EFFECT OF POLYGLECAPRONE 25 AND NYLON WITH CONTINUOUS

SMALL STITCH TECHNIQUE ON COLLAGEN TYPE I/III EXPRESSION

RATIO IN RATS
Abstract
Background: Surgical wound dehiscence is one of the most common complications
following abdominal wall incision with high rates of recurrence. The thread material and
the suturing methods used are also important factors in the incidence. The ratio of types 1
and 3 collagen is a factor that plays an important role in the process of wound healing.
Good wound healing will actually prevent complications such as surgical wound
dehiscence.
Objective: Surgical wound dehiscence is one of the most common complications following
abdominal wall incision. The ratio of collagen type 1 and 3 expression plays an important role in
the wound healing process. We aimed to compare the effect of polyglecaprone 25 and nylon on
the level of collagen type 1 and 3 ratio on the abdominal skin incision line of Wistar albino rat
(Rattus norvegicus).
Materials and Methods: Forty rats were divided into four groups. Groups 1 and 3 were sutured
with polyglecaprone 25. Groups 2 and 4 were sutured with nylon. Then, groups 1 and 2 were
euthanized on day 4th, while groups 3 and 4 were euthanized on day 7th. Collagen type 1,
collagen type 3 and the ratio of collagen type 1 and 3 were measured. Samples of skin (5 x 10
mm) were collected for analysis of collagen count using immunohistochemistry staining method.
The differences were then analyzed with independent t-tests.
Results: There were no significant differences of collagen type 1 and 3 ratio expressions in rats
stitched by continuous small stitch method with polyglecaprone 25 and nylon on day 4
[(0.68±0.16) vs (0.67±0.16) p= 0.853], but there were significant differences on day 7
[(1.43±0.47) vs (0.94 ±0.39) p= 0.021].
Conclusion: Collagen 1 and 3 ratio expressions in rats stitched by polyglecaprone 25 were
significantly higher than rats stitched by nylon. The use of polyglecaprone 25 is more
recommended in stitching the abdominal skin to reduce wound dehiscence complications.
Keywords: polyglecaprone 25, nylon, collagen type 1 and 3 ratio, continuous small stitch

