CONTENTS 1. INTRODUCTION 2. PLATELET BIOLOGY 3. HISTORY OF PRP 4. GROWTH FACTORS AND THEIR ROLE IN WOUND HEALING 5.

PLATELET-RICH PLASMA AND THIER PROCUREMENT TECHNIQUES 6. BASIC COMPONENTS OF PRP 7. APPLICATIONS OF PRP 8. ADVANTAGES OF USING PRP 9. PLATELET GROWTH FACTORS 10.BONE REGENERATION WITH PRP 11.CLINICAL APPLICATIONS 12.CLINICAL RESULTS WITH PLATELET RICH PLASMA 13.RISKS OF USING PLATELET RICH PLASMA GEL 14.CONCLUSION

INTRODUCTION
Tissue engineering is the emerging field of science aimed at developing techniques for the regeneration and reconstruction of new tissues to replace the damaged ones. The term Tissue Engineering was coined originally to denote the construction in the laboratory of a device containing viable cells and biologic mediators (e.g. growth factors and adhesives) in a synthetic or biologic matrix that could be implanted in patients to facilitate the regeneration of particular tissues.

After surgery, platelets begin to form a stable blood clot, releasing a variety of growth factors that induce and support healing and tissue formation. (Antoniades HN, Williams LT,Gonshor 2002,Dugrillon A, Eichler H, Kern S, Klter H 2002) A recently developed procedure can be used to create platelet-rich plasma (PRP), a concentrated suspension of growth factors that has been demonstrated to induce healing and regeneration of tissues, including those in the periodontal area (Camargo PM, Lekovic V, Weinlaender M, Vasilic N, Madzarevic M, Kenney EB 2002)

Platelet Morphology The identification of platelets as a class of blood corpuscles was described by Bizzozzero (1882)The importance of platelets for the formation of a hemostatic plug was first reported by Eberth and Schimmelbusch (1888). The platelets are very small bodies 2-4 m, consisting of cytoplasm encased within a membrane. Platelets are non-nucleated structures. Platelets contain mitochondria and Golgi apparatus. Several classes of granules are seen in the platelet viz, Alpha granules Dense granules Glycogen granules

Alpha granules on activation secrete a number of compounds. These granules contain a proteineous substance called thrombospondin, which binds platelet membrane receptors with fibrinogen or another platelet and probably thus paves way for
1.Mutual fixation of platelets 2.Fixation of platelet with blood clot. They have no nucleus for replication and will die off in five to nine days. Earlier, they were thought to contribute only for hemostatic process, but recently it has been proposed to actively extrude the growth factors involved in wound healing.

Laser Scan Microphotograph Showing Fibronylysis of Blood Clot

Whole Blood Clot

Peripheral blood smear. Scattered Platelets correlating to a peripheral blood platelet count of 225,000 per l

Complete growth factors are not released by platelet disruption or fragmentation. Instead, these growth factors are actively extruded through the cell membrane where histones and carbohydrate side chains are added to complete their unique chemistries and make them active growth factors (Robert E Marx, Eric R Carlson, Ralph M Eichtaedt, Steven R Schimmele, James E Strauss 1998).

GROWTH FACTORS

Growth factors are peptides [short sequences of amino acids], which transmit signals between cells and thereby modulate their activity. Growth factors have a very short half-life and are in very small amounts. A growth factor alters cell behavior at very low concentrations and operates over a short range usually micrometers or millimeters. Growth factors regulate cellular events in wound healing, such as proliferation, differentiation, chemotaxis and morphogenesis of tissues and organs. (Giannobile WV.1996, Carlson NE, Roach RB Jr. 2002, Schliephake H 2002). Growth factors may act in an autocrine, paracrine or endocrine manner. They are deposited in the extracellular matrix and are then released during matrix degradation.

Role of Growth Factors in the process of Wound Healing The process of wound healing can be divided into 3 different stages: 1. 2. 3. Biochemical activation, Cellular activation, and Cellular response.

