Nucleus Pulposus Mesenchymal Stem Cells Research Articles

Bicomponent hydrogel laden with TGF-β3-nucleus pulposus stem cells for disc degeneration repair

Degenerative intervertebral disc (IVD) disease, which cannot be completely treated with open or minimally invasive surgery, is primarily caused by nucleus pulposus (NP) degeneration. Meanwhile, NP regeneration requires the intricate coordination of stem cells, growth factors, and biomaterials. The current study sought to develop a regenerative approach for IVD degeneration. To this end, we used a lentiviral vector to introduce the transforming growth factor-β3 (TGF-β3) gene into NP mesenchymal stem cells (NPMSCs) to construct genetically modified seed cells. In addition, bicomponent gelatin methacrylate/hyaluronic acid methacrylate (GH) hydrogels were synthesized based on gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) precursors as carriers for TGF-β3-NPMSCs delivery. In vitro differentiation of the loaded stem cells into NP-like structures was improved by the hydrogels' superior mechanical and cytocompatibility features. Moreover, the stem cell-loaded GH hydrogel was delivered to NP tissue via a minimally invasive approach to construct an injectable “transgenic cell-hydrogel” in situ microenvironment, which attenuated IVD degeneration, preserved the integrity of the NP tissue, and sped up extracellular matrix synthesis in a rat model. Collectively, the results of this study demonstrate the potential of this novel therapeutic strategy as an effective regenerative approach for IVD degeneration.

Melatonin alleviates oxidative stress-induced injury to nucleus pulposus-derived mesenchymal stem cells through activating PI3K/Akt pathway

BackgroundThe changes in the microenvironment of degenerative intervertebral discs cause oxidative stress injury and excessive apoptosis of intervertebral disc endogenous stem cells. The purpose of this study was to explore the possible mechanism of the protective effect of melatonin on oxidative stress injury in NPMSCs induced by H2O2. MethodsThe Cell Counting Kit-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of melatonin. ROS content was detected by 2′7′-dichlorofluorescin diacetate (DCFH-DA). Mitochondrial membrane potential (MMP) was detected by the JC-1assay. Transferase mediated d-UTP Nick end labeling (TUNEL) and Annexin V/PI double staining were used to determine the apoptosis rate. Additionally, apoptosis-associated proteins and PI3K/Akt signaling pathway-related proteins were evaluated by immunofluorescence, immunoblotting and PCR. ECMs were evaluated by RT‒PCR and immunofluorescence. In vivo, X-ray, Magnetic resonance imaging (MRI) and Histological analyses were used to evaluate the protective effect of melatonin. ResultsMelatonin had an obvious protective effect on NPMSCs treated with 0–10 μM melatonin for 24 h. In addition, melatonin also had obvious protective effects on mitochondrial dysfunction, decreased membrane potential and cell senescence induced by H2O2. More importantly, melatonin could significantly reduce the apoptosis of nucleus pulposus mesenchymal stem cells induced by H2O2 by regulating the expression of apoptosis-related proteins and decreasing the rate of apoptosis. After treatment with melatonin, the PI3K/Akt pathway was significantly activated in nucleus pulposus mesenchymal stem cells, while the protective effect was significantly weakened after PI3K-IN-1 treatment. In vivo, the results of X-ray, MRI and histological analyses showed that therapy with melatonin could partially reduce the degree of intervertebral disc degeneration. ConclusionOur research demonstrated that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of nucleus pulposus mesenchymal stem cells induced by oxidative stress via the PI3K/Akt pathway, which provides a novel idea for the therapy of intervertebral disc degeneration. The translational potential of this articleThis study indicates that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of NPMSCs through activating the PI3K/Akt pathway. Melatonin might serve as a promising candidate for the prevention and treatment of Intervertebral disc degeneration disease (IVDD) in the future.

Growth differentiation factor 5 inhibits lipopolysaccharide-mediated pyroptosis of nucleus pulposus mesenchymal stem cells via RhoA signaling pathway.

