Wharton's Jelly Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Intervertebral Disc Degeneration Under Inflammatory Stress in an In Vitro 3D Culture System

Abstract

This study explores the therapeutic potential of extracellular vesicles (EVs) derived from Wharton's Jelly mesenchymal stem cells in an in vitro 3D model of intervertebral disc degeneration under inflammatory stress. The treatment with WJ-MSC-EVs enhanced nucleus pulposus cell proliferation, viability, and extracellular matrix synthesis while reducing oxidative stress and catabolic gene expression. These results support the promise of WJ-MSC-EVs as a novel, cell-free strategy for disc regeneration in inflammatory conditions.

Keywords: exosome; extracellular vesicles; intervertebral disc; intervertebral disc degeneration; intervertebral disc regeneration; low back pain; mesenchymal stromal cells.

FIGURE 1

Characterization of WJ‐MSC‐EVs. (A) Particle size distribution of WJ‐MSC‐EVs measured with NTA. (B) TEM showed the typical EV morphology in WJ‐MSC‐EVs (left, scale bar: 200 nm; right box, scale bar: 20 nm). (C) WB analysis of EV protein markers CD63, CD9, and TSG101. (D) Representative images of hNPCs incubated with PBS or PKH26‐labeled WJ‐MSC‐EVs (red). hNPC nuclei were stained with DAPI (blue). Magnification: 20× scale bar: 100 μm. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole, EVs, extracellular vesicles, hNPCs, human nucleus pulposus cells, NTA, nanoparticle trafficking analysis, PBS, phosphate‐buffered saline, TEM, transmission electron microscopy, WB, Western blot, WJ‐MSCs, Wharton's Jelly mesenchymal stromal cells. Reproduced with permission from Tilotta et al. [14].

FIGURE 2

Characterization of WJ‐MSCs. (A) Surface markers (CD90, CD105, CD73, and SSEA3) detected through flow cytometric analysis. (B) Multilineage differentiation of WJ‐MSCs towards the osteogenic, adipogenic, and chondrogenic lineages was confirmed by Alizarin Red, Oil red O, and Alcian Blue staining (left and center, scale bars: 50 μm; right, scale bar: 100 μm). Abbreviation: WJ‐MSCs, Wharton's Jelly mesenchymal stromal cells. Reproduced with permission from Tilotta et al. [14].

FIGURE 3

WJ‐MSC‐derived EVs promoted hNPC proliferation and viability. (A) Cell content significantly increased after treatment with 10 μg/mL WJ‐MSC‐EVs at day 4, 10, and 14, as compared with the IL‐1β control group (n = 4). Data were analyzed through two‐way ANOVA with Dunnett's multiple comparisons test, where each group was compared with IL‐1β. Within each timepoint, values were analyzed using unpaired t tests with a false discovery rate approach. (B) After 24 h, the WJ‐MSC‐EV treatment significantly reduced IL‐1β‐pre‐treated hNPC death at 100 μg/mL at flow cytometry (n = 4). Data were analyzed through two‐way ANOVA with Dunnett's multiple comparisons test, where each group was compared with IL‐1β. (C) Live/Dead staining showed reduced cell death in hNPCs treated with 10 and 100 μg/mL WJ‐MSC‐EVs (n = 4). Magnification: 20×, scale bar: 100 μm.*p < 0.05, **p < 0.01, compared to the control group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 compared to the IL‐1β group. Abbreviations: AM, acetoxymethylester; hNPCs, human nucleus pulposus cells; WJ‐MSC‐EVs, Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles.

FIGURE 4

(A) NOS2 mRNA levels were significantly reduced by 10 μg/mL WJ‐MSC‐EVs (n = 5). EVs led to a significant decrease in nitrite release in cell supernatant (B) after 7 days in groups treated with 10 μg/mL EVs compared to the IL‐1β group (n = 6). Data were analyzed through one‐way ANOVA with Dunnett's multiple comparisons test, where each group was compared with IL‐1β. **p < 0.01 compared to the control group, ^# p < 0.05, ^## p < 0.01 compared to the IL‐1β group. Abbreviations: HNPCs, human nucleus pulposus cells; NOS2, nitric oxide synthase 2; WJ‐MSC‐EVs, Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles.

FIGURE 5

WJ‐MSC‐EVs enhanced ECM synthesis in IL‐1β‐treated hNPCs. (A) GAG/DNA ratio in hNPCs after WJ‐MSC‐EVs treatment demonstrated an increase in all experimental groups, although significant only in the 10 μg/mL WJ‐MSC‐EV group. Data were analyzed through one‐way ANOVA with Dunnett's multiple comparisons test, where each group was compared with IL‐1β. (B) Representative images of Alcian Blue staining of hNPC alginate beads are shown. Blue color indicates proteoglycans. Arrowheads point to proteoglycan deposits. Upper rows, scale bar: 500 μm; bottom boxes, scale bar: 100 μm. Data are expressed as GAG/DNA ratio percent variation between the control and experimental groups (n = 5). *p < 0.05, ***p < 0.001 compared to the control group, ##p < 0.01 compared to the IL‐1β group. Abbreviations: GAG, glycosaminoglycans; hNPCs, human nucleus pulposus cells; WJ‐MSC‐EVs, Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles.

FIGURE 6

WJ‐MSC‐EVs maintained the hNPC phenotype and reduced catabolic gene expression levels. WJ‐MSC‐EV treatment resulted in a significant decrease of ADAMTS5 (A), MMP1 (B), MMP13 (C) and IL6 (D) mRNA levels, while increasing ACAN (E), SOX9 (F) and KRT19 (G) gene expression. Results were normalized based on GAPDH expression and calculated as fold change compared to the controls. Data were analyzed through one‐way ANOVA with Dunnett's multiple comparisons test, where each group was compared with IL‐1β. *p < 0.05, ***p < 0.001, ****p < 0.0001 compared to the control group, ^# p < 0.05, ^## p < 0.01 compared to the IL1‐β group. Abbreviations: ACAN, aggrecan; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; hNPCs, human nucleus pulposus cells; IL, interleukin; MMP, matrix metalloproteinase; NOS, nitric oxide synthase; SOX, SRY‐box transcription factor; WJ‐MSC‐EVs = Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles.