Pharmaceutical Biology

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Comparison of collagen content in skin wounds

evaluated by biochemical assay and by computer-
aided histomorphometric analysis

Guilherme F. Caetano, Marcio Fronza, Marcel N. Leite, Ary Gomes & Marco
Andrey Cipriani Frade

To cite this article: Guilherme F. Caetano, Marcio Fronza, Marcel N. Leite, Ary Gomes & Marco
Andrey Cipriani Frade (2016) Comparison of collagen content in skin wounds evaluated by
biochemical assay and by computer-aided histomorphometric analysis, Pharmaceutical Biology,
54:11, 2555-2559, DOI: 10.3109/13880209.2016.1170861

To link to this article: https://doi.org/10.3109/13880209.2016.1170861

Published online: 14 May 2016.

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PHARMACEUTICAL BIOLOGY, 2016
VOL. 54, NO. 11, 2555–2559
http://dx.doi.org/10.3109/13880209.2016.1170861

RESEARCH ARTICLE

Comparison of collagen content in skin wounds evaluated by biochemical assay

and by computer-aided histomorphometric analysis
Guilherme F. Caetanoa*, Marcio Fronzab*, Marcel N. Leitea, Ary Gomesc and Marco Andrey Cipriani Fradea
a
Division of Dermatology, Department of Internal Medicine, Ribeir~ao Preto School of Medicine, University of S~ao Paulo (USP), Ribeir~ao Preto, SP,
Brazil; bDepartment of Pharmacy, University of Vila Velha, Vila Velha, Espirito Santo, Brazil; cDepartment of Biology, University of Vila Velha, Vila
Velha, Espirito Santo, Brazil

ABSTRACT ARTICLE HISTORY

Context: The quantification of total collagen is of major importance in a wide range of research areas, Received 23 October 2015
including the study of cutaneous wound healing and new drugs trials. Revised 22 January 2016
Objective: The total collagen content in skin biopsies was compared by biochemical hydroxyproline assay Accepted 21 March 2016
and by two computer-aided histomorphometric analyses of histological sections. Published online 13 May 2016
Materials and methods: Two methods were used to evaluate collagen formation: the hydroxyproline KEYWORDS
assay, as the gold standard and histomorphometric image analysis of the filled areas by corresponding Cutaneous wound healing;
stained collagen fibres, using picrosirius and Gomori’s trichrome staining. The image analyses were deter- Gomori’s trichrome;
mined by digital densitometry recognition using computer-aided ImageJ software. One-way ANOVA, simple hydroxyproline; picrosirius
linear regression and ANCOVA were applied for the statistical analysis and correlation. red
Results: In a simple linear regression analysis carried out on the 14th day period after the induction of
skin injury, three techniques, picrosirius red (F ¼ 33.57, p ¼ 0.00), Gomori’s trichrome (F ¼ 81.61, p ¼ 0.00)
and hydroxyproline content (F ¼ 16.85, p ¼ 0.00) were able to detect collagen production. After scale
adjustment, there were no significant differences among either the slopes (F ¼ 1.17, p ¼ 0.32) or the inter-
cepts (F ¼ 0.69, p ¼ 0.51) of the estimated regression lines. It seems that a highly significant correlation
exists between the histomorphometrical analysis and hydroxyproline assay.
Discussion and conclusion: The morphometric analysis proved to be adequate and can be used as a sim-
ple, rapid, low-cost technology for evaluating total collagen in cutaneous wound specimens, compared
with the gold standard hydroxyproline assay.

