Authors: Smith JK, Bumgardner JD, Courtney HS, Smeltzer MS, Haggard WO, The University of Memphis, Memphis, TN
Title: Pliable Antibiotic In Situ Loaded Chitosan Film for Infection Prevention: A Preliminary In-vitro Characterization Evaluation
Purpose: This preliminary research study manipulates two integral chitosan variables to develop a pliable chitosan film able to be antibiotically loaded immediately before use and applied to a fracture fixation device for complex would infection prevention.
Methods: The potential of chitosan film to maintain its mechanical properties and prevent musculoskeletal infections will be determined by evaluating the in vitro antibiotic (ABx) elution and activity against staphylococcus aureus as well as the films adhesion and degradation properties. Chitosan powder at 61, 71 and 80% (abbreviated 61, 71 and 80) degree of deacetylation (DDA) was dissolved into solution using lactic (LA) or acetic acid (HAc) and then cast into films. Prior to testing dried chitosan films were ABx loaded in situ for 1 minute using daptomycin (D) or vancomycin (V). Chitosan film variations previously shown to absorb the highest quantities of ABx were used in elution testing. ABx elution was carried out over 72 hours (hr) in 50ml of phosphate buffered saline (PBS). ABx quantification of the samples was determined using high-pressure liquid chromatography, normalized to the chitosan film weight. Elution samples were used to perform ABx activity testing by placing the samples into a staphylococcus aureus broth and measuring the turbidity after 24hr. Testing the films for their adhesive strength to implant grade 316L stainless steel (SS) and Titanium 6-4 (Ti) was performed by fixing the rehydrated chitosan film between two of the metal alloy fixtures and determining the force required to separate the alloys from one another. The degradation of in situ ABx loaded chitosan films was determined by weight loss over time. An initial film weight was determined and after ABx loading the films were placed in 25ml solution of 100µg/ml lysozyme and PBS. Every 20hr, for 100hr, the chitosan films were dried, weighed, and placed into a fresh 25ml lysozyme and PBS solution. All of the data is presented as the mean ± standard deviation and analyzed initially by one-way ANOVA and then the Student t-test to determine significant differences. Statistical significance occured when p < 0.05.
Results: ABx elution results indicated that the 72hr elution's approximate ABx release for each individual film variations remained in the same range. Over the 72hr period, 80% DDA with HAc solvent variations eluted vancomycin at a higher average elution rate and 80% DDA with LA solvent variations eluted daptomycin at a higher average rate. ABx activity was determined using turbidity assays, and overall, the ABx eluate samples inhibited Staphylococcus aureus growth. Adhesion testing indicated the adhesive strength and the results are shown in figure 1. Generally, LA film variations had a higher adhesive strength than HAc variations and Ti71LA had significantly higher adhesive strength than most other alloy/film combinations. The degradation study indicated that variations with higher DDA degraded more than those with a lower DDA as in figure 2. Film variations with 61% DDA degraded to a lesser extent and with no significant difference between ABx loaded and non-loaded variations. When ABx loading had an effect, it was to decrease the film degradation rate. After approximately 60hr, the degradation rate slowed considerably in all variations.
Discuassion and Conclusion: Previous work in film swelling ratio, mechanical and ABx uptake studies determined the variations best suited for this research. The ability of 80LA film variations to adhere to fracture fixation alloys and elute large amounts ABx shows chitosan film's promising potential for traumatic injury infection prevention. ABx activity against Staphylococcus aureus was successfully shown to be unaffected by the film variations, loading, and elution processes. At 80% DDA, chitosan of this source holds the additional benefit of biodegradability which eliminates the need for its removal through secondary surgery. Further investigation into the interactions between the chitosan matrix, ABx structure, and acid solvent would yield information towards optimizing the film for a wider range of ABx and extended elution period. Additional studies are needed to ensure biocompatibility and establish a method for use in fracture device stabilization before in vivo evaluation commences. This preliminary in vitro research determined that a novel in situ loaded, chitosan film has potential as an adjunctive device for musculoskeletal extremity wound treatment where bacterial contamination is likely and orthopaedic fixation devices and implants are used.
