Cartilaginous Endplate Research Articles

Comparative immunolocalisation of perlecan, heparan sulphate, fibroblast growth factor-18, and fibroblast growth factor receptor-3 and their prospective roles in chondrogenic and osteogenic development of the human foetal spine

A comparative immunolocalisation study of perlecan, HS, FGF-18 and FGFR-3 in the 12-20-week gestational age human foetal spine was undertaken to identify spatiotemporal associations between these components to provide insights into prospective roles in spinal development. Comparative immunolocalisations of matrix and cell associated components in Histochoice-fixed paraffin-embedded human foetal spinal tissues. The 12-14-week-old human foetal spine was a predominantly cartilaginous structure with the discs displaying a relative paucity of proteoglycan compared to the adjacent cartilaginous vertebral rudiments, notochordal remnants were also observed. HS and perlecan had a widespread distribution throughout the spine at 12 weeks, however, FGF-18 was only localised to the outer AF margins and hypertrophic cell condensations in the vertebral bodies. This contrasted with HS distributions at 14-20 weeks, which were prominent in the developing intervertebral disc (IVD). Ossification centres were also evident centrally within the vertebral rudiments surrounded by small columns of hypertrophic chondrocytes which expressed FGFR-3 and FGF-18 and upregulated levels of perlecan. FGF-18 also had a prominent localisation pattern in the developing IVD and the cartilaginous endplate while FGFR-3 was expressed throughout the disc interspace. This suggested roles for perlecan, FGF-18 and FGFR-3 in chondrogenic and osteogenic events which drive discal development and ossification of the vertebral bodies. The above data supported a role for FGF-18 in discal development and in the terminal osteogenic differentiation of chondroprogenitor cell populations, which promote vertebral ossification during spinal development.

Back pain during growth

It is wrong to believe that back pain only burdens adults: the yearly incidence during growth ranges from 10-20%, continuously increasing from childhood to adolescence. Rapid growth-related muscular dysbalance and insufficiency, poor physical condition in an increasingly sedentary adolescent community or - vice versa - high level sports activities, account for the most prevalent functional pain syndromes. In contrast to adults the correlation of radiographic findings with pain is high: the younger the patient, the higher the probability to establish a rare morphologic cause such as benign or malignant tumours, congenital malformations and infections. In children younger than 5 years old, the likelihood is more than 50%. The following red flags should lower the threshold for a quick in-depth analysis of the problem: Age of the patient <5 years, acute trauma, functional limitation for daily activities, irradiating pain, loss of weight, duration >4 weeks, history of tumour, exposition to tuberculosis, night pain and fever. High level sport equals a biomechanical field test which reveals the biologic individual response of the growing spine to the sports-related forces. Symptomatic or asymptomatic inhibitory or stimulatory growth disturbances like Scheuermann disease, scoliosis or fatigue fractures represent the most frequent pathomorphologies. They usually occur at the disk-growth plate compound: intraspongious disk herniation, diminuition of anterior growth with vertebral wedging and apophyseal ring fractures often occur when the biomechanical impacts exceed the mechanical resistance of the cartilaginous endplates. Spondylolysis is a benign condition which rarely becomes symptomatic and responds well to conservative measures. Associated slippage of L5 on S1 is frequent but rarely progresses. The pubertal spinal growth spurt is the main risk factor for further slippage, whereas sports activity - even at a high level - is not. Therefore, the athlete should only be precluded from training if pain persists or in case of high grade slips. Perturbance of the sagittal profile with increase of lumbar lordosis, flattening of the thoracic spine and retroflexion of the pelvis with hamstrings contractures are strong signs for a grade IV olisthesis or spondyloptosis with subsequent lumbosacral kyphosis. Idiopathic scoliosis is not related to pain unless it is a marked (thoraco-) lumbar curve or if there is an underlying spinal cord pathology. Chronic back pain is an under recognised entity characterised by its duration (>3 months or recurrence within 3 months) and its social impacts such as isolation and absence from school or work. It represents an independent disease, uncoupled from any initial trigger. Multimodal therapeutic strategies are more successful than isolated, somatising orthopaedic treatment. Primary and secondary preventive active measures for the physically passive adolescents, regular sports medical check-up's for the young high level athletes, the awareness for the rare but potentially disastrous pathologies and the recognition of chronic pain syndromes are the cornerstones for successful treatment of back pain during growth.

Historical Schmorls Node: Time to Differentiation and Renaming (A Mini- Review)

“Schmorl’s nodes” (SN) are lytic lesions at endplates which are believed to be due to the herniation of the nucleus pulposus through the cartilaginous endplate into the body of a vertebral endplates. Our observations disclose the fact that all Schmorl’s nodes are not single entities and a subset of SNs seems to be a discrete clinical condition with similar (but not identical) imaging manifestations. They share many features of seronegative spondyloarthritis. We called them “Owlia’s nodes”.

Morphology of the Cartilaginous Endplates in Human Intervertebral Disks with Ultrashort Echo Time MR Imaging

To image human disk-bone specimens by using conventional spin-echo (SE) and ultrashort echo time (TE) techniques, to describe the morphology at magnetic resonance (MR) imaging, and to identify tissue components contributing to high signal intensity near the cartilaginous endplates (CEPs). This study was exempt from institutional review board approval, and informed consent was not required. Five cadaveric lumbar spines (mean age, 61 years ± 11) were prepared into six sample types containing different combinations of disk, uncalcified CEP, calcified CEP, and subchondral bone components and were imaged with proton density-weighted SE (repetition time msec/TE msec, 2000/15) and ultrashort TE (300/0.008, 6.6, echo-subtraction) sequences. Images were evaluated to determine the presence of intermediate-to-high signal intensity in regions excluding the bone marrow. Logistic regression was used to determine which tissue components were significant predictors of the presence of signal intensity for each MR technique. On ultrashort TE MR images, intact disk/uncalcified CEP/calcified CEP/bone samples exhibited bilaminar intermediate-to-high signal intensity in the region near the CEP, consistent with the histologic appearance of uncalcified and calcified CEPs. Conversely, proton density-weighted SE images exhibited low signal intensity in this region. Results of logistic regression suggested that the presence of uncalcified CEP (P = .023) and calcified CEP (P = .007) in the sample were strong predictors of the presence of signal intensity on ultrashort TE images, whereas the disk was the only predictor (P < .001) of signal intensity on proton density-weighted SE images. Ultrashort TE imaging, unlike proton density-weighted SE imaging, enabled direct visualization of the uncalcified and calcified CEP. Evaluation of the morphology and identification of sources of signal intensity at ultrashort TE MR imaging provides opportunities to potentially aid in the understanding of degenerative disk disease.

