Keywords
scalp - healing - ascorbic acid - Wistar rats
Palavras-chave
escalpe - cicatrização - ácido ascórbico - ratos Wistar
Introduction
The healing process is common to all wounds, regardless of the agent that caused it,
it is systemic and dynamic and is directly related to the general conditions of the
organism. It consists of a perfect and coordinated cascade of cellular, molecular,
and biochemical events that interact for tissue reconstitution to occur.
Tissue damage, the initial stimulus for the healing process, puts blood elements in
contact with collagen, synthesized by fibroblasts, and other substances in the extracellular
matrix, causing platelet degranulation and activation of the coagulation and complement
cascades. With this, the release of several vasoactive and chemotactic mediators that
guide the healing process by attracting inflammatory cells to the wound region occurs.
According to the literature, ascorbic acid acts as an electron donor for the proline
hydroxylation process, during collagen synthesis, a fact that leads to suspicion of
its increased demand in tissue repair processes.
Methodology
The present research was performed in the vivarium and in the laboratory of operative
technique and experimental surgery at the institute of medical research (IPEM, in
the Portuguese acronym) of Faculdade Evangélica Mackenzie do Paraná (FEMPAR). Thirty
male Wistar rats were used, divided into 3 groups, 10 from the control group (GI),
10 from the group treated with oral vitamin C (GII), and 10 from the group treated
with subcutaneous vitamin C (GIII).
Vitamin C was administered to animals from GII and GIII, from the 3rd to the 7th postoperative day, totaling 10 days of its administration, at a dose of 100 mg/kg/day.
On the 4th day of the study, the rats were submitted to a surgical procedure that consisted
of a 2-cm incision of the skin of the animals' heads and sutures with simple stitches.
After a determined period, the rats were killed and subjected to the collection of
material for study using the picrosirius red technique to assess collagen types I
and III, the degree of healing by hematoxylin and eosin (HE), and the rate of wound
contraction on subsequent days. The results were described by means, medians, minimum
and maximum values, and standard deviations. For the comparison of the three groups,
the one-way analysis of variance (one-way ANOVA) model or the Kruskal-Wallis non-parametric
test was used. The condition of normality of the variables was assessed by the Shapiro-Wilk
test. Values of p < 0.05 indicated statistical significance. The data were analyzed with the computer
program IBM SPSS Statistics for Windows, v.20.0. (IBM Corp., Armonk, NY, USA).
All ethical parameters were respected, and this research was approved by the Ethics
Committee on the Use of Animals of Faculdade Evangélica Mackenzie do Paraná (CEUAs
/ FEMPAR).
Results
A significant difference was found between the three groups in terms of the rate of
contraction ([Tables 1] and [2]). There was a significant difference between the control group and the groups treated
with oral vitamin C (p = 0.001) and subcutaneous vitamin C (p < 0.001). No significant difference was found between the two groups treated with
vitamin C (p = 0.227), the rates observed are shown in [Graph 1].
Table 1
Comparison of percentage values of operating wound contraction rate in each group
and among all groups
|
Variable
|
Group
|
N
|
Average
|
Median
|
Minimum
|
Maximum
|
Standard deviation
|
p*-value*
|
|
Contraction rate
|
Control
|
10
|
20.8
|
0.0
|
0.0
|
62.5
|
26.5
|
|
|
(%)
|
Oral vitamin C
|
10
|
64.9
|
75.0
|
0.0
|
81.3
|
23.4
|
0.001
|
|
Subcutaneous vitamin C
|
10
|
70.0
|
75.0
|
25.0
|
83.3
|
18.4
|
|
* Kruskal-Wallis non-parametric test, p < 0.05.
Table 2
Comparison of the contraction rate in relation to each group
|
Compared groups
|
p-value*
|
|
Control versus oral vitamin C
|
0.001
|
|
Control versus subcutaneous vitamin C
|
< 0.001
|
|
Oral vitamin C versus subcutaneous vitamin C
|
0.227
|
[Figs. 1], [2], and [3] demonstrate the evolution of the wound through the photos obtained on the 3rd, 5th, and 7th days of the study. It is possible to notice the reduction in the size of the wound
and the gradual disappearance of the crust and granulation tissue.
