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posthydrothermal treatments
Journal of Alloys and Compounds 778 (2019) 566e575
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Preparation of HA/Gelatin coatings on C/C composites via modified electrocrystallization/posthydrothermal treatments Xiong Xin-bo a, Ling Liu a, Ni Xin-Ye b, *, Ma Jun a, Zeng Xie-rong a a b
Shen Zhen University, College of Materials, Shen Zhen Key Laboratory of Special Functional Materials, Shen Zhen, 518086, China Second People's Hospital of Changzhou, Nanjing Medical University, Changzhou, 213003, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 July 2018 Received in revised form 8 November 2018 Accepted 16 November 2018 Available online 17 November 2018
Electrocrystallized calcium phosphate (CP) coatings generally show a porous structure on carbon/carbon composites (C/Cs) and poor adhesive strength that limit their application in orthopedics. To overcome this disadvantage, ultrasonic and ice bath were employed in the electrocrystallization process of preparing CP/gelatin coating with a thickness of 50 mm on C/Cs. The CP/gelatin coating was hydrothermally treated in aqueous ammonia and sodium silicate solutions, respectively, to obtain an ion-doped hydroxyapatite(HA)/gelatin coating on C/Cs. Results showed that the modified electrocrystallized process could achieve a compactly stacked flaky structure coating of brushite and gelatin on C/Cs, whose adhesive strength could reach a critical load of 6.3 N. HA/gelatin coating that contains nanoneedle crystals could be achieved after performing the ammonia hydrothermal treatment, but its adhesive strength decreased to 4.1 N. Further hydrothermal treatment of the HA/gelatin coating in the sodium silicate solution obtained a Na, Si-doped HA coating on C/Cs, which could reach a critical load as high as 7.72 N. This value is equivalent to shear strength of 47 MPa. The ion-doping and hydrothermal self-repair processes enhanced the adhesion strength of the Si,Na-HA coating on C/Cs. In vitro experiments showed that the Si, Na-HA/gelatin coating exhibited better cell compatibility than C/Cs and HA/gelatin coating. © 2018 Elsevier B.V. All rights reserved.
Keywords: Electrocrystallization Hydrothermal treatment Hydroxyapatite Gelatin Coating
1. Introduction Biomedical metals and ceramics, such as titanium alloy, zirconium oxide, silicon nitride, and carbonaceous materials, are the most widely used materials for clinical bone implantation [1,2]. However, these biomedical materials have higher rigidity than human bones, which causes stress shielding after their implantation into a human body [3]. Stress shielding results in bone adsorption around the implants and increases the risk of catastrophic failure of the implants during the service life of prostheses [4]. Thus, concerted effort has been devoted to design and engineer biocompatible materials with elastic modulus close to that of human bones, such as b Tie28Nbe35.4Zr [5], poly(ether-etherketone) [6] and carbon fiber composites [7]. Among these materials, carbon-fiber-reinforced carbon composites (C/Cs) are considered potential orthopedic implant materials because of their high strength and toughness, excellent biocompatibility, good
* Corresponding author. E-mail address: [email protected] (N. Xin-Ye). https://doi.org/10.1016/j.jallcom.2018.11.202 0925-8388/© 2018 Elsevier B.V. All rights reserved.
anticorrosion, and especially adjusted rigidity, which is capable of matching that of the human bones [8,9]. Nevertheless, C/Cs are bioinert materials that cannot bond to surrounding tissues. Moreover, releasing carbon debris from C/Cs often causes problems due to friction damage during surgical operations. The debris deposits in the neighborhood of implants and gains access to lymphatic nodes and skin, thereby resulting in the “black skin effect” [10,11]. Thus, bioactive calcium phosphates (CPs), such as monetite, brushite, hydroxyapatite (HA), and their composite coatings, are often applied to C/C surfaces to overcome these limitations. CP coatings were fabricated on C/Cs by several technologies, such as high temperature spraying methods (plasma spraying [12] and supersonic atmospheric plasma spraying [13]), induction heating technologies [14,15], electrocrystallization, electrophoretic deposition [16e18], and chemical bath. Among these methods, plasma spraying is the most popular for commercial preparation of HA coatings in orthopedics. However, a high spray temperature leads to impurity phases, such as tricalcium phosphate, calcium oxide, and internal stress, which deteriorate the in vivo mechanical performance. Induction heating methods combined with
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posthydrothermal treatment produces a dramatically bonded HA coating on C/Cs [19]. Nevertheless, these technologies are only suitable for the preparation of CP coatings on symmetric implants. CP coatings cannot be easily deposited on complex surfaces. Electrocrystallization methods are often used in recent studies to deposit CPs into C/C substrates because of the following advantages: (1) low temperatures that favor the achievement of coatings with low residual stresses, (2) ability to coat porous, geometrically complex, or non-line-of-sight surfaces, (3) easy regulation of required thickness, composition, crystal size, and microstructure of deposits, and (4) availability and low cost. However, the low bonding strength between electrocrystallized CP coating and C/Cs hampers their practical application in the field of orthopedics. The reason for this limitation arises from the discretely separated crystals of the as-deposited CP coatings, which show a porous structure due to the evolution of H2 gas near the surface of the C/C cathode during deposition process. To address this drawback, some of the strategies to avoid deposition of weak CP crystals into carbon materials or Ti substrate include chemical treatment [20], introduction of ultrasonic wave into electrolytes [21], employment of pulse power source [22], or addition H2O2 into electrolyte to inhibit the H2 evolution [23]. However, the coatings achieved through these methods still constitute amounts of discretely separated CP crystals, thereby resulting in a soft coating that is easy to scratch off. Another method is the use of reinforcements that can enhance the bonding performance of CP coatings on C/C to circumvent the adverse effects caused by H2 evolution, such as ion doping (Na, Si, F), reinforcement of nanotube, fiber or belt of carbon or silicon carbide, and integration of collagen with CPs [18,24e26]. Nevertheless, no sufficient data has demonstrated the direct existence of a good adhesion between reinforced materials and CPs. Employing electrocrystallization technologies for the preparation of highperformance CP coatings on C/C substrates seems difficult. Gelatin is a natural biological material derived from the alpha-1 type I collagen, which can be absorbed by the human body [27]. Gelatin stimulates transplanted tissue to generate new collagen [28] and promotes cell adhesion, proliferation, and differentiation [29]. Thus, it can also be used as an implant material for bone repair in tissue engineering [30]. Documents showed that the incorporation of gelatin into CP coatings on Ti alloy via electrodeposition could improve adhesive performance. The as-achieved hydroxyapatite coating on the Ti surface could achieve a bonding strength Medvecký reported the use of as high as 5.6 ± 1.8 MPa [31]. L. electrocrystallization to deposit a brushite/gelatin film on graphite substrates, which show a tensile strength of approximately 7 MPa. After annealing at 850 C in argon, this brushite/gelatin film transformed into an HA coating that contains some CaO. The existence of CaO impurities can enhance the dissolution of the coating and is detrimental to long-term implantation. Additionally, adhesion strength between the HA and graphite substrate has not been reported [32]. A novel route is developed in the present study to prepare an adhesive HA coating on C/C via electrocrystallization. First, an ultrasonic and ice bath is employed to the electrolyte. Gelatin, which served as a morphology regulator, is electrocrystallized together with CP into H2O2-treated C/C cathode surfaces to obtain a compact composite coating. This compact composite coating is then converted into an HA/gelatin coating by ammonia hydrothermal treatment. Finally, the HA/gelatin coating is hydrothermally treated to obtain a Si, Na-doped HA/gelatin coating on C/C in a sodium silicate solution. With the synergetic effects of the abovementioned tactics, the as-product HA/gelatin coating on C/Cs could reach a critical load value as high as 7.72 N, which could meet the requirements of implants in orthopedics.
