Bioactive, nanostructured Si-substituted hydroxyapatite coatings on titanium prepared by pulsed laser deposition Julietta V. Rau,1 Ilaria Cacciotti,2,3 Sara Laureti,4 Marco Fosca,1 Gaspare Varvaro,4 Alessandro Latini5 1
Istituto di Struttura della Materia, Consiglio Nazionale delle Ricerche, Via del Fosso del Cavaliere, 100-00133 Rome, Italy di Roma “Niccolo Cusano”, Via Don Carlo Gnocchi, 3-00166 Rome, Italy Universita 3 di Roma “Tor Vergata”, UdR INSTM-“Roma Tor Vergata”, Dipartimento di Ingegneria dell’Impresa, Universita Via del Politecnico, 1-00133 Rome, Italy 4 Istituto di Struttura della Materia, Consiglio Nazionale delle Ricerche, 00016 Monterotondo Scalo (RM), Italy 5 di Roma “La Sapienza”, Piazzale Aldo Moro, 5-00185 Rome, Italy Dipartimento di Chimica, Universita 2
Received 14 May 2014; revised 27 October 2014; accepted 2 December 2014 Published online 30 December 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33344 Abstract: Aims: The aim of this work was to deposit siliconsubstituted hydroxyapatite (Si-HAp) coatings on titanium for biomedical applications, since it is known that Si-HAp is able to promote osteoblastic cells activity, resulting in the enhanced bone ingrowth. Materials and Methods: Pulsed laser deposition (PLD) method was used for coatings preparation. For depositions, Si-HAp targets (1.4 wt % of Si), made up from nanopowders synthesized by wet method, were used. Results: Microstructural and mechanical properties of the produced coatings, as a function of substrate temperature, were investigated by scanning electron and atomic force microscopies, X-ray diffraction, Fourier transform infrared spectroscopy, and Vickers microhardness. In the temperature range of 400–600 C, 1.4–1.5 mm thick Si-HAp films, presenting composition similar to that of the used target, were depos-
ited. The prepared coatings were dense, crystalline, and nanostructured, characterized by nanotopography of surface and enhanced hardness. Whereas the substrate temperature of 750 C was too high and led to the HAp decomposition. Moreover, the bioactivity of coatings was evaluated by in vitro tests in an osteoblastic/osteoclastic culture medium (aModified Eagle’s Medium). Conclusions: The prepared bioactive Si-HAp coatings could be considered for applications in orthopedics and dentistry to improve the osteointegration of C 2014 Wiley Periodicals, Inc. J Biomed Mater Res bone implants. V Part B: Appl Biomater, 103B: 1621–1631, 2015.
Key Words: pulsed laser deposition, titanium, silicon-substituted hydroxyapatite coatings, physico-chemical properties, bioactivity
How to cite this article: Rau JV, Cacciotti I, Laureti S, Fosca M, Varvaro G, Latini A. 2015. Bioactive, nanostructured Sisubstituted hydroxyapatite coatings on titanium prepared by pulsed laser deposition. J Biomed Mater Res Part B 2015:103B:1621–1631.