Comparative immunolocalisation of fibrillin-1 and perlecan in the human foetal, and HS-deficient hspg2 exon 3 null mutant mouse intervertebral disc

The aim of this study was to examine the comparative localisations of fibrillin-1 and perlecan in the foetal human, wild-type C57BL/6 and HS-deficient hspg2Δ³⁻/Δ³⁻ exon 3 null mouse intervertebral disc (IVD) using fluorescent laser scanning confocal microscopy. Fibrillin-1 fibrils were prominent components of the outer posterior and anterior annulus fibrosus (AF) of the foetal human IVD. Finer fibrillin-1 fibrils were evident in the inner AF where they displayed an arcade-type arrangement in the developing lamellae. Relatively short but distinct fibrillin-1 fibrils were evident in the central region of the IVD and presumptive cartilaginous endplate and defined the margins of the nuclear sheath in the developing nucleus pulposus (NP). Fibrillin-1 was also demonstrated in the AF of C57BL/6 wild-type mice but to a far lesser extent in the HS-deficient hspg2Δ³⁻/Δ³⁻ exon 3 null mouse. This suggested that the HS chains of perlecan may have contributed to fibrillin-1 assembly or its deposition in the IVD. The cell-matrix interconnections provided by the fibrillin fibrils visualised in this study may facilitate communication between disc cells and their local biomechanical microenvironment in mechanosensory processes which regulate tissue homeostasis. The ability of fibrillin-1 to sequester TGF-β a well-known anabolic growth factor in the IVD also suggests potential roles in disc development and/or remodelling.

Nanoindentation of intervertebral disc tissues localised by SHG imaging

The research on mechanical properties of cartilaginous endplate (EP) seems to be more and more actual at present. EP is a layer of hyaline cartilage between the intervertebral d c (IVD) and vertebr...

Characterization of the annulus fibrosus–vertebral body interface: identification of new structural features

Current surgical treatments for degenerative intervertebral disc disease do not restore full normal spinal movement. Tissue engineering a functional disc replacement may be one way to circumvent this limitation, but will require an integration of the different tissues making up the disc for this approach to be successful. Hence, an in-depth characterization of the native tissue interfaces, including annulus insertion into bone is necessary, as knowledge of this interface is limited. The objective of this study was to characterize the annulus fibrosus-vertebral bone (AF-VB) interface in immature (6-9 months old) and mature (18-24 months old) bovine discs, as well as to define these structures for normal adult human (22 and 45 years old) discs. Histological assessment showed that collagen fibers in the inner annulus, which are predominantly type II collagen, all appear to insert into the mineralized endplate zone. In contrast, some of the collagen fibers of the outer annulus, predominantly type I collagen, insert into this endplate, while other fibers curve laterally, at an ∼ 90° angle, to the outer aspect of the bone, and merge with the periosteum. This is seen in both human and bovine discs. Where the AF inserts into the calcified zone of the AF-VB interface, it passes through a chondroid region, rich in type II collagen and proteoglycans. Annulus cells (elongated cells that are not surrounded by proteoglycans) are present at this interface. This cartilage zone is evident in both human and bovine discs. Type X collagen and alkaline phosphatase are localized to the interface region. Age-associated differences in bovine spines are observed when examining the interface thickness and the matrix composition of the cartilaginous endplate, as well as the thickness of the mineralized endplate. These findings will assist with the design of the AF-VB interface in the tissue engineered disc.

Diversity of intervertebral disc cells: phenotype and function

The intervertebral disc (IVD) is a moderately moving joint that is located between the bony vertebrae and provides flexibility and load transmission throughout the spinal column. The disc is composed of different but interrelated tissues, including the central highly hydrated nucleus pulposus (NP), the surrounding elastic and fibrous annulus fibrosus (AF), and the cartilaginous endplate (CEP), which provides the connection to the vertebral bodies. Each of these tissues has a different function and consists of a specific matrix structure that is maintained by a cell population with distinct phenotype. Although the healthy IVD is able to balance the slow matrix turnover of synthesis and degradation, this balance is often disturbed, leading to degenerative disorders. Successful therapeutic management of IVD degeneration requires a profound understanding of the cellular and molecular characteristics of the functional IVD. Hence, the phenotype of IVD cells has been of significant interest from multiple perspectives, including development, growth, remodelling, degeneration and repair. One major challenge that complicates our understanding of the disc cells is that both the cellular phenotype and the extracellular matrix strongly depend on disc maturity and health and as a consequence are continuously evolving. This review delineates the diversity of the cell types found in the intervertebral disc, with emphasis on human, but with reference to other species. The cells of the NP appear rounded and express a proteoglycan-rich matrix, whereas the more elongated AF cells are embedded in a collagen fibre matrix and the CEPs represent a layer of cartilage. Even though all disc cells have often been referred to as 'intervertebral disc chondrocytes', distinct phenotypical differences in comparison with articular chondrocytes exist and have been reported recently. The availability of more specific markers has also improved our understanding of progenitor cell differentiation towards an IVD cell phenotype. Ultimately, new cell- and tissue-engineering approaches to regenerative therapies will only be successful if the specific characteristics of the individual tissues and their context in the function of the whole organ, are taken into consideration.

Quantification of Intervertebral Disk Cartilaginous Endplate Morphology using MRI

Introduction The IVD cartilaginous endplate (CEP) is a 450 mm to 800 mm thick layer of hyaline cartilage,2 which functions as a mechanical barrier between the pressurized nucleus pulposus and the vertebral endplate, and as a gateway for nutrient diffusion to the avascular disk.1–3 With age and degeneration, the CEP becomes thinner, calcifies, and may have reduced porosity,4 and it has been implicated as a contributor to degeneration.1–3 As the CEP is not well imaged on MRI, key geometric parameters of the CEP have not been obtained noninvasively. Thus, the objective of this study was to measure geometric properties of the CEP with MRI. Materials and Methods Lumbar motion segments ( n = 24), from 11 human cadavers (age 64.9 ± 10.0 years) were imaged on a Siemens 7T MRI scanner using a custom RF coil. Disk degeneration was graded via mid-sagittal T2 mapping. Full volume imaging of each disk at 200 mm isotropic resolution was achieved via 3D FLASH (TR/TE = 9/3.7 ms) in 6 minutes (Fig. 1). Geometric parameters (area, circumference, anterior-posterior width, lateral width) were measured in the axial and mid-sagittal planes for both the CEP and the entire disk using OsiriX software. Disk height was measured using custom-written Matlab code.5 Evaluation of CEP thickness was done on a mid-sagittal image of each disc. Semiautomated image analysis was conducted using custom-written Matlab code in which signal intensity profiles across the CEP thickness were captured at the center of the CEP and at ± ¼ and ± ¾ of its A-P length. Comparisons between superior and inferior CEP geometry were established using paired, two-tailed t-tests ( p &lt; 0.05). Data, except thickness, was normalized by dividing CEP geometry by its corresponding disk geometry to account for differences in disk sizes between levels. A one-way ANOVA was used to evaluate differences between disk level and CEP geometry. CEP thickness measurements were evaluated using a two-way ANOVA with repeated measures, where the factors were disk level and anterior-posterior disk location. A posthoc Bonferonni test was performed when significance was detected. Linear regression analysis was performed to compare CEP geometry with T2 mapping values and subject age. Results Directly measured whole disk and CEP geometry for each level are provided in the Table. The mean CEP thickness in the mid-sagittal plane across all disk levels and locations was 0.50 ± 0.20 mm. No significant differences were found in CEP thickness with respect to the superior or inferior endplate or disk level ( p &gt; 0.2). There was no significant difference in CEP A-P width, lateral width, or circumference area with respect to superior/inferior location ( p &gt; 0.7) or disk level ( p &gt; 0.6) except between L1-L2 and L4-L5 ( p &lt; 0.05) (Fig. 2A-C). L1-L2 A-P width, lateral width, and circumference area were larger than in L4-L5 by 14, 18, and 28%, respectively. CEP thickness varied significantly with respect to anterior-posterior location for every level ( p &lt; 0.005) (Fig. 2D). The minimum thickness was at the center of the disk (superior = 0.39 ± 0.10 mm, inferior = 0.38 ± 0.11 mm). The thickness at the center was 19.9 and 42.4% less than that of the ± ¼ and ± ¾ A-P locations. In the linear regression analysis, no correlation was found between CEP geometries and T2 mapping or age. Conclusion This study noninvasively visualized the CEP morphology and quantified CEP circumference, area, A-P width, lateral width, and thickness using an MRI 3D FLASH sequence. The measured disk geometries in this study agree well with previous literature.5,6 CEP geometries were not linearly correlated with age or T2 mapping, which is correlated with disk degenerative grade. The lack of correlation between CEP geometries and either age or T2 mapping as a marker of degeneration was not completely expected, since it has been discussed that CEP thickness decreases with degeneration.1–3 Additional data and increased sample size may provide more insight into the potential role of the CEP in the degenerative cascade. Acknowledgment NIH grant R01 AR050052. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Urban JPG. Arthritis Research Therapy 2003;5:120 Roberts S. Spine 1989;14:166 Moore RJ. European Spine Journal 2000;9:92 Bernick S, Spine 1982;7:97 O'Connell GD. Spine 2007:32:328 Elliott DM. Spine 2004;29:713

