Current Pharmaceutical Biotechnology, 2012, 13, 1173-1184 1173

Applications of Leukocyte- and Platelet-Rich Plasma (L-PRP) in Trauma

Surgery

Ting Yuan1, Shang-Chun Guo2, Pei Han1, Chang-Qing Zhang1* and Bing-Fang Zeng1

1
Department of Orthopedics, Shanghai Sixth Peoples Hospital, Shanghai Jiaotong University, Shanghai, China;
2
Institute for Microsurgery Extremities, Shanghai Sixth Peoples Hospital, Shanghai Jiaotong University, Shanghai,
China

Abstract. Leukocyte- and platelet-rich plasma (L-PRP) contains high concentrations of platelet, leukocytes and other bio-
activities, which play an prominent role in both bone and soft tissue healing processes. Large numbers of studies provide
evidence for application of L-PRP in experiments and clinical practice. It has been identified to improve cellular chemo-
taxis, proliferation and differentiation, angiogenesis, and production of extracellular matrix, but also responsible for stimu-
lating defense mechanisms against infections. L-PRP is now playing an increasing role in the management of patients with
traumatic injuries. However, most studies are only anecdotal or case reports, and then larger controlled studies are needed.
This article introduces the reader to L-PRP properties and L-PRP applications in trauma surgery, including applications of
L-PRP in bone healing, acute soft tissue wound healing, and repairing of acute muscle, tendon, ligament, nerve and carti-
lage injury caused by trauma.
Keywords: Blood platelet, growth factor, trauma, fracture, wound healing, platelet-rich plasma.

1. INTRODUCTION rich material may also have increased concentrations of leu-

Leukocyte- and platelet-rich plasma (L-PRP) is a volume
of plasma fraction of autologous blood having platelet and
leukocyte concentrations above baseline. It has been utilized
in surgery for much of the past 2 decades for its healing
properties attributed to the increased concentrations of
growth factors and bioactive proteins. When platelets are
activated by thrombin, alpha granules contained in platelet
release a number of growth factors, such as platelet-derived
growth factor (PDGF), transforming growth factor-beta
(TGF-
), insulin-like growth factor (IGF), epidermal growth
factor (EGF), and vascular endothelial growth factor
(VEGF), which play an prominent role in both bone and soft
tissue healing processes [1, 2]. In addition, L-PRP contains
high concentration of leukocytes, which contribute to local
debridement and exhibit bactericidal activities in acute and
chronic wounds [3]. Up to now, L-PRP has been safely used
and documented in various fields including: maxillofacial
surgery, dentistry, neurosurgery, ophthalmology, otorhino-
laryngology, wound healing, cosmetic, cardiothoracic, sports
medicine, and orthopedics.
L-PRP has been described in the literature under different
names and abbreviations, such as platelet-rich plasma (PRP)
[4], autologous platelet concentrate (APC) [5, 6], platelet
concentrate (PC) [7, 8], platelet-rich concentrate (PRC) [9,
10], platelet gel (PG) [11], platelet-rich fibrin (PRF) [12, 13],
autologous growth factors (AGF) [14, 15], and platelet-
leukocyte gel (PLG) [16]. Some authors define platelet-rich
materials as only platelets whereas others note that platelet-
*Address correspondence to this author at the Department of Orthopedics,
Shanghai Sixth Peoples Hospital affiliated to Shanghai Jiaotong University,
No. 600 Yishan Road, 200233, Shanghai, PR China; Tel.: 00 86 21 6436
9181; Fax: 00 86 21 64 701361; E-mail: yuanting3000@yahoo.com.cn
kocytes, fibrin and some bioactive proteins [17]. In view of
the general state of confusion, it is necessary to use a pre-
cisely defined terminology for platelet-rich preparations. In
terminology chapter the authors presented that it might be
more appropriate to apply the term L-PRP, for this osteoin-
ductive biomaterial is rich in platelets, leukocytes and related
active substances.
In recent years, L-PRP has been showing its advantages
in the fields of trauma surgery for facilitating bone and soft
tissue healing experimentally and clinically. In experimental
studies, L-PRP has been identified to improve cellular che-
motaxis, proliferation and differentiation, angiogenesis, and
production of extracellular matrix, but also responsible for
stimulating defense mechanisms against infections [18-20].
Clinically, thanks to the development of commercially L-
PRP preparation devices, L-PRP can be produced easily and
safely in clinical practice, requiring only approximately 15-
30 minutes. The recent technologic advances have enabled
the administration of L-PRP to move from the hospital set-
ting into outpatient and ambulatory surgical centers, even
into physicians offices. Clinical studies also show the bene-
ficial effects of L-PRP, which include more rapid reepithe-
lialization and bone formation, reduced need for blood trans-
fusion, reduction in postoperative swelling, bruising, and
pain; shorten hospital stay and early return to mobility [21-
24].
Although the majority of studies about L-PRP have
yielded excellent outcomes, most are only anecdotal or case
reports, and then larger controlled studies are needed. This
article introduces the reader to L-PRP properties and L-PRP
applications in trauma surgery.

1873-4316/12 $58.00+.00 2012 Bentham Science Publishers

1174 Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. 7 Yuan et al.

