Platelet-rich Plasma: Is It Ready to Use?

JDSOR
10.5005/jp-journals-10039-1078
Platelet-rich
Plasma: Is It Ready to Use?
Review article
Platelet-rich Plasma: Is It Ready to Use?
1
Prerna Agarwal, 2RG Shiva Manjunath, 3Hirak Bhattacharya, 4Himanshu Pratap Singh
ABSTRACT
platelet-rich plasma: What is in a name?
For over 20 years, autologous blood products, such as plateletrich plasma (PRP) have been employed as a means to facilitate
the healing process. Platelet-rich plasma has been advocated
as a way to introduce increased concentrations of growth factors
and other bioactive molecules to injured tissues in an attempt
to optimize the local healing environment. This article reviews
the basic principles involved in creating PRP and outlines the
specific effects of these growth factors, both in vitro and in vivo,
on periodontal wound healing.
Platelet-rich plasma has been classically (Table 1)
described as ‘a volume of plasma that has a platelet count
above baseline (of whole blood)’.7 Although this definition
would suggest a pure mixture of plasma (the acellular,
liquid portion of blood that contains proteins involved
in the clotting mechanism as well as other bioactive
molecules that play a significant role in wound repair)
and platelets (and their associated growth factors and
cytokines), the generic term ‘PRP’ has recently expanded
to include a variety of final products. These products
can vary markedly not only in the final concentration
of platelets they produce but also in the amount of red
blood cells and/or white blood cells that are included
in the final preparation. In addition, some techniques
for creating PRP actively initiate the clotting cascade as
part of the process, creating a fibrin scaffold. Because,
the inclusion of these additional blood components
may affect the indication(s), potency, and efficacy of the
final PRP product, the generic classification ‘PRP’ does
not allow distinction between the different systems and
protocols.8 Therefore, to more precisely delineate these
various products based on their leukocyte and fibrin
content, categories, such as pure PRP, leukocyte rich PRP
(L-PRP), pure platelet-rich fibrin, and leukocyte- and
platelet-rich fibrin have been proposed.8
Keywords: Healing, Platelet-rich plasma, Regeneration.
How to cite this article: Agarwal P, Manjunath RGS,
Bhattacharya H, Singh HP. Platelet-rich Plasma: Is It Ready to
Use? J Dent Sci Oral Rehab 2015;6(3):115-120.
Source of support: Nil
Conflict of interest: None
Healing of tissues: Down the lane
The ultimate goal of periodontal therapy is not only
to prevent periodontal disease progression but also to
regenerate the lost dentition’s supporting structures,
such as cementum, periodontal ligament, and bone to a
diseased root surface where appropriate.1,2 But, at best,
surgeons attempt to remove the known obstacles to
healing, such as infection, instability, foreign bodies, etc.
A review of the literature related to wound healing
shows that debridement and primary closure was the ‘hot
topic’ of the 1950s. In the 1980s, the three seminal works
by Knighton,3 Hunt,4 and Marx et al5 marked a paradigm
shift by focusing attention on actively promoting healing
rather than just removing the obstacles to it. First,
introduced clinically by Knighton’s platelet-derived
wound healing factor (PDWHF), 6 and then through
topical recombinant human platelet-derived growth
factor bb (PDGFbb) (Regranex, Ortho McNeal) and
today’s platelet-rich plasma (PRP), platelets have found to
be the pivotal cells that initiate all human wound healing.
1,4
Senior Lecturer, 2Professor and Head, 3Professor
1-3
Department of Periodontics, Institute of Dental Sciences
Bareilly, Uttar Pradesh, India
4
Department of Oral Surgery, Institute of Dental Sciences
Bareilly, Uttar Pradesh, India
Corresponding Author: Prerna Agarwal, Senior Lecturer
Department of Periodontics, Institute of Dental Sciences, Bareilly
Uttar Pradesh, India, e-mail: [email protected]
Methods of preparation
At least 16 commercial PRP preparation systems are
currently available (Table 2). A sample of peripheral
venous blood is drawn and immediately spun in a
centrifuge to separate the erythrocytes from the platelets
and leukocytes. The increased density of the erythrocytes
causes them to sink to the bottom of the centrifuge tube
more rapidly than do the platelets and leukocytes. Further
concentration then isolates PRP from platelet-poor
plasma. Commercially available PRP kits concentrate
platelets in the final injectate up to 9 times the normal
concentration found in whole blood. The resultant
volume of PRP and the final platelet and leukocyte
concentrations differ among preparation systems.9
How many platelets are enough?
