Antimicrobial activity of pure platelet-rich plasma against microorganisms isolated from oral cavity

In the past 20 years, the use of autologous platelet concentrates (PCs) has gained great popularity in a variety of medical fields such as dentistry, oral surgery, orthopedics, sports medicine, dermatology, ophthalmology, cosmetic and plastic surgery. The rationale for their use stems from the fact that platelets store and release, upon activation, growth factors such as PDGF, TGF-β, EGF, VEGF, IGF-1, FGF, HGF and other molecules that modulate the wound healing response in both hard and soft tissues. In addition, anti-inflammatory properties of PCs have been pointed out associated with a marked reduction of postoperative pain and swelling [1–3].

Recently, the clinical and in vitro antibacterial effect of human PCs has been reported against bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Klebsiella pneumoniae while no activity has been found against Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter cloacae, Bacillus cereus and Bacillus subtilis[4–12].

The mechanism of the antibacterial effect of PCs is not yet fully understood. Existing evidence suggests that platelets may play multiple roles in antimicrobial host defense: they generate oxygen metabolites, including superoxide, hydrogen peroxide and hydroxyl free radicals; [13–15] they are capable of binding, aggregating, and internalizing microorganisms, which enhances the clearance of pathogens from the bloodstream; they participate in antibody-dependent cell cytotoxicity functions to kill protozoal pathogens; finally, platelets release an array of potent antimicrobial peptides [16, 17].

Several techniques are available for the production of PCs, leading to products with different biological characteristics. The various PCs can be classified into four main categories, depending on their leucocyte and fibrin content: pure platelet-rich plasma (P-PRP), pure platelet-rich fibrin (P-PRF), leukocyte- and platelet-rich plasma (L-PRP) and leukocyte- and platelet-rich fibrin (L-PRF). [18] L-PRP and L-PRF might contain substantial amount of white blood cells. The respective effects of platelets and leucocytes in PCs have not been elucidated yet, and the contribution of leucocytes to the observed overall effect remains unclear [19]. Therefore in this study we decided to use a widely documented technology developed in 1999 by Anitua that allows the production of leukocyte-poor platelet concentrate [20].

The aim of this study was to evaluate in vitro the antibacterial effect of P-PRP against microorganisms colonizing the oral cavity such as Enterococcus faecalis, Candida albicans, Streptococcus agalactiae, Streptococcus oralis and Pseudomonas aeruginosa.

As expected, the P-PRP produced was leukocyte-depleted (0,34 ± 0,27) × 10³/μl. In order to obtain the minimum platelet concentration ranges of P-PRP capable of inhibiting bacterial growth, we calculated the mean MIC of the 5 strains tested for each microorganism.

MIC values observed for ATCC bacterial strains fell into the same platelet concentration ranges as those of the corresponding clinical isolates.

MBC tests showed that C. albicans was never killed by P-PRP, while the other microorganisms were killed at concentrations 3–4 times the MIC.

The regenerative potential of PCs has been explored considerably during the last two decades. On the contrary, in the available literature only few reports can be found about their antimicrobial effects.

To date, the components responsible for the antimicrobial activity of PCs remain poorly understood, in particular because these materials are a complex mixture of platelets, white blood cells and plasma. The respective impact of the plasma and cellular components has not been studied in detailyet. Several antimicrobial factors have been proposed, including platelet antimicrobial proteins and peptides of the innate immune defense, or platelet α-granules components, such as complement and complement-binding proteins. [17, 21–26] Direct interaction of platelets with microorganisms and participation in antibody-dependent cell cytotocity and white blood cells in direct bacterial killing, release of myeloperoxidas, activation of the antioxidant responsive element and antigen-specific immune response have also been suggested. [12, 15, 27] The role of leucocytes within PCs is a matter of intense debate. Some authors have suggested that inclusion of white blood cells in PCs may help to improve the stability of the scaffold and increase the antimicrobial potential. [18] However, Anitua et al. [20] results showed that a further leucocyte dose did not significantly improve the antimicrobial properties of P-PRP. It is also possible that the additional leukocyte content might increase the inflammatory response at the site because of the metalloproteases, pro-inflammatory proteases and acid hydrolases secreted by white blood cells [28].

