Cytocompatibility Evaluation of Etched Zirconia Ceramics Using Human Osteoblast-Like MG 63 Cells

Ⅰ. INTRODUCTION

Tooth-colored, biocompatible ceramic materials have been introduced as a possible candidate for implants. In the 1980s, an implant was made of aluminum oxide (Al₂O₃). It was later withdrawn from the market owing to its high clinical fracture risk (Schulte W. 1984). Because of this critical weakness, tooth-colored zirconia have been suggested as an alternative materials in esthetically focused implant treatment (Kohal RJ et al. 2004).

In the 1970s, Garvie et al. and Gupta et al. reported that the fracture toughness of zirconia increased by tetragonal to monoclinic martensitic transformation toughening, which was considered revolutionary in the field of ceramics. Pure zirconia exists in three phase, namely, monoclinic, tetragonal, and cubic, depending on the temperature. Upon cooling from high temperature, a reverse transformation, which is accompanied by a substantial increase in volume (∼4.5%), from the tetragonal phase to monoclinic phase begins at approximately 950℃. This phenomenon can cause catastrophic failure. The increasing volume of zirconia via phase transformation can be stabilized at room temperature by the addition of oxides such as CaO, MgO, Y₂O₃ or CeO₂ (Kelly JR & Denry I, 2008). Yttria (Y₂O₃) is used as a general stabilizer for maintaining the tetragonal phase of zirconia (zirconium dioxide, ZrO₂) at room temperature. Using yttria (Y₂O₃) as a stabilizer, partially stabilized yttria tetragonal zirconia polycrystals (Y-TZP) possessed high fracture resistance owing to their energy absorption properties during the martensitic phase transformation (Lin JD & Duh JG, 2002). Zirconia ceramics have good mechanical properties such as high fracture toughness (900-1200 MPa), hardness (1200 Vickers), low thermal conductivity, and excellent resistance to corrosion (Piconi C & Maccauro G, 1999). However, the use of Y-TZP at low temperature (100-400℃) is limited by the low temperatures degradation (LTD, often referred to as “aging”). In 1981, Kobayashi et al. reported that partially stabilized zirconia polycrystals (PSZ) in a 250℃, wet environment was susceptible to LTD owing to deterioration in the mechanical properties. The speed of LTD of zirconia increased in the presence of water, and the consequences of this aging process are surface degradation with grain particle away, microcracks, and strength degradation (Swab JJ, 1991). Since LTD rapidly progresses at ∼250℃, it was not initially regarded as an issue under the physiologic conditions in the body. Nevertheless, some failures in its clinical use in hip joints were reported (Maccauro G et al. 2004). Similarly, the oral cavity is a moist and dynamic environment that can adversely affect the mechanical properties of zirconia.

There have been efforts to prevent deterioration of zirconia as a result of LTD, including the addition of stabilizers such as niobium oxide (Nb₂O₅) (Kim DJ et al. 1995). Such attempts led to stable lattices and low oxygen vacancy concentrations in (Y, Nb)-TZP, resulting in phase stability of tetragonal zirconia (Raghavan S et al. 2001). The substitution of Nb⁵⁺ for Zr⁴⁺ annihilates the oxygen vacancies formed by the Y³⁺ substitution, which reduces vacancy diffusion. Kim et al. (Kim DJ & Jung HJ, 1998) reported that 3Y-TZP doping with niobium oxide (Nb₂O₅) increased fracture toughness and decreased ionic conductivity, resulting in low temperature phase stability.

Based on these observations, we used 3Y-TZP co-doped with Nb₂O₅ in order to overcome the limitations of zirconia, which is vulnerable to LTD. Acid-etching method to increase surface roughness of zirconia is selected in our study. Zirconia has been shown to be as biocompatible as titanium (Ichikawa Y et al. 1992). Also, zirconia implants have shown biologic responses that are similar to those induced by titanium implants in vivo studies (Gahlert M et al. 2007). Therefore, Y-TZP is considered a potential alternative material to a titanium.

Hence, we assessed the surface topographies of etched (Y, Nb)-TZP and cytocompatibility of acid etching (Y, Nb)-TZP by evaluating cell attachment and proliferation and initial differentiation using human osteoblast-like MG 63 cells.

