This experimental study was performed at the Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University, USA. Thirty murine and ten human capsules (both of female gender, respectively) around microtextured breast implants were examined with SEM, light microscopy, and TEM at different points in time: day 15, day 30, and day 90 in murine capsules and Baker IV in human capsules. Cell invasion and collagen fiber alignment were assessed.
Animals
All animals were treated humanely, and protocols used were approved a priori by Institutional Animal Care and Use Committee at Stanford University (IACUC) and Stanford University's Administrative Panel on Laboratory Animal Care (Protocol No. 28410) according to National Institutes of Health and institutional guidelines. Six-week old female wild-type C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed in sterile micro-insulators. Food and water were provided ad libitum in accordance with institutional guidelines of Stanford University animal care. Ten mice per group were used per each experiment and the experiments were run three times to verify findings.
Human Breast Tissue
Human breast capsules were received in FFPE (Formalin Fixed Paraffin Embedded) blocks from the Institute of Pathology, University Hospital Regensburg, Germany. Approval was given by the local ethic committee in Regensburg (Reference No.: 15-101-0024).
Scanning Electron Microscopy (SEM)
For SEM analysis, specimens (breast implants and explanted capsular samples) were rinsed in PBS, fixed overnight in 4% Paraformaldehyde with 2% Glutaraldehyde in 0.1 M Sodium Cacodylate Buffer (pH 7.4), rinsed in the same buffer and post-fixed for 1 hour with 1% aqueous OsO4. After dehydration in an ascending ethanol series (50%, 70%, 90%, 100% (twice); 5 minutes each), small tissue (capsule) pieces were critical point dried (CPD) with liquid CO2 in a Tousimis Autosamdri-815B apparatus (Tousimis, Rockville, MD), while implants which exceeded the CPD chamber size were treated with HMDS (hexamethyldisilazane) for 30 minutes (2 x 15 minutes), before overnight drying in a desiccator. All specimens were mounted onto 15 - 50mm circular aluminum stubs (Electron Microscopy Sciences, Hatfield, PA), and sputter-coated with 50Å of gold-palladium using a Denton Desk II Sputter Coater (Denton Vacuum, Moorestown, NJ). Scanning Electron Microscopy (SEM) images captured with either a Hitachi S3400-N Variable Pressure SEM (Hitachi High-Tech, Dallas, TX) operated at 10kV accelerating voltage and using secondary electron detection or a Zeiss Sigma Field Emission SEM (Carl Zeiss Microscopy, Pleasanton, CA) operated at 3-5kV accelerating voltage and using InLens Secondary Electron (SE) and Everhard Thornly (SE2) detection. While SEM procedure can cause dehydration artifacts that could negatively impact the tissue processing, histology with respective SEM images of the same sections was performed to ensure that the collagen alignment and hydration state match the fibrotic profile observed in tissue.
Transmission Electron Microscopy (TEM)
Samples were fixed in Karnovsky’s fixative: 2% Glutaraldehyde (EMS Cat# 16000) and 4% Formaldehyde (EMS Cat# 15700) in 0.1M Sodium Cacodylate (EMS Cat# 12300), pH 7.4, for 1 hour, chilled, and sent to Stanford’s CSIF on ice. They were then warmed to room temperature (RT) in cold 1% Osmium tetroxide (EMS Cat# 19100) for 1 hour while rotating in a hood, washed 3x with ultrafiltered water, then en bloc stained overnight in 1% Uranyl Acetate at 4°C while rotating. Samples were then dehydrated in a series of ethanol washes for 30 minutes each at 4°C beginning at 50%, 70%, 95% where the samples were then allowed to rise to RT, changed to 100% twice, then Propylene Oxide (PO) was applied for 15 minutes. Samples were infiltrated with EMbed-812 resin (EMS Cat#14120) mixed 1:2, 1:1, and 2:1 with PO for 2 hours each and were subsequently left in 2:1 resin to PO overnight, rotating at RT in a hood. Samples were then placed into EMbed-812 for 2 to 4 hours before being placed into molds with labels and fresh resin, oriented, and placed into 65°C oven overnight.
Sections were taken between 75 and 90nm, picked up on formvar/Carbon coated slot Cu grids, stained for 30 seconds in 3.5% Uranyl Acetate/ 50% Acetone, followed by staining in 0.2% Lead Citrate for 3 minutes. Sections were observed in the JEOL JEM-1400 120kV with photos being taken with a Gatan Orius 832 4k X 2.6k digital camera with 9µm pixel size.
Histology
Mice were sacrificed with CO2 asphyxiation and cervical dislocation. Murine capsule (tissue) was harvested on day 15, day 30 and day 90 after placing the silicone implant and fixed in 4% paraformaldehyde overnight at 40°C. The samples were fixed in 4% paraformaldehyde in phosphate buffered solution/saline (PBS) overnight, washed twice with PBS and dehydrated in a 30% sucrose solution for 24 hours. Samples were processed routinely and embedded in paraffin. Sections were cut at 1µm and 3µm serially for histology staining. Sections were stained with toluidine blue, which has a high affinity for acidic tissue components, such as tissue rich in DNA and RNA. Standardized protocols were used for these stainings with no modifications. Each section was visualized under light microscopy at 5×, 10× and 20× (Leica microscope, Leica DM 4000B; Leica Microsystems, Buffalo Grove, Ill) and photographed using the Leica DFC 500 camera (Leica, Allendale, NJ).
Immunohistochemistry
For immunohistochemistry paraffin-embedded slides were de-paraffinized and washed twice with 0.25% Triton X-100 diluted in tris buffered saline (TBS) at room temperature for 5 minutes each. The slides were then blocked with 5% anti-donkey and 5% anti-goat antibodies to reduce non-specific binding of the secondary antibodies for 2 hours at room temperature. Primary antibodies (rabbit anti mouse Col 1 (Abcam, ab34710) and rat anti mouse F4/80 (Abcam, ab6640)) diluted 1:500 in TBS with 1% bovine serum albumin (BSA) were added to the sections and allowed to incubate at 40°C overnight. After incubation, the slides were washed twice with 0.25% Triton X-100 diluted in TBS at room temperature for 2 minutes each. The secondary antibodies (goat ant-rat AF488 (Thermofisher, A-11006) and (donkey anti rabbit AF594 (Thermofisher, A-21207)) diluted 1:500 with TBS with 1% BSA were added at room temperature for 1 hour. During this time the sections were kept in the dark to prevent photo-bleaching. The slides were rinsed twice with TBS at room temperature for 5 minutes each. Following the secondary stain, the slides were stained with DAPI (DAPI, Biolegend, B222486) diluted 1:1000 in TBS at room temperature for 10 minutes. The slides were rinsed twice with TBS at room temperature for 5 minutes each. Aqueous mounting media (Fluoromount G, eBioscience, E099088) was used to mount the tissues. Slides were imaged using an SP8 inverted confocal microscope. ImageJ software (US National Institutes of Health) was used to reconstruct and quantify the confocal images.