Pan-pan Ming†
ab,
Shui-yi Shao†ab,
Jing Qiu*ab,
Ying-juan Yuab,
Jia-xi Chenab,
Jie Yangab,
Wen-qing Zhuab,
Ming Lib and
Chun-bo Tangab
aJiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Nanjing, PR China. E-mail: qiujing@njmu.edu.cn; Tel: +86 25 85031834
bDepartment of Oral Implantology, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing, PR China
First published on 17th January 2017
Objectives. The aim of this study was to evaluate the corrosion behavior and cytocompatibility of a Co–Cr and two Ni–Cr dental alloys before and after the pretreatment with a biological saline solution. Methods. A commercial cobalt–chromium (Co–Cr) and two nickel–chromium (Ni–Cr) dental alloys [beryllium (Be)-free and Be-containing] were selected and pretreated with a biological saline solution containing 3% bovine serum albumin (BSA) for 72 h. Before and after the pretreatment, alloy specimens were examined for surface element compositions using X-ray photoelectron spectroscopy (XPS). Corrosion behavior and metal ion release were measured by electrochemical corrosion and immersion tests in artificial saliva respectively. The released metal ions in the exposed biological saline solution were also detected after the pretreatment for 72 h. 3T3 fibroblasts were cultured and exposed to specimens. After 4 hours of incubation, cell morphology and spreading were observed under a laser scanning confocal microscope. Cell proliferations were evaluated using CCK-8 assay after culturing for 1, 3 and 6 days. Results. After the pretreatment with a biological saline solution, the corrosion potential (Ecorr) and breakdown potential (Ebr) of the three dental alloys increased significantly, which corresponded with evident decreases of Co, Ni and Be levels on the alloy surfaces measured by XPS. The corrosion current (Icorr) and the polarization resistance (Rp) of the three alloys were not significantly affected by the pretreatment, corresponding to the slight reductions of Cr, molybdenum (Mo) and oxygen (O) levels on the surfaces of alloys except the Be-containing alloy. For the three alloys, the pretreatment obviously decreased the metal ions release in artificial saliva. There were a large amount of metal ions, including Co, Ni and Be ions, released from the alloys in the exposed biological saline solution after the pretreatment for 72 h. The results of in vitro study demonstrated that the pretreatment up-regulated cell spreading and proliferation on the three metallic substrates. Conclusions. The pretreatment with a biological saline solution optimized the surface elemental compositions of a Co–Cr and two Ni–Cr dental alloys by removing more labile metal elements from the alloy surfaces in form of cations. The corrosion susceptibility and metal ion release of the three dental alloys decreased significantly after the pretreatment, which led to improvements of their cytocompatibility.
However, corrosion behavior of dental alloys occurs in the oral cavity due to the complex biological and electrolyte environment. The metal ions that release during the corrosion processes are toxic to the surrounding tissues, and the biological response to particular metal ions can be evaluated in vitro using cell culture techniques.2 Nickel and cobalt ions are the primary elements released from Ni-based and Co-based alloys; the other elements are released at much lower concentrations.3,4 Our recent studies found that the corrosion rate and metal ions (Co and Ni) release of both Ni-based and Co-based dental alloys increased significantly after a simulated porcelain firing, which, in turn, increased the cytotoxicity of these base-metal alloys.5–7 Therefore, the released ions (Co and Ni) in the gingival crevicular fluid jeopardize periodontal health and cause gingival inflammation or discoloration.8,9
Since a large number of casting alloys used for clinical applications in restorative dentistry, current studies have been focused on the cytotoxicity and cytocompatibility of such metallic biomaterials. Nelson et al.10,11 have proposed the utility of a short-term pre-exposure of dental alloys for 72 or 168 h to either saline solution, saline/bovine serum albumin (BSA) solution or cell culture medium. Among three biological solutions, preconditioning with the saline/BSA solution had best effect to reduce cytotoxicity of the tested precious metal alloys. Sandrucci et al.12 also reported that the pretreatment with saline/BSA solution or cell culture medium for 72 h reduced the released Ag+ and Cu2+ from dental precious alloys and improved their cytocompatibility in vitro. The rationale for these procedure have been based on the possibility of the preconditioning treatment to remove more ionizable metal elements from alloy surfaces, as to decrease alloy cytotoxicity.
