The NFATc1/P2X7 receptor relationship in human intervertebral disc cells.

A comprehensive understanding of the molecules that play key roles in the physiological and pathological homeostasis of the human intervertebral disc (IVD) remains challenging, as does the development of new therapeutic treatments. We recently found a positive correlation between IVD degeneration (IDD) and P2X7 receptor (P2X7R) expression increases both in the cytoplasm and in the nucleus. Using immunocytochemistry, reverse transcription PCR (RT-PCR), overexpression, and chromatin immunoprecipitation, we found that NFATc1 and hypoxia-inducible factor-1α (HIF-1α) are critical regulators of P2X7R. Both transcription factors are recruited at the promoter of the P2RX7 gene and involved in its positive and negative regulation, respectively. Furthermore, using the proximity ligation assay, we revealed that P2X7R and NFATc1 form a molecular complex and that P2X7R is closely associated with lamin A/C, a major component of the nuclear lamina. Collectively, our study identifies, for the first time, P2X7R and NFATc1 as markers of IVD degeneration and demonstrates that both NFATc1 and lamin A/C are interaction partners of P2X7R.

[Effects of Oxidative Stress on Mitochondrial Functions and Intervertebral Disc Cells]. / 氧化应激对线粒体功能及椎间盘细胞的影响.

Intervertebral disc degeneration is widely recognized as one of the main causes of lower back pain. Intervertebral disc cells are the primary cellular components of the discs, responsible for synthesizing and secreting collagen and proteoglycans to maintain the structural and functional stability of the discs. Additionally, intervertebral disc cells are involved in maintaining the nutritional and metabolic balance, as well as exerting antioxidant and anti-inflammatory effects within the intervertebral discs. Consequently, intervertebral disc cells play a crucial role in the process of disc degeneration. When these cells are exposed to oxidative stress, mitochondria can be damaged, which may disrupt normal cellular function and accelerate degenerative changes. Mitochondria serve as the powerhouse of cells, being the primary energy-producing organelles that control a number of vital processes, such as cell death. On the other hand, mitochondrial dysfunction may be associated with various degenerative pathophysiological conditions. Moreover, mitochondria are the key site for oxidation-reduction reactions. Excessive oxidative stress and reactive oxygen species can negatively impact on mitochondrial function, potentially leading to mitochondrial damage and impaired functionality. These factors, in turn, triggers inflammatory responses, mitochondrial DNA damage, and cell apoptosis, playing a significant role in the pathological processes of intervertebral disc cell degeneration. This review is focused on exploring the impact of oxidative stress and reactive oxygen species on mitochondria and the crucial roles played by oxidative stress and reactive oxygen species in the pathological processes of intervertebral disc cells. In addition, we discussed current cutting-edge treatments and introduced the use of mitochondrial antioxidants and protectants as a potential method to slow down oxidative stress in the treatment of disc degeneration.

Wharton's jelly-derived multifunctional hydrogels: New tools to promote intervertebral disc regeneration in vitro and ex vivo.

The degeneration of intervertebral disc (IVD) is a disease of the entire joint between two vertebrae in the spine caused by loss of extracellular matrix (ECM) integrity, to date with no cure. The various regenerative approaches proposed so far have led to very limited successes. An emerging opportunity arises from the use of decellularized ECM as a scaffolding material that, directly or in combination with other materials, has greatly facilitated the advancement of tissue engineering. Here we focused on the decellularized matrix obtained from human umbilical cord Wharton's jelly (DWJ) which retains several structural and bioactive molecules very similar to those of the IVD ECM. However, being a viscous gel, DWJ has limited ability to retain ordered structural features when considered as architecture scaffold. To overcome this limitation, we produced DWJ-based multifunctional hydrogels, in the form of 3D millicylinders containing different percentages of alginate, a seaweed-derived polysaccharide, and gelatin, denatured collagen, which may impart mechanical integrity to the biologically active DWJ. The developed protocol, based on a freezing step, leads to the consolidation of the entire polymeric dispersion mixture, followed by an ionic gelation step and a freeze-drying process. Finally, a porous, stable, easily storable, and suitable matrix for ex vivo experiments was obtained. The properties of the millicylinders (Wharton's jelly millicylinders [WJMs]) were then tested in culture of degenerated IVD cells isolated from disc tissues of patients undergoing surgical discectomy. We found that WJMs with the highest percentage of DWJ were effective in supporting cell migration, restoration of the IVD phenotype (increased expression of Collagen type 2, aggrecan, Sox9 and FOXO3a), anti-inflammatory action, and stem cell activity of resident progenitor/notochordal cells (increased number of CD24 positive cells). We are confident that the DWJ-based formulations proposed here can provide adequate stimuli to the cells present in the degenerated IVD to restart the anabolic machinery.

