Hemostatic Tough Adhesives seal tissue and control hemorrhage

Domain: neuroscience
Published: 2026-04-08
Editorial outcome: major_revision

Abstract
Hemorrhage from internal organs remains a critical challenge in both trauma care and surgical procedures, as existing hemostatic adjuncts frequently fail to provide consistent and effective bleeding control, particularly under conditions of active bleeding or impaired coagulation. Here we develop and evaluate a Hemostatic Tough Adhesive (HTA) in controlled preclinical models of traumatic solid organ injury and compare its performance against leading commercially available hemostatic agents. The HTA consistently outperformed its counterparts, achieving 100% hemostasis in both liver and spleen injuries within an in vivo preclinical porcine model. In contrast, existing adjuncts exhibited variable and often incomplete efficacy. Beyond immediate hemostasis, the HTA demonstrated prolonged stability and biocompatibility during the postoperative wound healing phase. Notably, the HTA exhibited tissue surface adhesion energy several orders of magnitude greater than that of current hemostatic products, indicating its potential utility for surgical and trauma-related bleeding management.

Introduction
Intraoperative blood loss remains a critical challenge, with bleeding-related complications and/or blood transfusions occurring in approximately 30% of inpatient surgical procedures across various specialties1. Surgical complications account for one quarter of all transfusions in the U.S. each year2. Major bleeding is also associated with higher hospitalization costs, in some surgical specialties incurring additional costs exceeding $17,000 per case1. Additionally, bleeding-related complications extend hospital stays, with an average increase of 6.0 days and up to 9.6 days in certain specialties1. These factors collectively increase the risk of major perioperative complications, contribute to greater resource utilization, and place a significant burden on healthcare systems. Furthermore, approximately 40% of trauma-related deaths are caused by uncontrolled bleeding3. Severe or massive intraoperative bleeding has a combined mortality rate of over 20%4 that is further exacerbated by an aging population with comorbidities and anticoagulant use3-5. This problem is compounded in lower resource environments, such as rural areas6, on the battlefield7, in disaster zones8, safety-net healthcare settings9, and developing countries10. Achieving and maintaining hemostasis and preserving organ integrity is paramount to performing safe surgery, particularly when internal organs are repaired, manipulated, divided, or transplanted. Organs, such as the liver, spleen, kidneys, pancreas, and lungs, are particularly vulnerable to disruption, increasing the risk of coagulopathy and excessive hemorrhage11.

Current standard-of-care strategies to control surgical bleeding involve a combination of manual pressure, vascular ligation or embolization, electrosurgical coagulation, or application of surface hemostatic adjunct materials. Despite several products developed to address this pressing need8,12-14, the success of existing commercial products used to achieve hemostasis remains limited, and often require surgical techniques – such as vascular ligation, or thermal coagulation – to achieve reliable hemostasis. Common hemostatic adjuncts fall into three categories based on their effects on clotting: passive, active, and structural. Passive hemostatic adjuncts, which serve as a matrix to facilitate clot formation, are readily available for use, but these materials lose their hemostatic functions in the setting of coagulopathies associated with trauma15, when patient are on anticoagulant medications16, or in patients with inherited bleeding dyscrasias. Active hemostatic adjuncts, which chemically augment clot formation, often have short working periods prior to setting, provide only temporary hemostasis without long-term structural support, may increase the risk of re-bleeding and the need for additional interventions, and often do not display favorable physical characteristics in critical surgical scenarios17. Structural adjuncts promote rapid hemostasis through a combined sealant and adhesive effect via crosslinking, but existing agents may compromise normal dynamic organ function due to their rigidity and have been linked to significant cellular toxicities17,18. While hydrogel-based structural adjuncts have shown promise, they have yet to demonstrate robust tissue adhesion, often failing cohesively, which has limited their capacity to withstand high pressures and flow rates associated with hemorrhage.

The ideal material for treating solid organ hemorrhage while maximally preserving organ tissue would effectively reapproximate wound edges without excessive tension, adequately compress the affected organ to generate tamponade, promote localized thrombosis without adverse systemic effects, be rapidly applicable during emergency surgery, and reliably adhere in the presence of blood or other bodily fluids. Here, we explore a biocompatible hydrogel material synthesized from alginate and polyacrylamide, which, when combined with a chitosan-based tissue-facing adhesive, exhibits favorable characteristics and high adhesion to multiple tissue surfaces. Here we demonstrate that this system, named a Hemostatic Tough Adhesive (HTA), is a promising candidate for treating hemorrhage from internal organs (Figure 1). We designed a translational research study to evaluate the intrinsic performance of the HTA, in both permanent and degradable forms whose different properties may have different therapeutic benefits, and compare its effectiveness against commercially available hemostatic adjuncts, both on fresh cadaveric tissue and during live porcine surgery for the repair of iatrogenic lacerations.

Results
Hemostasis Tough Adhesives demonstrate high tissue adhesion and failure resistance
Compared to commercially available surgical hemostatic adjuncts, the HTA demonstrated significantly higher resistance to failure during burst pressure testing while adhered to ex vivo porcine pericardial tissue (Figure 2a) and tensile mechanical properties (Supplementary Figure 1). Additionally, adhesion strength testing on ex vivo porcine skin showed that HTA achieved an average interfacial toughness of 2500 J/m2, significantly surpassing existing adjuncts (Figure 2b). HTAs demonstrated strong and reliable adhesion across various tissue types (Figure 2c,d), with interfacial toughness ranging from 1000-5000 J/m2,as measured ex vivo by peel testing. Notably, HTA adhered effectively in both wet and dry environments, as well as in the presence of blood, in part due to the hemorepellent properties of the chitosan (Figure 2e). Hydrogel swelling post implantation has been examined in several studies suggesting minimal additional swelling after post synthesis wash steps19-21.

HTAs can withstand pressures in the supraphysiologic range
HTAs successfully bridged 1.5 cm linear defects in ex vivo porcine aortic tissue, maintaining intravascular hemostasis throughout prolonged cycles that mimicked physiological blood pressure using a peristaltic pump (Supplementary Figure 2a). Additionally, HTAs withstood supraphysiologic intravascular pressures until failure, demonstrating resilience comparable to BioGlue ex vivo (Supplementary Figure 2b) and in vivo (Supplementary Figure 2c).

Hemostatic Tough Adhesives control hemorrhage from solid organs during live surgery
To test whether the HTA possesses favorable properties for use as a hemostatic adjunct, HTAs were applied to intraperitoneal and retroperitoneal tissues in pigs during laparotomy (Figure 3a). Similar to bench performance testing, in vivo blinded and randomized controlled testing revealed robust adhesion to wet and actively bleeding solid organs compared to standard of care (Figure 3b, Supplementary Movie 1). Following biopsy injury, HTAs achieved 100% control of bleeding to liver and spleen, while commercially available hemostatic adjuncts demonstrated variable performance (Figure 3c). Acute evaluation of biocompatibility found that the HTA did not impact CBC blood panels (Supplementary Figure 3). The HTA exhibited excellent adhesion and hemostatic roles on actively bleeding kidney (Supplementary Figure 4) and adhesion to intraperitoneal surfaces, including the bladder (Supplementary Figure 5). Following euthanasia, the HTA was found to remain in place for at least two weeks on solid organs (Supplementary Figure 6a). Unlike commercial technologies which are easily displaced within 10 minutes following application, with none remaining in place after closure, the HTA revealed robust adhesion to liver, spleen, and kidney tissues (Supplementary Figure 6a) while the opposite slide was frictionless (Supplementary Figure 6b). Both the permanent and degradable formulations of the HTA demonstrated decreased bleeding scores during the index operation to liver and spleen tissues, unlike several commercial products (Supplementary Figure 7,8). Hemostatic control and adhesion of the HTA was independent of coagulation-competence, as manipulated via intravenous delivery of heparin (Supplementary Figure 9). Based on clinical feedback, certain faster setup adhesives are desirable while still maintaining working time. For these reasons, we tested a recently published analog22,23that can adhere within 60 seconds. A dried HTA was found to enable hemostasis within 1-minute of compression, thus enabling rapid hemostasis compared to standard of care (Supplementary Figure 10).

