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-β 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-β 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

The impact of signaling factors on intervertebral disc degeneration and regeneration - studies on disc and mesenchymal stem cells from chronic low back pain patients

INTRODUCTION: Chronic low back pain (LBP) is associated with degeneration of the intervertebral discs (IVDs). Increased expressions of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and matrix metalloproteinases (MMPs) in degenerated IVDs lead to loss of proteoglycan and extracellular matrix (ECM) which affect the viability of the disc cells (DCs). Treatment approaches using growth factors, cell therapy and extracellular vesicles (EVs) derived from human mesenchymal stem cells (hMSCs) could improve current treatment models by directly influencing the IVD degeneration processes. AIMS: To explore the effects of growth factors, hMSC derived signaling peptides and small EVs (sEVs) on degenerated DCs in terms of cell viability and ECM production and to investigate the impact of stress hormone cortisol on DCs and hMSCs in in vitro models. METHODS: DC and hMSC isolation from patients’ tissue, cell cultures in monolayer and 3D pellets, cell viability assay, histological staining, and immunohistochemistry were carried out. In Study I, hMSCs were encapsulated in a hydrogel and stimulated with bone morphogenetic growth factor 3 (BMP-3), or IL-1β pre-treatment followed by BMP-3 stimulation. In situ hybridization was used to investigate the gene expressions of COL2A1 and OCT4. In Study II, the effects of cortisol at physiological and increased levels were studied on DCs and hMSCs in the 3D pellet model. Apoptosis assays were carried out and immunohistochemistry was used to evaluate cytokine expressions. Study III was a follow-up study of Study I in a 3D pellet model investigating the effect of BMP-3 and pre-treatment on DCs, hMSCs and co-culture (DCs and hMSCs in 1:1 ratio). In Study IV, the effects of hMSC conditioned media (CM) and connective tissue growth factor (CTGF) were investigated on DC pellets. The constituents of CM were further identified using mass spectrometry analysis. In Study V, the concentration of MMP-1 was quantified by enzyme-linked immunosorbent assay in disc tissue. Furthermore, the ability of CM to mitigate the effects of MMP-1 at different concentrations was studied. In Study VI, small EVs were isolated with differential centrifugation, and further characterized using flow cytometry, nanoparticle tracking analysis, and western blot. DC pellets were then stimulated with sEVs and cell proliferation, ECM production, apoptosis, lactate dehydrogenase activity, cytokine and chemokine secretions were evaluated. RESULTS: Pre-treatment of IL-1β followed by BMP-3 enhanced chondrogenic differentiation in hMSCs in the hydrogel model (Study I) as well as in the 3D model (Study III). BMP-3 promoted chondrogenesis in DC pellets while a stronger effect was observed in co-culture (Study III). Study II demonstrated that exposure to cortisol even at physiological concentration restricted proliferation and compromised chondrogenesis in both DCs and hMSCs. CM from hMSCs enhanced viability and ECM production in DCs and mass spectrometry analysis revealed more than 120 peptides with high relative abundance (Study IV). Study V demonstrated that CM has the ability to mitigate the effect of MMP-1 on DCs, however, the potency of CM decreased with increased concentration of MMP-1. Lastly, Study VI demonstrated that sEVs enhanced cell proliferation while suppressed apoptosis. Early and increased ECM production was also observed in the DCs with sEVs treatment. CONCLUSION: Signaling factors from hMSCs have positive effects on DCs and can mitigate the degenerative properties of pro-inflammatory cytokines and enzymes known to be present in the degenerated IVDs. Further, pain- induced stress regulated by cortisol may be a contributing factor of IVD degeneration

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