Degenerative Human Nucleus Pulposus Cells Research Articles

Collagen II enrichment through scAAV6-RNAi-mediated inhibition of matrix-metalloproteinases 3 and 13 in degenerative nucleus-pulposus cells degenerative disc disease and biological treatment strategies.

Intervertebral disc (IVD) degeneration damaging the extracellular matrix (ECM) of IVDs is the main cause of spine-associated disorders. Degenerative disc disease (DDD) is a multifaceted disorder, where environmental factors, inflammatory cytokines and catabolic enzymes act together. DDD starts typically due to imbalance between ECM biosynthesis and degradation within IVDs, especially through unbalanced degradation of aggrecan and collagen II in nucleus pulposus (NP). Current treatment approaches are primarily based on conservative or surgical therapies, which are insufficient for biological regeneration. The disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) and matrix metalloproteinases (MMPs) are the key proteolytic enzymes for degradation of aggrecan and collagens. Previously, high expression levels of ADAMTS4, ADAMTS5, MMP3 and MMP13, which are accompanied with low levels of aggrecan and collagen II, were demonstrated in degenerative human NP cells. Moreover, self-complementary adeno-associated virus type 6 (scAAV6) mediated inhibitions of ADAMTS4 and ADAMTS5 by RNA-interference (RNAi) could specifically enhance aggrecan level. Thus, MMPs are apparently the main degrading enzymes of collagen II in NP. Furthermore, scAAV6-mediated inhibitions of MMP3 and MMP13 have not yet been investigated. Therefore, we attempted to enhance the level of collagen II in degenerative NP cells by scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13. MRI was used to determine preoperative grading of IVD degeneration in patients. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting MMP3 or MMP13; and analysed by fluorescence microscopy, FACS, MTT assay, RT-qPCR, ELISA and western blotting. scAAV6-shRNRs have no impact on cell viability and proliferation, despite high transduction efficiencies (98.6%) and transduction units (1383 TU/Cell). Combined knockdown of MMP3 (92.8%) and MMP13 (90.9%) resulted in highest enhancement of collagen II (143.2%), whereby treatment effects were significant over 56days (p < 0.001). Conclusively, scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13 help to progress less immunogenic and enduring biological treatments in DDD.

Overactivation of NF-kB pathway can induce apoptosis by down-regulating glycolysis in human degenerative nucleus pulposus cells

Intervertebral disc herniation, a prevalent condition in spinal surgery that frequently results in low back pain and lower limb dysfunction, significantly impacting patients' quality of life. Several factors, including spine biomechanics, biology, nutrition, injury, and abnormal inflammatory responses, have been associated with the development of intervertebral disc herniation. Among these factors, abnormal inflammatory responses have received considerable attention as a crucial mediator of both clinical symptoms and disease progression during the intervertebral disc herniation process. However, the underlying mechanisms of inflammation-induced intervertebral disc herniation remain inadequately explored. The NF-κB (Nuclear Factor-κB) pathway plays a central role in regulating the expression of proinflammatory cytokines. Research on intervertebral disc herniation has suggested that NF-κB can activate the NLRP3 inflammasome, thereby exacerbating intervertebral disc degeneration. Targeting the NF-κB pathway has shown promise in alleviating disc degeneration and associated pain. Previous research indicated that the upregulation of the NF-κB pathway, achieved through the inhibition of A20 (zinc finger protein A20), accelerated intervertebral disc herniation. In the present study, we observed that increased activation of NF-κB pathway activation suppressed the glycolysis process in nucleus pulposus cells (NPCs), leading to NPC apoptosis. Conversely, inhibition of the NF-κB pathway overactivated promoted the restoration of glycolysis and reversed NPC apoptosis, especially when treated with Lipopolysaccharide (LPS).

Single-cell RNA-seq analysis reveals the Wnt/Ca2+ signaling pathway with inflammation, apoptosis in nucleus pulposus degeneration

