Disc Cells Research Articles

CRISPR-Cas9-mediated loss of function of β-catenin attenuates intervertebral disc degeneration

Intervertebral disc degeneration is a very common medical condition causing pain and disability, and it cannot be reversed by available treatment options. Here we report that targeting β-catenin, a pivotal factor associated with disc degeneration, ameliorates disc degeneration in a mouse model of disc injury. Degenerative changes in the disc in response to disc injury include decompression of nucleus pulposus (NP), replacement of notochordal cells in the NP by chondrocyte-like cells, and disorganization of annulus fibrosus (AF). Importantly, downregulation of β-catenin through intradiscal injection of CRISPR-Cas9-expressing adeno-associated virus significantly mitigated all these pathological changes, by preserving notochordal cells and attenuating chondro-osteogenesis in the NP, as well as maintaining the AF structure. Moreover, β-catenin loss-of-function decelerated the rapid induction of catabolic reactions in disc matrix and attenuated pain-related neural events during disc degeneration. Thus, our data demonstrate that targeting β-catenin in disc cells through CRISPR-Cas9 has multifaceted therapeutic effects on disc degeneration, and we suggest that β-catenin plays a fundamental role in the remodeling and degenerative processes of the disc. In addition, this study proposes that CRISPR-Cas9 is a useful tool for identifying new drug targets and developing therapeutic strategies for disc degeneration.

The Endplate Role in Degenerative Disc Disease Research: The Isolation of Human Chondrocytes from Vertebral Endplate-An Optimised Protocol.

Background: Degenerative disc disease is a progressive and chronic disorder with many open questions regarding its pathomorphological mechanisms. In related studies, in vitro organ culture systems are becoming increasingly essential as a replacement option for laboratory animals. Live disc cells are highly appealing to study the possible mechanisms of intervertebral disc (IVD) degeneration. To study the degenerative processes of the endplate chondrocytes in vitro, we established a relatively quick and easy protocol for isolating human chondrocytes from the vertebral endplates. Methods: The fragments of human lumbar endplates following lumbar fusion were collected, cut, ground and partially digested with collagenase I in Advanced DMEM/F12 with 5% foetal bovine serum. The sediment was harvested, and cells were seeded in suspension, supplemented with special media containing high nutrient levels. Morphology was determined with phalloidin staining and the characterisation for collagen I, collagen II and aggrecan with immunostaining. Results: The isolated cells retained viability in appropriate laboratory conditions and proliferated quickly. The confluent culture was obtained after 14 days. Six to 8 h after seeding, attachments were observed, and proliferation of the isolated cells followed after 12 h. The cartilaginous endplate chondrocytes were stable with a viability of up to 95%. Pheno- and geno-typic analysis showed chondrocyte-specific expression, which decreased with passages. Conclusions: The reported cell isolation process is simple, economical and quick, allowing establishment of a viable long-term cell culture. The availability of a vertebral endplate cell model will permit the study of cell properties, biochemical aspects, the potential of therapeutic candidates for the treatment of disc degeneration, and toxicology studies in a well-controlled environment.

Pentosan Polysulfate, a Semisynthetic Heparinoid Disease-Modifying Osteoarthritic Drug with Roles in Intervertebral Disc Repair Biology Emulating the Stem Cell Instructive and Tissue Reparative Properties of Heparan Sulfate.

