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Low back pain is a common clinical symptom of intervertebral disc degeneration (IVDD), which seriously affects the quality of life of the patients. The abnormal apoptosis and senescence of nucleus pulposus cells (NPCs) play important roles in the pathogenesis of IVDD. PHLDA2 is an imprinted gene related to cell apoptosis and tumour progression. However, its role in NPC degeneration is not yet clear. Therefore, this study was set to explore the effects of PHLDA2 on NPC senescence and apoptosis and the underlying mechanisms. The expression of PHLDA2 was examined in human nucleus pulposus (NP) tissues and NPCs. Immunohistochemical staining, magnetic resonance imaging imaging and western blot were performed to evaluate the phenotypes of intervertebral discs. Senescence and apoptosis of NPCs were assessed by SA‐β‐galactosidase, flow cytometry and western blot. Mitochondrial function was investigated by JC‐1 staining and transmission electron microscopy. It was found that the expression level of PHLDA2 was abnormally elevated in degenerated human NP tissues and NPCs. Furthermore, knockdown of PHLDA2 can significantly inhibit senescence and apoptosis of NPCs, whereas overexpression of PHLDA2 can reverse senescence and apoptosis of NPCs in vitro. In vivo experiment further confirmed that PHLDA2 knockdown could alleviate IVDD in rats. Knockdown of PHLDA2 could also reverse senescence and apoptosis in IL‐1β‐treated NPCs. JC‐1 staining indicated PHLDA2's knockdown impaired disruption of the mitochondrial membrane potential and also ameliorated superstructural destruction of NPCs as showed by transmission electron microscopy. Finally, we found the PHLDA2 knockdown promoted Collagen‐II expression and suppressed MMP3 expression in NPCs by repressing wnt/β‐catenin pathway. In conclusion, the results of the present study showed that PHLDA2 promotes IL‐1β‐induced apoptosis and senescence of NP cells via mitochondrial route by activating the Wnt/β‐catenin pathway, and suggested that therapy targeting PHLDA2 may provide valuable insights into possible IVDD therapies. [ABSTRACT FROM AUTHOR]

Mechanical environment worsening is an important predisposing factor that accelerates intervertebral disc degeneration (IDD), but its specific regulatory mechanisms remain unclear. In this study, we reveal the molecular mechanisms of WTAP/YTHDF2‐mediated m6A modification in abnormal stress‐induced intervertebral disc (IVD) matrix degradation. WTAP expression in human nucleus pulposus cells was elevated under tension. Similarly, high WTAP expression was detected in severe degenerated human and rat nucleus pulposus tissues. Functionally, WTAP was found to increase the TIMP3 transcript methylation level under tension, resulting in YTHDF2 recognition, binding, and induction of its degradation. Reduction in TIMP3 caused increases in active matrix metalloproteinases, ultimately inducing extracellular matrix degradation in nucleus pulposus cells. Macroscopically, this promotes IDD. Additionally, in vitro and in vivo inhibition of WTAP expression or TIMP3 overexpression significantly increased stress resistance in the nucleus pulposus, thereby alleviating IDD. Our results show that abnormal stress disrupts IVD matrix stability through WTAP/YTHDF2‐dependent TIMP3 m6A modification. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration (IDD) poses a significant health burden, necessitating a deeper understanding of its molecular underpinnings. Transcriptomic analysis reveals 485 differentially expressed genes (DEGs) associated with IDD, underscoring the importance of immune regulation. Weighted gene co‐expression network analysis (WGCNA) identifies a yellow module strongly correlated with IDD, intersecting with 197 DEGs. Protein–protein interaction (PPI) analysis identifies ITGAX, MMP9 and FCGR2A as hub genes, predominantly expressed in macrophages. Functional validation through in vitro and in vivo experiments demonstrates the pivotal role of FCGR2A in macrophage polarization and IDD progression. Mechanistically, FCGR2A knockdown suppresses M1 macrophage polarization and NF‐κB phosphorylation while enhancing M2 polarization and STAT3 activation, leading to ameliorated IDD in animal models. This study sheds light on the regulatory function of FCGR2A in macrophage polarization, offering novel insights for IDD intervention strategies. Key points: This study unveils the role of FCGR2A in intervertebral disc (IVD) degeneration (IDD).FCGR2A knockdown mitigates IDD in cellular and animal models.Single‐cell RNA‐sequencing uncovers diverse macrophage subpopulations in degenerated IVDs.This study reveals the molecular mechanism of FCGR2A in regulating macrophage polarization.This study confirms the role of the NF‐κB/STAT3 pathway in regulating macrophage polarization in IDD. [ABSTRACT FROM AUTHOR]

Accumulation of advanced glycation end products (AGEs) causes apoptosis in human nucleus pulposus cells (NPCs), contributing to intervertebral disc degeneration (IVDD). The purpose of this study was to determine the roles of thioredoxin‐interacting protein (TXNIP) in the mechanisms underlying AGE‐induced apoptosis of NPCs. TXNIP was silenced or overexpressed in HNPCs exposed to AGEs. Glycolysis was assessed using extracellular acidification rate (ECAR), ATP level, GLUT1, and GLUT4 measurements. AGEs, TXNIP, GLUT1, and GLUT4 levels in IVDD patients were measured as well. In NPCs, AGEs reduced cell viability, induced apoptosis, inhibited glycolysis, and increased TXNIP expression. Silencing TXNIP compromised the effects of AGEs on cell viability, apoptosis, and glycolysis in NPCs. Furthermore, TXNIP overexpression resulted in decreased cell viability, increased apoptotic cells, and glycolysis suppression. Furthermore, co‐treatment with a glycolysis inhibitor improved TXNIP silencing's suppressive effects on AGE‐induced cell injury in NPCs. In IVDD patients with Pfirrmann Grades II–V, increasing trends in AGEs and TXNIP were observed, while decreasing trends in GLUT1 and GLUT4. AGE levels had positive correlations with TXNIP levels. Both AGE and TXNIP levels correlated negatively with GLUT1 and GLUT4. Our study indicates that TXNIP plays a role in mediating AGE‐induced cell injury through suppressing glycolysis. The accumulation of AGEs, the upregulation of TXNIP, and the downregulation of GLUT1 and GLUT4 are all linked to the progression of IVDD. [ABSTRACT FROM AUTHOR]