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2. Materials and methods 2.1. Coating preparation Bulk C/Cs, which came from Shanghai University, were cut into many sheets with a size of 1.0 cm 1.0 cm 0.6 cm. These sheets were washed in an ultrasonic bath for 30 min using distilled water and absolute ethyl alcohol to remove debris on the surface. The sheets were hydrothermally modified with a 2M H2O2 solution at 410 K for 24 h. The solution was taken out and ultrasonically cleaned with distilled water before being air dried for CP coating deposition. A CP/gelatin coating was deposited into the C/C substrate via electrocrystallization in an ice bath applied by an ultrasonic wave. A graphite plate served as the anode, whereas C/C sheets were the cathode. Two electrodes, which were 20 mm apart, were connected to a regulated direct power supply. An ultrasonic wave with a frequency of 25 KHz was applied into a 500-ml beaker with 200 ml of electrolyte. The electrolyte consisted of 0.02 M of calcium nitrate, 0.012 M of ammonium dihydrogen phosphate, and 0.5 g/L of gelatin solution. The duration and current density for the CP/gelatin coating deposition was 60 min and 8 mA/cm2, respectively. After deposition, the resultant CaP/gelatin coatings were hydrothermally treated in a 5 vol% ammonia aqueous solution at 493 K for 24 h. The samples were further hydrothermally treated in a 5 wt% sodium silicate aqueous solution at 493 K for 24 h. Finally, the second-step samples were taken out and ultrasonically washed for 5 min, and then dried at 393 K for the subsequent in vitro experiments and performance characterizations. 2.2. Characterization methods The phase, morphology, composition, and high-resolution image of the coatings were characterized via D8 advance X-ray diffractometry (XRD) (Bruker-Axs Co., Karlsuuhe, Germany) and field emission scanning electron microscopy (Se3400N, Tokyo, Japan), as well as environmental transmission electron microscopy (ETEM) at 80e300 kV (Titan ETEM G2 80-300, USA). To identify gelatin in the electrodeposited coating, the decomposition profile of 6 mg of scraped dried samples was thermogravimetrically analyzed (Q50, TA Instrument, New Castle, DE, USA). Dried samples were placed in the pan and heated from 100 C to 600 C under a nitrogen atmosphere at a heating rate of 10 C min1, and weight loss was recorded as a function of temperature. The functional groups of the coatings were analyzed using a Fourier transform infrared spectrometer (FTIR) (Equinox55). X-ray photo electron spectroscopy (XPS) (AxisUltra) measurements were used to identify the elemental composition and valence analyses of the coatings. The bonding strength of the coatings on C/C substrates was determined by an s-3400N scratch tester (CSM, Anton Paar Co. Ltd., Switzerland) fitted with a Rockwell C 0.2 mm-diamond stylus with a preload of 1 N. Finally, the scratch trace of the coatings was observed via stereomicroscopy. 2.3. Cell biology experiments In vitro cell biocompatibility experiments of the HA/gelatin and Si,Na-HA/gelatin coatings on C/C were performed using murine bone marrow-derived mesenchymal stem cells. Naked C/C samples were used as the control group. All samples were placed in a 12well culture plate, wherein each well was filled with 2 ml of culture medium containing 2 104 murine bone marrow-derived mesenchymal stem cells per milliliter. The medium for culturing mesenchymal stem cells contained 10% of fetal bovine serum, 100 mg/ml of streptomycin, 100 units/ml of penicillin, and 1% of
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growth factor. The resultant plates were placed in an incubator that contains 5% of CO2 at 300 K. The cells can naturally fall onto the coating surfaces during the culture process. The culture medium was absorbed after a 12-h culture. Then, the samples that adhered to the cells were transferred to a new 12-well culture plate. These cells were recultured by adding the culture medium to the wells. After inoculation for 2, 4, and 6 days, some samples were continuously cultured for 4 h by adding 200 ml of 5 mg/ml MTT reagents to the culture medium. Finally, the culture medium in the wells was absorbed and oscillated for 15 min by adding 2 ml of DMSO. Afterward, 200 ml of DMSO was collected and added into a 96-well plate for absorbency tests. The absorbency value A was measured with an enzyme-labeling measuring instrument (Thermo Multiskan GO Co. Lt). Wavelength was set at 570/630 nm, and the testing values of absorbency were averaged. Some samples were first cleaned using PBS, and then fixed using a 10 vol% formaldehyde solution for 15 min at room temperature. The fixed samples were then subjected to stain with fluorescence using a 5 mg/ml propidium iodide solution (Sigma Company) for 15 min. The fixed samples cleaned with PBS were placed under an inverted fluorescence microscope (FM) (Olymlusix71) for cell morphological observations. Other samples were treated with a 200 ml of 0.2% Triton X-100 solution for 15 min. The enzymatic activity of alkaline phosphatase (ALP) was measured when cells on the samples cracked. The remaining samples were also cleaned with PBS after 2 days. Then, the mesenchymal stem cells of mice were fixed with a 2.5% glutaraldehyde aqueous solution and were dehydrated in turns for 20 min with concentrations of 30%, 50%, 70%, 90%, and 95% ethyl alcohol aqueous solutions. Finally, isoamyl acetate was used to convert ethyl alcohol in the cells. The as-achieved samples were observed via scanning electron microscopy (SEM) (JSM-6360LA, Hitachi Co. Ltd., Japan). 3. Results and discussion 3.1. Morphology and composition analyses Generally, the reported electrocrystallized CP coatings on C/C were composed of amounts of discretely separated flaky or needle crystals due to the evolution of H2 during deposition process. This process resulted in a soft porous coating that is easy to scratch off. Thus, the current strategies, including the application of ultrasonic wave into the deposited electrolyte, ice bath, and addition of gelatin to CPs, was expected to increase the compactness of the asproduced coating, which further improved the bonding performance of CP coatings on C/C. SEM observations of the cross-section profiles in all coatings showed a thickness of 50 mm as shown in Fig. 1 (a). No changes in the thickness for different-type coatings were observed. The TG curves of the achieved coatings are shown in Fig. (b). The electrodeposited CP coating revealed a weight loss of 16.8%, which indicates that gelatin accounts for 16.8% of the weight of the coating. After two hydrothermal treatments, the weight loss of the coatings for the first and second treatments is 3.4% and 1.9%, respectively. The large weight loss before and after the first hydrothermal treatment may be correlated with the dissolution and recrystallization of brushite phases in the coating during the conversion of brushite into HA [33]. Moreover, the second hydrothermal treatment showed a lower weight loss than the first hydrothermal treatment, which could be related with the infiltration of Si and Na ions into HA crystals, thereby increasing the weight of coating. The morphologies of the coatings prepared via electrocrystallization (EC), ultrasonic-assisted electrocrystallization (UAE), UAE/ammonia hydrothermal treatment (AHT), UAE/AHT/sodium