This review highlights the attributes of pentosan polysulfate (PPS) in the promotion of intervertebral disc (IVD) repair processes. PPS has been classified as a disease-modifying osteoarthritic drug (DMOAD) and many studies have demonstrated its positive attributes in the countering of degenerative changes occurring in cartilaginous tissues during the development of osteoarthritis (OA). Degenerative changes in the IVD also involve inflammatory cytokines, degradative proteases, and cell signaling pathways similar to those operative in the development of OA in articular cartilage. PPS acts as a heparan sulfate (HS) mimetic to effect its beneficial effects in cartilage. The IVD contains small cell membrane HS proteoglycans (HSPGs) such as syndecan, and glypican and a large multifunctional HS/chondroitin sulfate (CS) hybrid proteoglycan (HSPG2/perlecan), that have important matrix-stabilizing properties and sequester, control, and present growth factors from the FGF, VEGF, PDGF, and BMP families to cellular receptors to promote cell proliferation, differentiation, and matrix synthesis. HSPG2 also has chondrogenic properties and stimulates the synthesis of extracellular matrix (ECM) components and expansion of cartilaginous rudiments, and has roles in matrix stabilization and repair. Perlecan is a perinuclear and nuclear proteoglycan (PG) in IVD cells with roles in chromatin organization and control of transcription factor activity, immunolocalizes to stem cell niches in cartilage, promotes escape of stem cells from quiescent recycling, differentiation and attainment of pluripotency and migratory properties. These participate in tissue development and morphogenesis, ECM remodeling and repair. PPS also localizes in the nucleus of stromal stem cells, promotes development of chondroprogenitor cell lineages, ECM synthesis and repair and discal repair by resident disc cells. The availability of recombinant perlecan and PPS offers new opportunities in repair biology. These multifunctional agents offer welcome new developments in repair strategies for the IVD.

Investigation into the anti-inflammatory properties of metformin in intervertebral disc cells.

IntroductionIntervertebral disc degeneration (IDD) is closely related to heightened inflammation in the annulus fibrosis (AF) and nucleus pulposus (NP) cells in the intervertebral disc. An imbalanced matrix homeostasis has been shown to contribute to disc degeneration and associated discogenic low back pain. Metformin, a diabetes medication, has been noted to exhibit anti‐inflammatory properties through upregulation of the AMPK pathway, leading to various anti‐inflammatory‐related responses in hepatocytes. However, it is still unclear how metformin influences disc cellular response to inflammatory stress and the corresponding mechanism. Hence, the objective of this study is to elucidate the effects of metformin on expression of key pro‐inflammatory, catabolic, and anabolic factors within rat AF cells in response to inflammatory stimulation and mechanical tensile stress.MethodsFive Fischer 344 rats were sacrificed and their spines isolated. AF cells were cultured and plated in flexible silicone membrane‐based six‐well plates. Wells were split into eight groups and subjected to metformin, IL‐1β, mechanical stretch, and combined treatments. Relative gene expressions of MMP‐13, COX‐2, iNOS, AGC, and Col1 were assessed with quantitative real‐time polymerase chain reaction (qRT‐PCR), and downstream prostaglandin E2 (PGE2) production was quantified with enzyme‐linked immunosorbent assay (ELISA). NF‐kB nuclear translocation was also quantified.ResultsMetformin in the presence of the combined stress treatments (M + IL/S) significantly increased Col1, COX‐2, and MMP‐13 gene expression, decreased PGE2 production compared to IL/S conditions alone. Metformin treatment of cultured rat annulus fibrosus cells significantly reduced the nuclear translocation of NF‐κB after 4 h of IL‐1β treatment from 43.1% in case of IL‐1β treatment down to 26.2% in the case of metformin + IL‐1β treatment.DiscussionThe lack of metformin‐mediated suppression of inflammatory response in the nonstretch groups indicates that metformin may be enacting its effects through a stretch‐dependent manner. These results suggest a foundation for pursuing further research into metformin's potential role as an anti‐inflammatory agent for curtailing intervertebral disc degeneration.

Partial reprogramming strategy for intervertebral disc rejuvenation by activating energy switch.

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

Senolytics: Eliminating Senescent Cells and Alleviating Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is the main cause of cervical and lumbar spondylosis. Over the past few years, the relevance between cellular senescence and IVDD has been widely studied, and the senescence-associated secretory phenotype (SASP) produced by senescent cells is found to remodel extracellular matrix (ECM) metabolism and destruct homeostasis. Elimination of senescent cells by senolytics and suppression of SASP production by senomorphics/senostatics are effective strategies to alleviate degenerative diseases including IVDD. Here, we review the involvement of senescence in the process of IVDD; we also discuss the potential of senolytics on eliminating senescent disc cells and alleviating IVDD; finally, we provide a table listing senolytic drugs and small molecules, aiming to propose potential drugs for IVDD therapy in the future.

