Short-Term Dynamic Unloading of Bovine Tail Discs in Culture Partially Mitigates Induced Degeneration After One-Strike Trigger.

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

Intervertebral disc (IVD) degeneration is the major cause of back pain. Notch signaling is activated in annulus fibrosus (AF) and nucleus pulposus (NP) tissues of degenerated IVDs, and induced by IL1-β and TNF-α in NP cells. However, the role of Notch activatin in the pathogenesis of IVD degeneration is largely unknown. In this study, we overexpressed the Notch1 intracellular domain (NICD1) in AF, NP, and chondrogenic ATDC5 cells via adenoviruses. Overexpression of NICD1 activated transcription of Notch signaling target genes in AF, NP, and ATDC5 cells, and caused cell type-specific effects on expression of matrix anabolic and catabolic genes. Activation of Notch signaling promoted expression of matrix catabolic genes and inhibited expression of matrix anabolic genes in both AF and ATDC5 cells, whereas its activation suppressed expression of matrix catabolic genes (including Mmp3, Mmp13, Adamts4, and Adamts5) and attenuated TNF-α and inflammatory macrophage-induced Mmp13 expression in NP cells. Consistently, sustained activation of Notch1 signaling in postnatal IVDs in mice severely disrupted growth plate and endplate cartilage tissues, but did not overly affect NP tissues. Together, these data indicated that activation of Notch signaling exerted differential and cell type-specific effects in intervertebral discs, and specific Notch signaling regulation may be considered during the treatment of IVD degeneration.

Nucleus pulposus (NP) cell proliferation plays a key role during the process of intervertebral disc degeneration (IDD). S-phase kinase-associated protein-2 (Skp2) has been proved as an important regulator for cell growth factors in vitro. Nonetheless, whether Skp2 attenuates IDD by mediating NP cell proliferation still remains unclear. Therefore, the aim of this study was to explore how Skp2 affected NP cell proliferation and the potential mechanism in vitro. In this study, we first collected different degenerated human NP samples and isolated NP cells from these tissues. NP cell degenerated model was established with IL-1β, and the cells were transfected with lentivirus to achieve Skp2 overexpression. Besides, SKPinC1 was used to suppress Skp2 expression in vitro. Western blot, RT-PCR, and immunocytofluorescence were applied to detect genetic differences among groups. Furthermore, cell viability and cell cycle were determined by CCK-8 assay and flow cytometry, respectively. Skp2 expression decreased significantly in degenerated disc samples (p<0.05). IL-1β stimulation significantly promoted NP cell degeneration, which could be reversed by Skp2 overexpression (p<0.05). Meanwhile, Skp2 in IDD significantly inhibited the expression level of medial p27 and promoted cell cycle by CDK2 activation (p<0.05). In addition, Skp2 suppression affected NP cell proliferation in vitro. NP cells exhibited significantly inhibited proliferation ability when down-regulated the expression level of Skp2. Our findings provided a more meritorious viewpoint of Skp2 in NP cell proliferation. Furthermore, the above results suggested that Skp2 was a novel target in the treatment of IDD.

Thursday, September 27, 2018 2:05 PM–3:05 PM Section on Biologics and Basic Research Abstract Presentations: 124. NF-κB dependent unfolded protein response in the survival and proliferation of nucleus pulposus cells under TNF-α stimulation

IntroductionThe aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.MethodsReal-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.ResultsLDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.ConclusionsOur data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.

Objective To investigate the expression of Protease nexin-1 (PN-1) in Intervertebral Disc nucleus pulposus (NP) cells and the related signal pathways. Methods Human primary NP cells were isolated and cultured, and divided into control group, interleukine-1β (IL-1β), TNF-α group. Histological examinations were undertaken to observe the morphologic changes of NP cells during the process of differentiation at day 3, 7, 14 and 21. MMPs expression in NP cells were accessed by Western blot. Then, NP cells were cultured in medium containing 10 ng/ml IL-1β and different concenration of PN-1 (0, 10, 50, 100 ng/ml) for 24h. Protein were extracted, and The expression level of Interleukin-1 receptor-associated kinase 1 (IRAK1) , p38 mitogen-activated protein kinase (p38 MAP Ki-nase), phosphos-P38 MAP Kinase (P-P38) wereanalyzed by using Western blot. Results Histologically, under induction with IL-1β, both morphological and biological properties of NP cells were changed, cell proliferation became slowly, cell density was sparse within disordered arrangement, and obvious cell necrosis appeared. The results also showed the degenerative process of NP cells, MMPs production increased and Agg and Col II protein decreased significantly. PN-1 administration could inhibit the activation of MMP3,MMP9 and MMP13 induced by IL-1β, and enhance the expression of AGG and Col2. IL-1β administration could stimulate Nuclear factor κB (NF-κB) signal pathway through the up-regulated expression of IRAK1 and P-P38, while PN-1 could inhibit the inductive effects by suppress the expression of IRAK1 and the phosphorylation of P-38, thereby, inhibit the degeneration of NP cells. Conclusion PN-1 is involved during the degeneration of NP cells. PN-1 administration could antagonize IL-1β-induced proteases and inhibit the activation of MMPs, and potentially preserve NP cells, and reverse its degeneration via IRAK1/NF-κB signaling pathway. Key words: Intervertebral disc degeneration; Interleukin-1 Receptor-Associated Kinases; NF-kappa B

