Name: prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp
Uploaded: 2013-04-09T12:00:00
Description: Watch this Scientific Journal Video about PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP at JoVE.com

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.

1. Preparation and Activation of PRP
1.1 Blood draw
<ol>
	<li>Anesthetize rabbit by inhalation of isoflurane (2% in O2 for induction and 1% for maintenance).</li>
	<li>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 &mu;m sterile filter.</li>
	<li>Sterilize the rabbit ear using 70% ethanol.</li>
	<li>Dr...

<ol>
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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...

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.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bovine thrombin</td>
 <td>King Pharmaceuticals, Inc </td>
 <td>60793-215-05</td>
 <td>Thrombin (bovine origin)</td>
 </tr>
 <tr>
 <td>Calcium chloride</td>
 <td>King Pharmaceuticals, Inc</td>
 <td>60793-215-05</td>
 <td>10% calcium chloride</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>E7023</td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Baxter</td>
 <td>1001936060</td>
 <td></td>
 </tr>
 <tr>
 <td>Mueller Hinton broth</td>
 <td>Becton, Dickinson and Company</td>
 <td>275710</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>Tri-sodium citrate</td>
 <td>Sigma-Aldrich</td>
 <td>W302600</td>
 <td></td>
 </tr>
 <tr>
 <td>Tryptic soy agar</td>
 <td>Fisher Scientific</td>
 <td>R01202</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Kendro Laboratory Products</td>
 <td>750043077</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe filter</td>
 <td>Millipore</td>
 <td>SLGP033RS</td>
 <td></td>
 </tr>
 </tbody>
</table>

prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp

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.

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...

Watch this Scientific Journal Video about PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP at JoVE.com

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

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.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

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T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...