Low-temperature biomimetic synthesis of β-tricalcium phosphate by altering pH

Article abstract of DOI:10.1080/15533174.2011.652272

Biomimetic synthesis of β-tricalcium phosphate (β-TCP) in polyvinyl alcohol is done at a much lower temperature of 450°C compared with conventional synthesis. By varying the _pH of the reaction in a patented process for hydroxyapatite synthesis, the authors synthesized β-TCP with controlled particle morphology at a very low temperature. The synthesized powder has been structurally characterized and cell studies with mesenchymal stem cells shows good adhesion.

Full text of DOI:10.1080/15533174.2011.652272

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Synthesis and Reactivity in Inorganic, Metal-Organic,
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Low-Temperature Biomimetic Synthesis of β-Tricalcium
Phosphate by Altering pH
Avijit Guha a & Suprabha Nayar a
a Biomaterials Lab , National Metallurgical Laboratory , Jamshedpur , India
Accepted author version posted online: 22 Jun 2012.Published online: 25 Feb 2014.
To cite this article: Avijit Guha & Suprabha Nayar (2014) Low-Temperature Biomimetic Synthesis of β-Tricalcium Phosphate
by Altering pH, Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 44:6, 789-792, DOI:
10.1080/15533174.2011.652272
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Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 44:789–792, 2014
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1553-3174 print / 1553-3182 online
DOI: 10.1080/15533174.2011.652272
Low-Temperature Biomimetic Synthesis of β -Tricalcium
Phosphate by Altering pH
Avijit Guha and Suprabha Nayar
Biomaterials Lab, National Metallurgical Laboratory, Jamshedpur, India
Ca9(HPO4) (PO4)5(OH) is treated above 900◦C it transforms
into the β-TCP as shown by the following:
Biomimetic synthesis of β-tricalcium phosphate (β-TCP) in
polyvinyl alcohol is done at a much lower temperature of 450◦C
compared with conventional synthesis. By varying the pH of the
reaction in a patented process for hydroxyapatite synthesis, the au-
thors synthesized β-TCP with controlled particle morphology at
a very low temperature. The synthesized powder has been struc-
900−1200◦C
Ca9(HPO4)(PO4)5(OH) −−−−−→ 3Ca3(PO4)2 + H2O
High thermal decomposition often leads to agglomeration
turally characterized and cell studies with mesenchymal stem cells and grain growth, thus causing a reduction in the surface area
shows good adhesion.
and porosity, and a subsequent loss of bioactivity. In situ syn-
thesis of calcium phosphate nanoparticles in PVA matrix and
its subsequent sintering at low temperature keeps the porosity
and nanostructure intact. By varying the pH of the reaction,
decomposition occurs at a much lower temperature, yielding
macroporous β-TCP nanoparticles.
Keywords β-Tricalcium phosphate, pH, polyvinyl alcohol
INTRODUCTION
Calcium phosphates (CaP) have excellent biocompatibility
and have multiple applications in the field of tissue engineering,
of which hydroxyapatite (HA) and β-TCP are very well stud-
ied.[1,2] HA is the most stable phase, and osteoconductive and
least soluble in the physiological environment.[3] β-TCP, on the
other hand, is one of the most resorbable phases and thus a more
promising material in terms of faster healing.[4,5] The variation
in atomic Ca/P ratio in the synthesized calcium phosphate leads
to differences in biodegradability and bioactivity.[6–8] Although
all calcium phosphates were initially considered as biocom-
patible but when used for clinical trials, they encounter many
problems such as poor adhesion, biocompatibility, and weak
mechanical strength.[9,10] Reports suggest that incorporation of
β-TCP particles into polyvinyl alcohol (PVA) matrix improves
the biocompatibility, osteoconductivity, and bone bonding ca-
pacity of these materials,[11] though there are many reports on
the bonding mechanism of β-TCP with living bone, the issue is
still debatable.[12,13]
EXPERIMENTAL
Calcium nitrate [Ca(NO3)2.4H2O] and diammonium hydro-
gen phosphate [(NH4) HPO4] (both from Merck, India) were
2
taken as Ca- and P-source, respectively. A 250 mL of 0.4M
calcium salt solution was mixed with equal volume of aqueous
solution of PVA (molecular weight 1 was 25000 and degree
of hydrolysis was 85–89%, from Qualigens, India) containing
3.5 g polymer. The system was incubated for 24 h at 30◦C.
Stoichiometric 356 mL volume of 0.156M diammonium salt
solution (pH > 8) was added to the previous mixture and mixed
thoroughly. The entire mixture was adjusted to pH 7 with or-
thophosphoric acid (H3PO4) and aged for one week at 30◦C; the
precipitate obtained was washed with deionized water and oven
dried at 80◦C (S-1). The powder was calcined in a tubular fur-
nace in air at a temperature 450◦C for 2 h. Sample synthesized
with Ca/P molar ratio of 1.50 has been designated as S-2. Ex-
periments are repeated three times to ensure the reproducibility
of phase formation.
There are many established techniques for β-TCP nanopar-
ticles synthesis, which involves sintering at high tempera-
tures, around 900–1200◦C.[14–21] When calcium-deficient HA
Materials Characterization
To identify the phase of the synthesized materials, X-ray
diffraction (XRD) was carried out using (Siemens, Germany,
D500) using Cu Kα radiation at 30 KV and 25 mA scanned
for diffraction angles: 2θ = –20◦ to 80◦ at room tempera-
ture. To determine average crystallite size, Full width at half
maxima (FWHM) was calculated according to Scherrer’s equa-
tion with the shape factor K (0.9). Relative volume fractions
of different crystalline phases formed were determined by the
Received 2 February 2011; accepted 19 December 2011.
The authors thank CSIR for providing the scholarship for research
work and also Dr. Sinha for valuable suggestions.
Address correspondence to Avijit Guha, Materials Lab, Na-
tional Metallurgical Laboratory, Jamshedpur 831007, India. E-mail:
bintuguha@indiatimes.com
Color versions of one or more of the figures in the article can be
found online at www.tandfonline.com/lsrt.
789

