Comparison of Antimicrobial Activity against Porphyromonas gingivalis between Advanced Platelet-Rich Fibrin and Injectable Platelet-Rich Fibrin

Platelet-rich fibrin (PRF) obtained via low-speed centrifugation has antimicrobial properties. This study was conducted to evaluate the effectiveness of advanced platelet-rich fibrin plus (A-PRF+) and injectable platelet-rich fibrin (I-PRF), obtained from patients with different periodontal states, against Porphyromonas gingivalis. A-PRF+ and I-PRF samples were obtained from venous blood of 60 subjects divided equally into three groups: periodontitis, gingivitis, and healthy gingiva groups. The antibacterial experiments evaluated biofilm inhibition, mature biofilm impact, and time-kill kinetics. The percent reduction in biofilm-growing and mature biofilm bacteria ranged from 39% to 49% and 3% to 7%, respectively. In the time-kill kinetics assay, PRF from the periodontitis group was more effective as an antimicrobial than that from the gingivitis and healthy gingiva group (p < 0.001); I-PRF was more effective than A-PRF+ (p < 0.05) and both of them showed peak antibacterial activity after 12 h of exposure. Both A-PRF+ and I-PRF exhibited antibacterial properties against P. gingivalis, but I-PRF appeared to be more effective. The PRF obtained from the different groups appeared to have different degrees of antimicrobial efficacy.


Introduction
Periodontal disease is an infammatory disease of the toothsupporting tissues, e.g., gingiva, periodontal membrane, and alveolar bone, induced by oral microbes, dental plaque, or dental bioflm [1]. Te presence of certain bacterial species, isolated as fve microbial complexes found together in the subgingival bioflm, is implicated in the occurrence of periodontal disease. Among these bacteria, Porphyromonas gingivalis (P. gingivalis), which is able to combine with other strains of bacteria, has previously been shown to play a crucial role in periodontal pathology [2]. P. gingivalis is one of the most prevalent bacteria in periodontitis and is also recognized as a keystone pathogen in peri-implantitis, a periodontal disease, which is why in the treatment of periodontal disease more and more antimicrobial strategies focus on restricting the microbial growth and activity of this bacterium [3]. P. gingivalis is a Gram-negative oral anaerobe and is considered a main etiological factor in periodontal diseases, producing several virulence factors and extracellular proteases such as lipopolysaccharide, fmbria, and gingipain that result in destruction of the periodontal tissue [4].
Te main therapeutic strategies of periodontal treatment are mechanical debridement by scaling and root planing, and periodontal surgery to remove dental plaque, and hopefully all the associated bacteria at the sites of infection. However, one of the major problems associated with surgical procedures and wound-healing processes is the potential risk of bacterial contamination and infection [5]. Tus, control of infections plays a key role in successful wound healing and avoiding chronic conditions. Because of the adverse efects stemming from the development of resistant bacteria, the use of postoperative systemic antibiotics following dental surgery has become controversial [6]. Recently, platelet concentrates have been used for the successful treatment of periodontal disease based on their regenerative potential and anti-infammatory action and reported activity against various bacteria strains [2], which could make them an alternative to systemic antibiotics. PRF is a second-generation platelet concentrate comprising a complex network of microfbrins with entrapped platelets and leukocytes [7]. Notably, the leukocytes in PRF are known to exhibit antimicrobial activity [2].
Te use of low-speed centrifugation to obtain new types of PRF has previously been explored, having the advantage of producing a high number of cells such as leukocytes [8]. I-PRF (injectable PRF) is available in injectable form and coagulates a few minutes after injection [2] while A-PRF+ (advanced PRF) has shown superior properties when compared with standard PRF [8]. Tese two types of PRF have great potential for further clinical application because they are simple to prepare and use and do not require the addition of anticoagulants [6] and also have an enriched antimicrobial content [7]. A-PRF has outstanding advantages, including the increased release of proteins and growth factors, proliferation of fbroblasts, its ability to migrate, and expression of growth factors [8]. I-PRF contains various components such as antimicrobial proteins, complement binding proteins, and antimicrobial peptides [7].
It has been demonstrated that the quality of PRF depends on several patient-related factors, especially their periodontal condition [9]. However, this issue has not been given proper attention in previous studies, in which only subjects without periodontal problems were selected to provide samples [5,7]. An in vitro study reported that periodontal diseases including periodontitis with evidence of infammatory responses could alter the amount of growth factors released by PRF [10]. To clarify this issue, the subjects recruited for our current study were expanded to include patients with gingivitis and periodontitis, in addition to subjects with healthy gingiva. Tis wider sampling was expected to provide novel data.
Tus, the aim of the present study was to provide in vitro evidence of the antimicrobial properties of new generations of PRF, including I-PRF and A-PRF+ that were obtained from patients with diferent periodontal conditions, against P. gingivalis.

