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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Streszczenie

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.

In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.

PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.

For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

Wprowadzenie

Implant-associated infection is a significant clinical complication. Staphylococcus aureus (S. aureus) is one of the most common microorganisms isolated from implant-associated infections. It is capable of producing a biofilm that covers the surfaces of implants and may lead to antibiotic-resistant infection 1,2. Treatment of implant-associated infection frequently requires long-term hospitalization for repeated debridements and prolonged parenteral antibiotic therapy. In antibiotic resistant cases, removal of the implant may be necessary. The rising resistance of bacteria to antibiotics has also been referred to by the Centers for Disease Control and Prevention (CDC) as "one of the world's most pressing health problems." In time, without the development of new and effective antimicrobial treatments, it is possible that multi-drug resistant pathogens will be untreatable with conventional antibiotics. Prevention of implant-associated infection is therefore important and novel prophylactic agents or approaches are needed for preventing such infections.

Platelet-rich plasma (PRP) is a concentration of autologous blood that contains over 30 growth factors which can help with bone and bone graft healing 3-5. The application of PRP to enhance bone regeneration and soft tissue maturation has been increasingly reported in clinics because of its high concentration of various growth factors released by platelets.

Several characteristics of PRP indicate that PRP may also have antimicrobial properties 6-9. PRP contains a large number of platelets, a high concentration of leukocytes (which may possess host-defense actions against bacteria and fungi), and multiple antimicrobial peptides 7,8,10. In a recent study of a large cohort of cardiac surgical patients, it was revealed that the intraoperative use of PRP-gel during wound closure significantly decreased the incidence of superficial and deep sternum infection 11. For these reasons and observations, we hypothesized that PRP, besides its well-studied healing-promoting properties, has antimicrobial properties. The potential advantages of using PRP to prevent infection may include: (i) PRP is less likely to induce resistance compared to conventional antibiotic treatments. (ii) PRP also has properties that promote healing which may have a synergistic effect on infection prevention; PRP's healing-promoting properties could provide a seal to prevent bacterial attachment thereby reducing the odds for infection as pathogens and human cells are racing for implant surfaces 12,13. (iii) PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.

Our long-term goal is to use PRP as a new approach to prevent implant-associated infections. The aim of this study was to prepare PRP using a twice centrifugation approach, to examine PRP's in vitro antimicrobial properties, and to describe the protocols for evaluating such antimicrobial properties.

Protokół

1. Preparation and Activation of PRP

1.1 Blood draw

  1. Anesthetize rabbit by inhalation of isoflurane (2% in O2 for induction and 1% for maintenance).
  2. Draw 2 ml 0.129 M tri-sodium citrate (an anticoagulent solution) into a 20 ml syringe. The tri-sodium citrate solution is prepared by dissolving 1.897 g tri-sodium citrate in 50 ml distilled H2O and filtering with a 0.22 μm sterile filter.
  3. Sterilize the rabbit ear using 70% ethanol.
  4. Draw blood (e.g. 5 ml) from the rabbit ear vein via a butterfly needle (25 G) connected to the syringe.
  5. Mix the blood with the tri-sodium citrate solution by gentle agitation. The volume ratio of blood and tri-sodium citrate solution is 9:1.

1.2 PRP preparation (Figure 1)

  1. Transfer the anticoagulated blood to a 50 ml plastic centrifuge tube. Take an aliquot of 10 μl blood to determine the baseline platelet count using hemocytometry.
  2. Centrifuge the blood at 300 x g for 10 min at room temperature (RT) in a centrifuge with a swing-out rotor. Set the acceleration and brake velocity to low (Figure 1).
  3. After centrifugation, the blood is separated into three layers. The bottom layer is mainly red blood cells, the middle layer (commonly referred to as the "buffy coat") is composed of concentrated platelets and leukocytes, and the top layer is mainly plasma, which is the liquid component of blood, and platelets (Figure 1). Carefully transport the centrifuge tube to a cell culture hood; do not disturb the layers. Transfer all of the plasma, buffy coat, and 2-3 mm thick red blood cell layer into a 15 ml plastic tube using a 1 ml plastic pipettor.
  4. Centrifuge the transferred sample a second time at 3,000 x g for 15 min at RT. The top layer (supernatant) is considered platelet poor plasma (PPP) and is transferred to a new tube.
  5. Obtain PRP by adjusting the platelet concentration in the remaining blood sample using PPP to obtain 2.0 x 106 platelets/μl (determined by hemocytometry).

1.3 PRP activation

  1. Prepare PRP activation solution by dissolving 5,000 IU bovine thrombin with 5 ml 10% calcium chloride to the working concentration of 1,000 IU/ml.
  2. Add the activation solution to PRP and PPP, and mix the solution by repeatedly pipetting to form PRP- and PPP-gels. The volume ratio of the activation solution to PRP or PPP is 1:4.

