Degenerative Nucleus Pulposus Research Articles

OA29 Epigenetic regulation of IL-4 promoter targeting p300 attenuating intervertebral disc degeneration and pain by inhibiting IL-1β

Abstract Background/Aims Low back pain (LBP) is a prevalent condition primarily caused by intervertebral disc degeneration (IVDD), which imposes a substantial social and medical burden. Effective targeted treatments to prevent the progression of IVDD and alleviate LBP are still lacking. p300, a critical member of the histone acetyltransferase (HAT) family, is involved in the regulation of inflammation. This study aims to investigate the role of p300/IL-4 in IVDD and pain and elucidate the underlying molecular mechanism. Methods Human intervertebral disc samples were collected and graded based on the Pfirrmann grading scheme. A rat model of IVDD was established through posterior disc puncture, and various treatments were administered. Human nucleus pulposus cells (NPCs) were isolated and cultured, followed by assessments including qRT-PCR, Western blotting, immunofluorescence, and histopathological analyses. The mechanical and thermal hyperalgesia were assessed using von Frey hair tests and the plantar test, respectively. Chromatin immunoprecipitation (ChIP) and ChIP-qPCR were performed, and lentivirus transduction was used for p300 knockdown. Results We investigated the role of CREB binding protein (CBP)/p300 in IVDD. Degenerated nucleus pulposus (NP) tissues from IVDD patients exhibited a significant downregulation of p300 at both nucleic acid and protein levels, while CBP levels remained unchanged. Manipulating p300 expression in NPCs regulated matrix production, with p300 overexpression enhancing aggrecan and collagen II expression and knockdown leading to increased matrix-degrading proteins. In a rat IVDD model, activating p300 function with CTPB (p300-specific activator) alleviated IVDD and pain, while inhibiting p300 with C646 exacerbated IVDD and pain. Transcriptome analysis implicated IL-4 as a downstream effector of p300, and IL-4 demonstrated protective effects against IVDD and pain. Mechanistically, p300 was found to acetylate H3K27 at the IL-4 promoter, promoting its expression. Conclusion The novel findings of this study suggest that the p300/IL-4/IL-1β axis may represent a promising therapeutic target for IVDD and LBP. These results propose a hypothesis that, early in the inflammatory phase, NPCs may initiate a self-protective mechanism through p300, aiding itself in preserving a favourable metabolic microenvironment. This mechanism is primarily mediated by the expression of IL-4, which has the capacity to counteract IL-1β, rather than TNF-α. Disclosure H. Cui: None. Z. Zheng: None.

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

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

Ephrin B2 (EFNB2) potentially protects against intervertebral disc degeneration through inhibiting nucleus pulposus cell apoptosis

Nucleus pulposus (NP) cell apoptosis is a significant indication of accelerated intervertebral disc degeneration; however, the precise mechanism is unelucidated as of yet. Ephrin B2 (EFNB2), the only gene down-regulated in the three degraded intervertebral disc tissue microarray groups (GSE70362, GSE147383 and GSE56081), was screened for examination in this study. Subsequently, EFNB2 was verified to be down-regulated in degraded NP tissue samples. Interleukin-1 (IL-1β) treatment of NP cells to simulate the IDD environment indicated that IL-1β treatment decreased EFNB2 expression. In degenerative NP cells stimulated by IL-1β, EFNB2 knockdown significantly increased the rate of apoptosis as well as the apoptosis-related molecules cleaved-caspase-3 and the Bax to Bcl-2 ratio. EFNB2 was found to promote AKT, PI3K, and mTOR phosphorylation; the PI3K/AKT signaling role was investigated using the PI3K inhibitor LY294002. EFNB2 overexpression significantly increased PI3K/AKT pathway activity in IL-1β-stimulated NP cells than the normal control. Moreover, EFNB2 partially alleviated NP cell apoptosis induced by IL-1β, reduced the cleaved-cas3 level, and decreased the Bax/Bcl-2 ratio after the addition of the inhibitor LY294002. Additionally, EFNB2 overexpression inhibited the ERK1/2 phosphorylation; the effects of EFNB2 overexpression on ERK1/2 phosphorylation, degenerative NP cell viability, and cell apoptosis were partially reversed by ERK signaling activator Ceramide C6. EFNB2 comprehensively inhibited the apoptosis of NP cells by activating the PI3K/AKT signaling and inhibiting the ERK signaling, obviating the exacerbation of IDD. EFNB2 could be a potential target to protect against degenerative disc changes.

