Saini K, Chopra P, Sheokand V. Journey of Platelet Concentrates: A Review. Biomed Pharmacol J 2020;13(1).

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Manuscript received on :29-May-2019
Manuscript accepted on :17-Jan-2020
Published online on: 11-02-2020

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Reviewed by: M Mohan Varma
Second Review by: Exbrayat Jean-Marie 
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Komal Saini, Vidushi Sheokand and Priyanka Chopra

Department of Periodontology, SGT University, Gurugram

Corresponding Author E-mail : drpriyankachopra79@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/1875

Abstract

One of the important action of platelets is their role in haemostasis and healing of wound. Now they are gaining popularity in Dentistry in periodontal regeneration. Earlier fibrin glue was introduced as sealant, later the platelet-rich plasma (PRP); first generation of platelet concentrates was utilized in various fields of Dermatology from chronic ulcer management to trichology and also in aesthetics. Choukroun et al. in Francein 2000’s introduced the second generation of platelet concentrates (PRF)Platelet Rich Fibrin. PRF have comparatively several advantages over traditionally prepared PRP. In this review we are focusing on why Platelet Concentrates are so important in Healing and Regeneration and we will also discuss the journey of fibrin glue from PRP to PRF, i-PRF, t-PRF, L-PRF etc.

Keywords

Platelet Concentrates; PRP; PRF; Wound Healing

Indroduction

Periodontal repair and regeneration is the ultimate goal in treatment of Periodontal defects. Various biomaterials, natural as well as synthetic have been tried since many years to accelerate wound healing of both soft and hard tissue. Naturally occurring materials also known as “autologous biomaterials” are present in the body and provides signalsfor repair, regeneration and healing. Similarly, synthetically generated alloplastic materials have also shown promising result in many fields of regenerative dentistry but the drawback is that they may exhibit foreign body reaction¹. One of greatest disadvantage of synthetic materials considered for regeneration is that most of them are avascular in nature, to improve the acceptance the concentration of autologous biomaterials is increased to the wound site for enhancing healing.² It has been noted that the bone graft material combined with bloodis known to promote neo-angiogenesis, new bone formation when compared to bone grafts alone .³Two important autologous biomaterials, platelets and fibrin  are believed to play important role in promoting the healing of wound and regeneration.

Platelet concentrates had travelled a long way from introduction of platelet concentrates as a source of blood proteins enriched with growth factors to promote wound healing to introduction of fibrin glue, PRP, PRF, A-PRF, t-PRF, i-PRF, PRF lysates and CGF in recent years.⁴ Platelet concentrates used these days are very much efficient than predecessors in terms of biological efficacy and method of preparation.

Histologically diameter of platelets varies from1 to 4 μm; they are colourless, non-nucleated with moderately refractive bodies. Precursor cell of platelet are megakaryocytes, which are extremely large hematopoietic cells present in bone marrow.These megakaryocytes fragment into the small disc shape structures called platelets either in blood stream or in bone marrow itself; From where they squeeze through capillaries. Normal concentration of the platelets in blood is between 150,000 and 300,000 per mm³.⁵ The average lifespan of a platelet is few days approximately 5 to 10 days. Spleen act as a reservoir for platelets, when needed released by sympathetically-induced contractions of splenic muscle.⁶

Role of platelets in wound healing: A proinflammatory biochemical environment of wound impairs the healing affiliated to increased protease activity, whichdecreases the concentration of variousGrowth Factors (GFs). As a rich source of Growth Factors Platelet concentrates areused as an interesting alternative for the treatment for wounds and also have mitogenic, angiogenic, and chemotactic properties.⁷Platelets exert its effects mainly due to three different granules namely α (alpha) granules, lysosomes and dense granules.These granules secretes the growth factors namely Platelet derived growth factor(PDGF), vascular endothelial growth factor (VEGF),Fibroblast Growth Factor (FGF), Insulin like growth factor (ILGF),transforming growth factor (TGF) and epidermal growthfactor (EGF).⁸PDGF accelerates the wound heling because of the effect on mitosis, angiogenesis and promote the release of other growth factors. TGF play important role in promoting chemotaxis and along with the Insulin ILGFhave role in activation of theOsteoblast. Platelets releases these growth factors within 10 min after clotting and approximately more than 95% of thesepresynthesized growth factor are released within first hour. When direct action of platelets diminish, macrophages arrives through vascular ingrowth stimulated by the platelets and then theybecome responsiblefor woundhealing regulation by secreting their own factors.⁹So the platelets act as pace setter for wound healing. 