HTAs provide durable hemostasis and facilitate safe wound healing during the critical postoperative phase
Five animals were included in the survival portion of the study, all of them survived for the full two-week defined postoperative period. Animals treated with both non-degradable and degradable HTA were included in this study. For these survival implant studies, injury-only control groups were not included due to concerns from our IACUC regarding the risks of prolonged bleeding and associated animal welfare implications. During laparotomy at the end of the study, the injury sites were identified in all organs, with permanent HTAs present in 100% of application sites. The degradable HTAs were no longer present, as expected since they were designed to degrade within 42 days. None of the animals experienced bleeding-related complications following surgery. After euthanasia, the organs were recovered for laboratory analysis.

Histological evaluation revealed no polymorphonuclear cells in the spleen or liver, suggesting an absence of acute inflammation or infection at the time of tissue sampling (Figure 4, Supplementary Figure 11). However, mild lymphocyte presence was noted in both organs, indicating a low level of chronic or subacute inflammation, with an average lymphocyte score of 2/4.

Methods
Ethical Statement
The in vivo portion of the experiment was approved by the Institutional Animal Care & Use Committee at CBSET, Inc. (Protocol Number: 100309) and the Animal Research Committee at Boston Children’s Hospital/Harvard Medical School (Protocol #00001490).

Hydrogel Synthesis
The HTA was synthesized by mixing one syringe containing a 10 mL solution of 2% sodium alginate [MVG or LF20/40], (Pronova, Novamatrix Norway)] and 12% acrylamide (Sigma, A8887) in HBSS (Gibco), 36 µl of 2% N,N’-methylenebis(acrylamide) (Sigma, M7279), and 8 µl of TEMED (Sigma, T7024), with a second syringe containing 226 µl of 6.6% ammonium persulfate (Sigma, A9164), and 191 µl of 0.75M calcium sulfate dihydrate (Sigma, 31221). The gel was cast into molds (80x15x1.5 mm3) sealed on both sides with glass and left to crosslink for 24h. After 24h, tough gel strips were removed from molds and stored in sealed plastic bags at 4ºC.
For degradable tough gels, 30 µL of the degradable PEGDA (Mn: 250Da, Sigma, 475629) crosslinker stock solution (100 mg of PEGDA in 1 mL of HBSS buffer) was mixed with 10 mL of the alginate monomer mixture prior to syringe mixing with calcium sulfate dihydrate, TEMED, and ammonium persulfate. For in vivo experiments, gel strips were prepared as 15 × 40 mm patches; these may be further trimmed to the desired dimensions as required.

Hydrogel Mechanical Properties Testing
Pure shear tests were carried out to measure the matrix toughness. The free ends of HTA and the substrate were attached to acrylic pieces with cyanoacrylate (Krazy Glue), to which the machine grips were attached. A mechanical testing system (Instron 3342, 50 N load cell) was used to apply unidirectional tension, while recording the force and the extension. The loading rate was kept constant at 100 mm/min. From the stress-stretch curves, the matrix maximum stretch, maximum stress, and fracture toughness were calculated using custom MATLAB code.

Tough Adhesive Application to Tissue
Ultrapure Chitosan HCl (54046) was obtained from Heppe Medical Chitosan (HMC), Halle, Germany. Chitosan (2%wt/vol) with or without coupling reagents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma E6383) and sulfated N-hydroxy-succinimide (sNHS, Thermofisher, PG82071) (12 mg/mL) were quickly mixed in water by vortexing. A final concentration of 12 mg/ml EDC/sNHS was used in adhesion experiments with coupling reagents. This mixture was applied to the surface of the tough gel (50 μL/cm2) (0.5 mL total/sample) and compressed to tissue surfaces.

Ex Vivo Tissue Testing
Fresh porcine tissue was prepared for benchtop testing at room temperature by removing adjacent connective tissue using standard surgical instruments. A 1x HBSS solution was used to maintain hydration of the tissue. Samples of skin, liver, spleen, and aorta were utilized. Adhesion to tissues with HTA was performed utilizing the previously described technique. Products used for comparison, including Evarrest™, Tachosil™, Surgiflo™, Floseal™, and Surgicel SNoW®, were applied to tissues based on the manufacturer’s guidelines. Aortic burst pressure testing was performed by applying the investigative materials over 1.5 cm longitudinal incisions on samples of aorta, which were then attached to a peristaltic pump (MasterFlex #1230016) perfused with starched and dyed water. A pressure gauge attached to the circuit measured the maximum pressure before failure of the investigative material.

In Vivo Hemostasis Study Design
Our study was an in vivo porcine survival study comparing hemostasis between the investigative agent (HTA) and existing commercial agents advertised and clinically used for controlling bleeding during operative surgery, using a validated protocol for defining successful hemostasis.

In Vivo Surgical Procedure and Hemorrhage Model Overview
A total of 7 live female Yorkshire pigs were used for the solid organ hemostasis trial; 4 were non-survival studies and 3 were survival studies. Only female animals were used, and sex was not evaluated as a biological variable due to the limited sample size and primary focus on acute and chronic hemostatic performance. Personnel present at each index operation included, at minimum, a veterinary specialist, surgeon, research scientist, and data collector. Pigs were anesthetized and intubated, prepped and draped in a sterile fashion. Systemic anticoagulation in the form of intravenous heparin at a dose of 50-200 IU/kg was given to the first non-survival animal with target activated clotting time (ACT) of 200 to 350 seconds. A midline ventral laparotomy was made to expose the abdominal organs, and the abdominal wall was retracted. A 6 mm circular biopsy punch was brought into the field for use in each injury. Following the defined surgical study for each animal the laparotomy was closed or the pig was euthanized.

Anesthesia
The swine were premedicated with telazol 4.4 -6.6 mg/kg, xylazine 1.2-2.2 mg/kg IM, and atropine 0.04 mg/kg IM, after which anesthesia was maintained with isoflurane (2% to 3%) and oxygen. Bupivacaine was available for local analgesia at a dose of 3mg/kg. For perioperative analgesia, buprenorphine 0.01 mg/kg via intravenous or intramuscular administration was utilized. Postoperatively, banamine 1-2 mg/kg via intravenous or intramuscular administration as needed was given, and a transdermal fentanyl patch with dosage of 1-4 µg/kg for 72 hours was available as needed.

Definitions
Successful hemostasis was defined as no visual active blood loss out of the injury site observed during the 2-minute post-application time interval. Failure to achieve hemostasis was defined as ongoing bleeding despite additional pressure application, and/or material application, etc. to control bleeding from interfering with other subsequent injury treatments. Breakthrough bleeding was defined by blood actively running out of the treatment site.

