Chitosan Membranes Stabilized with Varying Acyl Lengths Release Cis-2-Decenoic Acid and Bupivacaine at Controlled Rates and Inhibit Pathogenic Biofilm.

BACKGROUND: Adherence of complex bacterial biofilm communities to burned tissue creates a challenge for treatment, with infection causing 51% of burn victim deaths. This study evaluated the release of therapeutics from wound care biomaterials and their antimicrobial activity against pathogens Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. METHODS: Electrospun chitosan membranes (ESCMs) were fabricated and acylated with chain lengths ranging from 6-10 carbons then loaded with 0.15 mg of anti-biofilm agent, cis-2-decenoic acid (C2DA), and 0.5 mg of local anesthetic, bupivacaine. RESULTS: Combinations of therapeutics released from modified ESCMs at a cumulative amount of 45-70% of bupivacaine and less than 20% of C2DA. Results from bacterial studies suggest that this combination reduced biofilm 10-fold for S. aureus, 2-fold for Acinetobacter baumannii, and 2-3-fold for Pseudomonas aeruginosa by 24 hours. Additionally, dual loaded groups reduced planktonic Staphylococcus aureus ~4-fold by 24 hours as well as Acinetobacter baumannii ~3-fold by 48 hours. CONCLUSIONS: The combination of therapeutics used has a significant role in biofilm prevention for selected strains via direct contact or diffusion in aqueous solutions.

2023 International Consensus Meeting on musculoskeletal infection: Summary from the treatment workgroup and consensus on treatment in preclinical models.

In vitro and in vivo studies are critical for the preclinical efficacy assessment of novel therapies targeting musculoskeletal infections (MSKI). Many preclinical models have been developed and applied as a prelude to evaluating safety and efficacy in human clinical trials. In performing these studies, there is both a requirement for a robust assessment of efficacy, as well as a parallel responsibility to consider the burden on experimental animals used in such studies. Since MSKI is a broad term encompassing infections varying in pathogen, anatomical location, and implants used, there are also a wide range of animal models described modeling these disparate infections. Although some of these variations are required to adequately evaluate specific interventions, there would be enormous value in creating a unified and standardized criteria to animal testing in the treatment of MSKI. The Treatment Workgroup of the 2023 International Consensus Meeting on Musculoskeletal Infection was responsible for questions related to preclinical models for treatment of MSKI. The main objective was to review the literature related to priority questions and estimate consensus opinion after voting. This document presents that process and results for preclinical models related to (1) animal model considerations, (2) outcome measurements, and (3) imaging.

Cis-2-Decenoic Acid and Bupivacaine Delivered from Electrospun Chitosan Membranes Increase Cytokine Production in Dermal and Inflammatory Cell Lines.

Wound dressings serve to protect tissue from contamination, alleviate pain, and facilitate wound healing. The biopolymer chitosan is an exemplary choice in wound dressing material as it is biocompatible and has intrinsic antibacterial properties. Infection can be further prevented by loading dressings with cis-2-decenoic acid (C2DA), a non-antibiotic antimicrobial agent, as well as bupivacaine (BUP), a local anesthetic that also has antibacterial capabilities. This study utilized a series of assays to elucidate the responses of dermal cells to decanoic anhydride-modified electrospun chitosan membranes (DA-ESCMs) loaded with C2DA and/or BUP. Cytocompatibility studies determined the toxic loading ranges for C2DA, BUP, and combinations, revealing that higher concentrations (0.3 mg of C2DA and 1.0 mg of BUP) significantly decreased the viability of fibroblasts and keratinocytes. These high concentrations also inhibited collagen production by fibroblasts, with lower loading concentrations promoting collagen deposition. These findings provide insight into preliminary cellular responses to DA-ESCMs and can guide future research on their clinical application as wound dressings.

Titanium coated with 2-decenoic analogs reduces bacterial and fungal biofilms.

