Platelet Rich Plasma

INTRODUCTION: In the late 1970s, the importance of growth factors within the wound healing cascade were identified. Studies, however, have shown that a single growth factor applied into a wound is not as effective as multiple growth factors. This is not surprising, as the wound healing cascade requires multiple growth factors for different stimulatory and inhibitory functions at different phases over long periods of time within the different stages of the wound healing cascade. From prior research on surgical and nonsurgical wounds it became apparent from a physiological perspective that the goal of treatment is combination therapies utilizing a multitude of growth factors known to be instrumental in tissue proliferation and remodeling. This understanding has led to the need for bio-tissue engineering strategies, which can provide simultaneous multiple growth factor therapies. Over the past few years different emerging technologies have developed leading to the current use of Platelet Rich Plasma (PRP). PRP is obtained from autologous blood by sequestering and concentrating platelets by gradient density centrifugation.

HISTORY : In 1994 Tayapongsak et al introduced the novel idea of adding autologous fibrin adhesive(AFA) to cancellous bone during mandibular continuity reconstructions. They identified early radiographic consolidation in 33 cases; they attributed this to enhanced osteoconduction afforded to the osteocompetent cells in the graft by virtue of the fibrin network developed by AFA. They also reported the remarkable adhesive advantage of binding cancellous particles during graft placement . Robert Marx is considered the pioneer in the field of PRP and bone grafting .

RATIONALE: It is now well known that platelets have many functions beyond that of simple hemostasis2. Platelets contain a rich source of multiple growth factors within the alpha

granules, which can be activated and subsequently secreted after activation with thrombin. The processing of PRP involves the sequestration and concentration of platelets, and, therefore the many growth factors they contain. The strategy is to amplify and accelerate the effect of growth factors contained in platelets, which are the universal initiators of almost all wound healing5. Blood clot initiates soft tissue healing and bone regeneration. Natural blood clot contains 95% RBC, 4% platelets and 1% WBC; whereas PRP clot contains 95% platelets, 4% WBC and 1% RBC. By taking advantage of all the natural regeneration pathways, and using all the known and as yet to be identified growth factors in platelets, autologous PRP, which is nontoxic and nonimmunoreactive, accelerates wound healing pathways. CLINICAL APPLICATIONS1 : I. BONE REGENERATION : 1. Sinus lift grafting 2. Ridge augmentation 3. Repair of bone defects created by removal of teeth or small cyst 4. Ridge preservation techniques 5. Periodontal defects 6. Closure of cleft lip and palate defects 7. Repair of oro-antral fistulas 8. Craniofacial reconstruction

II. SOFT TISSUE REGENERATION 1. Periosteal and connective tissue flaps 2. Free connective tissue and gingival grafts 3. Root coverage procedures 4. Controlling soft tissue healing and tissue maturity

CONTRAINDICATIONS 1. Unexplained anaemia where Hg is <12.5 g% 2. Thrombocytopenia < 100,000 / cu. mm

3. Diagnosed and treated anaemia Hg < 10.0 g% 4. Patients who have metastatic disease 5. Presence of tumour in the wound bed 6. History of platelet dysfunction 7. Active wound infection and sepsis requiring systemic antibiotics 8. Patients with poor prognosis associated with other disease process 9. Patient with bovine sensitivity 10. Patients with religious beliefs that prevent the use of blood.

PROCUREMENT OF PRP Platelet-rich plasma is developed from autologous blood with a cell separator. This cell separator, can sequester and concentrate platelets during surgery without interfering with or slowing down the actual surgical procedure. A trained and certified technician or nurse can accomplish the harvest of PRP in 20 to 30 minutes. Each 100ml of blood will yield about 10cc PRP. PRP will have platelet count approx 5 times that of peripheral blood.

The commercially available plasma concentrating cell separators are : 1. ELMD 500 (Medtronic Eletromedic, Autotransfusion System, Parker, CO ) 2. HSPCS ( Harvest Technologies, Plymouth, MA ) 3. 3i PCCS (3i/Implant Innovations, Palm Beach Gaerdens, FL ) Which Office PRP Device Is Best?1 Following are the key features that characterize effective PRP machines: 1. FDAclearance. 2. Complete or nearly complete automation 3. Consistent platelet concentration yield of 4 to 6 times baseline per 6-mL volume (approximately 1 million platelets/mL). 4. Maintenance of platelet viability and activity after processing. 5. Amount of autologous blood required (120 mL or less).

There are different methods ; 1. Method using 55cc of blood6: Blood is taken from anticubital region with a 21- guage butterfly. There are 6 blood collection tubes which are yellow-top 10ml tubes, containing 1.5ml acid citrate dextrose solution (trisodium citrate, citric acid and dextrose), and permits a draw of 8.5ml of blood/tube . Each tube is inverted several times to ensure mixing of blood and anticoagulant. The yellow-top tubes are placed in a centrifuge. The first centrifugation is for 10min at 1,300 rpm. The result is a separation of the whole blood into a lower red blood cell (RBC) region and upper straw-colored plasma region. This plasma contains a relatively low concentration of platelets (platelet poor plasma; PPP) in the uppermost region and a higher platelet concentration in the boundary layer, often called the buffy coat. The test tubes are placed in a suitable rack and their tops removed. An equal number of 10ml red-top blood collection tubes, containing no anticoagulant, are placed into the rack and their tops removed. A 5ml syringe is used to draw the straw colored plasma from the blood collection tube, moving the needle from top downward as the draw continues. The draw stops when reaching the the RBC layer or into the first 1 to 2 ml of that layer. Clinical experience indicates that the first 1mm of the RBC layer contains the larger and more recently synthesized platelets. The contents are then expressed into the red-top tube. The same procedure is performed for the other tubes. The red-rubber tops are then placed back on the tubes, and the tubes are inserted into the centrifuge for the 10min rotation at 2,000 rpm. Their tops are then removed. The contents of each tube consist of an upper portion of clear yellow supernatant serum, containing fibrinogen, and a very low concentration of platelets; and the bottom layer often red tinged consist of highly concentrated PRP. The previously used 5ml syringe are now inserted into the tube as far as they can go, and liquid is drawn out until the syringe draws air. This leaves approx 1.5ml of serum and PRP in the tube. The PRP solution of all tubes are transferred to a sterile container.

