Journal of Materials Science & Technology, 2016, 32(9): 956-965
doi: 10.1016/j.jmst.2016.07.012
Effect of Amino-, Methyl- and Epoxy-Silane Coupling as a Molecular Bridge for Formatting a Biomimetic Hydroxyapatite Coating on Titanium by Electrochemical Deposition
Guoxin Tan1,*,, Kongyou Ouyang1, Hang Wang1, Lei Zhou2, Xiaolan Wang2, Yan Liu1, Lan Zhang3, Chengyun Ning2
1 School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, China
2 College of Materials Science and Technology, South China University of Technology, Guangzhou 510641, China
3 State Key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University, Xi'an 710049, China
The objective of this study was to determine the role of functional groups of silane coupling on bioactive titanium (Ti) surface by electrochemical deposition, and calcium phosphate (CaP) coating, as well as bone cell adhesion and proliferation. Methyl group (—CH3), amino group (—NH2), and epoxy group (—glyph name—C(O)C) were introduced onto the bioactive Ti surface using self-assembled monolayers (SAMs) with different silane coupling agents as molecular bridges. The effect of the surface functional groups on the growth features of the CaP crystals was analyzed (including chemical compositions, element content, minerals morphology and crystal structure etc.). CH3-terminated SAMs showed a hydrophobic surface and others were hydrophilic by contact angle measurement; NH2-terminated SAMs showed a positive charge and others were negatively charged using zeta-potential measurement. Scanning electron microscopy results confirmed that flower-like structure coatings consisting of various pinpoint-like crystals were formatted by different functional groups of silane coupling, and the CaP coatings were multicrystalline consisting of hydroxyapatite (HA) and precursors. CaP coating of CH3-terminated SAMs exhibited more excellent crystallization property as compared to coatings of —NH2 and —C(O)C groups. In vitro MC3T3-E1 cells adhesion and proliferation were performed. The results showed that CaP coatings on silane coupling functionalized surfaces supported cell adhesion and proliferation. Thus, these functional groups of silane coupling on Ti can form homogeneous and oriented nano-CaP coatings and provide a more biocompatible surface for bone regeneration and biomedical applications.
Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10].
The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far.
A self-assembled monolayer (SAM) technique that employs multiple steps has been used to investigate the effect of functional groups on apatite formation on Ti-based substrates in biomineralization[21]. For example, Toworfe et al.[22] reported amine (—NH2), carboxyl (—COOH) and hydroxyl (—OH) functionalized surfaces for biomineralization and OH-terminated surface enhanced CaP nucleation and growth. And —OH and —CH3 functional groups also exhibited different behaviors in terms of hydroxyapatite formation[23]. These results suggest that functional groups are quite beneficial for guiding the aggregation of Ca2+, PO43-, OH- and prenucleation clusters, and then promoting apatite minerals formation with diverse micro or nano structures[24] and [25].
In this study, the functional groups (—CH3, —NH2 and —C(O)C) were introduced on titanium surface by silane coupling agent used as a “bridging molecule.” We hypothesize that the functional groups of the Ti surface can influence interactions between mineral and Ti interface in electrodeposition process, thereby regulating the crystallinity characteristics of electrochemically deposited CaP coatings. The objective of this study was to determine the effect of the functional groups in electro-nucleation, crystallization and growth of calcium phosphate on bioactive titanium surface, and interaction with bone-forming cells in vitro of electrochemically deposited CaP coatings.
2. Experimental
2.1. Morphology and structure
Square Ti samples of approximately 25 mm × 25 mm were prepared from 99.7% medically pure titanium sheet (0.1 mm thick) (Baoji Qichen New Material Technology Co., Ltd., China). These were rinsed in absolute ethyl alcohol, then in acetone for 10 min, and finally in distilled water for 10 min. The samples were subsequently immersed in a 7:3 (v/v) mixture of concentrated H2SO4 (Sigma-Aldrich, USA) and 30% H2O2 (Sigma-Aldrich, USA) (piranha solution) for 30 min at 60 °C (named pTi)[26]. The samples were sonicated with distilled water for 10 min, and then dried in N2 atmosphere at room temperature.
2.2. Functionalization of pTi substrates using SAMs
Octadecyltrichlorosilane (OTS) (90%, Sigma-Aldrich, USA), 3-aminopropyltriethoxysilane (APTES) (98%, Sigma-Aldrich, USA) and glycidoxypropyltrimethoxysilane (GPTMS) (97%, Aladdin, China) organosilanes were grafted onto the titanium oxide surfaces. The titanium substrates were performed under a closed container environment, by immersing into a solution of the silane solution in an organic solvent (toluene: 99.8% anhydrous, Sigma-Aldrich) for 24 h at 25 °C. The concentrations (v:v) of the organic solvent and the silanes were optimized for each SAM type, i.e. OTS: 1% toluene, APTES: 2% toluene, and GPTMS: 5% toluene (named Ti-OTS, Ti-APTES, Ti-GPTMS respectively). After being immersed for 24 h, the samples were sonicated serially in three different solvents (toluene, absolute ethyl alcohol and ultrapure water) for 10 min, in order to enhance the organization of the silane monolayers on the surfaces and remove any physisorbed multilayers.
The calcium phosphate deposition experiment was carried out in the presence of SAMs sample by electrochemical deposition. An electrochemical cell in a standard reactor was configured to work with two electrodes, using platinum strings for counter electrode[10]. A negative potential was applied to the working electrode, where pTi samples of 4 cm2 were placed. Calcium phosphate coatings on titanium were obtained from stirred aqueous solution of 4.2 × 10-3 mol/L CaCl2 (AR, 96%, Aladdin, China) and 2.5 × 10-3 mol/L NaH2PO4 (AR, Aladdin, China), pH = 6.0, at 80 °C. The electrodeposition was performed at the current density of 2.5 mA/cm2 and deposition time of 45 min. Then the samples were dried in N2 atmosphere at room temperature (named pTi-CaP, Ti-OTS-CaP, Ti-APTES-CaP, Ti-GPTMS-CaP, respectively).
2.4. Cell adhesion
In vitro biocompatibility of the CaP coated SAMs sample with MC3T3-E1 mouse osteoblasts (ATCC CRL-2593) was evaluated using cell adhesion experiment. Cell were seeded directly on the samples (sterilized by ultraviolet-radiation for at least 2 h prior to the cell seeding process) at a density of 2 × 104 cells/mL in 24-well cell culture plates (500 µL/well) (Corning, USA) and incubated in the α-modified Eagle's minimum essential medium (α-MEM) (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 °C[27]. After incubation for 24 h, the cell viability was expressed in the percentage as following: the typical cellular adhesion and spread morphologies on the CaP coated different pTi surface were evaluated by scanning electron microscopy (SEM) after Pt/Au sputtering.
2.5. Cell proliferation
A 500 µL cell suspension was seeded on each specimen at a density of 2 × 104 cells/mL and cultured in α-MEM with 10% FBS. The cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (98%, Aladdin, China) assay on 1, 3, 5 and 7 days. Briefly, at each prescribed time point, the specimens were rinsed gently three times with PBS, 40 µL MTT (5 mg/mL) solution and 400 µL α-MEM without FBS were added every well, and the specimens were incubated at 37 °C for 4 h to form formazan crystals[28]. Then the formazan was dissolved in dimethyl sulfoxide (DMSO) (99.9%, Sigma-Aldrich, USA) and shocked for 10 min, and the optical density (OD) was measured at 570 nm using a microplate reader (Multiskan FC, Thermo, USA).
2.6. Materials characterization
Fourier-transformed infrared (FTIR) spectra of surface titanium following each reaction step, recorded in an ATR, were obtained using FTIR (Vector 33, Germany Bruker Co.). ATR-FTIR spectra were obtained for every films type, with each spectrum averaged from 10 scans collected from 600 to 4000 cm-1 at 4 cm-1 resolution.
Contact angle measurements using ultrapure water and diiodomethane respectively were made on the samples using a goniometer setup built in house comprising a Gilmont microsyringe (OCA15, Germany Dataphysics Co.) and a CCD camera. The images captured were analyzed from this software to evaluate the contact angles. Four spots were measured on each sample and averaged. All the measurements were made immediately after sample preparation in order to minimize contamination.
For a solid-liquid system, the contact angle was related to the surface tension of the liquid and solid by Owens-Wendt-Kaeble's equations (1), (2) and (3).
where θ is the contact angle; and
and
are the dispersion and polar surface energy of the solid and the liquid, respectively, and γSγS is the sum surface energy of the solid phases. Using archival surface force values of
with ultrapure water and diiodomethane, the values of γSγS could be calculated[29].
Zeta-potential is generated when liquid is forced to flow directly through a small gap formed by two sample surfaces with pressure. Charge carrier bound in double layer will be removed. The potential can be measured between two electrodes. The zeta-potential measurement was performed by using a solid surface zeta potentiometer (Anton Paar GmbH, Graz, Austria). For each sample, two zeta-potential/pH value functions had been measured in 1 × 10-3 mol/L KCl solution. For statistical reasons, three streaming potentials were measured at each pH value. The mean value of these data was used to calculate the potential/pH function.
The CaP coated samples were sputtered with Pt/Pd and FE-SEM (ZEISS Ultra 55, Germany) which used for SEM observations and chemical analysis by energy dispersive spectroscopy (EDS) under an accelerating voltage of 5 kV and 20 kV respectively. Meanwhile, the working distance ranged from about 5 mm to 10 mm.
X-ray photoelectron spectra (XPS) were recorded using a Kratos Axis Ultra system with monochromatized Al Ka radiation. Measurements were carried out using a takeoff angle of 15° with respect to the sample surface with a typical analyzed area of 1 mm2. Survey scans over a binding energy range of 0-1200 eV were taken for each sample with a constant detector pass energy range of 160 eV, followed by a high-resolution XPS measurement (pass energy 40 eV) for quantitative determination of binding energy and atomic concentration.
Crystallographic features of the CaP minerals formed on the pTi surface, and the functionalized surfaces were determined by X-ray diffraction (XRD) (D8 Advance, Bruker, Germany) useful for thin film applications, on a Rigaku diffractometer with a Cu Kα irradiation (18 kW HV generator, PW at 40 kV and 40 mA). The samples were scanned in the Bragg angle, 2θ, in a range of 10-70° at a scan rate of 1°/min, with a sampling interval of 0.02°. Crystallite sizes of CaP coatings on different surfaces could be calculated by Scherrer formula (4) with the full width at half maximum (FWHM).
where D(hkl) is crystallite size estimated using reflection (hkl) and λ is the X-ray wavelength (0.15418 nm) in this study, k is a constant related to crystallite shape, normally taken as 0.89. β corresponds to the experimentally determined full width at half of the maximum intensity of the peak in radians. The conversion of β(hkl)(rad-2θ) from β(hkl) (degree-2θ) was obtained using equation (5);
Based on Scherrer formula, the CaP crystallite size was determined upon using the XRD data emanating from the (002) and (211) reflection crystallographic plane peaks. Average size was directly obtained from Jade 5.0 software with all crystallographic plane peaks[30].
Transmission electron microscopy (TEM) investigations were performed under a JEOL JEM 2100 HR TEM operating at 200 kV (LaB6 filament). The CaP coatings were scraped off by blade, then dispersed in absolute ethyl alcohol and added dropwise onto a 3 mm diameter carbon coated copper TEM grid. TEM images were recorded by using a Gatan Model 694 Slow Scan CCD camera, where the morphology and the particle size of the dried and sintered CaP samples were observed and analyzed.
2.7. Statistical analysis
All experiments were conducted at least three times and all values were reported as the mean ± standard deviation. Statistical analysis was carried out using Student's t-test (assuming unequal variance). The statistical difference between two sets of data was considered significant when *p <0.05 and **p <0.01.
3. Results and Discussion
3.1. Schematic illustration of CaP nucleation
In order to illustrate the mechanism between CaP nucleation and growth with bridge molecule influencing on pTi surface, Fig. 1 shows the hypothetical schematic diagram of the SAMs and electrochemical deposition CaP coatings on the titanium surface (using pTi as cathode). It is obvious that hydroxyl groups were generated on titanium surface after treating with piranha solution, which could be stably bonded titanium surface with silane coupling as a molecular bridge (forming Si—O bond)[31]. On the one hand, the large amount of hydroxyl ions was generated through hydrogen evolution reaction of initial stage in electrochemical deposition, induced calcium phosphate ions rapid nucleation of initial precipitates[10]. On the other hand, in the case of the functional groups on silane coupling, the initial products that were assigned to HA precursors were rapidly precipitated as soon as the calcium and phosphate solutions were electrolyzed. The large amount of nuclei facilitated the heterogeneous nucleation during deposition, leading to precipitation and formation of apatite on the SAMs surface[15] and [21]. The nucleation of calcium phosphate particles on functionalized surface would yield diverse regular shape and relatively small size. The hydrogen evolution occurred at cathode interface and produced OH-, which could combine other ions in the electrolytes, such as Ca2+ and PO43-, thus causing nucleation of hydroxyapatite and its precursors. Meanwhile, the functionalized pTi surface charge and wettability did not resemble that of the bioactive pTi. The positive charges surface (—NH2 group) could attract negative charges (OH-, PO43-) while negative charges surface (—CH3 and —C(O)C group) could attract positive charges (Ca2+). Therefore, NH2-terminated surface (Ti-APTES) as a molecular bridge with positive charge had priority to attract OH- and PO43-, consequently Ca2+ was attracted to cathode surface and gradually formed hydroxyapatite[32]. Moreover, negatively charged surface (potential absolute value: pTi < Ti-OTS < Ti-GPTMS) had priority to attract Ca2+, but the number of negative charge on pTi was less than Ti-OTS (—CH3 group) and Ti-GPTMS (—C(O)C group), hydroxyl ions was produced by hydrogen evolution reaction firstly combined with Ca2+ on pTi surface. On the Ti-OTS surface, OH- and PO43- combined with Ca2+ at the same time, and because of coulombic repulsion between negative charge and anions, OH- and PO43- successively combined with Ca2+ that induced CaP minerals nucleation and growth. Consequently, the bridging molecule on pTi surface possessed different interfacial properties with nanoscale CaP particles that showed homogeneous and orderly surface morphology. This hypothesis will be discussed and confirmed in the following section.
Fig. 1.
Schematic illustration of electro-nucleation, crystallization and growth for CaP coatings on different functionalized Ti surfaces (—CH3, —NH2 and —C(O)C groups) by ECD.
3.2. Surface properties characterization of SAMs
In order to prove the SAMs being successfully grafted on pTi surface, ATR-FTIR spectra was used to analyze representative groups in this work. Fig. 2 shows the FTIR spectra corresponding to samples modified by different SAMs. Distinctly, pTi surface had marked peak in the range of 3200-3500 cm-1 which contributed to O—H vibration, because Ti was oxidized in H2SO4/H2O2 solution. After OTS was grafted, the stretching vibrations of OH groups in pTi were shifted from 3250 cm-1 to 3620 cm-1 in Ti-OTS, indicating that the hydrophilic OH groups were consumed by the OTS coating. Meanwhile, different SAMs grafted on pTi surface, and the intensity of H—OH vibration peak at 1637 cm-1 decreased compared to pTi surface[22]. Additionally, once the SAMs bonded with pTi surface, the new peaks located around 1056 cm-1 attributed to siloxane group (Si—O) from OTS, APTES and GPTMS, while 2929 and 2855 cm-1 attributed to methylene group (—CH2)[33], indicating that silane coupling agent as a molecular bridge had been grafted onto the surface of titanium substrate. The symmetric bending vibration of —NH2 in Ti-APTES appeared at 1536 cm-1[34]. Meanwhile, the presence of the peak at 910 cm-1 suggested that epoxy group (—C(O)C) in GPTMS was associated with pTi surface[35]. It was showed that the intensity of siloxane group (Si—O) at 1056 cm-1 after 1% OTS treatment decreased compared to 2% APTES and 5% GPTMS, and the absorption bands of Ti-OTS at 2929 and 2855 cm-1 were most obvious due to the presence of methylene group in octadecyl chain.
Fig. 2.
