Effect of advanced and injectable platelet-rich fibrins against Aggregatibacter actinomycetemcomitans in subjects with or without periodontal diseases

Abstract

Background/purpose: Data for comparing effects of advanced platelet-rich fibrin (A-PRF+) and injectable platelet-rich fibrin (i-PRF) against Aggregatibacter actinomycetemcomitans (Aa) in subjects with differently periodontal conditions are scarce. This study aimed to compare the antimicrobial capacity of A-PRF+ and i-PRF obtained from subjects with or without periodontal diseases against the pathogenic bacteria Aa.

Materials and methods

The number of red blood cells, platelets, and white blood cells on the blood samples of 60 individuals, including healthy subjects (n = 20), patients with gingivitis (n = 20), and patients with periodontitis (n = 20), were analyzed before preparing A-PRF+ and i-PRF. In addition, the in vitro antibacterial effect of the two platelet concentrates was evaluated by using the agar diffusion test and a minimum inhibitory concentration (MIC) experiment.

Results

I-PRF exhibited a significantly better antibacterial effect than A-PRF+ within the gingivitis and periodontitis groups, with a more expansive zone of inhibition and a lower MIC. Among the studied groups, the A-PRF+ and i-PRF collected from the periodontitis group inhibited Aa significantly more compared with the gingivitis and healthy groups.

Conclusion

Although both A-PRF+ and i-PRF exhibited an antibacterial effect against Aa through the zone of inhibition and MIC tests, in the gingivitis and periodontitis groups, i-PRF exhibited better antibacterial activity than A-PRF+, and PRF products from the periodontitis group had greater effects against Aa than PRF products from the two other groups.

Introduction

Periodontal infection is a progressive obliteration of the intricate periodontal tissues, spurred on by the formation of bacterial biofilm and the host's inability to clear these networks. The cause of gum disease and periodontitis is bacterial plaque, predominantly gram-negative species like Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Tannerella forsythia, and Treponema denticola.¹ Periodontitis causes immunoregulatory changes in the body and researchers have proposed a relationship between periodontitis and other fundamental illnesses.² Many studies have demonstrated changes in the inflammatory response of patients with periodontal disease, based on blood parameters, compared with healthy patients.3, 4, 5 According to Nicu, platelets and leukocytes may be more sensitive to stimulation by periodontal pathogens.⁶ Removing bacterial biofilm by non-surgical and surgical debridement remains the gold standard to treat periodontitis, in association with antimicrobial agents to eradicate all the pathogens and stimulate lesion healing. In recent years, besides eliminating pathogens, there has been an increase in regeneration of periodontal tissues by natural agents, especially biocompatible materials).

The use of platelet concentrates (PCs) in dentistry has seen a steady increase in regenerative therapy over the past decade. Platelet-rich fibrin (PRF) is the most widely used preparation among the different PCs.⁷ In 2001, Choukroun et al. first prepared PRF as an autologous source of platelets without the addition of thrombin or anticoagulants.⁸ Recently, the effects of PRF have been documented in several systematic reviews, demonstrating its long-term impact on tissue wound healing.^7,9 Two of the main documented advantages of PRF include the fact that it contains leukocytes, host immune cells that can fight infection.¹⁰ In addition, PRF is created by high centrifugation speeds, allowing a fibrin clot to form, which acts as a three-dimensional scaffold to help heal bone and gingival tissues.¹¹

Many clinicians have now pointed to the potential use of modified PRF, prepared by modifying centrifugation forces. These products are called advanced PRF (A-PRF+), first described in 2014, and injectable PRF (i-PRF), first described in 2015.^12,13 Much like traditional PRF, A-PRF+ and i-PRF have more leukocytes and provide greater stimulation of growth factor release.¹⁴ However, to date, a limited number of studies have described the antibacterial effect of these new PRFs,15, 16, 17 and only Karde has described this property of i-PRF.¹⁸ In addition, in previous studies PRF had been collected from the blood of healthy volunteers and used commercial bacterial strains for analysis.15, 16, 17^,19,20 A question that remains is whether A-PRF+ and i-PRF obtained from healthy subjects, patients with gingivitis, and patients with periodontitis exhibit different in vitro antibacterial activity differently. Another question is whether there is a difference between A-PRF+ and i-PRF in the ability to inhibit Aa. Eagle proposed that the antimicrobial effect of PRF depends on the characteristics of the patient's blood.⁹ To our knowledge, there have been no reports comparing the antibacterial capacity of these autologous materials collected from healthy subjects, patients with gingivitis, and patients with periodontitis against periodontal pathogens. Hence, we analyzed in vitro antibacterial of A-PRF and i-PRF collected from different patient groups against Aa, a prominent periodontal pathogen.