Liver Injuries
Any lobe of the liver could be used for injuries. A biopsy punch was utilized to create a parenchymal injury, after which the devitalized tissue plug was removed using Metzenbaum scissors. The tissue was then allowed to bleed freely for 30 seconds. In non-survival pigs, blood collection was attempted using gauze during this time, which were weight pre- and post- bleeding to determine blood volume lost. A visual assessment of the rate of bleeding was made by the data collector, and an attempt at manual control of bleeding with pressure and gauze was made by the surgeon and monitored by the data collector. The data collector then generated a score on the VIBe bleeding severity scale, which was later cross-tabulated with the SPOT bleeding score15. After confirmation and scoring of bleeding, the research scientist randomly assigned a hemostatic agent to be applied to the injury by the surgeon. The agent was held with manual pressure for 3 minutes, after which the site was observed for hemostasis. The time to hemostasis and volume of applied agent were recorded. Following the declaration of hemostasis, the site was observed for 2 additional minutes to evaluate breakthrough bleeding. If initial hemostasis was not achieved within the first 60 seconds, additional agent could be applied. If hemostasis did not occur after 3 minutes of observation, the treatment was considered a failure. If breakthrough bleeding (re-bleeding) was observed during the 2-minute observation period following the declaration of hemostasis, this was also noted as a failure.

Spleen Injuries
A series of bleeding sites was prepared for testing by creating multiple 6 mm parenchymal injuries with a biopsy punch, similar to the injuries made on the liver. The tissue plug within the defect was then amputated from the deep tissue attachment with scissors and removed with forceps. The severity of bleeding from biopsy style injuries was graded on the VIBe bleeding severity scale, which was later cross tabulated with the SPOT bleeding score15. After confirmation of consistent bleeding from each injury site, the injury was blotted with a surgical sponge or gauze to remove pooled blood. The adhesive was applied and the time to hemostasis and volume of adhesive applied were recorded. Following the declaration of hemostasis, an additional 2-minute observation period could be utilized to ensure no breakthrough bleeding. If initial hemostasis was not achieved within 60 seconds, additional adhesive could be applied. If hemostasis did not occur after 3 minutes of observation, the treatment was considered a failure. If breakthrough bleeding (re-bleed) was observed during the 2-minute observation period following the declaration of hemostasis, this was also noted as a failure.

Kidney Injuries
A series of bleeding sites were prepared for testing by creating incision injuries using a 6 mm biopsy punch. After confirmation of consistent bleeding from each injury site, the injury was blotted with a surgical sponge or gauze to remove pooled blood. The adhesive was then applied, and a stopwatch (or other appropriate timing device) was immediately started. The time to hemostasis and volume of adhesive was recorded. Following the declaration of hemostasis, an additional 2-minute observation period could be utilized to ensure that no breakthrough bleeding occurred. If initial hemostasis was not achieved within 60 seconds, additional adhesive could be applied. If hemostasis had not occurred after 3 minutes, this was noted as a failure, but the total observation time was still documented. Hemostasis was defined as no visual active blood loss observed during the 2-minute post-application observation time interval. Failure to achieve hemostasis was treated if needed with manual pressure application, and/or material application, etc. to control bleeding from interfering with other subsequent injury treatments.

Aortic Injuries
The abdominal aorta was exposed by retracting other abdominal organs. The exposed aortic sections could be superior or inferior to the renal arteries. The diameter of the untouched aorta was measured just prior to the defect creation. A vascular clamp was used to isolate a section of the aorta but not completely obstructing flow. After the clamp was placed, the aorta was incised 2cm in the longitudinal axis to create the defect. Once the defect was created, a repair was performed using conventional surgical techniques and sutures, and the adhesive was applied and allowed to adhere for 5 minutes prior to release of the clamp. The clamp was then removed and the aorta was observed for 15 minutes to ensure no bleeding. If severe active bleeding as noticed after the clamp release or during the 15-minute observation, additional adhesive, manual pressure, vascular clamping, or other surgical attempts could be applied to control the bleeding and would be documented.
At the beginning of the 15-minute observation, the repair sites and the distal and proximal regions were evaluated, and the success to achieve hemostasis was determined as absence of bleeding or only presence oozing, at 0 minute. The result was documented. At 1, 3, 5, 10, and 15-minute time points, the sites were re-evaluated and the results were documented, if no active bleeding or any additional intervention occurred.

Survival Study
The pigs that had been selected for the survival portion of the study were evaluated immediately for recovery from the operation and thereafter for the pre-determined period of two weeks. Behavior assessments of the animals as well as wound assessments were conducted daily to monitor for possible postoperative complications.

Post-mortem analysis
Data was collected during the study period and analyzed.

Histology
Representative uninjured and injured sections were stained with hematoxylin and eosin following standard procedures. To assess biocompatibility, the authors involved in the animal surgery (D.O.K., P.S.K., F.O.O., and B.R.F.) selected tissue regions of interest (ROIs) containing the surgical site and bioadhesive implantation for each animal. A board-certified histopathologist, blinded to the treatment group, assessed at least 5 high powered fields in each of the designated ROIs for biocompatibility with a semiquantitative evaluation of inflammatory cells, necrosis, and tissue response (i.e. neovascularization, fibrosis, and fatty infiltrate).

