Acta Biomaterialia 10 (2014) 701–708 Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat Blood-aggregating hydrogel particles for use as a hemostatic agent Adam M. Behrens a, Michael J. Sikorski a, Tieluo Li b, Zhongjun J. Wu b, Bartley P. Griffith b, Peter Kofinas a,⇑ a b Fischell Department of Bioengineering, University of Maryland, 2330 Jeong H. Kim Engineering Building, College Park, MD 20742, USA Department of Surgery, University of Maryland School of Medicine, Medical School Teaching Facility Building Room 434F, 10 South Pine Street, Baltimore, MD 21201, USA a r t i c l e i n f o Article history: Received 9 July 2013 Received in revised form 27 September 2013 Accepted 24 October 2013 Available online 1 November 2013 Keywords: Hemostatic Hydrogel Hemostasis Polymer a b s t r a c t The body is unable to control massive blood loss without treatment. Available hemostatic agents are often expensive, ineffective or raise safety concerns. Synthetic hydrogel particles are an inexpensive and promising alternative. In this study we synthesized and characterized N-(3-aminopropyl)methacrylamide (APM) hydrogel particles and investigated their use as a hemostatic material. The APM hydrogel particles were synthesized via inverse suspension polymerization with a narrow size distribution and rapid swelling behavior. In vitro coagulation studies showed hydrogel particle blood aggregate formation as well as bulk blood coagulation inhibition. In vivo studies using multiple rat injury and ovine liver laceration models demonstrated the particles’ ability to aid in rapid hemostasis. Subsequent hematoxylin and eosin and Carstairs’ method staining of the ovine liver incision sites showed significant hemostatic plug formation. This study suggests that these cationic hydrogel particles form a physical barrier to blood loss by forming aggregates, while causing a general decrease in coagulation activity in the bulk. The formation of a rapid sealant through aggregation and the promotion of local hemostasis through electrostatic interactions are coupled with a decrease in overall coagulation activity. These interactions require the interplay of a variety of mechanisms stemming from a simple synthetic platform. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction The body’s natural response to injury comprises three stages: the formation of a platelet plug, an enzymatic cascade resulting in formation of fibrin and the dissolution of the clot and healing of the wound site [1–3]. These stages are referred to as primary hemostasis, secondary hemostasis and fibrinolysis, respectively. The body’s natural mechanisms are not able to control massive hemorrhaging caused by major trauma or surgery, resulting in the requirement of hemostatic intervention. Depending on the type of injury and the capabilities at the site of treatment, different hemostatic approaches are utilized. In the operating room, hemorrhage control is imperative as many procedures including cardiovascular, hepatic, orthopedic and spinal have a high incidence of severe blood loss, requiring use of a hemostatic [4]. Storage and handling capabilities at hospitals allow for the use of costly and biologically active hemostatic agents. These hemostatic formulations typically include one or more coagulation cascade or hemostatically active proteins such as thrombin, fibrin and collagen and are applied topically as a patch [5,6], matrix [7,8] or liquid [9,10]. Non-protein materials based on polyethylene glycol [11] or oxidized cellulose [8,12] are also widely used. Although generally effective, issues including ⇑ Corresponding author. cost, storage and handling illustrate the need for inexpensive hemostatics with comparable efficacy [13]. Current approaches have investigated the use of topically applied self-assembling systems using polypeptides or hydrophobically modified chitosan with varying successes [14,15]. Most approaches require direct access to the injury site. To circumvent this, intravenous hemostatics such as recombinant factor VIIa (NovoSeven) have been investigated for clinical use [16,17]. Further exploration into the use of intravenous hemostatic agents that augment primary hemostasis by utilizing artificial platelets has shown promising results but may be difficult or costly to produce on a large scale [18,19]. Our research focuses on investigating the use of topically applied cationic hydrogel particles as a hemostatic agent. We have synthesized and characterized hydrogel particles capable of aiding in rapid hemostasis by promoting the formation of a robust hemostatic plug and forming a physical barrier to blood loss through electrostatic interactions and swelling. In vitro, the particles locally form hydrogel/blood aggregates, while delaying coagulation in any excess blood present. This is potentially important as the material will not cause, and may even inhibit, thrombotic events at distal sites while locally acting as a hemostatic through the interplay of multiple mechanisms. This paper reports on the synthesis and characterization of the particles, including potentiometric titration, to determine the extent of protonation, and temporal swelling behavior to investigate serum uptake. In vitro coagulation studies, E-mail address: kofinas@umd.edu (P. Kofinas). 