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Update on platelet-rich plasma

Article  in  Current Orthopaedic Practice · November 2011

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 S P E C I A L
Sports Medicine
Update on platelet-rich plasma
Jeffrey M. DeLonga, Knut Beitzelb, Augustus D. Mazzoccab, David Sheparda,
Brandon L. Rollera and Bryan T. Hanypsiaka
ABSTRACT
Numerous studies have demonstrated a positive clinical response to
platelet-rich plasma injections. However, few randomized, clinically
controlled trials have been published. An equal number of
publications have shown no effect. As of yet, no deleterious effects
have been identified. A literature search of articles pertaining to
platelet-rich plasma reveals thousands of publications but is
remarkable for how much remains unknown. This review attempts
to use the latest published research to answer the most frequently
asked questions surrounding platelet-rich plasma and to provide a
fundamental understanding for critically evaluating further platelet-
rich plasma studies.
Keywords
platelet rich plasma, platelet-rich plasma, PRP
WHAT IS PLATELET-RICH PLASMA?
P
latelet-rich plasma (PRP) can be defined as an
autologous blood product containing a platelet
concentration higher than the baseline level of
150,000--350,000 platelets/mL (mean ¼ 250,000) within
whole blood.1,2 Platelets play a central role in tissue
regeneration.3 They function in hemostasis, construction
of new connective tissue, and revascularization.4--7
What Bioactive Factors are Involved?
To date, over 1500 unique regulatory proteins and growth
factors have been identified in platelets.8 The individual and
synergistic roles of each of these substances have not yet
been determined. These bioactive factors promote and
accelerate the healing process and are stored and released
by platelet alpha and dense granules.9--11
Alpha granules are reservoirs for numerous growth factors,
cytokines, chemokines, and various coagulation and adhe-
sion proteins.9,12 Current focus has been on growth factors
a
Arthrex, Inc, Naples FL
b
University of Connecticut, Storrs, CT
Financial Disclosures: Brandon Roller is an employee of Arthrex. Augustus
D. Mazzocca is a consultant and has received support from Arthrex. Bryan
Hanypsiak is an employee of Arthrex.
The other authors declare no conflicts of interest.
Correspondence to Bryan T. Hanypsiak, MD, Arthrex, 1370 Creekside
Blvd., Naples, FL 34108
Tel: þ 239 566 5880; fax: þ 239 552 2316;
e-mail: bryan.hanypsiak@arthrex.com
1940-7041 r 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
Volume 00  Number 00  ’’ 2011
F O C U S
released by alpha granules including vasculoendothelial
(VEGF), transforming beta (TGF-b), platelet derived (PDGF),
platelet derived epidermal (PDEGF), fibroblast (bFGF),
epidermal (EGF), and hepatocyte growth factors (HGF).
Less emphasized in the literature but possibly of equal
importance in the healing process are bioactive factors released
by dense granules. These include substances such as serotonin,
histamine, calcium, adenosine diphosphate (ADP), adenosine
triphosphate (ATP), and dopamine (catecholamine).9,12 It is
theorized that pain relief experienced by patients after PRP
injection may result from the release of factors, such as
serotonin and catecholamines from dense granules.13
What is the Role of Platelets in Healing?
The healing process can be divided into three phases. Phase
1 (hemostasis and inflammation) is triggered by tissue injury
and lasts for 2--5 days. During phase 1, platelets become
activated when they encounter injured tissue and adhere to
the exposed collagen, aggregating to form a clot.14,15
Degranulation of platelets occurs and the release of growth,
bioactive, and hemostatic factors results in inflammation.
Platelets release 70--95% of the stored growth factors in the
first 10 minutes after tissue injury and additional growth
factors continue to be secreted for 7--9 days.9,16 Phase 2
(proliferation) begins 2 days after injury and can last for 3
weeks. This phase involves formation of blood vessels,
collagen deposition via fibroblasts, wound contraction and
continued release of small amounts of growth factors.9,12
Phase 3 (remodeling) follows and involves maturation of
collagen and formation of scar tissue and can take more
than a year to complete.9,12,17--20
HOW IS PRP PREPARED?
PRP can be obtained in two forms: plasma-based and buffy
coat preparations. Both begin with whole blood but differ in
the centrifugation process, which isolates and concentrates
different blood cell components.21
Plasma-based PRP
Plasma-based methods work to isolate only plasma and
platelet components. Protocols for these preparations leave
some platelets behind and focus on intentionally excluding
leukocytes, which are thought to be detrimental to the
healing process. The main goal of these systems is to capture
only platelets during the centrifugation, thus, a slower and
shorter spin regime is used. This yields platelet concentra-
tions typically around 2--3  baseline whole blood levels
(Table 1).
