Title Page

Quantification of growth factors in advanced platelet-rich fibrin and

concentrated growth factors and their clinical efficiency as adjunctive

to the GTR procedure in periodontal intrabony defects

Lihong Lei*, Yuanyuan Yu*, Jiayin Han*, Danhui Shi*, Weilian Sun*, Diya Zhang†, Lili

Chen*

*
Department of Periodontics, Second Affiliated Hospital, School of Medicine, Zhejiang

University, Hangzhou 310009, China

†
Dental Department, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University,

Hangzhou 310016, China;

***** Lihong Lei and Yuanyuan Yu contributed equally to this work*****

This is the author manuscript accepted for publication and has undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/JPER.19-0290.

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Correspondence

Lili Chen, MD, Department of Periodontics, Second Affiliated Hospital, Zhejiang University,

School of Medicine, No. 88 Jiefang Road, Hangzhou, China. Tel: 86-571-87767068, Fax:

86-571-87022776, E-mail: chenlili_1030@zju.edu.cn

4,029 Words; 6 figures; 44 references; 3 Supplementary figures; 5 Supplementary tables

Short Running Title: Laboratory and periodontal use of A-PRF and CGF

Key findings: A-PRF and CGF have the ability to release growth factors over time from their

respective platelet formulations, and the adjunctive use of A-PRF and CGF showed a definite

clinical and radiographic evidence of regeneration along with satisfactory healing.

Abstract:

Background： The development of platelet concentrated biomaterials has gained increasing

awareness for regenerative medicine. With different protocol, derivatives such as advanced

platelet-rich fibrin (A-PRF), injected PRF (I-PRF) and concentrated growth factors (CGF) have been

demonstrated effectively in pre-clinical and clinical studies. The aim of this study was to compare the

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level of growth factors releasing from A-PRF and CGF, and their clinical efficacy in the regenerative

management of intrabony defects (IBDs). Methods: Thirty-two blood samples were collected from 8

healthy donors and assessed for PDGF-AB, VEGF, BMP-2 and TGF-β1 release at indicated times. In

addition, the clinical records of forty-five patients (15 per group) who had undergone guided tissue

regeneration (GTR) with or without A-PRF/CGF were retrieved. The probing pocket depth (PPD) and

clinical attachment level (CAL) were recorded preoperatively and 6 months postoperatively.

Intrabony component (IC) depth, radiographic bone level (RBL) and bone defect filling were assessed

radiographically. Results: A-PRF had a looser fibrin network than the CGF but presented larger

amounts of growth factors with a more sustained release period. Although there was no difference in

PPD reduction, CAL gain, RBL height change and defect filling (%) between A-PRF and CGF group,

both achieved a more favorable clinical result in IC height reduction and defect filling (%) than the

control. Conclusion: A-PRF and CGF have the ability to stimulate a continual and steady release of

total growth factors over a 14-day period. A-PRF and CGF show a similar effectiveness in periodontal

bone regeneration with a potential benefit of improving GTR outcomes in IBD treatment.

Key Words: Platelet-rich fibrin, Growth Factors, Periodontitis, Guided Tissue Regeneration

，Bone Substitutes

Introduction:

Over the past several decades, platelet concentrates (PCs) obtained from autogenous blood

extraction have emerged as potential regenerative biomaterials, such as platelet-rich plasma (PRP)

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and plasma rich in growth factors (PRGF).1 In 2001, Choukroun et al introduced the second PC

generation –platelet-rich fibrin (PRF), which induces a significant release of cytokines including

platelet derived growth factor (PDGF), transforming growth factor (TGF)-β and vascular endothelial

growth factor (VEGF) within its three-dimensional fibrin meshes.2-6 Since then, PRF has been

extensively utilized in dentistry for a variety of procedures demonstrating its effectiveness for tissue

regeneration.1,7-9 In 2014, by decreasing centrifugation speeds from 2700 rpm (750g) to 1300 rpm