Materials and methods

Subjects and platelet-rich fibrin preparation

Blood samples were collected from volunteers after they had provided their informed consent. Sixty subjects were divided into three groups with 20 individuals per group. Group 1 included healthy subjects without periodontal disease, group 2 included patients with severe gingivitis, and group 3 comprised patients with moderate and severe chronic periodontitis, according to the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.²¹ The Ethics Committee approved all the procedures used in this study at the Hospital of Odonto-Stomatology in Ho Chi Minh City, Vietnam (reference number: 536/BVRHM). All participants were nonsmokers and in good general health. They also had no symptoms of infection and had not taken antibiotics and anti-inflammatory drugs in the 3 months prior to the experiments. The procedure described by Choukroun and co-workers was used to prepare A-PRF+.⁸ Blood was centrifuged at 1300 rpm for 8 min in sterile plain glass-based vacuum tubes. i-PRF was prepare from blood drawn into plastic tubes without any coatings and without anticoagulant. It was centrifuged at around 700 rpm for 3 min. The upper separated liquid portion, specifically the 0.3–0.5 mL layer above the red blood cell layer, contains enriched i-PRF.²²

Bacterial preparation

Aa that we previously cultivated from the subgingival plaque of patients with periodontitis was grown in Wilkin–Chalgren anaerobic agar (CM0619, Oxoid Ltd., Hants, United Kingdom) supplemented with fetal bovine serum (F7524, Sigma–Aldrich, Missouri, United States) at 37 °C under anaerobic conditions for 72 h.²³ For practical use, the bacterial suspension was diluted to 1 × 10⁶ or 1 × 10⁵ colony-forming units (CFU) mL⁻¹.

Inhibition ring test or agar diffusion test

A-PRF + clots were obtained by centrifugation in 10-mL glass tubes. The clots were weighed and divided into 50 μg pieces to determine their antibacterial ability. Five hundred microliters of Aa (1 × 10⁶ CFU mL⁻¹) was cultured on blood agar plates. The A-PRF+ membrane on the surface of the blood agar plate was in direct contact with Aa. The samples were incubated anaerobically at 37 °C for 24 h. Chlorhexidine 0.2% (CHX, C9294, Sigma–Aldrich, Missouri, United States) was used as a positive control. After 24 h, the zone of inhibition was measured using ImageJ software (National Institute of Health, Maryland, United States). For i-PRF, after spreading a volume of Aa inoculum over the entire agar surface, many holes 6–8 mm in diameter were punched aseptically with a tip and filled with 50 μL of i-PRF. The dishes were then incubated anaerobically at 37 °C for 24 h. The zone of inhibition was measured as described above (see Fig. 1).

Figure 1.

Zone of inhibition of A-PRF+ and i-PRF. After 24 h of incubation, an inhibition zone appeared around the A-PRF clot (left)/i-PRF (right) and chlorhexidine-infused disk.

Minimum inhibitory concentration

Different volumes (50, 100, 200, and 400 μL) of i-PRF and A-PRF+ clots were prepared and placed into 24-well plates. The specific part of the A-PRF+ clot was determined by floating this clot in serum solution and calculating the displaced liquid. Four hundred fifty microliters of culture medium was added to each well. Next, 50 μL of bacterial solution (1 × 10⁶ CFU mL⁻¹) added to each well, and the plates were incubated anaerobically at 37 °C for 24 h. CHX was used as a positive control, and pure culture medium was used as a negative control. After 24 h, the contents of the well were replaced with 50 μL of resazurin 0.015%, and the change of color of resazurin was used to determine the minimum inhibitory concentration (MIC).

