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Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating
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Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating Amirsalar Khandan a, Majid Abdellahi b,∗, Neriman Ozada a, Hamid Ghayour b a b
Mechanical Engineering department, Eastern Mediterranean University, North Cyprus, Gazima˘gusa, TRNC, Mersin 10 Turkey Advanced Materials Research Center, Faculty of Materials Engineering, Najafabad branch, Islamic Azad University, Najafabad, Isfahan, Iran
a r t i c l e
i n f o
Article history: Received 12 April 2015 Revised 29 September 2015 Accepted 5 October 2015 Available online xxx Keywords: Nanocomposite Coating Electrophoretic deposition Micro-hardness Wettability Apatite formation
a b s t r a c t The aim of the present study is to prepare the bovine hydroxyapatite-diopside (bHA-Di) nanocomposite coating using electrophoretic deposition (EPD) technique and then investigation of the bioactivity, wettability and hardness of the produced coating samples at different percentage of Di. For this purpose the HA powders is prepared from the bovine bones by a simple new method and then Di powders that have already synthesized by the use of mechanical milling method, is injected into the HA matrix. At the end a suspension of the bHA-Di nanocomposite is created and coated on the titanium (Ti) implants by the EPD technique. The sintered nanocomposite coating are then sintered and analysed. X-ray diffraction (XRD), Brunauer–Emmett– Teller (BET), scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and differential scanning calorimetric (DSC), energy dispersive spectroscopy (EDS) and inductive coupled plasma atomic emission spectroscopy (ICP-AES) are used as instruments for gathering and analysing the experimental results. According to the results obtained, the produced composite coating with 30 wt. % Di has favourable bioactivity, good wettability and high hardness. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Metallic materials such as stainless steels [1], cobalt alloys [2], titanium and titanium alloys [3] are most commonly used for load bearing implants and internal fixation devices. One of the advantages of titanium (Ti) based implants versus other types of implants is the formation of a fibrous tissue layer between the implant and surrounding tissue [4]. Besides, these implants have superior mechanical properties, chemical stability, and biocompatibility under in vivo conditions [5–8]. However, titanium is a bioinert material and the absence of bone bonding usually translates by the need to retrieve implants after a period of 15–20 years [9,10]. Alongside the other methods proposed [11], typically, a strategy to impart bioactivity to prosthetic implants is to coat their surface with bioactive materials such as hydroxyapatite (HA), a form of calcium phosphate that composes most of the bone mineral phase. A variety of surface coating methodologies such as ion beam assisted deposition, plasma spray deposition, electrophoretic deposition (EPD), pulsed laser, physical vapour deposition, micro-arc oxidation, magnetron sputtering, sol-gel derived coatings, etc. are being currently employed to deposit Ca–P on metallic implants [12].
∗
Corresponding author. Tel.: +989132894596; fax: +9835843046. E-mail address: [email protected], [email protected] (M. Abdellahi).
Unfortunately, the application of HA as a bioactive coating is limited due to its brittleness, high cost, high dissolution and difficult synthesising method [13]. Despite the poor mechanical properties of HA, its unique biological properties lead researchers think about work on improving its properties rather than completely replacing it with other biomaterials. Investigations on compositing silicate phases with calcium phosphate phases like HA, are increasingly growing and the obtained results have shown a significant improvement in mechanical properties of the final product as well as apatite forming abilities in simulated body fluid (SBF) [13,14]. Several novel calcium silicate-based ceramics like apatite/wollastonite [13], wollastonite [14], akermanite [15], baghdadite [16], zeolites [17], forsterite [18,19], and hardystonite [20] have been discovered to bond with the bone and teeth. Di (diopside - CaMgSiO6 ) as a silicate phase could be a material of attention because of the excellent mechanical and biological properties in comparison with pure HA [21]. In our recent work [21], the deposition of nanostructured HA-Di composite coatings on the surface of Ti based implants was investigated. The effect of high-energy ball milling parameters and subsequent thermal treatment on the structural and morphological features of the bHA-Di Nano composite was discussed in order to propose suitable conditions for the minimum surface roughness. Moreover the effects of EPD voltage, Di content and process temperature on the surface roughness was also studied. Furthermore, the chemical stability of the coating samples was also assessed.
http://dx.doi.org/10.1016/j.jtice.2015.10.004 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004
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In this work, HA-Di as a new silicate biocomposite was deposited on titanium plates by EPD technique using isopropanol as the suspending medium. Besides, in vitro bioactivity behaviour as well as bone-like apatite formation were performed by immersing the produced samples in SBF at various times. The possible coating mechanisms for the produced composite were also discussed. The substrate with composite coating was also tested for micro-hardness properties and the wettability of nanocomposite of the coating samples was also assessed. Results showed that the produced composite coating with 30 wt. % has favourable bioactivity, good wettability and high hardness. In the case of bioactivity results showed an optimum-constant pH of 7.6 (near the blood pH) after a long time (here 21 days). In the case of hardness, this work reported fabrication of a new coating with a microhardness range of 2–3 (GPa) and finally in the case of wettability the results derived from the experimental studies showed a minimum values of 35–40° for the contact angle which is suitable for biomaterials applications especially orthopaedic coating. 2. Materials and methods 2.1. Sample preparation The metal specimen’s commercially available titanium (Ti) was selected as substrate, since it is commonly used for biomedical applications. A surgical titanium alloy (Isfahan steel, IR) was cut into 10 × 10 × 2 mm3 dimensions, followed respectively by the polishing, etching in acids (40% HF, 10% HNO3 , and 50% H2 O), cleaning in acetone for 30 min and drying in the oven at temperature 110 °C for 2 min to get uniform surface roughness. According to our previous study [21], images which were obtained from the 2D AFM analysis of the NHA-Di coating after its deposition on Ti alloy specimen surface, indicated that the “Roughen” diagram fluctuates between the range of 0.03 and 0.01 (μm) under the conditions that the Di content reaches 30 wt. %. The visual observation in our previous work [21] indicated that the sample washed in acid had better surface bonding with the nanocomposite. 2.2. Di preparation A high energy planetary mill with a ball to powder weight ratio (BPR) of 10, and a rotational speed of 600 rpm was also used in order to produce Di Nano-powders. Talc (Merck 99% purity), Calcite (Merck 99% purity) and Silica (Merck 99% purity) at the stoichiometric conditions were used as starting materials. The sampling was performed every 2 h and the obtained samples of 2, 4, 8 and 10 h were analysed by the XRD method. The obtained nanopowder sample was then sintered at 1350 °C for 10 h. 2.3. Bovine hydroxyapatite preparation For preparing bovine hydroxyapatite (bHA) powder, bovine bones were first boiled for 2 h to remove the attached flesh and fat and then were dried at 110 °C for 2 h to eliminate their moisture. To prevent blackening with soot during heating, bones were cut into small pieces with 10 mm thickness, and heated at 550 °C (bone ash) for 2 h in air to evaporate organic substances. The resulting black bone ash was heated for 3 h at 850 °C. This synthesis process is called thermal decomposition of bone resource to fabricate bHA. The bHA powders obtained were again ball milled for 0.5 h to homogenize the product. 2.4. Fabrication of composite A bHA-Di Nano-composite powder with different percentages of Di (10 wt. %, 20 wt. % and 30 wt. %), was prepared via ball milling
