Among the materials available for implant applications in dental and orthopedic restoration, titanium and its alloys are largely used due to their special characteristics such as high corrosion resistance, superior mechanical properties, and good biocompatibility [1
The use of Ti implants in reconstructive surgery is in many cases affected by some Ti surface properties, such as wear, hardness, and mainly slow osseointegration. Therefore, diverse methods have been developed to improve surface properties of Ti implants, such as morphological modifications (regarding roughness, morphology, etc.) by mechanical, chemical, and physical methods, or deposition of organic or inorganic coatings on the Ti surface [2
]. Many studies indicate that Ti implants can be coated with calcium phosphate (e.g., hydroxyapatite) layers by various deposition methods such as electrodeposition, plasma spray, high-frequency magnetron sputtering, biomimetic precipitation, etc. [4
]. In this way, the bioactivity, biocompatibility, and corrosion resistance of the Ti-based implants are improved.
The hydroxyapatite Ca10
is a calcium phosphate ceramic found in nature, but can be obtained by various methods. In the human body, it is the main inorganic component of bone and dental dentin and enamel [9
]. Synthetic hydroxyapatite can be obtained by various methods, the most used being wet methods (chemical precipitation, sol-gel, hydrothermal, biomimetic, or electrodeposition) [10
]. Hydroxyapatite has many medical applications, especially as a basic dental or bone material in reconstruction and repair surgery. The use of hydroxyapatite as a bone substitute material is unfortunately limited due to its poor mechanical properties, especially its brittleness and inflexibility. Therefore, many studies have focused on obtaining polymeric or metal composite implants containing hydroxyapatite in order to improve the osseointegration process [11
]. Additionally, the crystal structure of the hydroxyapatite allows the substitution of Ca2+
ions with different foreign ions (Na+
, etc.) in small quantities and this substitution can increase the osteoblast adhesion and enhance the properties of the hydroxyapatite as biomaterial with medical applications [13
The bismuth (Bi) compounds, especially Bi(III), are widely applied in catalysis, pharmaceutical, and medical fields. The main medical applications of bismuth compounds are noticed in radiographic, anticancer, and antimicrobial studies [18
]. Some bismuth salts are used for medical purposes to treat gastrointestinal, infectious, or dermatological diseases. Additionally, bismuth alloys may be used in the realization of bone and dental devices. Materials containing bismuth show high radiopacity and can thus be used as contrast materials in bone and dental restorative cements to obtain better imaging information in X-ray analysis (e.g., computed tomography).
Studies on hydroxyapatite doped with bismuth are very few [20
]. In our previous investigations we have demonstrated the possibility of synthesizing Bi-doped hydroxyapatite nanopowders [22
]. Instead, the Bi-doped nanohydroxyapatite deposited as a thin film on the surface of the titanium implants has not been studied before by anyone.
Therefore, this research presents the possibility to obtain, by a biomimetic technique, Bi-doped nanohydroxyapatite coatings on the surface of the titanium implants. A supersaturated calcification solution (SCS) modified by adding an appropriate quantity of bismuth salt was used to achieve biomimetic Bi-doped nanohydroxyapatite coatings. By dipping a titanium sample in the SCS solution, the hydroxyapatite nucleation on the titanium surface takes place in a short time, which then grows over time uniformly covering the metallic surface. By adding an additional source of bismuth to the SCS solution, the incorporation of bismuth ions into the hydroxyapatite lattice is facilitated. These Bi-doped nanohydroxyapatite coatings were characterized and tested for their radiopacity and bactericidal behavior.
2. Materials and Methods
Calcium chloride dihydrate (CaCl2∙2H2O), monosodium phosphate monohydrate (NaH2PO4∙H2O), sodium bicarbonate (NaHCO3), bismuth (III) nitrate pentahydrate (Bi(NO3)3∙5H2O), sodium hydroxide (NaOH), ethanol and acetone were acquired from Sigma-Aldrich (Germany) and used without further purification.
