MC3T3-E1 cell response to stainless steel 316L with

Materials Science and Engineering C 56 (2015) 22–29

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

MC3T3-E1 cell response to stainless steel 316L with different surface treatments Hongyu Zhang a, Jianmin Han b,⁎, Yulong Sun a, Yongling Huang c, Ming Zhou a a b c

State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing 100084, China Dental Materials Laboratory, National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing 100081, China Jinghang Biomedicine Engineering Division, Beijing Institute of Aeronautical Material, Beijing 100095, China

a r t i c l e

i n f o

Article history: Received 30 October 2014 Received in revised form 29 April 2015 Accepted 9 June 2015 Available online 12 June 2015 Keywords: MC3T3-E1 Cell morphology Cell proliferation Stainless steel Surface treatment

a b s t r a c t In the present study, stainless steel 316L samples with polishing, aluminum oxide blasting, and hydroxyapatite (HA) coating were prepared and characterized through a scanning electron microscope (SEM), optical interferometer (surface roughness, Sq), contact angle, surface composition and phase composition analyses. Osteoblast-like MC3T3-E1 cell adhesion on the samples was investigated by cell morphology using a SEM (4 h, 1 d, 3 d, 7 d), and cell proliferation was assessed by MTT method at 1 d, 3 d, and 7 d. In addition, adsorption of bovine serum albumin on the samples was evaluated at 1 h. The polished sample was smooth (Sq: 1.8 nm), and the blasted and HA coated samples were much rougher (Sq: 3.2 μm and 7.8 μm). Within 1 d of incubation, the HA coated samples showed the best cell morphology (e.g., flattened shape and complete spread), but there was no significant difference after 3 d and 7 d of incubation for all the samples. The absorbance value for the HA coated samples was the highest after 1 d and 3 d of incubation, indicating better cell viability. However, it reduced to the lowest value at 7 d. Protein adsorption on the HA coated samples was the highest at 1 h. The results indicate that rough stainless steel surface improves cell adhesion and morphology, and HA coating contributes to superior cell adhesion, but inhibits cell proliferation. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Three kinds of materials are generally used in biomedical engineering due to their excellent biocompatibility and mechanical properties, i.e., pure Ti or Ti-based alloy, CoCrMo alloy, and 316L surgical stainless steel. For bone contact component, Ti-based alloy is usually the material of choice partly due to its relatively low elastic modulus. Artificial joint prostheses (hip, spine, etc.) are made of Ti-based alloy, as well as CoCrMo alloy as a result of its excellent anti-wear and anti-corrosion characteristics [1,2]. Stainless steel is widely used in orthopedic field for manufacturing of temporary implant [3,4]. One of the major concerns following implantation of these biomedical devices is the interaction between the implant and the surrounding host tissues [5,6]. As it is the surface of the material that the body is initially exposed to, the physicochemical characteristics of the implant surface are considered of vital significance, influencing the survivorship and the long-term performance of the implants [7,8]. In the past few years, an amount of studies have been performed to investigate the molecular and cellular reactions of the host to the implanted prostheses with different surface treatments, such as composition [9], morphology/roughness [10,11], structure [12], and surface ⁎ Corresponding author. E-mail address: [email protected] (J. Han).

http://dx.doi.org/10.1016/j.msec.2015.06.017 0928-4931/© 2015 Elsevier B.V. All rights reserved.

