Ethics approval
The animals in this study were maintained at the Research Center for Animal Life Science, Shiga University of Medical Science. The experimental protocol was approved by the Institutional Animal Care and Usage Committee at Shiga University of Medical Science (Permit Number: 2019-7-10 (H3)) and was in accordance with the ARRIVE guidelines. The number of animals used in this study was kept to a minimum and all efforts to reduce animal suffering were made in accordance with the protocols established by the Research Center for Animal Life Science of Shiga University of Medical Science.
Preparation of the bioabsorbable implants
The PLLA mesh implant containing CS (PELNAC®, Gunze Ltd., Tokyo, Japan) was prepared in a prolate spheroidal shape as previously reported[13]. Each columnar mesh was 1 cm in diameter and 1 cm in height and was knitted using a 2–0 PLLA thread (Gunze Ltd.). One side of the mesh was closed using purse string sutures. After tightly packing the mesh with 40 mm x 20 mm x 3 mm CS with a porosity of 80–95%, the other end of the mesh was closed using purse string sutures. The polar diameter of the implant was approximately 18 mm, and the greatest equatorial diameter of the implant was approximately 7.5 mm. The implants had multiple 1.5-mm square openings.
Next, 12- 0.015-mm thick PGA multi-filaments were woven into an envelope-shaped bag. We prepared two flat PGA-woven bags that were 110 mm x 35 mm. Thirty pieces of the implant were packed into each PGA woven bag, and two pieces of the implant aggregate were shaped into a cylinder with a diameter of 2.4 cm and a height of 9.0 cm (Figure 5).
Animal experiment
1. Experimental design and operative procedure
A minipig, CLAWN miniature swine, was chosen for this study due to its large body surface and skin that is similar to human skin. One 10-month-old female CLAWN miniature swine was purchased from the Kagoshima Miniature Swine Research Center. After a 2-week preservation period, the minipig weighed 26.3 kg. The animal was cared for as outlined in the Public Health Services Policy on Humane Care and Use of Laboratory Animals. General symptoms such as feeding status and limping were assessed daily.
The minipig was not fed overnight prior to the implantation. Sedation was achieved via an intramuscular injection of 25 mg/kg ketamine (KETALAR®, DAIICHI SANKYO Co., Ltd., Tokyo, Japan) and 0.02 mg/kg medetomidine (DOMITOR®, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan). After sedation, a peripheral intravenous line and a tracheal tube were inserted. The minipig’s SpO2 was monitored, and the minipigs were anesthetized by the inhalation of a mixture of air and oxygen containing 2.0 - 2.5% isoflurane. After shaving the abdominal region, a 6-cm midline incision was made in the abdomen starting 1 cm caudal to the umbilicus (Figure 3a). Next, the fat tissue was incised, and a pocket was prepared over the fascia on each side of the abdomen. Once the implant aggregates were placed into the pockets, the fat and skin were closed with 2-0 blade nylon sutures (Nurolon: Johnson & Johnson K.K., Tokyo, Japan). After this study period, the minipig was used for an additional study.
2. Evaluation of the implant aggregates
The area of implantation and the appearance of the implant aggerates were evaluated under general anesthesia using MRI, ultrasound, and 3D surface imaging prior to implantation, immediately after implantation, and 1, 3, and 6 months after implantation. The tissue from just below the skin to the fascia, including the implant aggregate, on the right side of the abdomen was harvested six months after implantation. The implant aggregate in the left side of the abdomen was preserved for the next study.