Mesenchymal stem cell-derived exosomes are beneficial to suppressing inflammation and promoting autophagy in intervertebral disc degeneration.

Intervertebral disc degenerative diseases is one the main causes of lumbago, and its main pathological mechanism is intervertebral disc degeneration (IDD). As shown in previous reports, mesenchymal stem cell (MSC)-exosomes can slow down or even reverse degenerated nucleus pulposus (NP) cells in IDD. Thus, we attempted to clarify the specific role of MSC-exosomes underlying IDD progression. In the present study, the harvested particles were identified as MSC-exosomes. MSC-exosomes facilitated activation of autophagy pathway in AGE-treated NP cells. MSC-exosomes repressed inflammatory response in AGE-treated NP cells. Autophagy pathway activation enhanced inflammatory response in AGE-stimulated NP cells. Mesenchymal stem cell-exosomes facilitated autophagy pathway activation and repressed inflammation in IDD rats. Autophagy inhibition exerted a protective role against inflammatory response in IDD rats. In conclusion, MSC-exosomes represses inflammation via activating autophagy pathway, which provides a potential novel insight for seeking therapeutic plans of IDD.

Maslinic acid alleviates intervertebral disc degeneration by inhibiting the PI3K/AKT and NF-κB signaling pathways.

Intervertebral disc degeneration (IDD) is the cause of low back pain (LBP), and recent research has suggested that inflammatory cytokines play a significant role in this process. Maslinic acid (MA), a natural compound found in olive plants ( Olea europaea), has anti-inflammatory properties, but its potential for treating IDD is unclear. The current study aims to investigate the effects of MA on TNFα-induced IDD in vitro and in other in vivo models. Our findings suggest that MA ameliorates the imbalance of the extracellular matrix (ECM) and mitigates senescence by upregulating aggrecan and collagen II levels as well as downregulating MMP and ADAMTS levels in nucleus pulposus cells (NPCs). It can also impede the progression of IDD in rats. We further find that MA significantly affects the PI3K/AKT and NF-κB pathways in TNFα-induced NPCs determined by RNA-seq and experimental verification, while the AKT agonist Sc-79 eliminates these signaling cascades. Furthermore, molecular docking simulation shows that MA directly binds to PI3K. Dysfunction of the PI3K/AKT pathway and ECM metabolism has also been confirmed in clinical specimens of degenerated nucleus pulposus. This study demonstrates that MA may hold promise as a therapeutic agent for alleviating ECM metabolism disorders and senescence to treat IDD.

Bioinformatics analysis and experimental validation of key genes associated with lumbar disc degeneration and biomechanics

BackgroundLumbar disc degeneration (LDD) is an important pathological basis for the development of degenerative diseases of the lumbar spine. Most clinical patients have low back pain as their main symptom. The deterioration of the biomechanical environment is an important cause of LDD. Although there is a large amount of basic research on LDD, there are fewer reports that correlate biomechanical mechanisms with basic research. Our research aims to identify 304 key genes involved in LDD due to biomechanical deterioration, using a bioinformatics approach. We focus on SMAD3, CAV1, SMAD7, TGFB1 as hub genes, and screen for 30 potential target drugs, offering novel insights into LDD pathology and treatment options. MethodsThe Gene Cards, GenCLip3, OMIM and Drugbank databases were explored to obtain genes associated with biomechanics and LDD, followed by making veen plots to obtain both co-expressed genes. GO enrichment analysis and KEGG pathway analysis of the co-expressed genes were obtained using the DAVID online platform and visualised via a free online website. Protein interaction networks (PPI) were obtained through the STRING platform and visualised through Cytoscape 3.9.0. These genes were predicted for downstream interaction networks using the STITCH platform. Then, the GSE56081 dataset was used to validate the key genes. RT-PCR was used to detect mRNA expression of core genes in the degenerated nucleus pulposus (NP) samples and western bolt was used for protein expression. Lastly, the obtained hub genes were searched in the drug database (DGIdb) to find relevant drug candidates. ResultsFrom the perspective of biomechanics-induced LDD, we obtained a total of 304 genes, the GO functional enrichment and KEGG pathway enrichment analysis showed that the functions of these genes are mostly related to inflammation and apoptosis. The PPI network was constructed and four Hub genes were obtained through the plug-in of Cytoscape software, namely SMAD3, CAV1, SMAD7 and TGFB1. The analysis of key genes revealed that biomechanical involvement in LDD may be related to the TGF-β signaling pathway. Validation of the GSE56081 dataset revealed that SMAD3 and TGFB1 were highly expressed in degenerating NP samples. RT-PCR results showed that the mRNA expression of SMAD3 and TGFB1 was significantly increased in the severe degeneration group; Western blot results also showed that the protein expression of TGFB1 and P-SMAD3 was significantly increased. In addition, we identified 30 potential drugs. ConclusionThis study presented a new approach to investigate the correlation between biomechanical mechanisms and LDD. The deterioration of the biomechanical environment may cause LDD through the TGF-β signaling pathway. TGFB1 and SMAD3 are important core targets. The important genes, pathways and drugs obtained in this study provided a new basis and direction for the study, diagnosis and treatment of LDD.

Injectable pathological microenvironment-responsive anti-inflammatory hydrogels for ameliorating intervertebral disc degeneration

Chronic local inflammation and resulting cellular dysfunction of nucleus pulposus (NP) cells are important pathogenic factors of intervertebral disc degeneration (IDD). Injectable pathological microenvironment-responsive hydrogels hold significant potential for treating IDD by adapting to dynamic microenvironment of IDD. Herein, we proposed an injectable gelatin-based hydrogel drug delivery system that could respond to the pathological microenvironment of IDD for controlled release of anti-inflammatory drug to promote degenerative NP repair. The hydrogel system was prepared by conjugating phenylboronic acid-modified gelatin methacryloyl (GP) with the naturally extracted anti-inflammatory drug epigallocatechin-3-gallate (EGCG) through dynamic boronic esters. The hydrogel exhibited excellent degradability, injectability, antioxidant properties, anti-inflammatory effects, and biocompatibility. It also displayed responsive-release of EGCG under high reactive oxygen species (ROS) levels and acidic conditions. The hydrogel demonstrated remarkable cytoprotective effects on NP cells in both hyperactive ROS environments and inflammatory cytokine-overexpressed environments in vitro. In vivo studies revealed that the hydrogel injected in situ could effectively ameliorate the intervertebral disc degeneration by maintaining the disc height and NP tissue structure in a rat IDD model. The hydrogel system exhibited excellent biocompatibility and responsive-release of diol-containing drugs in pathological microenvironments, indicating its potential application as a drug delivery platform.

Downregulation of VEGFA accelerates AGEs-mediated nucleus pulposus degeneration through inhibiting protective mitophagy in high glucose environments

Intervertebral disc degeneration (IVDD) contributes largely to low back pain. Recent studies have highlighted the exacerbating role of diabetes mellitus (DM) in IVDD, mainly due to the influence of hyperglycemia (HG) or the accumulation of advanced glycation end products (AGEs). Vascular endothelial growth factor A (VEGFA) newly assumed a distinct impact in nonvascular tissues through mitophagy regulation. However, the combined actions of HG and AGEs on IVDD and the involved role of VEGFA remain unclear. We confirmed the potential relation between VEGFA and DM through bioinformatics and biological specimen detection. Then we observed that AGEs induced nucleus pulposus (NP) cell degeneration by upregulating cellular reactive oxygen species (ROS), and HG further aggravated ROS level through breaking AGEs-induced protective mitophagy. Furthermore, this adverse effect could be strengthened by VEGFA knockdown. Importantly, we identified that the regulation of VEGFA and mitophagy were vital mechanisms in AGEs-HG-induced NP cell degeneration through Parkin/Akt/mTOR and AMPK/mTOR pathway. Additionally, VEGFA overexpression through local injection with lentivirus carrying VEGFA plasmids significantly alleviated NP degeneration and IVDD in STZ-induced diabetes and puncture rat models. In conclusion, the findings first confirmed that VEGFA protects against AGEs-HG-induced IVDD, which may represent a therapeutic strategy for DM-related IVDD.

