EDS (JEOL JSM-6700F) was performed to evaluate the chemical composition of the stainless steel surfaces of the three groups. Compared to the machined stainless steel (Fig. 3A), the Ti-coated, MAOed group showed a completely different surface, with high peaks of Ti, oxygen, calcium, and phosphate (Fig. 3B). The MAO was able to cover contaminating alumina particles impacted by the grit blasting. This was also confirmed by the EDS spectrum results of the surface chemical characterization (Fig. 3C).
To prepare specimens for morphologic assessment using SEM, 0.5 mL SaOS-2 cells (5 × 104 cells/mL) in a medium containing 1% fetal bovine serum (FBS) were seeded on each of the 16 stainless steel samples. After 6 hours of incubation, the medium was removed. Phosphate-buffered saline (PBS) was added, and the cells were washed with PBS two to three times. After adding 2% glutaraldehyde-PBS solution, the cells were allowed to stabilize for 2 hours. The fixative solution was removed, and the cells were washed with distilled water three times. At 30-minute intervals, the cells were dehydrated through 50%, 70%, 90%, and 100% ethanol solutions. Finally, the ethanol was removed, and the cells were left at room temperature to evaporate the ethanol completely. We determined cell morphology using a SEM (JEOL JSM-6700F) after the test specimens had been coated with platinum.
For the cell proliferation assays, 0.5 mL SaOS-2 cells (5 × 104 cells/mL) in a medium containing 1% FBS were seeded on each of the 16 stainless steel samples and incubated (37°C, 5% CO2, 95% humidity) for 24, 48, 72, and 96 hours. The medium was exchanged with fresh medium before measuring cell proliferation using the CellTiter 96® nonradioactive cell proliferation assay (Promega Corp, Madison, WI) according to the manufacturer’s protocol. This assay is a colorimetric method for determining the number of viable cells. The amount of formazan formed can be measured by its absorbance at 450 nm using a plate reader and is directly proportional to the number of viable cells in the culture. Using this kit, 15 μL dye solution was placed on the stainless steel sample and the cells were incubated in 5% CO2 at 37°C for 4 hours. After 100 μL stop solution was added, 100 μL pipetted cells was transferred to a 96-well plate (Nunc A/S, Roskilde, Denmark). Cell proliferation was measured at 450 nm using a spectrophotometer (EL 312e; BioTek Instruments, Inc, Winooski, VT).
ALP activity was measured by seeding 0.5 mL SaOS-2 cells (5 × 104 cells/mL) in medium containing 1% FBS on each of the 16 stainless steel samples, followed by incubation for 7, 14, and 21 days. The medium was removed, and the cells were washed with PBS three times to remove as much of the serum in the culture fluid as possible. Next, 1 mL 0.02% Triton® X-100 was placed on the stainless steel sample to lyse the cells. Cytolytic solution was transferred into a 1.5-mL tube, and the cells were sonicated. The tube was centrifuged (14,000 rpm, 4°C, 15 minutes), and the supernatant was transferred to a new 1.5-mL tube. Then, 100 μL 1 mol/L Tris-HCl, 20 μL 5 mmol/L MgCl2, and 20 μL 5 mmol/L p-nitrophenyl phosphate were added to the supernatant. The mixture was left to react at 37°C for 30 minutes, and 50 μL 1 N NaOH was added to stop the reaction. Using p-nitrophenol as a standard, the absorbance was measured at 410 nm using a spectrophotometer. The measured ALP activity was expressed as the p-nitrophenol production quantity divided by the reaction time and the protein synthesis quantity, as measured by the Bio-Rad Protein assay kit (Bio-Rad Laboratories, San Jose, CA).
