Platelets Plasma

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Trends Biomater. Artif. Organs, 25(2), 86-90 (2011) http://www.sbaoi.org

Platelet-Rich Plasma: A Novel Bioengineering Concept

Palwinder Kaur, Puneet, Varun Dahiya1

Department of Prosthodontics, Swami Devi Dyal Dental College & Hospital, Barwala, Distt. Panchkula. Haryana1Department of Periodontics, D J Dental College, Modinagar.

Corresponding author:Dr. Palwinder Kaur, e-mail: [email protected]

Received 19 August 2010; Accepted 12 January 2011; Available online 4 May 2011

A promising biologic therapy, offering various applications in dentistry is the use of platelet rich- plasma (PRP). PRP has became anincreasingly popular clinical tool as an alternative source of growth factors for several types of dental procedures. These growthfactors are described as promoters of tissue repair mechanisms and remodelling. The application of PRP in dentistry include sinusgrafting, periodontal soft and hard tissue surgical procedures, ridge augmentation, dental implant osseous-integration etc. The useof PRP represents a relatively new concept of biotechnology that is becoming inseparable part of tissue engineering and cellulartherapy today. The purpose of this article is to update clinicians knowledge about the preparation of PRP, its most efficaciousmeans of application and reviews the current literature on this emerging treatment modality.

Introdcution

Enhancement of the regenerative process of human body

by utilizing the patients own blood, is a unique conceptto dentistry. Platelet- rich plasma (PRP) is coming up asa biological revolution in dental field. PRP represents asimilarity to the natural healing process, with theapplication of multiple growth factors.Growth factors arethe biologically active substances that are involved intissue-repair mechanism such as chemotaxis, cellproliferation, angiogenesis, extracellular matrixdeposition, and remodelling. Platelet- rich plasma (PRP)has been traditionally used as a vehicle of growth factors.A concentrated source of autologous platelets, PRPcontains and releases (through degranulation) at leastseven different growth factors (cytokines) that stimulatebone and soft tissue healing. Each growth factor has the

capability to induce a unique response in theenhancement of healing. Alpha granules are storage unitswithin platelets, which contain pre-packaged growthfactors in an inactive form. The main growth factorscontained in these granules are: PDGF (platelet derivedgrowth factor), TGF-1 (transforming growth factorbeta1), TGF-2 (transforming growth factor beta2), ILGF(insulin like growth factor). PDGF is involved in the woundhealing procedures for its effects on mitosis,angiogenesis, releasing of other growth factors. TGF-1and TGF-2 stimulate chemotaxis and mitogenesis ofthe osteoblasts (precursors and inhibition ofosteoclasts ( activity. ILGF has a role in theactivation of the osteoblasts ( precursors and the

activation of the osteoblasts responsible for the depositionof bone in the initial phases of bone regeneration. PRPis more that just a platelet concentrate; it also contains

the 3 proteins in blood known to act as cell adhesion

molecules for osteoconduction and as a matrix for bone,connective tissue, and epithelial migration. These celladhesion molecules are fibrin itself, fibronectin, andvitronectin.

The autologous nature of PRP differ it from recombinanthuman growth factors like rhPDGF, which are purelyhuman but are foreign growth factors to that person. [1]The use of PRP in place of recombinant growth factorshas several advantages, in that growth factors obtainedfrom platelets not only have their own specific action ontissues but also interact with other growth factors,resulting in activation of gene expression and proteinproduction. As an autologous preparation, PRP requires

no considerations like antibody formation, effectivelypreventing the risk of graft vs. host disease and leadingto better acceptance by patients. The short shelf life andinefficient delivery to target cells are other major concernsassociated with local administration of recombinanthuman growth factors.

Until now, the use of PRP use has been restricted to thehospital setting. This was mainly due to the cost ofseparating the platelets from the blood (thousands) andthe large amount of blood needed (one unit) to producea suitable quantity of platelets.. An easy, cost-effectiveway to obtain high concentrations of growth factors fortissue healing and regeneration is autologous platelet

storage via PRP.This new technology permits to safelyharvest and produce a sufficient quantity of platelets fromonly 8-10 ml of blood drawn from patients in dental office.


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Ease of procurement along with application of PRP in thesurgical dental practice, and its beneficial outcomes haspopularized it among the clinicians.

