Introduction
Platelet concentrates are products that result from the centrifugation of a blood
sample. They concentrate platelets, fibrin, and leukocytes (depending on the used
protocol) converting them into a clinical and useful form.
With the knowledge of the biological features of the concentrates, the initial protocols
evolved from the platelet concentrates of first generation that include platelet-rich
plasma (PRP) and plasma rich in growth factors (PGRF) to the second-generation concentrates.
These ones include protocols such as leukocyte-platelet-rich fibrin (L-PRF) and advanced
platelet-rich fibrin (A-PRF). The substantial evolution between the first- and second-generation
platelet concentrates is based on the principle that in the second-generation ones,
there is no blood manipulation with additives and they remove the anticoagulants from
the centrifugation protocol.[1 ]
The biological and clinical properties of these second-generation platelet concentrates
make them highly attractive to use in regenerative medicine. In the past decades,
they were used mainly in the dentistry field to speed revascularization of damaged
tissues and bone regeneration before implant placement. Other oral applications include
periodontology, maxillofacial surgery such as gingival recession, intrabony defects,
alveolar filling postextraction, and sinus lifting.[2 ] Nowadays, the autologous platelet concentrates are used not only in dentistry but
also for ulcers/skin necrosis, plastic and reconstructive surgery, and even musculoskeletal
lesions.[3 ]
The success of the platelet concentrates depends on the platelet concentration, the
number/type of leukocytes entrapped in the fibrin membrane, and also of the release
of bioactive molecules at the sites of injury that will trigger the regenerative process.[4 ] Growth factors released include vascular endothelial growth factor (VEGF), platelet-derived
growth factor (PDGF), transforming growth factor-β (TGF-β), epidermal growth factor
(EGF), insulin-like growth factor (IGF-I), hepatocyte growth factor (HGF), cytokines
such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-4 (IL-4), and interleukin-10
(IL-10), and chemotactic molecules such as chemokine ligand-5 (CCL-5) and eotaxin.
Thus, platelet concentrates such as A-PRF have all the biological features to induce
an optimal healing.[5 ]
Platelet Concentrates
Characteristics of Platelet-Rich Plasma, Plasma rich in Growth Factors, Platelet-Rich
Fibrin, and Advanced Platelet-Rich Fibrin
In modern tissue regeneration, the evolution of the platelet concentrates was directed
toward less time, better quality, and less invasiveness using autologous products
and reducing the risk of rejection from the patient. The fact that the second-generation
concentrates respect the European Directive 2004/03/CE can also account for another
characteristic that compares the effectiveness of first- and second-generation platelets,
there is no manipulation of the patients’ blood, so the protocol is also simple and
cheaper.[6 ]
[7 ]
Platelet-Rich Plasma
Inside the PRP family, a clarification is necessary to allow comparing with other
recent protocols. The protocol developed by Jo et al[8 ] uses the same amount of blood as the others of first- and second generations—9 mL.
It is considered a first-generation concentrate using calcium glutamate/bovine thrombin
to activate coagulation and an anticoagulant solution of citrate phosphate dextrose
adenine (CPDA). There is a first centrifugation (900 rpm/5 minutes) to separate the
red cells from the white cells, and three layers are obtained—a superior one rich
in platelet and white cells, a medium one of buffy coat, and an inferior layer with
the red cells. After collecting the two superior layers, a second centrifugation at
1500 rpm/15 minutes excludes the red cells. Both centrifugations use plastic tubes
and the final result is 2 mL of gel containing the ×4.2 increase in the initial number
of platelets.[8 ]
[9 ]
Plasma Rich in Growth Factors
Furthermore, the first-generation concentrate with anticoagulants (sodium citrate)
and coagulation activators (calcium chloride) and the final product is obtained after
an 1850 rpm/8 minutes centrifugation also in plastic tubes. After that, three layers
of blood include a superior one divided into platelet-poor plasma (PPP) and PRP, a
medium one with buffy coat, and the inferior one with the red cells. Depending on
the clinical use, we can have different forms of presentation and the most used in
regenerative medicine is the fibrin mesh induced by PPP that forms after 15 to 20
minutes.[10 ]
Leukocyte-Platelet-Rich fibrin
This is a second-generation platelet concentrate, in which the final product is obtained
by a total physiologic process. For Inchingolo et al,[11 ] the lack of “additives” allows the natural activation of the platelets and coagulation
when in contact with the tube. This last one is made from glass, not plastic because
according to Jianpeampoolpol et al,[12 ] the silica from the glass is a natural coagulation inducer. The fibrinogen initially
concentrated in the superior layer of the tube when in contact with the thrombin is
converted into fibrin that will imprison the platelets: a quick process between the
harvest and blood centrifugation. According to Marrelli and Tatullo,[13 ] PRF has a mesh of fibrin that allows cellular migration and a slow release of growth
factors essential for tissue regeneration. The final product is originated after centrifugation
at 2700 rpm/12 minutes with the fibrin mesh in the middle of the tube between the
noncellular and cellular plasma (with platelets, leukocytes, and fibrin).
Advanced Leukocyte-Platelet-Rich Fibrin
In this second-generation platelet concentrate, the fibrin mesh is obtained using
lower G forces (1500 rpm/14 minutes) that clinically translate in an increased concentration
of growth factors and neoangiogenic potential.[14 ]
[15 ] They also use glass tubes for centrifugation, and according to Dohan Ehrenfest et
al,[14 ] the changes in the speed and time in the centrifugation protocol result in a shorter
membrane, lighter, and narrower with the increased capacity of cell imprison. Changing
the centrifugation protocol to Choukroun's PRF translated in the change of the leukocyte
pattern in the fibrin-rich clot: the number of neutrophils increased.[16 ]
The main characteristics of all the platelet concentrates are summarized in [Table 1 ].
Table 1
Biological comparison between the first- and second-generation platelet concentrates
Platelet concentrate
PRP[8 ]
PRGF
L-PRF
A-PRF
Abbreviations:↑↑↑, increased concentration; √, small amount; √√, medium amount; √√√,
good amount; √√√√, very good amount; √√√√√, excellent amount; A-PRF, advanced platelet-rich
fibrin; CPDA, citrate phosphate dextrose adenine; L-PRF, leukocyte-platelet-rich fibrin;
PRGF, plasma rich in growth factor; PRP, platelet-rich plasma.
Generation
1
1
2
2
Anticoagulants use
Yes (CPDA)
Yes (sodium citrate)
No
No
Coagulation activation
Calcium glutamate/bovine thrombin
Calcium Chloride
No
No
Protocol (rpm/min) centrifuge tubes
900/5+1500/15 (plastic tubes)
1850/8 (plastic tubes)
2700/12 (glass tubes)
1500/14 (glass tubes)
Protocol cost
High
High
Low
Low
Fibrin membrane
–
Yes
Yes
Yes
Fibrin membrane formation
–
Induced
Physiological
Physiological
Leukocytes
Nondetermined
0%
50–65%
50–65% (↑↑↑neutrophils)
Growth factors
√
√√
√√√√
√√√√√
Bone regeneration
√
√√
√√√
√√√
Presentation form
Gel
Liquid
Clot
Supernatant
Fibrin membrane
Plugs (for alveolar filling)
Exudate
Fibrin membrane
Plugs (for alveolar filling)
Exudate
Fibrin membrane
Platelet Growth Factors
The biological properties of the platelet concentrate explore the platelet's function
in organism homeostasis, namely, the fibrin coagulation and tissue regeneration. Such
potential is due to growth factors such as VEGF, PDGF, TGF-β, EGF, IGF-I, and HGF.
After centrifugation, all these factors contribute to soft- and hard-tissue regeneration
and wound healing after tissue injury[17 ]
[18 ] ([Table 2 ]).
Table 2
Platelet growth factors properties
Growth factors
Biological action
Abbreviations: EGF, epidermal growth factor; HGF, hepatocyte growth factor; IGF-I,
insulin-like growth factor; PDGF, platelet-derived growth factor; TGF-β, transforming
growth factor-β; VEGF, vascular endothelial growth factor.
