Human Bone Marrow-Derived Mesenchymal Cell Reactions to 316L Stainless Steel: An in Vitro Study on Cell Viability and Interleukin-6 Expression

Purpose: Human bone marrow-derived mesenchymal cell (hBMC) reactions to 316L stainless steel (316L-SS) have never been evaluated. The objective of this study was to assess cell viability and interleukin-6 expression of hBMC cultures upon treatment with a 316L-SS implant. Methods: A cytotoxicity analysis was conducted with a 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium (MTT) assay after a period of 24, 48 and 72 hours of incubation. Expression of interleukin-6 was measured using enzyme-linked immunosorbent assay (ELISA). Results: Cell viability measurement was performed via IC50 formula. All treatment group showed a > 50 % cell viability with a range of 56,5 - 96,9 % at 24 hours, 51,8-77,3% at 48 hours and 70,1- 120 % at 72 hours. Interleukin-6 expression was downregulated subsequent to treatment with 316L-SS compared to the control group. Conclusion: We found that 316L-SS did not exhibit toxicity towards hBMC culture.


Introduction
Presently, 316L stainless steel (316L-SS) implants are still widely utilized in the field of medical practice, especially orthopedic surgery. It has excellent mechanical properties, corrosion resistance and is cost effective. 1 It is mandatory to evaluate biomedical safety before using such implants in humans. 2 As one of the dominant cellular components of medullary bone marrow, mesenchymal cells have important roles in several human tissue regeneration processes. They also possess the capability of producing several cytokines (include interleukin-6/IL-6) after contact with foreign material. 3,4 Human bone marrow mesenchymal cells (hBMC) reactions to 316L-SS have never been evaluated. Therefore, herein, we conducted an in vitro cytotoxicity study of 316L-SS on hBMC along with evaluating the expression of IL-6.

Human bone marrow-derived mesenchymal cell isolation and culture
Bone marrow samples were obtained from a patient experiencing total hip arthroplasty surgery with written informed consent and ethical approval from the Ethics Commission of Prof. Dr. R. Soeharso Orthopedic Hospital, Solo, Indonesia. During surgery, an approximately 10 ml of bone marrow suspension was harvested from the intramedullary canal of the femur. It was captured in a 20 ml tube (Falcon, BD Bioscience) containing the same volume of heparinized (10 U/mL) phosphate -buffered saline (PBS) to prevent clotting. The mixture of bone marrow and heparinized PBS was kept at 4°C prior to further processing in the laboratory. hBMC culturing was conducted in the Laboratory of Cell Culture at the Department of Physiology, Gadjah Mada University School of Medicine, Yogyakarta, Indonesia. Bone marrow was loaded into a centrifuge tube and centrifuged at 2500 rpm for 10 minutes at room temperature. The top layer of mononuclear cells was collected and washed with PBS twice and Dulbeco's Modified Eagle Media (DMEM) once. The isolated cells were suspended in DMEM, supplemented with 15% PBS, 1% antibiotic -mycotic (Sigma -Aldrich, Co, USA) and seeded into 25 cm 2 flasks. The cells were incubated at 37°C, 5% CO2. After three days, nonadherent cells were removed by washing twice with PBS and the new medium was applied. The cell density and morphology was monitored under an inverted microscope. When the primary cultured cells reached at least 80% confluence, they were harvested using 0.25% trypsin (Sigma -Aldrich, Co, USA) and subcultured. The third passage of cells was used for subsequent studies.

Immunohistochemistry
Confirmation of mesenchymal cell markers was carried out with immunohistochemistry (IHC) staining. Cells were fixed with 4% paraformaldehyde, blocked to prevent non-specific antibody binding and incubated with primary antibodies at 4°C overnight. Following PBS washing, anti-CD-44 and anti-CD-19 were used for human BMSC (®Bioss). All conditions were maintained in negative controls excepting that the primary antibodies were eliminated. Dishes were examined under a fluorescence microscope (Nikon Eclipse E400). CD-44 was employed as a positive marker of mesenchymal lineage cells and CD-19 as a positive marker of hematopoietic lineage cells ( Figure 1A, B).