silicate hydrothermal treatment (SHT) are shown in Fig. 1(c)e(j). Photos at 5 K or 10 K magnification (Fig. 1(c) and [d]) show that the directly electrocrystallized coating exhibited a loose and porous morphology, which resulted in easy exfoliation from C/Cs. Single addition of gelatin to the brushite coating could produce a continuous coating, eliminating the formation of the discretely separated crystals. Nevertheless, this process could not hinder the occurrence of pores resulting from the evolution of the H2 gas, which could be the reason for the poor adhesion of the achieved CP coating. Instead, the UAE coating, as shown in Fig. 1(e) and (f), constitutes compactly stacked, flake-like grains owing to the cavitation effects arising from the propagation of ultrasonic waves in the electrolyte. This coating is difficult to scratch off with nails, which indicates its good adhesion to C/C substrates. However, further observation at 10 K magnification showed gaps and cracks in the UAE coating, which is responsible for the blast effects of ultrasonic cavitations. After UAE was followed by AHT, the asproduced coating manifested a compact morphology with some long cracks, as shown in Fig. 1(c). In addition, short needle-like crystals were observed (Fig. 1(d)). However, after further hydrothermal treatment of the UAE/AHT coating in a sodium silicate solution, the cracks shortened, as presented in Fig. 1(e), which indicated that the cracks in the UAE/AHT coating underwent selfrepair to some degree. The crack self-repair is a result of high temperature and high water vapor pressure during the hydrothermal process. Moreover, coating observation under high magnification showed a needle-like morphology. After two hydrothermal treatments, no significant changes were found in the morphologies of the two HA coatings. Composition analyses conducted via EDS (Fig. 2) showed that all the coatings contain Ca, P, and O. The Ca/P atomic ratio was about 1.02, 1.65, and 1.60 for the UAE, UAE/AHT, UAE/AHT/SHT coatings, respectively. The Ca/P atomic ratios of these coatings were close to those of brushite and HA. Additionally, the USE/AHT/SHT coating contains Si and Na elements, whose atomic concentrations are 4.22% and 1.03%, respectively. 3.2. XRD analyses Fig. 3 shows the XRD diffraction patterns of the coatings on C/C. As shown in Fig. 3(a), the coating prepared by UAE, the diffraction peaks, excluding those of C/C substrates, were in accordance with powder brushite (JSPD09-0077). This result indicates that the UAE coating was composed of a brushite phase. Diffraction peaks could be assigned to hydroxyapatite (HA, JSPD 03-0747) after the ammonia hydrothermal treatment, as shown in Fig. 3(b). This process shows the presence of polycrystalline apatite. Further hydrothermal treatment in the sodium silicate solution also displayed the existence of HA, as presented in Fig. 3(c). However, a split peak was observed at (211). One split peak shifted a low degree, which indicates the existence of some HA crystals with a larger d-spacing. 3.3. FTIR analyses Fig. 4 shows the FTIR spectra of the coatings prepared on C/C, which shows that the peaks at 3483 and 1645 cm1 were generated by the OH of free water. By contrast, peaks at 1126, 1059, 660, and 573 cm1 were the characteristic peaks of PO3 4 . Absorption peaks at 1126 and 1059 cm1 were ascribed to SiO2 3 , which overlap with 2 3 those of PO3 4 . The peak overlaps of the SiO3 and PO4 groups were 2 attributed to the penetration of SiO3 into the HA lattice, thereby replacing PO3 partially. Furthermore, the peaks at 875 and 4 1428 cm1 corresponded to the absorption peaks of CO2 3 . Another possibility is that the peak at 1428 cm1 was generated by PeO(H) of HPO2 4 , which could be replaced by carbonate. The substitute of
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Fig. 1. Cross-section SEM photo: (a) TG profile and (b) SEM morphology of the coatings prepared via (c)/(d) EC, (e)/(f) UAE, (g)/(h) UAE/AHT, and (i)/(j) UAE/AHT/SHT.
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Fig. 4. FITR spectra of the as-prepared coatings on C/C substrates: (a) brushite/gelatin, (b) HA/gelatin, and (c) Si,Na-HA.
3.4. TEM analyses
Fig. 2. EDS spectra of the coatings prepared via (a)/(b) UAE, (c)/(d) UAE/AHT, and (e)/(f) UAE/AHT/SHT.
Fig. 3. XRD patterns of as-prepared coatings via (a) UAE, (b) UAE/AHT, and (c) UAE/ AHT/SHT.
To further reveal the nanostructures of the as-prepared coatings, photos of their TEM, high-resolution TEM, and electron diffraction are shown in Fig. 5. The TEM photos shown in Fig. 5(a) and (b) show that the brushite coating was composed of numerous nanoneedlesheet crystals. Generally, the brushite coating deposited by electrocrystallization comprised plate-like crystals. The difference in the morphologies of the two brushites indicated that gelatin could regulate the growth of brushite crystals. High-resolution transmission electron microscopy (HRTEM) results displayed one lattice fringe with a d-spacing of 0.247 nm that corresponds to the (022) crystal plane of brushite. The corresponding fast Fourier transmission (FFT) photo of HRTEM disclosed the existence of concentric rings composed of bright spots, which demonstrate its polycrystalline feature. After ammonia hydrothermal treatments, the two HA coatings revealed the presence of needle-like crystals, as shown in Fig. 5(d) and (e). The HRTEM photo (Fig. 5(e)) showed one lattice fringe with a d-spacing of 0.2811 nm, which is ascribed to the (211) plane of the HA coating. The corresponding FFT graph showed many bright spots, which showed a single-crystal feature, as shown in Fig. 5(f). Further sodium silicate hydrothermal treatment did not indicate change in the morphology of the as-achieved Si, Na-HA coating, as displayed in Fig. 5(h). However, one HRTEM image (Fig. 5(i)) of the Si, Na-HA coating showed a d-spacing of 0.2828 nm of the (211) plane, which is larger than that of the HA coating. This finding is consistent with that in the XRD analyses. The lattice that expands in the Si, Na-HA coating indicated that the infiltration of Na, Si ions into HA because of their larger ion radius compared with that of Ca and P ions. The FFT image showed many bright spots, which are results of a few Si, Na-HA single crystals, as presented in Fig. 5(j). 3.5. XPS analyses
PeO(H) with carbonate group is indicated by the formation of a btype HA coating [33,34]. The absorption peaks at 2922 and 1579 cm1 were produced by acyl amino, which was generated by the coupling of NeH stretching and NeH bending vibrations and CeN stretching vibration [35]. The presence of the acyl amino group indicated the formation of gelatin into the brushite coating on C/C substrates.
XPS was further employed to investigate the chemical structure and valence states of the achieved coatings, as presented in Fig. 6. The survey spectra in Fig. 6(a) indicated the coexistence of Ca, P, O, and C for all coatings. The Ca/P atomic ratio was calculated at 1.05, 1.69, and 1.62 for brushite/gelatin, HA/gelatin, and Si, Na-HA/ gelatin coatings, respectively, in accordance with the results of EDS analyses. The intensity of N1s peak was considerably weak for
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Fig. 5. TEM, HRTEM, and ED images of as-prepared coatings: (aec) brushite/gelatin; (def), HA/gelatin; (hej) Si,Na-HA/gelatin.