Another method1: Platelet separation and concentration starts with an aseptic and minimally traumatic phlebotomy technique for the withdrawal of a small volume of blood appropriate for the particular device to be used, usually 20 to 60 mL. A 19-gauge needle or larger should be used to avoid platelet disruption or activation in the lumen of a narrow needle. A large vein such as the wrist vein over the radius (ie, the beginning of the cephalic vein) or an antecubital vein should be chosen. So that the blood is immediately coagulated, the syringe should contain anticoagulant citrate dextrose A (ACD-A).

Ethylenediaminetetraacetic acid (EDTA), which is used in diagnostic blood laboratories, is not recommended for this purpose because it is damaging to platelet membranes. Citrate phosphate dextrose (CPD), which is used to store red blood cells, also is not recommended for this purpose because it does not support platelet metabolism as well as ACDA. Today, blood banks use only ACD-A as a platelet-preserving solution for platelet transfusion. When 20 mL of autologous blood is used, 2 mL of ACD-A should be placed in the syringe prior to blood withdrawal. When 60 mL of autologous blood is drawn, 7 mL of ACD-A should be used. Using the SmartPReP device by Harvest Technologies, the anticoagulated autologous blood is placed into the red-topped canister, which is then placed into the device. Although all PRP processing devices operate by centrifugation, only a few produce consistently high concentrations of viable bioactive platelets. Effective platelet separation and concentration are a product of gravitational forces (g forces) over time, usually measured in minutes (g minutes). To separate and concentrate platelets, the device must use two separate centrifugations, called spins. The first spin, known as the separation spin, separates the red blood cells from the rest of the whole blood (white blood cells, platelets, and plasma), This is followed by a concentration spin, which separates and compacts the platelets, white blood cells, and a small number of residual red blood cells from the plasma after 95 % or more of the red blood cells have been separated and sequestered into another compartment of the canister. Single-spin machines are incapable of separating and concentrating platelets to a therapeutic level. Because of the convex-concave shape of the red blood cell and the

relatively smaller size of the platelets, the smaller platelets get trapped in the concavity of the larger red blood cells and become compacted with them rather than being concentrated separately. The most effective PRP development occurs when a separation spin of about 1,000 g for 4 minutes (4,000 g minutes) is followed by a concentration spin of about 800 g for 8 to 9 minutes (6,400 to 7,200 g minutes) for a total of about 11,000 g minutes. This force application is about one third the value known to disrupt platelet cell membranes (30,000 g minutes). Each of the two spins must be timed precisely to gain consistent platelet separation and concentration. This is best accomplished by a fully automated device that avoids manual manipulations that can disrupt the platelet separation. In addition, the effectiveness of the device should be wholly independent of the patient's hematocrit. In fully automated systems, this is usually accomplished by a density-dependent floating shelf; in systems that are less automated, this is accomplished by the more work intensive but equally effective manual separation of the red blood cell fraction after the separation spin. After the completion of the concentration spin, a few residual red blood cells, along with nearly all of the white blood cells and platelets, will be compacted at the bottom of the PRP compartment and overlaid by a volume of plasma. Together, these will appear as a small layer of red blood cells surrounded by a thin white line (the so-called buffy coat) over a larger volume of straw-colored but mostly clear liquid, which is the plasma. Commonly described as a red blood cell button, this appearance of the PRP as it emerges from the machine is the sign of quality for the clinician. The younger platelets, which contain more growth factors, are larger and therefore centrifuge out in the upper layer of the RBC fraction. The red blood cell button indicates the presence of these younger and more complete platelets. Then the PPP layer is aspirated away leaving the small volume of plasma, which is then used to resuspend the concentrated platelets. This is accomplished by drawing up the remaining plasma volume into the syringe and then ejecting it down the walls and onto the bottom of the canister three times. Following this maneuver, the suspension of concentrated platelets, which now contains a small number of RBC and WBC in plasma and usually appears as a light red suspension, is the developed PRP.

2. Method using 400-450ml of blood2 : The cell separator withdraws 400 to 450 mL of autologous whole blood at a rate of 50 mL per minute, using a centrifugation speed of 5,600 rpm through a central vein catheter placed during surgery. As it withdraws the blood, it adds citrate phosphate dextrose at a ratio of 1 mL of citrate phosphate dextrose to 5 mL of blood, which achieves anticoagulation through calcium binding. As the blood is centrifuged, it is separated into its three basic components as a function of density. From the least dense to the most dense : 1) platelet-poor plasma (PPP) comes off first, 2) platelet-rich plasma (sometimes referred to as the Buffy coat) comes off second, 3) the more dense red blood cells (RBC) come off last. The PPP component is acellular plasma; it accounts for about 200 mL of volume and is returned to the patient. The RBC component, essentially packed red blood cells, accounts for about 180 mL of volume and is also returned to the patient. The PRP is plasma with a concentrated number of platelets and white blood cells. It accounts for about 70 mL of volume. Both PPP and PRP are plasma fractions. Therefore, they contain abundant fibrinogen and clotting factors. The formation of fibrin, although not itself a growth factor, will provide the natural osteoconductive matrix needed in bone regeneration. During the centrifugation process at 5,400 rpm, the PPP will be separated first. Once the PPP is collected, the centrifugation speed is slowed to 2,400 rpm to create a precise separation of the PRP from the red blood cells. Clinical experience indicates that the first 1mm of the RBC layer contains the larger and more recently synthesized platelets. Therefore, this layer of RBCs is included in the PRP. This will impart a red tint to the otherwise straw-colored PRP. STORAGE AND ACTIVATION OF PRP1 Developed PRP is anticoagulated and will remain in that state until a clotting process is initiated. PRP has been found to remain sterile and its platelets to remain viable

and bioactive for up to 8 hours when stored at room temperature. Therefore, it is recommended that the PRP remain anticoagulated until it is needed at the tissue site. Because it can be stored for up to 8 hours, the PRP will be effective even when used during a long procedure or when the procedure is delayed. However, storing PRP for more than 8 hours is not recommended since its viability has not been tested beyond that time frame, and refrigeration and/or freezing without cryopreservatives disrupts platelet membranes. Since the development of PRP requires only a small amount of blood, and the entire process can be completed in just 15 minutes or less, it is best to discard any unused PRP after 8 hours and develop a second batch of PRP. The ACD-A that is used as an anticoagulant in developing PRP inhibits clotting by binding calcium. Therefore, activation of the PRP requires replacement of calcium and initiation of the cascade of blood coagulation. This can be accomplished by adding 5 ml of a 10% calcium chloride (CaCI2) solution to 5,000 units of topical bovine thrombin. When used in very small volumes, this solution will clot the PRP into what is often termed a smart clot. To clinically apply PRP, the anticoagulated PRP solution is placed into a 10-ml syringe and the CaCI2 / thrombin solution is placed into a 1-ml syringe (ie, a tuberculin syringe). The two syringes are then placed into an ejection assembly that has a nozzle to combine the two solutions into what looks like a squirt gun. Upon pushing the lever of the injection assembly, each solution is expressed in a proportion of 10: 1 through the nozzle tip, which delivers the PRP to a precise location, and clotting occurs within 6 to 10 seconds. Alternatively, the PRP can be activated in its cup receptacle by adding just two drops of the CaCI2 / thrombin solution to it and then carrying the activated PRP clot to the tissue site. Another option is to aspirate the anticoagulated PRP into a syringe and then to aspirate the equivalent of two drops of the CaCl2-thrombin solution into the syringe, along with a small amount of air to be used as a mixing bubble. In 6 to 10 seconds, the clotted (activated) PRP can be expressed onto the tissue site. It is important to note that using more than 2 drops of the CaCl2-thrombin solution is counterproductive. A larger volume of this solution will not speed the clotting process but will actually slow it down

or inhibit clotting altogether by diluting the fibrinogen concentration, which is the ratelimiting factor in clot formation. If desired the combination of PRP and thrombin/CaCl2 solution may also be used to infuse resorbable barrier membrane. Alternatively, 2 to 3 ml of PRP can be spread onto a sterile flat surface and 1 or 2 drops of thrombin/CaCl2 solution added. This mixture is agitated for several seconds and then left for several minutes. Any excess PRP is gently removed with a sterile gauze. This autologous sterile membrane can be cut & shaped prior to placement in the operative site. The PRP can be activated by calcium alone, although this requires waiting at least 8-10min or more. PRP can be activated by using 1ml of autologous whole blood and some autogenous cancellous bone, both containing thrombin (which initiates clotting cascade). UNNECESSARY CONCERNS ABOUT BOVINE THROMBIN1 Because it is an autogenous preparation, PRP is completely free of any transmissible human diseases such as HIV, hepatitis, etc. It is therefore also accepted well by patients. Specifically concerns have been advanced about the use of bovine thrombin as the clot initiator. Bovine thrombin remains in standard use today in many surgeries and is the safe initiator of clotting for the development of PRP. A more rational clinical concern relates to the rare cases in which bovine thrombin was used as a hemostatic agent in open orthopedic, neurosurgical, and cardiovascular surgeries that later developed bleeding episodes. Fewer than 20 such cases have been reported, and each of these adverse events has been thoroughly investigated. The secondset bleeding episodes in these patients was due to antibodies not against bovine or human thrombin but against bovine factor Va, which was a contaminant in certain commercial preparations of bovine thrombin. These antibodies cross-reacted with human factor Va and produced coagulopathies as well as the rare bleeding episodes. Since 1997, the processing of bovine thrombin by GenTrac (Jones Medical Industries) has virtually eliminated contamination of bovine thrombin with bovine factor Va. Prior to 1997, levels of bovine factor Va in bovine thrombin reached 50 mg/ml; today they are less than 0.2 mg/ml, and no further cases related to this specific preparation have been reported. In

addition, the bovine thrombin preparations used in the cases reported were high in dosage (more than 10,000 units) and were applied directly to the open wounds, where absorption into the systemic circulation is certain. The use of bovine thrombin in PRP is low in dosage (less than 200 units), is topical (does not enter systemic circulation), and is alredy clotted when it comes into contact with human tissues. It is therefore not absorbed systemically but instead is subsequently engulfed and digested by macrophages that also digest the clot itself. Today, bovine thrombin prepared by adding 5 ml of 10% CaCl2 solution to the lyophilized bovine thrombin preparation is the standard for initiating clotting of PRP and activation of platelets. It will lead to rapid clotting (within 6 to 10 seconds) and form a cross-linked clot that will allow for convinent handling and the binding of particulate grafts. However, safer methods to consider could include the utilization of recombinant human thrombin, autologous thrombin, or extra-purified thrombin. Landsberg and coworkers (2000) described a new method to activate PRP gel with the ITA gelling agent (Natrex Technologies, Greenville, NC). They stated that this method could be used more safely as an alternative to bovine thrombin for gelling the PRP; however they did not describe the specific composition and mechanism of action of ITA. PRP IN PERIODONTAL DEFECT TREATMENT1 Several authors have shown superior bone regeneration in human intrabony defects when PRP was combined with several graft materials as compared with use of the same graft materials without PRP, including porous bovine bone, calcium sulfate, tricalcium phosphate, allogeneic bone, autogenous bone, and composites of autogenous and allogeneic bone. The surgical approach to the treatment of a periodontal defect requires a sulcular incision that extends well past the defect site to allow sufficient flap reflection that will enable the surgeon to visualize the entire defect and suture the flap over a well-supported bony base. The granulation tissue in the defect must be thoroughly debrided and the root surfaces planed. The defect may be irrigated with saline or Peridex (Teva) or washed with citric acid and then irrigated with saline to remove any acid residue. The authors