Quantification of Intervertebral Disk Cartilaginous Endplate Morphology using MRI

IntroductionThe IVD cartilaginous endplate (CEP) is a 450 mm to 800 mm thick layer of hyaline cartilage,2 which functions as a mechanical barrier between the pressurized nucleus pulposus and the vertebral endplate, and as a gateway for nutrient diffusion to the avascular disk.1–3 With age and degeneration, the CEP becomes thinner, calcifies, and may have reduced porosity,4 and it has been implicated as a contributor to degeneration.1–3 As the CEP is not well imaged on MRI, key geometric parameters of the CEP have not been obtained noninvasively. Thus, the objective of this study was to measure geometric properties of the CEP with MRI.Materials and MethodsLumbar motion segments (n = 24), from 11 human cadavers (age 64.9 ± 10.0 years) were imaged on a Siemens 7T MRI scanner using a custom RF coil. Disk degeneration was graded via mid-sagittal T2 mapping. Full volume imaging of each disk at 200 mm isotropic resolution was achieved via 3D FLASH (TR/TE = 9/3.7 ms) in 6 minutes (Fig. 1). Geometric parameters (area, circumference, anterior-posterior width, lateral width) were measured in the axial and mid-sagittal planes for both the CEP and the entire disk using OsiriX software. Disk height was measured using custom-written Matlab code.5Evaluation of CEP thickness was done on a mid-sagittal image of each disc. Semiautomated image analysis was conducted using custom-written Matlab code in which signal intensity profiles across the CEP thickness were captured at the center of the CEP and at ± ¼ and ± ¾ of its A-P length.Comparisons between superior and inferior CEP geometry were established using paired, two-tailed t-tests (p < 0.05). Data, except thickness, was normalized by dividing CEP geometry by its corresponding disk geometry to account for differences in disk sizes between levels. A one-way ANOVA was used to evaluate differences between disk level and CEP geometry. CEP thickness measurements were evaluated using a two-way ANOVA with repeated measures, where the factors were disk level and anterior-posterior disk location. A posthoc Bonferonni test was performed when significance was detected. Linear regression analysis was performed to compare CEP geometry with T2 mapping values and subject age.ResultsDirectly measured whole disk and CEP geometry for each level are provided in the Table. The mean CEP thickness in the mid-sagittal plane across all disk levels and locations was 0.50 ± 0.20 mm. No significant differences were found in CEP thickness with respect to the superior or inferior endplate or disk level (p > 0.2). There was no significant difference in CEP A-P width, lateral width, or circumference area with respect to superior/inferior location (p > 0.7) or disk level (p > 0.6) except between L1-L2 and L4-L5 (p < 0.05) (Fig. 2A-C). L1-L2 A-P width, lateral width, and circumference area were larger than in L4-L5 by 14, 18, and 28%, respectively.CEP thickness varied significantly with respect to anterior-posterior location for every level (p < 0.005) (Fig. 2D). The minimum thickness was at the center of the disk (superior = 0.39 ± 0.10 mm, inferior = 0.38 ± 0.11 mm). The thickness at the center was 19.9 and 42.4% less than that of the ± ¼ and ± ¾ A-P locations.In the linear regression analysis, no correlation was found between CEP geometries and T2 mapping or age.ConclusionThis study noninvasively visualized the CEP morphology and quantified CEP circumference, area, A-P width, lateral width, and thickness using an MRI 3D FLASH sequence. The measured disk geometries in this study agree well with previous literature.5,6CEP geometries were not linearly correlated with age or T2 mapping, which is correlated with disk degenerative grade. The lack of correlation between CEP geometries and either age or T2 mapping as a marker of degeneration was not completely expected, since it has been discussed that CEP thickness decreases with degeneration.1–3 Additional data and increased sample size may provide more insight into the potential role of the CEP in the degenerative cascade.Acknowledgment NIH grant R01 AR050052.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredUrban JPG. Arthritis Research Therapy 2003;5:120Roberts S. Spine 1989;14:166Moore RJ. European Spine Journal 2000;9:92Bernick S, Spine 1982;7:97O'Connell GD. Spine 2007:32:328Elliott DM. Spine 2004;29:713

Ultrashort Time-to-Echo MRI of the Cartilaginous Endplate: Technique and Association with Intervertebral Disk Degeneration

