Authors:  Derek L. Hill, DO, FAOAO1, Cody Pinger, PhD2, Erica L Noland MS, DVM, DACVP3, Kevin Morton, DO4, Alessandra M Agostinho Hunt, DDS, PhD5, Elizabeth Pensler, DO, FACOS6, Sarah Korn, BS7, Paul S Attar, PhD7, Ahmed Siddiqi, DO, MBA8  Clinical Associate Professor of Surgery  Michigan State University College of Osteopathic Medicine  Hill Orthopedics  derekhill08@gmail.com  Research Assistant  Institute for Quantitative Health Science and Engineering  Michigan State University   pingerco@msu.edu  Department of Pathobiology and Diagnostic Investigation  College of Veterinary Medicine  Michigan State University  nolande1@msu.edu  General Surgery Resident  Ascension Macomb-Oakland  Michigan State University College of Osteopathic Medicine  kdm920@gmail.com   Safety Technologist   Environmental Health and Safety Department  Michigan State University   alehunt@msu.edu  Vascular Surgeon  Pensler Vein and Vascular Surgical Institute  Warren, MI  epensler@yahoo.com BRIDGE PTS, Inc  San Antonio, TX  Orthopedic Surgeon Adult Reconstruction Fellow  Cleveland Clinic Foundation  Cleveland, OH  Asiddiqi89@gmail.com  Corresponding Author:  Ahmed Siddiqi, DO, MBA  Cleveland Clinic Foundation  9500 Euclid Avenue, Crile Building A4  Cleveland, OH, 44195  Mobile: 718 869 0048  Asiddiqi89@gmail.com  KEY POINTS:  Question: What is the antimicrobial efficacy of a novel dual-action solution combining activated-zinc and sodium chlorite against Staphylococcus aureus and Pseudomonas aeruginosa?  Findings: Our novel activated-zinc compound demonstrated 99.5-99.9% reduction in total bacteria, 99.9-99.98 % reduction in S. aureus, and 100% eradication of Pseudomonas 1 hour after exposure. A synergistic zinc chloride and sodium chlorite solution avoids the cytotoxicity of CHG and povidone-iodine at varying concentrations.  Meaning: This novel solution may provide another significant tool in the armamentarium to treat and/or prevent PJI and other wound infections. ABSTRACT:  Importance: Identifying the optimal agent for irrigation for periprosthetic joint infection remains challenging as there is limited data. The ideal solution should have minimal cytotoxicity while maintaining bactericidal activity.  Objective: The purpose of this study was 1.) to investigate the antimicrobial efficacy of a novel activated-zinc solution (CleanZinc™) against Staphylococcus aureus and Pseudomonas aeruginosa and 2.) to evaluate any untoward effect of the solution on local wound tissue at 24 hours after solution exposure in pig wound models.  Design: The study was conducted and reported in accordance to the ARRIVE guidelines. One female, commercially raised, Yorkshire-cross female pig was acquired 12 days prior to the procedure. Within 1-week prior to the procedure, a small loopful of test bacteria, Pseudomonas (pig-isolate) and S. aureus (ATCC-6538), were streaked and cultured on a non-selective agar. 8 wounds were controls (exposed to bacteria but untreated). 8 wounds were treated with CleanZinc™-1, and 8 with CleanZinc™-2. Wounds were dressed and monitored for 1 hour and 24 hours.  Setting: A porcine model study  Participant: 1 porcine model  Main Outcomes and Measures: The primary outcome was to determine bacterial load after 1 hour(s) of treatment/exposure to test compounds. The secondary outcome was to evaluate local wound necrosis and neutrophil infiltrate 24 hours after exposure to CleanZinc™ solution.   Results: After 1 hour, the wounds treated with CleanZinc™-1 and CleanZinc™-2 had mean recoverable total bacteria of 2.8 and 3.5 logCFUs per gram of tissue, respectively. After 1 hour, the wounds treated with CleanZinc™-1 and CleanZinc™-2 had mean recoverable S. aureus of 2.3 and 1.6 logCFUs per gram of tissue, respectively (p=0.009). After 1 hour, the wounds treated with CleanZinc™-1 and CleanZinc™-2 had mean recoverable Pseudomonas of 0.3 and 0.0 logCFUs per gram of tissue, respectively (p=0.000). After 24 hours of exposure to CleanZinc™-1 and CleanZinc™-2, there was no statistically significant increased necrosis (p=0.12, p=0.31, respectively) or neutrophilic infiltrate (CleanZinc™-2, p=0.12) when compared to controls.   Conclusions and Relevance: Our novel activated-zinc compound demonstrated 99.5-99.9% reduction in total bacteria, 99.9-99.98 % reduction in S. aureus, and 100% eradication of Pseudomonas 1 hour after exposure. This novel solution may provide another significant tool in the arsenal to treat and/or prevent PJI and other wound infections.  