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CNTs composites
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In vitro biocompatibility and ageing of 3Y-TZP/ CNTs composites E. Mohamed, M. Taheri, M. Mehrjoo, M. Mazaheri, A.M. Zahedi, M.A. Shokrgozar, F. Golestani-fard
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S0272-8842(15)01228-6 http://dx.doi.org/10.1016/j.ceramint.2015.06.112 CERI10851
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Cite this article as: E. Mohamed, M. Taheri, M. Mehrjoo, M. Mazaheri, A.M. Zahedi, M. A. Shokrgozar, F. Golestani-fard, In vitro biocompatibility and ageing of 3Y-TZP/CNTs composites, Ceramics International, http://dx.doi.org/10.1016/j.ceramint.2015.06.112 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro biocompatibility and ageing of 3Y-TZP/CNTs composites E. Mohameda, M. Taheria, M. Mehrjoob, M. Mazaheric,*, A.M. Zahedia, M.A. Shokrgozarb, F. Golestanifarda a
Department of Materials and Metallurgical Engineering, Iran University of Science and Technology, Tehran, Iran
b
National Cell Bank of Iran, Pasteur Institute, Tehran, PO Box 13169-43551, Iran
c
International Center for Young Scientists (ICYS), National Institute for Materials Science (NIMS), Tsukuba, Japan
* Corresponding author: ' " #(Tel/Fax: +41788222256
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1. Abstract The intrinsic brittleness of Tetragonal Zirconia Polycrystalline (TZP) materials has been the major barrier for their use in biocompatible orthopedic implants. Purpose of the current paper was to demonstrate that the material’s toughness/hardness could be significantly improved with addition of carbon nanotubes (CNTs) to monolithic TZP. To verify possible application of the composites as implants, biocompatible responses of 3Y-TZP/CNTs was clarified in comparison with 3Y-TZP. The vitality, mineralization, and attachment status of osteoblast cells were studied by performing in vitro tests such as MTT assay, ALP assay, and SEM, respectively. Lowtemperature degradation through 20 h of heat treatment at 134oC and 3 bar was studied to determine lifespan of the composites under humid atmosphere of human body conditions. Results showed suitable biocompatibility and negligible amount of low-temperature degradation. The synthesized TZP/CNTs composites, as a result, can be realized as adequate candidates for use in bone implants owing to their proper biocompatibility as well as a prolonged resistance against failure corroborating prominent mechanical properties compared to pure zirconia.
KEYWORDS: A. Sintering, B. Nanocomposites, C. Mechanical properties, E. Biomedical applications. 2. Introduction The great interest in the incorporation of carbon nanotubes (CNTs) into the ceramic materials, through the recent decade, stems from their extraordinary mechanical, electrical and functional properties, which are unparalleled while compared to several analogous alternatives. In particular, combination of a remarkable aspect ratio with the excellent stiffness and strength of CNTs has created them magnificent candidates to reinforce a long spectrum of materials such as metals, polymers and specifically ceramics. Many researchers have recently declared their success in the development of CNTs-ceramics with superior mechanical properties compared to the monolithic materials. Moreover, CNTs have also been reported to impart excellent mechanical properties to oxide ceramics specifically alumina and zirconia composites. Mukherjee et al. [1] have reported, for first time, that addition of CNTs into the alumina matrix increased the fracture toughness as much as three times. They have introduced crack deflection and CNTs’ pull-out as responsible mechanisms for such a huge improvement in the toughness. In addition, Mazaheri and his research group [2-3] have recently focused on the mechanical properties of Zirconia-CNTs ceramics. They have primarily, explored the effect of CNTs’ presence using mechanical spectroscopy tests. The above mentioned observation was also confirmed by the former publication of Taheri et al. [4] who reported a theatrical enhancement in the fracture toughness of monolithic 3Y-TZP material up to nearly 100%, through addition of 5wt.% CNTs. According to their results, CNTs should be recognized for proposing a great competence to suppress the accelerated grain growth in the zirconia matrix, while Taheri et al. have eventually derived composites with much smaller final grain size compared to their
monolithic counterparts. As they have discussed, CNTs are flexible enough to wrap around the grains and prevent grain coarsening of composites. This may be, particularly, one of the major underlying reasons behind the improved mechanical properties of CNTs reinforced composites. On the other hand, the very recently discovered biocompatibility of Ceramic-CNTs composites has shed light on the great applicability of these materials to stand out as a promising alternative utilized in bone implants. In particular, use of CNTs as the secondary reinforcing component of zirconia based biomaterials is anticipated to improve not only the overall mechanical properties of implants but also biocompatibility of bone grafting materials. Such an expectation arises from their extremely small size (about 1–10 nm in diameter) which spans within the range of small components of biological organs [5–10]. The controversial issue whether CNTs are toxic or not, has been argued in several previous publications. Smart et al. [11] reviewed biocompatibility and cytotoxicity of CNTs and CNTs-containing materials in different biomedical fields. They stated that despite relatively few studies on this topic are available; most of the results were either inconclusive or contradictory. The numerous previous researches have studied effective parameters on biocompatibility of CNTs under in vitro and in vivo conditions. Most of the abovementioned reports have confirmed bone compatibility and mineralization of CNTs [7-9, 12-23], while there are also available reports in the literature defining their toxicity in specific laboratorial conditions [6, 24–26]. Nowadays, TZP ceramics are widely used as to synthesize varied dental/orthopedic implants such as femoral ball heads. These implants are normally utilized for total hip replacement owing to their excellent biocompatibility, high fracture toughness, and low wear rates [27-28]. However, there are several case studies reporting an absolute failure of TZP ceramic implants due to crack propagation caused by low-temperature degradation of tetragonal to monoclinic
phase in humid atmosphere of in vivo conditions [29-32]. Based on the information available in the open literature, one of the most severe failures has been reported at the beginning of the year 2000, for zirconia hip joints [29]. Such a drastic incident gave rise to complementary research activities focusing on the major causes behind the abovementioned fracture and coming up with strategies to prevent similar failures. Accordingly, as discussed by several researchers, since under in vivo conditions, low-temperature degradation of biological organs or implants is known to be disseminated with an extremely slow rate, extensive experiments over several years are required to reveal the effect of transformation within the oral environment [33-35]. Many efforts, subsequently, were made to create simulative laboratorial conditions approximating interior body conditions in which implants are required to work for many years. Chevalier [33], for instance, recommended some modifications in the processing conditions through the ageing test of zirconia-based materials. He stated that an accelerated ageing profile should be conducted in steam at 134oC. It was also calculated that 1 h of autoclave treatment at 134oC presented theoretically the same effect as 3–4 years under in vivo conditions. The very first purpose of the current investigation is to review the successful experiences of the authors to incorporate zirconia based composites with CNTs and increase mechanical properties (specifically fracture toughness) of the host zirconia material. As the second approach, our objective is to propose the novel 3Y-TZP/CNTs composites as a promising alternative to substitute conventionally applied zirconia hip implants. In particular, the synthesized composites have been put through in vitro experiments to study the biocompatibility of these materials for use in body conditions and clarify their strength and fracture toughness for substituting bone tissue implants.
