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After transection the lumbar spinal cord of lizards forms a bridge of connective and nervous tissues between the severed proximal and distal ends of the cord. The types of proliferating cells activated in the injured spinal cord have been analyzed using light and ultrastructural immunolabeling for 5BrdU and nestin from 11 to 34 days after injury, when recovery of some hindlimb movements has occurred. At 11-22 days post-transection an intense proliferation of glial, immune and meningeal cells takes place. Nestin is almost absent in the normal spinal cord but becomes detectable at 11-34 days postinjury in ependymal and sparse glial cells located in the bridge region. At 11-22 days postinjury also numerous macrophages, lymphocytes, and some plasma cells appear proliferating during the intense inflammatory and antimicrobial phase. Phagocytosis within the injured spinal cord probably decreases inflammation and may indirectly promote axonal regeneration. Proliferating cells likely derive from precursor or stem elements of the reactive ependymal epithelium, but also from glial cells and meningeal fibroblasts. This is indicated by the presence of 5BrdU-long retaining labeling cells of glial and fibroblast types located in the stumps of the spinal cord and in the bridge. The present observations suggest that meningeal, ependymal, and numerous glial cells are the precursors of those forming the bridge region. Among glial cells, sparse oligodendrocytes myelinating the few axons present at 34 day after the injury also appear capable to proliferate. The myelinated axons are probably involved in the limited but important functional recovery of limb movements observed after 30-90 days postinjury., (© 2019 Wiley Periodicals, Inc.)

Purpose: We aimed to assess the reliability of measuring the fat content of the lumbar vertebral marrow and the paraspinal muscles using magnetic resonance imaging (MRI) mDIXON-Quant sequence., Methods: Thirty-one healthy volunteers were included. All participants underwent liver mDIXON-Quant imaging on a 3.0 T Philips MRI scanner by observer A. Within two weeks, observer B repeated the scan. After the examination, each observer independently measured the fat content of the third lumbar vertebra (L3), and the psoas (PS), erector spinae (ES), and multifidus (MF) muscles on central L3 axial images. After two weeks, each observer repeated the same measurements. They were blinded to their previous results. Reliability was estimated by evaluating the repeatability and reproducibility., Results: The repeatability of the fat content measurements of L3, PS, ES, and MF was high. The intraclass correlation coefficients of the fat content of L3, PS, ES, and MF were 0.997, 0.984, 0.997, and 0.995 for observer A and 0.948, 0.974, 0.963, and 0.995 for observer B, respectively. The reproducibility of the measurement of the fat content of L3, PS, ES, and MF was high, and the interclass correlation coefficients were 0.984, 0.981, 0.977, and 0.998, respectively., Conclusion: Using mDIXON-Quant imaging to measure the fat content of the lumbar vertebral marrow and paraspinal muscles shows high reliability and is suitable for use in clinical practice.

Background: The presence of bridging syndesmophytes (BS) in spinal osteotomy region serves traditionally as one critical determinant for selection of osteotomy techniques. While nowadays the proportion of kyphotic ankylosing spondylitis (AS) patients receiving pedicle subtraction osteotomy (PSO) with yet mobile neighboring disc has seen a substantial increase. Literatures investigating the clinical relevance of the presence of BS on kyphosis correction and maintenance following PSO are scarce., Methods: A total of 71 thoracolumbar kyphotic AS patients treated with single-level PSO at our hospital between September 2010 and August 2014 were retrospectively reviewed, 32 of whom were stratified into the BS group (BG). The operative corrections of multiple spino-pelvic sagittal parameters were assessed. Comparison of the contribution of adjacent disc wedging to total correction per PSO segment was made between the BS and non-BS groups (NBG). The correction loss were also evaluated and compared with a minimum 2-year follow-up., Results: A significantly younger age (30.97 ± 8.28 vs. 40.31 ± 8.44 yrs., p < 0.001), smaller pelvic incidence (PI) (43.03 ± 10.60 vs. 49.36 ± 9.75°, p = 0.011), greater wedging index of osteotomized vertebra (1.17 ± 0.16 vs. 1.09 ± 0.08, p = 0.011) and larger local kyphosis (19.59 ± 10.84 vs. 13.56 ± 8.50°, p = 0.013) was observed in NBG preoperatively. Patients in BG and NBG accomplished comparable amount of kyphosis correction per PSO segment (40.22 ± 7.09 vs. 43.85 ± 8.71°, p = 0.062). However, the contribution of adjacent disc wedging to total correction per PSO was significantly larger in NBG [8.10 ± 6.19 (18.5%) vs. 1.09 ± 2.88° (2.7%), p < 0.001]. By ultimate follow-up, the global kyphosis (18.26 ± 10.97 vs. 21.51 ± 10.89°, p < 0.05) and thoracic kyphosis (37.95 ± 11.87 vs. 42.87 ± 11.56°, p < 0.05) deteriorated significantly in the NBG but not BG, so was further pelvic retroversion as represented by increased pelvic tilt (19.46 ± 8.13 vs. 23.44 ± 8.19°, p < 0.05) and decreased sacral slope (23.02 ± 9.12 vs. 18.62 ± 10.10°, p < 0.05). Loss of corrections concerning contribution of adjacent disc wedging was also larger in NBG (1.41 ± 3.27 vs. 0.22 ± 1.49°, p < 0.05)., Conclusions: Our study might suggest that the evaluation and treatment methods of kyphotic AS patients needed to be fine-tuned with appropriate subgrouping by the presence of syndesmophytes with bamboo sign as they were potentially distinct groups with different PI, contributor of lordosing capability and prognosis that might require separate analysis.

Background: One of the major challenges in orthopedics is to develop implants that overcome current postoperative problems such as osteointegration, proper load bearing, and stress shielding. Current implant techniques such as allografts or endoprostheses never reach full bone integration, and the risk of fracture due to stress shielding is a major concern. To overcome this, a novel technique of reverse engineering to create artificial scaffolds was designed and tested. The purpose of the study is to create a new generation of implants that are both biocompatible and biomimetic., Methods: 3D-printed scaffolds based on physiological trabecular bone patterning were printed. MC3T3 cells were cultured on these scaffolds in osteogenic media, with and without the addition of Calcitonin Receptor Fragment Peptide (CRFP) in order to assess bone formation on the surfaces of the scaffolds. Integrity of these cell-seeded bone-coated scaffolds was tested for their mechanical strength., Results: The results show that cellular proliferation and bone matrix formation are both supported by our 3D-printed scaffolds. The mechanical strength of the scaffolds was enhanced by trabecular patterning in the order of 20% for compression strength and 60% for compressive modulus. Furthermore, cell-seeded trabecular scaffolds modulus increased fourfold when treated with CRFP., Conclusion: Upon mineralization, the cell-seeded trabecular implants treated with osteo-inductive agents and pretreated with CRFP showed a significant increase in the compressive modulus. This work will lead to creating 3D structures that can be used in the replacement of not only bone segments, but entire bones.

Background: Patients with an impaired renal function show a high incidence of bone and mineral disturbances. These 'chronic kidney disease - mineral and bone disorders' (CKD-MBD) range from high turnover osteoporosis to adynamic bone disease. Currently, the histomorphometric analysis of a bone biopsy taken from the iliac crest is viewed as the gold standard for CKD-MBD subtype differentiation. However, the clinical relevance of such a biopsy is questionable since iliac crest fractures are an extremely rare finding. Therefore, we aimed to elucidate if the histomorphometric parameter 'trabecular bone volume (BV/TV)' from the iliac crest is representative for other biopsy locations. We chose two skeletal sites of higher fracture risk for testing, namely, the tibial bone and the lumbar spine, to examine if the current gold standard of bone biopsy is indeed golden., Methods: Bone biopsies were taken from 12 embalmed body donors at the iliac crest, the proximal tibia, and the lumbar vertebral body, respectively. Masson-Goldner stained sections of methyl methacrylate embedded biopsies were used for trabecular bone volume calculation. Furthermore, exemplary μ-computed tomography (XtremeCT) scans with subsequent analysis were performed., Results: Median values of trabecular bone volume were comparable between all body donors with median (interquartile range, IQR) 18.3% (10.9-22.9%) at the iliac crest, 21.5% (9.5-40.1%) at the proximal tibia, and 16.3% (11.4-25.0%) at the lumbar spine. However, single values showed extensive intra-individual variation, which were also confirmed by XtremeCT imaging., Conclusions: Distinct intra-individual heterogeneity of trabecular bone volume elucidate why a bone biopsy from one site does not necessarily predict patient relevant endpoints like hip or spine fractures. Physicians interpreting bone biopsy results should know this limitation of the current gold standard for CKD-MBD diagnostic, especially, when systemic therapeutic decisions should be based on it.

