Hydroxyapatite Reinforced Coatings with Incorporated Detonationally ...

Hydroxyapatite Reinforced Coatings with Incorporated Detonationally Generated Nanodiamonds L. Pramatarovaa*, E. Pechevaa, R. Dimitrovab, T. Spassovc, N. Krastevad, T. Hikova, D. Fingarovaa and D. Miteve a

Institute of Solid State Physics, Bulgarian Academy of Sciences, Sofia, Bulgaria; *[email protected] b Institute of Organic Chemistry with Centre of Phytochemistry, Sofia, Bulgaria c Faculty of Chemistry, Sofia University, Sofia, Bulgaria d Institute of Biophysics, Bulgarian Academy of Science, Sofia, Bulgaria e Space Research Institute, Bulgarian Academy of Sciences, Sofia, Bulgaria

Abstract. We studied the effect of the substrate chemistry on the morphology of hydroxyapatite-detonational nanodiamond composite coatings grown by a biomimetic approach (immersion in a supersaturated simulated body fluid). When detonational nanodiamond particles were added to the solution, the morphology of the grown for 2 h composite particles was porous but more compact then that of pure hydroxyapatite particles. The nanodiamond particles stimulated the hydroxyapatite growth with different morphology on the various substrates (Ti, Ti alloys, glasses, Si, opal). Biocompatibility assay with MG63 osteoblast cells revealed that the detonational nanodiamond water suspension with low and average concentration of the detonational nanodiamond powder is not toxic to living cells. Keywords: hydroxyapatite; coatings; growth from solution; nanodiamond; morphology; biocompatibility. PACS: 87.85.J, 81.10.Dn, 81.05.Uw, 81.15.-z, 81.07.-b, 87.85.J, 87.85.jj

INTRODUCTION An important goal of materials science is the development of surfaces that integrate the materials properties with the functions of living cells. The development of materials that serve as substrates for adherent cells is important in a wide range of applications [1-5]. Calcium phosphate (CaP) ceramic materials, such as hydroxyapatite (HA), have been shown to enhance osseointegration to foreign surfaces; they do not form fibrous tissues, but instead an extremely thin, epitaxial bonding layer with existing bone [6-9]. Many studies have focused on the production of HA as coating or as bulk material. Bone tissue is an example of a natural, hierarchically organized composite, built from collagen fibres and crystalline HA. On the other hand, diamond is the hardest known material, it is biologically compatible, chemically inert and resistant to chemical corrosion and wear [10-12]. Detonationally generated nanodiamond (DND) is of great interest, because it is able to strengthen bone tissue, producing tougher and more flexible artificial bone implants [13-16]. DNDs are members of the diverse structural family of nanocarbons that includes many varieties based on synthesis conditions, post-synthesis processes, and modifications and they are quickly becoming key components in the production of high-strength composite materials. Recently DNDs gained world-wide attention due to their inexpensive large-scale synthesis based on the detonation of carbon-containing explosives, small primary particle size (4-5 nm) with narrow size distribution, facile surface fictionalization including bio-conjugation, as well as high biocompatibility. It is anticipated that the attractive properties of the DNDs will be exploited for the development of therapeutic agents for diagnostic probes, delivery vehicles, gene therapy, anti-viral and anti-bacterial treatments, tissue scaffolds, as well as an reinforcement material for composites. The ideal reinforcement material would impart mechanical integrity to the composites at high loadings, without diminishing its bioactivity [17,18]. The aim of our work is to utilize a biomimetic technology for production of composite layers (HADND), namely immersion of

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various solid substrates in a mixture of simulated body fluid (SBF) and DND and to study the process of HADND growth on the different surfaces.

MATERIALS AND METHODS Substrates Various solid substrates were used in our work: (i) titanium (Ti); (ii) amorphous Ti50Cu50 alloy (TiCu); (iii) amorphous Ti40Cu50Al10 alloy (TiCuAl); (iv) SiOx layer evaporated from SiO at low vacuum on Ti substrate (Ti/SiOx); (v) cover glass (CG); (vi) silica glass (SG); (vii) opal; (viii) silicon (Si).

Detonation Nanodiamond Synthesis DND particles (4-6 nm, density 3.2 g/cm3, content of diamond 97-99%) were synthesized by shock-wave propagation method through the detonation of explosives at high pressure and high temperature produced in the detonation. Subsequent purification from graphite by applying oxidation with potassium dichromate in sulphuric acid was carried out, and after several washings with hydrochloric acid and water, the as-obtained DND powder was dried [19]. The purification method generally leads to an oxidation of the DND surface, which was covered by carboxyl, and to a lesser extent by carbonyl and hydroxyl groups [15,16,20].