This study investigates the native presence and potential anabolic effects of interleukin (IL)-4 and IL-10 in the human intervertebral disc (IVD). Human nucleus pulposus (NP) cells cultured in 3D from trauma and degenerate IVDs and NP explants were stimulated with 10 ng/mL IL-4, IL-10, or each in combination with 1 ng/mL IL-1β stimulation. The role of IL-4 and IL-10 in the IVD was evaluated using immunohistochemistry, gene expression, and Luminex multiplex immunoassay proteomics (73 secreted) and phosphoproteomics (21 phosphorylated proteins). IL-4, IL-4R, and IL-10R expression and localization in human cartilage endplate tissue were demonstrated for the first time. No significant gene expression changes were noted under IL-4 or IL-10 stimulation. However, IL-1β stimulation significantly increased MMP3, COX2, TIMP1, and TRPV4 expression in NP cells from trauma IVDs. Combined IL-4 and IL-1β treatment induced a significant increase in protein secretion of IL-1α, IL-7, IL-16, IL-17F, IL-18, IFNγ, TNF, ST2, PROK1, bFGF2, and stem cell factor exclusively in NP cells from degenerated IVDs. Conversely, the secretome profile of explants revealed an IL-4-mediated decrease in CXCL13 following treatment with IL-1β. Combined IL-10 and IL-1β treatment increased neurotrophic growth factor secretion compared with IL-10 baseline. The NP cell phenotype affects the pleiotropic role of IL-4, which can induce a pro-inflammatory response in the presence of catabolic stimuli and enhance the effects of IL-1β in degenerated IVDs. Environmental factors, including 3D culture and hypoxia, may alter IL-4's role. Finally, IL-10's potential neurotrophic effects under catabolic stimuli warrant further investigation to clarify its role in IVD degeneration.

Bardoxolone methyl breaks the vicious cycle between M1 macrophages and senescent nucleus pulposus cells through the Nrf2/STING/NF-κB pathway

Dear Editor, We read with interest and appreciation the article by Park et al. [1] attempting to illustrate the role of reactive oxygen species (ROS) in notochordal cells under high glucose concentrations. The study indicated that high glucose-induced oxidative stress promotes autophagy through mitochondrial damage of rat notochordal cells in a dose- and time-dependent manner. In humans, most notochordal cells are larger than nucleus pulposus (NP) cells, containing vacuoles, and disappear within the first decade of life [2]. A previous study showed that notochordal cells will be first replaced by smaller chondrocyte-like NP cells and then replaced by cells with a fibrocartilagenous phenotype with age [3]. The chondrocyte-like NP cells are the major cells in the adult nucleus pulposus and excessive apoptosis of NP cells plays an important role in the development of intervertebral disc degeneration[4]. As we know, there are two main types of diabetes: type1 diabetes and type 2 diabetes. Type 2 diabetes accounts for 90–95 % percent of all diabetes cases, and it usually occurs in people aged over 40. Moreover, intervertebral disk disease occurs primarily in middle aged and older persons. Therefore, NP cells may be the main cells which are affected by hyperglycaemia in diabetic patients with degenerative disc diseases. However, it is still unknown whether ROS-mediated mitochondrial dysfunction also play a critical role in high glucose-induced NP cell injury. Therefore, we performed a study to investigate the question. In our study, rat NP cells showed chondrocyte-like morphology under phase-contrast microscope (Fig. 1A1). By immunohistochemical staining (Fig. 1A2) and immunofluorescence staining (Fig. 1A3), cells were demonstrated to exhibit collagen II protein expression. To determine the effects of high glucose concentrations on cell viability, cells were treated with two concentrations (0.1M and 0.2M) of high glucose for 24 hours. Hoechst 33258 nuclear staining showed that the majority of NP cells exhibited significant morphological features of apoptosis such as bright nuclear condensation or fragments after treatment with high glucose concentrations (Fig. 1B2 and 1B3). To further explore whether ROS-mediated mitochondrial dysfunction plays a causal role in high glucose-induced NP cell injury, the level of ROS and mitochondrial membrane potential in cells was detected by DCFH-DA assay and JC-1 assay, respectively. As anticipated, the intracellular ROS levels which exhibit a green fluorescence signal were obviously increased in both high glucose groups compared with the control group (Fig. 1c). Mitochondrial membrane potential (Δψm) is an important indicator of mitochondrial function. We observed that in both high glucose groups (Fig. 1D2 and 1D3), there was an obvious decrease in the Δψm with a decrease in red fluorescence (JC-1 polymers) which indicates the intact Δψm, and an increase in green fluorescence (JC-1 monomers), which indicates the disruption of Δψm. Fig. 1 The effect of high glucose concentrations on NP intracellular ROS production and mitochondrial membrane potential (ΔΨm). (a1) Morphology of NP cells observed by phase-contrast microscopy. (a2) NP cells identified by collagen II immunohistochemical ... Combined with the previous research by Park et al. [1], we can draw a conclusion that ROS-mediated mitochondrial dysfunction not only plays a critical role in notochordal cells but also in NP cells under high glucose concentration. Further, it confirmed our previous hypothesis that antioxidants may be a novel treatment option for diabetic patients with degenerative disc diseases [5].

