Objective:

Exosomes are nano-sized extracellular vesicles secreted by various cell types, playing a pivotal role in intercellular communication and the regulation of numerous biological processes, including inflammation, angiogenesis, and tissue regeneration. In recent years, mesenchymal stem cell (MSC)-derived exosomes have attracted considerable attention due to their ability to transfer bioactive molecules such as proteins, lipids, and RNAs to target cells, thereby modulating cellular functions without the risks associated with direct stem cell transplantation. On the other hand, low-frequency electromagnetic fields (LF-EMF) have been independently reported to enhance cell proliferation, migration, and wound closure through mechanisms involving calcium signaling and growth factor expression. Despite the individual therapeutic potentials of exosomes and LF-EMF, the synergistic effects of their combination on skin cells, particularly keratinocytes which are central to re-epithelialization during wound healing, have remained largely unexplored. Therefore, this experimental study was designed to investigate the combined effect of bone marrow stem cell-derived exosomes and LF-EMF on key cellular responses of keratinocytes, including apoptosis and the expression of inflammatory and anti-inflammatory genes.

Materials and Methods:

Bone marrow mesenchymal stem cells (BM-MSCs) were isolated from healthy adult rats and cultured under standard conditions until reaching 70–80% confluence. The cell culture supernatant was collected, and exosomes were isolated using a well-established ultracentrifugation protocol involving sequential centrifugation steps at 300×g, 2000×g, and 10,000×g to remove cell debris and large vesicles, followed by ultracentrifugation at 100,000×g for 70 minutes at 4°C. The resulting pellet, enriched with exosomes, was characterized in terms of size, morphology, and specific surface markers using dynamic light scattering (DLS), transmission electron microscopy (TEM), and Western blotting for CD63 and CD81. For the main experiment, keratinocytes (HaCaT cell line) were cultured and divided into four groups: control (untreated), exosome-treated alone, LF-EMF treated alone (exposed to a 50 Hz, 1 mT electromagnetic field for 1 hour daily for three consecutive days), and a combination group receiving both exosomes and LF-EMF under identical conditions. Following treatment, the rate of apoptosis among keratinocytes was quantitatively assessed using the Annexin V-FITC/PI double-staining assay analyzed by flow cytometry. Additionally, total RNA was extracted from each group, reverse transcribed into cDNA, and subjected to real-time PCR to measure the expression levels of five target genes: transforming growth factor beta 1 (TGFβ1), interleukin-18 binding protein (IL-18bp), interleukin-1 beta (IL-1β), interleukin-18 (IL-18), and tumor necrosis factor-alpha (TNF-α). Relative gene expression was calculated using the ΔΔCt method with GAPDH as the housekeeping gene.

Results:

The flow cytometric analysis of Annexin V staining revealed that the lowest percentage of apoptotic keratinocytes was observed in the combination group (exosomes + LF-EMF), indicating a significant protective synergy compared to either treatment alone. The reduction in apoptosis was statistically significant (p < 0.05) relative to control and individual treatment groups. Moreover, real-time PCR data demonstrated a remarkable upregulation of the anti-inflammatory and tissue-repair-related genes in the combination group. Specifically, the expression of IL-18bp, a natural inhibitor of IL-18-mediated inflammation, and TGFβ1, a key cytokine involved in extracellular matrix deposition and wound contraction, increased significantly compared to the other groups (p < 0.01). Conversely, the combination treatment led to a marked downregulation of pro-inflammatory cytokines: TNF-α, IL-1β, and IL-18 gene expression levels were substantially reduced compared to the control and single-treatment groups. These molecular changes indicate a shift from a pro-inflammatory to an anti-inflammatory, pro-healing environment within the keratinocyte population.

Conclusion:

The findings of this study provide strong evidence that the combined application of BM-MSC-derived exosomes and low-frequency electromagnetic fields exerts protective and immunomodulatory effects on keratinocytes. The significant reduction in apoptosis, coupled with the favorable modulation of inflammatory gene expression (upregulation of TGFβ1 and IL-18bp; downregulation of TNF-α, IL-1β, and IL-18), suggests that this dual strategy enhances cell survival and reduces excessive inflammation, both of which are critical for efficient wound healing. These results highlight the potential of combining exosome therapy with physical stimuli like LF-EMF as a novel, cell-free regenerative approach. Future studies should focus on identifying the specific molecular cargo within exosomes responsible for these synergistic effects and evaluating the efficacy of this combination in in vivo wound models. Overall, the exosome-LF-EMF combination represents a promising candidate for developing advanced wound healing therapies, especially for chronic or non-healing wounds.