Degenerative disc disease(DDD)is one of the most important causes of low back pain (LBP). Programmed death of human nucleus pulposus mesenchymal stem cells (NPMSCs) plays an important role in the progression of DDD. Growth differentiation factor-5 (GDF-5) is a protein that promotes chondrogenic differentiation, and has been reported to slow the expression of inflammatory factors in nucleus pulposus cells. Compared with those in normal rats, MRI T2-weighted images show hypointense in the central nucleus pulposus region of the intervertebral disc in GDF-5 knockout rats. We aimed to evaluate the role of GDF-5 and Ras homolog family member A (RhoA) in NPMSCs. We used lipopolysaccharide (LPS) to simulate the inflammatory environment in degenerative disc disease, and performed related experiments on the effects of GDF-5 on NPMSCs, including the effects of pyroptosis, RhoA protein, and the expression of extracellular matrix components, and the effects of GDF-5, on NPMSCs. In addition, the effect of GDF-5 on chondroid differentiation of NPMSCs was included. The results showed that the addition of GDF-5 inhibited the LPS-induced pyroptosis of NPMSCs, and further analysis of its mechanism showed that this was achieved by activating the RhoA signaling pathway. These findings suggest that GDF-5 plays an important role in inhibiting the pyroptosis of NPMSCs and GDF-5 may have potential for degenerative disc disease gene-targeted therapy in the future.

Fisetin regulates the biological effects of rat nucleus pulposus mesenchymal stem cells under oxidative stress by sirtuin-1 pathway.

Excessive oxidative stress has been accepted as one of the critical factors for intervertebral disc degeneration (IDD), which is associated with low back pain (LBP). Fisetin (Fis) is a bioactive flavonoid that possesses strong bioactive activity. In present study, we aimed to illuminate the role of Fis on nucleus pulposus mesenchymal stem cells (NPMSCs). NPMSCs were isolated and cultured from rat NP tissues and identified by flow cytometry and multilinear differentiation. The cytotoxicity of Fis, EX-527, and hydrogen peroxide (H2 O2 ) on NPMSCs was validated using Cell Counting Kit-8 tests. Cell apoptosis was tested by flow cytometry and TUNEL assay. Inflammatory mediators were assessed by Elisa tests, RT-PCR. Extracellular matrix (ECM) metabolism was measured by Western blot analysis and RT-qPCR. The expression of the SIRT1 was evaluated by Western blot analysis. NPMSCs were successfully isolated and cultured from rat NP tissues, and it has been identified by flow cytometry and multilinear differentiation. The results showed that Fis attenuated H2 O2 -induced apoptosis, inflammation, and ECM degradation of NPMSCs. Moreover, the above protective effects of Fis can be inhibited by EX-527, a unique SIRT1 inhibitor, indicating that SIRT1 may involve in the mechanism of Fis in protecting NPMSCs from oxidative stress. As a natural compound with little cytotoxicity on NPMSCs, Fis alleviate H2 O2 -induced apoptosis, inflammation, and ECM degradation by suppressing oxidative stress, this finding may add the theoretical basis for research on new treatment of IDD based on NPMSCs.

Bioactive hydrogel encapsulated dual-gene engineered nucleus pulposus stem cells towards intervertebral disc tissue repair

Intervertebral disc (IVD) degeneration is a pathological process that affects the molecular, cellular, and tissue levels, but neither conservative nor surgical treatments currently address its root causes. Recent developments in genetic engineering and tissue engineering technologies indicate that a biomedical treatment for IVD degeneration may be possible. Lentivirus has been utilized in this study as a gene vector to introduce insulin-like growth factor-1 (IGF-1) and transforming growth factor-β3 (TGF-β3) into nucleus pulposus mesenchymal stem cells (NPMSCs) for the first time. This resulted in the construction of dual-gene engineered seed cells that could differentiate into nucleus pulposus cells overexpressing both IGF-1 and TGF-β3 growth factors. A bicomponent polymer network (BCN) hydrogel based on gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) was prepared and used as a carrier to deliver genetically engineered NPMSCs to the target tissues. BCN hydrogels effectively upregulated the extracellular matrix expression and nucleus pulposus differentiation after being loaded with NPMSCs overexpressing IGF-1 and TGF-β3. The novel biotherapeutic modality presented could be used to repair degenerated IVD tissues, replenish seed cells with a rich potential, and promote extracellular matrix synthesis and nucleus pulposus differentiation, thereby providing a theoretical basis and experimental rationale for the etiological treatment of IVD degeneration.

Pretreatment of nucleus pulposus mesenchymal stem cells with appropriate concentration of H2O2 enhances their ability to treat intervertebral disc degeneration

