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Use of Platelet-Rich Fibrin in Facial Esthetics
Chapter · January 2020
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6/
USE OF
PLATELET-RICH
FIBRIN IN FACIAL
ESTHETICS
Richard J. Miron
Yufeng Zhang
Ana Paz
Masako Fujioka-Kobayashi
Catherine Davies
Platelet concentrates have seen a steady rise in use in various fields of
medicine as a natural autogenous source of growth factors derived from
human peripheral blood. While platelet-rich plasma (PRP) was proposed
as a first-generation platelet concentrate over three decades ago, over
the past 10 years platelet-rich fibrin (PRF) has been introduced as a more
natural platelet concentrate because of its removal of anticoagulants. Its
use has expanded into many fields of medicine, including in facial rejuvenation procedures, because of its superior wound healing capabilities. Over
the years, modifications in centrifugal protocols (known as the low-speed
centrifugation concept) have demonstrated that lower centrifugation
speeds and times lead to an increase in platelets and white blood cells,
which favors higher growth factor release, in vivo vascularization, and tissue
regeneration when compared to PRP. This has been further enhanced utilizing horizontal centrifugation. This chapter reviews the history of platelet
concentrates from PRP to PRF and highlights the recent advancements
made and scientific foundation for these centrifugation protocols leading
to liquid and extended PRF (e-PRF). Finally, the use of PRF in facial esthetics
and facial rejuvenation protocols is presented in the form of an injectable
growth factor complex capable of stimulating tissue regeneration as well
as a platelet concentrate utilized as a topical growth factor solution for
microneedling procedures.
79
6 / Use of Platelet-Rich Fibrin in Facial Esthetics
Acellular plasma (PPP)
Fibrin clot (PRF)
FIG 6-1
Red corpuscules base
Overview of PRF in Medicine
Recent years have witnessed a steady increase in the
use of platelet concentrates to generate supraphysiologic doses of blood growth factors for the regeneration of various human tissues. Their use extends into
multiple fields of medicine, including for the management of osteoarthritic knees, the repair of rotator cuffs,
facial rejuvenation procedures, and the regeneration of
various tissues found in the oral cavity.1 While autogenous PRP was developed as a first-generation platelet
formulation in the 1970s and 1980s,2,3 its incorporation
of anticoagulants such as bovine thrombin has been
shown to slow optimal wound healing.4,5 Naturally,
the first step during wound healing after trauma is the
formation of a blood clot followed by the entrapment
of platelets and leukocytes that initiate wound healing.
Because PRP includes anticoagulants, this clotting
cascade is slightly reduced, leading to less-than-optimal wound healing. Nevertheless, PRP has been
utilized across multiple areas of medicine as a bioactive regenerative agent that releases growth factors
and cytokines to the surrounding microenvironment.
This has been shown to speed tissue regeneration of
both soft and hard tissues.4,6–12
80
Layers produced after centrifugation of whole
blood. A PRF clot forms in the upper third of
glass tubes after centrifugation.
Due to the reported limitations of PRP, PRF was
proposed with the aim of eliminating the use of anticoagulants within the platelet formulation.13 Because
the anticoagulants are removed, blood naturally clots
during the 8- to 12-minute centrifugation period.
Following centrifugation, three layers are typically
found, including an upper platelet-poor plasma layer,
a platelet-rich plasma layer, and a red corpuscle base
layer (Fig 6-1). This second-generation platelet concentrate differs significantly from previous versions of
PRP because the platelet-rich layer is then able to clot,
forming what is known today as platelet-rich fibrin.
PRF contains a high concentration of platelets and
leukocytes entrapped within a fibrin matrix, thereby
significantly improving not only the host’s immune
system defense against incoming pathogens8,14–18
but also the secretion of growth factors and cytokines responsible for tissue regeneration over time.4,19
The most common growth factors found in platelet
concentrates are platelet-derived growth factor
(PDGF), transforming growth factor β (TGF-β), and
vascular endothelial growth factor (VEGF).4,19 While
each component possesses an individual role in tissue
regeneration, PDGF has since been commercially available as a recombinant growth factor under the trademark name GEM 21S (Lynch Biologics).
