Low back pain (LBP), as a leading cause of disability, is a common musculoskeletal disorder that results in major social and economic burdens. Recent research has identified inflammation and related signaling pathways as important factors in the onset and progression of disc degeneration, a significant contributor to LBP. Inflammatory mediators also play an indispensable role in discogenic LBP. The suppression of LBP is a primary goal of clinical practice but has not received enough attention in disc research studies. Here, an overview of the advances in inflammation-related pain in disc degeneration is provided, with a discussion on the role of inflammation in IVD degeneration and pain induction. Puncture models, mechanical models, and spontaneous models as the main animal models to study painful disc degeneration are discussed, and the underlying signaling pathways are summarized. Furthermore, potential drug candidates, either under laboratory investigation or undergoing clinical trials, to suppress discogenic LBP by eliminating inflammation are explored. We hope to attract more research interest to address inflammation and pain in IDD and contribute to promoting more translational research.

The inflammatory response is induced by the overexpression of inflammatory cytokines, mainly interleukin (IL)-1β, and is one of the main causes of intervertebral disc degeneration (IVDD). NLR pyrin domain containing 3 (NLRP3) inflammasome activation is an important source of IL-1β. As an anti-inflammatory neuroendocrine hormone, melatonin plays various roles in different pathophysiological conditions. However, its roles in IVDD are still not well understood and require more examination. First, we demonstrated that melatonin delayed the progression of IVDD and relieved IVDD-related low back pain in a rat needle puncture IVDD model; moreover, NLRP3 inflammasome activation (NLRP3, p20, and IL-1β levels) was significantly upregulated in severely degenerated human discs and a rat IVDD model. Subsequently, an IL-1β/NF-κB-NLRP3 inflammasome activation positive feedback loop was found in nucleus pulposus (NP) cells that were treated with IL-1β. In these cells, expression of NLRP3 and p20 was significantly increased, NF-κB signaling was involved in this regulation, and mitochondrial reactive oxygen species (mtROS) production increased. Furthermore, we found that melatonin disrupted the IL-1β/NF-κB-NLRP3 inflammasome activation positive feedback loop in vitro and in vivo. Melatonin treatment decreased NLRP3, p20, and IL-1β levels by inhibiting NF-κB signaling and downregulating mtROS production. Finally, we showed that melatonin mediated the disruption of the positive feedback loop of IL-1β in vivo. In this study, we showed for the first time that IL-1β promotes its own expression by upregulating NLRP3 inflammasome activation. Furthermore, melatonin disrupts the IL-1β positive feedback loop and may be a potential therapeutic agent for IVDD.

Growing evidence shows that the inhibitory effect of inflammatory cytokines on new bone formation by osteogenic precursor cells is a critical cause of net bone‐density reduction. Melatonin has been proven to be a potential therapeutic candidate for osteoporosis. However, whether it is capable of antagonizing the suppressing effect of inflammatory cytokines on osteogenic precursor cells is so far elusive. In this study, using the cell culture system of human bone marrow stromal cells and MC3T3‐E1 preosteoblasts, we recorded the following vital observations that provided insights of melatonin‐induced bone formation: 1) melatonin induced bone formation in both normal and inflammatory conditions; 2) Wnt4 was essential for melatonin‐induced bone formation in inflammatory stimulation; 3) melatonin‐ and Wnt4‐induced bone formation occurred via activation of β‐catenin and p38‐JNK MAPK pathways by interaction with a distinct frizzled LDL receptor‐related protein complex; 4) melatonin suppressed the inhibitory effect of NF‐κB on osteogenesis in a Wnt4‐dependent manner; and 5) melatonin induced Wnt4 expression through the ERK1/2‐Pax2‐Egr1 pathway. In summary, we showed a novel mechanism of melatonin‐induced bone formation in an inflammatory environment. Melatonin‐induced Wnt4 expression is essential for its osteoinductive effect and the inhibitory effect of NF‐κB on bone formation. Our novel findings may provide useful information for its potential translational application.—Li, X., Li, Z., Wang, J., Li, Z., Cui, H., Dai, G., Chen, S., Zhang, M., Zheng, Z., Zhan, Z., Liu, H. Wnt4 signaling mediates protective effects of melatonin on new bone formation in an inflammatory environment. FASEB J. 33, 10126–10139 (2019). http://www.fasebj.org

