Several studies have shown that type X collagen (COL X), a marker of late-stage chondrocyte hypertrophy, is expressed in mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients. We recently found that Naproxen, but not other nonsteroidal anti-inflammatory drugs (NSAIDs) (Ibuprofen, Celebrex, Diclofenac), can induce type X collagen gene (COL10A1) expression in bone-marrow-derived MSCs from healthy and OA donors. In this study we determined the effect of Naproxen on COL X protein expression and investigated the intracellular signaling pathways that mediate Naproxen-induced COL10A1 expression in normal and OA hMSCs. MSCs of OA patients were isolated from aspirates from the intramedullary canal of donors (50-80 years of age) undergoing hip replacement surgery for OA and were treated with or without Naproxen (100 μg/mL). Protein expression and phosphorylation were determined by immunoblotting using specific antibodies (COL X, p38 mitogen-activated protein kinase [p38], phosphorylated-p38, c-Jun N-terminal kinase [JNK], phosphorylated-JNK, extracellular signal-regulated kinase [ERK], and phosphorylated-ERK). Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the expression of COL10A1 and Runt-related transcription factor 2 gene (Runx2). Our results show that Naproxen significantly stimulated COL X protein expression after 72 h of exposure both in normal and OA hMSCs. The basal phosphorylation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) in OA hMSCs was significantly higher than in normal. Naproxen significantly increased the MAPK phosphorylation in normal and OA hMSCs. NSAID cellular effects include cyclooxygenase, 5-lipoxygenase, and p38 MAPK signaling pathways. To investigate the involvement of these pathways in the Naproxen-induced COL10A1 expression, we incubated normal and OA hMSCs with Naproxen with and without inhibitors of ERK (U0126), JNK (BI-78D3), p38 (SB203580), and 5-lipoxygenase (MK-886). Our results showed that increased basal COL10A1 expression in OA hMSCs was significantly suppressed in the presence of JNK and p38 inhibitors, whereas Naproxen-induced COL10A1 expression was suppressed by 5-lipoxygenase inhibitor. This study shows that Naproxen induces COL X both at transcriptional and translational levels in normal and OA hMSCs. Elevated basal COL10A1 expression in OA hMSCs is probably through the activation of MAPK pathway and Naproxen-induced COL10A1 expression is through the increased 5-lipoxygenase signaling.

Neurotrophins (NTs) are the major contributors of sensory axonal sprouting, neural survival, regulation of nociceptive sensory neurons, inflammatory hyperalgesia, and neuropathic pain. Intervertebral disc (IVD) cells constitutively express NTs. Their expression is upregulated by proinflammatory cytokines present in the IVD during degeneration, which can promote peripheral nerve ingrowth and hyperinnervation, leading to discogenic pain. Currently, there are no targeted therapies that decrease hyperinnervation in degenerative disc disease. Link N is a naturally occurring peptide with a high regenerative potential in the IVD. Therefore, the suitability of Link N as a therapeutic peptide for suppressing NTs, which are known modulators and mediators of pain, was investigated. The aim of the present study is to determine the effect of Link N on NTs expression, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), and their cognate receptors TrkA and TrkB as they are directly correlated with symptomatic back pain. Furthermore, the neurotransmitter (substance P) was also evaluated in human annulus fibrosus (AF) cells stimulated with cytokines. Human AF cells isolated from normal IVDs were stimulated with interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) in the presence or absence of Link N. NGF release in the media was evaluated by Western blotting. Total RNA was isolated and gene expression was measured using real‐time PCR. Gene expression of NGF, BDNF, TrkA, and TrkB significantly decreased in human disc cells stimulated with either IL‐1β or TNF‐α supplemented with Link N when compared to the cells stimulated only with IL‐1β or TNF‐α. NGF protein expression was also suppressed in AF cells coincubated with Link N and IL‐1β when compared to the cells stimulated only with IL‐1β. Link N can suppress the stimulation of NGF, BDNF, and their receptors TrkA and TrkB in AF cells in an inflammatory milieu. Thus, coupled with previous observations, this suggests that administration of Link N has the potential to not only repair the discs in early stages of the disease but also suppress pain.

Introduction Although there are different causes of low back pain, intervertebral disc (IVD) degeneration is one of them. In healthy discs, nociceptive nerve fibers and mechanoreceptors penetrate up to the outer annulus fibrosus (AF). In contrast, the endplates are heavily innervated. However, neither the inner AF nor the nucleus pulposus (NP) are innervated. Discogenic pain can occur due to degenerated discs acquiring cracks and fissures (annulogenic pain) or endplate damage (vertebrogenic pain) resulting in increased nerve fibers that penetrate the inner AF and NP. The increased expression of neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) has been identified in human and animal models of degenerating IVDs.1 Proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) have been shown to trigger the expression of NGF, BDNF, and substance P in human NP and AF cells. In related studies, bone morphogenetic protein (BMP) was shown to suppress innervation while overexpression of the BMP inhibitor noggin resulted in a significant increase in nerve fibers.2 However, the use of growth factors in clinical practice is limited by their high cost. Using synthetic peptides, such as link N, which stimulate BMP signaling, can circumvent this cost. The purpose of the present study was to evaluate the effect of link N on neurotrophins and substance P by human IVD cells stimulated with proinflammatory cytokines as well as in injured bovine IVDs. Materials and Methods Lumbar IVDs were obtained through organ donations within 6 hours after death. The procedure was approved by the local Research Ethics Board. Cells were isolated from NP and AF regions of the discs by sequential digestion with pronase followed by collagenase IA digestion for NP and collagenase II digestion for AF, respectively. They were cultured in monolayers and stimulated with TNF-α (100 ng/mL) and IL-1β (10 ng/mL) in the presence or absence of link N (1 μg/mL) for 48 hours. Total RNA was isolated and gene expression was measured using reverse-transcriptase polymerase chain reaction. The release of substance P into the culture media was measured after cells were stimulated by TNF-α (100-ng/mL) and IL-1β (10 ng/mL) in the presence or absence of link N (1 μg/mL) at different time points (1, 2, 4,and 24 hours). Coccygeal IVDs from the tails of adult bovine steers (20-25 months) were used for disc isolation. Four discs with cartilage end plates were isolated and were treated (control, capsaicin [1.5 μg/mL], punctured with a 16G needle, link N [10 μg/mL] treated) after preconditioning for 24 hours in complete DMEM. Disc culture media was collected at different time points for analysis. Substance P in the media was concentrated by solid phase extraction and was assayed by ELISA. Results Link N is known to induce proteoglycan synthesis by isolated disc cells and in degenerate rabbit and human discs, and to enhance chondrogenesis of MSCs in vitro. It is, however, not known if link N can suppress inflammatory me...