Cytocompatibility of Pure Metals and Experimental Binary Titanium Alloys for Implant Materials PDF

journal of dentistry 41 (2013) 1251–1258 Available online at www.sciencedirect.com ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/jden C...

journal of dentistry 41 (2013) 1251–1258 Available online at www.sciencedirect.com ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/jden Cytocompatibility of pure metals and experimental binary titanium alloys for implant materials Yeong-Joon Park a,*, Yo-Han Song a, Ji-Hae An a, Ho-Jun Song a, Kenneth J. Anusavice b a Department of Dental Materials and MRC for Biomineralization Disorders, School of Dentistry, Chonnam National University, Gwangju 500-757, South Korea b Department of Restorative Dental Sciences, College of Dentistry, University of Florida, Gainesville, FL 32610-0415, USA article info abstract Article history: Objective: This study was performed to evaluate the biocompatibility of nine types of pure Received 25 July 2013 metal ingots (Ag, Al, Cr, Cu, Mn, Mo, Nb, V, Zr) and 36 experimental titanium (Ti) alloys Received in revised form containing 5, 10, 15, and 20 wt% of each alloying element. 11 September 2013 Methods: The cell viabilities for each test group were compared with that of CP-Ti using the Accepted 13 September 2013 WST-1 test and agar overlay test. Results: The ranking of pure metal cytotoxicity from most potent to least potent was as follows: Cu > Al > Ag > V > Mn > Cr > Zr > Nb > Mo > CP-Ti. The mean cell viabilities for Keywords: pure Cu, Al, Ag, V, and Mn were 21.6%, 25.3%, 31.7%, 31.7%, and 32.7%, respectively, which Cytocompatibility were significantly lower than that for the control group ( p < 0.05). The mean cell viabilities Titanium alloy for pure Zr and Cr were 74.1% and 60.6%, respectively ( p < 0.05). Pure Mo and Nb demon- Implant strated good biocompatibility with mean cell viabilities of 93.3% and 93.0%, respectively. The WST-1 test mean cell viabilities for all the Ti-based alloy groups were higher than 80% except for Ti– Agar overlay test 20Nb (79.6%) and Ti–10V (66.9%). The Ti–10Nb alloy exhibited the highest cell viability (124.8%), which was higher than that of CP-Ti. Based on agar overlay test, pure Ag, Cr, Cu, Mn, and V were ranked as ‘moderately cytotoxic’, whereas the rest of the tested pure metals and all Ti alloys, except Ti–10V (mild cytotoxicity), were ranked as ‘noncytotoxic’. Significance: The results obtained in this study can serve as a guide for the development of new Ti-based alloy implant systems. # 2013 Elsevier LtdElsevier B.V. All rights reserved. include high specific strength, high resistance to corrosion, 1. Introduction greater biocompatibility, low modulus of elasticity, and high capacity to be osseointegrated with bone.1–3 However, the use Recently, commercially pure titanium (CP-Ti) has become of unalloyed CP-Ti requires further improvement to overcome widely used as a biomaterial for dental implants, orthopaedic its limitations including strength, hardness, wear resistance, implants, cardiovascular appliances, and implant-supported fatigue strength, and poor grindability. It is desirable also to dental crowns because of outstanding characteristics that decrease its elastic modulus as close as possible to that of bone * Corresponding author at: Department of Dental Materials, School of Dentistry, Chonnam National University, 300 Yongbong dong, Buk gu, Gwangju 500 757, South Korea. Tel.: +82 62 530 4871; fax: +82 62 530 4875. E-mail addresses: [email protected], [email protected] (Y.-J. Park). 0300-5712/$ – see front matter # 2013 Elsevier LtdElsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jdent.2013.09.003 1252 journal of dentistry 41 (2013) 1251–1258 tissue.4–11 Reports have focused on improved properties of CP- extracted solutions, which may contain several types of Ti for use as implant materials. In a study of Ti–Ag and Ti–Cu released metallic compounds.45–48 Most of the biocompatibili- alloys,12 Ti–20% Ag and Ti–5% Cu alloys exhibited better ty reports for metallic elements have been based on the use of grindability than pure Ti, which is a desirable property for metal salts or on extracted solutions containing the metal dental CAD/CAM alloys. The results from a previous study cations.45,49,50 Because the cytotoxicity evaluations of metallic utilizing experimental Ti–Au (5–20 wt% Au), Ti–Ag (5–20 wt% compounds are performed at different places by various Ag), and Ti–Cu (2–10 wt% Cu) alloys suggested that their methods using several kinds