Unorthodox localization of P2X7 receptor in subcellular compartments of skeletal system cells.

Identifying the subcellular localization of a protein within a cell is often an essential step in understanding its function. The main objective of this report was to determine the presence of the P2X7 receptor (P2X7R) in healthy human cells of skeletal system, specifically osteoblasts (OBs), chondrocytes (Chs) and intervertebral disc (IVD) cells. This receptor is a member of the ATP-gated ion channel family, known to be a main sensor of extracellular ATP, the prototype of the danger signal released at sites of tissue damage, and a ubiquitous player in inflammation and cancer, including bone and cartilaginous tissues. Despite overwhelming data supporting a role in immune cell responses and tumor growth and progression, a complete picture of the pathophysiological functions of P2X7R, especially when expressed by non-immune cells, is lacking. Here we show that human wild-type P2X7R (P2X7A) was expressed in different samples of human osteoblasts, chondrocytes and intervertebral disc cells. By fluorescence microscopy (LM) and immunogold transmission electron microscopy we localized P2X7R not only in the canonical sites (plasma membrane and cytoplasm), but also in the nucleus of all the 3 cell types, especially IVD cells and OBs. P2X7R mitochondrial immunoreactivity was predominantly detected in OBs and IVD cells, but not in Chs. Evidence of subcellular localization of P2X7R may help to i. understand the participation of P2X7R in as yet unidentified signaling pathways in the joint and bone microenvironment, ii. identify pathologies associated with P2X7R mislocalization and iii. design specific targeted therapies.

Intervertebral disc cell chondroptosis elicits neutrophil response in Staphylococcus aureus spondylodiscitis.

To understand the pathophysiology of spondylodiscitis due to Staphylococcus aureus, an emerging infectious disease of the intervertebral disc (IVD) and vertebral body with a high complication rate, we combined clinical insights and experimental approaches. Clinical data and histological material of nine patients suffering from S. aureus spondylodiscitis were retrospectively collected at a single center. To mirror the clinical findings experimentally, we developed a novel porcine ex vivo model mimicking acute S. aureus spondylodiscitis and assessed the interaction between S. aureus and IVD cells within their native environment. In addition, the inflammatory features underlying this interaction were assessed in primary human IVD cells. Finally, mirroring the clinical findings, we assessed primary human neutrophils for their ability to respond to secreted inflammatory modulators of IVD cells upon the S. aureus challenge. Acute S. aureus spondylodiscitis in patients was characterized by tissue necrosis and neutrophil infiltration. Additionally, the presence of empty IVD cells' lacunae was observed. This was mirrored in the ex vivo porcine model, where S. aureus induced extensive IVD cell death, leading to empty lacunae. Concomitant engagement of the apoptotic and pyroptotic cell death pathways was observed in primary human IVD cells, resulting in cytokine release. Among the released cytokines, functionally intact neutrophil-priming as well as broad pro- and anti-inflammatory cytokines which are known for their involvement in IVD degeneration were found. In patients as well as ex vivo in a novel porcine model, S. aureus IVD infection caused IVD cell death, resulting in empty lacunae, which was accompanied by the release of inflammatory markers and recruitment of neutrophils. These findings offer valuable insights into the important role of inflammatory IVD cell death during spondylodiscitis and potential future therapeutic approaches.