INTRODUCTION:  With the rising number of total joint arthroplasties (TJA) performed in the United-States (USA) annually,1,2 periprosthetic joint infection (PJI) and surgical site infection (SSI) remains the most common and devastating complications.3,4 Debridement and irrigation using multiple irrigating solutions has been the first-line of PJI management.5 Identifying the optimal irrigation agent remains challenging as there is limited data on superiority. Although irrigating solutions contain antiseptics, antibiotics, detergents and/or surfactants, the cytotoxicity of some components may increase wound healing complications.6 Commonly used antiseptic solutions including chlorhexidine gluconate (CHG), hydrogen peroxide, sodium hypochlorite and povidone-iodine have all been associated with local tissue damage.6,7   The ideal antiseptic solution has minimal cytotoxicity at its Minimum Bactericidal Concentration (MBC), which is defined as the concentration required to diminish the bacterial load by 99.9% (3-log).6,7 Zinc chloride (ZnCl2) solutions have been well-reported to have bactericidal properties in the dental literature, as it is a key ingredient in halitosis and dental biofilm oral solutions with minimal cytotoxicity.8,9 Additionally, sodium chlorite (NaClO2) is effective at raising oxidation-reduction potential in low concentrations and works synergistically with ZnCl2.10,11   There is no study, to our knowledge, that explores the efficacy of a dual-action antimicrobial solution combining activated-zinc (ZnCl2) with an agent that raises the oxidation-reduction potential (NaClO2) against common PJI/SSI organisms including Staphylococcus aureus and Pseudomonas aeruginosa. Therefore, the purpose of this study was 1.) to investigate the antimicrobial efficacy of a novel solution (CleanZinc™) against S. aureus and Pseudomonas and 2.) to evaluate any untoward effect on local wound tissue at 24-hours after exposure in pig wound models. We hypothesize that this novel therapeutic solution will be highly effective in decreasing bacterial inoculation burden on surgical wounds without clinically significant local tissue adverse reaction.  MATERIALS & METHODS:  Ethics:  One pathogen-free, female, commercially raised, Yorkshire-cross female pig (34.2 kg) was purchased from a USDA accredited vendor (Real Hog Farms, Marion,TX) 12 days prior to testing. The animal was housed at the test facility in a raised stainless-steel pen cage and identified by permanent marking on the ear with a corresponding designation on the cage card. The pig was fed an antibiotic-free diet (PMI Nutrition International, Brentwood, MO). The animal was cared for in accordance with the “Guide for the Care and Use of Laboratory Animals” published by National Research Council12 and approved by AAALAC International.13 The study was conducted and reported in accordance to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.14  Microorganisms:  Within a week prior to the procedure, a loopful of test bacteria, Pseudomonas (pig isolate) and S. aureus (ATCC-6538), was streaked from frozen collections and cultured on a non-selective agar plate (tryptic soy agar (TSA)). Bacteria were incubated overnight (18-24 hours) at 37°±3°C and visually examined for purity. All bacteria colonies demonstrated the same morphology and color. The day prior to the day of infection, a loopful of Pseudomonas and S. aureus from an 18-24-hour agar plate of TSA were inoculated into an appropriate volume (20 mL) of tryptic soy broth (TSB) in individual sterile tubes. Fusobacterium sp. was added to the bacterial cocktail given its synergistic effect with co-organism growth in mixed infections,15 but was not recovered for final analyses. Fusobacterium sp. was obtained from a frozen culture and grown under anaerobic conditions in 20mL TSB supplemented with 5% Vitamin K for 48 hours in an incubator at 37°±3°C without shaking prior to inoculation. Pseudomonas and S. aureus were incubated at 37°±3°C with shaking at 150-rpm (Thermo Scientific, MaxQ 4450) overnight.   On the morning of the procedure, the organisms were prepared to a final density of approximately 108±1 log10CFU/mL in phosphate buffered saline (PBS) using the following approach: The bacterial suspensions were centrifuged once at 8,000-rpm (Thermo Scientific, FiberLite F15-6X100) for 15-minutes. Overnight cultures were assumed to grow to a density of approximately 108CFUs/mL. After the supernatant was removed, the pellet was re-suspended into the same PBS volume. The microorganisms were mixed together in a ratio of 1:1:0.5 (Pseudomonas:Staphylococcus:Fusobacterium). Sample sets were plated on TSA, Mannitol Salt Agar (MSA), and Pseudomonas Isolation Agar (PIA) to determine total bacterial counts, S. aureus and Pseudomonas load in the biopsy specimens, respectively. Bacterial counts were expressed as log10CFU/g.  Antiseptic Solution (CleanZinc™):  Our unpublished data determined Mean Inhibitory Concentration (MIC) and MBC for ZnCl2 solution, and independently for NaClO2 solution.11 A synergistic benefit was found with the combination of the two solutions, with significantly lower MIC/MBC for each component when combined immediately before bacterial exposure. The combination of these 2 solutions is commercially known as CleanZinc™. We used these data to formulate ZnCl2 and NaClO2 solution concentrations for the current in-vivo study.  Solution-1 contained 70.0mM NaClO2 with 154.0mM NaCl (0.9%) in pH 7.5, buffered with 10.0mM benzoic acid/benzoate. A diluent buffer (Buffer-A) was prepared with 18MΩ distilled and deionized H2O(DDW) to contain 127mM NaCl and 10mM benzoic acid. The pH was raised to 12.0 to dissolve the benzoic acid into solution. HCl was sequentially added until pH 7.2. The buffer was subsequently filtered through a 0.2-micron sterile filter (Sigma Aldrich). Solid NaClO2 from Fisher Scientific (Hampton, New Hampshire) with a reported purity of 80% was weighed to make a 70.0mM sodium chlorite solution when diluted to 50mL. The sodium chlorite was diluted with Buffer-A and an adjusted pH 7.5 with a total 50mL volume. The process was repeated to make a total of 100mL. The amount of sodium chlorite needed for the solution was calculated based on the reported reagent percent purity (80%) with a final NaCl concentration of 154mM. The solution was sterile filtered twice through 0.2-micron filters and transferred into sterile 50mL polypropylene conical tubes (Greiner, Kremsmunster, Austria), under sterile conditions. The caps of the tubes were sealed with parafilm.  Two concentrations of the second solution were made to contain 70mM (Solution-2) and 50mM (Solution-3) zinc chloride (ZnCl2), respectively, and 154.0mM NaCl (0.9%) in pH 4.8 buffered with 10.0mM benzoic acid/benzoate. A diluent buffer (Buffer-B) was prepared and sterily filtered with DDW to contain 154mM NaCl and 10mM benzoic acid to a final 500mL volume with adjusted pH 4.8. Prior to use, ZnCl2 (ACS grade, Crystals, Columbus Chemical Industries Inc., Columbus, WI) was dried for 2-hours at 90°C. Dried ZnCl2 was then measured to make 70mM and 50mM ZnCl2 solution when diluted to 50mL. The ZnCl2 was diluted with Buffer-B and the pH was adjusted to 4.8 with HCl. The total volume of both solutions was 50mL each. This process was repeated to make a total of 100mL per solution, which were subsequently sterile filtered and packaged similar to Solution-1.   After 20 hours, the solutions were test-mixed in a 1:1 ratio, by mixing 2mL ZnCl2 solution with 2mL NaClO2 solution in a clean conical tube. The resulting solution pH was 5.40±0.5.s  Procedure:  The pig was premedicated with intramuscular injection of Atropine (0.05 mg/kg) for 15-minutes and anesthetized with intramuscular and inhaled medications (Tiletamine-Zolazepam, 4.4 mg/kg intramuscular and 0.5-5% Isoflurane mixed with oxygen). Twenty-four full-thickness wounds (twelve per side of spine) were made using a 1.5cm diameter trephine. Wounds were spaced 1.5cm apart side-to-side from head-to-tail. 8 wounds were designated as controls (bacterial inoculum without treatment), 8 treated with CleanZinc™-1 (Compound-1=Solution1+Solution3 in a 1:1 ratio), and 8 treated with CleanZinc™-2 (Compound-2=Solution1+Solution2 in a 1:1 ratio) (Figure-1). All 3 bacterial species were subsequently introduced into the wounds by direct placement of the PBS-bacterial solution.  Figure-1  Antiseptic Solution Treatment:  Benzoin was applied as an adhesive surrounding the wound prior to 2x2-inch Tegaderm application over the wounds. Immediately prior to treatment, CleanZinc™-1 and CleanZinc™-2 were mixed. CleanZinc™-1 or CleanZinc™-2 was injected into the wound through the Tegaderm with a 30-gauge needle, while a second needle aspirated the displaced air from the space. The fluid was placed directly into the void space of the 16-treatment wounds 60-minutes after inoculation. A second 4x4-inch Tegaderm was applied over all the wounds to ensure a complete seal (Figure-2). All wounds were further covered with a blue-absorbent pad (Simplicity Fluff Underpad; Cardinal Health). The pig was wrapped with a layer of elastic bandage (ELASTIKON) over the blue pad to prevent dressing movement underneath as the tertiary dressing.   