2. APPLICATIONS OF L-PRP IN TRAUMA SURGERY platelet concentration required for a positive L-PRP effect on
bone regeneration seems to span a very limited range. It
Application of L-PRP appears and develops as a modifi-
showed that the variability of platelet concentration in the L-
cation of fibrin glue (fibrin sealant or fibrin gel). In the early
PRP may indeed be responsible for the published variable
1990s, fibrin glue was developed as a biomaterial with he-
results [41].
mostatic and adhesive properties. After fibrin glue was intro-
duced, the strategic modification of the fibrin to include
platelets was reported [25]. With an increasing understand-
ing of the physiological roles of platelets in wound healing,
using platelets as therapeutic tools and use of L-PRP are be-
coming more widespread, which open new avenues in
trauma surgery and other fields.
L-PRP works mainly via the degranulation of the
gran-
ules in platelets, which contain the synthesized and prepack-
aged growth factors. For the platelets to become activated
and degranulate, and thus release these growth factors, an
exogenous application of thrombin and 10% calcium chlo-
ride would be often added [26, 27]. After activated by throm-
bin, L-PRP begins secreting these proteins within ten
minutes of clotting, with more than 95% of the growth fac-
tors secreted within one hour [4]. Therefore, L-PRP must be
developed in an anticoagulated state and should be used on
the wound within 10 minutes of clot initiation. Many cur- Fig. (1). L-PRP and thrombin and 10% calcium chloride are in-
rently available L-PRP preparation kits provide the suitabil- jected simultaneously through a dual syringe mixing system into the
ity, with using a dual syringe mixing system, which consists nonunion fracture site in operation.
of a 10-mL syringe housing the L-PRP and a 1-mL syringe
containing thrombin solution (usually bovine thrombin) and 2.1. L-PRP and Bone Healing
10% calcium chloride Fig. (1) [28, 29]. Both syringe plung-
Musculoskeletal injuries are the most common cause of
ers are connected to move in concert with both output ports
severe long-term pain and physical disability, and affect
connected to a dual spray applicator tip that allows both so-
hundreds of millions of people around the world. The years
lutions to be mixed as they are applied to the surgical bed.
2000-2010 have been termed the decade of bone and joint
Then L-PRP can be transformed into a platelet geland
as a global initiative to promote further research on preven-
easily used to cover or fill wound site.
tion, diagnosis, and treatment [42]. In the past years, scien-
The lifespan of platelets in a wound and their influence tists have been making great efforts to develop ways of im-
on growth factors is less than five days. As the platelets proving bone healing. In treating bone trauma, mechanical
come to the end of their life cycle, attracted macrophages stabilization has been a hallmark of orthopaedic fracture
replace the platelets as the primary source of growth factors care, and now osteobiologics are playing an increasing role
to continuously stimulate tissue regeneration [30]. It can be in the management of patients with traumatic injuries.
considered that PRP jump starts the cascade of regenera-
Bone healing process is divided into four distinct but
tive events leading to the formation of a mature tissue.
overlapping stages: hematoma creation, inflammation, callus
Although available data suggest that L-PRP may be valu- formation and remodeling. Following a fracture, vascular
able in enhancing soft tissue and osseous healing [31]. How- exposure and tissue damage at the fracture site result in
ever, some studies suggest no benefits from L-PRP [32-34], platelet aggregation and activation to form platelet plug and
even report that L-PRP inhibits bone formation [35, 36]. hematoma, and a large amount of growth factors will release
Marx pointed that studies that failed to find much benefit from platelet
granules to fracture site. In the inflammatory
from L-PRP have often been compromised by improper stage, inflammatory cells (macrophages, monocytes, lym-
preparation of the L-PRP [4, 37, 38]. Truly, not all currently phocytes, and polymorphonuclear cells), mesenchymal cells,
available commercial L-PRP preparation systems may be the and fibroblasts infiltrate the bone. Because of the mitogenic
same in their ability to produce the therapeutic L-PRP. The activity of the growth factors such as TGF-, IGF and
other important reason for the confusing effects of L-PRP PDGF, mesenchymal cells and fibroblasts proliferate at the
application may be based on different platelet concentra- fracture site, resulting in the formation of granulation tissue
tions. Different concentrations result in different even oppo- and ingrowth of vascular tissue. During the callus formation
site effects. Graziani et al. [39] compared the in vitro effect stage, soft callus form around the repair site. Then the callus
of different L-PRP concentrations on osteoblasts and fibro- ossifies, forming a bridge of woven bone between the frac-
blasts and showed that the maximum effect was achieved ture fragments. Bone remodeling occurs slowly by mechani-
with a concentration of 2.5 fold, with higher concentrations cal stress placed on the bone. As the fracture site is exposed
resulting in a reduction of cell proliferation and a suboptimal to an axial loading force, bone is generally laid down where
effect on osteoblast function. Similar observations were it is needed and resorbed from where it is not needed.
found by Weibrich [40], who indicated that advantageous
During the bone healing process, repairing cells, growth
biological effects occur when L-PRP with a platelet concen-
factors and scaffolds are the important elements. Among
tration of approximately 1,000,000/microl was used, and the
Applications of Leukocyte- and Platelet-Rich Plasma (L-PRP) in Trauma Surgery
these three elements, growth factors are the key component.
