Application of PRP (platelet-rich plasma) in surgical periodontal therapy: overview

Abstract Clinical and morphological manifestation of periodontitis is associated with persistent inflammation of the gingiva, loss of connective tissue attachment, formation of a periodontal pocket and loss of alveolar bone, which indicate variations in a wide range in different individuals. The main purpose of non-surgical therapy for periodontitis is to achieve long-term control of inflammation, and the ultimate goal of therapy is the regeneration of periodontal tissues affected by destruction. The unique anatomy and structure of the entire periodontal complex determine the course of more complex processes related to the restoration of periodontal structures affected by destruction, which include coordination of response by four different types of tissues: epithelial tissue, connective tissue, periodontal ligament and bone. Periodontal regeneration is defined as the regeneration of main tooth-supporting tissues: alveolar bone, periodontal ligament, cementum and attachment. Modern modalities for carrying out the periodontal regeneration include the use of bone substitutes, guided tissue regeneration with barrier membranes, treatments with flaps and a variety of additional components, such as: soft tissue grafts, root bio-modifiers and growth factors, the carrier of which is platelet-rich plasma (PRP). This mini-review provides an overview of PRP applied in surgical periodontal therapy. Data of the initial experiences with this method are included, as well as brief references on its usage in the clinical practice. There is no substitute material in the modern literature shown as the gold standard in the treatment of periodontal bone defects.


Introduction
Clinical and morphological manifestation of periodontitis is associated with persistent inflammation of the gingiva, loss of connective tissue attachment, formation of a periodontal pocket and loss of alveolar bone, which indicate variations in a wide range in different individuals. The formation of periodontal pockets of variable depth, the attachment loss, as well as horizontal or vertical bone loss are pathognomonic characteristics for diagnosing periodontitis. The main purpose of non-surgical therapy for periodontitis is to achieve long-term control of inflammation and the ultimate goal of therapy is the regeneration of periodontal tissues affected by destruction. Periodontal regeneration is not only associated with obtaining a new connective tissue attachment; it involves, as well, the formation of a new functional cementum with the incorporation of new collagen fibers to that part of the root surface that has lacks periodontal ligament due to periodontitis progression. The unique anatomy and structure of the entire periodontal complex determine the course of more complex processes related to the restoration of periodontal structures affected by destruction, which include coordination of response by four different types of tissues: epithelial tissue, connective tissue, periodontal ligament and bone. Periodontal regeneration is defined as the regeneration of the main tooth-supporting tissues: alveolar bone, periodontal ligament, cementum and attachment [1][2][3].