1.Biochemical activation involves the translation of mechanical injury into biochemical signals that can be understood by the body. The trigger that starts the cascades is the Hageman factor found in serum. When injury causes disruption of the microcirculation, plasma comes in contact with tissue proteins and the basement membrane. This activates the Hageman factor and circulating platelets. Finally, the activated Hageman factor in turn amplifies the initial response and results in cellular Activation.

2. The activation of clotting cascade produces 1) fibrin which helps in hemostasis, and 2) thrombin that causes the maximal release of platelet alpha () granules. The complement cascade produces many biologically active molecules including C5a, a potent chemoattractant for neutrophils and monocytes that is also important in wound repair. The kinin cascade results in the production of bradykinin

that causes microvascular dilatation at the wound periphery. The fibrin degradation products that result from the enzymatic breakdown of fibrin are themselves biologically active molecules that can cause monocyte migration and vasodilation. 3. The Cellular Activation stage results in the influx of cells into the wound. The first cellular response involves neutrophils, monocytes, and platelets. Platelets accumulate at the wound site in response to the initial injury, and in response to thrombin, release - granules that contain locally acting growth factors. These factors signal the local mesenchymal and epidermal cells to migrate, divide, and increase their collagen and glycosaminoglycans synthesis

Platelet Rich Plasma

Platelet rich plasma (PRP) was first introduced to Oral and Maxillofacial community by Whitman et al (1997). However, Robert Marx (1998) is considered as a pioneer worker in the field of PRP technology and its uses in bone regeneration.
PRP is a component of blood in which the platelets are concentrated in a limited volume of plasma. This autologous plasma is a rich source of growth factors and its application has been reported as an effective way to induce tissue repair and regeneration. This led to the development of autologous platelet gel PRP to be used in various surgical fields such as Head and Neck surgery, Neurosurgery, General Surgery, Oral and Maxillofacial Surgery, and Periodontics . Platelet-rich plasma procurement techniques The following techniques are employed to procure PRP: 1.Use of General-Purpose Cell Separators 2.Use of Platelet-concentrating Cell Separators

1.Use of General-Purpose Cell Separators

The clinical procedure for obtaining autologous PRP requires the use of an ultracentrifuge and gradient density cell separator, which can sequester and concentrate platelets from 450ml of blood. (Kassolis JD, Rosen PS, Reynolds MA 2000)
2. Use of Platelet-concentrating Cell Separators Recent advanced technologies permit the procurement of PRP using smaller volumes of blood, increasing the platelet concentrations and avoiding the need for RBC and PPP reinfusion as done when General-Purpose Cell Separators are used. (Lozada JL, Caplanis N, Proussaefs P, Willardsen J, Kammeyer G 2001). Two additional systems are now available commercially for office use by dental practitioners 1. The Platelet Concentrate Collection System [PCCS] (3i Implant Innovations, Palm Beach Gardens, Florida) and 2. The Curasan PRP kit (Curasan, Pharma Gmbh AG, Lindigstrab, Germany). Published reports (Weibrich G, Kleis WK, Appel TR, Ptzsch B, Mller J, von Lindern JJ, Berge SJ, Reich RH. 2002) indicated that these systems have greater ease of handling and shorter preparation time.

The Curasan Prp Kit (Gernot Weibrich, Wilfried K.G. Kleis ,Gerd Hafner 2002)

Use of the color-coded Curasan kit is described here to demonstrate the ease of preparing a small amount of PRP for use in reconstruction of periodontal and osseous defects, augmentation of extraction sockets, and connective tissue grafting procedures. Recent publications have indicated that PRP prepared from 8 to 10 mL of whole blood is sufficient for periodontal regenerative therapies. (Camargo PM, Lekovic V, Weinlaender M, Vasilic N, Madzarevic M, Kenney EB 2002, Lekovic V, Camargo PM, Weinlaender M, Vasilic N, Kenney EB. 2002, Weibrich G, Kleis WK, KunzKostomanolakis M, Loos AH, Wagner W. 2001 ).
Consists of the following parts: 1. A multifly set. 2. Two multiadaptors. 3. One 8.5 ml CPDA monovette. 4. One 9 ml monovette 5. One 7.5 ml monovette. 6. One 1ml syringe

1.