The annulus-endplate anchorage system plays a vital role in structurally linking the compliant disc to its adjacent much more rigid vertebrae. Past literature has identified the endplate as a region of weakness, not just in the mature spine but also in the immature spine. The aim of this structural study was to investigate in detail the morphological changes associated with annulus-endplate integration through different stages of maturity. Ovine lumbar motion segments were collected from two immature age groups: (i) newborn and (ii) spring lamb (roughly 3 months old); these were compared with a third group of previously analysed mature ewe samples (3-5 years). Sections from the posterior region of each motion segment were obtained for microstructural analysis and imaged in their fully hydrated state via differential interference contrast (DIC) optical microscopy. Selected slices were further prepared and imaged via scanning electron microscopy (SEM) to analyse fibril-level modes of integration. Despite significant changes in endplate morphology, the annular fibre bundles in all three age groups displayed a similar branching mechanism, with the main bundle splitting into several sub-bundles on entering the cartilaginous endplate. This morphology, previously described in the mature ovine disc, is thought to strengthen significantly annulus-endplate integration. Its prevalence from an age as young as birth emphasizes the critical role that it plays in the anchorage system. The structure of the branched sub-bundles and their integration with the surrounding matrix were found to vary with age due to changes in the cartilaginous and vertebral components of the endplate. Microscopically, the sub-bundles in both immature age groups appeared to fade into the surrounding tissue due to their fibril-level integration with the cartilaginous endplate tissue, this mechanism being particularly complex in the spring lamb disc. However, in the fully mature disc, the sub-bundles remained as separate entities throughout the full depth of their anchorage into the cartilaginous endplate. Cell morphology was also found to vary with maturity within the cartilaginous matrix and it is proposed that this relates to endplate development and ossification., (© 2016 Anatomical Society.)

Fresh platelet-rich plasma (PRP) accelerates bone union in rat model. However, fresh PRP has a short half-life. We suggested freeze-dried PRP (FD-PRP) prepared in advance and investigated its efficacy in vivo. Spinal posterolateral fusion was performed on 8-week-old male Sprague-Dawley rats divided into six groups based on the graft materials (n = 10 per group): sham control, artificial bone (A hydroxyapatite-collagen composite) -alone, autologous bone, artificial bone + fresh-PRP, artificial bone + FD-PRP preserved 8 weeks, and artificial bone + human recombinant bone morphogenetic protein 2 (BMP) as a positive control. At 4 and 8 weeks after the surgery, we investigated their bone union-related characteristics including amount of bone formation, histological characteristics of trabecular bone at remodeling site, and biomechanical strength on 3-point bending. Comparable radiological bone union was confirmed at 4 weeks after surgery in 80% of the FD-PRP groups, which was earlier than in other groups (p < 0.05). Histologically, the trabecular bone had thinner and more branches in the FD-PRP. Moreover, the biomechanical strength was comparable to that of autologous bone. FD-PRP accelerated bone union at a rate comparable to that of fresh PRP and BMP by remodeling the bone with thinner, more tangled, and rigid trabecular bone.

Intervertebral disc (IVD) degeneration is a common cause of low back pain. Testing potential therapeutics in the regeneration of the disc requires the use of model systems. Although several animal models have been developed to investigate IVD degeneration, they are technically challenging to prepare, expensive, present with limitations when performing biomechanical studies on the disc, and are impractical in large-scale screening of novel anabolic and scaffolding agents. An IVD organ culture system offers an inexpensive alternative. In the current paradigm, the bony endplates are removed to allow for nutrient diffusion and maintenance of disc cell viability. Although this is an excellent system for testing biologics, it results in concave cartilage endplates and, as such, requires special platens for loading purposes in a bioreactor as flat ones can overload the annular disc region leading to improper loading. Furthermore, the absence of bone makes it unsuitable for applying complex cyclic loading, a topic of interest in the study of chronic progressive degeneration, as multiaxial loading is more representative of daily forces encountered by the IVD. We have developed and validated a novel long-term IVD organ culture model that retains vertebral bone and is easy to prepare. Our model is ideal for testing potential drugs and alternate-based therapies, in addition to investigating the long-term effects of loading paradigms on disc degeneration and repair.

The hypoxic growth plate cartilage requires hypoxia-inducible factor (HIF)-mediated pathways to maintain chondrocyte survival and differentiation. HIF proteins are tightly regulated by prolyl hydroxylase domain-containing protein 2 (Phd2)-mediated proteosomal degradation. We conditionally disrupted the Phd2 gene in chondrocytes by crossing Phd2 floxed mice with type 2 collagen-α1-Cre transgenic mice and found massive increases (>50%) in the trabecular bone mass of long bones and lumbar vertebra of the Phd2 conditional knockout (cKO) mice caused by significant increases in trabecular number and thickness and reductions in trabecular separation. Cortical thickness and tissue mineral density at the femoral middiaphysis of the cKO mice were also significantly increased. Dynamic histomorphometric analyses revealed increased longitudinal length and osteoid surface per bone surface in the primary spongiosa of the cKO mice, suggesting elevated conversion rate from hypertrophic chondrocytes to mineralized bone matrix as well as increased bone formation in the primary spongiosa. In the secondary spongiosa, bone formation measured by mineralizing surface per bone surface and mineral apposition rate were not changed, but resorption was slightly reduced. Increases in the mRNA levels of SRY (sex determining region Y)-box 9, osterix (Osx), type 2 collagen, aggrecan, alkaline phosphatase, bone sialoprotein, vascular endothelial growth factor, erythropoietin, and glycolytic enzymes in the growth plate of cKO mice were detected by quantitative RT-PCR. Immunohistochemistry revealed an increased HIF-1α protein level in the hypertrophic chondrocytes of cKO mice. Infection of chondrocytes isolated from Phd2 floxed mice with adenoviral Cre resulted in similar gene expression patterns as observed in the cKO growth plate chondrocytes. Our findings indicate that Phd2 suppresses endochondral bone formation, in part, via HIF-dependent mechanisms in mice.

Protein kinase A (PKA) regulates osteoblast cell function in vitro and is activated by important bone mass modulating agents. We determined whether PKA activation in osteoblasts is sufficient to mediate a bone anabolic response. Thus, a mouse model conditionally expressing a constitutively active PKA (CA-PKA) in osteoblasts (CA-PKA-OB mouse) was developed by crossing a 2.3-kb α1 (I)-collagen promoter-Cre mouse with a floxed-CA-PKA mouse. Primary osteoblasts from the CA-PKA-OB mice exhibited higher basal PKA activity than those from control mice. Microcomputed tomographic analysis revealed that CA-PKA-OB female mice had an 8.6-fold increase in femoral but only 1.16-fold increase in lumbar 5 vertebral bone volume/total volume. Femur cortical thickness and volume were also higher in the CA-PKA-OB mice. In contrast, alterations in many femoral microcomputed tomographic parameters in male CA-PKA-OB mice were modest. Interestingly, the 3-dimensional structure model index was substantially lower both in femur and lumbar 5 of male and female CA-PKA-OB mice, reflecting an increase in the plate to rod-like structure ratio. In agreement, femurs from female CA-PKA-OB mice had greater load to failure and were stiffer compared with those of control mice. Furthermore, the CA-PKA-OB mice had higher levels of serum bone turnover markers and increased osteoblast and osteoclast numbers per total tissue area compared with control animals. In summary, constitutive activation of PKA in osteoblasts is sufficient to increase bone mass and favorably modify bone architecture and improve mechanical properties. PKA activation in mature osteoblasts is, therefore, an important target for designing anabolic drugs for treating diseases with bone loss.

Study Design: A descriptive in vitro study on isolation and differentiation of human mesenchymal stem cells (MSCs) derived from the facet joints and interspinous ligaments., Objective: To isolate cells from the facet joints and interspinous ligaments and investigate their surface marker profile and differentiation potentials., Summary of Background Data: Lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament are progressive conditions characterized by the hypertrophy and ossification of ligaments and joints within the spinal canal. MSCs are believed to play a role in the advancement of these diseases and the existence of MSCs has been demonstrated within the ligamentum flavum and posterior longitudinal ligament. The aim of this study was to investigate whether these cells could also be found within facet joints and interspinous ligaments., Methods: Samples were harvested from 10 patients undergoing spinal surgery. The MSCs from facet joints and interspinous ligaments were isolated using direct tissue explant technique. Cell surface antigen profilings were performed via flow cytometry. Their lineage differentiation potentials were analyzed., Results: The facet joints and interspinous ligaments-derived MSCs have the tri-lineage potential to be differentiated into osteogenic, adipogenic, and chondrogenic cells under appropriate inductions. Flow cytometry analysis revealed both cell lines expressed MSCs markers. Both facet joints and interspinous ligaments-derived MSCs expressed marker genes for osteoblasts, adipocytes, and chondrocytes., Conclusion: The facet joints and interspinous ligaments may provide alternative sources of MSCs for tissue engineering applications. The facet joints and interspinous ligaments-derived MSCs are part of the microenvironment of the human ligaments of the spinal column and might play a crucial role in the development and progression of degenerative spine conditions.