in which alcohol medium was used to obtain a homogeneous mixture. For this purpose, a two cup planetary ball mill of Retch Company and stainless steel balls with a distribution of 20, 15 and 10 mm were used. The milling time was set to 10 h. 2.5. Coating technique Electrophoretic deposition (EPD) involves the deposition of charged powder particles suspended in a liquid medium onto a conductive substrate of opposite charge by the application of a DC electric filed [18–19,22,23]. The EPD technique was carried out at room temperature (37 ± 1 °C). Suspensions of the bHA-Di nanocomposite at a total concentration of 10 g solid/100 mL were prepared in isopropanol, treated in an ultrasonic bath for 30 min and finally stirred with a magnetic stirrer for 120 min to produce samples with excellent dispersion of the particles. Deposition was carried out on substrates of 10 mm × 10 mm × 2 mm. Ti sample and graphite electrodes were used as the anode and cathode, respectively. According to our previous work [21], the optimum applied DC electric field (V/l) and deposition time for EPD process were founded to be 50 V for 10 min. The sintering of the nanocomposite coatings after EPD process was carried out at 850 °C for 2 h in a vacuum furnace (10−5 Torr) with heating rate 10 °C/min. The thicknesses of the coatings were measured with SEM technique, and only coatings with uniform characteristics were selected for further bioactivity, micro-hardness and wettability studies. 2.6. Bioactivity evaluation in the SBF Along the margins in the definition of “bioactivity” [24], the term “bioactivity” was coined to refer to those materials that can develop a direct, adherent, and strong bonding with the bone tissue. To evaluate the bioactivity of the materials, it has been proposed that materials that form an appetite on their surfaces in the SBF also can form the apatite in a living body and can bond to bone through the apatite layer. In other words, the apatite-forming ability in the SBF is a measure of in vivo bioactivity [25]. A cellular SBF was used for in vitro experiments. The SBF solution was prepared according to the procedure described by Kokubo and Takadama. Ion concentrations of SBF are similar to those in human blood plasma (Table 1). Coated samples were soaked in the cell SBF solution (pH 7.4) at 37 °C for 1, 7, 14 and 21 days at a solid/liquid ratio of 10 mg/mL, without refreshing the soaking medium. 2.7. Wettability evaluation Contact angle analysis was carried out by sessile drop of SBF using optical measurements (Olympus MX-50, FTA200 dynamic contact angle analyser, EMU, Turkey) CCD camera. The wettability measurement carried out at 20 ± 1 °C at humidity of 30 ± 5% RH. Gradual modification of wettability of the HA performed by a low-energy
Table 1 Comparison of nominal ion concentrations of SBF and human blood plasma. Ion +
Na K+ Mg2+ Ca2+ Cl– HCO3 – HPO4 2 – SO4 2 – pH
SBF [m.mol]
Human plasma [m.mol]
142.0 5.0 1.5 2.5 147.8 4.2 1.0 0.5 7.4
142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5 7.2–7.4
Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004
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electron beam irradiation, for different exposition times for initial time t = 400, 600, 800, 1000, and 1200 s. The theoretical contact angle was calculated as the following equation:
cos(θ ) = (γSV − γSL )/γLV
(1)
In which γSV , γSL and γLV are respectively interfacial surface tensions of solid–gas, solid–liquid and V liquid–gas. 2.8. Micro-hardness evaluation Micro-hardness examination was performed by using a microhardness tester (Mechanical department of Eastern Mediterranean University, EMU, and Turkey). The indentations on polished surfaces were generated by the Vickers micro-hardness tester with a diamond pyramid indenter, at a load of 30 mN and a loading time of 30 s. The differences in micro-hardness of the coats were determined with the differences in the depth obtained at the applied load. 2.9. Coatings characterization Phase structure analysis was carried out by X-ray diffraction (XRD) tool (Philips X’Pert-MPD diffractometer with Cu Kα radiation (λ1 = 0.15418 nm)) over the 2θ range of 15–55°, with generator settings 40 kV, 35 mA. The modified Scherrer equation can provide the advantage of decreasing the sum of absolute values of errors, (± ln β )2 , and producing a single line through the points to give a single value of intercept ln Kλ/L. The ultimate modified Scherrer equation is shown in Eq. 2.
β=
1 kλ kλ + ln ⇒ ln β = ln L cos θ L cos θ
6000 S(BET) × ρ
2.10. Inductive coupled plasma atomic emission spectroscopy (ICP-AES) analysis The concentrations of Ca, Si ions in SBF after soaking are tested using inductive coupled plasma atomic emission spectroscopy (ICPAES; Zaies 110394c). 3. Results and discussion Fig. 1 indicates the variation of XRD peaks for synthesizing Di nanopowder at three different times (4, 8, and 10 h) of milling process. As can be seen the Di nanopowder has been synthesized after 10 h milling. It is observable from Fig. 1 that the forsterite as an impurity has been formed after 8 h of milling which fully disappeared by the increase of milling time up to 10 h. The linear regression plot is obtained as y = –1.2106x – 5.4617 for Di powders. This is equivalent to ln β = ln (1/Cosϴ) + ln (kλ/L). From this line, the intercept is –5.225 and e−5.225 = Kλ/L and L = 30–40 nm. Therefore, Di crystalline size average is 30−40 nm. As can be seen in the Fig. 2, after sintering Di nanopowders at 10 h of milling, the average crystallite size is 20– 30 (nm) which almost confirm the modified Scherrer equation. The size distribution of particles was determined by PSA graph (Fig. 3). According to the average grain size of the Di (TEM image) one
(2)
Scanning electron microscopy (SEM) analysis evaluations were performed using a Philips XL30 (Eindhoven, The Netherlands) to monitor the morphology of the Di powder, and nanocomposite coating soaked in SBF to evaluate bioactivity and apatite formation and/or dissolution. All the samples were coated with gold (Au) using a high vacuum and 40 kV accelerates voltage. The Ca and P ions contents were measured from three spots, and the average was measured. The Ca/P ratio was determined using energy-dispersive X-ray spectroscopy (EDS) microanalysis (FEI Quanta 200 ESEM equipped with an EDS device). Transmission electron microscopy (TEM) technique (Philips CM 200: Eindhoven, The Netherlands) was utilized to evaluate the shape and size of prepared Di powder after sintering process. Thermogravimetric analysis (TGA) is carried out by increasing the temperature slowly and plotting weight against temperature. After the data is obtained, curve smoothing and other operations may be done. Observations of the TGA (Materials Department, Tehran University, Selb, Germany) were done on dried samples using an alumina crucible in air and a heating ramp rate of 10 °C/min, from 30 to 1100 °C. In this study TGA was conducted on Di to assess its water loss temperature. The PSA technique was used to recognize geometric shape of Di nanoparticles. During PSA analysis the temperature was 19.9 °C, duration used 70 s with ethanol as a dispersant [viscosity (cp) = 1.2000)]. The specific surface area of the prepared Di powder was calculated from the N2 gas adsorption isotherms using the single point BET technique. The particle size, D, is given by Eq. (3),
D=
Fig. 1. XRD patterns of the starting powders for producing Di at different milling time.
Fig. 2. TEM micrograph of Di powder milled for 10 h and sintered at 1350 °C for 10 h.
(3)
The porosity of composites was measured by Archimedes method according to the following equation:
Porosity =
( w2 − w1 ) × 100% ( w2 − w3 )
3
(4)
where W1 is the weight of sample in air, W2 is the weight of sample with water, and W3 is the weight of samples suspended in water.
