THE EFFECT OF PLATELET-RICH FIBRIN (PRF) ON SKIN FIBROBLAST MIGRATION AFTER

MITOMYCIN-C TREATMENT
Publication Manuscripts of A Plenary Paper
Presented as Partial Fulfillment of the Requirements to Graduate from the Clinical Specialist
Program in Surgical Sciences at the Faculty of Medicine, Gadjah Mada University

Submitted by:
Nur Eko Hadi Sucipto
Student ID Number: 11/323819/PKU/12626

DEPARTMENT OF SURGERY
FACULTY OF MEDICINE, GADJAH MADA UNIVERSITY
SARDJITO GENERAL HOSPITAL OF YOGYAKARTA
2015

Publication Manuscripts of A Plenary Paper

THE EFFECT OF PLATELET-RICH FIBRIN (PRF) ON SKIN FIBROBLAST MIGRATION AFTER
MITOMYCIN-C TREATMENT

By:
Nur Eko Hadi Sucipto
Student ID Number: 11/323819/PKU/12626
Presented in the seminar on 22 October 2015

Supervisor I

DR. dr. Ishandono Dachlan.,M.Sc.,Sp.B.,Sp.BP-RE(K)

CSID No. 19520214 197903 1 001
Supervisor II

DR. dr. Yohanes Widodo Wirohadidjojo, Sp. KK(K)

CSID No. 195509151981031004

The Effect of Platelet-Rich Fibrin (PRF) on Skin Fibroblast Migration after

Mitomycin-C Treatment
Nur Eko Hadi Sucipto*, Ishandono Dahlan**, Yohanes Widodo Wirohadidjojo***
*Resident of General Surgery of Gadjah Mada University/ Sardjito General Hospital of Yogyakarta
** Department Plastic Surgery of Gadjah Mada University/ Sardjito General Hospital of Yogyakarta
*** Department Dermatology of Gadjah Mada University/ Sardjito General Hospital of Yogyakarta

ABSTRACK

Background: Wound healing is a dynamic process involving several phases including

inflammation, proliferation, and remodeling. Skin fibroblast migration plays a crucial role
especially during proliferation and remodeling. Platelet-rich fibrin (PRF) is autologous fibrin
matrix containing high levels of growth factors and cytokines that are required for wound
healing. On the other hand, Mitomycin-C is able to inhibit wound healing through induction
of DNA damages that result in apoptosis and inhibition of cell proliferation.
Methods: Fibroblasts were incubated in appropriated culture medium with and without
10g/mL Mitomycin-C for 2 hours. After 2 times washing with PBS, fibroblasts were
incubated in culture medium containing 12.5%, 25%, and 50% of PRF, or without PRF as a
negative control. Scratch assays were performed to measure migration of Mitomycin treatedfibroblasts following with or without PRF treatment.
Results: Fibroblast migration after Mitomycin-C treatment without PRF treatment was
16,7725 8,12405%. After 12.5%, 25%, and 50% PRF, migration of fibroblasts were
19,14393,01785%, 27,09103,46624%, and 51,09071,86437%, respectively. Treatment of
PRF significantly increased fibroblast migration compared to the control, (P value = 0.000)
Conclusion: PRF treatment significantly increases fibroblast migration after treatment with
Mitomycin-C. Further study using in vivo setting is required to confirm our in vitro study.
Key Word: Fibroblast Migration, Platelet-Rich Fibrin (PRF), Mitomycin-C
INTRODUCTION

Wound healing is a very dynamic

process as it involves several phases
including inflammation, proliferation, and
remodeling,
wound
closure
can
immediately reduce the incidence of
infections and the patient morbidity rate
(Xian et al., 2014), and is an issue which
provides a challenge for a surgeon to
develop a variety of materials and
techniques in wound healing processes
after surgery (Eshghpour et al., 2012).
These wound healing processes are
influenced by a number of phases which
precede one another and recur, namely
inflammation,
proliferation
and
maturation. The inflammatory phase takes
place since a trauma begins and triggers