The purpose of this study was to provide a novel stochastic assessment of inhomogeneous distribution of bone mineral density (BMD) from the Dual-energy X-ray Absorptiometry (DXA) scans of human lumbar vertebrae and identify the stochastic predictors that were correlated with the microarchitecture parameters of trabecular bone. Eighteen human lumbar vertebrae with intact posterior elements from 5 cadaveric spines were scanned in the posterior-anterior projection using a Hologic densitometer. The BMD map of human vertebrae was obtained from the raw data of DXA scans by directly operating on the transmission measurements of low- and high-energy X-ray beams. Stochastic predictors were calculated by fitting theoretical models onto the experimental variogram of the BMD map, rather than grayscale images, from DXA scans. In addition, microarchitecture parameters of trabecular bone were measured from the 3D images of human vertebrae acquired using a Micro-CT scanner. Significant correlations were observed between stochastic predictors and microarchitecture parameters. The sill variance, representing the standard deviation of the BMD map to some extent, had significantly positive correlations with bone volume, trabecular thickness, trabecular number and connectivity density. The sill variance was also negatively associated with bone surface to volume ratio and trabecular separation. This study demonstrates that the stochastic assessment of the inhomogeneous distribution of BMD from DXA scans of human lumbar vertebrae can reveal microarchitecture information of trabecular bone. However, future studies are needed to examine the potential of stochastic predictors from routine clinical DXA scans in providing bone fragility information complementary to BMD., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Human nucleus pulposus cell (HNPC) apoptosis plays an important role in the development of intervertebral disc degeneration (IVDD). Our previous research revealed that among all of the dysregulated microRNAs in the degenerated nucleus pulposus tissues of patient with IVDD, miRNA-494 (miR-494) is the most significantly increased. However, the influence of miR-494 HNPC apoptosis has not been confirmed., Objective: This study was designed to evaluate the effect of miR-494 on the HNPC apoptosis induced by TNF-α and to explore the possible mechanism of this process., Methods: First, HNPCs were stimulated with TNF-α at different concentrations (0 ng/ml, 10 ng/ml, 50 ng/ml, or 100 ng/ml) for 0 h, 8 h, 16 h, or 24 h. Annexin V-PE/7-AAD assays and real-time quantitative PCR were used to detect the cell apoptosis rates and miR-494 expression. Second, we successfully knocked down endogenous miR-494 in HNPCs via lentiviral antigomiR-494 vector infection and then stimulated with TNF-α (100 ng/ml, 16 h). The rates of apoptosis and miR-494 expression were then detected again. Additionally, a dual-luciferase reporter assay and western blotting were used to determine whether JunD is a target of miR-494. Finally, western blotting was used to analyze the expression of cytochrome C., Results: We found that the rate of apoptosis increased with concentration, time (p < 0.05) and miR-494 expression (p < 0.05). The rate of apoptosis in the 100 ng/ml, 16 h group appeared to be suitable. After transfection, the apoptosis rate and miR-494 expression were significantly decreased in the antigomiR-494+TNF-α group compared to the controls (p < 0.05). We also revealed that JunD is a target of miR-494. Western blotting analysis demonstrated that treatment with the lentiviral antigomiR-494 vector resulted in increased expression of JunD (p < 0.05) and decreased expression of cytochrome C (p < 0.05)., Conclusion: These results indicated that miR-494 is a novel regulator of HNPC apoptosis induced by TNF-α. The knock-out of miR-494 expression protected the HNPCs from apoptosis via the up-regulation of JunD, which was possibly mediated via cytochrome C apoptotic signaling. These findings suggest that the miR-494/JunD signaling pathway might represent a novel therapeutic target for the prevention of IVDD., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Background: As important as the vertebral ligaments are in maintaining the integrity of the spinal column and protecting the contents of the spinal canal, a single detailed review of their histology and embryology is missing in the literature., Methods: A literature search using online search engines was conducted., Results: Single comprehensive reviews of the histology and embryology of the spinal ligaments are not found in the extant medical literature., Conclusions: This review will be useful to those who study or treat patients with pathology of the spine.

The pharmacological inhibition of anandamide (AEA) hydrolysis by fatty acid amide hydrolase (FAAH) attenuates pain in animal models of osteoarthritis (OA) but has failed in clinical trials. This may have occurred because AEA also activates transient receptor potential vanilloid type 1 (TRPV1), which contributes to pain development. Therefore, we investigated the effectiveness of the dual FAAH-TRPV1 blocker OMDM-198 in an MIA-model of osteoarthritic pain. We first investigated the MIA-induced model of OA by (1) characterizing the pain phenotype and degenerative changes within the joint using X-ray microtomography and (2) evaluating nerve injury and inflammation marker (ATF-3 and IL-6) expression in the lumbar dorsal root ganglia of osteoarthritic rats and differences in gene and protein expression of the cannabinoid CB1 receptors FAAH and TRPV1. Furthermore, we compared OMDM-198 with compounds acting exclusively on FAAH or TRPV1. Osteoarthritis was accompanied by the fragmentation of bone microstructure and destroyed cartilage. An increase of the mRNA levels of ATF3 and IL-6 and an upregulation of AEA receptors and FAAH in the dorsal root ganglia were observed. OMDM-198 showed antihyperalgesic effects in the OA model, which were comparable with those of a selective TRPV1 antagonist, SB-366,791, and a selective FAAH inhibitor, URB-597. The effect of OMDM-198 was attenuated by the CB1 receptor antagonist, AM-251, and by the nonpungent TRPV1 agonist, olvanil, suggesting its action as an "indirect" CB1 agonist and TRPV1 antagonist. These results suggest an innovative strategy for the treatment of OA, which may yield more satisfactory results than those obtained so far with selective FAAH inhibitors in human OA.

Gene expression analysis provides an effective methodology to identify clinically relevant genes implicated in intervertebral disc (IVD) pathology. The analysis of gene profile in mesenchymal stem cells (MSCs) from human herniated IVD (H-IVD) and degenerated IVD (D-IVD) has not yet been investigated. We present in this study a characterization of MSCs isolated from clinically categorized H-IVD and D-IVD disc samples. H-IVD-MSCs and D-IVD-MSCs showed multipotent mesenchymal differentiation ability, expressing positivity for adipogenic, osteogenic, and chondrogenic markers with an immunophenotypical profile representative of MSCs. FACS analyses revealed a higher expression of CD44 in D-IVD-MSCs compared to H-IVD-MSCs. Gene expression profile revealed that most genes under investigation displayed large variations and were not significantly different in the two types of analyzed IVD-MSCs. Conversely, the gene expression of osteopontin (OPN), a protein involved in bone matrix mineralization and extracellular matrix destruction, was found markedly increased (more than 400-fold) in D-IVD-MSCs compared to H-IVD-MSCs. Moreover, the OPN protein expression was detectable only in D-IVD-MSCs, and its levels were directly related with D-IVD severity. These findings suggest that an abnormal expression of OPN in D-IVD-MSCs occurs and plays a pivotal role in the pathophysiological process of human disc degeneration. We speculate that the regulation of the OPN pathway might be a therapeutic target to counteract disc degeneration.

Purpose: Decay due to diffusion in the internal field (DDIF) MRI allows for measurements of microstructures of porous materials at low spatial resolution and thus has potential for trabecular bone quality measurements. In trabecular bone, solid bone changes (osteoporosis) as well as changes in bone marrow composition occur. The influence of such changes on DDIF MRI was studied by simulations and in vivo measurements., Methods: Monte Carlo simulations of DDIF in various trabecular bone models were conducted. Changes in solid bone and marrow composition were simulated with numerical bone erosion and marrow susceptibility variations. Additionally, in vivo measurements were performed in the lumbar spine of healthy volunteers aged 23-62 years., Results: Simulations and in vivo results showed that 1) DDIF decay times decrease with increasing marrow fat and 2) the marrow fat percentage needs to be incorporated in the DDIF analysis to discriminate between healthy and osteoporotic solid bone structures., Conclusions: Bone marrow composition plays an important role in DDIF MRI: incorporation of marrow fat percentage into DDIF MRI allowed for differentiation of young and old age groups (in vivo experiments). DDIF MRI may develop into a means of assessing osteoporosis and disorders that affect marrow composition., (© 2013 Wiley Periodicals, Inc.)

Study Design: Histological features of the intervertebral disc (IVD)-endplate interface were analyzed., Objective: To define cartilaginous and bony vertebral endplate in commonly used laboratory animals and compare with that of the humans., Summary of Background Data: Endplates are crucial for the IVD nutrient supply: the IVDs have limited blood supply; most nutrients diffuse through endplates to nourish the discs. Various animal models of IVD and endplate degeneration have been used to study the etiology and treatments of spinal disorders. However, because humans are biped, the spine mechanics differ significantly from other mammals. Translation of animal research findings requires a characterization and comparison of the vertebral endplate in the respective species. In this study, we compared the endplate structure of laboratory animal species at the age range commonly used for modeling spine degeneration with that of an adult human., Methods: Mouse, rat, rabbit, goat, and human IVDs and the adjacent vertebral bodies were isolated from the lower lumbar spine. Tissues were stained with Alcian Blue, counterstained with hematoxylin and eosin., Results: Structure of the vertebral endplate varied significantly between the adult animal species and that of the humans. Growth plates persisted in all adult animals studied, whereas the growth plate is absent in the adult humans. In the mice and rats, the cartilaginous endplates are in continuation with the growth plates, with only a small bony center. Rabbits and goats have a bony layer between cartilaginous endplate and the growth plate. The human endplate consist of a cartilaginous layer and the bony endplate., Conclusion: Significant differences exist in histological features of the endplate across animal species and that of the humans. Consideration should be given when animal models are used to study IVD degeneration and surgical treatments., Level of Evidence: 5.