AIMS: Due to antibiotic tolerance of microbes within biofilm, non-antibiotic methods for prevention and treatment of implant-related infections are preferable. The goal of this work is to evaluate a facile loading strategy for medium-chain fatty-acid signaling molecules 2-heptycyclopropane-1-carboxylic acid (2CP), cis-2-decenoic acid (C2DA), and trans-2-decenoic acid, which all act as diffusible signaling factors (DSFs), onto titanium surfaces for comparison of their antimicrobial efficacy. METHODS AND RESULTS: Titanium coupons were drop-coated with 0.75 mg of DSF in ethanol and dried. Surface characteristics and the presence of DSF were confirmed with Fourier Transform infrared spectroscopy, x-ray photoelectron spectroscopy, and water contact angle. Antimicrobial assays analyzing biofilm and planktonic Staphylococcus aureus, Escherichia coli, or Candida albicans viability showed that planktonic growth was reduced after 24-h incubation but only sustained through 72 h for S. aureus and C. albicans. Biofilm formation on the titanium coupons was also reduced for all strains at the 24-h time point, but not through 72 h for E. coli. Although ∼60% of the loaded DSF was released within the first 2 days, enough remained on the surface after 4 days of elution to significantly inhibit E. coli and C. albicans biofilm. Cytocompatibility evaluations with a fibroblast cell line showed that none of the DSF-loaded groups decreased viability, while C2DA and 2CP increased viability by up to 50%. CONCLUSIONS: In this study, we found that DSF-loaded titanium coupons can inhibit planktonic microbes and prevent biofilm attachment, without toxicity to mammalian cells.

Evaluation of Magnesium-Phosphate Particle Incorporation into Co-Electrospun Chitosan-Elastin Membranes for Skin Wound Healing.

Major challenges facing clinicians treating burn wounds are the lack of integration of treatment to wound, inadequate mechanical properties of treatments, and high infection rates which ultimately lead to poor wound resolution. Electrospun chitosan membranes (ESCM) are gaining popularity for use in tissue engineering applications due to their drug loading ability, biocompatibility, biomimetic fibrous structure, and antimicrobial characteristics. This work aims to modify ESCMs for improved performance in burn wound applications by incorporating elastin and magnesium-phosphate particles (MgP) to improve mechanical and bioactive properties. The following ESCMs were made to evaluate the individual components' effects; (C: chitosan, CE: chitosan-elastin, CMg: chitosan-MgP, and CEMg: chitosan-elastin-MgP). Membrane properties analyzed were fiber size and structure, hydrophilic properties, elastin incorporation, MgP incorporation and in vitro release, mechanical properties, degradation profiles, and in vitro cytocompatibility with NIH3T3 fibroblasts. The addition of both elastin and MgP increased the average fiber diameter of CE (~400 nm), CMg (~360 nm), and CEMg (565 nm) compared to C (255 nm). Water contact angle analysis showed elastin incorporated membranes (CE and CEMg) had increased hydrophilicity (~50°) compared to the other groups (C and CMg, ~110°). The results from the degradation study showed mass retention of ~50% for C and CMg groups, compared to ~ 30% seen in CE and CEMg after 4 weeks in a lysozyme/PBS solution. CMg and CEMg exhibited burst-release behavior of ~6 µg/ml or 0.25 mM magnesium within 72 h. In vitro analysis with NIH3T3 fibroblasts showed CE and CEMg groups had superior cytocompatibility compared to C and CMg. This work has demonstrated the successful incorporation of elastin and MgP into ESCMs and allows for future studies on burn wound applications.

Simulated large joint fluid model for evaluating intra-articular antibiotic delivery systems: initial evaluation using antibiotic-loaded calcium sulfate beads.

Introduction: Local antimicrobial delivery via calcium sulfate (CaSO 4 ) beads is used as an adjunctive treatment for periprosthetic joint infection. There is limited clinical information describing the performance of antimicrobial-loaded CaSO 4 (ALCS) in large-scale applications. We developed a simulated large joint model to study properties of eluting ALCS. Methods: The in vitro testing platform was an adapted standardized model for tribological testing of prosthetic total hips and total knees (ASTM F732). The model was 70 mL total fluid volume, 25 % bovine serum, and 75 % phosphate-buffered saline, using ISO standard 14242-1 for human synovial fluid simulation. Four brands of CaSO 4 were evaluated. Each 10 mL of CaSO 4 was loaded with 1.2 grams (g) of tobramycin and 1 g of vancomycin powders. A 35 mL bead volume, equaling 175 beads, of each product was placed in incubated flasks. The test period was 6 weeks with scheduled interval fluid exchanges. Fluid samples were tested for antibiotic and calcium concentrations and pH. Results: Antibiotic elution showed an initial burst on Day 1, followed by a logarithmic reduction over 1 week. Tobramycin fully eluted within 2.5 weeks. Vancomycin showed sustained release over 6 weeks. Calcium ion concentrations were high, with gradual decrease after 3 weeks. All four CaSO 4 products were inherently acidic. Fluid became more acidic with the addition of antibiotics primarily driven by vancomycin. Discussion: Clinicians should be cognizant of tobramycin elution burst with ALCS in large loads. The main driver of acidic pH levels was vancomycin. We propose that joint complications may result from lowered fluid acidity, and we suggest clinical study of synovial pH.