Brief History of Platelet Concentrates: From PRP to PRF
A recent systematic review investigating soft tissue
regeneration found that over 20 regenerative procedures have utilized PRF successfully to stimulate
tissue regeneration in various fields of medicine and
dentistry;1,20 7 of these procedures took place in the oral
and maxillofacial region, while the remaining 13 were
medical procedures. In medicine, the most common
use of PRF has been for the successful management
of hard-to-heal leg ulcers, including diabetic foot ulcers,
venous leg ulcers, and chronic leg ulcers.21–25 Furthermore, PRF has been investigated for the management
of hand ulcers26 and facial soft tissue defects;27 for laparoscopic cholecystectomy;28 in plastic surgery for the
treatment of deep nasolabial folds, volume-depleted
midface regions, facial defects, superficial rhytids,
and acne scars;29 for the induction of dermal collagenesis;30 for vaginal prolapse repair31 and urethracutaneous fistula repair;32,33 during lipostructure surgical
procedures;34 as well as in the management of chronic
rotator cuff tears35 and acute traumatic ear drum perforations.36 This chapter is focused on the use of PRF in
facial esthetics.
Brief History of Platelet
Concentrates: From PRP to PRF
While the use of platelet concentrates has recently
gained tremendous momentum as a regenerative
autologous source of growth factors, it is important
to note that their use spans over three decades in
surgery.37 It was originally proposed that concentrated
platelets derived from autogenous sources could be
collected in plasma solutions later to be utilized in
surgical sites to potentially release supraphysiologic
doses of growth factors capable of promoting local
healing.38,39 Further work in the 1990s led to the popular working name platelet-rich plasma.3,11,40 Because
the goal of PRP preparation was to collect the largest
and highest number of platelets, PRP was fabricated
with a protocol lasting over 30 minutes of centrifugation and required the use of anticoagulants to prevent
clotting. The final composition of PRP contains over
95% platelets, cells known to be responsible for the
active secretion of growth factors involved in initiating
wound healing of various cell types, including osteoblasts, epithelial cells, and connective tissue cells.40,41
Following surgical application of PRP, several limitations were observed. The technique and the preparation required the additional use of bovine thrombin or
calcium chloride in addition to coagulation factors,
and these were found to drastically reduce the healing
process during the regenerative phase. Furthermore,
the protocol was technique sensitive, with several steps
that could sometimes last upward of 1 hour, making it
inefficient for everyday clinical purposes. In addition,
because PRP is liquid in nature, in many fields of medicine a scaffold was required to improve growth factor
release over time. Very recent data has shown that
growth factor release from PRP occurs very early in the
delivery phase, whereas an optimal preference would
be to deliver growth factors over an extended period of
time during the entire regenerative phase.4,42,43
These combined limitations therefore led to the
development of a second generation of platelet concentrates that eliminate anticoagulants, thereby facilitating
formation of a fibrin matrix that incorporates the same
set of growth factors and cells that can be released
slowly over time.44 Furthermore, PRF (which has also
been named leukocyte PRF or L-PRF) contains white
blood cells, which have been shown to be key contributors to wound healing (Fig 6-2). These cells in combination with monocytes, neutrophils, and platelets are
the main players in tissue wound healing and together
are able to further enhance new blood vessel formation
(angiogenesis) and tissue formation.16,45–48
Numerous studies have investigated the regenerative potential of PRF in various medical clinical applications. With respect to tissue engineering, it has long
been proposed that in order to maximize the regenerative potential of various bioactive scaffolds, three
components are essential to improve tissue repair: (1)
a 3D matrix capable of supporting tissue ingrowth, (2)
locally harvested cells capable of influencing tissue
growth, and (3) bioactive growth factors capable of
enhancing cell recruitment and differentiation within
the biomaterial surface. PRF encompasses all three of
these properties, whereby (1) the fibrin matrix serves as
81
6 / Use of Platelet-Rich Fibrin in Facial Esthetics
Cell
types
Provisional
extracellular matrix
• PDGF
• VEGF
• IGF
• EGF
• TGFB
• BMP2
Platelet
Leutocyte
• Fibrin matrix
including: f ibronectin
vitronectin
Red cell
the scaffold surface material; (2) cells including leukocytes, macrophages, neutrophils, and platelets attract
and recruit future regenerative cells to the treatment
sites; and (3) the fibrin acts as a reservoir of growth
factors that may be released over time (10 to 14 days).
These three components are described below.
Fibrin matrix
The removal of anticoagulants from the collected
host blood allows for the formation of a fibrin clot
during the centrifugation process. Naturally this technology requires a centrifuge and a collection system
present within the office; because anticoagulants are
not utilized, clotting forms rapidly, so centrifugation
must take place immediately after blood collection.
