Abstract
Objective
This study compared the potencies of the antifibrotic agents mitomycin C (MMC) and halofuginone (HFN) and investigated whether coadministration of these agents produces synergic effects in an animal skin wound model.
Subjects and methods
Twenty male Sprague-Dawley rats were used for this study. After a full-thickness excisional wound was made on the dorsum of each rat, each rat was treated with topical mitomycin, intraperitoneal HFN, or both. Wound surface areas were measured over time, and histologic analysis was performed after wounds healed completely.
Results
The groups treated with MMC alone, HFN alone, and a combination of the two all exhibited delayed wound healing compared with the untreated group. Histologically, fibrosis and matrix metalloproteinase–2 expression were significantly inhibited in the treated groups. However, there were no gross or histologic differences between the MMC-treated group, the HFN-treated group, and the combination-treatment group.
Conclusions
Both MMC and HFN inhibited excessive fibrosis. However, there was no significant difference in the antifibrotic effects of MMC and HFN on surgically induced skin wounds. Moreover, combination treatment with both MMC and HFN failed to confer an additional antifibrotic effect on skin wounds when compared with treatment with MMC or HFN alone.
1
Introduction
Wound healing after injury is among the most complex of biological processes and results in restoration of tissue integrity and function. The healing process occurs in 3 overlapping but distinct stages: inflammation, new tissue formation, and remodeling . During wound healing, fibroblasts produce extracellular matrix, predominantly in the form of collagen .
Although the adequate production of wound collagen is critical to achieving tensile strength in healing wounds, excessive collagen synthesis commonly leads to fibrotic scars, resulting in functional and cosmetic morbidities of organs. Thus, there has been considerable research devoted to reducing excessive scar formation.
Mitomycin C (MMC), isolated from Streptomyces caespitosus , is an antibiotic and antineoplastic agent that inhibits fibroblast proliferation and is used to prevent scar tissue formation . Halofuginone (HFN), isolated from the plant Dichroa febrifuga , is another antifibrotic agent that inhibits gene expression of collagen I- α and synthesis of collagen type I . Both MMC and HFN have been used as pharmacologic agents for modulating wound healing and preventing excessive collagen synthesis .
Although several studies have demonstrated the antifibrotic effects of MMC and HFN on wound healing separately, there have been no studies to date testing the coadministrative effects of these antifibrotic agents on wound healing.
The purpose of this study was to compare the potencies of MMC and HFN and to determine whether the coadministration of MMC and HFN would result in synergic effects compared with the application of MMC or HFN alone in an animal skin wound model.
2
Materials and methods
2.1
Study design and surgical technique
Twenty male Sprague-Dawley rats, with an average weight of 350 ± 50 g, were used for this study. Animals were randomly allocated to 4 groups with 5 animals per group. The 4 study groups were as follows: group A (MMC-treated group), group B (HFN-treated group), group C (MMC- and HFN-treated group), and group D (untreated control group).
The surgical procedure was performed under sterile conditions, and anesthesia was induced by intraperitoneal injection of ketamine (40 mg/kg) and xylazine (10 mg/kg). A full-thickness excisional wound with a square shape (4 cm 2 ) was made at 2 cm from the spine on the dorsum of each rat, so that 10 skin wounds were included in each group.
After the creation of skin wounds, rats were administered treatment according to their study group assignments. Cottonoid soaked in MMC solution at 0.4 mg/mL was topically applied to the skin wounds of group A rats for 5 minutes. This concentration of MMC was chosen based on the literature . Group B was given intraperitoneal HFN at 0.1 mg/(kg d) for 21 days after the induction of skin wounds . Group C was treated with topical MMC and intraperitoneal HFN identically to groups A and B. Group D received no treatment and served as a control.
To minimize variations in the surgical intervention, one surgeon performed all the procedures in a uniform fashion. All animals were housed until the cessation of study in an approved animal care facility. All experiments were performed with the approval of the Animal Experiment Committee of the Clinical Research Institute at Chungnam National University Hospital.
2.2
Gross analysis of wound healing
Wound surfaces were measured and photographed twice per week until the wounds epithelialized and contracted completely. To compare the serial changes of wound surface areas, the contours of the skin wounds were manually traced onto transparent acetate sheets. The tracings were subjected to planimetry, and the surface areas of wounds were assessed based on the acetate tracings.