PRP preparation

Platelet rich plasma (PRP), is essentially an increasedconcentration of autologous platelets suspended in a smallamount of plasma after centrifugation. Centrifugationprocess accelerates the sedimentation of the heavier cellssuch as white blood cells and red blood cells, whileplatelets (which sediment to a lower rate) remain floatingwithin the plasma fraction at the top side of the tubes.PRP can be prepared in a laboratory or surgical/dentalclinic from blood collected in the immediate preoperativeperiod. [2,3,4]

Basically, patients blood is collected ( approx. 50 cc)and centrifuged at varying speeds until it separates into 3layers: platelet poor plasma (PPP) on topmost layer, PRP

in middle, and red blood cells at the bottom. Usually 2spins are used. The first spin (Hard spin) separates theplatelet poor plasma (PPP) from the red fraction andplatelet rich plasma (PRP). The second spin (Soft spin)separates the red fraction from the PRP. This is achievedby simple bench centrifugation at high gravitational force.After centrifugation, material with the highest specificgravity (PRP) will be deposited at the bottom of the tube.Immediately prior to application, a platelet activator/agonist(topical bovine thrombin and 10% calcium chloride) isadded to activate the clotting cascade, producing a plateletgel. The whole process takes approximately 12 minutesand produces a platelet concentration of 35x that ofnative plasma. [5]Although the concentration of platelets

can vary depending on the method of extraction andequipment, studies have shown that clinical benefit canbe obtained if the PRP used has an increased plateletconcentration of 4x greater than normal blood.

Recent studies have demonstrated that PRP preparedfrom 8 to 10 ml of whole blood is sufficient for periodontalregenerative therapies. [6,7,8] However, in oral andmaxillofacial reconstruction, 8 to 500ml of whole bloodshould be drawn , so as to obtain the larger amounts ofPRP needed for larger surgical defects. The preparationand processing of PRP is similar in most of commercialsystems available, except anticoagulant used, speed andduration of centrifugation may vary with different systems.

Risk with PRP use

As the gel formation after isolation of PRP involve theuse of bovine thrombin, which may be associated withantibodies to the factors 5, 9 and thrombin, resulting inlife threatening coagulopathies. Bovine thrombinpreparations has factor 5, which can stimulate immunesystem if challenged by a foreign protein. Safer optionsinclude the use of recombinant human thrombin orautologous thrombin. Contraindications to the use of PRPusage are rare and may include platelet dysfunctionsyndrome, septicemia, anemia (Hg < 10 g/dL), criticalthrombocytopenia, hemodynamically unstable patients

and pregnancy.Therefore, the sequence of procedures,healing periods and possible risk and complications shouldbe clearly explained to patients prior to treatment.

Mechanism of action of PRP

It isessential for understanding the biologic rationale ofPRP to know the role of platelets in wound healing. PRPinitiate wound repair by releasing locally acting growthfactors. These growth factors aid healing by attracting un-

differentiated cells in the newly formed matrix andtriggering cell division. PRP works via the degranulationof the granules in platelets, which contain the synthesizedand prepackaged growth factors. The active secretion ofthese growth factors is initiated by the clotting process ofblood and begins within 10 minutes after clotting. Morethan 95% of the presynthesized growth factors aresecreted within 1 hour. Therefore, PRP must be developedin an anticoagulated state and should be used on the graft,flap, or wound, within 10 minutes of clot initiation.PRPenhances the early wound-healing cascade by theinteractions of activated platelet-released growth factorswith the extra cellular matrix with potential potent anabolicaffects. Platelets in PRP also play a role in host defence

mechanism at the wound site by producing signallingproteins that attract macrophages. [9]

Clinical application in dentistry

Utilizing the methods of enhancement of bone and softtissues regeneration with the use of PRP is becomingmore evident in many areas of dentistry such asperiodontics, oral implantology and oral and maxillofacialsurgery. These procedures are applicable for improvingthe contour of alveolar ridge in relation to ideal pontic,papilla esthetics for fixed partial prosthesis, healthy dento-alveolar complex for periodontal attachment and bone fordental implant placement. New bone formation is aprerequisite for the regeneration of tissues lost throughperiodontal disease and for the osseointegration ofimplants used in restorative dentistry. [10]

Periodontal regeneration is a complex process of body.The aim of regenerative periodontal procedures is toinduce regeneration at the alveolar bone and cementumand to develop a new functional periodontal ligament. Allthe mineralized and non mineralized components mustbe restored to their original position and architecture forregeneration of the periodontium to occur. Growth factorsexert regulatory effects on the homeostasis of theperiodontal tissues and they also have the ability ofmodifying the response of periodontal soft and hardtissues during the healing processes after their exogenous

application. [11] Inperiodontal surgery it has been usedin gingival grafting, crown lengthening and periodontalregeneration.