ATR-FTIR absorbance spectra of pTi, CH3-terminated, NH2-terminated and —C(O)C-terminated Ti surface. O—H peak of pTi, C—H peak of Ti-OTS, —NH2 peak of Ti-APTES and epoxide of Ti-GPTMS were obviously observed. (ν (O—H): 3200-3500 cm-1, ν (C—H): 2929 and 2855 cm-1, ν (Si—O): 1056 cm-1, δ (—N—H): 1536 cm-1 and ν (—C(O)C—): 910 cm-1).
Macroscopically, the wettability of the SAMs was probed by means of the contact angle measurement using ultrapure water and diiodomethane. Sessile drop method is a useful and convenient way to track the change in wettability on the SAMs surface[36]. The water and diiodomethane contact angles could be used for calculating interfacial tension on each surface. The contact angle on pTi with SAMs strongly depended on the surface functional groups. As shown in Fig. 3, the water contact angle of the pTi was 18.2 ± 1.1° by oxidation in piranha solution. However, the pTi surface grafted with OTS was made highly hydrophobic, the water contact angle increased from with OTS is hydrophobic to 109.8 ± 2.1° with the hydrophobic group alkyl (—CH2—). While APTES and GPTMS were grafted on the pTi, the water contact angles increased from 18.2 ± 1.1° to 52.1 ± 4.5° and 41.7 ± 2.6° respectively, which indicated higher hydrophobicity on the APTES and GPTMS surfaces than that on pTi. Diiodomethane liquid was employed to verify the oleophilic property of the pTi surface. In Fig. 3, the diiodomethane contact angles with different SAMs could also be observed, which indicated different lipophilicity surface. In general, a smaller water contact angle means a higher distribution of hydrophilic groups on the surface that provides a relatively higher adhesion tension[37]. The surface energies in Table 1 were calculated by equations (1), (2) and (3). Evidently, surface energy was decreased from 71.12 mJ/cm2 (pTi) to 37.50 mJ/cm2 (Ti-OTS), 56.00 mJ/cm2 (Ti-APTES) and 62.12 mJ/cm2 (Ti-GPTMS), respectively. The three functionalized pTi surface showed lower surface energy compared to the pTi surface due to the presence of functional groups. Therefore, higher surface energy (pTi) could not conduce to form nanoscale particles, and lower surface energy could benefit to nucleation and formation of nanoscale CaP particles.
Fig. 3.
Water and diiodomethane contact angles of different functionalized pTi surfaces. Ti-OTS (—CH3 group) presents hydrophobic surface and others (pTi, —NH2 and —C(O)C groups) present hydrophilic surface.
Table 1
Table 1
Surface energy on different surfaces of titanium
Table 1
Surface energy on different surfaces of titanium
In addition, superficial charges play an important role in electrostatic adsorption ions of electrolyte, especially in inducing CaP nucleation and growth[32]. The charges of SAMs surface in the designated pH area was shown in Fig. 4(a). With the increase of pH, zeta-potential obviously decreased. Additionally, as shown in Fig. 4(b), Ti-APTES surface (—NH2 group) was covered with positive potential and others (pTi, —CH3 and —C(O)C groups) were covered with negative potential at pH = 6. The absolute zeta-potential comparison sequence: Ti-GPTMS (-41.21 mV) < Ti-OTS (-32.54 mV) < pTi (-26.79 mV) < Ti-APTES (1.52 mV), reflected that surface functional groups could provide different adsorption and integration sites by electrochemical deposition. Surface functional groups as a molecular bridge displayed different surface properties. Therefore, the influences on CaP nucleation were expected to be different, which would be discussed in the following section.
Fig. 4.
pH dependence of the zeta-potential (a) and zeta-potential at pH = 6 (b) of different functionalized Ti surface. Ti-APTES (—NH2 group) surface covered with positive potential and others (pTi, —CH3 and —C(O)C groups) were negative potential at pH = 6.
3.3. Surface morphology and CaP element distribution of coatings on Ti surface
Fig. 5 shows SEM micrographs of CaP coating on pTi surface: (a) oxidized-treatment, (b) Ti-OTS, (c) Ti-APTES, and (d) Ti-GPTMS. All samples exhibited various surface morphologies by deposition in the electrolyte solution including Ca2+ and PO43- under identical process conditions. As shown in Fig. 5(e), pTi surfaces were covered with flower-like CaP precipitates clustered with submicro pinpoint-like crystals, whereas Ti-OTS surface (—CH3 group) exhibited flower-like CaP precipitates clustered with nano pinpoint-like crystals (Fig. 5(f)). It was clearly observed in Fig. 5(g) that flower-like CaP precipitates clustered with nano squame-like crystals were thickly covered on Ti-APTES surface (—NH2 group), and flower-like CaP precipitates clustered with nano flake-like crystals (Fig. 5(h)) on Ti-GPTMS surface (—C(O)C group). Apparently, dense and homogeneous CaP coatings were achieved on SAMs surface[26], and CaP coatings on pTi surface was sparse and disorderly. Moreover, in order to explore the Ca and P element distribution, element mapping scanning mode of EDS was used in this work. As showed in Fig. 6, the green points show Ca element and red points show P element. Compared with CaP coatings on functionalized pTi surface, there were sparsely Ca and P elements distributed on pTi surface (Fig. 6(a)). Meanwhile, Ti-APTES exhibited denser Ca and P distributions of coating (Fig. 6(c)) comparing with Ti-OTS (Fig. 6(b)) and Ti-GPTMS (Fig. 6(d)). In conclusion, the surface energy and surface potential showed that the surface functional groups as a molecular bridge determined the solid/ion cluster interfacial energies[22], then different functionalized pTi surface exhibited different nucleation abilities of calcium phosphate with diverse morphology.
Fig. 5.
SEM and EDS of CaP coatings on different surfaces with different functional groups ((a) pTi, (b) Ti-OTS (—CH3), (c) Ti-APTES (—NH2), and (d) Ti-GPTMS (—C(O)C)), and (e-h) were the magnified images of (a-d), respectively. pTi were covered with flower-like CaP precipitates clustered with submicro pinpoint-like crystals, whereas others exhibited diverse nano pinpoint-like crystals (Ti-OTS), nano squame-like crystals (Ti-APTES) and nano flake-like crystals (Ti-GPTMS).
Fig. 6.
EDS element mapping of CaP coatings on different surface ((a) pTi, (b) Ti-OTS, (c) Ti-APTES, (d) Ti-GPTMS); Ca and P elements of coatings distributed on pTi were the most sparse, while Ti-APTES surface (—NH2 group) were more dense than other surfaces (—CH3 and —C(O)C group).
3.4. Surface element composition analysis by XPS
In this study, the surface composition of CaP coatings deposited on titanium was confirmed by XPS spectra. To further investigatethe interaction between SAMs and HA, XPS analysis was carried out and the results are shown in Table 2 and Fig. 7. Table 2 confirms that the coatings surface consisted of Ca, P, Si and N elements. As shown in Fig. 7, a characteristic doublet peak located at 459.0 and 464.7 eV were attributed to Ti2p[38], showing that TiO2 was the main component after oxidation with piranha solution, but others did not significantly appear to Ti2p doublet peak at this position, which demonstrated that the coating on pTi surface was very thin (in accordance with the results of SEM and EDS). Meanwhile, all of them exhibited stronger O KLL (Auger), Ti LMM (Auger), Ti2s and O1s peaks on the corresponding position. Si2s and Si2p peaks could be observed at 153 eV and 102.2 eV (Table 2), indicating Si—O bonded on the pTi surface. N1s derived from the APTES silane of bridging molecule was detected at the binding energy of 399.6 eV in a total amount of 1.08 at.%. Moreover, their compositions in Table 2 were lower than other elements because CaP coatings were entirely covered on SAMs surface[39]. Functional groups caused an increase of Ca atoms on the pTi surface from 6.6 to 12.11 at.%, and P atoms on the pTi surface increased from 4.92 to 7.27 at.%. The binding energy of Ca and P elements were almost not changed (Table 2), indicating that functional groups did not affect their valence state. All of these demonstrated that the surface mineral coatings consisted of Ca2+, PO43- and OH-.
Table 2
Table 2
XPS spectral components of different CaP coatings surface
Samples
Elemental composition (at. %)
C
O
N
Ca
P
Si
pTi
38.37
48.65
-
6.6
4.92
-
Ti-OTS
34.68
45.80
-
11.90
6.31
1.31
Ti-APTES
32.28
48.66
1.08
10.08
6.89
1.01
Ti-GPTMS
34.75
44.66
-
12.11
7.27
1.21
Table 2
XPS spectral components of different CaP coatings surface
Fig. 7.
High-resolution XPS full spectra CaP coatings of pTi (a), Ti-OTS (b), Ti-APTES (c) and Ti-GPTMS (d) were distinctly obtained. N1s (399.6 eV) of APTES and Si2p (about 102.2 eV) of silane coupling agents were clearly observed. Ca2s, Ca2p, P2s and P2p peaks markedly appeared on spectra.
3.5. TEM analysis and XRD patterns
The results of the microstructure and crystal property can be obtained via TEM observation[40]. Fig. 8(a1-d1) shows a representative TEM image of diverse pinpoint-like CaP crystals on different pTi surfaces, where large numbers of uniform flower-like precipitates clustered with their functionalized nucleation sites exist. A submicro pinpoint-like crystal can be observed in Fig. 8(a1) on pTi surface; however, more obvious nano pinpoint-like crystals with SAMs are shown in Fig. 8(b1-d1). CaP coating on NH2-terminated surface presented much more weeny needle-like crystals compared with that of Ti-OTS (—CH3 group) and Ti-GPTMS (—C(O)C group). More details about the structure of the needle-like crystals were investigated by the selected area electron diffraction (SAED) pattern (inset of Fig. 8(a2-d2)). The corresponding SAED pattern that was taken from individual needle-like shape confirmed that the crystals were well-crystallized. The average size of the CaP nano-crystals accorded with the sizes calculated by equations (4) and (5) from XRD data (Table 3). The CaP nano-crystals were confirmed to be multicrystalline minerals from the rings of the SAED pattern (Fig. 8), which mainly consisted of HA and little HA precursors such as DCPA, DCPD, and OCP[41].
Fig. 8.
TEM images, SAED graphs and XRD patterns of CaP coatings on different Ti surface ((a) pTi, (b) Ti-OTS, (c) Ti-APTES, and (d) Ti-GPTMS). The CaP coatings were multicrystalline minerals that consisted of HA and precursors (DCPA, DCPD, and OCP).
Table 3
Table 3
Crystallite sizes of CaP coatings on different surface calculated by Scherrer formula with the full width at half maximum (FWHM)
Samples
(002)
(211)
Average size/nm
FWHM
Crystal size/nm
FWHM
Crystal size/nm
pTi
0.199
40.55
0.166
49.27
181.7
Ti-OTS
0.253
31.89
0.384
21.30
100.3
Ti-APTES
0.547
14.75
0.216
37.87
95.9
Ti-GPTMS
0.650
12.42
0.357
22.91
88.1
Table 3
Crystallite sizes of CaP coatings on different surface calculated by Scherrer formula with the full width at half maximum (FWHM)
Fig. 8(e) presents the X-ray diffraction (XRD) patterns of the CaP minerals deposited on functionalized pTi surface. In all patterns, well-defined 25.9° (002) and 31.8° (211) planes were observed, and 32.2° (112), 32.9° (300) and 45.3° (203) planes appeared on partial patterns. The major peaks were well matched with the crystalline data of HA (PDF card #54-0022), justified by the nature of the HA crystal structure. On the one hand, parts of HA precursors shown in Fig. 8(e), for instance OCP (PDF card #26-1056), DCPA (PDF card #09-0080) and DCPD (PDF card #09-0077), were partly formed in the process of electrodeposition and showed low crystallinity. Evidently, the peaks of pTi exhibited much stronger than that of other functional groups surface because of the sparse CaP coating on pTi. On the other hand, crystalline data of Ti-OTS surface (—CH3 group) was well matched with HA compared with those of other functional groups (—NH2 and —C(O)C group). Based on Scherrer formula, the CaP crystallite sizes were determined by the XRD data emanating from the (002) or (211) crystallographic planes, and average sizes of crystals were directly obtained from Jade 5.0 software. As shown in Table 3, CaP crystallite size on pTi surface was larger (submicron scale, 181.7 nm) compared with that on functionalized pTi surface, while CaP crystals growing on functionalized pTi surface with silane coupling were smaller (nanoscale, 100.3 nm, 95.9 nm and 88.1 nm, respectively). Additionally, these results were in accordance with the TEM measurement.
3.6. Cell interaction with CaP minerals
Cells are inherently sensitive to their surrounding microenvironments, such as bio-chemical signals and surface topography of the materials[20] and [42]. It is generally accepted that cells use filopodia for spatial sensing in their movement and spread on functional surfaces[43]. Fig. 9 shows the morphology of MC3T3-E1 cells on CaP coatings discs with functional groups after culturing for 24 h, and the filopodia of cells were observed on all pTi surfaces. However, the MC3T3-E1 cells displayed different shapes on the different morphology of CaP coatings. Cells on pTi-CaP and Ti-OTS-CaP showed an elongated form and stretched less filopodia (Fig. 9(a, b), and cytoskeleton appeared to stand off the surface on pTi-CaP and Ti-OTS-CaP. In addition, the portion of cytoplasmatic prolongations appeared and spread in all directions on Ti-APTES-CaP and Ti-GPTMS-CaP (Fig. 9(c, d). Shi et al.[44] reported that nano-HA particles could obviously stimulate osteoblastic adhesion compared with micro-HA particles. It was shown that the initial adhesion behavior of cells may be ascribed to the enhanced interfacial adhesion on HA nano-morphology by different molecular bridge. Moreover, in this study, nanoscale surface of HA seemed to exhibit the trend of spreading more filopodia.
Fig. 9.
Representative SEM images of MC3T3-E1 cells on the different CaP coating surface after culturing for 24 h ((a) pTi, (b) Ti-OTS, (c) Ti-APTES, (d) Ti-GPTMS). Cell density: 2 × 104 cells/mL.
The mechanism of the MTT assay involves that pale yellow MTT substance will be converted to dark blue formazan crystals only by the viable cells[45]. Therefore, MTT assay was employed to confirm the MC3T3-E1 cells adhesion and proliferation on pTi and different functionalized pTi surfaces with CaP coatings. The results of cell proliferation behaviors are shown in Fig. 10. The values of optical density (OD) were varied on different surfaces for 1, 3, 5 and 7 days of culturing time following a similar trend. After 1 day of culture, the cell numbers on Ti-OTS-CaP surface (—CH3 group) were higher than others (—NH2 and —C(O)C group), and the trend was almost similar during 3 days, except that Ti-GPTMS-CaP showed lower activity compared with pTi-CaP. Up to 5 days, all of the OD values significantly increased as opposed to OD values of 3 days. After 7 days of culture, OD value of pTi-CaP did not obviously increase, and the other three functionalized pTi surfaces exhibited a similar trend on preceding days. All these results demonstrated that there was a high yield of proliferative activity (non-toxicity) in the osteoblast cells after a long cell culture time. The Ti-OTS-CaP surfaces exhibited higher cell viability than others during cell culture in 7 days. Meanwhile, the trend of MTT assay on different functionalized surfaces was inconsistent with the SEM micrographs (Fig. 9). It is possible that HA nano-morphology can be conducive in improving interfacial adhesion of cells due to the provided space for filopodia[46]. According to Okada et al.[47], the smaller size of nanostructures would adversely affect osteoblasts. Therefore, the cellular response to HA substrates may be influenced by the balance in size between the dense micro/nano structure and the focal adhesions of the cells.
Fig. 10.
Viability/proliferation (MTT assay) of MC3T3-E1 cells on different CaP coatings surfaces after 1, 3, 5 and 7 days. Cell density: 2 × 104 cells/mL. The data are reported as the mean ± standard deviation (n = 4), *p <0.05, and **p <0.01 relative to controls (pTi). Error bars represent the SD of four independent samples.