This question has been elegantly answered by the work
of Haynesworth et al,10 who showed that the proliferation
Journal of Dental Sciences and Oral Rehabilitation, July-September 2015;6(3):115-120
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Prerna Agarwal et al
Table 1: Platelet-rich plasma classification8
P-PRP
Autologous conditioned
plasma (ACPTM)
Arthrex Inc.
(Naples, FL, USA)
Preparation rich in growth
factors (PRGF)
Biotechnology Institute (San
Antonio, Alava, Spain)
Leukocyte and platelet-rich
fibrin (L-PRF)
Choukroun’s PRF
(nonspecific system)
P-LRP
SmartPreP 2
P-PRF
Cascade
Harvest technologies (Plymouth,
MA, USA)
GPS III
Musculoskeletal transplant
foundation (Edison, NJ, USA)
PRFG scaffold
Biomet biologics
(Warsaw, IN, USA)
MagellanTM
Arteriocyte medical systems
(Cleveland, OH, USA)
SymphonyTM II
Depuy
(Warsaw, IN, USA)
Platelet concentrate collection
system (PCCS II)
3i/Implant innovations (Palm Beach
Gardens, FL, USA)
Angel
Sorin group (Milan, Italy)
GenesisCS
EmCyte (Fort Myers, FL, USA)
Cell saver 5
Haemonetics (Braintree, MA, USA)
Biotechnology Institute (San
Antonio, Alava, Spain)
FIBRINET PREM
Cascade medical enterprised
(Wayne, NJ, USA)
Vivostat PRF
Vivostat A/S (Alleroed, Denmark)
Table 2: Representative platelet-rich plasma therapy preparation systems9
Available
activator
Leukocyte
concentration (103/l)
(compared with whole
blood)
Fibrinogen
(mg/dl)
Volume of
blood (ml)
Final PRP
volume (ml)
Final platelet
concentration
(compared with
whole blood)
Autologous conditioned
plasma (Arthrex, Naples,
FL)
9
3–5
2–3X
None
N/A
N/A
Cascade (Musculoskeletal
tissue foundation, Edison,
NJ)
9 or 18
2 or 4
1.6X
Calcium
1.1 + 0.2
(6-fold)
283.8 ± 34.2
GPS III (Biomet, Warsaw,
IN)
27 or 54
3 or 6
2.1–9.3X
Calcium chloride/ 34.4 ± 13.6
thrombin
(5-fold)
286.0 ± 42.7
SmartPreP (Harvest
Technologies, Plymouth,
MA)
20 or 60
3 or 7
4.4–7.6X
Thrombin
N/A
N/A
Magellan (Arteriocyte Inc,
Cleveland, OH)†
30,45 or 60
Variable
2.8–14X
NA
11.0 ± 8.2
(2-fold)
277.4 ± 30.5
System
of adult mesenchymal stem cells and their differentiation
were directly related to the platelet concentration. They
showed a dose-response curve, which indicated that a
sufficient cellular response to platelet concentrations first
began when a 4- to 5-fold increase over baseline platelet
numbers was achieved. Because most individuals have
a baseline blood platelet count of 200,000 ± 75,000/μl, a
PRP platelet count of 1 million/μl as measured in the
standard 6-ml aliquot has become the benchmark for
‘therapeutic PRP.’
The importance of this knowledge is that our studies
have indicated that only the aforementioned FDA cleared
116
devices consistently achieve this therapeutic level of
platelet concentration, and hence growth factor release.