Fig. 3. PSA graph of Di powder milled for 10 h and sintered at 1350 °C for 10 h.
Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004
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Fig. 4. TGA and DSC curves of synthesized bHA and Di powders.
Fig. 5. SEM micrograph of Di powders prepared by mechanical milling for (a) 4 h, (b) 8 h, (c) 10 h, and (d) 10 h followed by sintering at 1350 °C for 10 h.
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A. Khandan et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2015) 1–9 Table 2 BET-surface area, pore volume, and pore size of the Di samples milled for 4, 8 and 10 h. Sample
BET surface are (m2 /g)
Pore vol. (cm3 /g)
Pore size (nm)
Di 4 h (a) Di 8 h (b) Di 10 h (c) 10 h Sintered (d)
55 49 17 14
0.26 0.19 0.15 0.06
18 17.2 21 26
can say that particles are semi-spheroid. The PSA graph shows that average grain size is about 40 nm. The BET based results also showed that the average grain size is lower than 50 nm. For BET calculations (Eq. 3), the measured value of specific surface area by BET analysis for Di powder was considered 50 m2 /g, ρDi = 3.4 (g/cm3 ) so D = 35 nm (Table 2). Fig. 4 shows the thermal decomposition curve of bHA and Di powders. For bHA (Fig. 4a), the TG profile indicates three different weight losses, related to three processes caused by the temperature increase from 50 °C to 1100 °C. First drop in the weight loss is seen at 50– 150 °C which is due to the evaporation (weight loss, W.L) of water,
5
then the second W.L that occurs between 200 and 450 °C, followed by the last W.L in the range 500–900 °C which is due to the combustion of the organic materials. Fig. 4b shows the TG–DSC curves for the prepared Di powder synthesized by the mechanical milling. It seems that an exothermic peak observed at around 600 °C is related to the crystallization. Fig. 5 shows the morphology of the obtained Di powders at various milling time. The morphology of the prepared powders shows that each of the sample is composed of several agglomerates with an irregular shape. As can be seen with increasing milling time, the agglomeration degree of powders has decreased (Fig. 5a–c). As it is shown in Fig. 5d the glassy surface has formed on the samples sintered at 1350 °C for 10 h. Several inter-particle porosity have created in the Di powder at 1350 °C which help to the formation of apatite layers during the biological activity of the samples soaked in SBF. Fig. 6(a–d) respectively show the SEM micrographs of the surface of the porous bHA-Di composite coated on Ti substrates after EPD process and subsequent SBF soaking for 21 days. As can be seen the amount of the apatite layers formation increases with increasing the amount of Di percentage. Generally, these apatite formations decrease the corrosion rate of the substrate and also increase the in vitro bioactivity of the implants. More powder agglomerations may lead to
Fig. 6. SEM photograph of composite coatings at different percentage (a) 0, (b) 10, (c) 20 and, (d) 30 of Di after immersion in the SBF solution for 21 days.
Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004
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Fig. 7. EDS micrographs of composite coatings at different percentage (a) 0, (b) 10, (c) 20 and, (d) 30 of Di after immersion in the SBF solution for 21 days.
the formation of the hard tissue that surrounds the implant, and is beneficial for the construction and formation of osseous tissue [26]. In addition, the Di bio-layer, with its porous microstructure obtains considerable sites for apatite formation. In this case, Fathi et al. [27] indicated that the degradation rate of the Di powder is lower than HA powders and the bone-like-apatite formation is higher in the case of Di. It is clear that the porosity and roughness of the surfaces are advantageous for tissue growth and cells on the bone implants [28]. As can be seen in the SEM images the porosities increase with increasing Di contents which leads to more bioactivity and wettability properties. So, it is expected that HA-Di nanocomposite coatings have shown better bioactivity behaviour than single HA coatings. In order to recognize the composition of the white mineral biolayers formed on the surface of Ti, the samples were characterized by EDS (Fig. 7). The EDS patterns indicate that the intensity of Mg and Si peaks Mg/Si increases by increasing Di content. This means that the silicate phase has completely combined with the HA matrix to produce calcium-silicate phases as previously mentioned. It is also notable that the Ca/P ratio of the coating without Di is 3.13, increases up to 3.65 in the soaking by adding 30 wt. % diopside (Fig. 7(a–d)). It should be noted that the apatite layers quickly grow with high calcium and phosphate ions concentration. As a mechanism for the apatite formation on the surface of coats one can say that the silanol (Si–OH) groups are effective for creating heterogeneous nucleation. These groups with specific structures reveal negative charges, and help in apatite formation via
creation of an amorphous calcium compound (for example calcium silicate). The apatite formation mechanism is schematically presented in Fig. 8. The SBF soaking process leads to release of the sodium ions (Na+ ) from the sample via exchange with H3 O+ ions. The event leads to the formation of a Ti–OH layer which has a negative charge and can react with the Ca2+ ions with the positive charge through an electrostatic force. The process continues with the combination of the positively charged surface with negatively charged phosphate ions (PO4 3− ) to form amorphous calcium phosphate. Upon the formation of the apatite nuclei, they spontaneously grow on the surface by consuming the calcium and phosphate ions from the surrounding SBF solution [29]. The magnification of scaffold structure fabricated in the sample containing 30 wt. % Di is shown in Fig. 9. As can be seen the scaffolds exhibited high porosity (up to 80%) with open pores of less than 100–300 μm average diameters. The trend of increasing of apatite formation with increase in the Di powder amounts as well as the variation of pH is also observed with ICP-AES analysis in Fig. 10. The diagram based evaluations show that after first week there is an upward trend in the pH value apart from the sample with 0% Di. As can be seen in Fig. 10a the weakest pH jump exists for the sample with 20 wt. % Di while the increase in the pH value is strongest for the sample containing 30 wt. % Di. The slow increase of pH value of the solution for the samples with 30 wt. % Di during the first 7 days and 7 last days indicates a relatively slow chemical dissolution and an improvement in the bioactivity behaviour. Further observations show that when the percentage
Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004
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Fig. 8. Schematic presentations of the process of bone-like apatite formation in the SBF solution.
Table 3 Contact angles provided at different exposure times and Di content. Diopiside (%)
0 10 20 30
Time (s)/Theta (°) 400 (s)
600 (s)
800 (s)
1000 (s)
1200 (s)
100° 95° 90° 80°
80° 85° 85° 70°
75° 80° 70° 65°
70° 70° 60° 55°
65° 55° 45° 40°
content. The pH variations can be attributed to two things, first, ion replacement between Mg of the coats and H+ in the SBF which leads increase in pH of the SBF and second, fabrication of (Si–OH) groups fabricated in the surface of the coats. The chemical substitutions of soaked nanocomposite in the SBF shown in the following reaction,
Si − O − Mg2+ +H+ +OH− − → Si − OH− +Mg2+ +OH− (in
Fig. 9. SEM micrograph of the scaffold structure fabricated in the sample containing 30 wt. % Di.
