Human Levels of MMP-1 in Degenerated Disks Can Be Mitigated by Signaling Peptides from Mesenchymal Stem Cells.

Degradation of extracellular matrix (ECM) in intervertebral disks (IVDs) during IVD degeneration plays a vital role in low back pain (LBP). In healthy IVDs, synthesis and degradation of ECM are kept in balance by matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs. MMPs are enzymes responsible for ECM degradation, and their expression levels are known to increase in degenerated disks. However, the exact pathophysiological concentration of MMP-1 in the degenerated disks of patients with chronic LBP has not been reported previously. Factors secreted by human mesenchymal stem cells (hMSCs) have shown positive results in cell therapy of degenerated disks. The aim of this study was to investigate the pathophysiological MMP-1 concentration (in ng/mL) in degenerated disk tissue and to evaluate if conditioned media (CM) from hMSCs could mitigate the effects of MMP-1 at the detected levels in a 3D in vitro disk cell (DC) pellet model. Tissue levels of MMP-1 were quantified in disk tissue collected from 6 chronic LBP patients undergoing surgery. DC pellet cultures were performed to investigate the effects of MMP-1 alone and the effects of conditioned media (CM) in the presence of MMP-1. MMP-1 was introduced in the pellets on day 14 at concentrations of 5, 50, or 100 ng/mL. The pellets were harvested on day 28 and evaluated for cell viability, proliferation, and ECM production. The mean concentration of MMP-1 in disk tissue was 151 ng/mL. Results from pellet cultures demonstrated a higher number of viable cells, glycosaminoglycan production, and ECM accumulation in the CM group even in the presence of MMP-1 compared to the controls. However, the level decreased with increasing MMP-1 concentration. The results demonstrated that CM has the ability to mitigate matrix degradation property of MMP-1 up to 50 ng/mL suggesting that CM could potentially be used to treat early stages of disk degeneration.

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.