Alcohol types

Impact on capsule formation for three different types of implant surface tomography

The animal experimentation was conducted after review and approval by the Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF) Institutional Animal Care and Use Committee (Approval Number: DGMIF-19073103 -01). This study was performed in accordance with ARRIVE guidelines and all experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Breast implants

The experiment was conducted using a specially fabricated hemispherical implant (Hansbiomed Co., Ltd., Seoul, Korea) with three different types of topographies (smooth surface, macrotexture and nanotexture) with a diameter of 2 cm and a volume of 2 cm3. The surface of three different implants is made of polydimethylsiloxane and does not contain any other material. Additionally, a location-determining tantalum marker was inserted into the implants to detect their location (Fig. 7).

Picture 7

Three types of implants and surgical implantation procedure. (A) Smooth, (B) macrotexture, and (VS) types of nanotexture: Three types of implants with different topographies which have tantalum directional marker specially made by Hans Daeduk Engineering Research Lab (Daejeon, Korea), hemispherical shape, 2cm diameter and 2cm volume3. (D) The design was made on the dorsal area of ​​the Wister rats, while the incision line was approximately 2 cm where the implant should be located. (E) Metzenbaum dissection was performed under the panniculus carnosus muscle. (F) The implant was placed under the panniculus carnosus muscle.

Surface topography measurement

The surface of each implant was confirmed using an electron microscope (scanning electron micrographs, S-4200, Hitachi, Tokyo, Japan). Shell samples with a diameter of 1.5 cm were acquired from each of three different types of implants (smooth surface, rough surface and nano-textured surface). The acquired samples were coated with Pt after washing with isopropyl alcohol and then dried. Surfaces measured at 50, 100 and 300 times under 10.0 kV and 100 times with an angle of inclination of 20° were analyzed.

In addition, the implant surface roughness was measured using a confocal 3D laser scanning microscope (LEXT OL5000, Olympus Corporation, Tokyo, Japan). Based on surface topography (smooth surface, rough surface and nano-textured surface), 2 cm2Medium-sized shell samples were acquired from each of three different implant types, which were then washed with isopropyl alcohol and dried. Then, a 2 mm × 2 mm area of ​​the samples was measured using 20 × lenses, and an analysis was performed on the roughness of the measured surface.

Animal experimentation

A total of 48 10-week-old Wistar rats (Orientbio, Seongnam, Gyeonggido, Korea) weighing 200-250 g were each subjected to general anesthesia by intraperitoneal administration of a mixture of 10 mg xylazine hydrochloride/kg ( Rompun®; BayerKorea, Korea) and 30 mg tiletamine/zolazepam/kg (Zoletil®50; Virbac, Korea). A 2 cm diameter incision was made in the back through which a hemispherical silicone implant was inserted under the panniculus carnosus.

Rates were categorized into a total of six experimental groups: Group A in which the smooth surface implant was used and capsule extraction was performed 4 weeks after implantation; group D at 12 weeks later; groups B and E in which the macrotextured surface implant was used and capsule extraction was performed 4 and 12 weeks after implantation, respectively; and groups C and F in which the nanotextured surface implant was used and capsule extraction was performed 4 and 12 weeks after implantation, respectively. Additionally, computed tomography (CT) (Quantum FX) was performed at 1, 2, 4, and 12 weeks post-implantation to determine changes in the location of the embedded implant over time. This animal experiment was approved by the Institutional Animal Care and Use Committee of the Daegu-Gyeongbuk Medical Innovation Foundation (Approval No. DGMIF-19073103-01) and was conducted in accordance with the recommendations of the committee.

Histological examination

Paraffin blocks were fabricated within 24 h of fixation of the extracted capsular tissues with the implants in 10% paraformaldehyde. Tissue sections with a thickness of 3 μm were made from all the samples. Then, hematoxylin and eosin (H&E) staining and Masson’s trichrome staining were performed. For analysis, digital images of the tissue samples were created using a slide scanner (Axio Scan. Z1; Carl Zeiss A/S, Birkeroed, Denmark). To measure capsule thickness during H&E staining, a 1200 × 800 μm area was defined in each digital image using Zeiss Zen Ver software. 2 (Carl Zeiss GmbH, Jena, Germany). Subsequently, all analyzes used 25 stained slides for each group and the average thickness of six parts at 200 μm intervals was calculated for each capsule. Additionally, the density and arrangement of collagen fibers in the capsules in Masson’s trichrome staining were confirmed using Zeiss Zen Ver software. 2. After selecting five different parts in the same image, the collagen-stained areas were measured using the ImageJ program (1.43 u; http://rsbweb.nih.gov/ij/). Finally, a comparative analysis was performed based on the aforementioned data.

Immunohistochemical examination

To analyze myofibroblasts, which are factors related to fibrosis, an anti-smooth muscle (SMA) antibody (ab12512; Abcam, Cambridge, MA, USA.) was used as the primary antibody and DAKO K4001 was used as the secondary antibody. . The extracted capsule tissues were fixed in 10% paraformaldehyde, immersed in 95% ethanol for 5 min, and then fixed by washing in distilled water. They were treated in hydrogen peroxide for 5 min, washed with Tris-buffered saline (TBS), cultured after addition of the primary antibody, and finally washed with TBS. The secondary antibody was then added, after which tissue samples were cultured. They were then analyzed after washing with distilled water and counterstaining with hematoxylin after staining with a substrate-chromogen solution.

For sample analysis of the anti-SMA antibody, digital imaging was performed on a tissue sample using a slide scanner (Axio Scan. Z1; Carl Zeiss A/S, Birkeroed, Denmark ), after which the degree of each expression was identified using Zeiss Zen software Ver. 2. and ImageJ program (1.43 u; http://rsbweb.nih.gov/ij/).

Western blot examination

Proteins were extracted from formalin-fixed paraffin-embedded (FFPE) tissue using the Qproteome FFPE Tissue Kit (QIAGEN, Mettmann, Germany) according to the manufacturer’s instructions. The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% skimmed milk powder in Tris-buffered saline [20 mM Tris–HCl and 137 mM NaCl (pH 7.6)] containing 0.1% Tween-20 (TBS-T buffer) for 2 h at room temperature. The membranes were then incubated with antibodies against TGF-beta 1 (Abcam, Cambridge, UK) or β-actin (Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. were washed three times with TBS-T buffer and incubated with secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. After the membranes were washed three times, protein bands were detected using chemiluminescence reagent (Thermo Scientific, Waltham, MA, USA). Bands were quantified using the ImageJ program (1.43u; http://rsbweb.nih.gov/ij/).

Location measurement

To confirm changes in the location of the integrated implant over time, micro-CT (Quantum FX) was performed 1, 2, 4, and 12 weeks after implantation. To determine location changes, the degree of rotation of each implant was measured. For this purpose, the tip of the compass inside the implant was marked using tantalum, while the angle of rotation was measured clockwise or counterclockwise. clockwise according to the points marked on the compasses during implantation. A degree of rotation not exceeding 180° has been selected.

statistical analyzes

The measured values ​​were statistically analyzed using SPSS 23.0 (IBM, Armonk, NY, USA) and presented as mean ± standard deviation. Two-way ANOVA and Tukey’s multiple comparisons post-hoc tests were used to determine significant differences between experimental groups, with P

Ethics approval for animal experiments

The animal experimentation was conducted after review and approval by the Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF) Institutional Animal Care and Use Committee (Approval Number: DGMIF-19073103 -01). This study was performed in accordance with ARRIVE guidelines and all experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.