VEGF
The VEGF was first mentioned by Ferrara and Gerber[19 ] as a signaling protein, angiogenic and with specificity for the vascular endothelial
cells, stimulating chemotaxis and endothelial cell proliferation. Nör et al[20 ] demonstrate that VEGF also regulates vascular permeability, the main process for
the beginning of angiogenesis and also induces bone tissue regeneration. Thus, Di
Alberti et al[21 ] wrote that it is the main growth factor for tissue regeneration in dentistry implants
application
PDGF
This growth factor concentrates mainly in the platelets α- granules, liberated during
the coagulation cascade. Its effect is dependent on other growth factors presence,
inducing fibroblast, macrophages, and other leukocytes chemotaxis.[22 ] According to Caplan and Correa,[23 ] the target of PDGF is mainly the bone tissue inducing osteoblast chemotaxis, vascular
regeneration, and fracture repairing. It is the first growth factor to be found in
a wound, responsible for collagen synthesis in the connective tissue
TGF-β
As referred by Miyazono,[24 ] this factor as a role in the growth, proliferation, adhesion, and apoptosis of several
cell types being a key player in the inflammatory process. It induces chemotaxis and
mitogenesis of undifferentiated cells to the place of repair activating fibroblasts,
osteoblasts, and chondroblasts proliferation. It induces the first step of repairing
and extracellular matrix healing[25 ]
EGF
The EGF promotes chemotaxis and mitogenesis of epithelial, mesenchymal cells and fibroblasts
also inducing tissue regeneration. It stimulates epithelial proliferation in peri-implantar
tissues inducing the formation of the peri-implant junctional epithelium. Thus, its
presence together with EGF from saliva increases in oral surgery[26 ]
IGF-I
As referred by Kurten et al,[27 ] IGF-I is synthetized by almost every tissue with increased concentrations in bone.
It mediates growth, differentiation, and cellular transforming and stimulates osteoblasts.
It is also involved in keratinocytes migration and wound healing[28 ]
HGF
This platelet growth factor regulates the migration and cellular morphogenesis having
an important role in wound repair through its interaction with the mesenchymal epithelium[10 ]
Leukocytes and the Inflammatory Process
As mentioned before, the first goal in tissue regeneration was to separate the plasmatic
components to obtain a rich-platelet concentrate. Afterward, it was observed that
besides platelets the inclusion of other components such as leukocytes could improve
the healing process. Thus, leukocytes release VEGF and TGF that, again, improve chemotaxis
and angiogenesis.[29 ]
[30 ]
[31 ] Nowadays, there are in the market first-generation protocols with or without leukocytes
and second-generation ones that always include white cells.
Literature results about the impact of leukocytes in the first-generation platelet
concentrates revealed heterogeneous results. On the one hand, there is lack of clinical
evidence of the positive impact of leukocytes, and on the other hand, some authors
claim that the leukocyte exclusion improves the healing process, reducing the inflammatory
response modulated by the secretion of metalloproteinases, hydrolases, and proteases.[32 ]
[33 ]
The second-generation platelet concentrates, namely, L-PRF and A-PRF emerged in the
market to overcome the limitations found in the first-generation concentrates. A complete
autologous concentrate with a higher leukocyte inclusion in a fibrin mesh (to act
as a scaffold) should increase its osteogenic and antimicrobial potential. Following
this research new developed protocols also seem to substantiate the positive biological
impact of the fibrin in the cells support and migration, inducing rapid tissue regeneration.
