Collagen, fibrinogen and thrombin biological addesive is effective in treating experimental liver injuries

Objective: to evaluate the effectiveness of a collagen-based adhesive associated with fibrinogen and thrombin in experimental liver injury in rats. Methods: the study included 30 Wistar rats randomly divided into three groups: A, B and C. All underwent standard liver traumatic injury. In group A the lesion was treated with the adhesive; in group B, with conventional absorbable suture; and in group C, there was no treatment. We analyzed the time of hemostasis, mortality, occurrence of adhesions and any histological changes. Results: there was no statistical difference in relation to mortality (p = 0.5820). The group treated with the adhesive showed the lowest hemostasis times (p = 0.0573, odds ratio 13.5) and lower incidence of adhesions (p = 0.0119). Microscopic histological alterations of Groups A and B were similar, with foreign body granuloma formation separating the adhesive material or the suture from the hepatic stroma. Conclusion: the adhesive of collagen associated with fibrinogen and thrombin was effective in the treatment of experimental hepatic injury, providing a lower incidence of adhesions between the liver and surrounding structures.


INTRODUCTION
T he surgical techniques to approach liver bleeding in- clude local compression, cauterization, bandages, sutures, resections and drainage 1,2 .For complex liver lesions accompanied by hemodynamic instability, laparotomy is indicated for bleeding control, with Pringle maneuver [2][3][4] , ligation of affected vessels and ducts as described by Patcher 2 and even damage control surgery 5 .
The development of a wide variety of hemostatic agents and tissue adhesives within the last 6 years has been offering surgeons the opportunity to use these products in order to achieve quicker and easier bleeding control.The seriousness and the difficulty in conducting certain cases of liver trauma brings the motivation for the search for new therapeutic alternatives, especially for bleeding control.The efficiency of the new hemostatic agents led to the hypothesis of testing the efficacy of the biological adhesive made of collagen associated with fibrinogen and thrombin, comparing it with the conventional suture in the treatment of experimental traumatic liver injury.

METHODS
This experimental study was approved by the Ethics Committee for Animal Use of the Faculty of Medicine of Jundiaí under number 81/110.We included 30 adult, male Wistar rats with a mean age of 3.55 months, weighing on average 442,80g (342g -527g).We randomly divided the animals into three groups, A, B and C, with ten subjects each.
All rats received premedication with atropine at a dose of 0.05mg/kg subcutaneously in the dorsal region and acepromazine 1mg/kg by the same route.After 15 minutes of premedication they received an association of Tiletamine and Zolazepan 20mg/kg intramuscularly.We initiated the surgical procedure after full action of anesthetic drugs, monitored by loss of corneal and eyelid reflexes and limbs bending.
All rats underwent laparotomy under aseptic technique, started from the xiphoid process with about three centimeters long.After opening the abdominal wall, we placed a small orthostatic retractor and identified the liver, the organ chosen for performing a Michelino Collagen, fibrinogen and thrombin biological addesive is effective in treating experimental liver injuries standardized trauma with a biopsy surgical instrument (Punch Keyes® -ABC instrumentos cirúrgicos, Brazil) of 5mm diameter, introduced 5 mm in depth in the parenchyma (Figure 1A).
Since then the animals received the treatment according to the group they belonged to.Group A: After 1 minute of bleeding, we treated the lesion with the collagen surgical adhesive associated with fibrinogen and thrombin, previously activated in 0.9% saline (Figure 1B), followed by cleaning of the cavity and abdominal wall closure.Group B: After 1 minute of bleeding, we performed the treatment of the lesion with suture of the liver parenchyma using 3-0 polyglactin-910, (Vicryl®-Ethicon, USA), with subsequent cleaning of the cavity and abdominal wall closure.Group C: control group, we did not carry out any treatment of the hepatic injury, only closing the abdominal wall.
In the experiments in groups A and B, we recorded the hemostasis times for further analysis.Postoperatively, all rats received analgesia with dipyrone drops added to water and diet with appropriate chow at will.
After eight weeks, we euthanized the surviving rats in a carbon dioxide chamber, with immediate necropsy for observation of intra-abdominal conditions and removal of the liver for histological analysis.The variables studied were the time to hemostasis, the occurrence of deaths, the occurrence of adhesions and any histological changes.The hemostasis time was the time required to control bleeding, recorded in groups A and B. For group C the hemostasis time was not recorded, since we closed the abdominal wall immediately after the liver injury.In the study design, we decided not to interfere in any way in the hemostasis of the control group injuries.We feared that, during the bleeding observation to record the time of hemostasis, if there was a bulkier bleeding, the researcher would feel motivated to compress or soak up the blood with gauze.Attitudes like these would interfere with the results, with a tendency to decrease the degree of adhesions.
We classified adhesions into five grades, adapting the classification described in 1964 by Mazuji, Kalambaheti and Pawar 7 : Grade 0: no adhesion; Grade I: adhesion between the liver injury site and the abdominal wall, small and irregular; Grade II: adhesion of the liver injury site with the abdominal wall and omentum, of moderate intensity and easy separation; Grade III: adhesion of the liver injury site with the abdominal wall, omentum and intestinal loop, intense and of difficult separation; Grade IV: adhesion of the liver wound site to any other region, very intense, homogeneous and difficult to separate.After analysis of the adhesions, were removed the rats livers and placed them in 10% formalin with subsequent preparation of slides with hematoxylin-eosin and picrosirius for microscopic analysis.