BackgroundIncreasing studies have shown degeneration of nucleus pulposus cells (NPCs) as an critical part of the progression of intervertebral disc degeneration (IVDD). However, there are relatively few studies on single-cell transcriptome contrasts in human degenerated NPCs. Moreover, differences in Wnt/Ca2+ signaling in human degenerated nucleus pulposus cells have not been elucidated. The aim of this study is to investigate the differential expression of Wnt/Ca2+ signaling pathway between normal and degenerated nucleus pulposus cells in humans and try to investigate its mechanism.MethodsWe performed bioinformatics analysis using our previously published findings to construct single cell expression profiles of normal and degenerated nucleus pulposus. Then, in-depth differential analysis was used to characterize the expression of Wnt/Ca2+ signaling pathway between normal and degenerated nucleus pulposus cells in humans.ResultsThe obtained cell data were clustered into five different chondrocytes clusters, which chondrocyte 4 and chondrocyte 5 mainly accounted for a high proportion in degenerated nucleus pulposus tissues, but rarely in normal nucleus pulposus tissues. Genes associated within the Wnt/Ca2+ signaling pathway, such as Wnt5B, FZD1, PLC (PLCB1), CaN (PPP3CA) and NAFATC1 are mainly present in chondrocyte 3, chondrocyte 4 and chondrocyte 5 from degenerated nucleus pulposus tissues. In addition, as a receptor that activates Wnt signaling pathway, LRP5 is mainly highly expressed in chondrocyte 5 of degenerated nucleus pulposus cells. Six genes, ANGPTL4, PTGES, IGFBP3, GDF15, TRIB3 and TNFRSF10B, which are associated with apoptosis and inflammatory responses, and are widespread in chondrocyte 4 and chondrocyte 5, may be closely related to degenerative of nucleus pulposus cells.ConclusionsSingle-cell RNA sequencing revealed differential expression of Wnt/Ca2+ signaling in human normal and degenerated nucleus pulposus cells, and this differential expression may be closely related to the abundance of chondrocyte 4 and chondrocyte 5 in degenerated nucleus pulposus cells. In degenerated nucleus pulposus cells, LRP5 activate Wnt5B, which promotes nucleus pulposus cell apoptosis and inflammatory response by regulating the Wnt/Ca2+ signaling pathway, thereby promoting disc degeneration. ANGPTL4, IGFBP3, PTGES in chondrocyte 4 and TRIB3, GDF15, TNFRSF10B in chondrocyte 5 may play an important role in this process.

Trigonochinene E promotes lysosomal biogenesis and enhances autophagy via TFEB/TFE3 in human degenerative NP cells against oxidative stress

BackgroundMacroautophagy (henceforth autophagy) is the major form of autophagy, which delivers intracellular cargo to lysosomes for degradation. Considerable research has revealed that the impairment of lysosomal biogenesis and autophagic flux exacerbates the development of autophagy-related diseases. Therefore, reparative medicines restoring lysosomal biogenesis and autophagic flux in cells may have therapeutic potential against the increasing prevalence of these diseases. PurposeThe aim of the present study was thus to explore the effect of trigonochinene E (TE), an aromatic tetranorditerpene isolated from Trigonostemon flavidus, on lysosomal biogenesis and autophagy and to elucidate the potential underlying mechanism. MethodsFour human cell lines, HepG2, nucleus pulposus (NP), HeLa and HEK293 cells were applied in this study. The cytotoxicity of TE was evaluated by MTT assay. Lysosomal biogenesis and autophagic flux induced by 40 μM TE were analyzed using gene transfer techniques, western blotting, real-time PCR and confocal microscopy. Immunofluorescence, immunoblotting and pharmacological inhibitors/activators were applied to determine the changes in the protein expression levels in mTOR, PKC, PERK, and IRE1α signaling pathways. ResultsOur results showed that TE promotes lysosomal biogenesis and autophagic flux by activating the transcription factors of lysosomes, transcription factor EB (TFEB) and transcription factor E3 (TFE3). Mechanistically, TE induces TFEB and TFE3 nuclear translocation through an mTOR/PKC/ROS-independent and endoplasmic reticulum (ER) stress-mediated pathway. The PERK and IRE1α branches of ER stress are crucial for TE-induced autophagy and lysosomal biogenesis. Whereas TE activated PERK, which mediated calcineurin dephosphorylation of TFEB/TFE3, IRE1α was activated and led to inactivation of STAT3, which further enhanced autophagy and lysosomal biogenesis. Functionally, knockdown of TFEB or TFE3 impairs TE-induced lysosomal biogenesis and autophagic flux. Furthermore, TE-induced autophagy protects NP cells from oxidative stress to ameliorate intervertebral disc degeneration (IVDD). ConclusionsHere, our study showed that TE can induce TFEB/TFE3-dependent lysosomal biogenesis and autophagy via the PERK-calcineurin axis and IRE1α-STAT3 axis. Unlike other agents regulating lysosomal biogenesis and autophagy, TE showed limited cytotoxicity, thereby providing a new direction for therapeutic opportunities to use TE to treat diseases with impaired autophagy-lysosomal pathways, including IVDD.