Objective To explore the proliferation of human degenerate nucleus pulposus and annular fibrosus cell in vitro and compare the different biological behavior between the two kinds of cell after degeneration,and provide the new theoretical basis for the prevention and treatment of degenerative disc disease.Methods The samples of intervertebral disc tissue were collected from patients with lumbar disc herniation.The degree of degeneration was assessed by the pathological diagnosis and degenerate nucleus pulposus and annular fibrosus cell were cultured by enzymatic digestion and identified.In each case,the control groups of the nucleus pulposus cells and annulus fibrosus cells were cultured to the fifth generation.The inoculation density of cells was 1×105.The cell morphology of each generation was observed,while the proliferation of cells was detected by flow cytometry after 48h culture with the same conditions.Results The degenerate nucleus pulposus cell and annular fibrosus cell were in good condition in vitro.The percentages of S phase cell and proliferation index (PI) were both on the rise with the subculture.The PI of nucleus pulposus cells reached the peak in the 3rd generation; The PI of annulus cells was the highest in the generation 5.The proliferation activity of degenerate nucleus pulposus cell in 2～4 generations was higher than the degenerate annular fibrosus cell within the same generations (P＜0.05).Conclusion Different proliferative characteristics of the degenerate nucleus pulposus and annular fibrosus cell confirmed that the disc degeneration is reversible.The response mechanisms to the degenerate micro-environment are completely different in vivo and affects the entire disc degeneration progress. Key words: Intervertebral discs degeneration; Nucleus pulposus cells; Annulus fibrosus cells; Proliferation index

Inflammation plays an important role in the pathogenesis of intervertebral disc (IVD) degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNF‐α) has shown markedly higher expression in degenerated human disc tissue compared with healthy controls. Anti‐inflammatory treatment targeting TNF‐α has shown to alleviate discogenic pain in patients with low back pain. Therefore, in vitro and ex vivo inflammatory models utilizing TNF‐α provide relevant experimental conditions for drug development in disc degeneration research.The current method article addressed several specific questions related to the model establishment.(a) The effects of bovine and human recombinant TNF‐α on bovine nucleus pulposus (NP) cells were compared. (b) The required dose for an inflammatory IVD organ culture model with intradiscal TNF‐α injection was studied. (c) The effect of TNF‐α blocking at different stages of inflammation was evaluated.Outcomes revealed that bovine and human recombinant TNF‐α induced equivalent inflammatory effects in bovine NP cells. A bovine whole IVD inflammatory model was established by intradiscal injection of 100 ng TNF‐α/ cm3 disc volume, as indicated by increased nitric oxide, glycosaminoglycan, interleukin 6 (IL‐6), and interleukin 8 (IL‐8) release in culture media, and upregulation of MMP3, ADAMTS4, IL‐8, IL‐6, and cyclooxygenase (COX)‐2 expression in NP tissue. However, results in human NP cells showed that the time point of anti‐inflammatory treatment was crucial to achieve significant effects. Furthermore, anticatabolic therapy in conjunction with TNF‐α inhibition would be required to slow down the pathologic cascade of disc degeneration.

BackgroundRecently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin‐1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.PurposeHere, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence‐activated cell sorting‐based methodology to sort and analyze Tie2+ NPCs.MethodsWe present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2+ NPCs from the IVD cell population during the isolation process. A cross‐species phylogenetic analysis of Tie2 across species is presented.ResultsOur protocols are efficient towards labeling and isolation of Tie2+ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species.ConclusionsCurrent identification of Tie2+ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2+ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.

