Attachment and proliferation of neonatal rat calvarial osteoblasts on Ti6Al4V: effect of surface chemistries of the alloy
T. M. Leea, E. Changb and C. Y. Yang, , c
a Institute of Oral Medicine, National Cheng Kung University, Tainan 701, Taiwan
b Department of Materials Science and Engineering, National Cheng Kung University, Tainan 701, Taiwan
c Department of Orthopedics, Medical Center, National Cheng Kung University, Tainan 701, Taiwan
Received 26 October 2002; accepted 19 June 2003. ; Available online 27 August 2003.
Abstract
This study examined the cell attachment and proliferation of neonatal rat calvarial osteoblasts on Ti6Al4V alloy as affected by the surface modifications. The modifications could alter simultaneously the surface chemistries of the alloy (elemental difference of Ti, Al, V, Cu and Ni about 300-600
m thick examined by EDS) as well as the XPS nano-surface characteristics of oxides on the metal surface (chemistries of oxides, amphoteric OH group adsorbed on oxides, and oxide thickness). Three materials including two from modifications and a control were examined. It is argued that a slight change of the nano-surface characteristics of oxides as a result of the modifications neither alters the in vitro capability of Ca and P ion adsorption nor affects the metal ion dissolution behavior of the alloy. This implies that any influence on the cytocompatibility of the materials should only be correlated to the effect of surface chemistries of the alloy and the associated metal ion dissolution behavior of the alloy. The experimental results suggest that the cell response of neonatal rat calvarial osteoblasts on the Ti6Al4V alloy should neither be affected by the variation of surface chemistries of the alloy in a range studied.
Author Keywords: Titanium; Surface modification; Cytocompatibility; Cell culture; Osteoblast
Article Outline
1. Introduction
Biocompatibility of metallic implants and bone in orthopedic surgery plays an important role in long-term survivor of the prosthetic implant fixation. An indication of biocompatibility is indicated in the bone cell response such as bone apposition and bone formation when the biomaterials are implanted. Recently, cell cultures were applied to investigate the biological response of bone-implant interface during early phase. Several factors could affect the biocompatibility of the metallic materials. Increasing documents have reported that the biocompatibility of implants in terms of bone cells response is in association with the surface topography, energy and roughness of the biomaterials [1, 2, 3, 4 and 5]. Another factor is attributed to the nano-surface characteristics of metal oxides grown on the metal surface. Albrektsson et al. and Kasemo mentioned that the biocompatibility of titanium implants is associated with the surface titanium oxide and not with the titanium metal [6 and 7]. The chemistries of oxides, amphoteric OH group adsorbed on oxides, and oxide thickness on the metal surface are believed to influence the biocompatibility, but few evidences were provided [8, 9 and 10]. On the other hand, it was argued that the variations of nano-surface characteristics of oxides on the metal surface (chemistries of oxides, amphoteric OH group adsorbed on oxides, and oxide thickness) in a range studied should not alter the in vitro adsorption of Ca and P ions on the Ti6Al4V alloy [11].
Another major concern of the metallic implant in biocompatibility is the toxicity of metal ion dissolution. Metal ion dissolution has been demonstrated to adversely affect the healing of bone [12, 13 and 14]. Recent study suggested that passivation treatments influence the early-stage ion dissolution rate of the titanium alloy [11]. To prove the significance of metal ion dissolution, it is considered that the problem should be substantiated by a cytocompatibility evaluation. In the present study, we changed the surface chemistries of the Ti6Al4V alloy (elemental difference of Ti, Al, V, Cu and Ni in metals) by the surface modifications. The neonatal rat calvarial osteoblastic cells were cultured on the alloy in a simulated body fluid to examine the factor of surface chemistries of the Ti6Al4V alloy on the cell response.
