PRF and Sticky Bone as Regenerative Materials in Oral Surgery

Abstract

Platelet-rich fibrin (PRF) as a biological scaffold is attracting clinicians’ attention, mainly because it promotes bone and soft tissue healing. As autologous material, PRF has many advantages over other platelet concentrates, such as Platelet-rich plasma (PRP) and Plasma rich in growth factors (PRGF). Among many benefits, simple preparation (centrifugation protocol) stands out because no additional anticoagulant is added to the tubes. This chapter aims to clarify the PRF membranes and sticky bone preparation together with other platelet concentrates. A few clinical cases will show how sticky bone is together with PRF membranes applicative in different oral surgery indications. Clinical and radiological check-ups demonstrated excellent therapeutic outcomes. Sticky bone and PRF membranes have regenerative potential and are advised to use in many oral surgery procedures.

1. Introduction

Modern times bring many challenges in different life spheres, and medical treatment is not an exception. As dentists, we encounter constant scientific as well as technological developments, and it is with great eagerness that we strive to be active users of these benefits, thereby actively advocating for patients’ best care. It is not with ease that a modern man would come to peace with the information that they are not eligible for a particular treatment; in dentistry, for instance, that may be the case when one does not have enough available bone for implants to be placed. This further motivates dentists to replace lost tissues by utilizing regenerative procedures. The principle of regenerative medicine and dentistry is founded on its interdisciplinarity as well as on the application of bioengineering techniques that enable the replacement of any lost tissue. In the field of oral surgery, a branch of dentistry, the most interesting tissue replacement is bone and soft (gingival) tissue replacement [1].

Of great help in regenerative procedures is the application of platelet concentrates that originate from the patient’s blood [2]. The role of platelet concentrates in regenerative dentistry is based on the fact that they contain growth factors and scaffolds. In this chapter, we will be discussing platelet-rich fibrin (PRF) and sticky bone, their preparation techniques, and usage indications. We will hereby also comment on other platelet concentrates.

2. A brief review of platelets and their role in the body

Blood is a liquid tissue that consists of plasma (55%), red blood cells (or erythrocytes 45%), platelets (thrombocytes), and white blood cells (leukocytes) that together account for less than 1%. In the organism, blood acts as a transporting medium, participates in coagulation, and serves as a medium for information transduction (e.g., hormones) [3].

In regenerative processes, the focus is placed on platelets. They originate from megakaryocytes in the blood marrow. Platelets are small, discoid-shaped plate-like cells that have no nucleus and have a lifespan of 8–12 days in a resting state. Apart from their role in hemostasis, they also have a role in inflammatory reactions, wound healing, host defense, and tumor biology [4].

Platelets contain three types of granules (alpha, dense, and lysosomes) that dictate platelets’ function. Among the three types of granules, the alpha granules are the most abundant and have an important role in regenerative and wound healing processes [4, 5, 6]. They contain adhesive proteins, growth factors, and clot-forming factors. The regenerative function of the alpha granules is based on mitogenic factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF)) [5, 6, 7].

In order for the granule release from a cell to occur, platelet activation is required. This process is mediated by platelet activating molecules and some of these molecules are produced by platelets (e.g., collagen, thrombin, thromboxane A, adenosine phosphate, P-selectin, and protease activator receptor–related molecules). On contrary, there are so-called inhibitors that act as platelet receptors’ inhibiting molecules, thereby preventing platelet activation (e.g., coagulation factors, aspirin, ADP receptor inhibitors).

Once a blood vessel is injured and there is a rupture at the level of the endothelial layer, platelets start releasing molecules, such as collagen, that activate them. Activated platelets bind to the injured site of the blood vessel with collagen. This is known as adhesion. In addition, activated platelets secrete great amounts of ADP and, at the same time, thromboxane A2 is being synthesized, which then initiates granule (alpha and dense) release, which, in return, results in platelet aggregation. In other words, white platelet clot formation, that is, a result of the processes we explained beforehand, has three stages: platelet adhesion, granule release, and platelet aggregation. Simultaneously with platelet activation, blood vessel injury initiates a coagulation cascade. It starts when blood interacts with tissue factors. This process entails a series of biochemical reactions that convert inactive blood plasma proteins to active proteolytic enzymes. In this way, the coagulation process—that starts with thrombin activation upon blood vessel injury—ends with the conversion of fibrinogen to fibrin. As a result, fibrin fibers permeate and secure the thrombus. In this way, the process of hemostasis is accomplished [4, 5, 7].