charge [13]. It is demonstrated that the osteoblastic cells tend to attach preferentially to blasted or plasma sprayed surfaces with a rougher topography, while the fibroblast cells favor smooth surfaces [14]. From a systematic review of these studies it is indicated that most research focuses on surface modifications of pure Ti or Ti-based alloy, and there are relatively fewer reports on the treatments of stainless steel [15]. A typical study was performed by McLucas et al. in 2006 [16], who investigated the interaction between endothelial cells and stainless steel with three different surface treatments (as received, polished and sandblasted with the surface roughness value of 95.8 nm, 40.8 nm, and 671.8 nm, respectively). They concluded that the sandblasted stainless steel samples resulted in endothelial cell injury and activation in vitro, indicating that surface roughness was an important surface property in the manufacturing of vascular implants. In addition, Hao et al. in 2005 treated stainless steel by means of CO2 laser irradiation, and compared the adhesion and proliferation behavior of human osteoblast cells on the untreated and the CO2 laser treated stainless steel samples [17]. They found that CO2 laser surface treatment enhanced human osteoblast cell adhesion and proliferation, which was attributed to an increase in the wettability (or a reduction of the contact angle) of the stainless steel samples. Nowadays, restenosis of the vascular stent [18] and initial instability of the hip femoral stem following implantation still cause much concern [19–21], which highlights the demanding requirement on gaining further knowledge of the interaction between

H. Zhang et al. / Materials Science and Engineering C 56 (2015) 22–29

stainless steel material and host tissues. From this point of view, a better understanding and control of cell adhesion and proliferation behavior on the stainless steel surface should be beneficial to develop the optimal surface treatment in a specific situation. Consequently, the present study aims to investigate the MC3T3-E1 cell response to stainless steel 316L with three different surface treatments in order to determine which kind of surface treatment is more effective to enhance cell adhesion and proliferation.

2. Materials and methods

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2.3. MC3T3-E1 cell response to stainless steel 316L samples 2.3.1. Cell culture Mouse osteoblast-like MC3T3-E1 cells were purchased from ATCC (Manassas, VA, USA), and cultured in α-MEM medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Hualisentai Co. Ltd, Beijing, China), 100 U/mL penicillin (Xinke Co. Ltd, Beijing, China), and 100 mg/mL streptomycin (Amresco, Solon, USA) at 37 °C in an atmosphere of 100% humidity and 5% CO2. At 80% confluency, the cells were detached by 0.25% trypsin-1 mM EDTA (Gibco BRL, Gaithersburg, MD, USA). The culture medium was renewed every 3 days.

2.1. Preparation of stainless steel 316L samples Medical-grade stainless steel 316L disks were purchased from Goodfellow Cambridge Ltd (Huntingdon, UK), and manufactured into square samples (size: 10 mm × 10 mm × 1 mm) by wire-electrode cutting. These raw samples were modified by three kinds of surface treatments resulting in various surface morphologies and compositions, i.e., polishing, aluminum oxide blasting, and hydroxyapatite (HA) coating. In detail, chemical mechanical polishing of the samples was performed on an automatic polisher machine (Struers, UK) using flexible polyurethane polishing pad and slurry specific for stainless steel material, following an initial wet-grinding process by silicon carbide abrasive paper (1200 grit). The aluminum oxide blasted samples were prepared by air-pressurized dry blasting employing 80-mesh aluminum oxide powder. The HA coated samples were obtained by plasma spraying with the use of 160-mesh HA powder. The plasma spraying conditions were as follows: temperature ~2000 °C, distance between the spraying gun and the sample ~100 mm, HA powder feed rate ~15 g/min.

2.2. Surface characterization of stainless steel 316L samples The surface morphology of the stainless steel 316L samples was examined through a Quanta 200 FEG scanning electron microscope (SEM, FEI, Eindhoven, Netherlands) associated with an energy dispersive X-ray analysis. The cross section of the HA coated sample was prepared by electrical discharge machining and then sputter-coated with a gold–palladium layer using a precision etching & coating system (Gatan 682, Pleasanton, USA) and investigated by the SEM in order to evaluate the thickness and homogeneity of the HA coating. In addition, the three dimensional (3D) surface topography of the samples was measured employing a microXAM-3D optical interferometer (KLA-Tencor Corp, California, USA), with a scanning area of 0.14 × 0.13 mm2. A total of six random positions on the sample surface were measured, and the surface roughness value (Sq) was calculated. The non-contact measurement mode of optical interferometry ensures that the original sample surface is not damaged. The surface wettability of the samples was characterized by static contact angle, which was determined based on sessile drop method using a contact angle system with image analysis (OCA-20, Dataphysics Instruments, Filderstadt, Germany). A pipette was used to ensure that each time a drop of ultrapure distilled water (approximately 2 μL) was placed on the sample. Six measurements were performed for each kind of surface treatment and eventually the average value of the static contact angle was calculated. The surface compositions of the polished and blasted samples were investigated by X-ray photoelectron spectroscopy (XPS) using an Escalab 250 Xi spectrometer (Thermo Scientific, USA) equipped with a monochromatized Al Kα X-ray source. All binding energy values were calibrated against the C1s peak at 285 eV. A D/max 2550 V X-ray diffractometer (XRD, Rigaku Corp, Tokyo, Japan) was used to examine the phase composition of the HA coating on the stainless steel 316L sample. The XRD pattern was collected over a 2θ range from 20° to 50° at a scanning speed of 2°/min.

