Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles promote nucleus pulposus cell anabolism in an in vitro 3D alginate‐bead culture model

Abstract

BackgroundIntradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ‐MSC)‐derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate‐bead culture model.MethodsAfter EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ‐MSC‐EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9‐dimethylmethylene blue assays. hNPCs in alginate beads were paraffin‐embedded and stained for histological analysis (hematoxylin–eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.ResultsTreatment with WJ‐MSC‐EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.ConclusionsWJ‐MSC‐derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.

Enzymatic chemonucleolysis for lumbar disc herniation—an assessment of historical and contemporary efficacy and safety: a systematic review and meta-analysis

“…In addition, there is very little literature reporting long-term imaging evaluations of discs treated with chemonucleolysis (Supplemental item 4 ), and long-term clinical outcomes and more objective assessments remain largely undetermined 88 . Therefore, future work may consider revising the enzymatic destruction of herniated disc tissue through supplementation with regenerative treatments 16 , 89 , 90 e.g., co-injection of cells 91 , 92 , biomaterials 93 , 94 , extracellular vesicles 95 , 96 , or platelet-rich plasma (PRP) 97 to support the restoration of the disc over time, particularly regarding younger patients 16 , 79 . For example, previous animal studies have shown the capacity of PRP products to reverse condoliase-mediated disc deterioration 98 .…”

Getting to the Core: Exploring the Embryonic Development from Notochord to Nucleus Pulposus

“…Alternative strategies aim to employ cell-free methods to utilize the signaling factors produced by regenerative cell populations [ 132 , 133 , 134 ], particularly their extracellular vesicles (EVs) [ 135 , 136 , 137 , 138 ]. Research demonstrates that EVs derived from NCs contain potent bioactive molecules capable of modulating cellular behavior and are potentially able to promote tissue repair [ 139 , 140 ].…”

Why clinical trials in disc regeneration strive to achieve completion: Insights from publication status and funding sources

“…In the last decade, the possibility to directly tackle the pathophysiology of IDD by replenishing the disc cell content via transplantation of de novo cells has gained significant momentum in the field 4 . Several preclinical studies have demonstrated the capacity of transplanted cells, especially mesenchymal stromal cells (MSCs), to limit, halt, or even reverse IDD both in vitro and in vivo, variably reporting significant enhancements of cell metabolism, matrix composition, and inflammation inhibition, eventually leading to structural restoration of degenerative discs 5–7 . Therefore, the increasing body of preclinical evidence has promoted the translation of cell therapy for IDD to the clinical setting, with multiple prospective clinical trials emerging over the last few decades 8 .…”