3. Platelet concentrates

Platelets are not limited to hemostatic processes, but they also influence tissue regeneration, enhance collagen synthesis, and trigger angiogenesis as well as the immune response by releasing growth factors and cytokines [6]. The concept of platelet concentrate preparation is based on the fact that manipulation of normal physiologic processes, such as hemostasis, enables us to obtain concentrated platelets together with a greater amount of growth factors that play a crucial role in wound healing. They do that by stimulating tissue regeneration and proliferation, initiating extracellular matrix deposition, and supporting cell differentiation. By obtaining platelet concentrates, we obtain autologous biomaterials that have an important role in regenerative procedures. To date, there are numerous patents related to platelet concentrates [6]. The first one to be invented was, however, the fibrin glue, and it serves as the precursor of platelet concentrates afterward. Later, PRF (platelet-rich fibrin), sticky bone, and plasma gel were invented. The preparation process is simple. In outline, it is required to draw an adequate amount of blood from a patient in vacuum test tubes with or without anticoagulants and centrifuge them according to a protocol of choice. The point is that with every novel method of platelet concentrate preparation, scientists have struggled to improve the earlier one. The main objective was to increase the number of growth factors and prolong their release time. Novel methods made easier the preparation process by eliminating anticoagulants and simplifying centrifuge protocols.

Further in this chapter, we discuss a brief review of the most important platelet concentrates.

3.1 Fibrin glue

The fibrin glue is a precursor to platelet concentrates, application of which in medicine started 50 years ago. It is packed in two bottles with different content. One bottle contains lyophilized human fibrinogen, while another one contains either bovine or human thrombin. The mandatory ingredients are calcium salts. Proper usage entails mixing the contents of the two bottles, whereby the coagulation process is imitated, and a gel-like formulation is formed (fibrin clot) that can further be used as a topical hemostatic, tissue adhesive (glue), as well as to join bone graft particles. Although they do have broad applications, fibrin glues have several disadvantages, among which is costly manufacture that makes them less popular in comparison to platelet concentrates [6].

3.2 Platelet-rich plasma

Platelet-rich plasma (PRP) belongs to the first generation of platelet concentrates that were put into practice by Robert Marx in 1998. PRP is an autologous human platelet concentrate in a small plasma volume. Precisely, it consists of 1 million platelets per 1 microliter in a total volume of 5 milliliters of plasma. Platelet activation in PRP leads to a release of a variety of growth factors that have an important role in the regulation and stimulation of healing.

The PRP preparation process can be divided into the following phases (Figure 1):

• Blood drawing into vacuum test tubes with the anticoagulant (3.2% sodium citrate)

• First centrifugation with the relative centrifugation force (rcf) of 200 g for 10 minutes

• Transferring the supernatant to a sterile test tube and performing the second centrifugation at 2500 g for 15 minutes

• Upon completion of the second centrifugation, removal of two-thirds of the supernatant should be performed as it represents the platelet-poor plasma (PPP)

• The rest should be gently shaken to obtain the PRP.

Figure 1.

The PRP preparation process. (A) Blood drawing into 3.2% sodium citrate vacuum test tubes. (B) First centrifugation. (C) Transferring the supernatant to a sterile test tube. (D) Second centrifugation. (E) Removal of two-thirds of the supernatant (PPP). (F) Final product platelet-rich plasma (PRP).

When prepared accordingly, the PRP is in a liquid state due to the anticoagulant presence and can be stored for up to 8 hours in sterile conditions. Before using the PRP, calcium chloride and bovine thrombin should be added, thereby activating platelets that trigger coagulation processes, which, in return, results in a PRP transition from liquid to gel-like state. Activated thrombocytes release the granules—growth factors and cytokines.

Activated growth factors interact with the cell membrane. They never enter the cell or the nucleus. Consequently, they do not have a mutagenic potential; rather, they stimulate physiological healing processes only [8, 9, 10].

When in a liquid state, PRP can be injected into a tissue, or it can be mixed with biomaterials that serve as a substitute for bone. In contact with tissue collagen, platelet activation ensues. Activated PRP transitions to a gel, so it can be used as a membrane, or it can be mixed with bone grafts to obtain a graft with a specific shape.

A disadvantage to PRP is a complicated preparation as well as the presence of foreign substances such as anticoagulants (sodium citrate) and procoagulants (calcium chloride and bovine thrombin). Each of these substances can have an antigenic effect albeit, according to Marx, bovine thrombin does not have any contact with systemic circulation and is used in small quantities.

3.3 Platelet rich in growth factors (PRGF)

Platelet-rich growth factors were first described in 1999 by Eduardo Anitua, who patented PRGF within Biotechnology Institute, BTI, Vitoria, Spain, a dental implant company. For PRGF to be made, it requires one centrifugation only coupled by multiple pipetting to ensure precise isolation of the centrifugation end-products. The result is a preparation rich in growth factors, however, with no leukocytes [10, 11].

The procedure requires the following equipment: PRGF system centrifuge, four calibrated test tubes with anticoagulants, Plasmatherm (heating device), micropipettes, and activator (calcium chloride). The preparation phases are the following (Figure 2):

• Drawing of 9 ml of patient’s blood in vacuum test tubes with anticoagulant (sodium citrate).

• Centrifugation in the PRGF system centrifuge at the rcf of 460 g for 8 minutes.

• At the end of the centrifugation, two layers can be seen: yellow (top) and red (bottom). The top yellow layer is composed of three fractions (top to bottom).

• Fraction 1 has a volume of 1 ml and is used for fibrine membrane preparation. This fraction is growth factor poor, hence the name: platelet poor in growth factors (PPGF).

