PENETRATION OF CEFOTAXIME INTO INTERVERTEBRAL DISCS REMOVED FROM PATIENTS UNDERGOING DISCECTOMY

the response of cells from the annulus fibrosus (AF) and nucleus pulposus (NP) to varying oxygen (O2) concentrations was examined when cultured in alginate. to study the effect of O2 concentration on AF and NP cells. AF and NP cells possess different metabolic profiles in situ. However, it is not clear whether this difference is maintained in in vitro culture conditions. AF and NP cells can respond differently in the different systems, which may differ from the in vivo environment in terms of nutrient supply and O2 levels. In vivo, O2 levels vary from 1% to 5% within the intervertebral disc, and there is evidence that disc cell metabolism can vary with O2 concentrations. an alginate scaffold was seeded with bovine AF or NP cells and maintained in culture for up to 18 days under different O2 concentrations. The sulfated glycosaminoglycan (GAG) content in the culture medium and the expression of aggrecan, type I (COL1A2) and II (COL2A1) collagen genes were analyzed at day 9 and day 18. in both NP and AF cells cultured either in normoxia (21% O2) or in hypoxia (5% and 1% O2), the GAG content of the culture medium increased with time, though the rate of increase was diminished in 5% O2. With a decrease in O2 levels, the expression of aggrecan mRNA increased in NP cells. There was little effect of O2 on aggrecan mRNA level in AF cells. However, there was a slight decrease with time. Interestingly, aggrecan mRNA levels did not reflect GAG release for either NP or AF cells. There was no effect with time or O2 levels on COL2A1 message in NP cells. The highest Aggrecan/COL2 message ratio for NP cells was with 1% O2, suggesting this to be the best condition for maintaining the NP phenotype. COL1A2 gene expression in NP and AF cells increased with time, but showed little change with O2 levels in NP cells. The highest COL2/COL1 ratio in NP cells was also observed with 1% O2. Finally, NP cells tended to remain localized in the alginate beads, whereas AF cells tended to migrate from the beads. both NP and AF cells showed little change in GAG production with O2 levels ranging from 1% to 21%. Disc cell metabolism is not impaired at low O2 concentrations, which appear beneficial to matrix composition. Furthermore, low oxygen may promote a gelatinous NP matrix, whereas increased oxygen levels may promote a fibrous matrix.

