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78

Trends Biomater. Artif. Organs, Vol 20(1), pp 78-83 (2006)


Anila S. and K. Nandakumar http://www.sbaoi.org

Applications of Platelet Rich Plasma for Regenerative Therapy in Periodontics

Anila S and K. Nandakumar

Department of Periodontics
Govt. Dental College, Trivandrum 695011

Introduction site through an oxygen gradient of 30-40 mm Hg


and drive the remaining bone regeneration process.
Regeneration of tooth-supporting structures
By day 14, complete revascularization of the graft
destroyed by periodontitis is a major goal of
is seen. Stem cells differentiated into osteoblasts
periodontal therapy. Periodontal regeneration is
- osteoid is being laid down. Early bone formation
perhaps one of the most complex to occur in the
is occurring. By four to six weeks, random cellular
body since at least six tissues are involved: the
bone, called woven bone, is formed which is
gingival epithelium, gingival connective tissue,
immature and disorganized. In phase two
periodontal ligament, tooth root surface
remodeling lamellar bone is formed, representing
cementum, alveolar bone and corresponding
a more organized bone.
vasculature. All these mineralized and
nonmineralized components must be restored What is Platelet Rich Plasma?
to their original position and architecture for
A recent innovation in dentistry is the preparation
regeneration of the periodontium to occur.
and use of platelet-rich plasma (PRP), which is
Growth factors are a class of naturally occurring
a component of blood in which the platelets are
proteins involved in three key cellular events in
concentrated in a limited volume of plasma.
tissue repair: mitogenesis, migration and matrix
Autologous platelet gel was first developed as
synthesis and remodeling1. A combination of
a byproduct of multicomponent pheresis.
growth factors may more effectively stimulate
The platelet count in PRP can exceed 2 million
formation of mineralized as well as
platelets per micro liter. A natural blood clot
nonmineralized tissues1. Platelets are rich in
contains 95% red blood cells, 5% platelets, less
growth factors that may contribute to an
than 1% white blood cells, and numerous
accelerated tissue regeneration process.
amounts of fibrin strands. A PRP blood clot
Sequence of Bone Regeneration contains 4% red blood cells, 95% platelets, and
1% white blood cells. It can be considered that
Platelets are responsible for initiation of
PRP jump starts the cascade of regenerative
regeneration of tissue from trauma. During repair
events leading to the formation of a mature graft
platelets become entrapped in a fibrin clot and
site2. The PRP obtained offers up to a 2.16-times
degranulate releasing two primary growth
increase in the maturation rate and substantially
factors: PDGF and TGF-B. PDGF binds to
greater density of a bone graft procedure3.
endothelial cells to initiate capillary ingrowth; and
TGF-B binds to osteoblasts and stem cells to Components of PRP,
initiate mitosis and stimulate osteoid
1. Growth Factors
production2.
2. WBC & phagocytic cells
The lifespan of platelets in a wound is less than
3. Native fibrogen concentration
five days. Macrophages are attracted into the graft
Applications Of Platelet Rich Plasma For Regenerative Therapy In Periodontics 79

