Materials Science and Engineering C 40 (2014) 253–259
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Biological evaluation of zirconia/PEG hybrid materials synthesized via sol–gel technique M. Catauro a,⁎, F. Papale a, F. Bollino a, M. Gallicchio b, S. Pacifico b a b
Department of Industrial and Information Engineering, Second University of Naples, Via Roma 29, 81031 Aversa, Italy Department Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy
a r t i c l e
i n f o
Article history: Received 4 October 2013 Received in revised form 12 December 2013 Accepted 3 April 2014 Available online 12 April 2014 Keywords: Sol–gel Organic/inorganic hybrid Bioactivity Biocompatibility PEG
a b s t r a c t The objective of the following study has been the synthesis via sol–gel and the characterization of novel organic– inorganic hybrid materials to be used in biomedical field. The prepared materials consist of an inorganic zirconia matrix containing as organic component the polyethylene glycol (PEG), a water-soluble polymer used in medical and pharmaceutical fields. Various hybrids have been synthesized changing the molar ratio between the organic and inorganic parts. Fourier transform spectroscopy suggests that the structure of the interpenetrating network is realized by hydrogen bonds between the Zr-OH group in the sol–gel intermediate species and both the terminal alcoholic group and ethereal oxygen atoms in the repeating units of polymer The amorphous nature of the gels has been ascertained by X-ray diffraction analysis. The morphology observation has been carried out by using the Scanning Electron Microscope and has confirmed that the obtained materials are nanostructurated hybrids. The bioactivity of the synthesized system has been shown by the formation of a hydroxyapatite layer on the surface of samples soaked in a fluid simulating the human blood plasma. The potential biocompatibility of hybrids has been assessed as performing indirect MTT cytotoxicity assay towards 3T3 cell line at 24, 48, and 72 h exposure times. © 2014 Published by Elsevier B.V.
1. Introduction The study of organic–inorganic nanocomposite networks, for biomedical applications, has recently become an expanding field of investigation [1]. At first glance, these materials are considered as biphasic, where the organic and inorganic phases are mixed at nanometer to micrometer scale. However, this class of materials is very interesting because their properties are not just the sum of the individual contributions of both phases but are derived from their synergy. The nature of the interface has been used to divide these materials into two distinct classes [2]. In class I, organic and inorganic compounds are embedded by means of weak bonds (hydrogen, van der Waals or ionic bonds) which give the cohesion to the whole structure. In class II materials, the phases are linked together through strong chemical bonds (covalent or ionic–covalent bonds). They result from a deep research work emerging from sol–gel and polymer chemists and present a large diversity in their structures and final properties. Sol–gel process has proved to be versatile and has been widely used in the preparation of organic/inorganic hybrid biomaterials [3], non-linear optical materials [4], and mesoporous materials [5]. The sol–gel chemistry is based on the hydrolysis and polycondensation of metal alkoxides ⁎ Corresponding author. Tel.: +39 0815010360; fax: +39 0815010204. E-mail address: [email protected] (M. Catauro).
http://dx.doi.org/10.1016/j.msec.2014.04.001 0928-4931/© 2014 Published by Elsevier B.V.
M(OR)x, where M = Si, Sn, Zr, Ti, Al, Mo, V, W, Ce and so forth. Those reactions are affected by the value of many parameters such as structure and concentration of the reactants, solvents, and catalysts as well as reaction temperature and rate of removal of by-products and solvents. Chemical reactions occur at room temperature and this determines two main advantages: the saving of energy and the possibility to incorporate an organic and notoriously thermolabile [6] phase. A further advantage of sol–gel technique is that it is able to synthesize high purity glasses and ceramics, because the starting raw materials are synthetic and not extractive as for conventional glass. Moreover, materials prepared by sol–gel process have shown to be more bioactive than those with the same composition but prepared with different methods. In fact, the surface analysis of sol–gel materials and coatings has shown the presence of hydroxyl groups that can promote the nucleation of calcium and phosphate and, therefore, the osteointegration when those materials are implanted [7]. The presence of organic components modifies the morphology and physical properties of the sol–gel products [8,9]. A variety of organic polymers has been introduced into inorganic networks to obtain hybrid or composite materials with or without covalent bonds between the polymer and inorganic components, respectively. Catauro et al. prepared, by means of sol–gel methods, several hybrids for drug delivery or biomedical applications by using different oxides, including CaO and/or SiO2 [10–13], TiO2 [14–16], and ZrO2 [17–19], and different polymer, e.g. poly (ε-caprolactone) [13,17–26] and poly
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(ether-immide) [16]. In all the cases, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and for some supported also by solid-state NMR measurements gave circumstantial evidence of the formation of class I hybrids, characterized by the presence of hydrogen bonds among the H-acceptor groups of the polymer, and the OH-groups (H-donator) of the inorganic phase. The aim of the present preliminary paper is the sol–gel synthesis and characterization of novel hybrid materials for biomedical applications, consisting of an inorganic zirconia matrix where a polymer, polyethylene glycol (PEG), is entrapped. The component choice is related to previous researches. Several papers show the use of zirconia ceramics and glasses as biomaterials [17, 27,28]. PEG is known also to be a versatile, biocompatible polymer [29,30] mainly used in polymer-based materials for drug delivery [31]. PEG-derived products have been investigated also as biomaterials for implants for orthopedic and dental applications [32], e.g. Kawakami et al. [33] showed that polyethylene glycol could be safely used as a base component of biomaterials for internal medical use, such as in root canal filling material. The obtained materials were characterized by means of several instrumental technique (FT-IR, XRD and SEM) and their in vitro assessment of cytotoxicity was carried out by means of MTT assay on NIH3T3 murine fibroblast cell line, one of the recommended and appropriate step for the biological evaluation of medical devices. Moreover, in order to evaluate if these materials can be used in dental or orthopedic applications (as implantable therapeutic systems or filling materials for bone or tooth repair), their in vitro bioactivity was investigated by soaking the samples in a Simulated Body Fluid (SBF) and observing by SEM microscopy the hydroxyapatite formation on the surface [34]. Indeed, as reported in the literature [35], the essential condition for glasses and glasses–ceramics to bond to living bone is the formation of a bone-like apatite layer on the surfaces.