4. Conclusion
In summary, chemically modified pTi surfaces of functional groups created by SAMs technology can induce CaP nucleation, crystallization and growth, with well-controlled nanoscale crystals through electrodeposition. Surface functional groups acting as nucleation sites induced the bio-activation of pTi substrate, causing the rapid precipitation of calcium and phosphate ions to form HA and precursors. pTi surface with —CH3, —NH2 and —C(O)C groups in silane coupling possessed higher order and homogeneous CaP coatings than pTi, which is attributed to the surface potential and lower surface energies of functional groups as a molecular bridge. In vitro cell adhesion and proliferation also demonstrated that all of these functional groups terminated CaP coatings displaying good cell viability tendency. The results highlight the importance of functional groups on the bioactivity of bone tissue interface. Functionalized SAMs are a crucial parameter that should be considered in the design of CaP-based nano materials for tissue regeneration applications.
Acknowledgments
This study was supported by the National Key Basic Research Program of China (No. 2012CB619100); the National Natural Science Foundation of China (No. 51541201, 51372087); the Science and Technology Planning Project of Guangdong Province, China (No. 2014A010105048); the Natural Science Foundation of Guangdong Province, China (No. 2015A030313493) and the State Key Laboratory for Mechanical Behavior of Materials, China (No. 20141607).
The authors have declared that no competing interests exist.
H.S.Alghamdi, R.Bosco, J.J. van den Beucken, X.F. Walboomers, J.A. Jansen. Biomaterials, 34(2013), pp. 3747-3757
This study hypothesized that modification of titanium implant surface, e.g. by the deposition of inorganic/organic coatings, can significantly improve the implant-bone response compared in osteoporotic vs. healthy conditions. After osteoporosis was induced in 15 female Wistar rats by ovariectomy (OVX) and confirmed by in vivo micro-CT analysis, implants coated with calcium phosphate (Cap) or collagen type-I and non-coated implants were placed into bilateral femoral condyles. Another 15 sham-operated rats served as controls. Twelve weeks after implantation, micro-CT bone volume (%BV) and histomorphometrical bone area (%BA) were lower around control implants in osteoporotic rats (BV = 60.4%, BA = 43.8%) compared to sham-operated rats (BV = 74.0%, BA = 62.0%). Interestingly, CaP and collagen type-I surface coatings enhanced bone-to-implant contact (%BIC) compared to non-coated implants in osteoporosis (51.9%, 58.2%) as well as in sham-operated (69.7%, 64.4%) groups. The study confirmed that an osteoporotic condition has a significant effect on the amount of bone present in close vicinity to implants. Evidently, the use of osteogenic surface coatings has a favorable effect on the bone implant interface in both osteoporotic and sham-operated conditions. (C) 2013 Elsevier Ltd. All rights reserved.
A.Sugawara, K.Asaoka, S.J. Ding. J. Mater. Chem. B Mater. Biol.Med, 1(2013), pp. 1081-1089
Amino acid profile is a key aspect of human milk (HM) protein quality. We report a systematic review of total amino acid (TAA) and free amino acid (FAA) profiles, in term and preterm HM derived from 13 and 19 countries, respectively. Of the 83 studies that were critically reviewed, 26 studies with 3774 subjects were summarized for TAA profiles, while 22 studies with 4747 subjects were reviewed for FAA. Effects of gestational age, lactation stage, and geographical region were analyzed by Analysis of Variance. Data on total nitrogen (TN) and TAA composition revealed general inter-study consistency, whereas FAA concentrations varied among studies. TN and all TAA declined in the first two months of lactation and then remained relatively unchanged. In contrast, the FAA glutamic acid and glutamine increased, peaked around three to six months, and then declined. Some significant differences were observed for TAA and FAA, based on gestational age and region. Most regional TAA and FAA data were derived from Asia and Europe, while information from Africa was scant. This systematic review represents a useful evaluation of the amino acid composition of human milk, which is valuable for the assessment of protein quality of breast milk substitutes.
A.Jillavenkatesa, R.A.Condrate. J. Mater.Sci, 33(1998), pp. 4111-4119
Hydroxyapatite , a calcium phosphate-based compound with numerous applications in the biological field was synthesized using the sol–gel processing route. The formation of hydroxyapatite and other compounds during the heat-treatment cycle were identified and characterized using thermal analyses and X-ray diffraction together with infrared and Raman spectroscopy. The influence of the addition of various organic alcohols (R–OH, R=CH3–, C2H5– and C3H8–) on the reaction results was studied. 08 1998 Kluwer Academic Publishers
<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">A new biocompatible multi-layered coating was developed by alternately depositing Ti and HA layers on Ti6Al4V substrates using radio frequency magnetron-assisted sputter processing to improve the interface properties between the coating and the substrate. The multi-layered coating consisted of an underlying Ti bond coat, the alternating layer, and an HA top-layer. Between the bond coat and the top layer, an alternating layer was created by means of gradually increasing Ti content with increasing depth from the HA top-layer. The experimental results indicated that the as-sputtered coating had quite uniform thickness and well bonded to the substrate. The multi-layered coating exhibited a better electrochemical behavior than monolithic HA coating. The XRD and low vacuum SEM results consistently indicated that highly crystalline coating was not appreciably dissolvable in simulated body fluid. The adhesion strength higher than 60 MPa did not change much even after 14 weeks of immersion. The multi-layered composite coatings had the advantages of high and non-declining adhesion strength and high resistance to SBF attack.</p>
X.Zheng, M.Huang, C.Ding.Biomaterials, 21(2000), pp. 841-849
One of the most important clinical applications of hydroxyapatite (HA) is as a coating on metal implants, especially plasma-sprayed HA coating applied on Ti alloy substrate. However, the poor bonding strength between HA and Ti alloy has been of concern to orthopedists. In this paper, an attempt has been made to enhance the bonding strength of HA coating by forming a composite coating with Ti. The bioactivity of the coating has also been studied. HA/Ti composite coatings were prepared via atmospheric plasma spraying on Ti-6Al-4V alloy substrates. The bond strength evaluation of HA/Ti composite coatings was performed according to ASTM C-633 test method. X-ray diffractometer and scanning electron microscopy were applied to identify the phases and the morphologies of the coatings. The bioactivity of HA/Ti composite coating was qualified by immersion of coating in simulated body fluid (SBF). The obtained results revealed that the addition of Ti to HA improved the bonding strength of coating significantly. In the SBF test, the coating surface was covered by carbonate-apatite, which was testified by X-ray photoelectron spectroscope, indicating good bioactivity for HA/Ti composite coating. The bioactivity of the coating has not been reduced by the addition of Ti.
Immobilizing organic-inorganic hybrid composites onto the implant surface is a promising strategy to improve host acceptance of the implant. The objective of this present study was to obtain a unique macroporous titanium-surface with the organic-mineral composite coatings consisting of gelatin methacrylate hydrogel (GelMA) and hydroxyapatite (HA). A 3-(trimethoxysilyl) propyl methacrylate (TMSPMA) layer was first coated onto the titanium surface, and surface was then covalently functionalized with GelMA using a photochemical method. Mineralization of the GelMA coating on the titanium surface was subsequently carried out by a biomimetic method. After 3-day mineralization, a large number of mineral phases comprising spherical amorphous nanoparticles were found randomly deposited inside GelMA matrix. The resulting mineralized hydrogel composites exhibited a unique rough surface of macroporous structure. The structure of the prepared GelMA/HA composite coating was studied by field emission scanning electron microscopy (FESEM), energy dispersive X-ray spectra (EDS), attenuated total refraction Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). Water contact angle measurement revealed the hydrophilicity properties of composite coatings. GelMA/HA on titanium after the TMSPMA treatment is very stable when tested in vitro with a PBS solution at 37掳C, due to the role of TMSPMA as a molecular bridge. It was expected that the macroporous GelMA/HA composite coatings might potentially promote and accelerate titanium (Ti)-based implants osseointegration for bone repair and regeneration.
W.Ye, X.X.Wang.Mater. Lett, 61(2007), pp. 4062-4065
ABSTRACT Homogeneous coatings were attained by electrochemical method in electrolytes containing Ca2+ and PO4361 ions with Ca/P ratio being 1.67. SEM observation showed that the hydroxyapatite (HAp,Ca10(PO4)6(OH)2) crystals prepared with higher concentration electrolyte (4 × 1061 2 M Ca2+) are ribbon-like with thickness of nanometer size, a morphology seldom reported previously. In an electrolyte of lower concentration (6 × 1061 4 M Ca2+), the HAp crystals formed are rod-like with a hexagonal cross section and diameter of about 70–80 nm. XRD patterns and IR spectra confirmed that the coatings consist of HAp crystals. TEM micrographs and SAD indicated that the longitude direction for both ribbon-like and rod-like crystal is [002], and the flat surface of the ribbon is (110). HRTEM showed that the ribbon-like crystal is a mixture of HAp and octacalcium phosphate (OCP, Ca8H2(PO4)6.5H2O).
H.Wang, N.Eliaz, Z.Xiang, H.P.Hsu, M.Spector, L.W.Hobbs.Biomaterials, 27(2006), pp. 4192-4203
<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">Three different implants, bare Ti–6Al–4V alloy, Ti–6Al–4V alloy coated with plasma-sprayed hydroxyapatite (PSHA), and Ti–6Al–4V alloy coated with electrochemically deposited hydroxyapatite (EDHA), were implanted into canine trabecular bone for 6 h, 7, and 14 days, respectively. Environmental scanning electron microscopy study showed that PSHA coatings had higher bone apposition ratios than those exhibited by bare Ti–6Al–4V and EDHA coatings after 7 days; however, at 14 days after implantation, EDHA and PSHA coatings exhibited similar bone apposition ratios, much higher than that for bare Ti–6Al–4V. The ultrastructure of the bone/implant interface observed by transmission electron microscope showed that the earliest mineralization (6 h—7 days) was in the form of nano-ribbon cluster mineral deposits with a Ca/P atomic ratio lower than that of hydroxyapatite. Later-stage mineralization (7–14 days) resulted in bone-like tissue with the characteristic templating of self-assembled collagen fibrils by HA platelets. Though adhesion of EDHA coatings to Ti–6Al–4V substrate proved problematical and clearly needs to be addressed through appropriate manipulation of electrodepositon parameters, the finely textured microstructure of EDHA coatings appears to provide significant advantage for the integration of mineralized bone tissue into the coatings.</p>
C.He, G.Xiao, X.Jin, C.Sun, P.X.Ma.Adv. Funct. Mater, 20(2010), pp. 3568-3576
We developed a straightforward, fast, and versatile technique to fabricate mineralized nanofibrous polymer scaffolds for bone regeneration in this work. Nanofibrous poly(l-) scaffolds were fabricated using both electrospinning and phase separation techniques. An electrodeposition process was designed to deposit on the nanofibrous scaffolds. Such scaffolds contain a high quality mineral coating on the fiber surface with tunable surface topography and chemical composition by varying the processing parameters, which can mimic the composition and structure of natural bone and provide a more biocompatible interface for bone regeneration.
R.Hu, C.Lin, H.Shi, H. Wang.Mater.Chem. Phys, 115(2009), pp. 718-723
In this work, the electrochemical deposition behavior of calcium phosphate coating from an aqueous electrolyte containing very dilute calcium and phosphorus species (Ca–P) was studied. The effects of three process parameters, i.e. temperature, current density and duration, were systematically investigated and the underlying mechanism was thoroughly analyzed. It was observed that the coating is mainly composed of hydroxyapatite (HA) in a wide range of temperature and current densities. The temperature had a significant effect on the deposition velocity. An apparent activation energy of 174.902kJ02mol 611 was subsequently derived, indicating the mass-transfer control mechanism for the coating formation. The current density was identified to be an important parameter for structure controllability. The results of DR-FTIR/Raman spectroscopic studies of the initial deposition phase strongly suggested that the HA coating was instantaneously and directly precipitated on the substrate; neither induction period nor precursor was detected in this dilute Ca–P electrolyte system. Finally, a phase diagram of the Ca–P electrolyte system was constructed, which offered a thermodynamic reason for the direct single-phase HA precipitation observed only in this system, but not in conventional concentrated systems.
X.Lu, Z.Zhao, Y. Leng.J.Cryst. Growth, 284(2005), pp. 506-516
Cathodic deposition of calcium phosphate (Ca–P) on titanium was studied under controlled atmospheres. Electrochemical deposition was conducted in a sealed electrochemical cell with a controlled flow of nitrogen, carbon dioxide or compressed air. The Ca–P phases of the depositions on titanium were examined by X-ray diffraction, Fourier transform–infrared spectroscopy, scanning electron microscopy and transmission electron microscopy. Experimental results showed that hydroxyapatite (HA) deposition occurs under a pure nitrogen atmosphere, while octacalcium phosphate (OCP) deposition occurs under a carbon dioxide atmosphere. The atmosphere in the electrochemical cell effectively maintained constant levels of pH value in the electrolyte during the deposition process. A HA/OCP layered structure on titanium was obtained by controlling the atmosphere. The electrochemical deposition process of Ca–P can be explained by the thermodynamic and kinetics theory of the Ca–P precipitation from an aqueous supersaturated solution.
Y.Parcharoen, P.Kajitvichyanukul, S.Sirivisoot, P. Termsuksawad.Appl.Surf. Sci, 311(2014), pp. 54-61
Nanotubes modification for orthopedic implants has shown interesting biological performances (such as improving cell adhesion, cell differentiation, and enhancing osseointegration). The purpose of this study is to investigate effect of titanium dioxide (TiO 2 ) nanotube feature on performance of hydroxyapatite-coated titanium (Ti) bone implants. TiO 2 nanotubes were prepared by anodization using ammonium fluoride electrolyte (NH 4 F) with and without modifiers (PEG400 and Glycerol) at various potential forms, and times. After anodization, the nanotubes were subsequently annealed. TiO 2 nanotubes were characterized by scanning electron microscope and X-ray diffractometer. The amorphous to anatase transformation due to annealing was observed. Smooth and highly organized TiO 2 nanotubes were found when high viscous electrolyte, NH 4 F in glycerol, was used. Negative voltage (鈭4V) during anodization was confirmed to increase nanotube thickness. Length of the TiO 2 nanotubes was significantly increased by times. The TiO 2 nanotube was electrodeposited with hydroxyapatite (HA) and its adhesion was estimated by adhesive tape test. The result showed that nanotubes with the tube length of 560nm showed excellent adhesion. The coated HA were tested for biological test by live/dead cell straining. HA coated on TiO 2 nanotubes showed higher cells density, higher live cells, and more spreading of MC3T3-E1 cells than that growing on titanium plate surface.
G.Ciobanu, O. Ciobanu.Mater.Sci. Eng. C Mater. Biol. Appl, 33(2013), pp. 1683-1688
This study uses an in vitro experimental approach to investigate the roles of collagen and vitamins in regulating the deposition of hydroxyapatite layer on the pure titanium surface. Titanium implants were coated with a hydroxyapatite layer under biomimetic conditions by using a supersaturated calcification solution (SCS), modified by adding vitamins A and D 3 , and collagen. The hydroxyapatite deposits on titanium were investigated by means of scanning electron microscopy (SEM) coupled with X-ray analysis (EDX), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and Fourier transformed infrared (FTIR) spectroscopy. The results obtained have shown that hydroxyapatite coatings were produced in vitro under vitamins and collagen influence.
L.T.de Jonge, S.C.Leeuwenburgh, J.G.Wolke, J.A.Jansen. Pharm. Res, 25(2008), pp. 2357-2369
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[17]
X.Chen, W.Wang, S.Cheng, B.Dong, C.Y.Li.ACS Nano, 7(2013), pp. 8251-8257
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M.S.Djošić, V.Panić, J.Stojanović, M.Mitrić, V.B.Mišković-Stanković. Colloids Surf. A Physicochem. Eng. Asp, 400(2012), pp. 36-43
Electrochemical deposition of calcium phosphate coatings on titanium was performed galvanostatically from the aqueous solution of Ca(NO 3 ) 2 and NH 4 H 2 PO 4 with the current densities between 5.0 and 10mAcm 612 , for different deposition times, at room temperature. The coatings were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The XRD results showed that dicalcium phosphate dihydrate (DCPD), brushite (CaHPO 4 ·2H 2 O) coatings were deposited. The influence of applied current density and the deposition time on the phase composition, crystallite domain size and the morphology of brushite coatings were investigated. It was shown that brushite coating of the greatest mass was obtained for the longest deposition time, while the increase in current density over 7mAcm 612 does not affect significantly the mass of brushite coatings. The finest crystallites, with the smallest crystallite domain size of 15.6nm, were deposited at the current density of 9.0mAcm 612 . Brushite coatings were fully converted to hydroxyapatite in simulated body fluid (SBF) which was confirmed by XRD and SEM. The crystallite domain size of HA coatings is controlled by applied current density for brushite coatings deposition; crystallization of HA at more porous brushite coatings, deposited at higher current density, caused the formation of smaller crystallites of hydroxyapatite.