INTRODUCTION
Titanium and its alloys are most commonly used as implant materials in modern orthopedics and odontoiatrics, due to their prominent mechanical properties, resistance, and acceptable biocompatibility.1–4 However metals are not suitable to induce the formation of a biologically functional material/bone tissue interface. Therefore, the metal surface properties can be improved by coating the implant with appropriate biomaterials in order to induce the cellular response, adhesion and proliferation, and angiogenesis during osseointegration. To achieve this goal, calcium phosphate-based materials can be applied, presenting excellent biocompatibility and bioactivity for the bone and dental repair and replacement applications. Specially, biocompatible and osteoconductive hydroxyapatite (HAp) has been extensively studied as coating material for prosthetics.5,6 It is known that natural bone
and dental tissue are composed of substituted HAp, these substitutions influencing significantly bioactivity, surface chemistry, solubility, and morphology of the material. Silicon, along with other ions (Na1, K1, Sr21, Ba21, Mg21, F2, Cl2, CO322 etc.) present in biological apatites,7,8 is essential for the extracellular matrix formation in bone and cartilage.9–11 Moreover, it was reported that Si incorporation into the HAp lattice leads to a significant increase in the activity of the osteoblast cells.11–13 On the basis of these considerations, the fabrication of Si-HAp coatings is of evident and actual interest. The plasma spraying method, commercially available for the HAp coatings production,14–17 is the only process approved by the Food and Drug Administration (FDA) USA for biomedical coatings.18 However, the drawbacks of this deposition technique are poor adhesion, formation of voids and cracks, non-uniform morphology, and structure and
Correspondence to: J. V. Rau; e-mail: [email protected]
C 2014 WILEY PERIODICALS, INC. V
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possible variation of composition. Furthermore, the delamination risks of the plasma sprayed coatings, associated to their excessive thickness (50–150 lm), should be overcome.19,20 For this reason, other deposition methods have been developed, such as magnetron sputtering,21 aerosol deposition,22 electrochemical23 and electrolytic deposition,24 sol–gel,25 biomimetic process,26 and pulse laser deposition (PLD).27,28 This latter technique allows to deposit coatings with the controlled adherence, crystallinity, and roughness and with the stoichiometry similar to that of the target material. Recently, PLD was used to prepare carbonate-substituted,29 fluorine-substituted,30 and iron-substituted HAp coatings.31 It is important to highlight that only recently researchers’ attention has been moved to the preparation of Sisubstituted HAp in the form of coating, being siliconsubstituted bioceramics mainly developed as granular fillers or in bulk form.32–35 It should be noted, in addition, that SiHAp films have been rarely produced by PLD technique27,36,37 and mainly starting from targets composed of mixtures of commercial HAp with different Si powder sources (i.e., metallic Si, synthetic silica, or biologically derived silica). In our previous work,38 Si-substituted HAp coatings were deposited by means of PLD technique using Si-HAp targets (1.4 wt % Si) prepared starting from the respective nanopowders synthesized by wet precipitation. Nanostructured crystalline films were grown onto Ti substrates heated at 600 C. In the present work, we report the results of a more extensive study regarding the influence of deposition conditions (i.e., substrate temperature) on the coating’s properties, and the evaluation of their in vitro bioactivity. Therefore, the main objective of this research was to investigate the coatings microstructure, composition, and mechanical properties as a function of substrate temperature, optimizing the deposition parameters in order to obtain reliable quality films. The characteristics of the deposited coatings were investigated by scanning electron (SEM-EDS) and atomic force (AFM) microscopies, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and Vickers microhardness. In vitro bioactivity tests were performed in an osteoblastic/osteoclastic culture medium (a-Modified Eagle’s Medium, a-MEM), in order to investigate the coating’s bioactive behaviour based on the evaluation of its ability to induce the precipitation of a calcium phosphate surface layer, similar to the mineral part of the hard tissue. MATERIALS AND METHODS
Preparation of Si-HAp target and of Ti substrates Silicon-substituted HAp powder (Si-HAp, 1.40 wt % Si) was synthesized by wet precipitation. The synthesis was carried out in a double-walled jacket reactor at 40 C, as extensively described elsewhere.32 A silicon content of 1.4 wt % was properly selected on the basis of previous investigations11,32,39–41 in order to obtain a monophasic HAp, thermally stable up to high temperature, and to prevent its decomposition with the forma-
1622
RAU ET AL.
