Non-compressible hemorrhage poses a significant challenge in hemostasis, leading to high mortality rates. Current methods like tourniquets and manual compression are often insufficient. Hemostatic sponges offer a promising solution, but improving both permeability (for rapid blood absorption and shape recovery) and mechanical properties (for sustained pressure on the wound) simultaneously has proven difficult. Existing techniques often compromise one property to enhance the other, limiting hemostatic efficacy and tissue regeneration. The limited pore interconnectivity in many hemostatic sponges restricts blood absorption, prolongs shape recovery, and hinders cell infiltration and vascularization. Increasing porosity to improve permeability typically weakens the sponge's mechanical strength, compromising its ability to maintain pressure on the wound. This research aims to overcome this limitation by developing a novel fabrication strategy that enhances both permeability and mechanical properties of a chitosan-based hemostatic sponge.

Various hemostatic materials, including sponges, adhesives, and powders, have been developed to address non-compressible hemorrhage. Shape-recoverable hemostatic sponges are particularly promising, as they restore their shape upon blood absorption, applying mechanical compression to the wound. However, existing methods to create porous structures, such as direct lyophilization and the use of foaming agents, often result in limited pore interconnectivity, slow shape recovery, and poor cell infiltration, hindering hemostatic efficacy and tissue repair. Improving permeability by increasing porosity typically reduces mechanical strength. Compaction and densification strategies have been explored to enhance mechanical properties, such as freeze-casting and directional stretching, but their application to hemostatic sponges with optimized permeability remains a challenge. Chitosan, with its biocompatibility, biodegradability, and pro-coagulant properties, is a widely used biomaterial in hemostatic agent formulation. Phase separation of chitosan solutions can create 3D networks, but these often lack sufficient interconnectivity and pore size for optimal hemostasis.

The study introduces a temperature-assisted secondary network compaction (TA-2ndNC) strategy to fabricate superporous chitosan sponges (spCS). The process involves initial phase separation of an acidic chitosan solution using an alkaline solution, followed by cooling to an optimal pre-freeze-drying temperature (Tprd) of 0 °C before freeze-drying. This temperature allows for controlled secondary polymer network reorganization and compaction, creating a highly interconnected porous structure. A control group (PCS) was created without the secondary compaction step, and additional controls (CS and epCS) were prepared using different Tprd values (-80°C and 20°C respectively) to demonstrate the importance of the selected temperature. The surface of the spCS was then modified with hydrophobic dodecyl chains to enhance blood coagulation (resulting in A-spCS). Various characterization techniques were employed to assess the microstructure, porosity, mechanical properties, blood absorption capacity, and shape recovery of the sponges. Micro-CT, SEM, porosity tests, XRD, and XPS were used for structural analysis. Mechanical testing assessed fatigue resistance, and water/blood absorption tests measured absorption capacity and rate. In vitro tests evaluated the pro-coagulant ability (using a blood clotting index (BCI) test, RBC and platelet adhesion assays), antibacterial properties (against S. aureus, E. coli, and P. aeruginosa), and biocompatibility (cell viability and hemolysis assays). In vivo studies utilized rat liver perforation and Bama minipig liver and spleen injury models to evaluate hemostatic efficacy. A rat liver regeneration model assessed the in situ tissue regeneration capacity. Cell migration experiments in vitro further evaluated the infiltration ability of cells into the A-spCS.

The TA-2ndNC strategy yielded superporous chitosan sponges (spCS) with significantly larger pore sizes and higher porosity (88.42 ± 6.18%) compared to controls (PCS, CS). spCS exhibited a 269% higher porosity than PCS and a 53% higher network density. The optimal Tprd of 0°C enabled controlled network compaction, resulting in improved fatigue resistance (95% maximum stress retention after 100 cycles at 85% strain) and rapid shape recovery (0.84 s for water, 4.0 s for blood). Alkylated spCS (A-spCS) demonstrated superior hemostatic efficacy in rat liver (hemostasis in 13 s) and minipig liver/spleen models (39 s for liver). A-spCS significantly outperformed commercial gauze, gelatin sponges, and chitosan powder in hemostasis time and total blood loss. In vitro, A-spCS showed the lowest BCI values, highest RBC and platelet adhesion, and potent antibacterial activity against all tested bacteria. Biocompatibility assays showed no significant cytotoxicity or hemolysis. In vivo, A-spCS promoted cell migration, vascular regeneration, and in situ tissue regeneration in the rat liver model. Compared with the control groups (CELOX, CS, and PCS), the A-spCS group showed a much larger tissue ingrowth area, cell number, capillary density, and LPC number in rat livers at four weeks post-hemostasis. In vitro cell migration experiments demonstrated that cells can effectively penetrate and grow within the A-spCS sponge.

The findings demonstrate that the TA-2ndNC strategy successfully addresses the challenge of simultaneously enhancing permeability and mechanical properties in hemostatic sponges. The superior performance of A-spCS compared to existing hemostatic materials highlights the potential of this fabrication method for clinical applications. The rapid shape recovery and enhanced blood absorption contribute to efficient hemostasis, while the improved mechanical properties ensure sustained pressure on the wound. The pro-coagulant, antibacterial, and biocompatible properties of A-spCS further enhance its effectiveness and minimize infection risk. The in vivo tissue regeneration results indicate A-spCS can serve as a scaffold to support cell migration and vascular development, promoting in situ tissue repair and reducing the need for hemostat removal. This is especially significant for non-compressible wounds where secondary bleeding is a major concern.

This study presents a novel temperature-assisted secondary network compaction strategy for fabricating superporous chitosan hemostatic sponges with significantly improved permeability and mechanical properties. The resulting A-spCS sponge demonstrates superior hemostatic efficacy, promotes in situ tissue regeneration, and exhibits excellent biocompatibility and antibacterial properties. This simple and effective fabrication method holds great promise for developing advanced hemostatic materials for treating non-compressible hemorrhage and promoting wound healing. Future research could explore different modifications to further enhance the performance of the sponge, evaluate its long-term effects in larger animal models, and conduct clinical trials to confirm its efficacy and safety in human patients.

While the study demonstrates significant improvements in hemostatic sponge properties and efficacy, further research is warranted. The animal models, although informative, may not fully reflect the complexity of human hemorrhage. Long-term in vivo studies are needed to assess the biodegradation and potential long-term effects of A-spCS. The relatively small sample size in some of the in vivo experiments should be addressed in future studies. Although the current study focuses on liver and spleen injuries, further research should evaluate the suitability of the A-spCS for other types of non-compressible wounds.