Besides, there is also an increase in VEGF, TGF production, and anti-inflammatory
cytokines that will mediate the inflammatory process.[34 ]
IL-4 drives Th cell differentiation in a Th2 pattern, inducing activation and proliferation
of B-cells. During the inflammatory process, it negatively regulates IL-1 and tumor
necrosis factor-α, inducing IL-10, another Th2 cytokine. It induces the antagonist
of IL-1 receptor contributing to the control of postsurgery pain.[1 ] IL-6, according to Kumar and Shubhashini,[35 ] is a pleiotropic cytokine produced by cells such as fibroblasts, endothelial cells,
monocytes, and macrophages, also inducing B- and T cell differentiation. It is involved
in the acute-phase response not only sharing some properties with IL-1 but also inducing
IL-1 receptor and a negative feedback in the inflammatory cascade.[36 ]
[37 ]
However, there is a lack of clinical studies validating the role of the leukocytes
and cytokines in the inflammatory process beginning with inflammation, then proliferation
and finally regeneration. There are not enough in vivo studies to support that, when
using the platelet concentrates, we are also using M2 (alternatively activated macrophages
[anti-inflammatory molecule producer])-polarized macrophages instead of M1 (classically
activated macrophages [pro-inflammatory molecules producer]).[38 ]
[39 ]
Tissue repairing requires a strategic collaboration of all leukocytes, epithelial
and endothelial cells, fibroblasts, and platelets. In Choukron's A-PRF, the number
of leukocytes includes more neutrophils that also can contribute to a pro- or anti-inflammatory
state of the macrophages modulating tissue regeneration.[16 ]
The Inclusion of Fibrin as a Scaffold
In platelet concentrates PGRF, L-PRF, and A-PRF, the fibrin mesh represents a big
innovation in regenerative tissue processing. The fibrin and the final products from
fibrinogen degradation stimulate neutrophil migration and increase chemotaxis, by
overexpression of the membrane receptors CD11c/CD18.[40 ]
In dental tissues regeneration, such as periodontal tissue, the fibrin scaffold supports
and acts as a carrier for cells. It is a natural polymer that interacts directly with
the cells/tissue, inducing an, almost natural, tissue regeneration. This is one of
the foundations of tissue engineering applications.[41 ]
Aggour and Abd El-Hady[42 ] observed in vitro that fibrin-based membranes are better scaffolds for the proliferation
of bone cells when compared with collagen membranes. This strong mechanical and polymerization
characteristic makes it a more suitable support for regeneration, increasing fibrinogen
concentrations, and inducing the formation of a stronger matrix. In PRP, the fibrin
mesh is quite “immature” with small diameter fibrils. This mesh supports platelets
during surgery but rapidly dissolves like glue, not having the same angiogenic and
chemotactic potential of a membrane.[40 ]
In PRGF, the use of an anticoagulant and a gel agent influences the velocity and polymerization
of the membrane, and as a result, it forms a tetra-molecular mesh condensate with
an increased quantity of fibrin. Thus, in PGRF, where the use of coagulation activator
modifies the thrombin concentrations, the thickening of the fibrin polymers results
in the formation of a rigid mesh that favors the sealing of the biological tissues
but impairs cellular migration and cytokine release.[7 ]
In PRF (L-PRF and A-PRF), there is no patient blood manipulation, and the fibrin matrix
formation is completely physiological creating a tridimensional mesh. With no manipulation,
the thrombin concentrations are lower favoring the cellular imprisonment and molecules
deliverance during 10 days.[43 ] These cells and the VEGF are crucial for vascular angiogenesis and pathogen elimination
avoiding possible contaminations. The spatial confirmation of the fibrin mesh also
supports the platelets and stem cells ideal for concentration at the injury/surgery
site[44 ] ([Fig. 1 ]).[37 ]
[40 ]
[45 ]
[46 ]
[47 ]
Fig.1 Fibrin: an autologous matrix.
Leukocyte-Platelet-Rich Fibrin and Advanced Platelet-Rich Fibrin: Recent Applications
All the studies until now reveal that the biological effects achieved depend on the
platelet concentration and the number/type of leukocytes entrapped in the fibrin membrane.
