scholarly journals Non-viral reprogramming of human nucleus pulposus cells with FOXF1 via extracellular vesicle delivery: an in vitro and in vivo study

2021 ◽  
Vol 41 ◽  
pp. 90-107
Author(s):  
S Tang ◽  
◽  
A Salazar-Puerta ◽  
J Richards ◽  
S Khan ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Viral Vectors ◽  
Extracellular Vesicle ◽  
Proteoglycan Synthesis ◽  
Nucleus Pulposus Cells ◽  
Keratin 19 ◽  
Altered Gene ◽  
Ivd Degeneration

Intervertebral disc (IVD) degeneration is characterized by decreased cellularity and proteoglycan synthesis and increased inflammation, catabolism, and neural/vascular ingrowth. Regenerative methods for IVD degeneration are largely cell-therapy-based or involve viral vectors, which are associated with mutagenesis and undesired immune responses. The present study used bulk electroporation and engineered extracellular vesicles (EVs) to deliver forkhead-box F1 (FOXF1) mRNA to degenerate human nucleus pulposus (NP) cells as a minimally invasive therapeutic strategy for IVD regeneration. Bulk electroporation was used to investigate FOXF1 effects on human NP cells during a 4-week culture in 3D agarose constructs. Engineered EV delivery of FOXF1 into human IVD cells in monolayer was determined, with subsequent in vivo validation in a pilot mouse IVD puncture model. FOXF1 transfection significantly altered gene expression by upregulating healthy NP markers [FOXF1, keratin 19 (KRT19)], decreasing inflammatory cytokines [interleukin (IL)-1β, -6], catabolic enzymes [metalloproteinase 13 (MMP13)] and nerve growth factor (NGF), with significant increases in glycosaminoglycan accumulation in human NP cells. Engineered EVs loaded with FOXF1 demonstrated successful encapsulation of FOXF1 cargo and effective uptake by human NP cells cultured in monolayer. Injection of FOXF1-loaded EVs into the mouse IVD in vivo resulted in a significant upregulation of FOXF1 and Brachyury, compared to controls at 7 d post-injection, with no evidence of cytotoxicity. This is the first study to demonstrate non-viral delivery of FOXF1 and reprogramming of human NP cells in vitro and mouse IVD cells in vivo. This strategy represents a non-addictive approach for treating IVD degeneration and associated back pain.

scholarly journals CB2-mediated attenuation of nucleus pulposus degeneration via the amelioration of inflammation and oxidative stress in vivo and in vitro

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Xiaoqiang Cheng ◽  
Jiayi Lin ◽  
Zhanghuan Chen ◽  
Yubo Mao ◽  
Xiexin Wu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nucleus Pulposus ◽  
Nucleus Pulposus Cell ◽  
Immunocytochemical Staining ◽  
Nucleus Pulposus Cells ◽  
Collagen Ii ◽  
Ivd Degeneration ◽  
Oxide Synthase

Abstract Background Nucleus pulposus cell (NPC) degeneration is widely accepted as one of the major causes of intervertebral disc (IVD) degeneration (IVDD). The pathogenesis of IVDD is complex and consists of inflammation, oxidative stress, and the loss of extracellular matrix (ECM). Cannabinoid type 2 receptor (CB2) has been shown to be involved in the pathological mechanism of a variety of diseases due to its anti-inflammatory effects and antioxidative stress capacity. Method In Vitro, H2O2 was used to induce degeneration of nucleus pulposus cells, mRNA and protein expression level was determined by RT-PCR and Western Blot, and Immunocytochemical staining were used to detect expression of collagen II, aggrecan, MMP3/13, superoxide dismutase 2 (SOD2) and inducible nitric oxide synthase (iNOS). In vivo, the potential therapeutic effect of CB2 was detected in the rat acupuncture model. Result In vitro, we found that the CB2 agonist (JWH133) treatment reduced the oxidative stress level in NPCs induced by hydrogen peroxide (H2O2) treatment. Furthermore, the expression of inflammatory cytokines was also decreased by JWH133 treatment. We found that collagen II and aggrecan expression was preserved, whereas matrix metalloproteinase levels were reduced. In vivo, we established a rat model by needle puncture. Imaging assessment revealed that the disc height index (DHI) and morphology of IVD were significantly improved, and the disc degeneration process was delayed by treatment of JWH133. Furthermore, immunohistochemical (IHC) staining revealed that JWH133 could inhibit the degradation of collagen II and decrease the expression of MMP3. Conclusions The experiment indicates the oxidative stress and inflammatory response of rat NPCs induced by H2O2 could be inhibited by activating CB2. This study reveals that CB2 activation can effectively delay the development of IVDD, providing an effective therapeutic target for IVDD.