Michelino Collagen, fibrinogen and thrombin biological addesive is effective in treating experimental liver injuries
We performed the statistical analysis with the presentation of tables of absolute (n) and relative (%) frequency distributions for all variables.
We analyzed the variables death, hemostasis time and occurrence of adhesions by the Fisher exact test.For the qualitative variable death, we used the Fisher's exact test because the conditions of application of the Chi-square test were not met.For the variable time of hemostasis, we compared the occurrence of shorter time, which was 2 minutes, between the two groups (Adhesive and Suture) using the Fisher exact test, since it is a qualitative variable, we also calculated the odds ratio, with its respective confidence interval.The significance level for the statistical tests was 5%.

Hemostasis Time
The overall average was 3.5 minutes (range 2-10).Group A had a mean time of 2.4 minutes (range 2-5).Group B showed an average time of 4.2 minutes (range 2-10).The distribution of the occurrence of hemostasis times for each group is shown in Figure 2A.
When we grouped and analyzed the results with time equal to two minutes and time longer than two minutes in groups A and B (Table 1), we obtained a borderline significance between them with the Fisher's exact test (p = 0.0573), and when we calculate the odds ratio, the result was 13.5 (range 1.2015.2),which means that group B was 13.5 times more likely to have hemostasis time more than two minutes.Therefore, this data shows statistical significance.

Adhesions
Group A had three rats with Grade 0 adhesions and six with Grade I. Group B had two rats with Grade I adhesions, three with Grade II and two with Grade III.
Group C had a rat with Grade I adhesions, four with Grade II and one with Grade III.No rat showed Grade IV adhesions.
Table 3 shows the distribution of occurrence of the degrees of adhesions in each study group.
When we performed the statistical analysis of variable adhesion, we found that group A had a lower incidence than group B, with statistical significance and analyzed by Fisher's exact test (p=0.0119).We observed a similar result when comparing Group A with Group C (p=0.0069).
When comparing Group B with Group C, we found no statistically significant difference (p=1.0000).

Histological changes.
The histological changes found in the livers' slides of rats from Group A were: reaction of foreign body with formation of histiocytes palisade, separating the amorphous material (adhesive) from the liver cells stroma (Figure 3); plasma cell infiltration and extravasation of bilirubin due to ductal injury.We also observed intense collagen deposition (Figure 4), with dense fibrosis.
The histological changes found in the livers' slides of rats from Group B were foreign body granulo-ma-type inflammatory reaction around the suture fragments, with giant cells and absent fibrosis.
The slides of the rats in group C showed extravasation of red blood cells, without formation of inflammatory tissue.

DISCUSSION
The induced liver injury tried to reproduce intermediate lesions that correspond to grade III lesions, compared to the liver trauma classification of the American Association for the Surgery of Trauma (AAST) 1,3,8 .
For the choice of tissue adhesive, we looked for a product that could take advantage of the bleeding barrier properties, offered by mechanical hemostatic agents, associated with direct action on blood clotting, offered by active hemostatic agents.So the choice fell on a combination of products, already on the market, represented by the combination of collagen combined with fibrinogen and thrombin [9][10][11][12][13] .This is a totally biological product, without the use of synthetic components.This adhesive was evaluated in clinical studies in support of hemostasis in different kinds of surgery, most often in elective situations, especially in parenchymatous organs, showing effectiveness in bleeding control [9][10][11][12][13] .
Frilling, in 2005, reported superiority of adhesive compared with argon beam during liver resection regarding time of hemostasis 12 .We obtained similar  and pigs 10 .It is noteworthy that in some animals with sutured injury, the longer time to achieve hemostasis was due to the difficulty of handling of the liver tissue, very friable in rats.
Like the collagen, fibrinogen and thrombin adhesive, other hemostatic agents are also cited as effective in the control of various types of bleeding.
Garza and Rumsey, in 1990, showed effectiveness in bleeding control with the use of fibrin glue in two patients suffering from liver trauma 15 .In the same year, Oschner et al. used this product in 26 victims of hepatic and splenic trauma, also with effective control of bleeding 16 .
Several experimental studies show the effectiveness of fibrin adhesive in controlling hepatic hemorrhage in dogs 17 , pigs 18,19 , rats 20 and rabbits 21 , with good adhesion to the injured liver, little local inflammatory reaction and few complications.In our study we obtained findings similar to those.
The occurrence of adhesions, which can be classified as a complication of surgical treatment, was statistically lower in the group treated with the adhesive compared with the one treated with suture (p = 0.0119).
This may be due to the fact that the animals treated with suture presented major bleeding and bruising at the site of injury, resulting in greater inflammatory reaction and consequent adhesions.
Frena and Martin 13 , in 2006, found no biliary fistulas with the use of this product in electives hepatectomies in humans, a fact that also occurred in our study, even when dealing with liver trauma, which increases the chance of this type of complication.
The mortality of the group treated with the adhesive (10%) showed no statistically significant difference from the group treated with suture (33.3%, p = 0.5820) and the control group (40%, p = 0.3034).In a study of 1,000 patients suffering from liver trauma led by Feliciano and Mattox between 1979 and 1984, they found that mortality was 10% 22 .In another study conducted by Saaiq et al. in Islamabad, Pakistan, between 2003 and 2010, the mortality rate was 9.73% 23 .Thus, mortality with the adhesive experimental use was similar to those found in liver trauma treatments conventionally performed in humans.
The presence of foreign-body inflammatory reaction found in the histological analysis of the livers of rats treated with the collagen adhesive associated

