Authors:  Derek L. Hill, DO, FAOAO1, Andre Castiaux, PhD2, Elizabeth Pensler, DO, FACOS3, Joseph Knue, BS4, Paul S Attar, PhD4, Ahmed Siddiqi, DO, MBA5  Clinical Associate Professor of Surgery  Michigan State University College of Osteopathic Medicine  Hill Orthopedics  derekhill08@gmail.com  Post Doctoral Researcher  St. Louis University   Andre.castiaux@slu.edu Vascular Surgeon  Pensler Vein and Vascular Surgical Institute  Warren, MI  epensler@yahoo.com  BRIDGE PTS, Inc  San Antonio, TX  joe.knue@bridgepts.com  paul.attar@bridgepts.com   Orthopedic Surgeon  Hackensack Meridian Health  Orthopedic Institute of Central Jersey  Manasquan, NJ  Asiddiqi89@gmail.com Corresponding Author:  Ahmed Siddiqi, DO, MBA  Hackensack Meridian Health  Orthopedic Institute of Central Jersey  Manasquan, NJ  Mobile: 718 869 0048  Asiddiqi89@gmail.com 

ABSTRACT:  Background: The search for the optimal agent for infection eradication in periprosthetic joint infection(PJI) remains challenging as there are limited efficacious and safe options. The ideal solution should have significant bactericidal and anti-biofilm activity to be able to eradicate infection with the preservation of prosthetic components. Therefore, the purpose of this study was to 1.) investigate the anti-biofilm efficacy of a novel activated-zinc solution against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus(MRSA) biofilm in vitro and 2.) compare its efficacy against two leading commercially available antimicrobial irrigants(CHG and 0.35% PI).   Materials: Modified Robbins Device, Pseudomonas aeruginosa and MRSA biofilms, 2-hour exposure to test articles.  Methods & Results: After exposing Pseudomonas on TSA and PIA media for 2 hours, activated-zinc demonstrated a respective 7.04 and 5.44 log reductions, chlorhexidine demonstrated a respective 1.07 and 0.38 log reductions, and PI demonstrated a respective 0.72 and 0.85 log reductions. After 2 hours exposure, activated-zinc had a mean recoverable MRSA was below our means of detection with at least 5 log reduction with possible eradication, chlorhexidine had recoverable MRSA of 3.37±2.20 log CFU representing 2.06-3.08 log reduction(99.1-99.999%), and PI had a recoverable MRSA of 1.11±2.05 log CFU representing 5.04-5.34 log reduction(99.99999%).  Conclusions: Our novel activated-zinc compound demonstrated 99.9998-99.99999% reduction in Pseudomonas biofilm and no detectable bacteria in MRSA biofilm. This novel solution may provide a significant tool in the arsenal to treat and/or prevent PJI and other wound infections. Future in-vivo studies are warranted to further demonstrate clinical utility, efficacy, and safety.  INTRODUCTION:  Despite technological advancements in surgical sterility, antibiotic utilization, surgical techniques, post-operative multi-disciplinary care, periprosthetic joint infection(PJI) and surgical site infection(SSI) rates after total joint arthroplasty (TJA)[1]. The reduction of infection risk and improvement of infection management has focused on optimizing modifiable patient factors including obesity, diabetes, malnutrition, smoking concomitantly with surgical factors including prophylactic and local antibiotics, skin preparation, operating room environment, duration of surgery, antibiotic cement, and wound irrigation [2]. Irrigation fluid is a potentially highly cost-effective modality for both prevention and definitive management of PJI by minimizing bacterial contamination [2–4]. Identifying the optimal irrigation agent, however, 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 [3–5]. Commonly used antiseptic solutions including chlorhexidine gluconate(CHG), hydrogen peroxide, sodium hypochlorite and povidone-iodine(PI) have all been associated with local tissue damage [3–6].   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)[3–6]. 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[7,8]. Additionally, sodium chlorite(NaClO2) is effective at raising oxidation-reduction potential in low concentrations and works synergistically with ZnCl2.[9,10] Unlike the commercially available antiseptic solutions for PJI, a number of studies have shown zinc oxide nanoparticles as an antibacterial agent that is bio-safe and non-toxic to human cells.[11] Activated zinc disrupt bacterial growth by interacting with the bacterial surface and by altering bacterial enzymes by displacing magnesium ions that are essential for enzymatic bacterial activity.[12] A recent study found zinc oxide nanoparticles to have substantial bactericidal affect with over three-log reduction in colonies of methicillin-resistant Staphylococcus aureus(MRSA) with minimal increase in reactive oxygen species or lipid peroxidation.[13] 