scholarly journals Injectable Hydrogel Combined with Nucleus Pulposus-Derived Mesenchymal Stem Cells for the Treatment of Degenerative Intervertebral Disc in Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-17 ◽  
Author(s):  
Feng Wang ◽  
Li-ping Nan ◽  
Shi-feng Zhou ◽  
Yang Liu ◽  
Ze-yu Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Rat Model ◽  
Injectable Hydrogel ◽  
Cell Scaffold ◽  
Ivd Degeneration

Stem cell-based tissue engineering in treating intervertebral disc (IVD) degeneration is promising. An appropriate cell scaffold can maintain the viability and function of transplanted cells. Injectable hydrogel has the potential to be an appropriate cell scaffold as it can mimic the condition of the natural extracellular matrix (ECM) of nucleus pulposus (NP) and provide binding sites for cells. This study was aimed at investigating the effect of injectable hydrogel-loaded NP-derived mesenchymal stem cells (NPMSC) for the treatment of IVD degeneration (IDD) in rats. In this study, we selected injectable 3D-RGD peptide-modified polysaccharide hydrogel as a cell transplantation scaffold. In vitro, the biocompatibility, microstructure, and induced differentiation effect on NPMSC of the hydrogel were studied. In vivo, the regenerative effect of hydrogel-loaded NPMSC on degenerated NP in a rat model was evaluated. The results showed that NPMSC was biocompatible and able to induce differentiation in hydrogel in vivo. The disc height index (almost 87%) and MRI index (3313.83±227.79) of the hydrogel-loaded NPMSC group were significantly higher than those of other groups at 8 weeks after injection. Histological staining and immunofluorescence showed that the hydrogel-loaded NPMSC also partly restored the structure and ECM content of degenerated NP after 8 weeks. Moreover, the hydrogel could support long-term NPMSC survival and decrease cell apoptosis rate of the rat IVD. In conclusion, injectable hydrogel-loaded NPMSC transplantation can delay the level of IDD and promote the regeneration of the degenerative IVD in the rat model.

scholarly journals Oxidative Stress Aggravates Apoptosis of Nucleus Pulposus Cells through m6A Modification of MAT2A Pre-mRNA by METTL16

2022 ◽  
Vol 2022 ◽  
pp. 1-15
Author(s):  
Peng-Bo Chen ◽  
Gui-Xun Shi ◽  
Tao Liu ◽  
Bo Li ◽  
Sheng-Dan Jiang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Animal Model ◽  
Pilot Study ◽  
Nucleus Pulposus ◽  
Intervertebral Disc Degeneration ◽  
Nucleus Pulposus Cell ◽  
Nucleus Pulposus Cells ◽  
Human Npcs

The process of intervertebral disc degeneration (IVDD) is complex, and its mechanism is considered multifactorial. Apoptosis of oxidative stressed nucleus pulposus cells (NPCs) should be a fundamental element in the pathogenesis of IVDD. In our pilot study, we found that the expression of MAT2A decreased, and METTL16 increased in the degenerative nucleus pulposus tissues. Previous studies have shown that the balance of splicing, maturation, and degradation of MAT2A pre-mRNA is regulated by METTL16 m6A modification. In the current study, we aimed to figure out whether this mechanism was involved in the aberrant apoptosis of NPCs and IVDD. Human NPCs were isolated and cultured under oxidative stress. An IVDD animal model was established. It showed that significantly higher METTL16 expression and lower MAT2A expression were seen in either the NPCs under oxidative stress or the degenerative discs of the animal model. MAT2A was inhibited with siRNA in vitro or cycloleucine in vivo. METTL16 was overexpressed with lentivirus in vitro or in vivo. Downregulation of MAT2A or upregulation of METTL16 aggravated nucleus pulposus cell apoptosis and disc disorganization. The balance of splicing, maturation, and degradation of MAT2A pre-mRNA was significantly inclined to degradation in the NPCs with the overexpression of METTL16. Increased apoptosis of NPCs under oxidative stress could be rescued by reducing the expression of METTL16 using siRNA with more maturation of MAT2A pre-mRNA. Collectively, oxidative stress aggravates apoptosis of NPCs through disrupting the balance of splicing, maturation, and degradation of MAT2A pre-mRNA, which is m6A modified by METTL16.

