Topical Combination Therapy in Wound Healing
Four rabbits in the control group and 3 rabbits in the intervention group died before study completion; these wounds were used in the comparison as long as the rabbits were alive, then the wounds in the rest of the rabbits were entered into the analysis. All the wounds were created in a similar manner, but widened a bit on their own based on the size and the posture of the rabbits during wound creation, so the final wounds were greater in size. The rabbits were randomly allocated to the treated or control group after wound creation, and Mann-Whitney test showed the wound area in the first day was not significantly different in the groups ( Table 1 ). The wounds in the treated group healed faster than those in the control group ( Table 1 , Figure 1 and Figure 2). After 3 days, there was a 30.1 ± 8.7 percent reduction in wound area in the treated group, while in the control group it was only 3 ± 13.4 percent (P = 0.0001). After 7 days, the wound closure in the treated group was 48.7 ± 11 percent, compared to 20.3 ± 18 percent in the control group (P = 0.0001). There was no wound infection in any of the rabbits.
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Figure 1.
Wound closure in the control and treatment groups.
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Figure 2.
Wound healing in the control and treatment groups.
In the study of skin sections stained with HE after 7 days, there was an intact epithelium and hair follicles in the healthy area. In this area of the dermis region, thick collagen fibers were stained red and fibrocytes were dense and fusiform; in comparison, the wounded area showed no sign of epithelium, hair follicles, or thick collagen fibers. Instead, the wounded area contained an abundance of red blood cells, fibroblasts, and inflammatory cells (Figure 3). Van Gieson staining showed collagen fibers as red with a yellow background, and the Van Gieson-stained slides clearly showed the different pattern of collagen distribution in the control and treated samples (Figure 3). In intact areas, collagen fibers were thick with fibrocyte within collagen bundles, but in wounded areas or healing regions, collagen fibers were in the form of thin and distributed fibers with fibroblasts between them. This specific staining verified the results deduced with HE slides.
(Enlarge Image)
Figure 3.
Examination of microscopic sections from skin samples (wounded and healthy region), stained with hemotoxylin-eosin shows a distinct pattern of collagen fibers, fibrocytes, fibroblasts and inflammatory cells distributed in the dermis region of healthy and wounded skin (x1000). The Van Gieson staining shows a further extension of epithelium in treatment group samples in comparison to the control group samples. In this figure, the healthy region of the sample is characterized by thick collagen fibers and hair follicles.
After 7 days, like the control samples, the treated slides clearly showed 2 healthy and 2 wounded areas. In the treated group, in addition to a thicker dermis, there was a growing epithelium in the wounded area, while in the control slides, there was no sign of the epithelium in the wounded area at this time (Figure 4).
(Enlarge Image)
Figure 4.
Examination of microscopic sections from skin samples (wounded and healthy region), stained with hemotoxylin-eosin shows a further extension of epithelium in treated samples in comparison to the control samples. The arrows show the border of wound and healthy area.
After 14 days, the control slides showed signs of growing epithelium in the wounded area. However, there was still dried blood in most of the wounded areas. In comparison to the control group slides, the slides from the treated group showed almost a complete epithelium on the wounded area. The dermis in the wounded area was thicker in the treated samples (Figure 4).
Microscopic examination of the control and treated slides after 21 days showed no significant differences in terms of thickness of the dermis or epithelium in the healing region. In all 3 stages, the Van Gieson stained slides made it easy to compare the distribution of collagen fibers and of the epithelium (Figure 3).
Results
Four rabbits in the control group and 3 rabbits in the intervention group died before study completion; these wounds were used in the comparison as long as the rabbits were alive, then the wounds in the rest of the rabbits were entered into the analysis. All the wounds were created in a similar manner, but widened a bit on their own based on the size and the posture of the rabbits during wound creation, so the final wounds were greater in size. The rabbits were randomly allocated to the treated or control group after wound creation, and Mann-Whitney test showed the wound area in the first day was not significantly different in the groups ( Table 1 ). The wounds in the treated group healed faster than those in the control group ( Table 1 , Figure 1 and Figure 2). After 3 days, there was a 30.1 ± 8.7 percent reduction in wound area in the treated group, while in the control group it was only 3 ± 13.4 percent (P = 0.0001). After 7 days, the wound closure in the treated group was 48.7 ± 11 percent, compared to 20.3 ± 18 percent in the control group (P = 0.0001). There was no wound infection in any of the rabbits.
(Enlarge Image)
Figure 1.
Wound closure in the control and treatment groups.
(Enlarge Image)
Figure 2.
Wound healing in the control and treatment groups.
In the study of skin sections stained with HE after 7 days, there was an intact epithelium and hair follicles in the healthy area. In this area of the dermis region, thick collagen fibers were stained red and fibrocytes were dense and fusiform; in comparison, the wounded area showed no sign of epithelium, hair follicles, or thick collagen fibers. Instead, the wounded area contained an abundance of red blood cells, fibroblasts, and inflammatory cells (Figure 3). Van Gieson staining showed collagen fibers as red with a yellow background, and the Van Gieson-stained slides clearly showed the different pattern of collagen distribution in the control and treated samples (Figure 3). In intact areas, collagen fibers were thick with fibrocyte within collagen bundles, but in wounded areas or healing regions, collagen fibers were in the form of thin and distributed fibers with fibroblasts between them. This specific staining verified the results deduced with HE slides.
(Enlarge Image)
Figure 3.
Examination of microscopic sections from skin samples (wounded and healthy region), stained with hemotoxylin-eosin shows a distinct pattern of collagen fibers, fibrocytes, fibroblasts and inflammatory cells distributed in the dermis region of healthy and wounded skin (x1000). The Van Gieson staining shows a further extension of epithelium in treatment group samples in comparison to the control group samples. In this figure, the healthy region of the sample is characterized by thick collagen fibers and hair follicles.
After 7 days, like the control samples, the treated slides clearly showed 2 healthy and 2 wounded areas. In the treated group, in addition to a thicker dermis, there was a growing epithelium in the wounded area, while in the control slides, there was no sign of the epithelium in the wounded area at this time (Figure 4).
(Enlarge Image)
Figure 4.
Examination of microscopic sections from skin samples (wounded and healthy region), stained with hemotoxylin-eosin shows a further extension of epithelium in treated samples in comparison to the control samples. The arrows show the border of wound and healthy area.
After 14 days, the control slides showed signs of growing epithelium in the wounded area. However, there was still dried blood in most of the wounded areas. In comparison to the control group slides, the slides from the treated group showed almost a complete epithelium on the wounded area. The dermis in the wounded area was thicker in the treated samples (Figure 4).
Microscopic examination of the control and treated slides after 21 days showed no significant differences in terms of thickness of the dermis or epithelium in the healing region. In all 3 stages, the Van Gieson stained slides made it easy to compare the distribution of collagen fibers and of the epithelium (Figure 3).
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