2.2. Coating Solutions
In this study, certain amounts of CaCl2
O, and NaHCO3
reagents were dissolved in 1 L of deionized water, under vigorous stirring, in order to obtain the supersaturated calcification solution (SCS), as presented elsewhere [24
]. In this solution, the ion concentrations were of 6.5 mmol/L Na+
, 10 mmol/L Ca2+
, 20 mmol/L Cl−
, 5 mmol/L H2
, and 1.5 mmol/L HCO3−
, and the Ca/P atomic ratio was 1.67 (Table 1
), as in biological hydroxyapatite [9
By adding a certain amount of Bi(NO3
O salt to the original SCS solution, the modified SCS solution (denoted Bi-SCS) was obtained. In this solution, the (Bi + Ca)/P and xBi
= Bi/(Bi + Ca) atomic ratios were of 1.67 and 0.01, respectively (Table 1
). The concentration of Bi in Bi-SCS solution is low (of about 1%), according to the therapeutic range mentioned in literature [25
2.3. Alkali-Thermal Treatment
The plates of commercially pure Ti (c.p. Ti) of 10 × 10 × 3 mm in size were polished using silicon carbide (SiC) paper and then cleaned in an ultrasonic bath with distilled water. Prior to alkali-thermal treatment all the samples were cleaned for 15 min in acetone, 10 min in ethanol, and 5 min in deionized water. Then, all samples were subjected to an alkaline treatment in 0.6 M NaOH solution at 160 °C in a pressure chamber for 24 h, at heating rates of 5 °C/min. Finally, the samples were rinsed for 5 min in deionized water and then activated by a thermal oxidation treatment at 600 °C for 3 h in a furnace with a heating rate of 5 °C/min.
2.4. Biomimetic Deposition
After alkali-thermal treatment, the Ti samples were subjected to a biomimetic treatment in SCS (or Bi-SCS) solution at 37 °C, as presented elsewhere [24
]. Periodically, these biomimetic solutions (SCS or Bi-SCS) were refreshed in order to keep the ion concentrations constant. After a certain period of time, the Ti samples were removed from the biomimetic solution (SCS or Bi-SCS), rinsed with deionized water, and then dried in an oven for 1 h at 37 ° C.
The Ti samples covered with undoped hydroxyapatite and Bi-doped hydroxyapatite layers were denoted HA-Ti and Bi-HA-Ti, respectively.
2.5. Sample Characterization
Scanning electron microscopy (SEM) measurements were performed using a QUANTA 200 3D microscope (FEI, Eindhoven, The Netherlands), equipped with an energy dispersive X-ray spectrometer (EDX).
X-ray photoelectron spectroscopy (XPS) measurements were performed using a PHI-5000 VersaProbe photoelectron spectrometer (Φ ULVAC-PHI, INC., Chigasaki, Japan), equipped with a hemispherical energy analyzer (0.85 eV binding energy resolution). A monochromatic Al Kα X-ray radiation (hν = 1486.7 eV) was used as excitation source, operating at 15 kV and 20 mA.
X-ray diffraction (XRD) measurements were performed using a X’PERT PRO MRD diffractometer (PANalytical, Almelo, The Netherlands), with CuKα (λ = 0.15418 nm) radiation, operating at 40 kV and 50 mA over a 2θ range from 2 to 70°.
The radiographs of the samples were obtained in a dental X-ray system (X-Mind™ AC, SATELEC, Mérignac, France). The samples were placed on an occlusal radiographic film and exposed along with a graduated aluminum (99.5% pure) step wedge with thicknesses varying from 1 to 10 mm in 1 mm increments. The radiographs were digitized using a desktop scanner (VistaNet/VistaScan PERIO PLUS, Bietigheim-Bissingen, Germany). The digitized images were then imported into the Gendex Dental Systems VixWin 2000 software ((1.11 /17 Apr 2005 version, Gendex Dental Systems Manufacturer, Des Plaines, IL, USA) where a tool was applied to identify equal-density areas in the radiographic images. The areas of the aluminum step wedge and the samples were selected to determine the radiopacity values of the samples which were expressed in terms of equivalent millimeters of aluminum (mm Al). Three measurements were made for each cited area and the means of these readings calculated.
2.6. Antibacterial Tests
The antibacterial activity of the samples was investigated against Gram-positive Staphylococcus aureus
and Gram-negative Escherichia coli
bacterial strains, by using standardized Kirby–Bauer disc diffusion method [26
]. The samples were planted in a Mueller–Hinton agar inoculated with Escherichia coli
or Staphylococcus aureus
bacteria, followed by incubation at 37 °C for 24 h. To evaluate the antibacterial activity of the samples, the total diameter (in mm) of the inhibition zone was measured for each sample. The antibacterial assessment was performed in duplicate and the average results were reported. The values are expressed as means ± standard deviations. Statistical analysis was performed using Student’s t
-test, with the significant level with a p
value of less than 0.05.