Cytotoxicity study of 316L-SS on hBMC
The resultant hBMC from primary culture was recultured on 96-well microplates, one 96-well microplate being prepared for each incubation period: 24, 48 and 72 hours. The 316L-SS implants were placed in the wells before seeding the stem cells. The implants consisted of two typespolished and non-polished. Implant polishing was with toluene liquid. The implants also had two different sizes -316L-SS with a diameter of 2 mm (size A) and 316L-SS with a diameter of 4 mm (size B). Both had 1 mm thickness. Each type and size of implant was duplicated into three (N = 3 per group). 1x10 4 cells were seeded in each well. After the seeding process, the plates were incubated at 37°C, 5% CO 2 for 24, 48 and 72 hours. We evaluated the cells at the end of each incubation period under an inverted microscope. Culture medium was also collected for IL-6 expression measurement with an immunoassay kit (Komabiotech, ELISA). Each well of the 96-well microplates evaluated for cytotoxicity had 50 μl solution containing 5 mg/ml of 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium (MTT) reagent in PBS added, and then the microplates were reincubated for 4 hours in CO 2 5% at 37°C. Colorimetric assay with MTT was applied to assess viability of the cells after 24, 48 and 72 hours of treatment with 316L-SS implants. Next, to each well was added with 50 μl of dimethyl sulfoxide (DMSO), and the microplate was then reincubated for 5 minutes at 37°C. The optical density of each well was measured using an ELISA reader at 620 nm wavelength. Optical density data were tabulated and analyzed using the t-test (SPSS v17, Chicago, USA) at a significance of 95%. Percentage of viable hBMC were calculated using the IC 50 formula: 5

Percentage of viable cells =
Optical density of experiment group − Optical density of control media Optical density of control cells − Optical density of control media × 100%

Microscopic evaluation and cytotoxicity study
The visual microscopic evaluation was performed on all study groups before carrying out the MTT assay using an inverted microscope. We found that hBMC attached to the implant in all experiment groups (Figure 2A, B). According to the MTT assay results, the control group had greater optical density compared to the treatment group for all (24, 48 and 72 hours) incubation periods. Statistical analysis revealed that the 24-hour incubation period for all implants did not cause a significant difference in cell optical density compared to controls. Yet, a significant difference (p<0.05) relative to the control group was observed at 48 hours for non-polished B and at 72 hours for polished A and B (Table 1). Statistical analysis was also performed to evaluate the cell optical density differences between the period of incubation for each implant in the control and treatment group. It was showed that neither in the control or treatment groups was a significant difference (p<0.05) observed between the 24-hour and 72-hour groups, and this was also true when comparing the 48-hour and 72hour groups. No significant differences between the 24hour and 48-hour groups of controls with all experimental groups were found. Cell viability measurement was performed with the IC 50 formula. At 24, 48 and 72 hours, all treatment group exhibited a > 50 % cell viability ( Table 2).  Comparison to the control group at same period of incubation: * p = 0.03, **p = 0.00, *** p = 0.00.

Expression of IL-6
There was a lower expression of IL-6 in hBMC compared to controls for all polished implants with all incubation periods. The situation was similar for the nonpolished implant group, except with 72 hours of incubation and the small non-polished implant. In addition, the polished implant induced lower expression of IL-6 compared to non-polished implants for all treatment groups (Figure 3). Martinesi et al 7 described a remarkable increase in IL-6 expression in human PBMC after 316L-SS treatment. However, they saw that 316L-SS only causes a slight difference in IL-6 expression with HUVEC culturing. Here, we found a lower expression of IL-6 by hBMC that received treatment by polished implants compared to non-polished implants. This finding may indicate that a polished implant has a more inert surface and brings about less of a cell reaction compared to non-polished implants.

Conclusion
In this study, we found 316L-SS did not exhibit any toxic effects toward human bone marrow-derived mesenchymal cells cultures. Polishing 316L-SS implants appeared to reduce cellular reactions.