• Fraction 2 has a volume of 0.5 ml and contains growth factors, hence the name: plasma with growth factors (PGF).

• Fraction 3 has a volume of 0.5 ml and represents plasma rich in growth factors (PRGF).

• Bellow Fraction 3 there is a thin layer of leukocytes that is not utilized.

Figure 2.

The PRGF preparation process. (A) Blood drawing into 3.2% sodium citrate vacuum test tubes. (B) BTI centrifuge (PRGF system centrifuge. (C) Micropipetting PPGF, PGF, and PRGF fractions. (D) Three fractions transferred into calibrated tubes and activated with CaCl₂ and heating device (Plasmatherm).

PRGF prepared in this way is activated by procoagulant calcium chloride, whereby 50 μl of calcium chloride is used in 1 ml of PRGF. It takes 6 minutes for PRGF to transition from a liquid to a gel-like state upon activation and, as such, it becomes an autologous biomaterial ready for use. Plasmatherm (a device used for heating) should be set at 37°C to accelerate the transition.

In contrast to the previous, this protocol requires one centrifugation only. Instead of using bovine thrombin, it requires using calcium chloride to activate coagulation, while coagulation acceleration is achieved by using Plasmatherm. The greatest difference between PRGF and other platelet concentrates is the absence of leukocytes in the final product that is used as a biomaterial. Anitua et al., however, consider this as an advantage as they argue that proinflammatory activity is thereby prevented. This question remains controversial since there are many conflicting opinions among scientists.

3.4 Platelet-rich fibrin (PRF)

Platelet-rich fibrin belongs to the second generation of platelet concentrates. It was patented by Joseph Choukroun in 2000. In contrast to the previous platelet concentrates, this one does not require the use of anticoagulants or bovine thrombin as procoagulants. The principle behind preparing this autologous biomaterial starts with physiologically triggered coagulation processes. The equipment required is a phlebotomy pack, vacuum test tubes, and a centrifuge with a fixed angle [12].

The authentic PRF protocol is the leukocyte (L-PRF) or Choukroun’s protocol (Figure 3), which contains the phases listed below:

• Drawing patient’s blood in 9–10 ml volumed vacuum test tubes without anticoagulants

• Prompt transport of the blood to the centrifuge is required followed by centrifugation at 2700 rpm for 12 minutes.

• After some time, platelets that come in contact with the sidewalls of test tubes become activated. This is when the coagulation starts.

• Fibrinogen, initially concentrated at the top of the test tube, is mixed with circulating thrombin and then transformed into fibrin. In this way, the fibrin clot, located in the middle of the test tube, is formed.

• There can be visualized two distinct layers in the test tube upon centrifugation: yellow (top) and red (bottom). There is acellular plasma at the top of the yellow layer, while below that, there is a fibrin clot or so-called PRF.

• It is a rule of thumb that test tubes are placed in a test tube holder after centrifugation is done. Also, the test tubes should be left open for 5 minutes for the clots to mature.

• The matured clots are then extrapolated from the test tubes and placed on the grid of the PRF box. A lid should then be placed on the top of the clots to serve as a weight for the remaining liquid to exit the clots. Such clots are then turned into membranes, hence the name: PRF membranes.

Figure 3.

L-PRF protocol. (A) Vacutainer with clot activator. (B) PRF clots. (C) Centrifuge with chosen protocol. (D) PRF membranes. (E) PRF plug.

Apart from the PRF membranes, PRF plugs can also be made from the PRF clots. For this purpose, PRF clots need to be put into cylinders of the PRF box. On the top, stainless steel weights need to be placed to eliminate the residual liquid. By using this approach, clots are turned into plugs or disks of desired height.

PRF clots contain 100% platelets and the growth factors from the patient’s blood sample. Additionally, they contain 65% leukocytes, which are the growth factor source, especially for PDGF and VEGF.

Besides platelets and leukocytes, PRF contains fibrin too.

There are multiple roles of fibrin in PRF:

• Stimulates angiogenesis—supports microvascularization

• Supports the immune system

• Covers wounds, stimulates epithelization, and enhances healing

• Helps leukocyte migration—a significant role in infected wounds

PRF membrane and PRF disks or plugs are also called solid PRF.

Numerous protocols have been invented to obtain advanced PRF forms. Most of them are based on the change in velocity and time required for centrifugation. The objective was to obtain PRF with the best characteristics possible—uniform platelet distribution along the clot and prolonged growth factor release.

In the text below, we will further be discussing different types of PRF.

3.4.1 Leukocyte-platelet-rich fibrin (L-PRF)

L-PRF is the original version of solid PRF. It was first prepared by centrifuging samples at rcf of 400 g for 12 minutes [13]. The end product was a PRF clot that was used for PRF membrane and plug preparation. The research has shown that the majority of the growth factors could be found at the bottom of a membrane or plug rather than them being evenly distributed across the structures. This can also be considered a disadvantage of L-PRF.