Fig. 1 Aspect of group I rat wound contraction (Control).
Fig. 2 Aspect of group II rat wound contraction (oral vitamin) for 7 days of observation.
Fig. 3 Aspect of Group III Rat Wound Contraction (Subcutaneous Vitamin C) DURING 7 DAYS
OF OBSERVATION. NOTE: Animals in the control group on days 3, 5, and 7 of the study.
Using the table adapted from the protocol created by Greenhalgh D. G. et al., the
lesions were classified from 1 to 4, with 1 being the worst and 4 being the best degree
of healing [Table 3],[4],[5],[6].
Table 3
Comparison of percentage values according to hematoxylin and eosin groups and classifications
|
HE (degree)
|
Group (treatment)
|
|
Control
|
Oral
|
Subcutaneous
|
|
|
0
|
−
|
−
|
−
|
|
−
|
−
|
−
|
|
1
|
−
|
−
|
−
|
|
−
|
−
|
−
|
|
2
|
3
|
−
|
−
|
|
30%
|
−
|
−
|
|
3
|
4
|
3
|
3
|
|
40%
|
30%
|
30%
|
|
4
|
3
|
7
|
7
|
|
30%
|
70%
|
70%
|
|
Total
|
10
|
10
|
10
|
Table 4
Comparison of the percentage values of healing assessed by hematoxylin and eosin
|
Hematoxylin and eosin (degree)
|
Group (treatment)
|
|
Control
|
Oral
|
Subcutaneous
|
|
0, 1, or 2
|
3
|
0
|
0
|
|
30%
|
0%
|
0%
|
|
3 or 4
|
8
|
10
|
10
|
|
80%
|
100%
|
100%
|
|
Total
|
10
|
10
|
10
|
The degree of scarring assessed by hematoxylin and eosin was higher in the groups
that received vitamin C, however, with no significant difference between the groups.
Table 5
Comparison of the percentage values of the degree of healing in the groups assessing
the statistical significance
|
Groups compared
|
P-value*
|
|
Control x oral
|
0.214
|
|
Control x subcutaneous
|
1
|
|
Oral x subcutaneous
|
0.472
|
Table 6
Comparison of the percentage values of the collagen I and Iii area in each group and
among all groups
|
Variable
|
Group
|
N
|
Average
|
Median
|
Min
|
Max
|
Standard deviation
|
P-value*
|
|
Collagen I area
|
Control
|
10
|
50.7
|
50.0
|
12.3
|
84.0
|
23.7
|
|
|
(%)
|
Oral vitamin C
|
10
|
48.5
|
45.3
|
31.7
|
82.0
|
15.0
|
0.292
|
|
Subcutaneous vitamin C
|
10
|
37.5
|
39.0
|
1.7
|
64.8
|
20.1
|
|
|
Collagen III area
|
Control
|
10
|
49.3
|
50.0
|
16.0
|
87.7
|
23.7
|
|
|
(%)
|
Oral vitamin C
|
10
|
51.5
|
54.7
|
18.0
|
68.3
|
15.0
|
0.292
|
|
Subcutaneous vitamin C
|
10
|
62.5
|
61.0
|
35.2
|
98.3
|
20.1
|
|
*one-way ANOVA, p < 0.05.
[Figs. 4], [5], and [6] show the histological variation of the degree of healing in relation to each group.
Fig. 4 Photomicrograph of the wound healing area in the Control group. LEGEND: (A) (HE 40x)
- Granulation tissue of the epithelium (B) (HE200x). NOTE: (A) (*) Epithelium partially
covering the surgical wound. (B) (#) Thin and immature granulation tissue, predominantly
inflammatory cells, with few fibroblasts, capillaries, and collagen deposition.
Fig. 5 Photomicrograph of the wound healing area of the subcutaneous vitamin C group. (A)
(HE 40x) - Granulation tissue of the epithelium (B) (HE200x). NOTE: (A) (*) Epithelium
fully covering the lesion. (B) (#) Medium-thickness granulation tissue, with few inflammatory
cells with a predominance of fibroblasts with collagen deposition. Neovascularization
present in good quantity.
