**2. Materials and methods**

#### **2.1. Sample preparation**

SR sheets with dimensions of 100 x 100 x 1 mm were prepared from a two-component silicone system. Both component A and component B were clinical-quality liquids provided by Chenguang Research Institute of Chemical Engineering, China. Three doses of carbon ions were implanted using the ion implanter, respectively. The doses are 1 × 10<sup>15</sup> ions/cm<sup>2</sup> (C1), 3 × 10<sup>15</sup> ions/cm<sup>2</sup> (C2), and 1 × 10<sup>16</sup> ions/cm<sup>2</sup> (C3). After that, SR and C-SR sheets were manufactured into disc-like samples with a diameter of 6 mm using a hole puncher and into square samples with dimensions of 10 mm × 10 mm × 1 mm. The disc-like samples were used in in vitro antibacterial adhesion tests, and the square samples were used for in vivo evaluation. All samples were sterilized with 75% alcohol overnight. In all experiments, SR served as the control.

#### **2.2. Surface characterization**

The mechanical properties were analyzed by shore A durometer and electronic universal testing machine to gain parameters of the shore hardness and tear strength. The physicochemical properties were analyzed by the FTIR, SEM, XRD, XPS, water contract angel, zeta potential, and AFM experiments. The surface zeta potentials of materials were measured with a Zeta Potential Analyzer (DelsaNano C, Beckman Coulter, Germany). The measurements were carried out in 1 mmol 1-L NaCl electrolyte solution and with standard particles for flat surface cell (Otsuka Electronic Co., Ltd, Japan). Each sample chooses five points and each point tests 20 times. After that, the samples were scanned by the Environment Control Scanning Probe Microscope (NanoNavi E-Sweep, NSK Ltd., Tokyo, Japan) and each sample was imaged with a 5 μm × 5 μm scanned area. The surfaces were analyzed by measuring the average surface roughness (Ra) of 5 randomly chosen images per sample from selected areas of 1 μm x 1 μm under Atomic Force Microscope (AFM) analysis software (NanoNavi II, SII Nano Technology Inc., Tokyo, Japan). Ra is defined as the average absolute deviation of the roughness irregularities from the mean line over one sampling length and gives a good general description of height variations. Three replicas were used.

### **2.3. Bacterial culture preparation**

and biological applications, ranging from tissue fillers to tubes for dialysis and blood pumps [2–4]. Silicone rubber and silicone rubber-based materials have been used as medical tissue implants in the field of plastic surgery for many years, but there remain reports of adverse reactions to long-term implants, such as capsular contracture [5, 6]. Moreover, various prosthetic materials that contain silicone rubber can easily move and can permanently damage the prostheses. In addition, certain materials made from silicone rubber, such as catheters, are widely used in medicine but have several limitations; for example, bacteria can readily colonize the surfaces of silicone rubber, facilitating infection and even causing patient death

In recent years, many attempts have been made to modify medical materials to reduce bacterial adhesion and to minimize adverse inflammatory or foreign body reactions [10–15]. Among these methods, the surface modification of biomaterials is an economical and effective method to achieve biocompatibility and biofunctionality while preserving the favorable characteristics of the biomaterial, such as particular mechanical properties and thermal stability. Surface modifications, such as ion implantation [16–18], sintering [19], electrochemical deposition [20], and the sol-gel coating method, are common [21]. Among these surface modification methods, ion implantation has become a notably useful method because of its ease of operation and convenience [22–25]. In this context, we implanted three different doses of carbon ions into silicone rubber and obtained carbon ion silicone rubber (C-SR). Our study was designed to evaluate the surface characteristics and the biocompatibility of carbon ion silicone rubber. We focused on bacterial adhesion, cytocompatibility, and fibrosis/fibrous capsule development. The long-term goal is to gain a better biomaterial for use by plastic surgeons.

SR sheets with dimensions of 100 x 100 x 1 mm were prepared from a two-component silicone system. Both component A and component B were clinical-quality liquids provided by Chenguang Research Institute of Chemical Engineering, China. Three doses of carbon ions were

disc-like samples with a diameter of 6 mm using a hole puncher and into square samples with dimensions of 10 mm × 10 mm × 1 mm. The disc-like samples were used in in vitro antibacterial adhesion tests, and the square samples were used for in vivo evaluation. All samples were

The mechanical properties were analyzed by shore A durometer and electronic universal testing machine to gain parameters of the shore hardness and tear strength. The physicochemical properties were analyzed by the FTIR, SEM, XRD, XPS, water contract angel, zeta potential, and AFM experiments. The surface zeta potentials of materials were measured with a Zeta

(C3). After that, SR and C-SR sheets were manufactured into

(C1), 3 × 10<sup>15</sup>

implanted using the ion implanter, respectively. The doses are 1 × 10<sup>15</sup> ions/cm<sup>2</sup>

sterilized with 75% alcohol overnight. In all experiments, SR served as the control.

in certain cases [5, 7–9].