Bacterial infection is one of the most serious complications impairing wound healing and tissue regeneration. Even when applying strict disinfection, bacteria can infiltrate and colonize the underlying tissues of the wound. The combination of proteolytic enzymes, toxin-rich bacterial exudates and chronic inflammation can alter growth factors and metalloproteinases, thereby affecting the cellular machinery needed for cell proliferation and wound healing [29, 30].

Developing approaches and strategies that may help to control or prevent the problem of wound infections would have considerable clinical, social and economic effects.

Our study has shown that P-PRP was active against microorganisms colonizing the oral cavity such as E. faecalis, C. albicans, S. agalactiae and S. oralis, but not against P. aeruginosa. Except for E. faecalis and P. aeruginosa, PCs have never been tested against such microorganisms.

E. faecalis is associated with different forms of periradicular disease, including primary extraradicular and post-treatment persistent infections. [31] Such microorganism possesses the ability to survive the effects of root canal treatment and persists as a pathogen in the root canals and dentinal tubules of teeth. Implementing methods to effectively eliminate E. faecalis from the dental apparatus is a challenge. We found that P-PRP was active at low platelet concentration ranges (1–2 orders of magnitude lower than the baseline blood values) against this microorganism, while Bielecki et al. [10] observed no activity of platelet concentrate. The reasons for this discrepancy may lie in the different protocol used for platelet concentrate production, which can lead to products with different biological characteristics, or in the different sensibility of the method (Kirby-Bauer disc-diffusion method) used to evaluate the susceptibility to platelet concentrate.

Oral candidosis is the most common fungal infection encountered in general dental practice. It manifests in a variety of clinical presentations and can occasionally be refractory to treatment. It is caused by commensal Candida species. While a large majority of healthy individuals harbor strains of Candida intraorally, only selected groups of individuals develop oral candidosis. The most commonly implicated strain is C. albicans, which is isolated in over 80% of oral candidal lesions. [32] In the present study, we observed that P-PRP was active against C. albicans at higher plateletconcentration ranges (same order of magnitude of the baseline blood values) than those effective against the other bacteria tested. This result is consistent with the findings of Tang et al. who tested in vitro antimicrobial activity of seven antimicrobial peptides isolated from human platelets, and noticed that they were more potent against bacteria than fungi [17].

S. agalactiae, S. oralis and P. aeruginosa are some of the many oral biofilm bacteria. We observed that P-PRP was active against S. agalactiae and S. oralis at platelet concentration ranges similar to the range which inhibited E. faecalis. On the contrary, we found no activity of P-PRP against P. aeruginosa at the concentrations used in this experiment. This result is in line with the findings of Bielecki et al. and Burnouf et al., who even observed that platelet concentrate induced growth of this microorganism, suggesting that platelet concentrate may induce a flare-up of infection from P. aeruginosa. [10, 11] The value of PCs in the presence of a co-existing infection with this bacterium is therefore uncertain.

In our study we also used standard ATCC bacterial strains, which may behave in a way different from isolates, in order to assure reliability of results and reproducibility of experimentation. Results were similar to those obtained with clinical isolates of bacteria.

In addition, we performed a MBC test. We found such test difficult to perform, as P-PRP coagulates at high concentrations. We observed that C. albicans was never killed, while the other microorganisms were killed at concentrations 3–4 times the MIC. Further studies are necessary to investigate the potential bactericidal effect of P-PRP. In this study we tested P-PRP in the formulation commonly used in dentistry and oral surgery (that is, plasma fraction activated with CaCl2 to form a solid coagulum) to assess the potentiality of the use of such preparation in routine clinical practice. Future research may be focused on the analysis of the contribution of individual P-PRP components by employing methods such as separation (e.g. by fractionation according to size) or inactivation (e.g. by exposure to modifying agents, such as specific proteases, or to physical factors, such as heat treatment).

In conclusion, PCs are safe autologous products, which can be easily prepared during surgery and possess an antibacterial activity. They could be potentially useful substances in the fight against postoperative infections and might represent the linking of osteoinductive and antimicrobial activity. Further research should investigate PCs antimicrobial capacity compared to antibiotics, their exact antibacterial spectrum and prove its efficacy in the in vivo situation. The influence of patients’ characteristics (sex, age, hematocrit, platelet count, drug assumption, etc.…) on antimicrobial activity should be also clarified.