However, the surface modifications could simultaneously alter the X-ray photoelectron spectroscopy (XPS) nano-surface characteristics of oxides on the metal surface in the processing. The chemistries of oxides and the adsorbed OH group could also influence the cell response of osteoblasts on titanium. As well, the oxide thickness could also change the kinetic metal ion dissolution [15 and 16], that in turn exerts an influence on the cell response or the cytocompatibility of the materials investigated. It was intended and designed in a scheme as discussed below that the cytocompabitility of the materials can be discussed in relation to the surface chemistries and metal ion dissolution of the tested alloys, without the interfering influence from the factor of nano-surface characteristics of metal oxides on the alloy.
2. Materials and methods
2.1. Experimental materials
Three kinds of experimental materials (12.7 mm
×2.0 mm disk plates) were used in this study: (a) Ti6Al4V alloy (T), (b) Ti6Al4V alloy with a surface modification of chemistries about 300
m prepared by vacuum brazing at 970°C for 2 h (B2) [17], and (c) Ti6Al4V alloy with a surface modification of chemistries about 600
m prepared by vacuum brazing at 970°C for 8 h (B8) [17]. During the brazing treatment, a foil of chemistries Ti15Cu15Ni (wt%) with a melting point of 934°C was used and placed on the experimental materials. The experimental materials were ground through successive silicon carbide papers to 1500 grit and polished by 1
m Al2O3, then finally subjected to ultrasonic wash in acetone and rinsed three times in double-distilled water. After polishing and cleaning, each group of specimen was passivated by aging in boiling de-ionized water for 24 h (A). After passivation treatment, all the specimens were subjected to sonication five times in de-ionized water and one time in absolute alcohol. The specimens were then packed in double-sealed autoclaving bags, steam sterilized at 121°C for 30 min, and dried at 121°C for 15 min. After the treatments, three groups of experimental specimens were prepared: (a) TA, (b) B2A, and (c) B8A, respectively.
2.2. Materials characterization
The details of the experimental materials have been investigated in our previous studies [11, 18 and 19]. The procedure is described as follows. The experimental materials were ground through successive silicon carbide papers to 1500 grit, then subjected to sonication in acetone and rinsed three times in double-distilled water. After passivation and autoclaving, the chemical composition of experimental materials were analyzed by scanning electron microscopy (SEM) equipped with energy-dispersive spectroscopy (EDS). Shallower surface chemical analyses by X-ray photoelectron spectroscopy (XPS) techniques (VG ESCALAB 210) were carried out for various specimens. Using the program supplied by the manufacture, the nano-surface composition, amphoteric OH content, and oxide thickness of specimens were analyzed and showed in Table 1 [11, 18 and 19]. The ion dissolution of specimens in Hank's solution with 8.0 m
ethylenediaminetetraacetic acid (EDTA) was performed with a surface area to solution volume ratio of 0.1 cm−1. After 1-day immersion, the incubated medium was poured from each bottle into a cleaned and labeled polypropylene tube which was then sealed. The collected solutions were analyzed for Ti, Al, V, Cu and Ni using the inductively coupled plasma-mass spectrometer (ICP-mass, Hewlett Packard Model 4500 series). In this study, the surface hydrophilicity of 1
m Al2O3 polished specimens was determined by the sessile drop technique (Face, Model CA-A, Tokyo, Japan). Five specimens of each group were performed to evaluate the water contact angle.