Participant Selection.
Sixty patients aged 20-65 years who attended the Department of Periodontology, Odonto-Maxillo-Facial Hospital in Ho Chi Minh City and volunteered to participate in the study were included in our study sample. All were nonsmokers with no history of infection or any antibiotic or anti-infammatory use for at least 3 months before the study. None of them gave a history of any anticoagulant or immunosuppressive therapy that might interfere with the natural coagulation process nor did they report any bleeding or clotting disorders. All of them were examined and diagnosed by a periodontist prior to being divided into three groups of participants (20 patients for each): Group 1: patients without gingival diseases such as gingival bleeding and swelling, whose periodontal pocket depth was under 3 mm. Group 2: patients who had severe gingivitis with gingival bleeding around over a half of their dentition. Group 3: patients with moderate or severe chronic periodontitis according to the classifcation of the American Association of Periodontology, 2017 [11]. All patients gave written informed consent before beginning the study. Te study protocol and consent procedure were approved by the Ethics Committee of Ho Chi Minh Odonto-Stomatology Hospital, Vietnam (no. 536/BVRHM), and were conducted in full accordance with the World Medical Association Declaration of Helsinki.

Sample Preparation.
Twenty milliliters of blood were collected from each patient and placed immediately in the Dou Quattro Choukroun PRF system (designed by PRO-CESS for PRF ® , Nice, France). For the A-PRF+, 10 ml of blood was placed in a sterile plain glass-based vacuum tube and centrifuged according to the A-PRF+ mode with a preset rotation speed of 1,300 rpm for 8 minutes [12]. To obtain I-PRF, the blood tube was centrifuged at 700 rpm for 3 min (60 × g) [13]. Te A-PRF+ clot was then washed and sectioned, and its volume was measured by placing it in an Eppendorf containing 0.5 ml of sterile 1X PBS and marking the rise in the liquid level. Te obtained A-PRF and I-PRF were immediately used in antimicrobial tests.

Tests Performed.
A sterile inoculating loop was used to transfer an inoculum of P. gingivalis, which was cultured from subgingival plaque of patients with periodontitis in our previous study [14], onto Petri dishes that contained Wilkins-Chalgren Anaerobe Agar (Oxoid LP0011B) supplemented with 5% laked sheep blood (culture medium). Petri dishes were incubated under anaerobic conditions with Gaspak kits (Merk, USA) at 37°C for 7 days. Sufcient inoculum was added to Petri dishes until the turbidity equaled 0.5 McFarland. Tis prepared a bacterial suspension and was used in the subsequent experimental assays of antimicrobial activity.