1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.10.029 702 A.M. Behrens et al. / Acta Biomaterialia 10 (2014) 701–708 in vivo efficacy studies on rat and ovine surgical models and characterization via hematoxylin and eosin (H&E) and Carstairs’ method staining of the incision site were then used to assess the bioactivity and efficacy of the material. 2. Materials and methods 2.1. Materials N-(3-aminopropyl)methacrylamide hydrochloride (APM) was purchased from Polysciences (Warrington, PA). Ammonium persulfate (APS), inhibitor removers, N,N,N0 ,N0 -tetramethylethylenediamine (TEMED), 1 phosphate buffered saline (PBS), poly(ethylene glycol) diacrylate average Mn 700 (PEGDA) and Tween 80 were purchased from Sigma–Aldrich (Milwaukee, WI). Human platelet poor plasma (pooled, sterile filtered, 4% (w/v) sodium citrate) was purchased from Vital Products (Boynton Beach, FL). Ovine blood (4% (w/v) sodium citrate) was purchased from Hemostat Laboratories (Dixon, CA). Deionized water (DI water) was obtained using a Millipore Super-Q water system (Billerica, MA). Sodium hydroxide (NaOH) was purchased from Mallinckrodt Baker (Phillipsburg, NJ). Acetone, silicone oil, paraffin and xylene were purchased from Fisher Scientific (Pittsburgh, PA). Plasticcapped glass vials were purchased from VWR Scientific (West Chester, PA). All materials were used as received except for PEGDA, which was passed through a column containing inhibitor removers before use. 2.2. Synthesis of hydrogel particles APM hydrogel particles were synthesized via inverse suspension polymerization. 1584.2 mg of APM and 84.4 ll of PEGDA were dissolved in 2.5 ml of DI water. This corresponds to 98.5% by mole APM and 1.5% PEGDA for a 3 M solution. The solution was titrated to pH 7 by the addition of 10 M NaOH. DI water was added to achieve a final volume of 3 ml, forming the prepolymer solution. 390 mg of APS was then dissolved in the prepolymer solution, then 500 ll immediately added to vials containing 2 ml of 5% (v/v) Tween 80 in silicone oil, stirring at 2000 rpm with a magnetic stirrer. 5 ll of TEMED was added to each vial and allowed to react for 24 h. The particles were washed with acetone until the supernatant was clear and isolated via vacuum filtration. The hydrogel particles were then washed in 350 ml of DI water for 72 h, changing the DI water every 24 h. This was done to ensure that any unreacted monomer, initiator, crosslinker or catalyst was removed. Finally, the hydrogels were washed again with 350 ml of acetone until they completely collapsed, vacuum filtered and stored in a vacuum desiccator for up to 3 months. Dry hydrogel particles were imaged using an Axioskop 50 microscope and AxioCam color camera from Zeiss (Germany). 200 measurements were taken (n = 200). 2.3. Fourier transform infrared spectrum A Fourier transform infrared (FTIR) spectrum of the hydrogel particles was obtained on a Thermo Nicolet NEXUS 670 FTIR, range 500-4000 cm1, equipped with the attenuated total reflectance accessory for liquids and powders. Washed and dried hydrogel particles were ground by mortar and pestle to obtain a fine powder before the measurement. 2.4. Potentiometric titration Potentiometric titrations were conducted using a Fisher Scientific Accumet Excel XL15 pH/mV/Temperature Meter with a Fisher Scientific Accumet Glass Body Standard Size Combination Elec- trode (mercury-free) at room temperature. First, the APM polymer (poly(APM)) was prepared. In a glass vial, 1584.2 mg of APM was dissolved in 2.5 ml of DI water. 10 M NaOH was added dropwise until the solution reached a pH of 7. The volume of the solution was then raised to 3 ml with DI water. 30 ll of TEMED and 390 mg of APS were dissolved into the solution. The solution was allowed to react at room temperature for 24 h. The prepared polymer was dissolved in 100 ml of deionized water and stirred at 1000 rpm using a magnetic stir bar. During titration, 1.0 M NaOH was added in 400 ll increments with a 30 s time interval between each addition until the pH of the solution exceeded 13.5. 2.5. Temporal swelling 50 mg of dry hydrogel particles were portioned into preweighed glass vials, followed by the addition of 2 ml of DI water, human source platelet poor plasma or 1 PBS, and placed on a bench top rotator. At time intervals of 1, 2.5, 5, 10, 30, 60, 90, 120, 150 and 180 min, vials were removed from the rotator and drained of excess fluid by pipette before being weighed. All samples were replicated (n = 9). Swelling per cent at each time point was calculated using the following formula: SP ¼ Mt  Md  100 Md where SP is the swelling per cent, Mt is the mass of swollen particles at time t and Md is the mass of dry particles. Water-swollen particles at equilibrium were imaged. The imaged particles were measured and the swelling percentage based on volume was calculated using the following formula: SP ¼ Vf  Vd  100 Vd where SP is the swelling per cent, Vf is the volume of swollen particles at equilibrium and V d is the volume of dry particles. 