Generation of the NFAH patch
The preparation of the NFAH was modified from our previously described method [9]. To generate the fibrin-agarose hydrogel (FAH), a 30 ml mixture was prepared as follows: 27 ml of human plasma and 0.5 ml of tranexamic acid (Amchafibrin 500mg, Rottapharm, Milan, Italy) were added to a 50-ml conical tube. A solution containing 0.6 ml of 10% calcium chloride (B. Braun, Melsungen, Germany), 0.4 ml DPBS (Merck KGaA, Darmstadt, Germany) and 1.5 ml of melted 2% type VII agarose (Merck KGaA) was then added. After careful mixing, 5 ml of this solution was added to each well of a 6-well plate (Corning, New York, NY) and the plate was incubated at 37°C for 2 hours to allow gelation. Upon completion of this process, the hydrogels were covered with DPBS and maintained in the incubator at 37°C for 24 hours prior to nanostructuring.
Nanostructuration is a mechanical biofabrication process in which hydrogels are subjected to compression and dehydration [15]. The fibrin-agarose hydrogel was placed between two 10-µm nylon mesh filters (NY-1009000; Merck KGaA) and compressed with extra thick western blotting filter paper (88620; Thermo Fisher Scientific, Waltham, MA) for at least 1 minute and 40 seconds under a 250 g square glass. The final patch was then cryopreserved as described below or maintained in Ringer’s lactate solution (Fresenius Kabi AG, Bad Homburg, Germany) at room temperature until use.
Cryopreservation
Manufactured NFAHs were immersed in 0.4 M trehalose solution for 2 hours at room temperature. Subsequently, the NFAHs were stored at -20°C for 3 months. Cryopreserved NFAH (Cryo-NFAHs) were then thawed at room temperature at the time of use.
Microscopy analysis
Structural analysis of the NFAHs was performed using light microscopy and scanning electron microscopy (SEM). Samples were fixed in formaldehyde for 24 h, dehydrated and embedded in paraffin. Subsequently, 4-µm-thick sections were cut on a microtome, deparaffinized in xylene, cleared in ethanol and stained using routine hematoxylin-eosin staining protocols (GHS316 and HT110116, Merck KGaA). Histological images were obtained, and the porosity of the NFAH was calculated using ImageJ software [18]. For SEM analysis, NFAH samples were fixed in 2.5% glutaraldehyde in cacodylate buffer followed by dehydration in increasing concentrations of acetone. After drying with liquid CO2 at high pressure, samples were sputter-coated with gold-palladium and analyzed using a Quanta 200 microscope in high vacuum mode (FEI, Eindhoven, The Netherlands).
For histopathological analysis of hepatic sections, the samples were fixed in paraformaldehyde, embedded in paraffin and cut at a thickness of 2.5-4 µm. Sections were stained using hematoxylin-eosin and the Trichrome Stain (Masson) Kit (HT15-1KT, Merck KGaA) following standard methods.
The following histological variables were assessed by light microscopy: hemorrhage, inflammation (acute and chronic), necrosis, foreign body reaction, fibrosis, re-epithelialization, fibrin and mesothelial membrane. The categorization and scoring of liver injury were performed according to the criteria shown in Supplementary Table S1.
Cytotoxicity
For the cytotoxicity assay, we adapted a previously described protocol [19] (Supplementary Figure S1A). Briefly, the hemostatic agents Cryo-NFAH, TachoSil®, Hemopatch™ (Baxter, Deerfield, IL) and Surgicel® (Ethicon, Johnson and Johnson MedTech, Summerville, NJ) were immersed in MSC medium consisting of DMEM low glucose (Merck KGaA) supplemented with 15% (v/v) FBS (Merck KGaA), 2 mM ultraglutamine (Lonza, Basel, Switzerland), 2.5 µg/ml amphotericin B (Merck KGaA) and 50 µg/ml gentamicin (Normon Laboratories, Madrid, Spain). After incubation at 37°C for 24 hours, the leachate was filtered through a 100-µm filter and a 0.22-µm filter, sequentially, to remove possible traces of the hemostatic agents. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) obtained from the Andalusian Public Health System Biobank were seeded at a concentration of 3000 cells/cm2 and incubated at 37°C and 5% CO2 in MSC medium. After 24 hours, the medium was replaced with the medium in which the hemostatic agents had previously been incubated. The leaching medium was changed every two days. On days 1, 3 and 7, cell counts were performed in duplicate, and apoptosis and necrosis were assessed by flow cytometry using the Anexin V-FITC Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). Briefly, UC-MSCs were incubated with Anexin V-FITC and propidium iodide as indicated by the manufacturer. Sample acquisition was performed on the MACSQuant Analyzer 10 flow cytometer and the results were analyzed with MACS Quantify 2.10 software (both from Miltenyi Biotec). Events (> 10,000 single cells) were analyzed. The experiment was performed in triplicate using three independent UC-MSC samples. The use of human UC-MSCs was approved by the Andalusian Biomedical Research ethical committee, in compliance with current ethical guidelines and regulations (application code S2300454 and PEIBA internal code: 2026-N-23).