Bioflm Inhibitory Assay.
For the A-PRF+ experiment, overnight bacterial cultures were prepared in TSB medium at 37°C. Aliquots of 200 μl A-PRF+ 40% and bacterial inoculum were placed in sterile polystyrene test tubes. Te control tubes contained only culture medium. All of the tubes were incubated anaerobically for 24 hours at 37°C. Ten, the nonadherent cells were washed twice with PBS while the adherent cells were fxed by adding methanol for 15 minutes before staining with crystal violet 0.1% for 5 minutes. Distilled water was used to wash away the excess stain, then, the test tubes were air-dried before adding 200 μl ethanol 95%. Te quantity of bioflm was determined by measuring absorbance in a microplate reader at the wavelength of 610 nm.
Te procedure for the I-PRF experiment was similar to that for A-PRF+, with aliquots of 150 μl I-PRF and inoculum placed in sterile 96-well polystyrene plates that had been pretreated by the manufacturer. Te control subgroup was incubated in culture medium. Te plates were incubated anaerobically for 24 hours at 37°C prior to being washed twice with PBS and then fxed with methanol for 15 minutes before staining with crystal violet 0.1% for 5 minutes. Distilled water and an air-dryer were used, and 200 μl ethanol 95% was added. A microplate reader was used to measure the absorbance in each well at the wavelength of 610 nm.

Mature Bioflm Impact Assay.
To visualize the impact of A-PRF+ and I-PRF on the mature bioflm, the test tubes or plates containing only bacterial inoculum were incubated anaerobically for 48 hours to allow a mature bacterial bioflm to form. Ten, the culture solution was removed and 200 μl A-PRF+ 40% was added to the test tubes and 150 μl I-PRF was added to the test plates. All of the tubes and plates were incubated anaerobically for 4 hours at 37°C before they were washed twice with PBS and then fxed with methanol for 15 minutes before staining with crystal violet 0.1% for 5 minutes. Distilled water was used to wash away the excess stain, then, the test tubes were air-dried before adding 200 μl ethanol 95%. Te quantity of bioflm was determined by measuring absorbance in a microplate reader at the wavelength of 610 nm.

Time-Kill Kinetics Assay.
Te time-kill kinetics assay was conducted using 800 μl A-PRF+ or I-PRF 40% in culture medium placed in sterile polystyrene test tubes. Culture medium without PRF was placed in the control tubes. An inoculum of 5.0 × 10 5 CFU/mL was also added and anaerobically incubated at 37°C. At 0, 3, 6, 12, 24, 36, and 48 hours of incubation, serial dilutions were performed (down to 10 −4 ) and 50 μl was plated onto Wilkins-Chalgren agar plates. Tese were incubated in an anaerobic chamber for a further 24 hours and colony counting was performed.

Statistical Analysis.
All experiments were performed in triplicate and repeated at least three times. Results are reported as the mean ± standard deviation. Analysis was performed using either one-way analysis of variance or repeated measures analysis. P values <0.05 were considered to be signifcant. For statistical analysis, SPSS v.23 software (IBM, New York City, NY, USA) was used.

Demographic Data.
Te demographic characteristics of the participants are presented in Table 1. A total of 60 people compliant with our research standards were divided equally into three groups depending on their periodontal condition, which included healthy gingiva, gingivitis and periodontitis as mentioned in the Methods section. Te mean age of the healthy gingiva, gingivitis, and periodontitis groups was 23.7 ± 5.2, 33.0 ± 7.4, and 50.9 ± 13.3, respectively, with signifcant diferences between groups (p < 0.001). Te gender ratio in the three groups did not difer signifcantly (p > 0.05).

Bioflm Inhibitory Assay.
Antimicrobial efcacy was demonstrated by the percentage reduction in the number of bacteria in the test groups compared to that in the control group, as shown in Table 2.
No diferences in antibacterial efect on bioflm formation of P. gingivalis were found between the various A-PRF+ samples derived from the blood of diferent patient groups. Te percent reduction in bacterial number in the periodontitis, gingivitis, and healthy gingiva groups was 42 ± 9%, 41 ± 9%, and 39 ± 9%, respectively. Te diference between groups was not signifcant (p > 0.05). Similarly, the percent reduction following exposure to the I-PRF samples was 49 ± 8%, 47 ± 10%, and 45 ± 10% in the periodontitis, gingivitis, and healthy gingiva groups, respectively. Likewise, there was no signifcant diference between the groups (p > 0.05).
In the periodontitis group, the percentage reduction in bacterial number following exposure to A-PRF+ was signifcantly lower than following exposure to I-PRF (p < 0.05). Overall, there was a trend for a lower percent reduction following exposure to A-PRF+ in the gingivitis and healthy gingiva groups relative to I-PRF, but the diferences were not signifcant (p > 0.05).

Mature Bioflm Impact Assay.
Te antimicrobial impact of PRF on mature bioflm of P. gingivalis was expressed as the percent reduction in bacterial number in the test groups compared to that in the control group, as shown in Table 3.
No diferences in antibacterial efect on mature bioflm of P. gingivalis were found between the various A-PRF+ samples that were derived from the blood of diferent patient groups (3 ± 5%, 3 ± 6%, and 5 ± 9%, in the periodontitis, gingivitis, and healthy gingiva groups, respectively; p > 0.05). Similarly, the percent reduction following exposure to I-PRF was 5 ± 5%, 5 ± 5%, and 7 ± 8% in the  Data are presented as the mean ± SD (SD, standard deviation). * Comparison between A-PRF+ and I-PRF, two-samplet test; * * comparison among three patient groups, one-way ANOVA. Statistical signifcance was set at p < 0.05.
Tere was a trend for a lower percentage reduction following exposure to A-PRF+ from all groups compared with exposure to I-PRF; however, the diferences were not signifcant (p > 0.05).

Time-Kill Kinetics
Assay. Te percent reduction in bacterial colony number in the test groups compared to that in the control group at the corresponding time-points (3, 6, 12, 24, 36, and 48 hours) under the infuence of A-PRF+ and I-PRF is presented in Table 4.
Te percent decrease in bacterial colony number varied signifcantly among the healthy gingiva, gingivitis, and periodontitis groups (p < 0.001). In particular, at 3 h, 6 h, 12 h, 24 h, and 36 h, the percent reduction was highest in the periodontitis group and lowest in the healthy gingiva group. However, there was no signifcant diference at 48 h (p > 0.05). Tese data are depicted in Figures 1 and 2.
Regarding the antimicrobial efcacy of A-PRF+ and I-PRF, there was a higher percent reduction following exposure to I-PRF than A-PRF+. In the group of subjects with healthy gingiva, the diference was not signifcant. In the gingiva group, signifcant diferences were found at 6 h, 12 h, 24 h, and 36 h while in the periodontitis group the diference was also signifcant at 3 h (p < 0.05).
As shown in Figures 1 and 2, the percent reduction of microbial colonies changed over time, increasing from 3 h to 6 h and peaking at 12 h, before gradually decreasing to the end of the observation period.