BackgroundNucleus pulposus mesenchymal stem cells (NPMSCs) transplantation is a promising treatment for intervertebral disc degeneration (IVDD). However, the transplanted NPMSCs exhibited weak cell proliferation, high cell apoptosis, and a low ability to resist the harsh microenvironment of the degenerated intervertebral disc. There is an urgent need to explore feasible methods to enhance the therapeutic efficacy of NPMSCs transplantation.ObjectiveTo identify the optimal concentration for NPMSCs pretreatment with hydrogen peroxide (H2O2) and explore the therapeutic efficacy of NPMSCs transplantation using H2O2 pretreatment in IVDD.MethodsRat NPMSCs were pretreated with different concentrations (range from 25 to 300 μM) of H2O2. The proliferation, reactive oxygen species (ROS) level, and apoptosis of NPMSCs were detected by cell counting kit-8 (CCK-8) assay, 5-ethynyl-2′-deoxyuridine (EdU) staining, and flow cytometry in vitro. The underlying signalling pathways were explored utilizing Western blotting. A rat needle puncture-stimulated IVDD model was established. X-ray, histological staining, and a multimode small animal live imaging system were used to evaluate the therapeutic effect of H2O2-pretreated NPMSCs in vivo.ResultsNPMSCs pretreated with 75 μM H2O2 demonstrated the strongest elevated cell proliferation by inhibiting the Hippo pathway (P < 0.01). Meanwhile, 75 μM H2O2-pretreated NPMSCs exhibited significantly enhanced antioxidative stress ability (P < 0.01), which is related to downregulated Brd4 and Keap1 and upregulated Nrf2. NPMSCs pretreated with 75 μM H2O2 also exhibited distinctly decreased apoptosis (P < 0.01). In vivo experiments verified that 75 μM H2O2-pretreated NPMSCs-transplanted rats exhibited an enhanced disc height index (DHI% = 90.00 ± 4.55, P < 0.01) and better histological morphology (histological score = 13.5 ± 0.5, P < 0.01), which means 75 μM H2O2-pretreated NPMSCs can better adapt to the environment of degenerative intervertebral discs and promote the repair of IVDD.ConclusionsPretreatment with 75 μM H2O2 was the optimal concentration to improve the proliferation, antioxidative stress, and antiapoptotic ability of transplanted NPMSCs, which is expected to provide a new feasible method to improve the stem cell therapy efficacy of IVDD.

MCP‑1/CCR2 axis inhibits the chondrogenic differentiation of human nucleus pulposus mesenchymal stem cells.

Intervertebral disc degeneration (IDD) creates a hostile environment with high osmotic pressure, high mechanical stress, hypoxia and a low pH, where cytokines such as TNF‑α and IL‑1β are highly expressed. The degenerating intervertebral disc has high local expression of monocyte chemoattractant protein‑1(MCP‑1), which is associated with the degree of degeneration. However, there are a few reports on the influence of MCP‑1 on nucleus pulposus‑derived stem cells(NPSCs). In the present study, a significant upregulation of MCP‑1 was observed in NPSCs cultured invitro with pro‑inflammatory cytokines. MCP‑1 significantly inhibited the migration and proliferation of NPSCs in a dose‑dependent manner as detected via Cell Counting Kit‑8, wound healing and Transwell assays. Western blotting and histological analysis demonstrated that MCP‑1 significantly reduced chondrogenic NPSC differentiation. Reverse transcription‑quantitative PCR and western blotting revealed that C‑Cchemokine receptor type2(CCR2) mRNA and protein expression levels were significantly enhanced by MCP‑1. Furthermore, MCP‑1 significantly inhibited the migration, differentiation and proliferation of NPSCs, which was effectively reversed by blocking CCR2 with the inhibitor RS504393. Overall, these results demonstrated that MCP‑1 may contribute to the inhibition of chondrogenic NPSC differentiation via MCP‑1/CCR2 chemotaxis signals, providing a potential therapeutic target for IDD.

Construction of tissue-engineered nucleus pulposus by stimulation with periodic mechanical stress and BMP-2

SummaryIntervertebral disc (IVD) degeneration, which is common among elderly individuals, mainly manifests as low back pain and is caused by structural deterioration of the nucleus pulposus (NP) due to physiological mechanical stress. NP mesenchymal stem cells (NPMSCs) around the IVD endplate have multidirectional differentiation potential and can be used for tissue repair. To define favorable conditions for NPMSC proliferation and differentiation into chondroid cells for NP repair, the present study simulated periodic mechanical stress (PMS) of the NP under physiological conditions using MSC chondrogenic differentiation medium and recombinant human BMP-2 (rhBMP-2). rhBMP-2 effectively promoted NPMSC proliferation and differentiation. To clarify the mechanism of action of rhBMP-2, integrin alpha 1 (ITG A1) and BMP-2 were inhibited. PMS regulated the BMP-2/Smad1/RUNX2 pathway through ITG A1 and promoted NPMSC proliferation and differentiation. During tissue-engineered NP construction, PMS can effectively reduce osteogenic differentiation and promote extracellular matrix protein synthesis to enhance structural NP recovery.