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Background

Wound healing remains a problem that still can potentially arise in every abdominal
surgery. Complications can occur such as surgical wound infection which may continue to
wound dehiscence even though all the proper prevention efforts are done 1. The risk of infection
in surgical wounds is determined by technical problems in the operation process, especially
bleeding, the amount of damaged tissue, and wound drainage, as well as other metabolic factors.
Surgical wound infection can be the beginning of surgical wound dehiscence which can be fatal
for some patients2.
The definition of abdominal surgical wound dehiscence is the separation of the
abdominal lining after surgery, which can occur completely or partially, without complete
healing of the skin with or without protrusion of the contents of the stomach out of the
abdominal cavity3. The incidence of these cases is 0.4% - 3.5% after major abdominal surgery
with a mortality rate of 10% -45%4. Costs incurred due to dehiscence treatment costs, re-
operation costs, complications handling costs and length of stay in the hospital will increase 5.
Wound dehiscence occurs in 0.4-1.2% of patients after elective laparotomy, but can account for
up to 12% in emergency procedures. The diagnosis of wound dehiscence is based on clinical
signs. Abdominal pain and excessive tenderness, as well as unexplained vomiting and peritoneal
exudation also indicate wound dehiscence. Radiographs can also confirm dehiscence in the tissue
in the abdominal cavity6.
The median incision is closed with continuous sutures and has a recommended thread
length to wound length ratio of at least 4. This ratio can be achieved if the interval between
stitches in the wound is reduced by small stitches. This study shows that the interval between
sutures affects wound healing7. In any clean abdominal surgery, the skin incision must be sutured
to restore the function of the skin, while also treating bleeding and reducing postoperative pain.
Various suture techniques and materials have been used for skin closure in laparotomy 8. The
quality of the surgical suture technique is very important to prevent complications in the median
incision wound during laparotomy9.
The outcome of postoperative wound healing is determined by the material and skin
closure technique. Thread material is considered appropriate if there is minimal foreign body
reaction and inflammation. Meanwhile, the suturing technique is considered adequate if it is able
to close the empty space in the subcutaneous tissue and does not cause tension on the wound
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edges8. The suturing techniques for skin incision wounds that have been used are simple (one -
one), continuous suturing techniques, and horizontal or vertical mats10.
Absorbable threads do not increase the risk of wound dehiscence in skin sutures but
actually reduce the risk of wound dehiscence than using non absorbable threads 11. Thread
material can be either natural or synthetic, which is absorbable and non-absorbable.
Polyglecaprone 25, polydioxanone, and polyglactin are categorized as absorbable threads.
Meanwhile, non-absorbable threads encompass polypropylene, nylon, and silk12.
Several suture techniques for median abdominal incision wound closure have been
studied in recent decades. The findings from a meta-analysis of stitching techniques have shown
that the definition of large stitching is the interval of 1 cm between stitches, while the small
stitching is an interval of 5-8 mm. Small stitch continuous suture technique can reduce incisional
hernia incidence and surgical wound dehiscence compared to large stitch13. Closure of a
laparotomy surgical wound ending with a small stitch technique caused significantly smaller
incisional hernias and fewer surgical wound infections than the large stitch 14. This finding made
the researchers interested in using small stitch as a sewing technique.
Monofilament threads are more resistant to microorganisms than multifilament thread.
Closure of the abdominal wall can use nylon, polypropylene, or polydioxanone (PDS) no.1.
Complications of postoperative suture infection and surgical wound pain using polydioxanone
are lower than using non absorbable sutures15. Gaikwad et al. in 2009 compared median
laparotomy closure using nylon and PDS. The PDS group had as many as 17% complications of
surgical wound dehiscence, while the nylon group had no surgical wound desicence16.
Collagen plays an important role in the healing process of postoperative wounds in the
skin. Collagen is the main component of the extracellular matrix and functions to maintain tissue
elasticity and tensility. There are two types of collagen that are of concern to researchers and
their functions in wound healing and distribution have been widely studied, namely collagen type
1 and collagen type 3. Type 1 collagen is strong collagen that is widely distributed in the human
body including fascia, skin, ligaments and fibrous tissue and responsible for the mechanical
resistance of the network. Meanwhile, collagen type 3 appears at the beginning of wound healing
with less amount and less strength. As the wound healing process occurs, type 3 collagen is
replaced by type 1 collagen which is stronger. The ratio of types 1 and 3 collagen forms the basis
of the biomolecular understanding of wound healing in the skin. Accordingly, the ratio of

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collagen type 1: 3 plays an important role as a parameter in postoperative wound healing 17. This
is the basis for researchers to make the collagen ratio 1: 3 as the dependent variable in this study.
The level of cellular activity of fibroblast cells can describe the process of wound healing
and tissue repair that occurs after the surgical incision18. Based on these studies, the researchers
aimed to investigate the effect of thread type with small stitch interval on wound closure after
abdominal skin incision on the level of collagen type 1 and 3 ratio in Wistar albino rats.

Methods
The research subjects were albino rats Wistar strain (Rattus norvegicus) weighing 170-
200 grams and aged 2-3 months. The Wistar strain albino mice were obtained from the
University experimental animal development unit by a laboratory officer. This research was
conducted at the Integrated Laboratory and Testing in March 2018-May 2018. The exclusion
criteria in this study were illness during the 7-day adaptation period, infection during treatment,
and death during the treatment. All of the rats were maintained in cages. One cage housed a total
of 5 rats. All rats were given food and drink everyday with standard protocol. The samples were
selected from the population using simple random sampling technique by drawing done by the
laboratory officer.
The total sample size was 40 individual subjects to maintain a high representative level in
the event of a drop out, the sample was widened to 10 individuals per group. Each mouse was
assigned a number marked on an anklet. The numbers were sorted from 1 to 40. Then a roll of
paper numbered 1 to 40 was made, rolled up and put into a bottle then shaken and drawn. The
first 10 numbers that came out were grouped as group 1, namely the group of mice sewn with
PG-25 thread and euthanized on the 4th day. The third 10 numbers that came out were grouped
as group 3, namely the group of rats sewn with PG-25 thread and euthanized on the 7th day. The
fourth group was sewn with nylon and euthanized on the 7th day.
All surgical procedures were performed under sterile conditions by animal laboratory
assistants. All rats were subjected to an incision in the skin with a linear shape measuring 6 cm in
the midline of the ventral linea alba. Sewing involved the myofascial layer and the skin was
closed separately. All sutures were placed at 5mm from the wound edge with a distance of 5mm
between the cuts. The technique of suturing the wound with continuous sutures and wound
closure material was using either PG-25 or Nylon thread. Researchers conducted a pre-research

