Introduction

Dental implants are relatively innovative and superior treatments in dentistry and are widely used in an increasing number of cases. Most of the techniques are evidence‐based and predictable. However, in many cases, the implant site is inadequate for poor bone quality and quantity.

For these reasons, tissue regeneration is frequently required in implant dentistry, and autologous platelet concentrates are promising and innovative therapeutic approaches.

Implant osseointegration is a concept widely supported by the literature. In 1990, Zarb and Alberktsson defined osseointegration from a clinical point of view as a rigid and clinically asymptomatic fixation process of an alloplastic material with bone subjected to functional load [1, 2].

Since then a lot is changing and a lot is still to come.

The most important aspects for successful osseointegration are the biological characteristics of the host site (the patient) and the macro‐ and microstructure of the implant. Dental implant surfaces have now achieved outstanding performances, which were previously unimaginable.

Traditional implant surfaces can be classified into two broad categories: smooth (machined) and rough (treated). It has now been demonstrated that implants with micro‐rough surfaces stimulate a greater and faster apposition of new bone than implants with smooth surfaces. The differentiation of osteoblasts and their response during osseointegration varies and depends on the implant surface in its nano‐ and microtopographic aspects. Various phenomena, such as clot formation, retention of the fibrin texture, and differentiation of the cell population, are influenced by surface topography.

The biomechanical phenomena that arise at the interface between biological tissues and the implant surface as well as the implant–prosthetic material are governed by the design (external micro and macrostructure), the patient’s response, the surgical technique, and by the conditions and/or times of loading. Exposure of the implant surface to air leads to the formation of an oxide layer, which constitutes a benevolent substrate in the interaction with body fluids, the first and fundamental mediator of all biological phenomena. The insertion of the implant and the consequent surgical trauma cause the interruption of the bone blood vessels, with subsequent bleeding: this determines the contact between the biological fluids of the host and the surface of the newly inserted implant. The absorption on the implant surface of ions and macromolecules of blood origin and fibrin is fundamental for the platelet adhesion itself and the consequent osteogenesis. This ensures an extremely high percentage of osseointegration, even in the most complex situations.

On the other hand, this means that the margins for further improvement of modern surfaces through mechanical or chemical procedures are very small. Major improvement can be achieved biologically by adding autologous growth factors, obtained by processing the patient’s venous blood, to the implant surface.

The study on tissue reparative processes has highlighted the fundamental role played by platelets (in this context), which are physiological reservoirs of growth factors and proteins. There are various platelet concentrates, such as platelet‐rich plasma (PRP), platelet‐rich fibrin (PRF), and concentrated growth factor (CGF), which reconstruct bone defects [3].

Numerous studies have shown that PRF provides positive results in tissue engineering [4]. Research by Sohn et al. has demonstrated the greater regeneration capacity of the CGF and its multi‐purpose use [5]. After a long phase of the study, our therapeutic choice was the use of the CGF, for the following reasons.

It is 100% autologous and biocompatible, requires a simple preparation, is easily identifiable, has a very high concentration of platelets in a fibrin network, has a presence of growth factors and no manipulation of the product is necessary when exclusively using autologous blood products, without the addition of other substances.

Platelets, in particular, contain biologically active proteins at high concentrations and support cell healing, growth, and morphogenesis [6]. In addition to platelets, CGF contains fibroblasts, leukocytes, and endothelial cells for angiogenesis and tissue remodeling; and provides a matrix for cell migration [7, 8]. CGF is a fibrin biomaterial rich in the growth factors obtained by centrifugation of venous blood, at alternating speeds based on a special device (Silfradent Co.) [8].

