Hyaluronan-Coated Aligned Collagen Hydrogel Promotes Stabilization of the Intervertebral Disc by Inducing Fibrosis in Annulus Fibrosus.

Decision letter: DIPPER, a spatiotemporal proteomics atlas of human intervertebral discs for exploring ageing and degeneration dynamics

The spatiotemporal proteome of the intervertebral disc (IVD) underpins its integrity and function. We present DIPPER, a deep and comprehensive IVD proteomic resource comprising 94 genome-wide profiles from 17 individuals. To begin with, protein modules defining key directional trends spanning the lateral and anteroposterior axes were derived from high-resolution spatial proteomes of intact young cadaveric lumbar IVDs. They revealed novel region-specific profiles of regulatory activities and displayed potential paths of deconstruction in the level- and location-matched aged cadaveric discs. Machine learning methods predicted a ‘hydration matrisome’ that connects extracellular matrix with MRI intensity. Importantly, the static proteome used as point-references can be integrated with dynamic proteome (SILAC/degradome) and transcriptome data from multiple clinical samples, enhancing robustness and clinical relevance. The data, findings, and methodology, available on a web interface (http://www.sbms.hku.hk/dclab/DIPPER/), will be valuable references in the field of IVD biology and proteomic analytics.

Intervertebral disc generation tissue engineering treatment platform: gelatin-methacryloyl hydrogel composite system.

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.

IntroductionMultiple therapies for treating intervertebral disc (IVD) degeneration are under development and range from injectable biologics to implantable devices. Treatments that show promise in ...

Painful intervertebral disc (IVD) degeneration (IVDD) involves chronic inflammation. Developing translational immunomodulatory strategies for IVDD is a priority with tumor necrosis factor alpha (TNFα) signaling an important target. TNFα binds to 2 receptors (TNFRs), with TNFR1 signaling promoting catabolism and apoptosis and TNFR2 signaling promoting anabolism and proliferation. This study developed translational strategies to evaluate and modulate TNFR1 and TNFR2 signaling in rat invivo and invitro IVDD models. We used blocking antibodies, the TNFR2-activator Atsttrin, and small molecule inhibitors of TNFR1 to discern distinct TNFR1 and TNFR2-effects on annulus fibrosus (AF) and nucleus pulposus (NP) cells and to identify effective strategies for modulating specific TNFRs. TNFR1 was significantly increased with IVDD invivo in the NP while TNFR2 was unaffected with very faint staining. TNFR1-specific small molecule inhibitors were effective in reducing catabolic effects of TNFα, highlighting the efficacy of this small molecule strategy for TNFR1 signaling modulation. Meanwhile, TNFR1 and TNFR2 inhibition invitro was not effective with blocking antibodies on NP or AF cells, likely due to species-specificity of available blocking antibodies. Further, TNFR2 activation with Atsttrin was similarly ineffective, likely due to extremely low TNFR2 levels in both AF and NP cells. TNFα receptor-specific signaling is important in rat IVDD invivo and invitro. TNFR1 inhibition was more effective with small molecules than using blocking antibodies. Low levels of TNFR2 in rat AF and NP cells and lack of efficacy of TNFR2-activator Atsttrin suggest native AF and NP cells have little capacity for TNFR2-dependent IVD repair.

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

IntroductionLow back pain, often due to intervertebral disk (IVD) degeneration, is an age-associated disorder that afflicts more than half of the population, above age 70. Discs are composite struc...

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.

Intervertebral disc (IVD) herniation is a common origin of disabled spine and low back pain (LBP) which cause enormous health care toll. The annulus fibrosus (AF), which constitutes the outer part ...

BackgroundIntervertebral disc (IVD) degeneration is suggested as a major cause of chronic low back pain (LBP). Intradiscal delivery of growth factors has been proposed as a promising strategy for IVD repair and regeneration. Previously, BMP-4 was shown to be more potent in promoting extracellular matrix (ECM) production than other BMPs and TGF-β in human nucleus pulposus (NP) cells, suggesting its applicability for disc regeneration.MethodsThe effects of BMP-4 on ECM deposition and cell proliferation were assessed in sheep NP and annulus fibrosus (AF) cells in a pellet culture model. Further, a nuclectomy induced sheep lumbar IVD degeneration model was used to evaluate the safety and effects of intradiscal BMP-4 injection on IVD regeneration. Outcomes were assessed by magnetic resonance imaging, micro-computed tomography, histological and biochemical measurements.ResultsIn vitro, BMP-4 significantly increased the production of proteoglycan and deposition of collagen type II and proliferation of NP and AF cells. Collagen type I deposition was not affected in NP cells, while in AF cells it was high at low BMP-4 concentrations, and decreased with increasing concentration of BMP-4. Intradiscal injection of BMP-4 induced extradiscal new bone formation and Schmorl's node-like changes in vivo. No regeneration in the NP nor AF was observed.ConclusionOur study demonstrated that although BMP-4 showed promising regenerative effects in vitro, similar effects were not observed in a large IVD degeneration animal model.The Translational Potential of This ArticleThe contradictory results of using BMP-4 on IVD regeneration between in vitro and in vivo demonstrate that direct BMP-4 injection for disc degeneration-associated human chronic low back pain should not be undertaken. In addition, our results may also shed light on the mechanisms behind pathological endplate changes in human patients as a possible target for therapy.

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

IntroductionLow back pain is a debilitating disorder affecting a multitude of people and is currently ranked sixth among costliest treatable chronic illnesses.1 Low back pain is often attributed to...

Tissue-Engineered Intervertebral Discs: In Vivo Outcome in the Rodent Spine

IntroductionAlthough there are different causes of low back pain, intervertebral disc (IVD) degeneration is one of them. In healthy discs, nociceptive nerve fibers and mechanoreceptors penetrate up...