In the field of implant dentistry, the most frequentlyencountered hinderences at the implantation site are lackof adequate bone available and proximity to anatomicstructures, such as the maxillary sinus and the inferioralveolar nerve canal. Advanced surgical procedures thatact as an adjunct in dental implants consist of sinusgrafting and guided bone regeneration. With theapplication of PRP in addition to autogenous grafts usedfor these procedures like sinus lifts, ridge augmentations,etc will promote and accelerate osseointegration process.

It is specially benefitted in maxilla , in cases of previousimplant failures, in type 4 bone, in osteoporotic women,etc. [12]The ability to predictably augment the alveolar


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ridge with deficient bone volume has greatly benefitedpatients with such challenging situations. Block grafts offerthe advantage of slow resorption and easy fixation in thesite of compromised bone quantity. So the use ofautogenous bone has been recommended as thepreferred bone graft, with its osteogenic, osteoinductiveand osteoconductive properties.

In the field of oral and maxillofacial surgery, PRP hasproven to improve the surgical results in a variety ofprocedures . Surgical sites enhancedwith PRP have beenshown to heal at rates two to three times that of normalsurgical sites. Because PRP also enhance soft tissuemucosal and skin healing, it is used in onlay grafts,particulate grafts, alveolar cleft palate repair, oral/nasalfistula repair, mucosal flaps for root coverage, post-operative hemostasis of bone graft donor sites, continuitydefects of the mandible and hemophiliacs undergoingsurgery. Block grafts with PRP may be successfully usedin healthy patients with deficient alveolar ridges, restoring

the original bony architecture to accommodate a dentalimplant. [13] Calcium sulfate-Platelet rich plasma is a novelbiomaterial able to induce osteogenesis in bone defectscreated by removal of teeth, small cysts, and in variousclinical situations. [14] Promoting soft tissue healing andrapid epithelialisation of skin also play a crucial role incosmetic dentistry. So the goal of accelerating healingthat we now gain with PRP by activating natural healingprocess hold a promising future in advancing years ofdentistry.

Advantages

PRP offers many advantages in clinical use: it decreasesthe chances of intraoperative and postoperative bleedingat the donor and the recipient sites, also facilitates morerapid soft-tissue wound healing, aids in the initial stabilityof the grafted tissue at the recipient sites (as a result of itscohesive and adhesive nature), may promote fastervascularisation of the healing tissue by delivering growthfactors and, in combination with bone replacementmaterials, that induces regeneration.

Benefits of PRP

PRP acting as a vehicle for growth factors offer severaladvantages like ;(1) PRP is a by-product of the patientsown blood; therefore, chances of infectious diseasetransmission is rare,(2) PRP can be easily generated in

the dental office while the patient is undergoing anoutpatient surgical procedure, like placement of dentalimplants, extraction defects, variety of grafting and softtissue procedures,oral surgery etc.,(3) as there is supersaturation of the wound with PRP, and thus growth factors,fasten tissue regeneration,(4) since PRP harvesting isdone with only 8-10 ml of blood, the patient need not bearthe expense of the harvesting procedure in hospital or atthe blood bank,(5) it is easy to handle and actuallyimproves the ease of application of bone substitutematerials by making them more gel-like,(6) preparationtime is relatively short and concentrate can be obtainedwithin 12 min.