Current Orthopaedic Practice 1
2 | www.c-orthopaedicpractice.com Volume 00  Number 00  ’’ 2011

TABLE 1. Plasma-based PRP systems

Centrifuge time Initial blood Final PRP Platelet WBC
Device Name (min) volume (mL) volume (mL) concentration concentration References
22
Arthrex/ACP 5 16 4--7 2--3  Minimal to
none
23--26
Cascade/MTF 6 (plasma) 9 4.5 (plasma) 1.3--1.7  Minimal to
Fibrinet 21 (clot) 2 (clot) (plasma) none
17,27,28
BTI/PRGF 8 9 2--3 2--3  Minimal to
none
PRP indicates platelet-rich plasma; WBC, white blood cell.

Buffy Coat-based PRP Why are Accuracy and Documentation Crucial?

Buffy coat-based methods isolate a platelet poor plasma
layer and a buffy coat layer, which contains both leukocytes
and erythrocytes. Protocols for buffy coat systems seek to
capture all available platelets during the centrifugation,
thus, high spin rates and long spin regimes are used, which
do not allow for exclusion of leukocytes and red blood cells.
The typical platelet concentration yields are between 3--8 
baseline levels (Table 2).
Protocols, number and length of spins, platelet, and
leukocyte concentrations vary between commercially available
devices (Tables 1 and 2). Differences in concentration of
platelets, and the presence or absence of white blood cells
(WBC) leads to different amounts of anabolic and catabolic
proteins released to the target tissues after injection.41 An
in vitro study performed by Castillo et al.23 tested three
different PRP separation systems, and each system yielded
similar platelet concentrations (B600,000/mL), red blood cells
(RBC), active TGF-b1, and fibrinogen levels. However, each
system produced significantly different levels of PFDF-AB,
PDGF-BB, VEGF, and WBC concentration.23 Although the
effects of these bioactive factors on tissue were not tested, it is
possible that these types of variations could result in consider-
able and dramatically distinct effects on healing.21 Lack of
standardization of PRP preparations may therefore lead to
inconsistent results in the literature.42 Attempts have been
made to classify the different types of PRP.43,44 The important
points that need to be addressed within these classifica-
tion systems and within literature are platelet concentra-
tion, leukocyte concentration and differential, and delivery
method.
To determine the efficacy of PRP from system to system and
patient to patient, it is critical to accurately quantify the
platelets and cell components delivered to the target tissue
site.45 Adequate re-suspension of PRP and proper sample
preparation within a laboratory setting are required for
accurate values to be obtained. Platelet clumping or lack of
even distribution will result in inaccurately low counts even
though the clinical effect may be adequate.45
PRP preparation appears to be extremely delicate. As
described, the speed and number of centrifugations will result
in varying platelet concentrations with possible morphology
alterations, which may potentially cause clumping.46 Abso-
lute platelet value measurements may tend to be more
consistent with one system compared to another. Further-
more, the material of the centrifuge tube also may signifi-
cantly affect platelet function. Michelson46 indicated in his
comprehensive book on platelets that polypropylene tubes are
superior to polystyrene and polycarbonate when used for
platelet preparation or storage. Additionally, he noted that
uncoated glass may artificially activate platelets and affect
results.46 A study conducted by Grottum et al.47 determined
the effect of PRP with polystyrene particles. Incubation of PRP
with polystyrene particles induced surface contact between
platelets and particles, uptake of particles, and changes in
platelet morphology. After injection into rabbits, they
identified increased platelet aggregation that may have
resulted from a reduction in platelet surface charge caused
by interaction with polystyrene.48
It also is important to realize that there is significant
variation in platelet count between individuals. In fact, a

TABLE 2. Buffy coat-based PRP systems

Centrifuge Initial blood Final PRP Platelet WBC
Device name time (min) volume (mL) volume (mL) concentration concentration References
29--33
Biomet GPS II/III 12--15 30 or 60 3 or 6 2--8  Increased over
baseline
25,32--35
Harvest SmartPReP 2/ 12--15 20 or 60 3 or 7--10 3--7  Increased over
DePuy Symphony II baseline
23,33,36,37
Arteriocyte/Medtronic 14--20 30 or 60 3--10 3--7  Increased over
Magellan baseline
38--40
Emcyte Genesis CS/ 12 30 or 60 3 or 10 7--10  Increased over
Exactech Accelerate baseline
PRP indicates platelet-rich plasma; WBC, white blood cell.