The blood sample is drawn into a citrated tube (Fig. 3).

If more than 8 mL is needed (e.g., for larger defects), more than one tube of blood should be drawn. 2. The sample tube is then spun in a standard centrifuge for 10 minutes at 2400 rpm (Fig. 4) to produce PPP.

3. The PPP is taken up into a syringe with a long cannula and an additional air-intake cannula (Figs. 5a and 5b).

4. A second centrifugation (15 minutes at 3600 rpm) is performed to concentrate the platelets. The second supernatant is also taken up by a long cannula and an air-intake cannula (Fig. 6).

5. For each 8 mL of blood, the volume of supernatant is about 0.60.7 mL

6. This is the PRP, to be used for the surgical procedure (Fig. 7).

At the time of the application, the PRP is combined with an equal volume of a sterile saline solution containing 10% calcium chloride (a citrate inhibitor that allows the plasma to coagulate) and 100 U/mL of sterile bovine thrombin (an activator that allows polymerization of the fibrin into an insoluble gel, which causes the platelets to degranulate and release the indicated mediators and cytokines). The result is a sticky gel that will be relatively easy to apply to the surgical defects. The PPP can be stored for use as a protective barrier over the wound (Fig. 7).

PRP Preparation Using PCCS System

Step 1: Remove packaging post from PCCS II tube before filling blood port. Load blood (5ml of citrate anticoagulant and 55ml of whole blood) into blood port. Place into centrifuge. Counter balance centrifuge with 60ml of sterile saline.

Step 2: Spin for 12 minutes at 3200rpm

Step 3: Connect volume/depth gauge to blood port. Push down until it stops. This traps the platelet concentrate (10% of the starting volume) in the middle section.

Step 4: Extract Platelet Poor Plasma (PPP), by connecting the 30ml syringe to the plasma port and withdraw.

Step 5: Shake tube vigorously for 30 seconds to suspend platelets

Step 6: Remove center post of volume/depth gauge by unscrewing and then pulling out. Connect 10ml syringe and withdraw PRP from middle section.

On analysis of these 2 methods, the PCCS method is better, not only on the basis of results, but also because of the ease of handling clinically. The Advantages Includes The end products have a higher platelet count, which is considered a criterion for the quality of PRP. The platelet collection efficiency is higher, so the surgeon can use more of the drawn thrombocytes. The volume of PRP produced by the PCCS method is sufficient for most dentoalveolar procedures, using Curasan method, up to 10 monovettes are required to produce an equal volume of PRP, even more would be necessary to achieve the same quality of platelets. This would increase the number of working steps during PRP production. The preparation time needed is shorter using PCCS than using the Curasan method. The PCCS is a needle free system. Once the doctor has drawn the blood, there is no risk of injury to the staff from contaminated needles. PRP preparation using PCCS is more standardized and needs less training, diminishing the possibility of mistakes by staff.

The PCCS system has been developed and licensed as a therapeutic instrument, whereas the components of the PRP kit are considered as diagnostic tools.

Why there is decrease of platelets in Curasan PRP kit? (Gernot Weibrich, Wilfried KG Kleis, Gerd Hafner 2002) One major difference is in the material from which the monovettes are made. In PCCS Monovettes are made up of polyvinyl chloride with some additional ingredients. In Sarstedt monovette of Curasan PRP kit Monovettes are made up of polyethylene and polypropylene. Some of the missing platelets might adhere to the inner wall of the monovettes. For this reason, BARTHELMAN 1969, Used siliconized glass tubes and needles to prepare 1.5ml of PRP out of 30 ml of whole blood. This first centrifugation was for 10 minutes at 100 rpm, and second was at 3000 rpm for 15 minutes. Resulting collection efficiency was 72 %, which equals that of PCCS system. Even using siliconized glass tubes, some platelets were missing [2%]. This would have been damaged probably during the preparation process.