The original PRF protocol was very simple: A blood
sample is collected without anticoagulant in 10-mL
tubes that are immediately centrifuged at about 700g
for 12 minutes. The absence of anticoagulant implies
the activation of most of the blood platelets in contact
with the tube walls and the release of coagulation
cascades within a few minutes. Fibrinogen is initially
82
Bioactive
molecules
FIG 6-2
Natural components of PRF include various cell types (platelets, leukocytes, and
red blood cells), a provisional extracellular matrix 3D scaffold fabricated from
autologous fibrin (including fibronectin
and vitronectin), as well as a wide array
of over 100 bioactive molecules, including most notably PDGF, VEGF, insulin-like
growth factor (IGF), epidermal growth
factor (EGF), TGF-β, and bone morphogenetic protein 2 (BMP-2). (Reprinted with
permission from Miron et al.1)
concentrated in the upper layer of the tube, before the
circulating thrombin transforms it into fibrin. A fibrin
clot is then obtained in the upper middle portion of
the tube, between the red blood cells at the bottom
of the tube and the acellular platelet-poor plasma at
the top (see Fig 6-1).
As previously stated, the success of the technique
is entirely dependent on the speed of blood collection
and its subsequent transfer to the centrifuge. Indeed,
without anticoagulants, the blood samples start to
coagulate and it takes a minimum of a few minutes of
centrifugation to concentrate fibrinogen in the middle
and upper part of the tube when utilizing glass tubes.
Quick handling is therefore the only way to separate
the blood layers efficiently prior to clot formation. By
driving out the fluids trapped in the fibrin matrix, practitioners will obtain very resistant autogenous fibrin
membranes that may be utilized in place of commercially available collagen membranes or other scaffolds
that have been utilized to treat defects such as large
diabetic foot ulcers, skin burns, and soft tissue defects
following surgery.
Brief History of Platelet Concentrates: From PRP to PRF
Major cell types in PRF
Platelets
Platelets are one of the cornerstone cells found in
PRF and the cells that were first collected in previous
versions of platelet concentrates (ie, PRP). Interestingly, in PRF platelets are theoretically trapped within
the fibrin network, and their 3D mesh allows their slow
and gradual release as well as associated growth
factors over time.4
Platelets are constantly being formed in the bone
marrow from megakaryocytes. They are discoidal and
anuclear structures by nature, and their life span is typically in the range of 8 to 10 days. Their cytoplasm contains
many granules whose contents are secreted at the time
of activation. Alpha granules contain many proteins,
both platelet-specific (such as β-thromboglobulin) and
non–platelet specific (fibronectin, thrombospondin,
fibrinogen, and other factors of coagulation, growth
promoters, fibrinolysis inhibitors, immunoglobulins,
etc), that have been shown to possess many functions during wound healing.49,50 Moreover, the platelet
membrane is a double-layer phospholipid into which
receptors for many molecules are inserted (collagen,
thrombin, etc) and act to improve wound healing.
Activation is fundamental to initiate and support
hemostasis because of aggregation at the injured
site and interactions with the various coagulation
mechanisms.49,50
Leukocytes
Leukocytes are the other major cell type found in PRF,
playing a prominent role in wound healing. In fact, a
major difference between PRF and previous generations of platelet concentrates is that the latter contain
very low quantities of leukocytes, if any at all. The literature related to platelet concentrates often ignores the
importance of leukocytes and monocytes on tissue
wound healing. Several studies have already pointed to
their key role, both for their anti-infection actions and
immune regulation.51–53 Apart from their anti-infection
effect, leukocytes produce large amounts of VEGF
and PDGF, among other growth factors. The number
of white blood cells in PRF has been further improved
with newer centrifugation protocols as discussed later
in this chapter.
Studies from the basic sciences have revealed the
potent and high impact of leukocytes on tissue regeneration.8,18 In addition to releasing growth factors and
playing a large role in immune defense, they also serve
as key regulators controlling the ability of regenerative
agents to adapt and modify to new environments.
Studies have shown that patients receiving PRF
reported less postoperative pain, less need for analgesics, more rapid wound closure, and reduced swelling.54 This is primarily explained by the clotting that
occurs in PRF, which traps cells and growth factors
capable of regenerating tissue in a natural way.
Major growth factors in PRF
Cytokines and growth factors have been observed
to be released in high numbers from platelet alpha
granules following clotting. They are active through
specific cell receptors and play a predominant role in
wound healing. Centrifugation time and speed affect
the density and release rate of growth factors from
PRF clots (see next section). The most commonly
reported growth factors found in PRF include the
following:
• PDGF: As the main growth factors derived from
platelets, PDGFs are essential regulators for the
migration, proliferation, and survival of mesenchymal cell lineages. According to the distribution of
their specific receptors, they are able to induce
stimulation in many cell types. For this reason,
PDGFs play a critical role in the mechanisms of
physiologic healing and have been commercially
available in a recombinant source (rhPDGF-BB)
and FDA approved for the regeneration of various
defects in medicine and dentistry. Interestingly,
PDGFs are naturally produced and accumulated
in high quantities in PRF clots and are considered
one of the most important released molecules over
time from PRF.
