Abstract
Objectives
SUS 304 stainless steels have been widely used in orthodontics and implants such as archwires, brackets, and screws. The purpose of present study was to investigate the biocompatibility of both the commercial microcrystalline biomedical 304 stainless steel (microcrystalline 304ss) and novel-fabricated nanocrystalline 304 stainless steel (nanocrystalline 304ss).
Methods
Bulk nanocrystalline 304ss sheets had been successfully prepared by microcrystalline 304ss plates using severe rolling technique. The electrochemical corrosion and ion release behavior immersion in artificial saliva were measured to evaluate the property of biocorrosion in oral environment. The cell lines of murine and human cell lines from oral and endothelial environment were co-cultured with extracts to evaluate the cytotoxicity and provide referential evidence in vivo.
Results
The polarization resistance trials indicated that nanocrystalline 304ss is more corrosion resistant than the microcrystalline 304ss in oral-like environment with higher corrosion potential, and the amount of toxic ions released into solution after immersion is lower than that of the microcrystalline 304ss and the daily dietary intake level. The cytotoxicity results also elucidated that nanocrystalline 304ss is biologically compatible in vitro, even better than that of microcrystalline 304ss.
Significance
Based on the much higher mechanical and physical performances, nanocrystalline 304ss with enhanced biocorrosion property, well-behaved in vitro cytocompatibility can be a promising alternative in orthodontics and fixation fields in oral cavity.
1
Introduction
Conventional polycrystalline SUS 304 stainless steel (generally with the grain size at micrometer scale) has been well used in orthopedics , orthodontics and dentistry because of its high stainless corrosion resistance, proper bio-mechanical behavior, favorable bio-affinity, as well as good processability and low cost. However, their inferior bio-functional performance and reduced local corrosion resistance such as pitting, stress and crevice corrosions under the internal environment with the chloride ion , amino acids, and various proteins were reported in clinical application. When implanted in body, traditional microcrystalline 304ss would release metal ions into the surroundings, which had been found to cause toxic effects on the tissues .
Nanostructured bulk metallic materials, with newly built nanocrystalline Ti alloys as examples, are proposed to be the next generation biomaterials for their unique well-behaved physiochemical performances and bio-related properties . Yet up to date, no detailed work has been done on nanocrystalline 304ss for its bio-evaluation. Thus, in this paper, nanocrystalline 304ss sheet fabricated from conventional microcrystalline 304ss plate by deep rolling treatment has been measured for the biocorrosion, ion release behavior in simulated saliva fluids and cellular responses, with microcrystalline 304ss as the counterpart.
2
Materials and methods
2.1
Materials preparation
Commercial biomedical SUS304 stainless steel was used and nanocrystalline 304ss was further fabricated as described in our previous work . All samples were mechanically polished up to 2000 grit, ultrasonically cleaned in acetone, absolute ethanol and distilled water in sequence, and then dried in open air.
2.2
Microstructural characterization and tensile test
X-ray diffractometer (XRD, Rigaku DMAX 2400) using Cu Kα radiation was employed for the identification of the constituent phases. The tensile test samples were machined to 1.5 × 1.5 × 40 mm 3 . The tensile tests were carried out at a strain rate of 4 × 10 −4 s −1 in an Instron 3365 universal test machine.
2.3
Electrochemical measurement
A three-electrode cell was used for electrochemical measurements. The counter electrode was made of platinum and the reference electrode was saturated calomel electrode (SCE). The exposed area of the working electrode to the solution was 4 mm 2 . All the measurements were carried out on an electrochemical workstation (CHI660C, China) at the temperature of 37 °C. The electrolyte was artificial saliva . The surface morphology of the samples after corrosion was characterized by environmental scanning electron microscopy (ESEM, AMRAY-1910FE).
2.4
Immersion test
The immersion test was carried out according to ASTM in artificial saliva solution. Experimental samples (10 × 10 × 2 mm 3 ) were immersed in 40 ml solutions and the temperature was kept at 37 °C by water bath. After different immersion periods (from 1 day to 9 weeks), the samples were removed from the solution, gently rinsed with distilled water, and dried at room temperature. The surface chemical information of experimental samples was detected by XPS (AXIS Ultra, Kratos). The inductively coupled plasma atomic emission spectrometry (Leeman, Profile ICP-AES) was employed to measure the concentrations of Fe, Cr, and Ni element ions which might be dissolved from the alloy samples.