Adenosine is known to regulate bone production and resorption in humans and mice. Type 1 equilibrative nucleoside transporter (ENT1) is responsible for the majority of adenosine transport across the plasma membrane and is ubiquitously expressed in both humans and mice. However, the contribution of ENT1-mediated adenosine levels has not been studied in bone remodeling. With the recent identification of the importance of adenosine signaling in bone homeostasis, it is essential to understand the role of ENT1 to develop novel therapeutic compounds for bone disorders. Here we examined the effect of ENT1 deletion on bone density using X-ray, dual energy X-ray absorptiometry and micro-computerized tomography analysis. Our results show that bone density and bone mineral density is reduced in the lower thoracic and lumbar spine as well as the femur of old ENT1 null mice (>7 months) compared to wild-type littermates. Furthermore, we found increased mRNA expression of tartrate-resistant acid phosphatase (TRAP), an osteoclast marker, in isolated long bones from 10 month old ENT1 null mice compared to wild-type mice. In addition, aged ENT1 null mice displayed severe deficit in motor coordination and locomotor activity, which might be attributed to dysregulated bone density. Overall, our study suggests that ENT1-regulated adenosine signaling plays an essential role in lumbar spine and femur bone density.

Purpose: The better understanding of vertebral mechanical properties can help to improve the diagnosis of vertebral fractures. As the bone mechanical competence depends not only from bone mineral density (BMD) but also from bone quality, the goal of the present study was to investigate the anisotropic indentation moduli of the different sub-structures of the healthy human vertebral body and spondylophytes by means of microindentation., Methods: Six human vertebral bodies and five osteophytes (spondylophytes) were collected and prepared for microindentation test. In particular, indentations were performed on bone structural units of the cortical shell (along axial, circumferential and radial directions), of the endplates (along the anterio-posterior and lateral directions), of the trabecular bone (along the axial and transverse directions) and of the spondylophytes (along the axial direction). A total of 3164 indentations down to a maximum depth of 2.5 µm were performed and the indentation modulus was computed for each measurement., Results: The cortical shell showed an orthotropic behavior (indentation modulus, Ei, higher if measured along the axial direction, 14.6±2.8 GPa, compared to the circumferential one, 12.3±3.5 GPa, and radial one, 8.3±3.1 GPa). Moreover, the cortical endplates (similar Ei along the antero-posterior, 13.0±2.9 GPa, and along the lateral, 12.0±3.0 GPa, directions) and the trabecular bone (Ei= 13.7±3.4 GPa along the axial direction versus Ei=10.9±3.7 GPa along the transverse one) showed transversal isotropy behavior. Furthermore, the spondylophytes showed the lower mechanical properties measured along the axial direction (Ei=10.5±3.3 GPa)., Conclusions: The original results presented in this study improve our understanding of vertebral biomechanics and can be helpful to define the material properties of the vertebral substructures in computational models such as FE analysis., (Copyright © 2013. Published by Elsevier Ltd.)

In rats treated with strontium ranelate, the ultimate load of intact bone is increased and associated with changes in microstructure and material level properties. Evaluation by micro-computed-tomography-based finite element analysis has shown that these changes independently contribute to the improvement of bone strength induced by strontium ranelate treatment. However, the mechanism by which Sr ion acts on bone material level properties remains unknown. The vertebrae of intact female rats were exposed overnight to 0.5, 1 or 2M chloride salt solutions of Sr, Ca and Ba. The latter two were used to assess the specificity of Sr. Bone material level properties were evaluated by measuring hardness, elastic modulus and working energy in a nanoindentation test. Wavelength dispersive X-ray spectroscopy provided semi-quantitative elemental analysis and mapping. Incubation with Sr rendered bone stiffer, harder and tougher. Among the divalent ions tested, Sr had the greatest effect. Sr affinity was also assessed on in vivo treated bone specimens. After in vitro exposure, the highest improvements were observed in ovariectomized rats. However, anti-osteoporotic treatments did not influence the capacity of Sr to modify bone material level properties. Our findings demonstrated that in vitro incubation with Sr selectively improved bone material level properties, which may contribute to the macroscopic increase of bone properties observed under Sr therapy., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Recent studies have shown that osteoblasts and osteoclasts express β2-adrenoceptor, and increased sympathetic nervous activity causes bone loss via an increase in osteoclastic bone resorption and a decrease in osteoblastic bone formation. We previously demonstrated that non-selective β-adrenoceptor antagonist propranolol at low doses (0.1 and 1mg/kg), but not at a higher dose (10mg/kg), prevented a decrease in bone mass and an increase in bone fragility in spontaneously hypertensive rat (SHR), an animal model of osteoporosis with hyperactivity of the sympathetic nervous system, without affecting blood pressure. In the present study, the dose effects of butoxamine, a selective β2-adrenoceptor antagonist, on bone metabolism were examined in SHR by analysis of microcomputed tomography, bone histomorphometry, biomechanical testing and plasma biochemistry. Treatment of SHR with butoxamine at 0.1, 1 and 10mg/kg (per os) for 12 weeks increased bone mass indices and biomechanical parameters of strength and toughness of the lumbar vertebrae, suggesting antiosteoporotic activity. Butoxamine dose-dependently decreased osteoclast number and surface per bone surface with decreases in plasma tartrate-resistant acid phosphatase-5b level, a biochemical index of osteoclastic activity. On the other hand, histomorphometry indices of bone formation and plasma osteocalcin concentration reflecting osteoblastic activity were increased in SHR treated with butoxamine at 0.1 and 1mg/kg, but not at 10mg/kg. These results suggest that β-adrenoceptor antagonists at a low dose may improve osteoporosis with hyperactivity of the sympathetic nervous system via β2-adrenoceptor blocking action, while they may have a somewhat inhibitory effect on osteoblastic activity at a high dose., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Purpose: To determine age-related diffusivity changes of the lumbar bone marrow by measurement of apparent diffusion coefficient (ADC) values., Materials and Methods: The local ethics committee approved this study and written informed consent was obtained. The study group comprised 88 individuals including 75 healthy volunteers and 13 patients (48 female, 40 male; mean age 36 years, range 0-84 years). The pediatric cases were recruited from patients. Echo-planar diffusion weighted imaging (DWI) was performed with b-values of 50, 400 and 800 s/mm(2). ADC-values were calculated and measured in the 1st and 2nd vertebral body of the lumbar spine. Correlation between age and ADC-values was analyzed with Spearman's rho test., Results: The ADC values of the vertebral bone marrow of the lumbar spine showed a significant negative correlation with age (rho=-0.398, p=0.001). The mean ADC values (×10(-3)mm(2)/s) in the age groups 0-29 years (mean age 18.0 years, n=42) and 30-88 years (mean age 51.6 years, n=46) were 0.54 ± 0.07 and 0.47 ± 0.08, respectively (p<0.001, T-test). No significant differences were found between children and young adults., Conclusion: Bone marrow ADC values of the lumbar spine show a linear decrease with growing age and thereby reflect the gradual changes of cell composition occurring during marrow conversion., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Study Design: Descriptive experimental study., Objective: The aim of this study was to investigate the effect of exercise on cell proliferation in different areas of the intervertebral disc (IVD) and recruitment of cells possibly active in regeneration of normal rat lumbar IVDs., Summary of Background Data: Little is known about the effects of physical exercise on lumbar IVD tissue. Recently, stem cell niches in the perichondrium area of the IVD were identified and cells in these niches have been suggested to be involved in the normal regeneration of the IVD., Methods: Thirty Sprague-Dawley rats were exposed to 5-bromo-2-deoxyuridine (BrdU) diluted in the drinking water during 14 days. Fifteen rats ran on a treadmill daily for 50 min/d, 5 d/wk (exercise group), and 15 nonexercised rats served as controls. Immunohistochemical analyses (anti-BrdU antibody) were performed at 9, 14, 28, 56, and 105 days after the start of the exercise protocol. BrdU positive cells were counted in the stem cell niche area, the peripheral region of epiphyseal cartilage area, and the annulus fibrous outer and inner areas. Data were analyzed by 2-way analysis of variance (significance level; P < 0.05)., Results: The BrdU positive cell numbers in the stem cell niche and annulus fibrous outer regions were increased in discs from the exercising group on days 14 (P < 0.01) and 105 (P < 0.05) and at day 14 (P < 0.01) in the peripheral epiphyseal cartilage region compared with controls., Conclusion: Physical exercise was shown to have positive effects on cell proliferation in IVDs, with involvement of various disc regions, indicating a differential response by disc tissue to exercise depending on anatomical location and tissue characteristics.

Objective: The aim of this study was to evaluate sequences that are established at lower magnetic field strengths for lumbar spine imaging at 7 Tesla (7 T) MR imaging., Materials and Methods: The lumbar spine of five healthy volunteers and a patient with spina bifida and meningocele were evaluated at 7 T. The examination included a T2-TSE (turbo spin echo), a 3D-DESS (double-echo steady-state sequence), a 3D-CISS (constructive interference in steady-state sequence), and a 3D-VIBE (volumetric interpolated breath hold examination) sequence. Imaging quality was evaluated by two raters on a three-level scale. The assessment included visualization of intraforaminal structures, the cauda equina, facet joints, and any abnormalities. Contrast ratios for intervertebral discs/vertebral bodies, vertebral bodies/cerebrospinal fluid (CSF) and CSF/spinal cord were calculated., Results: The 3D-VIBE sequence provided best differentiation between intraforaminal structures. Visualization of the facet joints was reliable with VIBE, DESS, and CISS. Individual nerve roots of the cauda equina could only be delineated with the 3D-CISS sequence. CISS and DESS provided good contrast between vertebral bodies and intervertebral discs. Contrast between CSF and vertebral bodies was most pronounced for the T2-TSE sequence. Sufficient contrast between CSF and the spinal cord was only achieved with the T2-TSE sequence. VIBE and DESS sequences demonstrated best the bony malformations. Visualization of the meningocele was only possible with the 3D-CISS sequence., Conclusion: At 7 T most structures of the lumbar spine were visualized with a combination of sequences. At present, imaging quality is not superior to 1.5 T or 3 T, precluding routine clinical use.

