comparison of the pain scores from individuals. After surgery, IVD tissues were obtained, and NP cells were isolated. The NP cells were cultured in two various osmotic media, including iso-osmotic media (293 mOsm/kg H2O) to account for the morbid environment of NP cells in IVD degeneration disease and hyper-osmotic media (450 mOsm/kg H2O) to account for the normal condition of NP cells in healthy individuals. The relative mRNA expression levels of CCL5, OPRL1, CXCL13, and SST were measured by quantitative real-time PCR in the in vitro analysis of the osmotic pressure experiments. Finally, correlation analysis and a neural network module were employed to explore the linkage between significant gene biomarkers and pain. A total of 371 DEGs were identified, including 128 downregulated genes and 243 upregulated genes. Furthermore, the four genes (CCL5, OPRL1, SST, and CXCL13) were identified as significant gene biomarkers of low back pain (P < 0.001) based on univariate linear regression, and CCL5 (odds ratio, 34.667; P = 0.003) and OPRL1 (odds ratio, 19.875; P < 0.001) were significantly related to low back pain through multivariate logistic regression. The expression of CCL5 and OPRL1 might be correlated with low back pain in patients with IVD degeneration disease caused by changes in the osmotic pressure of NP cells.

Temporomandibular joint imaging: current clinical applications, biochemical comparison with the intervertebral disc and knee meniscus, and opportunities for advancement.

Temporomandibular disorders encompass multiple pathologies of the temporomandibular joint that manifest as middle/inner ear symptoms, headache, and/or localized TMJ symptoms. There is an important although somewhat limited role of imaging in the diagnostic evaluation of temporomandibular disorders. In this manuscript, we provide a comprehensive review of TMJ anatomy, outline potentially important features of TMJ disc ultrastructure and biochemistry in comparison with the intervertebral disc and knee meniscus, and provide imaging examples of the TMJ abnormalities currently evaluable with MRI and CT. In addition, we provide an overview of emerging and investigational TMJ imaging techniques in order to encourage further imaging research based on the biomechanical alterations of the TMJ disc.

Age estimation based on different molecular clocks in several tissues and a multivariate approach: an explorative study.

Several molecular modifications accumulate in the human organism with increasing age. Some of these "molecular clocks" in DNA and in proteins open up promising approaches for the development of methods for forensic age estimation. A natural limitation of these methods arises from the fact that the chronological age is determined only indirectly by analyzing defined molecular changes that occur during aging. These changes are not linked exclusively to the expired life span but may be influenced significantly by intrinsic and extrinsic factors in the complex process of individual aging. We tested the hypothesis that a combined use of different molecular clocks in different tissues results in more precise age estimates because this approach addresses the complex aging processes in a more comprehensive way. Two molecular clocks (accumulation of D-aspartic acid (D-Asp), accumulation of pentosidine (PEN)) in two different tissues (annulus fibrosus of intervertebral discs and elastic cartilage of the epiglottis) were analyzed in 95 cases, and uni- and multivariate models for age estimation were generated. The more parameters were included in the models for age estimation, the smaller the mean absolute errors (MAE) became. While the MAEs were 7.5-11.0 years in univariate models, a multivariate model based on the two protein clocks in the two tissues resulted in a MAE of 4.0 years. These results support our hypothesis. The tested approach of a combined analysis of different molecular clocks analyzed in different tissues opens up new possibilities in postmortem age estimation. In a next step, we will add the epigenetic clock (DNA methylation) to our protein clocks (PEN, D-Asp) and expand our set of tissues.

Long non-coding RNA zinc finger antisense 1 expression associates with increased disease risk, elevated disease severity and higher inflammatory cytokines levels in patients with lumbar disc degeneration.

This study aimed to investigate the correlation of long noncoding RNA zinc finger antisense 1 (lncRNA ZFAS1) expression with disease risk, disease severity and inflammatory cytokines levels in lumbar disc degeneration (LDD) patients.83 LDD patients underwent surgery and 28 traumatized, non-LDD patients underwent lumbar disc surgery (controls) were consecutively enrolled in this case-control study. Lumbar disc tissue was obtained during surgery and herniated nucleus pulposus (HNP) was isolated to detect lncRNA ZFAS1 expression and inflammatory cytokines mRNA levels by RT-qPCR, and determine protein levels of inflammatory cytokines by western blot.HNP lncRNA ZFAS1 expression in LDD patients was up-regulated compared with controls (P < .001), and receiver operating characteristic (ROC) curve showed lncRNA ZFAS1 expression disclosed a good predictive value for LDD risk with area under curve (AUC) 0.753 (95% CI 0.646-0.859). And after adjustment by age, gender and body mass index (BMI), lncRNA ZFAS1 (P = .017) remained to be an independent predictive factor for higher LDD risk. In addition, lncRNA ZFAS1 expression was positively associated with Modified Pfirrmann Grade (P = .015). As to inflammatory cytokines, lncRNA ZFAS1 expression was observed to be positively correlated with TNF-α (P = .002), IL-1ß (P = .007) and IL-6 (P = .015) mRNAs expressions while reversely associated with IL-10 mRNA level (P = .014); and lncRNA ZFAS1 expression was also positively correlated with protein levels of TNF-α (P = .038) and IL-6 (P = .027) while reversely associated with IL-10 protein expression (P = .039).lncRNA ZFAS1 expression associates with increased risk, elevated disease severity and higher inflammatory cytokines levels in LDD patients.

