Targeted screening of inflammatory mediators in spontaneous degenerative disc disease in dogs reveals an upregulation of the tumor necrosis superfamily.

Background: The regulation of inflammatory mediators in the degenerating intervertebral disc (IVD) and corresponding ligamentum flavum (LF) is a topic of emerging interest. The study aimed to investigate the expression of a broad array of inflammatory mediators in the degenerated LF and IVD using a dog model of spontaneous degenerative disc disease (DDD) to determine potential treatment targets. Methods: LF and IVD tissues were collected from 22 normal dogs (Pfirrmann grades I and II) and 18 dogs affected by DDD (Pfirrmann grades III and IV). A qPCR gene array was used to investigate the expression of 80 inflammatory genes for LF and IVD tissues, whereafter targets of interest were investigated in additional tissue samples using qPCR, western blot (WB), and immunohistochemistry. Results: Tumor necrosis factor superfamily (TNFSF) signaling was identified as a regulated pathway in DDD, based on the significant regulation (n-fold ± SD) of various TNFSF members in the degenerated IVD, including nerve growth factor (NGF; -8 ± 10), CD40LG (464 ± 442), CD70 (341 ± 336), TNFSF Ligand 10 (9 ± 8), and RANKL/TNFSF Ligand 11 (85 ± 74). In contrast, TNFSF genes were not significantly affected in the degenerated LF compared to the control LF. Protein expression of NGF (WB) was significantly upregulated in both the degenerated LF (4.4 ± 0.5) and IVD (11.3 ± 5.6) compared to the control group. RANKL immunopositivity was significantly upregulated in advanced stages of degeneration (Thompson grades IV and V) in the nucleus pulposus and annulus fibrosus of the IVD, but not in the LF. Conclusions: DDD involves a significant upregulation of various TNFSF members, with tissue-specific expression profiles in LF and IVD tissues. The differential involvement of TNFSF members within multiple spinal tissues from the same individual provides new insights into the inflammatory processes involved in DDD and may provide a basis to formulate hypotheses for the determination of potential treatment targets.

Human 3D nucleus pulposus microtissue model to evaluate the potential of pre-conditioned nasal chondrocytes for the repair of degenerated intervertebral disc.

Introduction: An in vitro model that appropriately recapitulates the degenerative disc disease (DDD) microenvironment is needed to explore clinically relevant cell-based therapeutic strategies for early-stage degenerative disc disease. We developed an advanced 3D nucleus pulposus (NP) microtissues (µT) model generated with cells isolated from human degenerating NP tissue (Pfirrmann grade: 2-3), which were exposed to hypoxia, low glucose, acidity and low-grade inflammation. This model was then used to test the performance of nasal chondrocytes (NC) suspension or spheroids (NCS) after pre-conditioning with drugs known to exert anti-inflammatory or anabolic activities. Methods: NPµTs were formed by i) spheroids generated with NP cells (NPS) alone or in combination with ii) NCS or iii) NC suspension and cultured in healthy or degenerative disc disease condition. Anti-inflammatory and anabolic drugs (amiloride, celecoxib, metformin, IL-1Ra, GDF-5) were used for pre-conditioning of NC/NCS. The effects of pre-conditioning were tested in 2D, 3D, and degenerative NPµT model. Histological, biochemical, and gene expression analysis were performed to assess matrix content (glycosaminoglycans, type I and II collagen), production and release of inflammatory/catabolic factors (IL-6, IL-8, MMP-3, MMP-13) and cell viability (cleaved caspase 3). Results: The degenerative NPµT contained less glycosaminoglycans, collagens, and released higher levels of IL-8 compared to the healthy NPµT. In the degenerative NPµT, NCS performed superior compared to NC cell suspension but still showed lower viability. Among the different compounds tested, only IL-1Ra pre-conditioning inhibited the expression of inflammatory/catabolic mediators and promoted glycosaminoglycan accumulation in NC/NCS in DDD microenvironment. In degenerative NPµT model, preconditioning of NCS with IL-1Ra also provided superior anti-inflammatory/catabolic activity compared to non-preconditioned NCS. Conclusion: The degenerative NPµT model is suitable to study the responses of therapeutic cells to microenvironment mimicking early-stage degenerative disc disease. In particular, we showed that NC in spheroidal organization as compared to NC cell suspension exhibited superior regenerative performance and that IL-1Ra pre-conditioning of NCS could further improve their ability to counteract inflammation/catabolism and support new matrix production within harsh degenerative disc disease microenvironment. Studies in an orthotopic in vivo model are necessary to assess the clinical relevance of our findings in the context of IVD repair.