2.3.2. Cell morphology All the stainless steel 316L samples were sonicated, sterilized, and washed with phosphate-buffered saline solution (PBS, pH = 7.2). For each of the three surface treatments, a total of 8 samples were placed in the well of 24-well culture plate, following sterilization using 75% ethanol for 10 min and rinse using sterilized PBS for three times to remove the ethanol. Then, 800 μL culture medium was added to each well, and 200 μL cell suspension with a cell density of 5 × 104 cell/mL was seeded onto the sample surface. The culture plates were gently transferred to a CO2 incubator (FORMA 311, Thermo Scientific, Marietta, USA), and the MC3T3-E1 cells were allowed for cultivation in an atmosphere of 5% CO2/95% humidified air at 37 °C. After culturing for 4 h, 1 d, 3 d, and 7 d, the samples were taken out, rinsed thoroughly with PBS to remove the unattached cells, and fixed with 2.5% glutaraldehyde solution (G6257, Sigma, St. Louis, MO, USA) for 30 min. Subsequently, the fixed cells were dehydrated progressively in a graded series of ethanol (50%, 75%, 90%, and 99%) for 15 min, and the samples were transferred into t-butyl alcohol and freeze-dried [22]. Finally, the samples were sputter-coated with a gold–palladium layer employing the precision etching & coating system, and the cell morphology was observed using the SEM. Two separate samples were examined for each time point. 2.3.3. Cell proliferation The quantity of the attached cells on the sample surface was evaluated using MTT method (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) according to ISO 10993-5 [23]. It is a measure of cell metabolism and the amount of formazan produced is related to the number of living cells. The cells were seeded onto the sample surface as mentioned above, and the culture plates were placed in the CO2 incubator for cell cultivation. After culturing for 1 d, 3 d, and 7 d, the samples were taken out and rinsed twice using PBS to remove the unattached cells. Then 300 μL MTT solution was added to each well and the plates were further incubated for 2 h in the incubator at 37 °C. After that the MTT solution was removed, and 300 μL isopropanol was added in each well to dissolve the blue-violet insoluble formazan, which was produced during metabolization of MTT by the living cells. After 30 min, 100 μL the solution from each well was transferred to a 96-well plate, and the optical density was measured by an enzyme labeling instrument (Model 680, Bio-Rad Laboratories Inc., Tokyo, Japan) at an excitation wavelength of 570 nm, with 650 nm as the reference wavelength. Five separate samples for each time point were examined, and the final absorbance was shown as mean value ± standard deviation. The difference among the three surface treatments was evaluated using one-way analysis of variance (ANOVA), with the statistical significance set at p b 0.05. 2.3.4. Protein adsorption Protein adsorption on the stainless steel 316L samples was evaluated using bovine serum albumin (BSA, Sigma, St. Louis, MO, USA). Briefly, 1 mL standard BSA solution with a concentration of 1 mg/mL (protein/deionized water) was pipetted onto the surface of each sample, which was incubated in a sterile humidified condition at 37 °C