Introduction of the extracellular matrix, which are deposited in the damaged

Wound healing is a dynamic process aimed at restoring the struc-
ture of the injured tissue (Menke et al. 2008; Ranzato et al. 2011).
It can be divided into four overlapping phases: inflammation,
coagulation, tissue formation and tissue remodelling (Menke et al.
2008; Stroncek et al. 2009). The process begins immediately after
injury, with the release of platelet granules, cytokines and growth
factors, which are important in recruiting inflammatory cells to
the wound, an essential cellular influx for local debridement
(inflammatory phase) to degrade foreign particles and to provide
a provisional matrix to further proliferation of fibroblasts leading
to formation of granulation tissue (Gurtner et al. 2008;
Velnar et al. 2009).
The phase of new tissue formation is characterized by the for-
mation of granulation tissue. Intense fibroblast proliferation and
migration occur in order to synthesize the new extracellular
matrix in the damaged tissue area (Stroncek et al. 2009; Velnar
et al. 2009; Guo & Dipietro 2010). Once in the damaged tissue
area, fibroblasts synthesize new matrix elements such as proteo-
glycans, glycosaminoglycans and collagen, the main components
area to replace the initial provisional matrix, comprised initially of
fibrin (Campos et al. 2008; Velnar et al. 2009; Guo & Dipietro
2010). Collagen is the major structural component of granulation
tissue, strengthening the extracellular matrix. The amino-acid pro-
line is an important component of the collagen fibre, and hydrox-
yproline is a biochemical marker for collagen tissue, as a positive
indication of the progression of healing (Kokane et al. 2009;
Nayak et al. 2011).
Studies on new therapeutic healing substances have gained
importance in recent years because of the long treatment time
and high costs of medical care associated with extensive or long-
term wound therapy (Mulder et al. 2012; Moura et al. 2013).
Some substances can stimulate fibroblast migration and/or prolif-
eration, resulting in accelerated accumulation of extracellular
matrix produced by these cells during the wound healing process.
These events are important for tissue formation and the subse-
quent remodelling phase (Mulder et al. 2012).
Although there are studies in the literature on the quantifica-
tion of the new collagen formation during the wound healing pro-
cess by biochemical and/or morphometrical methods, reports

CONTACT Prof. Dr. Marco Andrey Cipriani Frade prof.marcoandrey@gmail.com Department of Internal Medicine, Division of Dermatology, Ribeir~ao Preto School
of Medicine, University of S~ao Paulo (USP), Av. Bandeirantes, 3900, Postcode 14049-900, Monte Alegre, Ribeir~ao Preto, SP, Brazil
*These authors contributed equally to this work.
ß 2016 Informa UK Limited, trading as Taylor & Francis Group
2556 G. F. CAETANO ET AL.

comparing the two methods are rare (Kliment et al. 2011; Minossi were mounted on glass slides, dewaxed, rehydrated and stained
et al. 2014). Therefore, the aim of this study was to compare, separately with Gomori’s trichrome (GT) and picrosirius red
quantitatively and objectively, the total area occupied by collagen (PR) for the analysis of collagenesis, with one glass slide to
fibres in cutaneous wound specimens in Wistar rats, using the each staining on all post wounding days.
biochemical hydroxyproline assay and computer-aided histomor-
phometric analysis.
Histomorphometric analysis of collagenesis by image
analysis
Materials and methods
The slides of both staining in each day were examined and photo-
Animals micrographed in a blinded fashion using a digital camera (LEICA
DFC 280, Wetzlar, Germany) attached to a light microscope
Protocols involving the use of animals were performed according
(LEICA DM 4000B, Wetzlar, Germany). Six images per slide were
to the ethical guidelines of the Brazilian College of Animal
captured at 100 magnification (three fields from the epidermis-
Experimentation (COBEA), and approved by the Ethics
upper dermis and three fields from the lower dermis–hypodermis)
Committee on Animal Experimentation (CETEA), of the
for both histological staining. The colour deconvolution ImageJ
Ribeir~ao Preto School of Medicine, University of S~ao Paulo
software was used to evaluate the percentage of blue staining for
(FMRP-USP), registry number 085/2011. Forty adult male
GT and of red staining for PR, in the whole area of each image,
Wistar rats (180–200 g), aged 7–8 weeks were obtained from the
with the dye indicating the presence of collagen fibres in the tis-
central Bioterium of the Medical School, Ribeir~ao Preto,
sue. The morphometric analysis corresponding to the blue and
University of S~ao Paulo (FMRPUSP). In order to prevent skin
red colours was measured as the percentage of total pixels in each
lesions from fighting males, which could interfere with wound
image, using the Threshold (ImageJ software), as described by
repair, the animals were housed individually, at least one week
Caetano et al. (2015). Data were reported as the pixel average of
before, and throughout the entire experimental period. The ani-
ten samples per day.
mals were maintained under standard laboratory conditions with
a 12 h light–dark cycle and free access to food and water.
Hydroxyproline content (HyP)
Surgical procedure and groups Collagenesis was also measured using a biochemical hydroxypro-
line assay, according to the procedures described in the literature,
Immediately before excisional wound induction, the animals were
but with minor modifications (Reddy & Enwemeka 1996; Reddy
weighed and deeply anesthetized by intraperitoneal (i.p.) adminis-
et al. 2008). Samples were incubated at 60  C in open microtubes
tration of a combination of ketamine (80 mg/kg) and xylazine
for 15 h to obtain the dry weight, cut using a Polytron tissue
(15 mg/kg). The animals backs were shaved and swabbed with
homogenizer with 6 N hydrochloric acid solution (100 lL per
70% ethanol for asepsis; two circular full-thickness excision
milligram of dry tissue) and incubated for 4 h at 130  C to pro-
wounds were made on the dorsum cervical region with a sterile
mote acid hydrolysis. Afterwards, the pH was adjusted to 7.0 at
histological punch (1.5 cm diameter) comprising all the skin
room temperature. Hydroxyproline standard solutions were pre-
layers. Samples of the excised skin were saved for subsequent
pared at concentrations of 1.0–100 lg/mL and 10 lL of standard
histological and biochemical assays, representing samples of basal
and samples were transferred to a 96-well microplate. Then, 90 lL
collagen content in the rat skin (day 0).
of 0.056 M chloramine-T solution and 100 lL of Ehrlich’s reagent
The postoperative periods were defined according to the day
were added to each well. The absorbance values were measured at
of euthanization, as per standard protocols (n ¼ 10 rats each post-
550 nm and compared to those of the standard curve to deter-
operative days) (Fronza et al. 2014; Caetano et al. 2015). Both
mine the concentration of hydroxyproline in the samples. The
wounds of each animal were treated with 0.9% saline solution
concentrations of hydroxyproline in tissue homogenates were
immediately after wound induction and, at the same time, each
determined per volume of HCl used, and finally per milligram of
day thereafter. The wounds of all the animals were covered with
dry tissue, based on a standard curve.
gauze and tape, which were changed daily.