Oxidative stress can lead to nucleus pulposus cell (NPC) apoptosis, which is considered to be one of the main contributors to intervertebral disc degeneration (IVDD). Procyanidin B2 is a natural antioxidant that protects against oxidative stress. However, whether procyanidin B2 protects NPCs from oxidative stress remains unknown. In this study, we demonstrated that procyanidin B2 could reduce tert‐butyl hydroperoxide‐induced reactive oxygen species in rat NPCs and attenuate rat NPC apoptosis. Further experiments revealed that procyanidin B2 upregulated the expression of both nuclear factor erythroid 2‐related factor 2 (Nrf2) and phosphorylation of protein kinase B (Akt). We then used silencing of Nrf2 and LY294002 to silence Nrf2 expression and block the phosphatidylinositol 3‐kinase (PI3K)/Akt pathway, respectively, and found that the protective roles of procyanidin B2 in NPCs were inhibited. Therefore, we demonstrated that procyanidin B2 alleviated rat NPC apoptosis induced by oxidative stress by upregulating Nrf2 via activation of the PI3K/Akt signaling pathway. This study provides a potential therapeutic approach for procyanidin B2 in IVDD, which might help in the development of new drugs for IVDD treatment. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disc degeneration (IDD) is a pathological process that occurs during the natural aging of intervertebral discs. Accumulating evidence suggests that noncoding RNAs (ncRNAs), including microRNAs and long ncRNAs (lncRNAs), participate in the pathogenesis and development of IDD. Herein, we examined the role of lncRNA MAGI2‐AS3 in the pathogenic mechanism of IDD. Material and Methods: To develop an IDD in vitro model, we treated human nucleus pulposus (NP) cells with lipopolysaccharide (LPS). Aberrant levels of lncRNA MAGI2‐AS3, miR‐374b‐5p, interleukin (IL)‐10 and extracellular matrix (ECM)‐related proteins in NP cells were examined using reverse transcription‐quantitative PCR and western blot analysis. LPS‐induced NP cell injury and inflammatory response were confirmed using the MTT assay, flow cytometry, Caspase3 activity, and enzyme‐linked immunosorbent assay. Dual‐luciferase reporter assay and rescue experiments were performed to confirm targets between lncRNA MAGI2‐AS3 and miR‐374b‐5p or miR‐374b‐5p and IL‐10. Results: LPS‐induced NP cells exhibited low levels of lncRNA MAGI2‐AS3 and IL‐10 expression, along with high miR‐374b‐5p expression. miR‐374b‐5p was a target of lncRNA MAGI2‐AS3 and IL‐10. LncRNA MAGI2‐AS3 ameliorated injury, inflammatory response, and ECM degradation in LPS‐treated NP cells by downregulating miR‐374b‐5p to upregulate IL‐10 expression. Conclusions: LncRNA MAGI2‐AS3 increased IL‐10 expression levels by sponging miR‐374b‐5p, which, in turn, alleviated LPS‐triggered decreased NP cell proliferation and increased apoptosis, inflammatory response, and ECM degradation. Therefore, lncRNA MAGI2‐AS3 may be a potential therapeutic target for IDD. [ABSTRACT FROM AUTHOR]

Background: Cell therapy is considered a promising strategy for intervertebral disc (IVD) regeneration. However, cell products often require long‐term cryopreservation, which compromises cell viability and potency, thus potentially hindering commercialization and off‐the‐shelf availability. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant, however, DMSO is associated with cytotoxicity and cell viability loss. This study aimed to investigate the effects of DMSO on human nucleus pulposus cells (NPC) and the role of oxidative stress in DMSO‐induced cytotoxicity. Furthermore, we examined the potential of antioxidant N‐acetylcysteine (NAC) supplementation to mitigate the negative effects of DMSO. Methods: NPC were exposed to various concentrations of DMSO with or without a freezing cycle. Cell viability, cell apoptosis and necrosis rates, intracellular reactive oxygen species (ROS) levels, and gene expression of major antioxidant enzymes were evaluated. In addition, NAC was added to cryopreservation medium containing 10% DMSO and its effects on ROS levels and cell viability were assessed. Results: DMSO concentrations ≤1% for 24 h did not significantly affect the NPC viability, whereas exposure to 5 and 10% DMSO (most commonly used concentration) caused cell viability loss (loss of 57% and 68% respectively after 24 h) and cell death in a dose‐ and time‐dependent manner. DMSO increased intracellular and mitochondrial ROS (1.9‐fold and 3.6‐fold respectively after 12 h exposure to 10% DMSO) and downregulated gene expression levels of antioxidant enzymes in a dose‐dependent manner. Tempering ROS through NAC treatment significantly attenuated DMSO‐induced oxidative stress and supported maintenance of cell viability. Conclusions: This study demonstrated dose‐ and time‐dependent cytotoxic effects of DMSO on human NPC. The addition of NAC to the cryopreservation medium ameliorated cell viability loss by reducing DMSO‐induced oxidative stress in the freeze–thawing cycle. These findings may be useful for future clinical applications of whole cells and cellular products. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration (IDD) is a common and chronic inflammatory disorder. α‐Mangostin exhibits a novel biological function against inflammation in various inflammatory diseases. Here, we aimed to explore the role of α‐mangostin in IDD using an in vitro cell model. Human nucleus pulposus cells (NPCs) were exposed to lipopolysaccharide (LPS) to induce inflammatory injury. Cell viability of NPCs was determined by CCK‐8 assay. ELISA was performed to examine the production of interleukin (IL)‐1β and IL‐18. Apoptotic cell death in NPCs was detected by TUNEL staining. The expression levels of apoptotic‐associated proteins were detected by western blotting. Nuclear factor‐kappa B (NF‐κB) activation was examined by determining the expression levels of p‐p65, p65, and nuclear p65. Results showed that treatment with α‐mangostin improved the viability of LPS‐treated NPCs. α‐Mangostin treatment also inhibited the LPS‐induced increase in expression levels of NLRP3, ASC and pro‐caspase‐1, as well as the production of IL‐1β and IL‐18 in NPCs. Moreover, treatment with α‐mangostin or NLRP3 inhibitor (MCC950) significantly decreased apoptotic cell death in NPCs, as compared with treatment with LPS. In addition, the expression levels of cleaved caspase‐3 and Bax were decreased, while Bcl‐2 expression was increased in α‐mangostin‐ or MCC950‐treated NPCs. Treatment with α‐mangostin also suppressed LPS‐induced increase of p‐p65/p65 and nuclear p65 levels. Moreover, inhibition of NF‐κB by PDTC aggravated the inhibitory effects of α‐mangostin on NLRP3 inflammasome activation and apoptosis in LPS‐induced NPCs. These findings suggested that α‐mangostin exerted a protective effect on NLRP3 inflammasome‐mediated apoptosis in LPS‐induced NPCs through regulating NF‐κB signaling. α‐Mangostin exhibits a novel biological function against inflammation in various inflammatory diseases. Herein, we aimed to explore the role of α‐mangostin in intervertebral disc degeneration (IDD) using an in vitro cell model. Our data showed that α‐mangostin exerted a protective effect on NLRP3 inflammasome‐mediated apoptosis in LPS‐induced NPCs through regulating NF‐κB signaling. This indicates that α‐mangostin may have a protective effect against IDD and may potentially serve as a clinical agent for IDD prevention and treatment. [ABSTRACT FROM AUTHOR]