Fig. 2. ZrO2/PEG after drying in air.
zirconium propoxide, Zr(OC3H7)4 (Sigma-Aldrich), as precursor of the inorganic matrix, polyethylene glycol (PEG, Mw = 400, SigmaAldrich) as organic component, and ethanol as solvent. Acetylacetone was also added to control the hydrolytic activity of zirconium alkoxide. In fact, it is a chelating agent able to form complexes, with monomeric alkoxides, stable towards hydrolysis reactions [36,37]. Fig. 1 shows the flow chart of hybrid synthesis by the sol–gel method. After gelation the gels were air-dried at 50 °C for 24 h to remove the residual solvent and were obtained transparent and glassy samples (Fig. 2).
2. Materials and methods 2.2. Materials characterization 2.1. Sol–gel synthesis The hybrids inorganic/organic materials with ZrO2/PEG molar ratio 6, 12, 24 and 50 were prepared by means of sol–gel process using
The presence of hydrogen bonds between organic and inorganic components of the hybrid materials was ascertained by Fourier transform infrared spectroscopy (FT-IR). Transmittance spectra were recorded in the 400–4000 cm−1 region using a Prestige 21 (Shimadzu, Japan) system, equipped with a DTGS KBr (Deuterated Tryglycine Sulphate with potassium bromide windows) detector, with resolution of 2 cm−1 (45 scans). KBr pelletized disks containing 2 mg of sample and 198 mg of KBr were made. The FTIR spectra were elaborated by Prestige software (IR solution). The nature of ZrO2 gel and ZrO2/PEG hybrid materials were ascertained by X-ray diffraction (XRD) analysis using a Philips diffractometer. Powder samples were scanned from 2Θ = 5° to 60° using CuKα radiation. The microstructure of the synthesized gels has been studied by scanning electron microscope (SEM) (Quanta 200, FEI, The Netherlands). 2.3. Study in vitro bioactivity In order to study their bioactivity, samples of the studied hybrid materials were soaked in SBF, a solution with ion concentrations nearly
Table 1 Prepared material gelification time.
Fig. 1. Flow chart of ZrO2/PEG gel synthesis.
Hybrid system
Gelification time
ZrO2 ZrO2:PEG ZrO2:PEG ZrO2:PEG ZrO2:PEG
6 days 6 days 7 days 8 days 8 days
= = = =
50 24 12 6
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and Energy Dispersive X-ray (EDX) (EDAX, USA) analysis. Taking into account that the ratio of the exposed surface to the volume solution influences the reaction, a constant ratio of 10 mm2 mL−1 of solution, was respected as in reference [39]. 2.4. Cell culture and cytotoxicity assessment
Fig. 3. Comparison between FTIR spectra of : a) ZrO2, b) ZrO2/PEG 6, c) ZrO2/PEG 12, d) ZrO2/PEG 24, e) ZrO2/PEG 50, f) PEG.