4
test before conducting the research. This pre-study test was done to determine whether the mice
treated according to the abdominal skin injury model would actually experience the abdominal
skin healing process, as evidenced by the histopathological test of preparations stained with DAB
(3,3'-Diaminobenzidine). The results of the histopathological test were read by a pathologist. All
histopathological changes in skin tissue were documented.
Each Wistar rat that was marked according to the treatment group was anesthetized with
ketamine at a dose of 60 mg/kgBW intramuscularly in the medial thigh. The rats were sedated,
then underwent an aseptic antiseptic procedure and continued with the incision according to the
wound model. After that, suturing the fascia and skin wounds was done according to the study
design. During the treatment process the mice were monitored for vital signs to ensure that they
were still breathing. After the rats were conscious and it was confirmed that they were still alive,
they were transferred to the cage according to the group and treated according to the specified
number of days, namely 4 days or 7 days. Each cage contained 5 mice. During the treatment, the
mice were given processed feed consisting of corn, bran and flour in accordance with the
laboratory standards used. Euthanasia used saturated chloroform which was put into a large jar
with a diameter of 40 cm and was airtight so that it did not endanger the researchers. Each
terminated mouse was put in a jar and died within about 1 minute. Each dead mouse was given
Chinese ink on the sutured incision line and then cut from the skin to the peritoneum with a size
of 1x1 cm then immediately put in a jar containing 10% formalin. In less than 3 days the samples
entered into the Anatomical Pathology laboratory for making paraffin blocks. Collagen
examination by immunohistochemical methods used ULTRATEK HRP Anti-polyvalent (DAB)
Staining Complete System. This method is most often used on paraffin pieces of objects that are
adequately fixed (at least 24 hours but ideally 1 or 2 weeks) in a neutral formaldehyde solution.
The result is that in a microscopic light field, brown collagen appears in the cytoplasm of cells in
the skin against a blue background. Prepared photos were taken at 400x magnification using a
SIGMA camera with three shots.
Measurement of collagen levels used the Adobe Photoshop CC 2017 based image
analysis program. This software can recognize, select and separate colors based on the RGB
spectrum, so by using this program categorical data can be converted into numeric.
The data obtained were presented as mean ± standard deviation (SD), then the data were
statistically analyzed using an independent t-test to compare the means of the two groups. The

5
results of statistical analysis with p <0.05 were defined as statistically significant results. All data
were processed using commercial software SPSS 21 (IBM Corp., Armonk, NY).
The research protocol was submitted to the Medical and Health Research Ethics
Committee of the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada,
Yogyakarta, Indonesia. Research was conducted after obtaining an ethical eligibility letter.
Before conducting the research, the writer submitted an ethical feasibility assessment to the
Medical and Health Research Ethics Committee accompanied by a copy of the proposal. Our
methods were reported in accordance with the Animals in Research: Reporting In Vivo
Experiments (ARRIVE) guidelines19.

Results
Observations were done on the mice with wounds in the skin of the abdomen sewn with
PG-25 thread and Nylon thread on the fourth and seventh days. Observations showed that there
are no complications such as hematoma, infection, seroma, or wound dehiscence. Euthanasia was
performed by asphyxiation and decapitation on the fourth and seventh days and then skin
samples were taken as large as 5x10 mm in two places to analyze the amount of collagen
expressions of type 1 and 3 using the immunohistochemical staining methods.

Figure1. Type 1 collagen expression (twisted brown designated red arrow) with PG-25 thread
with small continuous stitch.
6
Figure 2. Expression of type 1 collagen (twisted brown pointed red arrow) with Nylon thread
with small continuous stitch.

Figure 3. Expression of type 3 collagen (twisted brown designated red arrow) with PG-25 thread
with small continuous stitch.