CGF, associated with guided bone regeneration, has been used to accelerate new bone formation. Due to its special characteristics, including lack of immune reaction, the capability of accelerating tissue healing and vascularization, and anti‐swelling properties, CGF is widely used in implant surgery [9–11]. However, the interaction between CGF and the dental implant is not clear. The addition of autologous growth factors to the implant surface is hindered by titanium’s characteristics of extremely low wettability [12, 13]. This means that simply wetting the implant with autologous growth factors is of little use unless it is left in immersion for more than 30 minutes [13]. This makes the procedure difficult to include in the clinical routine. Because of these difficulties, the challenge of producing a biologically active surface remains. Our study experiences demonstrate a protocol that could produce a biologically active implant surface. The growth factors are incorporated onto the implant surface, using a dedicated implant ampoule, which enables the procedure to be carried out in a closed field and a second device (Roundup) to incorporate GF onto the surface in a few seconds. We verified the adhesion of CGF on the titanium implant surface and then quantified the release of the vascular endothelial growth factor (VEGF) from CGF and the liquid phase of CGF (LPCGF) permeated implants. Later on, we tested other platelet‐derived growth factors release, bone morphogenetic proteins, and stem cells. In the end, we evaluated the clinical outcome on patients.

This justifies the assertion that the first biologically active implant surface was produced, permeated with both fibrin, essential for hosting the cellular network, and growth factors, essential for creating intercellular communication and neo‐angiogenesis. The osseointegration process thus becomes bilateral, operating both from the bone toward the implant and the implant toward the bone. This reduces the healing time and enhances the physiological response by four to six times [14, 15].

The research of this activity was focused on the adhesion of autologous growth factors, extracted with innovative techniques from the patient’s venous blood, on the surface of the device, thus obtaining a biologically active implant surface. These growth factors are appropriately characterized to evaluate the synergistic effect from the point of view of their activity.

Growth Factors

An ideal bone graft in dentistry should be biomimetic and should have the ability to induce differentiation of the appropriate cells for bone formation; it should be cost‐effective and it should be able to achieve consistent and predictable results without being affected by different levels of technical ability of the clinician.

The dream of any surgeon would be to have a miraculous substance that could heal all wounds quickly and avoid the secondary complications of surgical procedures, such as edema, pain, dehiscences, infections, and hematomas.

The bad news is that this substance does not exist.

To meet these demands, research has focused on the use of bioactive molecules to induce bone formation and reduce post‐surgery complications.

The growth factors associated with repair and regeneration coordinate important cellular processes such as stimulation and inhibition of growth, development and differentiation, mitogenesis, chemotaxis, angiogenesis, and apoptosis [16, 17].

Growth factors can be distinguished in:

• Natural: Extracted from cells or tissues and then reprocessed;
• Synthetic: Completely synthesized in the laboratory.

The characteristic common to both is their exceptional reactivity even at very low concentrations.

At present, we consider only autologous growth factors.

Synthetic growth factors could tomorrow be an alternative as far as legislative and even medico‐legal problems pose today a serious difficulty to their clinical use. On the other hand, the mechanism of action of growth factors is only partly known and for this reason, the application of synthetic growth factors poses difficulties also from an ethical point of view.

What normally happens is that the degranulating platelets release growth factors, which stimulate cell proliferation, intercellular communication, and neoangiogenesis. They, therefore, give way to a process aimed at hemostasis and healing.

This is a well‐known biological process that we are going to enhance and accelerate through the on‐site application of the patient’s autologous growth factors. The safety and the manageability in the application of growth factors (GF) are out of the question since it is an autologous product to which no exogenous components are added.

There are different methods and protocols to obtain GF from a patient’s blood. The most used are Platelet Rich Plasma (PRP), Plasma Rich in Growth Factors (PRGF), Advanced Platelet‐Rich Fibrin (A‐PRF), and Concentrated Growth Factors (CGF).

Platelet‐rich plasma (PRP) is a concentrated preparation of platelets now termed “first‐generation platelet concentrate.” PRP is a rich source of growth factors and promoted significant changes in monocyte‐mediated pro‐inflammatory cytokine/chemokine release. Leukotriene A4 (LXA4) was increased in PRP, suggesting that PRP may suppress cytokine release, limit inflammation, and, thereby, promote tissue regeneration [17]. Platelet activation allows access to autologous growth factors, which by definition, are neither toxic nor immunogenic and are capable of accelerating the normal processes of bone regeneration. In general, a large body of PRP studies demonstrated that PRP stimulates the proliferation and differentiation of fibroblasts, osteoblasts, chondrocytes, and mesenchymal stem cells [18, 19]. PRP can thus be considered a useful instrument for increasing the quality of regenerated bone [20] and wound healing of injury‐associated soft tissue defects [21–25] for chronic non‐healing tendon injuries.

