1. Introduction

1.2. Improved PRF protocols- A-PRF, A-PRF+ and i-PRF

Depending on individual surgical requirements, PRF can be prepared in solid and/or liquid form (e.g. a-PRF, iPRF, a-PRF+) by altering the G-force during centrifugation (Table 1 and Table 2). Both matrices are rich in growth factors and the differences between different second-generation platelet concentrates have been shown to be small and probably clinically not significant [11,12]. The second-generation PRF concentrates such as advanced platelet rich fibrin (A-PRF), A-PRF+ and injectable PRF (i-PRF) use lower centrifugal “g-forces” to obtain higher growth factor release compared with the original PRF protocol. The slower centrifugation reduces cell loss and increases the concentration of leukocytes in the PRF matrix (mostly in the buffy coat layer). The effect is to increase the concentration of the total number of cells (neutrophils, lymphocytes, undifferentiated monocytes, and immune cells) as well as the growth factors that are actively involved in bone and soft tissue regeneration. Advanced PRF (A-PRF) is obtained using reduced g-forces; at 1,500 rpm (230g) for 14 min or at 1,300 rpm (200g) for 14 min [11] (see Table 1.)

Table 1. Protocols for producing different formulations (solid and liquid) of PRF [12].

Table 1. Protocols for producing different formulations (solid and liquid) of PRF [12].

PRF Preparation	Tube	RCF (g)	Time (mins)	Speed (rpm)	Evidence
Solid Matrix
L-PRF	Glass or Silica coated	408	12	2700	Choukroun, 2001 [7]
A-PRF	Glass or Silica coated	194	14	1500	Ghanaati et al, 2014 [14]
A-PRF+	Glass or Silica coated	145	8	1300	Fujioka-Kobayashi et al 2017 [15]
Liquid/Flowable matrix
i-PRF	Plastic (PET)	60	3	700	Miron et al 2017 [9]
C-PRF	Plastic (PET)	408	12	2700	Miron et al 2020 [10]

Table 2. Conversion table for calculating g-force from rpm and radius arm of the centrifuge device. (Adapted from Sigma Aldrich).

Table 2. Conversion table for calculating g-force from rpm and radius arm of the centrifuge device. (Adapted from Sigma Aldrich).

Radius (cm)	4	5	6	7	8	9	10	11	12	13
Speed (rpm)
1000	45	56	67	78	89	101	112	123	134	145
1500	101	126	151	176	201	226	252	277	302	327
2000	179	224	268	313	358	402	447	492	537	581
2500	280	349	419	489	559	629	699	769	839	908
3000	402	503	604	704	805	906	1006	1107	1207	1308

The spin protocols change between different centrifuge devices and can be calculated using a formula that is based on the radius of the rotor arm of the device and the speed of rotation (see Table 2) Generally, slower centrifugation protocols of 1300 rpm × 8 min produce more evenly distributed number of platelets compared with the original PRF protocol of 2700 rpm x 14 min. Furthermore, Kobayashi et al, 2016 have demonstrated much-sustained release of GFs from slower preparation of PRF (e.g. a-PRF) compared with the original PRP or PRF protocols [17,18]. Injectable-PRF (i-PRF) protocol (60g g-force at 3 mins) produces the highest concentration of leukocytes/platelets in a smaller volume of liquid matrix. Plastic (PET) tubes used in i-PRF protocol do not activate the coagulation process since they have hydrophobic surfaces with no coatings thus a liquid plasma concentrate rich in growth factors, platelets, and bioactive substances forms quickly at the top of the tube [13–16 and 18,19].