2.3
Histologic analysis of wound healing
Full-thickness skin biopsies were performed when the original wounds were completely healed. The harvested skin specimens were processed in uniform fashion for histologic examination, and Masson trichrome staining was used to assess the degree of fibrosis. The degree of fibrosis was rated on a subjective scale of 0 to 3+, with 0 representing no fibrosis; 1+, mild fibrosis; 2+, moderate fibrosis; and 3+, severe fibrosis.
Immunohistochemical staining for matrix metalloproteinase–2 (MMP-2) and transforming growth factor– β (TGF- β ), which play important roles in the process of tissue fibrosis during wound healing, was performed. Immunoreactivity was scored as 0, 1, 2, or 3, corresponding to negative, weak, moderate, or strong staining intensity. The specimens were evaluated by a pathologist blinded to the group assignments of the specimens. Five uninjured normal rat skins were used for assessing the histologic differences.
2.4
Statistical analysis
SPSS 12.0 for Windows (SPSS Inc, Chicago, IL) was used for statistical analysis. Data were expressed as mean ± SD. The Mann-Whitney U test was used for analysis of means between groups, and P values < .05 were considered statistically significant.
2
Materials and methods
2.1
Study design and surgical technique
Twenty male Sprague-Dawley rats, with an average weight of 350 ± 50 g, were used for this study. Animals were randomly allocated to 4 groups with 5 animals per group. The 4 study groups were as follows: group A (MMC-treated group), group B (HFN-treated group), group C (MMC- and HFN-treated group), and group D (untreated control group).
The surgical procedure was performed under sterile conditions, and anesthesia was induced by intraperitoneal injection of ketamine (40 mg/kg) and xylazine (10 mg/kg). A full-thickness excisional wound with a square shape (4 cm 2 ) was made at 2 cm from the spine on the dorsum of each rat, so that 10 skin wounds were included in each group.
After the creation of skin wounds, rats were administered treatment according to their study group assignments. Cottonoid soaked in MMC solution at 0.4 mg/mL was topically applied to the skin wounds of group A rats for 5 minutes. This concentration of MMC was chosen based on the literature . Group B was given intraperitoneal HFN at 0.1 mg/(kg d) for 21 days after the induction of skin wounds . Group C was treated with topical MMC and intraperitoneal HFN identically to groups A and B. Group D received no treatment and served as a control.
To minimize variations in the surgical intervention, one surgeon performed all the procedures in a uniform fashion. All animals were housed until the cessation of study in an approved animal care facility. All experiments were performed with the approval of the Animal Experiment Committee of the Clinical Research Institute at Chungnam National University Hospital.
2.2
Gross analysis of wound healing
Wound surfaces were measured and photographed twice per week until the wounds epithelialized and contracted completely. To compare the serial changes of wound surface areas, the contours of the skin wounds were manually traced onto transparent acetate sheets. The tracings were subjected to planimetry, and the surface areas of wounds were assessed based on the acetate tracings.
2.3
Histologic analysis of wound healing
Full-thickness skin biopsies were performed when the original wounds were completely healed. The harvested skin specimens were processed in uniform fashion for histologic examination, and Masson trichrome staining was used to assess the degree of fibrosis. The degree of fibrosis was rated on a subjective scale of 0 to 3+, with 0 representing no fibrosis; 1+, mild fibrosis; 2+, moderate fibrosis; and 3+, severe fibrosis.
Immunohistochemical staining for matrix metalloproteinase–2 (MMP-2) and transforming growth factor– β (TGF- β ), which play important roles in the process of tissue fibrosis during wound healing, was performed. Immunoreactivity was scored as 0, 1, 2, or 3, corresponding to negative, weak, moderate, or strong staining intensity. The specimens were evaluated by a pathologist blinded to the group assignments of the specimens. Five uninjured normal rat skins were used for assessing the histologic differences.
2.4
Statistical analysis
SPSS 12.0 for Windows (SPSS Inc, Chicago, IL) was used for statistical analysis. Data were expressed as mean ± SD. The Mann-Whitney U test was used for analysis of means between groups, and P values < .05 were considered statistically significant.
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