They attract repairing cells to injury site and bind to target
cells receptors to induce signal transduction to reach the nu-
cleus and stimulate proliferation and differentiation of mes-
enchymal stem cells. L-PRP, as a concentrated source of
growth factors, promotes bone healing through a multitude
of cytokinetic processes such as recruitment of MSC, in-
crease of extracellular matrix synthesis and neovasculariza-
tion [43-46]. In addition, L-PRP contain leukocytes and fi-
brin. Leukocytes are responsible for removal of tissue debris
and also play an important role in fracture healing processes
[47]. Fibrin helps to form clot, a 3-dimensional organization
of a fibrin network, which supports cytokine enmeshment
and cellular migration [48]. Furthermore, the fibrin matrix
serves as a scaffold for cell proliferation, cell differentiation
and ultimately for bone formation.
2.1.1. In Vitro Studies
L-PRP has been investigated in a number of in vitro stud-
ies to affect osteoblasts, osteoclasts, and mesenchymal os-
teoprogenitor stem cells [10, 49-59].
Lucarelli [60] found that L-PRP can enhance human
stromal and mesenchymal stem cell proliferation. The effect
was dose dependent and 10% L-PRP is sufficient to induce a
marked cell proliferation. Opera et al. [61] studied the effects
of platelet releasate collected from activated L-PRP on rat
bone marrow derived cells. Cultures of primary rat bone
marrow cells were overlaid with a fibrin matrix, and the
number of cells migrating within the three-dimensional ma-
trix and the leading front of migration were quantified. The
addition of platelet releasate to the top of the fibrin gels at
different time points caused a 25% increase in the leading
front of migration and a 3.5-fold increase in the number of
migrating cells. The results showed that L-PRP stimulated
the initial recruitment of BMCs to migration and had a mito-
genic effect on bone cells in proliferation. Similar results
were obtained in Grubers study [62], who examined the
effects of supernatants released from thrombin-activated
platelets on osteoclast-like cell formation in murine bone
marrow cultures and suggested that, aside from a mitogenic
effect, L-PRP application at the time of surgery enhanced
bone healing capacity.
L-PRP is a suitable carrier for MSCs transplantation in
tissue engineering because it coagulated immediately by a
minute introduction of thrombin and calcium. The combina-
tion of bone marrow cell (BMC) and L-PRP has a favoring
effect to accelerate bone healing and bone remodeling proc-
ess [9, 46, 63-66].
Angiogenesis plays a pivotal role in skeletal development
and bone healing. Blood vessels provide nutrients and oxy-
gen to bone, as well as the appropriate niche for self-
renewing osteoprogenitors [67]. Cenni [68] studied the ef-
fects of L-PRP on the human umbilical vein endothelial cell
(HUVEC) to express mRNA for osteogenic growth factors
and stimulate the migration of BMCs. The results showed
that L-PRP induced a remarkable increase in endothelial cell
proliferation, and mRNA expression of PDGF-B, intercellu-
lar adhesion molecule 1 (ICAM-1), and osteoprotegerin.
These findings supported that L-PRP contributes to bone
repair by favoring the pro-osteogenic function of endothelial
Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. 7 1175
cells, including the recruitment of osteoblast precursors and
the expression of adhesion molecules for monocytes and
marcrophages, while inhibiting their pro-osteolytic proper-
ties.
Ogino [69] studied the capability of promoting bone
healing of L-PRP from the aspect of bone resorption, and
demonstrated that L-PRP increased the secretion of osteopro-
tegerin and suppressed osteoclastogeneisis, therefore inhibit-
ing bone resorption. Sipe et al. [70] has even gone so far as
to indentify BMP-2, -4 and -6 within platelet lysates (dam-
aged platelets with microparticles of cytoplasmic and mem-
brane and soluble growth factors), suggesting the possibility
that this might contribute to the role of platelets in bone for-
mation repair [37].
However, the effect of L-PRP of on differentiation of
MSCs is still controversial. Several authors [71] investigated
the effect of L-PRP on rat BMCs and found that L-PRP
stimulates the proliferation but suppresses the differentiation.
Choi [72] reported that the viability and proliferation of al-
veolar bone cells were suppressed by high L-PRP concentra-
tions, but were stimulated by low L-PRP concentrations (1-
5%). In Ranlys study [35], the author found L-PRP de-
creased the osteoinductivity of demineralized bone matrix
implanted in immunocompromised mice, and the activities
of both demineralized bone matrix and L-PRP were donor-
dependent. Based on these confused study results, Hsu et al.
[73] investigated whether negative regulators exist in the L-
PRP and negatively affect cells proliferation. The author
observed that L-PRP contains an angiogenesis inhibitor,
thrombospondin-1 (TSP-1). TSP-1 has been found to inhibit
adhesion, proliferation, and tube formation of endothelial
cells in culture and block neovascularization of the chick
chorioallantoic membrane [74, 75]. So the reason of high
concentration of L-PRP decreasing proliferation of cells may
attribute to the high concentration of TSP-1 secreted from L-
PRP, which leads to the antiproliferative effect [73].
2.1.2. Animal Studies
There is a large variety of animal studies on L-PRP re-
search in the literature [76-80]. But the results still tend to be
confusing. Various different animal species and L-PRP
preparations may be the reasons. Especially in small animals,
therapeutic L-PRP is not always achieved because the diffi-
culty of collecting blood may damage platelets. Therefore,
negative outcome following the use of L-PRP in animal
studies should be interpreted with caution.
