CIVIL ENGINEERING 365 ALL ABOUT CIVIL ENGINEERING


Materials

The photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was purchased from Tokyo Chemical Industry, Tokyo, Japan. The other reagents including glycidyl methacrylate solution (GMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fibrin glue (Greenplast Q®, Green Cross Corp., Yongin, Korea), polyurethane (Medifoam® liquid bandage: MLB, Mundipharma, Seoul, Korea), Avitene™ (C. R. Bard, Inc., Murray Hill, NJ, USA) or standard gauze were used as control materials for the various analyses.

Preparation of the silk fibroin sealant (Sil-MAS)

Sil-MAS was synthesized according to our established protocol15. Bombyx mori (B. mori) cocoons were provided by the Rural Development Administration (Jeonju, Korea). Each silkworm cocoon was sliced into four pieces. Forty grams of sliced cocoons were boiled in 1 L of 0.05 M Na2CO3 solution for 30 min at 100 °C to remove sericins from the silk and then were washed with distilled water (DW) several times. The degummed silk was dried at room temperature, and 20 g of it was dissolved in 100 mL of 9.3 M lithium bromide solution (in DW) at 60 °C for 1 h to make regenerated SF solutions. Then, 6 mL (424 mM) of GMA was slowly added to the mixture with stirring at 300 rpm and 60 °C for 3 h. Next, the resulting solution was filtered through a rayon-polyester membrane (Miracloth, Calbiochem®, San Diego, CA, USA) and dialyzed against DW using 12–14 kDa cutoff dialysis tubes for 7 days. Finally, this methacrylated SF solution was frozen at −80 °C for 12 h and freeze-dried for 48 h. The lyophilized methacrylated SF (d-Sil-MAS) was stored at −80 °C for further use.

d-Sil-MAS was dissolved in DW at a concentration of 25% (w/v). Then, LAP filtered through a cellulose acetate syringe filter (0.2 µm, GVS Filter Technology Inc., Findlay, OH, USA) was added into the solution at a concentration of 0.3% (w/v) and mixed until fully dissolved using a planetary vacuum mixer (ARV-310, Thinky Corp., Tokyo, Japan). This solution was then strained through a nylon mesh cell strainer (40 µm, SPL Co., Pocheon, Korea) and finally pasteurized at 60 °C for 30 min. For in vitro and ex vivo testing, the sterilization process was skipped. The final solution was called liquid (l)-Sil-MAS. After stabilization at room temperature for 30 min, the l-Sil-MAS was irradiated with ultraviolet (UV) light for 10–30 s at a distance of 5 cm using a UV spot-curing machine (SP-LED-1, ARC UV Corp., Yongin, Korea) equipped with a filter of 365 nm. The spot diameter was 1.5 cm, and the light intensity was measured at 6 mW/cm2 by a UV meter. This crosslinked material was called c-Sil-MAS.

c-Sil-MAS samples for the mechanical test and in vivo biocompatibility test were 3D printed by a digital light processing (DLP) printer that was customized by professional manufacturers (NBRTech. Ltd, Chuncheon, Korea)15. Samples were designed by CADian3D (IntelliKorea, Seoul, Korea) depending on the test conditions. The following printing parameters were used: printing thickness, 50 μm; number of base layers, 3; curing time of base layer, 4 s; number of buffer layers, 1; and curing time of buffer layers, 3 s. Prints were carried out by repeating the process of projecting an image into the hydrogel followed by raising the Z-stage in an aseptic environment. After printing, the printed objects were rinsed with saline to remove uncrosslinked l-Sil-MAS surrounding the c-Sil-MAS before testing.

Characterization of Sil-MAS

To determine the successful synthesis of the sealant prepolymer, d-Sil-MAS was examined through proton nuclear magnetic resonance (1H NMR) spectroscopy at a frequency of 400 MHz using a Bruker DPX FT-NMR Spectrometer (9.4 T, Bruker Analytik GmbH, Karlsruhe, Germany). First, 5 mg of d-Sil-MAS was dissolved in 700 μL of deuterium oxide (D2O, Sigma-Aldrich) and then filtered using a nylon syringe filter (0.45 μm) before analysis. 1H NMR spectra were recorded for unsubstituted SF and GMA with 1:1 mol unsubstituted amines on SF: mol GMA.