Sa12b-Modified Functional Self-Assembling Peptide Hydrogel Enhances the Biological Activity of Nucleus Pulposus Mesenchymal Stem Cells by Inhibiting Acid-Sensing Ion Channels

Various hydrogels have been studied for nucleus pulposus regeneration. However, they failed to overcome the changes in the acidic environment during intervertebral disc degeneration. Therefore, a new functionalized peptide RAD/SA1 was designed by conjugating Sa12b, an inhibitor of acid-sensing ion channels, onto the C-terminus of RADA16-I. Then, the material characteristics and biocompatibility of RAD/SA1, and the bioactivities and mechanisms of degenerated human nucleus pulposus mesenchymal stem cells (hNPMSCs) were evaluated. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) confirmed that RAD/SA1 self-assembling into three-dimensional (3D) nanofiber hydrogel scaffolds under acidic conditions. Analysis of the hNPMSCs cultured in the 3D scaffolds revealed that both RADA16-I and RAD/SA1 exhibited reliable attachment and extremely low cytotoxicity, which were verified by SEM and cytotoxicity assays, respectively. The results also showed that RAD/SA1 increased the proliferation of hNPMSCs compared to that in culture plates and pure RADA16-I. Quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting demonstrated that the expression of collagen I was downregulated, while collagen II, aggrecan, and SOX-9 were upregulated. Furthermore, Ca2+ concentration measurement and western blotting showed that RAD/SA1 inhibited the expression of p-ERK through Ca2+-dependent p-ERK signaling pathways. Therefore, the functional self-assembling peptide nanofiber hydrogel designed with the short motif of Sa12b could be used as an excellent scaffold for nucleus pulposus tissue engineering. Moreover, RAD/SA1 exhibits great potential applications in the regeneration of mildly degenerated nucleus pulposus.

Sa12b Improves Biological Activity of Human Degenerative Nucleus Pulposus Mesenchymal Stem Cells in a Severe Acid Environment by Inhibiting Acid-Sensitive Ion Channels.

Sa12b is a wasp peptide that can inhibit acid-sensitive ion channels (ASICs). The biological effects of nucleus pulposus mesenchymal stem cells (NP-MSCs) have not been investigated. Therefore, this study investigated the effect of Sa12b on the biological activity of NP-MSCs through ASICs in the acidic environment of intervertebral disc degeneration (IVDD). In this study, NP-MSCs were isolated from the nucleus pulposus (NP) in patients who underwent lumbar disc herniation surgery, identified by flow cytometry and tertiary differentiation, and cultured in vitro in an acidic environment model of IVDD with a pH of 6.2. Proliferation, and apoptosis were observed after different Sa12b concentrations were added to P2 generation NP-MSCs. The Ca2+ influx was detected using flow cytometry and laser confocal scanning microscopy, and qPCR was used to detect the relative expression of stem cell–associated genes (Oct4, Nanog, Jag1, and Notch1), the relative expression of extracellular matrix (ECM)–associated genes (collagen II, aggrecan, and SOX-9), and the relative expression of genes encoding ASICs (ASIC1, ASIC2, ASIC3, and ASIC4). Western blotting was used to detect the protein expression of collagen II and aggrecan in different treatment groups. Cells isolated and cultured from normal NP were spindle-shaped and adherent, and they exhibited expansion in vitro. Flow cytometry results showed that the cells exhibited high expression of CD73 (98.1%), CD90 (97.5%), and CD105 (98.3%) and low expression of HLA-DR (0.93%), CD34 (2.63%), and CD45 (0.33%). The cells differentiated into osteoblasts, adipocytes, and chondrocytes. According to the International Society for Cellular Therapy criteria, the isolated and cultured cells were NP-MSCs. With an increase in Sa12b concentration, the cell proliferation rate of NP-MSCs increased, and the apoptosis rate decreased significantly, reaching the optimal level when the concentration of Sa12b was 8 μg/μl. When the Sa12b concentration was 8 μg/μl and contained the ASIC non-specific inhibitor amiloride, the Ca2+ influx was the lowest, followed by that when the Sa12b concentration was 8 μg/μl. The Ca2+ influx was the highest in the untreated control group. qPCR results showed that as the concentration of Sa12b increased, the relative expression of Oct4, Nanog, Jag1, Notch1, collagen II, aggrecan, and SOX-9 increased, while that of ASIC1, ASIC2, ASIC3, and ASIC4 decreased. The difference was statistically significant (p < 0.05). In conclusion, Sa12b can improve the biological activity of NP-MSCs in severely acidic environments of the intervertebral disc by reducing Ca2+ influx via AISC inhibition and, probably, the Notch signaling pathway. This study provides a new approach for the biological treatment of IVDD. Inhibition of AISCs by Sa12b may delay IVDD and improve low back pain.

