Journal Pre-proof

Platelet-Rich Fibrin (PRF) Membranes Accelerate Open Wound Healing

Better Than Amniotic Membranes: A Histological Study on the
Proliferation Phase

Rezmelia Sari, Grace Sekar Larasati, Nurul Ghoutsiyah

Kuncorowati, Ahmad Syaify

PII: S2213-9095(20)30014-8
DOI: https://doi.org/10.1016/j.wndm.2020.100190
Reference: WNDM 100190

To appear in: Wound Medicine

Received Date: 27 January 2020

Accepted Date: 3 June 2020

Please cite this article as: Sari R, Larasati GS, Kuncorowati NG, Syaify A, Platelet-Rich Fibrin
(PRF) Membranes Accelerate Open Wound Healing Better Than Amniotic Membranes: A
Histological Study on the Proliferation Phase, Wound Medicine (2020),
doi: https://doi.org/10.1016/j.wndm.2020.100190

This is a PDF file of an article that has undergone enhancements after acceptance, such as
the addition of a cover page and metadata, and formatting for readability, but it is not yet the
definitive version of record. This version will undergo additional copyediting, typesetting and
review before it is published in its final form, but we are providing this version to give early
visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal
pertain.

© 2020 Published by Elsevier.

Platelet-Rich Fibrin (PRF) Membranes Accelerate Open Wound Healing Better Than Amniotic
Membranes: A Histological Study on the Proliferation Phase

(Rezmelia Sari,1 Grace Sekar Larasati,2 Nurul Ghoutsiyah Kuncorowati,2 Ahmad Syaify1)

1
Department of Periodontics Faculty of Dentistry Universitas Gadjah Mada, Yogyakarta-Indonesia
2
Dentistry Study Program Faculty of Dentistry Universitas Gadjah Mada, Yogyakarta-Indonesia

Abstract
Background: Biological membranes are widely used as dressings to prevent contamination

of
during the open wound healing process. The amniotic membrane has been clinically
demonstrated to be an accelerator of wound healing. However, this membrane is fragile and

ro
technically sensitive. The platelet-rich fibrin membrane has good physical properties and
contains growth factors. However, few controlled studies have compared the effects of these
-p
membranes on wound healing, specifically during the proliferation phase. The proliferation
phase is an important stage in the development of more effective therapeutic interventions.
re
This study investigated the differences in the wound healing process during the proliferation
phase after applying platelet-rich fibrin and amniotic membranes. This quasi-experimental
lP

study used 36 rabbits that were divided into three groups. A 4 mm x 4 mm section of the labial
gingiva was de-epithelized using a scalpel. Gingival biopsies were taken on days one, three,
five, and seven and stained with hematoxylin and eosin and Mallory’s trichrome stain.
na

Observations were done in a double-blind manner. The number of fibroblasts and blood
vessels, as well as epithelial thickness were tested using two-way analysis of variance followed
by a post-hoc LSD test. Collagen density was tested with the Kruskal–Wallis test followed by
ur

the Mann–Whitney test using SPSS software.

Results: The number of fibroblasts, angiogenesis, collagen density, and epithelial
Jo

thickness were higher in group II than in the other groups (P<0.05).

Conclusions: Histological parameters measured during the proliferation phase showed
that the platelet-rich fibrin membrane may accelerate wound healing better than the amniotic
membrane.

Key-words: amniotic, biological membrane, platelet-rich fibrin, proliferation, wound healing

1
Correspondence: Rezmelia Sari, Department of Periodontics Faculty of Dentistry Universitas Gadjah Mada,
Yogyakarta Indonesia, rezmelia_sari@mail.ugm.ac.id, +6285292002449

1. Introduction
The healing of open wounds is relatively slow due to epithelial loss. Therefore, a
dressing is usually added to cover the wound so that postoperative complications do not
develop [1]. Stimulation during the proliferation phase is critical to obtain rapid and optimal
healing and particularly for restoring barrier function [2].
Various biological wound dressings have been established and studied to obtain the
desired product. The most popular ones are the amniotic membrane [3], platelet-rich plasma
membrane, platelet-rich fibrin (PRF) membrane [4], and the collagen membrane [5].