3. Experiments 3.1.
Composite preparation and mechanical testing
Commercially available, high purity 3 mol.% yttria stabilized zirconia (3Y-TZP) powder (Tosoh Co., Japan) and multi walled carbon nanotubes (Arkema, France) were selected as starting materials. According to the images (Fig. 1) of scanning electron microscopy (SEM, XLF-30, Philips, Netherlands), the starting YSZ powder consisted of spherical nanoparticles with an averaged diameter of 70 nm. The CNTs were synthesized by catalytic chemical vapor deposition. They have a length of about 10–20 µm, a diameter of about 10–20 nm and the number of walls is between 5 and 15. A detailed information on the nanotube synthesis is available in [36]. CNTs were distributed within 3Y-TZP matrix with 0 and 3 wt.% compositions, using a Turbula Mixer machine with zirconia balls for 24 h. Morphology of composites and dispersion of CNTs within the matrix at varied amounts has been analyzed by scanning and transmission electron microscopy (SEM and TEM, CM-20, Philips, Netherlands). Powder mixture samples were consolidated using Spark Plasma Sintering (SPS, FCT GmbH, Germany) under vacuum (10−2 mbar). The temperature was measured by means of an optical pyrometer focused on the upper graphite punch, at about 4 mm from the sample. The SPS was carried out with a fixed heating rate of 50 oCmin−1 under a constant applied pressure of 50 MPa with a soaking time of 2 min. Temperature in the isothermal stage of sintering depended on the powder composition. In particular, while monolithic zirconia was sintered at 1250oC to obtain rather dense (relative density>98%) specimens, for densification of the composites with 3wt. %
)
CNTs, the sintering temperature was needed to be increased up to 1350oC. The final sintered specimen size was 40 mm diameter pellets with thickness of about 7 mm. The densities of sintered samples were determined by Archimedes method in deionized water. The theoretical densities of the composites were calculated based on the rule of the mixtures. The density of graphite (2.25gcm−3) has been used for CNTs. Raman Spectroscopy (Dispersive Raman, Senterra (2009), Bruker, Germany) was exploited to characterize the specimens in terms of degree of damage to CNTs before and after the processing. Indentation tests were carried out with a diamond Vickers indenter under 20 kg loads with a dwell of 20s on carefully polished surfaces. The hardness (HV) was calculated from the diagonal length of the indentation using the following equation (Eq. (1)): HV = 1.854P/d2
(1)
Where P is the applied load and d is the mean value of the diagonal length. Fracture toughness (KIC) was determined by measuring the crack length emanating the indentation center indicated by C in the Eq. (2) [37]: KIC = 0.0016(E/HV)1/2(P/C3/2)
(2)
Where E is the Young modulus, HV is the Vickers hardness and P is the load. The crack lengths were measured immediately after indentation using a calibrated optical microscope. At least ten valid measurements were carried out for each sample to calculate the average.
3.2.
In Vitro assays 3.2.1
Cell culture *
In this study, osteoblast-like cell line MG 63 (NCBI C555, National Cell Bank of Iran) was used to study biological properties of the samples. After de-freezing the cells, they were transferred to a cell culture flask containing the culture medium (Roswell Park Memorial Institute (RPMI) medium) with 10 %(v/v) Fetal bovine serum (FBS-Serum, Germany), 100 U.ml−1 penicillin, and 100 g.ml−1 streptomycin. Then, the flask was placed into an incubator under general conditions (a humidified, 5% CO2, 95% air environment, and temperature 37.4oC). 3.2.2
MTT assay
In this study, in order to evaluate vitality and proliferation rate of the osteoblast cells cultured on samples, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-iphenyltetrazolium bromide) assays, with the following procedure, were conducted. In this regard, the samples were sterilized by autoclave and then were placed in a 24-well plate. Afterwards, 2×103 cells were seeded on surface of the samples, and then the plate was located in the incubator for 5 h. Thereafter, 1500 l medium culture containing 10 % FBS was added to each well. The plates were kept in the incubator for 7 and 14 days. Then, the culture medium was removed from each well and 1000 l of 0.5 mg.ml-1 MTT (Sigma, USA) solution was added and was placed into the incubator for 4 h. After 4 h, the solution on the cells was removed and 1000 l of isopropanol (Sigma, USA) was added to make the generated purple crystals get solved. In order to solve the purple crystals more efficiently, the plate was put in a shaker incubator for 15-20 min. In the following, 100 l of solution of each well was transferred into a 96-well microtiter plate (Nunc, Denmark). Then the optical density (OD) of formazan in the solution was measured at 545 nm using a two-wall microplate reader (STAT FAX 2100, USA) for quantification cells viability. The cells incubated in the culture medium without any sample were used as a control sample.
+
3.2.3
Alkalin Phosphatase Activity
The amount of soluble ALP, a marker of osteoblast activity, was assayed by measuring the conversion to p-nitrophenol from p-nitrophenyl phosphate. The sterilized samples were put in each well of 6-well plate. Then 2000 cells were seeded on samples surface and the plates were placed into the incubator for 4-5 h. After 7 and 14 days, 10 µL cell supernatant was added to 1000 µL of ALP’s kit (Pars azmun, Iran) according to the manufacturer’s protocol at 37oC. The ALP activity was measured spectrophotometrically by monitoring the absorbance of the solution at a wavelength of 405 nm. The control groups were similar to MTT assay. After the prescribed time period, the amount of Calcium/Phosphor and Phosphate/Phosphor ratio present in the acidic supernatant was spectrophotometrically quantified using a commercially available kit (Pars azmun, Iran). The data of MTT and ALP assays were analyzed using one-way ANOVA with LSD post hoc test using SPSS (version 16.0 SPSS Inc. Chicago IL). P values of less than 0.05 were considered statistically significant. 3.2.4
Cell adhesion
In order to investigate the cellular adherence, the sterilized samples were put in each well of 24well plate. Then, 4000 - 5000 cells in the volume of 50 l were seeded on each sample surface, and the plate was placed into the incubator for 4-5 h. Afterward, a specific amount of culture medium containing 10 % FBS was added. After 48 h, the solution on the samples was taken out and washed by phosphate-buffer saline (PBS). Finally, the cells were fixed with 2.5% glutaraldehyde. After 1h, the samples were washed with deionized water. After gold sputtering,
,
the cell adherence to the samples was observed by scanning electron microscopy (SEM-VEGA TESCAN).
3.3
Ageing
XRD results demonstrated that the initial monoclinic content of the samples prior to the heat treatment process was close to zero in all cases. The samples were hydrothermally treated in steam at 134oC and 3 bar absolute pressure for 20 h to evoke low temperature degradation (LTD). After completion of ageing, content of the monoclinic and tetragonal zirconia phases of each specimen was measured by X-ray diffraction (XRD, Cu K radiation, Philips PW3710, Netherlands) on diamond polished side of the samples. Weight fraction of the monoclinic phase, Xm, was calculated using the following formula [38]: § ¹· I ¨111¸ m + I (111) m © ¹ Xm = § ¹· I ¨111¸ m + I (111) m + I (111) t © ¹ (1) ¹
where I(111)m, I(111 )m and I(111)t are the integrated intensity from the monoclinic (111), ¹
monoclinic (111 ) and the tetragonal (111) peaks, respectively.
4
Results and Discussion 4.1.
Mechanical properties
Fig. 1a. displays SEM micrograph of a powder mixture from 3 wt.% CNTs dispersed within 3YTZP matrix. As can be observed, CNTs are adequately dispersed within the ceramic powder,
-
while least effects of damages, cuts and breakage can be traced along the CNTs’ bodies after the mixing process. In particular, as formerly explained in other publications [2–4], deriving a homogeneous dispersion of CNTs within a ceramic powder could be recognized as the major challenge of producing these composites. TEM image of the above powder mixtures (Fig. 1b.), not only verifies our successful dispersion process, but also confirms that CNTs have been exposed to minimal amount of damages through the mixing stage. In addition, adequate dispersion of CNTs can be also monitored by exploring fracture surface of a SPSed zirconiaCNTs sample. As can be observed in Fig. 2, nanotubes of carbon are homogeneously spread through the whole consolidated specimen, while SPS process seems to have exerted least of damages to the nanotubes’ body and maintained CNTs at the sintering temperature. Such an observation was subsequently confirmed by performing Raman spectroscopy to ensure safety and maintenance of CNTs. Fig. 3 compares Raman Spectroscopy pattern of pure carbon nanotubes with as-sintered sample. As can be observed, the major peaks in the as sintered composites are pretty close to those in pure CNTs. Moreover, calculation of ID/IG intensity ratio revealed a negligible increase as a result of processing. This verifies CNTs’ maintenance after the sintering stage and validates our manufacturing method.
