Hemostatic devices
All five hemostat devices used in this study (Cutanplast Standard and Fast gelatin sponge products, Cutanplast Standard and Fast gelatin powder products and Emosist ORC gauze) are manufactured by Mascia Brunelli S.p.A., Milan, Italy. All products are supplied in sterile packaging. Cutanplast Standard gelatin sponge is prepared by presoaking in sterile saline solution and squeezing slightly before use. Cutanplast Standard and Fast gelatin powder products are prepared by adding 6 mL of sterile saline solution and until a soft and moldable paste is obtained. Cutanplast Fast gelatin sponge and Emosist ORC gauze are ready to use dry.
Animal experiments
The experimental procedures performed in this study were conducted in accordance with Italian law and European Union Directive 2010/63/UE on the protection of animals used for scientific purposes. The study received ethical approval from the Italian Ministry of Health (Authorization number 367-2015-PR). The animals were obtained with permission from the Biotechnology Research Center for Cardiothoracic Applications (CRABCC, Rivolta d’Adda, Cremona, Italy).
Five female Landrace-Large White pigs (average weight 75–80 kg) were used. Porcine models of liver abrasion, incision, and punch biopsy have previously been used to assess the efficacy of gelatin and ORC hemostat products for diffuse and focused mild-to-moderate surgical bleeding [19-21, 25, 26]. Before the surgical procedures, the animals had an acclimation and observation period of 10 days, during which they were housed under standard conditions (a light-dark cycle of 12:12 hours, temperature of 20°C, and humidity of 50 ± 5%), with freely available food and water. Before the surgical procedures, animals were fasted for 10 hours.
Each pig received an intramuscular injection of ketamine 10 mg/kg and midazolam 0.5 mg/kg, followed by inhalation anesthesia with a mixture of isoflurane 4.5% and oxygen. When a sufficient level of anesthesia was achieved, animals were intubated and anesthesia was maintained with isoflurane (2.5%) mixed with 100% oxygen. When necessary, curarization was obtained with an atracurium bolus (2 mg/kg) and maintained with a 0.12 mg/kg/min infusion. Animals were mechanically ventilated with a tidal volume of 10 mL/kg, a positive end-expiratory pressure of 5 cmH20 and a respiratory rate required for normocapnia (pCO2 35–45 mmHg). Intraoperative analgesia was provided with tramadol (intravenous [IV] 1 mL/kg) and meloxicam (0.4 mg/kg). A catheter was placed in the auricular vein, and IV Ringer’s Solution was administered during the surgery. Vital signs and oxygenation were continuously monitored (electrocardiography, SpO2 and invasive blood pressure using a transducer connected to a catheter placed in the auricular artery). Upon completion of the procedures, animals were humanely euthanized with an IV overdose of potassium chloride under deep anesthesia.
Surgical procedures
All surgical procedures were performed under the same sterile conditions. Animals were placed in the ventrodorsal recumbant position and a mini-laparotomy was performed. The skin incision began below the xiphoid process and extended 15 cm in the cranioventral direction. The linea alba was opened, the liver was exposed, and the Balfour self-retaining abdominal retractor was used to maintain access to the liver and spleen. Throughout each procedure, the liver and spleen were kept moist with saline-soaked laparotomy sponges.
There were five types of surgical injury: liver abrasion, incision and puncture (Fig. 1), and spleen incision and puncture. In the liver abrasion model, a scalpel blade was used to abrade the liver surface to create 2.0 × 2.0 cm lesions, with application of the right amount of pressure needed to break through the visceral peritoneum and fibrous liver capsule to expose the liver parenchyma and cause sustained, uniform bleeding (mild hemorrhage or oozing). Incisions measuring 1.0 cm in length and 6‑7 mm deep were made with a scalpel. Puncture wounds (6–7 mm deep) were made with a 12-gauge needle. All five hemostat devices (Cutanplast Standard and Fast gelatin sponge products, Cutanplast Standard and Fast gelatin powder products and Emosist ORC gauze) were tested in the liver abrasion and incision injuries, but only the Cutanplast Standard and Fast sponge products and Emosist ORC gauze were tested in the liver puncture and spleen incision and puncture injuries. For each animal, there was one injury for each product tested in each surgical model. Therefore, in total, there were 13 liver injury sites (five abrasion, five incision and three puncture sites) and six spleen injury sites (three puncture and three incision sites) per animal.
