Development of a chitosan-based wound dressing with improved hemostatic and antimicrobial properties
Introduction
Uncontrolled hemorrhage remains the leading cause of prehospital trauma deaths in both the combat and civilian settings [1], [2]. A number of hemostatic agents have been developed that can arrest bleeding and stabilize the casualty before evacuation to definitive care. Of these, the HemCon chitosan dressing and the QuikClot zeolite powder are being used routinely in the battlefield, and case reports show that both were effective in reducing or stopping bleeding in >90% of applications [3], [4]. However, the application of QuikClot generates heat that can cause burn injuries, and some studies have found no significant survival benefit of either QuikClot or HemCon over gauze in more extreme animal models of hemorrhage [5], [6]. The challenge remains to develop more effective hemostatic dressings that can control bleeding that would otherwise lead to exsanguination.
Another challenge facing caregivers is the growing incidence of infection by antibiotic-resistant bacteria strains in combat trauma wounds [7], [8]. Although the use of broad spectrum antibiotics has been implicated in the selection of these resistant pathogens, antibiotic prophylaxis remains the standard of care since early surgical debridement to reduce wound bacteria bioburden may not be possible under combat conditions [9], [10]. To contain the problem of resistance, alternatives to antibiotics should be used to manage wound infection. Ionic silver is active against a wide range of pathogens including multi-drug resistant strains, and have a far lower propensity for resistance development [11], [12]. Topical silver thus offers a useful first line intervention to stop the progress of infection that can lead to septicemia and death, while reducing the risk of inducing resistant strains that will be difficult to treat after evacuation.
In view of the dual challenges of bleeding and contamination in combat wounds, we conceived a chitosan-based dressing with improved hemostatic and antimicrobial properties. Chitosan is an attractive biomaterial for wound care because of its biocompatibility and intrinsic hemostatic and antimicrobial properties [13]. We hypothesized that the addition of polyphosphate (PP) polymers to chitosan can lead to a more potent hemostat. Polyphosphate, a linear polymer of inorganic phosphate, is found in high concentrations in platelet dense granules, and polymers with ≥45 phosphate units in their chain have been shown to accelerate blood coagulation and delay fibrinolysis [14]. Since polyphosphate dissolves in water to form a polyanion, it can ionically interact with chitosan to form polyelectrolyte complexes (PECs) [15].
A series of chitosan-polyphosphate dressings were fabricated and their hemostatic efficacies evaluated against chitosan and gauze in vitro. After identifying the most effective hemostatic formulation (coded as ChiPP), silver nanoparticles were incorporated to enhance the dressing's antimicrobial activity, since chitosan's antimicrobial action is limited against certain species of bacteria and in non-acidic pH environments [16], [17]. The silver dressing (coded as ChiPP-Ag) was then compared with ChiPP in time kill assays against two common wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, followed by in vitro characterization of silver elution and cytotoxicity, before evaluation in a murine model of wound infection.
Section snippets
Materials
Chitosan (deacetylation degree of 75–85% and MW ∼190,000–310,000) was obtained from Sigma. Polyphosphate polymers with either 45 or 65 phosphate units per chain (abbreviated as PP45 and PP65, respectively) were also obtained from Sigma. Silver nanoparticles were obtained from American BioTech Labs (UT, USA). Sterile gauze was obtained from Melintex Pharma Co (China), and cut into 0.5 × 0.5 cm2 for experiments. HemCon and Acticoat Absorbent dressings were obtained from HemCon Medical Technologies
Whole blood clotting
In order to evaluate whether chitosan-polyphosphate PECs can increase the rate of blood clotting, whole blood was contacted with dressings for 10 min before hemolyzing RBCs that were not trapped in the clot that formed on the dressing surface. A higher absorbance value of the hemoglobin solution thus indicates a slower clotting rate. Chitosan with 6.7% w/w or 10% w/w PP65 or PP45 led to significantly lower absorbance values than chitosan (Fig. 1A). The clots formed on Chi-10%PP45 were also
Discussion
The first part of this study evaluated the hemostatic activity of chitosan-polyphosphate dressings. Both components have been shown to activate coagulation by different mechanisms: Chitosan's protonated amine groups attracted negatively-charged residues on red blood cell membranes, causing strong hemagglutination [23], [24], [25], [26]. Chitosan also adsorbed fibrinogen and plasma proteins, enhancing platelet aggregation [27], [28]. On the other hand, polyphosphate specifically activated the
Conclusions
In conclusion, a new ChiPP dressing has been developed that accelerated blood clotting, platelet adhesion, thrombin generation, and absorbed significantly more blood than chitosan. To our knowledge, these results provide the first evaluation and optimization of a chitosan-polyphosphate complex for hemostatic applications. Further investigation will more precisely delineate the mechanisms behind the improved hemostatic activity of ChiPP, and validate the findings in vivo. The ChiPP with
Acknowledgements
The authors would like to thank American BioTech Labs for donating the silver nanoparticles used in this study. We are also very grateful to Li-Li Tan (DSO National Laboratories, Singapore) for her assistance with histology.
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