Plasma: The Other ‘P’ in PRP
Perhaps the most consistent component of many of the
PRP products is the plasma component. Plasma, the
fluid portion of blood, is a remarkable liquid containing
numerous ions, inorganic and organic molecules, and
many of the same proteins found in platelets.11 Plasma
differs from serum in that plasma still contains fibrinogen
as well as a number of clotting factors. Therefore, when
plasma is exposed to thrombin (either by the addition of
JDSOR
Platelet-rich Plasma: Is It Ready to Use?
exogenous thrombin or by coming in contact with tissue
thromboplastin (also known as tissue factor), the clotting
cascade is initiated and platelets are activated. The
resulting formation of a fibrin clot provides a provisional
scaffold for cell migration as well as a reservoir of growth
factors.12 Although in vitro studies have documented
significant differences in cell proliferation between PRP
and platelet-poor plasma preparations,13 it is possible
that the plasma component of PRP actually plays a more
significant role in creating a proper local environment
for tissue repair.
Exogenous Thrombin: To Activate
or not to Activate
Platelet activation can be initiated by a number of methods,
such as shear forces caused by fluid flow, contact with a
variety of material including fibrillar collagen and basement membranes of cells, and thrombin. Most current
PRP protocols rely on bovine or autologous thrombin
with or without calcium chloride to congeal the solution
and activate the platelets. Gelation of the PRP solution is
necessary to localize the product (and thereby its effects)
onto the area of interest without loss into the surrounding
tissues. To minimize the risk of immune reaction associated with thrombin, some have used calcium chloride as
an activator by itself.14 An alternative activator recently
reported is type I human collagen.15 As previously mentioned, when PRP is injected into connective tissues, it
comes into contact with tissue thromboplastin (tissue
factor), which can activate platelets and initiate the formation of a fibrin scaffold. Therefore, the need for platelet
activation with exogenous thrombin before injection is
not clear.
Mechanism of PRP-related to Growth Factors
The growth factors secreted by the platelets (i.e. PDGFaa,
PDGFbb and PDGFab) usually have two active sites
and are, therefore, called dimers. They attach only to
cells that have receptors to accommodate them. These
1. Before signaling
receptors are on the surface membrane of the target cell
(Fig. 1). The growth factor never enters the target cell;
instead, it activates the membrane receptor, which has an
intracytoplasmic portion and, therefore, is often termed as
‘transmembrane receptor’. Two adjacent transmembrane
receptors are then brought within a critical distance of
each other to activate dormant intracytoplasmic signal
transducer proteins. A signal transducer protein, then
detaches from the transmembrane receptor and floats
in the cytoplasm toward the nucleus. In the nucleus, the
transducer protein unlocks a specific gene sequence for
a regulated cellular function, such as mitosis, collagen
synthesis, osteoid production, etc. The significance of this
process is that it explains why an exogenous application
of growth factors, even in the highest concentration
possible, cannot produce a sustained overreaction, such
as a hyperplasia, a benign tumor, or a malignant tumor.
Growth factors are not mutagenic; they are natural
proteins acting through normal gene regulation and
normal wound healing feedback control mechanisms.16
Effects of PRP Growth Factors on Cells Involved
in Periodontal Wound Healing
Periodontal wound healing involves gingival fibroblasts,
gingival epithelial cells, periodontal ligament fibroblasts
and osteoblasts, all of which are important for tissue
repair and hard-tissue regeneration. A series of wellorchestrated cell—cell interactions is initiated after injury.
Disruption of the vasculature as a result of injury leads
to fibrin formation and platelet aggregation. Several
growth factors are then released into the tissue from the
platelets and from the adjacent cells after injury, including
platelet-derived growth factor (PDGF), transforming
growth factor-alpha, transforming growth factor-beta
(TGF-beta) and insulin-like growth factor I (IGF-I). Bone
and cementum may also release growth factors during
wound healing (Table 3).
Periodontal and oral surgical techniques may involve
use of these factors in both soft and mineralized tissues.