Reciprocal Regulation of TRPS1 and miR-221 in Intervertebral Disc Cells.

Intervertebral disc (IVD), a moderately moving joint located between the vertebrae, has a limited capacity for self-repair, and treating injured intervertebral discs remains a major challenge. The development of innovative therapies to reverse IVD degeneration relies primarily on the discovery of key molecules that, occupying critical points of regulatory mechanisms, can be proposed as potential intradiscal injectable biological agents. This study aimed to elucidate the underlying mechanism of the reciprocal regulation of two genes differently involved in IVD homeostasis, the miR-221 microRNA and the TRPS1 transcription factor. Human lumbar IVD tissue samples and IVD primary cells were used to specifically evaluate gene expression and perform functional analysis including the luciferase gene reporter assay, chromatin immunoprecipitation, cell transfection with hTRPS1 overexpression vector and antagomiR-221. A high-level expression of TRPS1 was significantly associated with a lower pathological stage, and TRPS1 overexpression strongly decreased miR-221 expression, while increasing the chondrogenic phenotype and markers of antioxidant defense and stemness. Additionally, TRPS1 was able to repress miR-221 expression by associating with its promoter and miR-221 negatively control TRPS1 expression by targeting the TRPS1-3'UTR gene. As a whole, these results suggest that, in IVD cells, a double-negative feedback loop between a potent chondrogenic differentiation suppressor (miR-221) and a regulator of axial skeleton development (TRPS1) exists. Our hypothesis is that the hostile degenerated IVD microenvironment may be counteracted by regenerative/reparative strategies aimed at maintaining or stimulating high levels of TRPS1 expression through inhibition of one of its negative regulators such as miR-221.

Lessons learned from intervertebral disc pathophysiology to guide rational design of sequential delivery systems for therapeutic biological factors.

Intervertebral disc (IVD) degeneration has been associated with low back pain, which is a major musculoskeletal disorder and socio-economic problem that affects as many as 600 million patients worldwide. Here, we first review the current knowledge of IVD physiology and physiopathological processes in terms of homeostasis regulation and consecutive events that lead to tissue degeneration. Recent progress with IVD restoration by anti-catabolic or pro-anabolic approaches are then analyzed, as are the design of macro-, micro-, and nano-platforms to control the delivery of such therapeutic agents. Finally, we hypothesize that a sequential delivery strategy that i) firstly targets the inflammatory, pro-catabolic microenvironment with release of anti-inflammatory or anti-catabolic cytokines; ii) secondly increases cell density in the less hostile microenvironment by endogenous cell recruitment or exogenous cell injection, and finally iii) enhances cellular synthesis of extracellular matrix with release of pro-anabolic factors, would constitute an innovative yet challenging approach to IVD regeneration.

Extracellular matrix composition of connective tissues: a systematic review and meta-analysis.

The function of connective tissues depends on the physical and biochemical properties of their extracellular matrix (ECM), which are in turn dictated by ECM protein composition. With the primary objective of obtaining quantitative estimates for absolute and relative amounts of ECM proteins, we performed a systematic review of papers reporting protein composition of human connective tissues. Articles were included in meta-analysis if they contained absolute or relative quantification of proteins found in the ECM of human bone, adipose tissue, tendon, ligament, cartilage and skeletal muscle. We generated absolute quantitative estimates for collagen in articular cartilage, intervertebral disk (IVD), skeletal muscle, tendon, and adipose tissue. In addition, sulfated glycosaminoglycans were quantified in articular cartilage, tendon and skeletal muscle; total proteoglycans in IVD and articular cartilage, fibronectin in tendon, ligament and articular cartilage, and elastin in tendon and IVD cartilage. We identified significant increases in collagen content in the annulus fibrosus of degenerating IVD and osteoarthritic articular cartilage, and in elastin content in degenerating disc. In contrast, collagen content was decreased in the scoliotic IVD. Finally, we built quantitative whole-tissue component breakdowns. Quantitative estimates improve our understanding of composition of human connective tissues, providing insights into their function in physiology and pathology.

Crystallinity dependency of the time-dependent mechanical response of polyethylene: application in total disc replacement.