Cre/LoxP Genetic Recombination Sustains Cartilage Anabolic Factor Expression in Hyaluronan Encapsulated MSCs Alleviates Intervertebral Disc Degeneration.

(1) Background: Inexplicable low back and neck pain frequently results from spinal disc degeneration with an imbalanced intervertebral disc (IVD) cell homeostasis. We hypothesize that introducing MSC expressing a sustained cartilage-anabolic factor in the IVD may stimulate the mucoid materials secreted from the IVD cells, promote the MSC’s chondrogenesis and maintain the hydration content providing mechanical strength to decelerate the disc degeneration progression; (2) Methods: This study expressed a cartilage-anabolic factor runx1 by a baculoviral vector (BV) transduced MSCs through a Cre/LoxP gene editing and recombination system for sustained recombinant runx1 transcription factor production. The Cre/LoxP BV modified MSCs were encapsulated by hyaluronan hydrogel, due to its’ vital composition in ECM of a healthy disc and transplanted to a punctured coccygeal disc in rats through micro-injection, followed by X-ray radiography and histological analysis at the 4- and 12-weeks post-transplantation; (3) Results: Data reveals the Cre/LoxP BV system-mediated long-termed runx1 gene expression, possessing good biosafety characteristics in the in vitro cell transduction and in vivo MSCs transplantation, and maintained superior hydration content in the disc than that of mock transduced MSCs; (4) Conclusions: This proof-of-concept study fulfills the need of implanting therapeutic cells accompanied with microinjection in the disc, such as a discography and paves a road to manufacture composite hyaluronan, such as peptide modified hyaluronan as an MSC carrier for IVD regeneration in the future study.

Histological Changes of Cervical Disc Tissue in Patients with Degenerative Ossification.

ObjectiveTo explore the histological feature of the cervical disc degeneration in patients with degenerative ossification (DO) and its potential mechanisms. MethodsA total of 96 surgical segments, from cervical disc degenerative disease patients with surgical treatment, were divided into ossification group (group O, n=46) and non-ossification group (group NO, n=50) based on preoperative radiological exams. Samples of disc tissues and osteophytes were harvested during the decompression operation. The hematoxylin-eosin staining, Masson trichrome staining and Safranin O-fast green staining were used to compare the histological differences between the two groups. And the distribution and content of transforming growth factor (TGF)-β1, p-Smad2 and p-Smad3 between the two groups were compared by a semi-quantitative immunohistochemistry (IHC) method. ResultsFor all the disc tissues, the content of disc cells and collagen fibers decreased gradually from the outer annulus fibrosus (OAF) to the central nucleus pulposus (NP). Compared with group NO, the number of disc cells in group O increased significantly. But for proteoglycan in the inner annulus fibrosus (IAF) and NP, the content in group O decreased significantly. IHC analysis showed that TGF-β1, p-Smad2, and p-Smad3 were detected in all tissues. For group O, the content of TGF-β1 in the OAF and NP was significantly higher than that in group NO. For p-Smad2 in IAF and p-Smad3 in OAF, the content in group O were significantly higher than group NO. ConclusionHistologically, cervical disc degeneration in patients with DO is more severe than that without DO. Local higher content of TGF-β1, p-Smad2, and p-Smad3 are involved in the disc degeneration with DO. Further studies with multi-approach analyses are needed to better understand the role of TGF-β/Smads signaling pathway in the disc degeneration with DO.