Discussion
In the current study, the mean age of patients was the highest in the periodontitis group and the lowest in the healthy gingiva group, indicating that the severity of periodontal diseases increases with aging; in other words, age is a contributing factor related to periodontal disease, as indicated in a previous study [15]. In the current study, the mean age of periodontitis patients was around 50 years, which was signifcantly higher than that of the remaining groups. Tis result was relatively consistent with the previous study, which showed that patients in their fourth decades of life witnessed a remarkable increase in the prevalence of severe periodontitis [15]. Terefore, the elderly population's oral health should be given more attention.
Autologous platelet concentrates were initially used to stimulate tissue healing. PRF has recently been of particular interest to researchers and clinicians because of its high clinical applicability in the treatment of bony defects, dental implant surgery, and postextraction healing and its ability to reduce the rate of postsurgery complications [6]. Moreover, it has been suggested that PRF, which was found to have antimicrobial properties thanks to its leukocyte component [2], could be used as a supportive agent and even as an alternative to antibiotics, whose side efects could lead to the development of antibiotic-resistant bacteria [5,6]. In the current study, two kinds of PRF (A-PRF+ and I-PRF) were obtained following the "low-speed concept" for blood centrifugation, whereby lower centrifugation speeds were shown to result in higher amounts of growth factors and higher numbers of cells including leukocytes [10], which play an important role in PRF's antibacterial mechanism [2]. A recent study found that leukocytes are infuenced by the roles of platelets to modulate their behaviors, and enhance their ability to phagocytose and kill microorganisms by triggering diferent types of signaling pathways [16]. Te advantages of utilizing PRF as an autologous source of growth factors in comparison to other allograft or xenograft sources are that concerns regarding disease transmission and adverse immunogenic reactions are eliminated [10]. Te characteristics of platelet concentrate depend a lot on the generation step. By that time, the literature gave more modifcation to promote the regenerative potential of this biological material and focused on the analysis of each PRF protocol [17,18]. In this current study, we generated A-PRF+ and I-PRF with the initial protocol, and primarily, to obtain the highest volume of leukocytes and platelets for I-PRF, we applied the harvesting method of Miron et al., in which collected the cell accumulating just above the red blood cell layer [19]. However, we had not tried horizontal centrifugation, which generated, according to Fujioka-Kobayashi et al., higher concentrations than those caused by routine fxed-angle centrifugation [20]. It is potential for future research if the last one can express a higher antimicrobial potential.
In another hand, in the context of the potential development of antibiotic-resistant bacteria, and the fact that P. gingivalis has been found to have an efcient mechanism for the transfer of resistance factors [6,21], the antibacterial properties of PRF are particularly benefcial. After Aggregatibacter actinomycetemcomitans, P. gingivalis is regarded as the periodontal pathogen that has received the most research. Within this family of asaccharolytic, intermediate, and highly saccharolytic species, they were instantly linked to periodontal disease. Gingivalis can damage the endothelium and gingival mucosal epithelial cells. As a result of the bacterium using fmbriae, this fact can be found both above and inside the epithelial cells [22]. In this study, we used the bacteria sample directly cultured from subgingival plaque to investigate the antimicrobial of platelet concentrates instead of commercial strains. According to Yang et al., the results can be readily confrmed by and compared Table 3: Mature bioflm impact assay: the percent reduction in the number of bacteria (%) compared to the control under the infuence of diferent blood-derived products (A-PRF+ and I-PRF) from the three groups. Data are presented as the mean ± SD (SD, standard deviation). * Comparison between A-PRF+ and I-PRF, two-samplet test; * * comparison among three patient groups, one-way ANOVA. Statistical signifcance was set at p < 0.05.