Samples were prepared by a 3D printer for the rheological properties test (a disk with 2.5 cm diameter and 0.2 cm height), for the compression test (a disk with 1.12 cm diameter and 1 cm height), for the tensile test (a dumbbell shape sample of a concave column with 1.6 cm (w) × 0.7 cm (d) × 0.2 cm (h)), for the swelling rate (a disk with 2.5 cm diameter and 0.2 cm height) and for the in vitro degradation study (a square sample with 1 cm (w) × 1 cm (d) × 0.2 cm (h)). The rheological properties of l-Sil-MAS and c-Sil-MAS were measured at 25 °C using an Anton Paar MCR 302 (Anton Paar, Zofingen, Switzerland) rheometer. The mechanical strength of the hydrogel was obtained by a universal testing machine (UTM; QM100S, QMESYS, Gunpo, Korea) that was equipped with a 10 kgf load cell in an unconfined environment. The mechanical force was loaded at a rate of 5 mm/min until the hydrogel broke. For the swelling property of c-Sil-MAS, the hydrogels were immersed in DW or PBS, and then, the swelling ratio was estimated by measuring the length of the X and Y axes based on the initial status immediately after printing.

In vitro degradation16 of 3D-printed c-Sil-MAS was measured for 30 days using protease XIV (from Streptomyces griseus) (Pronase E®, Merck, Darmstadt, Germany) with an activity of 2 U/mL. Samples were immersed in 2 mL of PBS (pH 7.4) containing 2 U/mL protease enzyme and incubated at 37 °C. The enzyme solution was exchanged with freshly prepared solution every 3 days. The three samples were dried and weighed at predetermined time points. The degradation rate was calculated by the following formula:

$${mathrm{Remaining}};{mathrm{weight}};{mathrm{% }};{mathrm{ = }};{mathrm{Wt / Wi}};{mathrm{times}};{mathrm{100;}}$$

where Wi is the initial dry weight of the construct and Wt is the final weight after 0, 3, 6, 9, 12, 15, 18, and 30 days of incubation.

Adhesive mechanical tests17

The common treatment for all adhesive tests was as follows: All tests were conducted according to the ASTM guidelines modified in terms of the size of the specimen, strain rate, or area of sample treatment. The three mechanical tests relative to adhesion of Sil-MAS (n = 3) were performed using a UTM that was equipped with a 3 kgf load cell in an unconfined environment. MLB (n = 3) was used as a control. Fresh shaved rat skin from male SD rats (180–220 g) was prepared as a substrate tissue. This rat skin tissue was kept in PBS, and the moisture was wiped immediately before use. To attach rat skin tissue on glass slides and steel fixtures, instantaneous adhesives (LOCTITE401, Henkel, Rocky Hill, CN, USA) were used. Different quantities of sealants (l-Sil-MAS and MLB) and UV treatment times for each test were used for the experiments. All tests of physical properties were performed in a room under strictly controlled conditions (80% humidification, room temperature) to prevent drying out.

(1) In vitro lap shear test: The strength properties of sealants in lap shear by tension loading were analyzed according to the ASTM F2255–05 standard. Rat skin (2.5 cm × 2.5 cm) was attached on the edge of the short side of two glass slides (9 cm × 2.5 cm). Next, 300 µL of each sealant was uniformly placed on the skin. Then, the other tissue-coated slide was symmetrically covered with sealants. The Sil-MAS group was irradiated with UV light (20 s) from every direction, and the MLB group was allowed to dry at room temperature. These assembled slides were loaded in a UTM for shear testing by tensile loading with a strain rate of 2 mm/min. The point of detachment was fixed as the maximum load. Shear strength (kPa) was the maximum load (N) divided by the bonding area (0.5 cm2). To visualize the adhesive strength of Sil-MAS, 300 µL of l-Sil-MAS (2.5 cm × 2.5 cm) was applied between a 0.9 kg wrench and a plastic ruler, and after UV crosslinking (20 s), the ruler was lifted for 2 h.

(2) Pull-off adhesion strength: The strength properties of sealants in tension were quantified according to the ASTM F2258–05 standard. The test aims to measure the adhesive tensile strength of Sil-MAS in conjunction with the skin tissue and the force necessary to separate this bond. Two rat skin tissues (2 cm × 2 cm) were attached on the steel fixtures with 2 cm in each dimension. Then, 300 µL of each sealant was uniformly placed on the skin on the bottom side of the steel fixture. The gap between the fixtures was set at 2 mm. The Sil-MAS group was irradiated with UV light (20 s) from every direction, and the MLB group was allowed to dry at room temperature. A transient tensile test at a constant transducer speed of 5 mm/min was performed until complete sealant-skin substrate seal failure. The adhesion strength of sealants was determined by dividing the maximum load detected in the pull-off curve by the bonding area (4 cm2).