4. Vasoactive and chemotactic agents of PDGF are released after adhesion of platelets
5. High concentration of platelets to an injured site. The AA and BB isoforms
enhance proliferation of bone cells, increasing
Dental applications of PRP include, sinus lift the production of PDGF-AA in osteoblast cultures
procedures, onlay grafts, particulate grafts, by an autocrine process 4 in reconstructive
alveolar cleft palate repair, oral/nasal fistula periodontal studies in rats, in vivo application of
repair, post-operative hemostasis of bone graft PDGF increased bone regeneration in calvarial
donor sites, continuity defects of the mandible defects when a resorbable membrane was used
and hemophiliacs undergoing surgery. In as a carrier5.
periodontal surgery it has been used in gingival
grafting, crown lengthening and periodontal Transforming Growth Factor (TGF) - TGF
regeneration. regulates proliferation and differentiation of
multiple cell types. TGF found in platelets is
Contraindications for PRP usage are few and subdivided into TGF1 and TGF2, which are
include platelet dysfunction syndrome, critical the more generic connective tissue growing
thrombocytopenia, hemodynamically unstable factors involved with matrix formation influencing
patients and pregnancy. osteoblasts to lay down bone matrix through the
Role of Growth Factors In PRP process of osteogenesis. Also cells activated
by TGF1 and TGF2 include fibroblasts,
PRP is an autologous source of concentrated endothelial and osteoprogenitor cells,
suspension of the growth factors found in chondroprogenitor cells and mesenchymal stem
platelets. Activated PRP release growth factors, cells. A condoroprogenitor cell will further
enhancing wound healing and wound strength. differentiate and produce the matrix for cartilage.
Growth factors derived from platelets initiate A mesenchymal stem cell stimulated to mitosis
connective tissue healing, bone regeneration and provides wound-healing cells. In vitro, TGF- has
repair, promote development of new blood been observed to promote extracellular matrix
vessels, and stimulate the wound healing production in many cell types, such as
process. periodontal ligament fibroblasts. TGF-1, used
alone or in combination with PDGF-BB,
Periodontal wound healing involves gingival stimulates the proliferative activity of periodontal
fibroblasts, gingival epithelial cells, periodontal ligament fibroblasts. In a recent study involving
ligament fibroblasts and osteoblasts, all of which a canine model, the application of rhTGF-1 in
are important for tissue repair and hard-tissue conjunction with nonresorbable barrier
regeneration. A series of well-orchestrated cell- membrane greatly enhanced bone regeneration
cell interactions is initiated after injury. Disruption in oral osseous defects (after 2 months)6.
of the vasculature as a result of injury leads to
fibrin formation and platelet aggregation. Several Insulin Growth Factor(IGF) - IGF is also important
growth factors are then released into the tissue in wound healing, and stimulates both
from the platelets and from the adjacent cells proliferation and differentiated function in
after injury osteoblasts. IGF has 2 forms, II, and I each of
which has 2 single chain peptides. IGF binds to
Platelet Derived Growth Factor (PDGF) - PDGF the same receptors as insulin and is involved in
is a very powerful regulatory growth factor and a the development of many tissues, including the
sentinel growth factor that begins nearly all teeth. In the area of periodontal regeneration,
wound healing. PDGFs main function is to more research has been done on IGF-I. This
stimulate cell replication (mitogenesis) of form of IGF is chemotactic for periodontal
healing capable stems and premitotic partially ligament cells, and it has strong effects on
differentiated osteoprogenitor cells, which are periodontal ligament fibroblasts and protein
part of the connective tissue-bone healing synthesis. IGF-I stimulates bone formation by
cellular make-up. PDGF also causes replication proliferation and differentiation, and it is
of endothelial cells, causing budding of new synthesized and secreted by osteoblasts 7 .
capillaries (angiogenesis). PDGF exists in three Human patients treated with a combination of
forms: PDGFaa, PDGFbb, PDGFab. All isoforms 150 mg/ml each of recombinant human platelet-
80 Anila S. and K. Nandakumar