Michelino
Collagen, fibrinogen and thrombin biological addesive is effective in treating experimental liver injuries with fibrinogen and thrombin was similar to changes found in studies using fibrin glue in rats 24 , fibrin glue in rabbits 21 and polyglycolic acid mesh in pigs 25 .We did not find histological findings suggestive of necrosis of the liver tissue or vacuolar degeneration, as described with the use of cyanoacrylate 26 , or abscesses near the adhesive areas.The intense collagen deposition near identified the adhesive use areas (Figure 3) is an important fact, if we consider that collagen is essential for the injured tissue repair process 24 .
Conservative treatment of isolated liver trauma has been increasingly performed in recent decades, reaching levels of 80% in the present day 27 .This fact, associated with the development of less invasive therapies, such as angiography with embolization 28,29 , decreases the need for surgery to control liver bleeding.But in situations of hemodynamic instability or associated trauma in other organs, particularly in hollow viscus, the surgical treatment is often mandatory 1,3,8,22,27,28 .The liver surgical approach can be complex, requiring great skill and experience of the surgeon 2 .The conducted study showed that collagen adhesive associated with fibrinogen and thrombin was effective in the treatment of traumatic liver injury in rats and has the potential to be used by sur-geons during the same approach in humans.The ease of handling of the adhesive compared with suturing the liver tissue, leading to diminished bleeding control time and low occurrence of complications, are the main points favorable for this material.
There was no significant difference in mortality when comparing the adhesive use with the suture and the no treatment.The collagen adhesive associated with fibrinogen and thrombin was superior to suture regarding hemostasis time, reflecting easy handling of the material and effective bleeding control.The adhesive was also superior to suture and no treatment in relation to the occurrence of adhesions.When we analyzed the histological changes, we did not identify any damage to liver tissue related to the use of the adhesive.
We conclude that the treatment with collagen adhesive associated with fibrinogen and thrombin was effective in experimental hepatic injury, opening new perspectives for use in liver injury in humans.
Figure 2 show the distribution of the number of deaths in each group.When we compared, by the Fischer's exact test, group A with group B (p=0.5820), group A with group C (p=0.3034) and group B with group C (p=1.0000), we did not identify any difference with statistical significance.

Figure 2 .
Figure 2. A) distribution of injuries' repair times among the groups A and B.In the vertical axis, the number of rats, and in the horizontal, the repair times in minutes.B: distribution of deaths among the all groups.In the vertical axis, the number of deaths, and in the horizontal, the groups.
when evaluating the injury repair time with the use of adhesive compared with conventional suturing.The shorter hemostasis time obtained reflects the easy handling and the effectiveness of the material in bleeding control, a fact already identified with the use of collagen alone, as demonstrated by Mantovani, Vidal and Concon Filho14 , or when combined with fibrinogen and thrombin, as shown by experimental studies using dogs9

Figure 3 .
Figure 3. Photomicrograph of histological section stained with hematoxylin-eosin, showing a group A rat's liver.The black arrow points to the amorphous adhesive material; the green arrow points to the area of foreign-body inflammatory process granuloma, with histiocytes distributed in Palisade, separating the liver stroma from the adhesive material.

Figure 4 .
Figure 4. Photomicrograph of histological section of the biochemical reaction of Masson's trichrome, showing a group A rat's liver.The yellow arrow points to collagen fibers stained in red permeating lymphonuclear inflammatory cells and giant cells; the black arrow points to the hepatic parenchyma.

Table 1 .
Distribution of hemostasis times equal to two minutes and larger than two minutes in groups A and B.
p = 0.0573, Odds Ratio = 13.5,MichelinoCollagen,fibrinogen and thrombin biological addesive is effective in treating experimental liver injuries

Table 2 .
Distribution of deaths in each group.

Table 3 .
Distribution of adhesions degrees.