• TGF-1: TGF-βs encompass a vast superfamily of more than 30 members known as fibrosis
83
6 / Use of Platelet-Rich Fibrin in Facial Esthetics
FIG 6-3
Histologic observation of leukocytes following centrifugation. Resulting white blood cells have been shown to be contained in the layers between the plasma PRF layer and the red
blood cell clot. This finding demonstrated quite clearly that
the g-force was excessive, necessitating the development
of newer protocols aimed to improve the retention of leukocytes. (Reprinted with permission from Ghanaati et al.58)
agents.55,56 TGF-β1 constitutes the most powerful fibrosis agent among all cytokines and the
growth factor commonly released from autogenous bone during tissue repair and remodeling.56
In simpler terms, it induces a massive synthesis of
matrix molecules such as collagen-1 and fibronectin, whether by osteoblasts or fibroblasts. Thus,
although its regulation mechanisms are particularly complex, TGF-β1 can be considered as an
inflammation regulator through its capacity to
induce fibrous cicatrization.
• VEGF: VEGF was previously isolated as the most
potent growth factor leading to angiogenesis of
tissues.57 It has potent effects on tissue remodeling, and the incorporation of VEGF alone into various bone biomaterials has demonstrated increases
in new bone formation, thereby pointing to its rapid
and potent effects.57
Together these three properties of PRF
membranes—a 3D fibrin matrix, host cells,
and cytokine and growth factor release—
synergistically lead to a fast and potent
increase in tissue regeneration.
84
The Low-Speed Centrifugation
Concept
It is now known that the most important factor for
stimulation is not the amount of growth factors
released but the maintenance of a low and constant
gradient of growth factor delivery to the environment. As the use of PRF has seen a continuous and
steady increase in regenerative medicine, there
has been great interest in determining if the protocols can be optimized by modifying centrifugation
protocols. This hypothesis was derived from the fact
that cells within the original PRF matrix surprisingly
were found accumulated at the bottom of the PRF
matrix or at the bottom of the centrifugation tubes
outside the PRF clots (Fig 6-3).58 To briefly explain
this concept, as centrifugation speed is increased (ie,
the longer centrifugation takes place), or the higher
the relative centrifugal force (RCF) value utilized
(g-force), the more cells move toward the bottom
of the tube. Because PRF is obtained from the upper
layer of centrifugation tubes, it was hypothesized
that lower speeds may be more beneficial for obtaining a higher concentration of platelets, leukocytes,
and growth factors.
Liquid PRF and Heat-Treated PRF
3
*
**
a
— LS-PRF
TGF-b1 accumulated release
over time (pg/mL)
Cell migration
(fold change to control)
— LS+T-PRF
2
#
1
0
Control
PRP
PRF
LS-PRF
b
40,000
--- PRF
**
30,000
**
20,000
10,000
0
15 min 60 min
8h
#
#
#
1d
3d
10 d
FIG 6-4
Cell migration and TGF-β growth factor release resulting from the low-speed centrifugation concept over a 10-day period. In general, low-speed PRF (LS-PRF) significantly demonstrated the greatest ability for cell migration and highest growth factor release.
Furthermore, a reduction in speed and time (LS+T-PRF) further favored additional growth factor release. An asterisk denotes a
significant difference, a double asterisk denotes a value significantly higher than all other groups, and a number sign denotes a
value significantly lower than all other groups. (Data from Fujioka-Kobayashi et al.19)
This hypothesis was confirmed by a classic study by
Ghanaati et al, who showed that by decreasing centrifugation speeds, a more optimal formulation of PRF
could be achieved with a higher number of leukocytes
more evenly distributed throughout the PRF matrix.58
It is now recognized that the leukocytes were being
pushed out of the fibrin clots unnecessarily down to the
bottom of centrifugation tubes because of these high
centrifugation speeds and times. More recently, it has
been demonstrated that both centrifugation speed and
time could be reduced to further enhance growth factor
release and cell performance from PRF (Fig 6-4).19
One of the primary proposed reasons for a slower
release of growth factors over time is the ability of the
fibrin matrix to hold proteins within its fibrin network
as well as contain cells capable of further releasing
growth factors into their surrounding microenvironment.59–63 Therefore, if centrifugation protocols are
optimized to contain more cells (most notably leukocytes), then they will subsequently have the potential
to release more growth factors over a 10-day period
as well as contribute to tissue defense and biomaterial
integration, all factors necessary to further enhance
tissue regeneration.