The transcription factor p300 is reportedly involved in age‐associated human diseases, including intervertebral disc degeneration (IDD). In this study, we investigate the potential role and pathophysiological mechanism of p300 in IDD. Clinical tissue samples were collected from patients with lumbar disc herniation (LDH), in which the expression of p300, forkhead box O3 (FOXO3), and sirtuin 1 (Sirt1) was determined. Nucleus pulposus cells (NPCs) isolated from clinical degenerative intervertebral disc (IVD) tissues were introduced with oe‐p300, oe‐FOXO3, Wnt/β‐catenin agonist 1, C646 (p300/CBP inhibitor), or si‐p300 to explore the functional role of p300 in IDD and to characterize the relationship between p300 and the FOXO3/Sirt1/Wnt/β‐catenin pathway. Also, we established a rat IDD model by inducing needle puncture injuries in the caudal IVDs for further verification of p300 functional role. We found that p300 was downregulated in the clinical tissues and NPCs of IDD. Overexpression of p300 promoted the proliferation and autophagy of NPCs while inhibiting cell apoptosis, which was associated with FOXO3 upregulation. p300 could increase the expression of FOXO3 by binding to the Sirt1 promoter, and thus, contributed to inactivation of the Wnt/β‐catenin pathway. In vivo results further displayed that p300 slowed down the progression of IDD by disrupting the Wnt/β‐catenin pathway through the FOXO3/Sirt1 axis. Taken together, we suggest that p300 can act to suppress IDD via a FOXO3‐dependent mechanism, highlighting a potential novel target for treatment of IDD. [ABSTRACT FROM AUTHOR]

Rejuvenation of nucleus pulposus cells (NPCs) in degenerative discs can reverse intervertebral disc degeneration (IDD). Partial reprogramming is used to rejuvenate aging cells and ameliorate progression of aging tissue to avoiding formation of tumors by classical reprogramming. Understanding the effects and potential mechanisms of partial reprogramming in degenerative discs provides insights for development of new therapies for IDD treatment. The findings of the present study show that partial reprogramming through short‐term cyclic expression of Oct‐3/4, Sox2, Klf4, and c‐Myc (OSKM) inhibits progression of IDD, and significantly reduces senescence related phenotypes in aging NPCs. Mechanistically, short‐term induction of OSKM in aging NPCs activates energy metabolism as a "energy switch" by upregulating expression of Hexokinase 2 (HK2) ultimately promoting redistribution of cytoskeleton and restoring the aging state in aging NPCs. These findings indicate that partial reprogramming through short‐term induction of OSKM has high therapeutic potential in the treatment of IDD. [ABSTRACT FROM AUTHOR]

Background: This study was designed to verify whether enhancer of zeste homolog 2 (EZH2) affects intervertebral disc degeneration (IVDD) development through regulation of microRNA (miR)‐129‐5p/MAPK1. Methods: Initially, we collected lumbar nucleus pulposus (NP) tissue samples from patients with juvenile idiopathic scoliosis (n = 14) and IVDD (n = 34). We measured the expression of related genes in clinical IVDD tissues and a lipopolysaccharide (LPS)‐induced NP cell model. After loss‐ and gain‐of‐function assays, NP cell proliferation and senescence were examined. The targeting relationship between miR‐129‐5p and MAPK1 was explored by dual luciferase reporter gene and RNA immunoprecipitation (RIP) assays. The enrichment of EZH2 and H3K27me3 in miR‐129‐5p promoter was verified by chromatin immunoprecipitation (ChIP). Finally, an IVDD rat model was established to test the effects of transduction with lentiviral vector carrying miR‐129‐5p agomir and/or oe‐EZH2 in vivo. Results: miR‐129‐5p was underexpressed, and EZH2 and MAPK1 levels were overexpressed in lumbar nucleus pulposus from human IVDD patients and in LPS‐induced NP cells. miR‐129‐5p overexpression or silencing of MAPK1 promoted proliferation of NP cells, while inhibiting their senescence. EZH2 inhibited miR‐129‐5p through H3K27me3 modification in the miR‐129‐5p promoter. miR‐129‐5p could target the downregulation of MAPK1 expression. EZH2 overexpression increased the release of inflammatory factors and cell senescence factors, which was reversed by miR‐129‐5p agomir in vivo. Conclusions: Taken together, EZH2 inhibits miR‐129‐5p through H3K27me3 modification, which upregulates MAPK1, thereby promoting the development of IVDD. [ABSTRACT FROM AUTHOR]