Effect of Inflammation on the Osmotic Response of Nucleus Pulposus Cells

2012 ◽  
Author(s):  
Robert Maidhof ◽  
Neena Rajan ◽  
Nadeen O. Chahine
Keyword(s):  
Nucleus Pulposus ◽  
Biomechanical Properties ◽  
Nucleus Pulposus Cells ◽  
Cellular Mechanisms ◽  
Osmotic Response ◽  
Tnf Α ◽  
Disc Cells ◽  
Cytokine Stimulation ◽  
Ivd Degeneration

Intervertebral disc (IVD) degeneration is accompanied by elevated levels of pro-inflammatory cytokines, particularly IL-1β and TNF-α [1]. Disc cells from the nucleus pulposus (NPs) respond to cytokine stimulation with increased catabolic breakdown of the tissue, resulting in a positive feedback of disc integrity loss and further inflammation [2]. Previous studies by our group have examined the response of NP cells to Toll-Like Receptor-4 (TLR-4) activation through stimulation with lipopolysaccharide (LPS). TLR-4 is a pattern recognition receptor that is activated in innate immunity and by polysaccharide fragments from degenerated proteoglycans. TLR-4 activation by LPS results in stimulation of multiple cytokines by NP cells [3]. Moreover, we have shown that in vivo LPS injection results in catabolic changes in the IVD, including matrix breakdown, decrease in biomechanical properties and loss of disc height [4]. However, the specific cellular mechanisms for these catabolic changes remain to be elucidated.

Hypoxia promotes nucleus pulposus phenotype in 3D scaffolds in vitro and in vivo

2014 ◽  
Vol 21 (2) ◽  
pp. 303-309 ◽  
Author(s):  
Ganjun Feng ◽  
Li Li ◽  
Ying Hong ◽  
Hao Liu ◽  
Yueming Song ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Collagen Type ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Low Oxygen ◽  
Nucleus Pulposus Cells ◽  
Experimental Conditions ◽  
3D Scaffolds

Object The role of oxygen in disc metabolism remains a matter of debate. Whether the effect of hypoxic priming on the nucleus pulposus phenotype can be maintained in vivo is not clear. The goal of the present study was to test the hypothesis that priming in a low oxygen tension in vitro could promote a nucleus pulposus phenotype in vivo. Methods Bovine nucleus pulposus cells were seeded in 3D scaffolds and subjected to varying oxygen tensions (2% and 20%) for 3 weeks. The constructs were then implanted subcutaneously for 8 weeks. Changes in the extracellular matrix were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction, glycosaminoglycan (GAG) assay, DNA assay, collagen quantification, and histological and immunohistological analyses. Results Hypoxia resulted in greater production of sulfated glycosaminoglycan and higher levels of gene expression for collagen Type II, aggrecan, and SOX-9. Furthermore, after hypoxic priming, the subcutaneously implanted constructs maintained the nucleus pulposus phenotype, which was indicated by a significantly higher amount of glycosaminoglycan and collagen Type II. Conclusions Hypoxia enhanced the nucleus pulposus phenotype under experimental conditions both in vitro and in vivo. When used in combination with appropriate scaffold material, nucleus pulposus cells could be regenerated for tissue-engineering applications.

Mechanical stress-induced apoptosis of nucleus pulposus cells: an in vitro and in vivo rat model

2014 ◽  
Vol 19 (2) ◽  
pp. 313-322 ◽  
Author(s):  
Yi-Jie Kuo ◽  
Lien-Chen Wu ◽  
Jui-Sheng Sun ◽  
Ming-Hong Chen ◽  
Man-Ger Sun ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Nucleus Pulposus ◽  
Rat Model ◽  
Nucleus Pulposus Cells ◽  
Induced Apoptosis

Alpha-smooth muscle actin in pathological human disc nucleus pulposus cells in vivo and in vitro

2004 ◽  
Vol 12 (4) ◽  
pp. 430-438 ◽  
Author(s):  
Dawn Hastreiter ◽  
Jeannie Chao ◽  
QI Wang ◽  
Richard M. Ozuna ◽  
Myron Spector
Keyword(s):  
Smooth Muscle ◽  
Nucleus Pulposus ◽  
Smooth Muscle Actin ◽  
Muscle Actin ◽  
Nucleus Pulposus Cells ◽  
Alpha Smooth Muscle Actin

scholarly journals Mitochondrial NDUFA4L2 attenuates the apoptosis of nucleus pulposus cells induced by oxidative stress via the inhibition of mitophagy