of cell lines, the results cannot be hardness and tensile strength were higher than these compared directly to each other. Moreover, the mass release of properties for pure Ti.13 In another study, Ti–Cr alloys a particular element is not expected to be proportional to its exhibited greater flexure strength than CP-Ti, which was atomic percentage in the alloy, and the lability of an element associated with the strengthening effect of the v phase.5 The can be altered by other elements in the alloy.51,52 Thus, when flexure strength of the Ti–20Cr alloy was about 80% greater developing new Ti alloys, cytotoxicity assays for the fabricated than that for CP-Ti, and the elastic recovery capability was alloys and the constituent pure metals are useful to study the 460% greater than that of CP-Ti. The lower elastic modulus of effect of each component on the behaviour of cells. The in vitro CP-Ti and Ti alloys, compared with 316L stainless steel and Cr– cytotoxicity tests are relatively fast and they can be standard- Co alloys, is potentially advantageous for preserving the ized relatively easily. Thus, the in vitro tests can provide highly surrounding bone by minimizing the reduction in physiologi- reliable data and reproducible measurements even though cal stress within bone and a reduction in bone density. This their relevance to clinical practice is not always consistent.53 occurs because of a more favourable match of elastic modulus The evaluation of the bulk alloy cytotoxicity in a biological between the implants and bone.14–18 The elastic moduli of environment should be performed initially. However, to date, recently developed b-Ti alloys range from 55 to 85 GPa, which there is scant information about the cytotoxicity of Ti-alloy are much lower than those of 316L stainless steel, Cr–Co alloys, ingots. and CP-Ti.2 Manganese (Mn), molybdenum (Mo), and niobium The aim of this study was to evaluate the biocompatibility (Nb) have been investigated as b stabilizers for Ti alloys and of candidate Ti-alloys using well-characterized fibroblast-like studies have suggested that they can decrease the elastic cell lines. Candidate alloying elements with Ti (Ag, Al, Cr, Cu, modulus and increase other important mechanical properties Mn, Mo, Nb, V, and Zr) were evaluated. Titanium alloys with of Ti-based alloys.19–27 Biocompatibility tests for dental alloys varying elemental contents of alloying elements were evalu- including Ti alloys have been performed in various stud- ated for their cytotoxicity, consistent with the aim of ies.19,28–31 When metallic materials are implanted inside a developing new Ti-alloy systems as dental implant materials. body, they may corrode and/or wear. The released metal ions and/or debris can be toxic or irritating to surrounding tissues. The release of metallic ions during the destruction of the 2. Materials and methods passive film can cause side effects in the body. Even though the Ti–6Al–4V alloy is an established implant material in 2.1. Sample preparation orthopaedics, it is reported that ions associated with Ti–6Al– 4V alloy inhibit the normal differentiation of bone marrow Binary Ti–A alloys that varied in the concentrations of element stromal cells to mature osteoblasts in vitro.32 If biologically A (where A was Ag, Al, Cr, Cu, Mn, Mo, Nb, V, and Zr), in relevant molecules are released from the metallic biomaterial concentrations of 5, 10, 15 or 20 wt% in the Ti alloys, were and interact with biologically relevant molecules, biologically fabricated using vacuum arc melting under a high purity argon active organo-metallic and metallic salts can be formed.30,33–40 atmosphere on a water-cooled hearth (Table 1). To homoge- Thus, the biocompatibility of the metallic materials used for nize the alloys, the prepared ingots were melted seven times, implant treatment should be evaluated during the develop- and the alloy specimens were treated for 4 h at temperatures ment of Ti alloys. 