Introduction An association between cartilaginous endplate (CEP) defects and intervertebral disk (IVD) degeneration has been previously suggested in animal and cadaveric studies. There have been no previous reports in the literature that describe the use of ultrashort time-to-echo (UTE) magnetic resonance imaging (MRI) to assess the CEP in humans in vivo. As such, the purpose of this study was to report the feasibility of the UTE MRI technique to assess CEP defects in humans in vivo using and to assess their relationship with IVD degeneration. Materials and Methods Nine volunteer subjects (mean age 43.9 years; range: 22 to 61 years) were recruited, representing 54 IVDs and 108 CEPs. The subjects underwent T2-weighted and UTE MRI to assess for the presence and severity of IVD degeneration, and for the presence of CEP defects, respectively, from T12 to S1. Intervertebral disk degeneration was graded according to the Schneiderman et al classification on T2-weighted MRI. CEP defects were defined on UTE MRI as discontinuity of high-signal over 4 consecutive images and were independently assessed by two observers. Results Total 37% out of 108 (34.3%) CEPs had defects, which mainly occurred at T12/L1, L1/L2, and L4/L5 ( p = 0.008). Multivariate logistic regression revealed that lower BMI ( p = 0.009) and younger ( p = 0.034) individuals had a decreased likelihood of having CEP defects. A statistically significant association was found to exist between the presence of CEP defects and IVD degeneration ( p = 0.036).A higher prevalence of degenerated IVDs with CEP defects were found at L4/5 and L5/S1, while degenerated IVDs with no CEP defects were found throughout the whole lumbar region. Mean IVD degeneration scores of the L4/5 and L5/S1 levels with CEP defects were higher in comparison to those with no CEP defects. Conclusion Our study demonstrates the feasibility of using UTE MRI in humans in vivo to assess the integrity of the CEP. A statistically significant association was found to exist between the presence of CEP defects and IVD degeneration. In the lower lumbar region, more severe degeneration was found to occur in the IVDs with CEP defects than in those without defects. Such imaging technology may broaden the understanding of IVD degeneration and facilitate management options that target IVD repair/regeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Role of Type II Collagen Turnover in Disk Development and Degeneration

Introduction Intervertebral disks (IVD) are joint-like structures located between the vertebral bodies in the spine, providing movement and flexibility. Each is composed of three distinct parts. The central nucleus pulposus (NP) is highly hydrated and gelatinous, functioning as a shock absorber. It is surrounded laterally by a tough annulus fibrosus (AF), a fibrous lamellae structure that provides the disk with tensile strength. Both the NP and AF are sandwiched between two cartilaginous end-plates (EP), a continuous of the adjacent vertebral bodies that are thought to be important in regulating the nutrient and metabolite flow in and out of the disk. The disks along the spine efficiently dissipate load through the spine. The extracellular matrix (ECM) microenvironment is important for the maintenance of IVD cell phenotype and function. Changes in ECM compositions may lead to cellular changes and hence IVD degeneration. Collagen II is detected throughout the IVD and is the major fibrillar collagen in the NP, EP, and the inner AF. It forms a network that entraps proteoglycans such as aggrecan. Its degradation is initiated by collagenases that cleave at a specific site of the triple helix (PQG775↓776LAG). During disk degeneration, collagen II degradation is enhanced (Antonious et al, 1996). Conversely, we hypothesize that IVD degeneration would be prevented or significantly reduced if collagen II is protected from degradation. A mouse mutant with a genetically altered collagenase cleavage site of collagen II ( Col2a1cr) has been generated, and found to have an improved outcome in articular cartilage erosion after antigen induction of arthritis (Gauci, 2008). In this project, we studied the cellular and molecular changes in the IVDs in mice that are homozygous for the Col2a1cr allele ( Col2a1cr/cr), and study the impact in normal development and degenerative conditions. Materials and Methods Histological and molecular analyses of the IVDs were performed at two postnatal stages, 6 weeks and 3 months, using a multichromatic staining method (Leung et al, 2009). To induce disk degeneration, we will use the bilateral compressive loading method (Tanaka et al., 2011) in wildtype (Wt) and Col2a1cr/cr mice in a 8-week-old mice; joining vertebral bodies number 5 and 13 with a stainless steel wire forming a loop. The cellular and molecular changes in the IVDs will be compared after 4 weeks of compression. Results In the two time-points examined, histology analysis showed morphological changes in the EP and NP of Col2a1cr/cr mice. The bony end-plates at either ends of the vertebral bodies failed to form, whereas the cartilaginous end-plate is expanded in its place with hypertrophic chondrocyte-like cells (Fig. A). The number of cells in the NP is consistently higher in both 6 weeks and 3 months old Col2a1cr/cr mice, and the ECM composition changed with an enhanced Safranin O staining, indicating an altered proteoglycan composition with decreased collagen II turnover. Conclusion Our data suggested that collagen II turnover plays a role in the maintenance and differentiation of cells in the IVD. A normal microenvironment is important for the maintenance of IVD cell phenotype. The altered matrix composition may change the cell-matrix interaction and hence interfere with the proliferation or differentiation of cells in NP and EP. The changes in organization structure of the bony and cartilaginous end-plates could affect structural function as well as nutrition supply. Specific studies on cellular differentiation, cell proliferation, and survival will provide critical insights, while potential protective effect conferred from reduced collagen II degradation will be studied using the bilateral compressive loading. This mouse model provides the first insight in the role of collagen II turnover in tissue homeostasis of the IVD. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Antoniou J, Steffen T, Nelson F, Winterbottom N, Hollander AP, Poole RA, Aebi M, Alini M. The human lumbar intervertebral disk: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. Journal of Clinical Investment 1996;98(4):996–P533 Gauci SJ. Collagenase cleavage of type II collagen is essential for normal skeletal growth and development [PhD thesis]. University of Melbourne, 2008 Leung VYL, Chan WCW, Hung SC, Cheung KMC, Chan D. Matrix remodeling during intervertebral disk growth and degeneration detected by multichromatic FAST staining. Journal of Histochemistry and cytochemistry 2009;57(3): 249–256 Tanaka M, Sakai D, Hiyama A, Tamura F, Arai F, Nakajima D, Nakai T, Mochida J. Evidence of non-notochordal origin in chondrocyte-like cells of the nucleus pulposus appearing in early stage disk degeneration in the mouse model [abstract]. In: New Horizons in Intervertebral Disk Research; November 16–18, 2011, Philadelphia: PSRS; 2011. p.57. Abstract no S6.1

Fabrication of Multicomponent Tissue-Like Constructs-Toward Spinal Motion Segment Tissue Engineering

