Characterization of BellaGel SmoothFine ® Implant Surfaces and Correlation with Capsular Contracture

BellaGel SmoothFine® implant is a novel nanotextured silicone breast implant. The objective of this study was to characterize differences of BellaGel SmoothFine® surfaces with commercial available implant surfaces in terms of texture, topography, and wettability as well as the behavior of capsular contracture. The surface textures of breast implants from two different manufac-turers (Hans Biomed and Motiva) were evaluated. The implants utilized in this study were BellaGel Smooth®, BellaGel Textured®, BellaGel SmoothFine® or Motiva SilkSurface®. The shell textures of these implants were characte-rized using a scanning electron microscopy, three dimensional confocal laser scanning microscope, and contact angle goniometer. Silicone breast implants were emplaced beneath the panniculus carnosus muscle on the dorsum of Sprague Dawley rats and observed for up to 8 weeks postoperative days. The fibrous capsules around silicone implants were explanted for histological examination. BellaGel SmoothFine® exhibits a relatively flat, with little or no depth in the texturing, 5.96 ± 0.41 μm surface roughness, and a contact angle of 103.14 ± 2.06 BellGel SmoothFine® implant resulted in significant decreases in capsule thickness (P < 0.05) and collagen production (P < 0.05) at 8 weeks with respect to the BellaGel Smooth® and BellaGel Textured® implant groups. Significant (P < 0.05) decreases in inducible nitric oxide synthase, an inflammation marker, were observed in the BellGel SmoothFine®. Fibrous tissue formation markers (Vimentin and alpha-smooth muscle actin) were significantly reduced in BellaGel SmoothFine® surfaces versus BellaGel Smooth® surfaces (P < 0.05) or BellaGel Textured® groups (P < 0.05). Overall, these findings suggest that the nanotextured BellaGel SmoothFine® implant is associated with less breast implant derived capsular contracture than other surfaces.


Introduction
According to a 2013 report from the American Society of Plastic Surgeons, there are more than 200,000 women in the USA who had reconstructive or cosmetic breast augmentations. The medical literature describes that silicone gel-filled breast implants are linked with significant adverse health effects [1] [2]. The most common local complication associated with silicone gel-filled breast implant is capsular contracture, with a combined overall incidence of 10.6% [3] [4] [5] [6]. Capsular contracture is a multifactorial fibrotic foreign body reaction that promotes the hardening and tightening of the capsule at the contact site between the tissue and implant, which then causes dissatisfaction and pain after breast augmentation in addition to deformity and device failure [7]. Though the pathogenesis of capsular contracture has not been fully elucidated, a variety of causal associations including surface texture of the breast implant have been proposed to date [8].
The surface texture of the shell surrounding breast implant acts as the interface between the breast tissue and device [9] and its understanding is important in the field of implantation. They can markedly alter the pathophysiology and directly influence cellular biology, body tissues, and fibrous capsule development, specially the adherence of the tissue to the breast implant and the alignment of collagen fibers [8] [10] [11]. Traditionally, there are two main kinds of implants: smooth and textured implants. Smooth-surface implants are used worldwide; however, the prevalence of capsular contracture is higher with the smooth implants than others. Meta-analyses studies showed approximately 5 times increase in the contracture rate on smooth surface with respect to textured surfaces [12] [13]. Textured-surface implants, which can disrupt the contractile forces, were developed to minimize capsular contracture [8]. However, serious complications such as double capsule formation, late seroma, and anaplastic large cell lymphoma (ALCL) have been appeared for textured implants due to their aggressive texturization [14] [15].
Recently, numerous articles have proposed the use of nanometric surface topographies to induce specific cellular behavior like cell proliferation, attachment, migration, and differentiation, which affect the prevalence rate of capsular contracture [8]

Breast Implants
Each shell of implants was obtained from 4 different breast implant devices (Table 1).

3D Confocal Images
Physical properties of silicone breast implant surfaces including roughness, skewness, and kurtosis were observed by looking at their topographical features using a 3D confocal laser scanning microscope (LEXT OLS5000, Olympus Corporation, Tokyo, Japan). The experiments have been performed on 3 sample areas.

Wettability
Wettability assessment was carried out using a contact angle meter Phoenix-MT(T) (SEO, Suwon, Gyeonggido, Korea). The experiments were undertaken three times to ensure significance of the tests.

In Vivo Animal Experiment
Sixty Sprague-Dawley rats with a body weight of about 250 -300 g (Orientbio, Seongnam, Gyeonggido, Korea) were maintained in an exceedingly 12/12 light/dark cycle under a pathogen-free condition and given water ad libitum.
Animal care and experimental procedures were approved from the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital

Hematoxylin & Eosin Staining
Implants were excised in block with the surrounding tissue. Harvested specimens were fixed with 10% neutral buffered formalin and embedded in paraffin.
Sections (5 µm) of tissue samples were stained with hematoxylin and eosin (H & E) before dewaxing and dehydration for histological analysis. Each stained slide was examined at × 100 magnification using a microscope (Carl Zeiss, Germany).
The capsular thickness was calculated using Image J software (National Institutes of Health, Bethesda, MD, USA).

