Platelet-rich plasma: a novel bioengineering concept.
|Abstract:||A promising biologic therapy, offering various applications in dentistry is the use of platelet rich-plasma (PRP). PRP has became an increasingly popular clinical tool as an alternative source of growth factors for several types of dental procedures. These growth factors are described as promoters of tissue repair mechanisms and remodelling. The application of PRP in dentistry include sinus grafting, periodontal soft and hard tissue surgical procedures, ridge augmentation, dental implant osseousintegration etc. The use of PRP represents a relatively new concept of biotechnology that is becoming inseparable part of tissue engineering and cellular therapy today. The purpose of this article is to update clinician's knowledge about the preparation of PRP, its most efficacious means of application and reviews the current literature on this emerging treatment modality.|
|Publication:||Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 Society for Biomaterials and Artificial Organs ISSN: 0971-1198|
|Issue:||Date: April, 2011 Source Volume: 25 Source Issue: 2|
Enhancement of the regenerative process of human body by utilizing the patient's own blood, is a unique concept to dentistry. Platelet-rich plasma (PRP) is coming up as a biological revolution in dental field. PRP represents a similarity to the natural healing process, with the application of multiple growth factors. Growth factors are the biologically active substances that are involved in tissue-repair mechanism such as chemotaxis, cell proliferation, angiogenesis, extracellular matrix deposition, and remodelling. Platelet-rich plasma (PRP) has been traditionally used as a vehicle of growth factors. A concentrated source of autologous platelets, PRP contains and releases (through degranulation) at least seven different growth factors (cytokines) that stimulate bone and soft tissue healing. Each growth factor has the capability to induce a unique response in the enhancement of healing. Alpha granules are storage units within platelets, which contain pre-packaged growth factors in an inactive form. The main growth factors contained in these granules are: PDGF (platelet derived growth factor), TGF-[beta]1 (transforming growth factor betal), TGF-[beta]2 (transforming growth factor beta2), ILGF (insulin like growth factor). PDGF is involved in the wound healing procedures for its effects on mitosis, angiogenesis, releasing of other growth factors. TGF-[beta]l and TGF-[beta]2 stimulate chemotaxis and mitogenesis of the osteoblasts 'precursors and inhibition of osteoclasts' activity. ILGF has a role in the activation of the osteoblasts' precursors and the activation of the osteoblasts responsible for the deposition of bone in the initial phases of bone regeneration. PRP is 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 cell adhesion molecules are fibrin itself, fibronectin, and vitronectin.
The autologous nature of PRP differ it from recombinant human growth factors like rhPDGF, which are purely human but are foreign growth factors to that person.  The use of PRP in place of recombinant growth factors has several advantages, in that growth factors obtained from platelets not only have their own specific action on tissues but also interact with other growth factors, resulting in activation of gene expression and protein production. As an autologous preparation, PRP requires no considerations like antibody formation, effectively preventing the risk of graft vs. host disease and leading to better acceptance by patients. The short shelf life and inefficient delivery to target cells are other major concerns associated with local administration of recombinant human growth factors.
Until now, the use of PRP use has been restricted to the hospital setting. This was mainly due to the cost of separating the platelets from the blood (thousands) and the large amount of blood needed (one unit) to produce a suitable quantity of platelets... An easy, cost-effective way to obtain high concentrations of growth factors for tissue healing and regeneration is autologous platelet storage via PRP. This new technology permits to safely harvest and produce a sufficient quantity of platelets from only 8-10 ml of blood drawn from patients in dental office. Ease of procurement along with application of PRP in the surgical dental practice, and its beneficial outcomes has popularized it among the clinicians.
Platelet rich plasma (PRP), is essentially an increased concentration of autologous platelets suspended in a small amount of plasma after centrifugation. Centrifugation process accelerates the sedimentation of the heavier cells such as white blood cells and red blood cells, while platelets (which sediment to a lower rate) remain floating within the plasma fraction at the top side of the tubes. PRP can be prepared in a laboratory or surgical/dental clinic from blood collected in the immediate preoperative period. [2,3,4]
Basically, patient's blood is collected (approx. 50 cc) and centrifuged at varying speeds until it separates into 3 layers: platelet poor plasma (PPP) on topmost layer, PRP in middle, and red blood cells at the bottom. Usually 2 spins are used. The first spin ("Hard spin") separates the platelet poor plasma (PPP) from the red fraction and platelet rich plasma (PRP). The second spin ("Soft spin") separates the red fraction from the PRP. This is achieved by simple bench centrifugation at high gravitational force. After centrifugation, material with the highest specific gravity (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) is added to activate the clotting cascade, producing a platelet gel. The whole process takes approximately 12 minutes and produces a platelet concentration of 3-5x that of native plasma.  Although the concentration of platelets can vary depending on the method of extraction and equipment, studies have shown that clinical benefit can be obtained if the PRP used has an increased platelet concentration of 4x greater than normal blood.