Growth factors
As early as 1986, Knighton DR et al. [4] examined the ability of platelets to provide additional potential for better healing and regeneration of lost tissue by means of growth factors release from them (Table 1). This thesis was proven by the authors using platelet mass for the treatment of difficult-to-heal skin ulcers [4]. Since then, many authors have considered the application of growth factors as biologically reactive molecules that can stimulate cells accounting for tissue regeneration. Growth factors have been shown to promote cell proliferation, migration and metabolic activity, as well as to influence chemotaxis and the production of extracellular matrix proteins [9,[17][18][19].
Platelets are known to be the first cells to respond to the healing site and, in addition to this pro-coagulant effect, locally release a cocktail of "growth factors" from a-granules contained in them, such as: plateletderived growth factor (PDGF), transforming growth factor (TGF-b 1 and b 2 ), insulin-like growth factor (IGF-1 and 2) and vascular growth factor (VEGF), which The authors report in their study that PRF can stimulate cell proliferation of osteoblasts, gingival fibroblasts, periodontal ligament cells and suppress epithelial growth in an in vitro model. These results determined the use of PRF in periodontal surgery as an independent graft material for the treatment of periodontal intrabony defects. Dohan Ehrenfest DM et al. 2010 [14] The dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane. J Periodontol 2010; 81(4):546-555.
The authors found that 97% of platelets and more than half of leukocytes are implanted in the fibrin network. They observed a major cluster of cellular elements in the so-called "buffycoat".
Under an electron-microscope this layer is divided into two zones: the first zone is made up of thick fibrin fibers and little erythrocytes, and the second zone contains platelets and fibrin, which form a dense and well-organized 3 D fibrin network. Douglas TE et al. 2012 [15] Enzymatically induced mineraliza-tion of platelet-rich fibrin. J Biomed Mater Res A, 2012; 100 (5):1335-1346.
The authors in their in vitro study have shown that PRF under the action of alkaline phosphatase may be mineralized.
Solakoglu € O, Heydecke G, Amiri N et al.2020 [16] The use of plasma rich in growth factors (PRGF) in guided tissue regeneration and guided bone regeneration. A review of histological, Immunohistochemical, histo-morphometrical, radiological and clinical results in humans. Annals of Anatomy-2020,vol.231; september151528 The authors summarize that the use of platelet concentrates for tissue regeneration is increasingly used in periodontal and maxillofacial surgery. Data from the study show that the use of plasma rich in growth factors (PRGF) significant improves tissue healing, reduces postoperative complication, accelerates the processes of recovery of soft and hard tissues. It is also used in the treatment of various complications arising during the healing process.
promote healing processes by stimulating fibroblast proliferation, output regulation and differentiation of extra-cellular matrix proteins and local vascularization [6,20]. Being considered independently, each of the growth factors has a definite role in the healing and regenerative processes: Platelet growth factor (PDGF) -stimulates increased formation of cell adhesion molecule fibronectin that activates cell proliferation; influences the differentiation of osteoprogenitor cells, which affect the healing processes in connective tissue; binds to endothelial cells of blood vessels and stimulates local vascularization.
Transforming growth factor (TGF-b 1 and b 2 )mainly affects osteoblasts and stem cell proliferation associated with the process of osteogenesis. It is involved in stimulating fibroblast, osteoprogenitor and endothelial cells, as well as in the production of collagen.
Insulin-like growth factor (IGF-1 and 2) -stimulates the proliferation and differentiation of osteoblasts, as for the processes of periodontal regeneration IGF-1 turns out to be of greater significance -it stimulates the proliferation of fibroblasts and the synthesis of extracellular proteins. Insulin-like growth factor-1 has a chemotaxis effect on progenitor cells of the periodontal ligament, as well.