Buffy coat layer, at the interface, histologically contains a highly dense layer of leukocytes or white blood cells (WBC). When collecting the PRF clot the face should be carefully separated from the red zone of RBC to preserve the WBC layer [19,20]. The solid fibrin matrix (PRF membrane) can be sutured or applied as a wound dressing in conjunction with guided bone regeneration (GBR) technique or can be packed into a fresh extraction site (fibrin plug) or a large bony defect to enhance tissue repair as part of SA. PRF membranes remain solid and release large quantities of GFs for 7 to 14 days. Liquid PRF can be mixed with a bone substitute material (e.g. xenograft or allograft) to produce a coagulated mixture known as “sticky bone” (Figure 2). This mixture acts not only as a physical scaffold to support new bone formation but also releases bioactive substances such as cytokines and growth factors (e.g. VEGF, PDGF, TGF) to enhance tissue regeneration (see Table 3).

Table 3. Platelet Derived Growth Factors found in PRF that have direct role in early wound healing. These factors and cytokines released from processed platelets are involved in mesenchymal cell recruitment, cell differentiation and activation, angiogenesis, and wound healing processes.

Table 3. Platelet Derived Growth Factors found in PRF that have direct role in early wound healing. These factors and cytokines released from processed platelets are involved in mesenchymal cell recruitment, cell differentiation and activation, angiogenesis, and wound healing processes.

Growth factors	Functions
Transforming Growth factor (TGF)	Growth of endothelial vascular cells, cell recruitment and proliferation in wound healing. Inhibits osteoclast formation and bone resorption. Stimulates fibronectin and collagen production.
Epidermal Growth Factor (EGF)	Promotion of mesenchymal cell proliferation and differentiation, epithelial cell growth and angiogenesis
Vascular Endothelial Growth Fcator (VEGF)	Restores oxygen supply to the injured tissue. Promotes repair and growth of vascular endothelial cells, angiogenesis
Platelet Derived Growth Factors (PDGF)	Cell growth, proliferation of smooth muscle cells within vascular tissue, angiogenesis, collagen production Provokes proliferation of mesenchymal cell lineage, enablers macrophage chemotaxis
Insulin-like Growth Factor (IGF)	Cell proliferation, cell to cell communications, stimulates chemotaxis and activation of osteoblasts and bone formation, induces mitogenesis of mesenchymal cells
Fibroblast Growth Factor (FGF)	Tissue Repair, Cell Growth, Hyaluronic acid and Collagen production

More recently, Miron et al (2020) managed to further increase the platelet and leukocytes concentration by over 10-fold using a new method of harvesting platelet-rich fibrin (C-PRF) from the buffy coat portion of the platelet concentrate [22]. Tunali et al (2014) described titanium-prepared platelet-rich fibrin protocol (T-PRF) using an identical centrifugal protocol to A-PRF but using titanium tubes arguing that titanium would help to activate platelets more effectively [23]. However, the benefit of this technique remains to be demonstrated clinically. PRF has been used in conjunction with socket augmentation, alveolar ridge grafting, GBR procedures, maxillary sinus grafting, treatment of periodontal and peri-implantitis defects, and soft tissue grafting in dentistry successfully. Although a PRF membrane does not act as a true barrier membrane it contributes directly to repair and regeneration at all stages of wound healing. Clinical and histomorphometric research has shown significantly more bone regeneration within bone defects when PRF is combined with xenografts (demineralized bovine matrix/ Bioss®) or allografts (demineralized freeze-dried bone) [24,25,26,27]. The reported benefits also included no need for primary wound closure, less ridge resorption, and improved bone quality histologically and density radiologically with less incidence of pain and alveolitis [28].

1.3. Mode of action and biological effects of PRF:

Platelets and leukocytes are the main cells that are responsible for the biological activity of PRF (Pavlovic et al 2021, Quirynen and Pinto, 2022) [11,29]. Activated platelet concentrate (PRF) initiates an immune cell response via anti-inflammatory and pro-inflammatory processes that include leukocytes (neutrophils, monocytes and lymphocytes). In addition to the activated platelets and leukocytes, the fibrin network acts as a reservoir for platelet-derived growth factors (Table 1) that are known to be involved in all stages of wound healing including angiogenesis, cell recruitment, cell differentiation, mineralization, and tissue regeneration [30,31]. The GF and bioactive substances are stored in alpha, delta, and lambda granules within the platelets. In addition to GFs, PRF is also enriched with leukocytes and immune cytokines (e.g. interleukin) which are the main drivers of bone and soft tissue regeneration [32].