N.Dumelie, H.Benhayoune, D.Richard, D.Laurent-Maquin, G. Balossier.Mater.Charact, 59(2008), pp. 129-133
On the basis of these results, we conclude that an electrodeposited calcium phosphate coating on roughened titanium alloy substrate may act as a precursor for newly precipitated calcium phosphate in in vitro experiments independent of cellular activities.
S.H.Kim, Y.H.Jeong, H.C.Choe, W.A.Brantley.Surf. Coat. Technol, 259(2014), pp. 281-289
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[21]
Q.Liu, J.Ding, F.K.Mante, S.L.Wunder, G.R.Baran.Biomaterials, 23(2002), pp. 3103-3111
<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">Surface functional groups play important roles in nucleating calcium phosphate deposition on surgical titanium implants. In this study, various functional groups were introduced onto the surface of commercially pure titanium foils using a self-assembled monolayer (SAM) technique. An organic silane, 7-oct-1-enyltrichlorosilane (OETS) was used and OH, PO<sub>4</sub>H<sub>2</sub>, COOH groups were derived from its unsaturated double bond. Ti foils were first oxidized in concentrated H<sub>2</sub>SO<sub>4</sub>/H<sub>2</sub>O<sub>2</sub>. ESCA and contact angle measurements were used to characterize the SAM surfaces and confirm the presence of various functional groups. A fast calcium phosphate deposition experiment was carried out by mixing Ca<sup>2+</sup>- and (PO<sub>4</sub>)<sup>3−</sup>-containing solutions in the presence of the surface-modified Ti samples at pH 7.4 at room temperature in order to verify the nucleating abilities of these functional groups. SEM, Raman spectroscopy, XRD and ATR-FTIR results showed that poorly crystallized hydroxyapatite (HA) can be deposited on the SAM surfaces with PO<sub>4</sub>H<sub>2</sub> and –COOH functional groups, but not onto the SAM with –CHCH<sub>2</sub> and –OH. –PO<sub>4</sub>H<sub>2</sub> exhibited a stronger nucleating ability than that of –COOH. The oxidized Ti sample also showed some calcium phosphate deposition but to a lesser extent as compared to SAM surfaces with –PO<sub>4</sub>H<sub>2</sub> and –COOH. The pre-deposited HA can rapidly induce biomimetic apatite layer formation after immersion in 1.5 SBF for 18 h regardless of the amount of pre-deposited HA. The results suggested that the pre-deposition of HA onto these functionalized SAM surfaces might be an effective and fast way to prepare biomimetic apatite coatings on surgical implants.</p>
G.K.Toworfe, R.J.Composto, I.M.Shapiro, P.Ducheyne.Biomaterials, 27(2006), pp. 631-642
<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">Upon implantation, calcium phosphate (Ca–P) surfaces form on materials that are bone bioactive. In this study, the evolving surface characteristics associated with calcium phosphate precipitation are modeled using self-assembled monolayers (SAMs), in a one-step nucleation process. SAMs were used to create amine (–NH<sub>2</sub>), carboxyl (–COOH) and hydroxyl (–OH) functionalized surfaces by grafting 3-aminopropyltriethoxysilane, 3-triethoxysilylpropyl succinic anhydride and glycidoxypropyl tri-methoxysilane, respectively, onto oxidized silicon wafers. The SAM surfaces were characterized using ellipsometry to establish the presence of grafted molecules. On the surfaces incubated in simulated physiological fluids for 7 days, the thickness of Ca–P layer grew slowly over the first few hours, increasing strongly between 1 and 5 days and then slowed down again. FTIR showed the dependence of calcium phosphate morphology on the type of surface groups, with stronger P–O bands seen on the OH-terminated surface. SEM analysis showed dispersed Ca–P precipitates on the –COOH and –OH terminated surfaces after 1 day immersion. After 7 days, all SAM surfaces were covered with uniformly dispersed and denser Ca–P precipitates. The underlying Ca–P layer showed cracks on the –NH<sub>2</sub>-terminated surface. Rutherford backscattering spectrometry (RBS) data analysis confirmed that Ca/P ratio is in excellent agreement with the theoretical value of 1.67 for hydroxyapatite. X-ray diffraction (XRD) analysis also showed evidence of apatite formation on all the surfaces, with stronger evidence on the –OH-terminated surface. Highly porous Ca–P precipitates were observed on the SAM surfaces portrayed by the AFM scans with nanoscale RMS roughness. Thus, using highly controlled surface chemistry, under physiological conditions, in vitro, this study demonstrates that a hydroxylated surface enhances Ca–P nucleation and growth relative to other surfaces, thereby supporting the concept of its beneficial effect on bone tissue formation and growth.</p>
D.J.Wu, Z.X.Liu, C.Z.Gao, X.C.Shen, X.M.Wang, H. Liang.Surf.Coat. Technol, 228(2013), pp. S24-S27
The effect of chemical functional groups and condition of supersaturated solutions are two important factors to induce the nucleation of hydroxyapatite onto the substrates. Two kinds of self-assembled monolayers were prepared to create functionalized surfaces by grafting thioalcohol molecular containing hydroxyl and methyl as terminal groups respectively onto single silicon chips. Atomic force microscopy and X-ray photoelectron spectroscopy were used to analyze the topography of the surfaces and estimate the surface density. The results provided the evidences that our experiments were made with almost the same density of chemical functional groups. The surfaces were incubated in the supersaturated solutions (37掳C, pH=7.4) whose Ca 2+ and PO 4 3鈭 ion concentrations were 1.5 times higher than in simulated body fluid. With the condition mentioned above, this study demonstrates that the growth process and morphology of HAp crystals present great differences.
F.R.Maia, S.J.Bidarra, P.L.Granja, C.C.Barrias.Acta Biomater, 9(2013), pp. 8773-8789
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[25]
D.P.Liu, P.Majewski, B.K.O'Neill, Y. Ngothai, C.B. Colby. J. Biomed. Mater. Res. A, 77(2006), pp. 763-772
Commercial interest is growing in biomimetic methods that employ self assembled mono-layers (SAMs) to produce biocompatible HA coatings on Ti-based orthopedic implants. Recently, separate studies have considered HA for various SAM surface functional groups. However, these have often neglected to verify crystallinity of the HA coating, which is essential for optimal bioactivity. Furthermore, differing experimental and analytical methods make performance comparisons difficult. This article investigates and evaluates HA for four of the most promising surface functional groups: --OH, --SO(3)H, --PO(4)H(2) and --COOH. All of them successfully formed a HA coating at Ca/P ratios between 1.49 and 1.62. However, only the --SO(3)H and --COOH end groups produced a predominantly crystalline HA. Furthermore, the --COOH end group yielded the thickest layer and possessed crystalline characteristics very similar to that of the bone. The --COOH end group appears to provide the optimal SAM surface interface for nucleation and of biomimetic crystalline HA. Intriguingly, this finding may lend support to explanations elsewhere of why bone is such a potent nucleator of HA and is attributed to the 's glutamic acid-rich sequences.
G.Tan, Y.Tan, G.Ni, G.Lan, L.Zhou, P.Yu, J.Liao, Y.Zhang, Z.Yin, H.Wang, C. Ning.J.Mater. Sci. Mater. Med, 25(2014), pp. 1875-1884
To further enhance the biological properties of acid-etched microrough titanium surfaces, titania nanotextured thin films were produced by simple chemical oxidation, without significantly altering the existing topographical and roughness features. The nanotextured layers on titanium surfaces can be controllably varied by tuning the oxidation duration time. The oxidation treatment significantly reduced water contact angles and increased the surface energy compared to the surfaces prior to oxidation. The murine bone marrow stromal cells (BMSCs) were used to evaluate the bioactivity. In comparison, oxidative nanopatterning of microrough titanium surfaces led to improved attachment and proliferation of BMSCs. The rate of osteoblastic differentiation was also represented by the increased levels of alkaline phosphatase activity and mineral deposition. These data indicated that oxidative nanopatterning enhanced the biological properties of the microrough titanium surfaces by modulating their surface chemistry and nanotopography. Based on the proven mechanical interlocking ability of microtopographies, enhancement of multiple osteoblast functions attained by this oxidative nanopatterning is expected to lead to better implant osseointegration in vivo.
Y.Xiao, T.Gong, S.Zhou.Biomaterials, 31(2010), pp. 5182-5190
<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">A simple and effective approach was introduced to functionalize multi-walled carbon nanotubes (MWNTs) by <em>in situ</em> deposition of hydroxyapatite (HA) to improve their hydrophilicity and biocompatibility. Firstly, we prepared two types of pre-functionalized MWNTs: acid-oxidated MWNTs and covalently modified MWNTs by poly (ethylene glycol) (PEG). The influences of the acid-oxidated time, pre-phosphorylation, and PEGylation of MWNTs on <em>in situ</em> growth of HA were further investigated in simulated body fluid (SBF) with ionic concentration: 2, 5 and 10 times, respectively, at 37 °C for 24 h. The results exhibited that all these factors have positive effects on the HA crystals growth, especially the PEGylation of MWNTs plays a key role during the deposition. Finally, the methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate their cytotoxicity, which showed that the PEGylated MWNTs wrapped by HA crystals have the best biocompatibility.</p>
This study examined the in vitro cell-material interactions on four different types of titanium surfaces: a polished Ti surface, TiO 2 nanotube surfaces fabricated in a fluorinated glycerol solution (TN), fluorinated glycerol solution with 1wt% anionic surfactant sodium dodecyl sulphate (TN-SDS), and fluorinated glycerol solution with 1wt% cationic surfactant cetyl trimethyl ammonium bromide (TN-CTAB), respectively. The surfaces exhibited distinct surface morphologies and geometrical features. Surface energy calculation shows that TN surface enhances the hydrophilic character by significantly increasing the surface energy. The osteoblast cell growth behavior on the four different surfaces was examined using the MC3T3-E1 cell line for 1 day. When the anodized surfaces were compared for the cell-materials interaction, each of the surfaces showed different properties that affected the cell鈥搈aterial interactions. Proliferation of the cells was noticed with distinctive cell-to-cell attachment on the TN surfaces. Good cellular adhesion with extracellular matrix extensions between the cells was noticed in the TN samples. The TiO 2 nanotubes grown in the surfactant-assisted fluorinated electrolyte did not show significant cell growth on the surface and some cell death was observed. The cell adhesion, differentiation and alkaline phosphatase activity were more pronounced on the TN surface. The MTT assays also revealed an increase in living cell density and proliferation on the TN surfaces. Overall, a rough surface morphology and surface energy are important factors for better cell material interactions.
M.A.Martins, C.Santos, M.M.Almeida, M.E.Costa.J. Colloid Interface Sci, 318(2008), pp. 210-216
[Cited within:1]
[31]
E.Ajami, K.F.Aguey-Zinsou. J. Mater. Sci. Mater. Med, 22(2011), pp. 1813-1824
Enhanced biocompatibility of titanium implants highly depends on the possibility of achieving high degrees of surface functionalization for a low immune response and/or enhanced mineralization of bioa
L.Chen, J.M.McCrate, J.C. Lee, H. Li. Nanotechnology, 22(2011) 105708
The objective of this study is to evaluate the effect of hydroxyapatite (HAP) nanoparticles with different surface charges on the cellular uptake behavior and cell viability and proliferation of MC3T3-E1 cell lines (osteoblast). The nanoparticles' surface charge was varied by surface modification with two carboxylic acids: 12-aminododecanoic acid (positive) and dodecanedioic acid (negative). The untreated HAP nanoparticles and dodecanoic acid modified HAP nanoparticles (neutral) were used as the control. X-ray diffraction (XRD) revealed that surface modifications by the three carboxylic acids did not change the crystal structure of HAP nanoparticles; Fourier transform infrared spectroscopy (FT-IR) confirmed the adsorption and binding of the carboxylic acids on the HAP nanoparticles' surfaces; and zeta potential measurement confirmed that the chemicals successfully modified the surface charge of HAP nanoparticles in water based solution. Transmission electron microscopy (TEM) images showed that positively charged, negatively charged and untreated HAP nanoparticles, with similar size and shape, all penetrated into the cells and cells had more uptake of HAP nanoparticles with positive charge compared to those with negative charge, which might be attributed to the attractive or repulsive interaction between the negatively charged cell membrane and positively/negatively charged HAP nanoparticles. The neutral HAP nanoparticles could not penetrate the cell membrane due to their larger size. MTT assay and LDH assay results indicated that as compared with the polystyrene control, greater cell viability and cell proliferation were measured on MC3T3-E1 cells treated with the three kinds of HAP nanoparticles (neutral, positive, and untreated), among which positively charged HAP nanoparticles showed the strongest improvement for cell viability and cell proliferation. In summary, the surface charge of HAP nanoparticles can be modified to influence the cellular uptake of HAP nanoparticles and the different uptake also influences the behavior of cells. These results may also provide useful information for investigations of HAP nanoparticle applications in gene delivery and intracellular drug delivery.
C.Yang, K.Cheng, W.Weng, C. Yang.J.Mater. Sci. Mater. Med, 20(2009), pp. 667-672
[Cited within:1]
[34]
G.B.Varadwaj, S.Rana, K.M.Parida.Dalton Trans, 42(2013), pp. 5122-5129
[Cited within:1]
[35]
M.Bariana, M.S.Aw, M.Kurkuri, D. Losic.Int.J. Pharm, 443(2013), pp. 230-241
Diatomaceous earth (DE), or diatomite silica microparticles originated from fossilized diatoms are a potential substitute for its silica-based synthetic counterparts to address limitations in conventional drug delivery. This study presents the impact of engineered surface chemistry of DE microparticles on their drug loading and release properties. Surface modifications with four silanes, including 3-aminopropyltriethoxy silane (APTES), methoxy-poly-(ethylene-glycol)-silane (mPEG-silane), 7-octadecyltrichlorosilane (OTS), 3-(glycidyloxypropyl)trimethoxysilane (GPTMS) and two phosphonic acids, namely 2-carboxyethyl-phosphonic acid (2 CEPA) and 16-phosphono-hexadecanoic acid (16 PHA) were explored in order to tune drug loading and release characteristics of water insoluble (indomethacin) and water soluble drugs (gentamicin). Successful grafting of these functional groups with different interfacial properties was confirmed using X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Thermogravimetric analysis (TGA) was applied to determine the amount of loaded drugs and UV-spectrophotometry to analyse in vitro drug release from modified DE microparticles. Differences in drug release time (13鈥26 days) and loading capacity (14鈥24%) were observed depending on functional groups on the surface of DE microparticles. It was found that hydrophilic surfaces, due to the presence of polar carboxyl, amine or hydrolyzed epoxy group, favor extended release of indomethacin, while the hydrophobic DE surface modified by organic hydrocarbons gives a better sustained release profile for gentamicin. This work demonstrates that by changing surface functionalities on DE microparticles, it is possible to tune their drug loading and release characteristics for both hydrophobic and hydrophilic drugs and therefore achieve optimal drug delivery performance.