Table 1. SEM/EDS surface chemistries, XPS nano-surface characteristics of oxides, and metal ion dissolution rate of TA, B2A and B8A specimens
2.3. Cell culture
Osteoblasts were isolated by sequential trypsin-collagenase digestion on calvaria of neonatal (<4 days old) Sprague-Dawley rats as described elsewhere [18]. In short, the calvaria were excised under aseptic conditions and kept in the ice-cold phosphate-buffered saline (PBS). The fibrous layers of the periostea were mechanically removed. The calvaria were then incubated with the Dulbecco's modified Eagle medium (DMEM) for 2×10 min at 37°C and rinsed 2×5 min with the PBS. To diminish fibroblastic contamination and cell debris we preincubated the calvaria for 20 min with the enzyme solution (18000 unit/ml collagenase I, 0.125% trypsin, 0.004
ethylenediaminetetraacetic acid (EDTA), and 0.02% DNase I at 37°C) and discarded the supernatant. After continuous enzyme treatment (6×20 min), the third, fourth, fifth and sixth supernatants were centrifuged (10 min at 1000 r.p.m.; 250 g). The pellets were resuspended in the DMEM containing 10% fetal bovine serum and maintained in a humidified, 5% CO2/balance air incubator at 37°C. The phenotype and function of the osteoblasts were characterized by the presence of alkaline phosphatase by using a commercial diagnostic kit (Sigma Chemical Co., St Louis MO, Catalog No. 86-R). According to the manufacture's protocol, the cells were immersed in fixative solution, washed, immersed in alkaline-dye mixture, washed, counterstained in Mayer's Hematorylin solution, mounted and observed by optical microscopy (OM). The deposition of calcium phosphate mineral was determined by the von Kossa method [20 and 21]. Briefly, cells were fixed with 3% formaldehyde, washed, stained with 1.5% silver nitrate, washed, the 5% sodium thiosulfate added to remove the silver nitrate, counterstained in 0.5% safranin, and observed by OM. Subcultured cells were used for the experiments after two or three passages.
The sterilized disks were placed in 24-well culture plates. These tissue culture plates, with 12% poly hydroxyethylmethacrylate (HEMA) coating, held the biomaterial samples to ensure that the osteoblasts would grow on the biomaterials only and not the tissue culture wells [21]. For experiments, 1 ml of osteoblast cells suspension were seeded on the disks at a density of 75,000 cells/ml and flooded with the growth medium supplemented with 4% fetal bovine serum, 50
g/ml ascorbic acid and 10 m
-glycerophosphate. The culture medium was changed every 2 days during culture.
2.4. Cell attachment
Specimens were prepared for SEM after 3-, 12- and 48-h cultures. The medium was pipetted out from the dishes, and the plates rinsed several times with cacodylate buffer (pH 7.2), and fixed for 2 h with 2.5% glutaraldehyde in 0.1
cacodylate buffered at pH 7.2. The specimens were then post-fixed with 1% OsO4 in buffer for 1 h, treated with 1% tannic acid in buffer for 1 h, dehydrated in an ascending alcohol series, and immersed in HMDS (hemaethyldisilazane) for 10 min in lieu of critical point drying. Finally, after sputter coating with gold, the specimens were examined using a Hitachi S-2500 scanning electron microscope at an accelerating voltage of 25 kV.
2.5. Cell growth
The cell growth on each biomaterial sample was determined after 1-, 3-, 5-, and 15-day cultures. At the designated times (2×10 min), the osteoblasts were released from the biomaterial surface by addition of 0.05% trypsin with 1 m
EDTA in PBS. A second trypsinization was performed to ensure that any remaining cells had been removed from the surface. SEM examination of the disks used in the preliminary studies showed that following the two trypsinizations, all cells and matrix were removed from the surfaces of specimens (data not shown). Cell suspensions from both trypsinizations were combined and centrifuged at 250 g for 10 min. The supernatant was decanted, and the cell pellet was reconstituted with the DMEM for measurement with hemocytometer. Cell viability was determined by the trypan blue exclusion, then counted the cell number with a hemocytometer.
2.6. Statistical analysis
Each data point represents the mean±standard deviation (SD) of the three individual cultures. The analysis of one-way variance (ANOVA) was used to evaluate the significant differences between the cell growth on different kinds of materials. Differences were considered significant at p<0.05.
3. Results
3.1. Detailed descriptions of the experimental materials
The SEM/EDS analyses of the chemical composition (wt%) of TA, B2A, and B8A specimens after passivation and autoclaving treatment are shown in Table 1 [11]. Comparing with TA, the B2A specimen shows a lower chemical content of Al and V, with some additional Cu and Ni elements. Longer time of surface modification for B8A specimen results in a reduction of Cu and Ni contents compared to B2A specimen. TA specimen has a characteristic equiaxed
- plus
-phase microstructure. By XRD analysis, minor phase of Ti2Cu was observed in B2A and B8A specimens [17]. The amount of Ti2Cu decreases with increasing period of surface modification treatment from 2 to 8 h.
