Tantalum ion implantation
A 0.85-mm-thick ePTFE membrane (Meari Co., Ltd., Gyeonggi-do, Republic of Korea) and a 1-mm-thick silicone rubber consisting of clinical-grade silicone (Bistool Co. Ltd., Seoul, Republic of Korea) with dimensions of 10 × 10 mm was prepared for the experimental evaluation. All samples were ultrasonically cleaned in alcohol and deionized water for 5 min before processing with tantalum (Ta) coating. A Ta target (diameter 75 mm, thickness 5 mm, purity 99.99%, Kojundo Korea Co., Ltd., Gyeonggi-do, Republic of Korea) was placed in a DC magnetron sputter gun housing (Ultech Co. Ltd., Daegu, Korea). The vacuum chamber was pumped to 5 × 10−4 Pa using rotary and diffusion pumps. To generate a sufficient amount of Ta ions and neutral atoms, 25 W of target power was applied to the Ta sputtering gun, and a working pressure and temperature were maintained at 0.6 Pa and 25 °C, respectively, during the process. The samples were placed on a stainless steel plate parallel to the Ta target surface at a 100-mm distance. Ta ions and neutral atoms were implanted into the sample surfaces for 3 min using a high negative bias of 2000 V. For comparative purposes, only untreated ePTFE and untreated silicone rubber sheets were used for the control group. Thus, all of the samples in this study were divided into the following four groups: Ta-treated silicone implant (G1), untreated silicone implant (G2), Ta-treated ePTFE implant (G3), and untreated ePTFE implant (G4).
TEM and SEM observation
To observe the Ta-implanted regions on the implant surfaces, high-resolution transmission electron microscope images were collected using transmission electron microscopy (TEM) (JEM-2100F, JEOL, Japan) operated at 200 kV. The cross-sectional image of the Ta-coated implant surface was prepared and obtained using focused ion beam milling and field-emission scanning electron microscopy (FIB/FE-SEM) (AURIGA, Carl Zeiss, Germany). Prior to the milling process, protective layers containing platinum and carbon were coated onto the implant surfaces.
Cell viability
To evaluate the viability of osteoblasts (MG-63) and fibroblasts (NIH3T3), an EZ-Cytox assay (Daeil Lab Service Co. Std., Seoul, Korea) was performed according to the manufacturer’s protocol. MG-63 and NIH3T3 cells were plated at a density of 3 × 104 cells/mL on the implant materials and cultured in Dulbecco’s modified Eagle’s medium (DMEM, ATCC 30-2003) (Life Technologies Co., Grand Island, NY, USA) and Eagle’s minimum essential medium (EMEM, Gibco 11995) (Life Technologies Co.) with 10% fetal bovine serum (FBS) containing 1% penicillin/streptomycin at 37 °C under 5% CO2 in a humidified atmosphere. Implants were conditioned in a medium for 5 h before insertion into 24-well cell culture plates. After culturing for 24, 48, or 72 h, the culture medium was discarded, and the samples were washed three times with phosphate-buffered saline (PBS) and then incubated at 37 °C for another 4 h in fresh culture medium containing 10 μL of EZ-Cytox solution. To investigate the effects of the tantalum-treated or untreated silicone or ePTFE surface on the viability of MG-63 and NIH3T3, the absorbance values of each cell culture were measured by using a spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) at 490 nm. Each test was repeated four times (n = 6).
Animal study
For biohistological evaluation, an animal experiment was conducted. All experimental surgical procedures were performed in a specific pathogen-free unit. The animals, 6-week-old male healthy Sprague-Dawley rats, were kept in a room with a 12-h light/dark cycle and temperature that varied between 23 and 25 °C. Furthermore, the animals were housed in soft, sterile bedding that was free from antibacterial products. The animals had open access to food and sterile non-acidic water.
The rats were randomly assigned to one of the following groups:
G1 (n = 6): tantalum-treated silicone implant
G2 (n = 6): untreated silicone implant
G3 (n = 6): tantalum-treated ePTFE implant
G4 (n = 6): untreated ePTFE implant
The experiment was conducted over two time intervals: 4 and 8 weeks for the silicone implant material and 2 and 4 weeks for the ePTFE implant material. The animals were anesthetized using a ketamine/xylazine mixture (75–100 mg/kg ketamine + 5–10 mg/kg xylazine), which was administered intraperitoneally (IP) using a maximum dose of 10 mL/kg. The incision site, which was located behind the lambdoid suture, was shaved and painted using iodine swabs. An approximately 2-cm-long incision was made behind the lambdoid suture through the skin, subcutaneous tissue, deep fascia, and periosteum up to the calvarial bone. The soft tissue over the skull bone was reflected, and the implants were inserted into all animals in all four groups (Fig. 1). Skin apposition was achieved using subcutaneous sutures made from 4-0 Vicryl (Ethicon, Livingston, UK).
Histological evaluation
At the completion of the study, the rats were sacrificed using an overdose of an intraperitoneal ketamine/xylazine mixture. Histological samples, including implants and the surrounding tissues, were obtained carefully to prevent implant movement. The samples were fixed in buffered formalin for 24 h, dehydrated, and embedded in paraffin wax. Tissue sections were mounted on glass slides and stained with hematoxylin and eosin (H&E) for histopathological evaluation. Images were captured using a specialized system, SPOT RTTM-KE color mosaic, and digitized via SPOT software version 4.6 (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). For simplicity, images were studied at × 400 magnification. The soft tissue thickness between the implants and the bone, the extent of new bone formation, and the severity of the inflammatory reaction were measured and analyzed.
The sections were processed, placed on slides, and stained with hematoxylin and eosin stain. Histological evaluation was performed on each section to evaluate the inflammation, foreign body reaction, amount of soft tissue filling the gap between the implants and the calvarial bone, and newly formed bone along the superficial layer of the calvarium toward the implants. The inflammation and foreign body reaction were blindly evaluated by a board-certified pathologist. According to the method of Pinese et al., soft tissue measurements were taken at 13 random regions along the implant-bone gap and then averaged to compare Ta-treated and untreated implants [10]. To evaluate the newly formed bone, histological evaluation of the slides was conducted. The newly formed bone was evaluated and scored according to its quantity. The following scores were assigned, as appropriate: no bone (score = 0), little stumps of bone (score = 1), moderate bone with gaps (score = 2), and complete bone along the calvarium surface (score = 3) [11].
Statistical analysis
All data are expressed as the mean ± standard deviation (SD). Data analysis was conducted using the SPSS Statistics software ver. 25 (IBM, Armonk, NY, USA). Nonparametric data comparisons were performed using the Wilcoxon-Mann-Whitney U test. Statistical significance was set at p < 0.05.