Introduction Allogenic spinal motion segment transplantation demonstrated promising results in treating late stage disk degeneration and stimulated immense interests of tissue engineering in the whole spinal motion segment from autologous cell source and safe biomaterials, solving the donor availability problem and potentially alleviating the degeneration and immunogenicity problems. Bioengineering complex tissues such as spinal motion segment is extremely challenging because it involves the integration of multiple tissues with distinct structures and composition such as lamellar annulus fibrosus (AF), gel-like nucleus pulposus (NP), cartilaginous endplate, and bony vertebra. Although attempts have been made to fabricate biphasic structures such as lamellar AF structure wrapping around a NP core, or bony block with cartilaginous structure, good methods integrating different tissue parts to form a combined unit are not available. In this study, a bioreactor-based method integrating multicomponent spinal motion segment-like tissue constructs, together with in vitro mechanical and biological stimulation, was reported, as the first step towards functional spinal motion segment tissue engineering. Materials and Methods Rabbit mesenchymal stem cells (rMSC) were used to fabricate cell-collagen constructs. Constructs were cultured separately in chondrogenic and osteogenic differentiation medium for 21 days. A layer of rMSC-collagen solution was added between chondrogenic and osteogenic units for coculture with cyclic compression to form the combined osteochondral constructs with intact interface. Afterward, an acellular cylindrical collagen gel was formed in between two such combined osteochondral constructs before circumferencing the whole construct with rMSC-collagen tubular layers. The multicomponent constructs were subjected to periodic compression and torsion simultaneously. The constructs were evaluated histologically for multicomponent structures. Cell alignment in the tubular layers was quantified using an adhoc MatLab program while collagen alignment was evaluated using scanning electron microscopy (SEM). Moreover, the interfacial strength of the osteochondral units was also evaluated using a tensile test machine. Results The combined constructs were able to be dismounted with integrity from the supporting shafts of the bioreactor after 14 days of culture. Every component of the combined constructs remained intact and can be manipulated with forceps. This method proves the feasibility to proceed to bioreactor-based functional remodelling and engineering of spinal motion segment in the future. Alignment analysis of rMSC in the tubular layers of the combined constructs demonstrated that the cell aligned along different preferred direction under torsional stimulation as compared with that from the static loading control (Fig. 1). However, SEM demonstrated no significant collagen alignment after 14 days culture for all groups. Histological results showed good integration among different components while cells were able to migrate into the acellular collagen gel at the center of the combined constructs although the source of the migrated cells was not studied yet. In addition, the osteogenic and chondrogenic phenotype in the osteochondral units were maintained while the interfacial strength of the osteochondral units was significantly higher when both the biological and the compression stimulation were present during their fabrication. Conclusion A method fabricating a multicomponent tissue construct with integrity for future functional spinal motion segment tissue engineering is demonstrated. Torsional stimulation can be used to stimulate rMSC re-orientation within a collagen tubular structure. Osteogenic differentiated and chondrogenic differentiated rMSC were able to maintain the phenotype throughout the culture period and their interface stabilized by biological and mechanical stimulation. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Cheng HW, Luk KD, Cheung KM, Chan BP. In vitro generation of an osteochondral interface from mesenchymal stem cell-collagen microspheres. Biomaterials 2011;32(6):1526–1535. Au-Yeung KL, Sze KY, Sham MH, Chan BP. Development of a micromanipulator-based loading device for mechanoregulation study of human mesenchymal stem cells in three-dimensional collagen constructs. Tissue Engineering Part C Methods 2010;16(1):93–107.

Cadherin 2 as Human Notochordal Cell Marker and its Expression Pattern in Disk Degeneration

Introduction Cadherins are transmembrane glycoproteins that mediate calcium-dependent cell adhesion. Recent studies suggest that cadherins also have nonadhesive functions. Maturation of human intervertebral disk (IVD) as well as early events of IVD degeneration in a mouse model1 is associated with segregation of nucleus pulposus (NP) cells suggesting that disk degeneration may be associated with cell adhesion molecule activities. From our microarray data analysis and subsequent immunohistochemical assays, we have revealed specific expression of Cdh2 gene and its product, cadherin 2/N-cadherin, in rodent notochordal NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. Cadherin 2 belongs to the classic cadherin family. To date, the expression and function of cadherin 2 in IVD and its relationship to disk degeneration remain elusive. We aim to study the expression pattern of cadherin 2 in human IVD using aborted fetus and clinical samples, and to investigate its function in the rodent NP via a protein ablation strategy. Materials and Methods Degenerated lumbar disks (grade 3, Schneiderman's classification) ( n = 5) were obtained from 45 to 58-year-old patients. Scoliotic lumbar disks ( n = 5) from patients were obtained as nondegenerated samples. Lower lumbar spine of the aborted fetus in the second trimester were used as controls. Compartmental origin and degeneration status of the samples was validated by our established multicolor FAST staining method,2 in which the disk sections were sequentially stained with alcian blue, safranin-O, tartrazine, and fast green. Immunohistochemistry for cadherin 2 was performed on paraffin sections with antigen retrieval by hyaluronidase and citrate buffer method and counterstained by hematoxylin to visualize cell nuclei. The percentage of positive cells was calculated and analyzed by nonparametric u test. For protein ablation study, 4-month-old inbred Lewis rats were anesthetized and the tail IVDs were exposed for injection of rabbit anti-cadherin 2 antibody or control IgG ( n = 6 for each) into the NP via 34G hypodermic needle. By 2 weeks and 8 weeks after operation, the disks were harvested for histological examination. Results Human fetal NP cells showed homogeneous cadherin 2 expression. Annulus fibrosus (AF), cartilaginous endplates, and vertebral bodies showed no signal of cadherin 2. However, adult NP showed heterogeneous cadherin 2 expression, where only a subpopulation of the NP cells expressed the protein. Nondegenerated and degenerated NP exhibited different cell distribution with the later tended to contain cell clusters. The frequency of cadherin 2 expressing cells was significantly higher in the degenerated NP (51.9%) than in nondegenerated NP (20.0%). FAST staining assay showed that degenerated NP samples were intensely stained by safranin-O instead of alcian blue, validating a loss of sulfated acidic glycosaminoglycan due to degeneration. Cadherin 2 protein ablation study showed that the vacuolated notochordal cells in the NP partially transformed into elongated or rounded nonvacuolated cells, which are commonly presented in degenerative disks. Conclusion Human fetal NP mainly contains notochordal cells. In this study, we showed that cadherin 2 is expressed homogeneously in fetal NP suggesting cadherin 2 is a marker of notochordal cells. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Under this notion, our findings imply that a subpopulation (20%) of NP cells retain a phenotype resembling notochordal cells in mature human IVD, although morphologically they are not vacuolated. The higher frequency of cadherin 2 expressing cells in degenerated NP implicates that proliferation of cells of notochordal origin occurs during disk degeneration. Protein ablation study suggests that cadherin 2 has a regulatory function in maintenance of notochordal cells and a loss of its function may initiate disk degeneration. Overall, our study implies a function of cadherin 2 in NP homeostasis. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Yang F, et al. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. Journal of Pathology 2009;218(1):113–121 Leung VY, et al. Matrix remodeling during intervertebral disk growth and degeneration detected by multichromatic FAST staining. Journal of Histochemistry and Cytochemistry 2009;57(3):249–256

Cells in the Endplate Cartilage Contribute to the Inner Annulus Fibrosus in the Intervertebral Disk