derived growth factor-BB (rhPDGF-BB) and that they will not normally move across a distance
rhIGF-I in a methylcellulose vehicle experienced greater than 0.4mm (400 microns).
43.2% osseous defect fill, whereas the control
PRP also accelerate endothelial, epithelial, and
group (vehicle only) had 18.5% osseous fill8.
epidermal regeneration. They stimulate
Epidermal Growth Factor (EGF) - EGF is angiogenesis, enhance collagen synthesis,
responsible for cell differentiation and stimulates promotes enhanced soft tissue wound healing,
re-epitheliation, angiogenesis and collagenase decreased dermal scarring, enhanced
activity. Vascular Endothelial Growth Factors hemostatic response and reverse the inhibition
(VEGF) have potent angiogenic, mitogenic, and of wound healing caused by glucocorticoids.
vascular permeability-enhancing activities High leukocyte concentration in PRP has an
specific for endothelial cells. added antimicrobial effect. It provides a watertight
seal (necessary for dural closures) and
The short shelf life and inefficient delivery to
target cells are major concerns associated with augmented rate of extracellular matrix deposition
local administration of recombinant human resulting in earlier wound closure. As it is an
growth factors. The growth factors are expensive, autologous blood product there is no risk of
and many doses may be required to achieve infectious disease transmission or clerical
any therapeutic effect. An easy, cost-effective way errors, thus making it a safe product with no time
to obtain high concentrations of growth factors consuming visits to the blood bank for pre-
for tissue healing and regeneration may be donation. It provides an immediate surgical
autologous platelet storage via PRP. hemostatic agent that is biocompatible, effective
and safe. The native fibrinogen concentration
Major Benefits of PRP imparts a gelatinous adhesive consistency, for
Jump-starts osteogenesis by releasing growth ease of surgical application and results in
factors at the local site. Osteoblasts can be reduction of pain and infection.
enticed to move across a greater distance by PRP-Related Studies
creating a scaffold system (fibrin) that will assist
their movement. In vitro, platelet membranes have been shown
to stimulate the mitogenic activity of human
Early consolidation of the graft: the increased trabecular bone cells, thus contributing to the
amount of PDGF in the graft initiates regeneration of mineralized tissues.
osteocompetant cell activity at an enhanced
molecular rate. Another study assessed the efficacy of
demineralized bone powder alone or combined
Speeds up mineralization : because with PRP in enhancing the osseointegration of
mineralization on a graft site is a coupled dental implants in a dog model. Standard
phenomenon, osteogenesis must proceed in histomorphometric methods at 6 and 12 weeks
such a way that activation and bone formation is after surgery revealed a higher percentage of
greater then resorption. Mineralization of the bone contact with bone powder and PRP than
collagen matrix is speeded up due to PDGF with bone powder alone. The authors
being added right from the start in the mineralized
bone segment of the graft, instead of being concluded that bone defects around titanium
released from collagen. Use of PRP has shown implants could be treated successfully with bone
to improve the rate of bone formation by 1.62 to powder and that PRP may improve bone
2.18 times that of the controls formation9. Human studies have also shown that
PRP can be advantageously and easily applied
Improves trabecular bone density: there has in surgery. PRP was used in 20 patients
been reported in the literature a 15% to 30% undergoing cosmetic surgery, including face-lifts,
improvement in trabecular bone density when breast augmentations, breast reductions and
platelet rich plasma factor is added to the graft. neck lifts. The application of PRP yielded
Allows placement of implants into the grafted site adequate hemostasis if platelet-poor plasma (PPP)
at an earlier time by enhancing osteoconduction was also applied to create a seal to halt bleeding.
: osteoblasts are normally non-motile cells in It has the advantage of minimizing use of
Applications Of Platelet Rich Plasma For Regenerative Therapy In Periodontics 81

electrocautery so as to minimize the chance of discontinuous plasmapheresis method is