128 Ion Implantation - Research and Application

**2. Materials and methods**

(C2), and 1 × 10<sup>16</sup> ions/cm<sup>2</sup>

**2.1. Sample preparation**

**2.2. Surface characterization**

ions/cm<sup>2</sup>

Gram-negative *Escherichia coli* (ATCC 25922), Gram-positive *Staphylococcus aureus* (ATCC 25923), and *S. epidermidis* (ATCC 12228) were employed to bacterial experiments. The strains were streaked on blood agar plates from frozen stocks and grown for 24 h at 37°C in ambient air. The agar plates were then kept at 4°C until further use. For each experiment, one colony from an agar plate was inoculated into 10 ml of tryptone soy broth (TSB) and incubated for 24 h. The bacterial suspension was then added to 0.9% sterile sodium chloride to a final concentration of 1.5 x 10<sup>6</sup> colony-forming units per ml (CFU/ml), after which a McFarland standard was prepared (in practical terms, OD600 nm = 0.132). The samples were placed on 96-well culture plates and separately incubated in 200 μl of the bacterial suspension at 37°C for 1 or 24 h. After that, the plate colony-counting, fluorescence staining, and scanning electron microscopy (SEM) observation were conducted.

#### *2.3.1. Plate colony‐counting*

After incubation of the samples in the bacterial culture for 1 and 24 h, the bacteria on each sample were gently rinsed with PBS, respectively, and ultrasonically detached in 1 ml of PBS solution. The bacteria in the PBS were recultivated on agar plates for colony-counting. The antibacterial rates were determined based on the following relationship: antibacterial rate (%) = (CFU of control − CFU of experimental groups)/CFU of control x 100%. This assay was performed in triplicate.

#### *2.3.2. Fluorescence staining*

After incubation for 1 and 24 h, various samples were gently rinsed with PBS before staining the bacteria on the samples. The staining was performed by applying LIVE/DEAD® BacLight™ Bacterial Viability Kit (L7029, Molecular Probes®, OR, USA) for 15 min in darkness and was examined by laser scanning confocal microscopy (LEICA TCS SP5, Germany). The areas of green and red color in the pictures were then analyzed by using Image-Pro Plus version 6.0 (Media Cybernetics, Inc., USA) and then the proportion of red coloration based on the following relationship was calculated: red proportion (%) = red area/(green area + red area) x 100%.

### *2.3.3. Scanning electron microscopy (SEM) observation*

After bacterial incubation for 1 and 24 h, the samples were rinsed with PBS to remove free bacterial cells and then fixed in 2.5% glutaraldehyde for 3 h at room temperature. The samples were then progressively dehydrated in a series of ethanol solutions (15, 30, 50, 70, 80, 90, 95, and 100%) for 15 min each. After that, the specimens underwent critical point drying and coating with a thin conductive layer of Au. Finally, the morphology and adhesion of the bacteria on the various samples were determined by SEM (VEGA 2 SEM, TESCAN Inc., Brno, Czech Republic).

### **2.4. Animals and surgery**

This research was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Washington, DC: The National Academies Press, 2011), and all of the animal protocols were approved by the Institutional Animal Care and Use Committee of the Third Military Medical University, China. A total of 16 female Sprague-Dawley rats (weighing 160–200 g) were used (4 groups of 4 animals each), and all rats were housed under a 12-h light–dark cycle with free access to water and food. Prior to surgery, all of the rats were anesthetized with 3% pentobarbital sodium (1 ml/1000 g). The skin was swabbed with iodine, and four parallel incisions (10 mm) were performed. The material samples were implanted subcutaneously along the back region. The implants and their surrounding tissues were retrieved from each group by wide excision at 7, 30, 90, and 180 days after implantation and were then fixed in 4% paraformaldehyde solution. After that, HE and Masson's staining and immunohistochemistry were carried out.