(200g), a better formulation of PRF was created with a higher number of leukocytes more evenly

distributed throughout the softer clot.10,11 This new formulation of PRF was given the working name

advanced PRF (A-PRF). Ongoing studies have further confirmed that with slower spinning

protocols, A-PRF favours a higher growth factors release than prototypical PRF which in turn may

directly influence tissue regeneration by increasing fibroblast migration, proliferation and collagen

mRNA levels.12,13 Another interesting observation has been that with even lower speeds and a

shorter time (700 rpm, 60 g force for 3 min), a liquid version of PRF could be obtained.14 This new

formulation was given the working name injected PRF (I-PRF) due to its hypothesized ability to be

solely injected into defects or combined with various biomaterials similar to PRP but without any

anticoagulants.15,16 On the other hand, investigators introduced another modification - concentrated

growth factors (CGF). 17 Prepared by repeatedly alternating the centrifugation speed from 2400 rpm

(547g) to 3000 rpm (855g), CGF is characterized by containing abundant concentrated growth

factors within its markedly rigid fibrin.17,18 Several data suggest that it exhibits a positive effect on

accelerate the proliferation and osteogenic differentiation of periodontal ligament stem cells and

bone marrow cells. 19,20

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 Continual research has indicated that the specific fibrin architecture, cell and growth factor

contents are key characteristics of PRF and the modification of the protocol can lead to a different

biological clinical result.21 Since the preparation protocols share the same principle of clot formation,

it seems interesting to distinguish between the characteristics of A-PRF and CGF. Although the

growth factor contents in A-PRF and CGF clots and their bioactivities have been demonstrated

separately in numerous studies by independent group,6,12,13,22,23 the exact differences between these

two materials have not been clearly studied and published scientifically. In clinical settings, both

A-PRF and CGF have been applied as barrier membranes and/or combined with various biomaterials

such as collagen membranes and/or bone grafts to facilitate wound healing and tissue

regeneration.24,25 However, there is a lack of evidence evaluating the effectiveness of A-PRF and

CGF in the same clinical application.

Therefore, the aim of the present study was to compare the differences between A-PRF and

CGF in the area of periodontal regeneration. In the first part, as representatives of the growth factors

contained in A-PRF and CGF, PDGF-AB, VEGF, bone morphogenetic protein 2 (BMP-2) and

TGF-β1 were analysed quantitatively by enzyme-linked immunosorbent assay (ELISA), and the fibrin

architectures in A-PRF and CGF were evaluated by scanning electron microscopy (SEM). In the

second part, which featured a 6-month follow-up period, we originally evaluated the clinical and

radiographic effectiveness of guided tissue regeneration (GTR) combined with or without A-PRF

and CGF in the treatment of intrabony defects (IBDs) in patients with chronic periodontitis.

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Materials and methods:

Part I: Basic characteristics

Blood sample centrifugation

Peripheral blood samples were collected from 8 non-smoker volunteers who were free of any

systemic disease (age range of 23-29 years, 4 males and 4 females). None had a history of intake of

aspirin or other medications that might interfere with coagulation for the previous three months. The

study was approved by the Ethical Committee at the Second Affiliated Hospital of Zhejiang

University (ethical approval number: I2018001393) and was conducted in accordance with the

Helsinki Declaration of 1975, as revised in 2013. All participants provided written informed consent.

Four tubes of blood samples were obtained from each volunteer (32 tubes in total). The first sample

was collected in A-PRF tubes*; the second sample was collected in I-PRF tubes†; the third sample was

collected in CGF tubes‡; and the fourth sample was drawn into conventional vacuum plastic tubes§

for use as the control group. All tubes contained no anticoagulants and were centrifuged accordingly

to the protocols (see supplementary Table 1 in online Journal of Periodontology). Then, the fibrin

clots were collected in the middle of the tube between the red corpuscles at the bottom and the

acellular plasma at the top (Figure 1). After eliminating the red blood cell (RBC) fractions (Figure 2),

the resulting A-PRF, CGF and control group clots were placed on dry gauze to eliminate excess

*
A-PRF Plain Vacuum Tube, Process for PRF, Nice, France.