As centrifugation speeds have been drastically
decreased since the first version of PRF, it has been
observed that should protocols be spun even slower,
a liquid formulation of PRF could be obtained, prior to
clot formation. This new formulation was given the
working name injectable PRF or liquid PRF because
of its hypothesized ability to be injected into defects
or to be combined with other biomaterials, further
improving tissue regeneration. While ongoing research
is underway, this new formulation of liquid PRF has
been shown to contain an increase in leukocytes and
platelets, which have also been detected utilizing
lower centrifugation speeds with a centrifugation
time of 3 to 5 minutes.
Liquid PRF and Heat-Treated PRF
Liquid PRF was developed to act as a regenerative
agent that could be delivered in liquid form by drawing
blood and rapidly processing it in a specific centrifugation tube at a very low speed for an even shorter
centrifugation time (3–4 minutes). Here the objective
was to centrifuge the blood without anticoagulants or
additives yet maintain the ability to separate it into 2
85
6 / Use of Platelet-Rich Fibrin in Facial Esthetics
FIG 6-5
Newer centrifugation protocols allow production of a liquid
formulation of PRF found in the top 1- to 2-mL layer of centrifugation tubes following a 3- to 5-minute protocol. This liquid
can be collected in a syringe and reinjected into defect sites or
mixed with biomaterials to improve their bioactive properties.
layers (Fig 6-5). Produced on a horizontal centrifuge
with spin cycles of 5 minutes at 300 g, liquid PRF is
very rich in cells and growth factors.
This new formulation can be utilized for a variety of
procedures, including knee injections for the management of osteoarthritis, temporomandibular joint (TMJ)
injections for the management of TMJ disorders,
as well as various procedures in facial esthetics to
improve collagen synthesis naturally. The principle
behind liquid PRF remains the same—It contains a
larger proportion of leukocytes and blood plasma
proteins due to the low-speed centrifugation concept.
Because liquid PRF contains the highest proportion of
platelets and growth factors by volume, it remains the
optimal PRF formulation for small-volume injections
such as those used for facial esthetics. Upon injection,
liquid PRF will subsequently clot, facilitating a better
ability to maintain deficient volumes such as those
observed in facial wrinkles (eg, nasolabial folds). It
has been discovered that clotting occurs better with
slightly higher g-forces and/or centrifugation times.
86
FIG 6-6
Layer separation produced on a fixed-angle centrifuge. Note the uneven separation at the junction between the red blood cells and PRF.
Therefore, should the clinician desire to produce a
more dense fibrin scaffold (ie, to fill deeper facial
voids), a heat-treated PRF protocol may be utilized
to extend the resorption of PRF from 2–3 weeks to
4–6 months (extended PRF [e-PRF]). The protocols for
the production of e-PRF are highlighted in chapter 12.
In 2019, a breakthrough article demonstrated that
horizontal centrifugation allowed for better blood
separation than traditional centrifugation methods.64
Because all PRF centrifuges were developed using
fixed-angled rotors, one of the disadvantages was the
accumulation of cells along the outside glass walls
caused by high g-force (Fig 6-6). Furthermore, with
traditional centrifuges separation cannot occur effectively because larger cells (such as red blood cells)
typically trap and pull smaller platelets to the bottom
of PRF tubes (Fig 6-7). With horizontal centrifugation,
on the other hand, the separation of cell layers is linear
without accumulation of cells along the outer centrifugation tube wall (see Fig 6-7).
Liquid PRF and Heat-Treated PRF
Fixed-angle
centrifugation
Horizontal
centrifugation
Fixed-angle centrifuge
Horizontal centrifuge
G-force applied
G-force applied
Rotor axis
Rotor axis
RCF min
Due to the fixed-angle centrifuge, cells
accumulate in an
angled fashion
Horizontal centrifugation produces
a completely linear
separation
a
RCF max
RCF max
RCF min
b
FIG 6-7
Illustrations comparing fixed-angle and horizontal centrifuges. (a) Following centrifugation on fixed-angle centrifuges, blood
layers do not separate evenly, and as a result an angled blood separation is observed. In contrast, horizontal centrifugation
produces an even separation. (b) With fixed-angle centrifuges, separation of blood layers based on density is achieved due to
the difference in RCF-min and RCF-max. Note how even at the same RCF-min, the RCF-max on a horizontal centrifuge is much
greater, which favors more effective cell layer separation. Because of the large RCF values (about 200–700g), on a fixed-angle
centrifuge cells are pushed toward the back of centrifugation tubes and then downward or upward based on cell density. These
g-forces produce additional shear stress on cells as they separate along the walls of centrifugation tubes. In contrast, horizontal
centrifugation allows for the free mobility of cells to separate into their appropriate layers based on density, allowing for more
optimal cell separation as well as less trauma/shear stress on cells.