of Di is 0 wt. %, the pH has its lowest value at the end of the first week (Fig. 10a); however, when the amount of Di reaches 30 wt.%, the pH value reaches 7.6 (near the pH of blood) after 2 weeks and remains constant. Other samples containing lower amount of Di show a periodic variation of pH which is unfavourable for the body. Fig. 10b shows that by increasing incubation time, the apatite generation increases. As mentioned above, an interchange between the Ca2+ ions and the H3 O+ occurs in the SBF, which may enhance the formation of Si–OH groups on the coated surface leading to the increase of nucleation sites. Also, it is obvious that, the formation of the apatite layer is due to the release of Ca2+ ions from the powder and also formation of Si–OH groups on the surface. The apatite formation analysis using EDS confirms that the tiny cauliflowers is composed of large amount of Ca, P, and Mg elements as it is shown in Fig. 7. The amount of Mg2+ and Ca2+ ions increases progressively in the SBF solution (see blue and red curve in Fig. 10b), while that of P ions are in a reverse trend (see green curve in Fig. 10b). It should be noted that the concentration of the Ca ions at the end of the second week in the SBF solution is dependent on the Si–OH nucleation sites. In other words, as these nucleation sites increase, the adsorption of calcium ions into these sites increases, so one can say that the concentration of the Ca ions in the SBF solution decreases at the end of first week for all samples except those of 30% Di. Mg, Ca and P are the most important factors for fabrication of CaP in SBF solution, and lead to more precipitation of apatite layer into SBF. Significant increase in bioactivity is due to more percentages of Di nanopowders because of high amount of Si and Mg
SBF ) (1)
By increasing soaking time, and increasing negative load on the samples surface, calcium ions in the SBF reaches the interface. These Ca+2 s are gathered between the coat and SBF. The attached Ca ions on the surface are a reason for increase in pH of the SBF. The surface hydrophilicity was studied by measuring contact angles with a sessile drop of SBF deposited on a sample surface. The wettability of a flat surface is expressed by the contact angle Ɵ between a liquid drop (here SBF) and a solid surface (here bHA-Di composite) due to intermolecular interactions described by Young’s equation (Eq. 1). Depending on the value of the contact angle, surface properties are determined as hydrophobic (Ɵ > 90°) or hydrophilic (Ɵ < 90°). The surface properties such as roughness, chemical composition, surface charge, and porosity play a significant role in the enhancing of wettability and bioactivity of biomedical implants. In addition, cell proliferation and biomimetic growth on biomedical implants were influenced by wettability values [30,31]. Fig. 11 shows the gradual modification of wettability of the bHA30 wt. % Di composite at different exposure times. In this study we reported the use of a low-energy electron irradiation to achieve tunable wettability of the bHA-30 wt. % Di composite in a wide range of contact angles, from 30° to 70°. As can be seen in the Fig. 6, by increasing the exposure time of HA-30 wt. % Di in the SBF solution from 400 s to 1200 s, the contact angle decreases. Table 3 shows a summary of the variations in the exposure time as well as contact angles at different amounts of Di. As can be seen in this figure the best results have been reported for the sample of 30 wt. % Di. Fig. 12 shows the load–displacement curves of prepared HA-Di coatings with different amounts of Di at a load of 30 mN. Established model was used to calculate quantitative hardness and elastic modulus values for such data. The differences in the depth obtained at the applied load represent the differences in the hardness of the
Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004
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pH
NHA
NHA10D
NHA20D
NHA30D
8 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7 6.9 0
3
6
9
12
15
18
21
Time (days)
a
128 116
Ion concentraon (%)
104 92 80 68 56 44 32 20 0
3
6
9
12
15
18
21
Time (days) Ca
Mg
P
b Fig. 10. The changes in (a) pH values versus immersion time and (b) calcium ions concentration in the SBF solution.
Fig. 11. The variations of angles of the SBF solution in contact with the composite coating with increasing exposure times.
materials. As clearly observed, with increasing Di content in the composite coating, the penetration depth of the indenter has decreased. As a result, among all of the samples, the sample with the maximum amount of Di and the sample without any Di content have respectively the maximum and the minimum amount of hardness. The measured values of hardness are in agreement with those reported by other researchers for HA coatings [32–35]. Table 4 gives the elastic modulus and hardness values of composite coating with different amounts of Di. It should be noted that all previous studies on the
Table 4 Micro-harness and elastic module values of the coating samples at different percentage of Di. Sample
Hardness (MPa)
Elastic modulus (GPa)
Load (mN)
HA HA10Di HA20Di HA30Di
2652 3225 3350 3390
45 65 90 95
30 30 30 30
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Fig. 12. The microhardness, load-depth plots of four types of composite coatings with different amounts of Di.
construction and properties of biomaterials, have not offered any statistical analysis of their results. According to the works presented by Davim et al. [36,37], statistical analysis can provide a detailed description of the final results. 4. Conclusion In the present study, bHA-Di nanocomposite was coated on the titanium plates by the EPD technique using isopropanol as the suspending medium. Besides, the assessment of in vitro bioactivity behaviour as well as apatite formation was done by immersing the produced samples in the SBF solution at various times. The possible coating mechanisms for the produced composite were also discussed. The substrate with composite coating was also tested for micro-hardness properties and the wettability of nanocomposite of the coating samples was also assessed. According to the results obtained, the bHA-Di nanocomposite coating at 30 wt. % of Di had an optimum bioactivity behaviour, good wettability conditions and high hardness property. The results gathered for the bioactivity experiment showed an optimum-constant pH of 7.6 (near the blood pH) after a long time (here 21 days), despite the fact that the other samples with lower amount of Di showed an oscillatory behaviour in pH variations. In the case of hardness, this work reported fabrication of a new coating with a micro-hardness range of 2–3 (GPa) and finally in the case of wettability the results derived from the experimental studies showed a minimum value of 35–40° for the contact angle which is suitable for biomaterials applications especially orthopaedic coating. Reference [1] Shih CC, Shih CM, Su YY, Chang MS, Lin SJ. Characterization of the thrombogenic potential of surface oxides on stainless steel for implant purposes. Applied Surf Sci 2003;219(3):347–62. [2] Ratner BD, Hoffman AS, Schoen FJ, Lemons JE. Biomaterials science: an introduction to materials in medicine. Academic press; 2004. [3] Black J. Does corrosion matter? J Bone Joint Surg 1988;70:517–20. [4] Mohammed MT, Khan ZA, Siddiquee AN. Surface modifications of titanium materials for developing corrosion behavior in human body environment: a review. Proc Mater Sci 2014;6:1610–16. [5] Saldana L, Vilaboa N, Vallés G, González-Cabrero J, Munuera L. Osteoblast response to thermally oxidized Ti-6Al-4V alloy. J Biomed Mater Res Part A 2005;73:97–107. [6] Martinesi M, Bruni S, Stio M, Treves C, Borgioli F. In vitro interaction between surface-treated Ti-6Al-4V titanium alloy and human peripheral blood mononuclear cells. J Biomed Mater Res Part A 2005;74:197–207. [7] Rivera-Denizard O, Diffoot-Carlo N, Navas V, Sundaram PA. Biocompatibility studies of human fetal osteoblast cells cultured on gamma titanium aluminide. J Mater Sci- Mater in Medic 2008;19:153–8.
