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Fluoride doped bioceramic from egg shell: a promising biomaterial for bone tissue engineering.

Fluoride doped Ca-phosphate based bioceramic material has been successfully synthesized from egg shell for the first time by wet chemical method. The developed bioceramic material was characterized by XRD and EDS techniques. Bioactivity, osteoclastic response of the bioceramic revealed that it would be a promising material for bone tissue engineering.

Recently, synthetic bioceramic materials which are biocompatible, bioactive and osteoconductive are extensively used in orthopaedics and dentistry fields [1,2]. Among the bioceramic materials, Ca-phosphate (CaP) based bioceramics have been widely used in clinical applications. Particularly, owing to excellent bioactivity, biocompatibility and osteoconductivity, Ca-hydroxyapatite [HA, [Ca.sub.10][(P[O.sub.4]).sub.6][(OH).sub.2]] gets enormous attraction as an implant material [3]. In addition to this HA, two other forms of CaP, e.g. [alpha]-tricalcium phosphate ([alpha]-TCP) and [beta]-tricalcium phosphate ([beta]-TCP) are also used as biomaterials [3,4]. However, wider applications of synthetic HA are restricted to some extent due to its poor thermal stability, undesirable fast dissolution rates in-vivo and poor mechanical properties [5]. On the other hand, considerable dissolution of [beta]-TCP promotes the formation of continuous interfaces between CaP ceramics and bone. Hence, this biomaterial is also a preferable option to the researchers [6].

Flexible structure of HA admits several substitutions. Such modification facilitates better mechanical and physiological stabilities of HA [7]. Recently fluoride doped hydroxyapatite (FHA) has attracted much attention due to its extensive performance relating to the stability and for its preventive role in dental carries [4]. Moreover it has superior mechanical properties when sintered at high temperatures [8] and exhibits fairly significant stability coupled with biocompatibility [9]. Considering such attractive combination we synthesize FHA from egg shell (% of CaC[O.sub.3] ~95.0), for the first time.

A generalized wet chemical precipitation method [2] was followed to synthesize the FHA. Aqueous doping solution (NaF) of certain concentration (0.025 M) was first added to the egg shell solution maintaining the pH of the solution at ~10.0 with aqueous ammonia. The ratio of egg shell solution (i.e. source of Ca) and [(N[H.sub.4]).sub.2]HP[O.sub.4] (i.e. source of P) was retained at 1.66. The addition of phosphate precursor solution in ammonia (pH ~10.0) facilitated the formation of FHA according to the reaction:


where 0 [less than or equal to] x [less than or equal to] 1. A variation in concentration of NaF (0.05 M, 0.10 M and 0.20 M) was used to explore the concentration effect of doping solution.

To characterize the synthesized FHA samples, phase analysis by x-ray diffraction (XRD) technique was performed as the first step. Since the sintering temperature plays an imperative role in the formation of crystalline phase, the synthesized FHA samples were first oven dried at 110[degrees]C, then sintered at 600[degrees]C and 900[degrees]C to investigate the temperature effect. The XRD spectra of oven dried and calcined (at 600[degrees]C) samples had low intensity with broad peaks but clearly visualized the characteristic peaks of HA for (2 1 1), (1 1 2) and (3 0 0) planes (JCPDS file: 89-6439) [2]. This information conform the formation of HA in single phase but in amorphous and / or poor crystaline form. Figure 1 shows the typical XRD spectra (temp. range: 110[degrees]C, 600[degrees]C and 900[degrees]C) of FHA sample synthesized by using 0.025 M doping solution.

On the other hand, the XRD spectra of all the FHA samples calcined at 900[degrees]C, showed the presence of well crystallined hexagonal HA together with [beta]-TCP having rhombohedral structure (JCPDS file: 09-0169) [10]. The diffraction peaks particularly for (3 0 0) (0 2 10) and (2 2 0) planes reflected the presence of [beta]-TCP along with the FHA. The volume fraction ([X.sub.[beta]]) of [beta]-TCP was estimated using the following relationships [11] and summarized in Table 1.

[X.sub.[beta]] = [PW.sub.[beta]]/[1 + (P - 1)[W.sub.[beta]]] (2)

where [W.sub.[beta]] = [I.sub.[beta]]/([I.sub.[beta]] + [I.sub.HA]) (3)

[I.sub.[beta]] and [I.sub.HA] are the XRD intensity values of [beta]-TCP at [0 2 10] and HA at [2 1 1] reflections respectively. The coefficient P is the integral intensity ratio of HA at [2 1 1] to [beta]-TCP at [0 2 10]. The value of P was estimated as 2.275 using the XRD of the mixtures of standard HA and [beta]-TCP [11].

The most noteworthy detection of XRD spectra of FHA samples was the shift of 2[theta] positions for (3 0 0), (4 1 0) planes to higher diffraction angles and for (0 0 2), (0 0 4) planes to lower diffraction angles as compared with the JCPDS values for pure HA [2,12]. Such shifts indicated the substitution of OH- (ionic radius 1.68) by F- ion (ionic radius 1.36') in the HA structure [13] and is characteristic of fluoride doped HA formation. This substitution resulted the expected contraction of [alpha]-axis without distorting the c-axis in FHA [12]. The calculated lattice parameters and cell volumes (V) for FHA and [beta]-TCP as tabulated in Table 2, matched very well with the JCPDS values [4,12,13].