Most studies showed a positive effect of L-PRP in the
bone healing in animal models. Kasten et al. [81] investi-
gated the efficacy of L-PRP in bone healing of a critical-size
diaphyseal radius defect in a rabbit model. The bone defect
was filled with calcium-deficient hydroxyapatite (CDHA),
with the addition of allogenic L-PRP, MSC or both. L-PRP
yielded better bone formation than the empty CDHA scaf-
fold as determined by both histology and microcomputer
tomography (p<0.05) after 16 weeks. The effect was similar
to that of MSC on the CDHA scaffold. In Nairs study [77],
he observed the same results. The study was designed to use
autogenous L-PRP and BMCs in combination with a tripha-
sic ceramic (calcium silicate, hydroxyapatite and tricalcium
phosphate)-coated hydroxyapatite (HASi) to repair segmen-
1176 Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. 7
tal bone defects created in a goat femur model. The combina-
tion of the HASi with BMCs and L-PRP promoted the ex-
pression of many osteoinductive proteins, leading to faster
bone regeneration. Interestingly, compared to the bare HASi
group, the addition of BMCs and L-PRP leaded to faster
degradation of HASi. The author indicated the reason was
that BMCs promote the adhesion of osteoclasts on the mate-
rial by secreting osteoinductive proteins-like osteopontin. In
both of the above studies, however, there were no significant
differences in the performance in bone regeneration between
bone substitute + BMCs and bone substitute + BMCs + L-
PRP.
Kroese-Deutman [82] et al. evaluated the bone inductive
properties of L-PRP with titanium fiber mesh and autologous
bone chips in a 15-mm rabbit radial defect model. After 12
weeks, Histomorphometrical analysis showed that bone for-
mation was higher in the implants with L-PRP (PRP-Ti-
Bone: 378%) than in those without L-PRP (Ti-bone:
256% and Ti: 255%). In a domestic pig model, Thorwarth
[83] et al. used immunohistochemistry to show increased
osteocalcin, osteopontin, and BMP-2 production in defined
defects filled with collagen lyophilisat and L-PRP in com-
parison to autogenous bone. Osteocalcin is a sensitive
marker of the mineralization process in the later phase of
bone formation, and osteopontin was early marker of bone
formation. The results showed that addition of the L-PRP
increased BMP-2 expression after 2 week and promoted ear-
lier calcification of autogenous bone graft, indicating that L-
PRP has an osseopromotive effect.
Kawasumis study [84] was to investigate the effect of L-
PRP on the proliferation and differentiation of rat BMCs
using a rat limb-lengthening model. Rat BMCs embedded in
L-PRP gel were cultured for six days and transplanted into
the distraction gap. The results showed that L-PRP stimu-
lated the proliferation of rat BMCs, but did not promote os-
teoblastic differentiations. The combination of L-PRP and
BMCs had favorable effect on osteogenesis in the rat limb-
lengthening model.
Some contradictory results were reported in animal stud-
ies by Rabillard et al. [79], who examined the bone healing
properties of adding L-PRP to osteoconductive calcium
phosphate ceramic granules in a 2 cm bone defect of ulnar in
dogs. Radiographic and histological evaluations were per-
formed and showed that addition of L-PRP did not enhance
bone regeneration in critical-size bone defects. Sarkar et al.
[85] reported on the effect of L-PRP on new bone regenera-
tion in a critical size defect in the tibial diaphysis of sheep,
supplied either with L-PRP in a collagen carrier or with col-
lagen alone (controls). Bone volume, mineral density, me-
chanical rigidity and histology of the newly formed bone in
the defect did not differ significantly between the L-PRP
treated and the control group, and no effect of L-PRP upon
bone formation was observed. And whats more, Choi [86] et
al. found that adding L-PRP to autogenous bone graft de-
layed bone formation compared with the contralateral control
side.
2.1.3. Human Studies
Clinical use of L-PRP in trauma and orthopaedics has
increased and prevailed currently. For example, the Depart-
Yuan et al.
ment of Perioperative Blood Management of the Catharina
Hospital performs close to 1600 management procedures
annually, of which 60% are related to obtain whole blood
platelets to produce L-PRP [76]. Application of L-PRP can
decrease postoperative drainage, reduce narcotic require-
ment, and facilitate an early return to mobility. Postopera-
tively, patients may experience fewer complications, recover
more rapidly, and have a reduced hospital stay [87].
In human studies, L-PRP is often applied in formation of
gelatineous mass resulted by bovine thrombin and used in
combination with different bone matrices. Due to the sticky
structure of L-PRP gel, caused by the fibrin strands present
in the gel, bone matrices are kept together, avoiding un-
wanted migration of bone particles. And the gelatineous
form is easier to handle and place into the graft site [88].
Moreover, thrombin itself stimulates fibroblast proliferation
and synthesis of type IV collagen and some active mediators.
There are many articles describing the clinical use of L-
PRP with both mineral and organic bone matrices [89-91].
Marx et al. [30] firstly used L-PRP clinically to enhance
bone healing. He evaluated bone density and healing time
when L-PRP was added to autogenous bone to reconstruct
mandibular continuity defects and reported a 1.62- to 2.16-
fold increase in radiographic bone density, and an increase in
histologic and histomorphometric bone density. Subse-
quently, clinical use of L-PRP prevailed gradually for regen-
eration of bone. In the following years, the easily obtainable
L-PRP and its possible beneficial outcomes hold promise for
wide applications in trauma surgery. L-PRP has been pro-
posed as a primary or adjunctive treatment for fractures [92],
bone defect and bone nonunion [93], osteomyelitis [3], and
spine interbody fusion [23, 94, 95].