Introduction Intervertebral disk (IVD) consists of three major structural components; a gelatinous nucleus pulposus (NP) in the center surrounded by a lamellar structured annulus fibrosus (AF), sandwiched between two cartilaginous endplates (EPs). These structures act synergistically, facilitating motions of the spine and acting as shock absorbers along the spine. Degenerative signals that lead to changes in cell phenotype or extracellular matrix in any of these compartments may cause IVD degeneration. However, we do not fully understand the origin and differentiation of cells in the IVD. Recently, a cell lineage tracing study confirmed that the original of the NP cells are derived from the notochord in the development (Choi et al, 2008). However, the origin of AF cells is still not clear. A clear understanding of the cell pools contributing to disk function is important in the formulation of cell therapy and tissue engineering protocols. The lamellar AF can be divided into inner and outer portions which have distinct cell population and extracelluar matrix environment. Cells in outer AF are fibroblast-like cells producing collagen I, whereas cells in the inner AF are chondrocyte-like producing collagen II and aggrecan. The number of inner AF cells increases during development, and these cells need to be replenished with age. Histological analysis showed evidence a continuum of cells from end-plate region to the inner AF. We hypothesize that cells in this region of the EP act as progenitors, contributing to the formation of the AF and replenishment of cells in adult life. Cell tracing in mice was performed to assessment of source of the AF cells in the IVD. Materials and Methods Collagen X ( Col10a1 ) is expressed in chondrocytes adjacent to the inner AF. Therefore, we can mark these cells by crossing Col10a1-Cre mice with Rosa26-lacZ reporter mice. Thus, Cre recombinase expressed under the regulation of Col10a1 will activate the LacZ gene as a marker of these cells, and their fate can be traced following the LacZ expressing cells. We also used a conditional Cre (CreERt) that can localize to the nucleus only in the presence of tamoxifen, and injection of tamoxifen provides a temporal activation of Cre, allowing a pulse-chase tracing of cell fate, and all descendant cells from a pulse of tamoxifen will express LacZ throughout the life of the animal. Results In situ hybridization showed Col10a1 is expressed in cells located in the EP and the growth plate (GP) of the adjacent vertebral bodies (White dots; Fig. A), but not in the AF (*). Interestingly, Col10a1-Cre; Rosa26-lacZ mouse showed LacZ positive cells (Fig. B; pink) detected in the inner AF (*), suggesting these cells are derived from the Col10a1 expressing cells in the EP. To eliminate the possibility that Col10a1 is expressed at low level in the inner AF that was not detected, we performed cell-tracing using the Col10a1-CreERT mouse crossed with the reporter mouse, with a single tamoxifen injection in 3-week-old mice that showed Lac-Z positive cells in the EP 3 weeks later, with typical AF cell morphology. Conclusion Using genetic tools in mice, we provide the first direct evidence that cells from the EP contribute to the inner AF. Our findings suggest that Col10a1 expressing cells in the EP are plastic, acting as progenitors given rise to AF cells. This population of cells can be considered in cell therapy and tissue engineering for the repair and construction of the AF, respectively. Moreover, this genetic system in mice provides the tools to further investigate the biology and pathology of the IVD as more mouse models for disk degeneration are produced. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Choi KS, Cohn MJ, Harfe BD. Developmental Dynamics 2008;237:3953–3958

Changes in Biomechanical, Histological and Quantitative MRI Parameters in Lumbar Caprine Intervertebral Discs Subjected to Chondroïtinase-Induced Degeneration

Introduction Intervertebral disk (IVD) degeneration is one of the major causes of low back pain. In the onset of IVD degeneration, the nucleus pulposus of the disk loses proteoglycans, decreasing the discs' capability to retain water. This is considered to detrimentally affect its biomechanical properties, making the disk prone to further degradation. Quantitative magnetic resonance imaging (MRI) techniques, such as T2- and T1-rho mapping, provide valuable information on the matrix status (proteoglycan content and distribution) of healthy and degenerating IVDs. We have developed a model to culture and load large (lumbar) IVDs, in which we can simulate IVD degeneration by injection of chondroitinase ABC (cABC; an enzyme that cleaves proteoglycans). The purpose of the current study is to characterize the changes in biomechanical, histological, and quantitative MRI parameters of caprine lumbar IVDs subjected to intranuclear cABC-injection. Materials and Methods Lumbar IVDs (36 total) with cartilaginous endplates were dissected from the spines of mature goats under sterile conditions. IVDs were randomly assigned to receive either no injection or injection (29G needle) of P83 µL PBS, 0.25 U/mL cABC, or 0.5 U/mL cABC. All IVDs were cultured in a bioreactor for 21 days under simulated-physiological dynamic loading conditions. Before injection and directly after culture, IVDs were scanned with a high-field MRI scanner (9T). T1-, T2-, and T1-rho mapping sequences were used to quantify the relaxation time parameters. IVDs axial deformation behavior during loading was analyzed and IVD stiffness was calculated from the load and displacement data collected during culture in the bioreactor. Matrix integrity of IVDs was studied on transverse paraffin sections stained with Safranin-O to assess proteoglycan content and distribution and Masson's Trichrome to assess collagen composition and structure. Results All biomechanical properties of the IVDs injected with cABC showed significant changes over the culture period. During preloading of the IVDs, within the first 8 hours of culture, creep behavior differed significantly between groups. A cABC dose-dependent subsidence was observed. Over the total culture period, disk height was reduced in all groups. However, cABC-treated groups showed significantly more loss in height. These discs progressively lost height during the entire culture period and stiffness of IVDs increased gradually over time. When comparing the images and quantitative MRI data from day 0 and day 21, significant changes could be observed in T2 and T1-rho values in the nucleus region for both the 0.25U and 0.5U cABC group. In the annulus, the apparent diffusion coefficient (ADC) changed significantly in the anterior and lateral regions. On histological sections, we observed a clear loss in staining intensity in the nucleus on Safranin-O sections, especially in the 0.5U cABC injected IVDs, with blurring of the posterior demarcation zone between the nucleus and annulus. In Masson's Trichrome stained sections, a clearly defined lamellar structure in the anterior and lateral annulus could be observed in day 0 discs, which showed degradation in the cABC-injected IVDs at day 21. Conclusion cABC injection into the nucleus had a rapid and potent effect on the measured characteristics of caprine lumbar IVDs. Enzymatic cleavage of proteoglycans from the nucleus directly affected biomechanical properties of the discs and we observed dose-dependent degenerative changes. We also found comparative changes in matrix content and integrity as observed on histological section and quantified by MRI. This study shows the parallels between the functional and histological changes in a large lumbar IVD during the first stages of degeneration. We demonstrate that T1rho MRI measurements can be used to accurately quantify this early degenerative process. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Cadherin 2 as Human Notochordal Cell Marker and its Expression Pattern in Disk Degeneration

IntroductionCadherins are transmembrane glycoproteins that mediate calcium-dependent cell adhesion. Recent studies suggest that cadherins also have nonadhesive functions. Maturation of human intervertebral disk (IVD) as well as early events of IVD degeneration in a mouse model1 is associated with segregation of nucleus pulposus (NP) cells suggesting that disk degeneration may be associated with cell adhesion molecule activities. From our microarray data analysis and subsequent immunohistochemical assays, we have revealed specific expression of Cdh2 gene and its product, cadherin 2/N-cadherin, in rodent notochordal NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. Cadherin 2 belongs to the classic cadherin family. To date, the expression and function of cadherin 2 in IVD and its relationship to disk degeneration remain elusive. We aim to study the expression pattern of cadherin 2 in human IVD using aborted fetus and clinical samples, and to investigate its function in the rodent NP via a protein ablation strategy.Materials and MethodsDegenerated lumbar disks (grade 3, Schneiderman's classification) (n = 5) were obtained from 45 to 58-year-old patients. Scoliotic lumbar disks (n = 5) from patients were obtained as nondegenerated samples. Lower lumbar spine of the aborted fetus in the second trimester were used as controls. Compartmental origin and degeneration status of the samples was validated by our established multicolor FAST staining method,2 in which the disk sections were sequentially stained with alcian blue, safranin-O, tartrazine, and fast green. Immunohistochemistry for cadherin 2 was performed on paraffin sections with antigen retrieval by hyaluronidase and citrate buffer method and counterstained by hematoxylin to visualize cell nuclei. The percentage of positive cells was calculated and analyzed by nonparametric u test. For protein ablation study, 4-month-old inbred Lewis rats were anesthetized and the tail IVDs were exposed for injection of rabbit anti-cadherin 2 antibody or control IgG (n = 6 for each) into the NP via 34G hypodermic needle. By 2 weeks and 8 weeks after operation, the disks were harvested for histological examination.ResultsHuman fetal NP cells showed homogeneous cadherin 2 expression. Annulus fibrosus (AF), cartilaginous endplates, and vertebral bodies showed no signal of cadherin 2. However, adult NP showed heterogeneous cadherin 2 expression, where only a subpopulation of the NP cells expressed the protein. Nondegenerated and degenerated NP exhibited different cell distribution with the later tended to contain cell clusters. The frequency of cadherin 2 expressing cells was significantly higher in the degenerated NP (51.9%) than in nondegenerated NP (20.0%). FAST staining assay showed that degenerated NP samples were intensely stained by safranin-O instead of alcian blue, validating a loss of sulfated acidic glycosaminoglycan due to degeneration. Cadherin 2 protein ablation study showed that the vacuolated notochordal cells in the NP partially transformed into elongated or rounded nonvacuolated cells, which are commonly presented in degenerative disks.ConclusionHuman fetal NP mainly contains notochordal cells. In this study, we showed that cadherin 2 is expressed homogeneously in fetal NP suggesting cadherin 2 is a marker of notochordal cells. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Under this notion, our findings imply that a subpopulation (20%) of NP cells retain a phenotype resembling notochordal cells in mature human IVD, although morphologically they are not vacuolated. The higher frequency of cadherin 2 expressing cells in degenerated NP implicates that proliferation of cells of notochordal origin occurs during disk degeneration. Protein ablation study suggests that cadherin 2 has a regulatory function in maintenance of notochordal cells and a loss of its function may initiate disk degeneration. Overall, our study implies a function of cadherin 2 in NP homeostasis.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declaredYang F, et al. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. Journal of Pathology 2009;218(1):113–121Leung VY, et al. Matrix remodeling during intervertebral disk growth and degeneration detected by multichromatic FAST staining. Journal of Histochemistry and Cytochemistry 2009;57(3):249–256

Pathomechanism of Thoracic Disk Herniation: A Postmortem Histological Study

Introduction Kokubun et al histologically demonstrated that herniated masses in all cases of cervical disk herniation contained cartilaginous endplate (CEP) fragments (Spine 1996). They also revealed that the CEP-type of herniation was the predominant type of herniation in the cervical spine resulting from horizontal and vertical cleft formations of the disks at the final stage of the degeneration. On the other hand, it is generally understood that herniated tissues in the lumbar spine can consist of nucleus pulposus and anulus fibrosus, and that lumbar disk herniation does not necessarily happen at the final stage of disk degeneration. However, to our knowledge, the histological features of the herniated masses in thoracic disk herniation have not yet been clarified. In the present study, intervertebral disks from autopsy cases were examined histologically as to elucidate the pathomechanism of thoracic disk herniation. Materials and Methods Specimens of 240 intervertebral disks from T1-T2 to T12-L1 were obtained at autopsy from 20 individuals, 16 men and 4 women. Only spines without tumorous or infectious abnormalities were collected. The age at the time of death ranged from 38 to 93 years (average, 63 years). A 5-mm thick midsagittal slab was cut-off from each specimen, then fixed in 10% buffered formalin, decalcified in 4% edetic acid, and embedded in paraffin. The sections were sagittally cut and stained with hematoxylin and eosin, toluidine blue, and elastica-Masson and were examined with light microscopy. Results Thoracic disk degeneration A horizontal cleft traversing the fibers of the disks was observed in 92% of the disks examined. Total 21%, 34%, and 37% had a horizontal cleft that was less than one-third, one-third to two-third, and more than two-third of the anteroposterior diameter of each disk, respectively. One or more vertical clefts extending to the CEP were observed in 38% of the disks examined. Specimens showing discontinuous separation of fiber bundles that might be artifacts were excluded. A portion of the CEP was avulsing, reflected or detached from itself or the vertebral body (CEP fragmentation) in 23% of the disks examined. Gaps without interposing tissue, such as fibrocartilage or granulation, were excluded. Disk herniation penetrating the deep layer of the posterior longitudinal ligament was observed in 55% (11) of the autopsy cases and 9% (21) of the disks examined. The youngest age with disk herniation was 38 years whereas the oldest was 93 years. Multiple herniations were observed in five autopsy cases. The frequencies of the above abnormalities were analyzed relative to decades and disk levels. Abnormal features of horizontal cleft, vertical cleft, and CEP fragmentation were most frequently observed in the ninth decade. The frequency of disk herniation decreased with aging. The frequency of horizontal cleft was high at T1-2, T2-3, or T5-6. The frequency of vertical cleft as well as CEP fragmentation was high at T7-8, T8-9, T9-10, or T10-11. The frequency of disk herniation was nearly the same at each disk level except T4-5. Histology of the disk herniation The 21 disk herniations were histologically classified into two types. Six were CEP type in which herniated masses contained CEP fragmentation, observed frequently at T7-8, T8-9, T9-10, or T10-11. Remaining 15 herniations were nucleus pulposus (NP) type in which herniated masses consisted of nucleus pulposus and anulus fibrosus; these were observed with nearly the same frequency at each disk level. In all cases of CEP type herniations, all of the abnormal features of horizontal cleft, vertical cleft and CEP fragmentation were observed. On the contrary, in cases of NP type herniations, horizontal cleft, vertical cleft and CEP fragmentation were observed only in 60%, 47%, and 20%, respectively. Conclusion Thoracic disk herniation was histologically observed in 55% of the autopsy cases and in 9% of the disks, using midsagittal sections. Two types of disk herniation were observed; CEP type herniation containing cartilaginous endplate fragments occurs in the disks at their final stage of degeneration. NP type herniation consisting of nucleus pulposus and anulus fibrosus occurs in the disks at their early stage of degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Characterization of Human Endplate Biopsies: Preliminary Results on Cellular Composition, Metabolic Activity and Interactions with Disk Cells

Introduction Tissue alterations that result in an impaired endplate (EP) permeability are assumed to play a crucial role in the pathogenesis of intervertebral disk (IVD) degeneration. Therefore, disturbance of endplate permeability is one of the main problems that need to be solved for successful disk tissue engineering. However, the biological mechanisms underlying these alterations are poorly understood. To shed light on this aspect, a standardized analysis of EP structure and cellular composition is essential to understand the degeneration processes. Purpose of this study was the development of standardized protocols for a descriptive characterization of EP samples by histology, transmission electron microscopy (TEM), analysis of differentiation and metabolic activity of EP cells. In first preliminary experiments we also aimed to detect possible interactions of the EP with disk cells of the same donor patients in a bioassay with EP-conditioned media. The suitability of these methods for EP characterization and possible correlations with degenerative alterations is discussed. Materials and Methods Cartilaginous endplates obtained during spine surgery of total 27 human donors underwent the following procedures: (1) isolation of endplate cells, in vitro expansion and characterization of chondrogenic differentiation in 3D pellet cultures ( n = 6), (2) preparation of standardized EP-sections for histological and ultrastructural analyses ( n = 8), and (3) preparation of standardized endplate punches that were incubated for 24 hours in serum-free culture medium followed by determination of metabolic activity of endplate cells by MTT-test ( n = 13) and further analysis of conditioned media (CM) in a bioassay with NP cells of the same donor ( n = 2). Pellet cultures of EP cells were characterized for matrix protein expression by Alcian blue staining. Influences of CM on gene expression of matrix proteins, matrix degrading enzymes, and inflammation factors by NP cells were determined. Results Differentiation of endplate cells in pellet cultures varied strongly not only between the six individual donors, but also in different disk levels of the same patient. Part of the samples exhibited a very low proteoglycan production whereas others were strongly positive in the Alcian blue staining intensity suggesting high variations in the differentiation capacity of EP cells. Histological examination of the endplate samples allowed a characterization of both the endplates and an observation of the adjacent structures as the very thin endplate layer could not be clearly separated from adjacent tissue structures. High variations between tissue samples from different donors could be found. TEM images showed signs of degeneration both in the matrix and the EP cells whereas higher numbers of apoptotic cells and matrix degradation products could be found in stronger degenerated tissue samples. Standardized EP punches that were cultured for 24 hours in DMEM and incubated for 4 hours with MTT showed different staining intensities suggesting donor-specific variations in metabolic activity of EP cells. In first two experiments with CM of EP samples, a dose-dependent upregulation of inflammation-factors and matrix metalloproteinases (Interleukins IL-1β, IL-6, IL-8, MMP-3, and MMP13) by NP-cells of the same donors could be detected. The extent of this effect differed strongly between the two patients with different degeneration degree. Conclusion The present results suggest that a combination of different methods allows the characterization of EP tissue and possible interactions with the adjacent disk tissue. As the cartilaginous EP itself is very thin and with low cellularity, isolation of an almost homogeneous EP population is very difficult and the cell yield is very poor. Therefore, methods that directly analyze the cells in situ for example by laser capture micro-dissection might be more suitable to determine the gene expression profile of EP cells. Analysis of metabolic activity of standardized EP punches by MTT test is a promising approach provided that the samples are standardized with regard to diameter and height with no adjacent other tissue regions. TEM investigation is appropriate to investigate cellular details, but also with this method, the poor cellularity of EP tissue is a challenge. A very promising approach is the analysis of CM in a bioassay with NP cells of the same donor that allows the detection of possible interactions between both tissue areas. In ongoing investigations, we analyze higher numbers of EP samples with different degrees of degeneration to enable a correlation between the biological and clinical data of the patients. For this purpose, analyses of nondegenerated control samples are essential. In summary, all these methods together allow a holistic view on the mechanisms that contribute to the morphological and pathophysiological changes of the EP and potential interaction with the adjacent disk tissue. Acknowledgement Part of this research work was supported by the EU framework 7 project Geneodisc (FP7, 2007–2013) under grant agreement no. HEALTH-F2-2008-201626. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Optimization of Bioreactor Design for Normal Loading of Human Intervertebral Disks

Introduction Intervertebral disk (IVD) degeneration is a common cause of back pain, which has a negative impact on the quality of life of the patient and is costly to the health care system. It is crucial to understand the interplay between mechanobiology, disk composition, and metabolism to understand the underlying cause of disk degeneration and to be able to study ways to regenerate the degenerate disk. To address such questions, a bioreactor has been developed that facilitates organ culture of intact human disks in a controlled dynamically loaded environment. The bioreactor is used in combination with an isolation method which maintains the integrity of the intervertebral disks by preserving the noncalcified part of the cartilage endplate. In this study, stress profilometry was used to evaluate optimal loading platen design for the bioreactor. Materials and Methods Human lumbar IVDs were obtained through organ donations via Transplant Quebec. The spines were assessed by X-ray to evaluate the degree of degeneration. Intact disks were prepared by parallel cuts through the adjacent vertebral bodies close to the end plates, and the remaining bone and the calcified part of the cartilage endplates were removed using a high-speed bone burr. Pictures of the disk were taken after processing and surface area was calculated with ImageJ software to calculate the load to be applied to generate pressures of 0.3 MPa and 0.6 MPa. Two platen sets were tested, full coverage of the whole disk and partial coverage of only the nucleus pulposus (NP) region. The disks were mounted between two platens and stress profiles were recorded at 0.3 MPa and 0.6 MPa static load. Vertical (V) and horizontal (H) stress profiles were generated for anterior-posterior (AP) and lateral diameters of the disk. Results In young and healthy isolated disks, stress profiles for full coverage and partial covering platens were very similar (Fig. 1) to the stress profiles generated from the same disk with intact vertebral bone. The stress profiles showed uniform distribution of load over the entire diameter of the disk, even when only the central part was loaded. As expected, degenerate specimens showed an uneven load profile with regions of perturbation, when loaded with full coverage platens. Loading the degenerate disks with partially covering platens resulted in very uneven load profiles and in failure of the cartilaginous endplates at the higher load. Conclusion A critical step in the development of a disk organ culture system that can be subjected to load is the validation of the various components of the bioreactor. We found that the choice of platens did not affect the load distribution in young and healthy but seemed to be critical when loading degenerate disks. The uneven load profiles obtained in degenerate disks using full coverage platens resembles the in vivo profiles published by McNally et al The similarities indicate that we are able to mimic an in vivo environment in degenerate samples using full coverage platens. Our findings indicate that the choice of load platen is critical to provide in vivo load conditions when studying degenerate disks in organ culture. The bioreactor provides an experimental platform useful to evaluate the potential for biologic repair over a range of loading conditions. It also provides a system where the effect of load magnitude and frequency on the onset of degeneration can be evaluated in healthy disks. Such knowledge is important for patient advice on lifestyle in general and for following a biological repair procedure. This bioreactor system provides a very useful tool to study the mechanisms of both degeneration and regeneration of human intervertebral disks. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared