ClotFoam An Intracavitary Hemostatic Agent for Non-compressible Hemorrhage Hemorrhage resulting from traumatic injuries is a major cause of death in accidents, and the primary cause of death on the battlefield. Over 40% of the trauma cases admitted at hospitals in the USA are due to road traffic accidents. Hemorrhage is the primary cause of death on the battlefield in conventional warfare. The vast majority of these deaths occur in the field before the injured can be transported to a treatment facility Tissue adhesives and sealants have been developed to control bleeding; but, since all existing haemostatic agents for abdominal intracavitary bleeding are designed to be used in the operating room, not in an emergency at the site of accident or in the battlefield. Hemorrhage is often lethal and with early and effective hemorrhage control more lives can be saved then by any other measure.	ClotFoam® is a biomimetic complex polymer cross-linked in situ that can stop bleeding from severe intracavitary trauma and achieve hemostasis without compression and/or sutures outside the operating room. It is a product in a viscous liquid phase, composed of three parts. When delivered through a CO2 propellant, the solutions form a foamy gel that spreads throughout the cavity to promote adhesiveness and stimulate the coagulation cascade. The foam enhances the distribution of active clotting agents and provides a scaffold upon which the fibrin network can distribute and thereby adhere to and bind to lacerated tissue. ClotFoam allows non-invasive application and dissemination of the agent in the peritoneal or other body cavities; adheres and compress lacerated or wound tissue to prevent flow of blood; and maintains the necessary components over the wound to produce a platelet plug followed by a fibrin clot that stimulates the coagulatory cascade. Rapid formation of a strong hydrogel, effective cleavage, and rapid polymerization to produce a functional fibrin clot over lacerated bleeding tissue ensures that the sealant remains at the site of application without being washed away by blood: High tensile strength and adhesive strength characterize the clot produced by the agent. This biological device uses a new and a form of application suited for non-compressible hemorrhage. ClotFoam was developed with the support of the Defense Advanced Research Project Agency (DARPA), The National Heart, Blood, and Lung Institute(NHLBI) of the national Institutes of Health (NIH); the Maryland Technology Development Corporation ( TEDCO ), and the University of Maryland. Animal studies were conducted by Martin Bluth, MD PhD, at the animal facility of SUNY Downstate and by Grant Bochicchio, MD, PhD at the Trauma Center, and University of Maryland at Baltimore. Chemical research was conducted at Biomedica's laboratories located at UMBC TechCenter, in Baltimore Maryland and at the Biotech Center of the University of Maryland, Baltimore. Adhesion and Coagulation Properties Adhesion and tensile measurements (Intratissular adherence and clot strength) were conducted in Sprague-Dawley rats' liver tissue. Tensil measurements. We tested the force necessary to separate the liver preparations at elapsed times of 1, 5 and 10 minutes of exposure and contact, and compared these results with a standard fibrin sealant and with saline solution as control. The final clot strength (after 10 min) induced by ClotFoam is more than 200% stronger than a standard fibrin sealant in the intratissular adhesion secondary to the exposition of damaged tissue to the foam, Figure 1.(CLICK) Clot strength. Clot strength was tested in three groups: blood plus saline solution (B+S), blood alone (BA) and blood with the CLOTFOAM (B+Gel). There is a statistically significant (P = 0.001) difference when the blood was treated with CLOTFOAM as compared to blood alone or blood plus saline. (Figure 2).(Click) Effect of ClotFoam on Blood Loss After Grade III Liver Injury in Rats. Ten male Sprague-Dawley rats weighing 225-250 gr. were used in this study. Anesthesia was induced with Ketamine-Xylazine combination (5mg/Kg and 0.5mg/Kg) about 20-30 minutes prior to surgery. Laparotomy was performed, Grade IV liver injuries were induced in the larger left and right lobes. The injury was induced by clamping with a hemostatic clamp both lobules and causing injury through the parenquima of the liver and the underlying vessels of the two medial lobes. After the first penetration of the liver (Fig 3A ) (Click), the clamp was opened and repositioned to the animal's left inducing the second lesion including more than 80% of distance from the border to the suprahepatic vena cava (3B)(Click). After this repositioning, the liver was penetrated a second time. Further documentation of the liver injury was achieved by excision and inspection of the liver at the conclusion of the experimental period. The injuries were through and through, with one or more of the left medial lobar vein, right medial lobar vein, and portal hepatic vein lacerated. No concomitant damage to the common bile duct, caudal vena cava, or hepatic artery was noted. Animals were assigned randomly to receive either saline solution (Control) 4 ml or the ClotFoam agent 4 ml. Immediately after the injury was induced, the treatment or saline was administered through a needle in the peritoneal cavity (Figure 4). (click) The bleeding time was observed and recorded. Then the abdominal cavity was closed with 4-0 nylon to observe the animals for 90 minutes. After this period of time the animal was re-anesthetized and all the fluids in the abdominal cavity were collected to measure the intraperitoneal volume and calculate blood losses (Figure 5). (Click). After the collection of intraperitoneal liquid, the liver was excised to further corroborate of the lesions and analyze the clot developed in the injured areas. Data Analysis Data were subjected to tests for heterogeneity of variance and non-normality. Where these conditions were detected, the data were log transformed before analysis. Means and + SD (standard deviation) are reported. Results All injuries were through and through, and major vessels lacerated with no differences noted among treatments. Bleeding time in the control group showed a mean of 37 seconds ( +SD 7.5) while the amount of blood allows the observation. In contrast, the CLOTFOAM treated group arrested the bleeding within 4 seconds (+ 1.0). This difference showed statistical significance with p<0.008. Blood loss measurements in the control (untreated) group were 2.45 mL ( from 1.86-3.61) with a SD of 0.67 in the control group in contrast to the treated group which had a mean of 0.95 mL (0.72 - 1.78) with a SD of 0.55, T-test showed this difference to be statistically significant with a p value of 0.028 (fig 6). (CLICK) Additional Experiments In an additional set of experiments, we investigated the clot forming behavior to determine if the foam stops bleeding from a high pressure artery. In this pilot experiment we induced liver damage with six incisions in the liver border. Each incision was standardized in size (5 mm deep), and also cut free the left renal artery and vein. We applied the foam and or saline (5 ml of intravascular volume with saline-glucose solution) to 4 control and 4 treated animals and observed the formation of the clot and the cessation of blood flow from the vessels. After 10 minutes of observation the animals were closed to monitor survival. All the animals with no foam treatment died of hypovolemic shock within the next hour of bleeding; those animals never awoke from anesthesia. In contrast, all animals treated with CLOTFOAM to stop active bleeding, woke up from anesthesia and all survived for at least 48 hours. Observations: Livers were removed for observation of the lesions and clot forming behavior. It was found that in controls the damaged areas develop some clots but invariably they remain separated (Figure 7). (Click). In contrast, when ClotFoam was administered through a needle, the foam distributed uniformly into the peritoneal cavity. Livers showed the formation of very strong clots in the injured areas, with no adherence of the clot to the undamaged tissue (Figure 8).(click) These clots were strong enough to tightly bond the two pieces of lacerated tissue (Figures 9, 10). (Click) Clot Histology Clot formation was observed only in lacerated areas with no presence of clot or adhesion in undamaged tissue. Fig. 11(click) Fig. 12(click) Fig. 13(click) Experiments in Rabbits In previous experiments in vitro and in vivo in rats ClotFoam has demonstrated two key beneficial effects: the agent 1) stops intraperitoneal non-compressive bleeding and 2) has an intra-tissue adhesive effect. In this study we tested ClotFoam in eight male white New Zealand rabbits weighing 4 pounds. Animals were induced with a mixture of Ketamine-Xylazine and Buprenorfine then they were maintained under isofluorane anesthesia. The rabbits were shaved and a medial laparotomy was performed. The liver was exposed and grade IV liver damage was induced in the right and left lobes (Figure 14 and 15) and an intraperitoneal catheter (12-10 G) was positioned (Figure 3). The animal was allowed to bleed for 5 minutes and the abdominal cavity was closed during that time with 3-0 black silk. After a 5 minutes of bleeding, ClotFoam was administered through a catheter attached to a Y connection to two syringes containing solution A and solution B (20 ml of solution A and 20 ml of solution B total 40 ml) (Figure 16,17). The animals were observed for a period of 12 hours for survival. Resuscitation fluid to maintain a mean blood pressure of 60 mm/hg was not applied. The control animals received 50 ml of intraperitoneal saline solution. After 12 hours, intravenous euthanasia under anesthesia was performed; the intraperitoneal content was collected and measured by weighing all fluid in the abdominal cavity. This fluid was analyzed to determine hematocrit content. The liver was then removed and observed for lesions, and samples for histology were taken. Results Intraperitoneal content Due to the rise in oncotic pressure produced by ClotFoam, the average intraperitoneal fluid content was higher in the treated groups (AB = Clotfoam formulation 1, and AB2 =ClotFoam formulation 2) when compared to the control group, but the content of blood (BL) or blood loss of the recovered volume (RV) was lower in the treated group (Figures 18 and 19). Intraperitoneal volumes into real blood volumes were determined by a conversion formula using the hematocrit of the blood in comparison to the hematocrit of the collected volume. Figure 18. Blood loss (BL) was less in the treated groups when compared to the control group. The intraperitoneal recovered volume (RV) was higher in the treated group AB. This experiment indicates that while Formulation 1 (AB) reduced blood loss more effectively, it has an important effect on the oncotic pressure. Additional Studies With Resuscitation and Blood Pressure Normalization Under endotracheal general anesthesia, intraperitoneal non-compressible hemorrhage was induced, secondary to grade IV traumatic liver damage in 6 rabbits. Resuscitation fluid (0.9 % of NaCl 5-10 ml) was administered to all animals every 5 minutes to maintain the systolic blood pressure over at 50 mm/Hg. Continuous blood arterial pressure monitoring was performed as well as measurement of intraperitoneal bleeding and survival for 12 hours. Three animals were treated with ClotFoam, and controls where not treated.The results indicate: Animal 1 Treated with ClotFoam Total Bleeding 3.2 ml Survived for more than 12 hours(26 hs) Animal 2 Treated with ClotFoam Total Bleeding 7.2 ml Survived for more than 12 hours (18 hs.) Animal 3 Treated with ClotFoam Total Bleeding 4.0 ml Survived more than 12 hours ( 26 hs) Animals 4, 5, 6 (Control) Untreated Total median Bleeding 20 ml Survived less than 10 hours PART II- Animal Studies with Enhanced Viscoelastic Properties A reformulation strategy was designed for use in cases of more severe injury in grade IV in larger animal models. The goal was to enhance the viscoelastic and volume expansion characteristics of ClotFoam while maintaining its adhesive properties through optimization of the current prototype. The foam production was increased by 600% while gel time was reduced to 5 seconds upon mixing of the solutions. Rheological studies were performed to evaluate the viscoelastic profiles of ClotFoam new formulations amid gelation. Three (3) formulations with ability to remain at the site of application in cases of severe trauma without being washed away by blood were evaluated. Studies were performed in an in-vivo aorta-perforated model, a cava vein perforated model and a close cavity liver wound grade IV/V in the rat.  Aortic and cava vein close and open cavity model Experiments Experiments were performed simultaneously at the University of Maryland School of Medicine (UMB) by Grant V. Bochicchio MD, MPH, FACS, Associate Professor of Surgery, Director of Clinical and Outcomes Research R Adams Cowley Shock Trauma Center, Study and by Michael Kilbourne, MD approved by the IACUC UMB, and at The SUNY Downstate School of Medicine, Brooklyn New York, by Martin Bluth, MD. PhD. Experiments at UMB Methods: In this model, a midline laparotomy is made. The bowel is retracted away from the retroperitoneum and the aorta is identified. The aorta is clamped just below the renal arteries and just above the bifurcation of the iliac arteries, effectively gaining infrarenal proximal and distal aortic control. The infrarenal aorta is then pierced with a 25 gauge needle once on both the left and right sides of the vessel. After 6 seconds of uncontrolled bleeding, 500 microliters of ClotFoam is applied diffusely throughout the intraperitoneal cavity. After completion of foam application, time to hemostasis is measured. The abdomen is then closed. Immediately after injury, the rat is given Ringer's solution to maintain mean arterial pressure at about 70-80% of initial MAP (if possible) which is the current standard resuscitation technique for trauma patients. The rat is observed for 20 minutes. After 20 minutes, the animal is re-explored through the same midline incision. All of the blood is collected with pre-weighed gauze pads and total blood loss is calculated. Results: Seventeen animals underwent aortic injury. Animals were randomized into 4 different groups. The three ClotFoam groups each had a different formulation. The final group received no treatment. Survival was 100% at 30 minutes for all animals treated with ClotFoam. No animals survived the injury in the no treatment group. All pre-injury MAP were similar. Table 1 below summarizes the outcomes measured in each group. Table 1: Comparison of outcomes for aortic-injured animals with three different ClotFoam formulations and those without treatment: Resuscitation index is defined as the resuscitation MAP percentage of pre-injury MAP. P values for all outcomes and all formulations are < 0.001 compared to no agent. Outcome Form-1 (N=5) Form #2(N=5) Form 3(N=4) No agent(N=3) Time to hemostasis (s) 16.0 ± 6.0 12.2 ± 2.9 19.3 ± 2.5 N/A Total blood loss (ml) 7.0 ± 0.9 5.2 ± 0.5 7.6 ± 0.6 16.3 ± 0.3 Resuscitation index (%) 68.2 ± 6.2 68.8 ± 14.0 73.9 ± 5.7 26.8 ± 2.4 Resuscitation volume (ml) 10 ± 0 12 ± 4.5 13 ± 3.6 20.3 ± 2.5 Survival (min) 30 ± 0 30 ± 0 30 ± 0 18.3 ± 2.9 Cava Vein Model The second model is a liver/vena caval injury model. In this model, a small upper midline laparotomy is made. The left lobe of the liver and the vena caval are exposed and isolated. A small incision is created in the right lower quadrant and the ClotFoam applicator tip is placed through that incision so that the opening is intraperitoneal but remote from where the injury will take place. Next, the injury is created by sharply transecting the left liver lobe, and then creating a stab injury into the vena cava. The mini-laparotomy incision is rapidly closed with staples. ClotFoam is then injected into the closed abdominal cavity. Resuscitation, observation and blood loss measurements are collected, as mentioned above. Given the closed cavity, time to hemostasis is not measured (Figure 10). Eight animals underwent liver/vena caval injuries. Animals were then randomized into 6 different groups. The 5 ClotFoam groups each had a different formulation. The final group received no treatment. Survival was 100% at 30 minutes for all animals treated with ClotFoam. The single animal in the no treatment group died at 18 minutes from injury. All pre-injury MAP were similar. Table 2 below summarizes Figure 10 The applicator was placed into the intraperitoneal cavity. At the 20 minute mark after liver/caval injury, the animal was opened fully to expose the injured area. As shown, ClotFoam is present (white material) in the pooled blood of area B, demonstrating its ability to move to target tissue despite blind (and remote) intracavity application. Table 2: Comparison of outcomes for liver/cava-injured animals conducted at UMB with five different ClotFoam formulations at various dilutions and those without treatment. Resuscitation index is defined as the resuscitation MAP percentage of pre-injury MAP. Outcome Form 1 (N=4) Form 2 (N=4) Form 3 (=4) No agent (N=4) Total blood loss (ml) 5.1 4.5 4.2 14.7 Resuscitation index (%) 73.0 74.7 92.9 54.3 Resus citation volume (ml) 10 10 10 16 Survival (min) 30 30 30 18 Open and Close cavity experiments at SUNY Downstate Martin Bluth, MD, PhD Principal Investigator; Protocol approved by the IACUC SUNY DOWNSTATE Aortic Vein model Experimental Procedure Spague Dawley rats were monitored for blood pressure and mean arterial pressure (MAP) via femoral catheterization and A-line placement and subjected to laparotomy as previously described in our Phase I submission. The great vessels were dissected and visualized, and the abdominal aorta was punctured with a 27 gauge needle.Blood loss occurred under high pressure. Blood pressure and (MAP) levels dropped from baseline levels of approximately 151/110 (120) to 38/31 (34) after 10 seconds. At this point ClotFoam formulation was administered. Administration took place with the mixing of proprietary components under CO2 pressure into the abdominal cavity through the device shown in Figure 11 which covered the aortic lesion ( Figure 12 ). Figure 11. ClotFoam administration apparatus. Syringes of ClotFoam components. Figure 12. Stabilization of ClotFoam after aortic puncture.   Figure 13 . ClotFoam administration coats the aorta in addition to other abdominal organs in a foam like fashion. Within 2 minutes after ClotFoam administration, the BP and (MAP) began to rise to 82/46 (57). Normal saline was administered for volume resuscitation and BP (MAP) did not drop demonstrating lack of plug leakage. Ten minutes after ClotFoam Administration the BP and (MAP) approached baseline levels (133/124 [110]) and were maintained over 2 hours when animals were sacrificed, and tissue at the site of the lesion (abdominal aorta) was obtained for histology. Cava Vein Model: Spague Dawley rats were monitored for blood pressure and mean arterial pressure (MAP) via femoral catheterization and A-line placement and subjected to laparotomy as previously described. The great vessels were dissected and visualized ( Figure 14 ) and in this case the vena cava was punctured with a 27 gauge needle which inflicts a low pressure injury with substantial blood loss. ClotFoam was administered as described for the aortic puncture above, and similar blood pressure and MAP readings were obtained to the aortic insult. As shown in Figure 15, 16 , ClotFaom administration to the site of the vena cava leasion provides a stable cap of foam which prohibits any further blood loss. Figure 14. Visualization of great vessels (under cotton applicator). Figure 15. Rapid oozing occuring after vena cava puncture with a 27 gauge needle Figure16.Stabilization of ClotFoam after vena cava puncture. Close Cavity Model Sprague Dawley rats were monitored for blood pressure and mean arterial pressure (MAP) via femoral catheterization and A-line placement. In this approach no laparotomy was performed. Instead a #14 blade was used to slice through the upper right abdominal quadrant more closely mimicking a knife wound. The area of wound was determined by ultrasound. Prior to the insult a small incision (0.5-1.0 cm) was placed on the lower left abdomen ( Figure 18 ) to allow placement of the delivery apparatus ( Figure 19 ) into the abdominal cavity for ClotFoam administration. Figure 17. Preparation for ClotFoam delivery in a closed abdominal wound. Fig 18 . A small peritoneal opening (highlighted in circle) was fashioned prior to insult to allow for ClotFoam administration Figure 19. Demostration of liver laceration by knife wound with #14 blade. As shown in highlighted circle the blade effectively traversed through the liver. Similar drops in blood pressure and MAP occurred after the blade was used to puncture the liver. After 10 seconds ClotFoam was administered through the lower left abdominal port under manual pressure. Once again the blood pressure stabilized and hemostatsis was maintained through 2 hours when animals were sacrificed for analysis. Table 3: Summary of ClotFoam effects in three different hemorrhagic models: Data represent one of 3-4 experiments in each model. Details of each model are described in the text. Control represents puncture model with NS treatment alone. Numbers represent blood pressure and mean arterial pressure (MAP - in parenthesis) at baseline 10 seconds, 10 minutes and two hours post insult. NS – normal saline administration in ml. NA – not applicable (control animals died before 2 end point). TTH – total time to hemostatsis, measured as function of a sustained and maintained rise in blood pressure and MAP of 70. Model N# baseline 10 sec NS (ml) 10 min 2hrs TTH Survival Aortic 3 151/110 (120) 38/31 (34) 4.0 133/124 (110) 131/117 (124) 3 m all Venous 4 140/105 (115) 63/34 (41) 3.5 109/69 (83) 130/72 (97) 2 m all Puncture 4 162/102 (128) 66/41 (50) 4.2 131/69 (87) 135/87 (102) 2 m all Control Venous 3 150/112 (128) 50/37 (41) 9.7 75/38 (48) NA NA none Control aortic 3 153/108(119) 42/34 (33) 10.5 N/A N?A N/A none Clot Histology Liver section samples were collected from all animals at necropsy. Samples of liver, containing the wounded site, were preserved in 5% formalin and processed using standard histology techniques. Fixed tissue samples were embedded in paraffin wax (melting point 56°C) and sectioned at 2–3 µm. Glass-slide-mounted sections were then stained with hematoxylin and eosin (H&E). Two liver sections perpendicular to the resection site were evaluated per animal. Figure 20 ClotFoam adheres only to the wounded tissue Figure 21 Bonding of ClotFoam to tissue Figure 22 Section of lacerated inferior vena cava covered by fibrin clot following application of CloFoam Figure 23 Section of liver adjacent to area of laceration with fibrin clot covering the surface following application Conclusion All three ClotFoam formulations tested, proved capable of forming a crosslinked foam structure (gel) that rapidly disperses in the intraperitoneal spaces, inducing clot formation. When used in non-compressible intraperitoneal hemorrhage secondary to grade IV liver injuries in small animal models, ClotFoam significantly decreases the bleeding time and blood loss , and improves the adhesion between lacerated and damaged tissue. Considering that the total circulating volume in the animal model used is between 8-10 ml, the blood loss of ClotFoam treated animals amount to 12%, while the controls lost over 25% to 35% of their total blood volume. ClotFoam has a affinity for injured or damaged areas of tissue but does not produce any adhesions in other organs, and there is no presence of clots or adhesions in non-damaged organs. PART III Studies in Pigs Formulation # 9 developed in the course of experiments described