Introduction Immature nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with strong cell-cell interactions.1 With maturation and aging, NP cells lose their ability to form these cell clusters, with aging-associated changes in NP cell phenotype, morphology, and proteoglycan synthesis that may contribute to IVD degeneration.2-4 Therefore it is important to understand the mechanisms governing immature NP cell cluster behavior toward the goal of revealing factors that can promote immature, healthy NP cell phenotypes. Prior work has shown healthy, immature NP cells preferentially bind laminin proteins, exhibit potential for cell cluster formation, and maintain matrix production when cultured upon soft, laminin-containing substrates.2 N-cadherin has been identified as a cell-cell adhesion marker that is present in immature NP cells, but disappears with age.5-6 The goal of this study was to determine if N-cadherin was an important molecule in regulating cell-cell interactions in immature NP cell cluster formation and to test for a regulatory role in maintaining an immature NP phenotype for IVD cells in vitro. Materials and Methods Porcine nucleus pulposus (NP) cells were isolated from IVD of immature pigs (3-6 mos).2 Laminin-rich substrates were constructed from basement membrane extract (BME, 13.7 mg/mL, Trevigen) to generate soft (300 Pa) or stiff (BME-coated glass, > 2,900 Pa) substrates; similar substrates were constructed of “soft” and “stiff” type I collagen (col 1, 4 mg/mL, Sigma). NP cells were plated (65,000 cells/cm2) on the substrates with the following treatment conditions for up to 4 days: N-cadherin (40 μg/mL) blocking antibody, E-cadherin (20 μg/mL) blocking antibody or no treatment control. NP cells were stained for polymerized F-actin (phalloidin, Invitrogen), anti-N-cadherin (Abcam), or anti-E-cadherin (Abcam) antibodies with propidium iodide nuclei counterstain. Changes in matrix production were analyzed in collected media and recovered cells via biochemical assays for sGAG (DMMB assay) and DNA (picogreen). Additionally, gene expression for immature NP markers, N-cadherin, Brachyury-T, aggrecan and type I collagen, were analyzed via quantitative real-time PCR. Results NP cells cultured upon soft BME substrates maintained their rounded morphology at all times with formation of 3D cell clusters ( Fig. 1A ). On stiff BME and all col 1 substrates, NP cells spread out and attached with formation of numerous actin stress fibers. Higher N-cadherin expression was observed on soft BME compared with all other substrates; E-cadherin expression was absent in porcine NP cells on all substrates. Additionally, significantly higher matrix production was observed on soft BME substrates compared with soft col 1 ( Fig. 1B ), and higher gene expression for NP markers was observed on soft BME substrates compared with all other substrates ( Fig. 1C ). NP cell cluster formation, proteoglycan synthesis and NP marker gene expression on soft BME substrates was influenced by N-cadherin blocking antibody but not E-cadherin blocking antibody ( Fig. 1A -C). [Figure: see text] Conclusion Soft BME substrates promote NP cell clustering in vitro with an associated expression of N-cadherin. In NP cells, it appears that N-cadherin-mediated signaling helps promote matrix production and elevated expression for multiple molecular markers of an immature NP phenotype. Treatment with an N-cadherin blocking antibody resulted in loss of all features of the immature NP cell on soft BME substrates. These findings establish the importance of N-cadherin in mediating immature NP cell cluster formation and maintenance of the immature phenotype. Ongoing studies aim to understand the downstream signaling mechanisms governed by N-cadherin-mediated signaling that promote the immature NP phenotype. Acknowledgments This study was supported with funds from NIH R01EB002263, R01AR047442, R01AR057410, T32GM008555, North Carolina Biotechnology Center, and with the NSF Graduate Research Fellowship. Disclosure of Interest None declared References Trout JJ, Buckwalter JA, Moore KC, Landas SK. Ultrastructure of the human intervertebral disc. I. Changes in notochordal cells with age. Tissue Cell 1982;14(2):359–369 Gilchrist CL, Darling EM, Chen J, Setton LA. Extracellular matrix ligand and stiffness modulate immature nucleus pulposus cell-cell interactions. PLoS ONE 2011;6(11):e27170 Boos N, Weissbach S, Rohrbach H, Weiler C, Spratt KF, Nerlich AG. Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine 2002;27(23):2631–2644 Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine 1995;20(11):1307–1314 Minogue BM, Richardson SM, Zeef LA, Freemont AJ, Hoyland JA. Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes. Arthritis Res Ther 2010;12(1):R22 Minogue BM, Richardson SM, Zeef LA, Freemont AJ, Hoyland JA. Characterization of the human nucleus pulposus cell phenotype and evaluation of novel marker gene expression to define adult stem cell differentiation. Arthritis Rheum 2010;62(12):3695–3705

