BIOMINERALIZATION IN VITRO AND IN VIVO STUDIES OF A
NOVEL NANOHYDROXYAPATITE/SUPERHYDROPHILIC
VERTICALLY ALIGNED CARBON NANOTUBE NANOCOMPOSITES
Anderson O. Lobo¹, Marcus A.F. Corat2, Cristina P. Soares3 and Fernanda R. Marciano¹
¹Laboratory of Biomedical Nanotechnology, Universidade do Vale do Paraíba, São José dos Campos, São Paulo,
Brasil.
2
CEMIB, Universidade Estadual de Campinas, Campinas, Sao Paulo, Brazil.
3
Laboratório de Dinâmica de Compartimentos Celulares, Universidade do Vale do Paraíba, São José dos
Campos, São Paulo, Brazil.
E-mail: aolobo@univap.br and loboao@yahoo.com
ABSTRACT.
Vertically-aligned multi-walled carbon nanotubes (VAMWCNTs) are of particular interest in regenerative
medicine. Template-induced hydroxyapatite (HA) has wide-ranging prospects in applied fields of bone
regenerative medicine. Thus, a combination of these two excellent materials might be used in bone tissue
engineering applications. In this study, the HA/VAMWCNT nanocomposites were used as scaffolds to Human
osteoblast cells culture. VAMWCNT films were produced as a thin film, using a microwave plasma chamber at
2.45GHz on Ti substrates. Superhydrophilic VAMWCNT films were obtained by the incorporation of oxygencontaining groups using a pulsed-direct current plasma reactor with pure oxygen. The fabrication of
HA/VAMWCNT nanocomposites was performed with a direct electrodeposition of HA nanocrystals on the
VAMWCNT films. The biomineralization in vitro process of superhydrophilic HA/VACNT nanocomposites were
investigated using simulated body fluid (SBF). Human osteoblast adhesion was evaluated by scanning electron
microscopy. The biomineralization in vivo studies were carried out in bone defects of C57BL/6/JUnib rats.
XRD, SEM, and µEDX analyses were employed to investigate the structural characteristics, semi-quantitative
analysis, and morphology on the formation of HA/VAMWCNT nanocomposites before and after soaking 21 days
in SBF. Excepcional bone regeneration with lamelar bone formation after 60 days was found in the in vivo
studies. These findings make of these new HA/VAMWCNT nanocomposites very atractive for application in
bone tissue regeneration.
Key-words: Carbon nanotubes, Nanohydroxyapatite, Wet method, Nanocomposites, Tissue
regeneration.
1.
INTRODUCTION
Hydroxyapatite (Ca10(PO4)6(OH)2, HAp) is used as a biomaterial for the regeneration
of bone tissue because its chemical and crystallographic similarities with the main inorganic
component of natural bone (Sanosh, 2009). The close chemical similarity of HAp to natural
bone has led to extensive research efforts to use synthetic HAp as a bone substitute and/or
replacement in biomedical applications (Habraken, 2007). To date, many methods have been
developed to prepare HAp powders. The techniques include sol–gel (Bezzi, 2003),
homogeneous precipitation (Zhang, 2001), hydrothermal (Liu, 1997), etc.
Thus, HAp is classified as one of the best biocompatible and bioactive materials many
biological applications such as bone repair scaffolds (Sanosh, 2009). However, its poor
mechanical properties such as brittleness and low wear resistance have limited the use of bulk
HAp coating in implant applications (Goller, 2003). With the advances on biomaterials
research have suggested that the best osteoconductivity of HAp could be achieved if the
crystal was closer to the structure, size and morphology of biological apatite, so the
nanohydroxyapatite (nHAp) is of current interest.
Multi-walled carbon nanotubes (MWCNTs) are arousing interest from researchers in
biomedical area due to their exceptional combination of mechanical properties, chemical
properties, and biocompatibility [Qi, (2003)]. Hydrophilic surfaces are generally favorable to
cell attachment and biomineralization of bone tissues [Sawase, (2008)].
