Synthesis, Structural analysis, Mechanical, Antibacterial and Haemolytic

Activity of Mg2 + and Cu2 + co-substitutions in β-Ca 3 (PO4 )2

Ram Kishore Singh, S. Kannan

PII: S0928-4931(14)00636-5
DOI: doi: 10.1016/j.msec.2014.10.017
Reference: MSC 4950

To appear in: Materials Science & Engineering C

Received date: 16 July 2014

Revised date: 20 August 2014
Accepted date: 2 October 2014

Please cite this article as: Ram Kishore Singh, S. Kannan, Synthesis, Structural
analysis, Mechanical, Antibacterial and Haemolytic Activity of Mg2 + and Cu2 +
co-substitutions in β-Ca3 (PO4 )2 , Materials Science & Engineering C (2014), doi:
10.1016/j.msec.2014.10.017

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 ACCEPTED MANUSCRIPT

Synthesis, Structural analysis, Mechanical, Antibacterial and Haemolytic Activity of

Mg2+ and Cu2+ co-substitutions in β-Ca3(PO4)2

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Ram Kishore Singh and S. Kannan*
Centre for Nanoscience and Technology,

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Pondicherry University, Puducherry-605 014, INDIA

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Corresponding Author’s Address

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Dr. S. Kannan
Assistant Professor
Centre for Nanoscience and Technology,
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Pondicherry University, Puducherry-605 014, INDIA
E-mail: para_kanna@yahoo.com, skannan.nst@pondiuni.edu.in.
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Phone: 0091-413-2654973
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Abstract

The present study reports the synthesis, structural, mechanical and in vitro characterization of a series of

Mg2+ and Cu2+ co-substitutions in β-Ca3(PO4)2. Aqueous precipitation technique ensured the formation of

calcium deficient apatite with substituted ions in the as prepared conditions and their subsequent heat

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treatments at 1000 C resulted in the formation of crystalline β-Ca3(PO4)2. The combined substitutions of

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lower sized Mg2+ and Cu2+ in the structural lattice of β-Ca3(PO4)2 led to the significant contraction in

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a=b-axis and c-axis lattice parameters of β-Ca3(PO4)2. The results from Rietveld refinement confirmed

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the occupancy of Mg2+ and Cu2+ at the Ca(5) site of β-Ca3(PO4)2. The results from mechanical tests

proved that Mg2+ and Cu2+ substitutions are not hindering the mechanical properties of β-Ca3(PO4)2. The

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antibacterial and haemolytic results determined for the Mg2+ and Cu2+ co-substituted β-Ca3(PO4)2 ensured
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their good activity against the investigated microbes and also confirmed their blood biocompatibility.

Key words: β-Ca3(PO4)2, Co-substitution, mechanical, antibacterial, haemolysis.

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1. Introduction
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Bioactive calcium phosphate based ceramics, especially Hydroxyapatite [HAP, Ca10(PO4)6(OH)2] and β-
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Tricalcium phosphate [β-TCP, β-Ca3(PO4)2] are well known for their orthopaedic and dental applications
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due to their chemical and crystallographic similarities with natural bone and tooth material [1]. Since the

solubility activity product of β-TCP 2.51  10-30 is better than HAP 2.34  10-59, β-TCP encourages

faster bone growth and facilitates integration with bone tissue as it reabsorbs [2-6]. Successful clinical

trials of β-TCP are well established as a bone substitute bioceramic due to its favourable features of

excellent biocompatibility, osteoinductivity and safety in living tissue [7-10]. In vivo resorbability (more

than 95%) of β-TCP in a minipig-tibia model observed over a period of 86 weeks has been reported by

Wiltfang et al., [11]. Recent years have focussed extensive investigation on the substitution of cations

(i.e. Mg2+, Sr2+, Na+, K+, Cu2+, Zn2+) and/or anions (i.e. F-, SiO24- and CO32-) in the β-TCP structure as

these substituted ions play a pivotal role in the overall biological performance of β-TCP by inducing

significant changes in certain physical, chemical and mechanical properties of β-TCP [12-17]. Many
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studies have focused on single ionic substitution in β-TCP structure [12-15] whereas only few researchers

have involved in the development of coupled ionic substitutions in the β-TCP structure [16-20]. The

present study is an attempt to develop coupled ionic substitutions in β-TCP involving two important trace

elements namely magnesium (Mg2+) and copper (Cu2+).

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Mg2+ is the fourth most abundant cation in the human body detected in the range of 0.44 - 1.23 wt.% [21].

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Mg2+ concentrations in enamel, dentine and bone of the human body are respectively detected as 0.44,

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1.23 and 0.72 wt.% [22]. The characteristic role of Mg2+ in the prevention of possible risk factors for

osteoporosis in human [23] and its significant role in the calcification process, bone fragility and its

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indirect influence on mineral metabolism [24-26] are reported. Moreover, Mg2+ is also known for its

substantial role in the delaying effect of allotropic β- to α- transformation of Ca3(PO4)2 [27] at high
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temperatures.
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Recent years have witnessed the failure of implants due to the bacterial infections at the implant site thus

leading to the premature removal of implants causing surgical revisions [28-29]. In this context, the
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development of bioceramics with cationic dopants that possesses antibacterial properties is the subject of
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extensive investigation. In vitro and in vivo studies on the ionic (Ag2+, Cu2+ and Zn2+) doped bioceramics
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are reported to show good inhibitory activities against the investigated mircrobes [30, 31]. The

antibacterial activity of copper (Cu2+) is reported by many researchers [32, 33] and in addition Cu2+ is

known to possess other important biological features such as its ability to enhance cell activity, osteoblast

proliferation and its potential to improve the angiogenesis of bioactive scaffolds [34, 35] .

Considering the importance of two biologically important cations namely Mg2+ and Cu2+, the present

study is an attempt to develop of Mg2+ and Cu2+ co-substitutions in β-Ca3(PO4)2. Literature survey have

revealed the studies on either Mg2+ only or Cu2+ only substituted β-Ca3(PO4)2 and to the best of our

knowledge there are no reports on the development of combined substitutions of Mg2+ and Cu2+ in β-

Ca3(PO4)2. Thus the co-substitutions of Mg2+ and Cu2+ in β-Ca3(PO4)2 is expected to offer the combined
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advantages provided by Mg2+ and Cu2+ ions in β-Ca3(PO4)2. The powder synthesis was attempted by a

simple aqueous precipitation technique and the detailed structural characterization was undertaken to

investigate the effective role played by the substituted ions. Further, the mechanical behaviour,

antibacterial activity and haemocompatibility of the Mg2+ and Cu2+ co-substituted β-Ca3(PO4)2 materials

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were determined.

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2. Materials and Methods

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2.1 Powder preparation

A chemical precipitation technique was used to synthesize four different concentrations of Mg2+ and Cu2+

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doped β-Ca3(PO4)2. For this purpose, analytical grade Calcium nitrite tetrahydrate [Ca(NO3)2.4H2O],
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Magnesium nitrate hexahydrate [Mg(NO3)2.6H2O] and Copper nitrite hexahydrate [Cu(NO3)2.6H2O] and

di-ammonium hydrogen orthophosphate [(NH4)2HPO4] were taken as precursors for Ca2+, Mg2+, Cu2+

and PO43- ions respectively. A pure β-Ca3(PO4)2 (Ca/P molar ratio = 1.5) and four different
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concentrations of both Mg2+ (M2+) only and Cu2+ (M2+) only substituted β-Ca3(PO4)2 were also prepared
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for the effective comparison of the results. The concentration of the precursors were made in such a way
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that the total (Ca2+ + M2+)/P molar ratio was equal to 1.5 for all the compositions. The stock solutions
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containing cationic mixtures ( Ca2+, Mg2+and Cu2+) and (NH4)2HPO4 were prepared separately in a
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200 ml standard measuring flask using de-ionized water. The prepared cationic solution was

transferred to a beaker and placed it on a magnetic stirrer (TARSONS, INDIA) with an operating

temperature of 90°C and rotation speed of about 250 rpm. The separately prepared (NH 4)2HPO4

solution was now added to the continuously stirred cationic solution in a drop wise manner. After the

completion of the addition, a white gelatinous precipitate was formed and the pH of the solution mixture

was measured as 4.75. Now the pH of the solution mixture was raised to 8.0 by using

concentrated NH4OH solution and the resultant mixture was continuously stirred under constant

temperature conditions for 2 hours. The resultant mixture was now transferred to a stable condition at

room temperature for the precipitate to settle down. The solution was decanted and the precipitate

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was transferred to a Hot air oven (TECHNICO OVEN, INDIA) and dried at 120°C overnight. The

obtained dried precipitate was grounded well to fine powders this was considered as prepared powder.

2.2 Powder characterization

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The as prepared powders and the powders after heat treatment at different temperatures (MATRI-MC

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2265 A, INDIA) for a dwelling period of two hours were subjected to X-ray diffraction (XRD,

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RIGAKU, ULTIMA IV, JAPAN) to observe any change in the phase behaviour and crystallinity.

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XRD studies for all the powders were carried out using a high-resolution RIGAKU, ULTIMA IV,

diffractometer with Cu Kα radiation (γ = 1.5406 nm) produced at 40 kV and 30 mA to scan the

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diffraction angles (2θ) between 10o and 70° with a step size of 0.02° 2θ per second. Any slow scan for

Rietved refinement was done with a with a step size of 0.01° 2θ per second. Phase determinations were
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made using Standard ICDD (International Centre for Diffraction Data) card no. 00-009-0432 for HAP

and 00-009-0169 for β-TCP. X-ray fluorescence (XRF) spectroscopy was carried out for all the
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synthesized powders after heat treatment at 1000 oC using a BRUKER S4 PIONEER instrument. For this
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purpose, the powder samples were mixed homogeneously with boric acid powder in the proportion of ¼
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(by weight) for 15 minutes. The presence of Ca2+, P5+, Mg2+ and Cu2+, and other elements present at trace
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levels was confirmed using this technique. The Fourier transform infrared (FT-IR) spectra for the
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powders (NICOLET iS10, FT-IR spectrophotometer, USA) were recorded to determine the vibrational

modes in the infra-red region. For this purpose each powder was mixed with potassium bromide (KBr)

in the proportion of 1/150 (by weight) for 15 min and pressed into a pellet using a hand press.

Raman spectra for the powders were recoded using a confocal Raman microscope (RENISHAW, United

Kingdom). All the powder samples were excited at a wavelength of 785 nm by semiconductor diode laser

(0.5% of power) with a data acquiring time of 30 seconds. The preferred scans for all the powder samples

were done in the range of 100 to 1200 cm-1 and all the analysis were done at room temperature. The

surface morphology of the powders was determined by Scanning electron miccroscope (HITACHI,

Japan) equipped with an EDX instrument (Thermo Super Dry II, USA).

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2.3 Quantitative analysis by Rietveld refinement

Quantitative phase analysis for the pure β-Ca3(PO4)2 and all the different concentrations of either single or

coupled ionic substitutions of Mg2+ and Cu2+ in the β-Ca3(PO4)2 samples were performed by the Rietveld

method using the GSAS-EXPGUI software. The X-ray scans for each powder sample was prepared and

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measured three times independently in order to perform the refinement study. The initial refinement of

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stoichiometric β-Ca3(PO4)2 was done with the standard CIF (crystallography information file) from

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Yashima et al., 36. The structural parameters taken for refinement were given as the space group R3c,

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Z = 21, a = b=10.4352 Å and c = 37.4029˚ Å, and 18 independent atomic positions of five Ca positions

(three in site 18b and two in site 6a at one-half occupancy), three P positions (two in site 18b and one in

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site 6a), and ten O positions (nine in site 18b and one in site 6a). The CIF file of pure β-Ca3(PO4)2 refined
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from Yashima et al., 36 was taken as a standard for refining either single or combined substitutions of

Mg2+ and Cu2+ doped β-Ca3(PO4)2. In the first step of the refinement, all the structural parameters were
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fixed to the literature values. Then, during the successive refinement cycles, numerous parameters were
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allowed to vary according to the relative weight amount of the observed phases. The following refinement

sequence was used as a standard for all the structures: scale factor, zero shift, background as a Chebyshev
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polynomial of fifth grade, peak profile and lattice parameters. Fittings were performed using pseudo-
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Voigt peak profile functions and a preferred orientation along [001] was taken into account with the
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Marsh model. The atomic parameters such as position, isotropic temperature and occupancy factors were

also refined during the final step.

2.4 Mechanical Studies

The mechanical properties measurements of pure β-Ca3(PO4)2 and Mg2+ and Cu2+ co-substituted β-

Ca3(PO4)2 powders samples with all four concentration samples were carried out at room temperature

using a Nano indenter machine (CETR, USA). For this purpose, the as dried powders were initially heat

treated at 600 °C to eliminate all the volatile impurities followed by fine grounding and sieving with 10

µm sized sieve pan (RETSCH, GERMANY). The sieved powders were uniaxially pressed in the form of

pellets (13 mm diameter and 1 mm thickness) using an semi-automatic hydraulic press machine
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(KIMAYA ENGINEERS, INDIA) under the applied force of 200 MPa for 60 seconds. The pellets thus

obtained were heat treated at 1000 °C for the dwell time of 2 hours followed by fine polishing using

diamond paste with mesh size (3µ and 0.5µ in size) by using a Sylvester cloth. All the pellets were

cleaned in an ultrasonic bath for 10 min to remove polishing debris which will provide a fine surface

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finish that was needed for the nanoindentation testing.

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A single load of 200 mN was used for testing the samples. A triangular pyramid (BERKOVICH)

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diamond tip indenter with a radius of curvature of 50 nm was used. In the nanoindentation method, the

selected mechanical properties such as the Young's modulus and hardness were evaluated using a

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conventional depth-sensing test, where the measurement cycle (load, unload and displacement curves)
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consists of a loading segment followed by a dwell time at maximum load and finally an unloading

segment. All the pellets were analysed through single indent mode with 10 numbers of indents for each

pellets choosing random locations to analysed to investigate the effect of Mg2+ and Cu2+ co-substitution in
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pure β-Ca3(PO4)2 sample and average were taken for analysis.

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2.5 Antimicrobial tests

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Antimicrobial activity of β-TCP and all the four different Mg2+ and Cu2+ co-substituted β-TCP powder
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samples after heat treatment at 1000 oC were tested against both Staphylococus aureus (S. aureus) and

Escheria coli (E. coli) in a quantitative manner. Prior to antimicrobial testing, all the samples were

sterilized in an autoclave at 121 oC for 20 min. Fresh inoculums of S. aureus and E. coli were prepared

from broth (Müller-Hinton broth, High media, India) and were kept it at 37 oC for overnight incubation

and were used for further tests. The antibacterial tests were performed with some modifications of the

work reported by Stanic et. al., [37] and the details of experiments are explained as follows. A test tube

containing the mixture of 0.1 g sterilized sample and 9.9 ml sterile potassium hydrogen phosphate buffer

solution (pH 7.4) were subjected to thorough mixing for 5 min by using vortex. 0.1 ml of the overnight

incubated culture was transferred to each test tube and these tubes were kept in an incubator shaker at 37
o
C. For viable cell determination, 1 ml aliquots were taken from each test tube after a time interval of 0, 1,
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2 and 4 h. Müller-Hinton agar (High media, India) was used to prepare petri dishes. From the appropriate

dilution, 0.1 ml was spread on petri dishes and kept it for 24 h incubation at 37 oC. Viable microorganism

counts were made after the different time periods of 0, 1, 2, and 4 h. Average value of results were taken

after performing the experiment three times.

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The percentage of microorganism reduction (R) was calculated according to the equation (1) below:

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CO  C
R   100 % (1)

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CO

where Co is the mean number of bacteria on the control sample tube (CFU/sample) without sample and C

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is the mean number of bacterial colonies with different β-TCP samples.
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2.6. Haemolysis test

The haemolytic activity of β-TCP and all the four different Mg2+ and Cu2+ co-substituted β-TCP powder
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samples after heat treatment at 1000 oC were investigated according to the method described by Parnham
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et. al., and Yinghui et. al., [38, 39]. A constant amount of 0.1 g sterile sample was taken in a test tube and
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10 ml of sterile saline solution was added to it. 8 ml fresh blood was extracted from a healthy human and
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this blood sample was diluted with 10 ml sterile saline solution. A 0.2 ml of the diluted blood was added

to the each tube containing sterile saline sample and was incubated at 37 oC for 60 min followed by the
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collection of supernatant through the centrifugation of tubes at 7000 rpm for 10 min. The amount of free

haemoglobin was determined by measuring the absorbance of supernatant at 540 nm (UV-1800,

SHIMADZU, UV spectrophotometer, JAPAN). The hemolysis rate (HR) was calculated according to the

equation (2) given below:

Dt  Dnc
HR  100 % (2)
Dpc  Dnc

Where Dt, Dnc and Dpc are the corresponding sample absorbance, the negative control and the positive

control. The experiments were run in triplicate and average values were taken.

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3. Result and Discussion

3.1. Phase Evolution

Fig. 1 presents the FT-IR patterns and Figs. 2 and 3 presents the X-ray diffraction (XRD) patterns for the

Mg2+ and Cu2+ co-substituted calcium phosphate powders after heat treatment at different temperatures.

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The formation of poorly crystalline apatite phase characterized by the broad diffraction bands for the as

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dried powders was confirmed from XRD patterns. The characteristic feature of apatite phase in the as

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dried condition was also confirmed from the FT-IR patterns as it indicated the vibrational modes of PO4

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group at 488, 567, 601, 966 and 1036-1093 cm-1. The formation of calcium deficient apatite phase

Ca9(PO4)5(HPO4)OH in the as dried condition was ensured by the characteristic presence of HPO4

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group from the observed band at 871 cm-1 and for the presence of OH groups from the observed peaks at
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[636 cm-1 (liberational mode) and 3569 cm-1 (stretching mode)] in the FT-IR spectra. The broad band for

the presence of adsorbed water (in the region of 1628, and 3400-3600 cm-1) and adsorbed nitrates (1320-

1480 cm-1) during aqueous precipitation process make apatite phase poorly crystalline [14,15,27].
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Progressive heat treatments accompanied by the gradual loss of adsorbed water and nitrates from the as-

prepared powder became evident from the FT-IR patterns due to the observed decrease in the
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corresponding infrared peak resolution in the region around 1320-1480 and 3400-3600 cm-1. The FT-IR
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patterns obtained at 300, 500 and 700 C indicated a band at near about 720 cm-1, which attributes for the
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presence of pyrophosphate groups (P2O74-). This band disappeared after heating at 1000 C and this result

agrees with the condensation of HPO42- groups in calcium-deficient apatite that usually occurs in the

range 350-720 C according to the mechanism proposed by Mortier et. al., [40]. FT-IR patterns presented

in Fig. 1 also confirm the disappearance of peaks for OH group at 636 and 3569 cm-1 at 1000 C.

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1000 oC

700 oC
Transmittance (a.u)

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500 oC

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300 oC

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P2O74- NO32- As dried OH

H2 O

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H2 O
HPO42-

450 950 1450 1950 2450 2950 3450 3950

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Wave number (cm-1)
Fig.1. FT-IR spectra obtained for the Mg2+ and Cu2+ co-substituted β-TCP (1xMgCuβ-TCP)
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powder after heat treatment at different temperature conditions.

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The XRD patterns presented in Fig. 2 confirmed the formation of single phase β-TCP after heat treatment
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at 1000 C for the Mg2+ and Cu2+ co-substituted β-TCP powder sample and this transformation process at

1000 C are in accordance with the literature reports [15]. The formation of calcium deficient apatite
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phase in the as dried condition and its gradual conversion to yield single phase β-TCP at 1000 C during

the progressive heat treatments were found evident from the XRD patterns presented in Fig. 2. Thus the

heat treatment temperature plays an important role in the formation of β-Ca3(PO4)2. It is a well-

documented fact that during the synthesis of β-Ca3(PO4)2 through aqueous precipitation technique,

calcium-deficient apatite (Ca/P < 1.67) is formed immediately after the synthesis and heat treatment

beyond 780 C has led to the conversion of the calcium-deficient apatite to single phase β-Ca3(PO4)2 [40].

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1000 oC
Intensity (a.u)

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β-TCP (00-09-0169 )

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700 oC
500 oC

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300 oC
As dried
20 25 30 35 40 45 50
2 θ (degrees)
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Fig.2. XRD patterns obtained for the Mg2+ and Cu2+ co-substituted β-TCP (1xMgCuβ-TCP)
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powder after heat treatment at different temperature conditions.

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4X-MgCuβ-TCP
Intensity (a.u)

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3X-MgCuβ-TCP

2X-MgCuβ-TCP

1X-MgCuβ-TCP

Pureβ-TCP

20 25 30 35 40 45 50
2 θ (degrees)

Fig. 3 XRD patterns obtained for the different concentration of Mg2+ and Cu2+ co-

substituted β-TCP after heat treatment at 1000 oC

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The formation of single phase β-Ca3(PO4)2 for the Mg2+ and Cu2+ co-substituted samples at 1000°C were

confirmed by matching their corresponding XRD patterns (Fig. 3) with the standard ICDD card No.00-

09-0169 of β-Ca3(PO4)2. Thus the formation mechanism of β-Ca3(PO4)2 with the substituted Mg2+ and

Cu2+ ions is ensured according to the equation (3) below.

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C
9  ( x  y )Ca( NO3 ) 2  xCu ( NO3 ) 2  yMg ( NO3 ) 2  6( NH 4 ) 2 HPO4  8 NH 4OH 120
 
C
1000
  3Ca3( x / 3 y / 3) Cu x Mg y ( PO4 ) 2  H 2O

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Ca9( x y ) Cu x Mg y ( HPO4 )( PO4 ) 5 (OH ) (3)

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3.2. Impact of Mg2+ and Cu2+ ions in β-Ca3(PO4)2 structure

The structural details due to the influence of either single or coupled ionic substitutions of Mg2+ and Cu2+

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in β-Ca3(PO4)2 structure were determined through Rietveld refinement. The corresponding refined

diffraction patterns for pure β-Ca3(PO4)2 and Mg2+ and Cu2+ co-substituted β-Ca3(PO4)2 presented in Figs.
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4 and 5 confirmed the formation of crystalline β-Ca3(PO4)2 phase. The refined lattice data obtained for β-

Ca3(PO4)2 without any added Mg2+ and Cu2+ ions confirmed the formation of crystalline β-Ca3(PO4)2 with
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unit cell parameters a = b = 10.4427 (4), c = 37.404 (4), α = β = 90, and γ = 120 in the hexagonal setting
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(space group R3c). The refined values of pure β-Ca3(PO4)2 were taken as the starting values for the
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structural refinement of either single or coupled Mg2+ and Cu2+ substitutions in β-Ca3(PO4)2. The refined
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parameters that determine the quality of refinement for all the investigated compositions are presented in

Table 1. As can be seen from Table 1, the goodness of fit (GoF) values were relatively low, i.e., all were
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approximately around 3%, which is considered as acceptable according to the basic principle of GoF less

than 4% and Rwp less than 20%.

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Observed
Calculated
Brag Peak
Difference
Counts

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10 20 30 NU
40 50 60 70
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2 θ (degrees)

Fig. 4 Refined diffraction patterns for pure β- Ca3(PO4)2 after heat treatment at 1000 oC
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Observed
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Calculated
Brag Peak
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Difference
Counts

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10 20 30 40 50 60 70
2 θ (degrees)

Fig. 5 Refined diffraction patterns for pure 4xMgCuβ-TCP after heat treatment at 1000 oC

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Table 1 Refined parameters of pure β-Ca3(PO4)2 and Mg2+ and Cu2+ doped β-Ca3(PO4)2
a a
Sample Code cRp cRwp RBraggc χ 2b
Pure β-TCP 0.1261 0.1610 0.0662 1.652
1xMgβ-TCP 0.1282 0.1680 0.0725 1.786
2xMgβ-TCP 0.1296 0.1695 0.0752 1.834
3xMgβ-TCP 0.1386 0.1872 0.0866 2.241

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4xMgβ-TCP 0.1513 0.2048 0.1017 2.258

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1xCuβ-TCP 0.1131 0.1473 0.0741 1.273
2xCuβ-TCP 0.1063 0.1352 0.0693 1.503

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3xCuβ-TCP 0.1256 0.1696 0.0917 2.685
4xCuβ-TCP 0.1453 0.1915 0.0824 2.822

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1xMgCuβ-TCP 0.1225 0.1594 0.0758 1.692
2xMgCuβ-TCP 0.1231 0.1634 0.0776 1.963

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3xMgCuβ-TCP 0.1384 0.1854 0.1029 2.632
4xMgCuβ-TCP 0.1154 0.1506 0.0851 1.923
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where, cRpa and cRwpa, represents the Rietveld agreement factors. χ 2b
is a goodness of fit (GoF) indicator defined by
(Rwp/Rexp)2 in which Rexp is the expected weighted profile factor. RBraggc demonstrates the agreement between the observed and
computed reflection during refinement.
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Table 2 Elemental analysis of single and combined substitutions of Mg2+ and Cu2+ ions used

in the synthesis of β-Ca3(PO4)2

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Mg2+ only substituted β-Ca3(PO4)2 Cu2+ only substituted β-Ca3(PO4)2 Mg2+ and Cu2+ co-substituted β-Ca3(PO4)2
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mol. % of Mg2+ mol. % of Cu2+ mol. % of Cu2+ and Mg2+

Code Code Code
Planned Determined Planned Determined Planned Determined
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Pure β-TCP - - - - - - - -

0.630 each of 0.65 of Mg2+ &

1xMgβ-TCP 1.260 1.311 1xCuβ-TCP 1.260 1.165 1xMgCuβ-TCP
Mg2+ & Cu2+ 0.58 of Cu2+
1.260 each of 1.39 of Mg2+ &
2xMgβ-TCP 2.521 2.848 2xCuβ-TCP 2.521 2.754 2xMgCuβ-TCP
Mg2+ & Cu2+ 1.33 of Cu2+
1.890 each of 2.01 of Mg2+ &
3xMgβ-TCP 3.781 4.061 3xCuβ-TCP 3.781 4.184 3xMgCuβ-TCP
Mg2+ & Cu2+ 1.95 of Cu2+
2.522 each of 2.57 of Mg2+ &
4xMgβ-TCP 5.042 4.917 4xCuβ-TCP 5.042 5.159 4xMgCuβ-TCP
Mg2+ & Cu2+ 2.63 of Cu2+

The comparative experimental concentration of Mg2+ and Cu2+ ions in mol.% used during the synthesis

and elemental analysis (determined from an X-ray fluorescence technique after heat treatment at 1000 oC)

are reported in Table 2. The elemental analysis determined from the X-ray fluorescence (Table 2) also

confirmed the presence of Mg2+ and Cu2+ in β-Ca3(PO4)2 structure. The comparative graphical

14
 ACCEPTED MANUSCRIPT
representations of the refined lattice parameters for the combined substitutions of Mg2+ and Cu2+ in β-

Ca3(PO4)2 are presented in Fig. 6. From Fig. 6, it is evident that both the Mg2+ only and Cu2+ only

substituted β-Ca3(PO4)2 showed decreasing trend in the structural parameters with the corresponding

increase in the substitution level of Mg2+ and Cu2+ in the crystal lattice of β-Ca3(PO4)2. In a similar

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manner, the combined substitutions of Mg2+ and Cu2+ also indicated a decreasing trend in the structural

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parameters of β-Ca3(PO4)2 with their corresponding increase in β-Ca3(PO4)2. The reason for this observed

R
decreasing trend in the behaviour of the structural parameters could be explained as follows.

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10.420 37.380

10.410 37.360
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c-axis
37.340
10.400
37.320
a = b-axis (Å)

10.390
D

a = b-axis 37.300

c-axis (Å)
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10.380 37.280

10.370 37.260
P

37.240
10.360
CE

37.220
10.350 37.200
AC

10.340 37.180
0 1 2 3 4 5 6
Conc. of Mg2+ & Cu2+ (Mole %)

Fig. 6 Influence of a = b-axis and c-axis lattice parameters with respect to the combined
substitutions of Mg2+ and Cu2+ in β-Ca3(PO4)2
*x-axis denotes the combined concentrations of Mg2+ and Cu2+ determined from XRF

The ionic radii of Mg2+, Cu2+ and Ca2+ are respectively designated as 0.072 nm, 0.073 nm and 0.099 nm,

in which ionic radii of both Mg2+ and Cu2+ were found considerably lower than the value of Ca2+. Thus

the substitution of lower sized Mg2+ and Cu2+ at the Ca2+ site of β-Ca3(PO4)2 is expected to cause

significant contraction in the structural parameters of β-Ca3(PO4)2. Both the Mg2+ only and Cu2+ only

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substituted β-Ca3(PO4)2 caused a considerable reduction in the structural parameters of β-Ca3(PO4)2 and

as expected the combined substitution of Mg2+ and Cu2+ also caused a similar reduction in the structural

parameters with their simultaneous enhancement in the dopant concentration. The results obtained from

the present study are accordance with the previous reports which states the fact that combined

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substitutions of any two divalent cations in the crystal lattice of β-Ca3(PO4)2 depends on their respective

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ionic sizes [16, 17]. Moreover, the elemental analysis determined from the XRF (Table 2) also confirms

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the equal distribution of Mg2+ and Cu2+ in the powders. Hence, the decreasing trend in the structural

SC
parameters due to the combined substitutions of Mg2+ and Cu2+ in β-Ca3(PO4)2 are justified from the

present results.

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Site Occupancy of Mg2+ and Cu2+ in β-Ca3(PO4)2
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3.3.

The structure of β-Ca3(PO4)2 is reported to possess five different Ca2+ sites in which Ca (4) site is

threefold-coordinated with oxygen atoms and has a partial occupancy factor of 0.5. On the contrary, each
D
TE

of the Ca (1), Ca (2), Ca (3), and Ca (5) sites are fully occupied by one Ca atom and these positions are

coordinated with seven, six (or) eight, eight, and six oxygen atoms, respectively. Yashima et. al., 36
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classified β-Ca3(PO4)2 into two columns in which column A possess less density that comprises of Ca (4),
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Ca (5) and P (1) atoms and column B is possess high density that comprises of Ca (1), Ca (2), Ca (3), P
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(2) and P (3) atoms. Hence the substitution of foreign atom shows a preferred occupancy in the less dense

A column as stated by the literature reports 16-18. The refined occupancy factors for the pure β-

Ca3(PO4)2 obtained from the present investigation have confirmed the defective nature of the Ca (4) site

with an occupancy value of 0.51. The preferential distribution of Mg2+ and Cu2+ ions at the five

crystallographically-independent Ca2+ sites of β-Ca3(PO4)2 were analysed by refining the occupancy

factors of all the Ca2+ positions.

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0.120 0.014

0.100 0.012

0.010

Cu2+ at Ca (5) site

Mg2+ at Ca (5) site

0.080

T
0.008

IP
0.060
0.006

R
0.040

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Mg2+ 0.004

0.020 Cu2+
0.002

0.000
0 0.5 1 NU
1.5 2 2.5 3
0.000
MA
Conc. of Mg2+ and Cu2+ (Mole %)

Fig. 7 Graphical representation of the combined Mg2+ and Cu2+ occupancy at their
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respective preferred Ca (5) sites of the β-Ca3(PO4)2 structure

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*x-axis denotes the combined concentrations of Mg2+ and Cu2+ determined from XRF
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In the preliminary step, all the five Ca2+ sites were refined by including either Mg2+ or Cu2+ at each of the
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Ca2+ sites. Such kind of inclusions of a foreign atom in the form of Mg2+ or Cu2+ resulted in yielding

negative occupancy values for the Ca (1), Ca (2), Ca (3) and Ca (4) site, while the Ca (5) site resulted in a
AC

positive occupancy value during refinement. Thus the positive value at the Ca (5) site confirmed the

accommodation of Mg2+ or Cu2+ at the sixfold coordinated Ca (5) site of β-Ca3(PO4)2. This is based on

the fact that the shortest mean distances (2.238 and 2.287˚ A) in the structure were determined for the Ca

(5-O bonds, so that the Ca (5) site then can easily accommodate Mg2+ or Cu2+ as shown in Fig. 7. In the

final step, all the five Ca2+ sites were refined by simultaneous inclusion of both Cu2+ and Mg2+ at the Ca

(5) site while other Ca2+ sites were kept fixed. A linear increase in the occupancy values of Mg2+ and Cu2+

at the Ca (5) site were observed with their corresponding increase in the concentration levels in β-

Ca3(PO4)2 (Fig. 7). Hence the formation of a solid solution of β-Ca3(PO4)2 with the combined substitution

of Mg2+ and Cu2+ were evident from the present investigation. The four different investigated

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compositions of the combined Mg2+ and Cu2+ substitutions in β-Ca3(PO4)2 determined from the

occupancy values are expressed as follows:

  Ca2.99737Mg 0.00231Cu 0.00032( PO4 ) 2 (4)

  Ca2.98857Mg 0.00994Cu 0.00149( PO4 ) 2 (5)
  Ca2.98234Mg 0.01540Cu 0.00226( PO4 ) 2 (6)
  Ca2.96466Mg 0.03200Cu 0.00334( PO4 ) 2

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(7)

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3.4. Raman spectra

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Figs. 8 and 9 illustrate the Raman spectra for pure β-Ca3(PO4)2 and four different Mg2+ and Cu2+ co-

substituted β-Ca3(PO4)2 powders after heat treatment at 1000 C. The vibrational modes of β-Ca3(PO4)2

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were observed at 969 cm-1 (strong peak) and 948 cm-1 (shoulder peak), which corresponds to the
MA
symmetric P-O stretching bonds of a PO4 polyhedron (v1). The bands observed in the range of 407, 440

and 482 cm-1 was attributed to the vibrational modes of doubly degenerate symmetric P-O bending (v2). A

minor peak at 1016 cm-1 and a broad peak observed at 1047 cm-1 were accounted for the asymmetric
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triply degenerate P-O stretching mode (v3). Other spectral peaks observed in the region at 548, 589, 612

and 630 cm-1 was accounted for the vibrational modes of triply degenerate asymmetric P-O bending (v4).
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These observed peaks in the Raman spectrum has shown good agreement with the literature data for β-
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Ca3(PO4)2 [41-43]. A similar kind of observation with respect to the vibrational modes of pure β-
AC

Ca3(PO4)2 were witnessed for all the four different Mg2+ and Cu2+ co-substituted β-Ca3(PO4)2 powders

and this inference confirms the fact that rhombohedral structure of β-Ca3(PO4)2 was retained even after

the substitution of Mg2+ and Cu2+ in the crystal lattice of β-Ca3(PO4)2. However, a close inspection of the

Raman spectra for all the four different Mg2+ and Cu2+ co-substituted β-Ca3(PO4)2 powders had given a

strong indication for the influence of substituted ions in the structure of β-Ca3(PO4)2.

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4X-MgCu β-TCP
Intensity (a.u)

3X-MgCu β-TCP

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IP
2X-MgCu β-TCP

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1X-MgCu β-TCP

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969
948

1089
1047
Pure β-TCP

612
407
440

548
589
482

200 300 400 500 600NU 700 800 900 1000 1100
MA
Raman Shift (cm-1)

Fig. 8 Raman spectra for pure β-Ca3(PO4)2 powder and four different combined
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substitutions of Mg2+ and Cu2+ in β-Ca3(PO4)2 .

P TE
CE
Intensity (a.u)

AC

4X-MgCu β-TCP

3X-MgCu β-TCP

2X-MgCu β-TCP

1X-MgCu β-TCP

Pure β-TCP

900 920 940 960 980 1000

Raman Shift (cm-1)

Fig. 9 A short range Raman spectrum for pure β-Ca3(PO4)2 and four different combined
Mg2+ and Cu2+ substitutions in β-Ca3(PO4)2.

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2+ 2+
All the four Mg and Cu co-substituted β-Ca3(PO4)2 compositions exhibited similarities with pure β-

Ca3(PO4)2 in case of v2 v3 and v4 bending and stretching vibrations of P-O bonds. In case of v1 vibration,

the strong peak witnessed in the region at 969 cm-1 also exhibited good similarities for both Mg2+ and

Cu2+ co-substituted β-Ca3(PO4)2 compositions and pure β-Ca3(PO4)2. However, a gradual shift towards

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the higher wavelength (Fig. 9) for the shoulder peak observed at 948 cm-1 was noticed with a

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simultaneous increase in the concentrations of Mg2+ and Cu2+ in β-Ca3(PO4)2 structure. The shifting

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towards either lower or higher wavelengths due to the influence of substituted ions in β-Ca3(PO4)2 are

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reported before. Shifting towards higher wavelengths is attributed to the substitution of lower sized ions

44 whereas shifting towards lower wavelengths is attributed to the substitution of higher sized ions 45.

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Here the combined substitution of lower sized ions in the form of Mg2+ and Cu2+ led to the Raman shift
MA
towards higher wavelength and thus the results coincides well with the existing literature reports.

The morphology of all the powder samples after heat treatment at 1000 oC is presented in Fig. 10. A
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similar kind of particle morphology were observed for all the pure β-Ca3(PO4)2 and Mg2+ and Cu2+ co-

substituted β-Ca3(PO4)2 samples. However, an enhancement in the particle agglomerations as a function

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of dopant concentration was observed. Thus the enhancement in the particle agglomerations is mainly
CE

attributed to the increased presence of Mg2+ and Cu2+ in β-Ca3(PO4)2 samples. SEM result indicates that
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the scanning technique measures the agglomerate size of calcium phosphate powder, instead of particle

size.

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R IP
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Fig. 10 SEM micrographs for pure β-Ca3(PO4)2 and four different combined Mg2+ and Cu2+

substitutions in β-Ca3(PO4)2.

(a) pure β-Ca3(PO4)2 (b) 1X-MgCu β-TCP, (c) 1X-MgCu β-TCP, (d) 1X-MgCu β-
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TCP, and (e) 1X-MgCu β-TCP

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3.5. Mechanical Evaluation

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The continuous load versus depth data obtained from the nanoindentation experiments performed on pure
AC

β-Ca3(PO4)2 sample by applying 200 mN load is presented in Fig. 11. A good reproducibility of the

performed nanoindentation experiments for both pure β-Ca3(PO4)2 and all the four Mg2+ and Cu2+ co-

substitutions in β-Ca3(PO4)2 were observed. Young's modulus was determined from the load-

displacement data according to the Sneddon relationship [46] as given below;

1

  
1  2

1  i
2
 (8)
E ff E Ei

where E and ν are the corresponding Young's modulus and Poisson's ratio of test sample whereas Ei and

νi are the respective Young's modulus and Poisson's ratio of indenter tip. Eeff is the effective Young's

modulus obtained from the nanoindentation test based on the Oliver-Pharr's method [47].

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200

150
Load (mN)

T
100

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50

R
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0
0 1 2 3
Depth (um)

Fig. 11
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Load-displacement curves obtained from the nanoindentation tests performed on
MA
pure β-Ca3(PO4)2
D

Hardness of material is defined as the resistance of material against local surface deformation caused by
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an external load and it was calculated by dividing the normal load by the projected area of the surface

where load was imposed on. Hardness was determined according to the Briscoe et. al., [48] relation given
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below;

FN
HN  (9)
AC

where FN is the maximum indentation load and A is the projected area of the contact surface between the

specimen and the indenter. The calculated mean values with standard deviations for the Young's modulus

and the mean values with standard deviation for hardness (HN) obtained for pure β-Ca3(PO4)2 and all the

four different Mg2+ and Cu2+ co-substituted β-Ca3(PO4)2 samples are listed in Table 3.

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Table 3 Young's modulus and hardness data for pure β-Ca3(PO4)2 and four different Mg2+ and Cu2+

co-substituted β-Ca3(PO4)2 samples obtained from Nanoindentation.

Sample Young's modulus (GPa) Hardness (GPa)

Pure β-Ca3(PO4)2 44.70 ± 1.30 1.14 ± 0.08

1xMgCuβ-TCP 42.80 ± 2.42 1.13 ± 0.42

T
2xMgCuβ-TCP 46.50 ± 2.67 1.12 ± 0.17

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3xMgCuβ-TCP 43.24 ± 3.59 1.17 ± 0.12

R
4xMgCuβ-TCP 36.53 ± 2.60 1.05 ± 0.15

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There was no significant trend noticed for both the Young’s modulus and hardness values with respect to

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the increasing concentration of Mg2+ and Cu2+ co-substitutions in β-Ca3(PO4)2. The 4xMgCuβ-TCP

sample resulted in the low values of both Young’s modulus and hardness on comparison with the other
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investigated samples. However, all the observed range of values for all the investigated samples are in

accordance with the results reported for pure β-Ca3(PO4)2 [49, 50]. Thus the substitution of Mg2+ and Cu2+
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in β-Ca3(PO4)2 did not affect the mechanical properties of β-Ca3(PO4)2.

P TE

3.6 Antimicrobial activity

CE

Generally, two types of mechanisms are proposed for the antibacterial actions of any material: (i) the

change in membrane permeability of bacteria arises due to the interaction between the antimicrobial metal
AC

ions eluded from the antimicrobial agent and bacterial outer membrane and (ii) interaction between the

bacteria and the free radical generated from the antimicrobial agents. In the first case, antimicrobial metal

ions eluded from the antimicrobial agent affect the bacterial outer membrane, causing the formation of

irregularly shaped pits in the outer membrane. This structural change induces changes in membrane

permeability, which causes a progressive release of lipopolysaccharide molecules and membrane proteins,

and finally, cell death [51, 52]. In the second case, two mechanisms are proposed for the antimicrobial

activity by the free radicals: (i) formation of hydroxyl radicals (OH) and (ii) superoxide anions (O2-)

generated by a photocatalytic reaction carried out on samples containing metal ions. These generated

radicals are expected to degrade intracellular and extracellular structures that interfere with the electron

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transport system thereby destroying deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and

finally, cell death [53]. During the performance of experiments in a quantitative manner, it was observed

that there is effective reduction in microorganism colonies of the investigated species namely S. aureus

and E. coli by using 0.1 g of the powders that are listed in Table 4.

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IP
Table 4 Antimicrobial efficiency of Mg2+ and Cu2+ co-substituted β-Ca3(PO4)2 against S.

R
aureus and E. coli microbes.