S.Bodhak, S.Bose, A.Bandyopadhyay.Acta Biomater, 5(2009), pp. 2178-2188
<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">Our objective was to determine the role of surface charge and wettability on early stage mineralization as well as bone cell adhesion and proliferation on polarized HAp surface. To estimate the surface wettability, contact angles were measured in water, simulated body fluid (SBF) and Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham (DMEM). Experimental results show that HAp surface wettability and surface energy can be tailored by inducing surface charge without introducing any volumetric effects in the material. Increasing the surface charge increased the wettability and also the energy of HAp surfaces in all tested media. A maximum surface energy of 49.47 ± 3.76 mJ/m<sup>2</sup> was estimated for positively charged HAp surfaces polarized at 400 <sup>o</sup>C. The <em>in vitro</em> bioactivity of polarized HAp samples was evaluated by soaking in SBF and DMEM (cell media). Cell–materials interaction was studied by culturing with human fetal osteoblast cells (hFOB). <em>In vitro</em> results show that tailoring the combined effect of wettability and charge polarity on the HAp surface enable differential binding of inorganic ions (e.g., Ca<sup>2+</sup>, Cl<sup>−</sup>, Na<sup>+</sup>, HCO<sub>3</sub><sup>−</sup> etc) and organic cell adhesive proteins (e.g., fibronectin, vitronectin etc) with different surface properties, which results in accelerated or decelerated mineralization as well as cell adhesion and proliferation on polarized HAp surface.</p>
A facile and quick approach to prepare self-assembled monolayers of water-dispersible particles on the water surface is presented. Particle suspensions in alcohols were dropped on a water reservoir to form long-range ordered monolayers of various particles, including spherical solid particles, soft hydrogel particles, metal nanoparticles, quantum dots, nanowires, single-wall carbon nanotubes (SWCNTs), nanoplates, and nanosheets. A systematic study was conducted on the variables affecting the monolayer assembly: the solubility parameter of spreading solvents, particle concentration, zeta potential of the particles in the suspension, surface tension of the water phase, hardness of the particles, and addition of a salt in the suspension. This method requires no hydrophobic surface treatment of the particles, which is useful to exploit these monolayer films without changing the native properties of the particles. The study highlights a quick 2D colloidal assembly without cracks in the wafer scale as well as transparent conductive thin films made of SWCNTs and graphenes.
F.Gao, P.M.A.Sherwood. Surf. Interface Anal, 45(2013), pp. 742-750
X‐ray photoelectron spectroscopy in the core and valence band region was used to study the formation of hydroxyapatite films on the surface of titanium. The approach used achieves the adhesion of hydroxyapatite by the initial formation of a thin, mainly oxide‐free, etidronate film on the metal. In this approach, it was not possible to prepare hydroxyapatite films of any reasonable thickness on the titanium surface without prior treatment with etidronic acid. Because hydroxyapatite is a principal component of teeth and bones, it is likely that the coated metals will have desirable biocompatible properties. The hydroxyapatite film showed no changes when the film was exposed to air, water, and 165m sodium chloride solution as representative components of the environment of the film in the human body. These films formed on titanium may find application in medical implants. The thin hydroxyapatite and etidronate film on the metal show differential charging effects that caused a doubling of some of the spectral features. Copyright 08 2012 John Wiley & Sons, Ltd.
K.Schickle, R.Kaufmann, D.F.Duarte Campos, M. Weber, H. Fischer. J. Eur. Ceram. Soc, 32(2012), pp. 3063-3071
The bioinert character of alumina implants does not promote bone bonding, thereby strongly limits possible applications for this material. Bone bonding to the implant surface is significantly dependent on its chemical composition. We hypothesized that a bioinert ceramic can be functionalized, introducing active groups onto the surface by self-assembled monolayer. The surface of alumina was coated with a silica layer using physical vapour deposition or flame pyrolysis. Subsequently SAM was attached by dint of octenyltrichlorosilane. Through further chemical treatments OH- and COOH-functional groups were created on the surface. The evidence of the functional groups was proven by X-ray photoelectron spectroscopy and contact angle measurements. Thereby for the first time functional groups were successfully coupled to an inert alumina surface using the method of SAM. In a next step of development, SAM technique could be used to couple biological agents to inert ceramic implant surfaces aiming the possible improvement of osseointegration.
A.E.Porter, T.Buckland, K.Hing, S.M.Best, W. Bonfield.J.Biomed. Mater. Res. A, 78(2006), pp. 25-33
The significance of micrometer-sized strut porosity in promoting bone ingrowth into porous hydroxyapatite (HA) scaffolds has only recently been noted. In this study, silicon-substituted HA (0.8 wt percent Si-HA) with approximately 8.5 percent of the total porosity present as microporosity within the struts of the implant was prepared for high-resolution transmission electron microscopy (HR-TEM) via both ultramicrotomy and focused ion beam milling. Between the struts of the porous Si-HA, pores with varying shapes and sizes (1-10 mum in diameter) were characterized. Within the struts, the Si-HA contained features such as grain boundaries and triple-junction grain boundaries. Bone ingrowth and dissolution from a Si-HA implant were studied using HR-TEM after 6 weeks in vivo. Minor local dissolution occurred within several pores within the struts. Organized, mineralized collagen fibrils had grown into the strut porosity at the interface between the porous Si-HA implant and the surface of the surrounding bone. In comparison, deeper within the implant, disorganized and poorly mineralized fibers were observed within the strut porosity. These findings provide valuable insight into the development of bone around porous Si-HA implants.
R.A.Surmenev, M.A.Surmeneva, A.A.Ivanova.Acta Biomater, 10(2014), pp. 557-579
A systematic analysis of results available from in vitro, in vivo and clinical trials on the effects of biocompatible calcium phosphate (Cap) coatings is presented. An overview of the most frequently used methods to prepare CaP-based coatings was conducted. Dense, homogeneous, highly adherent and biocompatible CaP or hybrid organic/inorganic CaP coatings with tailored properties can be deposited. It has been demonstrated that CaP coatings have a significant effect on the bone regeneration process. In vitro experiments using different cells (e.g. SaOS-2, human mesenchymal stem cells and osteoblast-like cells) have revealed that CaP coatings enhance cellular adhesion, proliferation and differentiation to promote bone regeneration. However, in vivo, the exact mechanism of osteogenesis in response to CaP coatings is unclear; indeed, there are conflicting reports of the effectiveness of CaP coatings, with results ranging from highly effective to no significant or even negative effects. This review therefore highlights progress in CaP coatings for orthopaedic implants and discusses the future research and use of these devices. Currently, an exciting area of research is in bioactive hybrid composite CaP-based coatings containing both inorganic (CaP coating) and organic (collagen, bone morphogenetic proteins, arginylglycylaspartic acid etc.) components with the aim of promoting tissue ingrowth and vascularization. Further investigations are necessary to reveal the relative influences of implant design, surgical procedure, and coating characteristics (thickness, structure, topography, porosity, wettability etc.) on the long-term clinical effects of hybrid CaP coatings. In addition to commercially available plasma spraying, other effective routes for the fabrication of hybrid CaP coatings for clinical use still need to be determined and current progress is discussed. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
M.Motskin, D.M.Wright, K.Muller, N.Kyle, T.G.Gard, A.E.Porter, J.N.Skepper.Biomaterials, 30(2009), pp. 3307-3317
<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">Synthetic colloid and gel hydroxyapatite (HA) nanoparticles (NPs) were spray dried to form microparticles (MPs). These are intended for use as slow release vaccine vectors. The physico-chemical properties of gel and colloid NPs and MPs were compared to those of HA obtained commercially. Their cytotoxicity to human monocytes'-derived macrophages (HMMs) was assessed <em>in vitro</em> using a range of techniques. These included the MTT assay, LDH leakage and a confocal based live–dead cell assay. Cytotoxicity differed significantly between preparations, with the suspended gel preparation being the most toxic (31–500 μg/ml). Other preparations were also toxic but only at higher concentrations (>250 μg/ml). Transmission electron microscopy (TEM) and stereology showed variable cellular uptake and subsequent dissolution of the various forms of HA. We have demonstrated that HA particle toxicity varied considerably and that it was related to their physico-chemical properties. Cell death correlated strongly with particle load. The intracellular dissolution of particles as a function of time in HMM suggests that increased cytoplasmic calcium load is likely to be the cause of cell death. Some HA NPs eluded the phagocytic pathway and a few were even seen to enter the nuclei through nuclear pores.</p>
S.W.Myung, Y.M.Ko, B.H.Kim.Appl. Surf. Sci, 287(2013), pp. 62-68
This study examined the plasma surface modification of biomimetic hydroxyapatite (HAp) formed on a titanium (Ti) surface as well as its influence on the behavior of preosteoblast cells. Ti substrates pre-treated with a plasma-polymerized thin film rich in carboxyl groups were subjected to a biomimetic process in a simulated body fluid solution to synthesize the HAp. The HAp layer grown on Ti substrate was then coated with two types of plasma polymerized acrylic acid and allyl amine thin film. The different types of Ti substrates were characterized by attenuated total reflection Fourier transform infrared spectroscopy, energy dispersive spectroscopy and X-ray diffraction. HAp with a Ca/P ratio from 1.25 to 1.38 was obtained on the Ti substrate and hydrophilic carboxyl ( COOH) and amine ( NH 2 ) functional groups were introduced to its surface. Scanning electron microscopy was used to observe the surface of the HAp coatings and the morphology of MC3T3-E1 cells. These results showed that the COOH-modified HAp surfaces promoted the cell spreading synergistically by changing the surface morphology and chemical state. NH 2 modified HAp had the lowest cell spreading and proliferation compared to HAp and COOH-modified HAp. These results correspond to fluorescein analysis, which showed many more cell spreading of COOH/HAp/Ti surface compared to HAp and NH 2 modified HAp. A MTT assay was used to evaluate cell proliferation. The results showed that the proliferation of MC3T3-E1 cells increased in the order of COOH/HAp/Ti02>02HAp/Ti02>02NH 2 /Ti02>02Ti, corresponding to the effect of cell spreading for 6 days. The change in morphology and the chemical surface properties of the biomaterial via plasma polymerization can affect the behavior of MC3T3-E1 cells.
Z.Shi, X.Huang, Y.Cai, R.Tang, D.Yang.Acta Biomater, 5(2009), pp. 338-345
<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">Nano-hydroxyapatite (nano-HAP) may be a better candidate for an apatite substitute of bone in biomedical applications than micro-sized hydroxyapatite (m-HAP). However, size control is always difficult when synthesizing well-defined nano-HAP particles. In this study, nano-HAP particles with diameters of ∼20 nm (np20) and ∼80 nm (np80) were synthesized and characterized. The size effects of these nano-HAPs and m-HAP were studied on human osteoblast-like MG-63 cells in vitro. Our results demonstrate that both cell proliferation and cell apoptosis are related to the size of the HAP particles. Np20 has the best effect on promotion of cell growth and inhibition of cell apoptosis. This work provides an interesting view of the role of nano-HAPs as ideal biomedical materials in future clinical applications.</p>
W.Cui, X.Li, C.Xie, H.Zhuang, S.Zhou, J.Weng.Biomaterials, 31(2010), pp. 4620-4629
<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">Controlled nucleation and growth of hydroxyapatite (HA) crystals on electrospun fibers should play important roles in fabrication of composite scaffolds for bone tissue engineering, but no attempt has been made to clarify the effects of chemical group densities and the cooperation of two and more groups on the biomineralization process. The aim of the current study was to investigate into HA nucleation and growth on electrospun poly(<span class="smallcaps">dl</span>-lactide) fibers functionalized with carboxyl, hydroxyl and amino groups and their combinations. Electrospun fibers with higher densities of carboxyl groups, combination of hydroxyl and carboxyl groups with the ratio of 3/7, and combination of amino, hydroxyl and carboxyl groups with the ratio of 2/3/5 were favorable for HA nucleation and growth, resulting in higher content and lower crystal size of formed HA. Carboxyl groups were initially combined with calcium ions through electrostatic attraction, and the introduction of hydroxyl groups could modulate the distance between carboxyl groups. The introduction of amino groups may lead to the inner ionic bonding with carboxyl groups, but can accelerate phosphate ions to form HA through a chelate ring with the calcium ion and carbonyl oxygen. The biological evaluation indicated that the mineralized scaffolds acted as an excellent cell support to maintain desirable cell–substrate interactions, to provide favorable conditions for cell proliferation and to stimulate the osteogenic differentiation.</p>
H.M.Herath, L. DiSilvio, J.R.Evans.Mater. Sci. Eng. C Mater. Biol. Appl, 57(2015), pp. 363-370
ABSTRACT Zirconia-3mol% yttria ceramics were prepared with as-sintered, abraded, polished, and porous surfaces in order to explore the attachment, proliferation and differentiation of osteoblast-like cells. After modification, all surfaces were heated to 600°C to extinguish traces of organic contamination. All surfaces supported cell attachment, proliferation and differentiation but the surfaces with grain boundary grooves or abraded grooves provided conditions for enhanced initial cell attachment. Nevertheless, overall cell proliferation and total DNA were highest on the polished surface. Zirconia sintered at a lower temperature (1300°C vs. 1450°C) had open porosity and presented reduced proliferation as assessed by alamarBlue64 assay, possibly because the openness of the pores prevented cells developing a local microenvironment. All cells retained the typical polygonal morphology of osteoblast-like cells with variations attributable to the underlying surface notably alignment along the grooves of the abraded surface.
S.Okada, A.Nagai, Y.Oaki, J.Komotori, H.Imai.Acta Biomater, 7(2011), pp. 1290-1297
We controlled the performance of L929 mouse fibroblasts using various hydroxyapatite (HA) nanocrystals, such as nanofibers, nanoneedles, and nanosheets, to better understand the effects of size and shape of the HA nanocrystals on the cells. The cellular activity on nanofibers with a diameter of 50-100 nm was significantly enhanced relative to that on a flat HA surface because large amounts of the proteins needed for adhesion and proliferation could be stored in the substrate. On the other hand, initial adhesion and subsequent proliferation were inhibited on surfaces consisting of fine nanoneedles and nanosheets with a diameter/thickness of less than 30 nm due to the limited area available for the formation of focal adhesions. These facts indicate that fibroblast activity is highly sensitive to the surface topography. Therefore, size tuning of the nanoscale units composing the substrate is essential to enhance cellular performance. (C) 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
... Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10]. ...
1
2013
... Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10]. ...
1
2008
... Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10]. ...
1
1998
... Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10]. ...
1
2003
... Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10]. ...
1
2000
... Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10]. ...
1
2013
... Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10]. ...
1
2007
... Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10]. ...
1
2006
... Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10]. ...
3
2010
... Calcium phosphate (CaP) is a class of materials which clearly shows osteoconductive ability for improving and accelerating integration with bone tissue[1]. CaP coating has been extensively utilized to improve bioactivity and biocompatibility of implants surface[2]. Various methods have been developed to deposit CaP apatite on titanium implant surfaces, such as sol-gel processing, sputtering, plasma spraying, biomimetic mineralization and electrochemical methods[3], [4], [5], [6], [7] and [8]. Among them, electrochemical deposition (ECD) possesses manifold advantages in CaP coatings production, the most powerful of which are the mild operating conditions of using aqueous solutions at low temperatures, which do not affect the structure of implant, and can be applied to complex shapes[9] and [10]. ...
... The calcium phosphate deposition experiment was carried out in the presence of SAMs sample by electrochemical deposition. An electrochemical cell in a standard reactor was configured to work with two electrodes, using platinum strings for counter electrode[10]. A negative potential was applied to the working electrode, where pTi samples of 4 cm2 were placed. Calcium phosphate coatings on titanium were obtained from stirred aqueous solution of 4.2 × 10-3 mol/L CaCl2 (AR, 96%, Aladdin, China) and 2.5 × 10-3 mol/L NaH2PO4 (AR, Aladdin, China), pH = 6.0, at 80 °C. The electrodeposition was performed at the current density of 2.5 mA/cm2 and deposition time of 45 min. Then the samples were dried in N2 atmosphere at room temperature (named pTi-CaP, Ti-OTS-CaP, Ti-APTES-CaP, Ti-GPTMS-CaP, respectively). ...