IGF Signaling in Intervertebral Disc Health and Disease

Low back pain (LBP) is a common musculoskeletal symptom, which brings a lot of pain and economic loss to patients. One of the most common causes of LBP is intervertebral disc degeneration (IVDD). However, pathogenesis is still debated, and therapeutic options are limited. Insulin-like growth factor (IGF) signaling pathways play an important role in regulating different cell processes, including proliferation, differentiation, migration, or cell death, which are critical to the homeostasis of tissues and organs. The IGF signaling is crucial in the occurrence and progression of IVDD. The activation of IGF signaling retards IVDD by increasing cell proliferation, promoting extracellular matrix (ECM) synthesis, inhibiting ECM decomposition, and preventing apoptosis and senescence of disc cells. However, abnormal activation of IGF signaling may promote the process of IVDD. IGF signaling is currently considered to have a promising treatment prospect for IVDD. An in-depth understanding of the role of IGF signaling in IVDD may help find a novel approach for IVDD treatment.

Translational control of E2f1 regulates the Drosophila cell cycle

E2F transcription factors are master regulators of the eukaryotic cell cycle. In Drosophila, the sole activating E2F, E2F1, is both required for and sufficient to promote G1→S progression. E2F1 activity is regulated both by binding to RB Family repressors and by posttranscriptional control of E2F1 protein levels by the EGFR and TOR signaling pathways. Here, we investigate cis-regulatory elements in the E2f1 messenger RNA (mRNA) that enable E2f1 translation to respond to these signals and promote mitotic proliferation of wing imaginal disc and intestinal stem cells. We show that small upstream open reading frames (uORFs) in the 5' untranslated region (UTR) of the E2f1 mRNA limit its translation, impacting rates of cell proliferation. E2f1 transgenes lacking these 5'UTR uORFs caused TOR-independent expression and excess cell proliferation, suggesting that TOR activity can bypass uORF-mediated translational repression. EGFR signaling also enhanced translation but through a mechanism less dependent on 5'UTR uORFs. Further, we mapped a region in the E2f1 mRNA that contains a translational enhancer, which may also be targeted by TOR signaling. This study reveals translational control mechanisms through which growth signaling regulates cell cycle progression.

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.

The Role of Oxidative Stress in Intervertebral Disc Degeneration.

Intervertebral disc degeneration is a very common type of degenerative disease causing severe socioeconomic impact, as well as a major cause of discogenic low back pain and herniated discs, placing a heavy burden on patients and the clinicians who treat them. IDD is known to be associating with a complex process involving in extracellular matrix and cellular damage, and in recent years, there is increasing evidence that oxidative stress is an important activation mechanism of IDD and that reactive oxygen and reactive nitrogen species regulate matrix metabolism, proinflammatory phenotype, autophagy and senescence in intervertebral disc cells, apoptosis, autophagy, and senescence. Despite the tremendous efforts of researchers within the field of IDD pathogenesis, the proven strategies to prevent and treat this disease are still very limited. Up to now, several antioxidants have been proved to be effective for alleviating IDD. In this article, we discussed that oxidative stress accelerates disc degeneration by influencing aging, inflammation, autophagy, and DNA methylation, and summarize some antioxidant therapeutic measures for IDD, indicating that antioxidant therapy for disc degeneration holds excellent promise.

Differential gene expression analysis identified determinants of cell fate plasticity during radiation-induced regeneration in Drosophila.

Ionizing radiation (IR) is used to treat half of all cancer patients because of its ability to kill cells. IR, however, can induce stem cell-like properties in non-stem cancer cells, potentiating tumor regrowth and reduced therapeutic success. We identified previously a subpopulation of cells in Drosophila larval wing discs that exhibit IR-induced stem cell-like properties. These cells reside in the future wing hinge, are resistant to IR-induced apoptosis, and are capable of translocating, changing fate, and participating in regenerating the pouch that suffers more IR-induced apoptosis. We used here a combination of lineage tracing, FACS-sorting of cells that change fate, genome-wide RNAseq, and functional testing of 42 genes, to identify two key changes that are required cell-autonomously for IR-induced hinge-to-pouch fate change: (1) repression of hinge determinants Wg (Drosophila Wnt1) and conserved zinc-finger transcription factor Zfh2 and (2) upregulation of three ribosome biogenesis factors. Additional data indicate a role for Myc, a transcriptional activator of ribosome biogenesis genes, in the process. These results provide a molecular understanding of IR-induced cell fate plasticity that may be leveraged to improve radiation therapy.