2.1. MRI procedure
The minipig was placed on the examination table in the MRI room in the supine position. The mini pig's abdominal part was scanned by a wide-bore 3 Tesla (T) MRI scanner (Magnetom Verio 3T, Siemens Healthcare, Erlangen, Germany) using a dedicated 4ch large flex coil. The scan acquired WI and WE images in the transverse plane using T1-weighted Dixon imaging (TR/TE = 5.26/2.46 ms; flip angle = 10°; acquisition matrix = 352 × 172; field of view (FOV) = 285 × 350 mm2; slice thickness = 1.0 mm). During the scanning, breathing was controlled using an intravenous injection of 0.4 mg/kg rocuronium bromide (ESLAX®, MSD K.K., Tokyo, Japan). The obtained images were loaded to 3D slicer[27], a software package for the analysis of medical images, in order to calculate the volume of the implant aggregate and the volume of the newly-formed adipose tissue inside the implant aggregate.
2.2. Ultrasonography protocol
An ultrasonographic system (ACUSON S2000 HELX Evolution: Siemens Healthcare K.K., Tokyo, Japan) with a 9L4 probe were used to evaluate the tissue formation inside the implant aggregate.
2.3. Three-dimensional surface imaging protocol
Three-dimensional surface images were obtained using 3D imaging solution (Vectra H2: Integral Corporation., Tokyo, Japan). The volume change of the implantation area between pre- and post-implantation was assessed from the cranial edge of the umbilicus to the inguinal ligament (Figure 3b).
3. Histological assessment of the newly-formed tissues inside the implant aggregate
The harvested specimen was fixed with 4% paraformaldehyde phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The specimen was divided into four equal blocks along the long axis of the implant aggregate. The second block from the caudal side was embedded in optimum cutting temperature compound (Sakura Fine Technical Co. Ltd., Tokyo, Japan) and frozen in ethanol dry ice prior to oil-red-o staining. The three remaining blocks were paraffin-embedded for subsequent H&E, AZAN, and immunohistochemical staining. The 16-μm-thick frozen section from the central region of the tissues was prepared for oil-red-o staining. The 5-μm-thick sections from three aspects of the specimen were prepared for H&E, and the 5-μm-thick sections at the central aspects of the specimen were prepared for AZAN and immunohistochemical staining. Immunohistochemical staining of CD31 was performed to evaluate the formation of new capillaries in the newly-formed tissue. After the deparaffinization and rehydration of the 5-mm-thick paraffin sections, they were immersed in diluted target retrieval solutions (415211; Nichirei Biosciences Inc., Tokyo, Japan) and incubated for 20 min at 98° C. After being cooled to room temperature, the sections were rinsed once in distilled water and immersed in 3% hydrogen peroxide (FUJIFILM Wako Pure Chemical Industries Ltd.) and methanol (FUJIFILM Wako Pure Chemical Industries Ltd.) for 10 min to block endogenous peroxidase activities. The sections were then rinsed in distilled water and 50 mM Tris-HCI buffered saline (Takara Bio Inc., Kusatsu, Japan) with 0.05% Tween 20 (FUJIFILM Wako Pure Chemical Industries Ltd.) and 0.15 M NaCl (TBST). To block nonspecific protein binding, 3% bovine serum albumin (BSA) diluted with phosphate buffered saline (PBS) was applied for 60 min at room temperature. A monoclonal mouse anti-rabbit CD31 antibody (ab182981: Abcam plc., Tokyo, Japan) was applied as the primary antibody at a dilution of 1:10,000 using 1% BSA in PBS and incubated overnight at 4° C. The sections were rinsed in TBST. Next, a peroxidase-labeled secondary antibody (rabbit anti-goat simple stain MAX PO [R]; Histofine; Nichirei Biosciences Inc.) was applied for 30 min at room temperature. Sections were then rinsed with TBST and exposed to 3,30-diaminobenzidine tetrahydrochloride (Dako Japan Co., Ltd., Tokyo, Japan) for 5 min at room temperature and counterstained with hematoxylin. All microphotographs were obtained using a light microscope (IX83: Olympus corporation, Tokyo, Japan) at 40x magnification. The newly-formed tissues and the newly-formed adipose tissue inside each implant were evaluated on the microphotographs of the H&E stained-sections using ImageJ software version 1.53 g (National Institutes of Health, Bethesda, Maryland, USA). The data were expressed as mean and range.