Objective: To investigate the protective effect and mechanism of shikonin on human intervertebral disk degeneration. Methods: Human primary nucleus pulposus (NP) cells cultured in vitro were used for the experiments. The effects of different concentrations of shikonin (1, 2, 4, 8, and 16 µM) on the activity of lipopolysaccharide (LPS)‐induced NP cells were determined using the CCK‐8 assay, and the appropriate drug concentration was determined. The experiment was divided into the control, LPS, and LPS + shikonin groups. ELISA and Western blot were used to detect the expression of the inflammatory factors tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β. NP cell apoptosis was measured using Western blot and caspase 3 activity. Western blot and immunofluorescence assays were used to detect the protein expression of p‐P65 and P65 and the nuclear translocation of P65. Results: The CCK‐8 assay showed that shikonin had no cytotoxic effect on NP cells and increased the activity of LPS‐induced NP cells, especially at a concentration of 4 μM. Shikonin reversed the expression of the inflammatory cytokines TNF‐α and IL‐1β and apoptosis‐related molecules Bax, Bcl‐2, and cleaved caspase 3 in LPS‐induced NP cells. In addition, shikonin significantly decreased apoptosis and caspase‐3 activity in LPS‐induced NP cells. Furthermore, shikonin treatment significantly inhibited the expression of p‐P65 and nuclear translocation of P65, and nuclear factor‐kappa B (NF‐κB) pathway inhibitor Pyrrolidinedithiocarbamate ammonium (PDTC) significantly enhanced the anti‐inflammatory and antiapoptotic effects of shikonin in LPS‐induced NP cells. Conclusion: Shikonin significantly inhibited the inflammatory response and apoptosis of human primary NP cells, possibly through the NF‐κB pathway. [ABSTRACT FROM AUTHOR]

P53 is an apoptosis marker which is involved in determining nucleus pulposus (NP) cell fate. Little is known about P53 interaction with N‐Myc downstream‐regulated gene 2 (NDRG2) in intervertebral disc degeneration (IVDD). Here, we studied the role of the P53‐NDRG2 axis in IVDD. We found that NDRG2 was expressed in NP tissue obtained from patients with IVDD. The level of NDRG2 was positively related to the severity of IVDD, as determined by Pfirrmann grading. Subsequently, we overexpressed NDRG2 in human NP cells by adenoviral transfection and studied the effects of increased levels of NDRG2 on the viability and apoptosis of these cells. NDRG2 overexpression induced NP cell apoptosis and reduced viability in NP cells obtained from patient with IVDD. We also found that the level of P53 was elevated in NP cells from patients with IVDD and treatment with exogenous P53 upregulated NDRG2 in NP cells. Last, IVDD model was established in P53 knockout mice and the pathological changes in the intervertebral discs and NDRG2 expression were examined. P53 knockout can reduce the damage of NP tissues after IVDD surgery to some extent. Restoration of NDRG2 antagonized the effect of P53 knockout on IVDD. Collectively, this study suggests that elevated P53 in NP cells stimulates apoptosis of the cells by upregulating NDRG2 expression, thereby exacerbating IVDD. [ABSTRACT FROM AUTHOR]

Hyperglycemia can drive advanced glycation end product (AGE) accumulation and associated nucleus pulposus cell (NPC) dysfunction, but the basis for this activity has not been elucidated. Hypoxia‐inducible factor‐1α (HIF‐1α) is subject to cell‐type‐specific AGE‐mediated regulation. In the current study, we assessed the mechanistic relationship between AGE accumulation and HIF‐1α degradation in NPCs. Immunohistochemical staining of degenerated nucleus pulposus (NP) samples was used to assess AGE levels. AGE impact on NPC survival and glycolysis‐related gene expression was assessed via 3‐(4,5)‐dimethylthiazol(‐z‐y1)‐3,5‐di‐phenyltetrazolium bromide assay and quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR), while HIF‐1α expression in NPCs following AGE treatment was monitored via Western blot analysis and qRT‐PCR. Additionally, a luciferase reporter assay was used to monitor HIF‐1α transcriptional activity. The importance of the receptor for activated C‐kinase 1 (RACK1) as a mediator of HIF‐1α degradation was evaluated through gain‐ and loss‐of‐function experiments. Competitive binding of RACK1 and HSP90 to HIF‐1α was evaluated via immunoprecipitation. Increased AGE accumulation was evident in NP samples from diabetic patients, and AGE treatment resulted in reduced HIF‐1α protein levels in NPCs that coincided with reduced HIF‐1α transcriptional activity. AGE treatment impaired the stability of HIF‐1α, leading to its RACK1‐mediated proteasomal degradation in a manner independent of the canonical PHD‐mediated degradation pathway. Additionally, RACK1 competed with HSP90 for HIF‐1α binding following AGE treatment. AGE treatment of NPCs leads to HIF‐1α protein degradation. RACK1 competes with HSP90 for HIF‐1α binding following AGE treatment, resulting in posttranslational HIF‐1α degradation. These results suggest that AGE is an intervertebral disc degeneration risk factor, and highlight potential avenues for the treatment or prevention of this disease. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this paper is to investigate the effects of senescent nucleus pulposus cell (NPC)‐derived exosomes (SNPC‐Exo) and the roles of the P53/P21 pathway on the senescence of NPC. Methods: The senescent phenotypes of NPC were induced by interleukin‐1β treatment. SNPC‐Exo was extracted from the culture medium of senescent NPC and purified by differential centrifugation. The structure of SNPC‐Exo was identified by transmission electron microscopy and western blot analysis was used to determine the exosomal marker proteins CD63 and Tsg101. Western blot analysis was performed to determine the relative expression levels of P16, P21, and P53 in NPC. Senescence‐associated β‐galactosidase (SA‐β‐gal) staining was used to stain the senescent NPC and a phase contrast microscope was used to observe and count the SA‐β‐gal staining of NPC. The proliferation of SNPC‐Exo‐treated NPC was assessed using growth curve analysis and the colony formation assay. The cell cycle of SNPC‐Exo‐treated NPC was determined by flow cytometry. NPC were transfected with siRNA to knock down P53 and P21 expression. Results: Interleukin‐1β‐treated NPC had a higher percentage of SA‐β‐gal positive cells (45%) than the control group (20%) and showed an increase in the relative expression of P16, P21, and P53 (P < 0.05). SNPC‐Exo were positive for exosomal marker protein CD63 and Tsg 101 and negative for calnexin, and successfully internalized as previously described. SNPC‐Exo‐treated NPC showed an increase in the relative expression of P21 and P53 (P < 0.05). Compared with the control group, the SNPC‐Exo‐treated NPC showed a lower growth rate (3 times lower on the 5th day and 2 times lower on the 7th day), fewer colony‐forming units (12.0%), and a higher percentage of SA‐β‐gal‐positive NPC (50.0%). The SNPC‐Exo‐treated NPC contained more G1 phase cells (68.0%) and fewer S phase (15.5%) cells than the control group (53.0% in G1 phase, 33.5% in S phase). The expression of P21 and P53 significantly decreased in SNPC‐exo‐treated NPC after siRNA transfection (P < 0.05), followed by a higher growth rate (2 times higher on the 5th day and 1.5 times higher on the 7th day) and lower percentage of SA‐β‐gal‐positive NPC (22.5%). Moreover, the inhibition of the P53/P21 pathway promoted the SNPC‐Exo‐treated NPC to enter the S phase (from 15.5% to 25.3%). Conclusion: The inhibition of the P53/P21 pathway attenuated the senescence of NPC induced by SNPC‐Exo. [ABSTRACT FROM AUTHOR]

