Efficient, biosafe and tissue adhesive hemostatic cotton gauze with controlled balance of hydrophilicity and hydrophobicity

Massive bleeding from traumatic wounds is a leading cause of preventable death and disability, as significant blood loss triggers hypothermia, coagulopathy, acidosis, sepsis, and organ failure. Although cotton gauze remains the most widely used hemostatic material due to safety, low cost, and absorbency, its highly hydrophilic and porous nature causes excessive blood absorption before bleeding stops, potentially worsening outcomes. Prior attempts to enhance gauze hemostasis include loading procoagulant minerals (e.g., kaolin in QuikClot Combat Gauze), growing zeolites on fibers, and designing Janus or superhydrophobic composites; however, these approaches still allow substantial blood loss or suffer from particle detachment and seam leakage at the gauze/tissue interface. The research question is whether a cotton gauze with a controlled balance of hydrophilicity/hydrophobicity and wet tissue adhesiveness can rapidly stop bleeding with minimal blood loss and without re-bleeding. Inspired by mussel catechol chemistry, the study proposes lightly grafting a catechol with a long alkyl chain onto cotton fibers to control blood movement both at the gauze/tissue interface and within the gauze.

- QuikClot Combat Gauze (QCG) binds kaolin particles to activate factor XII and accelerate coagulation but may lose particles and risk distal thrombosis; still absorbs substantial blood. - Chabazite zeolite particles chemically anchored to cotton fibers improve hemostasis over QCG but still exhibit significant blood absorption (about 40% less blood loss than QCG in rabbit femoral artery injury models reported previously). - Wettability tuning via Janus fabrics (hydrophobic top, hydrophilic bottom) aims to limit diffusion, yet bottom cotton layer still permits warp/weft permeation and seam leakage. - Superhydrophobic PVDF/carbon nanofiber composites show fast hemostasis and reduced re-bleeding via fibrin acceleration and repellency but face challenges with moisture management and seam leakage. - Mussel-inspired catechol chemistry offers strong wet adhesion via hydrogen bonding and π–π interactions, suggesting potential to block seam leakage and modulate intragauze blood flow.

Materials and surface modification: - Cotton gauze cleaned with water and ethanol; dried under N2. Free radicals introduced by low-temperature N2 plasma (400 Pa, 80 W, 3 min). - Graft-to reaction: Plasma-treated gauze refluxed in 2.0 wt% USO (1,2-benzenediol-3-(7,9,13-pentadecatrienyl)) in ethanol at 70°C for 2 h. Washed with ethanol; dried at 80°C vacuum oven. Graft loading ~0.1 wt% (gravimetry). Analogous procedures for ABO-g-gauze (4-allyl-1,2-benzenediol) and HTMS-g-gauze (hexadecyltrimethoxysilane). - Catechol modification controls: USOFe-g-gauze prepared by Fe3+ chelation (0.1 M FeCl3, 10 min, 37°C); USOQu-g-gauze by oxidation to quinone (20 mM Tris-HCl pH 9.8, 10 min, 37°C). Characterization: - Solid-state 13C NMR to confirm grafting (new peaks 10–40 ppm aliphatic, 120–150 ppm aromatic in USO-g-gauze). - FTIR and XPS to verify grafting for ABO, HTMS, USO. C1s XPS shows increased C–C for USO-g-gauze. - SEM for fiber/yarn morphology; surface roughness after plasma/grafting. - Wettability: water contact angle (WCA), droplet diffusion with simulated body fluid (SBF) and fresh rat blood (200 µL). - Water vapor transmission rate (WVTR): beaker method at 37°C, 24 h. - Water absorption kinetics: 2×2 cm specimens in SBF; Wabs = (m2−m1)/m1×100%. - Moisture management test (AATCC 195): wetting time, spreading, absorption, one-way transport using M290 tester with 0.2 mL test solution. In vitro blood–material interactions: - Erythrocyte and platelet adhesion: Incubate gauze with rat whole blood or PRP (1:10 in PBS) at 37°C for 90 min; rinse, fix (2.5% glutaraldehyde), graded ethanol dehydration, SEM. Adhesion (peeling force): - Gauze (5×2.5 cm) placed on fresh rat femoral tissue for 10 min pre-transection; peeled with digital push–pull gauge; peak force recorded (n=3). In vivo hemostasis models and outcomes: - Rat femoral artery transection: Four-layer gauze stacks (12×2.5 cm). After 2 s bleeding, gauze applied or compressed (150 s). Record blood diffusion, hemostasis time, blood loss (n=6). Define hemostasis when stained area stops expanding and no seam seepage. - Rat liver laceration: 1 cm incision on left lobe; similar application/measurements (n=6). - Pig models (Bama miniature pigs, 1.6–1.8 kg): skin laceration (2 cm length × 1 cm depth) and femoral artery exposure/incision. For femoral artery, wounds wrapped 3 min; upon removal, assess re-bleeding; measure absorbed blood mass at 3 min as proxy for blood loss (n=3). Skin laceration blood loss measured. - Survival monitoring: 120 min post-hemostasis observation. - Biocompatibility: L929 fibroblast live/dead assay up to 3 days on gauze. Subcutaneous implantation in rats with histology (H&E, toluidine blue) at 3, 7, 14, 21 days; neutrophil and mast cell counts. Computational analysis: - DFT calculations of adsorption energies (ΔEads) between catechol of USO-g-gauze and 16 amino acids (keratin constituents), considering π–π stacking and H-bonding; analyze distances (1.69–1.80 Å) and ΔEads magnitudes. Statistics: - Data as mean ± SD; one-way ANOVA; significance at p<0.05, p<0.01.