†
I-PRF Plain Vacuum Tube, Process for PRF, Nice, France
‡
Vacuette, Greiner Bio-One, Kremsmunster, Austria

§
GD100A, Gongdong, Taizhou, China

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amounts of serum and transferred to 6-well dishes. After I-PRF centrifugation, the upper liquid layer

was collected as I-PRF (see supplementary Figure 1 in online Journal) and accordingly transferred to

6-well dishes. Each sample included 5 mL Dulbecco’s modified eagle’s medium (DMEM) and was

processed for further investigation.

Scanning Electron Microscopy (SEM) Analysis

To detect the microstructures of the main region in A-PRF and CGF clots, the red thrombus of

the A-PRF, CGF and control samples, which was located approximately 2 mm below the yellow

fibrin gel, was scraped off. The resulting region was washed in PBS, fixed with 2.5% neutralized

glutaraldehyde, dehydrated with a series of ethanol solutions and then examined by SEM** with an

accelerating voltage of 3.0 kV.

Protein quantification with enzyme-linked immunosorbent assay (ELISA)

To determine the amount of released growth factors at 1, 3, 7 and 14 days, samples were

incubated at 37°C to allow growth factors release into the culture media. At each time point, 5 mL of

the culture media was collected and replaced with 5 mL of additional culture media. At indicated

time points, the concentrations of PDGF-AB, VEGF, BMP-2 and TGF-β1 were quantified using

human Quantikine ELISA kits††. All results were finally referred to a 1 mL volume and then

expressed as the total weight of molecules in 5 mL DMEM.

**
SU8010, HITACHI, Japan

††
R&D Systems, Minneapolis, MN, USA

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Part II: Clinical application

Patient selection

A retrospective cohort study was further designed to evaluate the efficacy of A-PRF and CGF for

intrabony periodontal defects. The criteria for inclusion were as follows: (1) age 18-80 years, (2) good

plaque control ( full mouth bleeding on probing (BOP) and plaque control rate ＜20%) after

completing the initial periodontal therapy (including occlusal therapy), (3) At least one tooth on the

surgical area with probing pocket depth (PPD)≥5 mm and presence of Intrabony component (IC)≥3

mm site 6-8 weeks after the presurgical therapy, (4) the same operator performed all surgical

procedures (A-PRF+GTR, CGF+GTR or GTR surgery), and the postoperative antibiotic treatments

were the same (50 mg cefuroxime axetil and 500 mg ornidazole, twice daily for 5-7 days), (5) regular

re-examination every 3 months, (6) complete clinical and radiographic data of baseline, 3 months and

6 months post surgery. Exclusion criteria were as follows: (1) presence of systemic problems that

would contraindicate periodontal surgery such as diabetes mellitus, rheumatoid arthritis or

hypertension, (2) usage of medications known to interfere with platelet function within the last 3

months or antibiotics taken within 1 months prior to treatment, (4) pregnancy or lactation in females,

(5) smoking, drug abuse or alcohol abuse, (6) poor oral hygiene or previous lack of cooperation with

the maintenance programme. The sample size was calculated based on a similar study,26 and at least

45 subjects (15 per group) were required to achieve 90% power at a 0.05 significance level. In total,

45 patients who underwent periodontal regenerative surgery in the Department of Periodontics, the

Second Affiliated Hospital, Zhejiang University between March 2014 to June 2018 with complete

recording data were included. The protocol was approved by the Ethical Committee (No.