Fig 1. a) SEM b) TEM images of mixed zirconia nanopowders with 3 wt% CNTs
Fig 2. SEM fracture surface figure of 3Y-TZP/3 wt.% CNTs composite
.
Fig 3.Raman Spectroscopy pattern of pure carbon nanotubes (before processing) in comparison with as-sintered composite sample (after processing)
Incorporation of monolithic 3Y-TZP sample with carbon nanotubes have, presumably, led to some modifications in the sintering profile, microstructure and mechanical properties of the final synthesized composites. Based on the results shown in table 1, CNTs have increased the temperature required to consolidate the monolithic zirconia up to 50oC and 3Y-TZP/ CNTs samples could be hardly sintered to near full density state by lower sintering temperature than 1300oC. On the other hand, CNTs have been found to wrap around grains and prevent an accelerated grain growth in the final stage of sintering. Such an occurrence is expected to culminate in a frustrated consolidation as well as a finer microstructure for CNTs incorporated composites compared to their monolithic counterparts. Accordingly, along with a lower sintered fractional density for the composite, one can observe that the presence of CNTs in the 3Y-TZP/ CNTs composites gave rise to an effective suppression of grain boundary mobility. As a result, while the grain size of monolithic zirconia is around 145 nm, the averaged grain size of the composite increased no further than 109 nm. The greatest effect of nanotubes, however, should be realized when the incorporated composites display enhanced mechanical properties (hardness & fracture toughness) compared to the monolithic zirconia. Quote to table 1, although CNTs have not remarkably increased hardness values for the composites, the fracture toughness can be speculated to have undergone a manifested increment (about 75% improvement) as a result of CNTs incorporation. Details of mechanisms how CNTs enhance fracture toughness of the material can be found in other publications of the authors [2–4]. What should be taken into account in this concern is that while indentation test is not generally recognized as the best device to have precise calculations of
fracture toughness values, such an experiment, as numerically reported through several publications [2–4, 39–41], is appreciable as a reliable scheme to present comparative trends of mechanical properties. Moreover, the current results of fracture toughness values have been formerly confirmed by other publications of the authors through utilization of SEVNB (Single Edge V-Notched Beam) method [3].
Table 1. Sintering temperature and characteristics of sintered 3Y-TZP/ CNTs composite in comparison with monolithic zirconia. Sintering
Fractional density
Grain size
Indentation fracture toughness
Vickers hardness
temperature (oC)
(%)
(nm)
(MPa.m1/2)
(GPa)
1250
99.3
145
5.8 ± 0.28
12.1 ± 0.21
1300
98.7
109
10.1 ± 0.21
12.6 ± 0.21
Material composition
3Y-TZP 3Y-TZP/3 wt.% CNTs
4.2.
In vitro biocompatibility
Osteoblasts, the bone-forming cells, proliferate on the bone surface and secrete bone matrix proteins as well as producing them. Therefore, studying the performance of osteoblasts cells on 3Y-TZP/CNTs composites as a future scaffold biomaterial seems necessary. Cells mitochondria are able to reduce MTT into a formazan precipitate that can be solved in n-propanol and be quantified spectrophotometrically. This assay allowed monitoring the vitality and proliferation of osteoblast cells on the surface of samples after the 7 and 14 days (Fig. 4).
Fig.4. MTT assay results showing viability of osteoblasts cells on the surface of 1) 3Y-TZP 2) 3Y-TZP/CNTs samples after 7 and 14 days in comparison with control Sample. Higher optical density indicates greater amounts of viable osteoblasts. * p<0.05: compare to the control
According to the results of MTT assay (Fig.4), optical density in both specimens was similar to the control sample after 7 days. Increase in the optical density of both samples over time in comparison with the control sample showed that the proliferation rate of osteoblast cells on the surface of specimens was higher than that of the control sample. Furthermore, addition of CNTs to 3Y-TZP had no significant effect on vitality of cells in comparison with 3Y-TZP. Indeed, osteoblast cells were able to survive and grow in contact with 3Y-TZP/CNTs as well as biocompatible 3Y-TZP. The effect of CNTs on increasing the cell proliferation has been reported by several researchers. For example, Chłopek et al. [22] reported biocompatibility of CNTs similar to polysulfone, which is currently used in medicine, by studying response of osteoblast and fibroblast cells in contact with the surface of CNTs. Ogihara et al. [42] showed that addition of CNTs as a reinforcement to alumina ceramics enhanced proliferation of osteoblast cells. Some other publications showed that CNTs are able to promote proliferation of osteoblast cells [7, 9]. On the other hand, some reports showed that CNTs substrates decreased some cell lines survival [24, 25]. Some in vitro studies on CNTs demonstrated that CNTs are cytotoxic [24–26]. Despite all these argues in the case of toxicity of CNTs, severe effects of toxicity have not been observed through addition of 3 wt. % CNTs into biocompatible 3Y-TZP based on our observations (Fig.4).
Fig.5. ALP assay results showing Alkalin Phosphatase activity of osteoblasts cells attached to 1) 3Y-TZP 2) 3YTZP/CNTs samples after 7 and 14 days in comparison with control sample. *p<0.05: compare to the control
Fig.6. Ca/Phosphor ratio measured present in the acidic supernatant on the surface of 1) 3Y-TZP 2) 3Y-TZP/CNTs samples after 7 and 14 days in comparison with control sample showing calcium production. *p<0.05: compare to the control
Fig.7. Phosphate/Phosphor ratio measured present in the acidic supernatant on the surface of 1) 3Y-TZP 2) 3YTZP/CNTs samples after 7 and 14 days in comparison with control sample showing phosphate production. *p<0.05: compare to the control
Alkalinphosphtase (ALP), a noncollagenous protein, is the most common indicator of osteoblast activity, bone mineralization, and predictor of the formation of osteogenic cells [43, 44]. ALP activity of osteoblast cells attached to samples that should be recognized as a confirming factor for the occurrence of cell differentiation was measured after 7 and 14 days (Fig. 5). Furthermore, Calcium/Phosphor and Phosphate/Phosphor ratios present in the acidic supernatant on the surface of the samples after 7 and 14 days were calculated as the markers of mineralization as well (Fig.6 and Fig.7). According to Fig.5, after the very first week, ALP activity of cells in contact with 3Y-TZP was similar to that of the control sample, while it was apparently lower in 3Y-TZP/CNTs sample. This trend was maintained over time although ALP activity increased in all samples. Fig.6 and Fig.7 also showed that Calcium/Phosphor ratio and Phosphate/ Phosphor ratio were lower in 3Y
TZP/CNTs sample in the first week compared to 3Y-TZP and control samples, but after 14 days the results were approximately similar in both samples. It is well known that ALP can be expressed as a critical phonotypical factor for bone forming cells and initiation of matrix mineralization. In the present study, osteoblast cells were observed to be capable of producing ALP in contact with 3Y-TZP and 3Y-TZP/CNTs composites. Consequently, Calcium/Phosphor ratio and Phosphate/ Phosphor ratio could be regarded as an indicator for promotion of matrix formation, which is an essential factor for bone regeneration. There were several studies that evaluated the effects raised by the addition of CNTs on mineralization of osteoblast cells. Li et al. [23] studied in vitro biocompatibility of CNTs. They unveiled that CNTs increase differentiation and maturation of osteoblast cells related to bone formation by absorbing more proteins owing to their larger surface area. Ogihara et al. [42] showed that addition of CNTs as a reinforcement to alumina ceramics enhanced ALP activity and bone mineralization of osteoblast cells. In our present study, it can be inferred that although osteoblasts were observed to differentiate in 3Y-TZP/CNTs composites with a delayed onset compared to biocompatible 3YTZP, the mineralization triggered approximately in a simultaneous starting time after two weeks in both samples due to mineralization ability of CNTs, or the differences were insignificant.
