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Identification of differentially expressed proteins involved in fetal scarless wound healing using a rat model of cleft lip.

ABSTRACT: In early pregnancy, fetal skin wounds can heal quickly and undergo a transition period from scarless healing to scar formation. The aim of the present study was to identify potential biomarkers associated with scarless repair of cleft lips, in order to determine the intrinsic factors leading to scar formation in embryonic tissue. A stable model of cleft lip was established using microsurgery by constructing a wedge‑shaped cleft lip‑like defect in fetal rats at gestational age (GA) 16.5 and GA18.5. The GA16.5 and GA18.5 groups were used to model scarless healing and scar formation, respectively. The fetuses were returned to the uterus following surgery, then removed 72 h after the procedure. Macroscopic observation of the cleft defect and histological examination were carried out. Reverse transcription‑quantitative (RT‑q) PCR and parallel reaction monitoring (PRM) were used to detect mRNA and protein expression levels, respectively. The upper‑left lip completely healed 72 h after surgery in the GA16.5 group of fetal rats. However, this was not the case in the GA18.5 group. Histological examination indicated new follicles visible under the epidermis of the scarless group (GA16.5). Scarring was visible on the upper‑left cleft lip wound of the fetal rats in the GA18.5 group. The expression of some growth and pro‑inflammatory factors, including TNF‑α, were also different between two groups. Label‑free quantification was used to identified differentially expressed proteins and five differentially expressed proteins (Smad4, Fabp5, S100a4, S100a8 and S100a9) were identified. The relative expression of these molecules at the mRNA and protein levels were measured using RT‑qPCR and PRM. These molecules may represent potential biomarkers for the scarless repair of fetal rat cleft lip wounds.

SUBMITTER: Yan Y

PROVIDER: S-EPMC8240453 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Wound dressings composed of natural polymers, such as type I collagen, possess good biocompatibility, water holding capacity, air permeability, and degradability, and can be used in wound repair. However, due to the persistent oxidative stress in the wound area, the migration and proliferation of fibroblasts might be suppressed, leading to poor healing. Thus, collagen-containing scaffolds are not suitable for accelerated wound healing. Antioxidant N-acetyl cysteine (NAC) is known to reduce the reactive oxygen species (ROS) and has been widely used in the clinic. Theoretically, the carboxyl group of NAC allows loading of graphene oxide (GO) for sustained release and may also enhance the mechanical properties of the collagen scaffold, making it a better wound-dressing material. Herein, we demonstrated an innovative approach for a potential skin-regenerating hybrid membrane using GO incorporated with collagen I and NAC (N-Col-GO) capable of continuously releasing antioxidant NAC. Methods: The mechanical stability, water holding capacity, and biocompatibility of the N-Col-GO hybrid membrane were measured in vitro. A 20 mm rat full-skin defect model was created to evaluate the repair efficiency of the N-Col-GO hybrid membrane. The vascularization and scar-related genes in the wound area were also examined. Results: Compared to the Col only scaffold, N-Col-GO hybrid membrane exhibited a better mechanical property, stronger water retention capacity, and slower NAC release ability, which likely promote fibroblast migration and proliferation. Treatment with the N-Col-GO hybrid membrane in the rat wound model showed complete healing 14 days after application which was 22% faster than the control group. HE and Masson staining confirmed faster collagen deposition and better epithelization, while CD31 staining revealed a noticeable increase of vascularization. Furthermore, Rt-PCR demonstrated decreased mRNA expression of profibrotic and overexpression of anti-fibrotic factors indicative of the anti-scar effect. Conclusion: These findings suggest that N-Col-GO drug release hybrid membrane serves as a better platform for scarless skin regeneration.

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Identification of differentially expressed proteins involved in fetal scarless wound healing using a rat model of cleft lip.

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