Some investigations have been performed on the synthesis of nHA and carbon
nanotubes (CNTs) using various methods, such as simulated body fluid (SBF) (Aryal, 2006)
aerosol (Balania, 2007), aerosol deposition (Hahn, 2009). However, these methods consist in
dispersing CNTs in HA solution (Najafi, 2009) and a thermal treatment is necessary to obtain
crystalline HA. Vertically aligned multiwalled carbon nanotubes (VAMWCNTs) were
efficient templates to grown HA films using plasma-enhanced chemical vapor deposition and
radio-frequency sputtering deposition, however, the authors clearly showed that the Ca- and
P-rich layer consists of a carbonate-containing HA with disordered structure and thus poor
crystallinity (Niu, 2010).
We have recently shown a new method to obtain crystalline HA/VAMWCNT
composites using direct electrodeposition process (Lobo, 2010). Here, we have shown an
evolution of a recently published paper (Lobo, 2010) that introduced a new and fast method to
obtain HA/VAMWCNT nanocomposites. From this, we introduce a new biomimetic
mineralization routine employing the HA/VAMWCNT nanocomposites as highly stable
template materials.
The biomineralization was obtained after HA/VAMWCNT
nanocomposites soaking in a synthetic body fluid (SBF) know to favor the "in vitro" and "in
vivo" calcification process.
2.
MATERIALS AND METHODS
2.1. Synthesis of vertically aligned multiwalled carbon nanotubes
The VAMWCNT films were produced as thin films using a microwave plasma
chamber equipped with a 2.45GHz microwave generator (MWCVD) (Lobo, 2010). The
substrates were 10mm titanium (Ti) squares covered by a thin Fe layer (10nm) deposited by
an e-beam evaporator. The Fe layers were pre-treated to promote nanocluster formation,
which forms the catalyst for VAMWCNT films growth. The pre-treatment was carried out
during 5 min in a plasma of N2/H2 (10/90sccm) with a substrate temperature of around 760oC.
After pre-treatment, CH4 (14 sccm) was inserted in the chamber at a substrate temperature of
800oC for 2 min. The reactor was kept at a pressure of 30 Torr during the whole process.
2.2. VAMWCNT functionalized by polar groups
Functionalization of the nanotube tips by the incorporation of oxygen-containing
groups was performed in a pulsed-direct current plasma reactor with an oxygen flow rate of 1
sccm, at a pressure of 85 mTorr, -700 V and with a frequency of 20 kHz (Lobo, 2010). The
total time of the plasma etching was 120 seconds. The superhydrophilic properties are given
elsewhere (Lobo, 2010)..
2.3. nHA/VAMWCNTs nanocomposites fabrication
The electrodeposition of the HA crystals on the VAMWCNT films was performed
using 0.042 mol L-1 Ca(NO3)2.4H2O + 0.025 mol L-1 (NH4).2HPO4 electrolytes (pH 4.7)
(Lobo, 2010). The electrochemical measurements were carried out using a three-electrode cell
coupled to an Autolab PGSTAT302 instrument. Superhydrophilic VAMWCNT films were
used as the working electrode and the geometric area in contact with electrolytic solution was
0.27 cm2. A platinum coil wire served as the auxiliary electrode and an Ag/AgCl electrode
was used as the reference electrode. The nHA films were produced by applying a constant
potential of -2.0 V for 30 min while the solution temperature was maintained at 70oC (Lobo,
2010).
2.4. Bioactivity analyses of nHA/VAMWCNTs nanocomposites
A simulated body fluid (SBF) (5x) solution was used for in vitro bioactivity study
(Lobo, 2010). It was prepared by dissolution of NaCl, KCl, K2HPO4, CaCl2 H2O, MgCl2,
H2O, NaHCO3, Na2SO4, and Na2O3 SiO2; all of analytical purity in distilled and deionized
water. The pH of all solutions was adjusted to 6.2 at 37ºC with 1 M HCl and
tris(hidroxymetil)aminomethane. The solutions were kept in closed polyethylene containers.
nHA/VAMWCNTs nanocomposites were placed in a polyethylene recipient and were
immersed in 15mL of SBF. All the substrates in their respective recipient were put in an
incubator at 37ºC for 21 days. After this incubation period, the nHA/VAMWCNTs were
immersed in distilled and deionized water and finally dried at 70oC for 24 hours.