of
Biological membranes are used as post-surgical dressings for closed and open
wounds. The amniotic membrane provides better healing after vestibuloplasty and gingival

ro
depigmentation.This membrane is ethically approved, relatively easy to obtain, and promotes
epithelialization. It also has anti-inflammatory, analgesic, and antimicrobial effects, as well as
growth factors that help with healing [3,6]. This membrane is proven to promote quick and
-p
continuous healing. The dehydrated membrane is easy to transport and store, as it can last for
five years in ambient temperature [7]. However, the amniotic membrane is thin and brittle, so
re
it is relatively difficult to apply[3,6].
The PRF membrane is widely used as a wound dressing. This membrane is obtained
lP

from the patient's blood and is processed with simple materials and techniques [8]. Besides
growth factors and cytokines, PRF membranes are enriched with antibacterial properties [9].
However, the number of membranes that can be obtained islimited [10].
na

There are limited number of studies that compared the differences in the wound healing
process after applying amniotic and PRF membranes, specifically in the proliferation phase.
Interestingly, both membranes clinically affect gingival thickness. This study compared
ur

differences in the proliferation phase of wound healing after applying PRF and amniotic
membranes.
Jo

2. Subjects and Methods

2.1 Ethical Approval
This was a quasi-experimental study conducted at the Animal Experiment Unit of
Laboratorium Penelitian dan Pengujian Terpadu Universitas Gadjah Mada Yogyakarta,
Indonesia. Ethical clearance was obtained from the Ethics and Advocacy Unit of the Faculty

2
of Dentistry Universitas Gadjah Mada Yogyakarta, Indonesia (No. 001464/KKEP/FKG-
UGM/EC/2018).

2.2 Preparation of the PRF Membrane

The PRF was prepared according to the protocol established by Choukroun et al.
Xylene (Interchemine, Waalre, Netherlands) was applied to the skin of rabbit ears to dilate
the blood vessels. Three ml of blood was taken using a syringe and stored in Eppendorf tubes
(Hamburg, Germany) [11]. The blood was centrifuged at 2,700 rpm for 12 min [12]. The PRF
was removed with tweezers and compressed with a PRF box (Osung, Houston, TX, USA).
Then, the PRF membrane was immersed in the growth factor-containing releasate.

of
2.3 Animal experiments

ro
This study used 36 male rabbits (Oryctolagus cuniculus; age four to eight months;
weight 1,500–2,500 g), which were bred by the Laboratorium Pengujian dan Penelitian
Terpadu of Universitas Gadjah Mada. The rabbits were divided into three groups: group I (no
-p
membrane), group II (PRF membrane), and group III (amniotic membrane).
re
lP
na

Figure 1. Application of membranes: platelet-rich fibrin (A) and amniotic (B)

ur

All rabbits were acclimated for 1 week before the experiment. Each rabbit was given
Jo

a 4 mm x 4 mm wound, measured with a sliding caliper, in the area of the lower labial
gingiva using a de-epithelization method and a disposable sterile scalpel (Dentica, Laval, QC,
Canada). Before de-epithelization, the rabbits were anesthetized intramuscularly in the groin
area using ketamine hydrochloride (Ketamine 10% Inj.; 25 mg/kg) [13] combined with
xylazine HCL at the ratio of 1:1. The PRF releasate membrane and amniotic membrane
(Batan Research Tissue Bank, Jakarta, Indonesia) were sewn (Figure 1) using blue nylon 6-0
(Ailee, Seoul, Korea).

3
2.4 Histological preparation
The experimental animals were decapitated on days one, three, five, and seven . The
tissues were fixed in a 10% buffered formalin solution (Mediss, Bali, Indonesia) for 24 hours
to maintain cell structure. Then, the tissues were dehydrated in a graded alcohol series (70%,
80%, 95%, and 100%) for 1.5 hours, followed by immersion in xylol solution to obtain tissue
transparency. Then, the tissues were inserted into molds. Liquid paraffin (56–60°C) was
poured into the block mold and frozen in a freezer. The hardened paraffin block was cut to a
thickness of 3–4 μm using a microtome and placed in a water bath at a fixed temperature of
46°C.