As the blood is centrifuged, it is separated into three basic components (Samuel E. Lynch, Robert J Genco, Robert E. Marx Quinte 1999) From the least dense to the most dense they are 1. Platelet poor plasma. 2. Platelet rich plasma-sometimes also called as buffy coat 3. Dense red blood cells. 1. Platelet poor plasma - Top level of serum. - Contains Autologous fibrinogen - Poor in platelets. - Acellular plasma. - Accounts for about 200ml of volume. Usually returned back to the patients

2. Platelet rich plasma - Second level of serum. - The plasma with a concentrated number of platelets and white blood cells. - Contains autologous fibrinogen. - Accounts for 70ml of volume.

Demarcation line (Dietmar Sonnleitner, Peter Huemer, Daniel Y Sullivan 2000) It is a whitish layer on the top of the RBC cell fraction. Rich in platelets and WBCs. Red blood cells/blood cells Red colored fraction of the second level, containing mainly, - Packed red blood cells. - Platelets. Usually, the upper 6-7mm is very rich in fresh, young platelets. Below this level, platelet concentration decreases.
.

Platelet poor plasma Platelet rich plasma

PRP smear. Dense platelet concentration Correlating to a platelet count of 1,400,000 Per l in a 5 ml volume.

Thus both PPP and PRP are plasma fractions. They contain 1.Fibrinogen. 2. Clotting factors. In order to maximize the platelet concentration, a point is marked 6-8mm below the dividing line between the 2 phases i.e., blood cell component and the serum component. The entire blood cell component and serum component up to this point is pipetted out and into a fresh sterile vacurette without citrate. The pipetted material is again centrifuged for 15 minutes at 200 rpm and top yellow fragment [serum component] is removed. The remaining substance, approximately 0.5ml in quantity is the available platelet concentrate [PC]. The second centrifugation provides fraction of between 800011000 platelets in upper serum component. The first 1-3ml of RBC layer contains the larger and more recently synthesized platelets. Therefore, this layer of RBCs is included in the PRP. This will impart the red tint, to the otherwise straw-colored PRP.

Application of PRP
Firstly the clinical application of PRP for grafts requires initiating the coagulation process by using a mixture of 10 ml of 10 % calcium chloride mixed with 10,000 units of topical bovine thrombin.(Samuel E . Lynch, Robert J Genco, Robert E. Marx Quinte 1999). The ratio of PRP to calcium chloride activator is 10: 1(Tara L Aghloo, Peter K Moy, and Earl G Freymiller 2002). The specific protocol for PRP application requires an individual 10 ml syringe for each mix (Samuel E. Lynch, Robert J Genco, Robert E. Marx Quinte 1999) Each mix draws, in order 6ml of PRP 1ml of calcium chloride thrombin mixture. 1 ml of air to serve as mixing bubble.

The syringe is agitated for 6-10 seconds to initiate clotting.

The PRP gel developed by this manipulation is added to the graft in several mixes. A sterile new syringe is used at each mix because even a minute introduction of calcium chloride and thrombin into the PRP container will coagulate the remaining PRP.

The introduction of one or two mixes of PRP into the graft material before it is placed in the recipient bed distributes the PRP and therefore, its growth factors through out the graft. Additional mixes are layered on the graft surface once it is in place. The fibrin formation of the PRP process serves as fibrin glue and binds the otherwise loose cancellous marrow together, which assists the surgeon in sculpting the graft. The fibrin network acts as a scaffold for osteoconduction throughout the graft after the growth factors within the platelets initiate osteogenesis.