Hemocompatibility
An adaptation of a previously developed protocol [20] was used to assess hemolytic effects (Supplementary Figure S1B). First, human erythrocytes were isolated by centrifugation of whole blood obtained from healthy donors at 840 g for 20 minutes. After removing the plasma and buffy coat, a 1:25 dilution of erythrocyte concentrate was prepared in DPBS and 0.5 ml of this diluted erythrocyte solution was added to a 1.5-ml Eppendorf tube with a 0.25 cm2 square piece of hemostatic agent and incubated at 37°C for 1 hour in an orbital shaker at 50 rpm. The hemostatic agents were then centrifuged at 800 g for 15 minutes and the supernatants were collected. Finally, the free hemoglobin in supernatants was quantified using the Plasma/Low Hb System photometer (Hemocue AB, Ängelholm, Sweden). The hemostatic agents tested in this assay were Cryo-NFAH, TachoSil®, Hemopatch® and Surgicel®. Each sample was analyzed in triplicate. Erythrocytes lysed with distilled water and unlysed erythrocytes in DPBS were used as positive and negative controls, respectively. The utilization of blood specimens was approved by the Andalusian Biomedical Research ethical committee, in compliance with established ethical guidelines and regulations (application code S2100047 and PEIBA internal code: 0405-N-21).
In vitro coagulation test
The in vitro coagulation test was conducted as reported [19]. Three batches of NFAH and Cryo-NFAH were manufactured, and three samples were analyzed from each batch. Specifically, a 0.5 × 0.5 cm square piece of each hemostatic agent (NFAH and Cryo-NFAH) was placed in a 1.5-ml Eppendorf tube (Supplementary Figure S2). Pre-warmed human blood was supplemented with CaCl2 (B. Braun Medical S.A., Madrid, Spain) at a final concentration of 20 mM and homogenized by inversion. Immediately, 1 ml of this mixture was rapidly added to each tube and placed in a water bath at 37°C. The tubes were inverted every 30 seconds to check for coagulation. The coagulation time was established when the generated clot did not dislodge under the action of gravity for at least five seconds. A tube without hemostatic agent was included as a control for this test.
Animal protocol and hepatic resection
Liver resection in rats was performed as described [9]. Wistar rats (Rattus norvegicus) were anesthetized (80 mg/kg ketamine and 10 mg/kg xylazine) by subcutaneous injection and maintained with isoflurane inhalation. The liver was exposed by a longitudinal laparotomy and the median hepatic lobe was resected through a 1.5-cm incision. Circular hemostatic agents 24 mm in diameter were then applied and the time to hemostasis was assessed. Time to haemostasis was defined as the time from application to cessation of blood extravasation through the resection surface.
Rats were housed in individually ventilated cages with free access to food and water, and analgesia was administrated as required. Body temperature and weight were measured and a protocol for monitoring signs of pain according to Morton and Griffiths criteria [21]. Animals were euthanized by cardiac puncture under deep anesthesia and assessed for post-operative bleeding, incidence of hematoma, hemostatic patch migration and intra-abdominal unwanted adhesions. Blood samples were taken for measurement of inflammatory factors by enzyme-linked immunosorbent assay (ELISA). Intra-abdominal adhesions were scored as follows: 0, no adhesion; 1, thin adhesions separable by gravity; and 2, thick adhesions not separable by gravity. Sections of injured liver tissue attached to the hemostatic agent were collected for histopathological analysis.
A total of 17 male Wistar rats (200–250 g) were used in the Cryo-NFAH versus NFAH efficacy study and 30 rats were used in the Cryo-NFAH versus TachoSil® safety study.
Approval for animal experiments
All institutional and national guidelines for the care and use of laboratory animals were followed. Animal care and experimental procedures were conducted according to “Guide for the Care and Use of Laboratory Animals” published by the Ministry of Agriculture, Fisheries and Food (R.D. 53/2013, Law 32/2007) and European Communities Council Directive 2010/63/EU. The protocol was approved by the Research Ethics Committee of University Hospital Virgen Macarena and Virgen del Rocio (internal reference: 1131-N‐15). All authors complied with the ARRIVE guidelines.
ELISA
Blood samples were collected in EDTA tubes (BD Bioscience, Franklin Lakes, NJ), centrifuged at 1480 g for 5 minutes and then stored at -80°C in small aliquots to avoid repeated freeze-thaw cycles. Cytokine analysis was performed using the following commercially available ELISA kits: rat C-reactive protein (CRP) ELISA Kit (Catalog No. ELR-CRP, RayBiotech Inc., Norcross, GA), rat Interleukin 1 beta (IL-1β) ELISA Kit (Catalog No. E-EL-R0012, Elabscience, Houston, TX), and rat tumor necrosis factor alpha (TNF-α) ELISA Kit (Catalog No. CSB-E11987R, Cusabio Technology LLC, Houston, TX).
Statistical analysis
Data are presented as mean ± SEM. Significance was determine using the Mann–Whitney U test or the Kruskal-Wallis analysis of variance test with Dunn’s post hoc multiple comparison tests. Categorical variables were analyzed using the Chi square test or Fisher’s exact test. Significance variation in weight or temperature of animals after surgery was determined by a mixed effect model test. Differences were considered significant at p ≤ 0.05. All statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA).