**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) [18–20].

#### **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:


### *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 heatcompressed 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]:


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