Synthesis and Characterization of 2-Decenoic Acid Modified Chitosan for Infection Prevention and Tissue Engineering.

Chitosan nanofiber membranes are recognized as functional antimicrobial materials, as they can effectively provide a barrier that guides tissue growth and supports healing. Methods to stabilize nanofibers in aqueous solutions include acylation with fatty acids. Modification with fatty acids that also have antimicrobial and biofilm-resistant properties may be particularly beneficial in tissue regeneration applications. This study investigated the ability to customize the fatty acid attachment by acyl chlorides to include antimicrobial 2-decenoic acid. Synthesis of 2-decenoyl chloride was followed by acylation of electrospun chitosan membranes in pyridine. Physicochemical properties were characterized through scanning electron microscopy, FTIR, contact angle, and thermogravimetric analysis. The ability of membranes to resist biofilm formation by S. aureus and P. aeruginosa was evaluated by direct inoculation. Cytocompatibility was evaluated by adding membranes to cultures of NIH3T3 fibroblast cells. Acylation with chlorides stabilized nanofibers in aqueous media without significant swelling of fibers and increased hydrophobicity of the membranes. Acyl-modified membranes reduced both S. aureus and P.aeruginosa bacterial biofilm formation on membrane while also supporting fibroblast growth. Acylated chitosan membranes may be useful as wound dressings, guided regeneration scaffolds, local drug delivery, or filtration.

2-Heptylcyclopropane-1-Carboxylic Acid Disperses and Inhibits Bacterial Biofilms.

Fatty-acid signaling molecules can inhibit biofilm formation, signal dispersal events, and revert dormant cells within biofilms to a metabolically active state. We synthesized 2-heptylcyclopropane-1-carboxylic acid (2CP), an analog of cis-2-decenoic acid (C2DA), which contains a cyclopropanated bond that may lock the signaling factor in an active state and prevent isomerization to its least active trans-configuration (T2DA). 2CP was compared to C2DA and T2DA for ability to disperse biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa. 2CP at 125 µg/ml dispersed approximately 100% of S. aureus cells compared to 25% for C2DA; both 2CP and C2DA had significantly less S. aureus biofilm remaining compared to T2DA, which achieved no significant dispersal. 2CP at 125 µg/ml dispersed approximately 60% of P. aeruginosa biofilms, whereas C2DA and T2DA at the same concentration dispersed 40%. When combined with antibiotics tobramycin, tetracycline, or levofloxacin, 2CP decreased the minimum concentration required for biofilm inhibition and eradication, demonstrating synergistic and additive responses for certain combinations. Furthermore, 2CP supported fibroblast viability above 80% for concentrations below 1 mg/ml. This study demonstrates that 2CP shows similar or improved efficacy in biofilm dispersion, inhibition, and eradication compared to C2DA and T2DA and thus may be promising for use in preventing infection for healthcare applications.

Long-Term Controlled Release of Simvastatin from Photoprinted Triple-Networked Hydrogels Composed of Modified Chitosan and PLA-PEG Micelles.

Local delivery of active agents using injectable or implantable hydrogels for tissue and bone regeneration is a promising therapy, but it remains challenging for controlling dose and duration of release. Simvastatin (SMV), a hydrophobic drug, has shown potential for osteogenic stimulation. Secure loading of hydrophobic drugs by physical interactions is particularly difficult to establish in hydrophilic polymer matrices, and their sustained release over several months for long-term regeneration has rarely been reported. Additionally, mechanical properties of hydrogels must be improved for a sufficient support while maintaining eventual biodegradability. This study assesses the effect of controlled SMV release from 3D-printed triple-network hydrogels for osteogenic stimulation and characterizes their mechanical and biological properties as an implant. SMV is loaded into polymeric micelles of polylactide/poly(ethylene glycol) triblock copolymers (PLA-PEG-PLA) and mixed with N-methacryloyl chitosan and PEG dimethacrylate to fabricate hydrogels by photo-cross-linked 3D printing. The hydrogel properties and drug release profiles have shown significant dependance on the polymer compositions. The SMV release from the triple-polymer-network hydrogel has continued for 17 weeks of observation. Cytocompatibility of hydrogels with various formulations is confirmed. The tunable triple-network hydrogels loaded with SMV provide a potential therapeutic value for bone regeneration.