2. In vitro Antimicrobial Test of PRP Using Kill Curve Assay (Figure 2)

  1. Using a sterile inoculating loop, add several colonies of S. aureus from its overnight plate culture into 5 ml of Mueller Hinton broth (MHB) in a plastic tube. Vortex briefly and then incubate the sample for 2 hr at 37 °C. Next, the optical density of the bacterial media was determined using a spectrophotometer and adjusted to an optical density equal to ~1 x 108 CFU/ml based on the pre-determined standard curve.
  2. Make a 100x dilution using PBS to obtain 1 x 106 CFU/ml and place the inoculums on ice.
  3. Set up and label sterile, disposable 5 ml round-bottom polystyrene tubes, and prepare the following sample groups as indicated in Table 1 for a final volume of 2 ml in each tube.
  4. Add PRP, PPP, or PBS first to the polystyrene tubes, followed by the thrombin solution for activation (gel formation). Next, add MHB and then the S. aureus inoculums (1 x 106 CFU/ml) to obtain the final concentration of 1 x 105 CFU/ml.
  5. Incubate the tubes at 37 °C with orbital agitation at 150 rpm.
  6. At pre-determined time points (e.g. 0, 1, and 2 hr), mix the solutions in each tube via repeat pipetting (this step is important since bacteria may be trapped inside the PRP gel). Take 10 μl of sample, dilute serially with sterile 0.9% saline, and pipette a 100 μl aliquot of each dilution onto a Tryptic soy agar (TSA, with 5% sheep blood) plate for CFU counting.
  7. Culture the agar plates overnight at 37 °C, then count and record the plate colonies. Plot data on a logarithimic scale with time (hr) on the x-axis and CFU/ml on the y-axis.

Wyniki

PRP is reproducibly prepared using a twice centrifugation approach (Figure 1). PRP is found to present strong (up to 100-fold reduction in CFUs) in vitro antimicrobial properties against methicillin resistant S. aureus (MRSA) (Figure 3), which is commonly found in hospitals worldwide 14. Similarly, PRP has strong antimicrobial properties against methicillin sensitive S. aureus (MSSA), Group A Streptococcus, and Neisseria gonorrhoeae...

Dyskusje

Platelet-rich plasma has been increasingly used for clinical applications due to its healing-promoting properties 15-17. In the present study, PRP was presented as a new approach for infection prevention. PRP was found to have strong antimicrobial properties against MRSA, MSSA, Group A Streptococcus, and Neisseria gonorrhoeae. The major advantages of PRP, compared to conventional antibiotic treatments, for infection prevention include: (1) Current antibiotic therapies are facing challenges in...

Ujawnienia

The authors declare that they have no competing financial interests.

Podziękowania

The authors thank Therwa Hamza, John E. Tidwell, Nina Clovis, and Suzanne Smith for experimental assistance and Suzanne Smith for proofreading. The authors also thank John Thomas, PhD for providing the bacterial clinical isolates and John B. Barnett, PhD for his support and the use of the biological safety lab at the Department of Microbiology, Immunology and Cell Biology at West Virginia University. The authors acknowledge financial support from the Osteosynthesis and Trauma Care Foundation and National Science Foundation (#1003907). Microscope experiments and image analysis were also performed in the West Virginia University Imaging Facility, which is supported in part by the Mary Babb Randolph Cancer Center and NIH grant P20 RR016440.

Animal use for blood draws were approved by the West Virginia University Institutional Animal Care and Use Committee. All experiments were executed in compliance with all relevant guidelines, regulations, and regulatory agencies.

Materiały

NameCompanyCatalog NumberComments
Name of Reagent/MaterialCompanyCatalog NumberComments
Bovine thrombinKing Pharmaceuticals, Inc 60793-215-05Thrombin (bovine origin)
Calcium chlorideKing Pharmaceuticals, Inc60793-215-0510% calcium chloride
EthanolSigma-AldrichE7023
IsofluraneBaxter1001936060
Mueller Hinton brothBecton, Dickinson and Company275710
Phosphate-buffered salineSigma-AldrichD8662
Tri-sodium citrateSigma-AldrichW302600
Tryptic soy agarFisher ScientificR01202
CentrifugeKendro Laboratory Products750043077
Syringe filterMilliporeSLGP033RS

Odniesienia

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Keywords Platelet rich Plasma PRPAntimicrobial PropertiesImplant associated InfectionAntibiotic ResistanceOrthopaedic InfectionsMethicillin sensitive Staphylococcus AureusMethicillin resistant Staphylococcus AureusGroup A StreptococcusNeisseria GonorrhoeaeInfection PreventionIn Vitro Antimicrobial ActivityBiocompatibleTwice Centrifugation Approach

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