The experiments were carried out at the Biomedical Research Institution of Seoul National University Hospital (IACUC approval number 22-00770-S1A1). All experiments were performed in accordance with relevant guidelines and regulations. Fifteen 8-week-old male Sprague–Dawley rats were randomly divided into three groups according to the implant coverage technique: control (n = 5), whole wrapping (n = 5), and anterior tenting (n = 5) (Fig. 4).
Surgical procedure
Smooth-type hemispherical implants with a diameter of 1.5 cm were prepared. Non-meshed ADM (BellaCell HD, Hans Biomed Corp., Seongnam, Korea) with thickness of 1.8–2.99 mm was used. The animals were subjected to general anesthesia with 3% isoflurane. Xylazine (5 mg/kg) was intramuscularly administered. They were maintained under controlled ventilation with 1–1.5% isoflurane. A prophylactic dose of cefazolin (120 mg/kg/dose) was also administered intraperitoneally after adequate anesthesia was achieved. After shaving the dorsum of each animal, skin preparation was performed with betadine in a sterile fashion. We dissected two pockets at the back of each rat: one left and one right of the midline. Each animal received two implants below the panniculus carnosus. In the whole wrapping group, the implant was covered by the ADM ex vivo secured with absorbable sutures (4 − 0 Monosyn), and the unit was secured using a 3 − 0 Vicryl suture at the 6 o’clock position. In the anterior tenting group, the ADM was placed over the skeletal muscle of the back and secured using three cardinal sutures at 12, 3, and 9 o’clock positions with a 3 − 0 Vicryl suture to form a pre-muscle pocket. After implant insertion, the pocket was closed with a 3 − 0 Vicryl suture at the 6 o’clock position.
Radiation protocol
The aim of our radiation protocol was to simulate the effects of adjuvant irradiation following mastectomy. For experimental purposes, the recommended radiation dose typically ranges between 14 and 25 Gy.24 In clinical practice, the conventional radiotherapy protocol delivers a total dose of 50 Gy in 25 fractions of 2 Gy to the chest wall. By using the linear-quadratic concept, we can compare the tissue effects of different fractionation regimes. This allows us to calculate the biological effective dose (BED) and determine that our radiation protocol yields a similar BED to conventional radiation therapy.25 In our study, the total dose of 23.25 Gy was divided into three fractions of 7.75 Gy each, administered over a period of 5 days. One of the two implantation sites for each animal was randomly selected and irradiated externally using a 6-MeV electron beam collimated by a 6×6 cm cone at a source-to-surface distance of 100 cm (VitalBeam, Varian, USA) 3 weeks after surgery.
Applanation Tonometry
At the three-month follow-up after surgery, the animals underwent intraprosthetic pressure evaluation using applanation tonometry. Tonometry has been effectively utilized to measure the pressure within the mammary glands.26 Tonometry was performed as described by Moore in 1979.27 A glass disc with a diameter of 5 cm and thickness of 2.5 mm was placed on the skin at the location of the implant. The skin was marked with gouache paint and a sheet of paper was placed between the glass disc and the painted skin surface. The flattened area was calculated using ImageJ software. It is expected that a larger flattened area would indicate lower tension within the implant pocket.
Histopathologic analysis
The animals were then euthanized through exposure to high concentrations of carbon dioxide 3 months postoperatively. The tissues were removed by cutting the skin along the curved surface of the implant. In the control group, specimens were collected, including the panniculus carnosus. In the group where the implant was covered with ADM, specimens were obtained as a whole, including the ADM. The collected tissues were then fixed for histological examination. Histopathological analysis was performed on the capsules to assess capsule thickness, collagen density, myofibroblast, and inflammation. Each sample with the implant in place was fixed in 10% buffered formalin. After fixation, the capsules were separated from the implants and carefully embedded in paraffin blocks. All specimens were taken, including the whole aspect (anterior and posterior surfaces) of the capsule and ADM at the midline (Supplement Fig. S3). The properties of the capsule and ADM were examined by categorizing them based on their respective orientations. This involved examining the anterior surface, where the ADM was in contact with the skin flap, and the posterior surface, where the ADM interacted with the underlying skeletal muscle. The specimens were examined histologically for thickness and collagen density, and immunohistochemical staining was used to examine the α-smooth muscle actin (α-SMA) positive area, the CD3 positive cell count, and the F4/80 positive area. Three areas were analyzed for each anterior and posterior surface. To quantify the area of the region of interest, which was stained, ImageJ software was used for quantitative analysis. For the evaluation of the cellular capsule, a region of 0.01 mm2 was analyzed. Similarly, for the assessment of ADM, a region of 0.102 mm2 was analyzed.
(1) Hematoxylin and eosin staining
Sections (5-µm thick) were cut and stained with hematoxylin and eosin to examine their histopathological characteristics. Using the NIKON ECLPSE Ci-L microscope, the thickness of the total capsule and the cellular capsule were evaluated. The cellular capsule refers to the parallel layers of collagen fibers that forms at the interface between the ADM and the implant. In contrast, the total capsule refers to the complete implant pocket, including the ADM, which is excised as a whole during the surgical procedure (Supplement Fig. S4).
(2) Masson’s trichrome staining of collagen fibers
Another set of capsule samples was stained with Masson’s trichrome after fixation in 10% formalin solution for 24 h. Samples were sectioned into 5-µm sections. The collagen fibers were stained blue, the nuclei were stained black, and the background was stained red. The stained collagen fiber area was quantified per respective region as collagen density using ImageJ software.
(3) Immunohistochemistry for α-SMA, CD3 and F4/80
The level of myofibroblast involvement in the capsule and ADM was examined using the anti-SMA antibody (ab5694, Abcam, Cambridge, MA, USA) as the primary antibody. The CD3 antibody (ab5690, Abcam) was used as the primary antibody to determine the degree of inflammation by examining T cell count. The F4/80 antibody (ab300421, Abcam) was used as the primary antibody to determine the degree of macrophage infiltration. Tissue sections were cut and mounted on slides and then stained using the Discovery XT automated immunohistochemistry stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA).
Statistical Analysis
Data are expressed as mean ± SD for continuous variables. GEE were used to verify the interaction between the therapy factor (coverage technique and radiation) and result variables (tonometry, capsule thickness, collagen density, level of α-SMA and F4/80, and CD3 positive cell count). The interaction effect (Group * radiation status) was assessed, and if no significant interaction was found, the significance of each factor (Group and radiation status) was evaluated after removing the interaction effect. Statistical significance was considered at p < 0.05. In cases where the GEE analysis revealed statistical differences among the groups, post-hoc analysis was performed using Bonferroni correction (adjusted for p < 0.05). All analyses were performed using SAS statistical software (SAS system for Windows, version 9.4; SAS Institute, Cary, NC).