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for 1 h. Subsequently, the samples were rinsed twice employing deionized water to remove the non-adherent BSA. BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) was used in this study. 1 mL protein reaction solution was pipetted onto the samples, and after 30 min, 100 μL reaction solution was transferred to a 96-well culture plate. The amount of adsorbed protein (mg/mm2) was quantified using a microplate reader (Bio-Rad Laboratories Inc., Tokyo, Japan) at 562 nm. A total of five samples for each surface treatment were examined, and the final result was shown as mean value ± standard deviation. The significant difference between the samples was evaluated by one-way ANOVA, with the p value set at 0.05. 3. Results 3.1. Surface characterization The SEM micrograph and 3D surface morphology of the stainless steel 316L samples treated by chemical mechanical polishing, aluminum oxide blasting, and HA coating were shown in Fig. 1. It was obvious that the polished sample was quite smooth, with a surface roughness value (Sq) of 1.8 ± 0.1 nm. The blasted and HA coated samples seemed much rougher, with high peaks and deep valleys present on the surface. The surface roughness values (Sq) of these two samples were 3.2 ± 0.4 μm and 7.8 ± 0.7 μm, respectively. The SEM micrograph of the cross section of the HA coated stainless steel 316L sample was demonstrated

in Fig. 2, and it was clear that the thickness of the HA coating was about 150 μm. According to the surface morphology and the element distribution (P, Ca, Cr, and Fe) of the HA coating, it was indicated that the coating was homogeneous. In addition, it was demonstrated from the 2D surface topography of the samples as shown in Fig. 3 that the fluctuation in surface height was just 9.1 nm for the polished sample, while the heights between the tallest peak and the deepest valley for the blasted and HA coated samples were approximately 10 μm and 40 μm, respectively. The comparison of surface wettability characteristics of the stainless steel 316L samples was shown in Fig. 4. The polished sample exhibited the lowest static contact angle of 74.2 ± 2.1°, and the blasted and HA coated samples showed larger static contact angles, which were 90.2 ± 1.5° and 106.9 ± 1.6°, respectively. The typical XPS scans over a binding energy from 0 to 1200 eV for the polished and blasted samples were demonstrated in Fig. 5, from which it was indicated that surface compositions of these two treatments were similar, with the presence of Fe2p, Cr2p, O1s, C1s, and Mo3d peaks. The XRD pattern of the HA coating on the stainless steel 316L sample was shown in Fig. 6, and both hydroxyapatite and tri-calcium phosphate (TCP) were detected. 3.2. Cell morphology The general shape and growth pattern of MC3T3-E1 cells at various incubation periods (4 h, 1 d, 3 d, 7 d) on the stainless steel 316L samples

Fig. 1. The surface morphology and three dimensional (3D) surface topography of the stainless steel 316L samples with different surface treatments: (a) polishing, Sq = 1.8 nm; (b) blasting, Sq = 3.2 μm; (c) HA coating, Sq = 7.8 μm. Bar: 100 μm.


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Fig. 4. The static contact angle of the stainless steel 316L samples with different surface treatments: (a) polishing, 74.2°; (b) blasting, 90.2°; (c) HA coating, 106.9°.

Fig. 2. SEM micrograph of the cross section of the HA coated stainless steel 316L sample associated with element distribution of P, Ca, Cr, and Fe. Bar: 100 μm.

treated by chemical mechanical polishing, aluminum oxide blasting, and HA coating were demonstrated in Fig. 7. The seeding cells showed distinctively different morphologies, especially within 1 d of incubation. In detail, after 4 h of incubation, the cells exhibited round shape and almost no spread on the polished sample. As for the blasted sample, the cells showed polygonal or slightly spindle shape on the surface. With regard to the HA coated sample, the cells became flattened with complete spread on the surface. After 1 d of incubation, the cells on the polished sample showed polygonal shape but no complete spread on the surface, and pseudopod could be observed for most of the cells. The cells on the blasted and HA coated samples flattened to a spindle shape and interacted with each other. There was no obvious difference

with regard to the cell morphology for these two samples. After 3 d and 7 d of incubation, flattened MC3T3-E1 cells were observed on the surfaces of all stainless steel 316L samples, which demonstrated great cell density with numerous cell–cell interactions. 3.3. Cell proliferation The proliferation kinetics of MC3T3-E1 cells on the stainless steel 316L samples treated by chemical mechanical polishing, aluminum oxide blasting, and HA coating from 1 d to 7 d was shown in Fig. 8. It was obvious that after 1 d and 3 d of incubation, the absorbance value for the HA coated samples was significantly higher than that of the polished and blasted samples (p b 0.05). Additionally, there was no significant difference between the polished and blasted samples at these two incubation periods (p N 0.05), although the absorbance value was slightly larger for the blasted samples. After 7 d of incubation, both the

