Expandable Carboxymethyl Chitosan/cellulose Nanofiber Composite Sponge For Traumatic Hemostasis

BackgroundTraumatic hemorrhage remains one of the leading causes of mortality worldwide.Hemorrhage can cause pathological changes,eventually leading to organ failure and death.In both military and civilian contexts,penetrating injuries can result in massive hemorrhage,posing a significant challenge to survival.Practical hemostatic measures are essential in the pre-hospital environment to save lives.However,the hemostatic equipment commonly used,such as tourniquets,bandages,and gauze,are often inadequate in most hemorrhages at the torso and "junction" sites(armpit,groin,and neck).Moreover,they may cause secondary trauma and cannot fully meet the needs of the pre-hospital environment in the warzone.Therefore,there is an urgent need to develop a safe and effective hemostatic dressing.We designed a hemostatic pen that can be used for pre-hospital emergency hemostasis.Based on previous research,this study aims to develop an expandable hemostatic sponge that serves as fill material for the medical hemostatic pen.It is expected to offer a unique application prospect as a penetrating trauma hemostatic agent.Method1.Preparation and characterization of Cellulose nanofibersCellulose nanofibers(CNFs)were prepared by TEMPO-mediated oxidation combined with high-pressure homogenization.The physical and chemical properties of the prepared CNFs were characterized by Fourier transform infrared spectroscopy,thermogravimetric analysis,X-ray photoelectron spectroscopy,scanning electron microscopy,and transmission electron microscopy.2.Preparation and characterization of carboxymethyl chitosan/cellulose nanofiber spongeThe carboxymethyl chitosan/cellulose nanofiber sponge(CNF-CMCS)was prepared by the CO-NH cross-linking method.We characterized the CNF-CMCS’s internal chemical structure using Fourier transform infrared spectroscopy,thermogravimetric analysis,and X-ray photoelectron spectroscopy.Moreover,we analyzed the sponge’s physical and mechanical characteristics,including density,porosity,swelling,water absorption,water retention capacity,stress-strain analysis post-water absorption,and swelling process post-compression.3.Evaluation of the hemostatic effect of carboxymethyl chitosan/cellulose nanofiber spongeWe observed the adhesion and activation of sponges to red blood cells and platelets through in vitro coagulation time,whole blood coagulation index(BCI),and scanning electron microscopy.Moreover,we investigated the procoagulant properties of CNF-CMCS in vitro.We preliminarily established the hemostatic effect of CNF-CMCS on the animal level using a liver puncture model in mice and a femoral vein puncture model in rats.Furthermore,we evaluated CNF-CMCS’s hemostatic effect on deep trauma massive hemorrhage by creating a femoral artery and femoral vein transection model in rats.4.Evaluation of in vitro antibacterial activity of carboxymethyl chitosan/cellulose nanofiber spongeThe bactericidal and bacteriostatic efficacy of CNF-CMCS against commonly encountered wound infection strains,including Escherichia coli,Pseudomonas aeruginosa,and Staphylococcus aureus,were evaluated via direct contact and diffusion methods(bacteriostatic zone method).Furthermore,mupirocin,a water-soluble model drug,was loaded into CNF-CMCS.The drug release rate and antibacterial activity were investigated through high-performance liquid chromatography(HPLC)and bacteriostatic zone testing.5.Evaluation of biocompatibility of carboxymethyl chitosan/cellulose nanofiber spongeWe conducted a hemolysis test in vitro to investigate the blood compatibility of CNF-CMCS.We evaluated the effect of CNF-CMCS on the proliferation and morphology of mouse fibroblast cells(L929)using the MTT test,Calcein/PI staining,and cell morphology analysis.Additionally,we observed the host response of rats after subcutaneous implantation of CNF-CMCS to evaluate its biocompatibility in vivo.Results1.Cellulose nanofibers(CNFs)were successfully synthesized through TEMPO-mediated oxidation and high-pressure homogenization.The prepared CNFs exhibit abundant carboxylic acid