Thus, the release of bioactive molecules at the sites of injury will trigger the healing
and regenerative process.[4 ]
Recent in vitro studies show that L-PRF ability to potentiate angiogenesis and vasculogenesis
at the injury site is mediated not only by the liberation of bioactive molecules but
also by the presence of hematopoietic stem cells and endothelial progenitor cells.[39 ]
The second-generation concentrates L-PRF and A-PRF have a similar level of platelets
and cytokines. However, A-PRF releases a significantly higher quantity of growth factors
TGF-β, PDGF, VEGF, and chemotactic molecules CCL-5 and eotaxin, when compared with
traditional PRF.[43 ] Furthermore, A-PRF has an increased number of neutrophils. These inflammatory cells
contribute to monocyte/macrophage differentiation, despite the similar total amount
of leukocytes.[16 ] Thus, A-PRF is an excellent candidate for tissue repair and vessel formation.[5 ]
The main clinical applications of L-PRF and A-PRF include tissue regeneration in oral
and maxillofacial surgery (alone or with bone grafts). In regenerative medicine and
dentistry, several clinical studies showed better outcomes with PRF than open-flap
debridement, in intrabony periodontal defects. Furthermore, its use together with
bone substitutes such as nanohydroxyapatite had a therapeutic effect compared with
the substitutes alone.[48 ] Osteonecrosis of the jaws can also benefit from L-PRF application: necrotic bone
replacement by L-PRF was used as a physical barrier against microorganisms, preventing
secondary infections.[49 ] PRF can also act in the treatment of ulcers/skin necrosis, plastic surgery,[3 ] and even musculoskeletal lesions.[50 ]
In A-PRF, the release of TGF-β-1, PDGF, and VEGF and the presence of monocytes/macrophages
facilitate wound healing and tissue regeneration.[51 ] All of these biological factors were previously associated with a high success in
several dentistry areas as follows: periodontal reconstructive surgery,[52 ] sinus lift, and implants.[53 ] Furthermore, A-PRF can be used with freeze-dried bone allograft improving bone osteogenesis
and alveolar stability in the use of implants.[54 ]
Current clinical trials are evaluating the use of A-PRF in periodontal angular defects.
Thus, there are studies focusing the treatment of intrabony defects with PRF, it lacks
evidence of the benefit of A-PRF in the same patients.[55 ]
In orthopaedic surgery, the use of L-PRF with bone cell proliferation/differentiation
inducers such as bone morphogenetic proteins-2 also seems to be a benefit for tissue
regeneration.[56 ] Furthermore, Crisci et al study the use of L-PRF in ulcers in a diabetic foot and
found that treatment improved wound healing with no evidence of infection.[57 ] In other types of chronic ulcers such as pressure and venous leg ulcers, the use
of L-PRF has also improved healing and cicatrization.[3 ]
Conclusion
When analyzing the efficacy in clinical applications has shown in the literature,
second-generation platelet concentrates result in more natural protocols, lower costs,
and faster time for obtaining a valid clinical product when compared with the first-generation
protocols.
When comparing the biological properties, proliferation versus differentiation, L-PRF and A-PRF use a high number of leukocytes, and platelets and
growth factors promoting both proliferation and differentiation. L-PRF and A-PRF mimic
the physiological process, opposite to the first-generation concentrates that use
anticoagulants and coagulation activators that can reduce the positive leukocyte impact
in wound healing. The TGF-β, PDGF, VEGF, eotaxin, and CCL-5 released by the leukocytes
promote local vascularization and tissue repairing, mainly due to the control of the
inflammatory process by anti-inflammatory cytokines IL-4, IL-6, and IL-10 also having
an antimicrobial potential.
Furthermore, the fibrin scaffold has different physical and biological properties,
capturing a higher number of cells, and including stem cells that can differentiate
in other autologous cells. In A-PRF, there is an increase not only in the number of
neutrophils but also the time of growth factors liberation increases in 10 days. This
results in better cellular conduction, migration, angiogenesis, and natural healing
of the tissues patient. For these reasons, the potential benefits of leukocyte (mainly
inflammatory neutrophils) and fibrin are reciprocal, and they are mutual stimulation
“actors” in the healing process.
Nowadays, the L-PRF and A-PRF are used not only in dentistry has coadjuvants in tissue
regeneration before implant rehabilitation but also for chronic ulcers, musculoskeletal
lesions, plastic surgery, and even orthopaedic surgery. In the future, there is a
wide range of clinical applications due to the low costs and rapid healing properties
of these concentrates, but still, there is a vast research to be done translating
the laboratory results to the clinical practice.