Dallari et al. [96] reported a randomized, controlled trial
of L-PRP among patients undergoing a medial, opening-
wedge osteotomy of the proximal tibia. The authors com-
pared the osteogenic potential of lyophilized bone chips
(BC) combined with L-PRP gel, or with L-PRP gel and
BMCs, with that of lyophilized bone chips alone in the heal-
ing of a high tibial osteotomy. After six weeks, in compari-
son with Group bone chip alone, histomorphometry showed
significantly increased osteoblasts and osteoid areas in both
Group L-PRP+ BC (p=0.006 and p=0.03, respectively) and
Group L-PRP+ BC + BMCs (p=0.009 and p=0.001), as well
as increased bone apposition on the chips (p=0.007 and
p=0.001, respectively), which was greater in Group L-PRP +
BC + BMCs than in Group L-PRP + BC (p<0.05). Radio-
graphic osteointegration was significantly better in groups
with L-PRP than group without L-PRP at all stages of fol-
low-up. The study demonstrated that adding L-PRP or L-
PRP combined with BMCs to lyophilized bone chips in-
creases the osteogenetic potential of the lyophilized bone
chips and may be a useful tool in the treatment of patients
with massive bone loss.
As a carrier for cell transplantation, L-PRP, combined
with marrow cells, was often used to treat large bone defect.
In Kitohs study [97], L-PRP combined with BMCs was ap-
plied during distraction osteogenesis of 32 long bones (14
femora, 18 tibiae) in 17 patients while the BMCs alone group
consisted of 60 bones (25 femora,35 tibiae) in 29 patients.
The study concluded that transplantation of BMCs in asso-
Applications of Leukocyte- and Platelet-Rich Plasma (L-PRP) in Trauma Surgery
ciation to L-PRP could shorten the treatment period
(28.67.07 days/cm in Group L-PRP + BMCs vs 36.610.1
days/cm in Group BMCs, p=0.0019) and reduced the associ-
ated complications by accelerating new bone formation
(p=0.041).
In a retrospective study of lumbar spinal fusions, L-PRP
was used with autograft and coralline hydroxyapatite in all
posterior fusions, and autograft, coral and intradiscal spacer
in intradiscal fusions [98]. The result showed that L-PRP
promoted early maturation of osseous fusion.
Nonunion has been a challenging clinical problem in
trauma and orthopaedic field. In comparison to fresh frac-
ture, growth factor levels at the site of the nonunion are sig-
nificantly reduced. So, addition of L-PRP to the nonunion
site brings critical growth factors to the region, resulting in
the positive influence of nonunion resolution [92]. Bielecki
[93] firstly reported the treatment of nonunion with L-PRP in
2006. He injected L-PRP gel percutaneously into the
humeral diaphyseal nonunion gap. As a minimally invasive
treatment method, L-PRP injection offered the advantage of
decreased morbidity associated with the classic open grafting
techniques. During the follow-up period, the healing process
on roentgenograms and increase in bone mineral density
were observed. At the 8th week over 75% of the circumfer-
ence of the bone at the defect site had resolved and during
later visits remodeling of the union was observed on X-ray
films. In the next year, Bielecki [99] reported a case of in-
fected tibial nonunion with fistula treated by percutaneous L-
PRP injection. Microbiological examination was positive for
MSSA in the fistula. Five months after L-PRP application,
union occurred and the wound was healed. This report indi-
cated that L-PRP has antibacterial activity and repairing abil-
ity, which may resolve infection and nonunion simultane-
ously. In a study of 12 patients with delayed union and 20
with nonunion treated by percutaneous injection of L-PRP,
union achieved in all cases of delayed union on average 9.3
weeks (rang 5-12 weeks). In the nonunion group, union was
observed in 13 of 20 cases and the average time to union was
10.3 weeks (range 8-18 weeks) [100]. Sanchez et al. [101]
applied L-PRP to clinical nonunions and reported on their
retrospective case series of 16 aseptic supracondylar and
diaphyseal atrophic nonunions after failed surgical fixation
21 months previously. The author pointed L-PRP technology
was clinically safe and could enhance the healing of non-
hypertrophic nonunions. For patients with nonunion of bone
fracture, L-PRP may act as an alternative to surgical treat-
ment to improve healing.
In recent years, L-PRP was used to aid in treating osteo-
myelitis by some authors. Yuan [102] reported one case of
chronic osteomyelitis in bilateral femurs failed to four-year
conventional treatments. The application of L-PRP seemed
to prohibit the development of necrosis and reduce inflam-
matory extravasate, and prevent recurrence for a long time.
Because of the properties of antimicrobial and promoting
tissue regeneration, L-PRP may act as a novel therapeutic
agent for osteomyelitis [103, 104].
Similarly, contradictory results of human studies exist.
Weiner et al. [105] evaluated two groups undergoing lumbar
fusion with or without L-PRP. The fusion rate for the control
group (without L-PRP) was 91% (24 of 27 patients), and the
Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. 7 1177
L-PRP group was 62% (18 of 32 patients). The use of L-PRP
resulted in inferior rates of arthrodesis compared with auto-
genous bone graft alone.
2.2. L- PRP and Healing of Traumatic Soft Tissue Injuries
Trauma frequently causes acute soft tissue injury, such as
open cutaneous wound, acute stretching or tearing of muscle,
tendon and ligament. In the healing process of acute trau-
matic soft tissue wound, platelets are the main regulator of
the blood clot phase and the inflammatory phase, play an
essential role in the regeneration phase, and then influence
indirectly the remodeling phase [106]. So, application of L-
PRP could positively influence the rate and quality of wound
repair.