150 8C below the respective solidus temperatures and furnace Cytotoxicity of a biomaterial can be examined using either cooled at an approximate rate of 10 8C/min to 600 8C in a high a monolith of the material,41 a particulate form,42–44, or purity argon atmosphere followed by air cooling to room Table 1 – Materials used in the study. Raw material Specification Lot no. Manufacturer CP-Ti (Grade 2) Rod, 10 mm dia. ASTM B265 04RB-10 Daido Steel Co. Ltd., Nagoya, Japan Titanium Sponge 3 mm and down 99.9% 129Q001 Alfa Aesar, Ward Hill, USA Aluminium Foil, 1.0 mm thick, annealed, 99.99% 40762 Alfa Aesar, Ward Hill, USA Chromium Pieces, 2–3 mm thick, 99.995% 38494 Alfa Aesar, Ward Hill, USA Copper Shot, 13 mm dia., 99.99%, oxygen free 36686 Alfa Aesar, Ward Hill, USA Manganese Granules, 0.8–10 mm, 99.98% K21T034 Alfa Aesar, Ward Hill, USA Molybdenum Foil, 1.0 mm thick, 99.95% 36215 Alfa Aesar, Ward Hill, USA Niobium Sheet, 99.9% SH-NB04 GMH Stachow-Metall GmbH, Goslar, Germany Silver Silver shot, 1–5 mm, Premion1, 99.99% 12186 Alfa Aesar, Ward Hill, USA Vanadium Pieces, 99.7% 42775 Alfa Aesar, Ward Hill, USA Zirconium Foil, 0.02 mm thick Annealed, 99.8% 44752 Alfa Aesar, Ward Hill, USA  journal of dentistry 41 (2013) 1251–1258 1253 temperature. Prepared alloy buttons were cut into disks with a decolorization only under the specimen; 2 for a decolorization diameter of 10 mm and a thickness of 1.2 mm. The disks were zone not greater than 5 mm from the specimen; 3 for a polished by a sequence of coarser to finer abrasives succes- decolorization zone not greater than 10 mm from the speci- sively through 2000 grit SiC abrasive. They were ultrasonically men; 4 for a decolorization zone greater than 10 mm from the cleaned in acetone, ethanol, and distilled water. CP-Ti disks specimen; and 5 when the total culture was decolorized. Cell were used as a control group. lysis was defined as a loss of cell membrane integrity that was visible by light microscopy. Cell lysis was scored as follows: 0 2.2. Cell viability test when no cell lysis was detectable; 1 for less than 20% cell lysis; 2 for 20–40% cell lysis; 3 for >40% to 0.05 vs. CP-Ti). ‘noncytotoxic’. The results from the agar overlay test were consistent with those from the WST-1 test. 3.2. Cytotoxicity based on the agar overlay test The agar overlay test results are listed in Table 3. Decoloriza- 4. Discussion tion of the stained agar layer was observed around the positive control and pure Ag, Cu, Mn, and V. For pure Cr, even though The release of metal ions from metallic implants affects implant the decolorization zone appeared just under the sample, many biocompatibility and may cause various complications, such as cells in the decolorization zone were lysed, producing a cell response of 1/3. Pure Ag, Cr, Cu, Mn, and V were ranked as Table 3 – Cytotoxicity of pure metals and Ti-based alloys evaluated by the agar overlay test using L-929 cells. Samples DI LI Cytotoxicity Table 2 – Percent cell viability (%) on pure metal ingot vs. on CP-Ti after cell culture for 24 h based on the WST-1 Positive control (polyurethane) 3 3 Moderate test. Negative control (polyethylene) 0 0 None CP-Ti 0 0 None Samples Mean (SD) Pure Ag 2 4 Moderate CP-Ti 100.0a (19.0) Pure Al 0 0 None Pure Ag 31.7d (16.3) Pure Cr 1 3 Moderate Pure Al 25.3d (7.3) Pure Cu 3 3 Moderate Pure Cr 60.6c (20.9) Pure Mn 3 4 Moderate Pure Cu 21.6d (10.5) Pure Mo 0 0 None Pure Mn 32.7d (12.8) Pure Nb 0 0 None Pure Mo 93.3a,b (14.4) Pure V 3 2 Moderate Pure Nb 93.0a,b (7.6) Pure Zr 1 1 Mild Pure V 31.7d (8.9) Ti–10V 1 1 Mild Pure Zr 74.1b,c (23.3) Ti–(5,10,15,20)wt% Aa 0 0 None N = 7; SD = standard deviation. N = 5; DI = decolorization index; LI = lysis index. a By Kruskal–Wallis statistics: K = 54.916, p  0.001. All the tested Ti–(5, 10, 15, and 20)wt% A alloys, where A = Ag, Al, Means with the same letter are not significantly different Cr, Cu, Mn, Mo, Nb, V, and Zr, alloys (except Ti–10V alloy) showed a ( p > 0.05). cell response of ‘0/00 , demonstrating noncytotoxicity in the agar Duncan post hoc grouping: a > b > c > d. overlay test.56  journal of dentistry 41 (2013) 1251–1258 1255 osteolysis, allergic reactions, remote site accumulation, and, In the agar overlay test, pure Mn, V, Ag, and Cu showed a eventually, implant failure.59–62 The frequently observed cell response of 3/4, 3/2, 2/4, and 3/3, respectively, which unwanted biological effects of different metals require biologi- means that they were moderately cytotoxic to fibloblasts cal tests of metallic implants before they are used in humans. (Table 3). In the cell morphological study of Cortizo et al.,64 The cytotoxicity test serves as an efficient screening test to osteoblast-like cells exposed to Cu or Ag progressively died identify the behaviour of cells in the presence of biomaterials.37 after cell division was arrested. The authors reported that Cytotoxicity tests for metallic materials have been per- apoptosis was caused by Cu and Ag, as evidenced by the formed mainly with metallic compounds, which are formed observation of membrane blebs and apoptotic bodies. After a from the alloying