Material properties: - USO-g-gauze exhibits an instant WCA ~68.2°; HTMS-g-gauze is highly hydrophobic (WCA 132.6°). On USO-g-gauze, 200 µL water/blood droplets diffuse vertically over ~60 s with minimal lateral spreading. - WVTR (g m−2 d−1 at 37°C): cotton 1028, ABO-g 1023, HTMS-g 1021, USO-g 1015. - Water absorption at 5 min: cotton 323%, ABO-g 321%, HTMS-g 9.0%, USO-g 267% (USO 17.3% lower than cotton). - USO-g-gauze initially floats then sinks after ~30 min, indicating reduced initial hygroscopicity; moisture management indicates balanced wetting/spreading and one-way transport. - SEM: Greater erythrocyte and platelet aggregation on USO-g-gauze than cotton; platelets show pseudopods (activation). HTMS-g-gauze shows fewer adhered erythrocytes. Rat femoral artery transection (n=6): - Hemostasis time (s): HTMS-g 356; cotton 180; ABO-g 173; QCG 147; USO-g 34 (USO 77% faster than QCG). - Blood loss (g): HTMS-g 1.03; cotton 0.52; ABO-g 0.49; QCG 0.42; USO-g 0.13 (USO ~71% less than QCG). - Re-bleeding: frequent with cotton/ABO/QCG/HTMS; absent with USO-g. Rat liver laceration (n=6): - Hemostasis time (s): cotton 172; ABO-g 153; HTMS-g 344; QCG 96; USO-g 32 (USO 67% faster than QCG). - Blood loss (g): cotton 0.39; ABO-g 0.37; HTMS-g 0.98; QCG 0.13; USO-g 0.03 (USO 77% less than QCG). - USO-g outperforms Surgicel (hemostasis time ~198 s; blood loss ~0.47 g; seam seepage observed). Pig femoral artery (3 min wrapped, n=3): - Absorbed blood mass (g): USO-g 0.80; cotton 5.11; ABO-g 4.16; QCG 3.94; HTMS-g 8.22. USO-g blood loss is 15.6% of cotton and 20.4% of QCG; no re-bleeding upon gauze removal at 3 min. Pig skin laceration (n=3): - Blood loss (g): cotton 0.54; ABO-g 0.32; QCG 0.22; HTMS-g 0.71; USO-g 0.03 (USO ~94% lower than cotton; ~85.5% lower than QCG). Survival (rats, 120 min): - USO-g: 100% survival; QCG: 20%; cotton/ABO/HTMS: 0%. Adhesion and mechanism: - Peeling force on wet rat femoral tissue: HTMS-g 24 mN; USO-g 90 mN (about twice cotton; strongest among groups). ABO-g shows noticeable adhesion; catechol modification (Fe3+ chelation or oxidation to quinone) reduces adhesion and hemostatic efficacy. - DFT: Amino acids with aromatic rings (Phe, Tyr) show strong π–π + H-bond adsorption to catechol (ΔEads ~0.621–0.729 eV); others bind via double H-bonds (ΔEads ~0.570–0.639 eV). H-bond distances 1.69–1.80 Å. - Post-hemostasis SEM: USO-g shows thick erythrocyte layers filling macropores in the first two gauze layers; thickness up to ~220 µm; minimal penetration to 3rd/4th layers. Cotton shows sparse RBCs across all four layers. Biocompatibility: - In vitro cytocompatibility comparable to cotton gauze. - In vivo implantation: transient inflammatory response with neutrophils and mast cells decreasing to low levels by 14–21 days; similar profiles for cotton and USO-g. Free USO may cause dermatitis, but grafted USO showed no dermatitis in use.