3.4.2 Advanced platelet-rich fibrin (A-PRF)

Advanced platelet-rich fibrin came to light in 2014 [14]. It was a result of a newly introduced slow centrifugation concept, objective of which was to prevent cell loss and increase the number of viable cells. A-PRF has a greater number of leukocytes, platelets, neutrophils, and lymphocytes and has been suggested to have a prolonged growth factor release [13]. It is obtained by centrifuging samples at the rcf of 208 g for 14 minutes [13]. In comparison to the L-PRF membrane, the A-PRF membrane is shorter, has a greater cell-retaining capacity, and it releases larger amounts of growth factors [9, 15].

3.4.3 Advanced A-PRF+

A-PRF+ is a variant of the A-PRF obtained by decreasing the time needed to centrifuge samples while remaining a constant relative centrifugation force of 208 g. Instead of centrifuging samples for 14 minutes, this protocol requires one to do so for 8 minutes [16]. In comparison with the A-PRF and L-PRF, A-PRF+ has been shown to release even greater amounts of growth factors, especially TGF-β1, VEGF, PDGF, EGF, and IGF1 [13, 17].

3.4.4 Advanced liquid PRF (A-PRF liquid)

A-PRF liquid can be prepared by centrifuging samples at 1300 rpm for 5 minutes. The end-product is in a liquid state, which allows it to be mixed with bone grafts, whereby so-called sticky bone is obtained. It can also be used for large membrane preparation.

3.4.5 Injectable PRF (i-PRF)

Injectable PRF is a liquid form of PRF intended for a variety of purposes including but not limited to tissue injections, mixing with grafts to obtain sticky bone, skin injections in the facial region to achieve rejuvenation, joint injections, and large membrane preparation. Choukroun has patented several types of i-PRF:

3.4.5.1 i-PRF

i-PRF can be considered a universal protocol for injectable PRF. The protocol instructs blood sample centrifugation at 700 rpm for 3 minutes.

3.4.5.2 i-PRF M

This protocol is intended for males since they naturally have a greater number of erythrocytes. Consequently, the centrifugation time needs to be longer compared to protocols addressing i-PRF preparation for women to ensure larger quantities of centrifuged i-PRF. In short, it should be centrifuged at 700 rpm for 4 minutes.

3.4.5.3 i-PRF+

The i-PRF+ protocol requires centrifuging blood samples at 700 rpm for 5 minutes. Its purpose lies in obtaining a liquid PRF that is oftentimes used for facial esthetic procedures as well as in orthopedic surgeries.

A novel protocol introduced in 2019 (1300 rpm for 14 minutes) by Choukroun covered both solid and liquid PRF preparation and simplified the preparation process. Namely, this protocol allows us to place test tubes for solid (A-PRF) and liquid PRF (S-PRF, i-PRF+) immediately upon taking blood samples in the very same centrifuge and spinning them according to the same protocol. Consequently, this protocol amnesties us from using different protocols for solid and liquid PRF preparation.

3.5 Sacco’s protocol for obtaining concentrated growth factors in solid form (CGF) and concentrated growth factors in liquid form (LPCGF)

Sacco’s concept entails three major steps. The first is acceleration whereby samples are accelerated for 30 seconds from 0 to 2700 rpm. This is followed by a combination of four protocols (2 minutes 2700 rpm, 4 minutes 2400 rpm, 4 minutes 2700 rpm, and 3 minutes 3000 rpm). Lastly, samples are deaccelerated for 36 seconds (from 3000 rpm to 0). The difference between CGF and LPCGF is in the type of test tubes used. Solid CGF uses glass red cap tubes (PV 200R—Medifuge Blood Separator CGF P Cycle), while liquid LPCGF uses the red cap tubes (PV 200R) with added sodium heparin or blue cap tubes (PV 200P) with separator gel and sodium citrate (Medifuge Blood Separator CGF Cycle) [18, 19, 20].

3.6 BIO-PRF

BIO-PRF was introduced to the practice by Richard J Miron. In contrast to Choukroun’s concept, which uses a fixed centrifugation angle, Richard’s concept uses horizontal centrifugation [21]. This centrifugation method has already been known and has been in use for PRP production; however, it is only after the BIO-PRF protocol had been introduced that it became commercially available for PRF production. This is because the BIO-PRF protocol mandates the usage of horizontal centrifuge [21].

The advantage of horizontal centrifugation lies in the benefit of attaining greater platelet and leukocyte concentrations in both solid and liquid states of PRF. These cells are more evenly distributed across the PRF clot [21]. The concentration of released growth factors is greater. There is less cellular damage and the accumulation of erythrocytes on the sidewalls of test tubes is rare [21].

Miron’s BIO-PRF concept we use today encompasses four protocols:

• Solid PRF

• Liquid PRF

• C PRF

• ALB-PRF

3.6.1 Solid PRF

Solid PRF is prepared by horizontally centrifuging samples at rcf of 700 g for 8 minutes. This protocol is used for PRF membrane preparation from PRF clots.

3.6.2 Liquid PRF

Liquid PRF is also prepared by horizontally centrifuging samples at rcf of 300 g for 5 minutes. This method enables us to attain greater concentrations of both leukocytes and platelets.