(3) In vitro wound closure strength: The wound closure strength of the sealants was tested according to the ASTM F2458–05 standard, which is specifically focused on determining the wound closure strength of medical glue. The rat skin was cut into rectangular shapes 2.5 cm × 2.5 cm in size. The tissues were fixed onto two glass slides (9 cm × 2.5 cm) with 1.5 mm spaces between the slides. The center of the tissue was cut using a razor blade to mimic wound injury. Then, 400 µL of each sealant was uniformly applied onto the cut area (0.5 cm × 0.5 cm), and UV light was applied to crosslink the sealant from every direction. These assembled slides were placed carefully into the UTM by tensile loading with a strain rate of 5 mm/min. The point of tearing was defined as the wound closure strength (kPa).

Ex vivo porcine aorta burst pressure test

The burst pressure testing of Sil-MAS (n = 3) was conducted using a modified setup for determining vessel seal burst strength18. Briefly, fresh porcine aortas (diameter 1.8 cm) from a local slaughterhouse (Hongcheon, Korea) were immersed in saline, and the experiment was carried out within 10 h after the aorta harvest. The aorta was cut into 15 cm segments, and an infusion catheter (diameter 2 cm) was fitted in one opened side of the aorta. The orifice diameter of the tube was larger than that of the aorta so that the aorta covered the tube very tightly. The other opened side of the aorta was gripped by a curved mosquito clamp. The catheter was connected to a syringe pump (PILOT Anaesthesia 2, Fresenius Vial Corp., Brezins, France), which was connected to a patient monitor (MP1000NT PLUS, MEK Corp., Giheung, Korea) to verify the intraluminal pressure. Then, a small incision (3 and 9 mm length) was made by scalpel (#15) in the middle of the aorta, and 200 µL of l-Sil-MAS was placed onto the sectioned area and photocrosslinked by UV light (20 s).

Next, to test the subsidiary effect of Sil-MAS on suturing, the porcine aorta was cut in half using a scalpel. An aorta end-to-end anastomosis was performed using interrupted polyamide sutures (Blue Nylon, Ailee Corp., Busan, Korea). One milliliter of l-Sil-MAS was then applied to the anastomotic site and treated with UV light (20 s). The aorta sutured without sealant was used as a control. The above aortas were filled with PBS using a syringe pump through the other opened side of the aorta until the aorta was swelled by its diameter or leakage occurred at the suture site, and this point was set to zero mmHg. Then, additional PBS was injected into the aorta using the syringe pump to increase the infusion pressure. The aorta was swollen under continuous PBS injection, and the burst pressure (kPa) of Sil-MAS was recorded at the point of material burst, detachment or tissue burst.

Animal study

In vivo study using a rat model

Common treatment for all rat tests: The animal studies were carried out in accordance with guidelines and approval of the Institutional Animal Care and Use Committee (IACUC) of Hallym University in Korea (# Hallym 2018–11). Healthy male Sprague-Dawley (SD) rats each weighing 180–220 g were used (Samtako, Osan, Korea). After being raised under experimental conditions for 1 week, the rats were randomly divided into 1–3 groups depending on the analysis. Anesthesia was induced with 3% isoflurane in a 75:25 mixture of nitrous oxide and oxygen in a standard induction chamber with a gas vaporizer (Harvard Apparatus, South Natick, MA, USA) and maintained at 2%, administered via a small rodent respirator. Each group was tested in triplicate. For animal sacrifices, the rats were euthanized with a lethal dose of thiopental urethane.

(1) Wound closure animal study: To evaluate the adhesive property of Sil-MAS, the backs of rats (n = 3) were shaved. After disinfection, skin incisions 1.5 cm long and partial-skin thickness deep were made on the backs of rats by a surgical blade (#10). l-Sil-MAS (200 µL) was quickly applied to the wound, with fingers used to slightly hold both ends of the incision, and then crosslinked with UV light for 10 s. Next, sealing by Sil-MAS was confirmed by gently pulling the sealed site in the opposite direction of incision with two fingers.