Discussion

According to what has been found in literature, P.R.P hasgot an important role in stimulation and acceleration of

bone and soft tissue healing. The first clinical dental resultswith PRP were reported by Marx and others in 1998, whoused PRP to improve graft incorporation in mandibularreconstructions in patients who had received cancellousbone marrow grafts after tumor removal. Their datastrongly suggested that adding PRP to bone graftsaccelerated the rate and degree of bone formation.Mineralization of collagen matrix is speeden up due toPDGF that is present in the graft material , instead of beingreleased from collagen. The rate of bone formation alsohas improved from 1.62 to 2.18 times that of the controls.[15]

Improvement in trabecular bone density has beenreported in literature with the use of platelet rich plasmain conjunction with graft. The patients undergoing implantplacement after extraction, who received a mixture ofautologous bone and PRP, much better epithelization andcompact mature bone with well-organized trabeculae was

demonstrated compared to the control group. Anothermajor benefit of PRP is jump start osteogenesis byreleasing growth factors at the local site and earlyconsolidation of graft. This allows placement of implantinto the grafted site at an early time. Bone defects aroundtitanium implants could be treated successfully with bonepowder and PRP that improve bone formation. If a toothis extracted and the extraction site has poor healing, itmay delay the use of dental prosthetics or implants. Insuch case use of PRP may aid in speeding up healingand bone formation.PRP also allow earlier implant loadingand improved osseointegration when used incompromised bone such as osteoporotic bone and boneafter radiotherapy. [16] In the field of periodontics, growthfactors have regulatory effects on homeostasis of theperiodontal soft and hard tissues upon their application.In a recent in vitro study, significant mitogenic effect ofgrowth factors on human osteoblast-like and periodontalligament cells was demonstrated. In this study it was alsoevaluated that gender and sex of the donor have noinfluence in platelet-count or growth factor concentrationsin PRP. [17]Researchers are studying PDGFfor its useas an adjunct to regenerative therapy. Studies have foundthat PDGFwas the only growth factor that effectivelystimulated periodontal ligament fibroblast migration andproliferation without the added risk of the patientexperiencing ankylosis of the teeth. When used in clinicaltrials on beagles, PDGF-modulated guided tissueregeneration, or GTR, therapy was shown to effectively

aid in the regeneration of periodontal furcation defects. Itwas also shown in another study that PDGF-BB and TGF-1 exert mitogenic effect on human periodontal ligamentcells and human fibroblasts by promoting DNA synthesis.[18] In addition, PRP was used in the treatment of severeform of chronic periodontitis, with an improvement in thedepth reduction of gingival recession and clinicalattachment level. [19] Moreover, PRP has also shownencouraging results when used in periodontal defectscombined with bone allograft and guided tissueregeneration (GTR). Marx and coworkers also performeda monoclonal antibody study on the platelets sequesteredby the centrifugation process and on the harvested bonegraft material. The data indicated that PDGF and TGF

from the platelets had been absorbed by the graft, andreceptors for PDGF and TGF were present within theautogenous grafts They concluded that the addition of


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Platelet-Rich Plasma: A Novel Bioengineering Concept 89

PRP accelerated the rate and degree of bone formationand that bone autografts contained platelets and thus werepositive for receptors for the growth factors. Furthermore,it was noted that the PRP was an autologous preparationobtained at the time of the surgery, thus eliminatingconcerns about disease transmission, immunogenicreactions, and mislabelling of the sample. [20, 21]Parkand colleagues conducted similar studies on beagles,comparing the treatment of Class III furcation defects withPDGFand GTR vs. with GTR alone. The study indicateda statisticallygreater amount of bone and periodontalligament in sites treated with PDGF and GTR togetherthan in sites treated with GTR alone The newly formedbone filled 80 percent of the lesion at eight weeks and 87percent of the lesion at 11 weeks in the sites treated withPDGF and GTR, compared with 14 percent of the lesionat eight weeks and 60 percent at 11 weeks in sites treatedwithGTR alone. Additionally, the sites treated with PDGFand GTRseemed to have increased ratios of wanted tounwanted tissuetypes filling the wounds as compared with

sites treated withGTR alone.[22]