200 measurements were taken (n = 200). 2.6. Coagulation time 100 mg of dry hydrogel particles were portioned into glass or plastic vials. Refrigerated ovine blood was allowed to reach room temperature. 0.5 ml, 1 ml and 2 ml of ovine blood were added to glass or polypropylene vials containing hydrogel particles. The same volumes of ovine blood added to glass or polypropylene vials served as the controls. Samples in glass vials were recalcified to a 10 mM CaCl2 final concentration using a 0.2 M CaCl2 stock. Samples in plastic vials were recalcified to a 20 mM CaCl2 final concentration using a 0.2 M CaCl2 stock. The difference in final calcium concentration was established to ensure consistent coagulation times amongst the control groups, as determined by previous unpublished studies. Vials were then rotated for 1 min, and set up vertically on the lab bench. Vials were inverted every minute until the blood/hydrogel aggregate completely ceased to flow, and the time recorded. All experimental groups and controls were run in triplicate (n = 3). 2.7. Rat liver puncture and tail amputation Surgeries were completed on 2-4-month-old Sprague-Dawley rats (weight of 300–400 g). Anesthesia was induced with 4% isoflurane, and maintained with 2.5% isoflurane by mechanical ventilation. All animal experiments and care were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland School of Medicine and were in accordance with the ‘‘Guide for the Care and Use of Laboratory Animals’’ published by the National Institutes of Health (National Institutes of A.M. Behrens et al. / Acta Biomaterialia 10 (2014) 701–708 Health publication 85–23, revised 1996). Two injury models were used on each animal: liver puncture and tail amputation. Liver injury was accomplished by a puncture of 5 mm in depth with an 18 G needle. Tail amputation at 50% tail length was completed using surgical scissors. In the mass of blood loss experiments, pre-weighed gauze was applied for 5 min (n = 6 for each injury) with minimal pressure and the resulting mass immediately recorded. The experimental group consisted of the application of 150 mg of hydrogel particles with minimal pressure from a preweighed gauze for 5 min and the resulting mass immediately recorded (n = 5 for each injury). In an analogous experiment, time to hemostasis was recorded after checking for bleeding in 1 min intervals (n = 5 for each injury). At the conclusion of each experiment, rats were euthanized by exsanguination while under anesthesia. 2.8. Ovine liver laceration Surgeries were completed on adult Dorsett hybrid sheep (weight of approximately 65 kg). Anesthesia was induced with sodium thiopental (10 mg kg1) and maintained by mechanical ventilation with 2% isoflurane mixed with oxygen. A Draeger anesthesia monitor (North American Draeger, Telford, Pa) was used during surgery. An incision of 5 cm in length and 1 cm in depth was made with a surgical scalpel on the right lobe of the sheep’s liver, corresponding to a grade II liver laceration [20,21]. 1 g of dry hydrogel particles was poured onto the laceration, which was then compressed with gauze for 5 min. The gauze was then removed and the incision was inspected visually. The incision site was then washed with 50 ml of saline solution to remove free particles. Compression with gauze alone served as the control. Two incisions were performed on each animal’s liver, one serving as an experimental group and the other as the control group. If bleeding was not stopped, the incision was cauterized immediately in order to not hamper the validity of the second incision. The first incision was alternated between the experimental and control group. Immediately following the surgery the liver was excised, fixed in formalin for 24 h, placed in a histological cassette then dehydrated in graded ethanol solutions. Following fixation and dehydration, the sample was set in paraffin, cut into 5 lm sections and mounted on glass slides. The sections were then deparaffinized with xylene and rehydrated with a series of graded alcohol solutions. H&E and Carstairs’ method staining were completed on the 703 samples. Images were taken using an Axioskop 50 microscope and AxioCam color camera from Zeiss (Germany). Three animals were used in this study (n = 3). After the conclusion of each study, sheep were euthanized according to the American Veterinary Association’s (AVMA) Guidelines for Euthanasia of Animals. The sheep were administered a lethal dose of potassium chloride (2 mmol kg1) intravenously to stop the heart, followed by exsanguination while still under general anesthesia. 