Spheroid-Based Tissue Engineering Strategies for Regeneration of the Intervertebral Disc.

Degenerative disc disease, a painful pathology of the intervertebral disc (IVD), often causes disability and reduces quality of life. Although regenerative cell-based strategies have shown promise in clinical trials, none have been widely adopted clinically. Recent developments demonstrated that spheroid-based approaches might help overcome challenges associated with cell-based IVD therapies. Spheroids are three-dimensional multicellular aggregates with architecture that enables the cells to differentiate and synthesize endogenous ECM, promotes cell-ECM interactions, enhances adhesion, and protects cells from harsh conditions. Spheroids could be applied in the IVD both in scaffold-free and scaffold-based configurations, possibly providing advantages over cell suspensions. This review highlights areas of future research in spheroid-based regeneration of nucleus pulposus (NP) and annulus fibrosus (AF). We also discuss cell sources and methods for spheroid fabrication and characterization, mechanisms related to spheroid fusion, as well as enhancement of spheroid performance in the context of the IVD microenvironment.

Nose to Spine: spheroids generated by human nasal chondrocytes for scaffold-free nucleus pulposus augmentation.

Cell-based strategies for nucleus pulposus (NP) regeneration that adequately support the engraftment and functionality of therapeutic cells are still lacking. This study explores a scaffold-free approach for NP repair, which is based on spheroids derived from human nasal chondrocytes (NC), a resilient cell type with robust cartilage-regenerative capacity. We generated NC spheroids (NCS) in two types of medium (growth or chondrogenic) and analyzed their applicability for NP repair with regard to injectability, biomechanical and biochemical attributes, and integration potential in conditions simulating degenerative disc disease (DDD). NCS engineered in both media were compatible with a typical spinal needle in terms of size (lower than 600µm), shape (roundness greater than 0.8), and injectability (no changes in morphology and catabolic gene expression after passing through the needle). While growth medium ensured stable elastic modulus (E) at 5 kPa, chondrogenic medium time-dependently increased E of NCS, in correlation with gene/protein expression of collagen. Notably, DDD-mimicking conditions did not impair NCS viability nor NCS fusion with NP spheroids simulating degenerated NP in vitro. To assess the feasibility of this approach, NCS were injected into an ex vivo-cultured bovine intervertebral disc (IVD) without damage using a spinal needle. In conclusion, our data indicated that NC cultured as spheroids can be compatible with strategies for minimally invasive NP repair in terms of injectability, tuneability, biomechanical features, and resilience. Future studies will address the capacity of NCS to integrate within degenerated NP under long-term loading conditions. STATEMENT OF SIGNIFICANCE: Current regenerative strategies still do not sufficiently support the engraftment of therapeutic cells in the nucleus pulposus (NP). We present an injectable approach based on spheroids derived from nasal chondrocytes (NC), a resilient cell type with robust cartilage-regenerative capacity. NC spheroids (NCS) generated with their own matrix and demonstrated injectability, tuneability of biomechanical/biochemical attributes, and integration potential in conditions simulating degenerative disc disease. To our knowledge, this is the first study that explored an injectable spheroid-based scaffold-free approach, which showed potential to support the adhesion and viability of therapeutic cells in degenerated NP. The provided information can be of substantial interest to a wide audience, including biomaterial scientists, biomedical engineers, biologists and medical researchers.

Intervertebral Disc-on-a-Chip as Advanced In Vitro Model for Mechanobiology Research and Drug Testing: A Review and Perspective.