Platelet-rich plasma (PRP)
PRP (platelet-rich plasma), i.e. plasma rich in platelets, acts by degranulation of platelet a-granules, which contain a number of platelet-specific proteins (b-thromboglobulin) and platelet-nonspecific proteins (fibronectin, fibrinogen), coagulation factors, fibrinolysin, immunoglobulins and synthesized growth factors [21,22]. Their active secretion begins in the first few minutes. More than 95% of the growth factors are secreted during the first hour and therefore, the use of an anticoagulant is necessary. Its application should be carried out within 10 min from the coagulum activation. The isolated growth factors immediately bind to the outer surface of the cell membranes of graft cells or flap cells, by means of the so-called transmembrane receptors. They, in turn, have a role to activate an endogenous intrinsic signaling protein that induces the expression of a gene stimulus on cells, which is expressed in: cell proliferation, extracellular matrix formation, osteoid production and collagen synthesis [23][24][25].
Conventional periodontal therapy involves primarily non-surgical treatment, but also various surgical approaches. The most common effect of non-surgical therapy is the formation of a long connecting epithelium between the instrumental root surface and the adjacent alveolar bone. Histological evidence is provided that the results lead to reparation rather than true regeneration. More unfavorable and complicated ways of healing related to external root resorption and ankylosis are possible, as well [1].
Modern modalities for carrying out the periodontal regeneration include the use of bone substitutes, guided tissue regeneration with barrier membranes, treatments with flaps and a variety of additional components, such as: soft tissue grafts, root bio-modifiers and growth factors, the carrier of which is PRP [16,18,26,27]. There is no substitute material in the modern literature shown as the gold standard in the treatment of periodontal bone defects.
The introduction of PRP usage in oral and maxillofacial surgery was done by Whitman et al. [7], who proposed this method in 1997. The first clinical data from the use of PRP were reported by Marx et al. [8], who used PRP in the incorporation of a bone graft to reconstruct a patient's mandible after removal of a tumor formation. The outcomes obtained by the authors indicated that the addition of PRP significantly accelerated the process of formation of new bone structure (Table 1). Similar results were observed afterwards by other authors who used a combination of PRP with a bone allograft and a membrane to regenerate intraosseous bone defects [28][29][30][31][32][33][34][35].
Over the last few years, the literature data on combined use of bone substitutes and barrier membranes with growth factors have been increasing, as well as those on the higher regenerative potential related to growth factors, expressed in stimulating the formation of mineralized and non-mineralized tissues [9,18,23]. In the initial stages of healing process, platelet-derived growth factors in platelet-enriched plasma attract undifferentiated mesenchymal cells to the fibrin matrix and activate cell division. In this way, they activate tissue regeneration: by proliferation of connective tissue progenitor cells, stimulation of fibroblast and osteoblast activity, as well as angiogenesis [10,17,19]. Blood coagulation is considered to be the main link in initiating the healing processes of all soft tissues, as well as in bone regeneration. Normally it contains approximately 94% erythrocytes and 6% platelets, and less than 1% leukocytes in the fibrin network [20]. In contrast to blood coagulation, PRP was found to contain 5% erythrocytes, 94% platelets and 1% leukocytes.
The main components of platelet-rich plasma are: growth factors, leukocytes, phagocytes, native fibrinogen, vasoactive and chemotactic agents, as well as a high concentration of platelets [6,25].
Platelet-rich plasma is considered to be the most accessible in terms of obtaining these factors in a physiological manner. The use of PRP is essentially an autologous transplant treatment: the product applied is the patient's own plasma. Therefore, it is reckoned that there is no risk of sensitization, disease transmission or genetic intervention. PRP therapy is a safe method, without any risk of infection, rejection, with long-lasting effect and without known side effects [20].
Platelet-rich plasma (PRP) is usually gel-like and is obtained after centrifugation of autologous blood and subsequent mixing of the separated plasma layer (PPP) with a portion of the fibrin-rich underlying layer (PRF), which is centrifuged again to obtain a yellow supernatant serum to which sterile bovine thrombin and 10% calcium chloride, dissolved in saline, are added. The plasma obtained as a final product has a high concentration of platelets: 3-4 times higher concentration compared to baseline level [15,26,27,36]. The platelet count in PRP can exceed 2,000,000 in 1 mL of plasma, while the normal concentration of platelets in blood is 150,000 to 350,000 in 1 mL [26]. When platelet-rich plasma is used for treating mineralized and non-mineralized tissues, the fact that is relied on is that platelets are activated and release "growth factors" that can stimulate collagen and elastin synthesis from fibroblasts, improve blood supply and metabolism of damaged tissues by means of influencing the process of angiogenesis (formation of new blood vessels), and thus bring about healing and restoration of the affected tissues.
The antimicrobial effect of platelet-rich plasma is determined by the presence of a high concentration of leukocytes therein. The concentration of the growth factors and matrix glycoproteins (glycosaminoglycans) contained in plasma enhances substantially during the first 7 days and the success of recovery is considered to be proportional to the number of platelets in the blood clot, which have a significantly higher concentration at the end of the procedure of preparation of PRP compared to the beginning [15,19].
Data from clinical trials have shown that the combined use of bone substitutes and PRP result in better clinical indicators (attachment gain and depth reduction on probing) in the treatment of periodontal bone defects [17]. PRP has the advantage of affecting osteoprogenitor cells in bone and bone replacement materials (autogenous bone) and is applied in procedures, such as sinus lift, techniques of vertical and horizontal augmentation of alveolar bone, periodontal and peri-implant bone defects [8,35,[37][38][39][40][41][42][43][44].

Plasma rich in growth factors (PRGF)
In 1999, Anitua [44] described a new modification of PRP, called Plasma rich in growth factors (PRGF). In this modification, the author used a 10% solution to activate the polymerization of fibrinogen in fibrin. PRGF, unlike PRP, does not contain leukocytes and has a low platelet concentration. Studies with mono and polyclonal antibodies have indicated the presence of high concentration of growth factors [44]. The suspension obtained that way and used as a solution for injection, showed good osteoconductive properties and a lack of antigenic activity.