equal to those in human blood plasma [38]: Na+ 142.0, K+ 5.0, Ca2+ 2.5, 2− Mg2 + 1.5, Cl− 147.8, HCO− 1.0, SO24 − 0.5 mM. During 3 4.2, HPO4 soaking the temperature was kept fixed at 37 °C. The ability to form an apatite layer was studied by submitting reacted samples to SEM
Cytotoxicity was measured through MTT [3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] cell growth inhibition assay (indirect test) using NIH-3T3 murine fibroblast cell line. Investigated extracts were obtained from disks of the studied materials, which were previously immersed for 24 h in 3.5 mL of a complete culture medium at 37 °C under continuous stirring. The cell line, purchased from ATCC (American Type Culture Collection), was grown in RPMI 1640 high glucose medium supplemented with 10% fetal bovine serum, 50.0 U/mL penicillin, and 100.0 μg/mL streptomycin, at 37 °C in a humidified atmosphere containing 5% CO2. NIH-3T3 cell line was seeded in 96-multiwell plates at a density of 1.0 × 104 cells/well. After 24 h of incubation, cells were treated with 200 μL culture medium solutions containing 100 μL of extracts from ZrO2, and ZrO2/PEG (molar ratio 6, 12, 24, and 50) hybrid sol–gel materials. At 24, 48, and 72 h of incubation, cells were treated with 150 μL of MTT (0.50 mg/mL), dissolved in the culture medium, for 1 h at 37 °C in a 5% CO2 humidified atmosphere. The MTT solution was then removed and 100 μL of DMSO were added to dissolve the formazan originated. Finally, the absorbance at 570 nm of each well was determined by
Table 2 FTIR peak interpretation. Absorption bands ZrO2 matrix peaks
PEG peaks
−1
3417 cm 1585 and 1377 cm−1 1529 and 1280 cm−1 1425 cm−1 931 cm−1 654 cm−1 460 cm−1 422 cm−1 2930 cm−1 2849 cm−1 1454 cm−1 1250 cm−1 1104 cm−1 948 cm−1
Fig. 4. XRD of ZrO2 (A) and ZrO2/PEG (B).
Interpretation –OH vibrations of H-bonded H2O and Zr-OH AcAc bidentate binding C_O vibrations C\C vibrations Methyl C\H symmetric bending AcAc C\C\H bending mixed with C\C stretching Zr\OH Zr–O–Zr stretching Zr\OAcAc vibrations Methylene C\H symmetric stretching Methylene C\H asymmetric stretching Methylene C\H bending Alcohols C\O stretching Ethereal C\O\C stretching PEG C\C stretching
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using a Tecan SpectraFluor fluorescence and absorbance reader. Cell viability was expressed as percentage of mitochondrial redox activity of the cells treated with the extracts compared to an untreated control [40]. Tests were carried out performing twelve replicate (n = 12) measurements for three samples of each extract (in total: 12 × 3 measurements).
3. Results and discussion Gelation is the result of hydrolysis and condensation reactions according to the following reactions: ZrðOC3 H7 Þ4 þ nH2 O⇒ZrðOC3 H7 Þ4−n ðOHÞn þ nC3 H7 OH
Fig. 5. SEM micrograph of samples surface: A) ZrO2, B) ZrO2/PEG 6, C) ZrO2/PEG 12, D) ZrO2/PEG 24, E) ZrO2/PEG 50.
ð1Þ
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−ZrOH þ CH3 O−Zr−⇒ ≡ Zr−O−Zr ≡ þ C3 H7 OH
ð2Þ
≡ Zr−OH þ OH−Zr ≡ ⇒ ≡ Zr−O−Zr ≡ þ H2 O
ð3Þ
The reaction mechanism is generally accepted to proceed through a second order nucleophic substitution [41]. Acetylacetone controls the kinetics of those reactions (hydrolysis of monomeric alkoxides and then polymerization of hydrolysis products) behaving as chelator and forming stable towards hydrolysis complexes with zirconium propoxide [36,37, 42]. Gelification times of the hybrid systems are reported in Table 1. 3.1. Hybrid material characterization Fig. 3 shows the FT-IR spectra of ZrO2 (a), PEG/ZrO2 with molar ratio 6, 12, 24 and 50 (b, c, d, e) and PEG (f). These gels show the characteristic peaks of PEG and ZrO2 (Table 2). In the ZrO2 spectrum (Fig. 3(a)) all typical peaks of AcAc-containing zirconia sol–gel materials [43] are present. The broad intense band at 3417 cm−1 is due to vibrations of –OH groups which are hydrogenbonded with H2O and Zr-OH groups. The bands observed at 1585 and 1377 cm−1 are assigned to C_O vibrations of the AcAc bidentate binding. The bands at 1529 and 1280 cm−1 are attributed to C\C vibrations, the peak at 1425 cm−1 is due to methyl C\H symmetric bending [44], whereas the bands at 1186, 1026 and 931 cm− 1 are assigned to the C\C\H bending mixed with stretching C\C vibrations of AcAc [43]. The bands placed at 654 and 460 cm−1 are due to Zr\OH and Zr\O\Zr stretching respectively [45], whereas the peak at 422 cm− 1 to Zr\OAcAc vibrations [43]. In the PEG spectrum, the broad intense band in the region between 3200 and 3600 cm−1 is due to –OH stretching and indicates that in the pure polymer hydrogen bonds between –OH end groups and ether oxygen in the PEG chains occur [46]. Moreover, all typical PEG peaks, visible in the pure polymer spectra, were observed in the hybrid system spectra and their intensity increases with the amount of the polymer (Fig. 3 from curve b to e). In particular, the intensity of the methylene stretching bands increases together with methylene C\H bending of the polymer [47] at 1454 cm−1. The peaks due to alcohols C\O and ethereal C\O\C stretching at 1250 and 1104 cm−1 appear in curve c and also increase from c to e respectively. Moreover, the sharp AcAc C\C\H bend at 931 cm−1 was replaced with a little broader peak at 948 cm−1 attributed to PEG C\C stretching. At the same time the peaks in the regions 1520–1250 cm− 1, assigned to the AcAc bidentate binding, and 900–400 cm−1, typical of