recommend incubating the graft material in PRP while the defect is being debrided and irrigated. By this means, the cell adhesion molecules and the clotting nature of the PRP will bind the graft material into a working composite that will be easier to handle. Once the graft material is placed into the defect, the authors recommend placing a layer of activated PRP over the surface of the graft. In this fashion, the growth factors secreted by the platelets in PRP will directly contact the mucoperiosteal flap used to cover the graft and at the same time trickle down into the graft to accelerate both the soft tissue healing and the bone regeneration. Since one- and two-wall bony defects have a small surface area of native bone and hence a diminished host progenitor cell population contributing to osteoconduction, combining some autogenous bone into the graft composite is highly recommended. Three- and four-wall bony defects have a sufficient population of osteoprogenitor cells in close proximity so that they need only a small percentage of autogenous bone or none at all. In each situation, PRP has proven benefits, and in all such grafts, PRPs effect on the overlying flap will support capillary ingrowth and reduce dehiscence and sequestration of graft particles. It can also be expected to hasten bone regeneration in the defect and cause the formation of a more dense bone, even when the graft material contains no autogenous osteoprogenitor cells.

USE OF PRP IN SOFT TISSUE FLAPS DURING IMPLANT SURGERY PRP has a strong potential in implant flap surgery, regardless of the particular type of flap employed. Since the periosteum is likely to be compromised by previous disease and/or surgeries regardless of the flap design, PRP is recommended for all such flaps. PRP has been recommended for use in increasing the rate of bone deposition and quality of bone regeneration when augmenting edentulous sites with bone autografts, allografts, xenografts, or alloplasts in sinus lifting and in alveolar ridge augmentation procedures prior to or in conjunction with dental implant placement. Activated PRP is applied either to the undersurface of the flap or over the bone and the implant just prior to closure. This will accomplish a seal at the suture line, which will resist leakage. PRPs mechanism of action in this application is that the growth

factors will speed the rate of vascular ingrowth into the bone, thereby reducing the degree and risk of crestal bone loss, and the cell adhesion molecules will initially stabilize the flap and heal the closure, and then later serve as a matrix for osteoconduction and vascular ingrowth. PRP has also been recommended for use alone or in combination with bone grafts and barrier membranes in the treatment of peri-implant defects created as a consequence of immediate implant placement or as a result of peri-implantitis. Garg and coworkers proposed that resorbable barrier membrane materials be infused with PRP. They have proposed that this PRP-based membrane could serve as a short-acting biologic barrier, since all platelets contained in PRP will degranulate within 3 to 5 days, and their initial growth activity expires within 10 days. Although PRP is an option in stage 2 implant-uncovering procedures, it is not essential unless numerous implants are to be uncovered and the mucosa has been severely compromised by repeated surgeries, infections, previously failing grafting procedures, or radiation therapy.

FREE GINGIVAL GRAFTS WITH PRP The most common application for a free gingival graft is to increase the zone of attached tissue caused by recession or a high mucogingival attachment. For this procedure, keratinized mucosa is harvested from the palate in the area of the lateral palatal shelf adjacent to the molar and premolar teeth. Because of the rich vascularity and nerve density in this area, excessive postoperative bleeding and significant pain may be experienced by the patient. To combat this the use of activated platelet poor plasma (PPP) or even PRP is recommended for hemostasis. The authors recommend placing the harvested full-thickness gingival graft in activated PRP while the recipient site is being prepared. This will allow the growth factors in PRP to be secreted and to attach themselves to the membranes of the cells in the graft, while the cell adhesion molecules coat the deep surface of the graft. Both of these mechanisms will facilitate adhesion of the graft to the recipient site and then promote the capillary and connective tissue ingrowth necessary for complete survival of the graft.

PRP WITH CONNECTIVE TISSUE GRAFTS Today connective tissue grafting is often used to create or add bulk and contour to the labial gingiva around a natural tooth or an implant-retained crown. It may also be used for root coverage in cases of slight or moderate root exposure. The connective tissue used in this procedure is almost always harvested from the lateral palatal shelf opposite the molars and premolars. In contrast to free gingival grafts harvested from the same area, the connective tissue graft donor site is closed primarily and therefore has less postoperative bleeding and pain. Placing PRP in this donor site is thus optional since the rich vascularity of the tissue and the use of a primary closure usually result in rapid and uncomplicated healing. However, like the free gingival graft, the connective tissue graft should be incubated in activated PRP while the recipient site is prepared. This will allow the platelets to secrete their growth factors that will attach to the membranes of the cells in the graft as the graft's collagen fibrils become coated with the cell adhesion molecules in PRP. The root surface should be prepared using a saturated solution of citric acid, a solution of tetracycline hydrochloride, or ethylenediaminetetraacetic acid (EDTA). This removes any proteinaceous deposits from the root surface and etches the dentin, opening the dentinal tubules for maximum ingrowth of the graft and development of a firm fibrous attachment to the root. Once the root surface has been debrided and etched in this fashion, the authors recommend coating it with activated PRP. The soft tissue component of the recipient site is usually developed by means of an interproximal papilla-sparing sulcular incision and dissection. The purpose of this tunneling procedure is to elevate the mucosa from the periosteum and gain a volume space to accommodate the graft. The PRP-coated connective tissue graft is then pulled through the tunnel from one end to the other using a 3-0 silk suture. The mucosa is then coronally repositioned and sutured to the palatal mucosa to achieve maximum stabilization. This procedure will serve to increase the gingival contour for improved esthetics as well as to cover the exposed root. Overcontouring of the graft is unnecessary since the rapid revascularization promoted by PRP will maintain the viability of the transplanted cells and prevent shrinkage. Stabilizing the graft is critical for complete