Leukocytes regulate cell proliferation and cell differentiation and play a key in response to tissue injury at all stages of healing and regeneration, inflammatory proliferation, tissue remodeling and maturation. During the inflammatory phase neutrophils are recruited to remove bacteria and necrotic tissue. They also produce inflammatory cytokines and GFs which are essential for early phases of wound healing. Monocytes are the second type of leukocytes that are the precursors to macrophages. Normally they are provided from bone marrow. PRF matrix, in addition to platelets, provides a highly concentrated cell line of leukocytes which enhance all phases of tissue healing, angiogenesis, mesenchymal stem cell activation and tissue regeneration including osteogenesis. PRF matrix has also been shown to exhibit a strong antibacterial capacity to most oral pathogens [33]. Both liquid and solid matrices of PRF release growth factors up to 10 days after reaching a peak at day 7. [29,34]. Protocols for producing PRF variants are shown in Table 1.

Figure 1. PRF protocol: distinct stages from blood drawing, centrifugation, and clotting process. Buffy coat is a thin layer of highly concentrated leukocytes and platelets at the “face” of the PRF matrix bordering the RBC zone below. Studies have shown significantly higher release of GFs and cytokines from the cells in buffy coat layer.

Figure 1. PRF protocol: distinct stages from blood drawing, centrifugation, and clotting process. Buffy coat is a thin layer of highly concentrated leukocytes and platelets at the “face” of the PRF matrix bordering the RBC zone below. Studies have shown significantly higher release of GFs and cytokines from the cells in buffy coat layer.

PRF and its variants have been shown to enhance wound healing during SA. Alissa et al (2010), reported a denser trabecular pattern and less pain sensation along with better soft tissue healing when PRP was used for ridge augmentation. Anitua et al 2015 [35] reported that PRGF yielded good outcomes such as regenerated socket volume, bone density and soft tissue healing along with the percentage of new bone formation when PRGF was used in mandibular extraction sockets. However, when PRGF, which is depleted of leucocytes, is used the results cannot be translated to PRF due to their different properties. Production of PRGF requires the addition of CaCl solution followed by heat treatment. Others have reported beneficial SA results with increased crestal ridge width and a higher percentage of vital bone [36,37]. Wu et al 2012 found that PRF stimulates PDLF proliferation, as well as osteoblast and gingival fibroblast proliferation by 1.28-fold collectively (P<0.05)- thus increasing post-operative bone regeneration. PRF is also shown to reduce the overall wound healing time [38].

There is strong biological evidence to support the application of PRF as a bioactive material to enhance tissue regeneration and wound repair. Numerous biochemical studies have shown that PRF exhibits significant concentration of biologically active-matrix proteins and growth factors (GF) which are released slowly due to the three-dimensional architecture of glycoproteins in the fibrin clot (see Table 1). Furthermore, PRF is thought to play a role in an osteo-immune response that occurs as a result of an interplay between bone and the immune system. GFs are expressed in both matrices of PRF and contribute to enhancement of angiogenesis and early wound healing [39,40]. In an animal study Yuan et al (2021) applied PRF with a resorbable gel and found it to be effective in ridge preservation by facilitating blood clotting and promotion of angiogenesis and osteogenesis and recommended it as a cost-effective graft material for SA [41].