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zirconia matrix, change shape. The first alteration is probably due to new interaction between AcAc and the polymer while the second phenomenon, which is particularly evident for the band assigned to Zr\OH stretching at 654 cm−1, together with the simultaneous change of broad band at 3417 cm−1, suggests the formation of new hydrogen bonds probably between those –OH groups and both the ethereal oxygen atoms (H-bond acceptors) and alcohol groups (H-bond acceptors and donator) of the PEG chains. The nature and the microstructure of the ZrO2/PEG hybrid materials have been studied by X-ray diffraction (XRD) and SEM. The diffractograms in Fig. 4 shows that ZrO2 (panel A) and ZrO2/PEG (panel B) gels exhibit broad humps characteristic of amorphous materials. The diffractograms of all systems are not shown because they are similar. SEM micrographs of a ZrO2 gel sample and of ZrO2/PEG gel samples are shown in Fig. 5 and no appreciable difference between the morphology of the five amorphous materials can be observed. Moreover, the two phases of which the hybrid consists (organic and inorganic) are indistinguishable confirming that the synthesized materials are hybrid. 3.2. Bioactivity test The evaluation of the apatite deposition on material surface after soaking in SBF, of its morphology and the qualitative elemental analysis were carried out by SEM/EDX microscopy observations. The formation of apatite on the materials after soaking in SBF can be explained by the presence of Zr-OH groups on the surface of the materials. These groups, combined with the Ca2+ ions present in the fluid, originate the increase of positive charge on the surface. The Ca2+ ions combine with the negative charge of the phosphate ions to form amorphous phosphate, which spontaneously transforms into hydroxyapatite [Ca10(PO4)6(OH)2]. After 21 days of incubation, all ZrO2/PEG systems show a deposition of a similar amount of apatite on their surface. Fig. 6 (A and B) shows crystals of apatite on the surface of the samples after 21 days of soaking in SBF and panel C illustrates EDX analysis of crystals; the ratio between the atomic content of Ca and P is 1:6 which agrees with the chemical formula of hydroxyapatite. 3.3. Cytotoxicity of ZrO2/PEG hybrid materials NIH-3T3 murine fibroblast cell line was treated, for three different exposure times (24, 48, and 72 h), with extracts obtained by placing, for 24 h, disks of the investigated ZrO2/PEG hybrid materials, in a complete culture medium. In order to assess the influence of the extracts of
Fig. 6. SEM micrographs of samples after soak in SBF for 21 days. Panels A–B: hydroxyapatite crystal. Panel C: EDX analysis.
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the new sol–gel materials on mitochondria and cell proliferation, MTT cytotoxicity assay was performed. The quantitative measurement of extracellular reduction of the yellow colored water soluble tetrazolium dye to insoluble formazan crystals allowed to state that extracts obtained by the different ZrO2/PEG samples affect positively cell viability and proliferation of tested cells (Fig. 7). In fact, it was observed that all the samples tested were able to induce an increase in cell viability during the considered exposure period compared to the inorganic material when it was tested in the non-hybrid form. The presence of different amounts of polyethylene glycol favored the preparation of biomaterials with improved biocompatibility. Detected data allowed to observe that the effects were dose- and time-dependent. Recently, it was reported that dental ceramics are not equivalent in their in vitro biologic effect, even with the same class of material and most ceramics cause only mild in vitro suppression of cell functions to levels that would be acceptable on the basis of standards used to evaluate alloys and composites (b 25% suppression of dehydrogenases activity) [48]. Thus, our findings are in accordance with the development of biomaterials free from toxicity and potentially useful as bioceramics in dental biomedicine. 4. Conclusions Zirconia/polyethylene glycol (ZrO2/PEG), prepared via sol–gel process, was found to be an organic/inorganic hybrid material.