The formation of nuclear groups in the posterior horns of the human embryonic spinal cord was traced in serial sections of embryos of developmental stages 13 to 23 (32 to 56 postovulatory days). The following observations, new for the human, are presented: 1. The differentiation of the neural tube into 3 zones (germinal, mantle and marginal) is detected in the middle of the 5(th) week. 2. The primordia of the posterior horns are marked at stage 14 (33 days). 3. In the middle of the 7(th) week the nucleus proprius and substantia gelatinosa are discerned. 4. Differentiation of the nuclei within the posterior horns proceeds in the ventrodorsal and rostrocaudal gradients.

We have colocalized elastin and fibrillin-1 with perlecan in extracellular matrix of tensional and weight-bearing connective tissues. Elastin and fibrillin-1 were identified as prominent components of paraspinal blood vessels, and posterior longitudinal ligament in the human fetal spine and outer annulus fibrosus of the fetal intervertebral disc. We also colocalized perlecan with a synovial elastic basal lamina, where the attached synovial cells were observed to produce perlecan. Elastin, fibrillin-1 and perlecan were co-localized in the intima and media of small blood vessels in the synovium and in human fetal paraspinal blood vessels. Elastic fibers were observed at the insertion point of the anterior cruciate ligament to bone in the ovine stifle joint where they colocalized with perlecan. Elastin has not previously been reported to be spatially associated with perlecan in these tissues. Interactions between the tropoelastin and perlecan heparan sulfate chains were demonstrated using quartz crystal microbalance with dissipation solid phase binding studies. Electrostatic interactions through the heparan sulfate chains of perlecan and core protein mediated the interactions with tropoelastin, and were both important in the coacervation of tropoelastin and deposition of elastin onto perlecan immobilized on the chip surface. This may help us to understand the interactions which are expected to occur in vivo between the tropoelastin and perlecan to facilitate the deposition of elastin and formation of elastic microfibrils in situ and would be consistent with the observed distributions of these components in a number of connective tissues.

The goal of tissue engineering is to use substitutes to repair and restore organ function. Bioreactors are an indispensable tool for monitoring and controlling the unique environment for engineered constructs to grow. However, in order to determine the biochemical properties of engineered constructs, samples need to be destroyed. In this study, we developed a novel technique to nondestructively online-characterize the water content and fixed charge density of cartilaginous tissues. A new technique was developed to determine the tissue mechano-electrochemical properties nondestructively. Bovine knee articular cartilage and lumbar annulus fibrosus were used in this study to demonstrate that this technique could be used on different types of tissue. The results show that our newly developed method is capable of precisely predicting the water volume fraction (less than 3% disparity) and fixed charge density (less than 16.7% disparity) within cartilaginous tissues. This novel technique will help to design a new generation of bioreactors which are able to actively determine the essential properties of the engineered constructs, as well as regulate the local environment to achieve the optimal conditions for cultivating constructs.

Study Design: Vertebral growth plates at different postnatal ages were assessed for active intercellular signaling pathways., Objective: To generate a spatial and temporal map of the major signaling pathways active in the postnatal mouse lumbar vertebral growth plate., Summary of Background Data: The growth of all long bones is known to occur by cartilaginous growth plates. The growth plate is composed of layers of chondrocyets that actively proliferate, differentiate, die and, are replaced by bone. The role of major cell signaling pathways has been suggested for regulation of the fetal long bones. But not much is known about the molecular or cellular signals that control the postnatal vertebral growth plate and hence postnatal vertebral bone growth. Understanding such molecular mechanisms will help design therapeutic treatments for vertebral growth disorders such as scoliosis., Methods: Antibodies against activated downstream intermediates were used to identify cells in the growth plate responding to BMP, TGFβ, and FGF in cryosections of lumbar vertebrae from different postnatal age mice to identify the zones that were responding to these signals. Reporter mice were used to identify the chondrocytes responding to hedgehog (Ihh), and Wnt signaling., Results: We present a spatial/temporal map of these signaling pathways during growth, and differentiation of the mouse lumbar vertebral growth plate., Conclusion: During growth and differentiation of the vertebral growth plate, its different components respond at different times to different intercellular signaling ligands. Response to most of these signals is dramatically downregulated at the end of vertebral growth.

Periostin, a matricellular protein in the fasciclin family, is expressed in tissues subjected to constant mechanical stress. Periostin modulates cell-to-extracellular matrix interactions and can bind to collagen, fibronectin, tenascin-C and several integrins. Our objective was to evaluate whether periostin is expressed in the human intervertebral disc. Immunohistochemical localization of periostin was carried out in tissue of human lumbar discs and lumbar discs of the sand rat (Psammomys obesus). Human discs also were examined for periostin gene expression. Immunohistochemical localization demonstrated periostin in the cytoplasm of annulus and nucleus cells, and occasionally in the surrounding pericellular and interterritorial extracellular matrix. Periostin distribution in the human disc was distinctive. Outer annulus contained the highest proportion of periostin-positive cells (88.8%), whereas inner annulus contained only 61.4%. The nucleus pulposus contained the fewest periostin-positive cells (18.5%). There was a significant negative correlation between the percentage of cells positive for periostin in the inner annulus and subject age. Periostin gene expression in the human disc also was confirmed using molecular microarray analysis. Because work by others has shown that periostin plays an important role in the biomechanical properties of other connective tissues (skin, tendon, heart valves), future research is needed to elucidate the role of periostin in disc, loading, aging and degeneration.

Study Design: The mechanisms of apoptosis behind the formation of tissue reactions at the surface of the dorsal root ganglion (DRG) exposed to the nucleus pulposus were studied with special reference to the role of interleukin-6 (IL-6), using electron microscopy and immunohistochemistry in rats., Objective: To study the role of IL-6 on the DRG., Summary of Background Data: It has been reported that nucleus pulposus cells are capable to produce proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and IL-6. Recently, it was observed that local application of nucleus pulposus induced a characteristic tissue reaction at the surface of the DRG. This change was due to apoptosis of DRG neurons. However, the role of IL-6 is not known regarding the apoptosis of the DRG neurons., Methods: Recombinant IL-6 was applied between the L4 DRG and the dura to mimic a disc herniation of the L4-L5 disc in rats. The L4 DRGs were resected 24 hours after surgery. The sections were processed for immunohistochemistry using antisera to TNF-α. Furthermore, the sections of the specimens were observed using light and electron microscopy to confirm the induced apoptosis of the DRG neurons. The sections were also processed for immunohistochemistry, using antisera to single-stranded DNA (ssDNA) and Caspase 3., Results: TNF-α immunoreactivity was observed in the peripheral area of DRG at the site of the application of IL-6. Typical changes of the cell nuclei were observed in the DRG by light and electron microscopy, indicating the presence of apoptosis. The presence of ssDNA and Caspase 3 further enhanced the impression that there was apoptosis of the DRG neurons., Conclusion: IL-6 seemed to induce TNF-α at the surface of DRG exposed to IL-6 and to induce a characteristic reaction at the surface of the DRG. IL-6 may thus play an important role in nucleus pulposus-induced apoptosis of the DRG neurons as well as TNF-α.

Background: The bevel type and location of the distal orifice of the needle may have relevance for potential complications occurring during transforaminal epidural injection., Methods: We examined by scanning electron microscopy the structural aspects of spinal nerve root cuffs of 3 human cadavers, and 3 needle types used in transforaminal injections: 22-gauge Quincke spinal needles, 22-gauge blunt nerve block needles, and 20-gauge radiofrequency blunt needles. We made punctures in vitro in the spinal nerve root cuffs, and we studied the structures affected., Results: There is fat tissue within the nerve root with irregular distribution. In needles with a round tip, the distal orifice maintained an extraneural location after puncture of the nerve root cuff. The length of the needle required to introduce the distal orifice completely inside the nerve root cuff was variable, depending on the type: shortest for a Quincke needle (1.8 mm), 4.1 mm for the Epimed, and longest for radiofrequency needles (5.7 mm)., Conclusions: The layer of fat around nerve roots may prevent the contact of the needle tip with axons. The sharp needle tip entered the nerve root cuff more easily than the blunt tip in the cadaveric nerve root samples, measured in a qualitative manner. There is a need for clinical studies to ascertain if blunt needles may be safer than sharp needles for transforaminal injections.