Identifying the potential role of IL-1β in the molecular mechanisms of disc degeneration using gene expression profiling and bioinformatics analysis.

We performed a bioinformatics analysis to identify the key genes that were differentially expressed between degenerative intervertebral disc (IVD) cells with and without exposure to interleukin-1β and explore the related signaling pathways and interaction networks. The microarray data were downloaded from the Gene Expression Omnibus (27,494). Then, analyses of the gene ontology, signaling pathways, and interaction networks for the differentially expressed genes (DEGs) were conducted using tools including the Database for Annotation, Visualization, and Integrated Discovery, Metascape, Gene Set Enrichment Analysis, Search Tool for the Retrieval of Interacting Genes, Cytoscape, Venn method, and packages of the R computing language. A total of 260 DEGs were identified, including 161 upregulated and 99 downregulated genes. Gene Ontology annotation analysis showed that these DEGs were mainly associated with the extracellular region, chemotaxis, taxis, cytokine activity, and cytokine receptor binding. A Kyoto Encyclopedia of Genes and Genomes signaling pathway analysis showed that these DEGs were mainly involved in the of cytokine-cytokine receptor interaction, rheumatoid arthritis, tumor necrosis factor signaling pathway, Salmonella infection, and chemokine signaling pathway. The interaction network analysis indicated that 10 hub genes, including CXCL8, CXCL1, CCL20, CXCL2, CXCL5, CXCL3, CXCL6, C3, PF4, and GPER1 may play key roles in IVD degeneration. Bioinformatic analysis showed that CXCL8 and other nine key genes may play a role in the development of disc degeneration induced by inflammatory reactions and can be used to identify potential target genes for therapeutic applications in IVD degeneration.

Overexpression of BNIP3 in rat intervertebral disk cells triggers autophagy and apoptosis

Excessive apoptosis of intervertebral disk cells and intervertebral disk degeneration (IDD) is the prime cause of low back pain. B-cell lymphoma 2 (Bcl-2) and adenovirus E1B 19 kDa interacting protein 3 (BNIP3), a member of the Bcl-2 family, are involved in cell autophagy and apoptosis. The roles and mechanisms of BNIP3 in intervertebral disk cell autophagy and apoptosis are unclear. In this study, primary rat intervertebral disk cells were prepared to study the effect of BNIP3 overexpression on their autophagy and apoptosis. The cell counting kit (CCK)-8 assay showed that BNIP3 overexpression decreased cell viability. Real-time PCR and Western blotting showed that BNIP3 overexpression significantly upregulated the expression of autophagy-related proteins and pro-apoptotic proteins, including hypoxia-inducible factor-1?, apoptotic protease activating factor 1, caspase 3 and cleaved caspase 3, microtubule-associated proteins 1A/1B light chain 3 (LC3) and Beclin-1 while downregulating the expression of anti-apoptotic protein Bcl-2. Cell staining detection of autophagy and apoptosis showed that BNIP3 overexpression increased the autophagy and apoptosis of rat intervertebral disk cells. BNIP3 RNA interference revealed that the effects of BNIP3 overexpression can be reversed. These findings suggested that BNIP3 enhanced the autophagy and apoptosis in the rat intervertebral disk cells in vitro, which might promote IDD development.

Current Status of the Instructional Cues Provided by Notochordal Cells in Novel Disc Repair Strategies.