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I2018001343) and was conducted in accordance with the Helsinki Declaration of 1975, as revised in

2008. For detailed surgical procedure of A-PRF and CGF, see Figures 3 and 4. The GTR group

received the same type of periodontal regenerative surgery except for the omission of the A-PRF or

CGF.

Clinical measures and radiographic assessment

PPD and clinical attachment level (CAL) were recorded at baseline, 3 months and 6 months

after surgery with a manual periodontal probe‡‡. Cone beam computed tomography (CBCT)§§

images were also acquired within a week before the surgical procedure and post-surgery. The

DICOM data of CBCT images were then transferred to a software program***. And the reorientation

was completed for the initial and postoperative images of each defect to enable parallel

measurements. The most apical point of the bone defect (BD) and the most coronal aspect of the

defect-associated alveolar crest (AC) were analysed. The following parameters were estimated on

the images:27

 Radiographic bone level (RBL): from cemento-enamel junction(CEJ) to BD;

 IBD depth (intrabony component, IC): from AC to BD;

Afterwards, the percentage of defect filling and defect resolution were calculated by the following

formulas:28

‡‡
UNC-15, G. Hartzell and Son, Concord, CA.

§§
Veraviewepocs X550, MORITA, Japan.

***
Mimics 10.1, Materialise, Belgium

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 aseline – months
 DEFECT RESOLUTION (%) = aseline
1

aseline – months
 DEFECT FILL (%) = aseline
1

All surgical procedures were performed by the same operator (LHL), single examiner (YYY)

evaluated all clinical parameters and another single examiner (JYH) evaluated all radiographic

parameters on CBCT. The authors who performed the measurements on the participants and the

statistician (DHS) were blinded to the surgical procedures.

Statistical analysis

All tests were performed using a statistical software†††. The means ± standard deviation was

calculated on the basis that the individual volunteer and BD site were regarded as the unit of analysis.

P<0.05 was considered to be statistically significant. For the study of growth factors, the Friedman

test was performed, and when there was a significant difference, the Wilcoxon matched-pairs signed

rank test was used. For the clinical study, the intra-group comparisons between the baseline and

6th-month measurements were performed by the paired Student t-test. Inter-group comparisons were

performed by ANOVA, and when there was a significant difference, the Bonferroni test was used.

†††
SPSS version 20, IBM, USA

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Results:

Part I: Basic characteristics

The appearances of the A-PRF, CGF and control samples under the indicated centrifugal

conditions are shown in Figures 1 and 2. In comparison to the control, A-PRF and CGF were more

integral (Figure 1). In addition, among the three groups, CGF possessed the highest density, the

smoothest surface and the clearest demarcation between the fibrin layer and RBC layer (Figure 2).

SEM Analysis

Clear surface microstructures of A-PRF, CGF and the control were compared by SEM analysis

(Figure 5). A fibrin network constituted by thin and thick fibrillary elements was observed in all

types. The fibrin network structure varied from the buffy coat (BC) to the white part, with tight

meshes in proximity to the BC, and progressively became less compact far from the BC. Both CGF

and A-PRF clots were composed of thicker and more organized fibres than the control. In particular,

CGF clots contained more highly cross-linked fibrin fibres than A-PRF clots did. Multiple cells

including platelets and white cells were observed to form a cell aggregate trapped in the fibrin

network of both A-PRF and CGF.

Growth factor release from different blood fracture

All proteins were quantified for growth factors release at indicated time points and total

accumulated protein quantities (Figure 6 and Supplementary Table 2 in online Journal of

Periodontology). It was found that the total release of PDGF-AB was highest in all PCs, followed by

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TGF-β1, VEGF and MP-2. Subsequently, the significantly lowest level of BMP-2 was found

compared to that of the other growth factors. Compared to the control, A-PRF and CGF released all

growth factors at 1-3-fold higher levels. Compared to the other groups, the I-PRF group released the

lowest levels of almost all growth factors.