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Bioactivity of calcium phosphate coatings prepared by electrodeposition in a modified simulated body fluid. Mater Lett 2006;60(21):2573–7. [26] Sun H, Wu C, Dai K, Chang J, Tang T. Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics. Biomaterials 2006;27:5651–7. [27] Razavi M, Fathi MH, Savabi O, Razavi SM, Hashemibeni B, Yazdimamaghani M, et al. Comparative study on the biodegradation and biocompatibility of silicate bioceramic coatings on biodegradable magnesium alloy as biodegradable biomaterial. Bullet Am Phys Soc 2014;59:201. [28] Wu C, Zreiqat H. Porous bioactive diopside (CaMgSi2 O6 ) ceramic microspheres for drug delivery. Acta Biomater 2010;6:820–9. [29] Liu X, Chu PK, Ding C. Surface modification of titanium, titanium alloys, and related materials for biomedical applications. Mater Sci Eng R 2004;47:49–121. [30] Aronov D, Rosenman G. 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Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating Amirsalar Khandan a, Majid Abdellahi b,∗, Neriman Ozada a, Hamid Ghayour b a b
Mechanical Engineering department, Eastern Mediterranean University, North Cyprus, Gazima˘gusa, TRNC, Mersin 10 Turkey Advanced Materials Research Center, Faculty of Materials Engineering, Najafabad branch, Islamic Azad University, Najafabad, Isfahan, Iran
a r t i c l e
i n f o
Article history: Received 12 April 2015 Revised 29 September 2015 Accepted 5 October 2015 Available online xxx Keywords: Nanocomposite Coating Electrophoretic deposition Micro-hardness Wettability Apatite formation
a b s t r a c t The aim of the present study is to prepare the bovine hydroxyapatite-diopside (bHA-Di) nanocomposite coating using electrophoretic deposition (EPD) technique and then investigation of the bioactivity, wettability and hardness of the produced coating samples at different percentage of Di. For this purpose the HA powders is prepared from the bovine bones by a simple new method and then Di powders that have already synthesized by the use of mechanical milling method, is injected into the HA matrix. At the end a suspension of the bHA-Di nanocomposite is created and coated on the titanium (Ti) implants by the EPD technique. The sintered nanocomposite coating are then sintered and analysed. X-ray diffraction (XRD), Brunauer–Emmett– Teller (BET), scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and differential scanning calorimetric (DSC), energy dispersive spectroscopy (EDS) and inductive coupled plasma atomic emission spectroscopy (ICP-AES) are used as instruments for gathering and analysing the experimental results. According to the results obtained, the produced composite coating with 30 wt. % Di has favourable bioactivity, good wettability and high hardness. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Metallic materials such as stainless steels [1], cobalt alloys [2], titanium and titanium alloys [3] are most commonly used for load bearing implants and internal fixation devices. One of the advantages of titanium (Ti) based implants versus other types of implants is the formation of a fibrous tissue layer between the implant and surrounding tissue [4]. Besides, these implants have superior mechanical properties, chemical stability, and biocompatibility under in vivo conditions [5–8]. However, titanium is a bioinert material and the absence of bone bonding usually translates by the need to retrieve implants after a period of 15–20 years [9,10]. Alongside the other methods proposed [11], typically, a strategy to impart bioactivity to prosthetic implants is to coat their surface with bioactive materials such as hydroxyapatite (HA), a form of calcium phosphate that composes most of the bone mineral phase. A variety of surface coating methodologies such as ion beam assisted deposition, plasma spray deposition, electrophoretic deposition (EPD), pulsed laser, physical vapour deposition, micro-arc oxidation, magnetron sputtering, sol-gel derived coatings, etc. are being currently employed to deposit Ca–P on metallic implants [12].
∗
Corresponding author. Tel.: +989132894596; fax: +9835843046. E-mail address: [email protected], [email protected] (M. Abdellahi).
Unfortunately, the application of HA as a bioactive coating is limited due to its brittleness, high cost, high dissolution and difficult synthesising method [13]. Despite the poor mechanical properties of HA, its unique biological properties lead researchers think about work on improving its properties rather than completely replacing it with other biomaterials. Investigations on compositing silicate phases with calcium phosphate phases like HA, are increasingly growing and the obtained results have shown a significant improvement in mechanical properties of the final product as well as apatite forming abilities in simulated body fluid (SBF) [13,14]. Several novel calcium silicate-based ceramics like apatite/wollastonite [13], wollastonite [14], akermanite [15], baghdadite [16], zeolites [17], forsterite [18,19], and hardystonite [20] have been discovered to bond with the bone and teeth. Di (diopside - CaMgSiO6 ) as a silicate phase could be a material of attention because of the excellent mechanical and biological properties in comparison with pure HA [21]. In our recent work [21], the deposition of nanostructured HA-Di composite coatings on the surface of Ti based implants was investigated. The effect of high-energy ball milling parameters and subsequent thermal treatment on the structural and morphological features of the bHA-Di Nano composite was discussed in order to propose suitable conditions for the minimum surface roughness. Moreover the effects of EPD voltage, Di content and process temperature on the surface roughness was also studied. Furthermore, the chemical stability of the coating samples was also assessed.
http://dx.doi.org/10.1016/j.jtice.2015.10.004 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004
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In this work, HA-Di as a new silicate biocomposite was deposited on titanium plates by EPD technique using isopropanol as the suspending medium. Besides, in vitro bioactivity behaviour as well as bone-like apatite formation were performed by immersing the produced samples in SBF at various times. The possible coating mechanisms for the produced composite were also discussed. The substrate with composite coating was also tested for micro-hardness properties and the wettability of nanocomposite of the coating samples was also assessed. Results showed that the produced composite coating with 30 wt. % has favourable bioactivity, good wettability and high hardness. In the case of bioactivity results showed an optimum-constant pH of 7.6 (near the blood pH) after a long time (here 21 days). In the case of hardness, this work reported fabrication of a new coating with a microhardness range of 2–3 (GPa) and finally in the case of wettability the results derived from the experimental studies showed a minimum values of 35–40° for the contact angle which is suitable for biomaterials applications especially orthopaedic coating. 2. Materials and methods 2.1. Sample preparation The metal specimen’s commercially available titanium (Ti) was selected as substrate, since it is commonly used for biomedical applications. A surgical titanium alloy (Isfahan steel, IR) was cut into 10 × 10 × 2 mm3 dimensions, followed respectively by the polishing, etching in acids (40% HF, 10% HNO3 , and 50% H2 O), cleaning in acetone for 30 min and drying in the oven at temperature 110 °C for 2 min to get uniform surface roughness. According to our previous study [21], images which were obtained from the 2D AFM analysis of the NHA-Di coating after its deposition on Ti alloy specimen surface, indicated that the “Roughen” diagram fluctuates between the range of 0.03 and 0.01 (μm) under the conditions that the Di content reaches 30 wt. %. The visual observation in our previous work [21] indicated that the sample washed in acid had better surface bonding with the nanocomposite. 2.2. Di preparation A high energy planetary mill with a ball to powder weight ratio (BPR) of 10, and a rotational speed of 600 rpm was also used in order to produce Di Nano-powders. Talc (Merck 99% purity), Calcite (Merck 99% purity) and Silica (Merck 99% purity) at the stoichiometric conditions were used as starting materials. The sampling was performed every 2 h and the obtained samples of 2, 4, 8 and 10 h were analysed by the XRD method. The obtained nanopowder sample was then sintered at 1350 °C for 10 h. 2.3. Bovine hydroxyapatite preparation For preparing bovine hydroxyapatite (bHA) powder, bovine bones were first boiled for 2 h to remove the attached flesh and fat and then were dried at 110 °C for 2 h to eliminate their moisture. To prevent blackening with soot during heating, bones were cut into small pieces with 10 mm thickness, and heated at 550 °C (bone ash) for 2 h in air to evaporate organic substances. The resulting black bone ash was heated for 3 h at 850 °C. This synthesis process is called thermal decomposition of bone resource to fabricate bHA. The bHA powders obtained were again ball milled for 0.5 h to homogenize the product. 2.4. Fabrication of composite A bHA-Di Nano-composite powder with different percentages of Di (10 wt. %, 20 wt. % and 30 wt. %), was prepared via ball milling