The effects of rivaroxaban, an oral anticoagulant, on human IVD primary cultures.

Introduction: The present study aimed to investigate the potential effects of rivaroxaban, an oral anticoagulant that inhibits the effects of factor Xa, on intact intervertebral disc tissue cells and the extracellular matrix (ECM). Material and methods: Rivaroxaban was applied to primary human cell cultures prepared from tissues of the intervertebral disc. Comparative molecular analyses were performed on non-drug-treated control group samples. Descriptive statistics were presented as the mean ± standard deviation. An analysis of variance test was performed to determine whether there were significant differences in the mean across the groups. When differences across groups were observed, Tukey's honestly significant difference post-hoc test was used for multiple pairwise comparisons. The significance of the obtained data was determined statistically. The α significance value was < 0.05. Results: The cells in the control group and in the rivaroxaban-treated group were viable, healthy, and proliferated (p < 0.05). However, the expression levels of the chondroadherin gene (CHAD), cartilage oligo matrix protein (COMP), matrix metalloproteinase (MMP)-13, and MMP-19 genes were changed (p < 0.05). Conclusions: Although rivaroxaban does not suppress cell proliferation due to morphological, biological, and biochemical changes in the intervertebral disc tissue, it may change the expression of genes that are related to ECM maintenance.

Genetic Predictors of Early-Onset Spinal Intervertebral Disc Degeneration: Part Two of Two.

Understanding genetic indicators is a fundamental aspect to characterizing the pathophysiology of chronic diseases such as intervertebral disc degeneration (IVDD). In our previous spinal genetics review, we characterized some more common genetic influencers in the context of IVDD. In this second part of our two-part comprehensive spinal genetics review, we characterize the more infrequently studied genes that have pathophysiological relevance. In doing so, we aim to expand upon the current gene-library for IVDD. The genes of interest include: asporin, cartilage intermediate layer protein, insulin-like growth factor 1 receptor, matrix metallopeptidase 9, and thrombospondin 2. Findings show that these genetic indicators have trends and polymorphisms that may have causal associations with the manifestation of IVDD. However, there is a narrow selection of studies that use genetic indicators to describe correlations to the severity and longevity of the pathology. Nevertheless, with the continued identification of risk genes involved with IVDD, the possibilities for refined models of gene therapies can be established for future treatment trials.

Extracellular vesicles from human mesenchymal stem cells expedite chondrogenesis in 3D human degenerative disc cell cultures.

BACKGROUND: Extracellular vesicles (EVs) from human mesenchymal stem cells (hMSCs) are known to be mediators of intercellular communication and have been suggested as possible therapeutic agents in many diseases. Their potential use in intervertebral disc (IVD) degeneration associated with low back pain (LBP) is yet to be explored. Since LBP affects more than 85% of the western population resulting in high socioeconomic consequences, there is a demand for exploring new and possibly mini-invasive treatment alternatives. In this study, the effect of hMSC-derived small EVs (sEVs) on degenerated disc cells (DCs) isolated from patients with degenerative discs and chronic LBP was investigated in a 3D in vitro model. METHODS: hMSCs were isolated from bone marrow aspirate, and EVs were isolated from conditioned media of the hMSCs by differential centrifugation and filtration. 3D pellet cultures of DCs were stimulated with the sEVs at 5 × 1010 vesicles/ml concentration for 28 days and compared to control. The pellets were harvested at days 7, 14, and 28 and evaluated for cell proliferation, viability, ECM production, apoptotic activity, chondrogenesis, and cytokine secretions. RESULTS: The findings demonstrated that treatment with sEVs from hMSCs resulted in more than 50% increase in cell proliferation and decrease in cellular apoptosis in degenerated DCs from this patient group. ECM production was also observed as early as in day 7 and was more than three times higher in the sEV-treated DC pellets compared to control cultures. Further, sEV treatment suppressed secretion of MMP-1 in the DCs. CONCLUSION: hMSC-derived sEVs improved cell viability and expedited chondrogenesis in DCs from degenerated IVDs. These findings open up for new tissue regeneration treatment strategies to be developed for degenerative disorders of the spine.