The study addresses the central challenge that conventional cotton gauze stops bleeding by absorbing large volumes of blood, thereby causing additional blood loss and potential destabilization. By grafting a catechol-terminated long alkyl chain (USO) at low loading onto cotton fibers, the gauze achieves a controlled hydrophilic/hydrophobic balance and strong wet tissue adhesion. The catechol moieties form non-covalent adhesive interactions (hydrogen bonding and π–π stacking) with tissue proteins, creating a dam-like seal at the gauze/tissue interface that suppresses seam leakage. Concurrently, hydrophobic interactions among the alkyl chains slow lateral and vertical blood wicking within the gauze, restricting blood movement largely to the first one to two layers. This confinement concentrates erythrocytes and platelets at the wound site, generating thick primary clots that rapidly stop bleeding and remain stable upon gauze removal, preventing re-bleeding. Across rat and pig models, USO-g-gauze consistently reduced hemostasis time and blood loss compared with cotton gauze and QCG, and improved survival. The mechanism is physical and does not rely on altering coagulation pathways, suggesting potential utility in coagulopathic patients. The approach generalizes to other fabrics (e.g., chitosan nonwoven), indicating broad applicability.

A highly efficient hemostatic cotton gauze was created by lightly grafting a catechol-containing, long-chain hydrophobic molecule (USO) onto cotton fibers. The resulting gauze combines breathable fabric structure with wet tissue adhesion and moderated wettability to control blood movement at the interface and within the gauze. In multiple animal bleeding models (rat femoral artery and liver, pig femoral artery and skin), USO-g-gauze achieved markedly shorter hemostasis times, dramatically reduced blood loss, absence of re-bleeding, and improved survival versus standard cotton gauze and QCG. DFT and adhesion measurements support a mechanism based on non-covalent catechol–tissue bonding and hydrophobic modulation leading to rapid formation of thick erythrocyte clots. The gauze shows biocompatibility comparable to cotton and the strategy extends to chitosan fabrics. Future work could optimize graft density and chain length, assess performance under coagulopathy and anticoagulation, evaluate long-term safety and scaling, and conduct clinical translation studies.

- Hemostasis time in the pig femoral artery model was not measured directly because wounds were wrapped; absorbed blood mass at 3 minutes was used as a proxy. - Animal group sizes were modest (rats n=6 per group; pigs n=3), and only preclinical models were tested; clinical efficacy remains to be established. - While grafted USO showed acceptable biocompatibility, the free small-molecule catechol (USO) can cause contact dermatitis; comprehensive sensitization and long-term safety assessments are warranted. - The study focused on specific grafting conditions (~0.1 wt% USO); the effect of varying graft density/chain length and durability under field conditions needs evaluation.