in part II showed the optimal viscoelastic properties for application in military relevant animal models (pig). This formulation served also as the “gel matter” to study interactively the quality and formation of the fibrin clot. Formulation 9 was evaluated in the swine model. Studies were performed in liver grade III/IV wound open and close cavity model by Grant Bochicchio, MD, FACS, at the Thomas Miller Animal facility in Baltimore . Methods: Nine female Yorkshire crossbred swine, age 2.5 months, weighing 37 ± 2 kg, were used. All animals were maintained in an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited facility, and all experimental manipulations were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. Animals then underwent either grade 3 or 4 liver injuries via open laparotomy or by laparoscopy. For the purposes of this model, a grade 3 injury is defined as a 3 cm long full-thickness parenchymal laceration (created sharply by an 11 blade scalpel). After the liver was exposed, a spot in the middle of the liver was selected to produce the liver injury with a scalpel. The position was calculated by approximation to the suprahepatic vessels and some branches of the portal vein. The spot was marked with a marker. After the damage was induced, surgeons close the cavity,allowed for 30 seconds of massive bleeding before applying ClotFoam through a small perforation. A grade 4 injury was a 10 cm deep parenchymal injury with a specially designed high-speed drill with a cutting drill bit creating an injury akin to a penetrating gunshot (GSW) wound (Figure 24). Injuries were highly reproducible and severity was similar between the open and laparoscopic techniques. These injuries were consistent with the American Association for the Surgery of Trauma Organ Injury Scaling system. Figure 24: A) Schematic of high-speed liver injury drill. B) Close-up schematic of sharp rotary drill bit. A) B) Animals were randomized into 4 groups to date. Group 1 (n= 2) consisted of animals who underwent grade 3 liver injuries through an open midline laparotomy and had open cavity ClotFoam application. In this group the agent was visually directed to the liver injury. Group 2 (n= 2) consisted of animals who underwent grade 3 liver injuries through and open midline laparotomy and had closed cavity ClotFoam application. In this group the agent was administered into the peritoneal cavity blindly without direct injury visualization or direction. Group 3 (n= 2) consisted of animals underwent grade 4 liver injuries through an open midline laparotomy without ClotFoam treatment (open controls). Group 4 (n= 3) underwent grade 4 liver injuries through the laparoscopic technique without ClotFoam treatment (laparoscopic controls). In all groups, 90 cc of ClotFoam was used for treatment. The ClotFoam was delivered via mixing syringes (Figure 25) propelled into the abdominal cavity using pressurized carbon dioxide (approximately 50 psi). This created a homogenous hemostatic foam that actively clotted and sealed the injuries on both the visceral and diaphragmatic aspects of the liver. Figure 25: Applicator bore tip through which ClotFoam was administered. Fluid resuscitation with Lactated Ringer's (LR) was begun immediately after injury. LR was infused as necessary to re-establish a MAP within at least 80% of the preinjury MAP if possible. Resuscitation was continued for the entire observation period (1Hr.). Results Fourteen animals were used in the study to date. Eight animals were grade IV liver injuries (Group 1= 4 open cavity ClotFoam applications and Group 2= 3 closed cavity applications). End points for animals in Groups 1 and 2 are shown in Table 1. Trend of mean arterial pressures (MAPs) are seen in Figure 1. Table 4: Outcome measures for Grade 3 liver injuries treated with ClotFoam. Group 1 = open cavity, Group 2 = closed cavity. All values reported as mean ± SEM Group Survival Time (min) Total Blood Loss (ml) Fluid Requirement (ml) 1 (n= 4) 60 ± 0 300 ± 283 1500 ± 283 2 (n= 4) 60 ± 0 600 ± 212 2175 ± 742  Figure 26. Open Cavity Model Figure 27. Close Cavity Model Table 5: Outcome measures for Grade 4 liver injuries not treated with ClotFoam. Group 3 = open laparotomy, Group 2 = laparoscopic. All values reported as mean ± SEM   Group Survival Time (min) Total Blood Loss (ml) Fluid Requirement (ml) 3 (n= 2) 26 ± 3 1900 ± 424 3050 ± 70 4 (n= 3) 22 ± 11 1700 ± 200 2467 ± 569 Fig. 28: Trend of MAPs for Grade 4 liver injuries treated with ClotFoam. Group 3 = open laparotomy, Group 4 = laparoscopic.   Fig. 29. Controls: Five animals underwent grade 4 liver injuries (3 laparoscopic and 2 open) to validate the laparoscopic model against the established open model. These animals were not treated with ClotFoam. Endpoints are seen in Table 2 and Figure 2.