Extracellular Matrix From Decellularized Wharton's Jelly Improves the Behavior of Cells From Degenerated Intervertebral Disc.

Regenerative therapies for intervertebral disc (IVD) injuries are currently a major challenge that is addressed in different ways by scientists working in this field. Extracellular matrix (ECM) deriving from decellularized non-autologous tissues has been established as a biomaterial with remarkable regenerative capacity and its potential as a therapeutic agent is rising. In the present study, we investigated the potential of decellularized Wharton's jelly matrix (DWJM) from human umbilical cord to act as an ECM-based scaffold for IVD cell culturing. An efficient detergent-enzymatic treatment (DET) was used to produce DWJM maintaining its native microarchitecture. Afterward, immunofluorescence, biochemical assays and electron microscopy analysis showed that DWJM was able to produce sizeable 3D cell aggregates, when combined with human mesenchymal stromal cells isolated from WJ (MSCs) and IVD cells. These latter cells are characterized by the loss of their chondrocyte-like phenotype since they have been isolated from degenerated IVD and in vitro expanded to further de-differentiate. While the effect exerted by DWJM on MSCs was essentially the induction of proliferation, conversely, on IVD cells the DWJM promoted cell differentiation toward a discogenic phenotype. Notably, for the first time, the ability of DWJM to improve the degenerated phenotype of human IVD cells was demonstrated, showing that the mere presence of the matrix maintained the viability of the cells, and positively affected the expression of critical regulators of IVD homeostasis, such as SOX2, SOX9, and TRPS1 transcription factors at specific culture time. Our data are in line with the hypothesis that the strengthening of cell properties in terms of viability and expression of specific proteins at precise times represents an important condition in the perspective of guiding the recovery of cellular functionality and triggering regenerative potential. Currently, there are no definitive surgical or pharmacological treatments for IVD degeneration (IDD) able to restore the disc structure and function. Therefore, the potential of DWJM to revert degenerated IVD cells could be exploited in the next future an ECM-based intradiscal injectable therapeutic.

Actions of Prostaglandins on Human Nucleus Pulposus Metabolism Inferred by Cyclooxygenase 2 Inhibition of Cytokine Activated Cells.

OBJECTIVE: Low back pain is frequently treated with nonsteroidal anti-inflammatory drugs (NSAIDs), but little is known about intervertebral disc metabolism of the prostaglandins that are diminished by these drugs. Hence, this study aimed at delineating prostaglandin actions in cytokine activated disc cells by comparing the response of nucleus pulposus (NP) cells to the pro-inflammatory cytokine interleukin (IL)-1ß with and without cyclooxygenase 2 (COX-2) inhibition. METHODS: NP cells cultured in alginate beads were activated with IL-1ß ± the COX-2 inhibitor Sc-58125. Media harvested from cultured cells were analyzed for prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2α), IL-6, and matrix metalloproteinase (MMP)-3 by enzymelinked immunosorbent assay and nitric oxide by Griess Reaction. Gene expression along with proteoglycan, collagen, and total protein synthesis were also measured. RESULTS: IL-1ß increased culture media PGE2 and PGF2α, but decreased proteoglycan and collagen syntheses as well as mRNA expression of the matrix genes aggrecan, versican, collagen I, and collagen II. COX-2 inhibition partially rescued proteoglycan and collagen syntheses and collagen I mRNA, but decreased collagen II mRNA IL-1ß activated NP cells. COX-2 inhibition initially enhanced and subsequently reduced IL-1ß induced inducible nitric oxide synthase, without altering medium nitrite. IL-1ß induction of MMP-3 mRNA was increased by COX-2 inhibition at 24 and 48 hours. CONCLUSION: COX-2 inhibition alters the response of NP cells to IL-1ß, suggesting IL-1ß action on disc cells is mediated at least in part through COX-2 and its prostaglandins. COX-2 inhibition produces minimal effects on several key catabolic mediators, with the exception of MMP-3. Blocking COX-2 might be beneficial for maintaining disc matrix since it provides an overall rescue of IL-1 induced loss of matrix protein synthesis.