of
The paraffin block pieces were stained with hematoxylin-eosin. The histological
slides were dipped twice and dried sequentially in xylol solution, followed by immersion in

ro
100%, 90%, and 70% alcohol for three minutes each. The slides were rinsed with tap water
for one minute and dried. The slides were put into a hematoxylin-eosin or Mallory solution
for six to seven minutes and rinsed with tap water for one minute. The slides were dipped in
-p
an 80%, 90%, and 100% alcohol series ten times. Finally, the slides were dipped and dried
sequentially three times in xylol, for three minutes each. Lastly, the tissues were mounted
re
with a cover slip and adhesive. The slides were left to dry and were labeled with codes.
lP

2.5 Histological observations

The parameters used to determine the healing process of open wounds during the
proliferation phase were the number of fibroblasts, blood vessels, the density of collagen, and
na

epithelial thickness. Observations and calculations were carried out using a microscope
equipped with an Optilab © camera and 400x magnification with five fields of view.
Observations were carried out by three calibrated observers in a double-blind manner.
ur

Fibroblasts, blood vessels, and epithelial thicknesses were calculated and measured using
ImageRaster® software. Epithelial thickness data were obtained by calculating the average of
Jo

the maximum and minimum thickness. Collagen density was assessed by referring to the
assessment criteria as follows: A score of one indicated low collagen fiber density, a score of
two indicated moderate collagen fiber density, and a score of three indicated a high density of
collagen fibers [14].

2.6 Data analysis

4
 The numbers of fibroblasts and blood vessels, as well as the epithelial thickness were
tested using two-way analysis of variance (p<0.05) followed by a post-hoc LSD test. Collagen
density was tested with the Kruskal–Wallis (p<0.05) followed by the Mann–Whitney test using
SPSS software (SPSS Inc., Chicago, IL, USA).

3. Results
3.1 Number of fibroblasts
Table 1: Comparison of the numbers of fibroblast among the three groups on days one, three, five,
and seven
Number of Fibroblast (Mean ± SD) Intergroup Comparison
Day p
I II III I-II I-III II-III

of
1 4.96 ± 1.95 8.53 ± 3.10 7.09 ± 2.88 <0.05 0.085 0.265 0.437

3 8.42 ± 2.08 9.62 ± 4.74 8.82 ± 2.15 <0.05 0.452 0.648 0.756

ro
5 15.58 ± 3.30 23.16 ± 6.99 23.02 ± 2.67 <0.05 0.008* 0.008* 0.947

7 14.02 ± 4.34 19.64 ± 4.70 15.29 ± 4.48 <0.05 0.049* 0.275 0.049*
-p
I: no membrane; II: PRF releasate membranes; III: amniotic membranes; *statistically significant

Table 1 shows that both membrane groups tended to have a higher number of
re
fibroblasts than the no-membrane group on day one (p>0.05). All groups had a similar
number of fibroblasts on day three. The peak number of fibroblasts in all groups was reached
lP

on day five when the PRF releasate membrane group tended to have the highest number of
fibroblasts (p>0.05). The number of fibroblasts in all groups started to decline on day seven.
The PRF releasate membrane group had the highest number of fibroblasts on day seven
na

(p<0.05).
ur

3.2 Collagen density

Table 2: Comparison of collagen density among the groups on days one, three, five, and seven
Collagen Density (Median) Intergroup Comparison
Jo

Day Subject p
I II III I-II I-III II-III

1 1 1 2 1 <0.05 0.114 0.000 0.114

2 1 2 1

3 1 1 1

3 1 1 2 2 <0.05 0.025* 0.114 0.317

2 1 2 2

5
 3 1 2 1

5 1 2 2 2 <0.05 0.317 0.317 1.000

2 2 2 2

3 1 2 2

7 1 2 3 3 <0.05 0.025* 0.025* 1.000

2 2 3 3

3 2 3 3

I: no membrane; II: PRF releasate membranes; III: amniotic membranes; *statistically significant

According to Spearman’s correlation test, the correlation between observers was very
strong. Table 2 shows that the PRF membrane tended to have the highest collagen density on

of
day one (p>0.05). On day three, the PRF membrane group had significantly higher collagen
density compared to the no-membrane group. The collagen density in all groups was similar

ro
on day five. The collagen density of both membrane groups was significantly higher than the
no-membrane group on day seven.