tion of secondary phases, such as tricalcium phosphate, tetracalcium phosphate, calcium oxide, and calcium phosphate silicates. In fact, it has been reported that for high Si contents (i.e. >2 wt %), the thermal decomposition took place at lower temperature, leading to the formation of tricalcium phosphate and calcium phosphate silicates.11,40,41 Briefly, the powder was synthesized by titration of 10 g/L aqueous suspension of calcium hydroxide (Ca(OH)2, Aldrich 99.5%, MW 74.10) with a phosphoric acid solution (H3PO4, Aldrich 86.3%, MW 98.00), after having added tetraethyl orthosilicate (TEOS) (Si(OC4H9)4, Aldrich 99.99%, MW 208.33), as Si source, to the H3PO4 solution. Finally, the pH was corrected to 9.4 through the addition of concentrated NH4OH. The resultant precipitates were aged in mother liquors for 24 h, submitted to several washings with a NH4OH aqueous solution, vacuum filtered, and dried at 60 C (asdried samples). The obtained powder was thermally treated at 600 C for 1 h (heating and cooling rates of 10 C/min), uniaxially pressed at 400 MPa, and sintered at 1100 C for 1 h (heating and cooling rates of 10 C/min). The pure Ti substrates (1 3 1 cm2 quadrates) were sandblasted with a 60-grid SiC abrasive powder in order to obtain a nanometric surface roughness. Afterwards, the substrates were boiled in aqua regia for 30 min in order to remove any contaminant from the surface.
Pulsed laser deposition The Si-HAp films were deposited on Ti substrates in a PLD high vacuum chamber. Depositions were performed by focusing a pulsed KrF Lambda Physik excimer laser (k 5 248 nm, pulse duration 5 17 ns) on a sintered Si-HAp rotating target. The spot energy fluence was 2 J/cm2, and the pulse repetition rate was 5 Hz for a total of 7500 pulses. During depositions, the Ti substrate was heated at different temperatures (400 C, 500 C, and 750 C). Substrate and target were assembled in a frontal geometry at 4 cm of reciprocal distance. The PLD chamber was evacuated down to a base pressure of 1 3 1026 mbar prior to depositions. The film sample depositions were performed at 5 3 1024 mbar in a controlled dynamic pressure produced by a nitrogen gas flow, directly introduced into the chamber through a needle valve.
X-ray diffraction Diffraction patterns of both target and Si-HAp films were acquired with a Panalytical X’Pert Pro diffractometer. The instrument uses a Bragg-Brentano geometry and it is equipped with a Cu source (CuKa, k 5 0.154184 nm). Diffractograms were acquired using h–h (gonio) modality, range between 20 and 90 , with the optics so configured: incident beam: 1 slit; 15 mm mask; 0.04 rad soller slit; Ni filter; diffracted beam: 0.04 soller slit; 6.6 mm anti-scatter slit; ultra-fast RTMS detector (X’Celerator).
BIOACTIVE SI-SUBSTITUTED HYDROXYAPATITE COATINGS ON TITANIUM
ORIGINAL RESEARCH REPORT
The Rietveld refinement procedure was performed on the diffraction patterns of the target using the High Score Plus software by Panalytical. For the films, since the peaks of the substrate (Ti) were of much higher intensity in comparison to those of the films, only qualitative analysis of the diffractograms was performed, using the ICDD PDF-2 database.42 Fourier transform infrared spectroscopy The Jasco FTIR 470 Plus interferometer (Italy), equipped with an IRTRON IRT-30 microscope, was used to perform the FTIR spectroscopy analysis. A sequence of spectra was collected in the range of 4000–500 cm21, each spectrum acquired in the reflectance mode executing 250 scans at 8 cm21 resolution. Scanning electron microscopy The LEO 1450 Variable Pressure SEM apparatus, with the resolution of about 4 nm in vacuum conditions, was utilized for morphological studies of the deposited film samples. This SEM apparatus, working in the secondary and backscattered electron modes, is coupled with the energy dispersive X-ray spectroscopy (EDS) INCA 300 microanalysis system, allowing to perform qualitative/quantitative analysis of elements with 0.2% precision limit. Atomic force microscopy Surface texture of the deposited films was investigated acquiring AFM images by means of a non-commercial atomic force microscope, described in detail elsewhere.43 AFM images were taken in contact mode, at constant average distance. The measurements were performed in air, at constant 30–35% relative humidity. V-shaped (0.032 N/m spring constant) gold coated Si3N4 microlevers (Veeco Inst.) with an apical radius of 10–20 nm were applied. In the contact mode, the microscope operates in the weak repulsive regime, that is the interaction between tip and sample
Istituto di Struttura della Materia, Consiglio Nazionale delle Ricerche, Via del Fosso del Cavaliere, 100-00133 Rome, Italy di Roma “Niccolo Cusano”, Via Don Carlo Gnocchi, 3-00166 Rome, Italy Universita 3 di Roma “Tor Vergata”, UdR INSTM-“Roma Tor Vergata”, Dipartimento di Ingegneria dell’Impresa, Universita Via del Politecnico, 1-00133 Rome, Italy 4 Istituto di Struttura della Materia, Consiglio Nazionale delle Ricerche, 00016 Monterotondo Scalo (RM), Italy 5 di Roma “La Sapienza”, Piazzale Aldo Moro, 5-00185 Rome, Italy Dipartimento di Chimica, Universita 2
Received 14 May 2014; revised 27 October 2014; accepted 2 December 2014 Published online 30 December 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33344 Abstract: Aims: The aim of this work was to deposit siliconsubstituted hydroxyapatite (Si-HAp) coatings on titanium for biomedical applications, since it is known that Si-HAp is able to promote osteoblastic cells activity, resulting in the enhanced bone ingrowth. Materials and Methods: Pulsed laser deposition (PLD) method was used for coatings preparation. For depositions, Si-HAp targets (1.4 wt % of Si), made up from nanopowders synthesized by wet method, were used. Results: Microstructural and mechanical properties of the produced coatings, as a function of substrate temperature, were investigated by scanning electron and atomic force microscopies, X-ray diffraction, Fourier transform infrared spectroscopy, and Vickers microhardness. In the temperature range of 400–600 C, 1.4–1.5 mm thick Si-HAp films, presenting composition similar to that of the used target, were depos-
ited. The prepared coatings were dense, crystalline, and nanostructured, characterized by nanotopography of surface and enhanced hardness. Whereas the substrate temperature of 750 C was too high and led to the HAp decomposition. Moreover, the bioactivity of coatings was evaluated by in vitro tests in an osteoblastic/osteoclastic culture medium (aModified Eagle’s Medium). Conclusions: The prepared bioactive Si-HAp coatings could be considered for applications in orthopedics and dentistry to improve the osteointegration of C 2014 Wiley Periodicals, Inc. J Biomed Mater Res bone implants. V Part B: Appl Biomater, 103B: 1621–1631, 2015.
Key Words: pulsed laser deposition, titanium, silicon-substituted hydroxyapatite coatings, physico-chemical properties, bioactivity
How to cite this article: Rau JV, Cacciotti I, Laureti S, Fosca M, Varvaro G, Latini A. 2015. Bioactive, nanostructured Sisubstituted hydroxyapatite coatings on titanium prepared by pulsed laser deposition. J Biomed Mater Res Part B 2015:103B:1621–1631.
INTRODUCTION
Titanium and its alloys are most commonly used as implant materials in modern orthopedics and odontoiatrics, due to their prominent mechanical properties, resistance, and acceptable biocompatibility.1–4 However metals are not suitable to induce the formation of a biologically functional material/bone tissue interface. Therefore, the metal surface properties can be improved by coating the implant with appropriate biomaterials in order to induce the cellular response, adhesion and proliferation, and angiogenesis during osseointegration. To achieve this goal, calcium phosphate-based materials can be applied, presenting excellent biocompatibility and bioactivity for the bone and dental repair and replacement applications. Specially, biocompatible and osteoconductive hydroxyapatite (HAp) has been extensively studied as coating material for prosthetics.5,6 It is known that natural bone
and dental tissue are composed of substituted HAp, these substitutions influencing significantly bioactivity, surface chemistry, solubility, and morphology of the material. Silicon, along with other ions (Na1, K1, Sr21, Ba21, Mg21, F2, Cl2, CO322 etc.) present in biological apatites,7,8 is essential for the extracellular matrix formation in bone and cartilage.9–11 Moreover, it was reported that Si incorporation into the HAp lattice leads to a significant increase in the activity of the osteoblast cells.11–13 On the basis of these considerations, the fabrication of Si-HAp coatings is of evident and actual interest. The plasma spraying method, commercially available for the HAp coatings production,14–17 is the only process approved by the Food and Drug Administration (FDA) USA for biomedical coatings.18 However, the drawbacks of this deposition technique are poor adhesion, formation of voids and cracks, non-uniform morphology, and structure and
Correspondence to: J. V. Rau; e-mail: [email protected]
C 2014 WILEY PERIODICALS, INC. V
1621
possible variation of composition. Furthermore, the delamination risks of the plasma sprayed coatings, associated to their excessive thickness (50–150 lm), should be overcome.19,20 For this reason, other deposition methods have been developed, such as magnetron sputtering,21 aerosol deposition,22 electrochemical23 and electrolytic deposition,24 sol–gel,25 biomimetic process,26 and pulse laser deposition (PLD).27,28 This latter technique allows to deposit coatings with the controlled adherence, crystallinity, and roughness and with the stoichiometry similar to that of the target material. Recently, PLD was used to prepare carbonate-substituted,29 fluorine-substituted,30 and iron-substituted HAp coatings.31 It is important to highlight that only recently researchers’ attention has been moved to the preparation of Sisubstituted HAp in the form of coating, being siliconsubstituted bioceramics mainly developed as granular fillers or in bulk form.32–35 It should be noted, in addition, that SiHAp films have been rarely produced by PLD technique27,36,37 and mainly starting from targets composed of mixtures of commercial HAp with different Si powder sources (i.e., metallic Si, synthetic silica, or biologically derived silica). In our previous work,38 Si-substituted HAp coatings were deposited by means of PLD technique using Si-HAp targets (1.4 wt % Si) prepared starting from the respective nanopowders synthesized by wet precipitation. Nanostructured crystalline films were grown onto Ti substrates heated at 600 C. In the present work, we report the results of a more extensive study regarding the influence of deposition conditions (i.e., substrate temperature) on the coating’s properties, and the evaluation of their in vitro bioactivity. Therefore, the main objective of this research was to investigate the coatings microstructure, composition, and mechanical properties as a function of substrate temperature, optimizing the deposition parameters in order to obtain reliable quality films. The characteristics of the deposited coatings were investigated by scanning electron (SEM-EDS) and atomic force (AFM) microscopies, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and Vickers microhardness. In vitro bioactivity tests were performed in an osteoblastic/osteoclastic culture medium (a-Modified Eagle’s Medium, a-MEM), in order to investigate the coating’s bioactive behaviour based on the evaluation of its ability to induce the precipitation of a calcium phosphate surface layer, similar to the mineral part of the hard tissue. MATERIALS AND METHODS
Preparation of Si-HAp target and of Ti substrates Silicon-substituted HAp powder (Si-HAp, 1.40 wt % Si) was synthesized by wet precipitation. The synthesis was carried out in a double-walled jacket reactor at 40 C, as extensively described elsewhere.32 A silicon content of 1.4 wt % was properly selected on the basis of previous investigations11,32,39–41 in order to obtain a monophasic HAp, thermally stable up to high temperature, and to prevent its decomposition with the forma-
1622
RAU ET AL.