790
A. GUHA AND S. NAYAR
FIG. 3. High-resolution image shows growth in a preferred direction.
FIG. 1. XRD patterns of room temperature synthesized powder (S-1) and
calcined sample (S-2).
The powder was sterilized using UV and washed in Hanks’s
Balanced Salt Solution (HBSS). Cells were plated on cover
slips and observed for 7–10 days. Cell adherence and growth
was monitored every day and at the end of 10 days, cover slips
were fixed with 2% glutaraldehyde and processed for scanning
electron microscopy.
area under the diffraction peaks of the respective phases. Size
and shape of biphasic nanoparticles have been characterized
using transmission electron microscopy (TEM; CM 200, CX
Philips, Germany, at 160 KV JEOL 2100), the samples were
ultrasonically dispersed in double distilled water and then one
drop mounted on carbon coated copper grids. Thermogravi-
metric analysis of powders coupled with differential thermal
analysis (DTA) was performed up to 1000◦C (SDTQ600). A
transformation from calcium phosphate to β-TCP was also en-
sured by Fourier transform spectroscopy (FTIR-410 JASCO,
USA). The calcium phosphate molar ratio was analyzed by in-
ductively coupled plasma–optical emission spectrometry anal-
ysis (ICP-OES). Samples were designated as (S-1) room
temperature synthesized nanocomposite and (S-2) calcined
sample.
RESULTS AND DISCUSSION
It was observed that all the diffraction patterns matched with
the standard calcium hydrogen phosphate and β-TCP (JCPDS
file Nos. 65–2384 and 9–169) in both room temperature and
treated samples (Figure 1). The diffraction patterns of the room
temperature synthesized powder show the formation of a com-
plex mixture comprising of calcium hydrogen phosphate and
β-TCP, the 100% diffraction peak was identified and indexed as
(112) corresponding to calcium phosphate along with a smaller
FIG. 2. TEM micrograph of thermal treated sample.
FIG. 4. The FTIR spectra of room temperature and thermally treated samples.