Adhesion of cells to surface of biomaterials is known as a major factor of biocompatibility. It is postulated that the more compatible the surface, the greater amount of cells are expected to attach. SEM images in Fig. 8 a and b indicate how the osteoblast cells are attached to the surface of samples after 48 h.
)
Fig.8. Scanning electron microscopy images of cell attachment a) 3Y-TZP b) 3Y-TZP/CNTs after 48 h
Initial cell attachment on the surface of biomaterials, which is one of the most complicated and integral features of biomaterials, have been argued by several previous researches. Bachle et al. [45] introduced surface roughness as influential factor on initial cell attachment and the spread of the cells. On the contrary, Yamashita [46], reported that surface condition had no effect on initial attachment. Price et al. [18, 19], proved that the attachment area of osteoblasts increased in case of finer surface of the specimens and the presence of carbon nano fibers. As can be seen in Fig.8, after 48 h, layering of osteoblast cells, which is an indicator of cells differentiation and matrix mineralization was observed on the surface of both samples. Indeed, osteoblast cells had spread on the surface of both specimens and showed normal morphology over time due to CNTs’ small size (about 1–10 nm in diameter). A number of studies showed proper adhesion and function of bone-forming osteoblast cells on the surface of CNTs-containing material [17,20,21]. Correspondingly, our results demonstrated that the scaffold synthesized by 3Y-TZP/CNTs composites, is not only biocompatible but also it provides a favorable environment for attachment and growth of bone cells.
According to the results of MTT assay, ALP assay, and SEM images, one could state that proliferation, differentiation, maturation, and mineralization of osteoblast cells on 3Y-TZP/CNTs composite were improved over time. Tutak et al. [47] studied toxicity of CNTs on osteoblast cells. Despite all controversial publications in this case, they revealed a mechanism showing that CNTs induce toxicity in the first days of culture, then dead cells release proteins and growth factors that enhance activity of remaining cells to proliferate and differentiate. Usui et al. [8], *
who first developed CNTs as biocompatible materials, clarified that MWCNTs did not cause strong inflammatory reactions in contact with bone, and have good bone-tissue compatibility. Correspondingly, our results indicated that 3Y-TZP/CNTs composite could be recognized for proposing bone tissue compatibility similar to biocompatible 3Y-TZP.
4.3.
Ageing
As transformation of zirconia is a thermally activated process, we had no option other than to conduct accelerated ageing tests under hydrothermal conditions to forecast the long-term behavior of the material. The objective behind performing the abovementioned experiments was to study ageing procedures of 3Y-TZP and 3Y-TZP/CNTs composites. Fig.9 demonstrates XRD results of 3Y-TZP and 3Y-TZP/CNTs samples before and after heat treatment for 20 h. What can be noted is the absence of initial monoclinic phase content in both samples. After 20 h of heat treatment at 134oC under pressure of 3 bar (which simulates the conditions of more than 60 years of in vivo performance [34]), the content of monoclinic phase in 3Y-TZP and 3YTZP/CNTs composites were 13.2 and 5.4, respectively. Indeed, not only were the variations of monoclinic content lower than 10% in 3Y-TZP/ CNTs composite, accepted by chevalier’s modifications [33], but also the low-temperature degradation decreased by addition of CNTs to 3Y-TZP. In the constrained ZrO2(t) - ZrO2(m) reaction, differential free energy Gt-m, between these two states per unit volume of transformed material, can be determined using the [48]: Gt-m=-G0+Use+Us
(4)
where G0 is the chemical free energy, Use is the strain energy associated with the transformed particle and surrounding matrix which is usually less sensitive to temperature and composition +
changes, and Us is the change in energy associated with the surface of the inclusion. With the presence of CNTs in ZrO2 matrix, Use increases because of high elastic modulus of CNTs, while Gt-m increases correspondingly, so tetragonal phase is more stable. Therefore, the addition of CNTs has successfully resulted in stabilization of tetragonal phase. Reviewing the above theory, we can expect that the bone tissue implant produced from 3YTZP/CNTs composites are potentially capable of resisting against durable stresses for prolonged times of use and are exposed to postpone failures compared to pure zirconia. However, further factors to which this composite are exposed in the oral environment (e.g. cyclic mechanical and thermal loading) are not considered in this calculation; therefore ageing may probably proceed more rapidly under in vivo condition.
Fig.9. XRD patterns of 3Y-TZP and 3Y-TZP/CNTs before and after 20 h of heat treatment at 134oC, 3 bar.
5.
Conclusion
The objective of the current paper was to explore the effect of carbon nanotubes’ addition -as the secondary phase- on the biocompatibility and mechanical properties of conventional 3Y-TZP hip implants. In terms of mechanical properties, CNTs were found to result in enhanced fracture toughness for the CNTs-containing materials, compared to monolithic Zirconia. To investigate basic biocompatibility features of 3Y-TZP/CNTs composites, on the other hand, the synthesized specimens were subjected to in vitro test examinations. According to our results, CNTs
,
incorporated composites, while presenting no further improvement in bio compatibility, at least demonstrated no decrement compared to the conventional 3Y-TZP implants. Moreover, the amount of low temperature degradation of CNTs incorporated composites was compared to that of 3Y-TZP samples through heat treatment for 20 h. As a result, one could observe a lower amount of monoclinic phase variation in 3Y-TZP/CNTs, defining the competence of this novel composite to survive under in vivo conditions for much longer durations compared to pure zirconia without severe failure and toxicity. However, this composite should be subjected to in vivo tests in order to realize if the major requirements of clinical applications are fulfilled by using these composites. 6.
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Figure Captions: Fig 1. a) SEM b) TEM images of mixed zirconia nanopowders with 3 wt% CNTs Fig 2. SEM fracture surface figure of 3Y-TZP/3 wt.% CNTs composite Fig 3. Raman Spectroscopy pattern of pure carbon nanotubes (before processing) in comparison with as-sintered composite sample (after processing) Fig.4. MTT assay results showing viability of osteoblasts cells on the surface of 1) 3Y-TZP 2) 3Y-TZP/CNTs samples after 7 and 14 days in comparison with control Sample. Higher optical density indicates greater amounts of viable osteoblasts. * p<0.05: compare to the control Fig.5. ALP assay results showing Alkalin Phosphatase activity of osteoblasts cells attached to 1) 3Y-TZP 2) 3YTZP/CNTs samples after 7 and 14 days in comparison with control sample. *p<0.05: compare to the control Fig.6. Ca/Phosphor ratio measured present in the acidic supernatant on the surface of 1) 3Y-TZP 2) 3Y-TZP/CNTs samples after 7 and 14 days in comparison with control sample showing calcium production. *p<0.05: compare to the control Fig.7. Phosphate/Phosphor ratio measured present in the acidic supernatant on the surface of 1) 3Y-TZP 2) 3YTZP/CNTs samples after 7 and 14 days in comparison with control sample showing phosphate production. *p<0.05: compare to the control Fig.8. Scanning electron microscopy images of cell attachment a) 3Y-TZP b) 3Y-TZP/CNTs after 48 h Fig.9. XRD patterns of 3Y-TZP and 3Y-TZP/CNTs before and after 20 h of heat treatment at 134oC, 3 bar.