Degeneration of the intervertebral disc (IVD) is a leading source of chronic low back pain or neck pain, and represents the main cause of long-term disability worldwide. In the aim to relieve pain, total disc replacement (TDR) is a valuable surgical treatment option, but the expected benefit strongly depends on the prosthesis itself. The present contribution is focused on the synthetic mimic of the native IVD in the aim to optimally restore its functional anatomy and biomechanics, and especially its time-dependency. Semi-crystalline polyethylene (PE) materials covering a wide spectrum of the crystallinity are used to propose new designs of TDR. The influence of the crystallinity on various features of the time-dependent mechanical response of the PE materials is reported over a large strain range by means of dynamic mechanical thermo-analysis and video-controlled tensile mechanical tests. The connection of the stiffness and the yield strength with the microstructure is reported in the aim to propose a model predicting the crystallinity dependency of the response variation with the frequency. New designs of TDR are proposed and implemented into an accurate computational model of a cervical spine segment in order to simulate the biomechanical response under physiological conditions. Predicted in-silico motions are found in excellent agreement with experimental data extracted from published in-vitro studies under compression and different neck movements, namely, rotation, flexion/extension and lateral bending. The simulation results are also criticized by analyzing the local stresses and the predicted biomechanical responses provided by the different prosthetic solutions in terms of time-dependency manifested by the hysteretic behavior under a cyclic movement and the frequency effect.

Transport of Vancomycin and Cefepime Into Human Intervertebral Discs: Quantitative Analyses.

STUDY DESIGN: Simulation of antibiotics transport into human intervertebral disc with intravenous infusion. OBJECTIVE: The objective of this study was to quantitatively investigate antibiotic concentrations in the disc. SUMMARY OF BACKGROUND DATA: Intravenous infusion of antibiotics is typically used to treat intervertebral disc infection in clinics. However, it is difficult to evaluate the drug concentrations within discs in vivo. METHODS: A computational model was used in this study. The variation of drug charge with pH was considered in the model. Thirty-minute infusions of two commonly used antibiotics in clinic-vancomycin and cefepime-were numerically investigated. Spatial and temporal concentration distributions of these drugs in both nondegenerated and moderately degenerated discs were calculated. RESULTS: For intravenous infusion of 1 g vancomycin and 2 g cefepime in 30 minutes repeated every 12 hours, it was predicted that vancomycin concentration in the disc fluctuated between 17.0 and 31.0 times of its minimum inhibitory concentration (1 ug/mL) and cefepime concentration fluctuated between 1.1 and 4.2 times of its minimum inhibitory concentration (i.e., 8 ug/mL) in about 2 days. It was also found that vancomycin concentration in moderately degenerated disc was lower than that in the nondegenerated disc. CONCLUSION: This study provides quantitative guidance on selecting proper dosage for treating disc infection. The method used in this study could be used to provide quantitative information on transport of other antibiotics and drugs in discs as well. LEVEL OF EVIDENCE: N/A.

Ingrowth of Nociceptive Receptors into Diseased Cervical Intervertebral Disc Is Associated with Discogenic Neck Pain.

OBJECTIVE: To investigate the distribution of nociceptive nerve fibers in the cervical intervertebral discs of patients with chronic neck pain and determine whether these nociceptive nerve fibers are related to discogenic neck pain. METHODS: We collected 43 samples of cervical intervertebral discs from 34 patients with severe chronic neck pain (visual analog scale [VAS] ≥ 70 mm), 42 samples from 36 patients who suffered cervical spondylotic radiculopathy or myelopathy without neck pain or with mild neck pain (VAS ≤ 30 mm) and 32 samples from eight donators to investigate their innervation immunohistochemically using an antibody against neuropeptide substance P. RESULTS: The immunohistochemical investigation revealed that substance P-positive nerve fibers were obviously increased in number and deeply ingrown into the inner anulus fibrosus and even into the nucleus pulposus in the degenerative cervical discs of patients with severe neck pain in comparison with the discs of patients with cervical spondylotic radiculopathy or myelopathy and normal control discs (P<0.01). CONCLUSIONS: The current study may indicate a key role of nociceptive nerve fibers in the pathogenesis of neck pain of cervical disc origin.

Estrogen Deficiency Exacerbates Intervertebral Disc Degeneration Induced by Spinal Instability in Rats.