2.9. Statistical analysis All experimental groups and controls were performed in replicate, as referred to in each specific methods section. Where suitable, data were analyzed using ANOVA single factor analysis to demonstrate differences between groups, assuming a normal data distribution with a confidence of 95% (P < 0.05). Mean values and error bars are reported on each figure as well as relevant statistical relationships, aside from Fig. 3, which presents the raw particle size distribution. 3. Results 3.1. Synthesis and characterization hydrogel particles Hydrogel particles were synthesized via inverse suspension polymerization, as described in Fig. 1, with a narrow size distribution and spherical morphology (Fig. 2). A size distribution of the dry hydrogel particles can be seen in Fig. 3. Particle diameter ranges from 450 lm to 1250 lm, with an average value of 795 ± 9.97 lm (mean ± SE, n = 200). To confirm the chemical composition of the hydrogel, FTIR spectroscopy was used. The hydrogel particles exhibit the characteristic absorbance peaks of N–H (3329 cm1), –CH3 (2960 cm1), C@O (1620 cm1) and N–H (1522 cm1), as shown in the spectrum displayed in Fig. 4. The absence of a peak at 1640 cm1 corresponds to the complete conversion of C@C during the polymerization process. The pKa of the polymer that makes up the majority of the hydrogel was determined to be 9.82 by potentiometric titration. The equivalence point was determined by finding the maximum of the first derivative of pH with respect to volume. The pKa was then determined by the pH at half of the equivalence point. At a physiological pH of 7.4, this corresponds to greater than 99% protonation of the primary amine groups of poly(APM). Fig. 1. Synthetic scheme of hydrogel particles consisting of a copolymer of APM and PEGDA, showing chemical structures and the described inverse suspension polymerization. 704 A.M. Behrens et al. / Acta Biomaterialia 10 (2014) 701–708 Fig. 4. FTIR spectrum of hydrogel particles with characteristic peaks labeled. Fig. 2. Photograph of APM hydrogel particles after swelling to equilibrium in DI water (scale bar = 5 mm). Inset, optical micrograph of APM hydrogel particles after washing and drying procedures (scale bar = 500 lm). Fig. 5. Temporal swelling of hydrogel particles in DI water, PBS and plasma over 180 min, and (inset) over 10 min. Error reported as standard error (n = 9). Fig. 3. Size distribution of hydrogel particles by diameter. Top: dry; bottom: swollen to equilibrium in water (n = 200). Temporal swelling studies were conducted with DI water, 1 PBS (pH = 7.4) and human source platelet poor plasma over time scales pertinent to hemostasis. Water caused the hydrogel particles to swell greater than 1600% by mass at equilibrium. The PBS and plasma equilibrium percentages were smaller in magnitude at 1000% by mass (Fig. 5). On a time scale more imperative to hemostasis, hydrogels exposed to both DI water and PBS swelled to their maximum within 10 min, whereas plasma swelled to 80% of its maximal value in this time (Fig. 5). Equilibrium swelling in water was also measured by volume; the size distribution of the swollen particles can be seen in Fig. 3 and an image of the swollen particles in Fig. 2. Swollen particle diameter ranges from 1400 lm to 2900 lm with an average value of 2079 ± 22 lm. This corresponds to a mean swelling of 1687% by volume with an error of ±0.27%, as determined through standard error propagation. The slight discrepancy between the equilibrium or maximum swelling percentage calculated by mass and volume is due to the assumption of equal density in the calculation by mass. 3.2. Coagulation time Coagulation time was evaluated in glass and plastic vials to elucidate any direct effect on coagulation in vitro. In both cases, when the recalcified volume of blood was comparable to the swollen volume of particles, a coagulum occurred significantly faster than the control case (Fig. 6) through the formation of a blood/hydrogel aggregate. However, when the volume of recalcified blood was significantly larger than the amount of hydrogel particles, a delay in the coagulation of the excess blood was observed. Even in the cases of delay, an aggregate was quickly formed between a fraction of the blood and the particles. A.M. Behrens et al. / Acta Biomaterialia 10 (2014) 701–708 705 A surgical scalpel was used to induce bleeding that would not be stopped by compression. Upon contact with blood the hydrogel particles swelled at a sufficient rate to halt blood loss and effectively formed a sealant through aggregation. After 5 min the gauze was removed and the injury site was inspected, showing complete hemostasis (Fig. 8). The organ was then washed with saline solution, confirming that no blood loss was occurring. H&E staining and Carstairs’ method staining of the incision site after saline irrigation showed significant cell accumulation (Fig. 9). Pressure with gauze alone for 5 min resulted in no significant hemostatic effect and required cauterization to seal the injury site in all cases. 