Intervertebral disc degeneration is considered a major source of low back pain. We therefore examined an absorbable polyglycolic acid (PGA) biomaterial for its utility to support disc tissue regeneration. Microdiscectomy for lumbar disc herniation was performed in six patients. Intervertebral disc cells were isolated and in vitro cell expansion was accomplished using human serum and FGF2. In a fibrin-hyaluronan solution, disc cells were loaded on PGA scaffolds and cultured for 2 weeks. Formation of disc tissue was documented by histological staining of the extracellular matrix as well as gene expression analysis of typical disc marker genes. The use of human serum and FGF2 ensures efficient isolation and expansion of human disc cells. During this phase, dedifferentiation of the disc cells was observed. Subsequent 3D tissue culture of disc cells in PGA scaffolds, however, is accompanied by the induction of typical disc marker genes, resulting in tissue containing glycosaminoglycans and collagens. Propidium iodide/fluorescein diacetate (PI/FDA) staining documented that 3D assembly of disc cells in PGA scaffolds allows prolonged culture and high viability of disc cells. Disc cells from tissue of the nucleus compartment can be reliably isolated and expanded in vitro with FGF. In combination with a fibrin-hyaluronan solution and loaded on a PGA scaffold, disc cells from expansion culture commence a redifferentiation process. PGA-based scaffolds could be useful as temporal matrices for regenerative disc repair approaches., (Copyright © 2010 John Wiley & Sons, Ltd.)

Study Design: changes in cell density of endplate (EP), nucleus pulposus (NP), and anulus fibrosus (AF) during ageing were systematically investigated in defined regions of interest in complete human motion segments., Objectives: to elucidate cell density and total cell number in distinct anatomic regions of the intervertebral disc; to test effects of gender, level and age on cell density; and to correlate changes in cell density with histologic signs of disc degeneration., Summary of Background Data: the available information on the cell density within intervertebral discs and its age-related changes is sparse. This knowledge, however, is a crucial prerequisite for cell-based tissue engineering approaches of the intervertebral disc., Methods: in 49 complete cross-sections from lumbar motion segments (newborn to 86 years) from 22 specimens, cell density was determined by the Abercrombie method in EP, NP, and AF, and total cell number was counted per region of interest., Results: cell density in EP, NP, and AF decreased significantly from 0 to 16 years with the main changes occuring from 0 to 3 years for NP and AF. No significant variations were observed thereafter. We found a significant correlation of cell density and histologic degeneration score between 0 and 1, but not for scores >1. Gender and disc level did not influence cell density., Conclusion: This study provides data concerning the total number of cells in the various regions of the intervertebral disc for different age groups. This knowledge will be beneficial for cell-based treatment approaches, which may evolve in the future.

Study Design: To identify mesenchymal stromal cells (MSC) from degenerate human nucleus pulposus (NP) and compare them with bone marrow (BM) MSC., Objective: To test whether MSC obtained from NP and BM from the same subjects share similar biologic characteristics., Summary of Background Data: Recent studies have proposed biologic strategies for the treatment of intervertebral disc degeneration, including cell therapy. Bone marrow (BM) MSC could be an attractive approach to restore disc function, and there is evidence that NP may contain MSC-like cells., Methods: Tissue samples were obtained from degenerate lumbar NP and from iliac crest of the same 16 patients with degenerative disc diseases, undergoing discectomy and fusion procedures. MSC isolated from both sources were compared regarding their expansion time, immunophenotype, differentiation ability, and molecular analysis., Results: In all cases, MSC from NP were isolated and expanded. They fulfil nearly all morphological, inmunophenotypical, and differentiation criteria described by the International Society of Cell Therapy for MSC, with the exception that NP-MSC are not able to differentiate into adipocytes. Slight differences were observed with BM-MSC from the same subjects., Conclusion: The NP contains mesenchymal stem cells. These cells were quite similar to mesenchymal stem cells from BM, with the exception of their adipogenic differentiation ability. These findings suggest that we may treat intervertebral disc degeneration by cell therapy (MSC from BM) and by stimulating endogenous MSC from NP.

Objective: To determine whether intervertebral disc (IVD) cells express β-catenin and to assess the role of the WNT/β-catenin signaling pathway in cellular senescence and aggrecan synthesis., Methods: The expression of β-catenin messenger RNA (mRNA) and protein in rat IVD cells was assessed by using several real-time reverse transcription-polymerase chain reaction, Western blot, immunohistochemical, and immunofluorescence analyses. The effect of WNT/β-catenin on nucleus pulposus (NP) cells was examined by transfection experiments, an MTT assay, senescence-associated β-galactosidase staining, a cell cycle analysis, and a transforming growth factor (TGFβ)/bone morphogenetic protein (BMP) pathway-focused microarray analysis., Results: We found that β-catenin mRNA and protein were expressed in discs in vivo and that rat NP cells exhibited increased β-catenin mRNA and protein upon stimulation with lithium chloride, a known activator of WNT signaling. LiCl treatment inhibited the proliferation of NP cells in a time- and dose-dependent manner. In addition, there was an increased level of cellular senescence in LiCl-treated cells. Long-term treatment with LiCl induced cell cycle arrest and promoted subsequent apoptosis in NP cells. Activation of WNT/β-catenin signaling also regulated the expression of aggrecan. We also demonstrated that WNT/β-catenin signaling induced the expression of matrix metalloproteinases (MMPs) and TGFβ in NP cells., Conclusion: The activation of WNT/β-catenin signaling promotes cellular senescence and may modulate MMP and TGFβ signaling in NP cells. We hypothesize that the activation of WNT/β-catenin signaling may lead to an increased breakdown of the matrix, thereby promoting IVD degeneration.

Background: ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG) tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg) or drug vehicle twice weekly for 8 weeks., Results: In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H). High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals., Conclusions: In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within rat DRG tissue may be required to support the potentially differing cuproenzyme requirements of distinct subsets of sensory neurons, and could influence the transport and neurotoxicity of oxaliplatin.

Background Context: Spinal fusion is facilitated when the fusion site is augmented with autograft bone. Iliac crest, long the preferred source of autograft material, is the site of frequent complications and pain. Connective tissue progenitor cells (CTPs) aspirated from marrow provide a promising alternative to traditional autograft harvest. The vertebral body represents an even larger potential reservoir of progenitor cells than the ilium., Purpose: To test the hypothesis that a suitable concentration of osteoprogenitor cells can be aspirated from different depths of the vertebral body, maintaining progenitor cell concentrations comparable to the "gold standard," the iliac crest, even after sequential aspirations along the same transpedicular axis., Study Design: Prospective clinical investigation quantifying CTP concentrations within the vertebral body relative to depth of sequential aspirations., Patient Sample: Adult men and women undergoing elective posterior lumbar fusion and pedicle screw instrumentation (six men and seven women, mean age 56 years [range 40-74 years])., Outcome Measures: Cell count, CTP concentration (CTPs/cc marrow), and CTP prevalence (CTPs/million cells) were calculated for both individual and pooled aspirate samples., Methods: Thirteen patients were enrolled into an institutional review board-approved protocol studying transpedicular aspiration of marrow progenitor cells. Connective tissue progenitor cells were aspirated from four depths along the transpedicular axis of the vertebral body and quantified according to cell concentration and CTP prevalence. Histochemical analysis of alkaline phosphatase-positive colony-forming units (CFUs) provided the prevalence of vertebral CTPs relative to depth of aspiration, vertebral level, age, and gender., Results: Four 2.0cc aspirations were obtained from each pedicle of lumbar vertebrae selected for pedicle screw fixation (four 2.0cc aspirates from each of four pedicles). Aspirates of vertebral marrow demonstrated comparable or greater concentrations of CFUs compared with standards previously established for the iliac crest. Overall, the 208 aspirations from 26 vertebral bodies provided a mean CTP concentration of 741.5+/-976.2 CTPs per cubic centimeter of marrow, ranging from a mean concentration of 1316+/-1473 CTPs per cubic centimeter of marrow at superficial (30mm) aspirations to 439+/-557 CTPs per cubic centimeter marrow at deepest (45mm) aspiration depths (p<.00002). There were no significant differences relative to vertebral body level, side aspirated, or gender. An age-related decline in cellularity was suggested for vertebral body aspirates., Conclusions: The vertebral body is a potential marrow reservoir for aspiration of autograft osteogenic CTPs that can be used to augment spinal fusion. The cancellous bone within that portion of the vertebral body routinely cannulated during pedicle screw placement allows serial aspirations with only modest depletion of progenitor cell concentrations or dilution with peripheral blood. Connective tissue progenitor cell concentrations from all depths were comparable to the mean levels previously established for the iliac crest. The ability to simultaneously harvest progenitor cells for graft augmentation while preparing the pilot hole for pedicle screw fixation will expand the potential for cell harvest techniques for fusion augmentation and reduce the need for iliac crest harvest.

This report evaluates the spatial profile of blood vessel fragments (BVFs) and CD34(+) and CD117(+) hematopoietic stem and progenitor cells (HSPCs) in human cancellous bone. Bone specimens were sectioned, immunostained (anti-CD34 and anti-CD117), and digitally imaged. Immunoreactive cells and vessels were then optically and morphometrically identified and labeled on the corresponding digital image. The distance of each BVF, or CD34(+) or CD117(+) HSPC to the nearest trabecular surface was measured and binned in 50-microm increments. The relative concentration of HSPCs and BVFs within cancellous marrow was observed to diminish with increasing distance in the marrow space. On average, 50% of the CD34(+) HSPC population, 60% of the CD117(+) HSPC population, and 72% of the BVFs were found within 100 microm of the bone surfaces. HSPCs were also found to exist in close proximity to BVFs, which supports the notion of a shared HSPC and vessel spatial niche.