Recent studies have demonstrated that PRP prepared from 8 to 10 ml of whole blood is sufficient for periodontal regenerative therapies. [6,7,8] However, in oral and maxillofacial reconstruction, 8 to 500ml of whole blood should be drawn, so as to obtain the larger amounts of PRP needed for larger surgical defects. The preparation and processing of PRP is similar in most of commercial systems available, except anticoagulant used, speed and duration of centrifugation may vary with different systems.
Risk with PRP use
As the gel formation after isolation of PRP involve the use of bovine thrombin, which may be associated with antibodies to the factors 5, 9 and thrombin, resulting in life threatening coagulopathies. Bovine thrombin preparations has factor 5, which can stimulate immune system if challenged by a foreign protein. Safer options include the use of recombinant human thrombin or autologous thrombin. Contraindications to the use of PRP usage are rare and may include platelet dysfunction syndrome, septicemia, anemia (Hg < 10 g/dL), critical thrombocytopenia, hemodynamically unstable patients and pregnancy. Therefore, the sequence of procedures, healing periods and possible risk and complications should be clearly explained to patients prior to treatment.
Mechanism of action of PRP
It is essential for understanding the biologic rationale of PRP to know the role of platelets in wound healing. PRP initiate wound repair by releasing locally acting growth factors. These growth factors aid healing by attracting un-differentiated cells in the newly formed matrix and triggering cell division. PRP works via the degranulation of the granules in platelets, which contain the synthesized and prepackaged growth factors. The active secretion of these growth factors is initiated by the clotting process of blood and begins within 10 minutes after clotting. More than 95% of the presynthesized growth factors are secreted within 1 hour. Therefore, PRP must be developed in an anticoagulated state and should be used on the graft, flap, or wound, within 10 minutes of clot initiation. PRP enhances the early wound-healing cascade by the interactions of activated platelet-released growth factors with the extra cellular matrix with potential potent anabolic affects. Platelets in PRP also play a role in host defence mechanism at the wound site by producing signalling proteins that attract macrophages. 
Clinical application in dentistry
Utilizing the methods of enhancement of bone and soft tissues regeneration with the use of PRP is becoming more evident in many areas of dentistry such as periodontics, oral implantology and oral and maxillofacial surgery. These procedures are applicable for improving the contour of alveolar ridge in relation to ideal pontic, papilla esthetics for fixed partial prosthesis, healthy dento-alveolar complex for periodontal attachment and bone for dental implant placement. New bone formation is a prerequisite for the regeneration of tissues lost through periodontal disease and for the osseointegration of implants used in restorative dentistry. 
Periodontal regeneration is a complex process of body. The aim of regenerative periodontal procedures is to induce regeneration at the alveolar bone and cementum and to develop a new functional periodontal ligament. All the mineralized and non mineralized components must be restored to their original position and architecture for regeneration of the periodontium to occur. Growth factors exert regulatory effects on the homeostasis of the periodontal tissues and they also have the ability of modifying the response of periodontal soft and hard tissues during the healing processes after their exogenous application.  In periodontal surgery it has been used in gingival grafting, crown lengthening and periodontal regeneration.
In the field of implant dentistry, the most frequently encountered hinderences at the implantation site are lack of adequate bone available and proximity to anatomic structures, such as the maxillary sinus and the inferior alveolar nerve canal. Advanced surgical procedures that act as an adjunct in dental implants consist of sinus grafting and guided bone regeneration. With the application of PRP in addition to autogenous grafts used for these procedures like sinus lifts, ridge augmentations, etc will promote and accelerate osseointegration process. It is specially benefitted in maxilla, in cases of previous implant failures, in type 4 bone, in osteoporotic women, etc.  The ability to predictably augment the alveolar ridge with deficient bone volume has greatly benefited patients with such challenging situations. Block grafts offer the advantage of slow resorption and easy fixation in the site of compromised bone quantity. So the use of autogenous bone has been recommended as the preferred bone graft, with its osteogenic, osteoinductive and osteoconductive properties.