After passivation and autoclaving treatment, the results of XPS nano-surface characteristics (chemistries of oxides, amphoteric OH group adsorbed on oxides, and oxide thickness) of TA, B2A, and B8A specimens are displayed in Table 1 [18 and 19]. The XPS analyses mainly detect the nano-order surface oxides and ions adsorbed on the surface of titanium. Modifications have slightly changed the nano-surface characteristics of the experimental materials by XPS analysis, meanwhile Cu and Ni are not found in the nano-surface of B2A and B8A specimens as explained later.
The ICP metal ion dissolution rates of TA, B2A and B8A specimens are shown in Table 1 [18]. Examination of the data indicates that the ion dissolution rate of Ti and Al ions for TA specimen is apparently higher than that for B2A and B8A specimens, while the latter two specimens contain Cu and Ni ions in the simulated body fluid [18]. In Table 1 it is noted that some extra data experimented with similar techniques as Refs. [11 and 20] are provided.
The water contact angle values measured by the sessile drop method are shown in Table 1. The results indicate that TA shows the lower contact angle than B2A and B8A, while the significant difference was not found among the three experimental materials by the method of ANOVA.
3.2. Cell attachment
After 3-h culture, the osteoblast morphologies on the surface of specimens are observed by SEM (Fig. 1). As shown in Fig. 1(a) for TA surface, the osteoblast attaches to Ti6Al4V surface, and the filopodia of osteoblast extends radially from the central area in all directions. On B2A surface, the osteoblast is spherical in shape with early sign of filopodia extending to adjacent areas of the prepared surfaces ( Fig. 1(b)). On B8A surfaces, the osteoblast is capable of attachment, spherical in shape, with radially extended filopodia ( Fig. 1(c)).
Fig. 1. Scanning electron micrographs of osteoblasts cultured for 3 h on (a) TA, (b) B2A, and (c) B8A. On all three specimens, the cell is spherical in shape with early sign of filopodial extensions to specimen surfaces.
After 12-h culture, Figs. 2(a)-(c) represent the micrographs from SEM of osteoblasts cultured on all the three specimens. By observation of SEM, the osteoblasts appear to flatten, spread, and take on the morphology of the underlying respective specimens. Cells cultured on TA surface have the morphologies very similar to those cultured on B2A and B8A surfaces. After 48-h culture, Figs. 3(a)-(c) represent randomly taken SEM of osteoblasts which were cultured on TA, B2A, and B8A. When the plating time increase to 48 h, the osteoblasts are in more close contact with each other and start to form a monolayer that could be observed on all the three specimens.
Fig. 2. Scanning electron micrographs of osteoblasts cultured for 12 h on (a) TA, (b) B2A, and (c) B8A. On all three specimens, cells with a flat morphology appear adherent to the surface.
Fig. 3. Scanning electron micrographs of osteoblasts cultured for 48 h on (a) TA, (b) B2A, and (c) B8A. In comparison with 12 h, the extensive spreading of cells are observed on the surface of all the three specimens.
3.3. Cell growth
As shown in Fig. 4, the cell growths of osteoblasts on the surface of each group of specimens are evaluated with cell number after culturing for 1, 3, 5 and 15 days. The mean cell number per area increases significantly (p<0.05) over time for all specimens. After culturing for 1 day, there is no significant (p>0.05) difference in the mean cell number per unit area for all the three specimens. At 3-day culture, the mean cell numbers per unit area on B2A and B8A are similar to that on TA. No differences in cell number are evident among the TA, B2A and B8A groups. After 5- and 15-day culture, there are no statistical (p>0.05) differences in the mean unit cell number for all the three specimens.
