COMPARISON OF STITCH EFFECT USING POLYGLECAPRONE 25

AND NYLON WITH SMALL STITCH CONTINUOUS TECHNIQUE

TO COLLAGEN TYPE I AND III RATIOS ON SKIN INCISION LINE
OF ALBIAN RATS WISTAR STRAIN (Rattus norvegicus) : An
Experimental Study WC:33 words! with 230 characters??
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: 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 strain (Rattus
norvegicus).
Material 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 collagen type 1 and 3 ratio were measured. After
euthanasia, samples of skin (5 x 10 mm) were collected for analysis of collagen count using
immunohistochemistry staining method. The differences were then analyzed with Mann-
Whitney tests if the data were not normally distributed and independent t-tests when the data
were normally distributed.
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 after
euthanasia [(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.
WC:298
Keywords: polyglecaprone 25, Nylon, collagen type 1, collagen type 3, collagen type 1 and
3 ratio, continuous small stitch

1
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 done1. 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
increase5. 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 laparotomy8. The quality of the surgical suture technique is very important to
prevent complications in the median incision wound during laparotomy9.

2
 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 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 threads11. Thread
material can be either natural or synthetic, which is absorbable and non-absorbable.
Examples of absorbable threads are polyglecaprone 25, polydioxanone, and polyglactin.
Meanwhile, examples of non-absorbable threads are 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 stitch 13.
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
stitch14. This finding made the researchers interested in using small stitch as a sewing
technique.
Monofilament threads are more resistant to microorganisms than multifilament
thread8. 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
3
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 collagen type 1: 3 plays an important role as a
parameter in postoperative wound healing17. 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 incision 18. 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 sample size was determined using the Federer
formula (1963).citations#18-22missing??

4
 The minimum sample size according to the formula above was 6. To maintain a high
representative level in the event of a drop out, the sample was widened to 10 individuals per
group, so the total sample size was 40 individual subjects. 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 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
5
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. Independent t-test was used to test whether there was a difference in the mean
between the two groups. The independent t-test are used for normally distributed data.
Therefore, it is necessary to test the normality for each variable with the Kolmogorov-
Smirnov test. The results of statistical analysis with p <0.05 were defined as statistically
significant results. If the data distribution was not normal then the Mann-Whitney test was
performed.
Simple regression test was used in this study to determine the effect of one
independent variable on one dependent variable with a significance value of p <0.05.
Correlation test is used to determine the relationship of a variable which is stated by the
correlation coefficient which is getting closer to the value of 1 or -1 which means that the
6
relationship between the two variables is getting stronger. 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) guidelines23.

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 done 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.

7
Figure1. Type 1 collagen expression (twisted brown designated red arrow) with PG-25
thread with small continuous stitch.

Figure 2. Expression of type 1 collagen (twisted brown pointed red arrow) with Nylon
thread with small continuous stitch.

8
Figure 3. Expression of type 3 collagen (twisted brown designated red arrow) with PG-25
thread with small continuous stitch.

9
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.
The data were then analyzed statistically using the Independent T-test method if the data
were normally distributed, or Mann-Whitney if the data were not normally distributed.
The mean value of 1: 3 collagen ratios on the fourth day with PG-25 thread was higher
than Nylon but with very small difference. To find out whether the mean collagen ratios of 1: 3
in the skin of the Wistar strain albino rats on the fourth day after the sutured abdominal incision
using the small stitch interval, the continuous suture technique with PG-25 thread and Nylon
thread were significantly different, a statistical hypothesis test needed to be performed.
Because the dependent variable is a numeric variable and the independent variable is a
categorical variable with a total of two groups, with normally distributed and unpaired data, the
Independent T-test was chosen. The significance of the Levene test p = 0.581 (<0.05) indicated
homogeneous data.

10
 Table 1. The mean normality test for type 1 and type 3 collagen on the 4th and 7th day
Variable p*
Collagen type 1 day 4 0.276
Collagen type 3 day 4 0.078
Collagen type 1 day 7 0.746
Collagen type 3 day 7 0.187
* Shapiro-Wilks test

Table 1 shows that all the mean collagen type 1 and type 3 on day 4 and day 7 were
normally distributed (p> 0.05) so that the analysis of the results used the independent T-test.

Table 2. Difference between collagen type 1 and collagen type 3 levels 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 4th 0.074 -0.82 15.95
day
a. PG-25 Thread 50.43 ± 10.78
b. Nylon Thread 42.86 ± 6.57
Collagen type 3 4th 0.150 - 4.34 26.20
day
a. PG-25 Thread 75.73 ± 21.63
b. Nylon Thread 64.80 ± 7.78
Collagen type 1 7th 0.149 -2.44 14.84
day
a. PG-25 Thread 55.83 ± 8.90
b. Nylon Thread 49.63 ± 9.48
th
Collagen type 3 7 0.087 -39.25 2.98
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

11
 Collagen type 1 PG-25
Collagen type 1 Nylon
Collagen type 3 PG-25
Collagen type 3 PG-25

4th Day 7th Day

Figure 5. Graph of the Mean Difference of Collagen 1 and 3 on Day 4 and 7

Table 2 shows that on day 4, the mean collagen type 1 from suturing using PG-25 threads
was higher than suturing with nylon thread. On the 7th day, the mean collagen type 1 from
suturing using nylon thread was higher than suturing with PG-25 thread. The two differences
were not statistically significant (p >0.05).
The mean of collagen type 3 from suturing using PG-25 thread on the 4th day was higher
than that of nylon thread, but on the 7th day, the mean collagen type 3 was found to be higher
than the results of suturing using nylon thread than PG-25 thread. This difference was not
statistically significant (p >0.05).
Figure 5 shows the mean difference between collagen 1 and 3 values in rats sewn with
PG-25 and Nylon thread after decapitation on day 4 and day 7 using the continuous small stitch
method.