Exogenous Stimulation of Human Intervertebral Disc Cells in 3-Dimensional Alginate Bead Culture With BMP2 and L51P: Cytocompatibility and Effects on Cell Phenotype.

OBJECTIVE: Spinal fusion surgery is a common treatment modality for various pathologic conditions of the spine. The bone morphogenetic protein 2 (BMP2) analogue L51P acts as a general inhibitor of BMP antagonists, whereas it shows a weak affinity for BMP type I receptor. It is suggested that L51P applied in bone disorders might prevent side effects of highly concentrated BMP dosage applications in the order of milligrams. The objective of this study was to investigate the effects of L51P and BMP2 on intervertebral disc cells (IVDCs), i.e. on nucleus pulposus cells, on annulus fibrosus cells (AFCs), and on cartilaginous endplate cells (CEPCs), respectively, in 3-dimensional (3D) culture. METHODS: Low-passage primary IVDCs were cultured in 3D alginate bead culture and exposed to 100-ng/mL BMP2 and/or L51P for 21 days. Here, we analyzed glycosaminoglycan (GAG) and DNA content and further performed gene expression analysis for major matrix genes. RESULTS: AFCs and cartilaginous CEPCs stimulated with each 100-ng/mL L51P and BMP2, showed a significant upregulation in GAG (AFCs: p = 0.00347 and CEPCs: p = 0.0115) and DNA production (AFCs: p = 0.0182 and CEPCs: p = 0.0179) compared to control. CONCLUSION: These results allow first insights into the behavior of IVDCs upon L51P stimulation.

BMP-3 Promotes Matrix Production in Co-cultured Stem Cells and Disc Cells from Low Back Pain Patients.

Low back pain is one of the most common disorders and believed to be due to intervertebral disc degeneration. Transplantation of human mesenchymal stem cells (hMSCs) is suggested as potential treatment option. Bone morphogenetic growth factor 3 (BMP-3) promotes chondrogenesis and is proven effective in enhancing chondrogenesis in hMSCs pretreated with interleukin-1 beta (IL-1ß) in hydrogel model. Three-dimensional co-cultures of hMSCs and disc cells (DCs) have previously been demonstrated to result in increased proteoglycan production. The aim was to study the effects of BMP-3 on hMSCs, DCs, as well as hMSCs and DCs in co-culture in a pellet system, both as single treatment and after pretreatment of IL-1ß. Cell pellet cultures with hMSCs, DCs, and co-culture (1:1 ratio) were performed and stimulated with BMP-3 at 1 or 10 ng/mL concentrations. For pretreatment (PRE-T), cell pellets were first stimulated with IL-1ß, for 24 h, and then BMP-3. The pellets were harvested on day 7, 14, and 28. Results demonstrated that BMP-3 stimulation at 10 ng/mL promoted cell viability, proteoglycan accumulation, as well as chondrogenesis in all pellet groups compared to 1 ng/mL. Cellular proliferation and chondrogenic differentiation of hMSCs were best promoted by PRE-T at 10 ng/mL, whereas BMP-3 best enhanced chondrogenesis in DC and co-culture pellets at the same concentration. Impact Statement Current therapies for low back pain include pain modulation and surgery, which do not tackle the underlying cellular mechanisms of the degenerated intervertebral discs (IVDs). To develop an understanding of the degeneration process and to further reverse its course, the effects of growth factor and cytokine on the native cells of the IVDs were investigated, revealing the potency of bone morphogenetic growth factor 3 on disc cells (DCs) and combined culture of mesenchymal stem cells and DCs. These results may impact future strategies in development of cell therapies that could directly influence the IVD degeneration process, which might alter the treatment models of today.

Reciprocal Regulation of TRPS1 and miR-221 in Intervertebral Disc Cells.