3.3 Number of blood vessels (angiogenesis)

-p
re
Table 3: Comparison of blood vessels among the groups on days one, three, five, and seven

Number of Blood Vessels (Mean ± SD) Intergroup Comparison

lP

Day p
I II III I-II I-III II-III

1 0.29 ± 0.39 0.31 ± 0.24 0.33 ± 0.20 <0.05 0.922 0.844 0.922

3 0.27 ± 0.20 0.82 ± 0.21 0.44 ± 0.37 <0.05 0.011* 0.289 0.048*
na

5 1.13 ± 0.40 2.11 ± 0.98 1.35 ± 0.31 <0.05 0.000* 0.053* 0.000*

7 1.51 ± 0.45 2.69 ± 0.39 1.87 ± 0.68 <0.05 0.001* 0.134 0.007*
ur

I: no membrane; II: PRF releasate membranes; III: amniotic membranes; *statistically significant

Table 3 shows that all groups had almost the same number of blood vessels on day
Jo

one. Both the PRF and amniotic membrane groups had higher numbers of blood vessels than
the no-membrane group on all days. The PRF membrane group had significantly more blood
vessels on days three, five, and seven than those in the other groups (p<0.05).

3.4 Epithelial thickness

Table 4: Comparison of epithelial thickness among the groups
Day Epithelial Thickness Growth p Intergroup Comparison

6
 I II III I-II I-III II-III

1 0±0 7.08 ± 12.27 5.16 ± 8.9 <0.05 0.317 0.317 0.796

3 10 ± 2.63 70.54 ± 0.82 44.94 ± 14.85 <0.05 0.049* 0.049* 0.049*

5 154.62 ± 46.28 233.77 ± 23.27 159.81 ± 57.07 <0.05 0.049* 0.827 0.127

7 226.13 ± 20.34 280.09 ± 45.4 253.90 ± 66.16 <0.05 0.127 0.513 0.513
I: no membrane; II: PRF releasate membranes; III: amniotic membranes; *statistically significant

Table 4 shows that epithelial thickness was similar between the groups on day one. The
epithelial thickness of all groups increased on day three. The highest epithelial thickness was
found in the PRF membrane group (p<0.05) followed by the amniotic membrane group. All
groups showed increased epithelial thickness on day five. Epithelial thickness of the PRF

of
membrane group remained the highest compared to the others, and the difference between the
PRF group and the no-membrane group was significant (p<0.05). The epithelial thickness of

ro
all groups remained enhanced on day seven, but the differences between the groups were not
significant (p>0.05).

4. Discussion
-p
re
Wound healing is a complex and dynamic interaction between various cell types, the
extracellular matrix, cytokines, and growth factors. Wound healing consists of several distinct
lP

but overlapping phases, including hemostasis, inflammation, cell transition and proliferation,
wound contraction, and remodeling. Granulation tissue forms in open wounds and consists of
macrophages, fibroblasts, immature collagen, blood vessels, and basic substances [15].
na

This study showed that the highest numbers of fibroblasts, collagen fibers, and blood
vessels, as well as the thickest epithelium were found on the PRF membrane. This second-
generation fibrin membrane comes from the blood (autologous) and is rich in platelets and
ur

leukocytes. This membrane is relatively cheaper compared to other commercially available

membranes.
Jo

The PRF membrane contains growth factors obtained from cells trapped in a fibrin
matrix, allowing them to be released slowly as the matrix dissolves [16]. Growth factors are
signaling molecules that regulate cellular responses. After being secreted, growth factors
bind to membranes or receptors on cytoplasmic cells and activate wound healing cellular
pathways [17]. In addition, the PRF fibrin matrix is elastic and very strong because it consists
of a weak concentration of thrombin that requires equilateral conversion. This intersection of

7
fibers allows the formation of fine and flexible fibrin tissue that is adequate to support
cytokines and facilitate cellular communication [18].
Fibroblasts respond immediately to factors released by activated platelets in acute
wounds, i.e., platelet-BB derivatives or β-growth change factors, small mediators, such as
lysophosphatidic acid, proteases, such as urokinase-type plasminogen activators, and
chemokines, such as platelet factor-4 [19]. Together with granulocytes, macrophages, and
blood vessels, fibroblasts replace the provisional wound matrix formed by blood clots and
restore tissue structure and function that assembles in the form of granulation tissue [20].
Besides the increase in the number of fibroblasts, this study also showed that collagen
fibers in the PRF group were denser than those in the amniotic membrane group. Fibroblasts