Fig. 4. The growth of osteoblasts on different specimens. During all experimental periods, cells on different specimens do not show statistically different levels of cell number. Values are in mean±SD
TA,
B2A,
B8A.
4. Discussion
Initial interaction between host and implant involves the conditioning of the implant by serum and other tissue fluids [22 and 23]. This conditioning process leaves a layer of macromolecules (e.g., serum proteins, growth factors, and cytokines) and water on the surface of the implant, which then influence the behavior of cells on encountering the surface. Following these initial events, a series of cell-material interactions takes place that leads to the release of additional growth factors and chemotactic factors that modulate cellular activity in the surrounding tissue [24]. Schneider and Burridge have investigated the effect of serum on the behavior of osteoblasts on titanium surfaces, and found that the osteoblasts attach and spread well with serum, resulting in rapid formation of focal adhesions and their associated stress fibers [25].
The material properties, such as surface energy, chemistries of alloy, roughness and topography, are believed to be of critical importance for the adsorption of proteins. Other factors of nano-surface oxide film, such as chemistries of oxides, crystal structure, and oxide thickness, are possible to change the formation of amphoteric OH groups. The acidic-hydroxyl group tends to act as cation exchange sites, while the basic-hydroxyl groups may act as anion exchange sites [26, 27, 28, 29 and 30]. The amphoteric OH groups are possible to influence the adsorbed protein on materials. Meanwhile, the oxide layer of TiO2 is non-conducting but electrons can tunnel through the layer, leading to conformational changes and denaturing of proteins [31]. It has been suggested that titanium is not in direct contact with the biological milieu, rather there is a gradual transition from the bulk metal: growing near-stoichiometric oxide and suboxide, Ca, P and S substituted hydrated oxide, adsorbed lipoprotein and glycolipids, proteoglycans, collagen filaments and bundles to cells [29 and 32].
In our previous results, the effect of modification and passivation on the XPS nano-surface characteristics of oxides have been studied [21]. The effects of the same processing on the corrosion behavior of the alloy [17] and the associated ion dissolution behavior in Hank's solution with 8.0 m
ethylene diamine tetra-acetic acid solution [18] were examined. In Ref. [11] the effects of surface chemistries of the alloys and nano-surface characteristics of oxides on the non-elemental Ca and P ion adsorption (Ca2+ and HPO42−, respectively) have been studied. It was concluded that passivations influence the surface oxide thickness and the early-stage dissolution rate of the alloy. However, the questions whether the ion dissolution will cause an effect on the cytocompatibility of the materials was unanswered. The study on the problem of cytocompatibility is complicated since many factors could be involved in interpreting the cause and result of the experiment. In the present study, hence we designed an experimental scheme that integrates all the previous experimental results as shown in Table 1 that allows one to study and elucidate the factors that might be involved in the cytocompatibility of the materials. Surface modification changes both the surface chemistries of the alloy as well as the nano-surface characteristics of oxides on the metal surface. An important question in the following discussion must first be raised: which factors of the surface chemistries of the alloy or the nano-surface characteristics of oxides that should really cause an effect, if any, on the cytocompatibility of the alloy. Besides, the different surface preparation of specimens between characteristics (the previous studies) and cell responses (this study) should be noticed. Although the surface characteristics of experimental materials ground by 1500-grit silicon carbide papers were analyzed in the previous studies, the same passivated and autoclaved treatment for both of characteristics and cell responses was used. The results of Table 1 could be adopted to present the physical characteristics of specimens for cell culture. In the present study, we used the sessile drop method to evaluate the water contact angle of all three experimental materials, and the results showed that TA, B2A, and B8A perform almost the same surface hydrophilicity. This could provide the same surface characteristics for cell responses of three experimental materials, and also suggest that the surface hydrophilicity is mainly influenced by the nano-surface characteristics of oxide on alloy rather than surface chemistries in this study.