ObjectivesThe aim of this study was to identify the role of tenascin-C (TNC) in entheseal new bone formation and to explore the underlying molecular mechanism.MethodsLigament tissue samples were obtained from patients with ankylosing spondylitis (AS) during surgery. Collagen antibody-induced arthritis and DBA/1 models were established to observe entheseal new bone formation. TNC expression was determined by immunohistochemistry staining. Systemic inhibition or genetic ablation of TNC was performed in animal models. Mechanical properties of extracellular matrix (ECM) were measured by atomic force microscopy. Downstream pathway of TNC was analysed by RNA sequencing and confirmed with pharmacological modulation both in vitro and in vivo. Cellular source of TNC was analysed by single-cell RNA sequencing (scRNA-seq) and confirmed by immunofluorescence staining.ResultsTNC was aberrantly upregulated in ligament and entheseal tissues from patients with AS and animal models. TNC inhibition significantly suppressed entheseal new bone formation. Functional assays revealed that TNC promoted new bone formation by enhancing chondrogenic differentiation during endochondral ossification. Mechanistically, TNC suppressed the adhesion force of ECM, resulting in the activation of downstream Hippo/yes-associated protein signalling, which in turn increased the expression of chondrogenic genes. scRNA-seq and immunofluorescence staining further revealed that TNC was majorly secreted by fibroblast-specific protein-1 (FSP1)+fibroblasts in the entheseal inflammatory microenvironment.ConclusionInflammation-induced aberrant expression of TNC by FSP1+fibroblasts promotes entheseal new bone formation by suppressing ECM adhesion forces and activating Hippo signalling.

The positive effect of low-magnitude, high‑frequency (LMHF) vibration on implant osseointegration has been demonstrated; however, the underlying cellular and molecular mechanisms remain unknown. The aim of this study was to explore the effect of LMHF vibration on the adhesion and the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) cultured on hydroxyapatite (HA)-coated surfaces in an in vitro model as well as to elucidate the molecular mechanism responsible for the effects of LMHF vibration on osteogenesis. LMHF vibration resulted in the increased expression of fibronectin, which was measured by immunostaining and RT-qPCR. Stimulation of BMSCs by LMHF vibration resulted in the rearrangement of the actin cytoskeleton with more prominent F-actin. Moreover, the expression of β1 integrin, vinculin and paxillin was notably increased following LMHF stimulation. Scanning electron microscope observations revealed that there were higher cell numbers and more extracellular matrix attached to the HA-coated surface in the LMHF group. Alkaline phosphatase activity as well as the expression of osteogenic-specific genes, namely Runx2, osterix, collagen I and osteocalcin, were significantly elevated in the LMHF group. In addition, the protein expression of Wnt10B, β-catenin, Runx2 and osterix was increased following exposure to LMHF vibration. Taken together, the findings of this study indicate that LMHF vibration promotes the adhesion and the osteogenic differentiation of BMSCs on HA-coated surfaces in vitro, and LMHF vibration may directly induce osteogenesis by activating the Wnt/β‑catenin signaling pathway. These data suggest that LMHF vibration enhances the osseointegration of bone to a HA-coated implant, and provide a scientific foundation for improving bone-implant osseointegration through the application of LMHF vibration.