Hauser et al used PRF preparations in 168 post extraction sockets in fifty cardiac surgery patients without modification of their anticoagulant therapy (mean international normalized ratio =  3.16 ± 0.39). In all cases, no alveolitis or painful events were reported, and wound closure was complete at the time of suture removal one week after surgery. They proposed using PRF protocol as a reliable therapeutic option to avoid significant bleeding after dental extractions without the suspension of the continuous oral anticoagulant therapy in heart surgery patients [42]. In a case study, Chenchev et al 2017 reported the benefits of using a-PRF and i-PRF in conjunction with a bone substitute on augmentation of the alveolar ridge before implant placement [43]. In a rare split-mouth randomized controlled clinical trial Temmerman et al (2016) investigated the effect of using a preparation of platelet-rich fibrin as a socket-filling material on ridge preservation. In twenty-two patients in need of single bilateral and closely symmetrical tooth extractions in the maxilla or mandible, they reported significant differences (p < 0.005) in total width reduction between test (-22.84%) and control sites (-51.92%) at 1 mm below crest level and significant differences were found for socket fill between test (94.7%) and control sites (63.3%). They concluded that use of PRF was effective in preservation of horizontal and vertical ridge dimensions at three months after tooth extraction is beneficial [44].

In another split mouth RCT Castro et al (2021) studied the effect of different platelet-rich fibrin preparations (A-PRF+ and LPRF) and reported that although the mean horizontal and vertical changes at 1-mm below the crest (buccal and palatal side) were similar for the test and control sites (p > 0.05) both PRF matrices showed radiographically a significant superiority for the socket fill (L-PRF (85.2%) and A-PRF+ (83.8%) compared with the controls (67.9%) [44,37]. A SR by Al-Maawi (2021) of 20 RCT and controlled studies showed that PRF was effective in reducing post-operative pain, accelerating soft tissue healing and preventing dimensional bone loss, especially in the early time period of 2–3 months. Dimensional bone loss was significantly lower in the PRF group compared with the unaided wound healing after 8-15 weeks but not after 6 months. Socket fill was significantly higher in the PRF group compared with spontaneous wound healing. The authors concluded that based on the analyzed studies, PRF was most effective in the early healing period of 2-3 months after tooth extraction, and allowing a longer healing period may not provide any benefits. However, the authors found heterogeneity between the included studies and assessed a relatively high risk of bias in blinding of participant and personnel as well as blinding of outcome assessment [44,39]. In a multi-arm parallel randomized controlled clinical trial, Clark et al (2018) evaluated the efficacy of platelet rich fibrin (A-PRF) alone or mixed with freeze-dried bone allograft (FDBA) in improving vital bone formation and alveolar dimensional stability during SA, in fresh extraction sites [40].

In forty patients, non-molar extraction sites were randomized into one of four ridge preservation approaches: A-PRF, A-PRF+FDBA, FDBA, or blood clot They concluded that A-PRF alone or augmented with FDBA is a suitable biomaterial for ridge preservation [40]. This finding supports recommendations by Miron et al (2021) of using of PRF alone in intact premolar and molar sites to prevent dimensional changes, reduce pain and infection, and enhance wound healing. However, when the buccal wall of an extraction socket is missing, the use of a bone graft mixed with PRF is advisable. Bernnardo et al 2023, evaluated the use of platelet-rich fibrin (PRF) as a natural carrier for antibiotics delivery. Different antibiotics were added to PRF and their release and antimicrobial activity were analyzed against various microorganisms. Gentamicin and linezolid were released from PRF without affecting its physical properties and showed massive antibacterial activity against all microorganisms. Vancomycin interfered with PRF formation. PRF loaded with antibiotics allowed the release of effective concentrations of antimicrobial drugs and could potentially reduce the risk of post-operative infection after oral surgery. However, further studies are needed to confirm its efficacy as a topical antibiotic delivery tool [40,41,42]. The above-mentioned studies demonstrate the benefits of using PRF alone or in combination with bone substitute materials to promote healing and tissue regeneration. However, it should be noted that as there is a general lack of randomized clinical trials using standardized protocols for different PRF preparation and homogeneous outcome measures, the evidence for clinical efficiency of PRF and it’s variants, in bone regeneration in dental implantology, has not yet been fully established [44]. Future studies are urgently needed to demonstrate the optimum SA and PRF protocols using standardized protocols and outcome measures. In summary, there is emerging evidence that supports the biological and clinical benefits of using PRF during SA to enhance all stages of wound healing and tissue regeneration.