3.6.3 C PRF

C PRF is a concentrated PRF, hence its acronym. It is prepared by using a strong centrifugal force of 2000 g for 8 minutes. The result is a greater concentration of platelets, leukocytes, and monocytes in the so-called buffy coat, immediately above the red layer, in which volume ranges from 0.3 to 0.5 mL [22].

The difference between C PRF and i-PRF is in the concentration of platelets; C PRF has a 15 times greater concentration of platelets, while i-PRF has only 2–3 times greater concentration. A very similar situation can be found with leukocytes whose concentrations can be even 500% greater [22].

3.6.4. ALB-PRF (autologous albumin gel and liquid platelet-rich fibrin)

ALB-PRF was made to prolong the regenerative potential of a classical PRF membrane. Namely, the issue is that the PRF membrane becomes resorbed after 15 days at most. Consequently, its regenerative potential, that is, growth factors release, ceases. Due to fast resorption, PRF membranes are not applicable as independent barrier membranes for procedures such as GBR (guide bone regeneration) and GTR (guide tissue regeneration).

The idea to use heat for prolonging PRF membrane viability was introduced by Kawase et al. in 2015. In contrast to classical PRF membranes, Kawase’s heat-compressed PRF was visible even 3 weeks after an in vivo implantation [23]. Although the thermally processed PRF/PPP has had longer viability, its regenerative potential was compromised considering that no cell or growth factor molecule could survive undergoing processes of denaturation (thermal heating) [24]. This has motivated Mour et al. to modify the production protocol of a membrane that consists of a combination of concentrated growth factors and denaturized albumin gel (ALB-CGF) [20]. The protocol entails drawing 9 ml of blood into plastic test tubes without anticoagulants and other additives. This is followed by a centrifugation process according to the protocol for concentrated growth factors in a liquid state by using Medifuge (Silfradent) centrifuge (acceleration for 30 seconds from 0 to 2700 rpm then combination of 4 protocols: 2700 rpm for 2 minutes, 2400 rpm for 4 minutes, 2700 rpm for 4 minutes, 3000 rpm for 3 minutes followed by deacceleration that should last 36 seconds) [20]. The yellow top layer, formed upon centrifugation, consists of platelet-poor plasma (PPP) that can be collected by a syringe in a volume of 2 ml, liquid phase concentrated growth factors (LPCGF), and buffy coat that can also be collected by a syringe in a volume of 4 ml [20]. The PPP in the syringe is then transferred to a special machine that increases its temperature to 75°C for 10 minutes. The heating process leads toward albumin denaturation and albumin gel formation. This is then left in a sterile glass container. Once the gel has cooled down, LPCGF and a buffy coat are poured upon it. It takes 5 minutes for polymerization to finish after which the ALB-CGF membrane, the end-product, is obtained. It has a usage potential in guide tissue regeneration because it resorbs after 4–6 months [20, 24]. However, researchers could not prove that the ALB-CGF membrane can release growth factors in a prolonged manner [20].

Modernized protocol for ALB-PRF membrane consists of the following steps [24]:

• Drawing 9 ml of blood into plastic test tubes without anticoagulants and other additives

• Horizontal centrifugation by using Bio-PRF centrifuge at 700 g for 8 minutes. In this way, the top yellow layer becomes divided layers: the top

• Layer that is platelet-poor, and the bottom layer, which is platelet- and growth factor-rich.

• Use a syringe to evacuate 2 ml of PPP from the top by using an 18G needle

• Put a cap on a syringe and place it in the middle portion of the heating machine (Bio-Heat). The heating (75°C) should last for 10 minutes

• Test tubes with the residual liquid PRF should be kept in the Bio-Cool device to prevent clotting

• After heating the syringe with albumin gel, it should be cooled down in the Bio-Cool device for 1–2 minutes

• Cool ALB gel from the syringe should be poured into a container and shaped

• Use a syringe to evacuate the remaining 1–2 mL of C-PRF (buffy coat) and pour it over the ALB gel. It is required to wait for 15 minutes before usage

• The end product is an ALB-PRF membrane with improved characteristics (Figure 4).

Figure 4.

Modernized ALB-PRF protocol. (A) Blood sampling into PET tubes. (B) BIO-PRF centrifuge set on rcf of 700 g for 8 min. (C) Bio-Cool and Bio-Heat device. (D) Syringes with ALB-gel. (E) ALB-gel in a bowl. (F) C-PRF-buffy coat. (G) Immediately after pouring C-PRF into cooled ALB-gel. (H) ALB-PRF membrane.

Upcoming clinical studies will provide us with more benefits concerning the ALB PRF membrane.

3.7 Sticky bone (concentrated growth factors enriched bone graft matrix)

Sticky bone is a mixture of autologous fibrin glue and bone grafts (autografts, xenografts, allografts) [25]. Sticky bone is also known as a mineralized plasmatic matrix [26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38]. A bone graft obtained in this way is gelatinous in structure, which renders it for shaping. It is rich in growth factors, thereby accelerating tissue regeneration [39, 40]. It has many indications in oral and periodontal surgery as well as in implantology.

