**4. Platelet-Rich Plasma (PRP)**

The PRP as a concept can be represented as the volume fraction of blood plasma, where the platelet concentration is increased compared to the baseline serum level. In platelet rich plasma, besides platelet concentrate, small amounts of white blood cells are also present and other blood components as well. Ideal is a concentration of 1,407,640 cells in microliter (with a standard deviation of 320.100) [21]. This value corresponds to a number of platelets, approx‐ imately five times higher than the normal, nomber in the blood, which is usually in the range of 150,000 to 350,000 cells in microliter (approximately an average value of about 200,000 cells in microliter) [22].

Marx et al. suggest that in order to obtain effective treatment with PRP, the approximate number of platelets should be about 1,000x103 platelets in microliter in volume of a 5 ml plasma [23,24]. Jacobson et al. studied the influence of higher than five times the platelet concentration and suggested that angiogenesis is initiated at concentrations of platelet rich plasma from 1,500x103 platelets in microliter and continues up to 3,000x103 platelets in microliter. Interestingly, at concentrations of 5,000x103 platelets in microliter, inhibition of angiogenesis is observed. They reached the conclusion that additional laboratory studies are necessary to elucidate the optimal concentration for use with a particular pathology. Concentration of the platelets less than 300x103 platelets in microliter is called ''low'', 300 -800x103 platelets in microliter is considered ''moderate'', and >800x103 platelets in microli‐ ter is called ''high'' [7,25].

The presence of white blood cells in PRP may affect its use, independent of the concentration of the platelets [26]. The presence of the layer of leukocytes has led to the current classification, which distinguishes a clean (without leukocytes) PRP, identical to the platelet rich plasma of Anitua, and PRP with leukocytes. Also present in the classification the fibrin-rich gel known as a clean, without leukocytes, platelet-rich fibrin, and platelet-rich fibrin with leukocytes or Choukroun type [27,28].

Ehrenfest et al. categorized the kits based on their fibrin and leukocytes content.


**•** Platelet-rich and leukocyte-rich fibrin (L-PRF): Choukroun's PRF [27,29].

This classification, despite the various comments, is an important step towards the systemat‐ ization of knowledge, which allows for more objective comparisons of the results of treatment. It is necessary in future therapy with PRP to choose the allegedly most appropriate kit for the particular pathology [29].

PRP is an autologous therapy, which stimulates the healing of tissues and positively influences the recovery processes. It ensures a high concentration of platelets in a small volume of plasma. Once platelets are activated, platelet aggregation occurs and the contents of their solid granules and alpha granules is released [30,31]. In order to release the growth factors of the platelets, PRP must be activated to start the clotting cascade. The activation of platelets in vivo is done in three ways: by adenosine diphosphate, via membrane phospholipids system (arachidonic acid), and by inducing the presence of thrombin [32]. In hospital and clinical conditions, the conversion of prothrombin to thrombin takes place by means of calcium dichloride and thus platelets are activated. Another way is by means of autologous or bovine thrombin [25].

In order for the platelets and the PRP to be sufficiently functional, they should be activated as with tissue injury. As a result platelets release their contents and a cascade of events is initiated. Normal collagen is repaired. Collagen repair consists of the following phases: inflammation, proliferation, and remodelling [33,34]. Each of these stages is needed to restore the normal function of the tissue.

The first phase may last up to three days. The growth factors are released during this phase. Then fibroblasts penetrate and initiate the second phase of wound healing. At this phase fibroblast differentiation and neovascularization are observed. During the third phase collagen matures and strengthens as this process may last over a year [33-35].

