Effect of Link N on the Expression of Neurotrophins and Substance P Release by Human Intervertebral Disc Cells Stimulated with Proinflammatory Cytokines

Abstract

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...