Exosomes Derived from Yazd Human Foreskin Fibroblast #8 (YhFF#8) Cells Fascilitate Cutaneous Wound Healing in a Rat Model: An in Vitro and In Vivo study
Exosomes Derived from Yazd Human Foreskin Fibroblast #8 (YhFF#8) Cells Fascilitate Cutaneous Wound Healing in a Rat Model: An in Vitro and In Vivo study
Amirhossein Ahmadieh-Yazdi,1Behrouz Aflatoonian,2,*Fatemeh Hajizadeh-Tafti,3
1. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran 2. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran 3. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Introduction: Wound healing is a complex biological process involving multiple stages, including inflammation, tissue formation, and remodeling. Effective healing requires a coordinated response from various cell types, growth factors, and extracellular matrix components. Despite advances in medical treatments, impaired wound healing remains a significant challenge, particularly in cases of chronic wounds or severe tissue injury. Novel therapeutic approaches that can enhance and accelerate the wound healing process are urgently needed. Exosomes have recently emerged as promising candidates for regenerative medicine. These vesicles play crucial roles in intercellular communication by transferring bioactive molecules. Exosomes derived from stem cells and fibroblasts have been shown to promote tissue repair and regeneration, making them attractive for therapeutic applications in wound healing. Human foreskin fibroblasts are a readily available and ethically sourced cell type with high proliferative potential. In this study, we assessed the ability of exosomes derived from Yazd human foreskin fibroblast #8 (YhFF#8) cells to promote wound healing in a Wistar rat model. By employing both local injection and topical administration, we aimed to determine the efficacy of these exosomes in improving the wound healing process.
Methods: Exosomes were isolated using the Exocib Exosome Extraction Kit from conditioned medium of YhFF#8 cells, which were derived at the Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Iran. The exosomes were characterized through flow cytometry using CD9, CD81, and CD63 markers, as well as transmission electron microscopy (TEM) and dynamic light scattering (DLS) to determine their size. In vitro assays, including scratch, hemolysis, and MTT assays, were conducted to assess the exosomes' bioactivity.
For the in vivo study, 24 Wistar rats were divided into four groups: no treatment, PBS treatment, and exosome treatments with 50 μg and 100 μg doses. Both local injection and topical administration of exosomes were employed for wound treatment.
Results: The analysis revealed that the extracted exosomes had an average size of approximately 78.8 nm, with a polydispersity index (PDI) of 0.8, indicating a relatively uniform particle distribution. Flow cytometry confirmed the presence of surface markers CD9, CD81, and CD63, ensuring that the extracted particles were indeed exosomes. TEM analysis validated the correct morphology of the exosomes, further confirming their presence.
The concentration of exosomes, determined using the BCA assay, ranged between 400 and 1,000 μg/mL per extraction batch. The exosomes showed no cytotoxic effects at any tested dose on YhFF#8 and YhFF#18 cell line, as indicated by the MTT assay. Scratch assay results demonstrated that only the 100 μg/mL dose significantly enhanced cell migration at 24 and 48 hours. The hemolysis assay confirmed that the exosomes did not cause significant blood lysis at any dose, suggesting that intravenous administration or accidental entry into the bloodstream would not pose a safety risk.
In the in vivo study, wound areas were measured on days 0, 3, 7, 10, and 14 using ImageJ software, and the percentage of wound healing was calculated. All treatment groups showed significant wound healing compared to controls throughout the study period. Notably, on day 3, the lower dose group demonstrated better wound healing. Histological analysis on day 7 showed no significant differences between groups, but by day 14, epithelial tissue reorganization was significantly better in the treatment groups (P=0.001). Angiogenesis did not differ significantly between the groups, and granulation tissue formation showed no significant variation. However, Masson's trichrome staining indicated that fibrosis was significantly higher in the treatment groups on day 14 compared to the control groups (P=0.0018), with increased blue staining reflecting greater tissue fibrosis.
Conclusion: The findings of this study demonstrate that exosomes derived from human foreskin fibroblasts can effectively promote wound healing in a Wistar rat model. Both local injection and topical administration of exosomes enhanced the healing process, particularly at higher doses, without causing cytotoxicity or significant hemolysis. Histological assessments further revealed improved epithelial tissue reorganization and increased fibrosis in the treatment groups. These results suggest that exosome-based therapies hold significant promise for accelerating wound healing and may offer a novel approach for regenerative medicine applications. Future studies are needed to optimize dosage and delivery methods for clinical translation.