Small extracellular vesicle-enriched preparations derived from genetically modified multipotent mesenchymal stromal cells promote fibroblast proliferation and extracellular matrix remodeling in vitro

Impaired skin repair is associated with insufficient fibroblast activity and dysregulated extracellular matrix turnover. Extracellular vesicle-enriched preparations derived from multipotent mesenchymal stromal cells (MMSCs) may provide a cell-free approach to modulate these processes; however, the effects of preparations from genetically modified producer cells remain incompletely characterized. We evaluated preparations obtained from native MMSCs and from MMSCs separately transfected with OCT4/SOX2, HIF-1alpha, or Klotho expression plasmids. A 1:1:1 mixture of preparations from the three modified cell populations was examined as a combined treatment. Rat skin fibroblasts were exposed to the preparations for 24, 48, or 72 h. Fibroblast density and concentrations of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), collagen type I, and collagen type III were assessed. The combined preparation produced the largest observed increase in fibroblast density, reaching 112.63 +/−5.55 × 10^3 cells/cm2 at 72 h, compared with 40.60 +/−1.12 × 10^3 cells/cm2 for the native-MMSC preparation and 37.10 +/−3.01 × 10^3 cells/cm2 in the untreated control. At 24 h, MMP-1 and MMP-3 concentrations in the combined group were 140.0 +/−4.77 ng/mL and 144.0 +/−4.74 ng/mL, respectively, compared with 283.1 +/−25.2 ng/mL and 208.1 +/−39.2 ng/mL in controls. Collagen type III concentration increased to 1.95 +/−0.25 ng/mL at 72 h versus 1.15 +/−0.15 ng/mL in controls. These findings indicate that the combined preparation was associated with enhanced fibroblast proliferation and altered extracellular matrix remodeling in vitro. Interpretation is limited by triplicate technical replication, volume-based dosing, and incomplete vesicle characterization.