tion of secondary phases, such as tricalcium phosphate, tetracalcium phosphate, calcium oxide, and calcium phosphate silicates. In fact, it has been reported that for high Si contents (i.e. >2 wt %), the thermal decomposition took place at lower temperature, leading to the formation of tricalcium phosphate and calcium phosphate silicates.11,40,41 Briefly, the powder was synthesized by titration of 10 g/L aqueous suspension of calcium hydroxide (Ca(OH)2, Aldrich 99.5%, MW 74.10) with a phosphoric acid solution (H3PO4, Aldrich 86.3%, MW 98.00), after having added tetraethyl orthosilicate (TEOS) (Si(OC4H9)4, Aldrich 99.99%, MW 208.33), as Si source, to the H3PO4 solution. Finally, the pH was corrected to 9.4 through the addition of concentrated NH4OH. The resultant precipitates were aged in mother liquors for 24 h, submitted to several washings with a NH4OH aqueous solution, vacuum filtered, and dried at 60 C (asdried samples). The obtained powder was thermally treated at 600 C for 1 h (heating and cooling rates of 10 C/min), uniaxially pressed at 400 MPa, and sintered at 1100 C for 1 h (heating and cooling rates of 10 C/min). The pure Ti substrates (1 3 1 cm2 quadrates) were sandblasted with a 60-grid SiC abrasive powder in order to obtain a nanometric surface roughness. Afterwards, the substrates were boiled in aqua regia for 30 min in order to remove any contaminant from the surface.
Pulsed laser deposition The Si-HAp films were deposited on Ti substrates in a PLD high vacuum chamber. Depositions were performed by focusing a pulsed KrF Lambda Physik excimer laser (k 5 248 nm, pulse duration 5 17 ns) on a sintered Si-HAp rotating target. The spot energy fluence was 2 J/cm2, and the pulse repetition rate was 5 Hz for a total of 7500 pulses. During depositions, the Ti substrate was heated at different temperatures (400 C, 500 C, and 750 C). Substrate and target were assembled in a frontal geometry at 4 cm of reciprocal distance. The PLD chamber was evacuated down to a base pressure of 1 3 1026 mbar prior to depositions. The film sample depositions were performed at 5 3 1024 mbar in a controlled dynamic pressure produced by a nitrogen gas flow, directly introduced into the chamber through a needle valve.
X-ray diffraction Diffraction patterns of both target and Si-HAp films were acquired with a Panalytical X’Pert Pro diffractometer. The instrument uses a Bragg-Brentano geometry and it is equipped with a Cu source (CuKa, k 5 0.154184 nm). Diffractograms were acquired using h–h (gonio) modality, range between 20 and 90 , with the optics so configured: incident beam: 1 slit; 15 mm mask; 0.04 rad soller slit; Ni filter; diffracted beam: 0.04 soller slit; 6.6 mm anti-scatter slit; ultra-fast RTMS detector (X’Celerator).
BIOACTIVE SI-SUBSTITUTED HYDROXYAPATITE COATINGS ON TITANIUM
ORIGINAL RESEARCH REPORT
The Rietveld refinement procedure was performed on the diffraction patterns of the target using the High Score Plus software by Panalytical. For the films, since the peaks of the substrate (Ti) were of much higher intensity in comparison to those of the films, only qualitative analysis of the diffractograms was performed, using the ICDD PDF-2 database.42 Fourier transform infrared spectroscopy The Jasco FTIR 470 Plus interferometer (Italy), equipped with an IRTRON IRT-30 microscope, was used to perform the FTIR spectroscopy analysis. A sequence of spectra was collected in the range of 4000–500 cm21, each spectrum acquired in the reflectance mode executing 250 scans at 8 cm21 resolution. Scanning electron microscopy The LEO 1450 Variable Pressure SEM apparatus, with the resolution of about 4 nm in vacuum conditions, was utilized for morphological studies of the deposited film samples. This SEM apparatus, working in the secondary and backscattered electron modes, is coupled with the energy dispersive X-ray spectroscopy (EDS) INCA 300 microanalysis system, allowing to perform qualitative/quantitative analysis of elements with 0.2% precision limit. Atomic force microscopy Surface texture of the deposited films was investigated acquiring AFM images by means of a non-commercial atomic force microscope, described in detail elsewhere.43 AFM images were taken in contact mode, at constant average distance. The measurements were performed in air, at constant 30–35% relative humidity. V-shaped (0.032 N/m spring constant) gold coated Si3N4 microlevers (Veeco Inst.) with an apical radius of 10–20 nm were applied. In the contact mode, the microscope operates in the weak repulsive regime, that is the interaction between tip and sample