Chronological Evolution of Platelet Concentrates

The Journey starts from the development of platelet concentrates originating as key concept for fibrin adhesives development which were quite popular in 1970s in Europe. A list of various platelet concentrates evolved over the time is given in table 1.

Table 1: Platelet Concentrates Evolution and their Drawbacks

Table 2: Advancement in Platelet Concentrates and Procedure

Platelet concentrates;concept was first started in the field of hematology.¹⁵ In 1970s  the term PRP was coined to describe  plasma with a platelet count many folds above that of peripheral blood count, which was earlier used to treat patients with thrombocytopeniaby transfusion products .¹⁶ Later in same era Fibrin glue was made by polymerizing fibrinogen with thrombin and calcium. Fibrin Sealants are human plasma derivatives that mimic the final stages of blood coagulation, forming a fibrin clot. It is used as topical haemostatic and tissue sealing agent. However, due to low concentration of fibrinogen in donor plasma, the quality and stability of fibrin glue was suboptimal and these products were associated with a risk of cross infection.

Numerous research works were continued in 1975-1978 which suggested an enhanced healing concept by using blood extracts and called them as plateletfibrinogenthrombin mixtures. During this time period various author designated those products as “GelatinPlatelet Gel Foam”.¹⁷In 1986 Knighton et al¹⁸was the first to demonstrate that platelet concentrate successfully promote healing and termed it as “Platelet Derived Wound Healing Factors (PDWHF)”, which were successfully used initially for thetreatment ofulcers of skin. Whitman first introduced the Platelet Rich plasma (PRP) in field of oral and maxillofacial surgery.Through the activation of the platelets within the gel there is resultant release of thegrowth factors from secretory granules resulting in enhanced wound healing .¹²

PRP produced by double centrifugation process consisted of a soft spin at 1300 RPM for 10 minutes followed by which the blood would separate into red corpuscular base, buffy coat and the platelet poor plasma.The last two components were aspirated and re centrifuged at hard 2000 RPM for 10 minutes after which PRP was collected in the bottom of the tube (Figure 1). PRP gained popularity in sinus lifting procedures, Augmentation of Alveolar Ridge, Preservation of  Socket, Repair of Alveolar cleft Palate, Oral/nasal fistula repair, Intra bony defects, Reconstruction surgeries of jaws, various Soft tissue procedures likegingival grafts, Connective tissue  grafts etc. due to its property of accelerating the healing of soft tissues.¹⁹

Figure 1: Steps in preparation of Platelet Rich Plasma by Double Centrifugation28 Click here to View Figure

In 1999 at the same time period,Anitua et al formulated another platelet concentrate utilizing anticoagulants and coined the term Platelet Rich Growth Factor (PRGF). These platelet concentrates were no longer in practice today because of lack of the specific pipetting steps, ergonomy of kit and the significant issues were present with this technique.¹³Vivostat PRF (Alleroed, Denmark) a widely accepted technique for production of P-PRP was commercialized. However, it is misnamed as it is not a true PRF producing kit instead produces a PRP product. All the products were named as Platelet rich plasma (PRP) or also known as first generation of platelet concentrate.²⁰

Platelet Rich Gel (PRG) proposed to be more active than PRP by Cieslik-Bielecka²¹ and they defined it as fibrin matrix rich in platelets, leuko­cytes and relative active molecule which is biologicallymore activated. Sacco²² in 2006 give a new concept of concentrated growth factors. Preparation of concentrated growth factor required venous blood with rpm in range of 2400-2700. Sacco’s membrane shows a complex three-dimensional architecture which makes it’s a biomaterial rich in fibrin, platelets, leukocytes and growth factors very suitable for both patients and doctor. One of the complication associated with PRP handling is its liquid nature, which reduce its application as it required in combination with other biomaterials. The clinical benefits with PRP is limited because of short release of growth factors.Due to this limitation second-generation platelet concentrate termed PRF have been emerged.In 2000Choukroun et al¹⁴   introducedPlatelet rich fibrin labelled as PRF, based on the strong fibrin gel polymerization which is another form of Platelet concentrate in France. This is an important turnaround in the evolution of terminology.

According to Everts et al²³platelet concentrates has been found in two forms, which include nonactivated and activated forms(on the basis of leukocyte component). The inactivated form was called as “platelet-leukocyte rich plasma (P-LRP) and the activated form was called platelet-leukocyte-gel” (PLG). In 2010by Sohn²⁴concept of sticky bone was introduced in which autologous fibrin glue was mixed with bone graft. Later on,APRF (advanced PRF) introducedin 2014 by Choukroun et alwhich contain more monocytes.²⁵Later Titanium prepared PRF (T-PRF) by Tunali et al²⁶ introduced. In 2015 Mourao et al²⁷ gave detailed infotmation on pre­paration of i-PRF (injectable PRF). 