Platelet-rich fibrin (PRF)
In 2001, Choukroun J et al. [11] developed an even newer generation of autogenous platelet concentrate, without biological and chemical additives. This concentrate is called Platelet-rich fibrin (PRF) [11]. PRF is an autogenous fibrin matrix wherein platelets and leukocytes are concentrated together with their growth factors. PRF is defined by researchers as the second generation of PRP [45]. The fact that no additional anticoagulant is added results in the activation of a larger number of platelets upon contact with the tube walls during centrifugation, which, in turn, triggers the coagulation cascade within only a few minutes. Most platelets and leukocytes remain trapped in the fibrin clot and its 3 D structure is similar to naturally polymerized fibrinogen [14]. Natural polymerization of fibrinogen in PRF determines the formation of a very stable fibrin structure, which allows the formation of a stable fibrin membrane accordingly. The platelets and leukocytes trapped in it are in high concentration, and the leukocytes remain unchanged during centrifugation. Platelets in turn are activated, which leads to the significant incorporation of the released growth factors and other biologically active molecules inside the fibrin matrix [36].
The obtained fibrin clot consists of three main parts: yellow fibrin part, red part located at the opposite end of the clot (composed of erythrocytes) and an intermediate whitish layer (buffy coat) between them. This layer is electronically microscopically divided into two areas. The first area is composed of maturing fibrin network, thick fibrin threads and a small number of erythrocytes. The second one contains more platelets and fibrin, which form a dense network and the platelets in it are activated. In PRF, in contrast to PRP, the formation of a finer and more flexible fibrin network is observed, capable of supporting the intrinsic cytokines incorporated in it and cell migration [12,37]. Such a configuration of the fibrin network has been proven to provide better survival of platelet-derived growth factors, which are available for a longer period of time among other cells and have sufficient time to stimulate healing and regenerative processes.
The resulting 3 D structure gives greater strength and elasticity to the obtained fibrin matrix, which is confirmed by the clinically observed properties of the PRF membrane: elasticity, strength and ability to be sutured.
The issue that has given rise to much controversy is the significance of platelet-rich fibrin. For example, Tuan et al. [6] describe the role of fibrin in tissue restoration. Their study shows that fibroblasts can actively reorganize the fibrin matrix in order to initiate collagen synthesis [6] (Table 1). Van Hinsbergh et al. [10] examined the growth of human vascular endothelial cells in the 3 D structure of the fibrin matrix obtained from PRF. The authors' study points out that the resulting fibrin structure affects the range and stability of the capillary structures obtained in vivo (Table  1). Circulating mesenchymal stem cells from the blood enter the available fibrin matrix, then they differentiate into different cell types and this ensures a more rapid course of the healing processes in damaged tissues [10,36]. Kawamura and Urist [5] demonstrate that PRF can also act as a supporting matrix for bone morphogenic proteins (BMP), while other authors, Douglas et al. [15], prove in their in vitro study that it is possible for PRF to be mineralized by the action of alkaline phosphatase [5,12,15,46] (Table 1).
For the process of angiogenesis, an extracellular fibrin matrix is needed to allow the migration, division and phenotypic change of endothelial cells. Such angiotensin properties have the unique 3 D structure of the fibrin network obtained by PRF. Growth factors, such as FGF-b (Fibroblast grown factor -b), PDGF (Platelet-derived growth factor) and angiopoietin play a major role in angiogenesis, as well. These growth factors are included in the fibrin network, with FGF-b and PDGF showing high affinity for fibrin [14]. The importance of fibrin, fibronectin, PDGF and TGF-b for modulating fibroblast proliferation and fibroblast migration at the site of tissue damage has been demonstrated [14,22,26].
According to Choukroun et al. [47] PRF can be regarded as natural fibrin and a basis for the development of micro vascularization. It has the ability to direct epithelial cells to migrate on its surface and the content of leuxkocytes is of particular importance to ensure faster healing of affected tissues [47,48]. Tsai et al. [13] reported in their study that PRF can stimulate cell proliferation of osteoblasts, gingival fibroblasts, and periodontal ligament cells and inhibits epithelial outgrowth in an in vitro model. These results lead to the use of PRF in periodontal surgery as an independent graft material for treatment of periodontal infraosseous defects [13] (Table 1). In recent years, many authors have supported the idea that PRF should be regarded as a fibrin biomaterial with very high potential [36,[49][50][51].

Conclusions
It is of high significance to emphasize that the additional use of PRP or PRF is one of the latest innovations for tissue regeneration in surgery. This is associated with rapid onset of osteogenesis, early consolidation of soft tissue and bone grafts due to growth factors initiation of osteocompetent cellular activity and subsequent early mineralization of extracellular matrix proteins.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This review was supported by project "Stimulating research in areas with high achievements" -Medical University of Sofia, Contract @ D 237/2019.