A second option (Dietmar Sonnleitner, Peter Huemer, and Daniel Y Sullivan 2002) is to mix PRP with the fibrinogen component alone in a 1: 1 ratio. This mixture is allowed to flow onto a glass plate or a small flat cup and is consolidated by coating with thrombin. This creates a flat, membrane like structure. It is elastic and silicon like in consistency and is shaped with the scalpel. This can be used to cover fenestrations and small defects and to fill small bone cavities.

Another method of application (Dean Whitman, Ronald L Berry 1998) is to compact the graft material into a series of 3ml syringes by alternately adding the graft to the barrel of the syringe and compressing with the plunger until the syringe is full of compacted bone to the 2.5 ml level. Once the recipient site is deemed ready for the graft, the platelet gel is prepared. Approximately, 1ml of the gel is introduced into the barrel of the syringe, the plunger is replaced and the platelet gel is forced through the graft.

Using a no.10 scalpel blade, the end of the syringe is cut off and the graft is expressed from the syringe by again compressing the plunger. The bone graft that is prepared in this manner is cohesive enough to be easily compacted into the graft site using hand instrumentation. After the graft material is compacted into its final position more platelet gel is applied over it before soft tissue closure.

Advantages of using PRP

- Safe eliminates risks of clerical errors (no risk of using another patient's blood by mistake, uses autologous sources). - Convenience for patient- no visit to blood bank is necessary and it is easily and readily available. - Improved support for tissue handling. - More rapid mineralization of collagen in bone repair and grafted sites. - Earlier stability of graft sites. - Promotes enhanced soft tissue wound healing. - Ease of surgical application of bone grafts

- Growth factors and cytokines are brought to the site in a manner that would not occur with fibrin glue. - Provides for an immediate surgical hemostatic agent that is biocompatible effective and safe - Promotes a firm seal in minutes of application. - PRP is reabsorbed by the body within few weeks duration. - No potential for disease transmission as it is autologous.

There are over 30 known platelet growth factors to date.

Some of these are: 1. Platelet Derived Growth Factor (PDGF) 2. Transforming Growth Factor (TGF) 3.Insulin Growth Factors (IGF) 4.Epidermal Growth Factor (EGF) 5.Platelet-Derived Angiogenesis Factor (PDAF) 6.Vascular Endothelial Growth Factor (VEGF) 7.Fibroblast Growth Factor (FGF)

1. Platelet Derived Growth Factor-PDGF (Eduardo Anitua 1999, Marx RE 2001, Samuel E. Lynch, Robert J Genco, Robert E. Marx Quinte 1999) PDGF is a highly dimeric glycoprotein of 30 KD consists of two disulfide bonded poly- peptides [A&B] encoded by different genes (Schliephake H 2002) So named because it was first found in platelets, where it is stored in alpha granules, but it has also been isolated from a variety of tissues and cells including monocytes, macrophages, fibroblasts, endothelial cells and bone matrix (Eduardo Anitua 1999). It has a dimeric structure formed by 2 amino acid chains named A and B. These chains have a similarity in their structure of 60%; chain A is formed of 121 amino acids; while chain B is formed of 125 amino acids (Eduardo Anitua 1999).

Two genes are responsible for the encoding of PDGF: Chain A of the peptide is encoded in chromosome 7 and chain B is encoded in the long branch of chromosome 22. PDGF-BB and PDGF-AB are systemically circulating isoforms contained in alpha granules of platelets from where they are released after adhesion of platelets to injured sites of vessel walls.

There are approximately 0.06ng of PDGF per one million platelets, [or] stated in other words, there are 6 x 10-17 gm of PDGF or about 1200 molecules of PDGF , in every individual platelet (Marx RE 2001). Actions of PDGF (Schliephake H 2002) Growth and development It plays an important role in the development of many tissues and organs in growing embryo. Also involved in the development and growth of 1. CNS. 2. Myoblasts. 3. Blood vessels.

4. Mesangial cells of kidney glomeruli.

5. Alveolar muscle cells of the lung.

Disruption of PDGF signaling leads to perinatal lethality.