Numerous publications over the past 22 years, beginning with a seminal paper by Aguiar et al., have demonstrated the ability of notochordal cell-secreted factors to confer anabolic effects upon intervertebral disc (IVD) cells. Since this seminal paper, other scientific publications have demonstrated that notochordal cells secrete soluble factors that can induce anti-inflammatory, pro-anabolic and anti-cell death effects upon IVD nucleus pulposus (NP) cells in vitro and in vivo, direct human bone marrow-derived mesenchymal stem cells toward an IVD NP-like phenotype and repel neurite ingrowth. More recently these factors have been characterized, identified, and used therapeutically to induce repair upon injured IVDs in small and large pre-clinical animal models. Further, notochordal cell-rich IVD NPs maintain a stable, healthy extracellular matrix whereas notochordal cell-deficient IVDs result in a biomechanically and extracellular matrix defective phenotype. Collectively this accumulating body of evidence indicates that the notochordal cell, the cellular originator of the intervertebral disc holds vital instructional cues to establish, maintain and possibly regenerate the intervertebral disc.

DUSP-1 Induced by PGE2 and PGE1 Attenuates IL-1β-Activated MAPK Signaling, Leading to Suppression of NGF Expression in Human Intervertebral Disc Cells.

The molecular mechanism of discogenic low back pain (LBP) involves nonphysiological nerve invasion into a degenerated intervertebral disc (IVD), induced by nerve growth factor (NGF). Selective cyclooxygenase (COX)-2 inhibitors are mainly used in the treatment of LBP, and act by suppressing the inflammatory mediator prostaglandin E2 (PGE2), which is induced by inflammatory stimuli, such as interleukin-1β (IL-1β). However, in our previous in vitro study using cultured human IVD cells, we demonstrated that the induction of NGF by IL-1β is augmented by a selective COX-2 inhibitor, and that PGE2 and PGE1 suppress NGF expression. Therefore, in this study, to elucidate the mechanism of NGF suppression by PGE2 and PGE1, we focused on mitogen-activated protein kinases (MAPKs) and its phosphatase, dual-specificity phosphatase (DUSP)-1. IL-1β-induced NGF expression was altered in human IVD cells by MAPK pathway inhibitors. PGE2 and PGE1 enhanced IL-1β-induced DUSP-1 expression, and suppressed the phosphorylation of MAPKs in human IVD cells. In DUSP-1 knockdown cells established using small interfering RNA, IL-1β-induced phosphorylation of MAPKs was enhanced and prolonged, and NGF expression was significantly enhanced. These results suggest that PGE2 and PGE1 suppress IL-1β-induced NGF expression by suppression of the MAPK signaling pathway, accompanied by increased DUSP-1 expression.

Glycyrrhizin regulates rat TMJOA progression by inhibiting the HMGB1-RAGE/TLR4-NF-κB/AKT pathway.

To investigate the role of glycyrrhizin on the progression of temporomandibular joint osteoarthritis (TMJOA) and the underlying mechanism by regulation of the high‐mobility group box 1 (HMGB1) receptor for advanced glycation end products (RAGE)/toll‐like receptor 4 (TLR4)‐nuclear factor kappa B (NF‐κB)/protein kinase B (AKT) pathway. After a rat model of TMJOA was built by intra‐articular injection of monosodium iodoacetate, glycyrrhizin was intragastrically administered at low concentration (20 mg/kg) or high concentration (50 mg/kg). Micro‐computed tomography, histological and immunohistochemical analysis were used to reveal the progression of TMJOA. Rat TMJ chondrocytes and disc cells were cultured in inflammatory condition with different doses of glycyrrhizin. Western blot was used to evaluate the effect of glycyrrhizin on the HMGB1‐RAGE/TLR4‐NF‐κB/AKT pathway. Administration of glycyrrhizin alleviated cartilage degeneration, lowered the levels of inflammatory and catabolic mediators and reduced the production of HMGB1, RAGE and TLR4 in TMJOA animal model. Increased production of RAGE and TLR4, and activated intracellular NF‐κB and/or AKT signalling pathways in chondrocytes and disc cells were found in inflammatory condition. Upon activation, matrix metalloprotease‐3 and interleukin‐6 were upregulated. Glycyrrhizin inhibited not only HMGB1 release but also RAGE and TLR4 in inflammatory condition. Glycyrrhizin alleviated the pathological changes of TMJOA by regulating the HMGB1‐RAGE/TLR4‐NF‐kB/AKT signalling pathway. This study revealed the potential of glycyrrhizin as a novel therapeutic drug to suppress TMJ cartilage degradation.