2.5. Cellular adhesion on nHA/VAMWCNTs nanocomposites
The nHA/VAMWCNTs nanocomposites were sterilized for 24 h under UV irradiation
and were placed in individual wells of 24-well culture plates. The cells were maintained as
sub-confluent monolayer’s in minimum essential medium (MEM) with 1.5 mML-glutamine
adjusted to contain 2.2 g/L sodium bicarbonate 85%; fetal bovine serum (FBS) 15% (Gibco,
BRL), 100 units/mL penicillin–streptomycin (Sigma), and 25 mg/mL l-ascorbic acid (Sigma).
The incubation occurred within a CO2 (5%) atmosphere at 37ºC.
The adhesion of the human osteoblast cells on the HA/VAMWCNTs nanocomposites
were monitored up to 7 days. The cells were seeded in each well at a concentration of 5x105
cells/mL, supplemented with 10% FBS. The incubation was performed under a CO2 (5%)
atmosphere, at 37ºC. The attached cells on the substrate were fixed with a 3%
glutaraldehyde/0.1M sodium cacodylate buffer for 1 h and dehydrated in a graded ethanol
solution series (30%, 50%, 70%, 95%, and 100%) for 10 min each. The drying stage at room
temperature used a 1:1 solution of ethanol with hexamethyldisilazane. The samples were
coated by a thin gold film to improve the visualization. SEM images were performed for
morphological analysis of human osteoblast cell on biomineralized nHA/VAMWCNTs
nanocomposites.
2.6. "In vivo" study
Were used five nHA/VAMWCNTs nanocomposites. All the nanocomposites were
sterilized for 24 h under UV irradiation. After this, all the samples (separated by your groups)
were mixture with gelatina solution (8% in water). After this procedure, the solution
(separated by your respectively groups) were centrifuged at 1500 rpm per 7 min, and the
pellets were placed in individual wells of 24-well culture plates (diameter of 0.5mm) and
maintained under refrigeration at 9oC by 24 hours for complete solidification.
Adult, male, C57BL/6 rats (22-28g body weight, CEMIB/UNICAMP, Campinas,
Brazil) were used in this study. All animal procedures were in agreement with the Ethical
Principles for Animal Research established by the Brazilian College for Animal
Experimentation (COBEA). This project was approved by the Institutional Committee for
Ethics in Animal Research at the University of Campinas - UNICAMP, Brazil. Rats were
anesthetized with an intraperithonial injection of Avertin (2,2,2-tribromoetanol) with 2,5%
(225-240mg/kg). The hair over the calvarium was shaved and cleaned. After the skin was
incised, a full-thickness circular critically sized defect (diameter of 0.5 mm) was made in the
calvarium using an ultrasound equipment CVDent1000® equipped as a sterile CVDENTUS®
diamond tip under irrigation with sterile normal saline to avoid damage to the bone.
All the sterilized nanocomposites (with your respectively groups) disks of 0.5 mm
diameter was placed into the defect, the surgical site was irrigated with sterile normal saline,
and the wound was closed in layers with resorbable sutures. In one group of rats, the surgical
defect was prepared but no implant was placed. There were three rats in each treatment group.
All rats were examined daily for signs of infection or discomfort and water and food for 9
weeks.
2.7. Histological study
Nine weeks after implantation of the disks, rats were sacrificed by Halotano
inhalation. Block sections of the calvarium containing the disks were harvested from the
surgical sites and fixed in 10% neutral buffered formalin. Specimens were then dehydrated for
12 days with a graded series of alcohols. Tissue sections were stained with Stevenel’s blue
and Van Gieson’s picro fuchsin.
3.
RESULTS AND DISCUSSION
Figures 1a show the scanning electron microscopy (SEM) images of Fe nanoclusters
with diameters between 30-70 nm. Figures 1b show SEM images of the high density of the
VAMWCNT film grown on Fe nanoclusters. VAMWCNTs presented a length of 6-8 µm.