2.5
Cytotoxicity test
Cell lines of L-929, NIH3T3, ECV304 and MG63 were adopted to evaluate the cytotoxicity of experimental materials. All kinds of cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 100 U ml −1 penicillin and 100 μg ml −1 streptomycin at 37 °C in a humidified atmosphere of 5% CO 2 . The cytotoxicity tests were carried out by indirect contact. Extracts were prepared using DMEM serum free medium as the extraction medium with the surface area of extraction medium ratio 3 cm 2 /ml in a humidified atmosphere with 5% CO 2 at 37 °C for 72 h, then refrigerated at 4 °C before the cytotoxicity test. The control groups involved the use of DMEM medium as negative controls and DMSO as positive controls. Cells were incubated in 96-well cell culture plates at 5 × 10 3 cells/100 μl medium in each well and incubated for 24 h to allow attachment. The medium was then replaced with 100 μl of extracts. After incubating the cells in a humidified atmosphere with 5% CO 2 at 37 °C for 1, 2 and 4 days, respectively, the 96-well cell culture plates were observed under an optical microscope. After that, 10 μl MTT was added to each well. The samples were incubated with MTT for 4 h at 37 °C, then 100 μl formazan solubilization solution (10% SDS in 0.01 M HCl) was added into each well overnight in the incubator in a humidified atmosphere. The spectrophotometrical absorbance of the samples was measured by microplate reader (Bio-RAD680) at 570 nm with a reference wavelength of 630 nm.
2.6
Statistical analysis
The results were expressed as mean standard error, with n > 3 per group for all comparisons. Statistical significance was determined by two-way ANOVA. The level of significance was selected as P ≤ 0.05.
2
Materials and methods
2.1
Materials preparation
Commercial biomedical SUS304 stainless steel was used and nanocrystalline 304ss was further fabricated as described in our previous work . All samples were mechanically polished up to 2000 grit, ultrasonically cleaned in acetone, absolute ethanol and distilled water in sequence, and then dried in open air.
2.2
Microstructural characterization and tensile test
X-ray diffractometer (XRD, Rigaku DMAX 2400) using Cu Kα radiation was employed for the identification of the constituent phases. The tensile test samples were machined to 1.5 × 1.5 × 40 mm 3 . The tensile tests were carried out at a strain rate of 4 × 10 −4 s −1 in an Instron 3365 universal test machine.
2.3
Electrochemical measurement
A three-electrode cell was used for electrochemical measurements. The counter electrode was made of platinum and the reference electrode was saturated calomel electrode (SCE). The exposed area of the working electrode to the solution was 4 mm 2 . All the measurements were carried out on an electrochemical workstation (CHI660C, China) at the temperature of 37 °C. The electrolyte was artificial saliva . The surface morphology of the samples after corrosion was characterized by environmental scanning electron microscopy (ESEM, AMRAY-1910FE).
2.4
Immersion test
The immersion test was carried out according to ASTM in artificial saliva solution. Experimental samples (10 × 10 × 2 mm 3 ) were immersed in 40 ml solutions and the temperature was kept at 37 °C by water bath. After different immersion periods (from 1 day to 9 weeks), the samples were removed from the solution, gently rinsed with distilled water, and dried at room temperature. The surface chemical information of experimental samples was detected by XPS (AXIS Ultra, Kratos). The inductively coupled plasma atomic emission spectrometry (Leeman, Profile ICP-AES) was employed to measure the concentrations of Fe, Cr, and Ni element ions which might be dissolved from the alloy samples.
2.5
Cytotoxicity test
Cell lines of L-929, NIH3T3, ECV304 and MG63 were adopted to evaluate the cytotoxicity of experimental materials. All kinds of cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 100 U ml −1 penicillin and 100 μg ml −1 streptomycin at 37 °C in a humidified atmosphere of 5% CO 2 . The cytotoxicity tests were carried out by indirect contact. Extracts were prepared using DMEM serum free medium as the extraction medium with the surface area of extraction medium ratio 3 cm 2 /ml in a humidified atmosphere with 5% CO 2 at 37 °C for 72 h, then refrigerated at 4 °C before the cytotoxicity test. The control groups involved the use of DMEM medium as negative controls and DMSO as positive controls. Cells were incubated in 96-well cell culture plates at 5 × 10 3 cells/100 μl medium in each well and incubated for 24 h to allow attachment. The medium was then replaced with 100 μl of extracts. After incubating the cells in a humidified atmosphere with 5% CO 2 at 37 °C for 1, 2 and 4 days, respectively, the 96-well cell culture plates were observed under an optical microscope. After that, 10 μl MTT was added to each well. The samples were incubated with MTT for 4 h at 37 °C, then 100 μl formazan solubilization solution (10% SDS in 0.01 M HCl) was added into each well overnight in the incubator in a humidified atmosphere. The spectrophotometrical absorbance of the samples was measured by microplate reader (Bio-RAD680) at 570 nm with a reference wavelength of 630 nm.
2.6
Statistical analysis
The results were expressed as mean standard error, with n > 3 per group for all comparisons. Statistical significance was determined by two-way ANOVA. The level of significance was selected as P ≤ 0.05.
3
Results and discussions
3.1
Microstructural characterization of nanocrystalline and microcrystalline 304ss
Fig. 1 shows typical XRD patterns of the experimental samples for the identification of constituent phases, structures and grain size. Extended wider peaks can be clearly seen from the pattern of nanocrystalline 304ss than that from the microcrystalline 304ss at the same position. This demonstrates severe refinement of crystalline down to about 50 nm according to Scherrer equation , with agreement to the TEM results in our previous work .