In the field of oral and maxillofacial surgery, PRP has proven to improve the surgical results in a variety of procedures. Surgical sites enhanced with PRP have been shown to heal at rates two to three times that of normal surgical sites. Because PRP also enhance soft tissue mucosal and skin healing, it is used in onlay grafts, particulate grafts, alveolar cleft palate repair, oral/nasal fistula repair, mucosal flaps for root coverage, postoperative hemostasis of bone graft donor sites, continuity defects of the mandible and hemophiliacs undergoing surgery. Block grafts with PRP may be successfully used in healthy patients with deficient alveolar ridges, restoring the original bony architecture to accommodate a dental implant.  Calcium sulfate-Platelet rich plasma is a novel biomaterial able to induce osteogenesis in bone defects created by removal of teeth, small cysts, and in various clinical situations.  Promoting soft tissue healing and rapid epithelialisation of skin also play a crucial role in cosmetic dentistry. So the goal of accelerating healing that we now gain with PRP by activating natural healing process hold a promising future in advancing years of dentistry.
PRP offers many advantages in clinical use: it decreases the chances of intraoperative and postoperative bleeding at the donor and the recipient sites, also facilitates more rapid soft-tissue wound healing, aids in the initial stability of the grafted tissue at the recipient sites (as a result of its cohesive and adhesive nature), may promote faster vascularisation of the healing tissue by delivering growth factors and, in combination with bone replacement materials, that induces regeneration.
Benefits of PRP
PRP acting as a vehicle for growth factors offer several advantages like; (1) PRP is a by-product of the patient's own blood; therefore, chances of infectious disease transmission is rare, (2) PRP can be easily generated in the dental office while the patient is undergoing an outpatient surgical procedure, like placement of dental implants, extraction defects, variety of grafting and soft tissue procedures, oral surgery etc., (3) as there is super saturation of the wound with PRP, and thus growth factors, fasten tissue regeneration, (4) since PRP harvesting is done with only 8-10 ml of blood, the patient need not bear the expense of the harvesting procedure in hospital or at the blood bank, (5) it is easy to handle and actually improves the ease of application of bone substitute materials by making them more gel-like, (6) preparation time is relatively short and concentrate can be obtained within 12 min.
According to what has been found in literature, P.R.P has got an important role in stimulation and acceleration of bone and soft tissue healing. 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 tumor removal. Their data strongly suggested that adding PRP to bone grafts accelerated the rate and degree of bone formation. Mineralization of collagen matrix is speeden up due to PDGF that is present in the graft material, instead of being released from collagen. The rate of bone formation also has improved from 1.62 to 2.18 times that of the controls. 
Improvement in trabecular bone density has been reported in literature with the use of platelet rich plasma in conjunction with graft. The patients undergoing implant placement after extraction, who received a mixture of autologous bone and PRP, much better epithelization and compact mature bone with well-organized trabeculae was demonstrated compared to the control group. Another major benefit of PRP is "jump start" osteogenesis by releasing growth factors at the local site and early consolidation of graft. This allows placement of implant into the grafted site at an early time. Bone defects around titanium implants could be treated successfully with bone powder and PRP that improve bone formation. If a tooth is extracted and the extraction site has poor healing, it may delay the use of dental prosthetics or implants. In such case use of PRP may aid in speeding up healing and bone formation. PRP also allow earlier implant loading and improved osseointegration when used in compromised bone such as osteoporotic bone and bone after radiotherapy.  In the field of periodontics, growth factors have regulatory effects on homeostasis of the periodontal soft and hard tissues upon their application. In a recent in vitro study, significant mitogenic effect of growth factors on human osteoblast-like and periodontal ligament cells was demonstrated. In this study it was also evaluated that gender and sex of the donor have no influence in platelet-count or growth factor concentrations in PRP.  Researchers are studying PDGF for its use as an adjunct to regenerative therapy. Studies have found that PDGF was the only growth factor that effectively stimulated periodontal ligament fibroblast migration and proliferation without the added risk of the patient experiencing ankylosis of the teeth. When used in clinical trials on beagles, PDGF-modulated guided tissue regeneration, or GTR, therapy was shown to effectively aid in the regeneration of periodontal furcation defects. It was also shown in another study that PDGF-BB and TGF-a1 exert mitogenic effect on human periodontal ligament cells and human fibroblasts by promoting DNA synthesis.  In addition, PRP was used in the treatment of severe form of chronic periodontitis, with an improvement in the depth reduction of gingival recession and clinical attachment level.  Moreover, PRP has also shown encouraging results when used in periodontal defects combined with bone allograft and guided tissue regeneration (GTR). Marx and coworkers also performed a monoclonal antibody study on the platelets sequestered by the centrifugation process and on the harvested bone graft material. The data indicated that PDGF and TGF from the platelets had been absorbed by the graft, and receptors for PDGF and TGF were present within the autogenous grafts They concluded that the addition of PRP accelerated the rate and degree of bone formation and that bone autografts contained platelets and thus were positive for receptors for the growth factors. Furthermore, it was noted that the PRP was an autologous preparation obtained at the time of the surgery, thus eliminating concerns about disease transmission, immunogenic reactions, and mislabelling of the sample. [20, 21] Park and colleagues conducted similar studies on beagles, comparing the treatment of Class III furcation defects with PDGF and GTR vs. with GTR alone. The study indicated a statistically greater amount of bone and periodontal ligament in sites treated with PDGF and GTR together than in sites treated with GTR alone The newly formed bone filled 80 percent of the lesion at eight weeks and 87 percent of the lesion at 11 weeks in the sites treated with PDGF and GTR, compared with 14 percent of the lesion at eight weeks and 60 percent at 11 weeks in sites treated with GTR alone. Additionally, the sites treated with PDGF and GTR seemed to have increased ratios of wanted to unwanted tissue types filling the wounds as compared with sites treated with GTR alone. 