Effect of lentivirus-mediated growth and differentiation factor-5 transfection on differentiation of rabbit nucleus pulposus mesenchymal stem cells

BackgroundIntervertebral disc degeneration (IDD) is a natural progression of age-related processes. Associated with IDD, degenerative disc disease (DDD) is a pathologic condition implicated as a major cause of chronic lower back pain, which can have a severe impact on the quality of life of patients. As degeneration progression is associated with elevated levels of inflammatory cytokines, enhanced aggrecan and collagen degradation, and changes in the disc cell phenotype. The purpose of this study was to investigate the biological and cytological characteristics of rabbit nucleus pulposus mesenchymal stem cells (NPMSCs)—a key factor in IDD—and to determine the effect of the growth and differentiation factor-5 (GDF5) on the differentiation of rabbit NPMSCs transduced with a lentivirus vector.MethodsAn in vitro culture model of rabbit NPMSCs was established and NPMSCs were identified by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR). Subsequently, NPMSCs were randomly divided into three groups: a transfection group (the lentiviral vector carrying GDF5 gene used to transfect NPMSCs); a control virus group (the NPMSCs transfected with an ordinary lentiviral vector); and a normal group (the NPMSCs alone). FCM, qRT-PCR, and western blot (WB) were used to detect the changes in NPMSCs.ResultsThe GDF5-transfected NPMSCs displayed an elongated shape, with decreased cell density, and significantly increased GDF5 positivity rate in the transfected group compared to the other two groups (P < 0.01). The mRNA levels of Krt8, Krt18, and Krt19 in the transfected group were significantly higher in comparison with the other two groups (P < 0.01), and the WB results were consistent with that of qRT-PCR.ConclusionsGDF5 could induce the differentiation of NPMSCs. The lentiviral vector carrying the GDF5 gene could be integrated into the chromosome genome of NPMSCs and promoted differentiation of NPMSCs into nucleus pulposus cells. Our findings advance the development of feasible and effective therapies for IDD.

Exosomes miR-15a promotes nucleus pulposus-mesenchymal stem cells chondrogenic differentiation by targeting MMP-3

The physiology of the nucleus pulposus (NP) in intervertebral disc degeneration (IVD) has been studied widely. However, interactions involving nucleus pulposus -mesenchymal stem cells (NP-MSCs) are less understood. MicroRNA 15a (miR-15a) is known to target and modulate genes involved in cellular proliferation and apoptosis. This study aimed to understand the interactions and impact of miR-15a and NP-MSCs on chondrogenic differentiation and IVD degeneration. Exosomes secreted by NP cells were purified by differential centrifugation and identified by transmission electron microscopy and exosomal markers. Further, by co-culture these exosomes were re-introduced into the NP-MSC cells, which were confirmed by fluorescence confocal microscopy. NP-MSCs treated with exo-miR-15a increases aggrecan and collagen II mRNA and protein levels while decreasing mRNA and protein levels of ADAMTS4/5 and MMP-3/-13. Toluidine blue staining confirmed that chondrogenic differentiation was increased in NP-MSCs treated with exo-miR-15a. NP-MSCs treated with exo-anti-miR-15a inhibit aggrecan and collagen II expression while increasing ADAMTS4/5 and MMP-3/-13 expression and decreasing chondrogenic differentiation. Dual-luciferase reporter assays revealed that miR-15a directly targets MMP-3 and downregulates its expression. Overexpression of miR-15a increased proliferation and colony formation, whereas combinatorial overexpression with MMP3, suppressed miR-15a's effects. This was also evident through the decreased phosphorylation of PI3K and Akt, upregulation of Wnt3a and β-catenin in the presence of miR-15a, but overexpression of MMP3 indicated an opposite effect. Overall, these data demonstrate that exo-miR-15a promotes NP-MSCs chondrogenic differentiation by downregulating MMP-3 through PI3K/Akt and Wnt3a/β-catenin axis.

ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration.