Platelet‐rich fibrin (PRF), a self‐clotted preparation of PRP derivative, also overcame these matters. Blood samples collected in the absence of anticoagulants are immediately centrifuged to form fibrin clots. This simple preparation procedure has been widely accepted in various medical fields and spread worldwide. The developer of PRF further modified it to an advanced form (A‐PRF), which is expected to contain a relatively greater number of white blood cells (WBC) [26]. Because of low‐speed centrifugation, this fibrin clot is softer than that of the original PRF. On the other hand, concentrated growth factors (CGF), another modified form of PRF, are prepared by repeatedly switching the centrifugation speed and are characterized as a relatively stiffer fibrin clot [27]. Therefore, it has been anticipated that the difference in mechanical characteristics may produce a difference in the growth factor content.

Novel technologies (such as the cell concentrator Silfradent^®) make possible the extraction of the so‐called CGF. CGF is an autologous leukocyte‐rich and platelet‐rich fibrin (L‐PRF) biomaterial termed “second‐generation platelet concentrate.” CGF contains autologous osteoinductive platelet growth factors and an osteoconductive fibrin matrix [28]. It is also present in CGF: TGF‐b1, VEGF, and CD34 positive cells [29]. The application of CGF resulted in excellent healing of critical‐size bone defects in vivo [28], hair loss [30], and promissory in the periphery and myocardial ischemia [31].

Platelet count is the first variable to consider. Absolute platelet count varies depending on the platelet concentration in the subjects’ peripheral blood. PRP devices can be usually divided into lower (2.5–3 times baseline concentration) and higher (5–9 times baseline concentration) systems. It would seem intuitive that a higher platelet count would yield more growth factors and better clinical results; however, this has not yet been determined. Graziani et al. suggested that the optimal concentration of PRP was 2.5 x baseline and above this, there may be an inhibitory effect [32]. The optimal concentration was estimated at 2.5‐fold to stimulate soft tissues. However, 2.5/3‐folds upper baseline was optimal to stimulate bone regeneration [33, 34].

PRP containing white blood cells will have different biologic activity than PRP in which they are absent. The lower platelet count systems separate the whole blood into two components: one with the cellular components and the other consists of serum in which the platelets are suspended. The higher platelet count systems separate the whole blood into three fractions: the red cells, serum, and buffy coat. The buffy coat contains both platelets and WBC.

WBC can be further classified into different types. These include neutrophils, monocytes/macrophages, and lymphocytes. Their roles in tissue healing are different. Neutrophils are phagocytic and contain over 40 hydrolytic enzymes. Their activation leads to phagocytosis of debris and the release of oxygen‐free radicals and proteases. This release of toxic molecules from the neutrophils can lead to secondary damage to the muscle [35, 36]. Whether or not neutrophils have a negative or positive effect on acute or chronically injured soft tissue is unknown.

Macrophages are the tissue forms of the circulating monocytes. Their role is the removal of debris and they are primarily phagocytic. They also have a role in balancing the pro‐inflammatory and anti‐inflammatory aspects of the healing [36, 37]. Since it is not possible to fractionate different types of white blood cells out of PRP, it may be that the absence of macrophages is more detrimental to healing than any secondary damage inflicted by neutrophils.

The growth factor contents in PRF and CGF preparations and their bioactivities have been demonstrated in vitro studies by several independent scientific groups [22, 24–35], the regenerative effects of PRF/CGF are not solely due to fibrin clots. The major source of growth factors in PRF preparations is its exudate; however, as a minor source, growth factors are thought to be secured by fibrin fibers [25]. The angiogenic activity of PRF/CGF preparations in endothelial cell cultures and the chick embryo chorioallantoic membrane (CAM) assay [36], demonstrated that PRF/CGF preparations are somewhat more potent in angiogenesis than PRP preparations. The comparison of the growth factor contents in four types of PRP derivatives (PRP, PRGF, A‐PRF, and CGF) prepared from the same donors showed that both A‐PRF and CGF preparations contained TGF‐β1, PDGF‐BB, VEGF, IL‐1β, and IL‐6 at levels similar to or higher than PRP preparations [25]