PDGF-AB growth factor release from A-PRF, I-PRF, and CGF over time

A-PRF and CGF maintained high levels consistently over 14 days, whereas the levels of the

I-PRF and control gradually decreased. Therefore, compared to the control, A-PRF and CGF

demonstrated a significantly higher PDGF concentration on day 14 (Figure 6A). Correspondingly,

the total PDGF accumulated protein for I-PRF was lower than that for the other groups and lowest at

day 14 (Figure 6B).

TGF-B1, growth factor release from A-PRF, I-PRF, and CGF over time

The release of TGF-β1 in the A-PRF, CGF and control groups showed a comparable trend of

increasing at the early stage and then decreasing. After 7 days, A-PRF showed significantly higher

growth factor release than CGF (Figure 6C). The total TGF-β1 accumulated proteins over time

demonstrated that I-PRF was lowest in all modalities at days 3, 7 and 14 (Figure 6D). Higher levels

were found for A-PRF and CGF from days 7 to 14 when compared to control levels (Figure 6D).

VEGF growth factor release from A-PRF, I-PRF, and CGF over time

Once again, the release of VEGF from A-PRF, CGF and the control was increased at the early

stage and then decreased, while I-PRF kept the lowest VEGF levels over a period of 14 days.

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Compared with CGF, A-PRF demonstrated a higher release of VEGF on day 3 but a lower release on

day 7 (Figure 6E). The total release was higher for A-PRF than for all other modalities, while the total

release from I-PRF was lowest at days 3, 7 and 14 (Figure 6F).

BMP-2 growth factor release from A-PRF, I-PRF, and CGF over time

All groups had similar sustained low concentration over time for the release of BMP-2 (Figures

6G and 6H).

Part II: Clinical outcomes

Baseline measurements

Forty-five subjects were enrolled in this study: 23 were upper jaws, 19 were anterior teeth (14

incisors, 5 canines), and 26 were posterior teeth (5 premolars, 21 molars). The analysis of the baseline

characteristics of defects revealed no significant differences among the three groups for any of the

parameters at baseline (see supplementary Table 3 in online Journal of Periodontology). Primary

closure was obtained in all treated sites at the completion of surgery, and no adverse events were

recorded.

Changes in clinical and radiographic measurements

At the end of the study, both test and control groups presented with shallow residual pockets and

a substantial number of CAL gains and bone gains (see supplementary Tables 4 and 5 in online

Journal). Differences between baseline and 6 months were statistically significant in all groups in

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terms of PPD reduction, CAL gain, RBL gain and IC depth reduction. Treatment with GTR alone

resulted in a mean PPD reduction of 3.87±1.88 mm, supplementation with A-PRF grafting produced a

greater PPD reduction of 4.33±1.35 mm, and adjunctive use of CGF produced the largest PPD

reduction of 4.60±1.24 mm, but no significant difference was observed (p=0.415, NS). The CAL gain

for the A-PRF group (4.20±1.70 mm) and CGF group (4.40±1.40 mm) was slightly higher than that in

the control group (3.93±2.02 mm), but this difference also did not reach statistical significance

(p=0.760). In terms of bone changes, the mean RBL gain over 6 months amounted to 5.12±2.34 mm

for the CGF group and a slighter greater gain of 5.70±1.80 mm for the A-PRF group. The IC decrease

in the A-PRF group was 5.11±1.27 mm in comparison to 4.71±1.07 mm in the CGF group, both of

which were significantly greater than that in the control group (2.91±1.33 mm, p=0.001). In addition,

the percentage of bone filling of the IC was 61±25% in the control, which was significantly less than

that obtained by A-PRF (87±10%) and CGF (84±13%). No statistically difference between CGF and

A-PRF in all parameters was obtained.

Discussion:

In current study, compared with CGF clots, A-PRF clots appeared more fragile and not so clearly

separated from the RBC part. This finding was supported by the SEM analysis, which showed that the

surface microstructures of CGF clots contained more highly cross-linked fibrin fibers than A-PRF did.