cellular and vascular responses, and

afterwards the proliferative phase begins
wherein an increase of tensile strength
happens followed by the maturation or
remodeling phase in which at this moment
there is an increase in collagen formation
(Kendrick, 2000).
The wound healing processes may
be inhibited due to a number of factors.
Acute wound healing disorders will make
the wound become a chronic wound.
Histologically, chronic wounds indicate
aging fibroblasts with a low proliferation
ability (Christofalo et al., 2000).
Fibroblasts are cells synthesizing
the extracellular matrix involved in the
formation of the tissue structure, these

fibroblasts play a significant role in the

proliferative phase of wound healing, these
cells are responsible for forming collagen
type I and III, elastin, proteoglycans and
adjusting the tensile strength in wound
healing process (Porter, 2007).
Cell migration is a fundamental
cellular
process
for
the
normal
development and homeostasis of the
tissues and organs with characteristics in
physiological and pathophysiological
processes in vessels and inflammatory
diseases. This cell migration also plays a
role in vital wound healing processes,
those cells involved in this wound healing
are fibroblasts, in wound healing,
fibroblast migration serves to repair tissue
damage (Thampatty and Wang, 2007).
Mitomycin-C
is
one
of
chemotherapy drugs used widely to treat
cancer. The use of Mitomycin-C in in-vitro
research indicates a state where cell
proliferation is being inhibited, both for
tumor cells and healthy cells and causes
cellular aging which is similar to cell
morphology in chronic wounds. in vitro
research conducted by Nieto in 2007 on
human fibroblasts shows that the
administration of 10 g/ml (0.03 M) of
Mitomycin-C for 2 hours can inhibit
proliferation and trigger apoptosis (Nieto
et al., 2007). Another in vitro research
undertaken by Chen et al. in 2006,
suggests that the administration of 0.4
mg/ml of Mitomycin-C for 4 minutes to
fibroblasts can reduce normal dermal
fibroblast proliferation.
Platelet
concentrate
products
already begin to be used in the field of
surgery in recent years. The main principle
adopted is to create a platelet-rich
substance using its growth factor and to
apply it to wounds with a view to
stimulating the local healing processes.
Platelet-Rich Fibrin (PRF) is a new
product of platelet concentrate produced
through centrifugation of peripheral blood.
This PRF is easier and simpler to produce
than
previous
platelet
concentrate
products. This PRF contains many

cytokines and growth factors which can

stimulate wound healing processes (Zhao
and Ding, 2013; Khiste and Tari, 2013).
METHODOLOGY
This is in vitro research aimed at
assessing the speed of fibroblast migration
employing the scratch assay method using
the quasi-experimental design.
The research sample were cultured
fibroblasts of normal human skins taken
that are not exposed to direct sunlight,
these cultured fibroblasts were taken from
the fourth culture cultured in a medium of
complete Dulbecocos Modified Eagles
medium (DMEM)-SigmaTM containing 5%
of fetal bovine serum (FBS), 100/ml of
penicillin-streptomycin
(penstrep)
GibcoTM, 100mg/ml of ceftriaxone and
2.5g/ml of amphotericineB-FungsioneGibcoTM, with a total of 5x104 cells/ml.
Then, these cultured fibroblasts were
classified into 5 groups, namely: Group 1
was added with the culture medium, while
Groups II to V were added with
mitomycin-C and each group received the
following treatments: (1) added with the
culture medium, (2) added with 50% PRF,
(3) added with 25% PRF (4) added with
12.5% PRF.
The data obtained from the present
reseach were entered and processed using
SPSS to analyze the average differences of
each treatment group, using one-way
ANOVA with a significance level by
p<0.05.
RESEARCH FINDINGS
Research has been conducted using
a sample of 20 fibroblast wells in which
each well contained 15,000 cells. They
were divided into 5 treatment groups,
namely the positive control using
subcultured fibroblasts + 10% FBS, the
negative control using subcultured
fibroblasts + 15% FBS and 3 treatments
added with the subculture + Mitomycin-C
by 0.4 ml and addition of PRF by 50%,

25% and 12.5%. Afterwards, incubation

was performed as well as scratch assays
for 72 hours. Subsequently, fixation and
washing using PBS were carried out. After
that, fibroblast migration was assessed.

Chart. 1 The Chart Illustrating the Average

Migration in the Control Group

Table.1 A comparison between the effects

of the negative control, the positive
control, PRF by 12.5%, PRF by 25%, and
PRF 50% on the speed of fibroblast
migration
Treatment
Positive
Control
Negative
Control
PRF by
12.5%
PRF by 25%
PRF by 50%

The average fibroblast

migration speed (%)with
standard deviation

p-value

36.27492.00351
16.77258.12405

0.000

19.14393.01785
27.09103.46624
51.09071.86437

Description:
Positive control: a suspension of fibroblasts + FBS 10%
Negative control: a suspension of fibroblasts +
Mitomycin-C by 10g/mL + FBS by 1%
PRF by 50% + mitomycin-C : a suspension of fibroblasts
+ Mitomycin-C by 10g/mL + PRF by 50%
PRF by 25%+ mitomycin-C : a suspension of fibroblasts
+ Mitomycin-C by 10g/mL + PRF by 25%
PRF by 12.5% + mitomycin-C : a suspension of
fibroblasts + Mitomycin-C by 10g/mL + PRF by 12.5%

In the table above, it can be seen

that the average speed of fibroblast
migration treated with a negative control is
equal to 16.7725 8.12405%, the average
speed of fibroblast migration treated with a
positive control is equal to 36.2749
2.00351%, the average speed of fibroblast
migration treated with 12.5% PRF is equal
to 19.1439 3.01785%, the average speed
of fibroblast migration treated with 25%
PRF is equal to 27.0910 3.46624%, and
the average speed of fibroblast migration
treated with 50% PRF is equal to 51.0907
1.86437%.
The ANOVA test generates a pvalue by 0.000 (or less than 0.05) and thus
it can be concluded that there are
differences in the effects generated by the
negative control, the positive control,
12.5% PRF, 25% PRF, and 50% PRF on
the speed of fibroblast migration.

Based on the chart above, it is

shown that the average speed of cell
migration for the positive control which is
a group of cultured cells + 10% FBS is
36.27% while for the negative control
which is a group of cultured fibroblasts +
1% FBS + 10 g/ml of mitomycin is
16.77%.
Chart. 2 The Chart Illustrating the Average
Migration in the Groups Treated with
Mitomycin and Mitomycin + PRF

In terms of the average cell

migration for each treatment group, for the
treatment group of cultured fibroblasts +
10 mg/ml of mitomycin-C + 1% FBS, it
is equal to 16.77%; for the treatment group
of fibroblasts + mitomycin-C + 50% PRF,
it is equal to 51.09%; for the treatment
group of fibroblasts + 10 mg/ml of
mitomycin-C + 25% PRF, it is equal to
27.09% and lastly, for the treatment group
of fibroblasts + mitomycin-C + 12.5%
PRF, it is equal to 19.14%.
`

DISCUSSION
Cell migration is a fundamental
cellular
process
for
the
normal
development and homeostasis of the
tissues and organs with characteristics in
physiological and pathophysiological
processes in vessels and inflammatory
diseases. This cell migration also plays a
role in vital wound healing processes,
those cells involved in this wound healing
are fibroblasts, in wound healing,
fibroblast migration serves to repair tissue
damage (Thampatty and Wang, 2007). In
the present research, significant findings
are obtained suggesting the existence od
fibroblast migration speed compared with
the negative control, the positive controls
and for each treatment.
The treatment by administering
PRF by 50% indicates the greatest cell
migration speed compared with the
treatment by administering PRF by 25%
and 12.5%. There is a significant
difference in the speed of fibroblast
migration between the treatment groups
with the negative control, the positive
control, PRF by 12.5%, PRF by 25%, and
PRF by 50% (p < 0.05).
The addition of 10% FBS to
cultured fibroblasts to which the scratch
assay treatment (the positive control) was
given indicates wound healing such as in
acute wounds, while in cultured fibroblasts
added with mitomycin-C with scratch
assays indicates chronic wound healing.
The administration of PRF at a
concentration of 50% results in a fibroblast
migration speed that is significantly higher
than that of the positive control (51.0907
1.86437% compared with 36.2749
2.00351% with a p-value by 0.000),
meaning that the administration of PRF at
a concentration of 50% is better than the
administration of the positive control.
This indicates the likelihood for
healing
chronic
wounds
(histopathologically cellular aging comes
to pass) in fibroblasts with the addition of
mitomycin-C and PRF at a concentration
of 50% generates better cell migration

results than cultured fibroblasts with the

addition of 10% FBS (histopathologically
similar to the healing of acute wounds).
CONCLUSIONS
Treatments with the administration
of PRF at a concentration of 50%, 25%
and 12.5% have a significant difference in
the speed of fibroblast migration after the
administration of mitomycin-C depending
on the increase in the PRF level that is
used.
The
treatment
with
the
administration of PRF at a concentration
of 50% will bring a larger effect of
fibroblast migration than that of PRF at a
concentration of 25% or 12.5%. Likewise,
the treatment with the administration of
PRF at a concentration of 25% will bring a
larger effect of fibroblast migration than
that of PRF at a concentration of 12.5%.
The administration of PRF at a
concentration of 50% to cultured
fibroblasts with the addition of mitomycinC results in better cell migration than that
of cultured fibroblasts with the addition of
FBS by 10%, indicating higher likelihood
of chronic wound healing with the
administration of PRF by 50% than that of
acute wound healing.
SUGGESTIONS
It is necessary to undertake in vitro
research investigating the effect of PRF on
the speed of fibroblast migration, where
PRF is taken from the same individual
(autologous) with a larger number of
samples.
It is necessary to undertake
prospective in-vivo research to examine
the PRF level required to improve
fibroblast migration and to accelerate
chronic wound healing clinically

REFERENCES
1. Acharya P.S., Majumdar S., Jacob M.,
Hayden M., Mrass P., Weningger W.,
Assoian R.K., Pure E. 2008.
Fibroblast migration is mediated by
CD 44-dependent TGF activation,
Journal of Cell Science 121:13931402.
2. Chen T., Kunnavatana S.S., Koch R.J.
2006. Effect of mitomycin on normal
dermal
fibroblast.Laringoscope
11(4):514-517.
3. Chignon S.B., Georgiou C.A., Fontas
E. 2012. Efficacy of leukocyte and
platelet-rich fibrin in wound healing:
randomized controlled clinical trial.
PlastReconstrSurg 130(6):514-517.
4. Christofalo V.J., Volker C., Alen R.G.
2000. Use of the fibroblast modelsin
the
studyof
cellular
senescence.Methods in molecular
medicine: Aging Methods and
Protocol. Vol 38.Humana Press
Inc.Totowa.New Jersey
5. Dohan D.M., Choukroun J., Diss A.,
Dohan A.J., Mouhyi J., Gogly B.
2006. Platelet-rich fibrin (PRF): A
second-generation
platelet
concentrate. Part I: Technological
concept and evaluation. Oral Surg
Oral Med Oral Pathol Oral
RadiolEndol101: E37-44.
6. Eshghpour M, Majidi M.R., Nejat
A.H. 2012. Platelet-rich fibrin:
autologous fibrin matrix in surgical
Procedure: A case Report and Review
of literature. Iranian Journal of
Otorhinolaryngology No4. Vol.24.
Serial No 69
7. Flanagan V., Iwamoto S. 2000.The
Physiology of wound healing.Review
Journal of WoundCare.Vol 9, No 6
8. Gassling V.L., Acil Y., Springer I.N.,
Hubert N., Wiltfang J.2009. Plateletrich Plasma and Platelet-Rich Fibrin
in human cell culture.Oral Surg Oral
Med
Oral
Pathol
Oral
RadiolEndol108:48-55