Sticky bone can be prepared by using PRP, PRGF, and i-PRF. All these formulations are in a liquid state and are prepared according to different protocols. They differ in their activation mechanisms—transformation to activated platelet gel. To summarize, when in a liquid state, they mix with bone particles or grafts and upon activation they transform to a gel-like state, hence leaving us with a gelatinous mass that we can further shape.

3.7.1 Different sticky bone preparation protocols

3.7.1.1 Sticky bone with PRP

This method entails PRP preparation according to the already established protocol. This is followed by mixing PRP with bone graft. For the mixture to become gelatinous, it should be activated by thrombin of a bovine origin or from autogenic serum. To enhance this, calcium chloride can be added as well.

3.7.1.2 Sticky bone with PRGF

PRGF obtained according to the established protocol is mixed with a bone graft. The mixture is then activated by adding calcium chloride and heating.

3.7.1.3 Sticky bone with i-PRF

This method has two main advantages:

• It uses test tubes without anticoagulants for PRF preparation

• The protocol for i-PRF is simplified (only one centrifugation is required according to either Choukroun’s or Miron’s protocol for liquid PRF preparation).

To prepare sticky bone, already prepared i-PRF is mixed with a bone graft. Thanks to physiologic coagulation processes, the mixture becomes gelatinous after some time. To accelerate the coagulation process and to enhance density and firmness, adding 1–2 fibrin clots to the mixture can be considered (Figure 5).

Figure 5.

Making sticky bone. A) Adding i-PRF into the bone substitute. B) Gel starting to form. C) Gelling completed. D) Sticky bone.

Sticky bone can be also prepared in a way that uses scissors to cut one or two PRF membranes into pieces. This is followed by adding a required amount of bone graft, thereby making a mixture. This should be thoroughly mixed all together with a PRF exudate. The PRF exudate is a liquid that is segregated upon PRF clot compression in the process of PRF membrane preparation. It contains autologous thrombin that stimulates coagulation as well as gelatinization. This gel-like mass can further be shaped in the desired shape. Every few seconds, i-PRF should be added to it drop by drop. In several minutes, the mass becomes gelatinous, and the sticky bone formed in this is ready for use.

3.8 BIO-bone

Bio-bone is a variant of sticky bone. It has improved characteristics and was patented by Richard Miron. Bio-bone is, in fact, a combination of Alb-gel, bone graft, and injectable PRF. In other words, in this autologous biomaterial, bone graft and ALB-PRF membrane with prolonged viability (4–6 months) are found together.

A Bio-PRF preparation protocol is identical to the ALB-PRF membrane preparation protocol. However, there is one distinction: after pouring one part of a liquid PRF (CGF) over a cooled ALB-gel, a layer of bone graft (preferably allograft) should be added to it. Furthermore, the rest of the PRF (CGF) should be poured over this construction. It takes 15 minutes for this mass to become gelatinous and ready for use.

Thanks to the prolonged viability of the ALB-PRF membrane, sticky bone prepared in this way can be used in augmentative procedures without the usage of a collagenous membrane, consequently reducing the financial costs of the procedure.

4. PRF preparation equipment

4.1 Centrifuge

The concept of PRF preparation is based on full blood centrifugation, which results in blood components’ separation (for simpler understanding: red and yellow fractions). Relative centrifugation force (rcf) is the key in this process rather than rotations per minute (rpm) that represents a variable parameter dependable on centrifuge design and radius [41].

Nowadays, there are different protocols for PRF preparation that use centrifuges with different rotors, test tube angulation and design [42]. These protocols are based on the rpm values rather than rcf values. Although there are numerous articles addressing PRF, they cannot be adequately compared simply due to the heterogeneity in rcf reporting [41]. In other words, the very same protocol that utilizes different centrifuges provides us with different results. For this reason, it is highly important to use certified centrifuges such as the Intraspin device for L-PRF protocol (that has a fixed centrifuge angle of 33°), Duo Quatro centrifuge for A-PRF+ protocol (that has a fixed centrifuge angle of 40°), and Bio-PRF centrifuge for Bio-PRF protocol (that has horizontal centrifugation adjustment) (Figure 6) [42].

Figure 6.

Fixed angle and horizontal centrifuge.

4.2 Vacuum test tubes for platelet concentrate preparation procedures

In the process of obtaining platelet concentrates, vacuum test tubes (also known as vacutainer tubes) are used. There are two types of test tubes: glass- and plastic-made test tubes (Figure 7).

Figure 7.

Blood collection tubes. (A) 10 ml glass vacuum red cap tubes for PRF membranes. (B) 10 ml silica-coated plastic tube with no additive for PRF membranes. (C) 13 ml, 10 ml, and 9 ml PET tubes with no additive added for sticky bone, large PRF membranes, and facial esthetics.