Figures 1c show transmission electron microscopy (TEM) images of typical internal bamboolike structures of the VACNTs obtained by Fe catalyst. No contaminants from either metallic
particles or amorphous carbon were observed outside the tubes. Virtually all metallic particles
are enclosed by the VACNTs produced, because the MWCVD uses all catalyst nanoclusters.
The high atomic hydrogen concentration in the gas mixture efficiently removes residues of
the amorphous carbon.
Fig. 1: (a) SEM images of Fe nanoclusters, formed after pre-treatment; (c) SEM images of
the VAMWCNT films obtained by Fe catalyst; (c) TEM image of bamboo-like structure of a
VACNTs obtained with Fe nanoparticles.
The superhydrophilicity of the VAMWCNT films obtained after the oxygen plasma
treatment was a requirement for obtaining the HA crystals and consequently the excellent
bioactivity presented here. Figure 2 shows the field emission gun scanning electron
microscopy (SEM) images of the nHA/VAMWCNT films before (a) and after (b) 21 days of
SBF incubation. The left picture is a top view, the central picture is the top view at a higher
magnification, and the right picture is a tilted view to show the cross section details. In
general, nHA/VACNT nanocomposites are bioactive. Commonly, globular apatites, including
biological ones, occur when SBF is used in this biomimetic approach. The SEM images in
figure 2b show the massive growth of the apatite crystallite clusters with a globular-like shape
on the nHA/VAMWCNT nanocomposites. This cross section (Figure 2b) view shows the
biological apatites formed on and between the nHA/VAMWCNT nanocomposites. It is very
illustrative to define a completely densification among the nHA/VAMWCNT. These findings
clearly evidence the exceptional bioactivity of nHA/VAMWCNT nanocomposites. This
property is due to the complete densification and calcification of nHA/VAMWCNT
nanocomposites (more details about the calcification can be seen in figures 3 and 4). There is
an increase of the deposited apatite layer (thickness between 1.5-2.0 µm). Details of the
densification of superhydrophilic VAMWCNT films after 21 days incubation in SBF are
shown in figure 2d compared to nanocristals formed after nHA electrodeposition (Figure 2c).
Figure 2. (a) SEM image of the cross section of HA film on top of the VAMWCNT. (b)
FEG-SEM images show the bioactivity of HA/VAMWCNTs nanocomposites after soaking
for 21 days in SBF. The cross section shows the densification of HA/VAMWCNT
nanocomposites. (c) Details of HA nanocristals formed after electrodeposition process. (d)
Details of biological apatites formed on HA nanocristals after biomineralization process.
In figure 3, the elemental composition of the coating was investigated by µEDX
mapping analysis of biological apatites grown on nHA/VAMWCNTs before (a) and after 21
days (b) of soaking in SBF. The calcium (Ca) and phosphorus (P) content profiles (% wt) as a
function of depth (μm) for each group are shown. The Ca/P ratios determined from the
analyses were: (a) 1.13 (as-grown superhydrophilic nHA/VAMWCNT) and (b) 1.55 (after 21
days of soaking in SBF). In this data, all the values are near the biological apatites in bone
tissue (Elliott, 1998).
Fig. 3. Micro X-ray fluorescence spectrometer by energy-dispersive mapping of biological
apatites formed on (a) superhydrophilic HA/VAMWCNT films and (b) after 21 days of
soaking in simulated body fluid.
Figure 4 illustrates the XRD diffraction patterns of the superhydrophilic VAMWCNT,
nHA/VAMWCNT nanocomposites produced by electrodeposition technique, and
nanocomposites after the imersed in SBF for 21 days. The peaks of hydroxyapatite indexed
in JCPDS (09-0432) was used for comparison. Figure 3A presents reflections in 25.9° (002)
and three peaks in the region around 31.6° (triplet), characteristic of the planes (211), (112)
and (300), and 34.0° (200). These reflections were compared to standard powder nHA sample
(Joint Committee on Powder Diffraction Standards, JCPDS card number: 9-432). nHA has a
hexagonal space group of p63/m with a = 0.9430 nm and c = 0.6891 nm. The strong peaks
around of 31.5 and 37.04° can be associated with the presence of calcite. As has been
discussed, the formation of nHA with Ca/P greater than the theoretical value implies the
formation of calcite during the production process. This system may favor the formation of
calcite (Swanson, 1953). In this diffracttion, the peak intensity of calcite is due to the
biomineralization in vitro process. Other studies involving immersion of CNTs with some
type of functionalization and soaked in SBF also found phase of calcite on the surface of the
nanotubes after a certain immersion time (Aryal, 2006).