Statistics & Reproducibility
Based on past study,40 a power analysis was conducted to determine the minimum sample for all assays. Sample sizes for individual experiments are reported in the corresponding figure legends. Animals and injury sites were randomized to treatment groups at the time of experimentation using a random assignment procedure. Blinding was implemented where feasible; specifically, investigators responsible for outcome assessment were blinded to treatment allocation during data collection. All attempts at replication were successful, and representative results are shown where applicable. Statistical analyses were performed as described in the figure legends with exact P values reported where appropriate.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript entitled “Hemostatic Tough Adhesives Rapidly Seal Tissue and Control Hemorrhage”, the authors present a biocompatible hydrogel system (HTA) composed of alginate-polyacrylamide and a chitosan-based adhesive, designed to rapidly achieve hemostasis on solid organs even under active bleeding conditions. The authors perform a series of ex vivo and in vivo evaluations, demonstrating superior burst resistance, interfacial toughness, and sealing capacity compared to commercial hemostatic agents in porcine models. The HTA’s performance under coagulopathic conditions and its biocompatibility are particularly noteworthy. While this work holds clear translational potential, several key issues should be addressed before the manuscript is suitable for publication.\n1. In the Materials and Methods section, the authors describe mechanical testing (e.g., pure shear tests using Instron), but no corresponding data are presented in the Results section or figures. The absence of these data weakens the claim that HTA offers superior mechanical robustness under physiological or supraphysiologic conditions. More importantly, the authors do not match HTA mechanical parameters (e.g., modulus, fracture energy, elasticity) with those of the target tissues (e.g., liver, spleen).\n2. The manuscript claims that HTA provides effective sealing under active bleeding and in coagulopathic states (e.g., after heparinization), but offers minimal mechanistic explanation. The role of chitosan is briefly mentioned, but given its generally hydrophilic and mucoadhesive nature, it remains unclear how waterproofing or hemorepellence is achieved.\n3. None of the physical test images or histological figures (e.g., Figures 2–4, S9) include scale bars, making it difficult to assess dimensions or tissue-material interactions.\n4. The authors state that the HTA adheres in <3 minutes, but whether this qualifies as “rapid” compared to standard clinical practices is unclear. Moreover, there is no discussion of whether continuous active bleeding affects the adhesion process. The manuscript states that HTA can be “easily trimmed to fit wound geometry,” but this feature is neither visually demonstrated nor quantified.\n5. Several recent bioadhesive strategies (e.g., barnacle-inspired pastes, nanoparticle-crosslinked gels, topological adhesives) have reported similar or related functionalities, including sealing wet surfaces and coagulation-independent hemostasis.", "reviewer_2": "The authors report a high tough adhesive for wound closure and bleeding control. It is biocompatible, and its in vivo adhesion strength is impressively tough and durable. Its overall performance outperforms many commercialized hemostatic adjuncts. However, I have several concerns:\n1. A very similar tough wet tissue adhesive was reported in ref. #20. It showed strong adhesion to blood-wetted porcine skin,and was applied in the sealing of a bleeding trauma like a bleeding liver. Compared to ref #20, what's the novelty of this work?\n2. The adhesion energy is closely related to the cohesion and adhesion. However, the authors neither showed the mechanical properties of the adhesive patch, nor analyze the interfacial interaction. Pls supplement those results and analysis.\n3. L155, PAAm is a water swellable hydrogel. The water swelling and volumetric change of this HTA should be evaluated in SBF.\n4. For a swellable adhesive patch, its adhesion energy should decrease with swelling, and result in adhesion failure. Thus, the ex vivo adhesion strength of HTA to organ tissues should be quantitatively measured with soaking time in SBF.\n5. L155-156, 24h is not enough for the wound healing, how to achieve durable adhesion and anti-adhesion if it is in vivo used for a longer duration.\n6. It is unclear how the nonadherence is achieved for the patch when in vivo used for over 24 h.\n7. Why can HTA have strong and durable in vivo adhesion to organs for over 2-weeks?\n8. ref #29 and 30 are not related to the adhesive hydrogel of alginate and PAAm. Thus, the readers do not understand the unprecedented adhesion to tissue surfaces claimed by the authors. Pls make sure the right references are cited."}, "author_response": "Comment Response\nIn this manuscript entitled “Hemostatic We thank Reviewer 1 for their overall positive\nTough Adhesives Rapidly Seal Tissue and review of our study. In this revision we have\nControl Hemorrhage”, the authors present a taken steps to address concerns raised.\nbiocompatible hydrogel system (HTA)\ncomposed of alginate-polyacrylamide and a\nchitosan-based adhesive, designed to rapidly\nachieve hemostasis on solid organs even\nunder active bleeding conditions. The authors\nperform a series of ex vivo and in vivo\nevaluations, demonstrating superior burst\nresistance, interfacial toughness, and sealing\ncapacity compared to commercial hemostatic\nagents in porcine models. The HTA’s\nperformance under coagulopathic conditions\nand its biocompatibility are particularly\nnoteworthy. While this work holds clear\ntranslational potential, several key issues\nshould be addressed before the manuscript is\nsuitable for publication.\n1. In the Materials and Methods section, the We have now included tensile testing results\nauthors describe mechanical testing (e.g., in supplemental data. This data complements\npure shear tests using Instron), but no additional data we have published on the\ncorresponding data are presented in the correlations between mechanical properties of\nResults section or figures. The absence of the platform hydrogel system and soft tissue\nthese data weakens the claim that HTA offers adhesion (see Cintron Cruz+ 2022 Advanced\nsuperior mechanical robustness under Materials1).\nphysiological or supraphysiologic conditions.\nMore importantly, the authors do not match\nHTA mechanical parameters (e.g., modulus,\nfracture energy, elasticity) with those of the\ntarget tissues (e.g., liver, spleen).\n“Figure S1: Tensile mechanical testing of the\nHTA and standard of care products\ncomparing (a) toughness, (b) maximum stress,\nand (c) maximum stretch. Data shown as\nmean +/- s.d., as evaluated with a one-way\nANOVA with post hoc Dunnet’s test for\nmultiple comparisons.”\nWe thank the reviewer for pointing out the\nimportance of matching mechanical\nparameters of the adhesive to the target\ntissues. We would like to clarify two key\npoints:\nScope of adhesive design\nOur adhesive system (HTA) was not specified\nwith the intent of exactly matching the full\nmechanical spectrum (modulus, fracture\nenergy, elasticity) of each target organ/tissue\n(e.g., liver, spleen). Rather, our design goal\nwas to provide adequate adhesion,\nbiocompatibility, and functionality under\nphysiological loading conditions. The\nmechanical properties of the adhesive were\nchosen for overall performance and handling,\nnot to precisely mimic each tissue’s modulus\nor fracture toughness. Accordingly, the\nmanuscript did not treat the adhesive as a\ntissue‐mimic or phantom material, so the\nabsence of detailed modulus‐matching to each\norgan should not be interpreted as an oversight\nof a design requirement.\nMechanical‐property context: target tissue\nvs HTA\nWe agree that it is useful to compare the\nadhesive’s viscoelastic (storage/loss modulus)\nbehavior against typical soft tissue ranges to\nconfirm that the adhesive is in a feasible\nregime.\no As reviewed by Effects of extracellular\nmatrix viscoelasticity on cellular\nbehavior (Chaudhuri et al., 2020),2 a\nbroad survey of tissue (and ECM)\nmechanical data shows storage and loss\nmoduli of soft tissues (at ~1 Hz)\nspanning a range from ~10 Pa to ~10^5\nPa (i.e., tens of Pa to tens of kPa)\ndepending on tissue type, animal,\nmethod, frequency and measurement\nmode.\no For example, bovine liver in rheometer\nshear tests exhibited storage moduli of\n~300–900 Pa at 0.1 Hz, ~400–600 Pa at\ncorresponding loss moduli on the order\nof ~50–250 Pa (depending on\npreservation condition).\no Additionally, spleen exhibits\nsimilarly soft mechanics (in the\nhundreds of Pa to low kPa\nrange) under physiological\nconditions.