integration and a precise outcome. CORONALLY REPOSITIONED FLAPS AND ALLOGENEIC DERMIS WITH PRP FOR ROOT COVERAGE This technique combines the use of two materials-allogeneic human dermis (AlloDerm, LifeCell) and PRP-with a coronally repositioned flap to achieve a predictable outcome. In this approach, the allogeneic dermis is rehydrated in activated PRP (rather than the standard saline) while the root surface is prepared with a saturated citric acid solution or other agent and the full-thickness flap is reflected. The allogeneic dermis is then placed over the root surfaces (usually several adjacent roots are involved and treated simultaneously) and the bone. It is then trimmed to follow the curvature of the gingival margin and sutured through the interproximal spaces to the lingual or palatal gingival. The apical edge is often tacked to bone using the tacks designed to secure a barrier membrane. Activated PRP is applied to the affixed allogeneic dermis and to the undersurface of the mucoperiosteal flap before it is sutured. PRP's cell adhesion molecules-fibrin, fibronectin, and vitronectin assist in the initial adherence and act as a scaffold for the rapid incorporation of the allogeneic dermis to bone as well as to the coronally repositioned flap. As with each of the other soft tissue procedures, the growth factors in PRP will also promote capillary and connective tissue ingrowth into the allogeneic dermis from the underlying bone as well as from the overlying coronally repositioned flap. Therefore, the composite of allogeneic dermis, PRP, and a full-thickness mucoperiosteal flap heals to the repositioned location and achieves coverage even in the most advanced cases of root exposure.

COMPONENTS of PRP Studies of PRP have identified following important growth factors in the alpha granules of the sequestered platelets: 1. Platelet-derived growth factor (PDGF) 2. Transforming growth factor- 3. Platelet-derived Epidermal Growth Factor (PDEGF)

4. Platelet-derived Angiogenesis Factor (PDAF) or Vascular Endothelial Growth Factor (VEGF) 5. Insulin-like growth factor-1 (IGF1) 6. Platelet Factor 4 (PF 4) Also, Fibrin, fibronectin, and vitronectin are present in PRP, which of not growth factors, but they are cell-adhesion molecules. course are

Platelet-derived growth factor:

Platelet-derived growth factor is involved in nearly all wound healing. Although it is the primary growth factor in platelets, it is also synthesized and secreted by other cells, such as monocytes, macrophages, smooth muscle cells and endothelial cells. By virtue of the presence of platelets in blood clots, it is the first growth factor in the wound healing and leads toward revascularization, collagen synthesis, and bone regeneration. Platelet-derived growth factors are dispersed throughout the wound as platelets degranulate. It is a glycoprotein with a molecular mass of approximately 30 kd. It exists mostly as a heterodimer of two chains, termed A and B chains, of about equal size and molecular mass (approximately 14 to 17 kd). Homodimers of A-A and B-B chains are also present in human platelets and have the same effects on bone regeneration. There are approximately 0.06 ng of PDGF per 1 million platelets. This calculates to 6 x 10-17 g of PDGF, or about 1,200 molecules of PDGF, per individual platelet1. PDGF acts as a potent mitogen in serum for mesenchymal cells, including fibroblasts and smooth muscle cells. The effect of PDGF is dependent upon the presence of other growth factors, and it also serves as a powerful chemoattractant for smooth muscle cells, fibroblasts, macrophages and leukocytes. In addition to angiogenic properties, it stimulates collagen and matrix formation in vivo3. Its effects are mediated when the PDGF molecule binds to cell membrane receptors. This binding activates an internal cytoplasmic signal protein with a highenergy phosphate bond (kinase activity). This signal protein, in turn, activates the gene expression for the specific activities of mitosis, angiogenesis, macrophage activation and upregulation of other growth factors and cells.

Transforming growth factor : The TGF-s proven to exist in PRP are the TGF-1 and TGF-2 proteins, which are the most common members of the TGF- superfamily and are general growth and differentiating factors involved with connective tissue healing and bone regeneration. Both TGF- 1and TGF- 2 are proteins with molecular masses of approximately 25 kd. They, like PDGF, are synthesized by thrombocytes and found in platelets. They are also synthesized and found in macrophages, osteoblasts, and some other cells. TGF- is a fundamental regulatory molecule that acts by both autocrine and paracrine mechanisms. When released by platelet degranulation, or actively secreted by macrophages, they act as a paracrine growth factor on adjacent cells such as fibroblasts, marrow stem cells, endothelial cells and preosteoblasts. However, each of these target cells also has the ability to synthesize and secrete its own TGF- proteins to act on adjacent cells as a paracrine growth factor, and to act on its own cell membrane, as an autocrine growth factor, to direct, alter, or maintain a certain activity. Therefore, TGF- represents a growth factor mechanism that not only can initiate bone regeneration but also can sustain long-term healing and bone regeneration, including bone remodeling of a maturing bone graft. TGF- stimulates angiogenesis and the production of fibronectin,

glycosaminoglycans, and collagen in the connective tissue. The most important functions of TGF-1 and TGF-2 seem to be the chemotaxis and mitogenesis of osteoblast precursors and the ability to stimulate their deposition of the collagen matrix for connective tissue wound healing and bone formation. Additionally, TGF- inhibits osteoclast formation and bone resorption, therefore favoring bone formation over resorption. This local connective tissue response to TGF- in vivo is strongly anabolic and leads to fibrosis and angiogenesis.

Platelet-derived Epidermal Growth Factor: PDEGF was discovered by Cohen in 1972 and was the first growth factor described. It stimulates epidermal regeneration, angiogenesis, promotes wound healing by stimulating the proliferation of keratinocytes and dermal fibroblasts, and enhances the effects and production of other growth factors.