Introduction Back pain is strongly associated with degeneration of the intervertebral disk (IVD), which is associated with ongoing mineral deposition.1 The presence of calcium deposits and type X collagen (COL X) and the level of the indicators of calcification potential (alkaline phosphatase (ALP), Ca2 + ions and Pi) were consistently higher in degenerative and scoliotic discs. We also showed that in mesenchymal stem cells (MSCs), parathyroid hormone (PTH 1–34) inhibits the expression of COL X while promoting type II collagen (COL II) expression, thereby preventing endochondral ossification.2 In this study, we investigated the effect of PTH on expression of COLII, COLX, and ALP in human IVD cells and analyzed the potential mechanisms related to its effect. Materials and Methods Human lumbar IVDs from a donor without spinal pathology were obtained within 24 hours after death. Nucleus pulposus (NP) and annulus fibrosus (AF) tissues from the IVDs were digested and the corresponding NP and AF primary cells were isolated as previously described.3These cells were cultured in complete DMEM to 90% confluence. Then the cells were incubated overnight in serum-free medium followed by treatment with 100 nM PTH 30 minutes to 48 hours. Protein expression was analyzed by immunoblotting using specific antibodies to COL I and COL II (Abcam, Cambridge, MA), COL X, and PTH receptor 1 (Sigma Aldrich). Expression and phosphorylation of AKT and MAPKs was assessed by using specific corresponding antibodies (Cell Signaling, Danvers, MA). Alkaline phosphatase activity was measured colorimetrically using the StemTAG kit (Cell Biolabs, San Diego, CA, USA) and Ca2 + release from cells was measured using calcium assay kit (Cayman Chemical, Ann Arbor, MI, USA). Statistical analyses were done using one-way ANOVA, posthoc tests p < 0.05 was considered significant. Results Effect of PTH on PTH receptor 1 We confirmed the presence of PTH receptor 1 on the AF and NP cells and showed the effect of PTH on the PTH receptor 1 activation by incubating AF and NP cells with 100 nM PTH (1–34). Effect of PTH on collagen expression PTH increased the expression of COL-II significantly in AF cells after 6 hours of incubation and in NP cells COL-II expression increased in a time-dependent and sustained manner from 6 to 48 hours of incubation. Expression of COL-X was not altered by PTH in AF cells, whereas in NP cells it decreased significantly from 6 to 48 hours. Effect of PTH on MAPK signaling PTH causes ERK activation by phosphorylation within 30 minutes both in AF and NP cells (Fig. 1). PTH stimulated ERK phosphorylation was found to be sustained up to 6 hours followed by a slow decrease (Fig. 1). However, PTH led to JNK phosphorylation within 30 minutes of incubation and this declined thereafter over 48 hours. PTH stimulation of p38 MAPK phosphorylation was not altered in AF cells, whereas in NP cells there was a significant activation of p38 at 30 minutes by PTH. There was a decline in p38 phosphorylation below the control levels after 24 hours of incubation with PTH. Effect of PTH on AKT signaling Results show that PTH causes AKT ( ser 473) activation by phosphorylation at 1 hour in NP cells and has no effect in AF cells. Effect of PTH on Calcification markers The activity of alkaline phosphatase was significantly decreased by PTH after 24 hours of incubation in NP cells. In AF cells, no significant changes were observed, even though there was a trend to increase at 48 hours. PTH significantly increased calcium release into the medium at 48 hours in NP cells and no significant change was observed in AF cells Conclusion PTH has previously been shown to promote chondrogenesis and to inhibit the expression of COL-X in chondrocytes probably via the activation of MAPK signaling pathways.4 The present results demonstrate that PTH upregulates COL-II and downregulates COL-X in IVD cells, indicating that PTH has the potential of being able to stimulate disk repair and to improve nutrient supply in the degenerative disc. Our data also suggests that activation of MAPK pathway takes place much earlier than the alterations in COL-II or COL-X expression. Interestingly, COL-II expression inversely correlates with alkaline phosphatase activity in NP cells treated with PTH. Although understanding of IVD calcification would be of great value, not only for elucidation of its mechanism, but with an eye toward eventual therapeutic intervention. PTH can thus be used towards disk regeneration therapy. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Hristova, GI et al. Journal of Orthopaedic Research 2011; epub Mwale F et al. Tissue Engeneering Part A (2010)16:3449–3455 Chelberg MK et al. Journal of Anatomy 1995;186 (Pt 1): 43–53 Datta, NS et al. Cell Signalling 2010;22: 457–46

Objective: As a main cellular component within the disc, nucleus pulposus (NP) cells play important roles in disc physiology. However, little is known on the biologic hallmarks of human NP cells. Therefore, the present study aimed to address the features of human NP cells.Methods: Human NP samples were collected from normal cadavers, patients with scoliosis and disc degeneration as normal, disease control and degenerative NP, respectively. The NP samples were studied using transmission electron microscopy and TUNEL assay. Pre-digested NP samples were studied using flow cytometry with PI/Annexin V staining.Results: Both control and degenerative human NP consisted of mainly viable cells with a variety of morphology. Both necrosis and apoptosis were noted in human NP as forms of cell death with increased apoptosis in degenerative NP, which was further confirmed by the TUNEL assay. Phagocytic NP cells had the hallmarks of both stationary macrophages with lysosomes and NP cells with the endoplasmic reticulum. Annulus fibrosus cells have similar morphologic characteristics with NP cells in terms of cell nest, phagocytosis and intracellular organs. Moreover, NP cells with long processes existed in degenerative and scoliotic NP rather than normal NP. When cultured in glucose-free medium, NP cells developed long and thin processes.Conclusion: Human degenerative NP consists of primarily viable cells. We present direct and in vivo evidence that both human annulus fibrosus and NP cells have phagocytic potential. Moreover, NP cells with long processes exist in both scoliotic and degenerative NP with lack of glucose as one of the possible underlying mechanisms.