Products were prepared and applied according to prescribing information/instructions for use. Injuries were left to bleed for 10 sec before applying the hemostat products. Cutanplast gelatin sponge products and Emosist ORC gauze were cut into 3.5 × 3.5 cm squares, with the ORC gauze folded into a double layer before being trimmed to size. After presoaking in sterile saline solution and squeezing, Cutanplast Standard sponge was applied using slight pressure (Fig. 2a). Cutanplast Fast gelatin sponge was applied dry without pressure (Fig.2b). After preparing the Cutanplast Standard and Fast gelatin powder products (addition of 6 mL of sterile saline solution to the packaging bottle and mixing for 30 sec), the resulting paste was applied to the surgical wound using a spatula without any pressure (Fig. 2c and 2d). Emosist ORC gauze was applied dry without any pressure (Fig. 2e). For removal, gelatin sponge and ORC products could be peeled away from the injured area, and paste (powder) products could be washed away from the injury using a syringe of sterile water.
Assessment of bleeding and quantification of blood loss
Bleeding was evaluated using an adapted version of a validated intraoperative bleeding severity scale, which classifies bleeding into grades according to visual presentation, anatomic appearance, qualitative description and visually estimated rate of blood loss (Table 1) [27]. An additional parameter (time to hemostasis) was included in the scale. Qualitative parameters (visual presentation, anatomic appearance, and qualitative description) were evaluated 2 min after application of the hemostatic device. These parameters were graded on a 5-point Likert-type scale, ranging from 0 (spurting or gushing, central arterial- or venous-like bleeding of a life-threatening nature) to 4 (no bleeding) by the same three assessors for each pig. Hemostasis was defined as the absence of observable active bleeding or the absence of sustained soaking of bleed into the hemostatic material. If hemostasis was not achieved 2 min after application of the hemostatic device, the rate of blood loss was quantified using dry pre-weighted gauze to collect blood for 1 min. The hemostatic products were applied for the time necessary to stop bleeding for up to 10 min. This cut-off point for the evaluation of bleeding time was chosen based on examples of previous models of hepatic bleeding [25, 26, 28]. If required, new hemostats were applied thereafter.
Table 1 Adapted version of the validated intraoperative bleeding scale. Adapted with permission from Lewis KM, et al. Surgery. 2017 [27]
Grade
|
Visual presentation
|
Anatomic Appearance
|
Qualitative description
|
Time to Hemostasisa (min)
|
Rate of blood lossb (mL/min)
|
4
|
No bleeding
|
No bleeding
|
No bleeding
|
≤ 2
|
≤ 1
|
3
|
Ooze or intermittent flow
|
Capillary-like bleeding
|
Mild
|
> 2–5
|
> 1–5
|
2
|
Continuous flow
|
Venule and arteriolar-like bleeding
|
Moderate
|
> 5–8
|
> 5–10
|
1
|
Controllable spurting and/or overwhelming flow
|
Non central venous and arterial-like bleeding
|
Severe
|
> 8–10
|
> 10–50
|
0
|
Unidentified or inaccessible spurting or gush
|
Central arterial- or venous-like bleeding
|
Life-threateningc
|
> 10
|
> 50
|
aParameter added to the original four items of the intraoperative bleeding scale, which was designed and validated for use in clinical studies to generate labelling claims [27]
bVisual rate of blood loss (original item of the intraoperative bleeding scale) [27], unless hemostasis was not achieved 2 min after application of the hemostatic device, in which case the rate of blood loss was quantified using dry pre-weighted gauze to collect blood loss for 1 min
cSystemic resuscitation required (e.g., volume expanders, vasopressors, or blood products)
|
Statistical analysis
For each bleeding rating scale item, the grades obtained from the three assessors were averaged for each animal, and contributed to the final results, which were expressed as mean values ± standard deviation.