Intervertebral disc-intrinsic Hedgehog signaling maintains disc cell phenotypes and prevents disc degeneration through both cell autonomous and non-autonomous mechanisms

Intervertebral disc degeneration is closely related to abnormal phenotypic changes in disc cells. However, the mechanism by which disc cell phenotypes are maintained remains poorly understood. Here, Hedgehog-responsive cells were found to be specifically localized in the inner annulus fibrosus and cartilaginous endplate of postnatal discs, likely activated by Indian Hedgehog. Global inhibition of Hedgehog signaling using a pharmacological inhibitor or Agc1-CreERT2-mediated deletion of Smo in disc cells of juvenile mice led to spontaneous degenerative changes in annulus fibrosus and cartilaginous endplate accompanied by aberrant disc cell differentiation in adult mice. In contrast, Krt19-CreER-mediated deletion of Smo specifically in nucleus pulposus cells led to healthy discs and normal disc cell phenotypes. Similarly, age-related degeneration of nucleus pulposus was accelerated by genetic inactivation of Hedgehog signaling in all disc cells, but not in nucleus pulposus cells. Furthermore, inactivation of Gli2 in disc cells resulted in partial loss of the vertebral growth plate but otherwise healthy discs, whereas deletion of Gli3 in disc cells largely corrected disc defects caused by Smo ablation in mice. Taken together, our findings not only revealed for the first time a direct role of Hedgehog-Gli3 signaling in maintaining homeostasis and cell phenotypes of annuls fibrosus and cartilaginous endplate, but also identified disc-intrinsic Hedgehog signaling as a novel non-cell-autonomous mechanism to regulate nucleus pulposus cell phenotype and protect mice from age-dependent nucleus pulposus degeneration. Thus, targeting Hedgehog signaling may represent a potential therapeutic strategy for the prevention and treatment of intervertebral disc degeneration.

Nucleus pulposus cells regulate macrophages in degenerated intervertebral discs via the integrated stress response-mediated CCL2/7-CCR2 signaling pathway

Lower back pain (LBP), which is a primary cause of disability, is largely attributed to intervertebral disc degeneration (IDD). Macrophages (MΦs) in degenerated intervertebral discs (IVDs) form a chronic inflammatory microenvironment, but how MΦs are recruited to degenerative segments and transform into a proinflammatory phenotype remains unclear. We evaluated chemokine expression in degenerated nucleus pulposus cells (NPCs) to clarify the role of NPCs in the establishment of an inflammatory microenvironment in IDD and explored the mechanisms. We found that the production of C-C motif chemokine ligand 2 (CCL2) and C-C motif chemokine ligand 7 (CCL7) was significantly increased in NPCs under inflammatory conditions, and blocking CCL2/7 and their receptor, C-C chemokine receptor type 2(CCR2), inhibited the inductive effects of NPCs on MΦ infiltration and proinflammatory polarization. Moreover, activation of the integrated stress response (ISR) was obvious in IDD, and ISR inhibition reduced the production of CCL2/7 in NPCs. Further investigation revealed that activating Transcription Factor 3 (ATF3) responded to ISR activation, and ChIP-qPCR verified the DNA-binding activity of ATF3 on CCL2/7 promoters. In addition, we found that Toll-like receptor 4 (TLR4) inhibition modulated ISR activation, and TLR4 regulated the accumulation of mitochondrial reactive oxygen species (mtROS) and double-stranded RNA (dsRNA). Downregulating the level of mtROS reduced the amount of dsRNA and ISR activation. Deactivating the ISR or blocking CCL2/7 release alleviated inflammation and the progression of IDD in vivo. Moreover, MΦ infiltration and IDD were inhibited in CCR2-knockout mice. In conclusion, this study highlights the critical role of TLR4/mtROS/dsRNA axis-mediated ISR activation in the production of CCL2/7 and the progression of IDD, which provides promising therapeutic strategies for discogenic LBP.