Fig. 6. (a) XPS survey spectra of the coatings and (b) narrow spectrum of Si2p of the Si,Na-HA coating.
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all the coatings. The intensities of the achieved coatings could not be easily viewed after conducting hydrothermal treatments. This finding indicates the low ratio of gelatin in the coating, which further validates the TG analytical results. In addition, Si, Na elements could be observed in the coating subjected to the two hydrothermal treatments from the survey spectrum. The Si2p peak was further analyzed for demonstration of the incorporation of Na, Si into HA lattice, as shown in Fig. 6(b). The Si2p peak could be mainly deconvoluted according to a GUASS fitting into three peaks at 101.4 eV, 102.3 eV, and 103.2 eV, which represent SieO, [SiO3] SieO, and SieOH [36,37]. This results show the existence of Si elements in the form of orthosilicate and metasilicate groups [38]. Thus, a small amount of sodium silicate had also infiltrated the HA 4 coating, besides the substitution of PO3 4 by SiO4 . 3.6. Scratch tests The adhesive performance of the coatings was examined via a scratch test and optical microscopy. Fig. 7(a) shows the scratch curves and the corresponding sites of the as-prepared coatings. The figure shows that the critical loading for brushite/gelatin, HA/ gelatin, and Si,Na-HA/gelatin coatings was 6.3, 4.1 N, and 7.72 N, respectively. The corresponding shear stress was calculated at approximately 42.4 MPa for the brushite/gelatin coating, 34.2 MPa for the HA/gelatin coating, and 47.1 MPa for the Si,Na-HA/gelatin coating. These results are based on the following equation:
.
tc ¼ ðHS Lc =pÞ1=2 R;
performance. Although the current adhesive strength of the electrocrystallized coating was lower than that of our previously prepared HA coatings via induction heating, it is at par with high-temperature coatings on C/Cs. Examples include ZrSi2 coatings on SiC-coated C/Cs prepared via supersonic atmospheric plasma spraying, which showed an adhesive strength of 5.5, 8, 11, and 8.9 N at a power of 30, 35, 40 and 45 KW [42] and (2) SiC coatings with a thickness of 50 mm for C/CeZrCeSi with a critical load of 3.25 N [43]. Our fabricated coating could be comparable to coatings on Ti alloys, such as TiO2 (8 N) and TiO2/HA (10N) coatings prepared via microarc oxidation and electrophoresis [44] and HA coating (7 N) deposited via RF magnetron sputtering [45]. These comparisons indicated a good adhesive performance of our prepared coatings. In summary, we reckon that the application of electrocrystallization technologies can possibly fabricate a well-bonded CP coating on C/C substrates. Further studies are being conducted in our laboratory. 3.7. Response behaviors of in vitro osteoblast Fig. 8 shows the proliferation curves of the marrow mesenchymal stem cells after 2, 4, and 6 days of cultures. All samples showed the tendency of an increase in the cell coverage with increasing culture duration. The HA/gelatin/Si-HA coating showed the best cell proliferation capability. The variance analyses with the
(1)
where tc is the shear stress; R is the radius of diamond stylus; Lc is the critical load; Hs is the Shore scleroscope hardness of C/C substrate. The stress strengths of the HA and Si,Na-HA coatings on C/C are greater than that of the C/C implanted into a mouse bone for 20 weeks (2.44 MPa) [19], which is high enough for handling before implant and strong enough to survive in the living body. By contrast, the adhesive strength of the HA/gelatin coating on C/Cs is lower than that of the HA/gelatin coating, which could be due to the loss of gelatin weight caused by the dissolution and crystallization of brushite phases. However, the Si,Na-HA/gelatin coating showed a higher bonding strength than HA/gelatin, which can contribute to the dope strengthening effect of Na and Si ions [39,40], as well as the self-repairing effect of the coating due to hydrothermal treatment [41]. The scratch traces of the coatings are also depicted in Fig. 6(b). The coatings experienced uniform plastic deformation and showed no break off from C/C substrates until the critical load was reached, thereby indicating their outstanding mechanical
Fig. 8. Comparison of cell coverage rates on different coating surfaces.
Fig. 7. (a) Scratch curves and (b) scratch trace morphologies of as-achieved coatings on C/Cs via optical microscopy.
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SPSS11.0 soft show that after 2, 4, and 6 days of cultures. The Pvalues of Si,Na-HA/gelatin coating were calculated at 1.2 103, 2.1 104, 2.6 104, respectively, which are smaller than 5.0 102. Thus, the results of the proliferation difference of the Si,Na-HA/gelatin coating was statistically significant. Fig. 9 shows that typical FM photos of the samples after a 2-day culture showed a significant increase in the number of mesenchymal stem cells in sequence from C/C, HA/gelatin coating and then Si, Na-HA/gelatin coating. The SEM photos of mesenchymal stem cells on different coating surfaces after a 4-day culture are shown in Fig. 10. Likewise, the number of cells on the Si,Na-HA/ gelatin surface was higher than that on C/C and the HA/gelatin coating. The cells were adhered well to the surfaces of C/C, HA/ gelatin, and Si, Na-HA/gelatin coatings and stretch their pseudopods and attached to the sample surfaces. The cell number and stretching degree of the cell pseudopods of on the Si,Na/HA coating surface were the highest among these cells, Moreover, the cells on the gelatin/Si-HA coating surface spread continuously. ALP was generally used to characterize the mature degree of differentiation of osteoblast on the coating surface and the osteogenesis and mineralization ability of cells. Changes in the culture duration are shown in Fig. 11. The activity of ALP increased remarkably as the inoculation time increased. Of the three samples, Si,Na-HA/gelatin coatings similarly showed the best ALP performance. These results suggest that the gelatin/Si-HA was more helpful to the differentiation and maturing of cells, showing the best osteoblast compatibility. Similarly, the two-step hydrothermal treatments have no diverse effects on the cell biocompatibility of gelatin in the coatings. In conclusion, our prepared Si,Na-HA coating on C/C does not only possess good mechanical performance but also osteoblast compatibility, which exhibits good potential for clinical application as bone implant materials.
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Fig. 11. Changes in the activities of ALP on different surfaces with culture duration.
on a C/C surface through the application of an ultrasonic and ice bath to undergo electrocrystallization. Adhesive strength could reach a critical load of 6.3 N. The brushite/gelatin coating could be transformed into an HA/gelatin coating with nanoneedle crystals after the ammonia hydrothermal treatment. The adhesive strength of the coating decreased to a critical load of 4.1 N. Further hydrothermal treatment in the sodium silicate solution led to the achievement of a Na, Si-doped HA coating on C/Cs. Adhesive strength could achieve a critical load value as high as 7.72 N, which is equivalent to a shear strength of 47 MPa. In vitro experiments showed that the Si, Na-HA/gelatin coating exhibited better cell compatibility than the C/C and the HA/gelatin coatings.
4. Conclusions A compact brushite/gelatin coating was successfully prepared
Fig. 9. FM images of bone marrow stem cells on different surfaces on the second day.