of
support the production of collagen, which provides strength and structure to the granulation
tissue [15]. The denser collagen fibers in the PRF group may have been caused by the growth

ro
factors contained within the PRF membrane, which can be absorbed more easily by the
injured tissue area [21].
The day five results showed that the number of fibroblasts in both biological
-p
membrane groups was higher than that in the no-membrane group. In other words,
application of either membrane increased the number of fibroblasts. In vitro studies have
re
reported that PRF and amniotic membranes increase fibroblast proliferation at different times.
The PRF membrane directly induces fibroblast proliferation beginning in the first 24 hours,
lP

while the amniotic membrane doubles the proliferation of fibroblasts the next day [22]. This
induction of proliferation may be caused by transforming growth factor beta (TGF-β) content
in the amniotic membrane. The PRF membrane also contains platelet-derived growth factor
na

(PDGF) [23], while the amniotic membrane contains basic fibroblast growth factor (b-FGF)
[8]. These two growth factors accelerates the migration and proliferation of fibroblasts [24].
PDGF and FGF also affect epithelial proliferation, mesenchymal cells, and angiogenesis.
ur

Besides platelets, PDGF is also secreted by macrophages, endothelial cells, fibroblasts, and
keratinocytes [25].
Jo

This study showed that the numbers of fibroblasts in all groups decreased on day
seven. Previous studies have reported that apoptosis occurs on day seven, indicating the final
healing process [26]. Interestingly, the number of fibroblasts in the PRF membrane group
was higher than that in the other groups. At the beginning of an injury, damaged blood
vessels cause blood to well up in the tissues, and blood platelets release growth factors and
supporting chemokines attract immune cells while increasing the population of fibroblasts to
produce and excrete additional chemokines, such as interleukin-6 (IL)-6 and IL-8. These ILs
8
recruit neutrophils to the damaged site so that the inflammatory phase can begin earlier. In
addition, this process can also be caused by the physical structure of the PRF membrane,
which is a three-dimensional matrix that functions as a scaffold and a network for release of
growth factors on days seven to eleven [4]. In addition, this thicker physical property
facilitates clinical application because the membrane is stronger to protect the wound.
In this study, collagen density of both membranes was higher than the no-membrane
group on day seven. The TGF-β1 contained in the PRF and amniotic membranes increases
contractions and actin changes in mesenchymal gingival cells [15]. Although there were
significant differences in the number of fibroblast cells, collagen density did change on day
seven. This contradicts previous research stating that the proliferation of fibroblasts is parallel

of
to the synthesis of collagen fibers [27]. Although fibroblasts are known to induce collagen
production to provide strength and structure to granulation tissue, several things may have

ro
caused differences in the results of this study. First, a limitation of this study was the
qualitative observation of collagen [13], as we did not measure other factors that affect
collagen density. The qualitative measurements used in this study grouped collagen into three
-p
groups: a score of one indicated a low collagen fiber density, a score of two indicated
moderate collagen fiber density, and a score of three indicated high collagen fiber density.
re
This measurement was simpler and easier but does not describe the amount or intensity of
collagen fibers produced. In other studies, measurements of collagen density were carried out
lP

quantitatively using ImageJ [28]. Further research is needed to determine the characteristics
and role of fibroblasts during collagen deposition, particularly on day seven, to provide a
better explanation of the differences in these results.
na

The number of fibroblasts observed in this study paralleled angiogenesis. Previous

studies have shown that fibroblasts secrete angiogenic growth factors that induce
angiogenesis [29]. Angiogenesis is characterized by endothelial cell migration and capillary
ur

formation. This is a natural recovery response to the injured microcirculation [30].

Higher numbers of blood vessels were found in the PRF membrane group from days
Jo

one to seven. In addition to increasing the number of fibroblasts, PRF membranes stimulate
angiogenesis faster than amniotic membranes because they contain vascular endothelial
growth factor (VEGF), PDGF, and bFGF, whereas the amniotic membrane only secretes
VEGF and bFGF [31]. In theory, blood vessels are composed of bFGF, VEGF, PDGF, and
endothelial progenitor cells. After injury, platelets produce growth factors, such as VEGF,
bFGF, PDGF, TGF-α, and TGF-β [32], which are bound by receptors to endothelial cells of
old blood vessels. Then, the endothelial cells are activated and release proteolytic enzymes
9
that degrade the basal membrane of the old vessel, which allows endothelial cells to multiply,
grow, and migrate to the injured area. Endothelial cell migration also depends on the
formation of fibrin-fibronectin clots formed during secondary hemostasis [33]. The PRF
membrane provides a strong fibrin matrix, making it possible to facilitate endothelial cells
and increase angiogenesis in wounds with this membrane. PRF also has a smaller amount of
thrombin so that migration is optimal. Matrix metalloproteinases (MMPs) are needed to
dissolve the matrix of tissue around the blood vessels that form new buds. These buds
develop and mature by recruiting smooth muscle cells and pericytes to stabilize the vessel
wall [34]. In contrast, amniotic membranes are known to have anti-angiogenic factors, such
as tissue inhibitor of metalloproteinase-2 and thrombospondin, which interrupt angiogenesis

of
[35].
Angiogenesis is a very important process in wound recovery because blood vessels

ro
are the main source of nutrients and oxygen to support tissue regeneration. When used
together with bone graft material, the PRF membrane activates a matrix that supports
angiogenesis, becoming a net for stem cells and facilitating the coordination of
osteoprogenitor cells in the graft center [36].
-p
Interestingly, we observed controversial results on day three. The numbers of blood
re
vessels in the groups with membranes continued to increase, while those in the no-membrane
group decreased. The regression of angiogenesis in the no-membrane group may have been
lP

caused by a decrease in pro-angiogenic stimulation in the area around the wound [37].
The proliferation stage ends with epithelialization. Epithelialization is a series of
processes in which the damaged epithelial surfaceis covered. Epithelialization involves a
na

series of events in which keratinocytes migrate, multiply, and differentiate to restore the
barrier function [38]. Epithelial thickness in all groups increased from days one to five and
then decreased from days five to seven.
ur

Re-epithelialization can also be stimulated by various secreted substances largely

synthesized by macrophages, such as nitric oxide, cytokines and growth factors, including
Jo

epidermal growth factor (EGF), keratinocyte growth factor, insulin-like growth factor-1, and
nerve growth factor [39].
Re-epithelialization was completed by day seven. Interestingly, the thickness of the
PRF epithelium increased significantly beginning on day three, indicating that
epithelialization in the PRF membrane group occurred faster than that in the other groups.
This also agrees with previous clinical results reporting that PRF membranes protect the
surgical site and support soft tissue recovery. Rapid epithelialization assures comfort of the
10
patient and prevents an infection by restoring a complete epidermal barrier [40]. This occurs
because the PRF membrane contains TGF-β, which increases the distribution and
proliferation of keratinocytes, thereby increasing epithelialization. PDGF and EGF also
accelerate the distribution and proliferation of keratinocytes [41]. Different mechanisms
during this proliferation phase are expected to be considered for use in post-surgical
dressings, particularly after open wound surgery.
We conclude that the PRF membrane stimulated the proliferation phase of the open
wound healing process better than the amniotic membrane by enhancing fibroblast
proliferation, epithelial thickness, and collagen density, as well as inducing earlier
angiogenesis.

of
Financial Disclosure
This research was funded by the Dana Masyarakat Funding Faculty of Dentistry

ro
Universitas Gadjah Mada 2018.

5. Competing Interests
-p
The authors declare that they have no competing interests.
re
Acknowledgements
This research was funded by the Dana Masyarakat Funding Faculty of Dentistry
lP

Universitas Gadjah Mada 2018.

References
na

[1] Grover A, Singh A, Sidhu DS, 2018. A Prospective Randomized Trial of Open
Wound Treatment vs Occlusive Dressings in Elective Surgical Cases with Respect to
ur

Surgical Site Infections, J Clin Diagn Res. 9 (6) 26–9.

[2] Ning Xu Landen, Dongqing Li MS, 2016. Transition from inflammation to
Jo

proliferation: a critical step during wound healing, Cell Mol Life Sci. 3861–85. DOI
10.1007/s00018-016-2268-0.
[3] Kesting MR, Wolff KD, Hohlweg-Majert B SL, 2008. The role of allogenic amniotic
membrane in burn treatment, J Burn Care Res. 29 (6) 907–16.
[4] Khiste SV, Tari RN, 2013. Platelet-Rich Fibrin as a Biofuel for Tissue Regeneration,
ISRN Biomaterial. 1-6.