Current Orthopaedic Practice
single individual’s platelet count can vary considerably on
different days.2 For example, normal platelet count varies
between 150,000/mL and 350,000 /mL. An individual with a low
normal count receiving PRP with a concentration of 1,000,000/
mL would see an approximate 6.5 times increase. An individual
with a high normal count receiving PRP with a concentration
of 1,000,000/mL would see an approximate 3 times increase.
Documenting not only the absolute value but also the fold
change will help determine the key factor in triggering the
desired response with respect to platelet concentrations.
In addition to documenting the components of the PRP
end product, it also is important to record the mechanism of
delivery and if additional factors were also distributed at the
tissue site. For example, anticoagulants alter the pH of PRP;
understanding the effect of this alteration becomes impor-
tant especially since variations in pH affects in vitro
proliferation.49,50 The use of localized anesthetics also may
produce a negative effect on PRP treatment, possibly
reducing its maximum potential as represented by decreased
proliferation within in vitro studies.2,51
SHOULD WBC BE INCLUDED?
PRP systems that use the buffy coat contain an increased
concentration of WBCs. Although normal levels of WBCs
have a positive immunomodulatory effect, heightened levels
in some PRP preparations may have deleterious effects.
What are the Theoretical Positive Effects of WBC?
Platelet-rich leukocyte gels are thought to have potent
antimicrobial effects. PRP containing WBCs may help prevent
postoperative Staphylococcus aureus and Escherichia coli infec-
tions.52 These gels are not effective across the entire spectrum,
with Pseudomonas as the most notable exception.19
Other potential benefits of WBCs include removal of
necrotic tissue through cellular engulfment of tissue bypro-
ducts, the release of proteolytic enzymes that breakdown
necrotic tissue or release additional growth factors that may
play a supplementary role in eliminating dead tissue.53--55
What are the Theoretical Negative Effects
of WBC?
Specific WBCs, such as neutrophils and CD8 þ lymphocytes,
may be detrimental to PRP therapy. Neutrophils are not
crucial to the overall process of wound healing and collagen
synthesis.56 Infiltration of neutrophils causes release of
oxygen free radicals during inflammation and has been
shown to play a central role in ischemic tissue injuries.57
This may cause endothelial and subendothelial damage and
lead to excessive fibrosis that can exacerbate a pre-existing
injury and prevent healing.57 White blood cells also release
catabolic cytokines, matrix metalloproteinases (MMPs) and
Interleukin-b.58 These substances are regulators of inflam-
mation and degradative turnover of tissue.13,58
Neutrophils also may exacerbate muscle damage after an
injury.59--63 Stretch injury in animals was shown to produce
peak levels of oxygen free radicals, pain, and muscle fiber
tearing within the first 24 hours.57,64 Leukopenic animals with
www.c-orthopaedicpractice.com | 3
the same injuries had lower neutrophilic derived oxidants and
less tissue damage during the reperfusion phase.65
A number of studies have shown accelerated wound
closure in neutrophil deficient animals.12,56,66 Knockout
and knockdown studies suggest that depletion of one or
more of the inflammatory leukocyte cell lineages can
actually enhance healing. These studies concluded that no
single cell lineage is absolutely necessary for direct repair
and when some are absent, wounds may actually close faster
and with less scarring.66 Additional animal studies that were
able to deplete the CD8 þ lymphocyte line resulted in
increased wound maturation and cross-linking of collagen
when compared with baseline.67--69
It has been suggested that WBCs are detrimental in fracture
healing by suppression of bone formation and bone healing.
A study was performed to determine the effects of poly-
morphonuclear leukocytes in fracture healing. Transverse
femoral midshaft osteotomies had a significantly higher
bending moment 6 weeks after operation in neutrophenic
rats when compared with nonneutrophil deficient rats.70
While WBCs do provide an antimicrobial effect, it is
important to note that platelets can play a similar role. Tang
et al.71 isolated antimicrobial peptides from human platelets
and found microbial activity against E. Coli and concluded
that platelets have a direct antimicrobial effect when
activated.71 As described, WBCs will lead to the release of
destructive oxygen free radicals. It may be argued that these
will play a role in removing unwanted tissue, but these
radicals are nonspecific so they will destroy healthy tissue as
well. Platelets on the other hand release growth factors that
will activate the anti-oxidant response element, which
accelerates tendon proliferation and regeneration.72,73 In
addition, it has been well established that platelets release
chemokines that activate and attract macrophages to the
site of injury, which provides the desired function of
necrotic tissue debridement.74
WHAT IS THE OPTIMAL PLATELET
CONCENTRATION?