For the cell migration assay, we placed each of 10 stainless steel samples in a Transwell® chamber in 24-well plates (Costar, Cambridge, MA) and put 500 μL medium with 1% FBS in the lower chamber (Fig. 4). We prepared 0.5 mL SaOS-2 cells (5 × 104 cell/mL) in serum-free medium, added this cell suspension plus culture fluid to the upper chamber above the polycarbonate filter (8-μm pore size), and cultured the cells for 24 hours at 37°C in 5% CO2. We then removed the medium from the upper chamber and removed the membrane and nonmigrated cells using a swab. To count the migrated cells, we placed the chamber in a new well, put 200 μL extraction solution in the chamber, shook the plate on an orbital shaker for 10 minutes, transferred 100 μL of each sample to a 96-well plate, and measured the absorbance at 560 nm using a spectrophotometer (EL 312e).
We compared the mean cell proliferation assay and ALP activity of the cells on the three different surfaces using the Kruskal-Wallis test and compared the cell migration assay results using the Wilcoxon signed-rank test. Statistical analyses were performed using SPSS® software (Version 11.5; SPSS Inc, Chicago, IL).
The morphologic assessment of the cells after 6 hours of incubation using SEM showed the machined stainless steel (Fig. 5A) and Ti-coated, MAOed (Fig. 5B) surfaces were covered with small, slender osteoblastlike cells, whereas the grit-blasted, Ti-coated, MAOed surface was largely covered with lamellipodia from the osteoblastlike cells (Fig. 5C). Additionally, the lamellipodia largely covered and the filopodia extended into the micropores on the MAO surface. Thin cytoplasmic processes branched out from the filopodia and entered the interior of the micropores (Fig. 5C).
After 24-, 48-, 72-, and 96-hours incubations, the proliferations of the SaOS-2 cells on the Ti-coated, MAOed stainless steel and grit-blasted, Ti-coated, MAOed stainless steel surfaces were higher (p < 0.001) than that on the machined stainless steel surface, and the cell proliferation on the grit-blasted, Ti-coated, MAOed stainless steel surface was higher (p = 0.01) than that on the Ti-coated, MAOed surface (Fig. 6).
We observed no differences in the ALP activity of the SaOS-2 cells on the surfaces among the groups after 7-, 14-, and 21-day incubations (Fig. 7). The ALP value reached a maximum at 14 days (Fig. 7).
The migrations of the SaOS-2 cells on the Ti-coated, MAOed stainless steel and grit-blasted, Ti-coated, MAOed stainless steel surfaces were higher (p < 0.001) than that on the machined stainless steel surface, but the cell migration on the grit-blasted, Ti-coated, MAOed stainless steel surface was similar to (p = 0.609) that on the Ti-coated, MAOed stainless steel surface (Fig. 8).
To enhance the potential for stainless steel to osseointegrate, we introduced a Ti coating using electron beam deposition, which has been successfully used in the space, turbine, optical, biomedical, and auto industries. We then developed a modified coating process using grit blasting followed by Ti coating and MAO. We asked whether this grit-blasting, Ti-coating, MAO process could improve the morphology, proliferation, differentiation, and attraction ability of SaOS-2 cells to stainless steel in vitro compared with two other surface modifications, machined and Ti-coated, and MAOed.
We note several limitations of our study. First, this study assessed only in vitro effects on cell morphology, proliferation, and differentiation. Further studies are needed to determine whether Ti coating and MAO actually improve osseointegration, which has not yet been studied in vivo. Second, we used SaOS-2 cells, an osteosarcoma cell line, which may behave differently from primary osteoblasts, so caution must be exercised in drawing any conclusion regarding osseointegration. SaOS-2 cell line revealed a 2 to 3-fold greater mean doubling-time and a 15 to 20-fold higher saturation density than osteoblasts . However, to study interactions of osteoblastic cells with biomaterials, osteoblastlike cell lines from human osteosarcoma (SaOS-2) are often used [1, 3, 15, 16]. SaOS-2 cells in particular exhibited the most mature osteoblastic phenotype compared with other osteosarcoma-derived cells line . That is why we used SaOS-2 cells in this study. Third, a DNA study was not performed. A DNA study could enhance the impact of these findings by also assessing the levels of Type I collagen and osteocalcin. Further study is needed. Fourth, the assessment of biocompatibility was limited by considering only alkaline phosphatase activity and cell proliferation rate, and we did not perform the necessary normalization experiments.