Figure-2  Microbiology:  After 1 and 24-hour(s) of treatment/exposure, respectively, the center of the wound was sampled via a 6-mm biopsy punch (Integra Miltex Disposable Biopsy Punch). The biopsies were placed into a pre-weighed vessel (0.5mL sterile saline) and the tissue weights were calculated. The samples were homogenized individually, serially diluted, drop-plated and incubated to determine bacterial counts. Samples were placed on their aforementioned respective culture media.  Histopathology:  The wound sites were excised en-bloc on postoperative day one after all punch biopsies were taken and the pig was euthanized. The wounds were sutured onto a plastic mesh and fixed in 10% Neutral Buffered Formalin (NBF) (10-times tissue sample volume) for another 24 hours. After 24 hours, slices were made in the wounds and fresh formalin was added for further 48-hour penetration. Formalin-fixed specimens were processed into paraffin embedded blocks sectioned at 5µm and stained with hematoxylin and eosin. One section through the wound center including normal tissue on each side was examined and graded by a veterinary pathologist blinded to the type of treatment. Wound evaluation was graded using a novel necrosis scale: 0 = no to minimal necrosis, 1 = small foci of necrosis (focal to multifocal), 2 = moderate necrosis, 3 = massive necrosis. Degenerative neutrophils in the lining crust and surrounding tissue was also quantified: 0 = no to minimal neutrophilic infiltrate, 1 = mild neutrophilic infiltrate, 2 = moderate neutrophilic infiltrate, 3 = significant neutrophilic infiltrate.  Statistical Analysis:  All statistical analyses were performed using SPSS, version 25, (IBM Corporation; Armonk, NY). One-way analysis of variance (ANOVA) was used to determine statistically significant differences between three independent groups. Fisher exact test was used to evaluate differences between each group for degenerative neutrophils and for tissue necrosis. To combat the small numbers of each necrosis category, the data were dichotomized. “No necrosis” or “minimal necrosis” was considered to be a single category and compared to wounds with “moderate necrosis”. There was no massive necrosis in any wound samples for histopathologic analysis. Since all neutrophil infiltrates were categorized as either “moderate” or “significant”, the two outcomes were compared directly. P-values <0.05 were statistically significant.  RESULTS:  The target and actual bacterial inoculation levels are summarized in Table-1. Wound-A5 was removed from the data set and analysis due to continuous bleeding and the solution’s inability to make appropriate wound bed contact.  Table-1 Recoverable Total Bacteria:  After one hour, the wounds treated with CleanZinc™-1 and CleanZinc™-2 had mean recoverable total bacteria of 2.8 and 3.5 logCFUs/gram of tissue, respectively. At 1 hour, the untreated control wounds had recoverable mean total bacteria of 5.7 logCFUs/g. One-way ANOVA analysis indicated significant bacterial eradication compared to the control group (p=0.017) (Figure-3).  Figure-3  Recoverable Staphylococcus aureus:  After 1 hour, the wounds treated with CleanZinc™-1 and CleanZinc™-2 had mean recoverable Staphylococcus of 2.3 and 1.6 logCFUs/g, respectively. The untreated control wounds had mean recoverable S. aureus of 5.3 logCFUs/g at 1 hour. Both compounds had statistically significant reduction of S. aureus at 1 hour (p=0.009) (Figure-4).  Figure-4  Recoverable Pseudomonas aeruginosa:  After one hour, the wounds treated with CleanZinc™-1 and CleanZinc™-2 had mean recoverable Pseudomonas of 0.3 and 0.0 logCFUs/g, respectively. The untreated control wounds had mean recoverable Pseudomonas of 5.5 logCFUs/g at 1 hour. Both compounds had statistically significant increased reduction of Pseudomonas at 1 hour (p=0.000) (Figure-5). Figure-5  Histologic Findings:   Wound Necrosis:  After 24 hours of exposure to CleanZinc™-1 and CleanZinc™-2, there was no significant increased necrosis when compared to controls (p=0.12, p=0.31, respectively) (Table-2). None of the samples demonstrated massive necrosis.   Table-2  Neutrophilic Infiltrate:  After 24 hours of exposure to CleanZinc™-1, there was significant increased neutrophilic infiltrate when compared to controls (p=0.04). After 24 hours of exposure to CleanZinc™-2, there was no significant increase neutrophilic infiltrate versus controls (p=0.12) (Table-3). None of the samples demonstrated no or mild neutrophilic infiltrate. Table-3  DISCUSSION:  With an expected rise in revision arthroplasty and awareness for antibiotic stewardship given increasing multi-drug resistant organisms, the need for new antimicrobials in the battle against PJI is critical.27 Current antimicrobials’ ability to eliminate pathogens is frequently offset by their own toxicities. Beyond this, there is increasing reports of resistance to antimicrobial solutions or antibiotics after exposure.20,21 The optimal antiseptic solution has minimal cytotoxicity at its MBC to diminish bacterial load by 99.9%.6,7 Our novel zinc solution demonstrated 2.3-2.9 log (99.5-99.9%) reduction in total bacteria, 3.0-3.7 log (99.9-99.98%) reduction in S. aureus and 5.1-5.45 log (99.999-100%) eradication of Pseudomonas 1 hour after exposure. Both compound solutions demonstrated minimal to moderate local tissue necrosis, which was analogous to the control group.  Bacteria have distinct metabolisms that produce bacteria-specific volatile organic compounds (VOCs).22 Specifically, gaseous volatile sulfur compounds (VSC) are produced by most bacteria, especially gram-negative microorganisms.22 Zinc Chloride (ZnCl2) and Sodium Chlorite (NaClO2) mixture increases zinc ion activation, augments antimicrobial activity and eliminates gaseous VSCs directly.9,23,24 ZnCl2 and NaClO2 combination has been readily reported in dentistry literature. Zinc’s addition to mouthwashes and toothpastes controls dental biofilm, inhibits calculus production and reduces halitosis.8,25 NaClO2 increases oxidation-reduction potential, which increases host defense, decreases microbial virulence and increases antimicrobial effect26 when utilized synergistically with ZnCl2. This is the first study, to our knowledge, to demonstrate the efficacy of a dual-action solution beyond oral flora organisms in two common virulent PJI pathogens, S. aureus and Pseudomonas.  CHG is a common antiseptic available in a variety of concentrations (0.5%-4%) that is active against broad spectrum organisms responsible for PJI.27 Although multiple in-vitro and in-vivo studies have demonstrated CHG efficacy against gram-positive and gram-negative pathogens,7,28–30 other studies have shown its antibacterial effect offset by increased host tissue toxicity.31,32 Penn-Barwell et al.32 used 30-rat femur models contaminated with S. aureus and compared three CHG concentrations (0.5%, 0.05%, 0.005%) with 0.05% CHG then saline and a control group treated with saline alone and found no bacterial load difference between multiple CHG concentrations versus saline alone. Furthermore, some studies report antibiotic resistance after exposure to chlorhexidine,33,34 as well as Enterococcus faecium resistance to chlorhexidine itself.35,36 Other studies have also shown significant CHG cytotoxic effect. Alleyn et al.37 demonstrated that 0.12% CHG exposure for 3-minutes had significant fibroblast cytotoxicity relative to control (p<0.001). Liu et al.31 showed that clinically used CHG concentrations (2%) had prominent osteoblasts, fibroblasts and myoblast toxicity. Increased local tissue toxicity was also seen at concentrations (0.002%) far below that is used clinically. This is concerning as osteoblasts, fibroblasts and myoblasts play a pivotal role in wound healing, muscle repair, and osseointegration.31   Our ZnCl2 solution’s minimal to moderate local wound necrosis was non-inferior to the control group. Additionally, aside from its antibacterial properties, activated-zinc is a micronutrient essential to human health and regulation of every wound healing phase.38 Contrary to tissue toxicity, we expect our solution to have potential wound healing properties. Further in-vitro studies are needed to evaluate the effect of ZnCl2 on host tissue cells responsible for wound healing and osseointegration.  