Statistical analysis
Harvesting of the material for study
Data were expressed in scatter graphs and bar graphs expressed
The animals were euthanized on the 2nd, 7th, 14th and 21st post-
as mean ± standard error of mean (SEM) for the quantitative vari-
operative days with an overdose of anaesthetic, and the wounds/
ables performed using GraphPad software (San Diego, CA).
scars were cut with a sterile biopsy punch (1.5 cm diameter). One
Statistical correlation between the histomorphometrical analysis of
wound from each animal was frozen in liquid nitrogen and stored
collagenesis by image analysis and hydroxyproline content were
at 80  C for biochemical assay (hydroxyproline), and the other
determined using one-way ANOVA, simple linear regression, and
wound was used for the histological analysis.
ANCOVA run in Systat 12.0 (Wilkinson 2007). For both meth-
ods, the null hypothesis was rejected when p  0.05.
Histological processing
The wound biopsies were fixed with buffered formaldehyde Results
solution at 3.7% (pH 7.4) on days 0, 2, 7, 14 and 21 post-
Collagenesis by quantitative image analysis
wounding, and processed according to the standard routine
light microscope tissue protocols. The processed tissues were Collagen synthesis, deposition and degradation are important
embedded in paraffin, (GT) and serial sections 5.0 lm thick events that occur during the entire cutaneous wound-healing