A key early sign of degenerative disc disease (DDD) is the loss of nucleus pulposus (NP) cells (NPCs). Accordingly, NPC transplantation is a treatment strategy for intervertebral disc (IVD) degeneration. However, in advanced DDD, due to structural damage of the IVD and scaffold mechanical properties, the transplanted cells are less viable and secrete less extracellular matrix, and thus, are unable to efficiently promote NP regeneration. In this study, we evaluated the encapsulation of NPCs in a photosensitive hydrogel made of collagen hydrolysate gelatin and methacrylate (GelMA) to improve NP regeneration. By adjusting the concentration of GelMA, we prepared hydrogels with different mechanical properties. After examining the mechanical properties, cell compatibility and tissue engineering indices of the GelMA‐based hydrogels, we determined the optimal hydrogel concentration of the NPC‐encapsulating GelMA hydrogel for NP regeneration as 5%. NPCs effectively combined with GelMA and proliferated. As the concentration of the GelMA hydrogel increased, the survival, proliferation and matrix deposition of the encapsulated NPCs gradually decreased, which is the opposite of NPCs grown on the surface of the hydrogel. The controllability of the GelMA hydrogels suggests that these NPC‐encapsulating hydrogels are promising candidates to aid in NP tissue engineering and repairing endogenous NPCs. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase‐1 (HO‐1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO‐1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO‐1 expression was reduced in IDD tissues and replicative senescent NP cells. HO‐1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3‐MA pretreatment reversed the anti‐senescent and protective effects on the mitochondrial function of HO‐1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO‐1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO‐1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route. [ABSTRACT FROM AUTHOR]

The present study determines whether the in vivo injection of TGFβ1 and CTGF mediated by AAV2 to transfect nucleus pulposus cells in degenerative lumbar discs can reverse the biological effects of rhesus lumbar disc degeneration. A total of 42 lumbar discs obtained from six rhesus monkeys were classified into three groups: experimental group, control group, and blank group. Degenerative lumbar discs were respectively injected with double gene‐transfected human nucleus pulposus cells using minimally invasive techniques. Immumohistochemical staining, RT‐PCR, and western blot were performed to observe the biological effects of double gene‐transfected human nucleus pulposus cells in degenerative lumbar discs on rhesus lumbar disc degeneration. At 4, 8, and 12 weeks after the transplantation of nucleus pulposus cells, the expression levels of TGF‐ß1, CTGF, proteoglycan mRNA, and type‐II collagen were detected by RT‐PCR. The values of immumohistochemical staining and RT‐PCR in the experimental group increased at 8 weeks, decreased with time at 12 weeks, and remained greater than the values in the control group, and the differences were statistically significant (P < .05). The western blot revealed that the values in the experimental group decreased with time, but remained greater than those in the PBS control group and blank control group, and the differences were statistically significant (P < .05). The double gene‐transfection of human nucleus pulposus cells in degenerative lumbar discs mediated by rAAV2 can be continuously expressed in vivo after transplantation in lumbar discs of rhesus monkeys, and promotes the synthesis of proteoglycan and type II collagen, achieving the treatment purpose. [ABSTRACT FROM AUTHOR]

Intervertebral disc (IVD) consists of surrounding tissue annulus fibrosus and central nucleus pulposus, which are partially degenerative in scoliotic IVDs. Successful regeneration of scoliotic alterations requires cognition of critical paracrine mediators of cell‐to‐cell contact in the IVD. In this work, we hypothesized that transforming growth factor β (TGF‐β) is involved in the intercellular communication of nucleus pulposus cells (NPCs) and mesenchymal stem cells (MSCs). We observed that in cultured NPCs TGF‐β1 stimulated COL1A1 expression, encoding collagen I, and in MSCs stimulated COL1A1 and SOX9 expressions. We subsequently co‐cultured NPCs and MSCs together using direct and indirect transwell systems. The expression of miR‐140 and miR‐145 were decreased in co‐cultured NPCs. We observed that direct co‐culture system stronger than the indirect system decreased expression of three miRNA. The expression of COL1A1, ACAN, encoding aggrecan, and SOX9 genes was increased in MSCs co‐cultured with NPCs. Co‐cultures were incubated with two inhibitors of TGF‐β type I receptor: SB‐431542 and SB‐525334. In co‐cultured NPCs, SB‐431542 and SB‐525334 annulated downregulation of miR‐140 and miR‐145. In MSCs these inhibitors diminished stimulation of COL1A1, ACAN, and SOX9. We concluded that stimulation of COL1A1, ACAN, and SOX9 in co‐cultured MSCs and regulation of miR‐140 and miR‐145 in NPCs were TGF‐β‐dependent and TGF‐β is involved in the communication of NPCs and MSCs in co‐culture. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3023–3032, 2018. [ABSTRACT FROM AUTHOR]