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 resistant organism with biofilm including Pseudomonas aeruginosa and MRSA. Therefore, the purpose of this study was to 1.) investigate the antimicrobial efficacy of a novel activated-zinc solution against Pseudomonas and MRSA biofilm and 2.) compare its efficacy against the two most common commercially available antiseptic solutions, dilute chlorhexidine and povidone-iodine.   MATERIALS & METHODS: Microorganisms:  The bacteria used in this study were Pseudomonas aeruginosa(ATCC 27317) and MRSA(ATCC 33593). Within a week prior to the experiment, a small loopful of each test bacteria were streaked from plate culture or frozen bead collection and cultured on a Tryptic Soy Agar(TSA) plate. Both test bacteria were incubated overnight at 37±3°C and the cultures were examined visually for purity prior to use. All the bacteria colonies demonstrated the same colony morphology and color, respective to each species, and were deemed appropriate for experimentation. The day prior to the start of the experiment, a small loopful of each bacterium were, respectively, inoculated from the TSA plate into 30 mL of full-strength Tryptic Soy Broth(TSB) and vortexed in a sterile conical tube. The bacteria were incubated at 37±3°C with shaking at 150 rpm (Thermo Scientific, MaxQ 4450) overnight.  Biofilm Formation:  The Modified Robbins Device(MRD) was autoclaved prior to use for sterilization. The MRD was connected by sterile siliconized tubing (Tygon, Pittsburgh, PA) to a sterile 2 L reservoir flask filled with 1.5 L of half-strength TSB media and held at 37±3°C. The reservoir was seeded to create a 2% inoculum of P. aeruginosa and MRSA, separately, so that the reservoir delivered logarithmic-phase microbes to the MRD. To ensure adequate aeration of the cultures, the reservoir was connected to an Elite 800 fish tank pump (Rolf C. Hagen Corp., Mansfield, MA). Air was vigorously pumped through an air filter and bubbled through the media. The MRD sampling ports were loaded with PVC sampling coupons and inserted into the MRD prior to starting the flow of media. Each sampling coupon was even with the upper surface of the MRD fluid chamber to ensure laminar flow properties. Media containing logarithmic phase growth bacteria was continuously circulated through the MRD by means of a peristaltic pump (Master flex, Cole Parmer). The pump ran for approximately 22 to 24 hours total. The pump delivered approximately 60 mL per hour of media with logarithmic growth-phase bacteria through the MRD for six hours. During the first six hours of incubation, only the half-strength TSB media with 2% P. aeruginosa or MRSA were circulated through the MRD. After six hours of incubation, the initial biofilm was established. The reservoir containing the half-strength TSB with inoculum was swapped for a fresh reservoir containing fresh, sterile half-strength TSB. The fresh media was delivered at approximately 60 mL per hour to the growth vessel for the remaining approximately 16 to 18 hours. The fresh media flowed through the MRD and into a waste beaker. After overnight incubation, the sampling ports with coupons were removed from the MRD and rinsed with 10 mL of 0.9% sterile saline to wash off the planktonic bacteria. The sampling ports, including coupon with biofilm matrix, were transferred to a five-port static testing device fitted with one blank coupon. Each test articles were added to a five-port static testing device and incubated at 37°C ± 3°C using a time course assay. Only four sampling ports with coupons were placed into the five-port static testing device. The other port was covered with a sterile, blank sampling port with coupon. At the end of the incubation period, the sampling ports were removed and evaluated.  