Fig. 10. SEM photos of bone marrow stem cell on different surfaces on the fourth day.
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Preparation of HA/Gelatin coatings on C/C composites via modified electrocrystallization/posthydrothermal treatments Xiong Xin-bo a, Ling Liu a, Ni Xin-Ye b, *, Ma Jun a, Zeng Xie-rong a a b
Shen Zhen University, College of Materials, Shen Zhen Key Laboratory of Special Functional Materials, Shen Zhen, 518086, China Second People's Hospital of Changzhou, Nanjing Medical University, Changzhou, 213003, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 July 2018 Received in revised form 8 November 2018 Accepted 16 November 2018 Available online 17 November 2018
Electrocrystallized calcium phosphate (CP) coatings generally show a porous structure on carbon/carbon composites (C/Cs) and poor adhesive strength that limit their application in orthopedics. To overcome this disadvantage, ultrasonic and ice bath were employed in the electrocrystallization process of preparing CP/gelatin coating with a thickness of 50 mm on C/Cs. The CP/gelatin coating was hydrothermally treated in aqueous ammonia and sodium silicate solutions, respectively, to obtain an ion-doped hydroxyapatite(HA)/gelatin coating on C/Cs. Results showed that the modified electrocrystallized process could achieve a compactly stacked flaky structure coating of brushite and gelatin on C/Cs, whose adhesive strength could reach a critical load of 6.3 N. HA/gelatin coating that contains nanoneedle crystals could be achieved after performing the ammonia hydrothermal treatment, but its adhesive strength decreased to 4.1 N. Further hydrothermal treatment of the HA/gelatin coating in the sodium silicate solution obtained a Na, Si-doped HA coating on C/Cs, which could reach a critical load as high as 7.72 N. This value is equivalent to shear strength of 47 MPa. The ion-doping and hydrothermal self-repair processes enhanced the adhesion strength of the Si,Na-HA coating on C/Cs. In vitro experiments showed that the Si, Na-HA/gelatin coating exhibited better cell compatibility than C/Cs and HA/gelatin coating. © 2018 Elsevier B.V. All rights reserved.
Keywords: Electrocrystallization Hydrothermal treatment Hydroxyapatite Gelatin Coating
1. Introduction Biomedical metals and ceramics, such as titanium alloy, zirconium oxide, silicon nitride, and carbonaceous materials, are the most widely used materials for clinical bone implantation [1,2]. However, these biomedical materials have higher rigidity than human bones, which causes stress shielding after their implantation into a human body [3]. Stress shielding results in bone adsorption around the implants and increases the risk of catastrophic failure of the implants during the service life of prostheses [4]. Thus, concerted effort has been devoted to design and engineer biocompatible materials with elastic modulus close to that of human bones, such as b Tie28Nbe35.4Zr [5], poly(ether-etherketone) [6] and carbon fiber composites [7]. Among these materials, carbon-fiber-reinforced carbon composites (C/Cs) are considered potential orthopedic implant materials because of their high strength and toughness, excellent biocompatibility, good
* Corresponding author. E-mail address: [email protected] (N. Xin-Ye). https://doi.org/10.1016/j.jallcom.2018.11.202 0925-8388/© 2018 Elsevier B.V. All rights reserved.
anticorrosion, and especially adjusted rigidity, which is capable of matching that of the human bones [8,9]. Nevertheless, C/Cs are bioinert materials that cannot bond to surrounding tissues. Moreover, releasing carbon debris from C/Cs often causes problems due to friction damage during surgical operations. The debris deposits in the neighborhood of implants and gains access to lymphatic nodes and skin, thereby resulting in the “black skin effect” [10,11]. Thus, bioactive calcium phosphates (CPs), such as monetite, brushite, hydroxyapatite (HA), and their composite coatings, are often applied to C/C surfaces to overcome these limitations. CP coatings were fabricated on C/Cs by several technologies, such as high temperature spraying methods (plasma spraying [12] and supersonic atmospheric plasma spraying [13]), induction heating technologies [14,15], electrocrystallization, electrophoretic deposition [16e18], and chemical bath. Among these methods, plasma spraying is the most popular for commercial preparation of HA coatings in orthopedics. However, a high spray temperature leads to impurity phases, such as tricalcium phosphate, calcium oxide, and internal stress, which deteriorate the in vivo mechanical performance. Induction heating methods combined with
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posthydrothermal treatment produces a dramatically bonded HA coating on C/Cs [19]. Nevertheless, these technologies are only suitable for the preparation of CP coatings on symmetric implants. CP coatings cannot be easily deposited on complex surfaces. Electrocrystallization methods are often used in recent studies to deposit CPs into C/C substrates because of the following advantages: (1) low temperatures that favor the achievement of coatings with low residual stresses, (2) ability to coat porous, geometrically complex, or non-line-of-sight surfaces, (3) easy regulation of required thickness, composition, crystal size, and microstructure of deposits, and (4) availability and low cost. However, the low bonding strength between electrocrystallized CP coating and C/Cs hampers their practical application in the field of orthopedics. The reason for this limitation arises from the discretely separated crystals of the as-deposited CP coatings, which show a porous structure due to the evolution of H2 gas near the surface of the C/C cathode during deposition process. To address this drawback, some of the strategies to avoid deposition of weak CP crystals into carbon materials or Ti substrate include chemical treatment [20], introduction of ultrasonic wave into electrolytes [21], employment of pulse power source [22], or addition H2O2 into electrolyte to inhibit the H2 evolution [23]. However, the coatings achieved through these methods still constitute amounts of discretely separated CP crystals, thereby resulting in a soft coating that is easy to scratch off. Another method is the use of reinforcements that can enhance the bonding performance of CP coatings on C/C to circumvent the adverse effects caused by H2 evolution, such as ion doping (Na, Si, F), reinforcement of nanotube, fiber or belt of carbon or silicon carbide, and integration of collagen with CPs [18,24e26]. Nevertheless, no sufficient data has demonstrated the direct existence of a good adhesion between reinforced materials and CPs. Employing electrocrystallization technologies for the preparation of highperformance CP coatings on C/C substrates seems difficult. Gelatin is a natural biological material derived from the alpha-1 type I collagen, which can be absorbed by the human body [27]. Gelatin stimulates transplanted tissue to generate new collagen [28] and promotes cell adhesion, proliferation, and differentiation [29]. Thus, it can also be used as an implant material for bone repair in tissue engineering [30]. Documents showed that the incorporation of gelatin into CP coatings on Ti alloy via electrodeposition could improve adhesive performance. The as-achieved hydroxyapatite coating on the Ti surface could achieve a bonding strength Medvecký reported the use of as high as 5.6 ± 1.8 MPa [31]. L. electrocrystallization to deposit a brushite/gelatin film on graphite substrates, which show a tensile strength of approximately 7 MPa. After annealing at 850 C in argon, this brushite/gelatin film transformed into an HA coating that contains some CaO. The existence of CaO impurities can enhance the dissolution of the coating and is detrimental to long-term implantation. Additionally, adhesion strength between the HA and graphite substrate has not been reported [32]. A novel route is developed in the present study to prepare an adhesive HA coating on C/C via electrocrystallization. First, an ultrasonic and ice bath is employed to the electrolyte. Gelatin, which served as a morphology regulator, is electrocrystallized together with CP into H2O2-treated C/C cathode surfaces to obtain a compact composite coating. This compact composite coating is then converted into an HA/gelatin coating by ammonia hydrothermal treatment. Finally, the HA/gelatin coating is hydrothermally treated to obtain a Si, Na-doped HA/gelatin coating on C/C in a sodium silicate solution. With the synergetic effects of the abovementioned tactics, the as-product HA/gelatin coating on C/Cs could reach a critical load value as high as 7.72 N, which could meet the requirements of implants in orthopedics.
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2. Materials and methods 2.1. Coating preparation Bulk C/Cs, which came from Shanghai University, were cut into many sheets with a size of 1.0 cm 1.0 cm 0.6 cm. These sheets were washed in an ultrasonic bath for 30 min using distilled water and absolute ethyl alcohol to remove debris on the surface. The sheets were hydrothermally modified with a 2M H2O2 solution at 410 K for 24 h. The solution was taken out and ultrasonically cleaned with distilled water before being air dried for CP coating deposition. A CP/gelatin coating was deposited into the C/C substrate via electrocrystallization in an ice bath applied by an ultrasonic wave. A graphite plate served as the anode, whereas C/C sheets were the cathode. Two electrodes, which were 20 mm apart, were connected to a regulated direct power supply. An ultrasonic wave with a frequency of 25 KHz was applied into a 500-ml beaker with 200 ml of electrolyte. The electrolyte consisted of 0.02 M of calcium nitrate, 0.012 M of ammonium dihydrogen phosphate, and 0.5 g/L of gelatin solution. The duration and current density for the CP/gelatin coating deposition was 60 min and 8 mA/cm2, respectively. After deposition, the resultant CaP/gelatin coatings were hydrothermally treated in a 5 vol% ammonia aqueous solution at 493 K for 24 h. The samples were further hydrothermally treated in a 5 wt% sodium silicate aqueous solution at 493 K for 24 h. Finally, the second-step samples were taken out and ultrasonically washed for 5 min, and then dried at 393 K for the subsequent in vitro experiments and performance characterizations. 2.2. Characterization methods The phase, morphology, composition, and high-resolution image of the coatings were characterized via D8 advance X-ray diffractometry (XRD) (Bruker-Axs Co., Karlsuuhe, Germany) and field emission scanning electron microscopy (Se3400N, Tokyo, Japan), as well as environmental transmission electron microscopy (ETEM) at 80e300 kV (Titan ETEM G2 80-300, USA). To identify gelatin in the electrodeposited coating, the decomposition profile of 6 mg of scraped dried samples was thermogravimetrically analyzed (Q50, TA Instrument, New Castle, DE, USA). Dried samples were placed in the pan and heated from 100 C to 600 C under a nitrogen atmosphere at a heating rate of 10 C min1, and weight loss was recorded as a function of temperature. The functional groups of the coatings were analyzed using a Fourier transform infrared spectrometer (FTIR) (Equinox55). X-ray photo electron spectroscopy (XPS) (AxisUltra) measurements were used to identify the elemental composition and valence analyses of the coatings. The bonding strength of the coatings on C/C substrates was determined by an s-3400N scratch tester (CSM, Anton Paar Co. Ltd., Switzerland) fitted with a Rockwell C 0.2 mm-diamond stylus with a preload of 1 N. Finally, the scratch trace of the coatings was observed via stereomicroscopy. 2.3. Cell biology experiments In vitro cell biocompatibility experiments of the HA/gelatin and Si,Na-HA/gelatin coatings on C/C were performed using murine bone marrow-derived mesenchymal stem cells. Naked C/C samples were used as the control group. All samples were placed in a 12well culture plate, wherein each well was filled with 2 ml of culture medium containing 2 104 murine bone marrow-derived mesenchymal stem cells per milliliter. The medium for culturing mesenchymal stem cells contained 10% of fetal bovine serum, 100 mg/ml of streptomycin, 100 units/ml of penicillin, and 1% of
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growth factor. The resultant plates were placed in an incubator that contains 5% of CO2 at 300 K. The cells can naturally fall onto the coating surfaces during the culture process. The culture medium was absorbed after a 12-h culture. Then, the samples that adhered to the cells were transferred to a new 12-well culture plate. These cells were recultured by adding the culture medium to the wells. After inoculation for 2, 4, and 6 days, some samples were continuously cultured for 4 h by adding 200 ml of 5 mg/ml MTT reagents to the culture medium. Finally, the culture medium in the wells was absorbed and oscillated for 15 min by adding 2 ml of DMSO. Afterward, 200 ml of DMSO was collected and added into a 96-well plate for absorbency tests. The absorbency value A was measured with an enzyme-labeling measuring instrument (Thermo Multiskan GO Co. Lt). Wavelength was set at 570/630 nm, and the testing values of absorbency were averaged. Some samples were first cleaned using PBS, and then fixed using a 10 vol% formaldehyde solution for 15 min at room temperature. The fixed samples were then subjected to stain with fluorescence using a 5 mg/ml propidium iodide solution (Sigma Company) for 15 min. The fixed samples cleaned with PBS were placed under an inverted fluorescence microscope (FM) (Olymlusix71) for cell morphological observations. Other samples were treated with a 200 ml of 0.2% Triton X-100 solution for 15 min. The enzymatic activity of alkaline phosphatase (ALP) was measured when cells on the samples cracked. The remaining samples were also cleaned with PBS after 2 days. Then, the mesenchymal stem cells of mice were fixed with a 2.5% glutaraldehyde aqueous solution and were dehydrated in turns for 20 min with concentrations of 30%, 50%, 70%, 90%, and 95% ethyl alcohol aqueous solutions. Finally, isoamyl acetate was used to convert ethyl alcohol in the cells. The as-achieved samples were observed via scanning electron microscopy (SEM) (JSM-6360LA, Hitachi Co. Ltd., Japan). 3. Results and discussion 3.1. Morphology and composition analyses Generally, the reported electrocrystallized CP coatings on C/C were composed of amounts of discretely separated flaky or needle crystals due to the evolution of H2 during deposition process. This process resulted in a soft porous coating that is easy to scratch off. Thus, the current strategies, including the application of ultrasonic wave into the deposited electrolyte, ice bath, and addition of gelatin to CPs, was expected to increase the compactness of the asproduced coating, which further improved the bonding performance of CP coatings on C/C. SEM observations of the cross-section profiles in all coatings showed a thickness of 50 mm as shown in Fig. 1 (a). No changes in the thickness for different-type coatings were observed. The TG curves of the achieved coatings are shown in Fig. (b). The electrodeposited CP coating revealed a weight loss of 16.8%, which indicates that gelatin accounts for 16.8% of the weight of the coating. After two hydrothermal treatments, the weight loss of the coatings for the first and second treatments is 3.4% and 1.9%, respectively. The large weight loss before and after the first hydrothermal treatment may be correlated with the dissolution and recrystallization of brushite phases in the coating during the conversion of brushite into HA [33]. Moreover, the second hydrothermal treatment showed a lower weight loss than the first hydrothermal treatment, which could be related with the infiltration of Si and Na ions into HA crystals, thereby increasing the weight of coating. The morphologies of the coatings prepared via electrocrystallization (EC), ultrasonic-assisted electrocrystallization (UAE), UAE/ammonia hydrothermal treatment (AHT), UAE/AHT/sodium