First of all, the high concentration of platelets and the
adhesive effect of L-PRP help prevent or decrease surgical
bleeding. A study compared the hemostatic ability of PRP
and/or PPP with untreated control wounds. The results
showed that L-PRP reduced bleeding by 70% at 5 minutes
when compared to controls [107]. Secondly, high concentra-
tions of leukocytes and growth factors in L-PRP also play a
prominent role in traumatic wound healing.
2.2.1. Healing of Acute Cutaneous Wounds
Experimental studies have reported that with the applica-
tion of L-PRP, wound healing is substantially improved by
increasing collagen content, promoting angiogenisis and
increasing early wound strength [108]. The early wound
strength results from the ability of L-PRP to decrease the
inflammatory phase of wound healing and to increase early
deposition of glycosaminoglycan and fibronectin [109]. In an
in vitro study of cell culture with the addition of L-PRP, Kil-
ian et al. [110] found that the different growth factors con-
tained in L-PRP could work together to stimulate the migra-
tion and proliferations of human endothelial and MSCs. Ka-
kudo et al. [111] examined the efficacy of L-PRP on human
adipose derived stem cells and human dermal fibroblast. The
addition of activated L-PRP significantly promoted the pro-
liferation of both cell types. In addition, low PRP concentra-
tions (1% and 5%) were suitable for human adipose derived
stem cells proliferation, and 5% had a maximal effect on
human dermal fibroblasts.
In a animal study, L-PRP embedded with endothelial
progenitor cells (EPCs) and/or basal cell keratinocytes (KCs)
was used to treat 20 full-thickness wounds in a pig model
[112]. All L-PRP-treated wounds showed more vascular
structure (p<0.001), and the addition of EPCs further im-
proved neovascularization. L-PRP+KCs resulted in the high-
est reepithelialization rates (p<0.001).The author indicated
that L-PRP enhanced deposition of matrix molecules in the
inflammatory and proliferative phase of wound healing, and
the combination of L-PRP and autologous embedded EPCs
or KCs could improve extracellular matrix organization,
promote angiogenesis, and accelerate reepithelialization.
Clinically, Kazakos [113] performed a study to assess the
efficacy of L-PRP in the treatment of acute limb soft tissue
wounds in 59 patients, including open fractures, closed frac-
tures with skin necrosis and friction burns. One group was
treated with conventional dressing, changed every 2 days,
1178 Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. 7
and the other group was managed with local application of
L-PRP gel, which was repeated weekly. The clinical end-
points were the healing rate and/or the time required to bring
about adequate tissue regeneration in order to undergo re-
constructive plastic surgery. The rate of wound healing rate
was significantly faster in the L-PRP group at weeks 1, 2,
and 3 as measured by the reduction of the wound size at each
time point (p=0.003, p<0.001 and p<0.001, respectively).
The mean time to plastic reconstruction in the L-PRP group
was 21.3 days (40.6 days for the control group). The author
concluded that L-PRP gel treatment could aid in the man-
agement of acute trauma wounds as it induces faster wound
healing rates and lowers the time to plastic reconstructive
surgery. In Marxs article [4], he also showed the enhanced
and faster healing of acute wounds when it was treated with
L-PRP and bovine thrombin as compared to bovine thrombin
alone. And the comparison provided evidence that PRP
might reduce scar and promoted a greater melanocyte sur-
vival.
In a prospective, single-blind, pilot study [114] to com-
pare the healing of acute human skin wounds treated with
topical L-PRP vs conventional therapy (antibiotic ointment
and/or occlusive dressings), 80 full-thickness skin punch
wounds were made on the thigh of eight healthy volunteers.
On day 17, the percentage of closure was 81.12.5% for the
L-PRP-treated sites and 57.25.9% for the control sites. And
the L-PRP wound closure velocities were significantly faster
than those of the controls (p=0.001). Histologically, L-PRP
wounds formed earlier epithelialization and granulation.
2.2.2. Healing of Acute Muscle, Tendon and Ligament
Trauma
Healing of muscle injuries is dependent on proliferation
of myofibroblasts, local vascularity and regeneration of in-
tramuscular nerve branches, which may be enhanced by L-
PRP [115-117]. Several growth factors within L-PRP have
been evaluated to enhance muscle regeneration and im-
proved muscle force after injury [118]. In a case report, a
professional bodybuilder with acute adductor longus tear
underwent 1 injection of L-PRP weekly for 3 weeks and got
healed completely [119]. Sanchez et al. [120] reported in a
study, after ultrasound guided injections of L-PRP in 22
muscle injuries of 20 high-level professional athletes, full
recovery of functional capabilities was restored in as early as
half of the expected recovery time without any evidence of
excess fibrosis. There are, however, no randomized con-
trolled human studies supporting the use of L-PRP for mus-
cle injuries.
Tendon and ligament have a low metabolic rate that en-
tail slow healing after injury. However, it has been shown
that several platelet derived growth factors can stimulate
tendon repair after exogenous local application [121]. Some
equine and human cell studies also reported L-PRP enhanced
type I collagen gene expression in L-PPR-cultured tendon
cells, stimulated human tenocyte proliferation and total col-
lagen production, and slightly increased matrix metalloprote-
inase 3 (MMP-3) expression [122, 123]. Aspenberg et al.