Biologic effects PDGF stimulates specific target cells by binding to the cell surface receptors .This activates a cascade of events that leads to cellular proliferation by accelerating cell recycle and inducing quiescent cells into the proliferation phase of cell cycle. The mitogenic effect of PDGF is neutralized by TGF beta 1. In vitro, PDGF- AA & BB - enhance proliferation of multiple types of bone cells, i.e. both osteoblasts and osteoclast. PDGF increases the production of osteopontin but decreases the production of bone sialoprotein and osteocalcin in bone cells and also reduces alkaline phosphatase activity and mineralization particularly after long-term exposure. PDGF stimulates bone cell migration in dose dependant manner, thereby contributing to recruitment of bone cells during remodeling and repair.

Uses in periodontal reconstruction (Samuel E. Lynch, Robert J Genco, Robert E. Marx Quinte 2002, .Eduardo Anitua 1991): - Mitogenesis increase in the number of healing cells. - Angiogenesis generating new capillaries. - Up regulation of other growth factors and cells resulting in promotion of fibroblastic and osteoblastic functions. - Increases the rate of proliferation of stem cells.

2. Transforming Growth Factor- Beta [TGF-]

Transforming growth factor beta is a term applied to a superfamily of growth and differentiating factors, of which bone morphogenic proteins are members.

Transforming growth factor beta has a dimeric structure formed by two subunits of 112 amino acids. It has an origin in an extracellular proteolysis of a precursor containing 391 amino acids.It has a total weight of 25,000 Daltons. A gene located in the long branch of chromosome 19 is responsible for its synthesis. This factor has 3 different structures: (Eduardo Anitua 1999)
TGF- beta 1,TGF- beta 2,TGF- beta 3

TGF beta 1 structure is found abundantly in - Platelets. - Lymphocytes. - Neutrophils. TGF - beta 2 is found mainly in - Bone extracts. - Platelets. - Lymphocytes. - Neutrophils Actions (Schliephake H 2002) Growth and development: Loss of TGF beta activity or its receptors results in increased perinatal lethality. Important for the development of nervous system and plays a prominent role in regulation of neuronal precursors and neuronal differentiation

.TGF beta also forms substrates with glycosaminoglycans of different composition and is an important signal in regulation of integrins, thus affecting cellular behaviour in adhesion, aggregation and migration
In soft tissue healing, TGF beta acts as a fibrogenic factor that causes wound Contraction.

When released by platelet degranulation, or actively secreted by macrophages, they act on cells such as fibroblasts, marrow stem cells and pre-osteoblasts. TGF beta represents a growth factor mechanism that can only initiate bone formation but also can sustain long term healing and bone regeneration, including bone remodeling of maturing bone graft. The most important functions of TGF beta 1 and TGF beta 2 seem to be chemotaxis and mitogenesis of osteoblast precursors and the ability to stimulate the deposition of collagen matrix for connective tissue wound healing. Additionally, TGF beta inhibits osteoclasts formation and bone resorption, therefore favoring bone formation over resorption. These factors also favor bone formation by enlarging the rate of stem cell proliferation.

4. Insulin Like Growth Factor [IGF] (Schliephake H 2002, Samuel E. Lynch, Robert J Genco, Robert E. Marx Quinte 1999) Insulin like growth factors is single chain peptides that exist in two isoforms i.e. IGF I - 70 amino acids. IGF - II 67 amino acids.

These are usually thought of as growth factors secreted by osteoblasts during bone formation to increase numbers of osteoblasts and thereby accelerate bone deposition.
Both IGF I & II are relatively small proteins, with molecular masses of 7.5 KD and 7.7 KD respectively.