Protocol of PRF Preparations and its Advancement

Dr. Joseph Choukroun the research pioneer has led to the development of a second-genera­tion platelet concentrate, in this anticoagula­tion factors have not been utilized. A platelet concentrate without coagulation factors can be gathered (750 g) from the superficial layer of cen­trifugation tubes following single centrifugation cycle (2,700 rpm,12 minutes). This formulation was called Platelet rich fibrin and it contained a fibrin matrix after centrifugation. The composition of PRF include concentrate of white blood cells, platelets, and fibrin. It has been shown that the initially developed Platelet rich fibrin (also termed L-PRF) composed of 97% platelets and more than 50% leukocytes in a high-density fibrin network when compared to whole blood.²⁸

The major drawback of PRF is its preparation along with storage. The factor affecting the potential benefit of PRF includes its quick handling between the blood collection and the centrifugation as PRF preparation does not include any anticoagulants. One of the other important benefit include the dehydration which cause decreased growth factor content in PRF and leukocyte viability will be adversely affected altered its biologic properties. Platelet rich fibrin is obtained as a gel form which is not conducive to be injected. To overcome these limitations several modifications are done and newer forms of PRF were introduced.²⁹ 

Later the advanced PRF matrices in solid form was developed having the (LSCC) Low Speed Centrifugation Concept. This improved preparation protocol for advanced PRF (A-PRF) is reducing the applied RCF to 208 g. The structure of advanced fibrin clot is a more porous with a larger interfibrous space when compared to PRF. For formation of A-PRF, slower speed (1500 rpm) and more time (14 mins) is used in the sterile plain glass-based vacuum tubes.³⁰

A-PRF shows more neutrophilic granules in distal part mostly at red blood cells-buffer coat interface. Another modification of A-PRF has been suggested by Kobayashi & co-workers in 2016 where they have reduced the centrifugation time to 1300 rpm for 8 minutes. They called this modification as A-PRF+ and suggested that less time would result in a decrease in the amount of forces that the cells of the blood would be exposed to & hence, would increase the number of cells contained in the PRF matrix.³¹

The PRF so obtained is in gel form which is not conducive to be injected. To overcome this limitation i-PRF is introduced. For producing i-PRF, blood is drawn without anticoagulant in plastic tubes without any coatings and centrifuged at around 700 for 3 minutes.²⁷ The time is considerably shorter than other two protocols (i.e. L-PRF & A-PRF). This can be attributed to the fact that for i-PRF only the separation of blood components is desired which happens in initial 2-4 minutes. Plastic tubes are used for this process as it have a hydrophobic surface and do not efficiently activate the coagulation process. Hence, all the blood components that are required to form a good platelet concentrate (plasma containing all clotting factors & platelets) reach the top of the tube under the centrifugation force in the first 2-4 minutes. Currently, it has been used for mixing with bone grafts, which forms a gel-putty consistency with the graft particles incorporated in the graft. The graft thus formed has a good workable consistency, leads to decreased leaching of the graft as it is tightly encapsulated in the fibrin matrix.³²

Another modification of PRF is T-PRF i.e. Titanium -PRF, obtained by centrifugation of blood at 2800 RPM for 12 minutes in Titanium tubes. T-PRF provide more tightly woven and thicker fibrin than classic L-PRF, as has titanium better hemocompatibility compared to glass and potentially led to the formation of a more polymerized fibrin.²⁶ 

Conclusion

Platelet concentrates has been used in various application of dentistry since many years. Technological advancement in this field shows promising result in use of platelet rich fibrin (PRF) in Periodontal regeneration. Various studies have been conducted to determine the utilization of PRF in various procedures i.e periodontology, oral surgery, and implant dentistry and encouraging results obtained in both soft and hard tissue regeneration. Various factors like speed, duration of centrifuge, temperature, blood haematocrit influence the quality of fibrin scaffold. Lastly, the prominent role of leukocytes or fibrin in PRF scaffolds is discussed as potential avenues for future research. 

Acknowledgement

I express my profound sense of gratitude and sincere regards to Dr.Priyanka Chopra and Dr.Vidushi Sheokand for their meticulous supervision, valuable guidance, encouragement, and critical appreciation. I would also like to thank my husband Maj (Dr) Sachin Saini, my little daughter Vedika and all family members for their unconditional love and support during this work.

Financial support and sponsorship

Nill

Conflict of Interest

I declared that there is no conflict of interest with regard to publication of my research work. 

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