processes. Over the experimental period, we observed that the
cutaneous wounds showed a marked and robust increase in the
deposition and organization of collagen fibres, detected after
staining with PR and GT in the wound biopsy (Figures 1 and 2).
Figure 1 shows photomicrographs of each day of histological anal-
yses for GT, in which the collagen is represented by blue-coloured
tissue, and PR is represented by red-coloured tissue.
Histomorphometric analysis of collagenesis by image analysis
from the skin revealed an expected decrease in the collagen
fibres in the wounds on day 2, compared with day 0 (Figure
2). The density of collagen fibres on day 7 post-wounding were
higher than on day 2, and similar to the physiological levels
observed on day 0, increasing still further after 14 d.
Interestingly, on day 21, the concentration of collagen fibre
slightly decreased, as a result of tissue remodelling, and seemed
better organized and distributed compared to previous days
(Figures 1 and 2).
Figure 1. Collagen accumulation in wound areas at day 0, 2, 7, 14 and 21 after wounding. Representative photomicrograph of wounds tissue sections stained with
Gomori’s trichrome (GT) and picrosirius red (PR) staining (100). Note the collagen intensity and disposition of fibres stained blue and red, respectively.
Figure 2. Quantification of collagen accumulation in the wound specimen on days
0, 2, 7, 14 and 21 post-wounding. Collagen content was measured by digital
densitometry using ImageJ in sections of wound tissue stained with picrosirius red
staining (open bars) and Gomori’s trichrome (dashed bars). The results show the
collagen content in each specimen as a percentage of total area. The data repre-
sent the means ± SEM (n ¼ 10 wounds/group). *The statistical evidence (p < 0.05)
was analyzed by one-way-ANOVA.
PHARMACEUTICAL BIOLOGY 2557
Measurement of hydroxyproline content in the wounds
The results of the hydroxyproline concentration in the skin dis-
play similar pattern to those results observed in the histomorpho-
metrical analysis. At day 2 post-wounding, the hydroxyproline
content in the skin was significantly lower (p < 0.05) than on day
0. After 7 and 14 d, a continuous increase in hydroxyproline was
observed. Following the same fact as observed in the histological
slides, on day 21, the collagen content declined slightly, to levels
similar to those observed on day 0 (Figure 3).
Correlations between methods
Percentage data obtained from the image measures were trans-
formed into the arcsine of the square root of their respective pro-
portions. In order to adjust the scale for data comparisons, all
measurements were standardized through ranging (Zar 2010). The
Figure 3. Determination of collagen accumulation in wound biopsies at day 0, 2,
7, 14 and 21 after injury. Determination of wound hydroxyproline content as an
indicator of collagen levels in lg of hydroxyproline/mg of dry wound tissue. The
data represent means ± SEM (n ¼ 10 wounds/group). *Statistical evidence was
(p < 0.05) analyzed by one-way ANOVA.
2558 G. F. CAETANO ET AL.
assumption of normality for colour measurements in both the
staining and the biochemical techniques were verified by Lillefors
test, and the null hypothesis of normality was rejected for
p  0.05 (Zar 2010). After transformation and standardization of
the colour measurements, they were distributed normally for PR
(p ¼ 0.27) and GT staining (p ¼ 0.14). Differences in capability of
collagen detection among the three techniques were tested
through two-way analysis of variance (ANOVA), and the predict-
ability of collagen production over the period 2–14 d was verified
through simple linear regression taking collagen production as a
function of time in days of observation (Zar 2010). Differences
among the slopes and the intercepts of the estimated regression
lines were tested through analysis of covariance – ANCOVA.
Two-way ANOVA, simple linear regression and ANCOVA were
run in Systat 12.0 (Wilkinson 2007), and for both of them, the
null hypothesis was rejected when p  0.05).
In relation to collagen detection power, there were no signifi-
cant differences among the measurements obtained through the
three techniques in each treatment, from normal skin (day 0) or
injured skin, over a 21-d period of observation (FANOVA ¼ 0.02,
df ¼ 2;70, p ¼ 0.99, r ¼ 0.80, r2 ¼ 0.63). Even when the techniques
were contrasted without considered the healing phases, no signifi-
cant difference could be found among them (FANOVA ¼ 0.03,
df ¼ 2;72, p ¼ 0.92, r ¼ 0.80, r2 ¼ 0.63). The only significant differ-
ences found were due to the wound healing phase after injury
(FANOVA ¼ 124.14, df ¼ 1;74, p ¼ 0.00). However, the estimated
linear model had a non-expressive capability to explain the nat-
ural variance in the original data (r ¼ 0.16, r2 ¼ 0.02), since the
normal skin (day 0) and the skin 21 d after injury were outside
the linear trend in collagen production (Figure 4).
Figure 4. Correlation between methods. Collagen production measured by the
Hydroxyproline production assay (-•-), Picrocirius red (-~-) and Gomori’s trichrome
(- -•- -).
Table 1. Statistical parameters of the estimated regression lines for the three techniques used to assess collagen production in the regeneration
process.
Parameters Picrosirius red
Slope (CI 95%) 026 (0.017–0.036, p < 0. 01)
Intercept (CI 95%) 0.582 (0. 493–0. 672, p < 0. 01)
r 0.85
r2 0.72
In the simple linear regression analysis during 14 d after skin
injury (2, 7 and 14 d post-wounding), the three techniques, PR
(F ¼ 33.57, p ¼ 0.00), GT (F ¼ 81.61, p ¼ 0.00) and hydroxyproline
content (F ¼ 16.85, p ¼ 0.00), were able to detect a growing colla-
gen production (Table 1). After scale adjustment for comparisons,
there were no significant differences among either the slopes
(F ¼ 1.17, p ¼ 0.32), or the intercepts (F ¼ 0.69, p ¼ 0.51) of the
estimated regression lines. Therefore, the three techniques were
equally sensitive in detecting collagen synthesis (Table 1 and
Figure 4).
Discussion
In an animal model of cutaneous wound healing, it is important
to measure new collagen formation and its quality. Collagen is the
major component of the connective tissue. The wound-healing
processes depend on the right metabolism of collagen, which
comprises the regulatory production, deposition and subsequent
maturation of collagen. These events are of vital interest not only
in cutaneous wound healing but also in a number of clinically
important diseases that are characterized by an accumulation or
loss of tissue collagen. On one hand, excessive production of col-
lagen has been documented in many disorders, such as liver cir-
rhosis, lung fibrosis, and hypertrophic and keloid scars. On the
other hand, loss of tissue collagen has been observed in certain
disorders of the connective tissue, including rheumatoid arthritis
and wound/ulcer damaged tissues (Milner & Cawston 2005;
Wynn 2008; Shih et al. 2010).
In some clinical situations, such as tissue repair and wound
healing, the production and deposition of collagen during the ini-
tial stages of healing are required to heal the damaged tissues
(Frade et al. 2001, 2004). Hence, collagen plays a vital role in
maintaining structural integrity and in determining tissue func-
tion. Considering the important role of collagen in healing, there
is a demand for techniques for its evaluation and quantification
(Yung-Kai & Che-Yung 2010).
The analysis of hydroxyproline in biological tissues is consid-
ered the gold standard method for routine measurement of colla-
gen content in extracts of various tissue specimens (Reddy &
Enwemeka 1996). Despite the numerous assay procedures
described for hydroxyproline analysis, this assay has its limitations
in regard to specificity, sensitivity, reproducibility, accuracy and
practical approach. In general, the procedures are laborious and
involve many time-consuming steps (Dang et al. 2005; Ignat’eva
et al. 2007; Kliment et al. 2011).
In this study, the correlations between hydroxyproline concen-
trations and collagen content measured by digital densitometry in
wound tissue sections stained with PR and GT were equally sensi-
tive. In relation to collagen detection power, there were no signifi-
cant differences among the measurements obtained through the
three techniques in each blocked treatment, from normal skin
(day 0) or injured skin, over a 21-d long period of observation.
Even comparing the techniques without considering the healing
phases, no significant differences were seen among them.
Gomori’s trichrome Hydroxyproline
030 (0.023–0.037, p < 0. 01) 040 (0.019–0.061, p < 0. 01)
0.511 (0. 446–0.557, p < 0. 01) 0.483 (0.2913–0.674, p < 0. 01)
0.93 0.76
0.86 0.56