Fig. 7. ZrO2, ZrO2/PEG 6, ZrO2/PEG 12, ZrO2/PEG 24, and ZrO2/PEG 50 cell viability (CV, %) towards NIH 3T3 cell line at 24 h, 48 h, and 72 h exposure times. Values, reported as percentage vs. an untreated control, are the mean ± SD of measurements carried out on 3 samples (n = 3) analyzed twelve times.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Biological evaluation of zirconia/PEG hybrid materials synthesized via sol–gel technique M. Catauro a,⁎, F. Papale a, F. Bollino a, M. Gallicchio b, S. Pacifico b a b
Department of Industrial and Information Engineering, Second University of Naples, Via Roma 29, 81031 Aversa, Italy Department Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy
a r t i c l e
i n f o
Article history: Received 4 October 2013 Received in revised form 12 December 2013 Accepted 3 April 2014 Available online 12 April 2014 Keywords: Sol–gel Organic/inorganic hybrid Bioactivity Biocompatibility PEG
a b s t r a c t The objective of the following study has been the synthesis via sol–gel and the characterization of novel organic– inorganic hybrid materials to be used in biomedical field. The prepared materials consist of an inorganic zirconia matrix containing as organic component the polyethylene glycol (PEG), a water-soluble polymer used in medical and pharmaceutical fields. Various hybrids have been synthesized changing the molar ratio between the organic and inorganic parts. Fourier transform spectroscopy suggests that the structure of the interpenetrating network is realized by hydrogen bonds between the Zr-OH group in the sol–gel intermediate species and both the terminal alcoholic group and ethereal oxygen atoms in the repeating units of polymer The amorphous nature of the gels has been ascertained by X-ray diffraction analysis. The morphology observation has been carried out by using the Scanning Electron Microscope and has confirmed that the obtained materials are nanostructurated hybrids. The bioactivity of the synthesized system has been shown by the formation of a hydroxyapatite layer on the surface of samples soaked in a fluid simulating the human blood plasma. The potential biocompatibility of hybrids has been assessed as performing indirect MTT cytotoxicity assay towards 3T3 cell line at 24, 48, and 72 h exposure times. © 2014 Published by Elsevier B.V.
1. Introduction The study of organic–inorganic nanocomposite networks, for biomedical applications, has recently become an expanding field of investigation [1]. At first glance, these materials are considered as biphasic, where the organic and inorganic phases are mixed at nanometer to micrometer scale. However, this class of materials is very interesting because their properties are not just the sum of the individual contributions of both phases but are derived from their synergy. The nature of the interface has been used to divide these materials into two distinct classes [2]. In class I, organic and inorganic compounds are embedded by means of weak bonds (hydrogen, van der Waals or ionic bonds) which give the cohesion to the whole structure. In class II materials, the phases are linked together through strong chemical bonds (covalent or ionic–covalent bonds). They result from a deep research work emerging from sol–gel and polymer chemists and present a large diversity in their structures and final properties. Sol–gel process has proved to be versatile and has been widely used in the preparation of organic/inorganic hybrid biomaterials [3], non-linear optical materials [4], and mesoporous materials [5]. The sol–gel chemistry is based on the hydrolysis and polycondensation of metal alkoxides ⁎ Corresponding author. Tel.: +39 0815010360; fax: +39 0815010204. E-mail address: [email protected] (M. Catauro).
http://dx.doi.org/10.1016/j.msec.2014.04.001 0928-4931/© 2014 Published by Elsevier B.V.
M(OR)x, where M = Si, Sn, Zr, Ti, Al, Mo, V, W, Ce and so forth. Those reactions are affected by the value of many parameters such as structure and concentration of the reactants, solvents, and catalysts as well as reaction temperature and rate of removal of by-products and solvents. Chemical reactions occur at room temperature and this determines two main advantages: the saving of energy and the possibility to incorporate an organic and notoriously thermolabile [6] phase. A further advantage of sol–gel technique is that it is able to synthesize high purity glasses and ceramics, because the starting raw materials are synthetic and not extractive as for conventional glass. Moreover, materials prepared by sol–gel process have shown to be more bioactive than those with the same composition but prepared with different methods. In fact, the surface analysis of sol–gel materials and coatings has shown the presence of hydroxyl groups that can promote the nucleation of calcium and phosphate and, therefore, the osteointegration when those materials are implanted [7]. The presence of organic components modifies the morphology and physical properties of the sol–gel products [8,9]. A variety of organic polymers has been introduced into inorganic networks to obtain hybrid or composite materials with or without covalent bonds between the polymer and inorganic components, respectively. Catauro et al. prepared, by means of sol–gel methods, several hybrids for drug delivery or biomedical applications by using different oxides, including CaO and/or SiO2 [10–13], TiO2 [14–16], and ZrO2 [17–19], and different polymer, e.g. poly (ε-caprolactone) [13,17–26] and poly
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(ether-immide) [16]. In all the cases, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and for some supported also by solid-state NMR measurements gave circumstantial evidence of the formation of class I hybrids, characterized by the presence of hydrogen bonds among the H-acceptor groups of the polymer, and the OH-groups (H-donator) of the inorganic phase. The aim of the present preliminary paper is the sol–gel synthesis and characterization of novel hybrid materials for biomedical applications, consisting of an inorganic zirconia matrix where a polymer, polyethylene glycol (PEG), is entrapped. The component choice is related to previous researches. Several papers show the use of zirconia ceramics and glasses as biomaterials [17, 27,28]. PEG is known also to be a versatile, biocompatible polymer [29,30] mainly used in polymer-based materials for drug delivery [31]. PEG-derived products have been investigated also as biomaterials for implants for orthopedic and dental applications [32], e.g. Kawakami et al. [33] showed that polyethylene glycol could be safely used as a base component of biomaterials for internal medical use, such as in root canal filling material. The obtained materials were characterized by means of several instrumental technique (FT-IR, XRD and SEM) and their in vitro assessment of cytotoxicity was carried out by means of MTT assay on NIH3T3 murine fibroblast cell line, one of the recommended and appropriate step for the biological evaluation of medical devices. Moreover, in order to evaluate if these materials can be used in dental or orthopedic applications (as implantable therapeutic systems or filling materials for bone or tooth repair), their in vitro bioactivity was investigated by soaking the samples in a Simulated Body Fluid (SBF) and observing by SEM microscopy the hydroxyapatite formation on the surface [34]. Indeed, as reported in the literature [35], the essential condition for glasses and glasses–ceramics to bond to living bone is the formation of a bone-like apatite layer on the surfaces.