The advantage of using glass test tubes lies in the glass’ ability to stimulate coagulation [43]. Considering that a modern approach to PRF preparation entails the avoidance of additives and anticoagulants, empty glass test tubes are the ideal choice for PRF membrane and plug preparation. In case we opt for plastic test tubes, they should be equipped with a clot activator that accelerates the coagulation process. Such activators are micronized silica particles that coat the inside surface of vacutainers. If a test tube is coated with a clot activator, then its wall is blurred. Regardless of that, it has recently been reported that silica particles have side effect [44]. It is mostly related to a possible negative effect on tissue regeneration, cytotoxic effect, cell apoptosis, and PRF clot size [44, 45, 46]. Albeit no serious side effects have been reported up to date, it is recommended to use test tubes without any chemical additives [44, 45, 46].

Plastic vacuum test tubes without additives are made of polyethylene terephthalate (PET) plastic [47]. In contrast to glass and silica particle-coated plastic test tubes with a red cap that are hydrophile, these plastic test tubes are hydrophobic. In these test tubes, the yellow fraction (liquid PRF) remains in a liquid state for 30 minutes under the hermetic conditions upon centrifugation. In conclusion, these test tubes are used in liquid PRF, CGF, and sticky bone preparation. Liquid PRF nowadays is frequently used in facial esthetic procedures.

A correct test tube selection is of vital importance for obtaining safe and high-quality PRF products. Oftentimes this may be an issue considering that there are test tubes of low and ambiguous quality in the market. For this reason, clinicians are recommended to use test tubes from safe and trustworthy retailers.

4.3 Phlebotomy equipment

Phlebotomy equipment includes Esmarch’s tourniquet and blood collection butterfly needles with tube holders (Figure 8).

Figure 8.

Phlebotomy equipment. (A) Esmarch’s tourniquet. (B) Safety butterfly blood collection needle with holder.

4.4 PRF kit

Besides an appropriate centrifuge and test tubes, the basic PRF kit includes (Figure 9):

• Test tube holder

• PRF box (for PRF membrane and plug production)

• PRF hand tools (bone compactors, double-ended graft spoon, PRF pad, PRF forceps, scissors).

Figure 9.

Basic PRF kit.

5. Examples of PRF and sticky bone applications in oral surgery

There are many indications for PRF and sticky bone usage in dentistry. In this section, only indications related to oral surgery and implantology will be discussed further.

5.1 Immediate dental implant placement after tooth extraction: single-phase approach

If the extraction of a multi-rooted tooth was atraumatic—that is, with preservation of the interradicular septum—it is possible to place an implant altogether with empty alveoli augmentation. An example is shown in Figure 10. In this patient, there was an indication to extract the first mandibular molar tooth. Immediately after the atraumatic extraction, an implant was placed in the intact interradicular septum. The empty alveoli were augmented by a mixture of bone graft and PRF membrane pieces. As a bone replacement, Novocor plus bone was used. It is a natural bone grafting material consisting of natural coral granules—Madreporic Coral consisting of 98% aragonite calcium carbonate. A PRF membrane covering the structure was placed. This was followed by positioning of the apical mattress stitch and a primary suturing of the elongated flap. After an osteointegration period, the patient was readmitted for a prosthetic procedure that entailed screw-retained crown placement.

Figure 10.

Implant placement immediately after tooth extraction. (A) Tooth indicated for extraction. (B) Implant placed into inter-radicular septum. (C) PRF membrane. (D) A mixture of PRF membrane cut into pieces and bone substitute. (E) Grafting the empty alveolar socket. (F) PRF membranes adapted over graft. (G) OPG view of inserted implant (H) final prosthetics—screw-retained crown.

5.2 Implant placement after tooth extraction and residual alveolar ridge augmentation: two-phase approach

This example (Figure 11) represents a case of a patient with severe periodontitis (horizontal and vertical bone loss) affecting two molar teeth. In the first visit, extraction of the two molar teeth ensued followed by a thorough curettage. Additionally, bone augmentation with sticky bone was performed. As a bone graft, Novocor plus bone was used. Nine months later, one implant was placed in the augmented region.

Figure 11.

Implant placement 9 months after bone augmentation- two-phase approach. (A) Teeth 47 and 48 indicated for extraction. (B) Sticky bone. (C) Grafted area. (D) Clinical appearance 9 months after augmentation with sticky bone. (E) OPG view 9 months after augmentation with sticky bone. (F) OPG of the implant inserted into the augmented bone.

5.3 Single implant placement in lower jaw with an insufficient vertical and horizontal dimension

This case is presenting immediate implant placement in the lower jaw in the premolar region with a lack of bone height and width. The patient had complicated extraction of root 44, which led to greater bone loss. Implant placement was carefully planned with the help of a 3D CBCT scan. Since there was a lack of bone height toward the mental canal, we planned to leave part of the implant not covered by bone and above the crest line. Missing bone was augmented with the help of artificial bone Novocor plus in combination with platelet-rich fibrin creating “sticky bone.” The implant used in this case was 3P by BB dental, and later we placed a screw-retained metal-ceramic crown (Figure 12).

Figure 12.