4. Discussion Fig. 6. Coagulation time reported as per cent of control. Top: clotting time in glass vials. Bottom: in plastic vials. The symbol (*) denotes statistical significance from relevant control (P < 0.05). Error reported as standard error (n = 3). 3.3. Rat liver puncture and tail amputation Initial efficacy studies were performed on liver puncture and tail amputation rat models. In both injury models a significant decrease in mass of blood loss was seen (Fig. 7). The liver puncture and tail amputation models saw over a 300% and 500% reduction of blood loss, respectively, both statistically significant relative to the controls. Within the 5 min of application of the gauze control, four of six liver punctures continued to bleed and six of six tail amputations. In comparison, after 5 min of particle application zero of five liver punctures continued to bleed and two of five tail amputations. The hemostatic efficacy was also demonstrated in a time to hemostasis experiment where hydrogel application achieved hemostasis an average of over 1 min faster in the liver puncture model and 12 min faster in the tail amputation model vs. the gauze control (Fig. 7). Any hydrogel in contact with a bleeding surface formed a visible aggregate; in some instances incomplete coverage with the hydrogel particles led to a delay in time to hemostasis or an increase in mass of blood loss. This aggregation, also seen in the coagulation studies, forms a rapid sealant at the surface of the injury that allows hemostasis to be rapidly reached. 3.4. Ovine liver laceration An ovine liver laceration model was used to simulate significant surgical bleeding. This animal was chosen as a model to accurately replicate human vasculature size, blood pressure and coagulation system [22,23]. Liver lacerations and subsequent blood loss present major mortality risks due to the organ’s extensive vascularization, the inability of the liver’s structure to secure vasoconstriction and ineffective implementation of suture and compression techniques on the organ [24–26]. Hydrogel-based materials have been widely investigated for hemostatic use due to their functional flexibly and the ability to easily impart adhesive and absorptive properties [27–29]. This research utilizes cationic APM hydrogel particles’ absorptive an electrostatic properties. Hydrogel particles were fabricated through a controlled synthesis to yield a specific particle size with low polydispersity. The corresponding FTIR spectrum is consistent with hydrogels of similar composition [30]. The size of the particles (800 lm dry, 2000 lm swollen) will help decrease the likelihood of migration into the circulatory system, avoiding thromboembolic complications. Sub-micron-size particles have a high likelihood of systemic circulation that could cause thrombus formation at distal sites. Additionally, the hydrogel particles were stored dry up to 3 months in a vacuum desiccator with no evidence to suggest that they would be degraded or altered by typical environmental factors. Comparing the majority component of this hydrogel to chitosan, which has a pKa of 5.5–7.8 depending on the degree of acetylation [31], chitosan displays significantly less cationic character than poly(APM) in physiological conditions. The high degree of poly(APM)’s positive charge coupled with a low degree of crosslinking (1.5% by mole) of the hydrogel leads to rapid swelling behavior. This amount of crosslinking was chosen as a balance between mechanical properties and swelling capacity previously investigated [32,33]. The material therefore has the propensity to locally concentrate coagulation factors, red blood cells and platelets as it quickly absorbs water from serum. Additionally, the same behavior allows the particles to act as a physical barrier to blood loss. Hydrogel particles in PBS, and plasma, saw a decrease in time to maximum swelling and the maximum swelling per centage as compared to water. This can be attributed to the buffering capacity and ionic strength of both PBS and plasma. Swelling in plasma is further retarded by the presence of proteins and other biomolecules that cause osmotic pressure differences and present the possibility for more charge shielding. Although there is a measurable decrease in swelling in the more physiologically relevant serum, the degree of swelling and charge interaction proved to be sufficient in the subsequent animal modeling. These differences, however, deserve significant consideration when designing any biologically functional polymeric material [34]. Glass vials were used in the coagulation time studies to determine if there was any significant difference when a coagulation activating surface was present, as glass is known to activate the contact activation pathway [35]. Polypropylene