Statistical analysis

All experiments were performed in triplicate, and the results are presented as mean ± standard deviation (SD). SPSS v.20 software (IBM Ltd., Tokyo, Japan) was used to perform all statistical analyses. One-way analysis of variance (ANOVA) and the independent-samples T-test were used to determine statistical significance. P < 0.05 was considered statistically significant.

Results

Table 1 shows the patient characteristics. The mean patient age was significantly higher (P < 0.01) in the periodontitis group (50.9 ± 13.3 years) compared with the healthy group (23.7 ± 5.2 years) and the gingivitis group (33.0 ± 7.4 years). Table 2 shows the blood component characteristics of the three patient groups. All indexes presented typical values. There was a significant difference in the red blood cell count among the groups.

Table 1.

Patient characteristics.

Group	Age (years), mean ± standard deviation	Sex, n (%)
		Male	Female	P-valuea
Healthy	23.7 ± 5.2	11 (55)	9 (45)	0.812
Gingivitis	33.0 ± 7.4	12 (60)	8 (40)
Periodontitis	50.9 ± 13.3	13 (65)	7 (35)
P-valueb	<0.001

^aUsing the chi-square test.

^bUsing one-way ANOVA. Statistical significance was set at P < 0.05.

Table 2.

Blood cell index of the patient groups.

Group	Red blood cells ( × 1012 L−1)	White blood cells ( × 109 L−1)	Platelets ( × 109 L−1)
Healthy	4.69 ± 0.58	6.11 ± 1.83	238.2 ± 48.94
Gingivitis	4.49 ± 0.59	6.48 ± 2.13	267.35 ± 68.33
Periodontitis	4.23 ± 0.55	7.19 ± 1.67	270.75 ± 49.80
P-value	0.045	0.193	0.143

Data are presented as mean ± standard deviation; P: using one-way ANOVA. Statistical significance was set at P < 0.05.

Table 3 shows the zone of inhibition for A-PRF+ and i-PRF from different patient groups. In each group, i-PRF produced a wider zone of inhibition than A-PRF+. There were significant differences between A-PRF+ and i-PRF in the gingivitis and periodontitis groups. When comparing among the groups, the zone of inhibition of both A-PRF+ and i-PRF was significantly larger in the periodontitis group, followed by the gingivitis group, and then the healthy group.

Table 3.

Zone of inhibition (mm²) of A-PRF+ and i-PRF for each patient group.

Group	Positive control (CHX)	A-PRF+	i-PRF	P-value∗
Healthy	119.55 (12.01)	65.50 ± 9.48	70.05 ± 7.34	0.099
Gingivitis	120.04 (11.72)	66.28 ± 10.40	77.45 ± 9.70	0.001
Periodontitis	121.75 (8.14)	76.57 ± 10.80	83.33 ± 8.62	0.035
P-value∗∗		0.002	<0.001

Data are presented as mean ± standard deviation; CHX: Chlorhexidine; A-PRF+: advanced platelet-rich fibrin; i-PRF: injectable platelet-rich fibrin; ∗: differences between A-PRF+ and i-PRF, using independent-samples T-test; ∗∗: differences among the patient groups, using one-way ANOVA. Statistical significance was set at P < 0.05.

MIC values of A-PRF+ and i-PRF against Aa are presented according to the concentration of A-PRF+ and i-PRF in each well. The average MIC of A-PRF+ in the healthy, gingivitis, and periodontitis groups against Aa was 0.38 ± 0.15, 0.37 ± 0.07, and 0.35 ± 0.09, respectively. There was not a significant difference among the groups. In contrast, for i-PRF, the MIC was highest in the healthy group (0.35 ± 0.09). It was significantly higher than the MIC in the gingivitis group (0.28 ± 0.13) and the periodontitis group (0.26 ± 0.09). Furthermore, when comparing between A-PRF+ and i-PRF in each group, the MIC of i-PRF was significantly higher than the MIC of A-PRF+ in the gingivitis and periodontitis groups (Table 4).

Table 4.

Minimum inhibitory concentration of A-PRF⁺ and i-PRF against Aa.