Stem cell approaches have become an attractive therapeutic option for intervertebral disc degeneration (IVDD). Nucleus pulposus mesenchymal stem cells (NP-MSCs) participate in the regeneration and homeostasis of the intervertebral disc (IVD), but the molecular mechanisms governing these processes remain to be elucidated. Acid-sensing ion channels (ASICs) which act as key receptors for extracellular protons in central and peripheral neurons, have been implicated in IVDD where degeneration is associated with reduced microenvironmental pH. Here we show that ASIC1 and ASIC3, but not ASIC2 and ASIC4 are upregulated in human IVDs according to the degree of clinical degeneration. Subjecting IVD-derived NP-MSCs to pH 6.6 culture conditions to mimic pathological IVD changes resulted in decreased cell proliferation that was associated with cell cycle arrest and induction of senescence. Key molecular changes observed were increased expression of p53, p21, p27, p16 and Rb1. Instructively, premature senescence in NP-MSCs could be largely alleviated using ASIC inhibitors, suggesting both ASIC1 and ASIC3 act decisively upstream to activate senescence programming pathways including p53-p21/p27 and p16-Rb1 signaling. These results highlight the potential of ASIC inhibitors as a therapeutic approach for IVDD and broadly define an in vitro system that can be used to evaluate other IVDD therapies.

Elucidating the role of cell-mediated inflammatory cytokines on allogeneic mouse-derived nucleus pulposus mesenchymal stem cells.

In this present study, our aim was to evaluate the cell-mediated specific anti-donor antibody and its associated inflammatory cytokine secretion along with its succeeding effects on Nucleus pulposus-derived mesenchymal stem cells (NPMSCs). Tissue from the NP compartment of 12 normal mice was isolated, expanded in cell culture, and the cell phenotypes were confirmed by flow cytometry. Multipotent differentiation and its specific gene expression analysis were confirmed by reverse transcriptase PCR. T and B cells were monitored for both donor and recipient mouse and further analysis of anti-donor antibody secretion was confirmed by lymphocyte crossmatch. In conjunction with anti-donor-specific antibody analysis, the associated inflammatory cytokine expression was analyzed by ELISA. In co-culture, cell-mediated antibody secretion was elevated in T and B cells positive mouse group, when compared to control mouse group. Allogeneic-derived donor NPMSCs were found to be stimulated the secretion of pro-inflammatory cytokines and the level of pro-inflammatory cytokines showed reduced expression in control mouse serum. In co-culture group the concentration of the cell-mediated pro- and anti-inflammatory cytokines found to be increased. PRACTICAL APPLICATIONS: Mesenchymal stem cell exhibit good regeneration capacity for many types of disease, and the mechanism belongs to regeneration is not clear. In intervertebral disc, the nucleus pulposus-derived mesenchymal stem cells showed a better regeneration capacity. On the contrary, the NP cells-based therapy, the Mesenchymal stem cells showed expanded anabolic and reduced catabolic activity together with induced anti-inflammatory effect. In this study, the T & B cells were used to evaluate the anti-donor antibody secretion and also to study how it stimulates the production of anti-donor antibodies against the donor cells. Finally, it was found that T & B cells lead the synthesis of inflammatory cytokines are IL-1, IL-6, and TNF-α. From this study, the results proved that the cell-mediated pro- and anti-inflammatory cytokines to be monitored in allogeneic stem cells-based therapy of intervertebral disc degeneration.

Activation of SIRT1 promotes cartilage differentiation and reduces apoptosis of nucleus pulposus mesenchymal stem cells via the MCP1/CCR2 axis in subjects with intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a condition involving disruption of the bone tissue distribution. Nucleus pulposus mesenchymal stem cells (NPMSCs) and Sirtuin 1 (SIRT1) play important roles in bone diseases, therefore the aim of the present study was to evaluate the roles of SIRT1 and NPMSCs in IDD. First, NPMSCs were harvested from patients with IDD. Then, the NPMSCs were treated with a SIRT activator, and monocyte chemoattractant protein 1 (MCP1) and chemokine receptor 2 (CCR2) inhibitors. Indices related to NPMSC growth, proliferation, differentiation and apoptosis were measured. Subsequently, IDD rat models were established and were transfected with NPMSCs overexpressing SIRT1. NPMSC apoptosis and cartilage differentiation were detected in the rat IDD model. SIRT1 expression was found to be decreased, and the expression of MCP1 and CCR2 increased in NPMSCs of patients with IDD. The upregulation of SIRT1 and the downregulation of the MCP1/CCR2 axis promoted cartilage differentiation and reduced the number of apoptotic NPMSCs. Furthermore, MCP1 reversed the progression of the cartilage differentiation of NPMSCs and the inhibition of NPMSC apoptosis induced by SIRT1 overexpression. Moreover, the transplantation of rat NPMSCs overexpressing SIRT1 relieved IDD in rats. Therefore, SIRT1 overexpression improved cartilage differentiation and reduced the apoptosis of NPMSCs by inactivating the MCP1/CCR2 axis, thus attenuating IDD in rats.