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In vitro biocompatibility and ageing of 3Y-TZP/ CNTs composites E. Mohamed, M. Taheri, M. Mehrjoo, M. Mazaheri, A.M. Zahedi, M.A. Shokrgozar, F. Golestani-fard
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Cite this article as: E. Mohamed, M. Taheri, M. Mehrjoo, M. Mazaheri, A.M. Zahedi, M. A. Shokrgozar, F. Golestani-fard, In vitro biocompatibility and ageing of 3Y-TZP/CNTs composites, Ceramics International, http://dx.doi.org/10.1016/j.ceramint.2015.06.112 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro biocompatibility and ageing of 3Y-TZP/CNTs composites E. Mohameda, M. Taheria, M. Mehrjoob, M. Mazaheric,*, A.M. Zahedia, M.A. Shokrgozarb, F. Golestanifarda a
Department of Materials and Metallurgical Engineering, Iran University of Science and Technology, Tehran, Iran
b
National Cell Bank of Iran, Pasteur Institute, Tehran, PO Box 13169-43551, Iran
c
International Center for Young Scientists (ICYS), National Institute for Materials Science (NIMS), Tsukuba, Japan
* Corresponding author: ' " #(Tel/Fax: +41788222256
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1. Abstract The intrinsic brittleness of Tetragonal Zirconia Polycrystalline (TZP) materials has been the major barrier for their use in biocompatible orthopedic implants. Purpose of the current paper was to demonstrate that the material’s toughness/hardness could be significantly improved with addition of carbon nanotubes (CNTs) to monolithic TZP. To verify possible application of the composites as implants, biocompatible responses of 3Y-TZP/CNTs was clarified in comparison with 3Y-TZP. The vitality, mineralization, and attachment status of osteoblast cells were studied by performing in vitro tests such as MTT assay, ALP assay, and SEM, respectively. Lowtemperature degradation through 20 h of heat treatment at 134oC and 3 bar was studied to determine lifespan of the composites under humid atmosphere of human body conditions. Results showed suitable biocompatibility and negligible amount of low-temperature degradation. The synthesized TZP/CNTs composites, as a result, can be realized as adequate candidates for use in bone implants owing to their proper biocompatibility as well as a prolonged resistance against failure corroborating prominent mechanical properties compared to pure zirconia.
KEYWORDS: A. Sintering, B. Nanocomposites, C. Mechanical properties, E. Biomedical applications. 2. Introduction The great interest in the incorporation of carbon nanotubes (CNTs) into the ceramic materials, through the recent decade, stems from their extraordinary mechanical, electrical and functional properties, which are unparalleled while compared to several analogous alternatives. In particular, combination of a remarkable aspect ratio with the excellent stiffness and strength of CNTs has created them magnificent candidates to reinforce a long spectrum of materials such as metals, polymers and specifically ceramics. Many researchers have recently declared their success in the development of CNTs-ceramics with superior mechanical properties compared to the monolithic materials. Moreover, CNTs have also been reported to impart excellent mechanical properties to oxide ceramics specifically alumina and zirconia composites. Mukherjee et al. [1] have reported, for first time, that addition of CNTs into the alumina matrix increased the fracture toughness as much as three times. They have introduced crack deflection and CNTs’ pull-out as responsible mechanisms for such a huge improvement in the toughness. In addition, Mazaheri and his research group [2-3] have recently focused on the mechanical properties of Zirconia-CNTs ceramics. They have primarily, explored the effect of CNTs’ presence using mechanical spectroscopy tests. The above mentioned observation was also confirmed by the former publication of Taheri et al. [4] who reported a theatrical enhancement in the fracture toughness of monolithic 3Y-TZP material up to nearly 100%, through addition of 5wt.% CNTs. According to their results, CNTs should be recognized for proposing a great competence to suppress the accelerated grain growth in the zirconia matrix, while Taheri et al. have eventually derived composites with much smaller final grain size compared to their
monolithic counterparts. As they have discussed, CNTs are flexible enough to wrap around the grains and prevent grain coarsening of composites. This may be, particularly, one of the major underlying reasons behind the improved mechanical properties of CNTs reinforced composites. On the other hand, the very recently discovered biocompatibility of Ceramic-CNTs composites has shed light on the great applicability of these materials to stand out as a promising alternative utilized in bone implants. In particular, use of CNTs as the secondary reinforcing component of zirconia based biomaterials is anticipated to improve not only the overall mechanical properties of implants but also biocompatibility of bone grafting materials. Such an expectation arises from their extremely small size (about 1–10 nm in diameter) which spans within the range of small components of biological organs [5–10]. The controversial issue whether CNTs are toxic or not, has been argued in several previous publications. Smart et al. [11] reviewed biocompatibility and cytotoxicity of CNTs and CNTs-containing materials in different biomedical fields. They stated that despite relatively few studies on this topic are available; most of the results were either inconclusive or contradictory. The numerous previous researches have studied effective parameters on biocompatibility of CNTs under in vitro and in vivo conditions. Most of the abovementioned reports have confirmed bone compatibility and mineralization of CNTs [7-9, 12-23], while there are also available reports in the literature defining their toxicity in specific laboratorial conditions [6, 24–26]. Nowadays, TZP ceramics are widely used as to synthesize varied dental/orthopedic implants such as femoral ball heads. These implants are normally utilized for total hip replacement owing to their excellent biocompatibility, high fracture toughness, and low wear rates [27-28]. However, there are several case studies reporting an absolute failure of TZP ceramic implants due to crack propagation caused by low-temperature degradation of tetragonal to monoclinic
phase in humid atmosphere of in vivo conditions [29-32]. Based on the information available in the open literature, one of the most severe failures has been reported at the beginning of the year 2000, for zirconia hip joints [29]. Such a drastic incident gave rise to complementary research activities focusing on the major causes behind the abovementioned fracture and coming up with strategies to prevent similar failures. Accordingly, as discussed by several researchers, since under in vivo conditions, low-temperature degradation of biological organs or implants is known to be disseminated with an extremely slow rate, extensive experiments over several years are required to reveal the effect of transformation within the oral environment [33-35]. Many efforts, subsequently, were made to create simulative laboratorial conditions approximating interior body conditions in which implants are required to work for many years. Chevalier [33], for instance, recommended some modifications in the processing conditions through the ageing test of zirconia-based materials. He stated that an accelerated ageing profile should be conducted in steam at 134oC. It was also calculated that 1 h of autoclave treatment at 134oC presented theoretically the same effect as 3–4 years under in vivo conditions. The very first purpose of the current investigation is to review the successful experiences of the authors to incorporate zirconia based composites with CNTs and increase mechanical properties (specifically fracture toughness) of the host zirconia material. As the second approach, our objective is to propose the novel 3Y-TZP/CNTs composites as a promising alternative to substitute conventionally applied zirconia hip implants. In particular, the synthesized composites have been put through in vitro experiments to study the biocompatibility of these materials for use in body conditions and clarify their strength and fracture toughness for substituting bone tissue implants.