Garg and coworkers proposed that resorbable barriermembrane materials be infused with PRP. They haveproposed that this PRP-based membrane could serve asa short-acting biologic barrier, since all platelets containedin PRP will degranulate within 3 to 5 days, and their initialgrowth activity expires within 10 days. So PRP infusedinto resorbable barrier membranes tend to retard epithelialmigration and also provide localized growth factors toaccelerate hard and soft tissue maturation. Placement ofplatelet rich fibrin membrane in recession defects can beused to restore the functional properties of the labialgingiva of mandibular anterior teeth by repairing gingival

defects and re-establishing the continuity and integrity ofthe zone of keratinized gingiva. [23] PRP also enhancessoft tissue mucosal and skin healing, it is therefore usedin connective tissue grafts, palatal grafts, gingival grafts,mucosal flaps together for root coverage, skin graft donoretc. PRP has been a valuable adjunct in several oralsurgerical procedures ; they include ablative surgicalprocedures, associated oroantral or oronasal fistulas,mandibular reconstruction and surgical repair of alveolarcleft. In these surgical procedures, the adhesivenatureof PRP allows easier handling of graft material, morepredictable flap adaptation and seal than with primaryclosure alone. Along with this is the resultant release ofthe previously mentioned growth factors.

In an attempt to enhance the bone grafting procedures,clinicians have used different kinds of growth factors aspart of this innovative tissue-engineering concept. Platelet-

rich plasma has been proposed as potential stimulant ofbone and soft tissue healing. Kassolisand colleaguesreported that the successful use of PRP-augmenteddemineralized freeze-dried bone allografts for alveolaraugmentationor sinus lift procedures before implantplacement. [24] More recently Forum and associatesconcluded that when FDBA was used with PRP forsubantral sinus augmentation, results showed that theapplication of PRP will only result in accelerated new boneformation if target cells such as osteoblasts and osteocytesare present. It was also reported that, PRP when combinedwith inorganic bovine bone grafts, had no effect on defectmineralization at any time point. This can be one of thereasons that several studies using non vital graftingmaterial have failed to show any promoting effect. [25,26] The incorporation of PRP into the sinus graft has beenproposed as a method to shorten healing time, enhancewound healing, and improve bone quality. [27]

As the growth factors stimulate cellular proliferation, some

have shown concern that PRP might stimulate cancers.Actually no growth factor can provoke a cancer. All growthfactors act on cell membranes, not the cell nucleus.Growth factors activate an internal cytoplasmic signalprotein, to promotes a normal gene expression, ratheran abnormal gene expression. Growth factors are notmutagens , but are normal body proteins. The specificrelation of PRP and cancer is nothing more than the sameblood clot that would be in any normal wound, except thatit contains a greater number of platelets. [12] So far mostof published researches showed it is safe to use theproduct, however, future study in this area is certainlyneeded.

ConclusionThe emerging science of transfusion medicine, as itapplies to regenerative therapyand the formation of anautogenous platelet gel, still is a growing field in whichmany studies are yet tobe made. Although the growthfactors, ideal ratios of the components and exactmechanisms, still arebeing investigated, and more clinicalresearch with long-term results is needed. With thereduction in cost, the ease of applying PRP in dental clinicprovides a promising adjunct to dental surgical proceduresby promoting safe and natural healing. PRP with itsbeneficial outcomes will definitely revolutionize the surgicaldentistry in the near future. It is the responsibility of theclinician to gain a thorough understanding of thisbiotechnology and to use it correctly and wisely for thebenefit and well being of our patients, who trust ourjudgment.

References

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7. Lekovic V, Camargo PM, Weinlaender M, Vasilic N, Kenney EB. Comparison of platelet-rich plasma, bovine porous bone mineral,and guided tissue regeneration versus platelet-rich plasma and bovine porous bone mineral in the treatment of intrabony defects:a reentry study. J Periodontol 2002; 73(2):198205.

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in oral mucosal wound healing. Clin Oral Implants Res. 2007;18(1):133-9.10. Sodek J, McKee MD. Molecular and cellular biology of alveolar bone. Periodontol 2000 2000;24:99-126.11. Dereka XE, Markopoulou CE, Vrotsos IA. Role of growth factors on periodontal repair. Growth Factors 2006;24:260-7.12. Marx RE. Platelet-Rich Plasma (PRP): What Is PRP and What Is Not PRP? Implant Dentistry.2001;10(4)225-228.13. Anila S, K Nandakumar. Applications of Platelet Rich Plasma for Regenerative Therapy in Periodontics. Trends Biomater. Artif.

Organs 2006;20(1):78-83.14. Intini G, Andreana S, Intini FE, Buhite RJ, Bobek LA. Calcium sulfate and platelet-rich plasma make a novel osteoinductive

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