... In order to illustrate the mechanism between CaP nucleation and growth with bridge molecule influencing on pTi surface, Fig. 1 shows the hypothetical schematic diagram of the SAMs and electrochemical deposition CaP coatings on the titanium surface (using pTi as cathode). It is obvious that hydroxyl groups were generated on titanium surface after treating with piranha solution, which could be stably bonded titanium surface with silane coupling as a molecular bridge (forming Si—O bond)[31]. On the one hand, the large amount of hydroxyl ions was generated through hydrogen evolution reaction of initial stage in electrochemical deposition, induced calcium phosphate ions rapid nucleation of initial precipitates[10]. On the other hand, in the case of the functional groups on silane coupling, the initial products that were assigned to HA precursors were rapidly precipitated as soon as the calcium and phosphate solutions were electrolyzed. The large amount of nuclei facilitated the heterogeneous nucleation during deposition, leading to precipitation and formation of apatite on the SAMs surface[15] and [21]. The nucleation of calcium phosphate particles on functionalized surface would yield diverse regular shape and relatively small size. The hydrogen evolution occurred at cathode interface and produced OH-, which could combine other ions in the electrolytes, such as Ca2+ and PO43-, thus causing nucleation of hydroxyapatite and its precursors. Meanwhile, the functionalized pTi surface charge and wettability did not resemble that of the bioactive pTi. The positive charges surface (—NH2 group) could attract negative charges (OH-, PO43-) while negative charges surface (—CH3 and —C(O)C group) could attract positive charges (Ca2+). Therefore, NH2-terminated surface (Ti-APTES) as a molecular bridge with positive charge had priority to attract OH- and PO43-, consequently Ca2+ was attracted to cathode surface and gradually formed hydroxyapatite[32]. Moreover, negatively charged surface (potential absolute value: pTi < Ti-OTS < Ti-GPTMS) had priority to attract Ca2+, but the number of negative charge on pTi was less than Ti-OTS (—CH3 group) and Ti-GPTMS (—C(O)C group), hydroxyl ions was produced by hydrogen evolution reaction firstly combined with Ca2+ on pTi surface. On the Ti-OTS surface, OH- and PO43- combined with Ca2+ at the same time, and because of coulombic repulsion between negative charge and anions, OH- and PO43- successively combined with Ca2+ that induced CaP minerals nucleation and growth. Consequently, the bridging molecule on pTi surface possessed different interfacial properties with nanoscale CaP particles that showed homogeneous and orderly surface morphology. This hypothesis will be discussed and confirmed in the following section. ...
1
2009
... The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far. ...
1
2005
... The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far. ...
1
2014
... The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far. ...
1
2013
... The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far. ...
2
2013
... The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far. ...
... In order to illustrate the mechanism between CaP nucleation and growth with bridge molecule influencing on pTi surface, Fig. 1 shows the hypothetical schematic diagram of the SAMs and electrochemical deposition CaP coatings on the titanium surface (using pTi as cathode). It is obvious that hydroxyl groups were generated on titanium surface after treating with piranha solution, which could be stably bonded titanium surface with silane coupling as a molecular bridge (forming Si—O bond)[31]. On the one hand, the large amount of hydroxyl ions was generated through hydrogen evolution reaction of initial stage in electrochemical deposition, induced calcium phosphate ions rapid nucleation of initial precipitates[10]. On the other hand, in the case of the functional groups on silane coupling, the initial products that were assigned to HA precursors were rapidly precipitated as soon as the calcium and phosphate solutions were electrolyzed. The large amount of nuclei facilitated the heterogeneous nucleation during deposition, leading to precipitation and formation of apatite on the SAMs surface[15] and [21]. The nucleation of calcium phosphate particles on functionalized surface would yield diverse regular shape and relatively small size. The hydrogen evolution occurred at cathode interface and produced OH-, which could combine other ions in the electrolytes, such as Ca2+ and PO43-, thus causing nucleation of hydroxyapatite and its precursors. Meanwhile, the functionalized pTi surface charge and wettability did not resemble that of the bioactive pTi. The positive charges surface (—NH2 group) could attract negative charges (OH-, PO43-) while negative charges surface (—CH3 and —C(O)C group) could attract positive charges (Ca2+). Therefore, NH2-terminated surface (Ti-APTES) as a molecular bridge with positive charge had priority to attract OH- and PO43-, consequently Ca2+ was attracted to cathode surface and gradually formed hydroxyapatite[32]. Moreover, negatively charged surface (potential absolute value: pTi < Ti-OTS < Ti-GPTMS) had priority to attract Ca2+, but the number of negative charge on pTi was less than Ti-OTS (—CH3 group) and Ti-GPTMS (—C(O)C group), hydroxyl ions was produced by hydrogen evolution reaction firstly combined with Ca2+ on pTi surface. On the Ti-OTS surface, OH- and PO43- combined with Ca2+ at the same time, and because of coulombic repulsion between negative charge and anions, OH- and PO43- successively combined with Ca2+ that induced CaP minerals nucleation and growth. Consequently, the bridging molecule on pTi surface possessed different interfacial properties with nanoscale CaP particles that showed homogeneous and orderly surface morphology. This hypothesis will be discussed and confirmed in the following section. ...
1
2008
... The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far. ...
1
2013
... The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far. ...
1
2012
... The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far. ...
1
2008
... The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far. ...
2
2014
... The electrodeposition of CaP coating is normally performed in an aqueous solution containing calcium and phosphorus species[11]. In general, calcium phosphate coating deposited from these electrolytes usually contain four types of CaP precursors, such as brushite or dicalcium phosphate dihydrate (CaHPO4⋅2H2O, DCPD), monetite or dicalcium phosphate anhydrous (CaHPO4, DCPA), octacalcium phosphate (Ca8H2(PO4)6, OCP) and hydroxyapatite (Ca10(PO4)6(OH)2, HA)[12], [13], [14] and [15]. The crystallinity of bioactive ceramic is reported to comprise micro or nano structure, which has many important characteristics of the bone mineral phase, such as a carbonated, defective HA with small crystallite dimensions[16]. These characteristics maximally influence the rate of bone bonding and are very useful in the context of artificial grafts because of their effect on bone cell function[17]. The adjustment of pH, temperature and current density in electrochemical deposition process is well known to be able to regulate the characteristics of the CaP in the coating[18], [19] and [20]. However, the effects of functional group on titanium substrate properties (such as wettability, surface energy and surface potential etc.) by electrocrystallization of CaP coating have rarely been investigated so far. ...
... Cells are inherently sensitive to their surrounding microenvironments, such as bio-chemical signals and surface topography of the materials[20] and [42]. It is generally accepted that cells use filopodia for spatial sensing in their movement and spread on functional surfaces[43]. Fig. 9 shows the morphology of MC3T3-E1 cells on CaP coatings discs with functional groups after culturing for 24 h, and the filopodia of cells were observed on all pTi surfaces. However, the MC3T3-E1 cells displayed different shapes on the different morphology of CaP coatings. Cells on pTi-CaP and Ti-OTS-CaP showed an elongated form and stretched less filopodia (Fig. 9(a, b), and cytoskeleton appeared to stand off the surface on pTi-CaP and Ti-OTS-CaP. In addition, the portion of cytoplasmatic prolongations appeared and spread in all directions on Ti-APTES-CaP and Ti-GPTMS-CaP (Fig. 9(c, d). Shi et al.[44] reported that nano-HA particles could obviously stimulate osteoblastic adhesion compared with micro-HA particles. It was shown that the initial adhesion behavior of cells may be ascribed to the enhanced interfacial adhesion on HA nano-morphology by different molecular bridge. Moreover, in this study, nanoscale surface of HA seemed to exhibit the trend of spreading more filopodia. ...
2
2002
... A self-assembled monolayer (SAM) technique that employs multiple steps has been used to investigate the effect of functional groups on apatite formation on Ti-based substrates in biomineralization[21]. For example, Toworfe et al.[22] reported amine (—NH2), carboxyl (—COOH) and hydroxyl (—OH) functionalized surfaces for biomineralization and OH-terminated surface enhanced CaP nucleation and growth. And —OH and —CH3 functional groups also exhibited different behaviors in terms of hydroxyapatite formation[23]. These results suggest that functional groups are quite beneficial for guiding the aggregation of Ca2+, PO43-, OH- and prenucleation clusters, and then promoting apatite minerals formation with diverse micro or nano structures[24] and [25]. ...
... In order to illustrate the mechanism between CaP nucleation and growth with bridge molecule influencing on pTi surface, Fig. 1 shows the hypothetical schematic diagram of the SAMs and electrochemical deposition CaP coatings on the titanium surface (using pTi as cathode). It is obvious that hydroxyl groups were generated on titanium surface after treating with piranha solution, which could be stably bonded titanium surface with silane coupling as a molecular bridge (forming Si—O bond)[31]. On the one hand, the large amount of hydroxyl ions was generated through hydrogen evolution reaction of initial stage in electrochemical deposition, induced calcium phosphate ions rapid nucleation of initial precipitates[10]. On the other hand, in the case of the functional groups on silane coupling, the initial products that were assigned to HA precursors were rapidly precipitated as soon as the calcium and phosphate solutions were electrolyzed. The large amount of nuclei facilitated the heterogeneous nucleation during deposition, leading to precipitation and formation of apatite on the SAMs surface[15] and [21]. The nucleation of calcium phosphate particles on functionalized surface would yield diverse regular shape and relatively small size. The hydrogen evolution occurred at cathode interface and produced OH-, which could combine other ions in the electrolytes, such as Ca2+ and PO43-, thus causing nucleation of hydroxyapatite and its precursors. Meanwhile, the functionalized pTi surface charge and wettability did not resemble that of the bioactive pTi. The positive charges surface (—NH2 group) could attract negative charges (OH-, PO43-) while negative charges surface (—CH3 and —C(O)C group) could attract positive charges (Ca2+). Therefore, NH2-terminated surface (Ti-APTES) as a molecular bridge with positive charge had priority to attract OH- and PO43-, consequently Ca2+ was attracted to cathode surface and gradually formed hydroxyapatite[32]. Moreover, negatively charged surface (potential absolute value: pTi < Ti-OTS < Ti-GPTMS) had priority to attract Ca2+, but the number of negative charge on pTi was less than Ti-OTS (—CH3 group) and Ti-GPTMS (—C(O)C group), hydroxyl ions was produced by hydrogen evolution reaction firstly combined with Ca2+ on pTi surface. On the Ti-OTS surface, OH- and PO43- combined with Ca2+ at the same time, and because of coulombic repulsion between negative charge and anions, OH- and PO43- successively combined with Ca2+ that induced CaP minerals nucleation and growth. Consequently, the bridging molecule on pTi surface possessed different interfacial properties with nanoscale CaP particles that showed homogeneous and orderly surface morphology. This hypothesis will be discussed and confirmed in the following section. ...
3
2006
... A self-assembled monolayer (SAM) technique that employs multiple steps has been used to investigate the effect of functional groups on apatite formation on Ti-based substrates in biomineralization[21]. For example, Toworfe et al.[22] reported amine (—NH2), carboxyl (—COOH) and hydroxyl (—OH) functionalized surfaces for biomineralization and OH-terminated surface enhanced CaP nucleation and growth. And —OH and —CH3 functional groups also exhibited different behaviors in terms of hydroxyapatite formation[23]. These results suggest that functional groups are quite beneficial for guiding the aggregation of Ca2+, PO43-, OH- and prenucleation clusters, and then promoting apatite minerals formation with diverse micro or nano structures[24] and [25]. ...
... In order to prove the SAMs being successfully grafted on pTi surface, ATR-FTIR spectra was used to analyze representative groups in this work. Fig. 2 shows the FTIR spectra corresponding to samples modified by different SAMs. Distinctly, pTi surface had marked peak in the range of 3200-3500 cm-1 which contributed to O—H vibration, because Ti was oxidized in H2SO4/H2O2 solution. After OTS was grafted, the stretching vibrations of OH groups in pTi were shifted from 3250 cm-1 to 3620 cm-1 in Ti-OTS, indicating that the hydrophilic OH groups were consumed by the OTS coating. Meanwhile, different SAMs grafted on pTi surface, and the intensity of H—OH vibration peak at 1637 cm-1 decreased compared to pTi surface[22]. Additionally, once the SAMs bonded with pTi surface, the new peaks located around 1056 cm-1 attributed to siloxane group (Si—O) from OTS, APTES and GPTMS, while 2929 and 2855 cm-1 attributed to methylene group (—CH2)[33], indicating that silane coupling agent as a molecular bridge had been grafted onto the surface of titanium substrate. The symmetric bending vibration of —NH2 in Ti-APTES appeared at 1536 cm-1[34]. Meanwhile, the presence of the peak at 910 cm-1 suggested that epoxy group (—C(O)C) in GPTMS was associated with pTi surface[35]. It was showed that the intensity of siloxane group (Si—O) at 1056 cm-1 after 1% OTS treatment decreased compared to 2% APTES and 5% GPTMS, and the absorption bands of Ti-OTS at 2929 and 2855 cm-1 were most obvious due to the presence of methylene group in octadecyl chain. ...
... Fig. 5 shows SEM micrographs of CaP coating on pTi surface: (a) oxidized-treatment, (b) Ti-OTS, (c) Ti-APTES, and (d) Ti-GPTMS. All samples exhibited various surface morphologies by deposition in the electrolyte solution including Ca2+ and PO43- under identical process conditions. As shown in Fig. 5(e), pTi surfaces were covered with flower-like CaP precipitates clustered with submicro pinpoint-like crystals, whereas Ti-OTS surface (—CH3 group) exhibited flower-like CaP precipitates clustered with nano pinpoint-like crystals (Fig. 5(f)). It was clearly observed in Fig. 5(g) that flower-like CaP precipitates clustered with nano squame-like crystals were thickly covered on Ti-APTES surface (—NH2 group), and flower-like CaP precipitates clustered with nano flake-like crystals (Fig. 5(h)) on Ti-GPTMS surface (—C(O)C group). Apparently, dense and homogeneous CaP coatings were achieved on SAMs surface[26], and CaP coatings on pTi surface was sparse and disorderly. Moreover, in order to explore the Ca and P element distribution, element mapping scanning mode of EDS was used in this work. As showed in Fig. 6, the green points show Ca element and red points show P element. Compared with CaP coatings on functionalized pTi surface, there were sparsely Ca and P elements distributed on pTi surface (Fig. 6(a)). Meanwhile, Ti-APTES exhibited denser Ca and P distributions of coating (Fig. 6(c)) comparing with Ti-OTS (Fig. 6(b)) and Ti-GPTMS (Fig. 6(d)). In conclusion, the surface energy and surface potential showed that the surface functional groups as a molecular bridge determined the solid/ion cluster interfacial energies[22], then different functionalized pTi surface exhibited different nucleation abilities of calcium phosphate with diverse morphology. ...
1
2013
... A self-assembled monolayer (SAM) technique that employs multiple steps has been used to investigate the effect of functional groups on apatite formation on Ti-based substrates in biomineralization[21]. For example, Toworfe et al.[22] reported amine (—NH2), carboxyl (—COOH) and hydroxyl (—OH) functionalized surfaces for biomineralization and OH-terminated surface enhanced CaP nucleation and growth. And —OH and —CH3 functional groups also exhibited different behaviors in terms of hydroxyapatite formation[23]. These results suggest that functional groups are quite beneficial for guiding the aggregation of Ca2+, PO43-, OH- and prenucleation clusters, and then promoting apatite minerals formation with diverse micro or nano structures[24] and [25]. ...
1
2013
... A self-assembled monolayer (SAM) technique that employs multiple steps has been used to investigate the effect of functional groups on apatite formation on Ti-based substrates in biomineralization[21]. For example, Toworfe et al.[22] reported amine (—NH2), carboxyl (—COOH) and hydroxyl (—OH) functionalized surfaces for biomineralization and OH-terminated surface enhanced CaP nucleation and growth. And —OH and —CH3 functional groups also exhibited different behaviors in terms of hydroxyapatite formation[23]. These results suggest that functional groups are quite beneficial for guiding the aggregation of Ca2+, PO43-, OH- and prenucleation clusters, and then promoting apatite minerals formation with diverse micro or nano structures[24] and [25]. ...
1
2006
... A self-assembled monolayer (SAM) technique that employs multiple steps has been used to investigate the effect of functional groups on apatite formation on Ti-based substrates in biomineralization[21]. For example, Toworfe et al.[22] reported amine (—NH2), carboxyl (—COOH) and hydroxyl (—OH) functionalized surfaces for biomineralization and OH-terminated surface enhanced CaP nucleation and growth. And —OH and —CH3 functional groups also exhibited different behaviors in terms of hydroxyapatite formation[23]. These results suggest that functional groups are quite beneficial for guiding the aggregation of Ca2+, PO43-, OH- and prenucleation clusters, and then promoting apatite minerals formation with diverse micro or nano structures[24] and [25]. ...