Intervertebral disc (IVD), a moderately moving joint located between the vertebrae, has a limited capacity for self-repair, and treating injured intervertebral discs remains a major challenge. The development of innovative therapies to reverse IVD degeneration relies primarily on the discovery of key molecules that, occupying critical points of regulatory mechanisms, can be proposed as potential intradiscal injectable biological agents. This study aimed to elucidate the underlying mechanism of the reciprocal regulation of two genes differently involved in IVD homeostasis, the miR-221 microRNA and the TRPS1 transcription factor. Human lumbar IVD tissue samples and IVD primary cells were used to specifically evaluate gene expression and perform functional analysis including the luciferase gene reporter assay, chromatin immunoprecipitation, cell transfection with hTRPS1 overexpression vector and antagomiR-221. A high-level expression of TRPS1 was significantly associated with a lower pathological stage, and TRPS1 overexpression strongly decreased miR-221 expression, while increasing the chondrogenic phenotype and markers of antioxidant defense and stemness. Additionally, TRPS1 was able to repress miR-221 expression by associating with its promoter and miR-221 negatively control TRPS1 expression by targeting the TRPS1-3'UTR gene. As a whole, these results suggest that, in IVD cells, a double-negative feedback loop between a potent chondrogenic differentiation suppressor (miR-221) and a regulator of axial skeleton development (TRPS1) exists. Our hypothesis is that the hostile degenerated IVD microenvironment may be counteracted by regenerative/reparative strategies aimed at maintaining or stimulating high levels of TRPS1 expression through inhibition of one of its negative regulators such as miR-221.

Pathological Effects of Cortisol on Intervertebral Disc Cells and Mesenchymal Stem Cells from Lower Back Pain Patients.

In western countries, lower back pain (LBP) is one of the most common disorders, experienced by more than 80% of the population. Chronic LBP due to disc degeneration has been linked to ongoing inflammatory processes in the disc and endplates. Pain effects the body in different ways, inducing a general stress response in which the body responds by releasing the stress hormone cortisol. Little is known about the impact of pain-induced stress on the progression of disc degeneration. Thus, the effects of cortisol on disc cells (DCs) and human mesenchymal stem cells (hMSCs) were explored in vitro with the objective of investigating the repercussions of cortisol on these cell types involved in de- and regenerative mechanisms of the disc. DC and hMSC pellet cultures were exposed to cortisol at two concentrations (150 and 300 ng/mL) for 28 days to simulate pain-induced stress. Cell viability, histological staining, and GAG DNA, along with apo-ptotic assays were conducted. Detection of OCT4, SOX9, IL-1R, and CXCR2 expressions was performed by immunohistochemistry. With cortisol treatment, restricted cell proliferation and less GAG production in both DCs and hMSCs were observed. Suppression of the differentiation and immunomodulatory efficacy of hMSCs was also detected. Moreover, elevated expressions of IL-1R and CXCR2 were detected in both cell types. To conclude, constant exposure to cortisol even at a physiological level enhanced pathological cellular processes in both DCs and hMSCs, which further jeopardized chondrogenesis. This suggests that cortisol resulting from pain-induced stress is a contributing component of intervertebral disc degeneration and may negatively affect regenerative attempts of the disc.

Investigation of the Effect of Secreted Factors from Mesenchymal Stem Cells on Disc Cells from Degenerated Discs.