The role of the miR-4306/PAK6 axis in degenerative nucleus pulposus cells in human intervertebral disc degeneration

Intervertebral disc degeneration (IDD), characterized by degenerative changes that occur in intervertebral discs due to aging or structural injury, is thought to be the most common cause of lower back pain. Recent studies have shown that microRNAs (miRNAs) have a critical role in the etiopathogenesis of IDD. In the current study, we aimed to determine the role of miRNAs in mediating the underlying mechanisms associated with IDD. First, differentially expressed miRNAs (DEmiRNAs) were identified using the GEO database, and subsequently confirmed by RT-qPCR and in situ hybridization. We found that miR-4306 expression was significantly decreased in human nucleus pulposus (NP) tissues compared with healthy controls, and was negatively correlated with the patients' Pfirrmann grade. To determine the mechanism by which miR-4306 was involved in IDD pathogenesis, we examined the effects of overexpressing or silencing miR-4306 on extracellular matrix (ECM) synthesis/degradation, proliferation, autophagy and apoptosis of human degenerated NP cells isolated from IDD patients. Next, we used dual-luciferase reporter assays to demonstrate that miR-4306 interacted with the 3′-untranslated regions of p21-activated kinase 6 (PAK6) mRNA, resulting in significant suppression of PAK6 expression. This effect was abolished by miR-4306 binding site mutations. Using miR-4306/PAK6 gain-of-function and loss-of-function studies in human degenerated NP cells, we demonstrated that miR-4306 promoted NP cell proliferation, ECM synthesis and autophagy, while inhibiting apoptosis and ECM degradation via PAK6. Thus, our findings indicate that miR-4306, acting via PAK6, has an important role in IDD and can be used as a promising therapeutic target for the treatment of patients with IDD.

PHLPP1 deficiency protects against age-related intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration is strongly associated with low back pain and is highly prevalent in the elderly population. Hallmarks of IVD degeneration include cell loss and extracellular matrix degradation. The PH domain leucine-rich-repeats protein phosphatase (PHLPP1) is highly expressed in diseased cartilaginous tissues where it is linked to extracellular matrix degradation. This study explored the ability of PHLPP1 deficiency to protect against age-related spontaneous IVD degeneration. Lumbar IVDs of global Phlpp1 knockout (KO) and wildtype (WT) mice were collected at 5months (young) and 20 months (aged). Picrosirius red-alcian blue staining (PR-AB) was performed to examine IVD structure and histological score. The expression of aggrecan, ADAMTS5, KRT19, FOXO1 and FOXO3 was analyzed through immunohistochemistry. Cell apoptosis was assessed by TUNEL assay. Human nucleus pulposus (NP) samples were obtained from patients diagnosed with IVD degeneration. PHLPP1 knockdown in human degenerated NP cells was conducted using small interfering RNA (siRNA) transfection. The expression of PHLPP1 regulated downstream targets was analyzed via immunoblot and real time quantitative PCR. Histological analysis showed that Phlpp1 KO decreased the prevalence and severity of age-related IVD degeneration. The deficiency of PHLPP1 promoted the increased expression of NP phenotypic marker KRT19, aggrecan and FOXO1, and decreased levels of ADMATS5 and cell apoptosis in the NP of aged mice. In degenerated human NP cells, PHLPP1 knockdown induced FOXO1 protein levels while FOXO1 inhibition offset the beneficial effects of PHLPP1 knockdown on KRT19 gene and protein expression. Our findings indicate that Phlpp1 deficiency protected against NP phenotypic changes, extracellular matrix degradation, and cell apoptosis in the process of IVD degeneration, probably through FOXO1 activation, making PHLPP1 a promising therapeutic target for treating IVD degeneration.

Depleted Long Noncoding RNA GAS5 Relieves Intervertebral Disc Degeneration via microRNA-17-3p/Ang-2.

Intervertebral disc degeneration (IVDD) remains a clinical challenge and requires more effective therapeutic targets. Long noncoding RNAs (lncRNAs) have emerged as critical modulators of multiple biological processes, such as cell proliferation and extracellular matrix (ECM) remodeling. Accordingly, the current study sets out to explore the influence of the lncRNA growth arrest-specific 5 (GAS5) on IVDD and investigate the possible involvement of microRNA-17-3p (miR-17-3p)/Angiopoietin-2 (Ang-2) axis. Firstly, the expression patterns of GAS5, miR-17-3p, and Ang-2 were characterized by RNA quantification from the isolated human degenerative nucleus pulposus (NP) tissues. miR-17-3p was found to express at an abnormal low level while GAS5 and Ang-2 expressed at aberrant high level in the human degenerative NP tissues. Utilizing dual-luciferase reporter, RNA immunoprecipitation, and pull-down assays, GAS5 was found to competitively bound to miR-17-3p and further upregulate the expression of Ang-2, a target gene of miR-17-3p. Employing gain- and loss-of-function approaches, their expressions were altered in human degenerative nucleus pulposus cells (NPCs), followed by IL-1β treatment, in order to identify their roles in NP cell proliferation, apoptosis, and ECM metabolism. Silencing of GAS5 expression restrained the levels of cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, MMP3, MMP13, ADAMTS4, and ADAMTS5 and increased collagen II and aggrecan levels. In vitro experiments also revealed that GAS5 depletion inhibited apoptosis and ECM degradation in HDNPCs, while elevating the proliferation through downregulation of Ang-2 by increasing miR-17-3p. Furthermore, in vivo data further validated that either GAS5 silencing or miR-17-3p reexpression alleviated IVDD degree with the help of IVDD mouse models. Altogether, our findings substantiated that downregulation of GAS5 reduced NPC apoptosis and promoted ECM remodeling, ultimately ameliorating the IVDD via miR-17-3p-dependent inhibition of Ang-2. We hope our discoveries offer a fresh molecular insight that can aid the development of novel therapies against IVDD.

LncRNA MIR155HG functions as a ceRNA of miR-223-3p to promote cell pyroptosis in human degenerative NP cells.

Nucleus pulposus (NP) cell pyroptosis plays a critical role in the pathogenesis of intervertebral disk degeneration (IDD). MIR155 host gene (MIR155HG) is a long non-coding RNA with pro-inflammatory activity. However, very little is known about its role in NP cell pyroptosis. This study aimed to observe the impact of MIR155HG on cell pyroptosis and to explore the underlying mechanism in human degenerative NP cells. Our results demonstrated that MIR155HG expression was significantly increased in human degenerative NP tissue samples and showed a positive correlation with Pfirrmann score. Overexpression of MIR155HG through a lentiviral vector decreased miR-223-3p levels, up-regulated NLRP3 expression and induced cell pyroptosis in human degenerative NP cells. A ceRNA action mode was identified among MIR155HG, miR-223-3p, and NLRP3. The stimulatory effect of MIR155HG on human degenerative NP cell pyroptosis was significantly reversed by pretreatment with miR-223-3p mimic or NLRP3 siRNA. In summary, these data suggest that MIR155HG sponges miR-223-3p to promote NLRP3 expression, leading to induction of cell pyroptosis in human degenerative NP cells. Targeting MIR155HG could be a novel and promising strategy to slow down the progression of IDD.

LIPUS inhibits inflammation and catabolism through the NF\u2010\u03baB pathway in human degenerative nucleus pulposus cells

BackgroundLow-intensity pulsed ultrasound (LIPUS) is a safe and noninvasive rehabilitative physical therapy with anti-inflammatory effects. The current study investigated the effect of LIPUS on the inflammation of nucleus pulposus (NP) cells and its underlying mechanism.MethodsHuman NP cells were acquired from lumbar disc herniation tissue samples and cultured for experiments. Human NP cells were treated with LPS and then exposed to LIPUS (15 mW/cm2, 30 mW/cm2 and 60 mW/cm2) for 20 min daily for 3 days to determine the appropriate intensity to inhibit the expression of the inflammatory factors TNF-α and IL-1β. The gene and protein expression of aggrecan, collagen II, MMP-3 and MMP-9 was measured by real‐time PCR and western blotting, respectively. The activity of the nuclear factor‐kappa B (NF‐κB) pathway was examined by western blotting and immunofluorescence. After pretreatment with the NF-κB inhibitor PDTC, the expression of TNF-α, IL-1β, MMP-3 and MMP-9 was measured by real‐time PCR.ResultsLIPUS at intensities of 15 mW/cm2, 30 mW/cm2 and 60 mW/cm2 inhibited LPS-induced NP cell expression of the inflammatory factors TNF-α and IL-1β, especially at 30 mW/cm2. LIPUS significantly upregulated the gene and protein expression of aggrecan and collagen II and downregulated the gene and protein expression of MMP-3 and MMP-9 in LPS-induced NP cells. The NF‐κB signaling pathway was inhibited by LIPUS through inhibiting the protein expression of p-P65 and the translocation of P65 into the nucleus in LPS-induced NP cells. In addition, LIPUS had similar effects as the NF-κB inhibitor PDTC by inhibiting the NF-κB signaling pathway, inflammation and catabolism in LPS-induced human degenerative nucleus pulposus cells.ConclusionLIPUS inhibited inflammation and catabolism through the NF‐κB pathway in human degenerative nucleus pulposus cells.

Tanshinone IIA and Astragaloside IV Inhibit miR-223/JAK2/STAT1 Signalling Pathway to Alleviate Lipopolysaccharide-Induced Damage in Nucleus Pulposus Cells.