Western Blot Analysis
The capsule tissue around silicone breast implant was prepared using a RIPA buffer (Sigma Aldrich, MO, USA) that contained phosphatase inhibitor cocktail (BioPrince, Chuncheon, Gangwon, Korea). Samples were then denatured by heating for five min and immediately placed on ice. After centrifugation, aliquots containing approximately 60 μg protein were separated by gel electrophoresis. After electrophoresis, the protein was transferred from the gel onto nitrocellulose membranes and then the membranes were blocked in 5% skim milk for 2 h. After blocking, the membranes were subjected to western blotting with antibodies for iNOS, α-SMA, ARG1 (1:1000; Abcam, Cambridge, UK), Vimentin and β-actin (1:1000; Santa cruz, CA, USA) at 4˚C for overnight. The blot was incubated with secondary antibodies (1:5000 in TBST, rabbit for iNOS and α-SMA; mouse for ARG1 and β-actin) for 1 h for protein detection. Finally, proteins were detected using the enhanced chemiluminescence reagent (Amersham Co. Newark, NJ, USA) following the manufacturer's instruction. The density of protein bands was measured using the Image J (National Institutes of Health, USA). The relative quantities were normalized by β-actin.

Statistical Analysis
All values are reported as means ± S.E.M. (standard error of the mean). Statistic-al analyses were performed using SPSS statistical software (SPSS 11.5, Armonk, NY, USA). For all data, significant differences were determined using an unpaired t-test. For all analyses, P < 0.05 was defined as statistically significant.

Texturing Analysis of BellaGel SmoothFine ® Implant
BellaGel Smooth® texture was found a characteristic relatively flat appearance, with no height or no depth in the texturing and occasional surface irregularity

Surface Characterization of BellaGel SmoothFine ® Implant
The surface area per mm 2 from 4 breast implant devices ranged from 1.0 mm 2 for the BellaGel Smooth® to 4.62 mm 2 for the BellaGel Textured® (  Table 2 and Figure 2). The relatively large increased peak roughness value obtained for which is relatively less rough than the BellaGel Textured® surface (P < 0.001).
Motiva SilkSurface® contains nano-scale features with an average roughness of 3.05 μm ± 0.82 μm, this low roughness therefore would reduce the friction and particle lose (Table 2 and Figure 2).

Effect of BellaGel SmoothFine ® Implant on Capsule Formation
We

Effect of BellaGel SmoothFine ® Implant on iNOS and Arg-1 Expression
iNOS levels are crucial to quantify local inflammatory response. As seen in Fig

Discussion
The  BellaGel SmoothFine® has a low surface roughness value at a subcellular level, which implies less particle friction coefficients and no tissue ingrowth [24] [25]. A skewness value of 0.36 ± 0.19, a kurtosis value of 4.23 ± 0.68 and a contact angle of 103.14˚ ± 2.06˚, are known to show higher biocompatibility. Therefore, we expected that BellaGel SmoothFine® may have led to the blocking of fibrous capsule formation in the current study.
Collagenous capsules formation is an inevitable response to all kind of foreign bodies and is always occur after silicone breast implant insertion into the body. Externally, a capsule develops a relatively undetectable thin membrane surrounding the implant in those undergoing breast augmentations. However, a stronger foreign body reaction to the implant leads to more excessive hypocellular thicker capsule formation, which is rich in collagen and positively related to the contracture formation [26]. This can cause an abnormally hard feel of the implant and pain in the breast. Previous study reported that surface texture may predispose implants to excessive capsular formation [9] [27]. Smooth surfaces are known to be correlated with high prevalence of capsular contracture, because fibroblasts on the surface of smooth textured implant produce collagen fibers, Journal of Biomaterials and Nanobiotechnology which are align highly within the capsule next to the implant in response to a shearing motion within the implant pocket [28] [29]. The continual rubbing between a smooth-surfaced implant and its nonadherent capsule plays a key role in causing a thick capsule and an acute, active tissue response [30]. By contrast, textured surfaces disrupt certain collagen alignment of the surrounding capsule through inhibiting micromotion at the porsthesis/host interface. Therefore, textured surfaces induce decreased malposition and capsular contracture with respect to smooth surfaces [5] [8] [12] [28] [31] [32] [33]. However, additional studies showed no statistically significant reduction in the capsule formation [33]- [39]. In the present study, we conducted an experiment in which silicone implants were emplaced beneath the muscle layer in rat, and the capsules that developed surrounding the silicone implants were then investigated. Inflammatory reaction occurred when silicone breast implant inserted into the body, plays a vital role in the progression of capsular contracture, because it activates fibroblasts around capsules to cause excessive fibrosis and hypertrophic scar contracture [40] [41]. iNOS, a degradative enzyme, is an acute phase inflammatory factor and expressed by macrophages. They seem central to degrade the silicone breast implant through the production of nitric oxide. Significantly, iNOS is important in the pathogenesis of breast implant derived capsular contracture [42] [43]. In this work we found that the expression of iNOS was reduced on BellaGel SmoothFine® and Motiva SilkSurfaces® in comparison to Bel-laGel Smooth® and Textured® surfaces. The constant rubbing between a smooth surfaces implant and host tissue might induce significantly increased inflammatory response [30]. The reduction in frictional forces between the textured surfaces and host tissues may result in minimal inflammation with respect to smooth surfaces. However, frequent cracking of the collagen fibers on textured surfaces may cause persistent inflammation. BellaGel SmoothFine® is not rough enough to cause friction with the surrounding tissues; therefore, the initial inflammatory response was decreased.
Fibroblasts differentiate into myofibroblasts in contracted fibrous capsules and upregulate the expression of IL-8, TGF-β, TGF-β 1, α-SMA, collagen 1, and MMP12 as they differentiate into capsular myofibroblasts [21]. Myofibroblasts present in some conditions associated with contraction processes, such as tenosynovitis, Dupuytren's contracture, and fibrous capsules formed around implant [44]. Inside the body, fibroblasts and myofibroblasts are known to make a stiff extracellular matrix that remodels the original healthy tissue. An abundance of Journal of Biomaterials and Nanobiotechnology This results indicated that silicone breast implant with nano texture inhibited BI-induced capsular contracture by inhibiting the proliferation of fibroblast and myofibroblasts.
In conclusion, our study showed that variations in surface roughness of breast