2
2014
... Square Ti samples of approximately 25 mm × 25 mm were prepared from 99.7% medically pure titanium sheet (0.1 mm thick) (Baoji Qichen New Material Technology Co., Ltd., China). These were rinsed in absolute ethyl alcohol, then in acetone for 10 min, and finally in distilled water for 10 min. The samples were subsequently immersed in a 7:3 (v/v) mixture of concentrated H2SO4 (Sigma-Aldrich, USA) and 30% H2O2 (Sigma-Aldrich, USA) (piranha solution) for 30 min at 60 °C (named pTi)[26]. The samples were sonicated with distilled water for 10 min, and then dried in N2 atmosphere at room temperature. ...
... Fig. 5 shows SEM micrographs of CaP coating on pTi surface: (a) oxidized-treatment, (b) Ti-OTS, (c) Ti-APTES, and (d) Ti-GPTMS. All samples exhibited various surface morphologies by deposition in the electrolyte solution including Ca2+ and PO43- under identical process conditions. As shown in Fig. 5(e), pTi surfaces were covered with flower-like CaP precipitates clustered with submicro pinpoint-like crystals, whereas Ti-OTS surface (—CH3 group) exhibited flower-like CaP precipitates clustered with nano pinpoint-like crystals (Fig. 5(f)). It was clearly observed in Fig. 5(g) that flower-like CaP precipitates clustered with nano squame-like crystals were thickly covered on Ti-APTES surface (—NH2 group), and flower-like CaP precipitates clustered with nano flake-like crystals (Fig. 5(h)) on Ti-GPTMS surface (—C(O)C group). Apparently, dense and homogeneous CaP coatings were achieved on SAMs surface[26], and CaP coatings on pTi surface was sparse and disorderly. Moreover, in order to explore the Ca and P element distribution, element mapping scanning mode of EDS was used in this work. As showed in Fig. 6, the green points show Ca element and red points show P element. Compared with CaP coatings on functionalized pTi surface, there were sparsely Ca and P elements distributed on pTi surface (Fig. 6(a)). Meanwhile, Ti-APTES exhibited denser Ca and P distributions of coating (Fig. 6(c)) comparing with Ti-OTS (Fig. 6(b)) and Ti-GPTMS (Fig. 6(d)). In conclusion, the surface energy and surface potential showed that the surface functional groups as a molecular bridge determined the solid/ion cluster interfacial energies[22], then different functionalized pTi surface exhibited different nucleation abilities of calcium phosphate with diverse morphology. ...
1
2010
... In vitro biocompatibility of the CaP coated SAMs sample with MC3T3-E1 mouse osteoblasts (ATCC CRL-2593) was evaluated using cell adhesion experiment. Cell were seeded directly on the samples (sterilized by ultraviolet-radiation for at least 2 h prior to the cell seeding process) at a density of 2 × 104 cells/mL in 24-well cell culture plates (500 µL/well) (Corning, USA) and incubated in the α-modified Eagle's minimum essential medium (α-MEM) (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 °C[27]. After incubation for 24 h, the cell viability was expressed in the percentage as following: the typical cellular adhesion and spread morphologies on the CaP coated different pTi surface were evaluated by scanning electron microscopy (SEM) after Pt/Au sputtering. ...
1
2012
... A 500 µL cell suspension was seeded on each specimen at a density of 2 × 104 cells/mL and cultured in α-MEM with 10% FBS. The cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (98%, Aladdin, China) assay on 1, 3, 5 and 7 days. Briefly, at each prescribed time point, the specimens were rinsed gently three times with PBS, 40 µL MTT (5 mg/mL) solution and 400 µL α-MEM without FBS were added every well, and the specimens were incubated at 37 °C for 4 h to form formazan crystals[28]. Then the formazan was dissolved in dimethyl sulfoxide (DMSO) (99.9%, Sigma-Aldrich, USA) and shocked for 10 min, and the optical density (OD) was measured at 570 nm using a microplate reader (Multiskan FC, Thermo, USA). ...
1
2010
... where θ is the contact angle; and
and
are the dispersion and polar surface energy of the solid and the liquid, respectively, and γSγS is the sum surface energy of the solid phases. Using archival surface force values of
with ultrapure water and diiodomethane, the values of γSγS could be calculated[29].
...
1
2008
... Based on Scherrer formula, the CaP crystallite size was determined upon using the XRD data emanating from the (002) and (211) reflection crystallographic plane peaks. Average size was directly obtained from Jade 5.0 software with all crystallographic plane peaks[30]. ...
1
2011
... In order to illustrate the mechanism between CaP nucleation and growth with bridge molecule influencing on pTi surface, Fig. 1 shows the hypothetical schematic diagram of the SAMs and electrochemical deposition CaP coatings on the titanium surface (using pTi as cathode). It is obvious that hydroxyl groups were generated on titanium surface after treating with piranha solution, which could be stably bonded titanium surface with silane coupling as a molecular bridge (forming Si—O bond)[31]. On the one hand, the large amount of hydroxyl ions was generated through hydrogen evolution reaction of initial stage in electrochemical deposition, induced calcium phosphate ions rapid nucleation of initial precipitates[10]. On the other hand, in the case of the functional groups on silane coupling, the initial products that were assigned to HA precursors were rapidly precipitated as soon as the calcium and phosphate solutions were electrolyzed. The large amount of nuclei facilitated the heterogeneous nucleation during deposition, leading to precipitation and formation of apatite on the SAMs surface[15] and [21]. The nucleation of calcium phosphate particles on functionalized surface would yield diverse regular shape and relatively small size. The hydrogen evolution occurred at cathode interface and produced OH-, which could combine other ions in the electrolytes, such as Ca2+ and PO43-, thus causing nucleation of hydroxyapatite and its precursors. Meanwhile, the functionalized pTi surface charge and wettability did not resemble that of the bioactive pTi. The positive charges surface (—NH2 group) could attract negative charges (OH-, PO43-) while negative charges surface (—CH3 and —C(O)C group) could attract positive charges (Ca2+). Therefore, NH2-terminated surface (Ti-APTES) as a molecular bridge with positive charge had priority to attract OH- and PO43-, consequently Ca2+ was attracted to cathode surface and gradually formed hydroxyapatite[32]. Moreover, negatively charged surface (potential absolute value: pTi < Ti-OTS < Ti-GPTMS) had priority to attract Ca2+, but the number of negative charge on pTi was less than Ti-OTS (—CH3 group) and Ti-GPTMS (—C(O)C group), hydroxyl ions was produced by hydrogen evolution reaction firstly combined with Ca2+ on pTi surface. On the Ti-OTS surface, OH- and PO43- combined with Ca2+ at the same time, and because of coulombic repulsion between negative charge and anions, OH- and PO43- successively combined with Ca2+ that induced CaP minerals nucleation and growth. Consequently, the bridging molecule on pTi surface possessed different interfacial properties with nanoscale CaP particles that showed homogeneous and orderly surface morphology. This hypothesis will be discussed and confirmed in the following section. ...
2
2011
... In order to illustrate the mechanism between CaP nucleation and growth with bridge molecule influencing on pTi surface, Fig. 1 shows the hypothetical schematic diagram of the SAMs and electrochemical deposition CaP coatings on the titanium surface (using pTi as cathode). It is obvious that hydroxyl groups were generated on titanium surface after treating with piranha solution, which could be stably bonded titanium surface with silane coupling as a molecular bridge (forming Si—O bond)[31]. On the one hand, the large amount of hydroxyl ions was generated through hydrogen evolution reaction of initial stage in electrochemical deposition, induced calcium phosphate ions rapid nucleation of initial precipitates[10]. On the other hand, in the case of the functional groups on silane coupling, the initial products that were assigned to HA precursors were rapidly precipitated as soon as the calcium and phosphate solutions were electrolyzed. The large amount of nuclei facilitated the heterogeneous nucleation during deposition, leading to precipitation and formation of apatite on the SAMs surface[15] and [21]. The nucleation of calcium phosphate particles on functionalized surface would yield diverse regular shape and relatively small size. The hydrogen evolution occurred at cathode interface and produced OH-, which could combine other ions in the electrolytes, such as Ca2+ and PO43-, thus causing nucleation of hydroxyapatite and its precursors. Meanwhile, the functionalized pTi surface charge and wettability did not resemble that of the bioactive pTi. The positive charges surface (—NH2 group) could attract negative charges (OH-, PO43-) while negative charges surface (—CH3 and —C(O)C group) could attract positive charges (Ca2+). Therefore, NH2-terminated surface (Ti-APTES) as a molecular bridge with positive charge had priority to attract OH- and PO43-, consequently Ca2+ was attracted to cathode surface and gradually formed hydroxyapatite[32]. Moreover, negatively charged surface (potential absolute value: pTi < Ti-OTS < Ti-GPTMS) had priority to attract Ca2+, but the number of negative charge on pTi was less than Ti-OTS (—CH3 group) and Ti-GPTMS (—C(O)C group), hydroxyl ions was produced by hydrogen evolution reaction firstly combined with Ca2+ on pTi surface. On the Ti-OTS surface, OH- and PO43- combined with Ca2+ at the same time, and because of coulombic repulsion between negative charge and anions, OH- and PO43- successively combined with Ca2+ that induced CaP minerals nucleation and growth. Consequently, the bridging molecule on pTi surface possessed different interfacial properties with nanoscale CaP particles that showed homogeneous and orderly surface morphology. This hypothesis will be discussed and confirmed in the following section. ...
... In addition, superficial charges play an important role in electrostatic adsorption ions of electrolyte, especially in inducing CaP nucleation and growth[32]. The charges of SAMs surface in the designated pH area was shown in Fig. 4(a). With the increase of pH, zeta-potential obviously decreased. Additionally, as shown in Fig. 4(b), Ti-APTES surface (—NH2 group) was covered with positive potential and others (pTi, —CH3 and —C(O)C groups) were covered with negative potential at pH = 6. The absolute zeta-potential comparison sequence: Ti-GPTMS (-41.21 mV) < Ti-OTS (-32.54 mV) < pTi (-26.79 mV) < Ti-APTES (1.52 mV), reflected that surface functional groups could provide different adsorption and integration sites by electrochemical deposition. Surface functional groups as a molecular bridge displayed different surface properties. Therefore, the influences on CaP nucleation were expected to be different, which would be discussed in the following section. ...
1
2009
... In order to prove the SAMs being successfully grafted on pTi surface, ATR-FTIR spectra was used to analyze representative groups in this work. Fig. 2 shows the FTIR spectra corresponding to samples modified by different SAMs. Distinctly, pTi surface had marked peak in the range of 3200-3500 cm-1 which contributed to O—H vibration, because Ti was oxidized in H2SO4/H2O2 solution. After OTS was grafted, the stretching vibrations of OH groups in pTi were shifted from 3250 cm-1 to 3620 cm-1 in Ti-OTS, indicating that the hydrophilic OH groups were consumed by the OTS coating. Meanwhile, different SAMs grafted on pTi surface, and the intensity of H—OH vibration peak at 1637 cm-1 decreased compared to pTi surface[22]. Additionally, once the SAMs bonded with pTi surface, the new peaks located around 1056 cm-1 attributed to siloxane group (Si—O) from OTS, APTES and GPTMS, while 2929 and 2855 cm-1 attributed to methylene group (—CH2)[33], indicating that silane coupling agent as a molecular bridge had been grafted onto the surface of titanium substrate. The symmetric bending vibration of —NH2 in Ti-APTES appeared at 1536 cm-1[34]. Meanwhile, the presence of the peak at 910 cm-1 suggested that epoxy group (—C(O)C) in GPTMS was associated with pTi surface[35]. It was showed that the intensity of siloxane group (Si—O) at 1056 cm-1 after 1% OTS treatment decreased compared to 2% APTES and 5% GPTMS, and the absorption bands of Ti-OTS at 2929 and 2855 cm-1 were most obvious due to the presence of methylene group in octadecyl chain. ...
1
2013
... In order to prove the SAMs being successfully grafted on pTi surface, ATR-FTIR spectra was used to analyze representative groups in this work. Fig. 2 shows the FTIR spectra corresponding to samples modified by different SAMs. Distinctly, pTi surface had marked peak in the range of 3200-3500 cm-1 which contributed to O—H vibration, because Ti was oxidized in H2SO4/H2O2 solution. After OTS was grafted, the stretching vibrations of OH groups in pTi were shifted from 3250 cm-1 to 3620 cm-1 in Ti-OTS, indicating that the hydrophilic OH groups were consumed by the OTS coating. Meanwhile, different SAMs grafted on pTi surface, and the intensity of H—OH vibration peak at 1637 cm-1 decreased compared to pTi surface[22]. Additionally, once the SAMs bonded with pTi surface, the new peaks located around 1056 cm-1 attributed to siloxane group (Si—O) from OTS, APTES and GPTMS, while 2929 and 2855 cm-1 attributed to methylene group (—CH2)[33], indicating that silane coupling agent as a molecular bridge had been grafted onto the surface of titanium substrate. The symmetric bending vibration of —NH2 in Ti-APTES appeared at 1536 cm-1[34]. Meanwhile, the presence of the peak at 910 cm-1 suggested that epoxy group (—C(O)C) in GPTMS was associated with pTi surface[35]. It was showed that the intensity of siloxane group (Si—O) at 1056 cm-1 after 1% OTS treatment decreased compared to 2% APTES and 5% GPTMS, and the absorption bands of Ti-OTS at 2929 and 2855 cm-1 were most obvious due to the presence of methylene group in octadecyl chain. ...
1
2013
... In order to prove the SAMs being successfully grafted on pTi surface, ATR-FTIR spectra was used to analyze representative groups in this work. Fig. 2 shows the FTIR spectra corresponding to samples modified by different SAMs. Distinctly, pTi surface had marked peak in the range of 3200-3500 cm-1 which contributed to O—H vibration, because Ti was oxidized in H2SO4/H2O2 solution. After OTS was grafted, the stretching vibrations of OH groups in pTi were shifted from 3250 cm-1 to 3620 cm-1 in Ti-OTS, indicating that the hydrophilic OH groups were consumed by the OTS coating. Meanwhile, different SAMs grafted on pTi surface, and the intensity of H—OH vibration peak at 1637 cm-1 decreased compared to pTi surface[22]. Additionally, once the SAMs bonded with pTi surface, the new peaks located around 1056 cm-1 attributed to siloxane group (Si—O) from OTS, APTES and GPTMS, while 2929 and 2855 cm-1 attributed to methylene group (—CH2)[33], indicating that silane coupling agent as a molecular bridge had been grafted onto the surface of titanium substrate. The symmetric bending vibration of —NH2 in Ti-APTES appeared at 1536 cm-1[34]. Meanwhile, the presence of the peak at 910 cm-1 suggested that epoxy group (—C(O)C) in GPTMS was associated with pTi surface[35]. It was showed that the intensity of siloxane group (Si—O) at 1056 cm-1 after 1% OTS treatment decreased compared to 2% APTES and 5% GPTMS, and the absorption bands of Ti-OTS at 2929 and 2855 cm-1 were most obvious due to the presence of methylene group in octadecyl chain. ...
1
2009
... Macroscopically, the wettability of the SAMs was probed by means of the contact angle measurement using ultrapure water and diiodomethane. Sessile drop method is a useful and convenient way to track the change in wettability on the SAMs surface[36]. The water and diiodomethane contact angles could be used for calculating interfacial tension on each surface. The contact angle on pTi with SAMs strongly depended on the surface functional groups. As shown in Fig. 3, the water contact angle of the pTi was 18.2 ± 1.1° by oxidation in piranha solution. However, the pTi surface grafted with OTS was made highly hydrophobic, the water contact angle increased from with OTS is hydrophobic to 109.8 ± 2.1° with the hydrophobic group alkyl (—CH2—). While APTES and GPTMS were grafted on the pTi, the water contact angles increased from 18.2 ± 1.1° to 52.1 ± 4.5° and 41.7 ± 2.6° respectively, which indicated higher hydrophobicity on the APTES and GPTMS surfaces than that on pTi. Diiodomethane liquid was employed to verify the oleophilic property of the pTi surface. In Fig. 3, the diiodomethane contact angles with different SAMs could also be observed, which indicated different lipophilicity surface. In general, a smaller water contact angle means a higher distribution of hydrophilic groups on the surface that provides a relatively higher adhesion tension[37]. The surface energies in Table 1 were calculated by equations (1), (2) and (3). Evidently, surface energy was decreased from 71.12 mJ/cm2 (pTi) to 37.50 mJ/cm2 (Ti-OTS), 56.00 mJ/cm2 (Ti-APTES) and 62.12 mJ/cm2 (Ti-GPTMS), respectively. The three functionalized pTi surface showed lower surface energy compared to the pTi surface due to the presence of functional groups. Therefore, higher surface energy (pTi) could not conduce to form nanoscale particles, and lower surface energy could benefit to nucleation and formation of nanoscale CaP particles. ...