Involvement of autophagy in the maintenance of rat intervertebral disc homeostasis: an in-vitro and in-vivo RNA interference study of Atg5

In the largest avascular low-nutrient intervertebral disc, resident cells would utilize autophagy, a stress-response survival mechanism by self-digestion and recycling wastes. Our goal was to elucidate the involvement of autophagy in disc homeostasis through RNA interference of autophagy-related gene 5 (Atg5). In vitro, small interfering RNAs (siRNAs) targeting autophagy-essential Atg5 were transfected into rat disc cells. Cell viability with levels of autophagy including Atg5 expression, apoptosis, and senescence was assessed under serum starvation and/or pro-inflammatory interleukin-1 beta (IL-1β) stimulation. In vivo, time-course autophagic flux was monitored following Alexa Fluor® 555-labeled Atg5-siRNA injection into rat tail discs. Furthermore, 24-h temporary static compression-induced disruption of Atg5 siRNA-injected discs was observed by radiography, histomorphology, and immunofluorescence. In disc cells, three different Atg5 siRNAs consistently suppressed autophagy with Atg5 protein knockdown (mean 44.4% [95% confidence interval:-51.7,-37.1], 51.5% [-80.5,-22.5], 62.3% [-96.6,-28.2]). Then, Atg5 knockdown reduced cell viability through apoptosis and senescence not in serum-supplemented medium (93.6% [-0.8, 21.4]) but in serum-deprived medium (66.4% [-29.8,-8.6]) further with IL-1β (44.5% [-36.9,-23.5]). In disc tissues, immunofluorescence detected intradiscal signals for the labeled siRNA even at 56-d post-injection. Immunoblotting found 56-d autophagy suppression with prolonged Atg5 knockdown (33.2% [-52.8,-5.3]). With compression, Atg5 siRNA-injected discs presented radiographic height loss ([-43.9,-0.8]), histological damage ([-5.5,-0.2]), and immunofluorescent apoptosis ([2.2, 22.2]) and senescence ([4.1, 19.9]) induction compared to control siRNA-injected discs at 56d. This loss-of-function study suggests Atg5-dependent autophagy-mediated anti-apoptosis and anti-senescence. Autophagy could be a molecular therapeutic target for degenerative disc disease.

Innovative Biological Treatment Methods for Degenerative Disc Disease

Low back pain is the leading cause of work absences and years lived with disability, and it is often associated with degenerative disc disease. In recent years, biological treatment approaches such as the use of growth factors, cell injections, annulus fibrosus (AF) repair, nucleus pulposus replacement, and tissue-engineered discs have been explored as means for preventing or reversing degenerative disc disease. Both animal and clinical studies have shown promising results for cell-based therapy on the grounds of its regenerative potential. Clinical data also indicate that stem cell injection is safe when appropriately performed, albeit its long-term safety and efficacy are yet to be explored. Numerous challenges also remain to be overcome, such as isolating, differentiating, and preconditioning the disc cells, as well as managing the nutrient-deficient and oxygen-deficient micromilieu of the intervertebral disc (IVD). AF repair methods including devices used in clinical trials have shown success in decreasing reherniation rates and improving overall clinical outcomes. In addition, recent studies that combined AF repair and nucleus pulposus replacement have shown improved biomechanical stability in IVDs after the combined treatment. Tissue-engineered IVDs for total disc replacement are still being developed, and future studies are necessary to overcome the challenges in their delivery, efficacy, and safety.