Povidone-iodine is an antiseptic consisting of polyvinylpyrrolidone and 10% iodine.6,39 Although iodine has bactericidal properties, its aqueous low solubility requires polyvinylpyrrolidone to allow free iodine delivery to cell surfaces and cause inactivation.6,40,41 Therefore, povidone-iodine cytotoxicity is directly proportional to free iodine concentration.6 Similar to CHG, several in-vitro and in-vivo studies have shown povidone-iodine activity against PJI broad spectrum microorganisms. VanMeurs et al.7 assessed both the bactericidal and cytotoxic characteristics of five antiseptics at commercially available dosages and clinically relevant exposure times. Octenidine-dihydrochloride, CHG and povidone-iodine all had superior bactericidal properties against S. aureus and S. epidermis with povidone-iodine being the least cytotoxic at the MBC. Additionally, although used for several decades, povidone-iodine has raised concerns from an increasing rate of patient reported iodine allergies42,43 which inhibits its routine use. There are no reports, to our knowledge, of clinically significant ZnCl2 allergies.  Furthermore, Cichos et al.44 reported the MIC and time to death for povidone-iodine, CHG and vancomycin powder against seven bacteria strains including methicillin-resistant S. aureus (MRSA) and Pseudomonas. All seven bacterial isolates were eradicated by povidone-iodine at all times (0,3,30,60-minutes) with an average 0.63% MIC. These results are similar to our unpublished data demonstrating povidone-iodine MIC and MBC of 0.63% for both Pseudomonas and S. aureus. Interestingly, these povidone-iodine concentrations needed against Pseudomonas were more than double the effective doses against MRSA, while other studies have shown dilute povidone-iodine to be ineffective against Pseudomonas.45,46 Our results demonstrate CleanZinc™ to be highly effective against standardized strains of both S. aureus and Pseudomonas.   Aspects of this study need to be highlighted when translating the results clinically. First, the bacterial strains were inoculated into the pig wound 1 hour prior to solution contact; therefore, these results cannot be extrapolated for chronic wound infections or biofilm. However, the primary purpose of this pilot study was to simply investigate the antimicrobial effect of two different concentrations of ZnCl2/NaClO2 against two common PJI strains by simulating a large surgical wound inoculation. Future in-vitro and in-vivo studies will focus on this solution’s efficacy on chronic infections with biofilm. Second, the bacterial load utilized in the porcine wound may have been higher than the expected bacterial load in infected surgical wounds. Third, the findings of this single pig model may vary in human studies. However, domestic pigs are closely related to human in terms of anatomy, genetics and physiology.47 Finally, these results demonstrate excellent efficacy to eradicate bacterial inoculation in simulated surgical wounds, despite the fact that there was only solution contact with the wound bed and no irrigation or other mechanical manipulation of the wounds.  CONCLUSION:  Our novel activated-zinc compound, CleanZinc™, demonstrated 99.5-99.9% reduction in total bacteria, 99.9-99.98 % reduction in S. aureus, and 100% eradication of Pseudomonas 1 hour after exposure. ZnCl2 has been well reported to promote innate wound healing while maintaining antibacterial properties.38 A synergistic ZnCl2/NaClO2 solution obviates CHG and povidone-iodine cytotoxicity at varying concentrations. Future studies are needed to determine optimal solution concentration for PJI/SSI and efficacy against chronic infections, fungals, and biofilm. This novel solution may prove to be a significant tool in the armamentarium to treat and/or prevent PJI and other wound infections.  SOURCE OF FUNDING: This study was funded by AZ Solutions, LLC, and conducted at a private third-party preclinical testing service site (BRIDGE PTS, Inc., San Antonio, Texas).  ACKNOWLEDGEMENT & DISCLOSURES:  Derek L. Hill is sole owner of AZ Solutions, LLC. Paul Attar is owner of BRIDGE PTS, Inc. Sarah Korn is employed by BRIDGE PTS, Inc.  REFERENCES:   1. Sloan M, Premkumar A, Sheth NP. Projected volume of primary total joint arthroplasty in the u.s., 2014 to 2030. 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Clinical photo and schematic illustration of bovine model with 24 full-thickness wounds (twelve on each side of spine) using a custom-designed 1.5 cm-diameter trephine  Figure-2. Clinical photo of solution injection through Tegaderm using a 30-guage needle while a second needle was used to aspirate the displaced air from the space   Figure-3. Recoverable Log CFUs of Total Bacteria per Gram of Tissue at 1-Hour  Figure-4. Recoverable Log CFUs of Staphylococcus aureus per Gram of Tissue at 1-Hour  Figure-5. Recoverable Log CFUs of Pseudomonas per Gram of Tissue at 1-Hour