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BACKGROUND Mesenchymal Stem Cells (MSCs) under TNF-α stimulation (MSC-CM-T) can release numerous trophic and survival molecules that have a promising prospect in wound healing acceleration. However, the effective levels of MSC-CM-T in topical gel preparation to accelerate wound healing should be further explored. The aim of this study was to investigate the effects of MSC-CM-T in topical gel preparation in accelerating optimal wound healing through analyzing PDGF levels, wound closure rate percentages, and fibroblast density appearances. Twenty-four male Wistar rats were performed a full-thickness excision. The group studies were randomly assigned into four subgroups: control gel, control medium, and two treatment groups: MSC-CM-T topical gel at doses of 100 μL and 200 μL (T1 and T2, respectively). Wound closure rates were measured by standard caliper, platelet-derived growth factor (PDGF) levels were analyzed using ELISA on days 3 and 6, whereas the fibroblast density appearances were determined using hematoxylin-eosin staining. This study found a significant increase in PDGF levels in all treatment groups on days 3 and 6. These findings were in line with the increase of wound closure rates in all treatment groups on day 6, in which the high dose of MSC-CM-T was more effective in initiating the increase of wound closure rate. We also found the fibroblast density appearances on day 6 in the T2 group. We conclude that the topical gel of MSC-CM-T is more effective in accelerating wound closure healing through increasing PDGF levels and wound closure percentages and fibroblast density appearances in the skin defect animal models.
METHODS Umbilical cords (UCs) from 19-day pregnant rats were collected under general anaesthesia. Under aseptic condition, the cords were cut into smaller pieces and transferred into a T25 culture flask (Corning, Tewksbury, MA, USA) containing Dulbecco’s modified eagle medium (DMEM) (Sigma-Aldrich, Louis St, MO), supplemented with 10% fetal bovine serum (FBS) (GibcoTM Invitrogen, NY, USA), 1% penicillin (100 U/mL)/streptomycin (100 μg/mL) (GibcoTM Invitrogen, NY, USA) and 0,25% amphotericin B (GibcoTM Invitrogen, NY, USA). These flasks were incubated at 37° and 5% CO2. The medium was renewed every 3 days, and the cells were passaged after reaching 80% confluency. The MSCs-like at passage 5 were employed for the following experiments.
RESULTS MSCs-like was isolated and cultured from umbilical cord based on their plastic adherent capability under a standard culture condition. In this study, the cell morphology of MSCs exhibited typical monolayers of spindle-shaped, fibroblast-like cells, with adherent capability to the plastic flask (Figure 1A). Isolated cells were cultured for 2-3 weeks in a monolayer and used for characterization and differentiation analysis after the fifth passage. At the end of the fifth passage’s expansion, osteogenic differentiation assay on MSCs-like was performed by administering the standard and osteogenic medium for 21 days. Calcium deposition was visualized in red color using alizarin red solution (Figure 1B). Moreover, the MSCs-like surface antigens were analyzed using flow cytometry. In this study, we found
a high level of CD90 (96.7±1.3%), CD105 (67.1±0.5%) and CD73 (99.2±0.4%) (Figure 1C).
Keywords: MSCs, MSC-CM-T, wound healing, PDGF, Fibroblast.