Optimal platelet concentration in PRP has not been defined
and may vary based on tissue type and number of growth
factor receptors available. A reasonable goal in determining the
ideal concentration of platelets would be to bind available
growth factor receptors without oversaturation, triggering a
positive proliferative response without inducing negative feed-
back inhibition. Few studies have been conducted comparing
the healing effects of different PRP platelet concentrations.
Unfortunately, lack of adequate platelet concentration mea-
surement in most PRP studies makes validation and accurate
comparison of studies extremely difficult.
Does low Concentration Provide any Benefit?
< 1  [ < Baseline]
Platelet concentrations below baseline may not allow for a
sufficient cellular response.75 Often used as a control,
platelet-poor plasma (PPP) has only shown the slightest
4 | www.c-orthopaedicpractice.com
benefit. The minimum positive in vitro effect that has been
described may be caused by additional growth factors that
typically are found within plasma only and not within
platelets, such as insulin-like growth factor.
Is Moderate Concentration Effective?
> 1X-- < 4X [ > Baseline--750,000 Platelets/lL]
An often cited paper by Marx76 in 2001, defined the ‘‘working
definition of PRP’’ as having 1,000,000 platelets/mL’’, or roughly
4  baseline. This ‘‘definition’’ has not been supported in the
literature, and the author himself conducted a randomized
study that showed an average PRP platelet concentration of
3.4  over baseline accelerated the rate of bone formation in 88
bone graft recipients.77 Furthermore, an in vitro study
performed by Anitua et al.21 analyzed the maximal fibroblast
proliferation response for tissues isolated from skin, synovium,
and tendon when combined with different PRP platelet
concentrations. It was concluded that a PRP elicits a statistically
improved fibroblast proliferation within all three cell types, but
a platelet dose-response effect was not observed when compar-
ing a platelet concentration of 2  (400,000 platelets/mL) and
4  (760,000 platelets/mL).21
Graziani et al.78 evaluated the effect of plasma, 2.5  ,
3.5  , and 5.5  PRP on the function of osteoblasts and
fibroblasts. After a 72-hour incubation period, the 2.5 
(B550,000 platelet/mL) PRP group resulted in a statistically
significant increase for the osteoblast and fibroblast cell lines
when compared with plasma and the negative control. The
2.5  PRP group also had a mean proliferation rate that was
greater than the higher concentrated PRP groups. The
authors concluded that a platelet concentration of 2.5 
yielded optimal results.78
Torricelli et al.79 evaluated the effect of PRP for the
treatment of musculoskeletal overuse injuries in competi-
tion horses. With the placebo effect nullified, they found
that PRP with a platelet concentration of 750,000/mL
accelerated healing and allowed an earlier return to
competition. This study may provide a similar model for
the treatment of overuse injuries in human athletes.79
Numerous in vitro, in vivo, and clinical studies have been
conducted and published by Anitua et al. and Sanchez et al.
showing effectiveness of PRP with a platelet concentration of
2--3  .17,28,80--86 Sanchez et al.28 injected exogenously activated
PRP with a 3  platelet concentration into surgically repaired
human Achilles tendons. They found significant improvement
and an earlier return to sports as compared with the control
group.28 The same researchers performed an uncontrolled
retrospective case study treating 16 aseptic nonunions intra-
operatively with a moderate PRP platelet concentration (2--3  )
with a bone allograft and an autologous fibrin membrane. The
researchers reported that all nonunions treated operatively
healed after this procedure.85 Additional studies with a 2--3 
platelet concentration enhanced tendon graft ligamentization
during anterior cruciate ligament (ACL) surgery. The temporal
histologic changes at month 6 and at month 24 postopera-
tively showed increased connective tissue remodeling com-
pared with the untreated grafts.87
Volume 00  Number 00  ’’ 2011
Is High Concentration More Effective?
4X-- 6X [ > 750,000 to 1,800,000 Platelets/lL]
An in vitro study by Haynesworth et al.75 evaluated
the effects of different platelet concentrations (0.625  ,
1.25  , 2.5  , 5  , 10  ) on adult mesenchymal stem cells.