Study Design: Microarray gene expression profiling, quantitative gene expression analysis, and immunohistochemistry was used to investigate molecular variations between nucleus pulposus (NP) and anulus fibrosus (AF) of the dog intervertebral disc (IVD)., Objective: To identify specific molecules with differing expression patterns in NP and AF and compare their profile with articular cartilage (AC)., Summary of Background Data: Although experimental and animal studies have demonstrated the potential of cell based approaches for NP regeneration, there is still a deficiency of basic knowledge about the phenotype of IVD cells., Methods: Comparative microarray analysis of beagle lumbar NP and AF was performed. Molecules of interest were evaluated by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, comparing lumbar and coccygeal NP and AF and AC. To assess interspecies variations, genes that had been found differentially expressed in rat tissues were also investigated., Results: Forty-five genes with NP/AF signal log ratio > or = 1 were identified. Alpha-2-macroglobulin, cytokeratin-18, and neural cell adhesion molecule (CD56) mRNA were higher in NP compared to AF and AC, and desmocollin-2 mRNA was higher in NP than AF. The expression profiles were similar in lumbar and coccygeal discs, although certain variations were noticed. Interspecies differences between rat and dog were evident in the expression of several genes. Immunohistochemistry confirmed differences in gene expression at the protein level., Conclusion: This study reports on the expression of molecules that have not been described previously in IVD, in non-notochordal discs comparable with human. Interspecies differences were noted between rat and dog tissues, whereas variations between caudal and lumbar discs were less prominent. The NP of the beagle as a chondrodystrophoid dog breed is potentially more similar to the human than the NP of species whose discs do not naturally degenerate. Therefore, studies on appropriate species may contribute to a better understanding of the cell types residing in the IVD.

Object: Tissue engineering appears to be a promising strategy for articular cartilage regeneration as a treatment for facet joint arthritis. Prior to the commencement of tissue engineering approaches, design criteria must be established to determine the required functional properties of the replacement tissue. As characterization of the functional properties of facet joint cartilage has not been performed previously, the objective of this study was to determine the biomechanical, biochemical, and histological properties of facet joint cartilage., Methods: The in vitro testing was conducted using 4 lumbar spinal segments obtained from skeletally mature canines. In each specimen, articular cartilage was obtained from the superior surface of the L3-4 and L4-5 facet joints. Creep indentation was used to determine the compressive biomechanical properties, while uniaxial tensile testing yielded the Young modulus and ultimate tensile strength of the tissue. Additionally, biochemical assessments included determinations of cellularity, glycosaminoglycan (GAG) content, and collagen content, as well as enzymelinked immunosorbent assays for collagen I and II production. Finally, histological characterization included H & E staining, as well as staining for collagen and GAG distributions., Results: The means +/- standard deviation values were determined. There were no differences between the 2 spinal levels for any of the assessed properties. Averaged over both levels, the thickness was 0.49 +/- 0.10 mm and the hydration was 74.7 +/- 1.7%. Additionally, the cells/wet weight (WW) ratio was 6.26 +/- 2.66 x 10(4) cells/mg and the cells/dry weight (DW) ratio was 2.51 +/- 1.21 x 10(5) cells/mg. The GAG/WW was 0.038 +/- 0.013 and the GAG/ DW was 0.149 +/- 0.049 mg/mg, while the collagen/WW was 0.168 +/- 0.026 and collagen/DW was 0.681 +/- 0.154 mg/ mg. Finally, the aggregate modulus was 554 +/- 133 kPa, the Young modulus was 10.08 +/- 8.07 MPa, and the ultimate tensile strength was 4.44 +/- 2.40 MPa., Conclusions: To the best of the authors' knowledge, this study is the first to provide a functional characterization of facet joint articular cartilage, thus providing design criteria for future tissue engineering studies.

Study Design: Histologic experiments and biomechanical tests were performed in human cadaveric lumbar spine models., Objective: To determine (1) the anatomic structures of lumbar endplates, (2) the relationship between bone mineral density (BMD) and biomechanical properties of lumbar endplates, and (3) the influence of spinal level on the failure loads of lumbar endplates., Summary of Background Data: Previous works have shown that the posterolateral corners of the lumbar endplates are stronger than the anterior and central regions. Information on the microstructures of lumbar endplates and the effects of both BMD and spinal level on biomechanical properties of lumbar endplates would be valuable for spine surgeons and implant designers to avoid subsidence., Methods: Twenty fresh human cadaver lumbar vertebrae specimens were collected for HE staining and the sections through the specimens were examined under light microscope. The areas of the pore structures in the lumbar endplates were measured and analyzed by statistical methods. Sixty-five cadaver lumbar vertebrae were evaluated with dual energy radiograph absorptiometry and according to their BMD, all vertebrae were divided into 3 groups. Indentation tests were performed at 27 standardized test sites in endplates of these vertebrae using a 1.5-mm-diameter, hemispherical indenter with a rate of 12 mm/min. The failure load at each test site was determined using the load-displacement curve. Spearman analysis was used to evaluate the correlation between BMD and failure loads of lumbar endplates. Factorial analysis of variance was performed to reveal the effects of both BMD and spinal level on failure load distribution on the lumbar endplates., Results: (1) The peripheral regions were thicker than the central regions of lumbar endplates. The central regions were porous, as the fused trabeculae and peripheral regions had fewer and smaller pore structures. (2) The tests showed that there were significant differences in failure load between 3 groups (P < 0.01). BMD was positively correlated with the failure load of the lumbar endplate. (3) The strongest position of the lumbar endplate was the posterolateral region closest to the pedicles and the failure loads tended to increase from anterior to posterior regions of the lumbar endplates. (4) The failure load distribution did not change with the BMD decrease. (5) The differences of the failure loads between the lumbar segments were significant (P < 0.05). The failure loads of the lumbar endplates showed an increasing tendency from L1-L5 segments., Conclusion: The differences in the anatomic structures of different regions are the histologic foundation of biomechanical properties of lumbar endplates. It is necessary that BMD should be measured before operation and implants should be placed in posterior-lateral regions of lumbar endplates so that subsidence syndrome can be reduced as much as possible. In addition, the fact that the endplates of the upper lumbar segments with the lower strength have higher risk of subsidence should be concerned about by spine surgeons and implant designers.

We investigated histological and compressive properties of a calcium phosphate bone cement (BoneSource (CPC); Stryker Orthopaedics, Mahwah, New Jersey) plus carboxymethyl cellulose (CMC) using a sheep vertebral bone void model. Bone voids were surgically created in L3 and L5 in each of 40 sheep, and the voids were filled with the cement. Histological and radiographic evaluations were performed on one vertebral body from each animal at either: 0, 3, 6, 12, 24, or 36 months after surgery; mechanical testing was performed on operated and non-operated vertebral bodies from 35 sheep. Undecalcified sections were digitized, and the area of the original defect, new bone formation, empty space, fibrous tissue, and residual cement were quantified with histomorphometry. Decalcified sections were evaluated qualitatively. The cement was biocompatible, extremely osteoconductive and underwent steady resorption and replacement by bone and bone marrow. Histomorphometry showed variations in the rate of cement remodeling among animals in each time group, but on average, at 36 months the original defect area was occupied by approximately 14% bone, 82% cement, and 4% bone marrow. Even in animals that had greater resorption of cement, there was good bone ingrowth with no fibrous tissue. Compressive testing did not reveal a significant difference in the mechanical properties between vertebral bodies augmented with cement and non-augmented controls, irrespective of the postoperative time. BoneSource mixed with CMC had adequate osteoconductivity, biocompatibility, and adequate compressive strength. There was variability among animals, but histology suggests that considerable cement was still present in most samples after 36 months.

We tested the hypothesis that the dose of recombinant human bone morphogenetic protein-2 (rhBMP-2) required to induce spine fusion can be reduced by combination with mesenchymal stem cells (MSCs). Twenty-four adult rabbits underwent posterolateral intertransverse fusion at the L4-L5 level. The animals were divided into four groups based on the implant material: autologous iliac graft, Alginate-MSCs composite, Alginate-BMP-2-MSCs composite, and Alginate-BMP-2 composite. After 16 weeks, the rabbits were euthanized for radiographic examination, manual palpation, biomechanical testing, and histology. Radiographic union of 12 intertransverse fusion areas for the autogenous iliac graft, Alginate-MSCs, Alginate-BMP-2-MSCs, and Alginate-BMP-2 groups was 11, 8, 11, and 0, respectively. Moreover, manual palpation of six fusion segments in each subgroup found solid union to be 6, 1, 5, and 0, respectively. The average torques at failure of the first three groups were 2278 +/- 135, 1943 +/- 140, and 2334 +/- 187 N-mm, respectively. The failure torque did not differ significantly between the autograft and Alginate-BMP-2-MSCs groups; both groups were significantly higher than the Alginate-MSCs group. The results indicate that MSCs delivered with in vitro cellular doses of rhBMP-2 are more osteoinductive than MSCs without rhBMP-2. In combination with MSCs, a low dose (2.5 microg) of rh-BMP-2 could enhance bone formation and posterolateral spine fusion success in the rabbit model., ((c) 2008 Orthopaedic Research Society.)