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. So PRP infused into resorbable barrier membranes tend to retard epithelial migration and also provide localized growth factors to accelerate hard and soft tissue maturation. Placement of platelet rich fibrin membrane in recession defects can be used to restore the functional properties of the labial gingiva of mandibular anterior teeth by repairing gingival defects and re-establishing the continuity and integrity of the zone of keratinized gingiva.  PRP also enhances soft tissue mucosal and skin healing, it is therefore used in connective tissue grafts, palatal grafts, gingival grafts, mucosal flaps together for root coverage, skin graft donor etc. PRP has been a valuable adjunct in several oral surgerical procedures; they include ablative surgical procedures, associated oroantral or oronasal fistulas, mandibular reconstruction and surgical repair of alveolar cleft. In these surgical procedures, the adhesive nature of PRP allows easier handling of graft material, more predictable flap adaptation and seal than with primary closure alone. Along with this is the resultant release of the previously mentioned growth factors.
In an attempt to enhance the bone grafting procedures, clinicians have used different kinds of growth factors as part of this innovative tissue-engineering concept. Platelet-rich plasma has been proposed as potential stimulant of bone and soft tissue healing. Kassolis and colleagues reported that the successful use of PRP-augmented demineralized freeze-dried bone allografts for alveolar augmentation or sinus lift procedures before implant placement.  More recently Forum and associates concluded that when FDBA was used with PRP for subantral sinus augmentation, results showed that the application of PRP will only result in accelerated new bone formation if target cells such as osteoblasts and osteocytes are present. It was also reported that, PRP when combined with inorganic bovine bone grafts, had no effect on defect mineralization at any time point. This can be one of the reasons that several studies using non vital grafting material have failed to show any promoting effect. [25, 26] The incorporation of PRP into the sinus graft has been proposed as a method to shorten healing time, enhance wound healing, and improve bone quality. 
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 growth factors act on cell membranes, not the cell nucleus. Growth factors activate an internal cytoplasmic signal protein, to promotes a normal gene expression, rather an abnormal gene expression. Growth factors are not mutagens, but are normal body proteins. The specific relation of PRP and cancer is nothing more than the same blood clot that would be in any normal wound, except that it contains a greater number of platelets.  So far most of published researches showed it is safe to use the product, however, future study in this area is certainly needed.
The emerging science of transfusion medicine, as it applies to regenerative therapy and the formation of an autogenous platelet gel, still is a growing field in which many studies are yet to be made. Although the growth factors, ideal ratios of the components and exact mechanisms, still are being investigated, and more clinical research with long-term results is needed. With the reduction in cost, the ease of applying PRP in dental clinic provides a promising adjunct to dental surgical procedures by promoting safe and natural healing. PRP with its beneficial outcomes will definitely revolutionize the surgical dentistry in the near future. It is the responsibility of the clinician to gain a thorough understanding of this biotechnology and to use it correctly and wisely for the benefit and well being of our patients, who trust our judgment.
[1.] Carlson ER. Bone grafting the jaws in the 21st century: The use of platelet-rich plasma and bone morphogenetic protein. Alpha Omegan 2000;93:26-30.
[2.] Gonshor A. Technique for producing platelet-rich plasma and platelet concentrate: background and process. Int J Periodontics Restorative Dent 2002; 22(6):547-57.
[3.] Man D, Plosker H, Winland-Brown JE. The use of autologous platelet-rich plasma (platelet gel) and autologous platelet-poor plasma (fibrin
glue) in cosmetic surgery. Plast Reconstr Surg 2001;107(1):229-37.