Destruction of extracellular matrix (ECM), especially collagen II and aggrecan, is an essential feature of intervertebral disc degeneration (IDD). This project planned to elucidate the role of cartilage-derived morphogenetic protein-1 (CDMP-1) in the collagen II and aggrecan synthesis of nucleus pulposus (NP) cells under the IL-1β induced degeneration. We cultured human primary NP cells in the different concentrations of IL-1β medium and analyzed the CDMP-1 level. Recombinant human CDMP-1 protein was used to co-culture with IL-1β to investigate its effects on collagen II and aggrecan synthesis of NP cells. Additionally, the bone morphogenetic protein type IB receptor (ALK6) gene silenced and upregulated NP cells were used to evaluate the function of ALK6 in the CDMP-1 treated NP cells. Collagen II, aggrecan, MMP9, MMP13, and TIMP4 expression level were analyzed to assess the ECM stability of NP cells. CDMP-1 gene expression decreased in the IL-1β treated NP cells with a dose-dependent. Appropriate CDMP-1 protein supplement contributed to the collagen II and aggrecan production, the suppression of MMP9 and MMP13, and the upregulation of TIMP4. However, the silencing of ALK6 rejected the positive function of CDMP-1 on the collagen II and aggrecan; on the contrary, ALK6 upregulation magnified the CDMP-1 induced collagen II and aggrecan production. CDMP-1 is efficient in promoting the collagen II and aggrecan synthesis of NP cells, which is probably based on the mediation of ALK6.

The aberrant proliferation of nucleus pulposus (NP) cells has been reported to be implicated in the pathogenesis of intervertebral disc degeneration (IDD). Previous studies have demonstrated that microRNAs (miRNAs), which are a group of small noncoding RNAs, are critical regulators of cell proliferation in various pathologies. However, the role of miRNA‑96 (miR‑96) in the proliferation of NP cells remains to be determined. In the present study, reverse transcription‑quantitative polymerase chain reaction was used to investigate the expression of miR‑96 in NP tissues from patients with IDD and healthy tissues from patients with traumatic lumbar fracture as the control. A dual‑luciferase reporter assay was used to investigate whether AT‑rich interaction domain 2 (ARID2) may be a direct target gene for miR‑96. Furthermore, isolated NP cells from patients with IDD were transfected with miR‑96 mimics and ARID2‑targeting small interfering RNAs; cell proliferation, and the protein expression of Akt, phosphorylated Akt and ARID2 were examined, whereas the effects of an Akt inhibitor on NP cell proliferation were also evaluated. The present results demonstrated that miR‑96 expression was significantly upregulated in IDD samples, and the level of miR‑96 expression was positively associated with disc degeneration grade, which was evaluated by a modified Pfirrmann grading system. In addition, the current study identified ARID2 as a direct gene target of miR‑96. Furthermore, it was demonstrated that ARID2 mRNA expression was inversely correlated with the expression of miR‑96 in NP tissues. In addition, miR‑96 overexpression promoted NP cell proliferation and induced Akt phosphorylation, which led to increased cyclin D1 translation. Notably, overexpression of ARID2 or treatment with an Akt inhibitor decreased the effect of miR‑96 on NP cell proliferation. In conclusion, the results of the present study indicate that miR‑96 may promote the proliferation of human degenerated NP cells by targeting ARID2 via activation of the Akt pathway, and potentially serves as a therapeutic target for IDD.

The main pathological mechanism of intervertebral disc degeneration (IVDD) is the programmed apoptosis of nucleus pulposus (NP) cells. Oxidative stress is a significant cause of IVDD. Whether mitophagy is induced by strong oxidative stress in IVDD remains to be determined. This study aimed to investigate the relationship between oxidative stress and mitophagy and to better understand the mechanism of IVDD in vivo and in vitro. To this end, we obtained primary NP cells from the human NP and subsequently exposed them to TBHP. We observed that oxidative stress induced mitophagy to cause apoptosis in NP cells, and we suppressed mitophagy and found that NP cells were protected against apoptosis. Interestingly, TBHP resulted in mitophagy through the inhibition of the HIF-1α/NDUFA4L2 pathway. Therefore, the upregulation of mitochondrial NDUFA4L2 restricted mitophagy induced by oxidative stress. Furthermore, the expression levels of HIF-1α and NDUFA4L2 were decreased in human IVDD. In conclusion, these results demonstrated that the upregulation of NDUFA4L2 ameliorated the apoptosis of NP cells by repressing excessive mitophagy, which ultimately alleviated IVDD. These findings show for the first time that NDUFA4L2 and mitophagy may be potential therapeutic targets for IVDD.