Fig. 3. Two dimensional (2D) surface profile of the stainless steel 316L samples with different surface treatments: (a) polishing; (b) blasting; (c) HA coating.

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Fig. 5. XPS scans of the polished and blasted stainless steel 316L samples.

polished and blasted samples showed increased absorbance value in comparison with that after 3 d of incubation. However, it was interesting to observe that the absorbance value for the HA coated samples decreased to a much smaller level, which was significantly lower than that of the polished and blasted samples (p b 0.05). 3.4. Protein adsorption The adsorption of BSA on the stainless steel 316L samples treated by chemical mechanical polishing, aluminum oxide blasting, and HA coating at 1 h was shown in Fig. 9. It was clear that the polished and blasted samples demonstrated similar amount of BSA adsorption, which was significantly lower than that of the HA coated samples (p b 0.05). 4. Discussion It has been generally accepted that cell response to an artificial implant is determined by its surface properties including surface morphology, physicochemical composition, surface free energy, etc., which can be modified through a series of surface treatment techniques such as polishing, sandblasting, plasma spraying, and acid-etching [24,25]. It is considered that recent research in this area has been intensified to investigate pure Ti or Ti-based alloy, and the cell response to stainless steel with different surface treatments has been lacking [26,27]. In the present study, stainless steel 316L was modified by three kinds of surface treatments, of which aluminum oxide blasting and HA coating

resulted in similar surface morphology but different compositions, and polishing and aluminum oxide blasting led to similar composition but different surface morphologies. Therefore, the effects of surface morphology and surface composition on osteoblast-like MC3T3-E1 cell response were evaluated, respectively. Cell response to surface morphology has been extensively studied, and a general conclusion that can be deduced is that osteoblastic cells tend to attach to rougher surfaces with increased initial stability [28]. In the present study, it was indicated from the SEM observations that although there was no obvious difference as to cell morphology after 1 d of incubation between the polished (Sq = 1.8 nm) and blasted (Sq = 3.2 μm) samples, the rougher surface did improve initial cell adhesion. Additionally, the absorbance value was slightly larger for the blasted samples after 1 d and 3 d of incubation. However, the tendency reversed after 7 d of incubation, indicating that a highly polished surface may contribute to cell proliferation. Although it has been suggested that a surface roughness value of 1–2 μm (obtained by optical profilometry) is suitable for bone osseointegration [29], there is still no consensus with regard to the optimized surface roughness for a specific metallic biomaterial. The calculation of surface roughness value is considered to be arbitrary, which is influenced by a series of factors including measurement methodology, measurement area, as well as postmeasurement techniques such as leveling and filtration. Additionally, the samples with the same surface roughness value may have different surface morphologies, and correspondingly different cell responses. As a consequence, extensive research is required to gain an insight into the effect of surface roughness on cell response. HA is a frequently used surface coating material for orthopedic and dental prostheses due to its similarity to the mineral component of bone, which can enhance bone osseointegration to the implant by modifying the surface from “biological inert” to “bioactive” [30,31]. In the present study, it was shown from the SEM observations that, in comparison with the blasted samples, the HA coated samples demonstrated better cell morphology (e.g., flattened shape and complete spread), although there was no distinctive difference between them after 1 d of incubation. Additionally, the absorbance value was significantly higher for the HA coated samples after 1 d and 3 d of incubation, indicating superior cell attachment. The mechanism of this significant improvement may be attributed to the proteins, which can adsorb onto the surface of an orthopedic or dental prosthesis immediately following implantation, acting as a mediator in cell–material interaction [32–34]. In the present study, BSA adsorption on the HA coated samples was significantly higher than that of the polished and blasted samples. The result was consistent with the study performed by Kilpadi et al., in which it was demonstrated that HA bound more serum proteins, purified integrins, and osteoblast cells than the stainless steel substrate

Fig. 6. X-ray diffraction pattern of the HA coating on the stainless steel 316L sample. HA: hydroxyapatite; TCP: tri-calcium phosphate.