Ⅱ. MATERLALS AND METHOD

Ⅲ. RESULTS

1. Surface characteristics

1) Morphology and roughness

The surface morphologies and topographical images of the investigated specimen were observed using SEM (Fig. 1). The specimens of the untreated Zr group showed polygonal grains of varying radii were observed and no porosity. The surfaces of the Zr 1.5 h group showed varying sizes of polygonal grains and minuscule gaps between the grains. The surfaces of the Zr 2.0 h group showed sharp grains and porosity inside the grains. The surfaces of the Zr 2.5 h group showed irregular, sharp grains. The surfaces were not smooth, and deeper porosity and more irregular grains were observed for this group than for the Zr 2.0 h group.

Figure 1.

SEM and topographical measurement images by non-contact optical profilometry Of untreated Zr, Zr 1.5 h, Zr 2.0 h and Zr 2.5 h groups (at ×5000 magnification).

The surface roughness values were measured at a micron-scale by using optical profilometry (Table 1). The R_a values for the untreated Zr, Zr 1.5 h, Zr 2.0 h and Zr 2.5 h groups were 0.36, 0.55, 1.22 and 1.01 μm, respectively.

Table 1.

Surface roughness parameters of untreated Zr, Zr 1.5 h, Zr 2.0 h and Zr 2.5 h groups (n = 4)

2) structure

In XRD analysis, a high intensity tetragonal phase peak at 30.2° was dominant in the (Y, Nb)-TZP specimens of all the groups. The intensities of monoclinic phase peaks at 28.2° and 31.5° were minimal in all groups (Fig. 2).

Figure 2.

XRD analysis of zirconia specimens.(a) untreated Zr group, ( b) Zr 1.5 h group, (c) Zr 2.0 h group, (d) Zr 2.5 h group.

2. Cell morphology

The morphology of cell attachment and the spreading of human osteoblast-like MG 63 cell on different specimens were observed using SEM at 6 h and 24 h of incubation (Fig. 3).

Figure 3.

SEM images showing the morphology of attachment and spread of osteoblast-like MG 63 cells on untreated Zr, Zr 1.5 h, Zr 2.0 h and Zr 2.5 h groups at 6 h and 24 h of incubation.

After culturing for 6 h, SEM showed that the cells were in the process of adhering onto the surfaces of all the groups.

After culturing for 24 h, most cells of all groups exhibited a flattened shape, that is, cells started to spread onto all specimens. The cells of different specimens often appeared to be in contact with each other via extensions.

3. Cell proliferation

A cell proliferation assay (CCK-8 assay) of human osteoblast-like MG 63 cells seeded onto the zirconia specimens was performed for 1, 2, 3, 4, and 5 d(Fig. 4).

Figure 4.

Cell proliferation assay (CCK-8 assay) of osteoblast-like MG63 cells seeded onto zirconia specimen for 5 d. The optical density (OD) for each well was measured after 90 min from the initiation of CCK-8 reaction at 450 nm by using a 96-well plate reader. The results are presented as mean ± standard deviation (N = 3).*: Statistically significant differences as compared to untreated Zr (P < 0.05).†: Statistically significant differences as compared to Zr 1.5 h (P < 0.05).‡: Statistically significant differences as compared to Zr 2.5 h (P < 0.05).

The optical densities of all the groups were enhanced up to 5 d. At 5 d, optical density of the untreated Zr group was higher than that of the other experimental groups. On day 1, the optical density among the groups was statistically insignificant (P > 0.05). On day 2, the optical density of the untreated Zr group was statistically significant (P < 0.05), as compared to Zr 2.0 h and Zr 2.5 h groups. Also, the difference between the Zr 1.5 h and Zr 2.0 h groups was statistically significant (P < 0.05). On day 3, the optical density of the untreated Zr group was statistically significant (P < 0.05), as compared to Zr 1.5 h, Zr 2.0 h, and Zr 2.5 h groups. Also, the difference between Zr 1.5 h and Zr 2.5 h groups was statistically significant (P < 0.05). On day 4, the optical density of the Zr 2.0 h group was statistically significantly (P < 0.05) lower than that of any other groups and no statistical differences were observed among the other groups except the Zr 2.0 h group (P > 0.05). On day 5, only the difference between the untreated Zr group and the Zr 2.0 h group was statistically significant (P < 0.05).

4. Alkaline phosphatase activity

Detection of ALP was performed using an ALP kit (Sigma) and the quantitative analysis of ALP staining density was accomplished using the Image J program at 7 and 14 d (Figs. 5a and 5b).