The extent of fluoride doping (x) into the calcined (at 900[degrees]C) FHA was calculated using the mass % data of Ca and F as obtained by EDS (Energy dispersive spectroscopy). The calculated value of x (Table 3) revealed that the doping of F- ion increased as a function of initial concentration of NaF solution and a maximum of 99% doping was achieved.

The in vitro dissolution behaviour of FHA (calcined at 900[degrees]C) was investigated by facilitating an osteoclastic resorption condition maintaining a pH of ~4.5 [14]. The compacted FHA samples (20 mm diameter) were weighed and submerged into aqueous environment at pH 4.5 (pH was controlled with HN[O.sub.3]). The incubation temperature was kept steady at 37[degrees]C. The dissolution % of FHA as monitored after 2 and 5 days is shown in Table 4. It is clearly evident from the in vitro osteoclastic response, that the chemical stability of FHA samples are fairly significant even after the immersion in acid solution for 5 days. The % of dissolution varied within the range 0.05-0.19.

Particularly the acid resistance efficiency of FHA prepared by using 0.10 M and 0.20 M NaF (where % of F-incorporation in FHA was 48.9% and 99.0% respectively, Table 3) led to the conclusion that these FHA would be suitable for using in dentistry field, such as in tooth enamel which comprised 50% fluoridation (i.e. 50% of OH-groups are being relaced by F- ions in tooth enamel) [14]. On the other hand, the natural bone contains [less than or equal to] 1 wt% of fluorine [14]. Thus due to slightly higher solubility, FHA prepared by using 0.025 M and 0.05 M doping solution, may be used in bone applications where a little dissolution is a requisite to promote general remodelling of skeleton system [14].

Bioactivity is an important criterion of any biomaterials to be deveoped for bone tissue engineering. Thus the bioactivity of the developed FHA was investigated in synthetic body fluid (SBF), as described previously [15]. To monitor the bioactivity, again compact disks (20 mm diameter) of FHA were prepared and submerged into SBF at 37[degrees]C. The % of weight gained by FHA disks (after 3 and 10 days) was within 1.5-2.6 which is obviously an indication of its bioactive behaviour. However, further research on biocompatibility, osteoconductivity is going on in our group.

Received 9 July 2013; Accepted 19 August 2013; Available online 10 October 2013


We gratefully acknowledge the financial support of BCSIR.


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H. Kabir, F. Nigar, S. Ahmed *, S.F. Kabir (a), A.I. Mustafa *, M. Ahsan

Bangladesh Council of Scientific and Industrial Research, Dr. Qudrat-i-Khuda Road Dhanmondi, Dhaka 1205, Bangladesh

(a) Department of Applied Chemistry and Chemical Engineering, University of Dhaka, Dhaka, Bangladesh
Table 1: Observed % of volume fraction ([X.sub.[beta]]) of
[beta]-TCP in the FHA samples calcined at 900[degree]C

   FHA            FHA            FHA            FHA
(0.025 M)      (0.050 M)      (0.100 M)      (0.200 M)

  71.00          86.20          88.00          90.41

Table 2: Calculated lattice parameters for the synthesized FHA samples

                                              FHA            FHA
                                           (0.025 M)      (0.050 M)

                                           a = 9.34 ?     a = 9.37 ?
Lattice parameters for FHA                 c = 6.87 ?     c = 6.88 ?
                                           V = 1556 ?     V = 1564 ?
                                           a = 10.41 ?    a = 10.42 ?
Lattice parameters for [beta]-TCP          c = 37.37 ?    c = 37.40 ?
                                           V = 3507 ?     V = 3517 ?

                                              FHA            FHA
                                           (0.100 M)      (0.200 M)

                                           a = 9.38 ?     a = 9.34 ?
Lattice parameters for FHA                 c = 6.89 ?     c = 6.87 ?
                                           V = 1569 ?     V = 1556 ?
                                           a = 10.41 ?    a = 10.40 ?
Lattice parameters for [beta]-TCP          c = 37.38 ?    c = 37.35 ?
                                           V= 3508 ?      V = 3498 ?

Table 3: Extent of fluoride doping in FHA

FHA             FHA            FHA            FHA
(0.025 M)       (0.050 M)      (0.100 M)      (0.200 M)

0.324           0.349          0.489          0.99

Table 4: Dissolution % of FHA [[Ca.sub.10][(P[O.sub.4])sub.6]

FHA                       FHA
(0.025 M)                 (0.050 M)

2 days        5 days      2 days        5 days
0.15          0.17        0.16          0.19

FHA                       FHA
(0.100 M)                 (0.200 M)

2 days        5 days      2 days        5 days
0.05          0.10        0.06          0.07
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Title Annotation:Short Communication
Author:Kabir, H.; Nigar, F.; Ahmed, S.; Kabir, S.F.; Mustafa, A.I.; Ahsan, M.
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Oct 1, 2013
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