11
 [5] Sowjanya NP, Rao N, Bhushan NVVS, Krishnan G, 2016. Versitality of the use of
collagen membrane in oral cavity, J Clin Diagn Res. 10 (2) ZC30-ZC33.
[6] Kumar A, Chandra RV, Reddy AA, Reddy BH, Reddy C, Naveen A, 2015.
Evaluation of clinical, antiinflammatory and antiinfective properties of amniotic
membrane used for guided tissue regeneration: A randomized controlled trial, Dent
Res J. 12 (2) 127-135.
[7] Zelen CM, Serena TE, Fetterolf, DE, 2014. Dehydrated human anion/chorion
membrane allograft in patients with chronic diabetic foot ulcers: A long-term follow-
up study, Wound Medicine. 4 (2014) 1-4.
[8] Naik B, Karunakar P, Jayadev M, Marshal VR, 2013. Role of Platelet rich fibrin in
wound healing : A critical review, J Conserv Dent. 16 (4) 284–93.

of
[9] Borie E, Oliví DG, Orsi IA, Garlet K, Weber B, Beltrán V, 2015. Platelet-rich fibrin
application in dentistry : a literature review, Int J Clin Exp Med. 8 (5) 7922–9.

ro
[10] Cortese A, Pantaleo G, Borri A, Amato M, 2016. Platelet-rich fibrin (PRF) in implant
dentistry in combination with new bone regenerative technique in elderly patients, Int
J Surg Case Rep. 28 52–6.
-p
[11] Jianpeampoolpol B, Phuminart S, Subbalekha K, 2016. Platelet-rich fibrin formation
re
was delayed in plastic tubes, Br J Med Med Res. 14 (9) 1-9.
[12] Dohan EDM, Pinto NR, Pereda A, Jimenez P, Corso MD, Kang BS, et al., 2018. The
lP

impact of the centrifuge characteristics and centrifugation protocols on the cells,

growth factors, and fibrin architecture of a leukocyte- and platelet-rich fibrin (L-PRF)
clot and membrane, Platelets. 29 (2) 171-184.
na

[13] Carpenter JW, Mashima TY, Rupiper DJ, Exotic Animal Formulary, 2nd ed, St.
Louis, Elsevier Saunders, 2001.
[14] Tandelilin RTC, Sofro ASM, Santoso AS, Soesatyo MHNE, Asmara W, 2006. The
ur

density of collagen fiber in alveolus mandibular bone of rabbit after augmentation

with powder demineralized bone matrix post incisivus extraction, Maj Ked Gigi (Dent
Jo

J). 39 (2) 43-47.

[15] Hess CT, Clinical Guide to Skin and Wound Care, 7th ed, Ambler, Lippincott
Williams and Wilkins, 2013.
[16] Kokdere NN, Baykul T, Findik Y, 2015. The use of platelet-rich fibrin (PRF) and
PRF-mixed particulated autogenous bone graft in the treatment of bone defects: An
experimental and histomorphometrical study, Dent Res J. 12 418-24.

12
[17] Park JW, Hwang SR, Yoon IS, 2017. Advanced growth factor delivery systems in
wound management and skin regeneration, Molecules. 22 E1259.
[18] Mosesson MW, Siebenlist KR, Meh DA, 2001. The structure and biological features
of fibrinogen and fibrin, Ann N Y Acad Sci. 936 11–30.
[19] Nurden AT, 2011. Platelets, inflammation and tissue regeneration, Thromb Haemost
105 (Suppl.) S13-33.
[20] Schultz GS, Wysocki A, 2009. Interactions between extracellular matrix and growth
factors in wound healing, Wound Repair Regen. 17 (2) 153–162.
[21] Toffler M, Toscano N, Holtzclaw D, Del Corso M, Ehrenfest DM, 2009. Introducing
Choukroun's platelet rich fibrin (PRF) to the reconstructive surgery milieu, J Implant

of
Adv Clin Dent. 1 21–30.
[22] Sari R, Lastianny SP, Fadhilah AN, 2019. The effect of biological membranes to