Fig. 2. ZrO2/PEG after drying in air.
zirconium propoxide, Zr(OC3H7)4 (Sigma-Aldrich), as precursor of the inorganic matrix, polyethylene glycol (PEG, Mw = 400, SigmaAldrich) as organic component, and ethanol as solvent. Acetylacetone was also added to control the hydrolytic activity of zirconium alkoxide. In fact, it is a chelating agent able to form complexes, with monomeric alkoxides, stable towards hydrolysis reactions [36,37]. Fig. 1 shows the flow chart of hybrid synthesis by the sol–gel method. After gelation the gels were air-dried at 50 °C for 24 h to remove the residual solvent and were obtained transparent and glassy samples (Fig. 2).
2. Materials and methods 2.2. Materials characterization 2.1. Sol–gel synthesis The hybrids inorganic/organic materials with ZrO2/PEG molar ratio 6, 12, 24 and 50 were prepared by means of sol–gel process using
The presence of hydrogen bonds between organic and inorganic components of the hybrid materials was ascertained by Fourier transform infrared spectroscopy (FT-IR). Transmittance spectra were recorded in the 400–4000 cm−1 region using a Prestige 21 (Shimadzu, Japan) system, equipped with a DTGS KBr (Deuterated Tryglycine Sulphate with potassium bromide windows) detector, with resolution of 2 cm−1 (45 scans). KBr pelletized disks containing 2 mg of sample and 198 mg of KBr were made. The FTIR spectra were elaborated by Prestige software (IR solution). The nature of ZrO2 gel and ZrO2/PEG hybrid materials were ascertained by X-ray diffraction (XRD) analysis using a Philips diffractometer. Powder samples were scanned from 2Θ = 5° to 60° using CuKα radiation. The microstructure of the synthesized gels has been studied by scanning electron microscope (SEM) (Quanta 200, FEI, The Netherlands). 2.3. Study in vitro bioactivity In order to study their bioactivity, samples of the studied hybrid materials were soaked in SBF, a solution with ion concentrations nearly
Table 1 Prepared material gelification time.
Fig. 1. Flow chart of ZrO2/PEG gel synthesis.
Hybrid system
Gelification time
ZrO2 ZrO2:PEG ZrO2:PEG ZrO2:PEG ZrO2:PEG
6 days 6 days 7 days 8 days 8 days
= = = =
50 24 12 6
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and Energy Dispersive X-ray (EDX) (EDAX, USA) analysis. Taking into account that the ratio of the exposed surface to the volume solution influences the reaction, a constant ratio of 10 mm2 mL−1 of solution, was respected as in reference [39]. 2.4. Cell culture and cytotoxicity assessment
Fig. 3. Comparison between FTIR spectra of : a) ZrO2, b) ZrO2/PEG 6, c) ZrO2/PEG 12, d) ZrO2/PEG 24, e) ZrO2/PEG 50, f) PEG.