Control
PRP
Liquid PRF
a
FIG 6-8
Cell migration (% of control)
400
b
**
*
300
200
100
0
Control
PRP
Liquid PRF
(a and b) Migration assay of human skin fibroblasts cultured with liquid PRF and PRP after 24 hours. (Scale bars = 100 μm. An asterisk [*] denotes a significant difference between two groups at P < .05, and a double asterisk [**] denotes a value significantly
higher than all other treatment groups at P < .05). This assay was performed in triplicate with three independent experiments.
Regenerative potential of PRP vs liquid PRF
In a recent study, dermal skin fibroblasts were cultured
with either liquid PRF or PRP and investigated for their
ability to promote/influence cell viability, migration,
spreading, proliferation, and mRNA levels of known
mediators of dermal biology, including PDGF, TGF-β,
and fibronectin.65 All platelet concentrates were
nontoxic to cells, demonstrating high cell survival.
Skin fibroblasts migrated over 350% more in liquid
PRF when compared to the control and PRP (200%
increase) (Fig 6-8). Liquid PRF also significantly
87
6 / Use of Platelet-Rich Fibrin in Facial Esthetics
PDGF
6
TGF-b
5
**
3
2
*
1
0
3 days
7 days
Relative gene expression
c
0
*
2
1
d
0
3 days
3
*
2
1
3 days
7 days
FN1
**
**
4
*
3
*
5
FIG 6-9
b
COL1
6
3
Relative gene expression
a
4
4
Relative gene expression
Relative gene expression
**
5
7 days
**
2
*
1
0
3 days
Control
PRP
7 days
Liquid PRF
Expression of regeneration-related and extracellular matrix–-related genes of gingival fibroblasts cultured with PRP and liquid
PRF at 3 and 7 days: (a) PDGF, (b) TGF-β; (c) COL1, and (d) FN1. (An asterisk denotes a significant difference between two groups
at P < .05, and a double asterisk denotes a value significantly higher than all other treatment groups at P < .05). This assay was
performed in triplicate with three independent experiments.
induced greater cell proliferation at 5 days. While both
PRP and liquid PRF induced significantly elevated cell
mRNA levels of PDGF, it was observed that TGF-β,
collagen-1 (COL1), and fibronectin (FN1) mRNA levels
were all significantly highest in the fluid PRF group (Fig
6-9). Lastly, liquid PRF demonstrated a significantly
greater ability to induce collagen matrix synthesis
when compared to PRP (Fig 6-10). In conclusion, it
was found that liquid PRF has greater regenerative
potential on human skin fibroblasts.65 Furthermore,
because PRF tubes do not contain any additives, it is
further considered a more natural approach to tissue
regeneration, not to mention less expensive for the
clinician.
88
Collecting PRF from peripheral blood
In order to utilize PRP or PRF, it is important to be
familiar with phlebotomy techniques. Because with
PRF a short working time is required, it is advised that
prior to initiating any blood collection, the centrifuge
is set on the appropriate protocol, open and ready for
use (Fig 6-11). Because no anticoagulants are being
utilized, blood collection must occur rapidly (within
90 seconds ideally) and then centrifuged to maximize the regenerative potential of PRF. After blood
collection, blood tubes are added to a centrifuge (Fig
6-12). Following a 3- to 5-minute protocol, the liquid
PRF tubes are removed.
Liquid PRF and Heat-Treated PRF
Control
PRP
Liquid PRF
a
Collagen 1 staining intensity
(% of control)
250
b
*
200
150
100
50
0
Control
FIG 6-10
**
PRP
Liquid PRF
Immunofluorescent collagen type 1 (COL1) staining of skin fibroblasts cultured with PRP and liquid PRF at 7 days. (a) COL1 staining (green) merged with DAPI staining (blue). (Scale bars = 100 μm.). (b) COL1 staining quantification. (An asterisk denotes a
significant difference between two groups at P < .05, and a double asterisk denotes a value significantly higher than all other
treatment groups P < .05). This assay was performed in triplicate with three independent experiments.
a
FIG 6-11
(a) Clinical photograph of a BIO-PRF centrifuge. (b) Photograph demonstrating the horizontal centrifugation concept.