in which alcohol medium was used to obtain a homogeneous mixture. For this purpose, a two cup planetary ball mill of Retch Company and stainless steel balls with a distribution of 20, 15 and 10 mm were used. The milling time was set to 10 h. 2.5. Coating technique Electrophoretic deposition (EPD) involves the deposition of charged powder particles suspended in a liquid medium onto a conductive substrate of opposite charge by the application of a DC electric filed [18–19,22,23]. The EPD technique was carried out at room temperature (37 ± 1 °C). Suspensions of the bHA-Di nanocomposite at a total concentration of 10 g solid/100 mL were prepared in isopropanol, treated in an ultrasonic bath for 30 min and finally stirred with a magnetic stirrer for 120 min to produce samples with excellent dispersion of the particles. Deposition was carried out on substrates of 10 mm × 10 mm × 2 mm. Ti sample and graphite electrodes were used as the anode and cathode, respectively. According to our previous work [21], the optimum applied DC electric field (V/l) and deposition time for EPD process were founded to be 50 V for 10 min. The sintering of the nanocomposite coatings after EPD process was carried out at 850 °C for 2 h in a vacuum furnace (10−5 Torr) with heating rate 10 °C/min. The thicknesses of the coatings were measured with SEM technique, and only coatings with uniform characteristics were selected for further bioactivity, micro-hardness and wettability studies. 2.6. Bioactivity evaluation in the SBF Along the margins in the definition of “bioactivity” [24], the term “bioactivity” was coined to refer to those materials that can develop a direct, adherent, and strong bonding with the bone tissue. To evaluate the bioactivity of the materials, it has been proposed that materials that form an appetite on their surfaces in the SBF also can form the apatite in a living body and can bond to bone through the apatite layer. In other words, the apatite-forming ability in the SBF is a measure of in vivo bioactivity [25]. A cellular SBF was used for in vitro experiments. The SBF solution was prepared according to the procedure described by Kokubo and Takadama. Ion concentrations of SBF are similar to those in human blood plasma (Table 1). Coated samples were soaked in the cell SBF solution (pH 7.4) at 37 °C for 1, 7, 14 and 21 days at a solid/liquid ratio of 10 mg/mL, without refreshing the soaking medium. 2.7. Wettability evaluation Contact angle analysis was carried out by sessile drop of SBF using optical measurements (Olympus MX-50, FTA200 dynamic contact angle analyser, EMU, Turkey) CCD camera. The wettability measurement carried out at 20 ± 1 °C at humidity of 30 ± 5% RH. Gradual modification of wettability of the HA performed by a low-energy
Table 1 Comparison of nominal ion concentrations of SBF and human blood plasma. Ion +
Na K+ Mg2+ Ca2+ Cl– HCO3 – HPO4 2 – SO4 2 – pH
SBF [m.mol]
Human plasma [m.mol]
142.0 5.0 1.5 2.5 147.8 4.2 1.0 0.5 7.4
142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5 7.2–7.4
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electron beam irradiation, for different exposition times for initial time t = 400, 600, 800, 1000, and 1200 s. The theoretical contact angle was calculated as the following equation:
cos(θ ) = (γSV − γSL )/γLV
(1)
In which γSV , γSL and γLV are respectively interfacial surface tensions of solid–gas, solid–liquid and V liquid–gas. 2.8. Micro-hardness evaluation Micro-hardness examination was performed by using a microhardness tester (Mechanical department of Eastern Mediterranean University, EMU, and Turkey). The indentations on polished surfaces were generated by the Vickers micro-hardness tester with a diamond pyramid indenter, at a load of 30 mN and a loading time of 30 s. The differences in micro-hardness of the coats were determined with the differences in the depth obtained at the applied load. 2.9. Coatings characterization Phase structure analysis was carried out by X-ray diffraction (XRD) tool (Philips X’Pert-MPD diffractometer with Cu Kα radiation (λ1 = 0.15418 nm)) over the 2θ range of 15–55°, with generator settings 40 kV, 35 mA. The modified Scherrer equation can provide the advantage of decreasing the sum of absolute values of errors, (± ln β )2 , and producing a single line through the points to give a single value of intercept ln Kλ/L. The ultimate modified Scherrer equation is shown in Eq. 2.
β=
1 kλ kλ + ln ⇒ ln β = ln L cos θ L cos θ
6000 S(BET) × ρ
2.10. Inductive coupled plasma atomic emission spectroscopy (ICP-AES) analysis The concentrations of Ca, Si ions in SBF after soaking are tested using inductive coupled plasma atomic emission spectroscopy (ICPAES; Zaies 110394c). 3. Results and discussion Fig. 1 indicates the variation of XRD peaks for synthesizing Di nanopowder at three different times (4, 8, and 10 h) of milling process. As can be seen the Di nanopowder has been synthesized after 10 h milling. It is observable from Fig. 1 that the forsterite as an impurity has been formed after 8 h of milling which fully disappeared by the increase of milling time up to 10 h. The linear regression plot is obtained as y = –1.2106x – 5.4617 for Di powders. This is equivalent to ln β = ln (1/Cosϴ) + ln (kλ/L). From this line, the intercept is –5.225 and e−5.225 = Kλ/L and L = 30–40 nm. Therefore, Di crystalline size average is 30−40 nm. As can be seen in the Fig. 2, after sintering Di nanopowders at 10 h of milling, the average crystallite size is 20– 30 (nm) which almost confirm the modified Scherrer equation. The size distribution of particles was determined by PSA graph (Fig. 3). According to the average grain size of the Di (TEM image) one
(2)
Scanning electron microscopy (SEM) analysis evaluations were performed using a Philips XL30 (Eindhoven, The Netherlands) to monitor the morphology of the Di powder, and nanocomposite coating soaked in SBF to evaluate bioactivity and apatite formation and/or dissolution. All the samples were coated with gold (Au) using a high vacuum and 40 kV accelerates voltage. The Ca and P ions contents were measured from three spots, and the average was measured. The Ca/P ratio was determined using energy-dispersive X-ray spectroscopy (EDS) microanalysis (FEI Quanta 200 ESEM equipped with an EDS device). Transmission electron microscopy (TEM) technique (Philips CM 200: Eindhoven, The Netherlands) was utilized to evaluate the shape and size of prepared Di powder after sintering process. Thermogravimetric analysis (TGA) is carried out by increasing the temperature slowly and plotting weight against temperature. After the data is obtained, curve smoothing and other operations may be done. Observations of the TGA (Materials Department, Tehran University, Selb, Germany) were done on dried samples using an alumina crucible in air and a heating ramp rate of 10 °C/min, from 30 to 1100 °C. In this study TGA was conducted on Di to assess its water loss temperature. The PSA technique was used to recognize geometric shape of Di nanoparticles. During PSA analysis the temperature was 19.9 °C, duration used 70 s with ethanol as a dispersant [viscosity (cp) = 1.2000)]. The specific surface area of the prepared Di powder was calculated from the N2 gas adsorption isotherms using the single point BET technique. The particle size, D, is given by Eq. (3),
D=
Fig. 1. XRD patterns of the starting powders for producing Di at different milling time.