vials were also used to remove any activating surface effect. In both cases, coagulation time was dependent on the blood sample volume relative to the amount of hydrogel particles. In instances where hydrogel and blood sample volumes were comparable, immediate blood/hydrogel aggregate formation occurred. Excess blood sample volumes resulted in delay in coagulation, although local aggregate formation 706 A.M. Behrens et al. / Acta Biomaterialia 10 (2014) 701–708 Fig. 7. (A) Mass of blood loss study in Sprague-Dawley rat liver puncture and tail amputation models assessing the hemostatic ability of the hydrogel particles (n = 5 for each injury) vs. a gauze control (n = 6 for each injury). (B) Time to hemostasis for the same injury models (n = 5 for each group). (C) Photograph of tail amputation after 5 min application of gauze showing bleeding. (D) Photograph of tail amputation after 5 min application of hydrogel particles showing bleeding has stopped. In (A) and (B), the symbol (*) denotes statistical significance from relevant control (P < 0.05). Error reported as standard error. Fig. 8. A series of photographs taken during the liver laceration animal model. (1) Incision by scalpel, (2) significant bleeding, (3) hydrogel particles applied with pressure from gauze for 5 min and (4) gauze removal showing complete hemostasis. still persisted. This aggregate formation was not seen in the plasma swelling studies and therefore can be attributed to the presence of cellular bodies. Although the inhibition mechanism was not directly investigated, there have been reports of polyamine and cationic hemostatic inhibition through platelet and/or polyphosphate interactions [36,37]. This aggregate/inhibition mechanism could offer a local hemostatic effect while possibly mitigating risks of thrombotic complications elsewhere in the body. Conversely, this could have negative ramifications for situations with multiple injuries or longer-term implications if a similar material was left implanted. Future studies will include a comprehensive investigation of the inhibition mechanism, and whether it is a local phenomenon or if it could have systemic effects on coagulation. For the single injury models, the hydrogel aggregate formation at the site of injury was able to control bleeding. A.M. Behrens et al. / Acta Biomaterialia 10 (2014) 701–708 707 Fig. 9. Carstairs’ method stained (left) and H&E stained (right) micrographs, showing significant accumulation of red blood cells and platelets deep within the incision site (scale bars = 200 lm). 5. Conclusion Appendix A. Figures with essential colour discrimination The present study demonstrates facile synthesis of APM hydrogel particles that show promise as an effective hemostatic agent. Potentiometric titration, temporal swelling experiments and coagulation time experiments were used to predict the in vivo behavior of the hydrogel particles. Coupling high positive charge with a low crosslink density gives the hydrogel the ability to rapidly swell to over 1000% in size over time frames pertinent to hemostasis and cause localized aggregation, while retarding bulk blood coagulation. This mechanism allows the hydrogel to quickly block blood flow, as demonstrated in vivo, and may mitigate risk of distal thrombosis due to coagulation activation. The inhibition/aggregation mechanism elicits further investigation to assess the localization of the antithrombotic effect and to assess long-term implications of the inhibition. Hemostatic ability was exhibited in both the decrease in time to hemostasis and the mass of blood loss in multiple rat injury models as compared to a gauze control. Further validation of hemostatic efficacy on a physiological pertinent ovine liver laceration was shown. Subsequent staining of the ovine liver incision site showed significant hemostatic plug formation, alluding to the hydrogel’s ability to promote accumulation of red blood cells and platelets through electrostatic interactions. In its current form the material would need to be removed after application but future iterations will introduce degradable chemistries and different initiation/synthesis schemes. APM hydrogel particles have potential as a hemostatic material with a practical hemostatic mechanism that could induce hemostasis while mitigating risks associated with many approaches. Certain figures in this article, particularly Figures 2, 7–9, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi: http://dx.doi.org/10.1016/ j.actbio.2013.10.029. Acknowledgements We would like to thank the Warren Citrin Fellowship, and the Research and Innovation Seed Grant Program between the University of Maryland, Baltimore (UMB) and the University of Maryland, College Park (UMCP) for supporting this research. This material is based on work supported by National Science Foundation Grant No. DMR-1041535. We are also grateful to Mr Steven W. 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