Disc degeneration alters the structure and function of intervertebral discs and is the basis of spinal degenerative diseases. To establish the molecular mechanism of intervertebral disc degeneration caused by mechanical strain, this study examined the effects of different amplitude (3%, 9%, 19%) cyclic mechanical strain (CMS) at a low frequency (0.01 Hz) on the secretion of cartilage extracellular matrix, expression of inflammatory cytokines and catabolic proteases, and activation of NF-κB signaling pathway in human nucleus pulposus cells. We also investigated effects of low frequency and high amplitude (19%) CMS on degeneration of human nucleus pulposus cells in the presence or absence of p65 inhibitor, p65 silencing shRNA, or p65 overexpression. While 3% CMS did not significantly decrease aggrecan or type II collagen expression, or increase TNF-α, IL-1β, IL-6 expression, 9% and 19% CMS showed the significant effects. Low frequency and high amplitude (19%) CMS was found to promote p65 activation in human nucleus pulposus cells, and IL-1β was found to promote p65 nuclear translocation though IκB kinase phosphorylation. Furthermore, degeneration process of nucleus pulposus cells was found attenuated in the presence of p65 inhibitor or p65 silencing shRNA, but promoted with p65 overexpression. These data suggest that high amplitude and low frequency CMS could promote degeneration of human nucleus pulposus cells significantly via the NF- κB p65 pathway. Our findings have uncovered the effect of CMS on human nucleus pulposus cell degeneration and have identified a previously unknown intrinsic underlying mechanism. [ABSTRACT FROM AUTHOR]

Although mesenchymal stem cells ( MSCs) transplantation into the IVD (intervertebral disc) may be beneficial in inhibiting apoptosis of nucleus pulposus cells ( NPCs) and alleviating IVD degeneration, the underlying mechanism of this therapeutic process has not been fully explained. The purpose of this study was to explore the protective effect of MSC-derived exosomes ( MSC-exosomes) on NPC apoptosis and IVD degeneration and investigate the regulatory effect of mi RNAs in MSC-exosomes and associated mechanisms for NPC apoptosis. MSC-exosomes were isolated from MSC medium, and its anti-apoptotic effect was assessed in a cell and rat model. The down-regulated mi RNAs in apoptotic NPCs were identified, and their contents in MSC-exosomes were detected. The target genes of eligible mi RNAs and possible downstream pathway were investigated. Purified MSC-exosomes were taken up by NPCs and suppressed NPC apoptosis. The levels of miR-21 were down-regulated in apoptotic NPCs while MSC-exosomes were enriched in miR-21. The exosomal miR-21 could be transferred into NPCs and alleviated TNF-α induced NPC apoptosis by targeting phosphatase and tensin homolog ( PTEN) through phosphatidylinositol 3-kinase ( PI3K)-Akt pathway. Intradiscal injection of MSC-exosomes alleviated the NPC apoptosis and IVD degeneration in the rat model. In conclusion, MSC-derived exosomes prevent NPCs from apoptotic process and alleviate IVD degeneration, at least partly, via miR-21 contained in exosomes. Exosomal miR-21 restrains PTEN and thus activates PI3K/Akt pathway in apoptotic NPCs. Our work confers a promising therapeutic strategy for IVD degeneration. [ABSTRACT FROM AUTHOR]

Nucleus pulposus (NP) cells locate in the center of avascular intervertebral discs, and thus have presumably adapted to a hypoxic environment. The purpose of this study was to investigate the influences of hypoxic condition, during isolation-expansion of human NP cells, on the cellular proliferation and extracellular matrix (ECM) synthesis in later three-dimensional cultures. Human NP tissues were obtained from patients who underwent lumbar disc surgeries. Immediately after retrieval, NP tissues from each patient were divided into two aliquots for in vitro cultivation either under classical normoxic (21% O2) or hypoxic (3.5% O2) condition. After isolation-expansion processes, microtissues of NP cells were formed and the analysis was performed after one-week culture. Experiments of pretreatment with TGF-β1 or lovastatin were designed to investigate if the isolation-expansion conditions affect the responsiveness to later exogenous treatments. Hypoxic isolation-expansion stimulated NP cell proliferation during monolayer culture. Hypoxia also upregulated mRNA levels of SOX9 and HIF-1α but downregulated type X collagen as well as improved aggrecan and type II collagen synthesis. Although TGF-β1 had no substantial effect, lovastatin pretreatment showed a greater enhancement on type II collagen expression in hypoxic group. Normoxia negatively affected the biochemical composition of regenerated ECM attributable to downregulation of SOX9 and HIF-1α, while hypoxia enhanced cellular proliferation, improved matrix production, and maintained a functional phenotype of NP cells. Hypoxic isolation-expansion of human NP cells is important to achieve better regenerative cells for later cultivation or cell transplantation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1575-1582, 2017. [ABSTRACT FROM AUTHOR]

