Gel Bleed and Rupture of Silicone Breast Implants Investigated by Light-, Electron Microscopy and Energy Dispersive X-ray Analysis of Internal Organs and Nervous Tissue

C l i n M e d International Library Citation: Kappel RM, Boer LL, Dijkman H (2016) Gel Bleed and Rupture of Silicone Breast Implants Investigated by Light-, Electron Microscopy and Energy Dispersive X-ray Analysis of Internal Organs and Nervous Tissue. Clin Med Rev Case Rep 3:087 Received: December 29, 2015: Accepted: February 06, 2016: Published: February 09, 2016 Copyright: © 2016 Kappel RM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Volume 3 | Issue 1


Introduction
Some women, who have received silicone breast implants either for breast augmentation or breast reconstruction, develop health problems in different gradations over the years and a thorough explanation for this has yet to be given [1][2][3] this is because this kind of research can only be performed on living humans. In addition, as the existence of associated complaints is still largely denied and neglected by the various medical disciplines, they are not recorded in medical histories of the involved women and thus do not appear in meta analyses [4] So, only combining into absolute alcohol. Next, the slides were rinses in demineralized water. For the MORO staining an oversaturated colouring solution of Oil Red O (0.5 g In 100 ml 100% 1,2-propanediol) was used, which acts like a solvent. The silicone polymers, which likely have fatty rest groups, are stained by the oil red O, because the Oil Red O is better resolved in the silicone polymers than in the 1,2-propanediol. The oil Red O moves from the relatively polar solvent to the non-aquatic polymers of the silicone. The principle of this staining thus is the physical binding between the Oil Red O molecules and the silicone polymers. After 5 days, the slides were agitated and differentiated in 85% 1,2-propanediol. After rinsing in tap water, the slides were stained with Hematoxylin Mayer to counter stain the nuclei in the surrounding tissue. After 10 minutes of rinsing in tap water, the slides were dipped in a 1% acetous alcoholic solution for further differentiation. After this, the slides were rinsed for 4 minutes and dipped in saturated lithium carbonate solution (4 g lithium carbonate the liver, ribs, vertebrae and hypothalamus. In the end, this caused her death. For orientation purposes in this new type of study, a second case was introduced, acting as a 'positive control'. This patient had silicone implants for 14 years and upon removal both implants were ruptured. The peri-prosthetic capsule only could be harvested. Of both patients in this study the samples were processed (HE, MORO and Toluidine blue staining) and assessed by light microscopy. Subsequently, EDX measurements were performed to quantify the amounts of silicone as found in the TEM.

Light microscopy
The modified Oil Red O (MORO) is a histological staining, used to visualize silicone. This was initially performed on frozen sections to maximize detection as paraffin preparation can dissolve some of the silicone. Paraffin sections were deparaffinized before staining with MORO. This was done with xylene for 15 minutes and 3 dips Colon

Septum and ventricular wall
-: Negative or no findings; NE: Not embedded; + to +++: High amounts of silicon in 100 ml demineralized water). In the next and final step the slides were rinsed for 4 minutes in tap water and covered with gelatineglycerin, a water based cover medium. Positive staining is seen as bright and deep red staining.

Electron microscopy
The plastic (Epon) embedded tissues were fixated in a 2% glutaraldehyde 0.1 M sodium cacodylate buffer solution with a pH of 7.4 for a minimum of 4 to a maximum of 24 hours [8]. Removal of free non-reacted aldehydes was done by a washing step in 0.1 M sodium-cacodylate buffer solution with pH range of 5.0 -7.4. All tissues were dehydrated in an ethanol-propylene oxide series and manually imbedded in EPON, an epoxy resin. EPON polymerizes for 1 night at 45°C and for 2 days at 67°C in an incubator [9]. Semi-and ultra thin sections were obtained with a Reichert-Jung ultramicrotome by using a Histo diamond knife for 1 μm semi thin sections and an Ultra diamond knife for the 90-200 nm ultrathin sections. Semi thin 1 μm sections were stained with toluïdin blue. This staining gives a clear overview of the semi thin sections and can be used to visualize areas of interest to be viewed in the ultrathin TEM sections or measured with EDX. Ultra thin 90-200 nm sections were collected on membrane coated (polyvinyl formal in 1% ethylene dichloride) 3.05 mm 100 mesh copper grids. The 90 nm ultrathin cuts were additionally contrasted with 4% uranyl acetate solution for 30 minutes and lead citrate for 6 minutes so they obtained the desired contrast for morphological examination. The uncontrasted 200 nm ultrathin cuts were used for EDX measurements. Specimen were next studied with a Jeol (JEM-1200 EX II TEM/STEM) Transmission/ Scanning Electron Microscope operating at 64 kV with Energy Dispersive X-ray (EDX) equipment.