Astragaloside IV (AS IV) and tanshinone (TS IIA) are the main natural components of Salvia miltiorrhiza and Radix Astragali, respectively. The amalgam of TS IIA and AS IV has potential therapeutic value in many inflammation-related diseases. However, the aftereffect of TS IIA and AS IV for lumbar disc herniation is not clear. Although the function of miR-223 in the inflammation-related JAK/STAT pathway is unknown, it is particularly expressed in human degenerative nucleus pulposus cells. This study has investigated the efficacy of the combined application of TS IIA and AS IV in the treatment of intervertebral disc nucleus pulposus cells (NP cells) injured by lipopolysaccharide (LPS). After miR-223 inhibitor imitated NP cells, the state of the JAK family and STAT family was recognized by Western blotting (Western blot, WB) and reverse transcriptase quantitative polymerase chain reaction (qPCR). The shRNA lentivirus interference vector targeting the STAT family was constructed, and the NP cell line stably interfering with the STAT gene was established after transfection. The expression of TNF-α, IL-6, MMP-9, MMP-3, caspase-1, and caspase-3 was detected by lipopolysaccharide (WTNP cells), control virus NP cells, STAT downregulation NP cells, enzyme-linked immunosorbent assay (ELISA), Western blot, and qPCR, respectively. The cell survival rate was detected by flow cytometry and TUNEL staining reverse transcriptase-polymerase chain reaction (qPCR). NP cells were treated with TS IIA and AS IV which had been made into different concentrations, and then, the expression of miR-223, p-STAT1, and p-JAK families was detected by WB Western blotting and qPCR. MiR-223 selectively acts on JAK2/STAT1 pathway, increases the expression of TNF-α, IL-6, MMP-9, MMP-3, caspase3-1, and caspase-3, and induces apoptosis, which can be eliminated by silencing STAT1. TS IIA combined with AS IV could inhibit the expression of miR-223, p-STAT1, and p-JAK2 in NP cells, and they showed a dose-dependent tendency to p-STAT1 and p-JAK2. This study shows that miR-223 promotes the inflammatory response and induces cell injury of NP cells by acting on the JAK2/STAT1 pathway, and the combination of TS IIA and AS IV may protect NP cells by downregulating miR-223 and inhibiting the expression of JAK2 and STAT1.

Bioactive in situ crosslinkable polymer-peptide hydrogel for cell delivery to the intervertebral disc in a rat model

Degeneration of the intervertebral disc (IVD) is associated with significant biochemical and morphological changes that include a loss of disc height, decreased water content and decreased cellularity. Cell delivery has been widely explored as a strategy to supplement the nucleus pulposus (NP) region of the degenerated IVD in both pre-clinical and clinical trials, using progenitor or primary cell sources. We previously demonstrated an ability for a polymer-peptide hydrogel, serving as a culture substrate, to promote adult NP cells to undergo a shift from a degenerative fibroblast-like state to a juvenile-like NP phenotype. In the current study, we evaluate the ability for this peptide-functionalized hydrogel to serve as a bioactive system for cell delivery, retention and preservation of a biosynthetic phenotype for primary IVD cells delivered to the rat caudal disc in an anular puncture degeneration model. Our data suggest that encapsulation of adult degenerative human NP cells in a stiff formulation of the hydrogel functionalized with laminin-mimetic peptides IKVAV and AG73 can promote cell viability and increased biosynthetic activity for this population in 3D culture in vitro. Delivery of the peptide-functionalized biomaterial with primary rat cells to the degenerated IVD supported NP cell retention and NP-specific protein expression in vivo, and promoted improved disc height index (DHI) values and endplate organization compared to untreated degenerated controls. The results of this study suggest the physical cues of this peptide-functionalized hydrogel can serve as a supportive carrier for cell delivery to the IVD. Statement of significanceCell delivery into the degenerative intervertebral disc has been widely explored as a strategy to supplement the nucleus pulposus. The current work seeks to employ a biomaterial functionalized with laminin-mimetic peptides as a cell delivery scaffold in order to improve cell retention rates within the intradiscal space, while providing the delivered cells with biomimetic cues in order to promote phenotypic expression and increase biosynthetic activity. The use of the in situ crosslinkable material integrated with the native IVD, presenting a system with adequate physical properties to support a degenerative disc.

Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative disc cells following hyperbaric oxygen treatment

BackgroundMicroRNA (miRNA) plays a vital role in the intervertebral disc (IVD) degeneration. The expression level of miR-573 was downregulated whereas Bax was upregulated notably in human degenerative nucleus pulposus cells. In this study, we aimed to investigate the role of miR-573 in human degenerative nucleus pulposus (NP) cells following hyperbaric oxygen (HBO) treatment.MethodsNP cells were separated from human degenerated IVD tissues. The control cells were maintained in 5% CO2/95% air and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The mRNA and protein levels of Bax were measured. The proliferation of NPCs was detected using MTT assay. The protein expression levels of Bax, cleaved caspase 9, cleaved caspase 3, pro-caspase 9, and pro-caspase 3 were examined.ResultsBioinformatics analysis indicated that the 3′ untranslated region (UTR) of the Bax mRNA contained the “seed-matched-sequence” for hsa-miR-573, which was validated via reporter assays. MiR-573 was induced by HBO and simultaneous suppression of Bax was observed in NP cells. Knockdown of miR-573 resulted in upregulation of Bax expression in HBO-treated cells. In addition, overexpression of miR-573 by HBO increased cell proliferation and coupled with inhibition of cell apoptosis. The cleavage of pro-caspase 9 and pro-caspase 3 was suppressed while the levels of cleaved caspase 9 and caspase 3 were decreased in HBO-treated cells. Transfection with anti-miR-573 partly suppressed the effects of HBO.ConclusionMir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative NP cells following HBO treatment.

Development of a library of laminin-mimetic peptide hydrogels for control of nucleus pulposus cell behaviors

The nucleus pulposus (NP) of the intervertebral disc plays a critical role in distributing mechanical loads to the axial skeleton. Alterations in NP cells and, consequently, NP matrix are some of the earliest changes in the development of disc degeneration. Previous studies demonstrated a role for laminin-presenting biomaterials in promoting a healthy phenotype for human NP cells from degenerated tissue. Here we investigate the use of laminin-mimetic peptides presented individually or in combination on a poly(ethylene) glycol hydrogel as a platform to modulate the behaviors of degenerative human NP cells. Data confirm that NP cells attach to select laminin-mimetic peptides that results in cell signaling downstream of integrin and syndecan binding. Furthermore, the peptide-functionalized hydrogels demonstrate an ability to promote cell behaviors that mimic that of full-length laminins. These results identify a set of peptides that can be used to regulate NP cell behaviors toward a regenerative engineering strategy.

Verteporfin treatment controls morphology, phenotype, and global gene expression for cells of the human nucleus pulposus.

Cells of the nucleus pulposus (NP) are essential contributors to extracellular matrix synthesis and function of the intervertebral disc. With age and degeneration, the NP becomes stiffer and more dehydrated, which is associated with a loss of phenotype and biosynthetic function for its resident NP cells. Also, with aging, the NP cell undergoes substantial morphological changes from a rounded shape with pronounced vacuoles in the neonate and juvenile, to one that is more flattened and spread with a loss of vacuoles. Here, we make use of the clinically relevant pharmacological treatment verteporfin (VP), previously identified as a disruptor of yes‐associated protein‐TEA domain family member‐binding domain (TEAD) signaling, to promote morphological changes in adult human NP cells in order to study variations in gene expression related to differences in cell shape. Treatment of adult, degenerative human NP cells with VP caused a shift in morphology from a spread, fibroblastic‐like shape to a rounded, clustered morphology with decreased transcriptional activity of TEAD and serum‐response factor. These changes were accompanied by an increased expression of vacuoles, NP‐specific gene markers, and biosynthetic activity. The contemporaneous observation of VP‐induced changes in cell shape and prominent, time‐dependent changes within the transcriptome of NP cells occurred over all timepoints in culture. Enriched gene sets with the transition to VP‐induced cell rounding suggest a major role for cell adhesion, cytoskeletal remodeling, vacuolar lumen, and MAPK activity in the NP phenotypic and functional response to changes in cell shape.

Interleukin-17A Promotes Human Disc Degeneration by Inhibiting Autophagy Through the Activation of the Phosphatidylinositol 3-Kinase/Akt/Bcl2 Signaling Pathway