Group	A-PRF+	i-PRF	P-value∗
Healthy	0.38 ± 0.15	0.35 ± 0.09	0.397
Gingivitis	0.37 ± 0.07	0.28 ± 0.13	0.012
Periodontitis	0.35 ± 0.09	0.26 ± 0.09	0.004
P-value∗∗	0.623	0.026

Data are presented as mean ± standard deviation; A-PRF+: advanced platelet-rich fibrin; i-PRF: injectable platelet-rich fibrin; ∗: differences between A-PRF+ and i-PRF, using independent-samples T-test; ∗∗: differences among the patient groups, using one-way ANOVA. Statistical significance was set at P < 0.05.

Discussion

To our knowledge, this is the first study that has compared the antimicrobial effect of A-PRF+ and i-PRF obtained from healthy subjects and patients with periodontal disease. An outstanding question has been whether PCs collected from patients with different periodontal conditions have different antibacterial capacity that PCs from healthy subjects. Based on the agar diffusion assay, although A-PRF+ and i-PRF from each patient group created a clear zone of bacterial inhibition, i-PRF from the periodontitis group showed the largest bacteria-free zone. The MIC test produced the same result: i-PRF had a significantly lower MIC compared with A-PRF+.

In our study, the products from the periodontitis group showed significantly fewer red blood cells and a numerical increase (not significant) in white blood cells and platelets compared with the two other groups. This result is similar to the study by Kumar:²⁴ The platelet counts of 120 subjects divided into two groups of chronic periodontitis patients and healthy groups showed no significant difference between the groups. In addition to leukocytosis, Al-Rasheed and Papapanagiotu reported an increased platelet count in patients with periodontitis, and periodontal treatment reduced platelet levels.^2,25 Changes in whole blood may affect the properties of PRF after formation. Indeed, the characteristics of PRF vary among patient groups. Miron showed that PRF produced by the blood of female and elderly patients with periodontitis produced a larger PRF membrane size than that of men and young adults.⁷ Yajamanya et al. found that the fibrin network of PRF-based membranes is less dense as the patient age increases.²⁶ Zhan et al. showed a decrease in platelet size in patients with generalized aggressive periodontitis due to the consumption of large platelets at the site of periodontal inflammation.²⁷

In contrast, studies have shown that PRF membranes and fibrin clots led to a measurably significant expansion of human gingival fibroblasts at 24 h, and there was a similar pattern with more cells at 3 and 5 days.^28,29 Patients with persistent provocative illnesses have expanded foundational levels of pro-inflammatory cytokines and developmental factors. A new report measured the development factors set free from PRF, serum concentrations of several cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α) and complete blood count (CBC) in patients with periodontitis versus healthy subjects.³⁰ There was only a difference in the white blood cell count, which was higher in the periodontitis group and had no connection with the level of developmental factors in PRF.³⁰ Therefore, PRF collected from different patient groups may exhibit distinct antimicrobial effects against Aa.

Regarding the antibacterial ring experiment, the zone of inhibition measured by Image J software showed no Aa around A-PRF+ and i-PRF, with a larger zone of inhibition around i-PRF than A-PRF + for all three patient groups. This result is similar to the study by Kour et al., who suggested that PRF could exhibit antibacterial effects against Aa. However, the authors also showed that the sterile region of PRF and i-PRF is significantly smaller than platelet-rich plasma (PRP).¹⁵ This phenomenon may be due to sodium citrate, an anticoagulant used to produce PRP, which also confers antimicrobial activity.¹⁵ Karde evaluated PRP, i-PRF, and PRF and found a significantly greater zone of inhibition for i-PRF and PRF.¹⁸ On the other hand, Badade et al. suggested that PRF does not exhibit antibacterial activity against Aa.¹⁹ In addition, when independently investigating the antibacterial activity of the membrane and PRF extract, Castro et al. recorded the zone of inhibition for the PRF membrane and PRF exudate after 72 h of anaerobic incubation. They concluded that both components were not resistant to Aa.¹⁷