2. Growth factor activates membrane receptor
Fig. 1: Mechanism of action for growth factor signaling
Journal of Dental Sciences and Oral Rehabilitation, July-September 2015;6(3):115-120
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Prerna Agarwal et al
Table 3: Effect of growth factors produced by platelets and their average concentration in platelet-rich plasma (PRP)24
Growth factor
Effect
PRP concentration (SD)
PDGF
Macrophage activation and angiogenesis
Fibroblast chemotaxis and proliferative activity
Enhances collagen synthesis
Enhances the proliferation of bone cells
ab: 117.5 ng/ml (63.4)
bb: 9.9 ng/ml (7.5)
TGF-b
Enhances the proliferative activity of fibroblasts
Stimulated biosynthesis of type I collagen and fibronectin
Induces deposition of bone matrix
Inhibits osteoclast formation and bone resorption
b1: 169.9 ng/ml (84.5)
b2: 0.4 ng/ml (0.3)
PDEGF
Promotes wound healing by stimulating the proliferation of keratinocytes and
dermal fibroblasts
470 pg/ml (320)
PDAF
Induces vascularization by stimulating vascular endothelial cells
PF-4
Stimulates the initial influx of neutrophils into wounds
A chemoattractant for fibroblasts
A potent antiheparin agent
0.189 nmol/ml (0.07)
EGF
Cellular proliferation
Differentiation of epithelial cells
51 pmol/l (5)
VEGF
Angiogenesis
Migration and mitosis of endothelial cells
Creation of blood vessel lumen
Creates fenestrations
Chemotactic for macrophages and granulocytes
Vasodilation (indirectly by release of nitrous oxide)
PDGF: Platelet-derived growth factor; TGF-b: Transforming growth factor beta; PDEGF: Platelet-derived endothelial growth factor; PDAF:
Platelet-derived angiogenesis factor; PF-4: Platelet factor-4; EGF: Endothelial growth factor; VEGF: Vascular endothelial growth factor
For example, local application of growth factors is used
to promote healing, especially regeneration. Numerous
studies, including some dental research, have shown that
PDGF, TGF-beta and IGF-I are found in PRP and, because
of their impact on wound healing, the use of these factors
has led to promising results.12,14
Platelet-derived growth factor is a basic dimeric
glycoprotein with two disulphide-bonded polypeptides,
referred to as A and B chains. Three isoforms of PDGF are
possible: AA, BB and the heterodimeric AB. All isoforms of
PDGF are released after adhesion of platelets to an injured
site. In vitro, all isoforms have proliferative activity on
periodontal ligament fibroblasts.17 Platelet-derived
growth factor is also chemotactic for these fibroblasts,
and it promotes collagen and protein synthesis.
Insulin-like growth factor has two forms, I and II,
each of which has two single chain peptides. Both forms
of IGF are potent factors for survival of hematopoietic
cells, fibroblasts and the nervous system.18 They are
found in bone, and IGF-II is the most abundant growth
factor in bone matrix. This form of IGF is chemotactic for
periodontal ligament cells, and it has strong effects on
periodontal ligament fibroblasts and protein synthesis.
Insulin-like growth factor-I stimulates bone formation by
proliferation and differentiation,19 and it is synthesized
and secreted by osteoblasts. It also has dose-dependent
chemotactic effects on osteoblasts.20
Human patients treated with a combination of
150 mg/ml each of recombinant human platelet-derived
118
growth factor-BB (rhPDGF-BB) and rhIGF-I in a methylcellulose vehicle experienced 43.2% osseous defect fill,
whereas the control group (vehicle only) had 18.5% osseous fill.21
Transforming growth factor-beta is the name given to a
group of homodimeric proteins involved in the formation
and development of many tissues. Transforming growth
factor-beta enhances collagen gel construction in vitro,
and its effects are influenced by the combination of PDGF
and IGF. In addition, TGF-beta stimulates biosynthesis
of type I collagen and fibronectin and induces deposition
of bone matrix.22
In a recent review, Yao and Eriksson23 reported that
short shelf life and inefficient delivery to target cells are
major concerns associated with local administration of
recombinant human growth factors. The growth factors
are expensive, and many doses may be required to
achieve any therapeutic effect.23 In light of this PRP can
be used as a pool of concentrated growth factors.