ro
fibroblast proliferation: Platelet-rich fibrin releasate vs amniotic (Research report),
Dentino Jur Ked Gigi. 4 (1) 83-86
[23] Rana MP, Mehrotra N, 2016. Human Amniotic Membrane : Hope in Periodontal
Regeneration, Int J Recent Sci Res. 5 (4) 564–9.
-p
[24] Kiwanuka E, Junker J, Ericksson E, 2012. Harnessing growth factors to influence
re
wound healing, Clin Plas Surg. 39 239-48
[25] Darby IA, Laverdet B, Bonté F, Desmoulière A, 2014. Fibroblasts and myofibroblasts
lP

in wound healing, Clin Cosmet Investig Dermatol. 7 301–11.

[26] Miller MD, Krangel MS, 1992. Biology and biochemistry of the chemokines: a family
of chemotactic and inflammatory cytokines, Crit Rev Immunol. 12 17-46.
na

[27] Prasetyono TOH, 2009. General concept of wound healing, revisited. Med J Indones.
18 (3) 208–16.
[28] Guilherme F. Caetano. Fronza M, Leite MN, Gomes A, Frade MAC, 2016.
ur

Comparison of collagen content in skin wounds evaluated by biochemical assay and

by computer-aided histomorphometric analysis, Pharm Biol. 54 (11) 2555-2559.
Jo

[29] Blakaj A, Bucala R, 2012. Fibrocytes in health and disease, Fibrogenes Tissue Repair.
5 (1) S6.
[30] Broughton II G, Janis JE, Attinger CE, 2006. Wound healing: an overview, Plast
Reconstr Surg. 117 (Suppl.) 1eS–32eS.
[31] Bogic LV, Brace RA, Cheung CY, 2000. Cellular localization of vascular endothelial
growth factor in ovine placenta and fetal membranes, Placenta. 21 203–9.

13
[32] Nofikasari I, Rufaida A, Aqmarina CD, Failasofia, Fauzia AR, Handajani J, 2016.
Efek aplikasi topikal gel ekstrak pandan wangi terhadap penyembuhan luka gingiva,
Maj Ked Gi Ind. 2 (2) 53–9.
[33] Dipietro LA, 2016. Angiogenesis and wound repair : when enough is enough, J Leuk
Bio. 100 979–84.
[34] Honnegowda TM, Kumar P, Govindarama E, Udupa P, Kumar S, Kumar U, et al.,
2015. Role of angiogenesis and angiogenic factors in acute and chronic wound
healing, Plast Aesthet Res. 2 (5) 243–9.
[35] Akram L, Anne E, Albert R, Louise C, Petrovski G, Singh H, et al., 2018. In vitro
anti-angiogenic effects of cryo-preserved amniotic membrane and the role of TIMP2

of
and thrombospondin, J EuCornea. 1 (1) 3–7.
[36] Simonpieri A, Del Corso M, Sammartino G, Dohan Ehrenfest DM, 2009. The

ro
relevance of Choukroun's platelet-rich fibrin and metronidazole during complex
maxillary rehabilitations using bone allograft. Part II: Implant surgery, prosthodontics,
and survival, Implant Dent. 18 220–9.
-p
[37] Pastar I, Stojadinovic O, Yin NC, Ramirez H, Nusbaum AG, Sawaya A, et al., 2014.
Epithelialization in wound healing: A comprehensive review, Adv Wound Care. 3 (7)
re
445-464.
[38] Rousselle P, Montmasson M, Garnier C, 2019. Extracellular matrix contribution to
lP

skin wound re-epithelialization, Matrix Biol. 75–76 12–26.

[39] Barrientos S, Stojadinovic O, Golinko MS, Brem H,Tomic-Canic M, 2008. Growth
factors and cytokines in wound healing, Wound Repair Regen. 16 (5) 585–601.
na

[40] Azar DT, Refractive Surgery 3rd ed, St. Louis, Elsevier Health Sciences, 2019. p.
238.
[41] Raja, Sivamani K, Garcia MS, Isseroff RR, 2007. Wound reepithelialization
ur

Modulating keratinocyte migration in wound healing, Front Biosci. 1 (12) 2849–68.

Jo

14
 Jo
ur
na
lP
re
-p
ro
of

15