The tubes are inserted vertically, but once the device begins
to rotate the tubes swing out completely horizontally. This
favors better blood cell layer separation with higher platelet
and growth factor concentrations.
b
89
6 / Use of Platelet-Rich Fibrin in Facial Esthetics
a
b
c
d
e
f
h
i
FIG 6-12
Blood collection procedure for PRF. (a) First, a tourniquet is tied about 3 inches above
the elbow. (b) A vein light is then utilized to locate the vein. (c) An alcohol wipe is
used to disinfect the area. (d) A bandage is then typically attached to a nearby location (in this case, the practitioner’s glove) to speed use. (e) The butterfly needle
is then inserted into the vein at a 15- to 30-degree angle and parallel to the vein. (f)
Backflow is observed within the butterfly needle. (g) The collection tubes are then
inserted, and vials of blood are collected. (h) Following blood draw, a bandage is
placed over the puncture site and the butterfly needle removed. (i) Compression is
applied to the puncture site.
g
90
Liquid PRF and Heat-Treated PRF
j
k
FIG 6-12 (cont)
(j) PRF tubes are placed in the centrifuge. (k) Many butterfly needles come with a safety feature locking the needle after use.
When utilizing the liquid PRF formulation, it is important to NOT remove the lids and expose it to oxygen. This
exposure will further speed clotting, and because the
goal is to use liquid PRF as an injectable material, clotting should be
When utilizing the liquid PRF formulaavoided. (If left
tion, it is important to NOT remove the
unexposed to
lids and expose it to oxygen.
oxygen, the PRF
will typically
clot after 20 to 45 minutes, depending on the centrifugation tubes utilized.) Therefore, after centrifugation,
a 21- to 27-gauge needle (ideally 3 inches in length or
longer) is penetrated through the rubber portion of the
lid, and the liquid PRF is aspirated into the syringe (Fig
6-13a). Here it is important to draw up as much as the
liquid PRF as possible, remembering that the greatest
proportion of cells are found at the junction between
the liquid PRF and the red blood cell layer. Figure 6-13b
depicts a syringe filled with liquid PRF that may then
either be used for injections or be added to the skin
layer prior to microneedling.
a
b
FIG 6-13
(a) Collection of liquid PRF with a syringe. Note that the lid
should not be removed, because oxygenation will speed clotting
and reduce the working time of the clinician. (b) Syringe with
collected liquid PRF ready for future facial injection purposes.
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6 / Use of Platelet-Rich Fibrin in Facial Esthetics
PRF in Facial Esthetics
Until recently, the use of PRF in facial esthetics
paled in comparison to the use of PRP.66,67 In 2010,
Dr Anthony Sclafani performed several studies
investigating the ability of PRF to successfully fill
nasolabial folds.68 PRF has since been proven to
be a safe and effective growth factor concentrate
capable of improving facial rejuvenation. 29 More
recently, PRF has been combined with various
other treatment strategies such as dermal fillers
or nanofat grafting protocols to further improve
tissue regeneration.69,70 PRF has also been shown
to increase hair density in androgenetic alopecia.71
PRF follows the same logic as the previously
utilized PRP in that it acts by stimulating mitogenic activity in cells while being capable of rapidly
improving tissue recruitment of cells. From this
point of view, much literature now supports the
use of PRP in facial esthetics and for hair regeneration. Because of the superior preclinical outcomes
of PRF, its ability to provide a slower and more gradual release of growth factors over time is thought
to further enhance tissue regeneration of facial
tissues, though to date no comparative studies
exists.
Once liquid PRF is drawn into a syringe, it is
important to note that it will clot within 20 to
40 minutes if left in the syringe. Furthermore, if
exposed to oxygen, clotting will occur significantly
more rapidly. From here, PRF may be utilized as an
injectable device either into facial tissues or into
the scalp in a similar fashion as PRP. It may also be
utilized as an autogenous growth factor applied to
the face prior to or after microneedling, in a similar
fashion to PRP in the vampire facelift technique.72,73
It is important to understand that in the field of
esthetic medicine, only plastic tubes are utilized
in order to prevent coagulation into a PRF fibrin
matrix. In medicine and dentistry, the use of PRF
membranes typically favor glass tubes or silicacoated plastic tubes to promote faster clotting. Two
separate formulations of PRF are discussed within
the present chapter: a liquid injectable formulation
92
of PRF termed liquid PRF centrifuged at lower
speeds to maintain cells in the upper layer (300g
for 5 minutes) as well as a more dense e-PRF utilized
as a substitute for fillers (700g for 5 minutes).
These protocols are for a horizontal centrifuge; if
using a fixed-angle centrifuge, lower g-forces are
utilized (typically 60–300g) because of the reduced
distance from the radius at the maximum distance
of the tube (see Fig 6-7b). The main aim of treatment with PRF is to naturally improve the patient’s
cosmetic appearance by providing a natural regenerative therapy. Unlike fillers that simply fill defects,
PRF aims to actually restore and rejuvenate skin.
Specific protocols are described in future chapters, but the treating practitioner should always
remember that liquid PRF contains more cells and
growth factors and can be effectively utilized with
microneedling and for the regeneration of superficial
tissues. e-PRF utilizes a faster centrifugation cycle
and as a result contains more fibrin. This is useful
for filling larger voids such as pronounced nasolabial folds. In general, the growth factor release
from PRF has been observed at up to 10 to 14 days.
Because of the regenerative cycle found in skin, a
typical 14- to 28-day treatment cycle is typically the
initial treatment regimen. The authors recommend
an initial PRF therapy treatment plan with three to
four therapies evenly distributed once a month for
the first 3 to 4 months. Thereafter, maintenance can
be performed every 6 to 12 months as discussed
later in this book.
Liquid PRF therapy with microneedling
A common and effective procedure for facial regeneration is microneedling with a Dermapen (Fig 6-14),
as described in chapter 7. For such procedures, the
treating practitioner aims to deliver small doses of
liquid PRF subdermally via the microneedling tips
from a 0.25- to 2.5-mm depth. The protocol begins
with a layer of liquid PRF (most concentrated in cells
and growth factors) applied topically to the face.
Thereafter, the microneedling device pushes the
topical liquid PRF layer into the skin subdermally.
PRF in Facial Esthetics
a
b
FIG 6-14
(a and b) Topical application of liquid PRF on facial skin surfaces followed by microneedling penetration into the skin.
Following microneedling of the area, another layer
of liquid PRF is applied to fill all the microchannels created via needling (see chapter 7). This 20to 30-minute procedure is generally considered
mechanical skin stimulation. Maintenance is highly
recommended in order to prolong clinical results.
Mesotherapy by syringe injections using
liquid PRF and e-PRF
In this procedure, a needle carrying liquid PRF or e-PRF
is injected more deeply into the skin74 (Fig 6-15), as
described in chapter 8. This has been shown to additionally benefit deeper deficits in skin volume, and
the protocols may be mixed or optimized depending
on the goal (the greater the volume deficit, the more
e-PRF is required). This procedure typically uses both
liquid PRF and e-PRF utilizing varying needle sizes and
gauges, as discussed in chapters 8 and 9. It is also
possible to inject into the same area with both liquid
PRF (which contains a higher concentration of cells
and growth factors) and e-PRF (which favors stability
and the slower and more gradual release of growth
factors), together either simultaneously by premixing
or with subsequent injections into the same area. A
combination approach can also be utilized whereby
liquid PRF is premixed with a facial filler such as hyaluronic acid, as demonstrated in chapter 12. While this
procedure is considered more invasive, causing more
skin damage when compared to microneedling, the
results are more noticeable and superior to less-invasive procedures. Maintenance is once again highly
recommended to maintain the acquired results.
93
6 / Use of Platelet-Rich Fibrin in Facial Esthetics
a
b
c
d
FIG 6-15
(a to e) Use of liquid PRF for facial injections utilizing different-sized needles. (Courtesy of Dr Ana Paz.) This topic is covered in great detail in chapter 8.
e
Conclusion
One of the advantages of PRF as a regenerative strategy is that it does not specifically induce the proliferation or differentiation of one specific tissue type.
It can therefore be utilized with many regenerative
strategies either alone or in combination with other
biomaterials for a variety of procedures. Ongoing
94
research continues to investigate the amount of
volume augmentation that can be achieved utilizing
PRF. Furthermore, very recent research has shown that
the plasma layer can additionally be heated and used
thereafter as a much slower-resorbing “filler” when
compared to liquid PRF, for example for lip augmentation (see chapter 12).
References
PRF has proven to be a next-generation autogenous platelet concentrate with a broad future in facial
esthetics. It can be utilized as a completely safe regenerative modality harvested from the patient’s own
blood at lower costs and even higher safety when
compared to PRP. Furthermore, its ability to speed
tissue angiogenesis leads to significant increases
in soft tissue repair. Future avenues of research are
focused at precisely characterizing the exact indications and protocols of PRF that will effectively treat a
greater variety of facial wrinkles and/or defects.
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