Discogenic back pain is one of the most diffused musculoskeletal pathologies and a hurdle to a good quality of life for millions of people. Existing therapeutic options are exclusively directed at reducing symptoms, not at targeting the underlying, still poorly understood, degenerative processes. Common intervertebral disc (IVD) disease models still do not fully replicate the course of degenerative IVD disease. Advanced disease models that incorporate mechanical loading are needed to investigate pathological causes and processes, as well as to identify therapeutic targets. Organs-on-chip (OoC) are microfluidic-based devices that aim at recapitulating tissue functions in vitro by introducing key features of the tissue microenvironment (e.g., 3D architecture, soluble signals and mechanical conditioning). In this review we analyze and depict existing OoC platforms used to investigate pathological alterations of IVD cells/tissues and discuss their benefits and limitations. Starting from the consideration that mechanobiology plays a pivotal role in both IVD homeostasis and degeneration, we then focus on OoC settings enabling to recapitulate physiological or aberrant mechanical loading, in conjunction with other relevant features (such as inflammation). Finally, we propose our view on design criteria for IVD-on-a-chip systems, offering a future perspective to model IVD mechanobiology.

Novel Fast and Reliable Method for Nano-Erythrosome Production Using Shear Force.

PURPOSE: The production of nano-erythrosomes (NEs) by extrusion, which is considered the "gold standard", has several disadvantages such as difficult equipment assembly, long procedure time, variable pressure, and problems with sterility. An alternative approach, using ultrasound probe, has been shown to overheat the sample and have suboptimal results compared to the extrusion method. In our study, we propose, develop, and test a new method for the fabrication of NEs based on shear force and then compare it to the "gold standard" extrusion approach. METHODS: The new method consists of mechanical shear force disruption of the hemoglobin-depleted erythrocyte ghost membranes, with the aid of a rotor stator based tissue homogenizer. Using the same batches of erythrocyte ghost membranes, we compared NEs produced by shear force to NEs produced by the well-established extrusion approach. NEs were characterized for yield, size, encapsulation efficiency, morphology, and stability by flow cytometry (FC), transmission electron microscopy (TEM), and zeta potential analysis. RESULTS: The shear force based process was easier to set up, significantly faster, had better sterility control, and decreased variability between batches. The shear force method generated NEs with the desired size distribution (particles diameter ~125 nm), which were morphologically and functionally equivalent to the NEs produced by extrusion. NEs produced by shear force were stable in terms of counts, size, and fluorescence intensity for 3 weeks at +4°C. Moreover, they showed colloidal stability and minimal influence to centrifugal stress, turbulence shock, and hemolytic potential. CONCLUSION: The newly proposed shear force method allows faster, easier, and highly reproducible NEs production when compared to the conventional extrusion approach. The new setup allows simultaneous production of sterile batches of NEs, which have homogenous size distribution, good stability, and improved shelf life storage. The ability of the shear force method to process also high concentration samples indicates a future potential development of large-scale NEs production and industrial application, which has been a challenge for the extrusion method.

TRPV4 Inhibition and CRISPR-Cas9 Knockout Reduce Inflammation Induced by Hyperphysiological Stretching in Human Annulus Fibrosus Cells.

Mechanical loading and inflammation interact to cause degenerative disc disease and low back pain (LBP). However, the underlying mechanosensing and mechanotransductive pathways are poorly understood. This results in untargeted pharmacological treatments that do not take the mechanical aspect of LBP into account. We investigated the role of the mechanosensitive ion channel TRPV4 in stretch-induced inflammation in human annulus fibrosus (AF) cells. The cells were cyclically stretched to 20% hyperphysiological strain. TRPV4 was either inhibited with the selective TRPV4 antagonist GSK2193874 or knocked out (KO) via CRISPR-Cas9 gene editing. The gene expression, inflammatory mediator release and MAPK pathway activation were analyzed. Hyperphysiological cyclic stretching significantly increased the IL6, IL8, and COX2 mRNA, PGE2 release, and activated p38 MAPK. The TRPV4 pharmacological inhibition significantly attenuated these effects. TRPV4 KO further prevented the stretch-induced upregulation of IL8 mRNA and reduced IL6 and IL8 release, thus supporting the inhibition data. We provide novel evidence that TRPV4 transduces hyperphysiological mechanical signals into inflammatory responses in human AF cells, possibly via p38. Additionally, we show for the first time the successful gene editing of human AF cells via CRISPR-Cas9. The pharmacological inhibition or CRISPR-based targeting of TRPV4 may constitute a potential therapeutic strategy to tackle discogenic LBP in patients with AF injury.