groups on their surface and can be readily dispersed into individual fibers in water.2.After process optimization,we identified a suitable formula for CNF-CMCS and successfully incorporated a small amount of CNFs into the CMCS structure.The introduction of CNFs significantly impacted the physical and mechanical properties of CNF-CMCS,resulting in low density,high porosity,compressibility,rapid expansion after water absorption,strong water retention capacity,and high mechanical strength.Notably,CNF-CMCS2 and CNF-CMCS3 exhibit high mechanical strength even after water absorption,aligning with our prior design expectations.3.In vitro clotting activity test revealed that the prepared CNF-CMCS exhibited potent coagulant activity.CNF-CMCS significantly shortened the clotting time in vitro and facilitated the adsorption and fixation of red blood cells.CNF-CMCS also exhibited strong adhesion to red blood cells and platelets,promoting platelet aggregation and activation.4.In mouse liver puncture and rat femoral vein puncture models,treatment with CNF-CMCS significantly improved bleeding conditions,reducing bleeding time and volume.Additionally,the hemostatic efficacy of CNF-CMCS was evaluated in rat femoral artery and vein transection models,confirming its potential for stopping bleeding in deep trauma.Notably,CNF-CMCS2 and CNF-CMCS3 sponges were particularly effective in minimizing blood loss.5.The in vitro antibacterial experiments demonstrated that CNF-CMCS effectively kills Escherichia coli,Pseudomonas aeruginosa,and Staphylococcus aureus,exhibiting a broad-spectrum contact antibacterial activity.However,no diffusion antibacterial activity against the strains mentioned above was observed in the agar medium.CNF-CMCS rapidly released mupirocin,which enhanced its diffusive antibacterial activity.The CNFs content significantly impacted drug release in CNF-CMCS/Mupirocin,with an optimal amount of CNFs facilitating the release and activity of mupirocin.CNF-CMCS2/Mupirocin demonstrated a higher mupirocin release rate and anti-Staphylococcus aureus activity.6.In vitro and in vivo biocompatibility evaluations suggested that CNF-CMCS possessed outstanding blood compatibility.No significant effect on L929 cell proliferation or morphology was observed.Host response conducted in vivo indicated that CNF-CMCS did not elicit any unusual inflammatory reactions 12 hours after implantation.Moreover,CNF-CMCS2 and CNF-CMCS3 only caused minor neutrophil aggregation and infiltration 72 hours post-implantation,indicating good histocompatibility of the sponge.Conclusion1.CNFs were incorporated into the CMCS sponge via amide coupling catalyzed by EDC/NHS,yielding the successful preparation of CNF-CMCS.The prepared sponge possessed strong water absorption,high porosity,compressibility,and rapid expansion upon water absorption,aligning with the desired characteristics for efficiently concentrating blood,promoting blood coagulation,and producing a compression hemostatic effect upon entering the wound.2.In vivo and in vitro experiments confirmed the excellent procoagulant activity of CNF-CMCS,significantly reducing both bleeding time and volume with precise hemostatic efficacy.It demonstrated remarkable hemostatic efficacy for severe trauma bleeding,indicating significant potential for application in deep trauma hemostasis.3.In vitro antibacterial experiment has demonstrated that CNF-CMCS possesses good direct contact antibacterial activity but does not exhibit diffusive antibacterial activity.Upon loading with the water-soluble drug mupirocin,it can rapidly release the drug and gain significantly enhanced diffusion bacteriostatic activity.4.In vitro and in vivo biocompatibility studies showed that CNF-CMCS offered good biocompatibility.In summary,the developed CNF-CMCS demonstrates a definitive hemostatic effect,excellent biocompatibility,and extensive antibacterial activity,with remarkable hemostatic efficacy for deep trauma,thereby satisfying the anticipated research and design requirements as a filling material for medical hemostatic pens.It holds excellent prospects for transformation and application in the medical field.