4
International Journal of Biomaterials to those of other laboratories using these thoroughly researched bacterial strains. Yet, after several passages in the lab, these strains may lose some of their harmful characteristics. Terefore, to examine these plasma preparations' antibacterial properties, it could be necessary to use recent clinical isolates [5]. A-PRF+ and I-PRF, two of the latest kinds of PRF, have been demonstrated to have superior properties over other types of PRF [16]. However, few studies have been conducted to compare the antimicrobial features of these two kinds of PRF, which is why our study was carried out using a bioflm inhibitory assay, mature bioflm impact assay, and time-kill kinetics assay.
In the current study, both kinds of PRF were found to signifcantly reduce P. gingivalis bioflm formation in the bioflm inhibitory assay, with the percent reduction in bacterial number ranging from 39% to 49%. A study by Jasmine et al. also found a signifcant percent reduction in bioflm-producing bacteria but was conducted on other bacterial strains (S. epidermis and S. aureus) [7]. Meanwhile, the mature bioflm impact assay in our study found no clear impact of A-PRF+ and I-PRF on mature bioflm of P. gingivalis, with a percent reduction of only around 3% to 7%. A previous study showed the remarkable resistance of bioflm-growing and mature bioflm bacteria to antibiotic therapy and phagocytosis [23]. Mature bioflms were demonstrated to be more resistant to host immune defenses and the action of antibacterial agents [24]. Tis may be why PRF in this study had a greater impact on bacteria at the stage of forming bioflm rather than those at the stage of producing mature bioflm. Interestingly, no diference  between A-PRF+ and I-PRF was found in these two assays, although I-PRF was found to contain more growth factors in a previous study [2]. Specifcally, it was reported that the wide range of inhibitory and bactericidal activity of I-PRF is due to it containing platelets, fbrin, fbronectin, thrombin, HBD-3 peptide (antimicrobial peptide) myeloperoxidase, and white blood cells [7].
In the time-kill kinetics assay, I-PRF and A-PRF+ had a positive antimicrobial impact on P. gingivalis during the frst 48 h, with a peak at 12 h, when the percent reduction was as high as 92-95% in the periodontitis group. A previous study also showed that PRF demonstrated inhibitory activities against P. gingivalis for up to 36 h [5]. Te time-kill assay in the latter study was assessed after 50 h while our assay was assessed after 3, 6, 12, 24, 36, and 48 h. Our study results do not support previous research that found that the efects of PRF only lasted for 48 hours, during which growth factors were released at the highest concentration [2]. In our study, I-PRF had a signifcantly better antibacterial efect than A-PRF in the periodontal disease groups, consistent with recent studies [2,25]. I-PRF, which was obtained using a slower and shorter centrifugation spin, contains a higher number of regenerative cells and a higher concentration of growth factors than A-PRF [2]. In the current study, A-PRF+ was obtained after centrifuging blood samples at 1,300 rpm for 8 minutes while I-PRF was obtained after a lower and shorter centrifugation spin, at 700 rpm for 3 minutes. Although I-PRF has similar properties to A-PRF, it is obtained in an injectable liquid form without forming a PRF membrane. Te liquid form of platelet concentrates has better antimicrobial ability than the membrane form [8]. In a previous study, I-PRF had the maximum antibacterial activity followed by PRP, and the lowest antibacterial activity was shown by PRF when tested against P. gingivalis [8].
To the best of our knowledge, previous studies have rarely used PRF obtained from patients with periodontal diseases [2,5,7], instead collecting blood samples from healthy donors [5,7]. In other words, patients who had periodontal problems were excluded from the study sample, which is a limitation of those studies. It is worth mentioning that patient-related factors such as chronic periodontitis may afect the quality of PRF [9], as although there were no signifcant diferences in cell proliferation between the healthy and periodontitis groups, there was a trend for increased cell proliferation in the periodontitis group [9]. Tis could be because patients with chronic periodontitis have increased systemic levels of proinfammatory cytokines and growth factors, which may afect the quantity and quality of the PRF [10]. Moreover, possible connections between periodontitis and systemic diseases were demonstrated by evidence of systemic infammatory responses that could activate platelets to release growth factors, and possibly with infammation, where the healing response may be downregulated, with decreased amounts of growth factors from PRF [10]. To date, whether the quality of PRF is signifcantly dependent on periodontal disease status remains a controversial issue that needs further investigation. Although in a previous study, the impact of periodontal condition on PRF as an autologous source of growth factors was not confrmed, the authors noted their small number of subjects and large standard errors [10]. Our study is thus the frst to evaluate and compare three groups of patients with healthy gingiva, gingivitis, and periodontitis.
According to the results of our time-kill kinetics assay, PRF from periodontitis patients was more efective against P. gingivalis than that from the gingivitis or healthy gingiva groups. On the contrary, no signifcant diferences were found between groups in the bioflm inhibitory assay or mature bioflm impact assay. Tus, further research is needed on patient-related factors in relation to PRF to reach a clearer conclusion. Although a strength of our study was that it is the frst to assess classifed subjects based on their periodontal condition, the nature and features of PRF that were collected from diferent subjects such as their proinfammatory cytokine content and growth factor release were beyond the scope of this study. Tese aspects should be addressed in future studies.

Conclusions
PRF not only stimulates the healing process of periodontal tissue but also has signifcant antimicrobial efcacy against P. gingivalis. Te PRF samples that were obtained from patients with diferent periodontal disease status appeared to have diferent antimicrobial efcacy. Specifcally, the PRF from the periodontitis group was more efective than that from the gingivitis group, while PRF from the healthy gingiva group was the least efective. Of the two kinds of PRF used in the current study, I-PRF had better antibacterial properties than A-PRF+.

Data Availability
All the data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te author declares that there are no conficts of interest.