TNFα-reliant FSP1 up-regulation promotes intervertebral disc degeneration via caspase 3-dependent apoptosis

Intervertebral disc degeneration (IDD) is a common chronic inflammatory degenerative disease that causes lower back pain. However, the underlying mechanisms of IDD remain unclear. Ferroptosis suppressor protein 1 (FSP1) is a newly identified suppressor for ferroptosis. This study aims to investigate the role of FSP1 in IDD. Nucleus pulposus (NP) tissues in humans were collected and NP cells from rats were isolated to detect FSP1 expression pattern. The relationship between FSP1-mediated ferroptosis and apoptosis was identified using FSP1 inhibitor iFSP1. RNA sequencing was utilized to seek downstream molecules and related signaling pathways. Moreover, both exogenous recombinant FSP1 protein and endogenous small interfering RNA were implemented in this study to clarify the role of FSP1 in tumor necrosis factor-alpha (TNFα)-mediated NP cell apoptosis. Ultimately, the underlying mechanisms of FSP1-related signaling pathway in IDD were uncovered both in vitro and in vivo. As a result, FSP1 was up-regulated in human degenerative NP tissues and after TNFα stimulation. FSP1 inhibition by iFSP1 fails to trigger ferroptosis in NP cells while inhibiting TNFα-mediated apoptosis. Further experiments demonstrated that FSP1 was closely related to TNFα-reliant caspase 3 activation and mitochondrial damage. However, the exogenous addition of recombinant protein FSP1 does not induce cell death or intensify the efficacy of TNFα. Mechanically, FSP1 is involved in TNFα-mediated NF-κB signaling activation to accelerate the development of IDD. This study demonstrated that FSP1 promotes IDD through TNFα-reliant NF-κB signaling activation and caspase 3-dependent apoptosis. These findings suggested a novel therapeutic target for the treatment of IDD.

Hypo-osmolality activates Wnt/β-catenin mediated by AQP1 in nucleus pulposus cells degeneration.

The goals of this study were to investigate whether Wnt/β-catenin signaling plays a role in hypo-osmolality-related degeneration of nucleus pulposus (NP) cells, and if so, to define the mechanism underlying AQP1 in this effect. Human NP cells were cultured under hypo-osmotic (300/350/400 mOsm) and iso-osmotic (450 mOsm) conditions. The cell viability, AQP1, the expression of Wnt/β-catenin signaling, collagen II/I, and MMP3/9 were evaluated. To determine the effects of the Wnt/β-catenin signaling, we used the inhibitor and the activator of Wnt during the hypo-osmotic culture of NP cells. We also examined whether the silencing and overexpressing of the AQP1 gene would affect the Wnt/β-catenin expression in NP cells. Hypo-osmolality caused NP cell degeneration and activated the Wnt/β-catenin signaling but suppressed the AQP1 level. Inhibiting the Wnt/β-catenin signaling alleviated the hypo-osmolality-induced NP cell degeneration. On the contrary, activating Wnt/β-catenin aggravated the NP cell degeneration under hypo-osmotic conditions, which did not affect AQP1 expression. AQP1-overexpressed NP cells exhibited decreased Wnt/β-catenin signaling and alleviated cell degeneration under the hypo-osmotic condition. Besides, AQP1 silencing accelerated NP cell degeneration and activated Wnt/β-catenin expression compared with untreated control. Hypo-osmolality promotes NP cell degeneration via activating Wnt/β-catenin signaling, which is suppressed by AQP1 expression. The upregulation of AQP1 suppressed the Wnt/β-catenin signaling and alleviated the hypo-osmolality induced by the NP cell degeneration.

Poly-3-hydroxybutyrate-co-3-hydroxyvalerate(PHBV)-Polyethylene glycol 20k(PEG20k) as a promising delivery system for PT2399 in the treatment of disc degeneration.