3. Experiments 3.1.
Composite preparation and mechanical testing
Commercially available, high purity 3 mol.% yttria stabilized zirconia (3Y-TZP) powder (Tosoh Co., Japan) and multi walled carbon nanotubes (Arkema, France) were selected as starting materials. According to the images (Fig. 1) of scanning electron microscopy (SEM, XLF-30, Philips, Netherlands), the starting YSZ powder consisted of spherical nanoparticles with an averaged diameter of 70 nm. The CNTs were synthesized by catalytic chemical vapor deposition. They have a length of about 10–20 µm, a diameter of about 10–20 nm and the number of walls is between 5 and 15. A detailed information on the nanotube synthesis is available in [36]. CNTs were distributed within 3Y-TZP matrix with 0 and 3 wt.% compositions, using a Turbula Mixer machine with zirconia balls for 24 h. Morphology of composites and dispersion of CNTs within the matrix at varied amounts has been analyzed by scanning and transmission electron microscopy (SEM and TEM, CM-20, Philips, Netherlands). Powder mixture samples were consolidated using Spark Plasma Sintering (SPS, FCT GmbH, Germany) under vacuum (10−2 mbar). The temperature was measured by means of an optical pyrometer focused on the upper graphite punch, at about 4 mm from the sample. The SPS was carried out with a fixed heating rate of 50 oCmin−1 under a constant applied pressure of 50 MPa with a soaking time of 2 min. Temperature in the isothermal stage of sintering depended on the powder composition. In particular, while monolithic zirconia was sintered at 1250oC to obtain rather dense (relative density>98%) specimens, for densification of the composites with 3wt. %
)
CNTs, the sintering temperature was needed to be increased up to 1350oC. The final sintered specimen size was 40 mm diameter pellets with thickness of about 7 mm. The densities of sintered samples were determined by Archimedes method in deionized water. The theoretical densities of the composites were calculated based on the rule of the mixtures. The density of graphite (2.25gcm−3) has been used for CNTs. Raman Spectroscopy (Dispersive Raman, Senterra (2009), Bruker, Germany) was exploited to characterize the specimens in terms of degree of damage to CNTs before and after the processing. Indentation tests were carried out with a diamond Vickers indenter under 20 kg loads with a dwell of 20s on carefully polished surfaces. The hardness (HV) was calculated from the diagonal length of the indentation using the following equation (Eq. (1)): HV = 1.854P/d2
(1)
Where P is the applied load and d is the mean value of the diagonal length. Fracture toughness (KIC) was determined by measuring the crack length emanating the indentation center indicated by C in the Eq. (2) [37]: KIC = 0.0016(E/HV)1/2(P/C3/2)
(2)
Where E is the Young modulus, HV is the Vickers hardness and P is the load. The crack lengths were measured immediately after indentation using a calibrated optical microscope. At least ten valid measurements were carried out for each sample to calculate the average.
3.2.
In Vitro assays 3.2.1
Cell culture *
In this study, osteoblast-like cell line MG 63 (NCBI C555, National Cell Bank of Iran) was used to study biological properties of the samples. After de-freezing the cells, they were transferred to a cell culture flask containing the culture medium (Roswell Park Memorial Institute (RPMI) medium) with 10 %(v/v) Fetal bovine serum (FBS-Serum, Germany), 100 U.ml−1 penicillin, and 100 g.ml−1 streptomycin. Then, the flask was placed into an incubator under general conditions (a humidified, 5% CO2, 95% air environment, and temperature 37.4oC). 3.2.2
MTT assay
In this study, in order to evaluate vitality and proliferation rate of the osteoblast cells cultured on samples, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-iphenyltetrazolium bromide) assays, with the following procedure, were conducted. In this regard, the samples were sterilized by autoclave and then were placed in a 24-well plate. Afterwards, 2×103 cells were seeded on surface of the samples, and then the plate was located in the incubator for 5 h. Thereafter, 1500 l medium culture containing 10 % FBS was added to each well. The plates were kept in the incubator for 7 and 14 days. Then, the culture medium was removed from each well and 1000 l of 0.5 mg.ml-1 MTT (Sigma, USA) solution was added and was placed into the incubator for 4 h. After 4 h, the solution on the cells was removed and 1000 l of isopropanol (Sigma, USA) was added to make the generated purple crystals get solved. In order to solve the purple crystals more efficiently, the plate was put in a shaker incubator for 15-20 min. In the following, 100 l of solution of each well was transferred into a 96-well microtiter plate (Nunc, Denmark). Then the optical density (OD) of formazan in the solution was measured at 545 nm using a two-wall microplate reader (STAT FAX 2100, USA) for quantification cells viability. The cells incubated in the culture medium without any sample were used as a control sample.
+
3.2.3
Alkalin Phosphatase Activity
The amount of soluble ALP, a marker of osteoblast activity, was assayed by measuring the conversion to p-nitrophenol from p-nitrophenyl phosphate. The sterilized samples were put in each well of 6-well plate. Then 2000 cells were seeded on samples surface and the plates were placed into the incubator for 4-5 h. After 7 and 14 days, 10 µL cell supernatant was added to 1000 µL of ALP’s kit (Pars azmun, Iran) according to the manufacturer’s protocol at 37oC. The ALP activity was measured spectrophotometrically by monitoring the absorbance of the solution at a wavelength of 405 nm. The control groups were similar to MTT assay. After the prescribed time period, the amount of Calcium/Phosphor and Phosphate/Phosphor ratio present in the acidic supernatant was spectrophotometrically quantified using a commercially available kit (Pars azmun, Iran). The data of MTT and ALP assays were analyzed using one-way ANOVA with LSD post hoc test using SPSS (version 16.0 SPSS Inc. Chicago IL). P values of less than 0.05 were considered statistically significant. 3.2.4
Cell adhesion
In order to investigate the cellular adherence, the sterilized samples were put in each well of 24well plate. Then, 4000 - 5000 cells in the volume of 50 l were seeded on each sample surface, and the plate was placed into the incubator for 4-5 h. Afterward, a specific amount of culture medium containing 10 % FBS was added. After 48 h, the solution on the samples was taken out and washed by phosphate-buffer saline (PBS). Finally, the cells were fixed with 2.5% glutaraldehyde. After 1h, the samples were washed with deionized water. After gold sputtering,
,
the cell adherence to the samples was observed by scanning electron microscopy (SEM-VEGA TESCAN).
3.3
Ageing
XRD results demonstrated that the initial monoclinic content of the samples prior to the heat treatment process was close to zero in all cases. The samples were hydrothermally treated in steam at 134oC and 3 bar absolute pressure for 20 h to evoke low temperature degradation (LTD). After completion of ageing, content of the monoclinic and tetragonal zirconia phases of each specimen was measured by X-ray diffraction (XRD, Cu K radiation, Philips PW3710, Netherlands) on diamond polished side of the samples. Weight fraction of the monoclinic phase, Xm, was calculated using the following formula [38]: § ¹· I ¨111¸ m + I (111) m © ¹ Xm = § ¹· I ¨111¸ m + I (111) m + I (111) t © ¹ (1) ¹
where I(111)m, I(111 )m and I(111)t are the integrated intensity from the monoclinic (111), ¹
monoclinic (111 ) and the tetragonal (111) peaks, respectively.
4
Results and Discussion 4.1.
Mechanical properties
Fig. 1a. displays SEM micrograph of a powder mixture from 3 wt.% CNTs dispersed within 3YTZP matrix. As can be observed, CNTs are adequately dispersed within the ceramic powder,
-
while least effects of damages, cuts and breakage can be traced along the CNTs’ bodies after the mixing process. In particular, as formerly explained in other publications [2–4], deriving a homogeneous dispersion of CNTs within a ceramic powder could be recognized as the major challenge of producing these composites. TEM image of the above powder mixtures (Fig. 1b.), not only verifies our successful dispersion process, but also confirms that CNTs have been exposed to minimal amount of damages through the mixing stage. In addition, adequate dispersion of CNTs can be also monitored by exploring fracture surface of a SPSed zirconiaCNTs sample. As can be observed in Fig. 2, nanotubes of carbon are homogeneously spread through the whole consolidated specimen, while SPS process seems to have exerted least of damages to the nanotubes’ body and maintained CNTs at the sintering temperature. Such an observation was subsequently confirmed by performing Raman spectroscopy to ensure safety and maintenance of CNTs. Fig. 3 compares Raman Spectroscopy pattern of pure carbon nanotubes with as-sintered sample. As can be observed, the major peaks in the as sintered composites are pretty close to those in pure CNTs. Moreover, calculation of ID/IG intensity ratio revealed a negligible increase as a result of processing. This verifies CNTs’ maintenance after the sintering stage and validates our manufacturing method.