Although improvements of cytocompatibility of dental precious alloys after the pretreatment had been reported, few studies to date have evaluated the preconditioning effectiveness for non-precious dental alloys,10 in particular for Co–Cr and Ni–Cr alloys, which generally have higher cytotoxicity. Moreover, the changes in detail of surface elements and their influences on corrosion properties of dental alloys, which closely correlate with the cytocompatibility, have not been further studied so far. During recent years, electrochemical corrosion test had been proven to be advantageous for corrosion characterization of metallic biomaterials compared with traditional immersing test.6,13 Also, surface chemistry of metal oxides can be discriminated and analyzed by X-ray photoelectron spectroscopy (XPS) wide- and high-resolution spectra.14 Such information would promote the understanding of effects of the pretreatment process on dental alloys. Therefore, the aim of the present study was to evaluate the effects of the pretreatment with a biologic saline solution on the surface elements, corrosion properties and cellular responses of a Co–Cr and two Ni–Cr dental alloys.
Alloy | Composition (wt%) | |||||||
---|---|---|---|---|---|---|---|---|
Co | Ni | Cr | Mo | W | Al | Be | Other elements | |
a Information supplied by the manufacturers. | ||||||||
Wirobond C (Co–Cr) | 63.3 | — | 24.8 | 5.1 | 5.3 | — | — | Si, Fe, Ce each < 1 |
Stellite N9 (Ni–Cr) | — | 64 | 22.5 | 9.5 | — | 1 | — | Nb 1, Si 1, Fe 0.5, Ce 0.5 |
ChangPing (Ni–Cr–Be) | — | 76.5 | 14 | 4.5 | — | 2.5 | 2 | Fe 0.5 |
A total of 78 specimens (10 mm diameter and 3 mm thickness), 26 specimens for each alloy, were fabricated by a flame casting method using oxygen–propane gas mixture in accordance with the manufacturers' recommendations. The same technician made all castings in a dental laboratory. To simulate the application of a porcelain veneer, all specimens were subjected to a PFM firing cycle under vacuum in a dental porcelain furnace. Briefly, the specimens were degassed at 1010 °C under vacuum holding for 5 min, opaque fired at 980 °C under vacuum and air cooled, body fired at 970 °C under vacuum and air cooled, and finally glaze fired at 980 °C and air cooled.6 After firing, using a grinder-polisher machine (Beta, Buehler Ltd., Lake Bluff, IL, USA), the casting specimens were wet grinded with a series of silicon carbide papers (180, 400, 600, and 1200 grit) and then polished with 3 μm diamond suspension (Metadi, Buehler Ltd., USA). After that, the specimens were ultrasonically cleaned in ethanol, and then in de-ionized water.
Preconditioning treatment was performed by immersing each specimen into 3 ml of a biological saline solution containing 0.9% NaCl added with 3% (wt vol−1) bovine serum albumin (BSA, Sigma, St. Louis, MO, USA) and by leaving them there undisturbed for 72 h in a fully humidified air atmosphere containing 5% CO2 at 37 °C.11,12 Finally, the metal specimens were placed on sterile gauze to remove residual preconditioning solution, and ultrasonically rinsed in sterile water.
To compare the cell proliferation rates, the cells were exposed indirectly to the alloys. Three specimens of each alloy before and after the pretreatment were immersed in DMEM for 7 days. After that, the cells were seeded at 3 × 103 cells per cm2 into a 96-well culture plate containing the exposed medium and incubated in a 5% CO2 humidified environment at 37 °C, replacing the culture medium after 3 days. After 1, 3 and 6 days, the cell proliferation was investigated using a CCK-8 assay. The absorbance of each solution was measured by a microplate reader (Spectramax190, MD, USA) at 450 nm wavelength. All the cell experimental investigations were performed in triplicate compared with control cultures using medium not exposed to alloys.
The representative XPS high-resolution spectra of Wirobond C (Co–Cr), Stellite N9 (Ni–Cr) and ChangPing (Ni–Cr–Be) alloys are presented in Fig. 2. The complex peaks of Co 2p, Ni 2p and Be 1s exhibited binding energies that correlated with the presence of Co3O4, NiO and BeO as well as Co0, Ni0 and Be0, respectively. As shown in Fig. 2(a and b), Wirobond C exhibited reductions of Co3O4 and Co0 sub-peaks indicating decreases of Co3O4 and metallic Co levels after the pretreatment, which led to a decrease of Co 2p peak. The proportions of sub-peaks were similar in both conditions. As shown in Fig. 2(c–f), after the pretreatment, both Stellite N9 and ChangPing exhibited decreases of NiO and Ni0 sub-peaks indicating decreases of NiO and metallic Ni levels, which caused reductions of Ni 2p peaks. The sub-peaks in Ni 2p peaks revealed different proportions after the pretreatment. For Stellite N9, NiO obviously increased from 26.2% to 64.5%, whereas Ni0 evidently decreased from 73.8% to 35.5%. For ChangPing, NiO increased from 61.1% to 70.3%, while Ni0 decreased from 38.9% to 29.7%. As shown in Fig. 2(g and h), after the pretreatment, ChangPing also exhibited reductions of BeO and Be0 sub-peaks indicating decreases of BeO and metallic Be levels, which led to a decrease of Be 1s peak. The sub-peaks in Be 1s peak revealed different proportions after the pretreatment. BeO increased from 53.8% to 61.4%, whereas Be0 decreased from 46.2% to 38.6%.