Study Design: Intervertebral discs at different postnatal ages were assessed for active intercellular signaling pathways., Objective: To generate a spatial and temporal map of the signaling pathways active in the postnatal intervertebral disc (IVD)., Summary of Background Data: The postnatal IVD is a complex structure, consisting of 3 histologically distinct components, the nucleus pulposus, fibrous anulus fibrosus, and endplate. These differentiate and grow during the first 9 weeks of age in the mouse. Identification of the major signaling pathways active during and after the growth and differentiation period will allow functional analysis using mouse genetics and identify targets for therapy for individual components of the disc., Methods: Antibodies specific for individual cell signaling pathways were used on cryostat sections of IVD at different postnatal ages to identify which components of the IVD were responding to major classes of intercellular signal, including sonic hedgehog, Wnt, TGFbeta, FGF, and BMPs., Results: We present a spatial/temporal map of these signaling pathways during growth, differentiation, and aging of the disc., Conclusion: During growth and differentiation of the disc, its different components respond at different times to different intercellular signaling ligands. Most of these are dramatically downregulated at the end of disc growth.

Study Design: This study follows postnatal intervertebral disc (IVD) growth and differentiation in the mouse. OBJECTIVE.: To initiate use of the mouse as a model system for postnatal IVD differentiation and growth, and to serve as a basis for assaying changes caused by disease or genetic or experimental perturbation., Summary of Background Data: Lower back pain caused by disc degeneration is one of the most common clinical conditions worldwide. There is currently no completely effective treatment, largely because of a lack of basic knowledge of the molecular and cellular controls of disc growth, differentiation, and maintenance after birth., Methods: Conventional histology of decalcified IVDs, differential interference contrast, polarizing optics, immunocytochemistry, laser capture microscopy followed by molecular analysis of the dissected cells by reverse transcriptase polymerase chain reaction., Results: There is a single postnatal growth spurt in the mouse IVD, between birth and 9 weeks of age. Cell proliferation was found in the nucleus pulposus (NP) and anulus fibrosus (AF) only until 3 weeks of age. Most of the postnatal growth of the IVD is due to accumulating extracellular matrix. NP cell numbers decline steadily after 2 weeks of age, because of apoptosis. Laser capture microscopy was used to dissect NP cells from the disc, and showed that these cells express markers of the embryonic notochord. The postnatal AF appears initially as a continuous structure surrounding the NP. This structure differentiates, during the first 2 postnatal weeks, to form the mineralized, but nonossified endplate over the surfaces of the vertebral growth plates, and the mature fibrous AF (fAF) passing between adjacent vertebrae. The fact that the mature fAF and the endplate form from an originally continuous layer of cells explains the anatomic relationship between these 2 structures, in which the fAF inserts into the vertebral endplate., Conclusion: Growth of the IVD takes place during the first 9 postnatal weeks, although cell proliferation ceases after 3 weeks. After birth, the early postnatal IVD differentiates into 3 tissue types, the NP, the fAF between the vertebrae, and the mineralized endplates over the surfaces of the vertebrae.

Prostaglandin E2 receptor subtype 4 agonists (EP4A) and basic fibroblast growth factor (FGF2) stimulate bone formation, but their effects on bone resorption are controversial. To provide additional insight into the skeletal effects of EP4A and FGF2, their regulation of expression of genes associated with bone formation and resorption in aged ovariectomized (OVX) rats and in cultured mouse bone marrow cells was determined. RNA was isolated from lumbar vertebrae of OVX rats (16 months of age) treated daily for 3 weeks with FGF2 or EP4A and processed for quantitative real time-PCR analyses. mRNA expression for the receptor activator of NF-kappaB ligand (RANKL) and cathepsin K (CTSK), but not osteoprotegerin (OPG), were upregulated by both FGF2 and EP4A. Addition of FGF2 and EP4A to the medium of cultured mouse bone marrow cells increased the formation of tartrate resistant acid phosphatase (TRAP) positive cells, upregulated the expression of RANKL and CTSK, and downregulated expression for OPG. EP4A also increased the formation of actin rings, an indicator of osteoclast activation, in a dose dependent manner in osteoclasts cultured on bone slices and triggered the formation of pits as revealed by a pitting assay. Gene expression for osterix (OSX) and IGF-2, genes associated with bone formation, was significantly greater in FGF2-treated OVX rats compared with EP4A-treated OVX rats. These findings at the molecular level are consistent with previous tissue-level histomorphometric findings, and at the doses tested, support the contention that FGF2 has a stronger bone anabolic effect than EP4A. The results of these in vivo and in vitro analyses clarify the effects of FGF2 and EP4A on bone formation and resorption, and provide insight into differences in the efficacy of two potential bone anabolic agents for restoration of lost bone mass in the osteopenic, estrogen-deplete skeleton.

Study Design: Mechanically induced annular disruption of lumbar intervertebral discs followed by microstructural investigation., Objective: To investigate the role that elevated nuclear pressures play in disrupting the lumbar intervertebral disc's annulus fibrosus., Summary of Background Data: Compound mechanical loadings have been used to recreate clinically relevant annular disruptions in vitro. However, the role that individual loading parameters play in disrupting the lumbar disc's annulus remains unclear., Methods: The nuclei of ovine lumbar intervertebral discs were gradually pressurized by injecting a viscous radio-opaque gel via their inferior vertebrae. Pressurization was conducted until catastrophic failure of the disc occurred. Investigation of the resulting annular disruption was carried out using microcomputed tomography and differential interference contrast microscopy., Results: Gel extrusion from the posterior annulus was the most common mode of disc failure. Unlike other aspects of the annular wall, the posterior region was unable to distribute hydrostatic pressures circumferentially. In each extrusion case, severe disruption of the posterior annulus occurred. Although intralamellar disruption occurred in the mid annulus, interlamellar disruption occurred in the outer posterior annulus. Radial ruptures between lamellae always occurred in the mid-axial plane., Conclusion: With respect to the annular wall, the posterior region is most susceptible to failure in the presence of high nuclear pressure, even when loaded in the neutral position. Weak interlamellar cohesion of the outer posterior lamellae may explain why the majority of herniations remain contained as protrusions within the outer annular wall.

Study Design: Integrating theoretical and experimental approaches for annulus fibrosus (AF) functional tissue engineering., Objective: Apply a hyperelastic constitutive model to characterize the evolution of engineered AF via scalar model parameters. Validate the model and predict the response of engineered constructs to physiologic loading scenarios., Summary of Background Data: There is need for a tissue engineered replacement for degenerate AF. When evaluating engineered replacements for load-bearing tissues, it is necessary to evaluate mechanical function with respect to the native tissue, including nonlinearity and anisotropy., Methods: Aligned nanofibrous poly-epsilon-caprolactone scaffolds with prescribed fiber angles were seeded with bovine AF cells and analyzed over 8 weeks, using experimental (mechanical testing, biochemistry, histology) and theoretical methods (a hyperelastic fiber-reinforced constitutive model)., Results: The linear region modulus for phi = 0 degrees constructs increased by approximately 25 MPa, and for phi = 90 degrees by approximately 2 MPa from 1 day to 8 weeks in culture. Infiltration and proliferation of AF cells into the scaffold and abundant deposition of s-GAG and aligned collagen was observed. The constitutive model had excellent fits to experimental data to yield matrix and fiber parameters that increased with time in culture. Correlations were observed between biochemical measures and model parameters. The model was successfully validated and used to simulate time-varying responses of engineered AF under shear and biaxial loading., Conclusion: AF cells seeded on nanofibrous scaffolds elaborated an organized, anisotropic AF-like extracellular matrix, resulting in improved mechanical properties. A hyperelastic fiber-reinforced constitutive model characterized the functional evolution of engineered AF constructs, and was used to simulate physiologically relevant loading configurations. Model predictions demonstrated that fibers resist shear even when the shearing direction does not coincide with the fiber direction. Further, the model suggested that the native AF fiber architecture is uniquely designed to support shear stresses encountered under multiple loading configurations.

Study Design: Microstructural investigation of interlamellar connectivity., Objective: To reveal the macro and micro structure of the translamellar bridging network in the lumbar annulus., Summary of Background Data: Contrary to the view that there is minimal interconnection between lamellar sheets, experimental data reveal a significant contribution to the material behavior of the annulus from interactions between fiber populations of alternating lamellae. Recent microstructural studies indicate a localized rather than a homogeneous or dispersed mode of interconnectivity between lamellae., Methods: Anterior segments of ovine lumbar discs in 2 age groups were sectioned along the oblique fiber angle. A 3-dimensional picture of the translamellar bridging network is developed using structural information obtained from fully hydrated unstained serial sections imaged by differential interference contrast optics., Results: A high level of connectivity between apparently disparate bridging elements was revealed. The extended form of the bridging network is that of occasional substantial radial connections spanning many lamellae with a subsidiary fine branching network. The fibrous bridging network is highly integrated with the lamellae architecture via a collagen-based system of interconnectivity., Conclusion: This study demonstrates a far greater complexity to the interlamellar architecture of the disc annulus than has previously been recognized. Our findings are clearly relevant to disc biomechanics. Significant degrading of the translamellar bridging network may result in annular weakening leading potentially to disc failure. Most importantly this work opens the way to a much clearer understanding of the microanatomy of the disc wall.