ABSTRACT The main reason for intervertebral disc (IVD) degeneration is the decrease in the quantity and activity of IVD cells with subsequent reduction of the extracellular matrix (ECM). In this study, we investigated a cell-based repair strategy by injecting nucleus pulposus cells (NPCs) transduced with human bone morphogenetic protein (hBMP7) by adeno-associated virus-2 into the canine degenerative IVD to determine whether NPCs expressing hBMP7 could delay the degeneration of the IVD. Fourteen canines received annular punctures to induce disc degeneration. Eight weeks later, saline (group A), allogeneic NPCs (group B), or allogeneic NPCs transduced with hBMP7 (group C) were injected into the degenerative discs. Twelve weeks after the injection, MRI scan showed that the degeneration process of groups C was slower and less severe compared with that of groups B and C. The IVD stability in group C was superior to that in groups A and B in left-right bending and rotation. HE, safranin-O staining, and ELISA indicated that the degenerative degree of the IVD in group C was significantly milder than that in groups A and B. The study demonstrated that the implantation of NPCs-hBMP7 could effectively maintained the structural integrity, ECM, and biomechanical properties of the canine degenerated discs. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1311-1322, 2017. [ABSTRACT FROM AUTHOR]

Degeneration of intervertebral discs (IVDs) results in an overall alteration of the biomechanics of the spinal column and becomes a major cause of low back pain. In this study, an injectable hydrogel composite is fabricated and characterized as a potential scaffold for the treatment of degenerated IVDs. Crosslinking of type II collagen-hyaluronic acid (HA) hydrogel with 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) increases the gel stability against collagenase digestion and reduces water uptake in comparison with non-crosslinked gel. Cell viability assay exhibits the proliferation of human nucleus pulposus (HNP) cells in hydrogels. The cells in non-crosslinked gel and the gel crosslinked with a low concentration of EDC (0.1 mM) show superior cell viability and morphology compared with cells in gels crosslinked with higher concentration of EDC. Quantitative PCR assay demonstrates the gene expression of extracellular matrix (ECM) by cells cultured in the gels. The expression of ECM genes by HNP cells in the gels demonstrated the phenotypic change of the cells. This study suggests that the type II collagen-HA hydrogel and crosslinked hydrogel (0.1 mM EDC) are permissive matrix for the growth of HNP cells and can be potentially applied in NP repair. [ABSTRACT FROM AUTHOR]

ABSTRACT The cyclooxygenase 2 (COX-2) product, prostaglandin E2 (PGE2), acts through a family of G protein-coupled receptors designated E-prostanoid (EP) receptors that mediate intracellular signaling by multiple pathways. However, it is not known whether crosstalk between tumor necrosis factor-α(TNF-α)-PGE2-mediated signaling and Wnt signaling plays a role in the regulation of intervertebral disc (IVD) cells. In this study, we investigated the relationship between TNF-α-PGE2 signaling and Wnt signaling in IVD cells. TNF-α increased the expression of COX-2 in IVD cells. The EP receptors EP1, EP3, and EP4 were expressed in IVD cells, and TNF-α significantly increased PGE2 production. Stimulation with TNF-α also upregulated EP3 and EP4 mRNA and protein expression in IVD cells. The inductive effect of the EP3 and EP4 receptors on Topflash promoter activity was confirmed through gain- and loss-of-function studies using selective EP agonists and antagonists. PGE2 treatment activated Wnt-β-catenin signaling through activation of EP3. We conclude that TNF-α-induced COX-2 and PGE2 stimulate Wnt signaling and activate Wnt target genes. Suppression of the EP3 receptor via TNF-α-PGE2 signaling seems to suppress IVD degeneration by controlling the activation of Wnt signaling. These findings may help identify the underlying mechanism and role of Wnt signaling in IVD degeneration. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1756-1768, 2015. [ABSTRACT FROM AUTHOR]

ABSTRACT Human degenerative disc disease (DDD) is characterized by progressive loss of human nucleus pulposus (HNP) cells and extracellular matrix, in which the massive deposition are secreted by HNP cells. Cell therapy to supplement HNP cells to degenerated discs has been thought to be a promising strategy to treat DDD. However, obtaining a large quality of fully functional HNP cells has been severely hampered by limited proliferation capacity of HNP cells in vitro. Previous studies have used lipofectamine or recombinant adeno-associated viral (rAAV) vectors to deliver human telomerase reverse transcriptase (hTERT) into ovine or HNP cells to prolong the activity of nucleus pulposus cells with limited success. Here we developed a lentiviral vector bearing both hTERT and a gene encoding green fluorescence protein (L-hTERT/EGFP). This vector efficiently mediated both hTERT and EGFP into freshly isolated HNP cells. The expressions of both transgenes in L-hTERT/EGFP transduced HNP cells were detected up to day 210 post viral infection, which was twice as long as rAAV vector did. Furthermore, we observed restored telomerase activity, maintained telomere length, delayed cell senescence, and increased cell proliferation rate in those L-hTERT/EGFP transduced HNP cells. Our study suggests that lentiviral vector might be a useful gene delivery vehicle for HNP cell therapy to treat DDD. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:159-166, 2014. [ABSTRACT FROM AUTHOR]

Obesity is an important risk factor for intervertebral disc degeneration and leptin is a biomarker of obesity. However, the expression of leptin receptors has not been determined in disc tissue. It is not known whether leptin has a direct effect on the nucleus pulposus (NP) cells. To determine whether the NP tissues and cells express leptin receptors (OBRa and OBRb) and whether leptin affects the organization and the expression of major cytoskeletal elements in NP cells. Messenger RNA (mRNA) and protein levels of OBRa and OBRb were measured by real-time PCR and Western blot, respectively, in NP tissues and cells. Immunofluorescence and real-time PCR and Western blot were performed to investigate the effect of leptin on cytoskeleton reorganization and expression. Results show that mRNA and proteins of OBRa and OBRb were expressed in all NP tissues and cells, and that OBRb expression was correlated with patients' body weight. Increased expression of β-actin and reorganization of F-actin were evident in leptin-stimulated NP cells. Leptin also induced vimentin expression but had no effect on β-tubulin in NP cells. These findings provide novel evidence supporting the possible involvement of leptin in the pathogenesis of intervertebral disc degeneration. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 847-857, 2013 [ABSTRACT FROM AUTHOR]