1
2011
... Macroscopically, the wettability of the SAMs was probed by means of the contact angle measurement using ultrapure water and diiodomethane. Sessile drop method is a useful and convenient way to track the change in wettability on the SAMs surface[36]. The water and diiodomethane contact angles could be used for calculating interfacial tension on each surface. The contact angle on pTi with SAMs strongly depended on the surface functional groups. As shown in Fig. 3, the water contact angle of the pTi was 18.2 ± 1.1° by oxidation in piranha solution. However, the pTi surface grafted with OTS was made highly hydrophobic, the water contact angle increased from with OTS is hydrophobic to 109.8 ± 2.1° with the hydrophobic group alkyl (—CH2—). While APTES and GPTMS were grafted on the pTi, the water contact angles increased from 18.2 ± 1.1° to 52.1 ± 4.5° and 41.7 ± 2.6° respectively, which indicated higher hydrophobicity on the APTES and GPTMS surfaces than that on pTi. Diiodomethane liquid was employed to verify the oleophilic property of the pTi surface. In Fig. 3, the diiodomethane contact angles with different SAMs could also be observed, which indicated different lipophilicity surface. In general, a smaller water contact angle means a higher distribution of hydrophilic groups on the surface that provides a relatively higher adhesion tension[37]. The surface energies in Table 1 were calculated by equations (1), (2) and (3). Evidently, surface energy was decreased from 71.12 mJ/cm2 (pTi) to 37.50 mJ/cm2 (Ti-OTS), 56.00 mJ/cm2 (Ti-APTES) and 62.12 mJ/cm2 (Ti-GPTMS), respectively. The three functionalized pTi surface showed lower surface energy compared to the pTi surface due to the presence of functional groups. Therefore, higher surface energy (pTi) could not conduce to form nanoscale particles, and lower surface energy could benefit to nucleation and formation of nanoscale CaP particles. ...
1
2013
... In this study, the surface composition of CaP coatings deposited on titanium was confirmed by XPS spectra. To further investigatethe interaction between SAMs and HA, XPS analysis was carried out and the results are shown in Table 2 and Fig. 7. Table 2 confirms that the coatings surface consisted of Ca, P, Si and N elements. As shown in Fig. 7, a characteristic doublet peak located at 459.0 and 464.7 eV were attributed to Ti2p[38], showing that TiO2 was the main component after oxidation with piranha solution, but others did not significantly appear to Ti2p doublet peak at this position, which demonstrated that the coating on pTi surface was very thin (in accordance with the results of SEM and EDS). Meanwhile, all of them exhibited stronger O KLL (Auger), Ti LMM (Auger), Ti2s and O1s peaks on the corresponding position. Si2s and Si2p peaks could be observed at 153 eV and 102.2 eV (Table 2), indicating Si—O bonded on the pTi surface. N1s derived from the APTES silane of bridging molecule was detected at the binding energy of 399.6 eV in a total amount of 1.08 at.%. Moreover, their compositions in Table 2 were lower than other elements because CaP coatings were entirely covered on SAMs surface[39]. Functional groups caused an increase of Ca atoms on the pTi surface from 6.6 to 12.11 at.%, and P atoms on the pTi surface increased from 4.92 to 7.27 at.%. The binding energy of Ca and P elements were almost not changed (Table 2), indicating that functional groups did not affect their valence state. All of these demonstrated that the surface mineral coatings consisted of Ca2+, PO43- and OH-. ...
1
2012
... In this study, the surface composition of CaP coatings deposited on titanium was confirmed by XPS spectra. To further investigatethe interaction between SAMs and HA, XPS analysis was carried out and the results are shown in Table 2 and Fig. 7. Table 2 confirms that the coatings surface consisted of Ca, P, Si and N elements. As shown in Fig. 7, a characteristic doublet peak located at 459.0 and 464.7 eV were attributed to Ti2p[38], showing that TiO2 was the main component after oxidation with piranha solution, but others did not significantly appear to Ti2p doublet peak at this position, which demonstrated that the coating on pTi surface was very thin (in accordance with the results of SEM and EDS). Meanwhile, all of them exhibited stronger O KLL (Auger), Ti LMM (Auger), Ti2s and O1s peaks on the corresponding position. Si2s and Si2p peaks could be observed at 153 eV and 102.2 eV (Table 2), indicating Si—O bonded on the pTi surface. N1s derived from the APTES silane of bridging molecule was detected at the binding energy of 399.6 eV in a total amount of 1.08 at.%. Moreover, their compositions in Table 2 were lower than other elements because CaP coatings were entirely covered on SAMs surface[39]. Functional groups caused an increase of Ca atoms on the pTi surface from 6.6 to 12.11 at.%, and P atoms on the pTi surface increased from 4.92 to 7.27 at.%. The binding energy of Ca and P elements were almost not changed (Table 2), indicating that functional groups did not affect their valence state. All of these demonstrated that the surface mineral coatings consisted of Ca2+, PO43- and OH-. ...
1
2006
... The results of the microstructure and crystal property can be obtained via TEM observation[40]. Fig. 8(a1-d1) shows a representative TEM image of diverse pinpoint-like CaP crystals on different pTi surfaces, where large numbers of uniform flower-like precipitates clustered with their functionalized nucleation sites exist. A submicro pinpoint-like crystal can be observed in Fig. 8(a1) on pTi surface; however, more obvious nano pinpoint-like crystals with SAMs are shown in Fig. 8(b1-d1). CaP coating on NH2-terminated surface presented much more weeny needle-like crystals compared with that of Ti-OTS (—CH3 group) and Ti-GPTMS (—C(O)C group). More details about the structure of the needle-like crystals were investigated by the selected area electron diffraction (SAED) pattern (inset of Fig. 8(a2-d2)). The corresponding SAED pattern that was taken from individual needle-like shape confirmed that the crystals were well-crystallized. The average size of the CaP nano-crystals accorded with the sizes calculated by equations (4) and (5) from XRD data (Table 3). The CaP nano-crystals were confirmed to be multicrystalline minerals from the rings of the SAED pattern (Fig. 8), which mainly consisted of HA and little HA precursors such as DCPA, DCPD, and OCP[41]. ...
1
2014
... The results of the microstructure and crystal property can be obtained via TEM observation[40]. Fig. 8(a1-d1) shows a representative TEM image of diverse pinpoint-like CaP crystals on different pTi surfaces, where large numbers of uniform flower-like precipitates clustered with their functionalized nucleation sites exist. A submicro pinpoint-like crystal can be observed in Fig. 8(a1) on pTi surface; however, more obvious nano pinpoint-like crystals with SAMs are shown in Fig. 8(b1-d1). CaP coating on NH2-terminated surface presented much more weeny needle-like crystals compared with that of Ti-OTS (—CH3 group) and Ti-GPTMS (—C(O)C group). More details about the structure of the needle-like crystals were investigated by the selected area electron diffraction (SAED) pattern (inset of Fig. 8(a2-d2)). The corresponding SAED pattern that was taken from individual needle-like shape confirmed that the crystals were well-crystallized. The average size of the CaP nano-crystals accorded with the sizes calculated by equations (4) and (5) from XRD data (Table 3). The CaP nano-crystals were confirmed to be multicrystalline minerals from the rings of the SAED pattern (Fig. 8), which mainly consisted of HA and little HA precursors such as DCPA, DCPD, and OCP[41]. ...
1
2009
... Cells are inherently sensitive to their surrounding microenvironments, such as bio-chemical signals and surface topography of the materials[20] and [42]. It is generally accepted that cells use filopodia for spatial sensing in their movement and spread on functional surfaces[43]. Fig. 9 shows the morphology of MC3T3-E1 cells on CaP coatings discs with functional groups after culturing for 24 h, and the filopodia of cells were observed on all pTi surfaces. However, the MC3T3-E1 cells displayed different shapes on the different morphology of CaP coatings. Cells on pTi-CaP and Ti-OTS-CaP showed an elongated form and stretched less filopodia (Fig. 9(a, b), and cytoskeleton appeared to stand off the surface on pTi-CaP and Ti-OTS-CaP. In addition, the portion of cytoplasmatic prolongations appeared and spread in all directions on Ti-APTES-CaP and Ti-GPTMS-CaP (Fig. 9(c, d). Shi et al.[44] reported that nano-HA particles could obviously stimulate osteoblastic adhesion compared with micro-HA particles. It was shown that the initial adhesion behavior of cells may be ascribed to the enhanced interfacial adhesion on HA nano-morphology by different molecular bridge. Moreover, in this study, nanoscale surface of HA seemed to exhibit the trend of spreading more filopodia. ...
1
2013
... Cells are inherently sensitive to their surrounding microenvironments, such as bio-chemical signals and surface topography of the materials[20] and [42]. It is generally accepted that cells use filopodia for spatial sensing in their movement and spread on functional surfaces[43]. Fig. 9 shows the morphology of MC3T3-E1 cells on CaP coatings discs with functional groups after culturing for 24 h, and the filopodia of cells were observed on all pTi surfaces. However, the MC3T3-E1 cells displayed different shapes on the different morphology of CaP coatings. Cells on pTi-CaP and Ti-OTS-CaP showed an elongated form and stretched less filopodia (Fig. 9(a, b), and cytoskeleton appeared to stand off the surface on pTi-CaP and Ti-OTS-CaP. In addition, the portion of cytoplasmatic prolongations appeared and spread in all directions on Ti-APTES-CaP and Ti-GPTMS-CaP (Fig. 9(c, d). Shi et al.[44] reported that nano-HA particles could obviously stimulate osteoblastic adhesion compared with micro-HA particles. It was shown that the initial adhesion behavior of cells may be ascribed to the enhanced interfacial adhesion on HA nano-morphology by different molecular bridge. Moreover, in this study, nanoscale surface of HA seemed to exhibit the trend of spreading more filopodia. ...
1
2009
... Cells are inherently sensitive to their surrounding microenvironments, such as bio-chemical signals and surface topography of the materials[20] and [42]. It is generally accepted that cells use filopodia for spatial sensing in their movement and spread on functional surfaces[43]. Fig. 9 shows the morphology of MC3T3-E1 cells on CaP coatings discs with functional groups after culturing for 24 h, and the filopodia of cells were observed on all pTi surfaces. However, the MC3T3-E1 cells displayed different shapes on the different morphology of CaP coatings. Cells on pTi-CaP and Ti-OTS-CaP showed an elongated form and stretched less filopodia (Fig. 9(a, b), and cytoskeleton appeared to stand off the surface on pTi-CaP and Ti-OTS-CaP. In addition, the portion of cytoplasmatic prolongations appeared and spread in all directions on Ti-APTES-CaP and Ti-GPTMS-CaP (Fig. 9(c, d). Shi et al.[44] reported that nano-HA particles could obviously stimulate osteoblastic adhesion compared with micro-HA particles. It was shown that the initial adhesion behavior of cells may be ascribed to the enhanced interfacial adhesion on HA nano-morphology by different molecular bridge. Moreover, in this study, nanoscale surface of HA seemed to exhibit the trend of spreading more filopodia. ...
1
2010
... The mechanism of the MTT assay involves that pale yellow MTT substance will be converted to dark blue formazan crystals only by the viable cells[45]. Therefore, MTT assay was employed to confirm the MC3T3-E1 cells adhesion and proliferation on pTi and different functionalized pTi surfaces with CaP coatings. The results of cell proliferation behaviors are shown in Fig. 10. The values of optical density (OD) were varied on different surfaces for 1, 3, 5 and 7 days of culturing time following a similar trend. After 1 day of culture, the cell numbers on Ti-OTS-CaP surface (—CH3 group) were higher than others (—NH2 and —C(O)C group), and the trend was almost similar during 3 days, except that Ti-GPTMS-CaP showed lower activity compared with pTi-CaP. Up to 5 days, all of the OD values significantly increased as opposed to OD values of 3 days. After 7 days of culture, OD value of pTi-CaP did not obviously increase, and the other three functionalized pTi surfaces exhibited a similar trend on preceding days. All these results demonstrated that there was a high yield of proliferative activity (non-toxicity) in the osteoblast cells after a long cell culture time. The Ti-OTS-CaP surfaces exhibited higher cell viability than others during cell culture in 7 days. Meanwhile, the trend of MTT assay on different functionalized surfaces was inconsistent with the SEM micrographs (Fig. 9). It is possible that HA nano-morphology can be conducive in improving interfacial adhesion of cells due to the provided space for filopodia[46]. According to Okada et al.[47], the smaller size of nanostructures would adversely affect osteoblasts. Therefore, the cellular response to HA substrates may be influenced by the balance in size between the dense micro/nano structure and the focal adhesions of the cells. ...
1
2015
... The mechanism of the MTT assay involves that pale yellow MTT substance will be converted to dark blue formazan crystals only by the viable cells[45]. Therefore, MTT assay was employed to confirm the MC3T3-E1 cells adhesion and proliferation on pTi and different functionalized pTi surfaces with CaP coatings. The results of cell proliferation behaviors are shown in Fig. 10. The values of optical density (OD) were varied on different surfaces for 1, 3, 5 and 7 days of culturing time following a similar trend. After 1 day of culture, the cell numbers on Ti-OTS-CaP surface (—CH3 group) were higher than others (—NH2 and —C(O)C group), and the trend was almost similar during 3 days, except that Ti-GPTMS-CaP showed lower activity compared with pTi-CaP. Up to 5 days, all of the OD values significantly increased as opposed to OD values of 3 days. After 7 days of culture, OD value of pTi-CaP did not obviously increase, and the other three functionalized pTi surfaces exhibited a similar trend on preceding days. All these results demonstrated that there was a high yield of proliferative activity (non-toxicity) in the osteoblast cells after a long cell culture time. The Ti-OTS-CaP surfaces exhibited higher cell viability than others during cell culture in 7 days. Meanwhile, the trend of MTT assay on different functionalized surfaces was inconsistent with the SEM micrographs (Fig. 9). It is possible that HA nano-morphology can be conducive in improving interfacial adhesion of cells due to the provided space for filopodia[46]. According to Okada et al.[47], the smaller size of nanostructures would adversely affect osteoblasts. Therefore, the cellular response to HA substrates may be influenced by the balance in size between the dense micro/nano structure and the focal adhesions of the cells. ...
1
2011
... The mechanism of the MTT assay involves that pale yellow MTT substance will be converted to dark blue formazan crystals only by the viable cells[45]. Therefore, MTT assay was employed to confirm the MC3T3-E1 cells adhesion and proliferation on pTi and different functionalized pTi surfaces with CaP coatings. The results of cell proliferation behaviors are shown in Fig. 10. The values of optical density (OD) were varied on different surfaces for 1, 3, 5 and 7 days of culturing time following a similar trend. After 1 day of culture, the cell numbers on Ti-OTS-CaP surface (—CH3 group) were higher than others (—NH2 and —C(O)C group), and the trend was almost similar during 3 days, except that Ti-GPTMS-CaP showed lower activity compared with pTi-CaP. Up to 5 days, all of the OD values significantly increased as opposed to OD values of 3 days. After 7 days of culture, OD value of pTi-CaP did not obviously increase, and the other three functionalized pTi surfaces exhibited a similar trend on preceding days. All these results demonstrated that there was a high yield of proliferative activity (non-toxicity) in the osteoblast cells after a long cell culture time. The Ti-OTS-CaP surfaces exhibited higher cell viability than others during cell culture in 7 days. Meanwhile, the trend of MTT assay on different functionalized surfaces was inconsistent with the SEM micrographs (Fig. 9). It is possible that HA nano-morphology can be conducive in improving interfacial adhesion of cells due to the provided space for filopodia[46]. According to Okada et al.[47], the smaller size of nanostructures would adversely affect osteoblasts. Therefore, the cellular response to HA substrates may be influenced by the balance in size between the dense micro/nano structure and the focal adhesions of the cells. ...
, Kongyou Ouyang