Platelet-derived Angiogenesis Factor: PDAF has the capacity to induce vascularization in vivo. It stimulates vascular endothelial cells by direct or indirect actions, and it is involved in the process by which new blood cells invade devascularized tissue. Several cytokines and growth factors upregulate PDAF, including IGF-1, TGF- and , PDGF, basic fibroblast growth factor (bFGF), PDEGF and interleukin l (IL- l ). This factor is highly expressed by the induction of hypoxia.

Insulin-like Growth Factor-1: IGF-1 is a single-chain polypeptide hormone weighing 7,500 daltons. IGF-1 has 47% homology with insulin. It is thought of as secreted by osteoblasts during bone formation to increase numbers of osteoblasts and thereby accelerate bone deposition. Insulin-like growth factors are also deposited in bone matrix; when the bone matrix is resorbed, They each bind to a specific IGF cell-membrane receptor that excites kinase activity (formation of a high-energy phosphate bond) to a cytoplasmic signal protein and it stimulates cartilage growth, bone matrix formation, and replication of preosteoblasts and osteoblasts. IGF-1 may directly stimulate the cells it activates (autocrine factor) and increase the aIkaline phosphatase activity in osteoblastic cells. IGF-1 transcripts have been isolated from macrophages in wounds, suggesting that this growth factor may also act as a local messenger (paracrine factor). IGF-1 in combination with PDGF can enhance the rate and quality of wound healing.

Platelet Factor-4 PF-4 is a chemoattractant for neutrophils also released from alpha granules, which may be partially responsible for the initial influx of neutrophils into wounds. It also acts as a chemoattractant for fibroblasts and is a potent antiheparin agent3.

Fibroblast Growth factor (FGB) It stimulates angiogenesis, endothetlial cell proliferation, collagen synthesis, wound contraction, matrix synthesis, epithelialization.

Fibrin: In clinical use, calcium chloride and thrombin are added to PRP to activate the proteolytic cleavage of fibrinogen into fibrin. Fibrin formation initiates clot formation, which, in turn initiates wound healing. Cross-linking occurs as part of the clotting process and ensures random distribution of platelets and their growth factors throughout the wound. It serves as a scaffold for cell migration and entraps platelets. Fibrin clot stabilizes the early wound healing matrix.

MECHANISM OF PRP RELATED TO GROWTH FACTORS The growth factors secreted by the platelets attach only to cells that have receptors to accommodate them. These receptors are on the surface membrane of the target cell. The growth factor never enters the target cell; instead, it activates the membrane receptor, which has an intracytoplasmic portion and therefore is often termed a transmembrane receptor. Two adjacent transmembrane receptors are then brought within a critical distance of each other to activate dormant intracytoplasmic signal transducer proteins. A signal transducer protein then detaches from the transmembrane receptor and floats in the cytoplasm towards the nucleus. In the nucleus, transducer protein unlocks a specific gene sequence for a regulated cellular function, such as mitosis, collagen synthesis, osteoid production, etc. The significance of this process is that it explains why an exogenous application of growth factors, even in the highest concentration possible, cannot produce a sustained overreaction such as hyperplasia, a benign tumor or a malignant tumor. Growth factors are not mutagenic; they are natural proteins acting through normal gene regulation and normal wound-healing feed-back control mechanisms.

THE ROLE OF PLATELETS AND PRP IN BONE REGENERATION: The alpha granules contained in platelets, whether in a normal blood clot or in a PRP clot, begin degranulating within 10 minutes of clot development and secrete over 90% of their pre-packaged growth factors within 1 hour. The growth factors immediately bind to the transmembrane receptors of osteoprogenitor cells, endothelial cells, and

mesenchymal stem cells. The fibrin and fibronectin contained within the acellular portion of the clot and the vitronectin arising from the platelet alpha granules envelop the graft in an initial matrix. A cancellous cellular marrow graft, whether for a mandibular continuity defect, a sinus augmentation surgery, or a dental implant, is placed in a dead space filled with clotted blood. The dead space is hypoxic (PO2 of 5 to 10 mm Hg), acidotic (pH 4 to 6), and contains platelets, leukocytes, red blood cells, and fibrin in a complex network around the transferred osteocytes, endosteal osteoblasts, and marrow stem cells . The marrow stem cells, which are the primary bone-regenerating cells, normally exist in very small numbers (about 1 per 250,000 structural cells at age 35). Just outside the surgeon's periosteal-level closure, the tissue is normoxic (PO> of 45 to 55 mm Hg) at physiologic pH (pH 7.42) and contains a population of structural cells, healing-capable stem cells (also in very small numbers), and cut capillaries with clots and exposed endothelial cells. The initiation of bone regeneration starts with the release of PDGF, TGF- and IGF from the degranulation of platelets in the graft. The three isomeres of PDGF act as mitogens for osteoblast, endothelial cell, and mesenchymal stem cell proliferation. The two TGF isomeres accomplish a similar mitogenesis and angiogenesis but also promote osteoblastic differentiation of the mesenchymal stem cells. The VEGF promotes specific capillary ingrowth. Because of its increased concentration of platelets, the PRP thus initiates a greater and faster initial cellular response in the bone graft than the normal blood clot. The PDGF stimulates mitogenesis of the marrow stem cells transferred in the graft to increase their numbers by several orders of magnitude. It also begins an angiogenesis of capillary budding into the graft by inducing endothelial cell mitosis. The TGF- initially activates fibroblasts and preosteoblasts to initiate mitosis and increase their numbers as well as promoting their differentiation toward mature functioning osteoblasts . Continued TGF- secretion influences the osteoblasts to lay down bone matrix and the fibroblast to lay down collagen matrix to support capillary ingrowth. The IGF acts on the endosteal osteoblasts that line the trabeculae of grafted cancellous bone. Not to be overlooked is the meshed clot itself, which contains fibrin, fibronectin, and