We have read with interest the research article by Philip Jones and colleagues, in which they studied whether bovine nucleus pulposus (NP) cells were capable of behaving as phagocytes [1]. Collectively, they drew the conclusion that disc cells clearly can undergo phagocytosis, which has implications for the intervertebral disc in vivo. We would like to differ from the authors, however, regarding the implications of the role of NP cells in vivo in comparison with in vitro as competent phagocytes to ingest apoptotic cells. First, NP cells cultured in vitro in a monolayer may not reflect the same physiological status as they showed in vivo. In fact, the extracellular matrix of NP cells consists largely of water, collagen type II and aggrecan [2]. The cell concentration within the disc is relatively sparse, making up only about 1% of the disc volume. NP cells in vivo therefore distribute sparsely in the intervertebral disc with ample extracellular matrix surrounding. Intercellular communications might pointedly differ from those in cell culture conditions, in which NP cells contact directly and closely. From this point of view, it may appear an impossible mission for NP cells to clear apoptotic cells as phagocytes in vivo. Second, the authors might have omitted one important hallmark of NP cells – that is, immune privilege. In fact, there is accumulating evidence demonstrating that Fas ligand (CD178) is highly expressed in human, rat and rabbit normal NP cells [3,4]. In immune privileged organs, Fas ligand has been shown to act via the induction of apoptosis on invading Fas-positive activated T cells and thus protects the cells from immune attack. The question of whether macrophages take part in the interaction of NP cells with Fas ligand and consequently contribute to clearance of apoptotic cells, however, remains open. Whether the death of NP cells occurs by apoptosis or necroptosis also remains unclear [5]. At present, therefore, we may not exclude macrophages perhaps playing a role in the clearance of apoptotic cells. Third, despite the special avascular hallmark of intervertebral discs, cells in the center of the disc exist at low concentrations of oxygen. Oxygen concentrations as low as 1% have been measured in the centers of discs [6]. The oxygen concentration in Jones and colleagues' paper was 21%, however, which may not be consistent with the physiological conditions of NP cells. As a consequence, the conclusion the authors made on the basis of altered oxygen concentration and the subsequently changed cellular physiology may not be reliable. Taking these points together, a more appropriate NP cell culture system and the role of macrophages in immune privilege of NP cells should be further explored.

Introduction Our previous work have proved that Syndecan 4 (SDC4) plays an important role in TNFa and IL1b induced disc degeneration by promoting extracellular matrix degradation, Syndecan 2 (SDC2) and SDC4 both are members of SDC family, however, expression and role of SDC2 in intervertebral disc is still unclear. The role of this study is to investigate the expression pattern and regulation of SDC2 and its and role in intervertebral disc degeneration. Materials and Methods Nucleus pulposus (NP) cells and annulus fibrosis (AF) cells were cultured separately. mRNA were extracted and expression of SDC2 in NP and AF cells were examined by q-PCR method. Expression of SDC2 was examined by q-PCR in 4, 8, 24hs after NP cells were treated by TNFa (20mg/L), IL1-b(10mg/L) and TGF-b (20mg) retrospectively. NP cells were pretreated with p38, JNK and NFkB signaling pathway inhibitors before treatment with TNFa, then expression of SDC2 was examined by qPCR. Results SDC2 was expressed in both NP and AF cells. NP cells have a higher expression level than AF cells of SDC2 in mRNA level. TNFa and IL1b could inhibit SDC2 expression in NP cells robustly. What's more, SDC2 expression was induced after TGFb treatment in NP cells. Inhibition of SDC2 expression by TNFa could be reduced by p38 and NFkB pathway inhibitor. JNK inhibitor has no role in Inhibition of SDC2 expression by TNFa. Conclusion Inflammatory cytokines could inhibit SDC2 expression and induce SDC4 expression in NP cells, p38 and NFkB pathway participant in this process. The different regulation pattern of SDC2 and SDC4 might play important role in keeping intervertebral disc homeostasis. Disclosure of Interest None declared

Objective To investigate the role of PI3K/AKT signaling pathway in nucleus pulposus (NP) cells. Methods Nucleus pulposus (NP) cells were isolated from SD rat, and thereafter, passage three (P3) NP cells were divided into the following experimental groups: control, PI3K/AKT agonist IGF-1 (25 ng/ml, 50 ng/ml, and 100 ng/ml), and PI3K/AKT inhibitor LY294002 (5 μM, 10 μM, and 20 μM). Flow cytometry and BrdU cell proliferation assays were performed to assess apoptosis and the proliferation rate of NP cells. Western blot analysis was performed to examine the protein expression level of Col II, Col X, Aggrecan, and MMP13. Results PI3K/AKT inhibitor LY294002 increased the rate of apoptosis in NP cells when compared to the control and decreased the proliferation rate when compared to control. Moreover, LY294002 decreased the protein expression level of Col-II and Aggrecan in NP cells. At the same time, LY294002 increased the protein expression level of MMP13 and Col-X in NP cells. Through activating PI3K/AKT, IGF-1 increased the proliferation rate when compared to control and decreased the rate of apoptosis when compared to control. Additionally, IGF-1 decreased the protein expression level of MMP13 and Col-X and increased Col-II and Aggrecan in NP cells. Conclusion The inhibition of PI3K/AKT signaling pathway accelerated the apoptosis of NP cells and facilitated the extracellular matrix degradation. However, the activation of PI3K/AKT pathway partly prevented the NP cell from apoptosis and promoted their proliferation. Meanwhile, its activation also delayed the loss of extracellular matrix.