Histomorphometric analysis by histological sections has been
proven to be a simple, rapid assay that uses low-cost technology
and materials. However, there is a very important standardization
step during the quantification that researchers need to bear in
mind. In this study, the authors used the ImageJ software to work
on all the images and quantifications for both stained slides. The
colour deconvolution plugin from ImageJ software was used to
evaluate the blue and red staining percentage (collagen) in the
image area from GT and PR, respectively. The plugin recognizes
the image colours and decomposes them into another three basic
images. The blue image for GT and the red image for PR should
be used for the quantification. The morphometric analyses, corre-
sponding to the blue or red colours, were measured as the per-
centage of total pixels in each image using the ‘Threshold plugin’,
also from ImageJ software, as described previously by Caetano
et al. (2015). Two bars to adjust the colour intensity in each
image are created by the Threshold plugin. This is the crucial step
for obtaining the real result, because both bars need to be stand-
ardized prior to start the quantification, otherwise researchers
could obtain unrealistic data.
Perini et al. (2015) also used histomorphometric analysis and
the biochemical assay to determine collagen deposition in sam-
ples. The authors demonstrated the similarity of the two methods.
Pessoa et al. (2015) also used GT and PR staining to evaluate new
collagen in rat skin samples. The authors reported good efficacy
of the method to achieve realistic and satisfactory data.
Conclusion
In conclusion, a simple, convenient and reliable computer-aided
histomorphometric method for the determining total collagen in
cutaneous wound specimens has been proven to be comparable to
the gold standard hydroxyproline assay. The methods proved to
be equivalent, therefore, the choice of the methodology will
depend on the laboratory conditions and infrastructure. It is
important to note that both methods are considered useful tools
in clinical and biomedical research.
Disclosure statement
The authors report that they have no conflicts of interest.
Funding information
The authors gratefully acknowledge FAPESP, CAPES, FAEPA-
HCFMRP-USP, FAPES and CNPq for their financial support.
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