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Figure 4. Expression of type 3 collagen (twisted brown designated red arrow) with Nylon thread
with small continuous stitch.

Figures 1, 2, 3 and 4 show the expressions of microscopic images of abdominal skin

sutured at continuous small stitch intervals seen at 400X magnification. Positive expression
contained the cytoplasm of cells expressing collagen types 1 and 3 which appear dark brown.

Table 1. Comparison of collagen type 1 and type 3 expression between PG-25 and Nylon
threads on the 4th and 7th days
Variable Mean ± SD p* 95% CI
Lower Limit Upper Limit
Collagen type 1 0.074 -0.82 15.95
th
4 day
a. PG-25 Thread 50.43 ± 10.78
b. Nylon Thread 42.86 ± 6.57
Collagen type 3 0.15 - 4.34 26.20
th
4 day
a. PG-25 Thread 75.73 ± 21.63
b. Nylon Thread 64.80 ± 7.78
Collagen type 1 0.149 -2.44 14.84
7th day
a. PG-25 Thread 55.83 ± 8.90
b. Nylon Thread 49.63 ± 9.48
Collagen type 3 0.087 -39.25 2.98
7th day
a. PG-25 Thread 42.99 ± 15.32
b. Nylon Thread 61.13 ± 27.14
* Independent T-test; CI, confidence interval; SD, standard deviation
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 Table 1 shows that on the 4th day, the expression of collagen type 1 using PG-25 thread
(50.43 ± 10.78) was higher than suturing with nylon thread (42.86 ± 6.57) with p = 0.074. On the
7th day, the expression of collagen type 1 using nylon thread (49.63 ± 9.48) was lower than
suturing with PG-25 thread (55.83 ± 8.90) with p = 0.149. The two differences were not
statistically significant (p >0.05).
The expression of collagen type 3 using PG-25 thread on the 4 th day (75.73 ± 21.63) was
higher than that of nylon thread (64.80 ± 7.78) with p = 0.15. On the 7th day, the expression of
collagen type 3 (42.99 ± 15.32) was found to be lower than the results of suturing using nylon
thread (61.13 ± 27.14) with p = 0.087. These two differences were not statistically significant (p
>0.05).
The difference between the expression of collagen type 1 and 3 ratio between PG-25 and
nylon threads in the suturing of the abdominal skin incision line of the Wistar albino rats using
continuous small stitch technique on the 4th and 7th days is shown in Table 2.

Table 2. Comparison of Collagen Type 1 and 3 Ratio Expression on Day 4 and 7

Variable Mean ± SD p*

Mean of collagen 1:3 ratio day 4 0.853

a. PG-25 0.68 ± 0.16
b. Nylon 0.67 ± 0.16

Mean of collagen 1:3 ratio day 7 0.021

a. PG-25 1.43 ± 0.47
b. Nylon 0.94 ± 0.39

* Independent T-test; SD, standard deviation

Table 2 shows that the mean ratio of collagen type 1 and 3 on day 4 was found to be
higher in subjects stitched with PG-25 group than nylon group. However, statistically the
difference of the ratio expression was not statistically significant with p = 0.853 (p >0.05).

9
 Moreover, on day 7 the mean ratio of collagen type 1 and 3 was found to be higher in the
PG-25 group than nylon group and statistically the difference of the ratio was significant with p
= 0.021 (p <0.05).

Discussion
In this study, the expression of collagen type 1 on day 7 after abdominal incision in rats
sutured with PG-25 and Nylon was higher than the expression of collagen type 1 on day 4.
Meanwhile, collagen type 3 on day 7 sewn with PG-25 and Nylon were expressed higher than
collagen type 3 sewn with PG-25 and Nylon on day 4. The study result might indicate that the
expression of collagen type 1 was higher when using PG-25. This could be because the
transitional phase from hemostasis to the inflammatory phase with PG-25 was superior although
this difference was not statistically significant. A study in 2015 stated that the use of slow
absorbable and monofilament threads is recommended over fast absorbable and multifilament
threads14. Likewise, a meta-analysis study stated that the incisional hernia incidence decreased
significantly with the use of non-absorbable sutures 15. The anomalous phenomenon in the data
obtained in this study can be caused by the method of data collection during the transition period
of hemostasis and inflammation so that the synthesis of collagen type 1 and the transformation of
collagen type 3 into collagen type 1 was not completed, but the amount of collagen type 1 still
increased with the increasing days of wound healing. The current hypothesis is that fast-
absorbable threads are superior in the early phase of wound healing because they are absorbed
more quickly by the body, so that the foreign bodies that are recognized by the body will not last
long and the inflammatory process that interferes with the wound healing process will not last
long either.
This study result shows that over time there was an increase in the ratio of collagen type
1 to 3. An increase in the ratio of collagen type 1 to 3 was shown to significantly reduce the risk
of surgical wound dehiscence. Collagen type 1 is a strong collagen that is widely distributed in
the human body including fascia, skin, ligaments, and fibrous tissue and is responsible for tissue
mechanical resistance. Meanwhile, collagen type 3 appears at the beginning of wound healing
with less amount and less strength. As the wound healing process occurs, collagen type 3 is
replaced by collagen type 1 which is stronger18.