Disc degeneration often leads to a highly prevalent symptom known as low back pain. Healthy nucleus pulposus tissue exhibited a hypoxic environment devoid of blood vessels, while degenerated nucleus pulposus experienced hypoxic deterioration and the formation of new blood vessels. In this study, the expression of important genes like HIF-2α was found to vary between normal and degenerated nucleus pulposus cells when compared to the hypoxic surroundings. The aim of this study was to examine how HIF-2α is controlled in nucleus pulposus cells under hypoxic conditions and its role in angiogenic mechanisms. To assess the impact of gradual inhibition of HIF-2α on disc degeneration, we utilized PHBV-based synthetic materials loaded with inhibitors of HIF-2α. Specifically, we employed LPS and PT2399 loaded PHBV-PEG20k (PP20) to intervene with human nucleus pulposus cells. Additionally, we treated APD rat models with PT2399 loaded PP20 to evaluate its effects. The expression levels of target markers in nucleus pulposus cells were detected using PCR, WB, and immunofluorescence. Additionally, the effect of drugs on disc degeneration was identified through HE staining. The findings indicated that HIF-2α, CAIX, PPP1R15A, VEGFA, and EGLN3 could potentially serve as new indicators of disc degeneration. Additionally, HIF-2α might contribute to the progression of disc degeneration through involvement in angiogenesis and the regulation of hypoxia. Furthermore, the utilization of PT2399 loaded PHBV-PEG20k (PP20) could potentially offer a fresh alternative for treating disc degeneration.

REACTIVATION OF THE NUCLEUS PULPOUS CELLS BY PLATELET DERIVATIVES AS A NEW TOOL IN INTERVERTEBRAL DISC REGENERATION

Degenerative disc disease, associated to low back pain, afflicts more than 50% of humans, and represents a major healthcare problem, especially for the pathology initiation. Current treatments range from conservative strategies to more invasive surgical techniques, such as disc removal and vertebral fusion. In the Intervertebral Disease (IVD) the nucleus pulposus (NP) degeneration is a key factor for the pathology initiation. Several tissue engineering approaches aiming to restore the appropriate NP cell (NPCs) and matrix content, were attempted by using adult stromal cells either from bone marrow or adipose tissue, chondrocytes, notochordal cells and more recently also pluripotent stem cells. However, none was fully satisfactory since the NP acid and a-vascularized environment appeared averse to the implanted heterologous cells. Several studies demonstrated the efficacy of platelet derivatives such as platelet rich plasma (PRP) in promoting the regeneration of connective tissues. We investigated the efficacy of PRP on NPCs proliferation and differentiation with the goal to propose the direct stimulation of resident cells (stimulation of endogenous cells – less invasive surgical procedure) or the implantation of NPCs expanded in vitro in the presence of PRP as therapeutic agents in IVD degeneration.NPCs were isolated from small fragments of NP explants, cultivated in medium supplemented with PRP or FCS (standard condition control) and characterized by FACS analysis for the expression of the typical mesenchymal stem cells markers CD34, CD44, CD45, CD73, CD90 and CD105. NPCs cultured in PL showed a phenotypic profile like the cells cultured in FCS. However, compared to NPCs expanded in the presence of FCS, NPCs expanded in PRP showed a much better proliferation and differentiation capacity. NPCs differentiation was evaluated by the cell ability to produce an organized metachromatic cartilaginous matrix, confirmed by the positive immunohistochemical staining for chondrogenic markers.

FUNCTIONAL ACTIVITY OF MONOMERIC C-REACTIVE PROTEIN IN INTERVERTEBRAL DISC

Monomeric C reactive protein (mCRP) presents important proinflammatory effects in endothelial cells, leukocytes, or chondrocytes. However, CRP in its pentameric form exhibits weak anti-inflammatory activity. It is used as a biomarker to follow severity and progression in infectious or inflammatory diseases, such as intervertebral disc degeneration (IVDD). This work assesses for the first time the mCRP effects in human intervertebral disc cells, trying to verify the pathophysiological relevance and mechanism of action of mCRP in the etiology and progression of IVD degeneration.We demonstrated that mCRP induces the expression of multiple proinflammatory and catabolic factors, like nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and lipocalin 2 (LCN2), in human annulus fibrosus (AF) and nucleus pulposus (NP) cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signaling of mCRP.Our results indicate that the effect of mCRP is persistent and sustained, regardless of the proinflammatory environment, as it was similar in healthy and degenerative human primary AF cells. This is the first article that demonstrates the localization of mCRP in intravertebral disc cells of the AF and NP and that provides evidence for the functional activity of mCRP in healthy and degenerative human AF and NP disc cells.