Figure 5.

(a) Photographs showing staining density on ALP activity. MG 63 cells were stained for alkaline phosphatase (ALP) activity using an ALP kit (Sigma). Cells were cultured in osteogenic medium for 7 and 14 d. (b) Quantitative analysis of ALP staining density of MG 63 cells cultured on different groups at 7 and 14 d. The staining density was quantified using the Image J program. The results are presented as mean ± standard deviation (N = 3) .* Statistically significant differences as compared to untreated Zr (P < 0.05).†: Statistically significant differences compared to Zr 1.5 h (P < 0.05).‡: Statistically significant differences compared to Zr 2.5 h (P < 0.05).

At 7 d, differences of ALP staining density was not statistically significant (P > 0.05). At 14 d, the cells grown on the Zr 2.0 h group exhibited significantly higher ALP staining density than those on other groups (P < 0.05). Among others groups, the difference of ALP staining density were not statistically significant (P > 0.05).

Ⅳ. DISCUSSION

For analyzing the surface characteristics of (Y, Nb)-TZP specimens, SEM observation, surface roughness measurements, and XRD analysis were performed. With acid etching, minute gaps at grain boundaries started to form, and grains with rough surfaces and porosity between grains appeared in the SEM images . In our study, the surface roughness (Ra) measured by optical profilometry was 1.22 μm in the Zr 2.0 h group and 1.01 μm in the Zr 2.5 h group. In a previous in vitro study, the S_a value of zirconia discs etched in solutions of sodium hydroxide and potassium hydroxide for 30 h was 1.19 μm (Hempel U et al. 2010). In another in vitro study, the Ra value of Y-TZP abraded by air-borne particles and additionally acid etched with hydrofluoric acid for 5 minutes was 0.93 μm (Bächle M et al. 2007). Although we used a different approach for improving the surface properties, the profilometric measurements were similar. Hence, it was considered that etching of (Y, Nb)-TZP in a HF/H₂NO₃ solution is one of the ways to modify surface topographies.

The micro-structural characterization of (Y, Nb)-TZP was carried out using XRD analysis. Zirconium dioxide (ZrO₂) in Y-TZP exists in a tetragonal phase at room temperature, which contributes to the structural stability of Y-TZP by transformation toughening. In our study, XRD analysis of the (Y, Nb)-TZP specimen showed a high intensity tetragonal peak at 30.2°. The intensities of monoclinic phase peaks at 28.2° and 31.5° were minimal. With increasing acid-etching time, the XRD patterns of all experimental groups were similar to that of the untreated (Y, Nb)-TZP. These results indicate that use of the acid-etching method for modifying surface topographies did not adversely affect the phase stability of (Y, Nb)-TZP.

SEM observation of the cells after culturing for 6 h showed that the cells in all groups were in the process of adhesion. Furthermore, the cells of all groups after culturing for 6 h and 24 h demonstrated a similar morphologic appearance: partially spread onto the specimens and a flattened shape. Grinell (1978) reported that adhesion of cells to substrates followed a particular sequence of steps, that is, adsorption of serum proteins, contact of the rounded cell with the substrate, cell attachment to the substrate, and cell spreading on the substrate. In the present study, we did not analyze the early steps of osteoblast attachment onto zirconia ceramics. However, we observed close contact between the cells on the zirconia ceramics and flattened cells on the zirconia ceramics in SEM. These observations in the present study correspond with those in a previous study, which demonstrate that MC3T3-E1 cells on all the plates in the study appeared to attach and spread well after 6 h and 24 h incubation on the rough and smooth surfaces of Y-TZP (Ra 1.01 μm vs. 0.18 μm) (Yamashita D et al. 2009).

Cell proliferation was observed every day for 5 days in each group, and staining of MG 63 cells for ALP activity was observed under a phase-contrast microscope. Quantitative analysis was then performed using the Image J program. Optical density, which was determined using the CCK-8 assay, tended to increase in each group over 5 d. However, on day 1, the optical density of the osteoblast-like MG 63 cells was higher in the Zr 2.0 h group. On day 4, the optical density of the Zr 2.0 h group was significantly lower than that of the other groups (P < 0.05). However, the ALP staining density was the highest in the Zr 2.0 h group at 14 d (P < 0.05).