10
 The expression of collagen type 3 in rats sewn with PG-25 and Nylon continued to
gradually decrease from day 4 to day 7. The mean level of collagen type 3 appeared higher on
day 4 when compared to day 7. This finding might be due to collagen type 3 is formed first,
which then during maturation phase will form collagen type 1. Accordingly, on the seventh day
the amount of collagen type 3 was less than the fourth day. A study conducted in Germany
reached the conclusion that incisional hernia incidence would be influenced by an increase in
collage type 3 resulting in a decrease of the collagen type 1:3 ratio. This result might be related
to impaired collagen synthesis with poor tensile strength of collagen type 3. Compared to
collagen type 1 which is contained in tissues other than bone, collagen type 3 is an important
component of reticular fibers in the interstitial tissues of the lung, liver, dermis, spleen, and
vessels. These homodimeric molecules also frequently contribute to mixed fibrils with collagen
type 1 and are also abundant in elastic tissue 20. A study conducted in 2015 supported the
superiority of short stitch suture technique to prevent the incidence of incisional hernia in the
long term. This is in accordance with the suture method used in our study that imply the usage of
short stitch suture might have a beneficial effect on the outcome of abdominal wall closure21.
This study result concludes that in the transitional phase of hemostasis to the
inflammatory phase, PG-25 threads were superior in terms of higher collagen type 1:3 ratio on
day 4 although this difference was not statistically significant. Whereas, on the 7th day the
expression of collagen type 1: 3 ratio with PG-25 thread showed a significant difference. When
entering the proliferation phase, the decreased wound strength can increase the risk of an
incisional hernia so that the collagen synthesis process will be disrupted. A study in china
produced similar results in terms of a low collagen type 1 and 3 ratio that appears at the onset of
injury. A low collagen type 1 and 3 ratio is associated with higher flexibility in the wound. The
more a wound heals, the higher the ratio of collagen 1 and 3 indicates increased collagen type 1
in a healing wound. The longer the ratio will remain stable, this will result in the limited
sensitivity to growth factors so that there is no hypertrophic wound healing22.

Conclusion
Collagen 1 and 3 ratio expressions in rats stitched by polyglecaprone 25 were
significantly higher than rats stitched by Nylon. Our findings imply the usage of polyglecaprone
25 might have a beneficial effect on reducing wound dehiscence complications.There was no

11
significant difference in the value of the 1: 3 collagen ratio on the 4th day, but there was a
significant difference on the 7th day after in mice sutured with PG-25 and Nylon thread using the
continuous small stitch suture technique.

Conflict of Interest
There was no conflict of interest in this research.

Funding
This experiment was conducted with support from individual funding.

Data Statement
The data that support the findings of this study are available from the corresponding author upon
reasonable request.