Previous studies have suggested that interleukin (IL)-17A is a key factor that contributes to intervertebral disc degeneration (IDD), whereas autophagy has been shown to be a protective mechanism in IDD. However, the relationship between IL-17A and autophagy in IDD remains to be fully elucidated. This study sought to evaluate the association between IL-17 and autophagy and the potential mechanism through which IL-17A affects autophagy in IDD. Intervertebral disc specimens were collected from 10 patients with lumbar disc herniation. Human degenerated nucleus pulposus (NP) cells were cultured in the presence or absence of IL-17A treatment. Western blot and monodansylcadaverine staining were used to measure autophagy levels in human degenerated NP cells. Subsequently, phosphatidylinositol 3-kinase (PI3K)/Akt/Bcl-2 pathway inhibitors were used to reveal the potential mechanism. IL-17A treatment inhibited the autophagic activity in human NP cells in a time- and dose-dependent manner. Moreover, monodansylcadaverine staining showed that cells treated with IL-17A had significantly fewer changes in their autophagic vacuoles compared with control-treated cells. After IL-17A treatment, expression levels of PI3K, p-Akt, and Bcl-2 in NP cells were significantly increased. Further assays with PI3K/Akt/Bcl-2 inhibitors revealed that IL-17A suppressed autophagy in NP cells by activating the PI3K/Akt/Bcl-2 signaling pathway. These data suggest that IL-17A promotes IDD by inhibiting autophagy through activation of the PI3K/Akt/Bcl-2 signaling pathway and may offer new insights for targeted therapy of this disease.

Effects of pulsed electromagnetic fields on the A2A adenosine receptor in human degenerative nucleus pulposus cells

Objective To explore the expression of the A2A adenosine receptor and the inflammatory cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in human degenerative nucleus pulposus (NP) cells after they have been treated with a pulsed electromagnetic field (PEMF). Methods Human degenerative NP cells were cultured in vitro and treated using an 0.8mT PEMF with a pulse frequency of 50Hz. The pulse width was 150μs and the exposure time was 30min, repeated 5 times at 12 hour intervals. The expression of the A2A adenosine receptor in NP cells was determined using western blotting and reverse transcription polymerase chain reactions. The expression of the inflammatory cytokines IL-1β and TNF-α were detected using enzyme-linked immunosorbent assays (ELISA). The human degenerative NP cells were also treated with an antagonist and agonist of the A2A adenosine receptor, and the expression of IL-1β and TNF-α were also determined using ELISA. Results After the PEMF treatment the expression of the A2A adenosine receptor increased significantly, while the expression of IL-1β and TNF-α decreased significantly. However, the A2A adenosine receptor antagonist reversed the inhibitory effect of the PEMF on the expression of IL-1β and TNF-α, while the agonist played an opposite role. Conclusion A PEMF can significantly inhibit the expression of IL-1β and TNF-α in human degenerative NP cells, which could be related to up-regulation of the expression of the A2A adenosine receptor in those cells. Key words: Pulsed electromagnetic fields; Adenosine receptors; Nucleus pulposus cells; Inflammatory cytokines

Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells.

We investigated the effects of a fibrin-hyaluronic acid hydrogel (FBG–HA) and fibroblast growth factor 18 (FGF-18) for nucleus pulposus (NP) regeneration. Healthy bovine (n = 4) and human degenerated NP cells (n = 4) were cultured for 14 days in FBG-HA hydrogel with FGF-18 (∆51-mutant or wild-type) in the culture medium. Gene expression, DNA content, and glycosaminoglycan (GAG) synthesis were evaluated on day 7 and 14. Additionally, histology was performed. Human NP cells cultured in FBG-HA hydrogel showed an increase in collagen type II (COL2) and carbonic anhydrase XII (CA12) gene expression after 14 or 7 days of culture, respectively. GAG release into the conditioned medium increased over 14 days. Healthy bovine NP cells showed increased gene expression of ACAN from day 7 to day 14. Wild type FGF-18 up-regulated CA12 gene expression of human NP cells. Histology revealed an increase of proteoglycan deposition upon FGF-18 stimulation in bovine but not in human NP cells. The FBG-HA hydrogel had a positive modulatory effect on human degenerated NP cells. Under the tested conditions, no significant effect of FGF-18 was observed on cell proliferation or GAG synthesis in human NP cells.

Expression and effects of leukemia inhibitory factor on nucleus pulposus degeneration.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine. The present study aimed to determine the expression and effects of LIF on nucleus pulposus generation. Degenerated nucleus pulposus samples were obtained from animal models and patients with lumbar intervertebral disc herniation. Degradation scores of intervertebral discs were evaluated via magnetic resonance imaging (MRI) and histology, and the protein expression levels of LIF were detected. Furthermore, cultured primary human degenerated nucleus pulposus cells (DNPCs) were stimulated with various concentrations of recombinant human LIF protein (rhLIF), and aggrecan and collagen type II α1 (COL2α1) protein expression levels were detected by western blotting. In addition, aggrecan expression was determined by toluidine blue staining. The effects of rhLIF on proliferation and apoptosis of DNPCs were evaluated by Cell Counting Kit-8 and flow cytometry, respectively. The results revealed that the degradation scores of intervertebral discs were significantly associated with modeling time, as determined by MRI and histology. In addition, the protein expression levels of LIF were initially increased in patients with lumbar disc herniation and in rabbit models, particularly in the 2-week modeling group; however, its expression decreased with the progression of disc degeneration. Notably, LIF expression in each modeling group was higher than that in the control and 0 week modeling group. The in vitro study revealed that the protein expression levels of aggrecan and COL2α1 were significantly increased in response to rhLIF, in a dose-dependent manner, and statistical differences were identified between the treatment groups and control group. The results of toluidine blue staining were consistent with this finding. Although rhLIF had no effect on proliferation, it inhibited apoptosis of DNPCs in a concentration-dependent manner. In conclusion, LIF was upregulated during the process of intervertebral disc degeneration, and may promote the expression of extracellular matrix components. It may also be hypothesized that LIF acts as a potential protective factor by inhibiting apoptosis of DNPCs without affecting cell proliferation.