To sample the planktonic population, a 100 µL aliquot of the culture fluid was removed from the MRD (after the first 6 hours of incubation) and serially diluted in sterile saline. The dilutions were drop plated in duplicate. TSA plates were used to determine the total number of all bacteria. Pseudomonas Isolation Agar(PIA) plates were used to determine the number of Pseudomonas, as appropriate. Mannitol Salt Agar(MSA) plates were used to determine the number of Staphylococcus, as appropriate. The plates were incubated at 37±3°C overnight and colonies were counted. To sample the sessile population, a sample port was aseptically removed and rinsed under a stream of 10 mL of 0.9% sterile saline to remove any loosely adherent bacteria into a waste beaker. The sample coupon was removed from the sampling port, using sterilized forceps after loosening the screw, and then placed in a sterile Petri dish and 1 mL of 0.9% sterile saline was added. The sample coupon was then scraped with a sterile #10 scalpel blade. The coupon and sterile blade were added to a sterile 15cc polystyrene tube. An additional 1mL of sterile saline was used to rinse the plate along with any scrapings and placed into the tube (for a total of 2 mL sterile saline). The conical tubes were ultrasonicated using a low output ultrasonic (50-60Hz) bath (Branson, Shelton, CT) for ten minutes to remove any cells still attached to the surface and to disperse any aggregates of bacteria. Each sample was serially diluted in sterile saline. Each of the dilutions was drop plated in duplicate. TSA plates were used to determine the total number of bacteria. PIA or MSA plates were used as appropriate. The plates were incubated at 37±3°C overnight and colonies were counted. Bacterial counts were expressed as log10(CFU/g).  Antiseptic Solution:  Our unpublished data determined the Mean Inhibitory Concentration(MIC) and Mean Bactericidal Concentration(MBC) for ZnCl2 solution, and independently for NaClO2 solution.[10] A synergistic benefit was found with combination of the two solutions, with substantially lower MIC/MBC for each when combined immediately before bacterial exposure. We used these data to formulate ZnCl2 and NaClO2 solution concentrations for the current study.  A detailed procedure explaining the preparation of each solution can be found in Appendix 1. Solution-A was prepared to contain 70 mM sodium chlorite(NaClO2) and 154.0 mM-NaCl(0.9%), buffered to pH 7.5 with 10.0mM-benzoic acid/benzoate. Additionally, Solution-1 and Solution-2 were prepared to contain 50 and 70 mM ZnCl2, respectively, and 154.0 mM-NaCl(0.9%), buffered to pH 7.5 with 10.0mM-benzoic acid/benzoate. The chemical constituents of each solution can be found in Table-1.  All solutions were sterile filtered twice through 0.2-micron filters and transferred into sterile 50mL polypropylene conical tubes (Greiner, Kremsmunster, Austria), under sterile conditions and sealed with parafilm. After 20 hours, the solutions were test-mixed in a 1:1 ratio, by mixing 2mL-Solution-A with either 2mL- Solution 1 or 2 in a clean conical tube. The resulting solution pH was 5.40±0.5. Compounds 1A and 2A were prepared immediately before application by mixing Solution-A with either Solution-1 or Solution 2 in a 1:1 volumetric ratio.  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. P-values <0.05 were statistically significant. RESULTS: The target and actual bacterial inoculation levels are summarized in Table 2.   Recoverable MRSA: After 2 hours exposure, activated-zinc had a mean recoverable MRSA was below our means of detection with at least 5 log reduction with possible eradication, chlorhexidine had recoverable MRSA of 3.37±2.20 log CFU representing 2.06-3.08 log reduction(99.1-99.999%), and PI had a recoverable MRSA of 1.11±2.05 log CFU representing 5.04-5.34 log reduction(99.99999%). Recoverable Pseudomonas aeruginosa:  After exposing Pseudomonas on TSA and PIA media for 2 hours, activated-zinc demonstrated a respective 7.04 and 5.44 log reductions, chlorhexidine demonstrated a respective 1.07 and 0.38 log reductions, and PI demonstrated a respective 0.72 and 0.85 log reductions.  DISCUSSION:  Irrigation and debridement in treatment of PJI serves an integral role in eradication of bacterial burden and subsequent re-infection rates. Current antiseptic solutions’ ability to eliminate organisms is frequently concentration dependent and often offset with cytotoxicity against host tissue with theoretical wound healing concerns [3,4]. The ideal antiseptic solution should have minimal cytotoxicity while maintaining bactericidal activity at its Minimum Bactericidal Concentration(MBC), which is defined as the concentration required to diminish the bacterial load and biofilm by 99.9%(3 log) CFU compared with growth controls in the same conditions.