Provenance and peer review

Not commissioned, externally peer-reviewed

References
1. Aksamija G, Mulabdic A, Rasic I, Aksamija L. Evaluation of Risk Factors of Surgical
Wound Dehiscence in Adults After Laparotomy. Med Arch (Sarajevo, Bosnia
Herzegovina). 2016;70(5):369–72.
2. Szczęsny W, Szczepanek J, Tretyn A, Dąbrowiecki S, Szmytkowski J, Polak J. An
analysis of the expression of collagen i and III genes in the fascia of obese patients. J Surg
Res. 2015;195(2):475–80.
3. Van Ramshorst GH, Eker HH, Hop WCJ, Jeekel J, Lange JF. Impact of incisional hernia
on health-related quality of life and body image: A prospective cohort study. Am J Surg.
2012;204(2):144–50.
4. Khorgami Z, Shoar S, Laghaie B, Aminian A, Hosseini Araghi N, Soroush A.
Prophylactic retention sutures in midline laparotomy in high-risk patients for wound
dehiscence: A randomized controlled trial. J Surg Res. 2013;180(2):238–43.
5. Gomes A, Teixeira C, Ferraz R, Prudencio C, Gomes P. Wound-healing peptides for
treatment of chronic diabetic foot ulcers and other infected skin injuries. Molecules.
2017;22(10):1–18.
6. Slater NJ, Bleichrodt RP, Van Goor H. Wound dehiscence and incisional hernia. Surg
(United Kingdom). 2012;30(6):282–9.
7. Harlaar JJ, Van Ramshorst GH, Jeekel H, Lange JF. Effect of stitch length on wound
complications. Arch Surg. 2010;145(6):599.
8. Choudhary A, Bansal N, Chaudhari P. Closure of Pfannenstiel skin incisions in cesarean
sections: comparison of wound outcomes with interrupted mattress vs. subcuticular suture.
Int J Reprod Contraception, Obstet Gynecol. 2017;6(7):2964.
9. Israelsson LA, Millbourn D. Prevention of incisional hernias. How to close a midline

12
 incision. Surg Clin North Am. 2013;93(5):1027–40.
10. Al-Mubarak L, Al-Haddab M. Cutaneous wound closure materials: An overview and
update. J Cutan Aesthet Surg. 2013;6(4):178.
11. Sajid MS. Systematic review of absorbable vs non-absorbable sutures used for the closure
of surgical incisions . World J Gastrointest Surg. 2014;6(12):241.
12. Gurusamy KS, Davidson BR. Continuous versus interrupted skin sutures for non-obstetric
surgery. Cochrane Database Syst Rev. 2013;2013(2).
13. Deerenberg EB, Harlaar JJ, Steyerberg EW, Lont HE, Van Doorn HC, Heisterkamp J, et
al. Small bites versus large bites for closure of abdominal midline incisions (STITCH): A
double-blind, multicentre, randomised controlled trial. Lancet. 2015;386(10000):1254–60.
14. Muysoms FE, Antoniou SA, Bury K, Campanelli G, Conze J, Cuccurullo D, et al.
European Hernia Society guidelines on the closure of abdominal wall incisions. Hernia.
2015;19(1):1–24.
15. Hodgson NCF, Malthaner RA, Østbye T. The search for an ideal method of abdominal
fascial closure: A meta- analysis. Ann Surg. 2000;231(3):436–42.
16. Gaikwad V, Kapoor R, Thambudorai R. An ideal suture for midline abdominal closure?
Indian J Surg. 2009;71(3):128–32.
17. Radu P, Brǎtucu M, Garofil D, Pasnicu C, Iorga C, Popa F, et al. Molecular factors of
failure in incisional hernia surgery. Chir. 2013;108(2):193–8.
18. Gelse K, Pöschl E, Aigner T. Collagens - Structure, function, and biosynthesis. Adv Drug
Deliv Rev. 2003;55(12):1531–46.
19. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience
research reporting: The arrive guidelines for reporting animal research. PLoS Biol.
2010;8(6):6–10.
20. Klinge U, Si ZY, Zheng H, Schumpelick V, Bhardwaj RS, Klosterhalfen B. Abnormal
collagen I to III distribution in the skin of patients with incisional hernia. Eur Surg Res.
2000;32(1):43–8.
21. Fortelny RH, Baumann P, Thasler WE, Albertsmeier M, Riedl S, Steurer W, et al. Effect
of suture technique on the occurrence of incisional hernia after elective midline abdominal
wall closure: Study protocol for a randomized controlled trial. Trials. 2015;16(1):1–8.
22. Cheng W, Yan-Hua R, Fang-Gang N, Guo-An Z. The content and ratio of type I and III
collagen in skin differ with age and injury. African J Biotechnol. 2011;10(13):2524–9.

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