[4.] Anitua E. Plasma rich in growth factors: preliminary results of use in the preparation of future sites for implants. Int J Oral Maxillofac Implants 1999; 14(4):529-35.
[5.] Wang HL, Avila G. Platelet Rich Plasma: Myth or Reality? Eur J Dent. 2007 October; 1(14):192-194.
[6.] Camargo PM, Lekovic V, Weinlaender M, Vasilic N, Madzarevic M, Kenney EB. Platelet-rich plasma and bovine porous bone mineral combined with guided tissue regeneration in the treatment of intrabony defects in humans. J Periodontal Res 2002; 37(4):300-6.
[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 Periodontal 2002; 73(2):198-205.
[8.] Weibrich G, Kleis WK, Kunz-Kostomanolakis M, Loos AH, Wagner W. Correlation of platelet concentration in platelet-rich plasma to the extraction method, age, sex, and platelet count of the donor. Int J Oral Maxillofac Implants 2001; 16(5):693-9.
[9.] Lindeboom JA, Mathura KR, Aartman IH, Kroon FH, Milstein DM, Ince C. Influence of the application of platelet-enriched plasma 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. Periodontal 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 biomaterial for bone regeneration. J Transl Med 2007 Mar 7;5:13.
[15.] Marx RE, Clarison ER, Eichstaedt RM. PRP: Growth enhancement factor for bone grafts. Oral Srg, Oral Med, Oral Pathol Oral Radiol Endod 1998;85:638-646.
[16.] Kim SG, Kim WK, Park JC, Kim HJ. A comparative study of osseointegration of Avana implants in a demineralized freeze-dried bone alone or with platelet-rich plasma. J Oral Maxillofac Surg 2002;60(9):1018-25.
[17.] Dereka XE, Markopoulou CE, Mamalis A, Pepelassi E, Vrotsos IA. Time--and dose-dependent mitogenic effect of basic fibroblast growth factor combined with different bone graft materials: an in vitro study. Clin Oral Implants Res 2006;17:554-9.
[18.] Marcopoulou CE, Vavouraki HN, Dereka XE, Vrotsos IA. Proliferative effect of growth factors TGF-beta1, PDGF-BB and rhBMP-2 on human gingival fibroblasts and periodontal ligament cells. J Int Acad Periodontol 2003;5:63-70.
[19.] Martinez-Zapata MJ, Marti-Carvajal A, Sola I, et al. Efficacy and safety of the use of autologous plasma rich in platelets for tissue regeneration: a systematic review. Transfusion 2009;49:44-56.
[20.] Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR. Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:638-46.
[21.] 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 re-entry study. J Periodontal 2002;73:198-205.
[22.] Park JB, Matsuura M, Han KY, et al. Periodontal regeneration in class III furcation defects of beagle dogs using guided tissue regenerative therapy with platelet-derived growth factor. J Periodontol 1995;66:462-77.
[23.] Garg AK, Gargenese D, Peace I. Using platelet-rich plasma to develop an autologous membrane for growth factor delivery in dental implant therapy. Dental Implant Update 2000;11:41-3
[24.] Kassolis JD, Rosen PS, Reynolds MA. Alveolar ridge and sinus augmentation utilizing platelet-rich plasma in combination with freeze-dried bone allograft: case series. J Periodontal 2000;71:1654-61.
[25.] Froum SJ, Wallace SS, Tarnow DP, Cho SC. Effect of platelet-rich plasma on bone growth and osseointegration in human maxillary sinus grafts: three bilateral case reports. Int J Periodontics Restorative Dent. 2002;22:45-53.
[26.] Wiltfang J, Kloss FR, Kessler P, Nkenke E, Schultze-Mosgau S, Zimmermann R, et al. Effects of platelet-rich plasma on bone healing in combination with autogenous bone and bone substitutes in critical-size defects. An animal experiment. Clin Oral Implants Res. 2004;15:187-193.
[27.] Boyapati L, Wang HL. The role of platelet-rich plasma in sinus augmentation: a critical review. Implant Dent 2006. Jun; 15(2):160-170.
Palwinder Kaur, Puneet, Varun Dahiya (1)
Department of Prosthodontics, Swami Devi Dyal Dental College & Hospital, Barwala, Distt. Panchkula. Haryana
(1) Department of Periodontics, D J Dental College, Modinagar.
Corresponding author: Dr. Palwinder Kaur, e-mail: firstname.lastname@example.org
Received 19 August 2010; Accepted 12 January 2011; Available online 4 May 2011
|Gale Copyright:||Copyright 2011 Gale, Cengage Learning. All rights reserved.|