Expression and activity of hyaluronidases HYAL-1, HYAL-2 and HYAL-3 in the human intervertebral disc.

PURPOSE: Hyaluronic acid plays an essential role in water retention of the intervertebral disc (IVD) and thus provides flexibility and shock absorbance in the spine. Hyaluronic acid gets degraded by hyaluronidases (HYALs), and some of the resulting fragments were previously shown to induce an inflammatory and catabolic response in human IVD cells. However, no data currently exist on the expression and activity of HYALs in IVD health and disease. METHODS: Gene expression, protein expression and activity of HYALs were determined in human IVD biopsies with different degrees of degeneration (n = 50 total). Furthermore, freshly isolated human IVD cells (n = 23 total) were stimulated with IL-1ß, TNF-α or H2O2, followed by analysis of HYAL-1, HYAL-2 and HYAL-3 gene expression. RESULTS: Gene expression of HYAL-1 and protein expression of HYAL-2 significantly increased in moderate/severe disc samples when compared to samples with no or low IVD degeneration. HYAL activity was not significantly increased due to high donor-donor variation, but seemed overall higher in the moderate/severe group. An inflammatory environment, as seen during IVD disease, did not affect HYAL-1, HYAL-2 or HYAL-3 expression, whereas exposure to oxidative stress (100 µM H2O2) upregulated HYAL-2 expression relative to untreated controls. CONCLUSION: Although HYAL-1, HYAL-2 and HYAL-3 are all expressed in the IVD, HYAL-2 seems to have the highest pathophysiological relevance. Nonetheless, further studies will be needed to comprehensively elucidate its significance and to determine its potential as a therapeutic target. These slides can be retrieved under Electronic Supplementary Material.

Electrospray-Based Microencapsulation of Epigallocatechin 3-Gallate for Local Delivery into the Intervertebral Disc.

Locally delivered anti-inflammatory compounds can restore the homeostasis of the degenerated intervertebral disc (IVD). With beneficial effects on IVD cells, epigallocatechin 3-gallate (EGCG) is a promising therapeutic candidate. However, EGCG is prone to rapid degradation and/or depletion. Therefore, the purpose of this study was to develop a method for controlled EGCG delivery in the degenerated IVD. Primary IVD cells were isolated from human donors undergoing IVD surgeries. EGCG was encapsulated into microparticles by electrospraying of glutaraldehyde-crosslinked gelatin. The resulting particles were characterized in terms of cytocompatibility and anti-inflammatory activity, and combined with a thermoresponsive carrier to produce an injectable EGCG delivery system. Subsequently, electrospraying was scaled up using the industrial NANOSPIDER™ technology. The produced EGCG microparticles reduced the expression of inflammatory (IL-6, IL-8, COX-2) and catabolic (MMP1, MMP3, MMP13) mediators in pro-inflammatory 3D cell cultures. Combining the EGCG microparticles with the carrier showed a trend towards modulating EGCG activity/release. Electrospray upscaling was achieved, leading to particles with homogenous spherical morphologies. In conclusion, electrospray-based encapsulation of EGCG resulted in cytocompatible microparticles that preserved the activity of EGCG and showed the potential to control EGCG release, thus favoring IVD health by downregulating local inflammation. Future studies will focus on further exploring the biological activity of the developed delivery system for potential clinical use.

Expression and Activity of TRPA1 and TRPV1 in the Intervertebral Disc: Association with Inflammation and Matrix Remodeling.