equal to those in human blood plasma [38]: Na+ 142.0, K+ 5.0, Ca2+ 2.5, 2− Mg2 + 1.5, Cl− 147.8, HCO− 1.0, SO24 − 0.5 mM. During 3 4.2, HPO4 soaking the temperature was kept fixed at 37 °C. The ability to form an apatite layer was studied by submitting reacted samples to SEM
Cytotoxicity was measured through MTT [3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] cell growth inhibition assay (indirect test) using NIH-3T3 murine fibroblast cell line. Investigated extracts were obtained from disks of the studied materials, which were previously immersed for 24 h in 3.5 mL of a complete culture medium at 37 °C under continuous stirring. The cell line, purchased from ATCC (American Type Culture Collection), was grown in RPMI 1640 high glucose medium supplemented with 10% fetal bovine serum, 50.0 U/mL penicillin, and 100.0 μg/mL streptomycin, at 37 °C in a humidified atmosphere containing 5% CO2. NIH-3T3 cell line was seeded in 96-multiwell plates at a density of 1.0 × 104 cells/well. After 24 h of incubation, cells were treated with 200 μL culture medium solutions containing 100 μL of extracts from ZrO2, and ZrO2/PEG (molar ratio 6, 12, 24, and 50) hybrid sol–gel materials. At 24, 48, and 72 h of incubation, cells were treated with 150 μL of MTT (0.50 mg/mL), dissolved in the culture medium, for 1 h at 37 °C in a 5% CO2 humidified atmosphere. The MTT solution was then removed and 100 μL of DMSO were added to dissolve the formazan originated. Finally, the absorbance at 570 nm of each well was determined by
Table 2 FTIR peak interpretation. Absorption bands ZrO2 matrix peaks
PEG peaks
−1
3417 cm 1585 and 1377 cm−1 1529 and 1280 cm−1 1425 cm−1 931 cm−1 654 cm−1 460 cm−1 422 cm−1 2930 cm−1 2849 cm−1 1454 cm−1 1250 cm−1 1104 cm−1 948 cm−1
Fig. 4. XRD of ZrO2 (A) and ZrO2/PEG (B).
Interpretation –OH vibrations of H-bonded H2O and Zr-OH AcAc bidentate binding C_O vibrations C\C vibrations Methyl C\H symmetric bending AcAc C\C\H bending mixed with C\C stretching Zr\OH Zr–O–Zr stretching Zr\OAcAc vibrations Methylene C\H symmetric stretching Methylene C\H asymmetric stretching Methylene C\H bending Alcohols C\O stretching Ethereal C\O\C stretching PEG C\C stretching
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using a Tecan SpectraFluor fluorescence and absorbance reader. Cell viability was expressed as percentage of mitochondrial redox activity of the cells treated with the extracts compared to an untreated control [40]. Tests were carried out performing twelve replicate (n = 12) measurements for three samples of each extract (in total: 12 × 3 measurements).
3. Results and discussion Gelation is the result of hydrolysis and condensation reactions according to the following reactions: ZrðOC3 H7 Þ4 þ nH2 O⇒ZrðOC3 H7 Þ4−n ðOHÞn þ nC3 H7 OH
Fig. 5. SEM micrograph of samples surface: A) ZrO2, B) ZrO2/PEG 6, C) ZrO2/PEG 12, D) ZrO2/PEG 24, E) ZrO2/PEG 50.
ð1Þ
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−ZrOH þ CH3 O−Zr−⇒ ≡ Zr−O−Zr ≡ þ C3 H7 OH
ð2Þ
≡ Zr−OH þ OH−Zr ≡ ⇒ ≡ Zr−O−Zr ≡ þ H2 O
ð3Þ
The reaction mechanism is generally accepted to proceed through a second order nucleophic substitution [41]. Acetylacetone controls the kinetics of those reactions (hydrolysis of monomeric alkoxides and then polymerization of hydrolysis products) behaving as chelator and forming stable towards hydrolysis complexes with zirconium propoxide [36,37, 42]. Gelification times of the hybrid systems are reported in Table 1. 3.1. Hybrid material characterization Fig. 3 shows the FT-IR spectra of ZrO2 (a), PEG/ZrO2 with molar ratio 6, 12, 24 and 50 (b, c, d, e) and PEG (f). These gels show the characteristic peaks of PEG and ZrO2 (Table 2). In the ZrO2 spectrum (Fig. 3(a)) all typical peaks of AcAc-containing zirconia sol–gel materials [43] are present. The broad intense band at 3417 cm−1 is due to vibrations of –OH groups which are hydrogenbonded with H2O and Zr-OH groups. The bands observed at 1585 and 1377 cm−1 are assigned to C_O vibrations of the AcAc bidentate binding. The bands at 1529 and 1280 cm−1 are attributed to C\C vibrations, the peak at 1425 cm−1 is due to methyl C\H symmetric bending [44], whereas the bands at 1186, 1026 and 931 cm− 1 are assigned to the C\C\H bending mixed with stretching C\C vibrations of AcAc [43]. The bands placed at 654 and 460 cm−1 are due to Zr\OH and Zr\O\Zr stretching respectively [45], whereas the peak at 422 cm− 1 to Zr\OAcAc vibrations [43]. In the PEG spectrum, the broad intense band in the region between 3200 and 3600 cm−1 is due to –OH stretching and indicates that in the pure polymer hydrogen bonds between –OH end groups and ether oxygen in the PEG chains occur [46]. Moreover, all typical PEG peaks, visible in the pure polymer spectra, were observed in the hybrid system spectra and their intensity increases with the amount of the polymer (Fig. 3 from curve b to e). In particular, the intensity of the methylene stretching bands increases together with methylene C\H bending of the polymer [47] at 1454 cm−1. The peaks due to alcohols C\O and ethereal C\O\C stretching at 1250 and 1104 cm−1 appear in curve c and also increase from c to e respectively. Moreover, the sharp AcAc C\C\H bend at 931 cm−1 was replaced with a little broader peak at 948 cm−1 attributed to PEG C\C stretching. At the same time the peaks in the regions 1520–1250 cm− 1, assigned to the AcAc bidentate binding, and 900–400 cm−1, typical of