Simulated Body Fluid Preparation SBF is an aqueous solution that resembles the composition and ion concentrations of human blood plasma. It was prepared by dissolving the reagent grade chemicals NaCl, NaHCO3, KCl, K2HPO4-3H2O, MgCl2-6H2O, CaCl22H2O, and Na2SO4-10H2O in distilled water and buffering at pH 7.4 with TRIS buffer or HCl [21]. The DND particles were added to the SBF solution in a concentration of 0.5 g/l [22,23]. The various samples were immersed in the as obtained SBF-DND mixture at 37°C and pH 7.4 for 24 h, finally washed with distilled water and dried in air.

Biocompatibility Assay With Osteoblast-Like Cells Osteoblast-like cells MG-63 (ATCC number CRL 1427) were cultured in Dulbecco's modified Eagle medium (DMEM, Invitrogen France) supplemented with 10% fetal calf serum (FCS, Invitrogen, France) and 0.5% antibiotics (penicillin-streptomycin, Invitrogen, France). Cells were maintained at 37°C in a humidified incubator in the presence of 5% CO2. At confluence, cells were harvested with 0.1% trypsin-EDTA solution in phosphate-buffered saline (PBS, pH 7.4) at 37°C for 5 min, centrifuged to pellet the cells, and resuspended in DMEM with FCS and antibiotics. Cell morphology was visualized by vital staining with fluorescein diacetate (FDA) and phase-contrast images after incubation of the cells for 24 hours with DND suspended in water in varying concentrations.

Coating Characterization Structure of the coatings grown on the various solid surfaces was revealed by Fourier transform infrared spectroscopy (FTIR; Brucker-Vector 22 in the region of 4000-400 cm-1). Morphology and elemental composition of the HADND coatings were analyzed by scanning electron microscopy (SEM; JEOL-JSM 5510, 15 kV), coupled with energy dispersive X-ray spectroscopy (EDX).

RESULTS AND DISCUSSION FTIR spectra of the DND powder suspended in water (in a concentration of 0.5 g/l) and of the DND suspended in SBF (SBF-DND mixture) are shown in Fig. 1 (left graph). They revealed high intensity broad peak at 3440 cm-1 and a narrow peak at 1636 cm-1 due to the O-H stretching and bending vibrations of OH groups. The intensity of these peaks strongly decreases when a substrate is immersed into the SBF-DND mixture (spectrum 3 in Fig. 1). When substrate is introduced into a precipitated solution like the SBF solution in this work, a precipitation occurs according to the laws of crystal growth from solutions [24]. As a result, a coating is grown and its structure changes


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FIGURE 1. Left: FTIR spectra of the DND powder suspended in water (0.5 g/l) – spectrum 1; DND suspended in SBF (SBFDND mixture) – spectrum 2; SBF-DND mixture in the presence of a substrate – spectrum 3; Right: FTIR spectrum of the composite coating grown on cover glass substrate.

the FTIR spectrum – vibration due to the coating are already observed. Thus, spectrum 3 is dominated by the ν4 Р-О bending at 610 and 570 cm-1 and ν3 Р-О stretching in PO43- groups. The peak in spectrum 2 at 460 cm-1 is also assigned to Р-О vibration modes in PO43-. These peaks are characteristic for the formation of HA. Spectrum 3 revealed also C-O vibrational mode at 810 and 1424 cm-1, thus indicating CO3 incorporation in the HA structure [9]. Thus, it was concluded that the presence of a solid surface facilitated the growth of HA coating [24]. Evidence for the structural incorporation of the DND in the HA is given on Fig. 1, right graph, where FTIR spectrum of the composite coating grown on the CG substrate is presented. The presence of the DND is revealed by the CHx-CH3 vibrational modes in the region of 2980-2838 cm-1.

FIGURE 2. EDX measurements from the coatings grown on TiCuAl alloy (similar data was measured on the coatings grown on various substrates). Inset shows particles of the composite coating grown on the TiCuAl alloy (area 1) and on the substrate (area 2; thin coating is present according to the data).