Transient receptor potential (TRP) channels have emerged as potential sensors and transducers of inflammatory pain. The aims of this study were to investigate (1) the expression of TRP channels in intervertebral disc (IVD) cells in normal and inflammatory conditions and (2) the function of Transient receptor potential ankyrin 1 (TRPA1) and Transient receptor potential vanilloid 1 (TRPV1) in IVD inflammation and matrix homeostasis. RT-qPCR was used to analyze human fetal, healthy, and degenerated IVD tissues for the gene expression of TRPA1 and TRPV1. The primary IVD cell cultures were stimulated with either interleukin-1 beta (IL-1ß) or tumor necrosis factor alpha (TNF-α) alone or in combination with TRPA1/V1 agonist allyl isothiocyanate (AITC, 3 and 10 µM), followed by analysis of calcium flux and the expression of inflammation mediators (RT-qPCR/ELISA) and matrix constituents (RT-qPCR). The matrix structure and composition in caudal motion segments from TRPA1 and TRPV1 wild-type (WT) and knock-out (KO) mice was visualized by FAST staining. Gene expression of other TRP channels (A1, C1, C3, C6, V1, V2, V4, V6, M2, M7, M8) was also tested in cytokine-treated cells. TRPA1 was expressed in fetal IVD cells, 20% of degenerated IVDs, but not in healthy mature IVDs. TRPA1 expression was not detectable in untreated cells and it increased upon cytokine treatment, while TRPV1 was expressed and concomitantly reduced. In inflamed IVD cells, 10 µM AITC activated calcium flux, induced gene expression of IL-8, and reduced disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and collagen 1A1, possibly via upregulated TRPA1. TRPA1 KO in mice was associated with signs of degeneration in the nucleus pulposus and the vertebral growth plate, whereas TRPV1 KO did not show profound changes. Cytokine treatment also affected the gene expression of TRPV2 (increase), TRPV4 (increase), and TRPC6 (decrease). TRPA1 might be expressed in developing IVD, downregulated during its maturation, and upregulated again in degenerative disc disease, participating in matrix homeostasis. However, follow-up studies with larger sample sizes are needed to fully elucidate the role of TRPA1 and other TRP channels in degenerative disc disease.

p38 MAPK Facilitates Crosstalk Between Endoplasmic Reticulum Stress and IL-6 Release in the Intervertebral Disc.

Degenerative disc disease is associated with increased expression of pro-inflammatory cytokines in the intervertebral disc (IVD). However, it is not completely clear how inflammation arises in the IVD and which cellular compartments are involved in this process. Recently, the endoplasmic reticulum (ER) has emerged as a possible modulator of inflammation in age-related disorders. In addition, ER stress has been associated with the microenvironment of degenerated IVDs. Therefore, the aim of this study was to analyze the effects of ER stress on inflammatory responses in degenerated human IVDs and associated molecular mechanisms. Gene expression of ER stress marker GRP78 and pro-inflammatory cytokines IL-6, IL-8, IL-1ß, and TNF-α was analyzed in human surgical IVD samples (n = 51, Pfirrmann grade 2-5). The expression of GRP78 positively correlated with the degeneration grade in lumbar IVDs and IL-6, but not with IL-1ß and TNF-α. Another set of human surgical IVD samples (n = 25) was used to prepare primary cell cultures. ER stress inducer thapsigargin (Tg, 100 and 500 nM) activated gene and protein expression of IL-6 and induced phosphorylation of p38 MAPK. Both inhibition of p38 MAPK by SB203580 (10 µM) and knockdown of ER stress effector CCAAT-enhancer-binding protein homologous protein (CHOP) reduced gene and protein expression of IL-6 in Tg-treated cells. Furthermore, the effects of an inflammatory microenvironment on ER stress were tested. TNF-α (5 and 10 ng/mL) did not activate ER stress, while IL-1ß (5 and 10 ng/mL) activated gene and protein expression of GRP78, but did not influence [Ca2+]i flux and expression of CHOP, indicating that pro-inflammatory cytokines alone may not induce ER stress in vivo. This study showed that IL-6 release in the IVD can be initiated following ER stress and that ER stress mediates IL-6 release through p38 MAPK and CHOP. Therapeutic targeting of ER stress response may reduce the consequences of the harsh microenvironment in degenerated IVD.