#### *2.4.1. HE and Masson's staining*

The fixed tissues were sectioned (6 μm thick) and stained using a HE Staining Kit (C0105, Beyotime Inc., Shanghai, China). The thickness of the fibrotic capsule around each implant was determined at five equidistant points for statistical accuracy. Collagen deposition in the tissue around the implants was studied by Masson's trichrome staining, which was performed using a staining kit (MST-8003, Maixin Biological Technology Co., Ltd., Fujian, China). All procedures were performed based on the manufacturer's instructions.

#### *2.4.2. Immunohistochemistry*

Immunohistochemistry was performed on 4% paraformaldehyde-fixed cryostat sections of frozen tissue specimens. Endogenous peroxidase and nonspecific antibody binding were blocked with 3% H<sup>2</sup> O2 and 100% methanol, with a ratio of 1:5 and a blocking time of 30 min. Next, 0.02 M PBS was used for antigen retrieval while heating in a water bath, followed by treatment with 5% blocking reagent at 37°C for 30 min. The slides were then incubated at 4°C for 12 h with a primary antibody against CD68, CD4, TNF-α, α-SMA, or elastin (1:25) (Boster Biological Engineering Co., Ltd., Hubei, China). After washing in PBS, a secondary antibody was applied for 30 min. Visualization was achieved by adding 3, 3'-diaminobenzidine chromogen.

#### **2.5. Statistics**

*2.3.3. Scanning electron microscopy (SEM) observation*

Czech Republic).

**2.4. Animals and surgery**

130 Ion Implantation - Research and Application

*2.4.1. HE and Masson's staining*

*2.4.2. Immunohistochemistry*

O2

blocked with 3% H<sup>2</sup>

dine chromogen.

After bacterial incubation for 1 and 24 h, the samples were rinsed with PBS to remove free bacterial cells and then fixed in 2.5% glutaraldehyde for 3 h at room temperature. The samples were then progressively dehydrated in a series of ethanol solutions (15, 30, 50, 70, 80, 90, 95, and 100%) for 15 min each. After that, the specimens underwent critical point drying and coating with a thin conductive layer of Au. Finally, the morphology and adhesion of the bacteria on the various samples were determined by SEM (VEGA 2 SEM, TESCAN Inc., Brno,

This research was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Washington, DC: The National Academies Press, 2011), and all of the animal protocols were approved by the Institutional Animal Care and Use Committee of the Third Military Medical University, China. A total of 16 female Sprague-Dawley rats (weighing 160–200 g) were used (4 groups of 4 animals each), and all rats were housed under a 12-h light–dark cycle with free access to water and food. Prior to surgery, all of the rats were anesthetized with 3% pentobarbital sodium (1 ml/1000 g). The skin was swabbed with iodine, and four parallel incisions (10 mm) were performed. The material samples were implanted subcutaneously along the back region. The implants and their surrounding tissues were retrieved from each group by wide excision at 7, 30, 90, and 180 days after implantation and were then fixed in 4% paraformaldehyde solution. After that,

The fixed tissues were sectioned (6 μm thick) and stained using a HE Staining Kit (C0105, Beyotime Inc., Shanghai, China). The thickness of the fibrotic capsule around each implant was determined at five equidistant points for statistical accuracy. Collagen deposition in the tissue around the implants was studied by Masson's trichrome staining, which was performed using a staining kit (MST-8003, Maixin Biological Technology Co., Ltd., Fujian,

Immunohistochemistry was performed on 4% paraformaldehyde-fixed cryostat sections of frozen tissue specimens. Endogenous peroxidase and nonspecific antibody binding were

min. Next, 0.02 M PBS was used for antigen retrieval while heating in a water bath, followed by treatment with 5% blocking reagent at 37°C for 30 min. The slides were then incubated at 4°C for 12 h with a primary antibody against CD68, CD4, TNF-α, α-SMA, or elastin (1:25) (Boster Biological Engineering Co., Ltd., Hubei, China). After washing in PBS, a secondary antibody was applied for 30 min. Visualization was achieved by adding 3, 3'-diaminobenzi-

and 100% methanol, with a ratio of 1:5 and a blocking time of 30

China). All procedures were performed based on the manufacturer's instructions.

HE and Masson's staining and immunohistochemistry were carried out.

All data are expressed as the mean ± standard deviation (SD), and statistics were analyzed using SPSS statistical software. One-way ANOVA combined with multiple comparisons performed along with Tukey multiple comparison tests was utilized to determine the level of significance. In all of the statistical evaluations, *P* < 0.05 was considered significant.