Clinical Effects on Osseointegration
Osseointegration of dental implants arises from cell
migration, differentiation, bone formation, and bone
remodeling along the implant surface; each of these
processes is platelet and blood clot dependant.
During implant placement, the blood clot, or the
PRP that is placed into the drill site, coats the implant
surface as well as the microgap (about 25 μm wide)
that lies between the actual bone and the metal surface.
JDSOR
Platelet-rich Plasma: Is It Ready to Use?
Table 4: Studies showing clinical effects of PRP with various bone substitutes27
PRP therapy
PRP with autogenous bone
Researcher and year
of study
Marx et al (1998)
PRP with anorganic bone
mineral
Wiltfang et al (2003)
PRP with organic bone
matrix
Shanaman et al (2001)
PRP when used alone
Papli and Chen (2007)
Study protocol
Forty-four continuity bone grafts
to the mandible, placed without
PRP, were assessed against 44
grafts placed with PRP at 2, 4, and
6 month maturity intervals with
panoramic plain-film radiographs
A total of 45 sinus lifts were
performed, with half receiving PRP
and tricalcium phosphate
Performed alveolar ridge
augmentation on 3 patients,
primarily using freeze-dried
demineralized bone. The grafts were
mixed with PRP and were protected
with barrier
Compared the treatment of
infrabony defects by an intralesional
graft of PRP to guided periodontal
regeneration (GPR) using a
bioabsorbable barrier membrane
(MEM) over a 52 week period
Within this microgap are found the usual components:
platelets, red blood cells, white blood cells, and the cell
adhesion molecules of fibrin, fibronectin, and vitronectin.
In this situation, the cell adhesion molecules perform
the important roles of coating the implant surface and
bridging the microgap between the implant surface and
the bone.
The model for osseointegration demonstrates that
platelets degranulate and secrete their seven growth
factors. As a result, the osteoclasts and marrow stem
cells along the bony walls of the drill site proliferate and
migrate along the strands of fibrin and other cell adhesion
molecules spanning the microgap. As they migrate along
the surface of the fibrin strands, the marrow cells can pull
the fibrin strands off the implant surface. As the marrow
cells migrate along the fibrin starnds, they undergo
differentiation and produce osteoid.
Clinical Applications of PRP
Different applications of PRP include sinus lift procedures,
ridge augmentations, socket preservation, alveolar cleft
palate repair, oral/nasal fistula repair, intrabony defects,
furcation defects, jaw reconstruction surgeries, structural
defects of the mandible and specially in medically
compromised patients who undergo surgery have been
suggested.25 Some clinicians also use PRP applications in
soft-tissue procedures, like gingival grafts, subepithelial
connective tissue grafts, etc., because of its property of
increasing soft tissue healing.26 Platelet-rich plasma has
also found its application in medicine as a treatment
of skin ulcers, macular lesions, and corneal epithelial
Conclusion
Investigators assessed PRP grafts
to be 2.16 times more mature at 2
months, 1.88 times more mature at 4
months, and 1.62 times more mature
at 6 months. These differences were
statistically significant (p = 0.001).
The authors report 8% or 10% greater
new bone formation in PRP group
The authors concluded that ‘the
addition of PRP did not appear
to enhance the quality over that
reported in comparable guided bone
regeneration studies without PRP’
Their case series suggested that an
PRP graft achieves a similar CAL gain
and PD reduction to GPR using an
MEM over a 52 week period
defects. Recent studies have shown that combining PRP
with bone or bone substitutes present significant faster
radiographic maturation and denser bone regeneration
histologically. The results of few studies have been
summarized in the Table 4.
Conclusion
Today’s understanding of bone science recognizes the
pivotal role of growth factors in clinical bone grafting
success. Platelet-rich plasma is seen as an available and
practical tool for enhancing the rate of bone formation
and the final quality of bone formed. Undoubtedly, all
clinicians involved with bone grafting have high hopes
that PRP will eventually prove to be of great benefit in
bone graft healing. The conflicting results in today’s
literature make it overwhelmingly evident that more
research is needed before surgeons can feel confident in
recommending this procedure to their patients.
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