A similar phenomenon was reported by Bonazza et al,29 who demonstrated that CGF show a compact

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fibrin network architecture, while Dohan et al30 reported that A-PRF have a slimmer and more

disorganized fibrin network. Judging from other investigators’ data, it is thought that the alternated

speeds of CGF might affect the agglutination of platelets, fibrinogen, factor XIII, and thrombin which

facilitate the conversion of fibrinogen to fibrin and the polymerization of the fibrin. 22,23,31,32 This

highly cohesive fibrin matrix provides protection from plasmin degradation, resulting in higher fibrin

tensile strength and stability.17,33 On the other hand, the low-speed centrifugation recommended for

the A-PRF preparation could not clearly fractionate RBCs, leading to a looser structure with more

interfibrous space, which might be better for cell-loaded and tissue ingrowth. In a recent study,

Kobayashi et al13 demonstrated significantly higher levels of human fibroblast migration and

proliferation of A-PRF than PRF. Ghanaati et al6 reported more cells could be counted in the

fibrin-rich clot of A-PRF. Furthermore, when gingival fibroblasts were cultured with A-PRF, higher

mRNA levels of PDGF and TGF-β were detected.13 The results from the present study also showed

that A-PRF had an apparent advantage in growth factor contents. The total VEGF concentration in

A-PRF was significantly higher than that in CGF. In addition, A-PRF had a prominent advance in the

concentration of TGF-β1 after 7 days. We therefore hypothesize that the porous character of A-PRF

might provide it with the ability to hold more proteins within its fibrin network and further release

more growth factors into their surrounding micro-environment. As one previous report31 demonstrate

that two distinct mechanisms are involved in controlled release of growth factors in self-clotted fibrin

clots: growth factors adsorbed to fibrin fibers and growth factors caged in platelets aggregated on

fibrin fibers. The initial phase of growth factor release from fibrin clots is mainly attributed to simple

diffusion, while the late phase is probably due to degradation of fibrin fibers and the amount of

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platelets. In the late phase, the looser fibrin network of A-PRF might facilitate the platelets

accumulating and also further degradation with more growth factors releasing. On the contrary,

Masuki et al34 presented conflicting results that the amounts of TGF-β1, PDGF-BB and VEGF in

A-PRF and CGF showed no difference. Overall, the current study observed that both A-PRF and CGF

had a parallel, specific, slow release kinetic even 14 days after production with a general profile

characterized by a quick increase in the release during the first 3 days. And the concentrations of

growth factors in A-PRF and CGF were significantly higher than those detected in the control.

Although no difference was observed between A-PRF and CGF in the early stage, the relatively loose

fibrinogen structure of A-PRF might enhance the regenerative potential by increasing cells migration,

as well as the amount of growth factors caged, to promote a durable growth factors release in the late

stage of tissue healing. For the evaluation of BMP-2, the concentration was fairly low and almost

undetectable by ELISA. In another study, the author also demonstrated that no traces of BMP-2 could

be detected in the A-PRF.30 Interestingly, I-PRF formed a hydrogel-like small fibrin clot

approximately 15 min after centrifugation and gradually dissolved in the medium. This finding is

supported by a recent study which reveals I-PRF a three-dimensional fibrin network embedding

platelets, leukocytes, osteocalcin and type I collagen35. All growth factors investigated from I-PRF

(with the exception of BMP-2) showed the highest release on the first day and decreased thereafter.

These changes are consistent with the results observed by Varela et al35 who revealed a high release

rate of PDGF-AB and VEGF, followed by a gradual decrease with time. Our findings also corroborate

the findings from a previous study, which hypothesized that even after 10 days, an additional release

of growth factors could still be expected from I-PRF.36 The fast action of released cytokines found in

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I-PRF may be helpful to accelerate the recruitment of incoming progenitor cells in defect locations

and be a valuable approach in dental procedures.