Results
Droplets and plaques containing silicone were found in the random parts of the sampled tissues. They could be seen in the HE, MORO and Toluïdin blue through light microscopy. An overview of silicone containing organs and tissues of our patient and the findings in TEM/EDX analysis is presented in table 1. Table 1 is a summary of positive silicone containing material. The plaques are comprised of elemental silicon and titanium. The vacuoles (droplets) are comprised of elemental silicon. Thus we identified two variants of silicone containing material. These were also found in macrophages. Different stainings are the MORO silicone staining and Sudan Black/ Normal ORO fat stains which have been performed on a silicone positive paraffin control, consisting of a peri prosthetic capsule. Also electron microscopical investigation and EDX analysis was done on this positive control. Questions about the specificity of the MORO staining for silicone in freeze and paraffin cuts could thereby be established ( figure 1).
The Modified Oil Red O (MORO) staining was initially performed on 4 μm freeze cuts from frozen material of our patient, acquired in body autopsy. This first staining was performed to check if any positive material is present and stainable. Results of this first MORO staining show large quantities of positive stained material in a large range of tissues. (Table 1 and Figure 2).
To ensure that the frozen material from patient 1 also contains the silicone after treatment with different fixatives we performed MORO staining on different samples. As can be seen in all panels there is a significant amount of positive material present (Figure 3).
Also different lymph nodes were excised for histological examination and showed extensive foreign body reaction in the presence of many epithelioid multinucleated giant histiocytes and the presence of translucent material, suggestive for silicone. MORO positive stained material is located inside vacuoles and histiocytes ( Figure 4). Samples are cut approximately 100 and 200 nm thick for assessment in TEM and analysis by EDX. EDX analysis is performed on this lymph node material to see if large amounts of elemental silicon is measurable. (Figure 5).
EDX analysis reveals the true nature of the plaque like structures found in the toluïdin blue stain, elemental silicon and titanium were found in all the plaque like structures. The found component of elemental titanium represents the dens particles located inside the plaques. Also loose droplets of elemental silicon were found, situated partially in vacuoles, for instance in the thyroid gland, figure 6.
The plaque like structures found in the spinal cord of patient 1 are located inside the surrounding spinal cord tissue. The TEM micrographs demonstrate the plaque like structure and a higher magnification of a TEM micrograph show small particles inside the plaque, clearly visible around the asterisk. EDX analysis is performed

Discussion
Gel bleed is a phenomenon that is inherent to all types or models of silicone breast implants, regardless whether they are soft and round or cohesive anatomically shaped [10][11][12][13][14]. The bleed retardation layer in the late models, retards the bleeding, but does not abolish it. The bleeding silicone polymers behave like softeners and eventually weaken the silicone elastomeric shell of silicone breast implants, regardless of the brand [15]. This could be the reason why spontaneous ruptures occur. It goes without saying that implant rupture is an intensified form of gel bleed. Table 1 gives a good impression of the dissemination of the silicone polymers found throughout the body. This also gives an impression of the Si-presence, although the amount can be variable, depending on which part of the organ analysis had been performed. As a starting point, EDX-measurement was done on pure silicone gel. The average Si-net counts of 4 measurements was approximately 345,500 counts (data not shown). In contrast, EDX measurements in control tissues of patients without silicone breast implants, always levelled below 500 net counts. With that in mind we maintained a safe threshold of 1000 silicon net counts in the tissues examined. Counts below 1000 were considered negligible and can act as a negative control, whereas net counts beyond 1000 represent Silicon stacking and thus are of pathological interest. Epidemiological studies have not been able to show a statistical significant  it is nicely demonstrated that there are actually droplets present located inside vacuoles which appear to be partially washed out; (G) A higher magnification of a TEM micrograph were it is clearly seen that the droplets actually are located inside a vacuole and are part of the tissue. EDX analysis is performed on these droplets, figure D shows a TEM micrograph of the EDX measuring points performed on the thoracic spinal cord of patient 1; (F) Demonstrates EDX analysis Point 1 on a droplet found inside vacuolated spaces (spectra 1) = 22211 Si-counts. Point 3 is on the surrounding tissue (spectra 3) = 195 Si-counts (H). Original microscopic magnifications figure A and B 400x, resp. 200x, C-E and G; resp. 50 x, 2.5K and 10K.
connection between silicone breast implants and "silicone Implant Incompatibility Syndrome" [3]. The common conception seems to be that if the connection cannot be demonstrated epidemiologically, it has to be absent. Studies to elucidate the silicone issue, should at least document the health complaints that surface in some women with silicone breast implants and follow them for many years. However, women with health complaints are present and when the transplants were explanted they often report an improvement of their condition. What is also remarkable, is that hours after explantation surgery, the pre-operative complaints can first increase tremendously for days to weeks, before they subside. These two experiences with this group of patients clearly suggest that such a link exists.
In this study agglomerates of silicone in the form of plaques together with titanium or in the form of droplets are detected in several organs and nervous tissue. Titanium was frequently used in the sealing patches of the silicone breast implants and that could possibly be the source of the titanium in the plaques. In the lymph nodes there are signs of chronic inflammation, but this does not seem to be the case in the organs and nervous tissue, where the silicone material is either located within vessels or encapsulated in collagen. However, the presence of silicone embolism or more fibrosis within internal organs could interfere with proper functioning of its cell systems. In nervous tissue it could interfere with conduction of nerve impulses. Many of the patients with silicone related complaints demonstrate a more neurological form of the disease with ultimately loss of control of the lower extremities and walking difficulties. This phenomenon has sporadically been coined as pre-MS by neurologists. The patient in this study had those walking difficulties and became wheelchair bound. But other neurological phenomena also exist, like loss of clear thinking, tremors and psychological disturbances. This has to be investigated further.
Although strict Si-counts are not absolute in number, the samples that we have seen by electron microscopy, do reveal that the disseminated silicone material is everywhere in the body. The larger silicone polymer molecules can be detected in agglomerates. The smaller molecules of siloxane monomers and oligomers, may infiltrate on a cellular level and at random work their way through the multitude of biochemical intracellular pathways, stressing the cells and creating a variable syndrome of various health complaints.
In this way this syndrome may appear to be elusive and parade ISSN: 2378-3656 Kappel  over a long period of time as no more than the accelerated aging process, too subliminal for any epidemiological study to be detected, but which will in due course surface in susceptible women. This hypothesis can only be tested through further research.

Conclusion
Our study using light microscopy and EDX-analysis, clearly demonstrates that silicone material is present in patients who have been exposed to gel bleed from silicone breast implants for longer time, in all organs and nervous tissue in great amounts.