Fig. 2. TEM micrograph of Di powder milled for 10 h and sintered at 1350 °C for 10 h.
(3)
The porosity of composites was measured by Archimedes method according to the following equation:
Porosity =
( w2 − w1 ) × 100% ( w2 − w3 )
3
(4)
where W1 is the weight of sample in air, W2 is the weight of sample with water, and W3 is the weight of samples suspended in water.
Fig. 3. PSA graph of Di powder milled for 10 h and sintered at 1350 °C for 10 h.
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Fig. 4. TGA and DSC curves of synthesized bHA and Di powders.
Fig. 5. SEM micrograph of Di powders prepared by mechanical milling for (a) 4 h, (b) 8 h, (c) 10 h, and (d) 10 h followed by sintering at 1350 °C for 10 h.
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A. Khandan et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2015) 1–9 Table 2 BET-surface area, pore volume, and pore size of the Di samples milled for 4, 8 and 10 h. Sample
BET surface are (m2 /g)
Pore vol. (cm3 /g)
Pore size (nm)
Di 4 h (a) Di 8 h (b) Di 10 h (c) 10 h Sintered (d)
55 49 17 14
0.26 0.19 0.15 0.06
18 17.2 21 26
can say that particles are semi-spheroid. The PSA graph shows that average grain size is about 40 nm. The BET based results also showed that the average grain size is lower than 50 nm. For BET calculations (Eq. 3), the measured value of specific surface area by BET analysis for Di powder was considered 50 m2 /g, ρDi = 3.4 (g/cm3 ) so D = 35 nm (Table 2). Fig. 4 shows the thermal decomposition curve of bHA and Di powders. For bHA (Fig. 4a), the TG profile indicates three different weight losses, related to three processes caused by the temperature increase from 50 °C to 1100 °C. First drop in the weight loss is seen at 50– 150 °C which is due to the evaporation (weight loss, W.L) of water,
5
then the second W.L that occurs between 200 and 450 °C, followed by the last W.L in the range 500–900 °C which is due to the combustion of the organic materials. Fig. 4b shows the TG–DSC curves for the prepared Di powder synthesized by the mechanical milling. It seems that an exothermic peak observed at around 600 °C is related to the crystallization. Fig. 5 shows the morphology of the obtained Di powders at various milling time. The morphology of the prepared powders shows that each of the sample is composed of several agglomerates with an irregular shape. As can be seen with increasing milling time, the agglomeration degree of powders has decreased (Fig. 5a–c). As it is shown in Fig. 5d the glassy surface has formed on the samples sintered at 1350 °C for 10 h. Several inter-particle porosity have created in the Di powder at 1350 °C which help to the formation of apatite layers during the biological activity of the samples soaked in SBF. Fig. 6(a–d) respectively show the SEM micrographs of the surface of the porous bHA-Di composite coated on Ti substrates after EPD process and subsequent SBF soaking for 21 days. As can be seen the amount of the apatite layers formation increases with increasing the amount of Di percentage. Generally, these apatite formations decrease the corrosion rate of the substrate and also increase the in vitro bioactivity of the implants. More powder agglomerations may lead to
Fig. 6. SEM photograph of composite coatings at different percentage (a) 0, (b) 10, (c) 20 and, (d) 30 of Di after immersion in the SBF solution for 21 days.
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Fig. 7. EDS micrographs of composite coatings at different percentage (a) 0, (b) 10, (c) 20 and, (d) 30 of Di after immersion in the SBF solution for 21 days.
the formation of the hard tissue that surrounds the implant, and is beneficial for the construction and formation of osseous tissue [26]. In addition, the Di bio-layer, with its porous microstructure obtains considerable sites for apatite formation. In this case, Fathi et al. [27] indicated that the degradation rate of the Di powder is lower than HA powders and the bone-like-apatite formation is higher in the case of Di. It is clear that the porosity and roughness of the surfaces are advantageous for tissue growth and cells on the bone implants [28]. As can be seen in the SEM images the porosities increase with increasing Di contents which leads to more bioactivity and wettability properties. So, it is expected that HA-Di nanocomposite coatings have shown better bioactivity behaviour than single HA coatings. In order to recognize the composition of the white mineral biolayers formed on the surface of Ti, the samples were characterized by EDS (Fig. 7). The EDS patterns indicate that the intensity of Mg and Si peaks Mg/Si increases by increasing Di content. This means that the silicate phase has completely combined with the HA matrix to produce calcium-silicate phases as previously mentioned. It is also notable that the Ca/P ratio of the coating without Di is 3.13, increases up to 3.65 in the soaking by adding 30 wt. % diopside (Fig. 7(a–d)). It should be noted that the apatite layers quickly grow with high calcium and phosphate ions concentration. As a mechanism for the apatite formation on the surface of coats one can say that the silanol (Si–OH) groups are effective for creating heterogeneous nucleation. These groups with specific structures reveal negative charges, and help in apatite formation via
creation of an amorphous calcium compound (for example calcium silicate). The apatite formation mechanism is schematically presented in Fig. 8. The SBF soaking process leads to release of the sodium ions (Na+ ) from the sample via exchange with H3 O+ ions. The event leads to the formation of a Ti–OH layer which has a negative charge and can react with the Ca2+ ions with the positive charge through an electrostatic force. The process continues with the combination of the positively charged surface with negatively charged phosphate ions (PO4 3− ) to form amorphous calcium phosphate. Upon the formation of the apatite nuclei, they spontaneously grow on the surface by consuming the calcium and phosphate ions from the surrounding SBF solution [29]. The magnification of scaffold structure fabricated in the sample containing 30 wt. % Di is shown in Fig. 9. As can be seen the scaffolds exhibited high porosity (up to 80%) with open pores of less than 100–300 μm average diameters. The trend of increasing of apatite formation with increase in the Di powder amounts as well as the variation of pH is also observed with ICP-AES analysis in Fig. 10. The diagram based evaluations show that after first week there is an upward trend in the pH value apart from the sample with 0% Di. As can be seen in Fig. 10a the weakest pH jump exists for the sample with 20 wt. % Di while the increase in the pH value is strongest for the sample containing 30 wt. % Di. The slow increase of pH value of the solution for the samples with 30 wt. % Di during the first 7 days and 7 last days indicates a relatively slow chemical dissolution and an improvement in the bioactivity behaviour. Further observations show that when the percentage
Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004
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Fig. 8. Schematic presentations of the process of bone-like apatite formation in the SBF solution.