Electron beam deposition is a simple process in which a focused high-energy electron beam is directed to melt the evaporant material in a vacuum chamber . The evaporating material condenses on the surface of the substrates or components, resulting in the formation of a coating . During deposition, external heating is often applied to the substrate to enhance metallurgical bonding between the coating and the substrate. Electron beam deposition technology produces a new material with controlled microstructure and microchemistry in the form of coatings. This technology is being used to form coatings for many applications, including space, turbine, optical, biomedical, and auto industries. Coatings are often applied on components to extend their performance and life under severe environmental conditions, such as thermal, corrosion, wear, and oxidative stress. Performance and properties of the coating depend on its composition, microstructure, and deposition conditions [20, 24].
The cell morphology and proliferation assay indicated all the surfaces examined were cytocompatible. This is consistent with many studies indicating Ti and stainless steel 316L are biocompatible substrates for cell culturing . In our study, cell proliferation was elevated on the Ti-coated, MAOed groups compared with the machined group. Moreover, we noticed osteoblastlike cells on the grit-blasted, Ti-coated, MAOed stainless steel spread and formed lamellipodia on mirror-polished stainless steel surfaces comparable to those observed on the machined and Ti-coated, MAOed stainless steel surfaces. These findings indicate the grit-blasting, Ti-coating, MAO process can enhance cell integration on a stainless steel surface.
ALP activity is an indicator of osteogenic differentiation, bone formation, and matrix mineralization . Several studies have demonstrated the influence of surface roughness on ALP activity [5, 12, 25]. However, we did not find ALP activity increased with roughness parameters, in contrast to the results reported by Kim et al. . This discrepancy may be attributed to the use of SaOS-2 cell lines in this experiment, and SaOS-2 cells may lift off after achieving high confluence. ALP activity peaked at about 14 days in bone-derived cell cultures because the cells were slowly dividing over 3 weeks [8, 17, 25].
To assess whether the stainless steel attracted osteoblastlike cells, we used a Transwell® migration assay system. The cell migration results were higher with Ti-coated, MAOed stainless steel than with machined stainless steel; thus, these surface modifications enhanced attraction of osteoblastlike cells.
A stable oxide layer on the surface of metals is essential for good biocompatibility [7, 9, 11, 22]. On stainless steel, such an oxide layer cannot readily form, in contrast to Ti. Thus, electron beam Ti deposition was used to make a stable oxide layer on the stainless steel surface. We also wanted to improve the biocompatibility of the Ti-coated stainless steel, so we introduced the MAO process to make a uniform oxide layer on the stainless steel [11-14]. However, not all metals can be treated by the MAO process. For the MAO process, passivation at the anode side should occur, and the oxide layer formed on the metal surface should be stable in the working electrochemical environment. For these reasons, stainless steel cannot be MAOed . The minimum thickness required for stable MAO treatment is dependent on the applied voltage; with increasing voltage, the minimum required Ti coating thickness increases . For stable MAO treatments with 230 V, the Ti coating layer should be thicker than 3 μm . The coating thickness in this research was in the range of 3 to 5 μm.
To increase surface roughness, we modified the surface of the stainless steel 316L by grit blasting followed by Ti coating and MAO. We then assessed the advantages of MAO treatment. This modification did enhance the cell proliferation and migration compared to machined stainless steel. Moreover, this process can overcome the disadvantages of grit blasting, such as the release of blasting particles. In this method, the blasting material is also embedded in the surface, as it is with grit blasting. MAO is not a mechanical phenomenon, but a chemical bond to the surface of the Ti alloy, in which a stable oxide layer surrounds embedded particles .
We used the grit-blasting, Ti-coating, MAO process on stainless steel 316L. These surface modifications enhanced the biocompatibility (proliferation and migration of osteoblastlike cells) of stainless steel. This process is not unique to stainless steel; it can be applied to many metals and should also improve their biocompatibility. More generally, it should allow a broad range of materials for cementless arthroplasty components, which today are largely limited to Ti and cobalt-chromium alloys.
We thank Hyoun Ee Kim, Seung Soo Kim, and Shang Joong Lee for providing editorial assistance.
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