2. Materials and Methods

Figure 2. “Sticky bone” is produced by mixing PRF liquid fraction with a bone substitute material (BioOss® Geistlich).

Figure 2. “Sticky bone” is produced by mixing PRF liquid fraction with a bone substitute material (BioOss® Geistlich).

Figure 3. Semi-open minimally invasive socket augmentation technique with PRF fibrin: tooth extraction is carried out with a periotome to prevent fracture/loss of the thin buccal plate. A soft tissue flap is not raised to avoid damage to the periosteum and the blood supply. The socket is degranulated and irrigated with saline. The socket is augmented with sticky bone produced by mixing a bone substitute material (e.g. BioOss® Geistlich) and iPRF liquid. The graft is covered with a A-PRF fibrin which is sutured to the edges of the socket without any attempt to achieve primary closure. When using a “sticky bone” mixture of PRF and a biomaterial, it may not be essential to cover the extraction site with PRF fibrin membrane although evidence suggests that using 2 layers of PRF over the SA would be highly beneficial.

Figure 3. Semi-open minimally invasive socket augmentation technique with PRF fibrin: tooth extraction is carried out with a periotome to prevent fracture/loss of the thin buccal plate. A soft tissue flap is not raised to avoid damage to the periosteum and the blood supply. The socket is degranulated and irrigated with saline. The socket is augmented with sticky bone produced by mixing a bone substitute material (e.g. BioOss® Geistlich) and iPRF liquid. The graft is covered with a A-PRF fibrin which is sutured to the edges of the socket without any attempt to achieve primary closure. When using a “sticky bone” mixture of PRF and a biomaterial, it may not be essential to cover the extraction site with PRF fibrin membrane although evidence suggests that using 2 layers of PRF over the SA would be highly beneficial.

Results

Case study 1

In this case study the first author has used SA technique in conjunction with PRF and a particulate xenograft (BioOss® Gesitlich) to enhance fresh extraction site healing prior to delayed implant placements after 8 weeks. A 40-year male generally fit and well and no smoking history presented complaining of the poor aesthetics associated with his anterior teeth. He gave a recurring history of infection from these teeth with the last episode being 6 months earlier. Clinically his oral hygiene was fair to poor with marginal plaque deposits and probing depths generally up to 4mm. The upper incisor teeth, however, had probing depths extending to the root end with grade 2/3 mobility and recession (Figure 4a,b). A diagnosis of chronic periodontitis with possible perio-endo lesions on the upper anterior teeth made. The prognosis of the teeth was poor and after a course of initial treatment, the extraction of the upper anterior teeth was planned with interim replacement using an Essix retainer. Due to the labial bone defect, the decision was made to extract the teeth and carry out SA using a xenograft (Bioss Geistlich®) mixed with i-PRF. The extraction defect was treated with a double layer of A-PRF fibrin matrix membrane which was sutured to the mucosa surrounding the edges of the extraction sockets (Figure 4c,d).

The four incisor teeth were extracted using the minimally invasive extraction technique as described in part 1 of this paper, and all granulation tissue removed. The extraction sockets were augmented using a xenograft (Bio-Oss®/ Geistlich Biomaterials) mixed with i-PRF (see Table 1). Two Megagen Anyridge® implants were subsequently placed and restored with a 4-unit bridge after successful control of the periodontal disease. Good incorporation of graft particles surrounded by newly formed bone was noted at the time of implant placement.