The Pathobiology of the Meniscus: A Comparison Between the Human and Dog.

Serious knee pain and related disability have an annual prevalence of approximately 25% on those over the age of 55 years. As curative treatments for the common knee problems are not available to date, knee pathologies typically progress and often lead to osteoarthritis (OA). While the roles that the meniscus plays in knee biomechanics are well characterized, biological mechanisms underlying meniscus pathophysiology and roles in knee pain and OA progression are not fully clear. Experimental treatments for knee disorders that are successful in animal models often produce unsatisfactory results in humans due to species differences or the inability to fully replicate disease progression in experimental animals. The use of animals with spontaneous knee pathologies, such as dogs, can significantly help addressing this issue. As microscopic and macroscopic anatomy of the canine and human menisci are similar, spontaneous meniscal pathologies in canine patients are thought to be highly relevant for translational medicine. However, it is not clear whether the biomolecular mechanisms of pain, degradation of extracellular matrix, and inflammatory responses are species dependent. The aims of this review are (1) to provide an overview of the anatomy, physiology, and pathology of the human and canine meniscus, (2) to compare the known signaling pathways involved in spontaneous meniscus pathology between both species, and (3) to assess the relevance of dogs with spontaneous meniscal pathology as a translational model. Understanding these mechanisms in human and canine meniscus can help to advance diagnostic and therapeutic strategies for painful knee disorders and improve clinical decision making.

The potential of CRISPR/Cas9 genome editing for the study and treatment of intervertebral disc pathologies.

The CRISPR/Cas9 system has emerged as a powerful tool for mammalian genome engineering. In basic and translational intervertebral disc (IVD) research, this technique has remarkable potential to answer fundamental questions on pathway interactions, to simulate IVD pathologies, and to promote drug development. Furthermore, the precisely targeted CRISPR/Cas9 gene therapy holds promise for the effective and targeted treatment of degenerative disc disease and low back pain. In this perspective, we provide an overview of recent CRISPR/Cas9 advances stemming from/with transferability to IVD research, outline possible treatment approaches for degenerative disc disease, and discuss current limitations that may hinder clinical translation.

An Inflammatory Nucleus Pulposus Tissue Culture Model to Test Molecular Regenerative Therapies: Validation with Epigallocatechin 3-Gallate.

Organ cultures are practical tools to investigate regenerative strategies for the intervertebral disc. However, most existing organ culture systems induce severe tissue degradation with only limited representation of the in vivo processes. The objective of this study was to develop a space- and cost-efficient tissue culture model, which represents degenerative processes of the nucleus pulposus (NP). Intact bovine NPs were cultured in a previously developed system using Dyneema jackets. Degenerative changes in the NP tissue were induced either by the direct injection of chondroitinase ABC (1-20 U/mL) or by the diffusion of interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) (both 100 ng/mL) from the culture media. Extracellular matrix composition (collagens, proteoglycans, water, and DNA) and the expression of inflammatory and catabolic genes were analyzed. The anti-inflammatory and anti-catabolic compound epigallocatechin 3-gallate (EGCG, 10 µM) was employed to assess the relevance of the degenerative NP model. Although a single injection of chondroitinase ABC reduced the proteoglycan content in the NPs, it did not activate cellular responses. On the other hand, IL-1ß and TNF-α significantly increased the mRNA expression of inflammatory mediators IL-6, IL-8, inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (PTGS2) and matrix metalloproteinases (MMP1, MMP3, and MMP13). The cytokine-induced gene expression in the NPs was ameliorated with EGCG. This study provides a proof of concept that inflammatory NP cultures, with appropriate containment, can be useful for the discovery and evaluation of molecular therapeutic strategies against early degenerative disc disease.