9. Gurtner G.C. 2007. Wound healing:

Normal and abnormal.in Thorne C.H.
10. editor.Grabb and Smiths Plastic
Surgery.6th edition.Chapter 2. Page
:15-22. Lippincott Williams and
Wilkins, Philadelphia
11. Hulkower K.I., Herber R.L. 2011. Cell
migration and invasion assay as tools
for drug discovery. PharmaceuticsVol
3: 107-124
12. Hsu A., Mustoe T.A. 2004. Principles
of wound healing on fundamental
principle plastic surgery.
13. Kanukar B.N., Jayadeve I.M., Marshal
R. 2013. Role of Platelet-rich fibrin in
wound healing: A critical review.
Journal of ConservativeDentistry.Vol
16.Issue 4.
14. Kendrick S. 2000. Wound Healing
and Management.Care of Surgical
Patient. Page 69-72
15. Kiste S.V., Tari R.N. 2013. PlateletRich Fibrin as a Biofuel for Tissue
Regeneration.Biomaterials.Hindawi
Publishing Corporation
16. Kurniawan H.S. 2014.EfekPlateletRich
Fibrin
(PRF)
terhadapProliferasiSelFibroblasKulit
Normal
PascaPajananMitomycinC.BagianIlmuBedahUniversitasGadja
hMada Yogyakarta
17. Liang C., Park A.Y., Guan J.L. 2007.
Invitro scratch assay: a convenient and
inexpensive method for analysis of
cell migration in vitro. Nature
Protocol. Vol.2. no.2:329-332
18. McHugh P.J., Spanswick V.J., Hartley
J.A.2001. Repair DNA Interested
cross- link: molecular mechanism and
clinical
relevance.
The
lancet
Oncology2: 483-490
19. Missailidis S. 2008. Antitumour
Antibiotics.Anticancer Therapeutics.8
: 112-114. John Willey and Sons
Corp., West Sussex, UK.
20. Mosesson M.W., Siebenlist K.R., Meh
D.A.2001.
The
structure
and
biological features of fibrinogen and
fibrin.Ann NY AcadSci 936:11-30

21. Nieto A., Cabrera C.M., Catalina P.,

Cobo F., Barnie A., Cortes J.L., Jesus
A.B.,
Montes
R.,
Concha
A,2007.Effect of Mitomycin-C on
human foreskin fibroblast used as
feeder in human embryonic stem cells:
Immunocytochemistry MIB1 score
and DNA ploidy and apoptosis
evaluated
by
flow
cytometry.
Cell.BiolInt 31:269-278
22. Orsted.H.L.,Keast D., Lalande L.F.,
Megie M.F. 2013. Basic Principles of
wound healing.Wound Care Canada.
Vol 9. No2
23. Porter S. 2007. The Role of Fibroblast
in
Wound
Contraction
and
Healing.Wounds.UK vol.3 No1. 33-39
24. Rini S. 2014. EfekPlatelet-Rich Fibrin
(PRF)
TerhadapTimbunanKolagenFibroblas
PascaPajanandenganMitomycinC.BagianIlmuBedahUniversitasGadja
hMada Yogyakarta
25. Ribeiro F.A.Q., Guaraldo L., Borges
J.P., Zachi F.F.S., Eckley C.A.2004.
Clinical and Histological Healing of
surgical
wound
treated
with
Mitomycin-C.
Laringoscope.
114:148-152
26. Sinno H., Prakash S. 2013.
Complement and the wound healing
cascade: up date review. Plastic

SurgeryInternational.
Article
ID
146764
27. Steenvoorde P., Van Doorn L.P.,
Naves C, Oskam J.2008. Use of
autologous platelet-rich fibrin on hardto-heal
wound.Journal
of
WoundCare.Vol 17.No 2
28. Thampatty B.P., Wang J.C.2007.New
approach
to
studyfibroblast
migration.Cell Motility and the
Cytoskeleton
64: 1-5
29. Toffler M., Toscano N., Holtzclaw D.,
DelCarso
M.,
Ehrenfest
D.D.
2009.Introducing
Choukrouns
platelet-rich
fibrin
(PRF)
to
reconstructive surgery milieu. JIACD
1 (6): 21-32
30. Xian L.J., Chowdhury S.R., Saim,
Ruszyman. 2014. Concentrationdependent effect of Platelet-rich
plasma on keratinocyte and fibroblast
wound healing. International society
for cellular therapy.
31. Yarrow J.C., Parlman Z.E., Westwood
N.J., Mitchison T.J. 2004. A highthroughput cell migration assay using
scratch wound healing, a comparison
of image-based readout methods,
BMC Biotechnology 4: 21
32. Zhao Q.M., Ding Y.J., SiT. 2013.
Platelet-rich fibrin in plastic surgery.
OA Evidence-Based Medicine 1(1):3.