Preliminary hematologic studies revealed that while CGF formed a denser fibrin clot, A-PRF

was better-suited for the long-term release of growth factors. It remains interesting to know whether

these differences have an effect on clinical use. The outcomes of the following retrospective study

suggested that both PCs yielded significant improvement in clinical and radiographical parameters

between baseline and 6 months. Among the measured variables, the IC reduction in the A-PRF and

CGF treatment protocols was significantly better than that in the GTR group. The findings were also

in line with the bone filling (%), where A-PRF and CGF achieved more than 80% while GTR showed

61%. In fact, the magnitudes of the hard tissue dimensional changes observed in the current study are

comparable to those reported by others after classical regenerative treatment of IBDs using

bioresorbable membranes combined with the deproteinized bovine bone mineral.27,37,38 This finding is

also in agreement with those of previous investigations indicating that A-PRF and CGF are beneficial

for alveolar bone regeneration.39,33,40,41,42 However, there is minimal information regarding their

optimal clinical application in periodontal IBDs. This study represents the first retrospective study

comparing clinical effectiveness of A-PRF and CGF, indicating which both have the potential

application value as a desired GTR biomaterial by the promotion of vital bone formation at the treated

BD site. The advantage of adding A-PRF/CGF to bone substitutes might be interpreted as providing

good space maintenance with a proper resorption rate. Since delaying the degradation of

bovine-derived xenograft may impede osteogenesis by occupying the space available for new bone

formation,43 in collaboration with PCs, the amount of the xenograft usage was successfully reduced

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and therefore offers more space suitable for cell migration as well as new tissue ingrowth. As the

other important aspect, bone formation might also benefit from anti-infectious action and immune

regulation of the leukocytes and neutrophils clustered in the fibrin clot,44 as remarkably earlier soft

tissue healing was observed after applying the A-PRF and CGF membranes. It must also be noted that

a small quantity of vertical bone augmentation over the supra-alveolar defects was observed on CBCT

images or in some cases undergoing re-entry surgery (see supplementary Figure 2 in online Journal of

Periodontology), which is still a difficult area in the regeneration of horizontal bone loss.

Nevertheless, no difference was observed between A-PRF and CGF in clinical efficacy at the end of

the study. As a retrospective study, the greatest limitation of present research is not engaging the

principles of a randomized clinical trial design and risk of bias. Therefore, long term, prospective

randomized controlled trials with more samples is further needed to support the outcome of this study.

Conclusions:

The results from the present study demonstrated that A-PRF, CGF and I-PRF were able to

release growth factors over time from their respective platelet formulations. Interestingly, while

A-PRF and CGF have quite similar structure and release kinetics of gradually releasing of growth

factors up to a 14-day period, CGF formed a denser and highly cross-linked fibrin fiber and A-PRF

yields significantly higher growth factors release over time. The clinical and radiographic findings of

adjunctive use of A-PRF and CGF are consistent with regeneration and satisfactory healing. In these

limited number of advanced intrabony lesions, a trend that no difference between A-PRF and CGF

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was noted. Therefore, since both A-PRF and CGF can be easily procured and cost effective, they can

be utilized as a regenerative material during periodontal surgery.

Acknowledgements

This study was supported by the Second Affiliated Hospital, Zhejiang University, School of

Medicine. And it was financially supported by research program from Science and Technology

Project of Zhejiang Province, China (2017C33141), Natural Science Foundation of Zhejiang

Province, China (LY17H140002) and National Natural Science Foundation of China (81771072). We

would like to thank Prof. Rong (Center of Electron Microscopy, Zhejiang University) and Dr. Peng

(Hey Laboratory of Cancer Prevention and Intervention Ministry of Education, The Second Affiliated

of Medical College of Zhejiang University) for their excellent assistance in the study.

Conflict of Interest statement

The authors declare no other potential conflicts of interest with respect to the authorship and/or

publication of this article.