Table 3 Contact angles provided at different exposure times and Di content. Diopiside (%)
0 10 20 30
Time (s)/Theta (°) 400 (s)
600 (s)
800 (s)
1000 (s)
1200 (s)
100° 95° 90° 80°
80° 85° 85° 70°
75° 80° 70° 65°
70° 70° 60° 55°
65° 55° 45° 40°
content. The pH variations can be attributed to two things, first, ion replacement between Mg of the coats and H+ in the SBF which leads increase in pH of the SBF and second, fabrication of (Si–OH) groups fabricated in the surface of the coats. The chemical substitutions of soaked nanocomposite in the SBF shown in the following reaction,
Si − O − Mg2+ +H+ +OH− − → Si − OH− +Mg2+ +OH− (in
Fig. 9. SEM micrograph of the scaffold structure fabricated in the sample containing 30 wt. % Di.
of Di is 0 wt. %, the pH has its lowest value at the end of the first week (Fig. 10a); however, when the amount of Di reaches 30 wt.%, the pH value reaches 7.6 (near the pH of blood) after 2 weeks and remains constant. Other samples containing lower amount of Di show a periodic variation of pH which is unfavourable for the body. Fig. 10b shows that by increasing incubation time, the apatite generation increases. As mentioned above, an interchange between the Ca2+ ions and the H3 O+ occurs in the SBF, which may enhance the formation of Si–OH groups on the coated surface leading to the increase of nucleation sites. Also, it is obvious that, the formation of the apatite layer is due to the release of Ca2+ ions from the powder and also formation of Si–OH groups on the surface. The apatite formation analysis using EDS confirms that the tiny cauliflowers is composed of large amount of Ca, P, and Mg elements as it is shown in Fig. 7. The amount of Mg2+ and Ca2+ ions increases progressively in the SBF solution (see blue and red curve in Fig. 10b), while that of P ions are in a reverse trend (see green curve in Fig. 10b). It should be noted that the concentration of the Ca ions at the end of the second week in the SBF solution is dependent on the Si–OH nucleation sites. In other words, as these nucleation sites increase, the adsorption of calcium ions into these sites increases, so one can say that the concentration of the Ca ions in the SBF solution decreases at the end of first week for all samples except those of 30% Di. Mg, Ca and P are the most important factors for fabrication of CaP in SBF solution, and lead to more precipitation of apatite layer into SBF. Significant increase in bioactivity is due to more percentages of Di nanopowders because of high amount of Si and Mg
SBF ) (1)
By increasing soaking time, and increasing negative load on the samples surface, calcium ions in the SBF reaches the interface. These Ca+2 s are gathered between the coat and SBF. The attached Ca ions on the surface are a reason for increase in pH of the SBF. The surface hydrophilicity was studied by measuring contact angles with a sessile drop of SBF deposited on a sample surface. The wettability of a flat surface is expressed by the contact angle Ɵ between a liquid drop (here SBF) and a solid surface (here bHA-Di composite) due to intermolecular interactions described by Young’s equation (Eq. 1). Depending on the value of the contact angle, surface properties are determined as hydrophobic (Ɵ > 90°) or hydrophilic (Ɵ < 90°). The surface properties such as roughness, chemical composition, surface charge, and porosity play a significant role in the enhancing of wettability and bioactivity of biomedical implants. In addition, cell proliferation and biomimetic growth on biomedical implants were influenced by wettability values [30,31]. Fig. 11 shows the gradual modification of wettability of the bHA30 wt. % Di composite at different exposure times. In this study we reported the use of a low-energy electron irradiation to achieve tunable wettability of the bHA-30 wt. % Di composite in a wide range of contact angles, from 30° to 70°. As can be seen in the Fig. 6, by increasing the exposure time of HA-30 wt. % Di in the SBF solution from 400 s to 1200 s, the contact angle decreases. Table 3 shows a summary of the variations in the exposure time as well as contact angles at different amounts of Di. As can be seen in this figure the best results have been reported for the sample of 30 wt. % Di. Fig. 12 shows the load–displacement curves of prepared HA-Di coatings with different amounts of Di at a load of 30 mN. Established model was used to calculate quantitative hardness and elastic modulus values for such data. The differences in the depth obtained at the applied load represent the differences in the hardness of the
Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004
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pH
NHA
NHA10D
NHA20D
NHA30D
8 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7 6.9 0
3
6
9
12
15
18
21
Time (days)
a
128 116
Ion concentraon (%)
104 92 80 68 56 44 32 20 0
3
6
9
12
15
18
21
Time (days) Ca
Mg
P
b Fig. 10. The changes in (a) pH values versus immersion time and (b) calcium ions concentration in the SBF solution.
Fig. 11. The variations of angles of the SBF solution in contact with the composite coating with increasing exposure times.
materials. As clearly observed, with increasing Di content in the composite coating, the penetration depth of the indenter has decreased. As a result, among all of the samples, the sample with the maximum amount of Di and the sample without any Di content have respectively the maximum and the minimum amount of hardness. The measured values of hardness are in agreement with those reported by other researchers for HA coatings [32–35]. Table 4 gives the elastic modulus and hardness values of composite coating with different amounts of Di. It should be noted that all previous studies on the
Table 4 Micro-harness and elastic module values of the coating samples at different percentage of Di. Sample
Hardness (MPa)
Elastic modulus (GPa)
Load (mN)
HA HA10Di HA20Di HA30Di
2652 3225 3350 3390
45 65 90 95
30 30 30 30
Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004
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Fig. 12. The microhardness, load-depth plots of four types of composite coatings with different amounts of Di.
construction and properties of biomaterials, have not offered any statistical analysis of their results. According to the works presented by Davim et al. [36,37], statistical analysis can provide a detailed description of the final results. 4. Conclusion In the present study, bHA-Di nanocomposite was coated on the titanium plates by the EPD technique using isopropanol as the suspending medium. Besides, the assessment of in vitro bioactivity behaviour as well as apatite formation was done by immersing the produced samples in the SBF solution at various times. The possible coating mechanisms for the produced composite were also discussed. The substrate with composite coating was also tested for micro-hardness properties and the wettability of nanocomposite of the coating samples was also assessed. According to the results obtained, the bHA-Di nanocomposite coating at 30 wt. % of Di had an optimum bioactivity behaviour, good wettability conditions and high hardness property. The results gathered for the bioactivity experiment showed an optimum-constant pH of 7.6 (near the blood pH) after a long time (here 21 days), despite the fact that the other samples with lower amount of Di showed an oscillatory behaviour in pH variations. In the case of hardness, this work reported fabrication of a new coating with a micro-hardness range of 2–3 (GPa) and finally in the case of wettability the results derived from the experimental studies showed a minimum value of 35–40° for the contact angle which is suitable for biomaterials applications especially orthopaedic coating. Reference [1] Shih CC, Shih CM, Su YY, Chang MS, Lin SJ. Characterization of the thrombogenic potential of surface oxides on stainless steel for implant purposes. Applied Surf Sci 2003;219(3):347–62. [2] Ratner BD, Hoffman AS, Schoen FJ, Lemons JE. Biomaterials science: an introduction to materials in medicine. Academic press; 2004. [3] Black J. Does corrosion matter? J Bone Joint Surg 1988;70:517–20. [4] Mohammed MT, Khan ZA, Siddiquee AN. Surface modifications of titanium materials for developing corrosion behavior in human body environment: a review. Proc Mater Sci 2014;6:1610–16. [5] Saldana L, Vilaboa N, Vallés G, González-Cabrero J, Munuera L. Osteoblast response to thermally oxidized Ti-6Al-4V alloy. J Biomed Mater Res Part A 2005;73:97–107. [6] Martinesi M, Bruni S, Stio M, Treves C, Borgioli F. In vitro interaction between surface-treated Ti-6Al-4V titanium alloy and human peripheral blood mononuclear cells. J Biomed Mater Res Part A 2005;74:197–207. [7] Rivera-Denizard O, Diffoot-Carlo N, Navas V, Sundaram PA. Biocompatibility studies of human fetal osteoblast cells cultured on gamma titanium aluminide. J Mater Sci- Mater in Medic 2008;19:153–8.
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Please cite this article as: A. Khandan et al., Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.004