silicate hydrothermal treatment (SHT) are shown in Fig. 1(c)e(j). Photos at 5 K or 10 K magnification (Fig. 1(c) and [d]) show that the directly electrocrystallized coating exhibited a loose and porous morphology, which resulted in easy exfoliation from C/Cs. Single addition of gelatin to the brushite coating could produce a continuous coating, eliminating the formation of the discretely separated crystals. Nevertheless, this process could not hinder the occurrence of pores resulting from the evolution of the H2 gas, which could be the reason for the poor adhesion of the achieved CP coating. Instead, the UAE coating, as shown in Fig. 1(e) and (f), constitutes compactly stacked, flake-like grains owing to the cavitation effects arising from the propagation of ultrasonic waves in the electrolyte. This coating is difficult to scratch off with nails, which indicates its good adhesion to C/C substrates. However, further observation at 10 K magnification showed gaps and cracks in the UAE coating, which is responsible for the blast effects of ultrasonic cavitations. After UAE was followed by AHT, the asproduced coating manifested a compact morphology with some long cracks, as shown in Fig. 1(c). In addition, short needle-like crystals were observed (Fig. 1(d)). However, after further hydrothermal treatment of the UAE/AHT coating in a sodium silicate solution, the cracks shortened, as presented in Fig. 1(e), which indicated that the cracks in the UAE/AHT coating underwent selfrepair to some degree. The crack self-repair is a result of high temperature and high water vapor pressure during the hydrothermal process. Moreover, coating observation under high magnification showed a needle-like morphology. After two hydrothermal treatments, no significant changes were found in the morphologies of the two HA coatings. Composition analyses conducted via EDS (Fig. 2) showed that all the coatings contain Ca, P, and O. The Ca/P atomic ratio was about 1.02, 1.65, and 1.60 for the UAE, UAE/AHT, UAE/AHT/SHT coatings, respectively. The Ca/P atomic ratios of these coatings were close to those of brushite and HA. Additionally, the USE/AHT/SHT coating contains Si and Na elements, whose atomic concentrations are 4.22% and 1.03%, respectively. 3.2. XRD analyses Fig. 3 shows the XRD diffraction patterns of the coatings on C/C. As shown in Fig. 3(a), the coating prepared by UAE, the diffraction peaks, excluding those of C/C substrates, were in accordance with powder brushite (JSPD09-0077). This result indicates that the UAE coating was composed of a brushite phase. Diffraction peaks could be assigned to hydroxyapatite (HA, JSPD 03-0747) after the ammonia hydrothermal treatment, as shown in Fig. 3(b). This process shows the presence of polycrystalline apatite. Further hydrothermal treatment in the sodium silicate solution also displayed the existence of HA, as presented in Fig. 3(c). However, a split peak was observed at (211). One split peak shifted a low degree, which indicates the existence of some HA crystals with a larger d-spacing. 3.3. FTIR analyses Fig. 4 shows the FTIR spectra of the coatings prepared on C/C, which shows that the peaks at 3483 and 1645 cm1 were generated by the OH of free water. By contrast, peaks at 1126, 1059, 660, and 573 cm1 were the characteristic peaks of PO3 4 . Absorption peaks at 1126 and 1059 cm1 were ascribed to SiO2 3 , which overlap with 2 3 those of PO3 4 . The peak overlaps of the SiO3 and PO4 groups were 2 attributed to the penetration of SiO3 into the HA lattice, thereby replacing PO3 partially. Furthermore, the peaks at 875 and 4 1428 cm1 corresponded to the absorption peaks of CO2 3 . Another possibility is that the peak at 1428 cm1 was generated by PeO(H) of HPO2 4 , which could be replaced by carbonate. The substitute of
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Fig. 1. Cross-section SEM photo: (a) TG profile and (b) SEM morphology of the coatings prepared via (c)/(d) EC, (e)/(f) UAE, (g)/(h) UAE/AHT, and (i)/(j) UAE/AHT/SHT.
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Fig. 4. FITR spectra of the as-prepared coatings on C/C substrates: (a) brushite/gelatin, (b) HA/gelatin, and (c) Si,Na-HA.
3.4. TEM analyses
Fig. 2. EDS spectra of the coatings prepared via (a)/(b) UAE, (c)/(d) UAE/AHT, and (e)/(f) UAE/AHT/SHT.
Fig. 3. XRD patterns of as-prepared coatings via (a) UAE, (b) UAE/AHT, and (c) UAE/ AHT/SHT.
To further reveal the nanostructures of the as-prepared coatings, photos of their TEM, high-resolution TEM, and electron diffraction are shown in Fig. 5. The TEM photos shown in Fig. 5(a) and (b) show that the brushite coating was composed of numerous nanoneedlesheet crystals. Generally, the brushite coating deposited by electrocrystallization comprised plate-like crystals. The difference in the morphologies of the two brushites indicated that gelatin could regulate the growth of brushite crystals. High-resolution transmission electron microscopy (HRTEM) results displayed one lattice fringe with a d-spacing of 0.247 nm that corresponds to the (022) crystal plane of brushite. The corresponding fast Fourier transmission (FFT) photo of HRTEM disclosed the existence of concentric rings composed of bright spots, which demonstrate its polycrystalline feature. After ammonia hydrothermal treatments, the two HA coatings revealed the presence of needle-like crystals, as shown in Fig. 5(d) and (e). The HRTEM photo (Fig. 5(e)) showed one lattice fringe with a d-spacing of 0.2811 nm, which is ascribed to the (211) plane of the HA coating. The corresponding FFT graph showed many bright spots, which showed a single-crystal feature, as shown in Fig. 5(f). Further sodium silicate hydrothermal treatment did not indicate change in the morphology of the as-achieved Si, Na-HA coating, as displayed in Fig. 5(h). However, one HRTEM image (Fig. 5(i)) of the Si, Na-HA coating showed a d-spacing of 0.2828 nm of the (211) plane, which is larger than that of the HA coating. This finding is consistent with that in the XRD analyses. The lattice that expands in the Si, Na-HA coating indicated that the infiltration of Na, Si ions into HA because of their larger ion radius compared with that of Ca and P ions. The FFT image showed many bright spots, which are results of a few Si, Na-HA single crystals, as presented in Fig. 5(j). 3.5. XPS analyses
PeO(H) with carbonate group is indicated by the formation of a btype HA coating [33,34]. The absorption peaks at 2922 and 1579 cm1 were produced by acyl amino, which was generated by the coupling of NeH stretching and NeH bending vibrations and CeN stretching vibration [35]. The presence of the acyl amino group indicated the formation of gelatin into the brushite coating on C/C substrates.
XPS was further employed to investigate the chemical structure and valence states of the achieved coatings, as presented in Fig. 6. The survey spectra in Fig. 6(a) indicated the coexistence of Ca, P, O, and C for all coatings. The Ca/P atomic ratio was calculated at 1.05, 1.69, and 1.62 for brushite/gelatin, HA/gelatin, and Si, Na-HA/ gelatin coatings, respectively, in accordance with the results of EDS analyses. The intensity of N1s peak was considerably weak for
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Fig. 5. TEM, HRTEM, and ED images of as-prepared coatings: (aec) brushite/gelatin; (def), HA/gelatin; (hej) Si,Na-HA/gelatin.