[5,6] Our novel activated-zinc compound demonstrated 5.5-7 log(99.999-99.99999) reduction in Pseudomonas biofilm and 6 log reduction no detectable bacteriaafter 2-hour exposure. Dilute CHG demonstrated a non-clinically efficacious 0.38-1.1 log reduction(58-92%) in Pseudomonas biofilm with a better effect against MRSA biofilm(2-3 log reduction, 99.1-99.9%). Similarly, povidone-iodine demonstrated 0.72-0.85 log reduction(81-86%) in Pseudomonas biofilms and 5 log reduction(99.999%) against MRSA biofilm.   Zinc chloride(ZnCl2) and sodium chlorite(NaClO2) used in conjunction increases zinc ion activation, augments antimicrobial activity and eliminates gaseous volatile sulfur compounds produced by most gram-positive and gram-negative organisms directly.[8,14,15] Although ZnCl2 and NaClO2 combination has been readily reported in dentistry literature, its broad spectrum antimicrobial and antifungal efficacy has been increasingly reported.[16,17]  Zinc’s addition to dental hygiene products has been utilized against oral microorganism biofilm with inhibition of calculus production and halitosis.[7,18] NaClO2 increases oxidation-reduction potential, which increases host defense, decreases microbial virulence and increases antimicrobial effect[19] when utilized synergistically with ZnCl2. This is the first study, to our knowledge, to demonstrate the efficacy and superiority of a dual-action solution against two resistant virulent PJI pathogen biofilm, MRSA and Pseudomonas when compared with commercially available chlorhexidine and dilute povidone-iodine.  CHG is a cationic bisbiguanide that binds to negatively charged bacterial cell wall, altering the osmotic equilibrium of the bacterial cell.[20–22] Since chlorhexidine is water insoluble, the commercially available chlorhexidine is usually formulated with gluconic acid to form water soluble salts for surgical applications.[22] CHG is bacteriostatic by disrupting bacterial cell membrane at low concentrations(0.0002% to 0.5%) and is bactericidal by causing intracellular content coagulation at higher concentrations(>0.5%).[22,23] Smith et al.[24] investigated the optimal concentration of CHG and found that concentrations above 2% was needed to provided persistent decrease of biofilm burden. Although lower concentrations of CHG decreased biofilm, the authors reported a rebound growth of biofilm with prolonged incubation proposing that lower concentrations are likely to be ineffective in-vivo.[22,24]   Currently, a 0.05% CHG preparation in sterile water to prevent precipitation of the active compound (Irrisept®, Innovation Technologies, Inc, Lawrenceville, Georgia) is the only U.S. Food and Drug Administration-approved orthopedic intrawound additive irrigation solution.[25] CHG has been reported to have broad spectrum activity against a wide variety of organisms responsible for PJI including MSSA, MRSA, coagulase negative Staphylococcus, gram-negative bacteria, fungi and mycobacteria.[22] Although the results of our study demonstrate high efficacy against MRSA, commercially available CHG has suboptimal efficacy against a common virulent gram-negative PJI organism. Furthermore, studies have shown CHG antibacterial effect offset by increased host tissue toxicity.[26,27] Multiple in vitro studies found higher CHG concentrations (0.5% to 2%) to substantially reduce human fibroblast, myoblast, osteoblast, and stromal cell survival.[6,25,26] This is especially concerning as osteoblasts, fibroblasts and myoblasts play a pivotal role in wound healing, muscle repair, and osseointegration.[26]   Povidone iodine(PI) consists of approximately 1% available iodine in polyvinylpyrrolidone to make it hydrophilic for use in solution. The iodine itself acts as a potent oxidizer to cell membranes and intracellular components effectively inactivating them in a concentration dependent manner.[5] Therefore, the PI’s cytotoxicity is directly proportional to its iodine concentration delivered to the cell wall.[5] This mechanism of cell death has been shown to be effective against gram-positive and gram-negative bacteria, fungi, viruses, and protozoa.[28] Similar to CHG, several in vitro and in vivo studies have shown povidone-iodine activity against PJI broad spectrum microorganisms. VanMeurs et al.[6] 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.   Povidone-iodine is commercially available at 100 g/L (10%), which is both bactericidal and cytotoxic.