ASIC3 roles in mechanosensitive elongation of nucleus pulposus cells

Morphological changes of the nucleus pulposus (NP) cells occur concomitantly as part of the intervertebral disc (IVD) degeneration and excessive mechanical loading has been speculated as a significant key factor for contributing to such morphological changes. Therefore, we hypothesize that stress exerted on NP cells can cause a deformity of nucleus in response. The changes of cell morphology is observed in degenerative nucleus pulposus. One of the reasons for degeneration of NP is due to overloading of NP especially in the obese population. So the nucleus deformity caused by stress/force is of our study interest. To delineate the effects and role of mechanical stress, we developed a 3D assay using hydrogel cultures with a circular hole generated with needle indentation to simulate a local stress concentration along the edge of the hole. A stressed zone, encompassing 100 μm of range from the circular edge, is defined based on stress concentration calculation to enable quantitative analysis against the control zone. Our results demonstrated that the circular hole produces stress-induced morphological changes in NP cells. The tangential elongation of NP cells and their nucleus shape changes in the stressed zone are significantly increased compared to the non-stressed control zone. It is proposed that the cell elongation is a direct response to elevated stress within the stressed zone. Subsequently we found the stress induced morphological changes of the NP cells can be significantly reduced by inhibiting ASIC3. This suggests ASIC3 plays an important role of play in mechano-signaling of NP cells.

Regulating inflammation and apoptosis: A smart microgel gene delivery system for repairing degenerative nucleus pulposus

The progression of intervertebral disc degeneration (IDD) is attributed to the gradual exacerbation of cellular apoptosis and impaired extracellular matrix (ECM) synthesis, both of which are induced by progressive inflammation. Therefore, it is crucial to address the inflammatory microenvironment and rectify the excessive apoptosis of nucleus pulposus cells (NPCs) to achieve intervertebral disc (IVD) regeneration. In this study, we devised a smart microgel gene delivery system that incorporates functionalized gene nanoparticles (NPs) for the purpose of IVD regeneration. siGrem1 was loaded into the NPs to enhance their antiapoptotic ability and protective effects. Furthermore, the encapsulation of HADA further endows the NPs (referred to as HSGN) with targeted delivery and anti-inflammatory effects, as well as reactive oxygen species (ROS) scavenging capacities. To create an microenvironment-responsive microgel system, phenylboronic acid-functionalized microspheres (referred to as M.S.) were fabricated and dynamically loaded with the HSGN. This microgel system (MHSGN), which is highly biocompatible, enables the sustained release of siGrem1, effectively modulating inflammation, scavenging ROS, and alleviating apoptosis in NPCs. These multifunctional capabilities promote the restoration of metabolic homeostasis within the nucleus pulposus ECM, ultimately leading to delayed IDD.

Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

BackgroundIntervertebral disc degeneration (IVDD) is the major cause of low-back pain. Histone deacetylase 9 (HDAC9) was dramatically decreased in the degenerative nucleus pulposus (NP) samples of patients with intervertebral disc degeneration (IVDD) according to bioinformatics analysis of Gene Expression Omnibus (GEO) GSE56081 dataset. This study aims to investigate the role of HDAC9 in IVDD progression.MethodsThe contribution of HDAC9 to the progression of IVDD was assessed using HDAC9 knockout (HDAC9KO) mice and NP-targeted HDAC9-overexpressing mice by IVD injection of adenovirus-mediated HDAC9 under a Col2a1 promoter. Magnetic resonance imaging (MRI) and histological analysis were used to examine the degeneration of IVD. NP cells were isolated from mice to investigate the effects of HDAC9 on apoptosis and viability. mRNA-seq and coimmunoprecipitation/mass spectrometry (co-IP/MS) analysis were used to analyze the HDAC9-regulated factors in the primary cultured NP cells.ResultsHDAC9 was statistically decreased in the NP tissues in aged mice. HDAC9KO mice spontaneously developed age-related IVDD compared with wild-type (HDAC9WT) mice. In addition, overexpression of HDAC9 in NP cells alleviated IVDD symptoms in a surgically-induced IVDD mouse model. In an in vitro assay, knockdown of HDAC9 inhibited cell viability and promoted cell apoptosis of NP cells, and HDAC9 overexpression had the opposite effects in NP cells isolated from HDAC9KO mice. Results of mRNA-seq and co-IP/MS analysis revealed the possible proteins and signaling pathways regulated by HDAC9 in NP cells. RUNX family transcription factor 3 (RUNX3) was screened out for further study, and RUNX3 was found to be deacetylated and stabilized by HDAC9. Knockdown of RUNX3 restored the effects of HDAC9 silencing on NP cells by inhibiting apoptosis and increasing viability.ConclusionOur results suggest that HDAC9 plays an important role in the development and progression of IVDD. It might be required to protect NP cells against the loss of cell viability and apoptosis by inhibiting RUNX3 acetylation and expression during IVDD. Together, our findings suggest that HDAC9 may be a potential therapeutic target in IVDD.Graphical

Application and development of hydrogel biomaterials for the treatment of intervertebral disc degeneration: a literature review.

Low back pain caused by disc herniation and spinal stenosis imposes an enormous medical burden on society due to its high prevalence and refractory nature. This is mainly due to the long-term inflammation and degradation of the extracellular matrix in the process of intervertebral disc degeneration (IVDD), which manifests as loss of water in the nucleus pulposus (NP) and the formation of fibrous disc fissures. Biomaterial repair strategies involving hydrogels play an important role in the treatment of intervertebral disc degeneration. Excellent biocompatibility, tunable mechanical properties, easy modification, injectability, and the ability to encapsulate drugs, cells, genes, etc. make hydrogels good candidates as scaffolds and cell/drug carriers for treating NP degeneration and other aspects of IVDD. This review first briefly describes the anatomy, pathology, and current treatments of IVDD, and then introduces different types of hydrogels and addresses "smart hydrogels". Finally, we discuss the feasibility and prospects of using hydrogels to treat IVDD.

Extracellular vesicles loaded dual-network bioactive sealant via immunoregulation and annulus fibrosus repair for intervertebral disc herniation

Intervertebral disc herniation (IVDH) is a common manifestation of intervertebral disc degeneration (IVDD) characterized by inflammation that results in the rupture of the annulus fibrosus (AF) and herniation of the nucleus pulposus (NP). While current clinical research primarily focuses on regulating the degenerative NP, the crucial role of the AF in maintaining the mechanical stability and metabolic balance of the intervertebral disc (IVD) has been overlooked. Resolving immunoregulation and AF repair is imperative to effectively prevent recurrent herniation. Therefore, this study introduces a bioactive sealant (OD/GM/QCS-sEVs), which combines gelatin methacryloyl (GM) and oxidized dextran (OD) with quaternized chitosan (QCS) and incorporates small extracellular vesicles (sEVs). The developed sealant possesses injectability, self-healing capabilities, tissue adhesiveness, and mechanical stability, with an average adhesive strength of 109.63 kPa. In vitro experiments demonstrate that OD/GM/QCS-sEVs effectively seal AF defects while preserving mechanical properties comparable to those of a normal IVD. Additionally, the sealant releases sEVs through a pH-responsive mechanism, thereby modulating macrophage polarization to the M2 phenotype via the NF-κB signaling pathway. This mechanism facilitates immunoregulation and anti-inflammatory effects, and promotes stem cell differentiation into fibrocartilage. Animal experiments confirm the ability of OD/GM/QCS-sEVs to seal defects, prevent proteoglycan loss, inhibit IVDD development, and promote AF regeneration. Overall, OD/GM/QCS-sEVs hold promise as an innovative bioactive sealant for recurrent herniation by resolving immunoregulation and AF regeneration.