Fig. 4. Bioactivity characterization of nHA/VAMWCNT nanocomposites X-ray diffraction
patterns of the superhydrophilic VAMWCNT films, nHA/VAMWCNT nanocomposites
obtained by electrodeposition technique and soaked in SBF for 21 days.
The superhydrophilic VAMWCNT tips can be considered as active sites of defects in
grown crystals. These active sites of formed nHA crystals were sufficient to obtain excellent
biomineralization. It has been suggested that the carboxylate groups facilitate the initial
deposition of calcium ions, and the attraction of calcium ions is an important initial step in
calcium phosphates formation (Kawashita, 2003). With the increasing density of carboxylate
groups, the abundant supply of coordination sites available for complexation with calcium
ions leads to a very large number of nuclei for natural apatite deposition. The large amount of
nuclei facilitates the precipitation and formation of apatite on the superhydrophilic
HA/VAMWCNT films.
Figure 5 shows the SEM images of human osteoblasts adhesion on the crystalline
platelet-like HA surfaces after 7 days. The cells spread with no preferential direction,
acquiring a flat roughly circular form over the surface (Fig. 5a). Fig. 5b shows that cells
proliferate and evolutes to tissue formation. Very healthy cell behavior is observed on
HA/VAMWCNT composites showing active formation of membrane projections all over the
cell surfaces (Fig. 5b). The cells already occupy a considerable area of nHA/VAMWCNT
composite surface. Details about the cell adhesion is show in Fig. 5c.
Fig. 5. Human osteoblast cells adhesion on platelet-like HA/VACNT composites after 7 days.
Initial in vivo studies were performed using the novel nHA/VAMWCNT
nanocomposites. Fig. 6a show a picture of the local and Fig.6b show the histological analysis
after sixteen weeks of the implant. Details of the surgical defect is show in Fig 6a.
Fig. 6. Histological appearance of lamellar bone formation in response to nHA/VAMWCNTs
(a) and a picture of local surgery (b) implants in the calvarium rats at sixteen weeks after
surgery.
It was shown by the qualitative histological observation that there was a lot of new
lamellar bone formation (pink) in the HA/VAMWCNTs group and that the
nHA/VAMWCNTs were integrated entirely into new bone tissue at sixteen weeks after
surgery (Fig. 6b).
Biomimetic bone tissue engineering materials could be used to restore and improve
damaged bone. The objective of the study was the generation of nHA/VAMWCNT
nanocomposites to induce calcification "in vitro" and "in vivo". nHA/VAMWCNT
nanocomposites appears to be privileged candidates for inducing the mineralization of
biological apatites. However, further studies with in vitro and in vivo assays are under study
to better characterize the platelet-like nHA/VAMWCNT composites bioactivity and
calcification.
4. CONCLUSION
The great novelty of the current paper is the direct growth of crystalline HA on
VACNT, which shows a high mineralization without any thermal treatment, in only 30 min,
with bioactivity properties and exceptional cellular adhesion. These results also proved that
the control of the initial stage of high crystallinity of HA nucleation due to the HA/VACNT
composites. The high crystallinity influenced positively in the biomineralization by soaking in
SBF, which shows an excellent bioactivity compared to similar experiments shown in the
literature. Further studies on biomineralization are under way to establish better conditions to
decrease the biomineralization induction time. The consolidated composite obtained after
biomineralization is under study to test its mechanical properties. This new method ofHA
formation may provide an alternative way for the design and preparation of bioactive
nanomaterials with improvedmechanical properties and tailoredmicrostructure
and macrodimension control.
ACKNOWLEDGEMENT
We gratefully acknowledge funding by Fundacao de Amparo a Pesquisa do Estado de
Sao Paulo (FAPESP) under the grants 11/17877-7 and CNPq.