The interaction between co-cultured human nucleus pulposus cells and mesenchymal stem cells in a bioactive scaffold

Introduction Mesenchymal stem cells (MSCs) have been proposed for the purposes of tissue engineering and repair of intervertebral disk (IVD) tissue. These cells would be induced to differentiate into cells with a disc-cell like phenotype, as an alternative treatment for against disk degeneration. One major drawback is that there currently exists no simple way to distinguish a nucleus pulposus (NP) cell from its annulus fibrosus (AF) or hyaline cartilage counterparts. In addition, immature NP cells produce similar macromolecules to those of hyaline cartilage1; therefore, it is difficult to confirm MSCs differentiation into a disk cell phenotype. Our previous studies have shown that surfaces rich in primary amines, -NH2, can influence the adhesion properties of cells.2 In this study, we created reproducible NH2-rich surfaces by low-pressure plasma polymerization of ethylene-ammonia mixture (so-called “L-PPE: N”) to test the hypothesis that selective adhesion of disk cells can be used to distinguish between an NP and an AF phenotype. This would serve as a marker to distinguish an appropriate NP, generated by tissue engineering techniques, from other cartilaginous phenotypes that may be inappropriate for function in the disc. Materials and Methods Surface preparation L-PPE: N coatings approximately 100 nm thick were prepared as previously described.3 These deposits, which contain about 7.5% NH2, were deposited on poly(ethylene terephthalate) (PET) film in a low-pressure (L) capacitively coupled radio-frequency glow-discharge plasma reactor. However, half of the films' surfaces were masked, so as to remain uncoated, bare PET. Cell isolation and culture Adult bovine tails (2 to 3 years old) were obtained 2 hours after slaughter at a local abattoir. The IVDs were dissected from their adjacent vertebral bodies and separated into nucleus pulposus (NP) and AF. Both tissues of the AF and the NP were first weighed (wet weight) and then subjected to a pronase digestion (0.2% W/V), followed by a digestion of collagenase 1A (0.04% W/V) for NP tissue and collagenase II (0.04% W/V) for AF tissue. After isolation, the AF cells and the NP cells were separately cultured on the half-coated surfaces in DMEM high glucose, supplemented with 10% FBS and 1% penicillin/streptomycin in a concentration of 1 × 106 cells/mL. Regular polystyrene culture dishes (TCPS) were used as a control. After 4 days of culture, cells were washed with ddH20, fixed and stained with Safranin-O. Images were captured of both cell types before and after wash, using an optical microscope. Results AF cells adhered preferentially to the L-PPE: N-coated surface, but did not adhere to the bare part of the PET surface (Fig. 1). NP cells, however, indiscriminately adhered to both PET and L-PPE: N surfaces. Both NP and AF cells adhered to regular TCPS dishes after 4 days. After washing with ddH20, which was meant to remove any nonadherent cells, and to provide a clearer image to be captured, AF cells remained bound to L-PPE:N surfaces; however, NP cells were completely removed from both surfaces. Conclusion In this study, we show that AF cells adhered preferentially to the substrate's portion that was L-PPE: N-coated, whereas NP cells did not manifest any preference to coated or uncoated portions. This preferential attachment to an amine-containing surface can be used to distinguish NP from AF cells; together with the production of a high GAG-to-hydroxyproline (proteoglycan-to-collagen) ratio by NP cells, it could help in identifying an NP-like phenotype. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Mwale F, et al. European Cells and Materials 2004;8:58–64 Girard-Lauriault PL, et al. Macromolecular Bioscience 2009;9:911–921 Girard-Lauriault PL, et al. Plasma Processes and Polymers 2005;2:263–270