4.1. The role of nano-surface characteristics of oxides on in vitro cytocompatibility
4.1.1. Influence of oxides on Ca and P ion adsorption
Table 1 shows that the surface modifications have changed the surface chemistries of Ti6Al4V material by EDS chemical analysis; however, the treatments have only slightly altered the nano-surface chemistries of the oxides. Comparing the XPS chemistries of oxides with the corresponding chemistries of alloy for the same specimen shows that aluminum is preferentially oxidized while copper and nickel are not. The reason for not detecting copper and nickel by XPS on the surface of B2A and B8A specimens is rationalized as follows. Copper and nickel are electrochemically noble to aluminum and titanium [33], and copper and nickel containing Ti6Al4V material has been found electrochemically noble to the untreated Ti6Al4V alloy in an aqueous solution [17].
The influence of nano-surface characteristics of oxides (oxide chemistries, total OH content, and oxide thickness) on the cytocompatibility is considered herewith. First, since the surface modifications only slightly alter the nano-surface chemistries of oxides and the oxide thickness among the TA, B2A and B8A specimens as shown in Table 1, those two factors can be assumed not related to any variation of cytocompatibility. Modification has changed the amphoteric OH content slightly among the TA, B2A and B8A specimens as shown in Table 1. However, it has been demonstrated that the variation at this range has not changed the in vitro Ca and P ion adsorption in the Hank's EDTA solution [11]. Hence, in the present study we have assumed that the amphoteric OH content on the titanium oxides is irrelevant to any variation of cytocompatibility. In summary, the factors of nano-surface characteristics of oxides on the surface of TA and modified B2A and B8A specimens should not affect the in vitro cytocompatibility in terms of Ca and P ion adsorption.
4.1.2. Influence of oxides on cytocompatibility via indirectly affecting the metal ion dissolution
Furthermore, the nano-surface characteristics of oxides should also not affect the cytocompatibility of the experimental materials via indirectly affecting the metal ion dissolution rate of the experimental materials as explained below. Dissolution of metal ions can be attributed to many kinetic transport processes [28, 34, 35, 36, 37, 38 and 39]. Healy and Ducheyne argued that the dissolution kinetics is governed by the hydrolysis of surface oxides [29]. Examination of Table 1 shows that the nano-surface chemistries of oxides contain no Cu and Ni elements, while the ICP metal ion analyses indicates the existence of those elements in the Hank's EDTA solution. This implies that the dissolution comes from the transport of metal ions from the metal through the oxide layer, rather than the hydrolysis of the oxides per se. Hence, the factor of nano-surface chemistries of oxides should not affect any variation of cytocompatibility in the present study through indirectly affecting the metal ion dissolution. Metal ion dissolution rate, however, has been deduced related to the nano-surface oxide thickness [11]. Since the oxide thicknesses of the experimental materials are virtually the same as shown in Table 1, we have assumed that this factor is also not related to any change of cytocompatibility through indirectly affecting the metal ion dissolution rate.
4.2. Effect of surface chemistries of Ti6Al4V alloy on cytocompatibility
As stated the slight change in nano-surface characteristics of oxides of the modified Ti6Al4V should not alter the capability of in vitro Ca and P ion adsorption or cause a change in metal ion dissolution rate of the experimental materials. Based on the arguments we have made the following assumption. The cytocompatibility of Ti6Al4V and modified Ti6Al4V studied in the present study in terms of cell attachment and proliferation of neonatal rat calvarial osteoblasts on the surface of the experimental materials should not be correlated with the nano-surface characteristics of oxides of the materials.
Studies of biological behavior of metallic materials have shown that local adverse tissue reaction or allergic reactions could originate from the dissolution of metal ions from the implants [40, 14, 15, 16, 41, 42 and 43]. Table 1 shows that modifications might have changed the metal ion dissolution rate of the experimental materials. The Ti and Al ions are reduced in the modified alloys due to a change of nobility or electromotive force potential of the materials [17], while some Cu and Ni ions are induced by surface modifications due to a change of chemistries of the alloy. Thus, this study aimed to investigate the effect of surface chemistries of the alloy with similar nano-surface characteristics of oxides of the alloy on cytocompatibility. For the purpose, selective specimens were chosen from the previous studies as shown in Table 1 for further cytocompatibility examination.