Simvastatin loaded chitosan guided bone regeneration membranes stimulate bone healing.

BACKGROUND AND OBJECTIVE: Electrospun chitosan membranes (ESCM) modified with short-chain fatty acids have the ability to control the release of simvastatin (SMV), an anti-cholesterol drug with osteogenic potential, for guided bone regeneration (GBR) applications. This study evaluated in vivo osteogenic effects of rapid short release of SMV (4 weeks) vs long sustained release (8 weeks) from acetic anhydride (AA)-and hexanoic anhydride (HA)-modified ESCMs, respectively. METHODS: AA ESCMs loaded with 10 or 50 µg SMV and HA ESCMs loaded with 50 µg SMV were evaluated for biocompatibility and bone formation at 4 and 8 weeks, in 5 mm critical size rat calvarial defects, using histological evaluation and micro-CT analysis. RESULTS: No severe inflammatory response was noticed around the ESCMs. Less hydrophobic AA membranes showed signs of resorption by week 4 and were almost completely resorbed by week 8 whereas the more hydrophobic HA membranes resorbed slowly, remaining intact over 8 weeks. In micro-CT analysis, 10 µg SMV-loaded AA membranes did not show significant bone formation as compared to non-loaded AA membranes at either evaluation time points. 50 µg SMV-loaded AA membranes stimulated significantly more bone formation than non-loaded AA membranes by week 4 (%bone = 31.0 ± 5.9% (AA50) vs 18.5 ± 13.7% (AA0)) but showed no difference at week 8. HA membranes with 50 µg SMV showed significantly more bone formation as compared to corresponding non-loaded membranes by week 8 (%bone = 61.7 ± 8.9% (HA50) vs 33.9 ± 29.7% (HA0)), though such an effect was not significant at week 4. CONCLUSION: These results indicate that modified ESCMs may be used to control the release of SMV and promote bone healing in GBR applications.

Adjuvant antibiotic-loaded bone cement: Concerns with current use and research to make it work.

Antibiotic-loaded bone cement (ALBC) is broadly used to treat orthopaedic infections based on the rationale that high-dose local delivery is essential to eradicate biofilm-associated bacteria. However, ALBC formulations are empirically based on drug susceptibility from routine laboratory testing, which is known to have limited clinical relevance for biofilms. There are also dosing concerns with nonstandardized, surgeon-directed, hand-mixed formulations, which have unknown release kinetics. On the basis of our knowledge of in vivo biofilms, pathogen virulence, safety issues with nonstandardized ALBC formulations, and questions about the cost-effectiveness of ALBC, there is a need to evaluate the evidence for this clinical practice. To this end, thought leaders in the field of musculoskeletal infection (MSKI) met on 1 August 2019 to review and debate published and anecdotal information, which highlighted four major concerns about current ALBC use: (a) substantial lack of level 1 evidence to demonstrate efficacy; (b) ALBC formulations become subtherapeutic following early release, which risks induction of antibiotic resistance, and exacerbated infection from microbial colonization of the carrier; (c) the absence of standardized formulation protocols, and Food and Drug Administration-approved high-dose ALBC products to use following resection in MSKI treatment; and (d) absence of a validated assay to determine the minimum biofilm eradication concentration to predict ALBC efficacy against patient specific micro-organisms. Here, we describe these concerns in detail, and propose areas in need of research.

Characterization of trimethyl chitosan/polyethylene glycol derivatized chitosan blend as an injectable and degradable antimicrobial delivery system.