[124, 125] reported greater maturation when L-PRP was
used to treat Achilles tendon ruptures in a rat Achilles tendon
transaction model. Other researchers showed that repetitive
Yuan et al.
injection of L-PRP within Archilles tendon fascicles in a
sheep model triggered a healing response assessed by in-
creased cell number and angiogenesis [126, 127]. The basic
information gave insight into clinical application of L-PRP
in treating tendon injuries. Currently, L-PRP has been ap-
plied during the ruptured tendon surgical procedure and a
significant acceleration in functional recovery may be ob-
served compared with a matched group who had conven-
tional surgery [128]. In these conditions, L-PRP may offer an
alternative treatment over palliative or operative treatments
for tendon injury.
2.2.3. Healing of Acute Nerve Injury
Severe trauma often results in acute nerve injury, but few
studies have investigated the effect of L-PRP on nerve heal-
ing. In a prospective, randomized, and controlled animal
study aimed to investigate the effects of L-PRP on facial
nerve regeneration, L-PRP was compared with PPP and fi-
brin sealant, all with or without suture after nerve transac-
tion. The authors [129] found the best results for the return
of function occurred when the nerve ends were sutured to-
gether. At the same time, the data demonstrated a measurable
neurotrophic effect when L-PRP was present, with the most
favorable results seen with L-PRP added to suture in com-
parison with fibrin sealant or PPP. Sariguney [130] observed
similar results in a study of repairing rat sciatic nerve with L-
PRP. In this study, L-PRP helped to improve remyelinization
of the interrupted sciatic nerve when the epineural repair was
done with sutures.
Based on the above results, the application of L-PRP
might extend to the treatment of central nervous system in-
jury, especially for traumatic brain or spinal cord injury.
Theoretically, the properties of L-PRP also support this hy-
pothesis: the receptors of platelet derived cytokines have
been found localized widely on the surface of neurons and
glia not only in the peripheral nerve but also in the central
nervous system, especially after pathologic events, the high
levels of these receptors are expressed in the lesion areas of
central nervous system [131]. So, local application of L-PRP
can provide large numbers of cytokines to activate these re-
ceptors to regulate the gene transcription, and induce neural
progenitor cells proliferation, migration and differentiation to
replace the injured nerve. Up to date, there have been no
further experimental and clinical studies to provide data
about L-PRP treating central nerve injury, but L-PRP is
likely to act as an important method for treating nerve in the
coming years.
3. EFFICACY OF L-PRP IN REPAIRING CARTI-
LAGE CAUSED BY TRAUMA
Due to the limited regenerative capacity of cartilage tis-
sues, articular cartilage defect caused by various lesions re-
mains a problem to be resolved. Several growth factors, es-
pecially PDGF and TGF-
, have been proven to be effective
for cartilage and meniscal regeneration [132-134]. Some in
vitro studies suggest the effects of L-PRP for stimulating
chondrocytes [134, 135]. In the arthroscopic treatment of a
large avulsion of articular cartilage in the knee of an adoles-
cent soccer player using L-PRP, exciting results were ob-
served, complete articular cartilage healing was considerably
Applications of Leukocyte- and Platelet-Rich Plasma (L-PRP) in Trauma Surgery Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. 7 1179

accelerated, and the functional outcome was excellent, al- Up till now, there has been no standardized method for
lowing a rapid resumption of symptom-free athletic activity L-PRP preparation. The volume of whole blood drawn, the
[136]. Kon [137] compared the efficacy of L-PRP and visco- volume of L-PRP yield, and the concentration of platelets,
supplementation (hyaluronic acid injection) for treatment of can be adjusted through different centrifugal force and time,
severe chondropathies of the knee, and observed that L-PRP to meet clinical or experimental requirements.
injections provide more and longer efficacy than hyaluronic
acid injections in reducing pain, symptoms and recovering
articular function. Even though the results need to be con-
firmed in a large cohort of patients, this technique opens new
perspectives in the area of joint cartilage repair.

4. POTENTIAL PROBLEMS AND RISKS

4.1. Understand the mechanisms of manually producing
L-PRP
Although many L-PRP preparation devices have been
commercially available on the market in European and
American countries in recent years, L-PRP has to be pro-
duced manually in a large number of developing countries
today. They have no such automated devices for L-PRP
preparation. So, surgeons in these countries must completely
understand the mechanisms of producing L-PRP before they
apply L-PRP to patients. In fact, the mechanism of manual
L-PRP preparation is the same with that of automated de-
vices, separating platelets from whole blood through centri-
fugations based on different densities of blood components.
The best effective methods of preparing L-PRP should yield
platelets with high concentration and without lysing the
platelets or damaging them. After comparing centrifugations
based on varying the force and time, Landesberg et al. [138]
indicated that optimal L-PRP preparation should be with
both spins at 200g for 10 minutes, because centrifugations of
less than 5 minutes failed to achieve any significant platelet
enrichment, and forces of greater than 250g resulted in plate-
lets damage and forces of greater than 800g might reduce the
amount of TGF-
.