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zirconia matrix, change shape. The first alteration is probably due to new interaction between AcAc and the polymer while the second phenomenon, which is particularly evident for the band assigned to Zr\OH stretching at 654 cm−1, together with the simultaneous change of broad band at 3417 cm−1, suggests the formation of new hydrogen bonds probably between those –OH groups and both the ethereal oxygen atoms (H-bond acceptors) and alcohol groups (H-bond acceptors and donator) of the PEG chains. The nature and the microstructure of the ZrO2/PEG hybrid materials have been studied by X-ray diffraction (XRD) and SEM. The diffractograms in Fig. 4 shows that ZrO2 (panel A) and ZrO2/PEG (panel B) gels exhibit broad humps characteristic of amorphous materials. The diffractograms of all systems are not shown because they are similar. SEM micrographs of a ZrO2 gel sample and of ZrO2/PEG gel samples are shown in Fig. 5 and no appreciable difference between the morphology of the five amorphous materials can be observed. Moreover, the two phases of which the hybrid consists (organic and inorganic) are indistinguishable confirming that the synthesized materials are hybrid. 3.2. Bioactivity test The evaluation of the apatite deposition on material surface after soaking in SBF, of its morphology and the qualitative elemental analysis were carried out by SEM/EDX microscopy observations. The formation of apatite on the materials after soaking in SBF can be explained by the presence of Zr-OH groups on the surface of the materials. These groups, combined with the Ca2+ ions present in the fluid, originate the increase of positive charge on the surface. The Ca2+ ions combine with the negative charge of the phosphate ions to form amorphous phosphate, which spontaneously transforms into hydroxyapatite [Ca10(PO4)6(OH)2]. After 21 days of incubation, all ZrO2/PEG systems show a deposition of a similar amount of apatite on their surface. Fig. 6 (A and B) shows crystals of apatite on the surface of the samples after 21 days of soaking in SBF and panel C illustrates EDX analysis of crystals; the ratio between the atomic content of Ca and P is 1:6 which agrees with the chemical formula of hydroxyapatite. 3.3. Cytotoxicity of ZrO2/PEG hybrid materials NIH-3T3 murine fibroblast cell line was treated, for three different exposure times (24, 48, and 72 h), with extracts obtained by placing, for 24 h, disks of the investigated ZrO2/PEG hybrid materials, in a complete culture medium. In order to assess the influence of the extracts of
Fig. 6. SEM micrographs of samples after soak in SBF for 21 days. Panels A–B: hydroxyapatite crystal. Panel C: EDX analysis.
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the new sol–gel materials on mitochondria and cell proliferation, MTT cytotoxicity assay was performed. The quantitative measurement of extracellular reduction of the yellow colored water soluble tetrazolium dye to insoluble formazan crystals allowed to state that extracts obtained by the different ZrO2/PEG samples affect positively cell viability and proliferation of tested cells (Fig. 7). In fact, it was observed that all the samples tested were able to induce an increase in cell viability during the considered exposure period compared to the inorganic material when it was tested in the non-hybrid form. The presence of different amounts of polyethylene glycol favored the preparation of biomaterials with improved biocompatibility. Detected data allowed to observe that the effects were dose- and time-dependent. Recently, it was reported that dental ceramics are not equivalent in their in vitro biologic effect, even with the same class of material and most ceramics cause only mild in vitro suppression of cell functions to levels that would be acceptable on the basis of standards used to evaluate alloys and composites (b 25% suppression of dehydrogenases activity) [48]. Thus, our findings are in accordance with the development of biomaterials free from toxicity and potentially useful as bioceramics in dental biomedicine. 4. Conclusions Zirconia/polyethylene glycol (ZrO2/PEG), prepared via sol–gel process, was found to be an organic/inorganic hybrid material.
Fig. 7. ZrO2, ZrO2/PEG 6, ZrO2/PEG 12, ZrO2/PEG 24, and ZrO2/PEG 50 cell viability (CV, %) towards NIH 3T3 cell line at 24 h, 48 h, and 72 h exposure times. Values, reported as percentage vs. an untreated control, are the mean ± SD of measurements carried out on 3 samples (n = 3) analyzed twelve times.
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