The results of SEM observation indicate no significant difference can be distinguished among the morphology of osteoblasts on the TA, B2A and B8A specimens, implying that a change of the surface chemistries of the alloy in the present range studied could induce the similar cellular responses during the early phases. At early cell attachments (3 h), osteoblasts exhibit early signs of attachment and spreading on all surfaces (Fig. 1). In general, the cells are round in shape, and exhibit filopodial extensions, indicating cell spreading. Increasing the culture period to 12 h, the osteoblasts flatten and spread well on the three specimens (Fig. 2). At 48 h, the osteoblasts grow confluently on the surface of all the three specimens (Fig. 3). Folkman and Moscona [43] and Archer et al. [44] mentioned that one of the main regulators of proliferation rate in anchorage-dependent cells is shape. Their results indicate that cells in round configuration divide at a lower rate than the flattened and well-spread shapes. By SEM, similar cell morphologies are observed on the Ti6Al4V and the surface-modified specimens, respectively. Therefore, we suggest that the surface-modified Ti6Al4V exhibits similar cytocompatibility as Ti6Al4V in the present study.
There is no significant difference in the level of cell growth for TA, B2A and B8A specimens (Fig. 4). This result is compatible with the observation of cell morphology. After the same preparation of specimens, all the three different specimens preserve the similar surface roughness and topography, as well as the nano-surface characteristics of oxides. Thus, variation of the individual factor of surface chemistries of the alloy for TA, B2A and B8A specimens do not show an evidence to induce any different cellular response in terms of cell attachment and proliferation rates in 15 days. Previous study shows that the metal ion dissolution rate tends to decrease to near-equilibrium values after about 15 days for various kinds of surface-modified Ti6Al4V alloys [11]. This implies that the toxicity of metal ion dissolution should be paid more attention at the initial stage of implantation in this period [11 and 45]. It is also noted that increasing the time of modification treatment from 2 to 8 h has not induced any favorable response of osteoblasts, although the metal ion dissolution rate of B8A specimen is correspondingly reduced (Table 1).
In this study, specimen was washed in absolute ethanol, passivated by aging in boiling de-ionized water for 24 h, and the specimens were then packed in double-sealed autoclaving bags, steam sterilized at 121°C for 30 min, and dried at 121°C for 15 min. There is also a concern about the possibility of adsorbed endotoxins at the surface of specimens on cell culture. In clinical, degradation or wear particles generated from implanted medical devices induce a foreign body inflammatory reaction. Bi et al. have found that titanium particles with adherent endotoxin stimulate substantially more cytokine production and osteoclast differentiation in vitro, as well as osteolysis in vivo, than do titanium particles without adherent endotoxin [46 and 47]. However, Cho et al. have used the different pretreatment to inactive particle-associated endotoxin, and the results suggest that implant materials washed with 95% ethanol could remove the endotoxin contamination [48]. In this study, we used the absolute ethanol to wash specimens, followed by the passivated and autoclaving treatment, and these treatments could remove the endotoxin on the specimens. Moreover, we expect to explore the endotoxin test on specimens in subsequent studies.
The present study suggests that the variation of chemistries of Ti6Al4V alloy, with additional Cu and Ni but less Ti and Al contents, should not alter the cytocompatibility of the materials in terms of cell attachment and proliferation. Currently, various techniques including thermal evaporation, sputtering, and ion plating of materials are being used to produce the culture surfaces of biomaterials for biological research. However, these techniques would produce different metallographic structures, surface chemistries of the alloy and nano-surface characteristics of oxides compared with the base materials. Interpreting the effect of those factors on the in vitro and in vivo response of the materials presents difficulties. The problems are that those factors are not changed independently [8, 9, 10, 49 and 50]. In this study, we differentiate the effect of surface chemistries of the alloy from other factors to examine the sole effect of a variable on the cellular response. It is concluded that variation of surface chemistries of Ti6Al4V alloy will affect the metal ion dissolution rate of the alloy. However, the variation of surface chemistries of the alloy within the range studied in this work should not alter the cytocompatibility of the alloy. However, the effects of a larger variation for the both surface chemistries of the alloy as well as the nano-surface characteristics of oxides on cytocompatibility of the material need further studies.