REFERENCES
Aryal, S., Bhattarai, S.R., Bahadur, K.C.R., Khil, M.S., et al., (2006)" Synthesis and
characterization of hydroxyapatite using carbon nanotubes as a nanomatrix", Scripta
Materialia, 54, 131–135.
Balania, K., Anderson, R., Laha, Tapas, Andara, M., et al., (2007) "Plasma-sprayed carbon
nanotube reinforced hydroxyapatite coatings and their interaction with human osteoblasts in
vitro", Biomaterials, 28, 618-624.
Bezzi, G. Celotti, G., Landi, E., La Torretta, T.M.G., Sopyan, I., Tampieri, A. (1997), " A
novel sol–gel technique for hydroxyapatite preparation", Mater. Chem. Phys., 78, 816– 824.
Deck, C.P., Flowers, J., McKee, G.S.B. and Vecchio, (2007), K., "Mechanical behavior of
ultralong multiwalled carbon nanotube mats", J. Appl. Phys. 101, 023512-023521.
Elliott, J.C., (1998) "Recent Studies of Apatites and other Calcium Orthophospates. Calcium
Phosphate Materials", Fundamentals, Bres E., Hardouin P., Eds.; Sauramps Medical:
Monpellier, 1998, p. 25.).
Goller, G., Demirkiran, H., Oktar, F.N., Demirkesen, E. (2003) "Processing and
characterization of bioglass reinforced hydroxyapatite composites", Ceram Int. 29, 721-724.
Habraken ,W, Wolke, JGC, Jansen, JA. (2007), " Ceramic composites as matrices and
scaffolds for drug delivery in tissue engineering", Adv Drug Deliv Rev, 59, 234–248.
Hahn, B-D. Lee, J-M. Park, D-S, Choi, J-J, et al., (2009) "Mechanical and in vitro biological
performances of hydroxyapatite–carbon nanotube composite coatings deposited on Ti by
aerosol deposition", Acta Biomaterialia, 5, 3205-3214.
Kawashita, M., Nakao, M., Minoda, M., Kim, H., Beppu, T., Miyamoto, T., et al. (2003)
"Apatite-forming ability of carboxyl group-containing polymer gels in a simulated body
fluid", Biomaterials, 24, 2477-2484.
Liu, H.S., Chin, T.S., Lai, L.S., Chiu, S.Y., Chuang, K.H., Chang, C.S., Lui, M.T. (1997),"
Hydroxyapatite synthesized by a simplified hydrothermal method", Ceram. Int. 23, 19-25.
Lobo, A.O., Corat, M.A.F., Ramos, S.C., Matsushima, J.T., Granato, A.E.C., (2010) "Fast
Preparation of Hydroxyapatite/Superhydrophilic Vertically Aligned Multiwalled Carbon
Nanotube Composites for Bioactive Application", Langmuir, 26, 18308-18314.
Najafi, H., Nemati, Z.A., Sadeguian, Z., (2009) " Inclusion of carbon nanotubes in a
hydroxyapatite sol–gel matrix", Cer. Int., 35, 2987-2991.
Niu, L., Kua H., Chua, D.H.C., (2010) "Bonelike apatite formation utilizing carbon nanotubes
as template", Langmuir, 26, 4069–4073.
Sanosh, K.P., Chu, M.C.H. , Balakrishnan, A., Lee, Y.J., Kim, T.N., Cho S.J. (2009), "
Synthesis of nano hydroxyapatite powder that simulate teeth particle morphology and
composition", Current Applied Physics, 9(6), 1459–1462.
Sawase, T., Jimbo, R., Baba, K., Shibata, Y., et al., (2008) "Photo-induced hydrophilicity
enhances initial cell behavior and early bone apposition", Clin Oral Implan Res. 19, 491-497.
Swanson, F. (1953) "Natl. Bur. Stand." U.S. II, 51 Circ. 539.
Zhang, H.Q., Li, S.P., Yan, Y.H. (2001), "Dissolution behavior of hydroxyapatite powder in
hydrothermal solution", Ceram. Int. 27, 451– 454.