(2) Hemostasis and wound healing in an in vivo partial-thickness skin wound model: The rats were divided into three groups: the Sil-MAS group (n = 3), the Avitene™ group (n = 3) and the gauze group (n = 3). Commercial artificial dermis Avitene™ and standard gauze as controls were cut into 1 cm × 1 cm square shapes. After shaving and disinfection of the backs of rats, in the rostral-to caudal direction, three 1 cm (w) × 1 cm (d) × 0.3 cm (h) partial-thickness skin defects per animal were cut out with a homemade tool. Through this procedure, the epidermis and partial dermis were removed. One of the three wounds was covered with Avitene™, and another was covered with gauze. The other wound was sequentially treated with l-Sil-MAS (200 µL) and UV light (25 s). To secure the treatment, Surgifix® (BSN medical, Victoria, Australia) was wrapped around the animal from below the forelimbs to above the hindlimbs. The wound site was photographed on the 0, 3rd, 7th, 14th, and 21st days, and the remaining wound area was measured and converted to a percentage based on the initial wound area. After photography, rats were sacrificed, and full-thickness skin tissue was harvested at the initial wound size and divided into two equal portions for western blotting and histology analysis.

(3) Femoral artery hemorrhage: The hair of rats (n = 3) was removed over the right inguinal region. After disinfection, the skin and subcutaneous tissues were cut to expose the femoral vein and artery. Bleeding was produced by incision of the femoral artery and vein using a sterilized scalpel. After free bleeding was observed for 5 s, the damaged area was pressed gently with gauze for 3 s. The gauze was removed, and immediately, l-Sil-MAS (50 µL) and UV light (20 s) were sequentially applied to the wound.

(4) Subcutaneous implantation: For in vivo degradation tests and biocompatibility tests, samples (1.5 cm (w) × 1.5 cm (d) × 0.2 cm (h)) were printed out by a 3D DLP printer. The backs of rats were shaved and disinfected. Two incisions 1 cm long were created on the rostral side and caudal side on the dorsal midline of the backs of the rats. The separated subcutaneous pockets were made using Metzenbaum scissors. Printed c-Sil-MAS samples were placed in the subcutaneous pockets. At 2, 4, 10, and 16 weeks after implantation, the rats were euthanized, and the implanted c-Sil-MAS samples were harvested with the surrounding tissue. Explanted samples were washed with DW. For the degradation test (n = 3), excess tissue surrounding the samples was carefully removed. In vivo degradation was measured based on the sample size before and after implantation using a stereomicroscope (SMZ25, Nikon Co., Tokyo, Japan).

(5) Liver hemorrhage: For liver hemostasis surgery, rats were divided into two groups: the Sil-MAS group and the fibrin glue group. An abdominal midline incision was made after local disinfection. The liver was exposed and separated from the surrounding tissues by a retractor. Two of the three lobes of the liver were scraped out (0.5 cm deep and 0.5 cm diameter) using tissue forceps and a scalpel after punching with a tissue punch (diameter 0.5 cm). After free bleeding for 5 s, the damaged area was pressed gently with gauze for 3 s. Then, l-Sil-MAS (200 µL) and UV light (20 s) were sequentially applied to the wound immediately after removal of gauze. The fibrin sealant was prepared and applied to the wound according to the manufacturer’s instructions. At 1, 2, 4, or 8 weeks after surgery, the rats were euthanized, and the sealant-treated region, including peri-implant tissue, was resected from the rats for further histological analysis.

Laparoscopic surgery in a rabbit laceration model of liver and stomach serosa

Laparoscopic surgery was performed in a female New Zealand white rabbits weighing 2.8 kg. Anesthesia was induced with intramuscular ketamine (250 mg/5 mL, Huons, Seongnam, Korea) and Rompun injection (23.32 mg/mL, Bayer Korea, Seoul, Korea) and maintained with inhalational sevoflurane (2%) and oxygen mixed with room air (1.5 L/min) using a ventilator (Veterinary anesthesia ventilator, Solar Medical Tech, Taipei, Taiwan). The study was approved by the IACUC of Hallym University in Korea. A rabbit was secured to the table in the supine position. The CO2 insufflator and camera system with a 4 mm 0° endoscope (Karl Storz, Tuttlingen, Germany) was combined with the complete laparoscopic unit (MGB, Berlin, Germany). Three 5 mm laparoscopic trocars (Laport, Sejong Medical, Paju, Korea) were introduced into the abdominal cavity, and the insufflation pressure was established as 8–12 mmHg CO2 gas. The instruments used were endoscopic scissors, forceps, a suction–irrigation probe, a homemade endoscopic sealant device with fiber optics and a microsyringe pump. Multiple superficial or deep liver laceration wounds were made by endoscopic scissors. Bleeding liver laceration lesions were briefly pressed with gauze, and l-Sil-MAS (1 mL) and UV light (20 s) were applied by a homemade endoscopic sealant device. Rebleeding or leakage of blood at the lesions was checked using an endoscopic camera and forceps. In the same way, through laparoscopic operation, laceration of stomach serosa, Sil-MAS treatment, and checking the result were performed.