\no In our own data3 the adhesive\nexhibits a storage modulus of\n~10-20kPa and a loss modulus\nof ~2-8kPa at ~1 Hz (or\nrelevant frequency). These\nvalues place the adhesive\nmodulus within or above the\ntypical soft‐tissue viscoelastic\nrange (hundreds of Pa to low\nkPa). Thus we believe the\nadhesive does not present a\ndrastic mismatch (e.g., many\norders of magnitude stiffer)\nrelative to the target tissues.\no The viscoelastic modulus data\nprovide a useful benchmark\nshowing that the adhesive\nresides in the same ballpark as\nsoft tissues. Given the\nadhesive’s compliance and\nviscoelastic behavior, the risk\nof extreme mechanical\nmismatch (which might lead to\nstress‐concentrations or\ndelamination) is reduced.\nWe have included new lines in the revised\nmanuscript to: (i) clarify that modulus‐\nmatching was not a primary design spec for\nthe adhesive, and (ii) summarize the typical\nviscoelastic property ranges of liver and\nspleen (and similar soft organs) alongside our\nadhesive’s measured storage/loss moduli, to\nshow consistency of scale.\nLines 281-284: “The HTA was not designed to\nprecisely match the mechanical parameters of\nspecific target tissues, but rather to achieve\ncompliant, viscoelastic behavior suitable for\nsoft tissue adhesion. The measured storage\nmodulus (10–20 kPa) and tan δ (0.2–0.4)\ncorrespond to a loss modulus of ~2–8 kPa,\nwhich lies within the range reported for soft\norgans such as liver and spleen.2”\n2. The manuscript claims that HTA provides We have provided further explanation for the\neffective sealing under active bleeding and in mechanism of sealing under active bleeding in\ncoagulopathic states (e.g., after coagulopathic states. Unlike existing\nheparinization), but offers minimal hemostatic devices that generate adhesion\nmechanistic explanation. The role of chitosan through chemical coagulation by impregnating\nis briefly mentioned, but given its generally fibrin or thrombin, the HTA does not have\nhydrophilic and mucoadhesive nature, it similar requirements. Instead, the HTA can\nremains unclear how waterproofing or adhere before a clot is formed through tissue\nhemorepellence is achieved. adhesive mechanisms as previously\ndescribed.1 In short, the chitosan layer diffuses\ninto the gel and tissue surface and upon\nengagement with more basic substrates forms\nentanglements with itself as the chitosan\ndeprotonates, which does not depend on\ncoagulopathic states.1\nThe following lines have been added to reflect\nthis change:\nLines 225-231: “Unlike traditional hemostatic\ndevices that depend on fibrin or thrombin to\nform a clot for adhesion, the HTA adhesion\nfunctions through bridging polymer\ndeprotonation upon interaction with tissue\nsubstrates.1 This process promotes molecular\nentanglement with the tissue, creating strong\nadhesion even in the absence of a clot.\nConversely, conventional devices require a\nclot to form before they can adhere to tissue,\nwhereas the HTA adheres before clot\nformation occurs. As a result, the HTA offers\ngreater versatility and effectiveness, especially\nin conditions where clot formation is delayed\nor impaired.”\n3. None of the physical test images or We have now included scale bars in Figures 2-\nhistological figures (e.g., Figures 2–4, S9) 4, and S9.\ninclude scale bars, making it difficult to\nassess dimensions or tissue-material\ninteractions.\n4. The authors state that the HTA adheres in We thank the reviewer for raising this\n<3 minutes, but whether this qualifies as “rapid” important point. While most experiments\n“rapid” compared to standard clinical were tested using the 3-minute tamponade,\npractices is unclear. Moreover, there is no additional 1-minute experiments were\ndiscussion of whether continuous active performed. We have now included this data.\nbleeding affects the adhesion process. The For comparison, standard of care products are\nmanuscript states that HTA can be “easily indicated for 3 minutes of compression, as we\ntrimmed to fit wound geometry,” but this now indicate in the text.\nfeature is neither visually demonstrated nor\nquantified. Lines 167-168: “A dried HTA was found to\nenable hemostasis within 1-minute of\ncompression, thus enabling rapid hemostasis\ncompared to standard of care.”\nHTA is a soft hydrogel sheet that can be cut\ncleanly with standard surgical tools (scalpel or\nscissors). In this study we typically pre-\nprepared pieces to the defect dimensions;\nhowever, we also fabricate larger sheets that\ncan be rapidly trimmed at the time of use to\nmatch irregular wound shapes.\nLines 339-340: “For in vivo experiments, gel\nstrips were prepared as 15 × 40 mm patches;\nthese may be further trimmed to the desired\ndimensions as required.”\n5. Several recent bioadhesive strategies (e.g., We have included additional discussion on\nbarnacle-inspired pastes, nanoparticle- these other emerging strategies.\ncrosslinked gels, topological adhesives) have\nreported similar or related functionalities, Lines 214-225: “The HTA differs significantly\nincluding sealing wet surfaces and from other emerging technologies4-6 in several\ncoagulation-independent hemostasis key aspects. The fracture toughness and\nadhesion energy of the HTA is at least 10-\n1000x higher, making it more resistant to\nmechanical failure under stress, which is\ncrucial for medical applications requiring\nrobust adhesion. Unlike alternatives, the HTA\nis also transparent, making it more suitable\nfor clinical settings where visibility of\nunderlying tissues is important. In addition,\nthe HTA offers significant advantages in terms\nof storage and handling, as it remains stable\nat room temperature and does not require\nrefrigeration or complex preparation.\nMoreover, the HTA can be used effectively as\na patch to cover large defects, providing\nversatility in a range of clinical applications.\nFinally, the HTA allows for a longer working\ntime before adhesion begins, offering greater\nflexibility during application, giving medical\npractitioners more time to position and adjust\nthe adhesive. These distinctions make HTA a\nmore adaptable and reliable option for a wide\nrange of medical procedures.”\nReviewer 2\nComment Response\nThe authors report a high tough adhesive for We thank reviewer 2 for an overall positive\nwound closure and bleeding control. It is review of our study. Below are our responses\nbiocompatible, and its in vivo adhesion to the concerns raised and the steps taken to\nstrength is impressively tough and durable. address them.\nIts overall performance outperforms many\ncommercialized hemostatic adjuncts. The material system in ref 20 was the first\nHowever, I have several concerns: version of the hydrogel used as the initial\n1. A very similar tough wet tissue adhesive basis for the HTA. Here, however, we present\nwas reported in ref. #20. It showed strong a tougher version (see Cintron+ 2022\nadhesion to blood-wetted porcine skin,and Advanced Materials)1 and a degradable\nwas applied in the sealing of a bleeding system (Freedman+ 2021 Advanced\ntrauma like a bleeding liver. Compared to ref Materials)7, and evaluate its performance in\n#20, what's the novelty of this work? large animals. Previous studies only\nexamined small animals in the acute setting.\n2. The adhesion energy is closely related to We have now included mechanical property\nthe cohesion and adhesion. However, the data in supplemental.\nauthors neither showed the mechanical\nproperties of the adhesive patch, nor analyze “Figure S1: Tensile mechanical testing of the\nthe interfacial interaction. Pls supplement HTA and standard of care products\nthose results and analysis. comparing (a) toughness, (b) maximum stress,\nand (c) maximum stretch. Data shown as\nmean +/- s.d., as evaluated with a one-way\nANOVA with post hoc Dunnett’s test for\nmultiple comparisons.”\n3. L155, PAAm is a water swellable We have previously evaluated the swelling\nhydrogel. The water swelling and volumetric behavior of the hydrogel (see Liu+ 2025).8 We\nchange of this HTA should be evaluated in have included a reference to this in the paper.\nSBF.\nLines 128-130: “Hydrogel swelling post\nimplantation has been examined in several\nstudies suggesting minimal additional\nswelling after post synthesis wash steps.7-9”\n4. For a swellable adhesive patch, its We thank Reviewer 2 for raising this concern.\nadhesion energy should decrease with This study, similar to our previous studies (see\nswelling, and result in adhesion failure. Thus, Freedman + 2022 Nat BME; Lazow + 2021 J\nthe ex vivo adhesion strength of HTA to Ped Surg; Blacklow 2019 Science Advances;\norgan tissues should be quantitatively Wu STM)9-11 have all demonstrated multi-\nmeasured with soaking time in SBF. week adhesion in vivo. Furthermore, we have\nstudied adhesion longitudinally in previous\npapers.9,12,13 Although swelling may reduce\nadhesion, adhesion to tissue is still\nmaintained. We have included this\ninformation as follows:\nLines 287-291: “As demonstrated in our prior\nstudies9,12,13, the materials maintain multi-\nweek adhesion in vivo despite swelling. While\nswelling can modestly reduce adhesion\nenergy, the adhesion remains sufficient for\nrobust tissue integration over clinically\nrelevant timescales, as shown in our\nlongitudinal evaluations.”\n5. L155-156, 24h is not enough for the We thank Reviewer 2 for raising this\nwound healing, how to achieve durable important point. We have adjusted the text to\nadhesion and anti-adhesion if it is in vivo reflect that the materials remained in place for\nused for a longer duration. at least two weeks.\nAs most metrics for hemostasis are\nintraoperative within 10 minutes following\nbleeding, strong adhesion in the following\ndays has not been demonstrated in other\nhemostatic adjuncts. In our studies,\ncommercial products were typically displaced\nwithin 10-20 minutes. Thus, we also\ninvestigated performance out to 2-weeks. This\nwas consistent with previous studies in rats\nand rabbits.10\nLines 159-163: “Following euthanasia, the\nHTA was found to remain in place for at least\ntwo weeks on solid organs (Figure 4a).\nUnlike commercial technologies which are\neasily displaced within 10 minutes following\napplication, with none remaining in place\nafter closure, the HTA revealed robust\nadhesion to liver, spleen, and kidney tissues\n(Figure S5a) while the nonadherent slide was\nfrictionless (Figure S5b).’\n6. It is unclear how the nonadherence is We have removed the term ‘nonadherence’ to\nachieved for the patch when in vivo used for replace with ‘opposite’ for clarity. This was\nover 24 h. qualitatively examined through the presence\nof low friction on the surface.\n7. Why can HTA have strong and durable in We thank Reviewer 2 for raising this\nvivo adhesion to organs for over 2-weeks? important feature. This study, similar to our\nprevious studies (see Freedman + 2022 Nat\nBME; Lazow + 2021 J Ped Surg; Blacklow\ndemonstrated multi-week adhesion in vivo.\nFurthermore, we have studied adhesion\nlongitudinally in previous papers.9,12,13\nTo explain this further, we have added the\nfollowing lines to the manuscript:\nLines 232-249: \"This work adds to our\nprevious studies,9,10,12,14 as the HTA maintains\nhigh interfacial toughness (greater than 1000\nJ/m²) in vivo, allowing it to resist detachment\ndespite tissue motion, swelling, and\ndegradation. Unlike conventional hemostatic\nagents that rely on blood coagulation to\ngenerate adhesion, the HTA forms a stable\nand direct interface with tissues. The chitosan\nbridging polymer penetrates both the hydrogel\nmatrix and tissue, forming a topologically\nentangled interface that resists mechanical\ndisruption. In addition to this physical\ninterlocking, covalent amide bonds form via\ncarbodiimide chemistry between tissue\ncarboxyl groups and chitosan amine groups,\nfurther reinforce the interface. Interchain\nhydrogen bonding between deprotonated\nchitosan and ECM components also\ncontributes to bond strength. The hydrogel’s\ndouble-network structure, composed of\nreversible ionic crosslinks with alginate and a\ncovalently crosslinked polyacrylamide matrix,\ndissipates mechanical stress and prevents\ncrack propagation or delamination.\nElectrostatic interactions between the\npositively charged chitosan and negatively\ncharged tissue proteins further reinforce the\ninterface and can reform after disruption,\nmaintaining adhesion even during localized\ndegradation or swelling. These adhesive\nmechanisms are sustained by a minimal\ninflammatory response, which helps maintain\nthe contact between the hydrogel and tissue\nsurface. Together, these interfacial bonding\nmechanisms, combined with the hydrogel’s\ninherent mechanical toughness and\nbiocompatibility, contribute to the long-term\nstability of HTA adhesion in vivo.\"\n8. ref #29 and 30 are not related to the Thank you for your close attention to our\nadhesive hydrogel of alginate and PAAm. references. References 29 and 30 have been\nThus, the readers do not understand the removed from lines 101 and 102 to their\nunprecedented adhesion to tissue surfaces correct location to ensure appropriate\nclaimed by the authors. Pls make sure the citations."}, {"round": 2, "reviewer_comments": {"reviewer_2": "Good responses to the reviewers' questions and concerns.", "reviewer_3": "The reviewer finds that the original concerns and comments from Reviewer 1 were not fully addressed in the revised manuscript. While the authors provided additional data and descriptions to address the original concerns and comments on mechanical performance, other more fundamental comments were not fully addressed as summarized below:\n1. Mechanism of hemostasis\nThe provided additional description does not fully address the original concerns about how active and continuous bleeding can be managed during hemostasis achieving period of the present technology. The provided description by the authors is identical to the previous work on chitosan solution-based tough hydrogel adhesive on tissue adhesion not specifically for hemostasis in the presence of active bleeding.\n2. Rapid hemostasis\nAlthough the authors provided additional data on 1-min experiment in the revised manuscript, 1 minute is still technically unjustifiable or at least not usually considered as 'rapid' in the field of hemostasis given there are numerous technologies in the literature that achieves much less than 1 minutes (for example, 10 seconds or less). Based on the authors' response and provided data in the revised manuscript, the reviewer finds the description of 'rapid' is still unjustified and misleading.\n3. Novelty\nThe reviewer finds that this is the most fundamental and critical concern raised by the Reviewer 1 which is not addressed at all in the revised manuscript and the authors' response. After careful literature review, the reviewer finds that the current work's materials, method, and proposed mechanism of action are essentially identical to the previous work (Li et al,. Science 357, 378-381 (2019)) and several follow-up works from the same corresponding author. The authors' response does not address this concerns on novelty directly and somehow misleading by not clearly acknowledge the near-identical nature of the technology from the previously reported work to the current work. On top of the Reviewer 1's original concern and criticism, the reviewer further weighs in significant criticism on the lack of novelty and clear acknowledgement of the rather obvious similarities to the previous works from the same corresponding author.\nOn top of the Reviewer 1's original concerns and comments, the reviewer has additional major concerns on the work as summarized below:\n1. Comparison to Evarrest\nEverest is FDA-cleared topical adjunct hemostatic agent from JnJ. Based on FDA database of its safety and effectiveness summary as well as published pre-clinical data from JnJ (for example, Matonick et al., Journal of Investigative Surgery 27, 360-365 (2014)), Evarrest is effective in terms of achieving successful hemostasis within 3 minutes on splenic bleeding in porcine model. Hence, the reviewer is very confused and concerned about the authors' claim regarding ineffectiveness of Evarrest on splenic bleeding model as the standard and level of safety & effectiveness required for FDA clearance are largely considered higher and gold standard. These unclearly supported superiority claims in the current manuscript significantly adds strong skepticism from the reviewer whether the authors adopted proper bleeding model and study design to evaluate the safety and effectiveness of the control group as well as the test article."}, "author_response": "Comment Response\nReviewer #2 (Remarks to the Author) We thank Reviewer 2 for their positive review of\nGood responses to the reviewers' questions our revision.\nand concerns.\nReviewer #3 (Remarks to the Author) We thank Reviewer 3 for their careful review of\nThe reviewer finds that the original our study. We have tried to address additional\nconcerns and comments from Reviewer 1 foundational questions below and have tailored\nwere not fully addressed in the revised the paper to reflect these changes.\nmanuscript. While the authors provided\nadditional data and descriptions to address\nthe original concerns and comments on\nmechanical performance, other more\nfundamental comments were not fully\naddressed as summarized below:\n1. Mechanism of hemostasis We thank the reviewer for raising this point. To\nThe provided additional description does more clearly separate the mechanisms, we have\nnot fully address the original concerns provided an additional description specific to the\nabout how active and continuous bleeding role of HTA in managing active bleeding. The\ncan be managed during hemostasis primary mechanism of hemostasis is mechanical\nachieving period of the present technology. containment where HTA acts as a physical\nThe provided description by the authors is barrier that seals the bleeding site, creating a\nidentical to the previous work on chitosan confined environment. This effect is independent\nsolution-based tough hydrogel adhesive on of platelet aggregation, activation of clotting\ntissue adhesion not specifically for factors, or external hemostatic agents, allowing\nhemostasis in the presence of active HTA to function even in high-flow or\nbleeding. coagulopathic conditions.\nLines 249-252: “The mechanism of action for\nhemostasis is mechanical containment of the\nbleeding site. Containment results in\ncoagulation due to platelet aggregation which\ndoes not depend on hematologic coagulation\nfactors and remains effective even under active\nbleeding or coagulopathic conditions.”\n2. Rapid hemostasis We appreciate the reviewer’s comments\nAlthough the authors provided additional regarding the use of the term “rapid.” All FDA-\ndata on 1-min experiment in the revised cleared hemostatic products are currently\nmanuscript, 1 minute is still technically evaluated using a 3-minute compression\nunjustifiable or at least not usually standard, and achieving consistent hemostasis\nconsidered as 'rapid' in the field of within 1 minute represents a meaningful\nhemostasis given there are numerous improvement over this benchmark. Based on\ntechnologies in the literature that achieves clinical feedback, this reduction in time has been\nmuch less than 1 minutes (for example, 10 described as “rapid,” particularly since many\nseconds or less). Based on the authors' commercially available hemostats struggle to\nresponse and provided data in the revised achieve hemostasis even within 10 minutes. We\nmanuscript, the reviewer finds the would also like to clarify that the term “rapid” is\ndescription of 'rapid' is still unjustified and not intended to imply increased toughness,\nmisleading. adhesiveness, or long-term durability. At the\nsame time, we acknowledge recent academic\nreports demonstrating hemostasis on the order of\n10-20 seconds1-4, which is faster than the 60-\nsecond endpoint evaluated in the present study,\nand we recognize this as an important area of\nongoing advancement in the field.\n• <3 seconds: Wang+ 2023 Biomat Sci\nstudied PEGDA-di(cyanoacrylate)\npowder for hemostasis and adhesion\n• 12 seconds: Lee+ 2022 studied a PEG\nsuccinimidyl glutarate-based active\ncrosslinking hemostatic sealant for\nadhesion\n• 15 seconds: Liu+ 2024 studied a N-2(-\nhydroxy-3-tri-methylammonium propyl)\nchitosan chloride/phytic acid powder\n• 15 seconds: Yuk+ 2021 studied a\nmicroparticle poly(acrylic acid) grafted\nwith N-hydroxysuccinimide ester and\nchitosan for adhesion within 15s.\n• 20 seconds: Tiryaki+ 2025 examined an\nalgan hemostatic agent tested as a\npowder and liquid impregnated gauze\nsponge in rats to suggest hemostasis\nWhile the term “rapid” was intended to denote\nperformance that is faster than the current\nstandard of care, we appreciate the reviewer’s\nconcern regarding terminology and the\nimportance of precise communication. We have\nrevised the title accordingly to better reflect the\nscope and positioning of the work.\nLines 167-169: “Based on clinical feedback,\ncertain faster setup adhesives are desirable\nwhile still maintaining working time. For these\nreasons, we tested a recently published analog5,6\nthat can adhere within 60 seconds.”\nLines 327-330: “The faster setup may be further\naccelerated in future studies as other works have\nrecently proposed hemostatic properties in <15-\n20 seconds and will be tested against these\nproposed systems,….”\nTitle: “Hemostatic Tough Adhesives Seal Tissue\nand Control Hemorrhage”\n3. Novelty We thank the reviewer for raising important\nThe reviewer finds that this is the most questions regarding novelty and for the\nfundamental and critical concern raised by opportunity to clarify the relationship between\nthe Reviewer 1 which is not addressed at the present work and Li et al., Science (2017),7\nall in the revised manuscript and the as well as subsequent bioadhesive studies from\nauthors' response. After careful literature our laboratory.5,6,8 In response to Reviewer 1 in\nreview, the reviewer finds that the current Revision 1, we substantially expanded the\nwork's materials, method, and proposed manuscript to include detailed mechanical\nmechanism of action are essentially characterization, evaluation in coagulopathic\nidentical to the previous work (Li et al,. states, testing under accelerated hemostatic\nScience 357, 378-381 (2019)) and several conditions relative to the 3-minute standard, and\nfollow-up works from the same an updated discussion of recent bioadhesive\ncorresponding author. The authors' strategies.\nresponse does not address this concerns on\nnovelty directly and somehow misleading Reviewer 3 now raises a distinct question\nby not clearly acknowledge the near- focused on the novelty of the hemostatic tissue\nidentical nature of the technology from the adhesive (HTA) relative to Li et al. (2017) and\npreviously reported work to the current related prior work.7 While the present study\nwork. On top of the Reviewer 1's original builds upon the foundational tough hydrogel\nconcern and criticism, the reviewer further framework, several key advances distinguish this\nweighs in significant criticism on the lack work and collectively enable superior and\nof novelty and clear acknowledgement of clinically relevant performance. These include\nthe rather obvious similarities to the (i) incorporation of an enhanced adhesive\nprevious works from the same strategy first described in Cruz, Freedman, and\ncorresponding author. Mooney (2022, Advanced Materials)6, which\nwas previously evaluated primarily in benchtop\nmodels, now with in vivo data; (ii) integration of\na degradable adhesive chemistry introduced in\nFreedman et al. (2021)8 that has not been tested\nin large-animal hemostasis models; and (iii)\nimplementation of an accelerated adhesion\nmechanism, as described in Cruz and Freedman\net al. (2022),6 enabling faster tissue attachment\nand hemostasis under clinically relevant\nconditions.\nTaken together, these advances strengthen and\nsimplify the adhesion mechanism beyond that of\nLi et al. (2017),7 and their combined application\nin a large-animal hemostasis model represents a\nsubstantive and novel contribution. We also\nrecognize and explicitly acknowledge that our\nprior studies have applied the core tough\nhydrogel technology across multiple indications;\nhowever, the present work advances this\nplatform through new chemistry, kinetics, and in\nvivo validation that extend beyond earlier\nreports.\nLines 236-242: “While the present work builds\non the foundational tough hydrogel framework7,\nit integrates multiple advances that were not\npreviously combined or validated in large-\nanimal acute and survival studies. Specifically,\nthe hemostatic tissue adhesive incorporates\nenhanced and accelerated adhesion mechanisms\nand a degradable adhesive chemistry5,6,8,\nenabling faster and more reliable hemostasis\nunder clinically relevant conditions. The\nconvergence of these features, together with in\nvivo validation beyond prior studies, establishes\nthe novelty of the current approach.”\nOn top of the Reviewer 1's original We thank the reviewer for raising this important\nconcerns and comments, the reviewer has and insightful question. We were also initially\nadditional major concerns on the work as surprised by the limited efficacy observed for\nsummarized below: several existing hemostatic products; however,\nthis finding is consistent with a substantial body\n1. Comparison to Evarrest of clinical and regulatory literature that has\nEverest is FDA-cleared topical adjunct identified persistent unmet needs in surgical\nhemostatic agent from JnJ. Based on FDA bleeding control. Multiple randomized trials,\ndatabase of its safety and effectiveness systematic reviews, and expert consensus\nsummary as well as published pre-clinical statements have reported that many FDA-\ndata from JnJ (for example, Matonick et approved topical hemostatic agents provide\nal., Journal of Investigative Surgery 27, limited or no improvement in clinically\n360-365 (2014)), Evarrest is effective in meaningful outcomes, such as transfusion rates,\nterms of achieving successful hemostasis postoperative bleeding complications, or length\nwithin 3 minutes on splenic bleeding in of stay 9-16. While some products reduce time to\nporcine model. Hence, the reviewer is very hemostasis or intraoperative blood loss, these\nconfused and concerned about the authors' effects are often modest, highly procedure-\nclaim regarding ineffectiveness of Evarrest dependent, and supported by evidence of\non splenic bleeding model as the standard variable or low certainty.\nand level of safety & effectiveness required\nfor FDA clearance are largely considered Importantly, FDA approval for hemostatic\nhigher and gold standard. These unclearly agents is typically based on demonstration of\nsupported superiority claims in the current safety and achievement of hemostasis under\nmanuscript significantly adds strong defined testing conditions, rather than\nskepticism from the reviewer whether the superiority over standard care or consistent\nauthors adopted proper bleeding model and improvement in downstream clinical outcomes.\nstudy design to evaluate the safety and As a result, regulatory approval and residual\neffectiveness of the control group as well clinical limitations are not mutually exclusive,\nas the test article. and the widespread approval of these materials\nreflects acceptable safety and baseline efficacy\nrather than resolution of all bleeding-related\nchallenges. Our findings therefore align with\nprior evidence suggesting that existing products\nare useful in certain scenarios but leave\nsignificant room for improvement.\nAs Reviewer 3 noted, Matonick et al. reported\nthat TachoSil achieved 0% hemostatic success at\n3 minutes and only 4% at 10 minutes, despite\nTachoSil also being an FDA-approved product\nfor multiple bleeding indications17. Several\nmethodological differences are important to note\nwhen comparing that study with ours. Matonick\net al. employed an incisional injury (15 mm × 3\nmm depth) with a substantially larger patch (3 ×\n4.5 cm), extending approximately 1.5 cm beyond\nthe wound margins, whereas our study used a\ndeep excisional punch injury (6 mm diameter,\nnearly 1 cm depth), with a 15x40mm HTA\npatch. In addition, excess blood and fluid were\nremoved prior to application in Matonick et al,\nwhereas the HTA was applied directly to the\nbleeding surface in the present study, enabling\neffective placement in a wet environment – a\nkey differentiator in real-world hemorrhage\nscenarios\nIt is also important to note that bleeding\nassessment methodologies have evolved over\ntime. We employed the SPOT bleeding scale 18,\nwhich was introduced within the past five years\nand has since become a standard assessment\nframework used in FDA-reviewed studies.\nDifferences in injury models, application\nconditions, and bleeding scales are likely to\ncontribute to variability across studies and\nfurther underscore the need for improved and\nmore robust hemostatic technologies.\nSeparate from baseline performance, the longer-\nterm biocompatibility of implanted materials is\nimportant to recognize. We conducted an\nadditional review of publicly available FDA\ndocumentation for EVARREST to better\ncontextualize the performance and safety of\nexisting approved products. These FDA\ndocuments note several safety considerations\nobserved in studies conducted for EVARREST,\nincluding thrombotic events (with uncertain\netiology), re-bleeding at the treatment site,\npersistent inflammation, hemorrhage, and\nadhesion formation. The FDA review further\nhighlighted concerns regarding potential risks\nassociated with repeated or sequential use of\nEVARREST. Importantly, categories of adverse\nevents, including adhesion formation, re-\nbleeding, thrombotic complications, and\ninflammatory responses, were also reported in\nclinical evaluations of EVARREST. The FDA\nreview suggested that immune-mediated\nresponses may be more pronounced in\nimmunocompetent animal models, potentially\nreflecting xenogeneic components of the fibrin-\nbased material. These observations underscore\nlimitations of existing products, despite their\napproval, and further motivate the development\nof hemostatic materials with improved\nperformance and biocompatibility profiles.\nLines 234-248: “Although several topical\nhemostatic agents are FDA approved,\nregulatory clearance is generally based on\ndemonstration of safety and achievement of\nhemostasis under defined experimental\nconditions, rather than consistent improvement\nin downstream clinical outcomes or superiority\nover standard care. As highlighted by multiple\nrandomized trials, systematic reviews, and\nconsensus statements,1-3,9-13,17 many approved\nhemostats provide modest, procedure-dependent\nbenefits and often fail to reduce transfusion\nrequirements or postoperative bleeding\ncomplications when used routinely. Persistent\nbleeding-related complications in surgical\npractice therefore continue to motivate the\ndevelopment of materials with improved\nrobustness and reproducibility across injury\ntypes and physiologic conditions. Importantly,\nthe HTA outperforms standard of care in both\nnon-heparinized and heparinized conditions,\nindicating efficacy beyond native clot formation.\nBecause porcine coagulation is more efficient\nthan human coagulation19,20, animal models may\noverstate the benefit of coagulation-dependent\nadjuncts, while HTA’s tamponade-based\nmechanism may better translate to human\nsettings of trauma, infection, or medical\ncoagulopathy.”\nLines 248-255: “A previous study reported that\nEVARREST had superior performance to\nTachoSil, as TachoSil achieved 0% hemostatic\nsuccess at 3 minutes and only 4% at 10 minutes\n17. Matonick et al. employed an incisional injury\n(15 mm × 3 mm depth) with a substantially\nlarger patch (3 × 4.5 cm), extending\napproximately 1.5 cm beyond the wound\nmargins, whereas our study used a deep punch\ninjury (6 mm diameter, ~1 cm depth), with a\n15x40mm patch (<half the wound margin). In\naddition, excess blood and fluid were removed\nprior to application in Matonick et al, whereas\nthe HTA was applied directly to the bleeding\nsurface in the present study, enabling effective\nplacement in a wet environment.”\nLines 255-261: “Review of publicly available\nFDA documentation for commercial products\nsuch as EVARREST highlight safety\nconsiderations, including inflammation,\nadhesion formation, re-bleeding, and thrombotic\nevents, particularly in the context of repeat or\nchronic exposure. These observations do not\ndiminish the clinical utility of existing products\nbut rather contextualize their limitations and\ninform design priorities for next-generation\nhemostatic materials. Together, enhanced\nadhesive robustness and attention to\nbiocompatibility represent key differentiators of\nthe present approach.”"}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors provided response to the reviewer's comments. Most comments are meaningfully addressed but the comment and concern on Novelty still lingers. The authors' response provides deeper context on what's new in the current manuscript in view of previous works including Li et al., Science 357, 378-381 (2019). Based on the authors' response, the reviewer found that the current manuscript is technically combination of various improvements made in series of works from the same laboratory on top of the first work (Li et al., Science 357, 378-381 (2019)). As the authors pointed out in the response, the reviewer acknowledge that the large animal in vivo data is novel in the current manuscript as previous works did not specifically show hemostatic effectiveness in active bleeding lesions in clinically relevant injury models. This is not a typical level of innovation over the existing literature that the reviewer expects from works published in Nature Communications as a frequent reviewer, author, and reader of the journal."}, "author_response": "Comment Response\nReviewer #3 (Remarks to the Author): We thank Reviewer 3 for their positive review\nof our responses and additional comments.\nThe authors provided response to the\nreviewer's comments. Most comments are While the material design builds upon our\nmeaningfully addressed but the comment and prior mechanistic advances, the present work\nconcern on Novelty still lingers. The authors' represents the first integrated system validated\nresponse provides deeper context on what's in a clinically relevant large-animal active-\nnew in the current manuscript in view of bleeding model, establishing hemostatic\nprevious works including Li et al., Science efficacy under conditions that directly mimic\n357, 378-381 (2019). Based on the authors' real surgical and traumatic hemorrhage. We\nresponse, the reviewer found that the current believe this translational demonstration is a\nmanuscript is technically combination of substantial advance beyond prior reports,\nvarious improvements made in series of which lacked evidence of robust performance\nworks from the same laboratory on top of the in dynamic, high-flow injury environments.\nfirst work (Li et al., Science 357, 378-381\n(2019)). As the authors pointed out in the Lines 226-227: “The present work represents\nresponse, the reviewer acknowledge that the the first integrated system validated in a\nlarge animal in vivo data is novel in the clinically relevant large-animal active-\ncurrent manuscript as previous works did not bleeding model, establishing hemostatic\nspecifically show hemostatic effectiveness in efficacy under conditions that directly mimic\nactive bleeding lesions in clinically relevant real surgical and traumatic hemorrhage.”"}]

Decision letter