The author reported that proliferation and differentiation
increased continuously with increasing platelet concentra-
tion. Significant proliferation, as determined by the author,
was shown to begin at 5  and produced the highest cell
number at 10  .75 The author used PRP in a clot prepara-
tion, but determined platelet count before forming the clot.
It is difficult to determine with this protocol the absolute
number of activated platelets, and it is likely that the
effective concentration of platelets was substantially lower
than estimated. This study obtained the supernatant from
the clot for their culture work, leaving the leukocytes and
erythrocytes retained within the clot. Even though a buffy
coat PRP was used for the study, the components that were
released by platelets were the only thing evaluated through
this proliferation study. A study with a similar protocol was
conducted by Giusti et al.88 on endothelial cells who found
that the optimal concentration for cell proliferation was
1.5  106 platelets/mL. Similar to the previous study de-
scribed, the actual platelet and leukocyte concentration
would have been much less because of the cellular
components confined within the clot. However, what was
different for this study was that platelet concentrations
above the optimal amount yielded inferior results represent-
ing a threshold effect.
In contrast, not all results in the 4  --6  range have been
positive. An in vivo pig study identified no effect with the use
of 4  and 6.5  on the regeneration of bone,89 and
a rabbit study found that a mean concentration of 5.3 
(1,761,930 platelet/mL) did not lead to greater bone
remodeling.9 For these in vivo studies, a buffy-coat PRP
again was evaluated and the leukocytes were included in the
final PRP preparation.
What is the Effect of Super Concentration?
> 6  [ > 1,800,000 Platelets/lL]
Never has the phrase, ‘‘Too much of a good thing,’’ been
more adequately demonstrated than in the case of PRP
systems with super concentrations of platelets. Overwhelm-
ing the healing milieu with an excessive amount of platelets
may lead to apoptosis, growth factor receptor down-
regulation, and receptor desensitization, which may result
in a paradoxical inhibitory effect.75,90
Several studies have demonstrated this effect. Weibrich
et al.91 demonstrated that highly concentrated platelets
(6--11  ; 1,845,000--3,200,000 platelets/mL) had an inhibi-
tory effect on osteoblast activity when compared with the
more optimal lower concentrations.91 Choi et al.92 described
how PRP did not increase new bone formation in autologous
bone grafts in rats but concluded that the high concentra-
tion of platelets may have resulted in adverse effects on cell
behavior rendering an inadequate result.92 Also, as described
above, Giusti et al.88 and Graziani et al.78 were both able to
Current Orthopaedic Practice
demonstrate how a particular concentration of platelets
were able to yield an optimal proliferation effect. Yet, when
using concentrations above this optimal amount, the
proliferative results were mediocre in comparison.
Is Platelet Concentration Tissue Specific?
Different tissues types may require different platelet con-
centrations to achieve the optimal response to PRP admin-
istration. Receptor levels vary between tissues, therefore
ideal PRP concentrations may vary as well. This may explain
why a specified concentration produces significant effects in
one study with a specific tissue type and does not show an
effect in another study involving a different tissue. Anitua
et al.83 were able to demonstrate this when they studied the
effects of 2  and 4  PRP on tendon, dermal, and synovial
fibroblasts. They demonstrated different positive effects for
both platelet concentrations with respect to proliferation,
hyaluronic acid production, and secretion of angiogenic
growth factors.83 However, the positive effect was not
consistently the same for each tissue type. The authors
concluded ‘‘that the biological effects of preparations rich in
growth factors may depend on concentration of platelets
and on the anatomical source of the cells.’’
HOW IS PRP ACTIVATED?
In addition to the number of platelets, the type of activation
used may affect the outcome of PRP therapy. Different
activators and delivery methods also may influence end
results. For instance, Graziani et al.,78 Giusti et al.88 and
Haynesworth et al.75 used thrombin and calcium salt (i.e.,
CaCl or CaGluconate), whereas Anitua et al.17,28,80--86 and
Sanchez et al.28,80,82--86 preferred to use CaCl only. Addi-
tionally, Weibrich et al.91 did not report using any type of
exogenous clotting factor. Ultimately, researchers can
choose to have platelets activated endogenously or through
the addition of an exogenous clotting factor to any
commercially available system, thus, further giving support
for the need to accurately document not only the PRP
cellular components obtained from preparation but also the
delivery method used.
Is Exogenous Activation Necessary?
Exogenous activation requires the addition of an external
clotting factor. The type of activator used may significantly
influence growth factor kinetics. For instance, rapid exo-
genous activation may result in a more immediate release of
growth factors compared with a slower activation that
releases more growth factors over time.
What are the Effects of Thrombin Activation?
Exogenously activated thrombin is used intra-operatively
and results in rapid activation of platelets. This type of
activation and aggregation of platelets can provide localiza-
tion and prevent the diffusion of administered PRP during
open surgery.93,94 However, rapid activation may lead to
excessive condensing of the fibrin matrix and significant
www.c-orthopaedicpractice.com | 5
retraction of clots, which may be inferior with respect to cell
migration and growth factor enmeshment when compared
with less condensed physiologic activation.69,95--97 A rapid
activation also may lead to a decrease in the total amount of
growth factors available at the tissue site over time. Growth
factors have a short half-life (minutes to hours) and will
degrade before additional tissue receptors become available
if they are not immediately used upon release from a
platelet.98,99
Less than ideal results have been reported when PRP is
activated with thrombin. An in vivo study looked to
determine the effect of thrombin activation on PRP’s ability
to improve the osteoinductive nature of demineralized bone
matrix. The results showed that PRP prepared through a
buffy coat method significantly increased demineralized
bone matrix osteoinductivity, but this effect did not hold
true for the thrombin activated PRP group.100
What are the Effects of Activation by CaCl2?
Calcium Chloride added exogenously to PRP preparation in
lieu of bovine thrombin may result in the formation of a less
condensed fibrin matrix. The fibrin matrix may provide a
trapping mechanism for platelets resulting in smaller
amounts of thrombin formation endogenously, allowing a
slower release of growth factors over a 7-day period, which
may enhance cell migration and healing.97,101,102 Betoni-
Júnior et al.103 evaluated bone healing effects with the use of
a bone graft with PRP combined with CaCl2 and thrombin
compared with CaCl2 only. After histological analysis, both
methods presented good bone repair with similar maturity,
organization, and vascularization.103 The results from this
paper demonstrated that CaCl2 was an adequate activator of
PRP when used alone, but unlike the previous article
discussed, thrombin also yielded a positive effect.
What are the Benefits of Collagen Activation?
Tendon-derived type I collagen is equally effective as
thrombin in activating platelets and stimulating the release
of growth factors. An in vitro study of PRP prepared from
human donors was clotted using type I collagen or bovine
thrombin. Type I collagen resulted in similar release of PDGF
and VEGF between days 1--10 but a more extended and
overall greater release of TGF-b than thrombin. Clots formed
using type I collagen also exhibited far less retraction than
those formed with bovine thrombin.104 This same research
group performed a similar study using a 3  concentration
of PRP and compared the release profiles between thrombin
and a collagen sponge intended to function as a scaffold
delivery system for PRP. Once again it was noted that
thrombin results in a more immediate release of TGF and
PDGF, but the collagen scaffold produced an 80% greater 7-
day cumulative release of TGF.99 These studies help to
indicate that platelets react and activate to different stimuli
in very unique ways.
Another study was able to describe the importance of
collagen when analyzing chondrocyte proliferation in an
inflammatory cytokine-induced arthritic in vitro model.
This study analyzed PRP’s ability to induce chondrocyte
proliferation, chondrogenic gene expression, and matrix
6 | www.c-orthopaedicpractice.com
remodeling in the presence or absence of a collagen matrix.
This work highlighted the fact that the inclusion of a
collagen matrix will statistically improve the physiological
microenvironment for articular chondrocytes in the pre-
sence of PRP.8
Is Endogenous Activation More Appropriate?
Endogenous activation can be used as an alternative to
bovine thrombin activation. Endogenous activation has the
potential for slower aggregation of platelets and release of
growth factors by allowing collagen within the tissue to
operate as the activator providing a natural release pattern.99
Clot formation occurring after injection provides the benefit
of administration through a needle and may allow a more
precise delivery to, and within, the target tissue. Further-
more, slower activation by tissue collagen may provide
increased availability of growth factors and allow for more
accurate dosing.
Not only will tissue collagen cause activation of platelets,
simple agitation of platelets, such as centrifugation, as well
as needle-induced bleeding during PRP injection may
provide the appropriate endogenous clotting factors needed
for activation. A study reported that shear stress from
vigorous exercise will enhance shedding of procoagulant
particles from platelets and thus generate thrombin forma-
tion.105 The addition of clotting factors to PRP may not be
needed to significantly activate platelets. Perhaps, sufficient
platelet activation could occur through collagen exposure
and needle fenestration of a tendon before PRP injection.106
SHOULD DELIVERY TECHNIQUE BE
CONSIDERED?
The technique employed during delivery of PRP is another
fundamental variable that should be understood and
discussed. Individual tissues may have ideal anatomical
locations for delivery of PRP. For example, PRP injection into
the osseotendinous, midsubstance, or myotendinous zone
of a tendon may produce varying results.94 An in vivo rabbit
model study determined the effect of PRP on IGF-1
expression in the epitenon and endotenon of rabbit Achilles
tendons. At week 4, histological analysis revealed superior
expression of IGF-1 in the epitenon of the PRP group
compared with the saline group that produced superior IGF-
1 levels in the endotenon (P < 0.0001).12
The PRP delivery platform (liquid, spray, gel, or clot) also
should be considered. Whether the procedure is open or
closed and if slow or fast activation of platelets are required
are determining factors of which PRP delivery platform to
employ. For instance, an open procedure involving a
scaffold or hydrogel applied only to the injury surface may
result in a significantly different effect compared with direct
injection into a specific site.
Dynamic musculoskeletal ultrasound guidance also may
be beneficial to ensure accurate injection and prevent
diffusion of PRP from the desired treatment site. In support
of the use of dynamic guided imagery, Sanchez et al.107
reported a prospective muscle injury pilot study of 20 high-
level professional athletes who received PRP injections. At 6-
Volume 00  Number 00  ’’ 2011
months follow-up, the ultrasound-guided injection of PRP
enhanced healing and functional capacities 50% faster than
the control group.107 Additionally, a level 1 double-blind
randomized, placebo-controlled clinical study reported
that PRP did not increase tendon structure or alter the
degree of neovascularization in chronic midportion Achilles
tendinopathy compared with the saline control group.91
The author noted possible reasons for the lack of PRP effects,
including fluid leakage from the tendon from internal
hydrostatic pressure.
WHAT IS THE OPTIMAL DOSAGE FREQUENCY?
In addition to the concentration of platelets delivered, the
frequency and number of PRP treatments is another
important variable. Few studies have compared the effects
of single and multiple doses of PRP. Additionally, chronicity
of the injury, growth factor kinetics, and tissue type must be
considered in determining frequency of injections.
Should Single or Multiple Doses be Used?
Most PRP studies have only evaluated the efficacy of single
doses. An in vivo study determined the effect of a single
postoperative injection in a rat Achilles tendon transaction
model. The researchers reported that the treatment had an
effect on the early phases of healing because of the observed
increase in tendon regeneration and strength at days 3 and
5.108 An additional rat study determined the effect of PRP on
the initial mobilization of circulation-derived cells in the
healing of patellar tendon. The results showed that local
injection of PRP caused a significant increase in circulation-
derived cells in days 3 and 7 compared with the control
group.20 Identifying when PRP enhances these circulating
reparative cells (or simply delays the normal decline) may
indicate a more precise time period to administer treatment.
Few multiple dose treatments have been evaluated.
Mazzocca2 presented some pilot in vitro work in which
tenocytes were treated with either one, two or three doses of
PRP spaced out every 4 days. His work demonstrated a
progressive benefit with two doses yielding increased
proliferation compared with one dose, and three doses
yielding increased proliferation when compared with two.2
Hammond et al.109 reported a multiple dose treatment of
PRP enhanced injured tibialis anterior muscles in rats. Doses
of PRP were administered at days 0, 3, 5 and 7, and the
findings showed significant improvement at days 3, 7, and
14 for the PRP treated group.109 Centrally nucleated muscle
fibers (CNF) were used as a marker for myogenesis and the
CNFs for the PRP group peaked at day 14 and actually
resulted in more CNFs than the PPP and control group. A
possible reason for the improvement at day 3 may be
because of PRP’s role in decreasing inflammation in acute
tissue injuries.64,110 In a prospective study of 20 athletes
with chronic patellar tendinosis (jumper’s knee) who
received three PRP injections at 15-day intervals, statistically
significant improvements in all scores were noted at 6-
months follow-up.111
The answer to the question of appropriate dosage
frequency may lie in the understanding of cyclic factors.
Current Orthopaedic Practice
For instance, peak levels of growth factors in tissues could
indicate the optimal time for administering treatment.
Furthermore, an understanding of the plateau effects of
tissue regeneration also may provide further insight.