vitronectin. These cell-adhesion molecules act as a surface matrix for the vascular ingrowth, cell proliferation, and cell migration occurring during this phase. This matrix will also act as the initial scaffold for osteoid production that will signal the transition to the next stage. By the third day, capillaries can be seen to penetrate the graft. By 17 to 21 days, the capillary penetration of the graft is complete and the osteoprogenitor cells have vastly increased in number. Thus, the first phase of bone graft healing occurs during the first 3 weeks and is characterized by capillary ingrowth and rapid cellular metabolism, proliferation, and activity. This initial flurry of cellular activity is the direct result of PDGF, TGF-, and IGF primarily, as well as some other growth factors. The ratio of these mesenchymal stem cells to structural marrow cells is about 1:100,000 when a person is a teenager, 1:250,000 at age 35, 1:400,000 at age 50, and 1:1,200,000 at age 80. The human organism relies on growth factors to rapidly increase the numbers of these cells and promote their activity during a time of repair and healing. The life span of a platelet in a wound, and the direct influence of its growth factors, is fewer than 5 days. The extension of healing and bone regeneration activity are accomplished by two mechanisms. The first is the increase and activation of marrow stem cells into osteoblasts, which then secrete TGF- and IGF into the osteoid matrix. The second and more dominant mechanism seems to be through the chemotaxis and activation of macrophages that replace the platelets as the primary source of growth factors after the third day. The macrophage is attracted to the graft by actions of PDGF and by any oxygen gradient between the graft dead space and adjacent normal tissue that is greater than 20 mm Hg. In fact, the graft's inherent hypoxia (5 to 10 mm Hg) establishes the oxygen gradient of 30 to 40 mm Hg adjacent to the normal tissues, which have a P02 of 45 to 55 mm Hg. As PDGF fades in influence, macrophage-derived growth and angiogenic factors take over (days 5 to 7). However, macrophage-derived growth factors and angiogenic factors may actually be identical to PDGF, only synthesized by macrophages. The marrow stem cells will secrete TGF- and IGF to continue self-stimulation of bone formation as an autocrine response.

By 4 weeks, the revascularized graft eliminates the oxygen gradient needed to maintain macrophage activity. Thus the macrophage leaves the area, no longer required by a graft that is now self-sustaining even though immature, with woven osteoid bone rather than mature lamellar bone. Thus, the platelets and PRP act in the early biochemical first phase of a three-phase bone regeneration sequence, when the pivotal role of setting the rate and amount of bone regeneration takes place. In the second phase of bone regeneration, i:e between 3 and 6 weeks, the osteoprogenitor cells have proliferated and differentiated sufficiently to produce osteoid. Their production of osteoid consolidates the graft and forms a union to the adjacent native bone. During this time the completed capillary ingrowth matures by developing adventitial supporting cells around the vessels, making them much more capable of withstanding instability and mild function. The oxygen that these vessels supply to the graft reverses the hypoxia and thus down regulates the macrophage so that the wound does not "overheal" into a hyperplasia. Beginning at the 6th week, the osteoid undergoes an obligatory resorption-remodeling cycle. The weak and elastic osteoid is resorbed by osteoclasts, which release BMPs, ILG1, and ILG2, and these in turn induce adjacent osteoblasts and mesenchymal stem cells to differentiate and produce a more mature replacement bone that contains lamellar archietecture and Haversian systems not present in the osteoid. The third phase of bone regeneration continues throughout the lifetime of the graft as it settles into the normal resorption-remodelling turnover rate of the skeleton (about 0.7% per day). This is seen clinically and radiographically by the formation of mineralized dense bone.

( Pictures from Dental and Craniofacial Applications of Platelet Rich Plasma R. Marx and Arun Garg)

ADVANTAGES OF PRP:

Safety - Autologous blood product, with no risk of infectious disease transmission or clerical errors, thus making it a safe product

Convenient to procure - It is non-invasive (other than phlebotomy ) and painless procedure. No requirement for anaethesia, and outpatient (dental office/treatment room) performance. No time consuming visits to the blood bank for pre-donation. Sequester is done in the immediate preoperative period, and utilized perioperatively

Accelerate endothelial, epithelial, and epidermal regeneration Stimulate angiogenesis Enhance collagen synthesis Promotes enhanced soft tissue wound healing Decreased dermal scarring Provides for an immediate surgical hemostatic agent that is biocompatible, effective and safe with enhanced hemostatic response

Reverse the inhibition of wound healing caused by glucocorticoids High leukocyte concentration adding an antimicrobial effect The native fibrinogen concentration imparts a gelatinous adhesive consistency, for ease of surgical application

When mixed with crushed coral or crushed bone fragments it forms a putty ideal for packing or structural reconstructions (as in mandibular reconstructions, maxillofacial procedures, dental implants) and actually improves handling characteristics of bone grafts.

Augmented rate of extracellular matrix deposition, resulting in earlier wound closure.

CLINICAL RESULTS WITH PLATELET-RICH PLASMA: 1. PRP with Autogenous bone Marx et al (1998): Forty-four continuity bone grafts to the mandible, placed without PRP, were assessed against 44 grafts placed with PRP at 2-month, 4-month, and 6- month maturity intervals with panoramic plain-film radiographs. Independent investigators consistently assessed grafts with PRP growth factor additions to be more mature. They found platelet sequestration ability of the process and quantified the concentration as 338% of baseline platelet counts. Using a graft maturity index, whereby each investigator assessed the radiographic age of the against its actual age,

investigators assessed PRP grafts to be 2.16 times more mature at 2 months, 1.88 times more mature at 4 months, and 1.62 times more mature at 6 months. These differences were statistically significant (P = .001). Fennis et al (2001) performed a similar study in a goat model. Radiographs taken at 3, 6 and 12 weeks were blindly evaluated. In 4 of the 15 comparisions, the group with PRP showed statistically superior healing. Aghaloo et al (2002) used a rabbit model to evaluate autologous bone graft healing with PRP. In each of 15 rabbits, 4 cranial bone defects and grafted with either

autogenous bone, autogenous bone with PRP, or PRP alone or were left empty as a control. The study failed to show any significant benefit when PRP was used. Jakse et al (2003) performed bilateral sinus lift procedures on 12 sheep. Cancellous iliac crest bone was used alone in one sinus and with PRP in the other. They concluded that their results show a regenerative capacity of PRP of quite low potency. Butterfield et al (2003) performed a very similar study using the rabbit model. Their study revealed that addition of PRP had no significant effect on bone formation.