IGFs are involved in development of large number of organs such as prostate, mammary gland spleen, lung, brain, kidney, thymus and teeth. IGF is also important for skeletal growth and for skeletal mass maintenance. The presence of IGFs in the platelets would be expected to act on precursors of osteoblasts and on endosteal osteoblasts, which are the cells that produce the initial phase I bone in bone grafts. IGFs are therefore mitogenic to osteoblasts lineage cells and are also stimulators of bone formation from existing differentiated osteoblasts.

5. Cementum-Derived Growth Factor (CGF) Cementum-derived growth factor, a newly characterized growth factor, appears to be found exclusively in cementum. This growth factor has been shown to be mitogenic for both periodontal ligament fibroblasts and gingival fibroblasts. It has been suggested that CGF may promote the migration and growth of progenitor cells present in adjacent structures to the dentin matrix and participates in their differentiation into cementoblasts.

Bone Regeneration With PRP (Robert E Marx, Eric R Carlson, Ralph M Eichtaedt,
Steven R Schimmele, James E Strauss 1998, Samuel E . Lynch, Robert J Genco, Robert E. Marx Quinte 1999) A cancellous cellular marrow graft is placed in 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 , red cells and fibrin in a complex network around the transferred osteocytes, endosteal osteoblasts, and marrow stem cells Just outside the surgeons periosteal level closure, the tissue is normoxic at physiologic pH of 7.42 and contains a population of structural cells , healing-capable stem cells and cut capillaries with clot

The initiation of bone regeneration starts with the release of PDGF, TGF beta and IGF from the degranulation of platelets in the graft. The PDGF stimulates mitogenesis of the marrow stem cells transferred in the graft to increase their numbers. It also begins an angiogenesis of capillary budding into the graft by inducing endothelial cell mitosis. The TGF beta initially activates fibroblasts and prefibroblasts to mitose and increase their numbers, as well as promoting their differentiation towards mature functioning osteoblasts. Continued TGF beta secretion influences the osteoblasts to lay down bone matrix and the fibroblast to lay down collagen matrix to support capillary ingrowth. These activities begin immediately on wound closure

By third day, capillaries are seen to penetrate the graft.

Complete capillary regeneration of the graft is seen by day 14 to day 17.This initial flurry of cellular activity, though it is also the result of some other growth factors, it is primarily the direct result of PDGF and TGF beta. Chemotaxis and activation of macrophages, which replace the platelet as the primary source of growth factors after third day.
The macrophage is attracted by the actions of PDGF and by any oxygen gradient [between the graft dead space and adjacent non toxic tissue] greater than 20 mm Hg. As the influence of PDGF fades out macrophage derived growth factor and angiogenic factors take over [5 to 7 days].

2. Formation Of Bone

The initial bone formation arises from the endosteal osteoblasts that line the cancellous bone surfaces. These cells survive transplantation because of their surface location, which allows them to absorb nutrients directly until revascularization takes place.
Because these are already differentiated osteoblasts, they begin osteoid formation directly on the cancellous bone surface while the transplanted stem cells undergo PDGF and TGF beta directed mitosis and guided osteoblasts lineage differentiation. This initial bone is disorganized woven bone with no haversian systems and little structural integrity termed as phase I bone and develop over the first 4 weeks of the graft. The phase I bone will then begin an obligatory resorption replacement sequence, which will eventually lead to a mature bone with lamellar architecture and haversian systems termed phase II bone. This mature bone, with a developed endosteum and periosteum, is a self-maintaining bone of full structural integrity

3. Maturation of regenerated bone The actual maturation of the graft from a disorganized woven bone into a mature lamellar bone with haversian systems involves IGF and bone morphogenetic proteins. As bone matrix is formed and then mineralized by osteoblasts, IGF and BMP are laid down within the bone matrix. These acid insoluble proteins are then released by the osteoclastic resorption of normal bone remodeling, which progresses at a rate of 0.7 % per day in normal bone but may occur as rapidly as 5 % to 8 % per day in a maturing bone graft. The released BMP and IGF link bone resorption to new bone formation by acting on adjacent stem cells and preosteoblasts to induce their proliferation and differentiation into functioning osteoblasts, which actively secrete bone matrix. Thus, the graft cycle progresses from a cellular transplant, which is placed in a complex biochemical environment, to a mature functional bone, which is self-maintaining through the normal resorption remodeling cycle