Disc degeneration is strongly associated with back pain and herniation that increase the costs of health care. The degeneration of intervertebral disc (IVD) could be divided into 5 stages. In the first and second stages, there are no significant symptoms but could be traced by magnetic resonance imaging or computed tomography-scan. Generally, no aggressive treatment would be processed in the clinics. Recent studies indicated that overproduction of reactive oxygen species (ROS) may accelerate the degenerative process of IVD and associate with apoptosis of nucleus pulposus (NP) cells and degradation of extracellular matrix. Ferulic acid (FA) is an excellent antioxidant and relatively stable in air. FA has been proven to have ability to prevent ROS-induced diseases. The object of the study was aimed to evaluate the possible therapeutic effect of FA on hydrogen peroxide (H2O2)-induced oxidative stress NP cells and the feasibility of use the thermosensitive chitosan/gelatin/glycerophosphate (C/G/GP) hydrogel as a sustained release system of FA for early treatment in IVD degeneration. In the study, NP cells were harvested from the IVD of New Zealand rabbits. The results showed that 500 μM of FA might be the threshold to treat NP cells without cytotoxicity. Post-treatment of FA on H2O2-induced oxidative stress NP cells significantly up regulated the expression of aggrecan, type II collagen and BMP-7 and down regulated the expression of MMP-3 in mRNA level. Post-treatment of FA on H2O2-induced oxidative stress NP cells could restore the production of sulfated glycosaminoglycans (GAGs) and inhibit the apoptosis caused by H2O2. The results showed that the release of FA from C/G/GP hydrogel could decrease the H2O2-induced oxidative stress. Post-treatment of FA-incorporated C/G/GP hydrogel on H2O2-induced oxidative stress NP cells showed up-regulation of aggrecan and type II collagen and down-regulation of MMP-3 in mRNA level. The results of sulfated GAGs to DNA ratio and alcian blue staining revealed that the GAGs production of H2O2-induced oxidative stress NP cells could reach to normal level. The results of caspase-3 activity and TUNEL staining indicated that FA-incorporated C/G/GP hydrogel decreased the apoptosis of H2O2-induced oxidative stress NP cells. The results showed that FA was successfully immobilized on C/G/GP hydrogel by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) crosslinking method. The gelation temperature of the FA-immobilized C/G/GP hydrogel was 31.80 degree celsius under neutral pH. Post-treatment of FA-immobilized C/G/GP hydrogel on H2O2-induced oxidative stress NP cells showed down-regulation of MMP-3 and up-regulation aggrecan and type II collagen in mRNA level. The sulfated GAGs production of H2O2-induced oxidative stress NP cells could be increased to the normal level in the post-treatment of FA-immobilized C/G/GP hydrogel group. The results of caspase-3 activity and TUNEL staining showed that the apoptosis of H2O2-induced oxidative stress NP cells could be inhibited by post-treatment of FA-immobilized C/G/GP hydrogel. From the results of the study, FA could be used as a therapeutic molecule for NP regeneration and FA-incorporated C/G/GP hydrogel might be potentially applied as a long-term release system. The immobilization of FA on C/G/GP hydrogel could significantly prolong the release period of FA. These results suggest that combination of FA and thermosensitive C/G/GP hydrogel can treat NP cells from the damage caused by oxidative stress and may apply in minimally invasive surgery for NP regeneration in the future.

Objective To study the viscoelastic properties of nucleus pulposus (NP) cells from human in vitro. Methods NP was obtained from discarded NP tissue of 3 scoliosis patients aged from 13 to 16 years. Pancreatin and collagenase type Ⅱ were used to digest NP and cells were isolated from NP. Type Ⅱ collagen immunofluorescence and Fan seaing were used to identify NP cells. The micropipette aspiration test was used in combination with a three-parameter viscoelastic solid model to measure the mechanical properties of NP cells. Results The mean diameter of the digested NP cells was ( 15.40 ± 1.83) μm. In response to a prescribed pressure, the NP cells exhibited viscolastic solid creep behavior, which was characterized initially by a jump in displacement followed by a monotony decreasing rate of deformation that generally reached an equilibrium. NP cells were deformed to a length as much as 2 times the radius of the micropipette without completely entering the micropipette. The viscolastic parameters were k1 (0. 101 ±0. 052) kPa, k2 (0. 353 ± 0. 199) kPa, and μ ( 3. 034 ± 1. 843 ) kPa· s, respectively. Only the k1 was positively correlated to the cell diameter (r =-0. 389, P ＜ 0. 05 ). Conclusion Human normal NP cells behave as a typical viscolastic solid creep. Micropipette aspiration technique is a valid method for the study on biomechanics of NP cells. Key words: Nucleus pulposus cells; Biomechanics; Viscoelastic properties