Intervertebral disc (IVD) degeneration is a common disorder of the lower spine. Since it is caused by loss of cellularity, there is interest in the comprehension of the cellular phenotypes. This study aimed to verify if stem cells isolated from nucleus pulposus of intervertebral discs (NPs-IVD), which may express neurogenic properties, may be implicated in IVD disease. NPs-IVD isolated from 14 human pathological discs were cultured under mesenchymal and neural differentiation. An induction of the neural markers GFAP, NF, MAP2, O4, and a decrement of the expression of the immature neural markers β-tubulin III, Nestin, NG2, occurred within the neural differentiation. The expression of TrkA and p75NGFR, the receptors of NGF, was not correlated with neural induction; in contrast, TrkB, the BDNF receptor, increased and was co-expressed with acid sensing ion channel 3 (ASIC3). In the same condition, neuroinflammatory markers were over-expressed. We confirm our hypothesis that stem cells within IVD degeneration acquire neurogenic phenotype, causing the induction of markers related to inflammatory condition. These cells could promote the enrolment of neurotrophines in adaptation to the acidic microenvironment in degenerative conditions. These data could improve our knowledge about IVD cellularity and eventually lead to the development of pharmacological therapies. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1470-1477, 2012 [ABSTRACT FROM AUTHOR]

To acquire the capacities for matrix production and preservation of an expanded volume within a damaged intervertebral disc (IVD), cells isolated from human nucleus pulposus (NP) tissues must undergo several passages in monolayer culture. However, chondrocytes and IVD cells in monolayer culture undergo 'dedifferentiation,' characterized by decreased synthesis of type II collagen and increased synthesis of type I collagen, thereby compromising the properties of regenerative tissues. The present study was undertaken to ascertain whether lovastatin reverses 'dedifferentiation' of human NP cells during monolayer expansion. Expression of genes encoding type II collagen and transcription factor SOX9 in these cells was upregulated by lovastatin, with maximal stimulations observed at 5 µM, whereas type I collagen gene expression was suppressed by the drug, with maximal inhibitions observed at 5-10 µM. At lovastatin concentrations ≥1 µM, expression of genes encoding the bone morphogenetic proteins BMP-2 and BMP-7 was also significantly enhanced. Furthermore, the number of NP cells exhibiting a rounded shape and positive staining for S-100 protein and type II collagen protein increased during treatment with lovastatin. These findings strongly support the induction by lovastatin of 'redifferentiation' of human NP cells during their expansion in monolayer culture. [ABSTRACT FROM AUTHOR]

Tissue microenvironment plays a critical role in guiding local stem cell differentiation. Within the intervertebral disc, collagen type II and nucleus pulposus (NP) cells are two major components. This study aimed to investigate how collagen type II and NP cells affect adipose tissue-derived stem cells (ASCs) in a 3D environment. ASCs were cultured in collagen type I or type II hydrogels alone, or co-cultured in transwells with micromass NP cells for 4 and 14 days. ASCs seeded in collagen type II gels acquired dentritic cell shapes, and orchestrated cell density-dependent gel contraction rates. Up-regulation of collagen type X, but not of other chondrogenic markers was observed at day 4, irrespective of the hydrogel type. Strikingly, in co-cultures with NP cells, more pronounced differentiation of ASCs along the cartilaginous lineage was observed (up-regulation of collagen IIA, IIB and aggrecan gene expression, as well as stronger alcian blue staining), when ASCs were embedded in collagen type II in comparison with type I hydrogels. Interestingly, strong cellular condensations/aggregations were observed in ASC-seeded type II, but not type I gels, and this aggregation was markedly delayed when the same gels were co-cultured with NP cells. The NP cell-mediated inhibition of ASC aggregation in collagen type II gels coincided with down-regulation of integrin subunit α2 gene expression. We conclude that soluble factors released by NP cells can direct chondrogenic differentiation of ASCs in collagen hydrogels, and that combination with a nucleus-mimicking collagen type II microenvironment enhances differentiation towards a more pronounced cartilage/NP lineage relative to collagen type I hydrogels. [ABSTRACT FROM AUTHOR]

The intervertebral disc (IVD) is a highly hydrated tissue, the rich proteoglycan matrix imbibes water, enabling the disc to withstand compressive loads. During aging and degeneration increased matrix degradation leads to dehydration and loss of function. Aquaporins (AQP) are a family of transmembrane channel proteins that selectively allow the passage of water in and out of cells and are responsible for maintaining water homeostasis in many tissues. Here, the expression of all 13 AQPs at gene and protein level was investigated in human and canine nondegenerate and degenerate IVDs to develop an understanding of the role of AQPs during degeneration. Furthermore, in order to explore the transition of notochordal cells (NCs) towards nucleus pulposus (NP) cells, AQP expression was investigated in canine IVDs enriched in NCs to understand the role of AQPs in IVD maturation. AQP0, 1, 2, 3, 4, 5, 6, 7, and 9 were expressed at gene and protein level in both nondegenerate and degenerate human NP tissue. AQP2 and 7 immunopositivity increased with degeneration in human NP tissue, whereas AQP4 expression decreased with degeneration in a similar way to AQP1 and 5 shown previously. All AQP proteins that were identified in human NP tissue were also expressed in canine NP tissue. AQP2, 5, 6, and 9 were found to localize to vacuole‐like membranes and cell membranes in NC cells. In conclusion, AQPs were abundantly expressed in human and canine IVDs. The expression of many AQP isotypes potentially alludes to multifaceted functions related to adaption of NP cells to the conditions they encounter within their microenvironment in health and degeneration. The presence of AQPs within the IVD may suggest an adaptive role for these water channels during the development and maintenance of the healthy, mature IVD. This study identified the expression of many AQP family within the human and canine IVD. AQPs are present in both NC cells during maturation and NP cells during degeneration. Alterations in AQP expression and the disc environment are shown as ↓ = decrease and ↑ = increase. [ABSTRACT FROM AUTHOR]