Clinical Applications (Marx RE 2001)

1. Augmentation grafting of the maxillary sinus. 2. Ridge preservation. 3. Extraction sockets.

4. Mucogingival grafts.
5. Marginal tissue recession root coverage. 6. Periodontal defect. 7. Immediate implant placement. 8. Failing implants. 9. Coating implants and osteotomy sites. 10. Particulate and block grafts.

11. Biological membranes and sealants

Clinical Results With PRP

The first clinical dental results with PRP were reported by Marx and others in 1998, who used PRP to improve graft incorporation in mandibular reconstructions in patients who had received cancellous bone marrow grafts after tumour removal. Their data strongly suggested that adding PRP to bone grafts accelerated the rate and degree of bone formation.
Anitua 1999 studied 20 healthy patients for whom an extraction was indicated because of a non treatable tooth with vertical fractures or severe periodontal disease and who were contemplating subsequent implant placement . The patients received a mixture of autologous bone and PRP, whereas the control group received only autologous bone. Those who received PRP demonstrated much better epithelialization and compact mature bone with well-organized trabeculae. In 2000, Kassolis and others used PRP with freeze-dried bone allograft for sinus elevation or ridge augmentation (or both) for 36 implant placements. On histological evaluation of the biopsy specimens12 months later, numerous areas of osteoid and bone formation were observed around the freeze-dried bone allograft particles, with no evidence of inflammatory cell infiltration.

De Obarrio and others (de Obarrio JJ, Aruz-Dutari JI, Chamberlain TM, Croston A 2000) incorporated PRP into a combination technique involving bone allograft and guided tissue regeneration as periodontal therapy for intrabony defects in humans. They observed significant gain in clinical attachment and filling of the treated defects Petrungaro in 2001 published a case series in which PRP, sub epithelial connective tissue grafts and collagen membranes were used to cover gingival recessions. The therapy was successful in all cases.

In 2002, Lekovic and others compared a combination of bovine porous bone mineral (BPBM), PRP and guided tissue regeneration with the combination of PRP and BPBM for the treatment of intrabony defects in humans. The group determined that the combination of PRP and BPBM provided additional regenerative effect in guided tissue regeneration.

Gingival Recession

Intra Bony defects

In Implant Dentistry

In Open Sinus

Risks of Using Platelet Rich Plasma Gel (Regina Landsberg, Mark Moses,
Margaret Karpatkin 1998)
Preparation of the PRP involves the isolation of the platelet rich plasma after which gel formation is accelerated using calcium chloride and bovine thrombin. It has been found that, the use of bovine thrombin may be associated with the development of antibodies to the factors V, XI and thrombin resulting in the risk of life threatening coagulopathies. It has been reported that one death has occurred due to serious coagulopathy after exposure to bovine topical thrombin. Furthermore, the coagulopathies are extremely difficult, to treat. Bovine thrombin preparations have been shown to contain factor V resulting in stimulation of the immune system when challenged with a foreign protein. Factor V deficiency after thrombin exposure is most likely caused by cross reactivity of antibovine factor V antibodies with human factor V. The risks associated with the use of bovine topical thrombin have led many surgeons neurosurgery and cardiovascular surgery to avoid its use.

CONCLUSION
The availability to enhance regenerative process of the human body through utilizing patients own blood, is something now easily available and is well substantiated in literature. The application of the PRP offers the dental patient that is something safe from outside disease transmission , or immunologic reactions Although the growth factors and the mechanisms involved are still poorly understood. More well designed and properly controlled studies are needed to provide solid evidence of PRPs capacity for impact on wound healing, soft-tissue reconstruction and (in combination with bone grafts) augmentation procedures, especially in oral and periodontal therapy.