In our study, the sterile area was significantly greater in the periodontitis group. Consistently, the MIC was significantly lower for the products obtained from the periodontitis group. According to Table 4, the mean MIC value of A-PRF + hovered around 35%–38%, while this value of i-PRF was 26%–35%. This result was higher than a previous study by Jasmine et al., who reported MIC of i-PRF against Staphylococcus aureus, Staphylococcus epidermis, S. epidermis ATCC 35984, and S. epidermis ATCC 12228 of 16%, 16%, 16%, and 8%, respectively.¹⁶ Those microbes are gram positive without an outer lipid membrane, so they might be more sensitive to antibacterial agents. The outer layer of gram-negative microbes, such as Aa, is the primary driver of protection from numerous antimicrobials, including β-lactams, quinolones, colistin, and other bactericidal agents. Most biocides should cross the external layer to arrive at their target. Hence, any adjustment in the outer layer by microbes—for example, an adjustment of hydrophobicity or a change in the porin channel—can lead to obstruction.¹⁶ The MIC results showed that i-PRF could inhibit Aa better than A-PRF+ (Table 4), and the lowest MIC was found in the products obtained from the periodontitis group. Hence, when generating A-PRF+ and i-PRF, patients with periodontitis have better Aa antibacterial ability than PCs obtained from healthy human blood. In the case of gingivitis, the difference was not significant. Cheng suggested that the local gingival inflammatory response is not reflected by apparent changes in the frequencies of primary blood immune cell subsets.³¹

As mentioned above, there was a change in blood cells between the healthy and periodontitis groups. The increased platelets and white blood cells may have increased the antibacterial activity of the PCs. Platelets produce oxygen metabolites and antimicrobial peptides, which target bacterial cells, and also help to limit the spread of microorganisms in the bloodstream.³² Besides, plasma contains a supplementary framework, which can promote bacterial cell lysis and leukocyte enlistment for humoral immunity against pathogens.³³ The alpha granules of platelets are believed to contain proteins with antibacterial potential.³⁴ They also contain complement and complement-binding proteins, which facilitate the removal of microorganisms from the circulatory system.³³ In addition, Papapanagiotu emphasized that there was increased platelet activation in patients with periodontitis compared with controls, denoted by increased sP-selectin and sCD40 platelets.²⁵ The authors emphasized that in addition to monocytes, platelets in patients with periodontitis were more susceptible to activation than healthy patients.³⁵ Brousseau-Nault et al. demonstrated that local and systemic elevation of platelet factor 4 (PF4) is associated with periodontitis. At the same time, circulating PF4 levels were doubled in patients with severe periodontitis compared with controls.³⁶

Leukocytes are notable white blood cells that have an extensive bactericidal capacity. Upon substantial contamination, actuated neutrophils move to the disease site and deliver active antibacterial substances to advance the phagocytosis of unfamiliar microorganisms. Leukocytes produce an assortment of antimicrobial peptides and compounds, including lactoferrin, defensins, BPI azurocidin/heparin-restricting protein, cathelicidins, phospholipase A2, and calprotectin.³⁷ However, the role of leukocytes in PCs remains controversial. According to some authors, an increase in the concentration of leukocytes in PCs can improve substrate stability, increase antibacterial potential, and modulate the inflammatory response.³⁸ In our study, there was a numerical (but not significant) increase in white blood cells and platelets in the periodontitis group that may have contributed to the antibacterial activity against Aa. Furthermore, PRF was prepared by manipulating patient blood without any other additives, so if PRF of patients with periodontitis exhibits a better capacity to fight infection, then these patients would receive a greater benefit to improve their periodontal condition.¹⁴

There is not a lot of proof for the antibacterial action of i-PRF against periodontal microorganisms especially in patients with periodontal diseases because i-PRF has only been described in recent years. In our study, even though both PRF items had an antibacterial effect against Aa, i-PRF had a greater impact than A-PRF+ and could serve as a potential periodontal treatment. According to Jasmine, there is heterogeneity between PRF and i-PRF regarding the fibrin matrix architecture (shape and pore size) and fibrin content (diameter, width, roughness, and smoothness of fibrin fibers). This disparity may alter the concentrations of platelets and leukocytes. The dissimilarity in the topography of the PRF and i-PRF matrixes and the cells entrapped in the fibrin network may influence the quality of the PRF, in terms of wound healing and, to a greater extent, the antimicrobial effect.³⁹

Within the limits of this study, in the gingivitis and periodontitis groups, i-PRF exhibited better antibacterial activity than A-PRF+, and PRF products of patients with periodontitis had a greater effect against Aa than the two other patient groups. However, additional studies with a larger population are required to confirm the link between the antimicrobial effects of PRFs for different patient groups.

Declaration of competing interest

The authors have no conflicts of interest relevant to this article.