The effect of mechanical stretching stress on the proliferation, apoptosis of degenerative disc nucleus pulposus cells and expression of extracellular matrix

Objective To observe in vitro the effect of mechanical stress at different intensities on the proliferation, apoptosis and extracellular matrix of degenerative human nucleus pulposus cells. Methods The cells were isolated and cultured in vitro, and divided into a control group, a low-intensity group, a medium-intensity group and a high-intensity group. The low-, medium- and high-intensity groups were stretched mechanically by 1000 μ, 2000 μ or 4000 μ respectively for 6 hours using a four-point bending system, while the control group was not stressed. Flow cytometry was used to explore any changes in the cell cycle and the proliferation index (PI). The expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (BCL-2)/Bax, collagen II and aggrecan were measured using real-time quantitative polymerase chain reactions. Results The mechanical stretching significantly influenced proliferation, apoptosis and the extracellular matrices compared with the control group. The PI and PCNA expression increased at first and then decreased gradually with the exercise intensity. Compared with the control group, the mRNA expression level of Bcl-2/Bax increased significantly to 1.53 times that of the control group after 1000 μ stretching, but to only 0.71 times that of the control group at 2000 μ and 0.43 times at 4000 μ. The gene expression of collagen II increased significantly by 1.1 times and that of aggrecan by 1.3 times after 1000 μ stress stimulation compared with the control group (P≤0.05). However, the expression of collagen II and aggrecan was inhibited significantly at 2000 and 4000 μ , with the lowest levels at 4000 μ (P≤0.05). Conclusion Stretching at different intensities has different effects on the proliferation, apoptosis and extracellular matrix of human pulposus cells with degenerate nuclei. Key words: Mechanical stress; Stretching; Degenerate nuclei; Pulposus cells; Proliferation; Apoptosis; Extracellular matrices

Inhibition of microRNA-30d attenuates the apoptosis and extracellular matrix degradation of degenerative human nucleus pulposus cells by up-regulating SOX9

Accumulating evidence has suggested that microRNAs are critical regulators of intervertebral disc degeneration (IDD). The excessive apoptosis and extracellular matrix degradation of nucleus pulposus (NP) cells contribute to the initiation of IDD. However, the precise regulatory role of miRNAs in NP cell apoptosis and extracellular matrix degradation remains largely unknown. MicroRNA-30d (miR-30d) has been reported to be involved in regulating apoptosis and bone homeostasis. In this study, we aimed to investigate the role of miR-30d in regulating apoptosis and the extracellular matrix degradation of NP cells, along with the potential underlying molecular mechanism. Herein, our results showed that miR-30d was significantly increased in degenerative NP tissues compared with normal controls. Functional experiments showed that the inhibition of miR-30d promoted the viability and reduced the apoptosis of NP cells in vitro. Moreover, miR-30d inhibition increased the expression of type II collagen and aggrecan and inhibited the expression of matrix metalloproteinase. In contrast, the overexpression of miR-30d showed the opposite effects. Bioinformatics analysis, the dual-luciferase reporter assay, real-time quantitative PCR and western blot analysis showed that miR-30d directly targeted the 3′-untranslated region of SRY-related high mobility group box 9 (SOX9) and negatively regulated SOX9 expression. Correlation analysis showed that miR-30d expression was inversely correlated with SOX9 expression in degenerative NP tissues. Moreover, siRNA-mediated silencing of SOX9 expression significantly blocked the protective effects of miR-30d inhibition against NP cell apoptosis and extracellular matrix degradation. Overall, these results demonstrate that the inhibition of miR-30d attenuates the apoptosis and extracellular matrix degradation of degenerative human NP cells by up-regulating SOX9, suggesting a potential therapeutic target for IDD.