5. Summary
In our previous studies, the surface modifications were applied on Ti6Al4V alloy, which resulted in surface layers about 300-600
m thick on the alloy. This modified layer is characteristic with lower Ti, Al and higher Cu, Ni elements. A study of the corrosion behavior of the materials shows that the modified alloy exhibits a more corrosion-resistant property, due to a change of nobility of the modified alloy by Cu and Ni elements in the alloy [17]. The nano-surface characteristics of oxides on the alloy (chemistries of oxides, amphoteric OH group adsorbed on oxides, and oxide thickness) were studied as a function of various passivation methods [19]. The metal ion dissolution behavior of the modified and unmodified alloys in Hank's EDTA solution was reported [18]. Consistent with the previous study [17], the results from Ref. [20] suggests that nobility by Cu and Ni elements in Ti6Al4V decreases the Ti and Al ion dissolution, while additional Cu and Ni ions are found in the simulated body fluid. The influence of nano-surface characteristics of oxides for modified and unmodified Ti6Al4V on the adsorption of Ca and P species in the simulated body fluid was studied in Ref. [11]. The study suggested that the change of nano-surface characteristics of oxides in a range studied should not alter the capability of Ca and P adsorption. Nor should the characteristics influence the metal ion dissolution behavior of the materials, unless the oxide thickness is significantly changed. However, variation of surface chemistries of the alloy alters the electromotive force potential of the metal and affects the corrosion and ion dissolution rate. This effect might exert an effect on the cytocompatibility of the material [11].
In the present study, the surface chemistries of Ti6Al4V were varied by surface modifications for 2 and 8 h as in the previous works. The experimental materials were passivated in boiling de-ionized water for 24 h. The cytocompatibility of the materials was studied in terms of osteoblast cell attachment and proliferation. The purpose of the study aimed to elucidate the influence of surface modification, particularly the individual influence of surface chemistries of the alloy, on the osteoblastic response. Variation of surface chemistries of the alloy by surface modification results in lower Ti and Al and correspondingly additional Cu and Ni elements on the surface of the materials. Other factors such as surface roughness and topography of Ti6Al4V and modified Ti6Al4V remained similar by the same surface preparation procedures of the materials.
It is argued in the present study that the cytocompatibility of Ti6Al4V and modified Ti6Al4V studied in terms of cell response of neonatal rat calvarial osteoblasts should not be correlated with the nano-surface characteristics of oxides on the materials. Modifications have not changed the nano-surface chemistries of oxides and amphoteric OH group adsorbed on the alloy to the extent to significantly alter the Ca and P ion adsorption. Meanwhile, by designing an experimental scheme which keeps the oxide thickness a virtual invariant, any variation of this factor on cytocompatibility can be excluded. Hence, any change of nano-surface characteristics of oxides should not exert an effect on the metal ion dissolution rate of the alloy, which might otherwise indirectly affect the cytocompatibility. Hence the cytocompatibility of the materials can be evaluated and interpreted solely by the effect of surface chemistries and the associated metal ion dissolution of the alloy. It is concluded that the variation of surface chemistries of Ti6Al4V alloy in the present range studied will affect the corrosion and metal ion dissolution rate of the alloy. However, this variation should not significantly alter the cytocompatibility of the alloy by a statistical analysis.
Acknowledgements
This study was supported by Grant NSC-90-2218-E-242-003 from the National Science Council, Taiwan.
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