Low back pain is experienced by a large number of people in western countries and may be caused and influenced by many different pathologies and psychosocial factors including disc degeneration. Disc degeneration involves the increased expression of proinflammatory cytokines and matrix metalloproteinases (MMPs) in the disc environment, which leads to the loss of extracellular matrix (ECM) and the viability of the native disc cells (DCs). Treatment approaches using growth factors and cell therapy have been proposed due to the compelling results that growth factors and mesenchymal stem cells (MSCs) can influence the degenerated discs. The aim of this study was to investigate the effects of conditioned media (CM) from human MSCs (hMSCs) and connective tissue growth factor (CTGF) and TGF-ß on disc cells, and hMSCs isolated from patients with degenerative discs and severe low back pain. The aim was also to examine the constituents of CM in order to study the peptides that could bring about intervertebral disc (IVD) regeneration. DCs and hMSC pellets (approx.. 200,000 cells) were cultured and stimulated with hMSC-derived CM or CTGF and TGF-ß over 28 days. The effects of CM and CTGF on DCs and hMSCs were assessed via cell viability, proteoglycan production, the expression of ECM proteins, and chondrogenesis in 3D pellet culture. To identify the constituents of CM, CM was analyzed with tandem mass spectrometry. The findings indicate that CM enhanced the cellular viability and ECM production of DCs while CTGF and the control exhibited nonsignificant differences. The same was observed in the hMSC group. Mass spectrometry analysis of CM identified >700 peptides, 129 of which showed a relative abundance of ≥2 (CTGF among them). The results suggest that CM holds potential to counter the progression of disc degeneration, likely resulting from the combination of all the substances released by the hMSCs. The soluble factors released belong to different peptide families. The precise mechanism underlying the regenerative effect needs to be investigated further, prior to incorporating peptides in the development of new treatment strategies for low back pain that is potentially caused by IVD degeneration.

MicroRNA-221 silencing attenuates the degenerated phenotype of intervertebral disc cells.

The aim of this study was to investigate the role of an antichondrogenic factor, MIR221 (miR-221), in intervertebral disc degeneration (IDD), and provide basic information for the development of a therapeutic strategy for the disc repair based on specific nucleic acid based drugs, such as miR-221 silencing. We established a relatively quick protocol to minimize artifacts from extended in vitro culture, without selecting the different types of cells from intervertebral disc (IVD) or completely disrupting extracellular matrix (ECM), but by using the whole cell population with a part of resident ECM. During the de-differentiation process miR-221 expression significantly increased. We demonstrated the effectiveness of miR-221 silencing in driving the cells towards chondrogenic lineage. AntagomiR-221 treated cells showed in fact a significant increase of expression of typical chondrogenic markers including COL2A1, ACAN and SOX9, whose loss is associated with IDD. Moreover, antagomiR-221 treatment restored FOXO3 expression and increased TRPS1 expression levels attenuating the severity grade of degeneration, and demonstrating in a context of tissue degeneration and inflammation not investigated before, that FOXO3 is target of miR-221. Data of present study are promising in the definition of new molecules useful as potential intradiscal injectable biological agents.

Inhibitory Effects of Human Primary Intervertebral Disc Cells on Human Primary Osteoblasts in a Co-Culture System.

Spinal fusion is a common surgical procedure to address a range of spinal pathologies, like damaged or degenerated discs. After the removal of the intervertebral disc (IVD), a structural spacer is positioned followed by internal fixation, and fusion of the degenerated segment by natural bone growth. Due to their osteoinductive properties, bone morphogenetic proteins (BMP) are applied to promote spinal fusion. Although spinal fusion is successful in most patients, the rates of non-unions after lumbar spine fusion range from 5% to 35%. Clinical observations and recent studies indicate, that the incomplete removal of disc tissue might lead to failure of spinal fusion. Yet, it is still unknown if a secretion of BMP antagonists in intervertebral disc (IVD) cells could be the reason of inhibition in bone formation. In this study, we co-cultured human primary osteoblasts (OB) and IVD cells i.e., nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC), to test the possible inhibitory effect from IVD cells on OB. Although we could see a trend in lower matrix mineralization in OB co-cultured with IVD cells, results of alkaline phosphatase (ALP) activity and gene expression of major bone genes were inconclusive. However, in NPC, AFC and CEPC beads, an up-regulation of several BMP antagonist genes could be detected. Despite being able to show several indicators for an inhibition of osteoinductive effects due to IVD cells, the reasons for pseudarthrosis after spinal fusion remain unclear.

Human nucleus pulposus intervertebral disc cells becoming senescent using different treatments exhibit a similar transcriptional profile of catabolic and inflammatory genes.