The protective effects of PI3K/Akt pathway on human nucleus pulposus mesenchymal stem cells against hypoxia and nutrition deficiency

BackgroundTo study the effects of hypoxia and nutrition deficiency mimicking degenerated intervertebral disc on the biological behavior of human nucleus-derived pulposus mesenchymal stem cells (hNP-MSCs) and the role of PI3K/Akt pathway in the process in vitro.MethodshP-MSCs were isolated from lumbar disc and were further identified by their immunophenotypes and multilineage differentiation. Then, cells were divided into the control group, hypoxia and nutrition deficiency group, the LY294002 group, and insulin-like growth factor 1 (IGF-1) group. Then cell apoptosis, the cell viability, the caspase 3 activity, and the expression of PI3K, Akt, and functional genes (aggrecan, collagen I, and collagen II) were evaluated.ResultOur work showed that isolated cells met the criteria of International Society for cellular Therapy. Therefore, cells obtained from degenerated nucleus pulposus were definitely hNP-MSCs. Our results showed that hypoxia and nutrition deficiency could significantly increase cell apoptosis, the caspase 3 activity, and inhibit cell viability. Gene expression results demonstrated that hypoxia and nutrition deficiency could increase the relative expression of PI3K and Akt gene and inhibit the expression of functional genes. However, when the PI3K/Akt pathway was inhibited by LY294002, the cell apoptosis and caspase 3 activity significantly increased while the cell viability was obviously inhibited. Quantitative real-time PCR results showed that the expression of functional genes was more significantly inhibited. Our study further verified that the above-mentioned biological activities of hNP-MSCs could be significantly improved by IGF1.ConclusionsPI3K/Akt signal pathway may have protective effects on human nucleus pulposus-derived mesenchymal stem cells against hypoxia and nutrition deficiency.

Puerarin Relieved Compression-Induced Apoptosis and Mitochondrial Dysfunction in Human Nucleus Pulposus Mesenchymal Stem Cells via the PI3K/Akt Pathway.

Puerarin (PUR), an 8-C-glucoside of daidzein extracted from Pueraria plants, is closely related to autophagy, reduced reactive oxygen species (ROS) production, and anti-inflammatory effects, but its effects on human nucleus pulposus mesenchymal stem cells (NPMSCs) have not yet been identified. In this study, NPMSCs were cultured in a compression apparatus to simulate the microenvironment of the intervertebral disc under controlled pressure (1.0 MPa), and we found that cell viability was decreased and apoptosis level was gradually increased as compression duration was prolonged. After PUR administration, apoptosis level evaluated by flow cytometry and caspase-3 activity was remitted, and protein levels of Bas as well as cleaved caspase-3 were decreased, while elevated Bcl-2 level was identified. Moreover, ATP production detection, ROS, and JC-1 fluorography as well as quantitative analysis suggested that PUR could attenuate intercellular ROS accumulation and mitochondrial dysfunction. Besides, the rat tail compression model was utilized, which indicated that PUR could restore impaired nucleus pulposus degeneration induced by compression. The PI3K/Akt pathway was identified to be deactivated after compression stimulation by western blot, and PUR could rescue the phosphorylation of Akt, thus reactivating the pathway. The effects of PUR, such as antiapoptosis, cell viability restoration, antioxidation, and mitochondrial maintenance, were all counteracted by application of the PI3K/Akt pathway inhibitor (LY294002). Summarily, PUR could alleviate compression-induced apoptosis and cell death of human NPMSCs in vitro as well as on the rat compression model and maintain intracellular homeostasis by stabilizing mitochondrial membrane potential and attenuating ROS accumulation through activating the PI3K/Akt pathway.

Pioglitazone Protects Compression-Mediated Apoptosis in Nucleus Pulposus Mesenchymal Stem Cells by Suppressing Oxidative Stress

Excessive compression, the main cause of intervertebral disc (IVD) degeneration, affected endogenous repair of the intervertebral disc. Pioglitazone (PGZ) is the agonist of peroxisome proliferator-activated receptor γ, which has been widely used in the treatment of diabetes mellitus. The present study aim at investigating whether pioglitazone has protective effects on compression-mediated cell apoptosis in nucleus pulposus mesenchymal stem cells (NP-MSCs) and further exploring the possible underlying mechanism. Our results indicated that the isolated cells satisfied the criteria of MSC stated by the International Society for Cellular Therapy. Besides, our research revealed that pioglitazone could protect cell viability, cell proliferation of NP-MSCs and alleviated the toxic effects caused by compression. The actin stress fibers was suppressed obviously under compression, and pioglitazone alleviated the adverse outcomes. Pioglitazone exerted protective effects on compression-induced NP-MSCs apoptosis according to annexin V/PI double-staining and TUNEL assays. Pioglitazone suppressed compression-induced NP-MSCs oxidative stress, including decreasing compression-induced overproduction of reactive oxygen species (ROS) and malondialdehyde (MDA), and alleviated compression-induced mitochondrial membrane potential (MMP) decrease. Ultrastructure collapse of the mitochondria exhibited a notable improvement by pioglitazone in compression-induced NP-MSCs according to transmission electron microscopy (TEM). Furthermore, the molecular results showed that pioglitazone significantly decreased the expression of apoptosis-associated proteins, including cyto.cytochrome c, Bax, cleaved caspase-9, and cleaved caspase-3, and promoted Bcl-2 expression. These results indicated that pioglitazone alleviated compression-induced NP-MSCs apoptosis by suppressing oxidative stress and the mitochondrial apoptosis pathway, which may be a valuable candidate for the treatment of IVD degeneration.