Fig. 2 Representative XPS high-resolution spectra of Co 2p, Ni 2p, and Be 1s for Wirobond C (Co–Cr), Stellite N9 (Ni–Cr), and ChangPing (Ni–Cr–Be) alloys before and after the pretreatment. |
Alloy | Condition | Corrosion parameters (n = 3) | |||
---|---|---|---|---|---|
Ecorr (mV) | Icorr (μA cm−2) | Rp (kΩ cm−2) | Ebr (mV) | ||
a Wirobond C: Co–Cr alloy; Stellite N9: Ni–Cr alloy; ChangPing: Ni–Cr–Be alloy; Ecorr: open circuit potential; Icorr: corrosion current; Rp: polarization resistance; Ebr: breakdown potential. *Indicates a statistical difference between the unpretreated and pretreated conditions (P < 0.05). | |||||
Wirobond C | Unpretreated | −76.7 (15.7) | 0.31 (0.09) | 203.7 (82.8) | 810.7 (127.6) |
Pretreated | −45.2 (3.5)* | 0.24 (0.12) | 268.9 (193.2) | 1273.3 (55.1)* | |
Stellite N9 | Unpretreated | −185.7 (32.0) | 0.34 (0.24) | 193.4 (54.1) | 797.7 (13.7) |
Pretreated | −63.4 (5.1)* | 0.27 (0.05) | 190.4 (71.8) | 1123.3 (86.2)* | |
ChangPing | Unpretreated | −204.7 (17.7) | 0.41 (0.13) | 179.7 (59.4) | 514.3 (15.0) |
Pretreated | −64.3 (7.2)* | 0.32 (0.11) | 213.6 (75.7) | 876.0 (205.9)* |
Fig. 5 Total metal ions released into the biological saline solutions exposed to Wirobond C (Co–Cr), Stellite N9 (Ni–Cr), and ChangPing (Ni–Cr–Be) alloys after the pretreatment for 72 h. |
Co–Cr and Ni–Cr dental alloys depend on a surface oxide layer for corrosion resistance in the oral environment.21,22 Metal oxides on the alloy surfaces are formed spontaneously because of the rapid O uptake from the atmosphere after polishing.23 In this study, the oxides of Co, Ni, Cr, Mo, and other alloying elements on three alloy surfaces were found by XPS (Fig. 1). Previous studies had reported that the surface oxide layers formed on Co-based and Ni-based alloys were composed predominantly of Cr oxides and less Co and Ni oxides respectively.24–26 Regarding the surface oxide layer, the outer layer was Co-rich or Ni-rich oxides mixed with metallic Co or Ni, and the predominant inner one was compact Cr-rich oxides.26,27 As is well known, the compact Cr oxides act as a vital role in the corrosion resistance for non-precious dental alloys in the electrolyte.5–7,28 After the pretreatment, in the current study, the XPS examination of Wirobond C (Co–Cr) and Stellite N9 (Be-free Ni–Cr) alloys demonstrated evident reductions in the levels of Co and Ni respectively, which originated from the reductions of metallic Co or Ni and their oxides, as shown in the XPS survey and high-resolution spectra (Fig. 1 and 2). This result indicated that the pretreatment reduced the metallic Co or Ni and delayed the growth of the outer oxide layer on the two alloy surfaces. However, after the pretreatment, the XPS data revealed little, if any, changes in the relative amounts of Cr and O on both alloy surfaces, which indicated that the inner Cr-rich oxide layer retained compact and continuous. Thus, for Wirobond C and Stellite N9, changes of the surface oxide layer involved primarily in its outer part after the pretreatment. The optimized outer oxide layer containing less metallic Co or Ni, which are more liable, might lead to the decreases of corrosion susceptibility. This presumption was confirmed by the results of corrosion tests shown in Table 2, where both Wirobond C and Stellite N9 exhibited significantly higher Ecorr and Ebr values after the pretreatment, and, though not significant, Icorr values decreased slightly.