elements.45–49 Sometimes they are exam- longer metal exposure, signs of necrosis became more ined in particulate form,42–44 which react directly with cells. In prevalent. In our agar overlay test results, the cells contacting rare cases such as in the present study, the cytotoxicity tests pure Mn, V, Ag, and Cu through agar overlay for 1 d changed to were performed using metallic ingots.25,41 Even with the same a globular shape and most of the cells showed blebbing at 200 types of component metals, their mechanical, physico- magnification, demonstrating their apoptosis, while cells chemical, and corrosion properties are significantly affected under negative control and against CP-Ti maintained their depending on the compositional variations of the alloys.51,52,5 cell morphology. However, decolorization at the perimeter of Therefore, besides testing with a related metallic salt solution, the samples was weaker than that of the positive control, biocompatibility testing of the metal ingot itself is necessary which demonstrates that some of the cells were damaged, and for the development of new metallic biomaterials. cells experiencing necrosis were scarcely seen. Because the cytotoxicity evaluation of metallic materials In the WST-1 test, when Ag, Cu, Mn, and V were alloyed has been performed with various metallic forms in several with Ti, the cell viability for Ti-based alloys increased labs by several methods using several types of cell lines, the drastically by a factor of three or more (Fig. 2). As shown in results cannot be compared directly with each other.30,34–40 Table 3, apoptosis of L929 cells was evident for pure Ag, Cr, Cu, Even though there are various test methods for evaluating the Mn, and V, whereas the viability of L929 cells was maintained cytotoxicity of biomaterials, the agar overlay and MTT tests for all tested Ti alloys, except for Ti–10V (cell response 1/1). are well established for assessing the cytocompatibility of Based on a study of the MG-63 osteoblast cell viability (%) of biomaterials.25,31,38 The tests are accurate and easy to perform, pure Ti and Ti–Mn, the cell viabilities of Ti–5Mn, Ti–8Mn, Ti– and the detailed procedure is well described in ISO stan- 12Mn, and pure Mn were 89%, 86%, 72%, and 50%, respectively, dards.56 demonstrating that only a very high concentration of Mn In the present study, we investigated the reaction of (12 wt% in that study) inhibits cell proliferation.25 In our study, fibroblast-like cells to nine types of pure metals and 36 types of the cell viabilities of Ti–Mn alloys using L929 cells were Ti alloys (5, 10, 15, or 20 wt% of each alloying element) using 99.9  20.8%, 83.3  10.7%, 109.3  28.3%, 97.8  19.9%, and the WST-1 test. The WST-1 assay has advantages over the MTT 32.7  12.8% for Ti–5Mn, –10Mn, –15Mn, –20Mn, and pure Mn, assay; it is simple to perform, it provides a more effective respectively. We hypothesize that this fluctuation in cell signal, and the toxicity of the assay procedure itself to cells is viability was derived from altered electrochemical character- decreased.54 istics that correspond to the variation in the microstructural In the WST-1 cell proliferation assay, the degree of cleavage phase characteristics that was associated with the composi- of a tetrazolium salt by mitochondrial dehydrogenases to form tional change. To verify the influence of these factors on formazan in viable cells was evaluated. The greater the biocompatibility, a further investigation of the corrosion rate number of metabolically active cells, the greater was the and dissolved metal ion species and microstructural phase amount of formazan produced. By detection of the formazan analysis are required. level in the cells, the cytotoxicity was measured. The It has been reported that Al3+ can significantly suppress the generation of the dark-yellow formazan was colorimetrically expression of alkaline phosphatase, osteocalcin, and osteo- measured at 450 nm and was directly correlated to the cell pontin genes of the osteoblastic cell line, whereas Ti4+, V3+, number. and Cr3+ display few inhibitory effects in cytocompatibility The pure metals tested in this study using L-929 cells for tests.65 In our WST-1 tests, the cell viability of pure Al was metal ingots demonstrated different degrees of cytotoxicity. 