damage to the adjacent nerves10. limited because of high cardiovascular stress
to the recipient, known health risks and high
The first clinical dental results with PRP were
production costs13. The processed blood can be
reported by Marx and others in 1998, who used
autotransfused to the patient.
PRP to improve graft incorporation in mandibular
reconstructions in patients who had received * Manual PRP systems
cancellous bone marrow grafts after tumor
End User can manually recover the maximum
removal 3. Their data strongly suggested that
amount of platelets. Clearly the best way to
adding PRP to bone grafts accelerated the rate
produce a true PRP product. up to Counts of 4 to
and degree of bone formation. When patients
10 times the patients baseline can be achieved
contemplating subsequent implant placement
using a manual system. Since there is no re-
after extraction received a mixture of autologous
suspending (dilution) of platelets, the final
bone and PRP, much better epithelization and
product has a high concentration of platelets.
compact mature bone with well-organized
trabeculae was demonstrated compared to the Eg: Curasan ( Pharma Gmbh AG, Germany),
control group11. 3I PCCS (3i Implant Innovations, Florida),
Medtronic Sequestra , Haemonetics Cell Saver
Combination of PRP and freeze-dried bone graft
has been suggested as an alternative A simple chairside technique
therapeutic method for implants placements. for PRP procurement
PRP in combination with bone allograft and
Recent publications have indicated that PRP
guided tissue regeneration as periodontal
prepared from 8 to 10 ml of whole blood is
therapy for intrabony defects in humans resulted
sufficient for periodontal regenerative
in significant gain in clinical attachment and filling
therapies14. We are procuring PRP with the help
of the treated defects, as revealed by 2-year
of a general purpose tabletop laboratory
follow-up.
centrifuge by the following method. It is simple
Procurement and cost-effective method for producing PRP in
an in-office environment. Patients are selected
PRP can be obtained in various ways in the
based on the absence of any blood
dental office. Techniques for PRP preparation
abnormalities or use of anti-coagulants. 10 ml
vary from using 10 cc of a patients blood and
blood is withdrawn from the anticubital region
spinning it in a lab centrifuge, to using a unit of
with a 10ml syringe and transferred to a container
blood that is put through a cell separator, that
containing 1.4ml anticoagulant (Citrate
sequesters and concentrates the platelets12.
phosphate dextrose solution). It is then
Systems for procurement, centrifuged for 10 mins at 1300 rpm. The result
is a separation of whole blood into a lower red
* One touch automated PRP systems (Eg:
blood cell (RBC) region and upper straw-colored
Harvest SmartPREP)
plasma region as shown in figIB. There is
It provides simplicity in operation and may relatively high concentration of platelets found in
provide a good platelet count before plasma re- the boundary layer between these two regions.
suspension. This system requires 50ml blood The upper straw colored plasma layer (platelet
for procurement and do not sense the Plasma/ poor plasma;PPP) and 1-2 mm of the top part of
Blood Interface and hence may yield low platelet the RBC layer is aspirated and transferred into
count another container and again centrifuged for 10
mins at 2000 rpm. This results in an upper portion
* Plasmapheresis
of clear yellow supernatant serum and the bottom
Requires approx. 450ml of blood from which 20- red tinged layer consisting of highly concentrated
60cc of PRP obtained. This method of cell PRP as shown in figIC. The upper clear layer is
separation is used only when large quantities of aspirated until 1.5ml of serum is left.
PRP are required. The use of platelet The contents of the tube is mixed well and
concentrates obtained from blood banks by the transferred into a sterile container.
82 Anila S. and K. Nandakumar

A. Intrabony defect B. PRP placed


A. Whole blood B. After initial centrifuge

C. Defect site sutured

C. After final centrifuge D. PRP with up to 90% platelet recovery

Figure 1 : Procurement of platelet rich plasma


D. PRP + Bone graft E. Barrier membrane placed
over defect

Figure 2 : Application of Platelet rich plasma

Pre-operative probing depth Intrabony Defect

Pre-operative probing depth Intrabony defect

PRP placed Defect site sutured


Resorbable collagen
PRP placed barrier membrane placed

PRP

Defect site sutured


Figure 3
Figure 4
At the time of the application, the PRP is
combined with an equal volume of a sterile of the fibrin into an insoluble gel, which causes
saline solution containing 10% calcium chloride the platelets to degranulate and release the
(a citrate inhibitor that allows the plasma to indicated mediators and cytokines). This results
coagulate) and 100 U/ml of sterile bovine in formation of a sticky gel that is relatively easy
thrombin (an activator that allows polymerization to apply to the surgical defects.
Applications Of Platelet Rich Plasma For Regenerative Therapy In Periodontics 83

Technique of application in periodontal Conclusion


intrabony defects
PRP is a new application of tissue engineering
The periodontal defect site is opened and debrided. and a developing area for clinicians and
The PRP gel is placed into the defect site and researchers. It is a storage vehicle for growth
sutured as shown in figure II(B,C). A barrier factors, especially PDGF and TGF-b, both of
membrane can also be place over the defect to which influence bone regeneration. Although the
augment the results (figure IIE). PRP can be mixed growth factors and the mechanisms involved are
with a graft material and placed in the defect (figure still poorly understood, the ease of applying PRP
IID). PRP results in early consolidation and take in the dental clinic and its beneficial outcomes,
up of the graft. All these treatment modalities have including reduction of bleeding and rapid
shown good clinical results. Figure III presents a healing, hold promise for further procedures.
case report in which PRP was used alone; and Most important, this autologous product
figure IV presents a case where PRP was used in eliminates concerns about immunogenic
combination with a resorbable collagen barrier reactions and disease transmission. PRP may
membrane. become a routine treatment modality for
periodontal regeneration in future.
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