Abstract. The potential effects of intermittent parathyroid hormone (1-34) ] administration on bone formation have previously been investigated. A number of studies have suggested that the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway is associated with PTH-induced osteogenic differentiation. However, the precise signaling pathways and molecular mechanism by which PTH (1-34) induces the osteogenic differentiation of bone mesenchymal stromal cells (BMSCs) remain elusive. The purpose of the present study was to investigate the mechanism underlying the effect of intermittent PTH (1-34) application on the proliferation and osteogenic differentiation of BMSCs. BMSCs were randomly divided into four groups, as follows: Osteogenic medium (control group); osteogenic medium and intermittent PTH (1-34); osteogenic medium and intermittent PTH (1-34) plus the adenylyl cyclase activator forskolin; and osteogenic medium and intermittent PTH (1-34) plus the PKA inhibitor H-89. A cell proliferation assay revealed that PTH (1-34) stimulates BMSC proliferation via the cAMP/PKA pathway. Furthermore, reverse transcription-quantitative polymerase chain reaction, alkaline phosphatase activity testing and cell examination using Alizarin Red S staining demonstrated that PTH (1-34) administration promotes osteogenic differentiation and mineralization, mediated by the cAMP/PKA pathway. Crucially, the results of western blot analyses suggested that PTH (1-34) treatment and, to a greater degree, PTH (1-34) plus forskolin treatment caused an increase in phosphorylated cAMP response element binding protein (p-CREB) expression, but the effect of PTH on p-CREB expression was blocked by H-89. In conclusion, the current study demonstrated that intermittent PTH (1-34) administration regulates downstream proteins, particularly p-CREB, in the cAMP/PKA signaling pathway, to enhance the proliferation, osteogenic differentiation and mineralization of BMSCs.

Pathological new bone formation is a typical pathological feature in ankylosing spondylitis (AS), and the underlying molecular mechanism remains elusive. Previous studies have shown that the calcium-sensing receptor (CaSR) is critical for osteogenic differentiation while also being highly involved in many inflammatory diseases. However, whether it plays a role in pathological new bone formation of AS has not been reported. Here, we report the first piece of evidence that expression of CaSR is aberrantly upregulated in entheseal tissues collected from AS patients and animal models with different hypothetical types of pathogenesis. Systemic inhibition of CaSR reduced the incidence of pathological new bone formation and the severity of the ankylosing phenotype in animal models. Activation of PLCc signalling by CaSR promoted bone formation both in vitro and in vivo. In addition, various inflammatory cytokines induced upregulation of CaSR through NF-jB/p65 and JAK/Stat3 pathways in osteoblasts. These novel findings suggest that inflammation-induced aberrant upregulation of CaSR and activation of CaSR-PLCc signalling in osteoblasts act as mediators of inflammation, affecting pathological new bone formation in AS.

Previous studies have indicated that growth differentiation factor 6 (GDF6) is a potential candidate for intervertebral disc (IVD) degeneration (IDD) treatment.Here, we investigated the effect of GDF6 on IDD by examining changes in disc structure and the expression of inflammatory and pain-related factors. A rat posterior disc puncture model of single segments and three consecutive segments was constructed, and GDF6 or phosphate-buffered solution was administered via intradiscal injection 1 or 2 weeks after surgery. Magnetic resonance imaging showed a clear degeneration signal in the punctured disc, which was inhibited by GDF6.Histological staining revealed that GDF6 did not significantly improve the structure of IVDs in rats 8 weeks after puncture surgery, but it had an inhibitory effect on expression of the tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β in the IVD. Furthermore, GDF6 was found to protect the morphology and structure of the IVD 32 weeks after surgery. Mechanical and thermal hyperalgesia tests suggested that GDF6 injection can significantly improve mechanical and thermalstimulated pain behavior in rats and inhibit the expression of inflammatory factors TNF-α and IL-1β and the pain factor calcitonin gene-related peptide in the dorsal root ganglion. A rat protein array test indicated that GDF6 could reduce the expression of cytokines IL-6, intercellular cell adhesion molecule-1, matrix metalloproteinase-13, IL-1β, and TNF-α and increase the expression of tissue inhibitor of metalloproteinases 1, Transforming growth factor-beta 2, IL-10, and resistin in a TNF-α-induced IDD cell model. Thus, our study demonstrates that GDF6 can improve the structure of the IVD, inhibit the expression of inflammatory and pain-related factors, and improve pain behavior in rats. Clinical Significance: To establish further preclinical research and clinical trials, comprehensive data are needed to validate the regenerative properties of GDF6. Ideally, a regenerative