Fig. 6. (a) XPS survey spectra of the coatings and (b) narrow spectrum of Si2p of the Si,Na-HA coating.
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all the coatings. The intensities of the achieved coatings could not be easily viewed after conducting hydrothermal treatments. This finding indicates the low ratio of gelatin in the coating, which further validates the TG analytical results. In addition, Si, Na elements could be observed in the coating subjected to the two hydrothermal treatments from the survey spectrum. The Si2p peak was further analyzed for demonstration of the incorporation of Na, Si into HA lattice, as shown in Fig. 6(b). The Si2p peak could be mainly deconvoluted according to a GUASS fitting into three peaks at 101.4 eV, 102.3 eV, and 103.2 eV, which represent SieO, [SiO3] SieO, and SieOH [36,37]. This results show the existence of Si elements in the form of orthosilicate and metasilicate groups [38]. Thus, a small amount of sodium silicate had also infiltrated the HA 4 coating, besides the substitution of PO3 4 by SiO4 . 3.6. Scratch tests The adhesive performance of the coatings was examined via a scratch test and optical microscopy. Fig. 7(a) shows the scratch curves and the corresponding sites of the as-prepared coatings. The figure shows that the critical loading for brushite/gelatin, HA/ gelatin, and Si,Na-HA/gelatin coatings was 6.3, 4.1 N, and 7.72 N, respectively. The corresponding shear stress was calculated at approximately 42.4 MPa for the brushite/gelatin coating, 34.2 MPa for the HA/gelatin coating, and 47.1 MPa for the Si,Na-HA/gelatin coating. These results are based on the following equation:
.
tc ¼ ðHS Lc =pÞ1=2 R;
performance. Although the current adhesive strength of the electrocrystallized coating was lower than that of our previously prepared HA coatings via induction heating, it is at par with high-temperature coatings on C/Cs. Examples include ZrSi2 coatings on SiC-coated C/Cs prepared via supersonic atmospheric plasma spraying, which showed an adhesive strength of 5.5, 8, 11, and 8.9 N at a power of 30, 35, 40 and 45 KW [42] and (2) SiC coatings with a thickness of 50 mm for C/CeZrCeSi with a critical load of 3.25 N [43]. Our fabricated coating could be comparable to coatings on Ti alloys, such as TiO2 (8 N) and TiO2/HA (10N) coatings prepared via microarc oxidation and electrophoresis [44] and HA coating (7 N) deposited via RF magnetron sputtering [45]. These comparisons indicated a good adhesive performance of our prepared coatings. In summary, we reckon that the application of electrocrystallization technologies can possibly fabricate a well-bonded CP coating on C/C substrates. Further studies are being conducted in our laboratory. 3.7. Response behaviors of in vitro osteoblast Fig. 8 shows the proliferation curves of the marrow mesenchymal stem cells after 2, 4, and 6 days of cultures. All samples showed the tendency of an increase in the cell coverage with increasing culture duration. The HA/gelatin/Si-HA coating showed the best cell proliferation capability. The variance analyses with the
(1)
where tc is the shear stress; R is the radius of diamond stylus; Lc is the critical load; Hs is the Shore scleroscope hardness of C/C substrate. The stress strengths of the HA and Si,Na-HA coatings on C/C are greater than that of the C/C implanted into a mouse bone for 20 weeks (2.44 MPa) [19], which is high enough for handling before implant and strong enough to survive in the living body. By contrast, the adhesive strength of the HA/gelatin coating on C/Cs is lower than that of the HA/gelatin coating, which could be due to the loss of gelatin weight caused by the dissolution and crystallization of brushite phases. However, the Si,Na-HA/gelatin coating showed a higher bonding strength than HA/gelatin, which can contribute to the dope strengthening effect of Na and Si ions [39,40], as well as the self-repairing effect of the coating due to hydrothermal treatment [41]. The scratch traces of the coatings are also depicted in Fig. 6(b). The coatings experienced uniform plastic deformation and showed no break off from C/C substrates until the critical load was reached, thereby indicating their outstanding mechanical
Fig. 8. Comparison of cell coverage rates on different coating surfaces.
Fig. 7. (a) Scratch curves and (b) scratch trace morphologies of as-achieved coatings on C/Cs via optical microscopy.
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SPSS11.0 soft show that after 2, 4, and 6 days of cultures. The Pvalues of Si,Na-HA/gelatin coating were calculated at 1.2 103, 2.1 104, 2.6 104, respectively, which are smaller than 5.0 102. Thus, the results of the proliferation difference of the Si,Na-HA/gelatin coating was statistically significant. Fig. 9 shows that typical FM photos of the samples after a 2-day culture showed a significant increase in the number of mesenchymal stem cells in sequence from C/C, HA/gelatin coating and then Si, Na-HA/gelatin coating. The SEM photos of mesenchymal stem cells on different coating surfaces after a 4-day culture are shown in Fig. 10. Likewise, the number of cells on the Si,Na-HA/ gelatin surface was higher than that on C/C and the HA/gelatin coating. The cells were adhered well to the surfaces of C/C, HA/ gelatin, and Si, Na-HA/gelatin coatings and stretch their pseudopods and attached to the sample surfaces. The cell number and stretching degree of the cell pseudopods of on the Si,Na/HA coating surface were the highest among these cells, Moreover, the cells on the gelatin/Si-HA coating surface spread continuously. ALP was generally used to characterize the mature degree of differentiation of osteoblast on the coating surface and the osteogenesis and mineralization ability of cells. Changes in the culture duration are shown in Fig. 11. The activity of ALP increased remarkably as the inoculation time increased. Of the three samples, Si,Na-HA/gelatin coatings similarly showed the best ALP performance. These results suggest that the gelatin/Si-HA was more helpful to the differentiation and maturing of cells, showing the best osteoblast compatibility. Similarly, the two-step hydrothermal treatments have no diverse effects on the cell biocompatibility of gelatin in the coatings. In conclusion, our prepared Si,Na-HA coating on C/C does not only possess good mechanical performance but also osteoblast compatibility, which exhibits good potential for clinical application as bone implant materials.
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Fig. 11. Changes in the activities of ALP on different surfaces with culture duration.
on a C/C surface through the application of an ultrasonic and ice bath to undergo electrocrystallization. Adhesive strength could reach a critical load of 6.3 N. The brushite/gelatin coating could be transformed into an HA/gelatin coating with nanoneedle crystals after the ammonia hydrothermal treatment. The adhesive strength of the coating decreased to a critical load of 4.1 N. Further hydrothermal treatment in the sodium silicate solution led to the achievement of a Na, Si-doped HA coating on C/Cs. Adhesive strength could achieve a critical load value as high as 7.72 N, which is equivalent to a shear strength of 47 MPa. In vitro experiments showed that the Si, Na-HA/gelatin coating exhibited better cell compatibility than the C/C and the HA/gelatin coatings.
4. Conclusions A compact brushite/gelatin coating was successfully prepared
Fig. 9. FM images of bone marrow stem cells on different surfaces on the second day.
Fig. 10. SEM photos of bone marrow stem cell on different surfaces on the fourth day.
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