2. PRP with Anorganic Bone Mineral Kim et al (2001) grafted surgically created cranial defects of rabbit with Bio-Oss with or without PRP. Digitized plain films and computed tomography scans both showed significantly greater with the use of PRP at both 1 and 2 months. Aghaloo et al (2002) created 4 rabbit cranial defects in each of 15 rabbits. They were grafted with either Bio-Oss, Bio-Oss with PRP, or autogenous bone or were left empty as a control. Histomorphometric evaluation showed that the addition of PRP significantly increased the % of bone formation over that of Bio-Oss alone at all three time periods(1, 2 and 4 months). However, in this study, the autogenous bone was still significantly better than either Bio-Oss or Bio-Oss with PRP. Furst et al (2003) performed bilateral sinus lifts on 12 minipigs, using Bio-Oss alone on one side and Bio-Oss mixed with PRP on the other. They concluded that when combined with hydroxyapatite, PRP was not demonstrably superior to HA alone. Ouyang and Qiao (2006) evaluated the effectiveness of PRP as an adjunct to bovine porous bone mineral (BPBM) graft in the treatment of human intrabony defects. They found that the treatment with a combination of PRP and BPBM led to a significantly favorable clinical improvement in periodontal intrabony defects compared to using BPBM alone. Further studies are necessary to assess the long-term effectiveness of PRP, and a larger sample size is needed. You et al (2007) studied the influence of platelet-rich plasma (PRP) used as an adjunct to Bio-Oss for the repair of bone defects adjacent to titanium dental implants. Their results indicate that when PRP is used as an adjunct to Bio-Oss in the repair of

bone defects adjacent to titanium dental implants, PRP may decrease periimplant bone healing.

3. PRP with Organic bone Matrix Shanaman et al (2001) performed alveolar ridge augmentation on 3 patients, primarily using freeze-dried demineralised bone. The grafts were mixed with PRP and were protected with barrier. The authors concluded that the addition of PRP did not appear to enhance the quality or quantity of new bone formation over that reported in comparable guided bone regeneration studies without PRP. Kim et al (2002) placed dental implants into the iliac crest of dogs and created bone defects around the most superficial implant threads. These defects were grafted with freeze-dried demineralized bone powder, with or without the addition of PRP. More direct bone-to-implant contact was seen in the group with PRP. Palmisano et al (2002) used the canine model to investigate the effect of PRP when combined with mineralized bone. They showed no benefit with the addition of PRP. Harris et al (2003) used the canine model to investigate the effect of PRP when combined with mineralized bone. They showed no benefit radiographically or histologically with the addition of PRP. Dudziak and Block (2003) extracted teeth and performed an alveolectomy on 6 dogs. 12 weeks later, ridge augmentation was performed with mineralized bone with either PRP or PPP. When the specimens were evaluated histologically 12 weeks later, neither group showed evidence of osteoconduction. Dori et al (2007) clinically compare treatment of deep intra-bony defects with natural bone mineral(NBM)+PRP+GTR with NBM+GTR. Their results have shown that the use of PRP has failed to improve the results obtained with NBM+GTR.

4. PRP when used alone When PRP has been placed into bone defects without other grafting materials, the results are again inconclusive.

Anitua (1999) reported improved epithelization and bone density when PRP was placed into extraction sockets. Mancuso et al (2003) showed a lower rate of alveolar osteitis, less pain, and more dense radiographic bone healing when PRP was placed into third molar extaction sockets. Farrell et al (2002) found no enhanced bone formation when inferior border mandibular defects in dogs were treated with PRP. Aghaloo et al (2002) similarly showed no benefit for the use of PRP alone in rabbit cranial defects compared with otherwise untreated defects. Zechner et al (2003) and Schlegel et al (2003)
3

evaluated PRP alone when

inserted before implant placement in pigs and showed a slight increase in initial osseointegration. Papli and Chen (2007) compared the treatment of infrabony defects by an intralesional graft of PRP to guided periodontal regeneration (GPR) using a bioabsorbable barrier membrane (MEM) over a 52-week period. Their case series suggested that an PRP graft achieves a similar CAL gain and PD reduction to GPR using an MEM over a 52-week period. A larger, controlled clinical trial is needed to evaluate further the efficacy of autologous platelet-rich plasma for the treatment of infrabony defects. CONCLUSION : Todays understanding of bone science recognizes the pivotal role of growth factors in clinical bone grafting success. PRP is seen as an available and practical tool for enhancing the rate of bone formation and the final quality of bone formed. Undoubtedly, all clinicians involved with bone grafting have high hopes that PRP will eventually prove to be of great benefit in bone graft healing. Much is still unknown about PRP, and an adequate body of research should precede a widespread use of this adjunctive material. The literature review demonstrates a lack of scientific evidence to support the current use of PRP in combination with bone grafts during augmentation procedures. However at this early stage of investigation, the results are far from conclusive. While it has been recognized that these types of studies represent the starting point, they are not definitive. The conflicting results in todays literature make it overwhelmingly evident

that more research is needed before evidence-based surgeons can feel confident in recommending this procedure to their patients.