PURPOSE: Chronic low back pain has been associated with intervertebral disc (IVD) degeneration, which is characterized by the accumulation of extracellular matrix (ECM)-degrading proteases and inflammatory molecules in the degenerate tissue. IVD degeneration could be the outcome of natural organismal ageing and/or of the exposure of the disc to cumulative stressful environmental stimuli and is accompanied by an increased population of senescent cells in the tissue. On the other hand, senescent cells are known to secrete proteolytic enzymes and inflammatory molecules, which can contribute to ECM catabolism. The aim of this study was to investigate the transcriptional profile of selected metalloproteinases (MMPs) and inflammatory mediators in human nucleus pulposus IVD cells that became senescent using three different approaches: serial subculturing, exposure to ionizing radiation and p16INK4a overexpression. METHODS: Gene expression was assessed using quantitative RT-PCR and protein levels were determined by western blot analysis. The proliferative potential of the cells, as well as the percentage of senescent cells in the population were estimated by nuclear BrdU incorporation and by senescence-associated ß galactosidase staining, respectively. RESULTS: All senescent cells showed a similar regulation of MMP-1, -2, -3, -9, interleukin (IL) 6, IL8 and interferon γ at the level of transcription, with only some quantitative differentiations observed in p16INK4a-overexpressing cells. CONCLUSIONS: Data described here suggest that senescent cells may have similar functions in IVD homeostasis, irrespective of the origin of senescence induction.

CCAAT/enhancer binding protein ß regulates the expression of tumor necrosis factor-α in the nucleus pulposus cells.

Tumor necrosis factor alpha (TNF-α) is important in the process of intervertebral disc (IVD) degeneration because of its ability to regulate other inflammatory mediators in autocrine and paracrine fashions. The mechanism responsible for the cell type-specific regulation of TNF-α is not well known. CCAAT/enhancer binding protein ß (C/EBP ß) is one of the transcriptional factors that is implicated in TNF-α expression. However, it is not known whether cross talk occurs between C/EBP ß and the TNF-α pathway in IVD cells. The expression and effect of the C/EBP ß mRNA and protein in rat IVD cells was assessed using real-time reverse transcription polymerase chain reaction, immunohistochemical, and immunofluorescence analyses. We present data that show that the C/EBP ß mRNA and protein were expressed in rat and human IVDs in vivo. We also found that the expression of TNF-α is regulated by the transcription factor C/EBP ß in rat NP cells. The TNF-α promoter was suppressed completely in the presence of the ERK inhibitor PD98059 and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190, but not in the presence of the JNK inhibitor SP600125. In addition, gain and loss of function analyses showed that the expression of TNF-α was regulated by C/EBP ß through the MAPK pathways. These findings showed that C/EBP ß acts as a potent pro-inflammatory mediator by inducing the TNF-α gene at the transcription and protein levels via the ERK1/2 and p38 pathways in rat NP cells. Our findings may open a new avenue toward the understanding of the cellular and molecular mechanisms of IVD cells. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:865-875, 2016.

Thalidomide represses inflammatory response and reduces radiculopathic pain by inhibiting IRAK-1 and NF-κB/p38/JNK signaling.

Intervertebral disc (IVD) disease, the most common cause of disc failure and low back pain, is characterized by age-related changes in the adult disc. In this study we aimed to analyze the potential of thalidomide for the treatment of IVD disease, through identifying and explaining its anti-inflammatory and anti-catabolic activity in both in vitro IVD cell culture and in vivo animal model. Inflammatory response was induced by IL-1ß, then the activity and expression of inflammatory mediators and pathways were assessed in the presence or absence of thalidomide. The p38 inhibitor SB203580 was also used to investigate the involvement of the MAPK pathway in the observed effects. Moreover the analgesic properties of thalidomide were analyzed by the von Frey filament test in Sprague-Dawley rats. Our results indicated that thalidomide significantly inhibited the expression of pro-inflammatory mediators and matrix metalloproteinases in vitro, as well as radiculopathic pain in vivo, most probably by modulation of the activity of IRAK-1 and its downstream effectors p38, JNK and NF-κB. Our current study strongly supports the potential of thalidomide for the treatment of pain and inflammation in degenerative disc disease.