25.3  7.3% compared with that of the control group. This was The ranking of pure metal cytotoxicity, from most to similar in cytotoxicity level compared with those of pure Ag, least potent, was as follows: Cu > Al > Ag > V > Mn > Cr > Cu, Mn, and V (Fig. 1). However, neither the decolorized zone Zr > Nb > Mo > CP-Ti (Table 2, Fig. 2). This result differs to nor cell lysis was observed for pure Al in the agar overlay test some extent from the ranking reported from other studies.63 (Table 3). When analysing these data, one must consider that Yamamoto et al.63 evaluated the cytotoxicity of 43 metal salts the agar overlay test requires substances to diffuse through using two kinds of culture cells, and reported that the intensity the agar layer before they can be detected as toxic, and there is of the metal salts’ cytotoxicity were quite similar between no direct contact of the cells with the material.66 Thus, the MC3T3-E1 and L-929 cells. Based on their results using L-929 differentiation of the cytotoxicity between tested material cells, they reported that the cytotoxicity order from most groups can be possible only by use of a series of materials with to least toxic was as follows: V3+ > Ag+ > Cr3+ > Cu2+ > Mn2+ > widely different toxic properties. In our study, the WST-1 test Nb5+ > Al3+ > Mo5+. The differences in rankings between the seemed to be more sensitive for detecting the cytocompat- present and previous study are related to the difference in the ibility of materials compared with the agar overlay test, and form of the tested samples, i.e., metal ingots vs. dissolved pure Al seemed to have a detrimental influence on the metal ions. metabolic function of the cells, even though the cell 1256 journal of dentistry 41 (2013) 1251–1258 membranes remained intact when they contacted the pure Al the type of component metallic elements but also to their ingot. stability in the biological environment that may vary Li et al.30 reported that a 72-h extracted solution of Zr metal considerably because of differences in their microstructures (99.8% pure) in the form of powder or bulk was noncytotoxic. and electrochemical properties. Several reports have demonstrated the favourable cell viability property of Zr.67,68 However, in our study, the cell viability for pure Zr and Cr (purity of 99.8% and 99.995%, 5. Conclusions respectively) displayed moderate cell viabilities of 74.1  23.3% and 60.6  20.9%, respectively, which demon- This study employed WST-1 and agar overlay tests to analyse strates a significant difference when compared with that of the reaction of fibroblast-like cells to nine types of pure metal CP-Ti ( p < 0.05, Table 2). Based on the results of our agar ingots and 36 experimental titanium alloys that contained 5, overlay test, pure Zr was mildly cytotoxic (cell response 1/1). 10, 15, and 20 wt% of alloying elements. The ranking of pure However, pure Cr showed a moderate cytotoxicity (cell metal cytotoxicity from most potent to least potent was as response 1/3), as demonstrated by the appearance of the follows: Cu > Al > Ag > V > Mn > Cr > Zr > Nb > Mo > CP-Ti. decolorization zone just under the sample and the presence of In the agar overlay test, pure Mn, V, Ag, and Cu were numerous lysed cells in the decolorization zone (Table 3). The moderately cytotoxic. However, the other pure metals and all differences between the present and previous results may be tested Ti alloys, except Ti–10V, were noncytotoxic. The Ti–10V associated with the experimental form of the metal, i.e., metal alloy exhibited mild cytotoxicity. The cell viabilities for all of ingots vs. metallic salts or extracts. Based on our results in the Ti-based alloy groups were higher than 80%, except for Ti– which a Cr ingot was used as well as on previous results using 20Nb (79.6  7.8%) and Ti–10V (66.9  22.0%). Among the tested metallic salts,69 the cytotoxicity of pure Cr prompted consid- Ti alloys, the Ti–10Nb alloy demonstrated the highest cell eration of its use. However, when pure Cr or Zr were alloyed viability (124.8  16.9%), which was even greater than that of with Ti, cell viability increased up to a level similar to that of CP-Ti. Even though Ti and the Ti alloys tested herein, except CP-Ti ( p > 0.05), and those Ti-alloys were ranked as ‘non- for the Ti–10V group, were biocompatible, our cytotoxicity test cytotoxic’ based on the agar overlay test (Table 3, Fig. 2). results indicate that Ti alloys containing Ag, Al, Cr, Cu, Mn, V, A previous investigation of the effects of Al, Cr, Cu, Mo, Nb, and Zr need to be investigated further for their long-term V, and Zr ions at concentrations ranging from 0.05 to 5.0 mM safety in a biological corrosive environment. 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