Advanced local delivery systems are needed as adjunctive treatments for severe injuries with high infection rates, such as open fractures. Chitosan systems have been investigated as antimicrobial local delivery systems for orthopaedic infection but possess mismatches between elution and degradation properties. Derivatives of chitosan were chosen that have enhanced swelling ratios or tailorable degradation properties. A combination of trimethyl chitosan and poly(ethylene glycol) diacrylate chitosan was developed as an injectable local delivery system. Research objectives were elution of antimicrobials for 7 days, degradation as open fractures heal, and cytocompatibility. The derivative combination eluted increased active concentrations of vancomycin and amikacin compared to the non-derivatized chitosan paste, 6 vs. 5 days and 5 vs. 4 days, respectively. The derivative combination degraded slower than non-derivatized paste in an enzymatic degradation study, 14 vs. 3 days, which increased antimicrobial delivery duration. Cytocompatibility of the combination with fibroblast and pre-osteoblast cells exceeds the cell viability standard set in ISO 10993-5. Combination paste requires an increased ejection force of 9.40 N (vs. 0.64 N), but this force was within an acceptable injection force threshold, 80 N. These preliminary results indicate combination paste should be further developed into a clinically useful adjunctive local delivery system for infection prevention.

2018 international consensus meeting on musculoskeletal infection: Summary from the biofilm workgroup and consensus on biofilm related musculoskeletal infections.

Biofilm-associated implant-related bone and joint infections are clinically important due to the extensive morbidity, cost of care and socioeconomic burden that they cause. Research in the field of biofilms has expanded in the past two decades, however, there is still an immense knowledge gap related to many clinical challenges of these biofilm-associated infections. This subject was assigned to the Biofilm Workgroup during the second International Consensus Meeting on Musculoskeletal Infection held in Philadelphia USA (ICM 2018) (https://icmphilly.com). The main objective of the Biofilm Workgroup was to prepare a consensus document based on a review of the literature, prepared responses, discussion, and vote on thirteen biofilm related questions. The Workgroup commenced discussing and refining responses prepared before the meeting on day one using Delphi methodology, followed by a tally of responses using an anonymized voting system on the second day of ICM 2018. The Working group derived consensus on information about biofilms deemed relevant to clinical practice, pertaining to: (1) surface modifications to prevent/inhibit biofilm formation; (2) therapies to prevent and treat biofilm infections; (3) polymicrobial biofilms; (4) diagnostics to detect active and dormant biofilm in patients; (5) methods to establish minimal biofilm eradication concentration for biofilm bacteria; and (6) novel anti-infectives that are effective against biofilm bacteria. It was also noted that biomedical research funding agencies and the pharmaceutical industry should recognize these areas as priorities. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Development and Evaluation of an Injectable Chitosan/ß-Glycerophosphate Paste as a Local Antibiotic Delivery System for Trauma Care.

Complex open musculoskeletal wounds are a leading cause of morbidity worldwide, partially due to a high risk of bacterial contamination. Local delivery systems may be used as adjunctive therapies to prevent infection, but they may be nondegradable, possess inadequate wound coverage, or migrate from the wound site. To address this issue, a thermo-responsive, injectable chitosan paste was fabricated by incorporating beta-glycerophosphate. The efficacy of thermo-paste as an adjunctive infection prevention tool was evaluated in terms of cytocompatibility, degradation, antibacterial, injectability, and inflammation properties. In vitro studies demonstrated thermo-paste may be loaded with amikacin and vancomycin and release inhibitory levels for at least 3 days. Further, approximately 60% of thermo-paste was enzymatically degraded within 7 days in vitro. The viability of cells exposed to thermo-paste exceeded ISO 10993-5 standards with approximately 73% relative viability of a control chitosan sponge. The ejection force of thermo-paste, approximately 20 N, was lower than previously studied paste formulations and within relevant clinical ejection force ranges. An in vivo murine biocompatibility study demonstrated that thermo-paste induced minimal inflammation after implantation for 7 days, similar to previously developed chitosan pastes. Results from these preliminary preclinical studies indicate that thermo-paste shows promise for further development as an antibiotic delivery system for infection prevention.

Evaluation of Antibiotic-Releasing Triphasic Bone Void Filler In-Vitro.