Fig. 6 Photomicrograph of the wound healing area of the oral vitamin C group. (A) (HE 40x)
- Granulation tissue of the epithelium (B) (HE200x). NOTE: (A) (*) Epithelium fully
covering the lesion with little crust formation. (B) (#) Thick and vascularized granulation
tissue, predominance of fibroblasts and large collagen deposition.
The results indicate that there is no significant difference between the three groups
in relation to the area of collagen I and the area of collagen III ([Graphic 2] and [Graphic 3]).
[Fig. 7] shows the greater emphasis of type-I collagen fibers in relation to type-III collagen
fibers in a control group rat. In the groups that received vitamin C, [Fig. 8] and [9], especially in the group that received it subcutaneously, [Fig. 9], the percentage of type-III collagen stands out in relation to type-I collagen.
Fig. 7 Photomicrography of type-I and -III collagen fibers. 400x in histological staining
with pricosirius red in an animal in the control group (GI) on the tenth day.
Fig. 8 Photomicrography of type I and III collagen fibers. 400x in histological staining
with pricosirius red in an animal in the group treated with oral vitamin C (GII) on
the tenth day.
Fig. 9 Photomicrography of type-I and -III collagen fibers. 400x in histological staining
with picrosirius red in an animal in the group treated with subcutaneous vitamin C
(GIII) on the tenth day. Type-I collagen represented by the symbol #. Type-III collagen
represented by the symbol *
Discussion
In our study, the highest amount of type-III collagen was identified in the groups
that received vitamin C, however, with no significant difference. The rate of contraction
of the surgical wound was higher in the groups treated with vitamin C, with a significant
difference between groups I and II (p = 0.001), and between groups I and III (p < 0.001). No significant difference was found between the groups that were treated
with vitamin C (p = 0.227).
The dose of ascorbic acid was chosen based on previous studies, which verified that
these are the minimum concentrations capable of affecting wound healing and that can
be used in humans without leading to toxic and harmful effects when administered.
The surgical technique was chosen, as it is a technique that is easy to reproduce
and standardize, based on previous works, which opted for the incision in the cranial
region, of ∼ 20 mm in length. The technique used to assess the intensity of the inflammatory
process was HE, which is considered the main means of analysis when the objective
of the study is the epithelial tissue.
Regarding the intensity of healing, the most advanced form was found in groups II
and III in relation to the control group, that is, in the group that did not receive
vitamin C supplementation, there was a greater degree of inflammation and a more pronounced
granulation tissue.
The present study was based on previous works, cited in the bibliographic reference,[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55] for the choice and organization of groups, surgical technique, dose of ascorbic
acid, technique for evaluating the inflammatory process and intensity of healing,
which observed a greater number and better arrangement of fibroblasts in animals in
groups II and III when compared with the control group, since the use of ascorbic
acid maintains an adequate concentration of the vitamin in the skin, which stimulates
the proliferation of dermal fibroblasts.
The macroscopic evaluation was necessary since the wound contraction process is the
fourth phase of the healing process and consists of the centripetal movement of the
edges. The phase that precedes the contraction of the wound is that of proliferation,
responsible for the closure of the lesion itself, and it is divided into three subphases,
which are reepithelization, fibroplasia, and angiogenesis. Finally, the remodeling
phase follows that of contraction of the wound and is the last stage of healing.
Conclusion
Ascorbic acid supplementation achieved more effective cranial healing compared with
the group that did not receive vitamin C supplementation. There was no significant
difference in healing between the groups that received oral or subcutaneous vitamin
C.
Regarding the rate of wound contraction, there was a significant difference between
the control group and the groups treated with oral vitamin C (p = 0.001) and subcutaneous vitamin C (p < 0.001). No significant difference was found between the two groups treated with
vitamin C (p = 0.227).
The degree of healing assessed by HE was higher in the groups treated with vitamin
C, but without significant difference between oral and subcutaneous.
The amount of type-III collagen was higher in the groups that received vitamin C,
with a significantly greater difference in the group that received it subcutaneously.
Graph 1 Contraction rate of the wound in percentage observed on day 7 of treatment.
Graph 2 Collagen I area (%) between the groups on the seventh day of treatment.
Graph 3 Collagen III area (%) between groups on the seventh day of treatment.