As for ChangPing (Be-containing Ni–Cr) alloy, after the pretreatment, the XPS results exhibited reductions in the levels of elements, especially Ni and Be (Fig. 1). Furthermore, as shown in the XPS high-resolution spectra for Ni 2p and Be 1s (Fig. 2), the percentage for metallic Ni decreased from 38.9% to 29.7% and that for metallic Be from 46.2% to 38.6%. It was reported that the corrosion resistance of Ni–Cr alloys was reduced by as little as 0.6 wt% Be content.29,30 In the present study, the ChangPing alloy had a 2.0 wt% Be content, which was considered to be negative for its corrosion resistance. The small atomic radius of Be facilitated its migration to the alloy surface.25 Due to the surface migration of Be, Be-containing Ni–Cr alloys were reported to form the Ni–Be eutectic phase that induced the development of non-homogeneous Cr surface oxides and depleted areas of Cr, which were less corrosion resistant.31 Therefore, after the pretreatment, although the surface oxides of the ChangPing alloy decreased, evident reductions of surface metallic Ni and Be might weaken the adverse effect of Ni–Be eutectic phase on the surface distribution of Cr oxides. The optimization, leading to less segregation and more homogeneous of Cr surface oxides, was positive for the alloy's corrosion resistance. This result was supported by the corrosion data shown in Table 2, where ChangPing exhibited higher Ecorr and Ebr values after the pretreatment, and, though not statistical, Icorr value decreased and Rp value increased slightly.
In the present study, the pretreated Wirobond C (Co–Cr), Stellite N9 (Ni–Cr) and ChangPing (Ni–Cr–Be) alloys released less metal ions into the artificial saliva compared with the same alloys without the pretreatment (Fig. 4). These findings were in consistent with the XPS and corrosion results. After the pretreatment, the reduced surface levels of alloying elements, in particular for metallic Co, Ni, and Be, led to the decreases of the alloys' corrosion susceptibility, which, in turn, caused less dissolution of metal elements into the artificial saliva. Also, it was found that the tested alloys released a large amount of metal ions into the biological saline solution when underwent the pretreatment (Fig. 5). This was in accordance with Nelson et al.,10,11 who found high concentrations of Ag and Cu in the solution containing NaCl and BSA employed for preconditioning precious dental alloys. Nevertheless, in this study, little Cr ions dissolved in the biological saline solution and were still below the detection limit, indicating that the pretreatment almost had little influence on the tested alloys' surface Cr oxides. Together with the XPS analysis, this could confirm that the biological saline solution provoked the release of metal ions except Cr within the pretreatment process. Consequently, it is reasonable to assume that the pretreatment could be considered a useful method to remove ionizable metal elements, such as Co, Ni and Be, from surfaces of the three dental alloys.
As far as cellular responses were concerned, the pretreatment with a biological saline solution enhanced the cytocompatibility of Wirobond C (Co–Cr), Stellite N9 (Ni–Cr), and ChangPing (Ni–Cr–Be) alloys in this study. In the assessment of biomaterial cytocompatibility, cell adhesion and proliferation rate are two of the most studied biological parameters on in vitro experimental models with cell lines.32–34 Furthermore, it is well known that cell proliferation is strictly correlated to cell capability to adhere to the substrate; hence it seemed appropriate to investigate both two biological aspects in relationships to the presence of different dental alloys during performing the current study. We observed that the pretreatment influenced the biological behavior of fibroblast cultures on the alloys. Cells spread more evenly and extended more pseudopodia in the presence of the preconditioned alloys in comparison with those on the unconditioned alloys (Fig. 6). This evidence, together with the increased cell proliferation rates (Fig. 7), could confirm the effectiveness of the pretreatment for these base-metal dental alloys, in parallel with previous studies, which reported higher cell viability in the presence of noble-metal dental alloys preconditioned with protein-containing biological solutions.10–12 Cytotoxicity of dental alloys, especially base-metal alloys, had been obviously correlated with their metal ion release,35–37 which could induce adverse physiological effects in adjacent oral tissues, including gingival inflammation or discoloration. In our work, the biological saline solution encouraged the release of more labile metal elements in the pretreatment phase, which significantly decreased subsequent cytotoxicity of the three dental alloys. Thus, this observation of avoiding or limiting successive metal ion release and improving the cytocompatibility implied the possible utilization of the preconditioning treatment before clinical applications of these dental alloys.
Our investigation primarily assessed the effects of the pretreatment on the in vitro cytocompatibility of the dental alloys. However, the exact mechanism of the effectiveness and its biocompatibility evaluation still require further in vitro and in vivo studies.
Footnote |
† These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2017 |