Fig 1. a) SEM b) TEM images of mixed zirconia nanopowders with 3 wt% CNTs
Fig 2. SEM fracture surface figure of 3Y-TZP/3 wt.% CNTs composite
.
Fig 3.Raman Spectroscopy pattern of pure carbon nanotubes (before processing) in comparison with as-sintered composite sample (after processing)
Incorporation of monolithic 3Y-TZP sample with carbon nanotubes have, presumably, led to some modifications in the sintering profile, microstructure and mechanical properties of the final synthesized composites. Based on the results shown in table 1, CNTs have increased the temperature required to consolidate the monolithic zirconia up to 50oC and 3Y-TZP/ CNTs samples could be hardly sintered to near full density state by lower sintering temperature than 1300oC. On the other hand, CNTs have been found to wrap around grains and prevent an accelerated grain growth in the final stage of sintering. Such an occurrence is expected to culminate in a frustrated consolidation as well as a finer microstructure for CNTs incorporated composites compared to their monolithic counterparts. Accordingly, along with a lower sintered fractional density for the composite, one can observe that the presence of CNTs in the 3Y-TZP/ CNTs composites gave rise to an effective suppression of grain boundary mobility. As a result, while the grain size of monolithic zirconia is around 145 nm, the averaged grain size of the composite increased no further than 109 nm. The greatest effect of nanotubes, however, should be realized when the incorporated composites display enhanced mechanical properties (hardness & fracture toughness) compared to the monolithic zirconia. Quote to table 1, although CNTs have not remarkably increased hardness values for the composites, the fracture toughness can be speculated to have undergone a manifested increment (about 75% improvement) as a result of CNTs incorporation. Details of mechanisms how CNTs enhance fracture toughness of the material can be found in other publications of the authors [2–4]. What should be taken into account in this concern is that while indentation test is not generally recognized as the best device to have precise calculations of
fracture toughness values, such an experiment, as numerically reported through several publications [2–4, 39–41], is appreciable as a reliable scheme to present comparative trends of mechanical properties. Moreover, the current results of fracture toughness values have been formerly confirmed by other publications of the authors through utilization of SEVNB (Single Edge V-Notched Beam) method [3].
Table 1. Sintering temperature and characteristics of sintered 3Y-TZP/ CNTs composite in comparison with monolithic zirconia. Sintering
Fractional density
Grain size
Indentation fracture toughness
Vickers hardness
temperature (oC)
(%)
(nm)
(MPa.m1/2)
(GPa)
1250
99.3
145
5.8 ± 0.28
12.1 ± 0.21
1300
98.7
109
10.1 ± 0.21
12.6 ± 0.21
Material composition
3Y-TZP 3Y-TZP/3 wt.% CNTs
4.2.
In vitro biocompatibility
Osteoblasts, the bone-forming cells, proliferate on the bone surface and secrete bone matrix proteins as well as producing them. Therefore, studying the performance of osteoblasts cells on 3Y-TZP/CNTs composites as a future scaffold biomaterial seems necessary. Cells mitochondria are able to reduce MTT into a formazan precipitate that can be solved in n-propanol and be quantified spectrophotometrically. This assay allowed monitoring the vitality and proliferation of osteoblast cells on the surface of samples after the 7 and 14 days (Fig. 4).
Fig.4. MTT assay results showing viability of osteoblasts cells on the surface of 1) 3Y-TZP 2) 3Y-TZP/CNTs samples after 7 and 14 days in comparison with control Sample. Higher optical density indicates greater amounts of viable osteoblasts. * p<0.05: compare to the control
According to the results of MTT assay (Fig.4), optical density in both specimens was similar to the control sample after 7 days. Increase in the optical density of both samples over time in comparison with the control sample showed that the proliferation rate of osteoblast cells on the surface of specimens was higher than that of the control sample. Furthermore, addition of CNTs to 3Y-TZP had no significant effect on vitality of cells in comparison with 3Y-TZP. Indeed, osteoblast cells were able to survive and grow in contact with 3Y-TZP/CNTs as well as biocompatible 3Y-TZP. The effect of CNTs on increasing the cell proliferation has been reported by several researchers. For example, Chłopek et al. [22] reported biocompatibility of CNTs similar to polysulfone, which is currently used in medicine, by studying response of osteoblast and fibroblast cells in contact with the surface of CNTs. Ogihara et al. [42] showed that addition of CNTs as a reinforcement to alumina ceramics enhanced proliferation of osteoblast cells. Some other publications showed that CNTs are able to promote proliferation of osteoblast cells [7, 9]. On the other hand, some reports showed that CNTs substrates decreased some cell lines survival [24, 25]. Some in vitro studies on CNTs demonstrated that CNTs are cytotoxic [24–26]. Despite all these argues in the case of toxicity of CNTs, severe effects of toxicity have not been observed through addition of 3 wt. % CNTs into biocompatible 3Y-TZP based on our observations (Fig.4).
Fig.5. ALP assay results showing Alkalin Phosphatase activity of osteoblasts cells attached to 1) 3Y-TZP 2) 3YTZP/CNTs samples after 7 and 14 days in comparison with control sample. *p<0.05: compare to the control
Fig.6. Ca/Phosphor ratio measured present in the acidic supernatant on the surface of 1) 3Y-TZP 2) 3Y-TZP/CNTs samples after 7 and 14 days in comparison with control sample showing calcium production. *p<0.05: compare to the control
Fig.7. Phosphate/Phosphor ratio measured present in the acidic supernatant on the surface of 1) 3Y-TZP 2) 3YTZP/CNTs samples after 7 and 14 days in comparison with control sample showing phosphate production. *p<0.05: compare to the control
Alkalinphosphtase (ALP), a noncollagenous protein, is the most common indicator of osteoblast activity, bone mineralization, and predictor of the formation of osteogenic cells [43, 44]. ALP activity of osteoblast cells attached to samples that should be recognized as a confirming factor for the occurrence of cell differentiation was measured after 7 and 14 days (Fig. 5). Furthermore, Calcium/Phosphor and Phosphate/Phosphor ratios present in the acidic supernatant on the surface of the samples after 7 and 14 days were calculated as the markers of mineralization as well (Fig.6 and Fig.7). According to Fig.5, after the very first week, ALP activity of cells in contact with 3Y-TZP was similar to that of the control sample, while it was apparently lower in 3Y-TZP/CNTs sample. This trend was maintained over time although ALP activity increased in all samples. Fig.6 and Fig.7 also showed that Calcium/Phosphor ratio and Phosphate/ Phosphor ratio were lower in 3Y
TZP/CNTs sample in the first week compared to 3Y-TZP and control samples, but after 14 days the results were approximately similar in both samples. It is well known that ALP can be expressed as a critical phonotypical factor for bone forming cells and initiation of matrix mineralization. In the present study, osteoblast cells were observed to be capable of producing ALP in contact with 3Y-TZP and 3Y-TZP/CNTs composites. Consequently, Calcium/Phosphor ratio and Phosphate/ Phosphor ratio could be regarded as an indicator for promotion of matrix formation, which is an essential factor for bone regeneration. There were several studies that evaluated the effects raised by the addition of CNTs on mineralization of osteoblast cells. Li et al. [23] studied in vitro biocompatibility of CNTs. They unveiled that CNTs increase differentiation and maturation of osteoblast cells related to bone formation by absorbing more proteins owing to their larger surface area. Ogihara et al. [42] showed that addition of CNTs as a reinforcement to alumina ceramics enhanced ALP activity and bone mineralization of osteoblast cells. In our present study, it can be inferred that although osteoblasts were observed to differentiate in 3Y-TZP/CNTs composites with a delayed onset compared to biocompatible 3YTZP, the mineralization triggered approximately in a simultaneous starting time after two weeks in both samples due to mineralization ability of CNTs, or the differences were insignificant.