Yuan et al. [139] studied the mechanisms of manually Fig. (2). After the first spin, PPP was divided into three equal parts
preparing L-PRP through comparing four different centrifu- and red cell was divided into two equal parts to measure platelet
gations in terms of centrifugation time and force. These four concentration.
centrifugations are quoted from published literatures [138,
4.2. Possibility of Carcinogenic Effect of L-PRP
140-143], and have been approved to be effective to yield
therapeutic L-PRP. One was single-spin centrifugation and There has been some concern about a possible carcinogenic
three are double-spin. The results showed that single-spin effect related to the stimulation of cellular proliferation and
centrifugation would not separate and concentrate platelets to the local presence of L-PRP in high concentrations. Actually,
a therapeutic level. It yielded a disappointingly low platelet no growth factor can provoke carcinogenesis. Because all
counts. After the first spin, whole blood in container is cen- growth factors contained in L-PRP are known to act on cell
trifuged into its three basic components: red blood cells, L- membranes, not the cell nucleus. They are not mutagens and
PRP, and platelet-poor plasma (PPP). Because of differential promote a normal gene expression, not an abnormal gene
densities, the red blood cell layer forms at the lowest level, expression [144].
the platelet concentrate layer in the middle, and the PPP
layer at the top. The authors divided the volume of PPP frac- 4.3. Risks of Bovine Thrombin
tion into three equal parts from top to bottom, and divided
the red cell fraction into two equal parts accordingly Fig. (2). Another consideration is that L-PRP is frequently acti-
Platelet concentrations of each layer were measured respec- vated by bovine thrombin when used. Many surgeons are
tively (Table 1). The study results showed that the most of hesitant to use a bovine thrombin in human trauma surgical
platelets stay in the lowest layer of PPP and the top layer of procedures as numerous reports have documented the devel-
red cell, especially in the latter. Excessive centrifugal force opment of anti-bovine antibodies cross reacted with human
leads to more platelets sinking into the bottom layer of red clotting factors, which led to clinical syndromes that range
cell, and this layer would be discarded after the first spin. from severe postoperative bleeding to high rates of thrombo-
Removal of the bottom layer of red cell will reduce the re- sis [145-150]. Up to now, however, no related complications
covery of platelets. have been reported. Based on the potential risks mentioned
above, and in order to overcome this problem, autologous
1180 Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. 7
Table 1. After the First Spin, Platelet Concentration Yielded in Each Layer
Platelet Count (1,000/
l) 1500g 6 Mins
Whole blood 217.5
Upper layer of PPP 3.9
Middle layer of PPP 7.4
Lowest layer of PPP 97.5
Upper layer of red cell 833.4
Lower layer of red cell 204.4
thrombin seems to be the logical choice, but the preparation
of autologous thrombin is cumbersome. In the recent years, a
commercial autologous thrombin kit has been developed
which produces autologous thrombin out of platelet-poor
plasma. In addition, using recombinant human thrombin may
eliminate this risk. Some studies show that calcium sulfate is
also able to induce activation of L-PRP [151], as well as acts
as a delivery system for the platelet-released growth factors
[152]. On the other hand, Dohan [48] suggested no use of
anticoagulant citrate dextrose and bovine thrombin, because
the thrombin solution leads to high speed polymerization,
which will influence the mechanical and biologic properties
of L-PRP gel. Instead, the author supported platelets polym-
erize naturally and slowly during centrifugation without bo-
vine thrombin. It would form a platelet-rich fibrin (PRF), he
called the second-generation platelet concentrate, favorable
to cytokine enmeshment and cellular migration.
Han [78] et al. compared the osteoinductivity of L-PRP
on demineralized bone matrix used with bovine thrombin
activation and without thrombin activation. In vivo, L-PRP
stimulated chondrogenesis on day 14 and osteogenesis on
day 28 and 56, whereas thrombin-activated L-PRP inhibited
such events. Thus, the application of bovine thrombin also
must be further studied and considered in clinical applica-
tions.
4.4. Clinical Contraindications of L-PRP
So far, there have been no major health problems arisen
through the therapeutic use of L-PRP in published literature.
Obviously, contraindications to the administration of L-PRP
include platelet dysfunction, critical thrombocytopenia, in-
fection, anemia and sensitivity to bovine thrombin. And we
need to know that many of these are not absolute contraindi-
cations.
5. CONCLUSIONS
L-PRP is now playing an increasing role in the manage-
ment of patients with traumatic injuries. The enhancement of
bone and soft tissue healing by its placement at the site of
tissue injury or surgery is supported by basic science and
clinical studies.
Because it is an autogenous preparation, L-PRP is inher-
ently safe and therefore free from concerns over transmissi-
Yuan et al.
200g 10 Mins 215g 10 Mins
213.5 220.0
68.8 65.4
74.3 74
312.7 309.7
674.6 682.2
68.1 71.2
ble diseases such as HIV, hepatitis, and West Nile fever. In
recent years, L-PRP is well accepted by patients.
Although a lot of studies provide evidence for application
of L-PRP, some important questions about the biologic
mechanisms of L-PRP still remain unclear, such as the inter-
actions between proteins, whether leukocytes have a positive
or negative influence, whether dense granules contained in
L-PRP also play a role in tissue modulation and regenera-
tion, and the molecular basis of how L-PRP can reduces
pain.
Conflicting data exist in clinical and experimental re-
search on the efficacy of L-PRP treatment. Many studies are
anecdotal or case reports, the sample sizes are frequently
small, limiting the generalization of the findings. Therefore,
further investigation of high-level studies with control group
comparison are needed to definitively determine the role of
L-PRP, and standardized dosing and composition of L-PRP
is necessary in order to compare the data from different stud-
ies.
CONFLICT OF INTEREST
None declared.
ACKNOWLEDGMENTS
This work was supported by a grant from the National
Natural Science Foundation of China (Grant No. 30801221)
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