2019 ◽  
Vol 51 (11) ◽  
pp. 1-16 ◽  
Author(s):  
Wen-Ning Xu ◽  
Huo-Liang Zheng ◽  
Run-Ze Yang ◽  
Tao Liu ◽  
Wei Yu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Intervertebral Disc Degeneration ◽  
Nucleus Pulposus Cells ◽  
Pathological Mechanism ◽  
First Time ◽  
The Relationship

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

scholarly journals The role of altered glycosylation in human nucleus pulposus cells in inflammation and degeneration

2020 ◽  
Vol 41 ◽  
pp. 401-420
Author(s):  
K Joyce ◽  
◽  
IL Mohd Isa ◽  
A Krouwels ◽  
L Creemers ◽  
...  
Keyword(s):  
Cell Migration ◽  
Nucleus Pulposus ◽  
In Vitro Model ◽  
Lectin Histochemistry ◽  
Nucleus Pulposus Cells ◽  
Altered Glycosylation ◽  
Vitro Model ◽  
Ivd Degeneration

Intervertebral disc (IVD) degeneration causes low-back pain through disc compression, prolapse and herniation. Inflammation of the IVD and subsequent degeneration produce altered glycosylation profiles in several animal models of IVD injury and ageing, although the function of this altered glycosylation pattern in a human is unknown. Altered N-glycome, specifically sialylated and fucosylated N-glycosylation motif expression, might play a role in inflammation and disease progression. Healthy (foetal and adolescent idiopathic scoliosis) and degenerated (lumbar degeneration) human IVD glycosylation patterns were studied using lectin histochemistry. Small-molecule fluorinated sugar analogues (3Fax-Peracetyl Neu5Ac; 2F-Peracetyl-Fucose) were used to inhibit sialylation and fucosylation in an in vitro model of inflammation, to investigate their effects on the glycosignature, cell metabolism, extracellular matrix synthesis and cell migration. The effects of interleukin (IL)-1β, tumour necrosis factor (TNF)-α and IL-6 on glycosylation in human nucleus pulposus cells were investigated by lectin histochemistry, PCR and enzyme-linked immunosorbent assay (ELISA). In the in vitro model of IVD degeneration, cytokine-induced inflammation-induced hypersialylation was observed, as indicated by Sambucus nigra I binding. However, this modification was inhibited by the sialyltransferase inhibitor. Inhibition of sialylation and fucosylation modulates cell migration and protein translation of catabolic enzymes in response to inflammation. The altered patterns of glycosylation in human tissue in degeneration was consistent with previous IVD studies in murine, bovine and ovine models. The present study was the first functional investigation of glycosylation in human degenerated IVD, elucidating the role of the glycome in disease progression and identified potential therapeutic targets for future regenerative therapies.

scholarly journals Recent Advances in CRISPR/Cas9 Delivery Strategies

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 839 ◽  
Author(s):  
Bon Ham Yip
Keyword(s):  
Viral Vectors ◽  
Insertional Mutagenesis ◽  
Gene Editing ◽  
Ex Vivo ◽  
Extracellular Vesicle ◽  
Delivery Methods ◽  
Delivery Strategies ◽  
In Vivo Delivery

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the field of gene editing. Continuous efforts in developing this technology have enabled efficient in vitro, ex vivo, and in vivo gene editing through a variety of delivery strategies. Viral vectors are commonly used in in vitro, ex vivo, and in vivo delivery systems, but they can cause insertional mutagenesis, have limited cloning capacity, and/or elicit immunologic responses. Physical delivery methods are largely restricted to in vitro and ex vivo systems, whereas chemical delivery methods require extensive optimization to improve their efficiency for in vivo gene editing. Achieving a safe and efficient in vivo delivery system for CRISPR/Cas9 remains the most challenging aspect of gene editing. Recently, extracellular vesicle-based systems were reported in various studies to deliver Cas9 in vitro and in vivo. In comparison with other methods, extracellular vesicles offer a safe, transient, and cost-effective yet efficient platform for delivery, indicating their potential for Cas9 delivery in clinical trials. In this review, we first discuss the pros and cons of different Cas9 delivery strategies. We then specifically review the development of extracellular vesicle-mediated gene editing and highlight the strengths and weaknesses of this technology.

Sign in / Sign up

Export Citation Format

Share Document