Adhesion of cells to surface of biomaterials is known as a major factor of biocompatibility. It is postulated that the more compatible the surface, the greater amount of cells are expected to attach. SEM images in Fig. 8 a and b indicate how the osteoblast cells are attached to the surface of samples after 48 h.
)
Fig.8. Scanning electron microscopy images of cell attachment a) 3Y-TZP b) 3Y-TZP/CNTs after 48 h
Initial cell attachment on the surface of biomaterials, which is one of the most complicated and integral features of biomaterials, have been argued by several previous researches. Bachle et al. [45] introduced surface roughness as influential factor on initial cell attachment and the spread of the cells. On the contrary, Yamashita [46], reported that surface condition had no effect on initial attachment. Price et al. [18, 19], proved that the attachment area of osteoblasts increased in case of finer surface of the specimens and the presence of carbon nano fibers. As can be seen in Fig.8, after 48 h, layering of osteoblast cells, which is an indicator of cells differentiation and matrix mineralization was observed on the surface of both samples. Indeed, osteoblast cells had spread on the surface of both specimens and showed normal morphology over time due to CNTs’ small size (about 1–10 nm in diameter). A number of studies showed proper adhesion and function of bone-forming osteoblast cells on the surface of CNTs-containing material [17,20,21]. Correspondingly, our results demonstrated that the scaffold synthesized by 3Y-TZP/CNTs composites, is not only biocompatible but also it provides a favorable environment for attachment and growth of bone cells.
According to the results of MTT assay, ALP assay, and SEM images, one could state that proliferation, differentiation, maturation, and mineralization of osteoblast cells on 3Y-TZP/CNTs composite were improved over time. Tutak et al. [47] studied toxicity of CNTs on osteoblast cells. Despite all controversial publications in this case, they revealed a mechanism showing that CNTs induce toxicity in the first days of culture, then dead cells release proteins and growth factors that enhance activity of remaining cells to proliferate and differentiate. Usui et al. [8], *
who first developed CNTs as biocompatible materials, clarified that MWCNTs did not cause strong inflammatory reactions in contact with bone, and have good bone-tissue compatibility. Correspondingly, our results indicated that 3Y-TZP/CNTs composite could be recognized for proposing bone tissue compatibility similar to biocompatible 3Y-TZP.
4.3.
Ageing
As transformation of zirconia is a thermally activated process, we had no option other than to conduct accelerated ageing tests under hydrothermal conditions to forecast the long-term behavior of the material. The objective behind performing the abovementioned experiments was to study ageing procedures of 3Y-TZP and 3Y-TZP/CNTs composites. Fig.9 demonstrates XRD results of 3Y-TZP and 3Y-TZP/CNTs samples before and after heat treatment for 20 h. What can be noted is the absence of initial monoclinic phase content in both samples. After 20 h of heat treatment at 134oC under pressure of 3 bar (which simulates the conditions of more than 60 years of in vivo performance [34]), the content of monoclinic phase in 3Y-TZP and 3YTZP/CNTs composites were 13.2 and 5.4, respectively. Indeed, not only were the variations of monoclinic content lower than 10% in 3Y-TZP/ CNTs composite, accepted by chevalier’s modifications [33], but also the low-temperature degradation decreased by addition of CNTs to 3Y-TZP. In the constrained ZrO2(t) - ZrO2(m) reaction, differential free energy Gt-m, between these two states per unit volume of transformed material, can be determined using the [48]: Gt-m=-G0+Use+Us
(4)
where G0 is the chemical free energy, Use is the strain energy associated with the transformed particle and surrounding matrix which is usually less sensitive to temperature and composition +
changes, and Us is the change in energy associated with the surface of the inclusion. With the presence of CNTs in ZrO2 matrix, Use increases because of high elastic modulus of CNTs, while Gt-m increases correspondingly, so tetragonal phase is more stable. Therefore, the addition of CNTs has successfully resulted in stabilization of tetragonal phase. Reviewing the above theory, we can expect that the bone tissue implant produced from 3YTZP/CNTs composites are potentially capable of resisting against durable stresses for prolonged times of use and are exposed to postpone failures compared to pure zirconia. However, further factors to which this composite are exposed in the oral environment (e.g. cyclic mechanical and thermal loading) are not considered in this calculation; therefore ageing may probably proceed more rapidly under in vivo condition.
Fig.9. XRD patterns of 3Y-TZP and 3Y-TZP/CNTs before and after 20 h of heat treatment at 134oC, 3 bar.
5.
Conclusion
The objective of the current paper was to explore the effect of carbon nanotubes’ addition -as the secondary phase- on the biocompatibility and mechanical properties of conventional 3Y-TZP hip implants. In terms of mechanical properties, CNTs were found to result in enhanced fracture toughness for the CNTs-containing materials, compared to monolithic Zirconia. To investigate basic biocompatibility features of 3Y-TZP/CNTs composites, on the other hand, the synthesized specimens were subjected to in vitro test examinations. According to our results, CNTs
,
incorporated composites, while presenting no further improvement in bio compatibility, at least demonstrated no decrement compared to the conventional 3Y-TZP implants. Moreover, the amount of low temperature degradation of CNTs incorporated composites was compared to that of 3Y-TZP samples through heat treatment for 20 h. As a result, one could observe a lower amount of monoclinic phase variation in 3Y-TZP/CNTs, defining the competence of this novel composite to survive under in vivo conditions for much longer durations compared to pure zirconia without severe failure and toxicity. However, this composite should be subjected to in vivo tests in order to realize if the major requirements of clinical applications are fulfilled by using these composites. 6.
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Figure Captions: Fig 1. a) SEM b) TEM images of mixed zirconia nanopowders with 3 wt% CNTs Fig 2. SEM fracture surface figure of 3Y-TZP/3 wt.% CNTs composite Fig 3. Raman Spectroscopy pattern of pure carbon nanotubes (before processing) in comparison with as-sintered composite sample (after processing) Fig.4. MTT assay results showing viability of osteoblasts cells on the surface of 1) 3Y-TZP 2) 3Y-TZP/CNTs samples after 7 and 14 days in comparison with control Sample. Higher optical density indicates greater amounts of viable osteoblasts. * p<0.05: compare to the control Fig.5. ALP assay results showing Alkalin Phosphatase activity of osteoblasts cells attached to 1) 3Y-TZP 2) 3YTZP/CNTs samples after 7 and 14 days in comparison with control sample. *p<0.05: compare to the control Fig.6. Ca/Phosphor ratio measured present in the acidic supernatant on the surface of 1) 3Y-TZP 2) 3Y-TZP/CNTs samples after 7 and 14 days in comparison with control sample showing calcium production. *p<0.05: compare to the control Fig.7. Phosphate/Phosphor ratio measured present in the acidic supernatant on the surface of 1) 3Y-TZP 2) 3YTZP/CNTs samples after 7 and 14 days in comparison with control sample showing phosphate production. *p<0.05: compare to the control Fig.8. Scanning electron microscopy images of cell attachment a) 3Y-TZP b) 3Y-TZP/CNTs after 48 h Fig.9. XRD patterns of 3Y-TZP and 3Y-TZP/CNTs before and after 20 h of heat treatment at 134oC, 3 bar.
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