STUDY DESIGN: An experimental animal study of osteoporosis (OP) and intervertebral disc degeneration (IDD). OBJECTIVE: The aim of this study was to clarify the effects of estrogen deficiency and supplement on cervical IDD induced by bilateral facetectomy in rats. SUMMARY OF BACKGROUND DATA: The relationship between IDD and OP is still controversy with the wide prevalence in aged people. METHODS: Seventy-two Sprague-Dawley female rats were randomly divided into ovariectomy (OVX) group, facet joints resection of C4-6 (FR), FR-OVX group, estrogen replacement therapy (ERT, based on the FR-OVX group) group, and sham group. Specimens of C4-6 segment were harvested at 12 and 24 weeks. The microstructures of C5 vertebrae, vertebral endplate lesions and calcification, and IDD of C5/6 disc were evaluated by micro-computed tomography (micro-CT) and histology. The protein and gene levels of aggrecan, Col2α1, matrix metalloprotease (MMP)-3, and MMP-13 in the C5/6 and C4/5 discs were measured. RESULTS: Microstructures of C5 vertebral body were weakened significantly after ovariectomy, while restored effectively with estradiol supplementation. The facetectomy led to significant IDD, and the IDD was aggravated when combined with OVX. The IDD of the ERT group was alleviated effectively and similar to that of the FR group in intervertebral disc height, vertebral endplate lesions and calcification, and disc degeneration scores. In addition, the estrogen supplement maintained the extracellular matrix by decreasing MMP-3 and MMP-13, and increasing aggrecan and Col2α1 expression. CONCLUSION: The present study demonstrated that estrogen deficiency exacerbated IDD induced by spinal instability, while estrogen supplementation alleviated the progression of disc degeneration related to osteoporosis. LEVEL OF EVIDENCE: N/A.

New evidence for structural integration across the cartilage-vertebral endplate junction and its relation to herniation.

BACKGROUND CONTEXT: The cartilaginous and bony material that can be present in herniated tissue suggests that failure can involve both cartilaginous and vertebral-endplates. How structural integration is achieved across the junction between these two distinct tissue regions via its fibril and mineral components is clearly relevant to the modes of endplate failure that occur. PURPOSE: To understand how structural integration is achieved across the cartilaginous-vertebral endplate junction. STUDY DESIGN: A micro- and fibril-level structural analysis of the cartilage-vertebral endplate region was carried out using healthy, mature ovine motion segments. METHODS: Oblique vertebra-annulus-vertebra samples were prepared such that alternate layers of lamellar fibers extended from vertebra to vertebra. The endplate region of each sample was then decalcified in a targeted manner before being loaded in tension along the fiber direction to achieve incomplete rupture within the region of the endplate. The failure regions were then analyzed with differential interference contrast microscopy and scanning electron microscopy. RESULTS: Microstructural analysis revealed that failure within the endplate region was not confined to the cement line. Instead, rupture continued into the underlying vertebral endplate with bony material still attached to the now unanchored annular bundles. Ultrastructural analysis of the partially ruptured regions of the cement line revealed clear evidence of blending/interweaving relationships between the fibrils of the annular bundles, the calcified cartilage and the bone with no one pattern of association appearing dominant. These findings suggest that fibril-based structural cohesion exists across the cement line at the site of annular insertion, with strengthening via a mechanism somewhat analogous to steel-reinforced concrete. The fibrils are brought into a close intermingling association with interfibril forces mediated via the mineral component. CONCLUSIONS: This study provides clear evidence of structural connectivity across the cartilaginous-vertebral endplate junction by the intermingling of their fibrillar components and mediated by the mineral phase. This is consistent with the clinical observation that in some disc herniations bony material can be still attached to the extruded soft tissue.

Changes in human intervertebral disc biochemical composition and bony end plates between middle and old age.

OBJECTIVE: This study evaluates molecular, nutritional and biochemical alterations in human intervertebral discs between middle and old age. METHODS: Twenty-eight human lumbar intervertebral discs from donors were evaluated and separated into two groups: Middle-aged (35-50 years old, relatively non-degenerate discs of Pfirrmann grades 1-3, n = 15) and Old-aged (≥80 years old, all degenerate Pfirrmann grade 4 or 5, n = 13). Parameters which might be expected to to be related to nutrient supply and so the health of disc cells (eg the porosity of the vertebral endplate, cell viability and cell density) and to disc extracellular composition (ie quantification of glycosaminoglycan disaccharides and hyaluronic acid molecular weight) and collagen organization, were analyzed. Three regions of the intervertebral disc (anterior annulus fibrosus, nucleus pulposus, and posterior annulus fibrosus) were examined. RESULTS: The old-aged group showed a decrease in content of sulphated and non-sulphated glycosaminoglycans relative to middle-aged and there were also alterations in the proportion of GAG disaccharides and a decrease of collagen fiber size. Hyaluronic acid molecular weight was around 200 kDa in all regions and ages studied. The anterior annulus differed from the posterior annulus particularly in relation to cell density and GAG content. Additionally, there were changes in the bony endplate, with fewer openings observed in the caudal than cranial endplates of all discs in both groups. CONCLUSIONS: Results show the cranial vertebral endplate is the main vascular source for the intervertebral discs. Hylauronic acid molecular weight is the same through the intervertebral disc after age of 50 years.