Figure 4. Extraction sockets of periodontally involved upper incisor teeth which were augmented with a xenograft (BioOss ®, Geistlich Biomaterials) mixed with i-PRF and closed with double layer of A-PRF membrane using vicryl ® sutures.

Figure 4. Extraction sockets of periodontally involved upper incisor teeth which were augmented with a xenograft (BioOss ®, Geistlich Biomaterials) mixed with i-PRF and closed with double layer of A-PRF membrane using vicryl ® sutures.

Figure 5a. Radiographs showing socket augmentation dense radiopacity and contour preservation, 8 weeks after SA.

Figure 5a. Radiographs showing socket augmentation dense radiopacity and contour preservation, 8 weeks after SA.

Figure 5b. Control periapical radiograph shows the stability of marginal bone support around the two Megagen Anyridge® implants in function. Although the implant: crown ratio appears to be unfavourable in this radiograph, the internationally recognized restorative guidelines were used in selection of implant lengths.

Figure 5b. Control periapical radiograph shows the stability of marginal bone support around the two Megagen Anyridge® implants in function. Although the implant: crown ratio appears to be unfavourable in this radiograph, the internationally recognized restorative guidelines were used in selection of implant lengths.

3.2. Case Study 2

UR1 was removed with SA using BioOss® and A-PRF. Eight weeks post-SA, a CBCT scan revealed outstanding preservation of the alveolar bone shape with radiographically dense bone. A Megagen Anyridge® implant was inserted eight weeks post-SA. Before the implants were inserted, the implant bed was drenched with i-PRF solution using a one-time use syringe. A periapical X-ray indicated successful implant integration and preservation of the newly formed bone at the UR1 extraction site during a subsequent follow up see (Figure 6 and Figure 7 and Table 4).

Figure 6. Extraction of UR1 and SA using BioOss ® and A-PRF. A CBCT was taken 8 weeks after SA showing excellent maintenance of alveolar bone contour with radiologically dense bone.

Figure 6. Extraction of UR1 and SA using BioOss ® and A-PRF. A CBCT was taken 8 weeks after SA showing excellent maintenance of alveolar bone contour with radiologically dense bone.

Figure 7. A Megagen Anyridge® implant was placed at 8 weeks after SA. Implant bed was soaked with i-PRF solution using a disposable syringe just prior to insertion of the implants. Periapical radiograph shows good implant integration and maintenance of the newly generated bone within the extraction site at UR1 at a follow up appointment.

Figure 7. A Megagen Anyridge® implant was placed at 8 weeks after SA. Implant bed was soaked with i-PRF solution using a disposable syringe just prior to insertion of the implants. Periapical radiograph shows good implant integration and maintenance of the newly generated bone within the extraction site at UR1 at a follow up appointment.

The success of the two case studies presented in this paper were evaluated using specific primary and secondary outcome measures seen inable 4 summarises the results.

Table 4. Outcomes of case studies presented in our study.

Table 4. Outcomes of case studies presented in our study.

Case No	Outcomes:
Case No:	SA	BQn	BQ	PS	Surgery BQ	Secondary Grafting	Time since SA	Early Loading	CBS
Case 1	open SA	Full contour	excellent	high	D2-3	None	8 weeks	6 weeks	No crestal bone loss
Case 2 implant 1	open SA	Full contour	excellent	high	D2-4	None	9 weeks	7 weeks	No crestal bone loss
Case 2 implant 2	open SA	Full contour	excellent	high	D2-5	None	10 weeks	8 weeks	No crestal bone loss

Table 4: SA: Socket Augmentation, BQn: Bone quality/ Full contour preservation, BQ: excellent/good/poor; PS: Primary Stability on implant placement High/medium/Low; Surgery BQ: bone density at surgery D1, D2, D3, D4; Secondary Grafting: Need for additional grafting at the time of implant placement; SA: Time since SA (weeks); Early Loading: Time since implant placement (weeks); CBS: Crestal bone stability.

4. Discussion

Conclusion

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