EDX measurements from the coatings (Fig. 2) revealed main signals of O, Ca and P, as well as signals from the substrate (Ti, Cu and Al in the case of Ti alloys, Si in the case of glass, Si and opal substrates). Additional elements as Mg, K and Na, as well as C due to DND incorporation in the composite coating were also detected. Mg, K and Na are present in small quantities in natural bone, yielding a lower crystallinity along with the CO3 groups [9]. Fig. 2 shows the EDX measurements from the coatings grown on TiCuAl alloy; similar data was measured on the coatings

FIGURE 3. SEM image on a particle in the HADND composite coating grown for 2 h on the cover glass substrate (left); particle in the HA coating grown for 2 h on the cover glass substrate (right)


FIGURE 4. SEM images of the HADND composite coating grown for 24 h on Ti (upper left), TiCu (upper right), TiCuAl (down left) and Ti/SiOx (down right) substrates.

grown on various substrates. Inset in the figure shows particles of the composite coating grown on the TiCuAl alloy (area 1) and on the substrate (area 2; thin coating is present according to the data). All signals are much more intensive in area 1 due to higher thickness of the composite there. The composite coating was investigated in the initial hours of its growth. The HADND coating grown on the CG substrate after 2 h in the SBF-DND mixture (Fig. 3, left) revealed the formation of porous particles with sphere-like shape, more compact then the particles in a pure HA coating grown for 2 h on the same substrate (Fig. 3, right). Further, SEM revealed different morphology of the composite coatings grown after immersion of the various substrates in the SBF-DND mixture for 24 h. Ti and TiCu substrates (Fig. 4, upper left and right, respectively) induced the formation of randomly distributed on the surface deposited without any specific shape. White spherelike particles typical for the coatings obtained by a biomimetic approach and formation of porous areas similar to the structure of natural bone were observed on the TiCuAl substrates (Fig. 4, down left). Dense homogeneous coating with platelike crystals and small white aggregates were formed on the Ti/SiOx substrates (Fig. 4, down right).

FIGURE 5. SEM images of the HADND composite coating grown for 24 h on CG (upper left), SG (upper right), opal (down left) and Si (down right) substrates.


Very porous homogeneous coating with adhering compact white aggregates with no definite shape covered the CG substrate (Fig. 5, upper left). Porous coating with small aggregates was also found on the SG and opal substrates (Fig. 5, upper right and down left, respectively), while very compact coating was observed on the Si substrate (down right). The differences of the morphology of the grown HADND composite coating on the various samples are most probably due to the different chemistry of the substrates.

FIGURE 6. Overall morphology of MG63 cells (FDA staining, magnification 20х): control sample of cell culture medium without DND (upper left); SBF-DND mixture with low concentration of DND (upper right); denser SBF-DND mixture (down left); very dense SBF-DND mixture (down right).

Cell morphology was visualized by vital staining with FDA (Fig. 6) and phase-contrast images (Fig. 7) after incubation of MG63 osteoblast cells for 24 hours with DND suspended in water in varying concentrations (low, average and high). Changes in the pH of the cell culture medium with the addition of the DND were not observed. Both FDA and phase-contrast images revealed that with increasing of the DND density, the number of vital cells

FIGURE 7. Overall morphology of MG63 cells (phase contrast images, magnification 20х) : control sample of cell culture medium without DND (upper left); SBF-DND mixture with low concentration of DND (upper right); denser SBF-DND mixture (down left); very dense SBF-DND mixture (down right).


decreased, however overall cell morphology did not change significantly. As we can see from the FDA staining (Fig. 6) osteoblastic cells were well spread and there were no rounded cells, which is typical for an unfavorable environment. However, phase-contrast images (Fig. 7) showed slightly vacuolation and swelling of the cells, which could mean cell necrotization as a result of a cytotoxic effect of the high DND concentration.

CONCLUSIONS In the present work, studies have been carried out to study the effect of the substrate chemistry on the morphology of the grown HADND composites. When DND is added to the SBF solution, the morphology of the grown HADND composite particles is porous but more compact then that of pure HA particles. It was shown that DND stimulate the HA growth with different morphology on the various substrates (Ti, Ti alloys, glasses, Si, opal). HADND composite coatings obtained by a biomimetic approach, i.e. immersion in a mixture of SBF and DND revealed mostly formation of porous areas similar to the structure of natural bone and white sphere-like particles or white aggregates with no definite shape on the various samples. Cell culture experiments reveal that the DND water suspension with low and average concentration of the DND powder is not toxic to living cells.

ACKNOWLEDGMENTS This work was supported by grants TK-X-1708/2007 with the Bulgarian Ministry of Education and Science and NIF 02-54/2007 X-1507 with the Agency of Innovation of Bulgaria. NATO grant CBP.EAP.RIG.982693 and ANNA project (№ 026134(RI3), Research Infrastructure Action under the FP6 are also acknowledged.

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