SC
Sample Microorganism Time (h) Mircroorganism
reduction R%(h)

NU
0 1 2 4 1 2 4
6
S. aureus 2.0 × 10
Pure β-TCP 6.7 × 105 7.9 × 105 8.9 × 105 66.5 60.5 55.5
MA
1xMgCuβ-TCP 9.2 × 104 1.1 × 105 1.3 × 105 95.4 94.5 93.5
4 4 4
2xMgCuβ-TCP 9.3 × 10 7.8 × 10 7.2 × 10 95.4 96.1 96.4
3xMgCuβ-TCP 6.7 × 104 5.4 × 104 6.9 × 104 96.6 97.3 96.6
D

4 4 4
4xMgCuβ-TCP 4.7 × 10 5.1 × 10 5.8 × 10 97.6 97.5 97.1
E. coli 2.3 × 106
TE

Pure β-TCP 8.5 × 105 9.1 × 105 1.4 × 106 63.0 60.4 39.1
1xMgCuβ-TCP 1.1 × 105 9.3 × 104 1.2 × 105 95.2 95.9 94.8
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4 4 5
2xMgCuβ-TCP 9.2 × 10 9.7 × 10 1.2 × 10 96.0 95.8 94.8
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3xMgCuβ-TCP 8.4 ×104 7.8 × 104 9.2 × 104 96.3 96.6 96.0
4 4 4
4xMgCuβ-TCP 7.2 × 10 4.2 × 10 5.1 × 10 96.9 98.2 97.8
AC

Table 4 indicates the antibacterial effect for both pure β-Ca3(PO4)2 and Mg2+ and Cu2+ doped β-Ca3(PO4)2

powders. An overall reduction in cell number for all the tested samples were observed which might be

due to some adhesion of microorganism cells to the β-Ca3(PO4)2 particles. However, a rapid reduction in

the efficiency of pure β-Ca3(PO4)2 against the investigated species was noticed as the time proceeds.

Moreover, the efficiency of Mg2+ and Cu2+ doped β-Ca3(PO4)2 powders against the investigated microbes

started to enhance with the simultaneous increase in the dopant concentrations in β-Ca3(PO4)2. Further, all

the Mg2+ and Cu2+ doped β-Ca3(PO4)2 powders indicated better efficiency on comparison with the pure β-

Ca3(PO4)2. This is due to the fact that Mg2+ and Cu2+ doped β-Ca3(PO4)2 powders facilitated the contact

of Cu2+ ions with cell wall and thus enabling their damaging effect to the bacterial cell [32, 33] . From the

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results obtained from antimicrobial activity tests, all the Mg2+ and Cu2+ doped β-Ca3(PO4)2 powders

confirmed their efficiency in countering the investigated S. aureus and E. coli microbes.

3.7 Haemolysis Test

T
The prerequisite for the success of any synthetic implant is its compatibility with the blood cells that

IP
surrounds the implant site and hence the material haemolysis is considered an important factor [54].

R
Interaction of bio-ceramics such as HAP and β-TCP occur with cellular system in vivo and some of these

SC
interactions may lead to damage cells and stimulate platelet activation, coagulation and thrombus

formation [55, 56]. The data obtained from the haemolysis tests are presented in Table 5. A haemolysis

NU
rate lower than 5% observed for the pure β-Ca3(PO4)2 and four different Mg2+ and Cu2+ doped β-
MA
Ca3(PO4)2 powders justify their non-haemolytic activity and give a strong support for medical application.

No significant trend in the haemolysis rate with respect to the dopant concentration was observed;

however, 4xMgCuβ-TCP sample had indicated greater hemolysis than any other samples. Further
D
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research at relevant human cells and in vivo studies in animals are necessitated to evaluate the safety

usage of ionic doped β-Ca3(PO4)2 for medical applications.

P
CE

Table 5 Haemolysis rate by pure β-Ca3(PO4)2 and all Mg2+ and Cu2+ co-substituted β-
AC

Ca3(PO4)2 samples

Sample Optical density at 540 nm Hemolysis rate (%)

Distilled water 1.036 Positive control

Saline 0.014 Negative control

Pure β-TCP 0.045 3.00

1xMgCuβ-TCP 0.044 2.93

2xMgCuβ-TCP 0.051 3.62

3xMgCuβ-TCP 0.049 3.42

4xMgCuβ-TCP 0.053 3.82

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4. Conclusions

In this study, we have shown that Mg2+ and Cu2+ can be co-substituted at the crystal lattice of β-Ca3(PO4)2

by a simple aqueous precipitation technique. It was observed that co-substitution of lower sized Mg2+ and

Cu2+ ions for the higher sized Ca2+ led to the significant contraction in the unit cell parameters of β-

T
Ca3(PO4)2 structure and the both the substituted ions were found occupied at the Ca (5) site of β-

IP
Ca3(PO4)2. There was no significant influence noticed on the hardness and Young’s modulus of β-

R
Ca3(PO4)2 due to the substitution of Mg2+ and Cu2+ ions. Mg2+ and Cu2+ co-substituted β-Ca3(PO4)2

SC
demonstrated good activity against the investigated microbes and the activity was found dependent as a

function of the dopant concentration. A haemolysis rate less than 5% was determined for all the

NU
investigated samples. Thus the high antimicrobial activity and hemocompatibility of Mg2+ and Cu2+ co-

substituted β-Ca3(PO4)2 can be considered as a promising candidate material for implant applications,
MA
reconstructive surgery and bone tissue engineering.
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References

[1] J. Dumbleton, M. T. Manley, J Bone Joint Surg. 86 (2004) 2526-40

P

[2] R. Tang, W. Wu, M. Haas, G.H. Nancollas, Langmuir 17 (2001) 3480.

CE

[3] W. Wu, R. Tang, M. Haas, G.H. Nancollas, J. Colloid Interface Sci. 244 (2001) 347.
AC

[4] H. McDowell, W.E. Brown, J.R. Sutter, Inorg. Chem. 10 (1971) 1638

[5] L. L. Hench, J. Am. Ceram. Soc. 74 (1991) 1487-510.

[6] D. F. Williams, In: D. F. Williams, editor. Biocompatibility of tissue analogs, Vol II. Boca

Raton, FL: CRC Press; 1985

[7] H. Yuan, J.D. de Bruijn, Y. Li, J. Feng, Z. Yang, K. de Groot, X. Zhang, J. Mater. Sci. Mater.

Med. 12 (2001) 7-12.

[8] R. D. A. Gaasbeek, H. G. Toonen, R.J. van Heerwaarden, P. Buma, Biomaterials 26 (2005)

6713-6720.

[9] R. Z. LeGeros, Clin. Orthop. Relat. Res. 395 (2002) 81-87.

26
 ACCEPTED MANUSCRIPT
[10] S. Kotani, Y. Fujita, T. Kitsugi, T. Nakamura, T. Yamamuro, C. Ohtssuki, J. Biomed. Mater.

Res., 25 (1991) 1303-1315.

[11] J. Wiltfang, H. A. Merten, K. A. Schlegel, S. S. Mosgau, F. R. Kloss, S. Rupprecht, P.

Kessler, J. Biomed. Mater. Res. 63B (2002) 115-122.

T
[12] K. Kawabata, T. Yamamoto, A. Kitada, Phys. B 406 (2011) 890-894.

IP
[13] F. J. G. Mayer, S. Cuisinier, S. Gdalya, I. Popov, J. Inorg. Biochem. 102 (2008) 311-317.

R
[14] S. Kannan, J. M. Ventura, J. M. F. Ferrreira, Ceram. Inter. 33 (2007) 637-641.

SC
[15] S. Kannan, S. Pina, J. M. F. Ferreira. J. Am. Ceram. Soc. 89 (2006) 3277-3280.