Additionally, identification of growth factor receptor regula-
tion and desensitization may provide an idea of the ideal
window for treatment. Finally, acute versus chronic condi-
tions would most likely require different frequency and
timing of dosage.
CLINCAL PUBLICATIONS
PRP or Cortisone
PRP has been used as an alternative to corticosteroids to
promote angiogenesis in the early phase of tendon heal-
ing.108,112 Peerbooms et al.113 and Gosens et al.114 performed
a level 1 double blind, randomized, controlled trial with a
1-year113 and 2-year114 follow-up of 100 patients with lateral
epicondylitis on the effect of PRP compared with cortico-
steroid injection. They concluded that treatment with
PRP reduced pain and function significantly more
(P < 0.0001) than corticosteroid injection at 1-year113 and
2-year114 follow-ups.
Patients, previously unresponsive to nonsurgical treat-
ments (including corticosteroids) also have shown improve-
ment in pain and function with PRP therapy. A
nonrandomized study administered a single treatment of
PRP to patients with chronic epicondylar tendonopathy
(longer than 6 months). They reported a 25% decrease in
pain, as measured by the visual analog scale (VAS) and a
modified version of the American Shoulder and Elbow
Surgeons (ASES) assessment survey at 6-month follow-up.115
PRP or Hyaluronic Acid
The efficacy of PRP also has been compared with hyaluronic
acid (HA). A nonrandomized study evaluated 60 patients
with knee osteoarthritis (OA) who received three weekly
injections. Half of the group received PRP injections and the
other half received HA injections as a control. Pain was
measured using the Western Ontario and McMaster Uni-
versities (WOMAC) questionnaire with a 33% improvement
for the PRP group compared with 10% for the HA group at 5
weeks (P ¼ 0.004).17 Additionally, intra-articular PRP was
shown to significantly enhance HA secretion (P > 0.05) and
may have provided a better balance of angiogenesis in OA
synovial cells.82 Another study compared the efficacy of PRP
with high-moloecular and low-molecular weight HA in 150
patients affected by both early and severe OA of the knee.
Three injections were administered every 14 days, and 6-
month follow-up revealed statistically significant improve-
ment (P < 0.05) in function and quality of life as measured
by the International Knee Documentation Committee
(IKDC) and Euroqol Visual Analog Scale (EQ-VAS) scores in
the PRP group compared with the HA group. The study
showed that autologous PRP injections may result in longer
lasting and better efficacy for recovering articular function
and reducing symptoms in patients affected by knee
degeneration than HA injections.116
www.c-orthopaedicpractice.com | 7
AUTHORS’ OPINION
After a careful review of the currently available literature, it
is the authors’ recommendation that a plasma-based,
moderately concentrated PRP preparation be used for
standard injections. This type of system will eliminate the
possible harmful effects of leukocytes, particularly neutro-
phils, and provide a sustained release of growth factors from
endogenously activated platelets. These preparations allow
for rapid PRP preparation from a single spin, are cost
effective, and provide platelet concentrations in the
2 --3  range, sufficient for clinical improvement in most
studies.
High concentrations of platelets may likely flood the
system, leading to increased scar tissue formation and
potential delay in healing.
With regard to frequency of dosing, it seems reasonable to
base this on the average growth factor half-life, again to
provide a constant elevated level, rather than a sudden flood
of factors. A gap of 3--7 days between injections is supported
in the literature.
The number of injections provided should be based on the
chronicity of the injury. Acute tendinous and ligamentous
injuries should be treated with 1--3 injections, while chronic
and arthritic conditions should receive 3--5 injections.
Literature supporting this recommendation is largely anec-
dotal, and treatment should be customized to the individual
patient.
Injections with PRP offer a safe and effective treatment
and have not shown any deleterious effects in either clinical
of basic science research. PRP compares favorably to
cortisone, with its chronic deleterious effects, and hyaluro-
nic acid, whose mechanism of action and long-term
outcomes remain undefined.
Local anesthetic has shown a deleterious effect when
combined with PRP injections, and should be used only on
the skin if desired.49
CONCLUSION
Improving a healing process developed over millions of
years of evolution is a daunting task. While PRP may be the
key to jump starting the body’s reparative processes,
investigations into the mechanism of action, indications,
optimal dosage and frequency of application are in their
infancy. With a lack of clinically relevent level 1 evidence,
recommendations for use of PRP are based largely on
opinion. As the popularity of PRP continues to rise, future
studies will answer these basic questions. Until then, PRP
will remain a safe, effective alternative treatment for acute
and chronic injuries.
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