## **5. Cascade of wound healing**

platelets also release various substances depending on the stimuli, by which they are activated [16,18]. Alpha granules contain many substances with directly opposing activities. This implies the existence of a mechanism for specific release of only a specific content of the granule, which is possible, but this is still not well studied [19,20]. Inflammation, immunity, and tissue

The PRP as a concept can be represented as the volume fraction of blood plasma, where the platelet concentration is increased compared to the baseline serum level. In platelet rich plasma, besides platelet concentrate, small amounts of white blood cells are also present and other blood components as well. Ideal is a concentration of 1,407,640 cells in microliter (with a standard deviation of 320.100) [21]. This value corresponds to a number of platelets, approx‐ imately five times higher than the normal, nomber in the blood, which is usually in the range of 150,000 to 350,000 cells in microliter (approximately an average value of about 200,000 cells

Marx et al. suggest that in order to obtain effective treatment with PRP, the approximate number of platelets should be about 1,000x103 platelets in microliter in volume of a 5 ml plasma [23,24]. Jacobson et al. studied the influence of higher than five times the platelet concentration and suggested that angiogenesis is initiated at concentrations of platelet rich plasma from 1,500x103 platelets in microliter and continues up to 3,000x103 platelets in microliter. Interestingly, at concentrations of 5,000x103 platelets in microliter, inhibition of angiogenesis is observed. They reached the conclusion that additional laboratory studies are necessary to elucidate the optimal concentration for use with a particular pathology. Concentration of the platelets less than 300x103 platelets in microliter is called ''low'', 300 -800x103 platelets in microliter is considered ''moderate'', and >800x103 platelets in microli‐

The presence of white blood cells in PRP may affect its use, independent of the concentration of the platelets [26]. The presence of the layer of leukocytes has led to the current classification, which distinguishes a clean (without leukocytes) PRP, identical to the platelet rich plasma of Anitua, and PRP with leukocytes. Also present in the classification the fibrin-rich gel known as a clean, without leukocytes, platelet-rich fibrin, and platelet-rich fibrin with leukocytes or

Ehrenfest et al. categorized the kits based on their fibrin and leukocytes content.

**•** Platelet-rich and leukocyte-rich plasma (L-PRP): Curasan, Regen, Plateltex,

**•** Pure platelet-rich plasma (P-PRP): cell separator PRP, Vivostat PRF, or Anitua's

recovery are some of the most characteristic features of platelets [7].

**4. Platelet-Rich Plasma (PRP)**

176 Immunopathology and Immunomodulation

in microliter) [22].

ter is called ''high'' [7,25].

Choukroun type [27,28].

**•** SmartPReP, PCCS, Magellan, or GPS PRP; **•** Pure platelet-rich fibrin (P-PRF) Fibrinet;

**•** PRGF;

Wound healing is a complex process that comprises of a sequence of events, starting from the time of injury and lasting several months, which can be divided into three phases: inflamma‐ tion, proliferation, and remodeling [10,36,37].

In the inflammatory phase is observed at the beginning of the cascade with the activation and aggregation of platelets and the formation of a fibrin matrix. With platelets degranulation, cytokines are released that guide the healing process. Leukocytes are attracted by the cytokines through hemostasis and migrate to the damaged area. The first leukocytes involved and responsible for the initial wound cleaning are the neutrophils, which eliminate the bacterial and cellular waste [36,38,39].

During the proliferative phase, which spans through the next few days, monocytes migrate to the wound area under the influence of chemical signals from growth factors. The differenti‐ ating of macrophages by circulating monocytes is observed. Platelet signaling and modulation function are also performed by the macrophages. Gradually, the damaged area is impover‐ ished of platelets. The macrophages clean the area through phagocytosis and secrete factors responsible for the formation of granulation tissue from the fibroblasts. During this phase, angiogenesis also begins under the influence of growth factors and thrombin. With the recovery of vascular endothelial cells new vessels also develop. Endothelial cells are activated by thrombin, while a process of interaction is also ensured, which restricts the intensity of the formation of new vessels [40]. In this phase mesenchymal stem cells also emerge and their differentiation is performed in specific tissues such as bone, cartilage, or vascular tissues being determined by chemical signals [36,38,39].

At the time of the remodeling phase, contraction of the collagen and convergence of the edges of the wound is observed. Cell density and vascularization is reduced, excess matrix is removed, and the collagen fibrils are aligned along the stress lines, which increases the strength of the newly-formed tissue [10]. Accumulated granulation tissue remodels or slowly trans‐ forms into a specific tissue such as skin or bone [36,38,39].