[5,6] Some studies suggest that PI at lower concentrations is very effective and causes minimal damage to healthy tissue.[29,30] Other reports suggest PI’s toxicity against healthy host tissue is greater than its bactericidal effectiveness regardless of concentration.[30–33] A recent in-vitro analysis compared 0.35% PI, 10% PI and 1:1 combination of 10% PI and 4% hydrogen peroxide against 24-hour and 72-hour MSSA biofilm growing on plastic, cement and porous titanium.[34] The authors found 10% PI and 10% PI/Hydrogen peroxide combination to be effective across all three surfaces with minimal effectiveness of 0.35% PI. However, due to concerns for host tissue toxicity, 0.35% dilute PI is most commonly utilized in primary and revision TJA for the prevention and management of acute and chronic PJI.[35–37]   Similar to commercially available CHG, our study demonstrated 0.35% dilute PI to have adequate anti-biofilm efficacy against MRSA but poor efficacy against Pseudomonas aeruginosa. Prior studies have also reported PI ineffectiveness against potent gram-negative bacteria. Cichos et al.[38] reported the MIC and time to death for povidone-iodine, CHG and vancomycin powder against seven bacteria strains including 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. 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.[39,40] This further questions the widespread adoption of both CHG and PI in routine prevention and management of PJI as gram-negative organisms represent up to 25% of arthroplasty infections.[41] Our novel solution had complete biofilm eradication of highly resistant gram-positive and gram-negative pathogens to near undetectable levels.  Aspects of this study need to be highlighted when translating the results clinically. First, future in vitro and in vivo studies will focus on this solution’s efficacy against other bacterial and fungal biofilms. Second, the results of this biofilm model study may vary in human investigation. Third, this study did not directly compare tissue toxicity. However, the current literature has demonstrated CHG and PI cytotoxicity at varying solution concentrations while zinc oxide has been well reported to be non-toxic in human tissue.  CONCLUSION:  Our novel activated-zinc compound demonstrated 99.9% reduction of Pseudomonas biofilm and 100% eradication of MRSA biofilm after 2 hours of exposure. Dilute CHG demonstrated a non-clinically efficacious 58-92% reduction in Pseudomonas biofilm with a better effect against MRSA biofilm(99.1-99.9%). Similarly, povidone-iodine demonstrated 81-86% reduction in Pseudomonas biofilms and 99.9% reduction against MRSA biofilm. Furthermore, 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 other resistant bacterial and fungal biofilms. This novel solution may provide a significant tool in the arsenal to treat and/or prevent PJI and other wound infections. Future in vivo studies are warranted to further demonstrate clinical utility, efficacy, and safety. 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] McMaster Arthroplasty Collaborative Group. Risk Factors for Periprosthetic Joint Infection Following Primary Total Hip Arthroplasty. J Bone Jt Surg 2020;102:503–9. doi:10.2106/JBJS.19.00537.  [2] Wood T, Ekhtiari S, Mundi R, Citak M, Sancheti PK, Guerra-Farfan E, et al. 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J Bone Jt Surg - Ser A 2014. doi:10.2106/JBJS.M.00313.  [7] Almoudi MM, Hussein AS, Abu Hassan MI, Mohamad Zain N. A systematic review on antibacterial activity of zinc against Streptococcus mutans. Saudi Dent J 2018. doi:10.1016/j.sdentj.2018.06.003.  [8] Choi EK, Lee HH, Kang MS, Kim BG, Lim HS, Kim SM, et al. Potentiation of bacterial killing activity of zinc chloride by pyrrolidine dithiocarbamate. J Microbiol 2010. doi:10.1007/s12275-009-0049-2.  [9] Kleinberg I, Codipilly M. Compositions to Control Oral Microbial Oxidation-Reduction Eh Levels, 2002.  [10] Bridge PTS, Inc., San Antonio, TX. Data on file with AZ Solutions, LLC, Bloomfield Hills, MI. 2019.  [11] Sirelkhatim A, Mahmud S, Seeni A, Kaus NHM, Ann LC, Bakhori SKM, et al. Review on zinc oxide nanoparticles: Antibacterial activity and toxicity mechanism. Nano-Micro Lett 2015. doi:10.1007/s40820-015-0040-x.  [12] Tavassoli Hojati S, Alaghemand H, Hamze F, Ahmadian Babaki F, Rajab-Nia R, Rezvani MB, et al. 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Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms. FEMS Microbiol Lett 2008. doi:10.1111/j.1574-6968.2007.01012.x.  [18] Lynch RJM. Zinc in the mouth, its interactions with dental enamel and possible effects on caries; A review of the literature. Int Dent J 2011. doi:10.1111/j.1875-595X.2011.00049.x.  [19] Kim NH, Park TH, Rhee MS. Enhanced bactericidal action of acidified sodium chlorite caused by the saturation of reactants. J Appl Microbiol 2014. doi:10.1111/jam.12484.  [20] Lim KS, Kam PCA. Chlorhexidine - Pharmacology and clinical applications. Anaesth Intensive Care 2008. doi:10.1177/0310057x0803600404.  [21] Weinstein RA, Milstone AM, Passaretti CL, Perl TM. Chlorhexidine: Expanding the Armamentarium for Infection Control and Prevention. Clin Infect Dis 2008. doi:10.1086/524736.  [22] George J, Klika AK, Higuera CA. Use of Chlorhexidine Preparations in Total Joint Arthroplasty. J Bone Jt Infect 2016. doi:10.7150/jbji.16934.  [23] Mcdonnell G, Russell AD. Antiseptics and disinfectants: Activity, action, and resistance. Clin Microbiol Rev 1999;12:147–79. doi:10.1128/cmr.12.1.147.  [24] Smith DC, Maiman R, Schwechter EM, Kim SJ, Hirsh DM. Optimal Irrigation and Debridement of Infected Total Joint Implants with Chlorhexidine Gluconate. J Arthroplasty 2015;30:1820–2. doi:10.1016/j.arth.2015.05.005.  [25] Kavolus JJ, Schwarzkopf R, Rajaee SS, Chen AF. Irrigation Fluids Used for the Prevention and Treatment of Orthopaedic Infections. J Bone Jt Surg - Am Vol 2020;102:76–84. doi:10.2106/JBJS.19.00566.  [26] Liu JX, Werner J, Kirsch T, Zuckerman JD, Virk MS. Cytotoxicity evaluation of chlorhexidine gluconate on human fibroblasts, myoblasts, and osteoblasts. J Bone Jt Infect 2018. doi:10.7150/jbji.26355.  [27] Penn-Barwell JG, Murray CK, Wenke JC. Comparison of the antimicrobial effect of chlorhexidine and saline for irrigating a contaminated open fracture model. J Orthop Trauma 2012. doi:10.1097/BOT.0b013e31826c19c4.  [28] Zamora JL. Chemical and microbiologic characteristics and toxicity of povidone-iodine solutions. Am J Surg 1986. doi:10.1016/0002-9610(86)90477-0.  [29] Doughty D. A rational approach to the use of topical antiseptics. J Wound, Ostomy Cont Nurs 1994. doi:10.1097/00152192-199411000-00008.  [30] Rabenberg VS, Ingersoll CD, Sandrey MA, Johnson MT. The bactericidal and cytotoxic effects of antimicrobial wound cleansers. J Athl Train 2002.  [31] PA F, DS P, Becker D, Lewis D, GT R. A relative toxicity index for wound cleansers. Wounds A Compend Clin Res Pract 1993.  [32] Borenfreund E, Puerner JA. A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J Tissue Cult Methods 1985. doi:10.1007/BF01666038.  [33] Maklebust J. Using wound care products to promote a healing environment. Crit Care Nurs Clin North Am 1996. doi:10.1016/s0899-5885(18)30331-9.  [34] Premkumar A, Nishtala S, Bostrom MP, Carli A. In-Vitro Analysis of Anti-Biofilm Effect of Intraoperative Irrigation Solutions. AAHKS 2020.  [35] World Health Organization. WHO Global guidelines for the prevention of surgical site infection. Who 2018:6–7.  [36] Blom A, Cho JE, Fleischman A, Goswami K, Ketonis C, Kunutsor SK, et al. General Assembly, Prevention, Antiseptic Irrigation Solution: Proceedings of International Consensus on Orthopedic Infections. J Arthroplasty 2019. doi:10.1016/j.arth.2018.09.063.  [37] Goswami K, Austin MS. Intraoperative povidone-iodine irrigation for infection prevention. Arthroplast Today 2019;5:306–8. doi:10.1016/j.artd.2019.04.004.  [38] Cichos KH, Andrews RM, Wolschendorf F, Narmore W, Mabry SE, Ghanem ES. Efficacy of Intraoperative Antiseptic Techniques in the Prevention of Periprosthetic Joint Infection: Superiority of Betadine. J Arthroplasty 2019. doi:10.1016/j.arth.2019.02.002.  [39] Michalová K, Moyes AL, Cameron S, Juni BA, Obritsch WF, Dvorak JA, et al. Povidone-iodine (betadine) in the treatment of experimental Pseudomonas aeruginosa keratitis. Cornea 1996. doi:10.1097/00003226-199609000-00014.  [40] Association for Research in Vision and Ophthalmology. E, Tran P, Pham P, Hamood A, Mitchell K, Reid TW. 5% Betadine solution in not effective in inhibiting the growth of different Gram Negative and Gram Positive Pathogens in vitro. Invest Ophthalmol Vis Sci 2014.  [41] Hsieh PH, Lee MS, Hsu KY, Chang YH, Shin HN, Ueng SW. Gram-negative prosthetic joint infections: risk factors and outcome of treatment. Clin Infect Dis 2009;49:1036–43. doi:10.1086/605593. FIGURE LEGENDS:  Figure 1: Diagram of Modified Robbins Device (MRD) Table 1. Solution Compositions  Table 2. Bacteria Cultures and Populations