Western blotting

Twenty milligrams of frozen tissue (frozen with liquid nitrogen) was mixed with 1 mL of ice-cold RIPA buffer (with protease, dephosphorylase inhibitor cocktail) and homogenized using a hand homogenizer (Super Fast Prep-2TM, MP Biomedicals, Santa Ana, CA, USA). Then, the mixture was incubated at 4 °C for 2 h with agitation, and the supernatants were collected by centrifugation for 20 min. The protein concentration in the supernatants was measured using a Bradford assay (Bio-Rad, Hercules, CA, USA). The same amount of denatured protein sample was then loaded and separated on an SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Nonspecific binding was blocked using 10% skim milk for 2 h at room temperature. Then, the membrane was incubated with primary antibodies (1:1000) overnight at 4 °C. Primary antibodies for vimentin, fibronectin, VEGF, and cyclin D1 were purchased from Abcam (Cambridge, UK). β-Actin antibody was supplied by Sigma-Aldrich and used at 1:5000. Afterward, the membrane was incubated at room temperature for 1 h with species-matched secondary antibodies (goat anti-rabbit and goat anti-mouse IgG) (Enzo Life Sciences, Farmingdale, NY, USA). Finally, bands were detected by a chemiluminescence system (Fusion FX, Vilber Lourmat). Experiments were conducted using tissue samples from three different rats.

Histology

The harvested samples were fixed with 4% paraformaldehyde for 24 h, dehydrated in graded alcohols and xylene and then embedded in paraffin. Tissue samples five microns thick were prepared and stained with hematoxylin and eosin (HE) to analyze the re-epithelialization and inflammatory response and Masson’s trichrome (MT) to detect collagen production. Furthermore, to check tissue compatibility, samples harvested from liver and subcutaneous tissue at each time point were immunostained with anti-CD68 (Abcam, Cambridge, MA, USA) and anti-CD31 (Novus Biologicals, Centennial, CO, USA) primary antibodies and then with DyLight® 550-conjugated (Bethyl Laboratories, Montgomery, TX, USA) and FITC-conjugated secondary antibodies, respectively. The slides were mounted by Vectashield antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Stained sections were analyzed under a fluorescence microscope (Eclipse 80i, Nikon Co.).

Cytocompatibility assay and cell migration assay

To test the cytotoxicity of Sil-MAS depending on the UV treatment time and wound healing effect of Sil-MAS in vitro, the elution test method was applied. Two milliliters of l-Sil-MAS was prepared in 6-well plates, and UV treatment was administered for 10, 20, and 30 s. Then, 10 mL of a growth medium consisting of Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin was added and incubated in a 5% CO2 incubator at 37 °C for 72 h. This medium (Sil-MAS conditioned medium) was extracted and sterilely filtered with a cellulose acetate syringe filter (0.2 µm) to preserve all living cells. NIH/3T3 mouse fibroblast cells (Koram Biotech, ATCC distributor, Seoul, Korea) were cultured in the above growth medium. All cultures were maintained in a 5% CO2 incubator at 37 °C with the medium changed every three days. Cells were detached using trypsin-EDTA 0.25%, and 5,000 cells/well were plated in 24-well plates. After stabilization of cell attachment, the culture medium was replaced with 200 µL of the filtered Sil-MAS conditioned medium and cultured for 3 days without medium changes. The cell viability depending on culture time was examined by CCK-8 assay (Dojindo Molecular Technology, Rockville, MD, USA) according to the manufacturer’s protocol. As a positive control group, 200 µL of SF solution prepared at a concentration of 25% (w/v) in the growth medium was added to cells.

For the wound healing assay in vitro, NIH/3T3 cells were cultured until the confluence reached approximately 80–90%. A scratch was created using 200 µL micropipette tips. Immediately after the scratch, floating cells were removed by PBS washing, and 200 µL of Sil-MAS conditioned medium and growth medium (control) was added to each well. While cells were being cultured, images were captured at 3 h intervals for 15 h with a digital camera (EOS 100D, Canon, Tokyo, Japan) through an optical microscope (Eclipse 80i, Nikon Co.). The rate of migration was quantified through the total distance that the NIH3T3 cells moved toward the center of the wound from the edge of the wound within 15 h.

Statistical analysis

Samples for each group were assessed in triplicate to analyze the statistical data. The data are presented as the mean value ± standard deviation (s.d.) (*p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001). Curve fitting was performed using Prism, version 6.0 software (GraphPad, San Diego, CA, USA), and one-way ANOVA followed by the Tukey multiple comparisons test was used to assess significant differences among multiple groups.



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