Stability of (-)-epigallocatechin gallate and its activity in liquid formulations and delivery systems.

(-)-Epigallocatechin gallate (EGCG) has become a popular disease-preventive supplement worldwide because it may aid in slowing down the onset of age-related diseases such as cancer, diabetes and tissue degeneration. As largely demonstrated in cell culture studies, EGCG possesses antioxidant properties and exhibits favorable effects on gene expression, signal transduction and other cell functions. However, only limited effects have been observed in experimental animals and human epidemiological studies. The inconsistency between the biological activity of EGCG in cell cultures and in vivo can be attributed to its low stability, which not only decreases its bioavailability but also leads to the formation of degradation products and prooxidant molecules with possible side-effects. Understanding EGCG degradation kinetics in solution and in vivo is crucial for its successful clinical application. Ambient conditions (pH, temperature, oxygen) can either enhance or decrease the stability of EGCG, thus influencing its biological activity. Usage of stabilizers and/or encapsulation of EGCG into particulate systems such as nanoparticles or microparticles can significantly increase its stability. In this review, the effects of ambient conditions, stabilizers and encapsulation systems on EGCG stability, activity and degradation rate are illustrated.

The Natural Polyphenol Epigallocatechin Gallate Protects Intervertebral Disc Cells from Oxidative Stress.

Oxidative stress-related phenotypic changes and a decline in the number of viable cells are crucial contributors to intervertebral disc degeneration. The polyphenol epigallocatechin 3-gallate (EGCG) can interfere with painful disc degeneration by reducing inflammation, catabolism, and pain. In this study, we hypothesized that EGCG furthermore protects against senescence and/or cell death, induced by oxidative stress. Sublethal and lethal oxidative stress were induced in primary human intervertebral disc cells with H2O2 (total n = 36). Under sublethal conditions, the effects of EGCG on p53-p21 activation, proliferative capacity, and accumulation of senescence-associated ß-galactosidase were tested. Further, the effects of EGCG on mitochondria depolarization and cell viability were analyzed in lethal oxidative stress. The inhibitor LY249002 was applied to investigate the PI3K/Akt pathway. EGCG inhibited accumulation of senescence-associated ß-galactosidase but did not affect the loss of proliferative capacity, suggesting that EGCG did not fully neutralize exogenous radicals. Furthermore, EGCG increased the survival of IVD cells in lethal oxidative stress via activation of prosurvival PI3K/Akt and protection of mitochondria. We demonstrated that EGCG not only inhibits inflammation but also can enhance the survival of disc cells in oxidative stress, which makes it a suitable candidate for the development of novel therapies targeting disc degeneration.

The effect of serotonin-noradrenaline reuptake inhibitor duloxetine on the intervertebral disk-related radiculopathy in rats.

INTRODUCTION: Neuropathic pain, commonly related to intervertebral disk (IVD) degeneration, responds poorly to standard pain treatments. Serotonin-noradrenaline reuptake inhibitors (SNRIs) have been reported to reduce neuropathic pain; however their effect on radiculopathy induced by lumbar disk herniation remains unclear. The aim of this study was to investigate the effect of SNRI duloxetine in rat model of IVD-related neuropathic pain. MATERIALS AND METHODS: Effects of SNRI duloxetine were tested in Sprague-Dawley rats (n = 135). Neuropathic pain was induced by applying autologous nucleus pulposus (NP) on the left L5 dorsal root ganglion (DRG). Duloxetine in concentrations 0.4 mg/kg (low dose) and 1.2 mg/kg (high dose) or saline were administered orally for 10 days. Von Frey test was carried out on post-operative days 2, 7, 14, 21, and 28 to test pain sensitivity. Immunohistochemistry of L5 DRG and L5 segment of spinal cord (SC) was performed on days 7 and 21 to examine expressions of tumor necrosis factor alpha (TNF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and ionized calcium-binding adapter molecule 1 (Iba1). On days 14, 21, and 28, expressions of TNF in DRG as well as NGF and BDNF in SC were tested by immunoblotting. Sham-operated rats and naive rats were used as controls. RESULTS: Duloxetine in both concentrations significantly improved pain threshold from postoperative day 21 onward, compared to the NP + saline group (p < 0.05). High-dose duloxetine significantly inhibited the expression of TNF in DRG (day 28, p < 0.05). Both duloxetine concentrations reduced the expression of NGF in SC (day 21, p < 0.05), but the expression of BDNF remained unchanged. CONCLUSION: SNRI duloxetine inhibited neuropathic pain in rats possibly via down-regulating TNF, NGF, and microglia activation. We conclude that duloxetine, and most likely other SNRIs, may be used for the management of lumbar neuropathic pain.

Expression and regulation of toll-like receptors (TLRs) in human intervertebral disc cells.

PURPOSE: Although inflammatory processes play an essential role in painful intervertebral disc (IVD) degeneration, the underlying regulatory mechanisms are not well understood. This study was designed to investigate the expression, regulation and importance of specific toll-like receptors (TLRs)--which have been shown to play an essential role e.g. in osteoarthritis--during degenerative disc disease. METHODS: The expression of TLRs in human IVDs was measured in isolated cells as well as in normal or degenerated IVD tissue. The role of IL-1ß or TNF-α in regulating TLRs (expression/activation) as well as in regulating activity of down-stream pathways (NF-κB) and expression of inflammation-related genes (IL-6, IL-8, HSP60, HSP70, HMGB1) was analyzed. RESULTS: Expression of TLR1/2/3/4/5/6/9/10 was detected in isolated human IVD cells, with TLR1/2/4/6 being dependent on the degree of IVD degeneration. Stimulation with IL-1ß or TNF-α moderately increased TLR1/TLR4 mRNA expression (TNF-α only), and strongly increased TLR2 mRNA expression (IL-1ß/TNF-α), with the latter being confirmed on the protein level. Stimulation with IL-1ß, TNF-α or Pam3CSK4 (a TLR2-ligand) stimulated IL-6 and IL-8, which was inhibited by a TLR2 neutralizing antibody for Pam3CSK4; IL-1ß and TNF-α caused NF-κB activation. HSP60, HSP70 and HMGB1 did not increase IL-6 or IL-8 and were not regulated by IL-1ß/TNF-α. CONCLUSION: We provide evidence that several TLRs are expressed in human IVD cells, with TLR2 possibly playing the most crucial role. As TLRs mediate catabolic and inflammatory processes, increased levels of TLRs may lead to aggravated disc degeneration, chronic inflammation and pain development. Especially with the identification of more endogenous TLR ligands, targeting these receptors may hold therapeutic promise.

Analysis of nuclear nestin localization in cell lines derived from neurogenic tumors.

Nestin is a class VI intermediate filament protein expressed in the cytoplasm of stem and progenitor cells in the mammalian CNS during development. In adults, nestin is present only in a small subset of cells and tissues, including the subventricular zone of the adult mammalian brain, where neurogenesis occurs. Nestin expression has also been detected under such pathological conditions as ischemia, inflammation, and brain injury, as well as in various types of human solid tumors and their corresponding cell lines. Furthermore, nestin was recently found in the nuclei of glioblastoma, neuroblastoma, and angiosarcoma cells and it was proved to interact directly with the nuclear DNA in neuroblastoma cells. Here, we perform the first study of the intracellular distribution of nestin in cell lines derived from neurogenic tumors. Using immunodetection methods, we examined nestin expression in tumor-derived cell lines obtained from 11 patients with neuroblastoma, medulloblastoma, or glioblastoma multiforme. Besides its standard cytoplasmic localization, nestin was present in the nuclei of two neuroblastoma cell lines and one medulloblastoma cell line. Nestin was only present in the nuclei of cells with diffuse cytoplasmic staining for this protein, and the proportion of cells positive for nestin in nuclei, as well as the intensity of staining, varied. The presence of nestin in the nuclei was confirmed by both transmission electron microscopy and Western blotting. Our results indicate that the presence of nestin in the nuclei of tumor cells is not very rare, especially under in vitro conditions.