Table 3. Normality test of mean collagen type 1: 3 ratio

Variable p*
Mean ratio of collagen type 0.607
1:3 on day 4
Mean ratio of collagen type 0.514
1:3 on day 7
*Shapiro-Wilk test

12
 Table 3 shows that the mean ratio values of collagen type 1 and type 3 on day 4 and day 7
were normally distributed (p> 0.05), therefore statistical analysis used the independent T-test.
The difference in the mean ratio of type 1 and type 3 collagen 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 after euthanasia is shown in Table 4.

Table 4. The Difference in Average Ratio of Type 1 and 3 Collagen on Day 4 and 7

Variable Mean ± SD p*

Mean of collagen 1:3 ratio day 4 0.853

a. PG-25
b. Nylon 0.68 ± 0.16
0.67 ± 0.16
Mean of collagen 1:3 ratio day 7 0.021
a. PG-25
b. Nylon 1.43 ± 0.47
0.94 ± 0.39
* Independent T-test; SD, standard deviation

Table 4 shows that the mean ratio of collagen types 1 and 3 on day 4 was found to be
higher in subjects stitched with PG-25 threads than nylon thread. However, statistically the
difference in the ratio was not statistically significant (p >0.05).
Whereas on day 7 the mean ratio of collagen type 1 and 3 was found to be higher in the
PG-25 group than nylon thread and statistically the difference in the ratio was significant (p
<0.05). The table above shows that the mean difference in the collagen ratio of 1: 3 after
euthanasia in the Wistar albino rats sewn with PG-25 and Nylon was not statistically significant
with p = 0.853 (p <0.05).

13
 1.6

1.4

1.2

0.8

0.6

0.4

0.2

0
Day 4 Hari ke 7

PG-25 Nylon

Figure 6. Graph of Mean Difference of Collagen Ratio 1: 3 on Day 4 and 7 with PG-25
and Nylon thread

Discussion
Collagen 1 level on day 7 after abdominal incision in Wistar albino rats sutured with PG-
25 thread and Wistar strain albino mice sutured with Nylon thread was higher than collagen level
1 of albino Wistar rats sewn with PG-25 thread and Wistar strain albino rats sutured with Nylon
thread on day 4 after abdominal incision. Collagen 3 levels on day 7 after abdominal incision in
Wistar albino rats sewn with PG-25 thread and Wistar albino rats sewn with Nylon thread were
higher than collagen levels 3 albino Wistar rats sewn with PG-25 thread and Wistar strain albino
rats sutured with Nylon thread on day 4 after abdominal incision. These results show that over
time there was an increase in the ratio of collagen 1 to collagen 3. An increase in the ratio of
collagen 1 to collagen 3 was shown to significantly reduce the risk of surgical wound dehiscence.
Collagen 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, type 3 collagen is replaced by type 1 collagen
which is stronger18

14
 The difference changed in the mean of collagen 1 in mice sewn with PG-25 (50.43 ±
10.78) and Nylon (42.86 ± 6.57) threads on the 4th day, while on the 7th day the mean difference
of collagen 1 in mice which were sewn with PG-25 (55.83 ± 8.90) and Nylon (49.63 ± 9.48)
threads. It appears that the collagen 1 mean was higher when using PG-25 thread. This could be
because the transitional phase from hemostasis to the inflammatory phase with PG-25 strings
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, in a similar study which 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 1
and the transformation of collagen 3 into collagen 1 was not completed, but the amount of
collagen 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.
The mean difference of collagen 3 in rats sewn with PG-25 thread (75.73 ± 21.63) and
Nylon (64.80 ± 7.78) on day 4 continued to change, becoming on the seventh day the mean
difference of collagen 3 in sewn mice with PG-25 (42.99 ± 15.32) and Nylon (61.13 ± 27.14).
The mean of collagen 3 appeared higher on day four when compared to day seven. This finding
may happen because collagen 3 is formed first, which then during maturation will form collagen
1 over time. Accordingly, on the seventh day the amount of collagen 3 was less than the fourth
day20. Collagen type 3 is widely distributed in collagen 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 21. Collagen
3 is formed at the beginning of the wound, then the maturation of collagen 3 becomes collagen 1.
The mean difference of collagen ratio was 1: 3 at day 4 in mice sutured with PG-25 (0.68
± 0.16) and nylon thread (0.67 ± 0.16). We can conclude that in the transitional phase of
hemostasis to the inflammatory phase, PG-25 threads were superior although this difference was

15
not statistically significant p = 0.853 (p >0.05). Whereas, on the 7th day the mean difference of
collagen ratio 1: 3 with PG-25 thread (1.43 ± 0.47) and Nylon thread (0.94 ± 0.39) showed a
significant difference with p = 0.021 (p <0.05). 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 disrupted15. A low collagen 1 and 3 ratio is associated with higher
flexibility in the wound. A low collagen 1 and 3 ratio appears at the onset of injury. The more a
wound heals, the higher the ratio of collagen 1 and 3 indicates increased collagen 1 in a healing
wound. The longer the ratio will remain stable, this will cause limited sensitivity to growth
factors so that there is no hypertrophic wound healing22.

Conclusion
There was no significant difference in the value of the 1: 3 collagen ratio on the 4th day
after euthanasia, but there was a significant difference on the 7th day after euthanasia 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

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16
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