Single implant placement in lower jaw with an insufficient vertical and horizontal dimension. (A) Clinical appearance of the area where the implant should be inserted. (B–D) CBCT planning. (E and F) The implant threads are exposed. (G) Sticky bone. (H–J) Augmentation with sticky bone. (L) Flap repositioned and sutured.

5.4 Lateral sinus lift with sticky bone coupled with a simultaneous implant placement

Tooth loss results in bone resorption. In the transcanine sector of the maxilla, the most characteristic consequence of tooth loss is the lowering of the maxillary sinus, which imposes unfavorable conditions for implant placement. In the cases when the indication for implant placement exists, whereby the height of the remaining bone is less than 5 mm, a lateral sinus lift procedure is done. In this example (Figure 13), lateral sinus lift with sticky bone and PRF membrane usage coupled with three dental implant placements was done in a single visit. At the end of the osteointegration process, which went without any complications, three single screw-retained crowns were placed.

Figure 13.

(A) Insufficient bone height due to maxillary sinus pneumatization. (B) Incision line. (C and D) Lateral window approach. (E) Three implants inserted after sinus lift. (F) PRF membranes placed over sinus membrane. (G) Sticky bone. (H) Collagene membrane. (I) Flap repositioned and sutured. (J) OPG immediately after implant placement. (K) Three screw-retained solo prosthetic crowns. (L) Intraoral view.

5.5 Apicoectomy with a huge cystectomy in the maxilla

This case (Figure 14) represents a huge radicular maxillary cyst encompassing three teeth (11, 21, and 22). The cyst has destroyed a part of the vestibular and palatine bone with its expansive growth. Once endodontic treatment of the affected teeth had been done, apicoectomy with cystectomy was performed. The bony defect was filled with a sticky bone, which was prepared with a xenograft (BioSS). This was covered with PRF membranes and a collagen membrane. The flap was repositioned and sutured. Three months after the surgery, the OPG X-Ray (Figure 12L) shows excellent signs of bone regeneration.

Figure 14.

Apicoectomy with large cystectomy in the maxilla. (A) Arrows pointing to large radiolucency in the maxilla. (B) Large radiolucency on CBCT image- axial view. (C) Clinical appearance. (D) Cyst destroyed vestibular cortical lamella. (E) Palatal flap raised. (F) Enucleated cyst. (G) Sticky bone. (H) Bone cavity filled with sticky bone. (I) PRF membranes. (J) Collagene membranes. (K) Flap repositioned and sutured. (L) OPG 3 months after the surgery.

5.6 Apicoectomy with extraoral fistula excision on the face

This is a very interesting case of a patient with an extraoral fistula on the face that has dental etiology (Figure 15). The patient had been mistreated for a longer period as the facial pathology had been considered a dermatologic condition. The examination by the oral surgeon set the correct diagnosis. It was an infection of a dental etiology of tooth 13 that resulted in a periapical lesion, which was not treated and has consequently resulted in an extraoral fistula. The therapeutic approach entailed endodontic treatment of the tooth 13. During the treatment, the extraoral fistula on the face was regressing. Upon competition of definite root obturation of the tooth 13, apicoectomy ensued and was followed by the removal of the periapical bony lesion as well as excision of the canal of the fistula. For the oral wound to heal well, two PRF membranes were placed. Throughout the opening on the face, additional two PRF membranes were extraorally placed. Three months after the surgery, it can be noted (Figure 13L) that intraoral healing was excellent with almost unnoticeable scar extraorally.

Figure 15.

Large extraoral fistula of dental origin. (A) Extraoral fistula. (B) Tooth 13 with periapical radiolucency causes the infection. (C) Definitive canal obturation. (D) The appearance of fistula during endodontic treatment. (E) Intraoral view. (F) Periapical lesion. (G) Root resected. (H) PRF membranes placed over the operating wound. (I) Flap repositioned and sutured. (J) Sinus tract. (K) Extraoral fistula was extirparted, two PRF membranes were placed in the hole, and the wound was sutured. (L) Almost invisible scar on the face.

6. Conclusion

Platelet concentrates were discovered by manipulating normal physiological processes such as hemostasis. All known platelet concentrates are high in growth factors and so have tremendous potential in regeneration processes. PRF, BIO-PRF, Alb-PRF, and sticky bone are newer generations of platelet concentrates that will influence the direction of regenerative dentistry. In the future, we may expect a greater number of clinical trials that will investigate the full potential of novel platelet concentrates.

Conflict of interest

The author declares no conflict of interest.

Notes/thanks/other declarations

I would like to thank my parents, Prof. dr Halid Sulejmanagic and Prim. dr Ajsa Ismailovic Sulejmanagic, as well as the Private Dental Clinic “Sulejmanagic,” for their support during my professional and scientific development.

I’d like to thank my friend and colleague Armin Klancevic DMD for drawing Figures 1 and 2 for this chapter.

I would like to express my gratitude to Richard Miron DDS, MSc, PhD, Dr. med. dent for donating BIO-PRF equipment to the Faculty of Dental Medicine with Clinics University of Sarajevo Bosnia and Herzegovina, which enabled me to gain new experiences in the field of novel platelet concentrates such as ALB-PRF.