Bone void fillers (BVFs) containing calcium sulfate, tricalcium phosphate (TCP), and hydroxyapatite can be loaded with antibiotics for infection treatment or prevention under surgeon-directed use. The aim of this study was to characterize the handling and elution properties of a triphasic BVF loaded with common antibiotics. BVF was mixed with vancomycin and/or tobramycin to form pellets, and the set time was recorded. A partial refreshment elution study was conducted with time points at 4, 8, and 24 h, as well as 2, 7, 14, 28, and 42 days. Effects on dissolution were evaluated in a 14-day dissolution study. Set time increased to over 1 h for groups containing tobramycin, although vancomycin had a minimal effect. Pellets continued to elute antibiotics throughout the 42-day elution study, suggesting efficacy for the treatment or prevention of orthopedic infections. BVF containing vancomycin or tobramycin showed similar dissolution at 14 days compared to BVF without antibiotics; however, BVF containing both antibiotics showed significantly more dissolution.

Ciprofloxacin and Rifampin Dual Antibiotic-Loaded Biopolymer Chitosan Sponge for Bacterial Inhibition.

Complex extremity wounds in Wounded Warriors can become contaminated with microbes, which may cause clinical outcomes resulting in amputation, morbidity, or even fatality. Local delivery of multiple or broad-spectrum antibiotics allows practicing clinicians treatment solutions that may inhibit biofilm formation. Propagation of vancomycin-resistant Staphylococcus aureus is also a growing concern. The development of vancomycin-resistant S. aureus has become a critical challenge in nosocomial infection prevention in the USA, but to date has seen little occurrence in osteomyelitis. As an alternative, locally delivered ciprofloxacin and rifampin were investigated in a preclinical model for the prevention of biofilm in complex extremity wounds with implanted fixation device. In vitro assays demonstrated ciprofloxacin and rifampin possess an additive effect against Gram-negative Pseudomonas aeruginosa and were actively eluted from a chitosan sponge based local delivery system. In an in vivo orthopedic hardware-associated polymicrobial model (S. aureus and Escherichia coli) the combination was able to achieve complete clearance of both bacterial strains. E. coli was detected in bone of untreated animals, but did not form biofilm on wires. Results reveal the clinical potential of antibiotic-loaded chitosan sponges to inhibit infection through tailored antibiotic selection at desired concentrations with efficacy towards biofilm inhibition.

Local control of polymicrobial infections via a dual antibiotic delivery system.

BACKGROUND: Contaminated traumatic open orthopedic wounds are frequently complicated by polymicrobial contamination and infection. In high-risk wounds, the standard of care comprises debridement and irrigation combined with antibiotics which can be applied directly or combined with systemic antibiotics. Recently, bioabsorbable chitosan sponges have been shown to be an effective single-agent delivery device for local antibiotics with and without negative pressure wound therapy (NPWT). Severely contaminated orthopedic wounds, however, are often complicated by polymicrobial infections, necessitating multiple antibiotic agents. As such, the purpose of this study was to determine if a chitosan sponge would provide a suitable delivery vehicle for multiple antibiotics for the treatment of a polymicrobial infection in a large animal polytraumatic extremity wound model. METHODS: A complex polytraumatic extremity wound was created in 11 adult male Boer goats. Each wound was contaminated with a bioluminescent strain of S. aureus (1 ml of 108 colony forming units/ml) and of P. aeruginosa (1 ml of 108 CFU/ml) which are genetically engineered to allow quantification with a photon-counting camera. Six hours following initial wound creation and contamination, wounds were debrided and irrigated with low-pressure normal saline. The animals were randomized into one of two treatments: wet-to-dry dressings alone or a commercially available chitosan sponge loaded with 1 g vancomycin and 1.2 g of tobramycin. Each animal was then recovered and reimaged 48 h later for total bacteria content; tissue samples were taken from the wound bed to determine relative bacterial colonization. RESULTS: All animals in the chitosan sponge group saw significant reductions in overall bacterial load of S. aureus and P. aeruginosa (p = 0.001). The bioluminescence was also significantly reduced compared to the wet-to-dry dressing group (p = 0.0001). Furthermore, whereas the antibiotic sponge group displayed near complete eradication of bacteria, the wounds treated with the wet-to-dry dressings alone displayed a significant 2-log increase in total bacteria at 48 h p = 0.0001). S. aureus was the predominant species found in the wounds, comprising 95 and 99% of all bacteria found in the chitosan sponge and wet-to-dry, respectively. CONCLUSION: Dual antimicrobial therapy loaded in a chitosan sponge is an effective way to reduce polymicrobial infections traumatic extremity wound.