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Figures legends:

Figure 1. Blood sample after centrifugation

Appearance of the A-PRF, CGF, and I-PRF clots prepared using the relative recommended tubes.

After centrifugation, three blood fractions were identified in the A-PRF, CGF and control group: (1)

the upper layer, representing the liquid phase of plasma, (2) the lower layer, at the bottom of the

tube, consisting of free RBCs, and (3) the middle layer, a fibrin-rich gel with aggregated cells and

growth factors that were indicated by the BC. For I-PRF, the blood was separated into two layers;

this liquid formulation of PRF was collected in the top 1 mL layer of centrifugation tubes, and had to

be utilized within 15 min prior to fibrin clot formation.

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Figure 2. The clot preparation of A-PRF, CGF and control samples

Each clot consists of three parts: the upper white part (WP), the lower red part (RP), and the middle

BC. In comparison to the control (C), both A-PRF(A) and CGF(B) were more integral. However,

compared with the CGF clots, the A-PRF clots appeared obviously larger, more fragile and not so

clearly separated from the RBC part.

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Figure 3. A-PRF treatment of the maxillary right premolar of a 45-year-old male.

Presurgical CBCT images show an advanced IBD of the maxillary right premolar (E). After defect

debridement (A), the A-PRF clots were o tained from the patient’s lood and one of which was

subsequently cut into small pieces while the other was compressed to a membrane. The BD was

grafted with A-PRF pieces + deproteinized bovine bone mineral (B) and covered with A-PRF

membrane and bioabsorbable collagen membranes (C). Flaps were sutured with a 4-0 suture (D). 6

months postsurgery CBCT suggests favourable improvement of the area with a gain of 4.94 mm in

IC height (F).

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Figure 4. CGF treatment of the maxillary right incisor of a 27-year-old female.

Presurgical CBCT images show an advanced IBD of the maxillary right incisor (G). After defect

debridement, the relevant intraosseous component of the defect and the extension prevalent on the

mesial and buccal (A) and palatal (B) sides was exposure. The CGF clots were obtained from the

patient’s lood and one of which was subsequently cut into small pieces while the other was

compressed to a membrane. Then, the BD was grafted with CGF+ deproteinized bovine bone

mineral (C) and covered with CGF and bioabsorbable collagen membranes (D). Flaps were sutured

with 4-0 sutures (E, F). The CBCT image taken 6 months postsurgery suggests favourable

improvement of the area with a gain of 4.47 mm in IC height (H).

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Figure 5. SEM analysis of the self-clotted A-PRF, CGF and control.

For reference, the control group was analysed and presented lightly polymerized fibrin with slim

fibrin fibres and some visible cells. A-PRF and CGF showed a more strongly polymerized fibrin

matrix with thick fibrin fibres, where multiple cell elements, including RBCs, WBCs and platelets

were aggregated and trapped. Moreover, A-PRF exhibited a looser structure with more interfibrous

space than CGF. Note: (A) Scale bar=10.0 µm, magnification (x5000); (B) Scale bar=5.00 µm,

magnification (x10,000).

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Figure 6. ELISA protein quantification of PDGF-AB (A), TGF-β1 (C), VEGF (E) and BMP-2

(G) at each time point over a 14-day period. Total accumulated growth factors released over a

14-day period for PDGF-AB (B), TGF-β1 (D), VEGF (F) and BMP-2 (H).

Error bars represent the mean ± SE for n=8.

For each time point （A, C, E, G）: *p < 0.05, statistically significant difference between groups; †

p < 0.05, statistically significant higher than all other groups; ‡p < 0.05, statistically significant

lower than all other groups.

For total accumulated protein quantities（B, D, F, H）: §p < 0.05, statistically significant difference

compared to the control; ‖p < 0.05, statistically significant higher than all other groups, ¶p < 0.05,

statistically significant lower than other groups.

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