NANOCOMPOSITES
791
absorbance band at 451 cm−1 is assigned to doubly degenerate
ν2 O-P-O bending moment, ad the band at 556.72 cm−1 is due
to PO34− vibration modes. The band at 873.96 cm−1 is due to
P-O(H) stretching in HPO−4 2 group.[22–25] Similarly, absorbance
bands at 1026 and 1076 cm−1 confirm the presence of P-O
stretching mode. The absorbance band at 1429 cm−1 correspond
to a –CH2 cm−1 stretching band, confirming the presence −o1f
polymer in the synthesized samples. Another band at 2917 cm
corresponds to a –CH- stretching mode. The band at 1632 cm−1
also corresponds to –OH vibration mode (Figure 3).[26–28]
The pH value of the reaction during synthesis is mainly con-
trolled by H3PO4 and (NH4) 2HPO4, responsible for the phase
formation.[29,30] Addition of H3PO4 solution to the mixture re-
sults in excess PO34− ions, which dominates the pH drop from
10 to 7. At a lower pH, a large number of protonated phos-
phate ions (i.e., H2PO41− and HPO24−) are present, which enters
the crystal lattice and probably forms a Ca vacancy that is sus-
ceptible to heat treatment. On the other hand, the increase in
the reaction pH promotes the dissociation of H3PO4 and (NH4)
2HPO4 and enhances the formation of PO34−. Usually in the wet
chemical synthesis high pH is required for the precipitation of
stoichiometric and the thermally stable phase. Apatite crystals
with calcium have a phosphate molar ratio of 1.63–1.33 that
involves Ca and OH vacancies and can substitute some PO43−
FIG. 5. Thermal graph of as synthesized powder.
peak (102). The other peaks (125), (217), (128), (131), (1.1.15),
(214), (128), 042), (404), (327), (120), and (2.0.2o) correspond
to β-TCP. After the sample was treated at 450◦C for 2 h, the
two peaks of calcium phosphate were not observed. All major
diffraction peaks of the powder sample were identified and in-
dexed as (214), (210), (2.1.10), (226), (1.0.16), (3.0.12), (238),
and (2.0.20) peaks of β-TCP, with an average crystallite size of
40 nm.
TEM images confirmed the formation of nanosized crys-
talline β-TCP particles of needle shape and uniform size (24 nm
length and 14 nm width; Figure 2). The lattice fringes were ob-
served in micrographs that indicated the growth in a preferred
direction (Figure 2).
groups with HPO24−
.
Thermal stability studies of sample shown in Figure 4. The
thermograms of samples exhibit temperature difference in the
peaks in the region of 200◦C and 450◦C.[31,32] It is attributed
to the removal of adherent water in the precipitated powder.
Endothermic peaks in the range of 200–400◦C are due to re-
versible water loss from the lattice of apatite structure caused
by the rearrangement of oxygen atoms in the lattice and also it
shows the decomposition of PVA molecules from the sample.
The total weight loss of the sample was observed around (8%) at
450◦C where the PVA decomposition as well as transformation
occurred.
The FTIR spectra of both samples are almost similar except
the fact that absorbance at 3444 cm−1 corresponding to hydroxyl
groups in the thermally treated sample shows a decrease. The
Mesenchymal cells (MSC) were isolated from the bone mar-
row; these cells were seeded onto the β-TCP nanocomposites.
The culture was incubated for seven days in the CO2 incubator
with intermittent change of media. After seven days nanocom-
posites containing cells were examined under SEM. A large
number of cells adhered on the surface of the composite and
proliferated (Figure 5).
CONCLUSION
We have successfully synthesized β-TCP in situ in a PVA
matrix at a low temperature. Addition of H3PO4 along with
(NH4)2HPO4 ions in the solution not only decreases the pH
but also the excess HPO34− ions cause an atomic imbalance in
the reaction. The presence of high amount of HPO34− ions in
the solution decreases the Ca vacancies in the lattice, which
renders the material unstable and forms calcium deficiency that
is thermally unstable.
FIG. 6. The cells adhered on the surface of the composite and proliferated.

792
A. GUHA AND S. NAYAR
17. Raynaud, S.; Champion, E.; Bernache-Assollant, D.; Thomas, P. Calcuim-
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