Objective: Intervertebral disc degeneration (IVDD) is very common in the elderly, so it is particularly important to find appropriate prevention or treatment. The aim of this study was to explore the effect of dexmedetomidine (DEX) on the degeneration of nucleus pulposus (NP) cells and its mechanism. Methods: We established a mouse model of IVDD and cultured mouse NP cells and treated them with IL-1β and DEX. The effect of DEX on NP cells was determined by detecting the extracellular matrix of NP cells, changes in ROS levels and inflammatory mediators. LY294002, a PI3K inhibitor, is used to inhibit the activity of the PI3K/Akt signaling pathway. The effect of DEX on the PI3K/Akt signaling pathway was determined by studying the effects of DEX on PI3K/ Akt signaling pathway-related molecules and the effect of LY294002 on NP cells degeneration. DEX significantly increased the disc height index and attenuated IVDD in mice. Results: DEX significantly inhibited the expression of MMP3/9 in NP cells, effectively inhibiting the degradation of extracellular matrix. In addition, oxidative stress levels and inflammatory levels in NP cells are also attenuated by DEX. The expression of PI3K, Akt and p-Akt was significantly increased in DEX-stimulated NP cells, indicating that DEX increased the activity of the PI3K/Akt signaling pathway. DEX promotes PI3K/Akt signaling pathway, inhibits oxidative stress and inflammatory of NP cells, thereby slowing the degeneration of NP cells. Conclusion: DEX promotes PI3K/Akt signaling pathway, inhibits oxidative stress and inflammatory of NP cells, thereby slowing the degeneration of NP cells.

IntroductionNucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.MethodsMicroarrays were conducted on bovine AC, AF and NP cells, using Affymetrix Genechip® Bovine Genome Arrays. Differential expression levels for a number of genes were confirmed by quantitative real time polymerase chain reaction (qRT-PCR) on bovine, AC, AF and NP cells, as well as separated bovine NP and notochordal (NC) cells. Expression of these novel markers were further tested on normal human AC, AF and NP cells, and degenerate AF and NP cells.ResultsMicroarray comparisons between NP/AC&AF and NP/AC identified 34 NP-specific and 49 IVD-specific genes respectively that were differentially expressed ≥100 fold. A subset of these were verified by qRT-PCR and shown to be expressed in bovine NC cells. Eleven genes (SNAP25, KRT8, KRT18, KRT19, CDH2, IBSP, VCAN, TNMD, BASP1, FOXF1 & FBLN1) were also differentially expressed in normal human NP cells, although to a lesser degree. Four genes (SNAP25, KRT8, KRT18 and CDH2) were significantly decreased in degenerate human NP cells, while three genes (VCAN, TNMD and BASP1) were significantly increased in degenerate human AF cells. The IVD negative marker FBLN1 was significantly increased in both degenerate human NP and AF cells.ConclusionsThis study has identified a number of novel genes that characterise the bovine and human NP and IVD transcriptional profiles, and allows for discrimination between AC, AF and NP cells. Furthermore, the similarity in expression profiles of the separated NP and NC cell populations suggests that these two cell types may be derived from a common lineage. Although interspecies variation, together with changes with IVD degeneration were noted, use of this gene expression signature will benefit tissue engineering studies where defining the NP phenotype is paramount.