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Fig. 7. SEM observation of MC3T3-E1 cells on the polished, blasted, and HA coated stainless steel 316L samples at 4 h, 1 d, 3 d and 7 d of incubation. Magnification: 1000×.

[35]. However, it was noted that after 7 d of incubation, the absorbance value of the HA coated samples was significantly lower than that of the polished and blasted samples, which was consistent with the SEM observations as shown in Fig. 7, i.e., there seemed a reduced number of MC3T3-E1 cells present on the HA coated samples. This may be related to the change of the micro-environment for cell proliferation. In the present study, HA coating on the stainless steel 316L sample is prepared by plasma spraying, and the melted HA particles deposited on the metal substrate are quenched with a high cooling rate, resulting in the formation of amorphous calcium phosphate compound, metastable compound, and the reduction of crystallinity, as indicated in Fig. 5 and previous studies [36,37]. It has been shown that a decreased crystallinity of the HA coating could promote adhesion of osteoblast cells as a result of the higher biosolubility of amorphous calcium phosphate

compound and metastable compound [38]. This may be the reason contributing to the superior MC3T3-E1 cell attachment on the HA coated stainless steel samples that was observed in the present study. However, an excessive dissolution of the amorphous calcium phosphate compound and metastable compound is considered to be detrimental for the long term stability and reliability of the HA coated implant, and it has been shown that a lower crystallinity of the HA coating can elevate the pH value of the culture medium, resulting in a cytotoxic effect that inhibits proliferation of the attached cells [39]. In addition, Yamada et al. reported the formation of thin bone tissue and reduced bone mass around HA coated titanium implant, and attributed the reduction of cell proliferation rate to the elevation of local pH value [40]. Consequently, in the present study, the change of the micro-environment may have caused the significantly reduced absorbance value of the HA

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and cell attachment, but inhibits cell proliferation; and (3) HA coating processing with optimized control of crystallinity may represent the most favorable surface treatment for 316L surgical stainless steel. Acknowledgment This study was supported by the National Natural Science Foundation of China (Grant no. 51375008). References

Fig. 8. Cell proliferation kinetics of MC3T3-E1 cells on the polished, blasted, and HA coated stainless steel 316L samples at 1 d, 3 d, and 7 d of incubation. *p b 0.05, indicates a statistically significant difference between the compared surface treatments.

coated samples after 7 d of incubation, in comparison with that of the polished and blasted samples. Another potential reason may be attributed to the confluence of the MC3T3-E1 cells after a prolonged culture time (7 d), resulting in the decrease of the absorbance value. While the present study focuses on the MC3T3-E1 cell response to the stainless steel 316L samples with different surface treatments, we will investigate this issue in further study. One limitation of the present study is that additional tests on the amount of MC3T3-E1 cells (e.g., viability assay, total DNA) are not performed, and the MTT assay, as a representative of cell metabolic activity, does not necessarily indicate better cell viability. An increase in cell metabolic activity represents that the cells are either more active on the culturing material or there are more in terms of cell number, which warrants future investigations. 5. Conclusions In the present study, three kinds of surface treatments are used to modify stainless steel 316L, and MC3T3-E1 cell response to the samples is investigated based on cell morphology and cell proliferation. The following three conclusions can be drawn: (1) the rougher surface shows improved cell adhesion and cell morphology in comparison with the polished surface; (2) HA coating results in superior cell morphology

Fig. 9. Adsorption of bovine serum albumin on the polished, blasted, and HA coated stainless steel 316L samples at 1 h. *p b 0.05, indicates a statistically significant difference between the compared surface treatments.

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