The inflammatory cytokine TNF-α regulates the biological behavior of rat nucleus pulposus mesenchymal stem cells through the NF-κB signaling pathway in vitro.

Nucleus pulposus (NP) mesenchymal stem cells (NPMSCs) are a potential cell source for intervertebral disc (IVD) regeneration; however, little is known about their response to tumor necrosis factor-α (TNF-α), a critical inflammation factor contributing to accelerating IVD degeneration. Accordingly, the aim of this study was to investigate the regulatory effects of TNF-α at high and low concentrations on the biological behaviors of healthy rat NPMSCs, including proliferation, migration, andNP differentiation. In this study, NPMSCs were treated with different concentration of TNF-α (0-200 ng/mL). Then we used annexin V/propidium iodide flow cytometry analysis to detect the apoptosis rate of NPMSCs. Cell Counting Kit-8, Edu assay, and cell cycle test were used to examine the proliferation of NPMSCs. Migration ability of NPMSCs was detected by wound healing assay and transwell migration assay. Pellets method was used to induce NP differentiation of NPMSCs, and immunohistochemical staining, real-time polymerase chain reaction, and Western blot analysis were used to examine the NPC phenotypic genes and proteins. The cells were further treated with the nuclear factor-κB (NF-κB) pathway inhibitor Bay 11-7082 to determine the role of the NF-κB pathway in the mechanism underlying the differentiation process. Results showed that treatment with a high concentration of TNF-α(50-200 ng/mL) could induce apoptosis of NPMSCs, whereas a relatively low TNF-α concentration (0.1-10 ng/mL) promoted the proliferation and migration of NPMSCs, but inhibited their differentiation toward NP cells. Moreover, we identified that the NF-κB signaling pathway is activated during the TNF-α-inhibited differentiation of NPMSCs, and the NF-κB signal inhibitor Bay 11-7082 could partially eliminate the adverse effect of TNF-α on the differentiation of NPMSCs. Therefore, our findings provide important insight into the dynamic biological behavior reactivity of NPMSCs to TNF-α during IVD degeneration process, thus may help us understanding the underlying mechanism of IVD degeneration.

Umbilical cord mesenchymal stem cell conditioned medium restored the expression of collagen II and aggrecan in nucleus pulposus mesenchymal stem cells exposed to high glucose.

Diabetes can cause intervertebral disc degeneration by accelerating apoptosis and senescence of nucleus pulposus mesenchymal stem cells (NPMSCs). The aim of this study was to determine the effect of umbilical cord mesenchymal stem cells (UCMSCs) conditioned medium on high glucose (HG) induced degradation of NPMSCs produced extracellular matrix. NPMSCs were isolated from the inner intervertebral disc tissue using type XI collagenase digestion. According to Annexin V/propidium iodide (PI) flow cytometry analysis; HG leads to an increase in the rate of NPMSCs apoptosis. HG injury also resulted in a marked decrease in the percentage of cells in G0/G1 phase and an increase in cells in S and G2/M phases, indicating that HG induces cell cycle arrest of NPMSCs. Treatment with MSC-CM abolished the effect of HG on cell senescence. HG also significantly inhibited collagen II and aggrecan expression in NPMSCs. After MSC-CM treatment, the expression of these two extracellular matrix components was restored. Exposure to HG resulted in phosphorylation of p38 MAPK, while the levels of total p38 MAPK were not affected. When treated with MSC-CM, phosphorylated p38 MAPK levels of NPMSCs were lower than those without CM treatment. Our data also showed that p38 MAPK inhibitor SB203580 can attenuated phosphorylation of p38 MAPK and resumed the collagen II and aggrecan expression in NPMSCs. In summary, this study demonstrated that MSC-CM has the potential to alleviate HG induced extracellular matrix degradation via the p38 MAPK pathway.