Introduction Integrity of the nucleus pulposus (NP) has been implicated in the function and homeostasis of intervertebral disc (IVD). Understanding the regulation of NP cells would contribute to their engineering and development of therapeutics for treating IVD degeneration. Studies in mouse models indicate that early events of IVD degeneration involve segregation of the notochordal NP cell clusters,1 suggesting that disc degeneration may be associated with cell adhesion molecule activities. Cadherins are transmembrane glycoproteins that mediate calcium dependent cell adhesion.2 Based on microarray analysis, we have revealed specific expression of Cdh2 gene, encoding cadherin 2/N-cadherin, in rodent NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. To date, the function of cadherin 2 in IVD and its relationship to IVD degeneration remain elusive. We hypothesize that cadherin 2 has a regulatory role in NP cells and that a deregulation of its activities has adverse effects on IVD homeostasis. We aimed to study the expression pattern of cadherin 2 in rodent discs during development, aging, and degeneration, and to investigate its function in the NP via a gene and protein ablation strategies. Materials and Methods Animal experiments were approved by local ethics committee. The vertebral columns of wild-type C57BL/6N mice were collected at different ages ( n = 4): embryonic day (E) 12.5, E14.5, postnatal day 0 (P0), 3 months old, 6 months old, 1 year old, 1.5 and 2 years old. Progressive disc degeneration was induced by annulus puncture of 4-month-old inbred Lewis rats ( n = 6) with 25G needle, and the discs were harvested after 2-, 4-, and 8-weeks of operation. Lumbar IVD of scoliosis patients and lower lumbar spine of the aborted fetus in the second trimester were used as controls. Immunohistochemistry was performed on paraffin sections to study the cadherin 2 expression pattern. 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 rabbit IgG ( n = 6) into the NP via 34G hypodermic needle. Cadherin 2 gene (Cdh2) was knocked out in the NP using notochord-specific Foxa2-Cre recombination strategy. Disc height was measured and expressed as disc height index (%DHI). The IVD were harvested by 2 and 8 weeks after operation (protein ablation) and from P0 and 1 month-old mutants (gene knockout) for histological analysis. Results By immunofluorescence, cadherin 2 was weakly detected in murine embryonic notochord and newborn NP. At 3- and 6-month old, strong cadherin 2 signals were specifically detected as foci along the cell-cell junctions of the vacuolated NP cells (notochordal cells). Annulus fibrosus showed no signals. In aged IVD, the notochordal cells were replaced by small chondrocyte-like cells with lower expression of cadherin 2. In puncture-induced degenerative rodent discs, the notochordal NP cells were replaced by rounded chondrocyte-like cells, showing reduced level of cadherin 2 expression. Human NP showed heterogeneous cadherin 2 expression. By injecting cadherin 2 antibody into rat NP to perturb its function in vivo, cadherin 2 expression was reduced along with reduction of disc height. Compared with the IgG injection control, cadherin 2 antibody ablation group showed a transformation of the notochordal NP cells into less vacuolated chondrocyte-like phenotype with upregulation of collagen II. Cdh2 conditional knockout mice (CKO) showed absence of cadherin 2 and a loss of vacuolated phenotype in the NP cells (Fig. 1A), displaying significantly smaller body size by 1-month old (Fig. 1B). Moreover, the mutant exhibited irregular annulus organization and reduced disc height in IVD (V). Conclusion Our study suggests cadherin 2 as a marker of notochordal NP cells. The lower expression of cadherin 2 in the aged and puncture-injured rodent IVD substantiates its association with IVD degeneration. 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. Our parallel in vivo studies of gene/protein ablation support that cadherin 2 is essential to the maintenance of a vacuolated phenotype of the notochordal NP cells, and that a loss of cadherin 2 function may initiate degenerative changes in the IVD. In summary, our study implicates an important role of cadherin 2 in notochordal cell function and regulating IVD homeostasis. Acknowledgments This work is funded by the Area of Excellence grant (AoE/M-04/04) and the General Research Fund (HKU763712M) from the Research Grant Council of Hong Kong. Disclosure of Interest None declared References Yang F, Leung VY, Luk KD, Chan D, Cheung KM. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. J Pathol 2009;218(1):113–121 Leckband D, Prakasam A. Mechanism and dynamics of cadherin adhesion. Annu Rev Biomed Eng 2006;8:259–287

The goal of this investigation was to study the expression and regulation of beta1,3-Glucuronosyltransferase-I (GlcAT-I), a key enzyme regulating GAG synthesis in cells of the intervertebral disc. There was a robust expression of GlcAT-I in the nucleus pulposus in vivo. Treatment with the calcium ionophore ionomycin resulted in increased GlcAT-I expression, whereas GlcAT-I promoter constructs lacking TonE site or a mutant TonE were unresponsive to the ionophore. Experiments using TonEBP and DN-TonEBP constructs showed that TonEBP positively regulated GlcAT-I promoter activity. ChIP analysis confirmed binding of TonEBP to the promoter. We further validated the role of TonEBP in controlling GlcAT-I expression using mouse embryo fibroblasts from TonEBP null mice. GlcAT-I promoter activity in null cells was significantly lower than the wild type cells. In contrast to wild type cells, treatment with ionomycin failed to increase GlcAT-I promoter activity in null cells. We then investigated if calcineurin (Cn)-NFAT signaling played a regulatory role in GlcAT-I expression. Inhibition of Cn following ionomycin treatment did not block GlcAT-I and tauT, a TonEBP-responsive reporter activity. GlcAT-I promoter activity was suppressed by co-expression of Cn, NFAT2, NFAT3, and NFAT4. Moreover, following ionomycin treatment, fibroblasts from CnAalpha and CnAbeta null mice exhibited robust induction in GlcAT-I promoter activity compared with wild type cells. Results of these studies demonstrate that calcium regulates GlcAT-I expression in cells of the nucleus pulposus through a signaling network comprising both activator and suppressor molecules. The results suggest that by controlling both GAG and aggrecan synthesis, disc cells can autoregulate their osmotic environment and accommodate mechanical loading.