Stein et al. (1990) proposed a model of the relationship between proliferation and differentiation during the initial period of the development of a rat osteoblast. This relationship had an osteoblast developmental sequence with three distinct periods: proliferation, extracellular matrix maturation, and mineralization (Lian JB & Stein GS, 1992). Considering our results for the Zr 2.0 h group, although cell proliferation was relatively low, this group showed the highest ALP staining density, which is consistent with Stein’s model, in which proliferation is down-regulated by maturation of the extracellular matrix (ECM) and induction of ALP was initiated at the end of proliferation. ALP is an early differentiation marker for osteoblasts, and it supplies the cell with inorganic phosphate for subsequent calcium phosphate accumulation. The phosphate, which is released from β-glycerophosphate by ALP, is necessary for the formation of calcium phosphate nodules around osteoblasts in vitro.

The effect of increasing surface roughness on the cellular response of osteoblast has not concluded (Lincks J et al. 1998, Cooper LF et al. 1999). In a previous in vitro study, cell attachment and proliferation on Ti disks decreased on rough surfaces in comparison with smooth surfaces (Boyan BD et al. 2003). However, in another in vitro study, it was reported that the number of attached osteoblasts on rough surfaced titanium was higher than that on smooth surfaced titanium (Pae A et al. 2011).

Since Y-TZP has been introduced as an alternative biomaterial to Ti implants, the role of the zirconia surface topography on the response of osteoblasts has been the focus of several in vitro studies in recent years. In one in vitro study, it was demonstrated that zirconia with two different topographical surfaces (sandblasted vs. etched: S_a 1.13 μm vs. 1.11 μm) has a more pronounced effect on the adhesion, proliferation, and differentiation of the cells, compared with titanium and that topographical differences of zirconia have a minor effects on osteoblast biology (Hempel U et al. 2010). In another in vitro study, roughened Y-TZP (sandblasted vs. sandblasted/etched: R_a 0.85 μm vs. 0.93 μm) was found to be an appropriate substrate for the proliferation and spreading of osteoblastic cells (Bächle M et al. 2007). According to the majority of studies, surfaces with an S_a of ≥1 μm are considered rough, whereas those with an S_a of ≤1 μm are described as smooth, with the average surface roughness of titanium (Sykaras N et al. 2000). In this present study, the roughness value of (Y, Nb)-TZP after acid-etching over time ranged from 0.55 μm to 1.22 μm. Although the current results are different from the SEM image of SLA (sandblast, large grit, acid-etching) surface roughness, which shows a waviness by the sandblasting and micro-roughness by the acid-etching in titanium, the roughness values of (Y, Nb)-TZP after acid-etching for 1.5 h, 2.0 h and 2.5 h was 0.55 μm, 1.22 μm and 1.01 μm, respectively. The roughness value of (Y, Nb)-TZP with the untreated Zr group was 0.36 μm. On day 5, the optical density of the untreated Zr group was significantly higher than that of the Zr 2.0 h group (P < 0.05); the optical density of the other groups were not statistically significant as compared to the untreated Zr group (P > 0.05). Cellular proliferation of the Zr 2.0 h group with an R_a value of 1.22 μm was somewhat lower than that of the other groups at 5 d, but the staining density of ALP, an early differentiation marker of osteoblast, was significantly higher than that of the other groups (P < 0.05) at 14 d. It suggests that the rough surface of Zr 2.0 h group may stimulate the ALP activity causing by down regulation of proliferation in human osteoblast-like MG 63 cells. Thus the increase of ALP activity means initiation of cellular differentiation (Lian JB & Stein GS, 1992)

On the basis of our results, we postulate that staining density of ALP is relative low by stage of proliferation up to 7 days, since then ECM maturation contribute to down-regulate of proliferation and staining density of ALP, early differentiation marker, was remarkably higher in the Zr 2.0 h group at 14 days. Hence, roughened (Y, Nb)-TZP showed higher staining density of ALP than relatively smooth (Y, Nb)-TZP.

Ⅴ. CONCLUSION

This in vitro study evaluated the response of osteoblast cells on etched (Y, Nb)-TZP specimens. XRD analysis showed the structural stability of the dominant tetragonal phase after acid-etching zirconia ceramics in a HF/H₂NO₃ solution. Using niobium oxide as a stabilizer compensate for the LTD of Y-TZP, (Y,Nb)-TZP with adequate etch times served appropriate substrates for the proliferation and initial differentiation of osteoblast cells in the present study.

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