[16] S. Kannan, F. Goetz-Neunhoeffer, J. Neubauer, S. Pina, P.M.C. Torres, J.M.F. Ferreira, Acta

Biomater. 6 (2010) 571-576.

NU
MA
[17] P. Nandha Kumar, M. Boovarasan, Ram Kishore Singh, S. Kannan, RSC Adv. 3 (2013)

22469-22474.

[18] S. Quillard, M. Paris, P. Deniard, R. Gildenhaar, G. Berger, L. Obadia, J.-M. Bouler, Acta
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Biomaterials 7 (2011) 1844-1852.

[19] N. Matsumoto, K. Sato, K. Yoshida, K. Hashimoto, Y. Toda. Acta Biomater. 5 (2009) 3157-
P

3162.
CE

[20] A. Ewald, C. Kappel, E. Vorndran, C. Moseke, M. Gelinsky, U. Gbureck. J. Biomed. Mater.

AC

Res. Part A 100 A (2012) 2392-2400.

[21] I. Cacciotti, A. Bianco, M. Loombardi, L. Montanaro, J. Eur. Ceram. Soc. 29 (2009) 2969-

2978.

[22] W. L. Suchanek, K. Byrappa, P. Shuk, R. E. Riman, Biomaterials 90 (2004) 4647-4657.

[23] R. K. Rude. J. Bone. Miner. Res. 13 (1998) 749-58.

[24] A. Bigi, E. Foresti, R. Gregoriani, A. Ripamonti, N. Roveri, J. S. Shah. Calcif. Tissue. Int. 50

(1992) 439-444.

[25] J. Althoff, P. Quint, E. R. Krefting, H. J. Hohling. Histochemistry 74 (1982) 541-542.

[26] L. A. Martini. Nutr. Rev. 57 (1999) 227-229.

27
 ACCEPTED MANUSCRIPT
[27] S. Kannan, A. F. Lemos, J. H. G. Rocha, J. M. F. Ferreira. J. Am. Ceram. Soc. 89 (2005)

2757-2761.

[28] D. Campoccia, L. Montanaro, C. R. Arciola, Biomaterials 27 (2006) 2331-2339.

[29] P. Hardy, R. Kania, S. Verliac, A. Lortat-Jacob, J. Benoit, Eur. J. Orthop. Surg. Traumatol. 7

T
(1997) 63-67.

IP
[30] W. Chen, Y. Liu, H. S. Courtney, M. Bettenga, C. M. Agrawal, J. D. Bumgardner, J. L. Ong,

R
Biomaterials 27 (2006) 5512-5517.

SC
[31] F. Heidenau, W. Mittelmeier, R. Detsch, M. Haenle, F. Stenzel, G. Ziegler, H. Gollwitzer, J.

Mater. Sci. Mater. Med. 16 (2005) 883-888.

[32]
NU
E. A. Neel, I. Ahmed, J. Pratten, S. N. Nazhat, J. C. Knowles, Biomaterials 26 (2005) 2247-
MA
2254.

[33] N. Matsumoto, K. Sato, K. Yoshida, K. Hashimoto, Y. Toda, Acta Biomater. 5 (2009) 3157-

3164.
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TE

[34] A. Ewald, C. Kappel, E. Vorndran, C. Moseke, M. Gelinsky, U. Gbureck, J. Biomed. Mater.

Res., Part A, 100 (2012) 2392-2400.

P

[35] C. Gerard, L. J. Bordeleau, J. Barralet, C. J. Doillon, Biomaterials 31 (2010) 824-831.

CE

[36] M. Yashima, A. Sakai, T. Kamiyama, A. Hoshikawa, J. Solid State Chem. 175 (2003) 272-
AC

277.

[37] V. Stanic, S. Dimitrijevi, J. Anti-Stankovi, M. Mitri, B. Joki, S. Rai cevi, Appl. Surf. Sci. 256

(2010) 6083-6089.

[38] K. P. Tank, K. S. Chudasama, V. S. Thaker, M. J. Joshi, J. Nanopart. Res. 15 (2013) 1644.

[39] L. Yinghui, C. Xiguang, W. Qizhao, W. Ye, Z. Jing, L. Chengsheng, L. Chenguang, M.

Xianghong, Y. Lejun, J. Wuhan Univ. Technol. 22 (2007) 695-700.

[40] A. Mortier, J. Lemaitre, P. G. Rouxhet, Thermochim. Acta 143 (1989) 265-282.

[41] P. N. de Aza, C. Santos, A. Pazo, S. de Aza, R. Cusco, L. Artus, Chem. Mater. 9 (1997) 912-

915.

[42] A. Jillavenkatesa, R. A. Condrate, Spectrosc. Lett. 31 (1998) 1619-1634.

28
 ACCEPTED MANUSCRIPT
[43] R. Cusco, F. Guitian, S. de Aza, L. Artus, J. Eur. Ceram. Soc. 18 (1998) 1301-1305.

[44] B. Choudhury, M. Dey, A. Choudhury, Int. Nano Lett. 3 (2013) 25-28.

[45] C. H. Yang, Z. Q. Ma, Appl. Opt. 51 (2012) 5438-5441.

[46] L.N. Sneddon, Int .J. Eng. Sci. 3 (1965) 47-51.

T
[47] W.C. Oliver, G.M. Pharr, J. Mater. Res. 19 (2004) 3-20.

IP
[48] B. J. Briscoe, P. D. Evans, S. K. Biswas, S. K. Sinha, Tribol. Int. 29 (1996) 93-104.

R
[49] C.X. Wanga, X. Zhoub, M. Wang, Mater. Character. 52 (2004) 301-307.

SC
[50] D. S. Metsger, M. R. Rieger, D. W. Foreman. J. Mater. Sci. Mater. Med. 10 (1999) 9-17.

[51] Y. Y. Ki, H. B. Jeong, H. P. Jae, H. Jungho, Sci. Total. Environ. 37 (2007) 3572-3575.

[52]
NU
N. A. Amro, L. P. Kotra, K. Wadu-Mesthrige, A. Bulychev, S. Mobashery, G. Liu, Langmuir
MA
16 (2000) 2789-2796.

[53] K. Sunada, T. Watanabe, K. Hashimoto, J. Photochem. Photobiol. A (2003) 227-233.

[54] B. Xiao, M. Karren, M. R. Christopher, A. Rabiei, Acta Biomater. 6 (2010) 2264-2273.

D
TE

[55] Z. W. Huang, T. T. Ding, J. Sun. Adv. Mater. Res. 378 (2012) 711-714.

[56] V. Stanic, D. Janackovi, S. Dimitrijevic, S. B. Tanaskovic, M. Mitric, M. S. Pavlovic, A.

P

Krstic, D. Jovanovic, S. Raicevic, Appl. Surf. Sci. 257 (2011) 4510-4518.

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Highlights

 Mg2+ and Cu2+ co-substitutions in β-Ca3(PO4)2 were obtained through aqueous precipitation

technique.

 Co-substitutions of Mg2+ and Cu2+ led to the significant reduction in the lattice parameters of β-

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Ca3(PO4)2.

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 Mechanical properties of β-Ca3(PO4)2 were found stable even after the substitutions of Mg2+ and

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Cu2+ at its crystal lattice.

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 In vitro tests ensured better antibacterial behavior and good hemolytic activity of Mg2+ and Cu2+

co-substituted β-Ca3(PO4)2.

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