Structural characterization of nanocrystalline hydroxyapatite produced from waste egg shell.
Calcium phosphate based natural and synthetic ceramic materials, particularly, hydroxyapatite (HAP) and tricalcium phosphate (TCP) are extensively used for medical purposes that includes bone filling materials, biocoating of prosthesis, fabrication of implants, coating on prostheses (1). However, among these bioceramics, HAP, [Ca.sub.10][(P[O.sub.4]).sub.6][(OH).sub.4], is an important inorganic material in both biology and chemistry  and due to its excellent bioactive, biocompatible nature and bone bonding ability with surrounding tissues, it is widely used in tissue engineering and bone replacement (3,4,5). It is well known that human bone is a hybrid composition of inorganic (~70% apatitic calcium phosphate with a Ca/P ratio 1.66) and organic (~30% collagen) materials. The submicroscopic crystal of calcium phosphate in bone resembles the crystal structure of synthetic hydroxyapatite, [HA, [Ca.sub.10][(P[O.sub.4]).sub.6][(OH).sub.2]]. A number of synthesis techniques using various sources of Ca and P have been developed which includes wet chemical method (precipitation), sol-gel method, hydrothermal synthesis procedure (6), continuous precipitation, thermal deposition and solid state reaction method (7,8,9). However, among these methods, wet chemical precipitation route is popular because of its simple, cheap and easy application in industrial production (10,11,12).
The eggshell is a waste material after its use as food or hatching. India currently ranks fourth in world in egg production with an annual production of 17,32,500 tons of egg. By taking 11% of the weight, nearly it comes around 1,90,000 tons of eggshell waste (13). Consumption of eggs in Kolkata is also estimated around 5 million per day. So we can see that such a vast amount of egg is produced and in turn consumed by the people. So this large amount of eggshells is wasted after using the egg. The eggshells constitute the 11% of the total weight of the egg and are composed by calcium carbonate (94%), calcium phosphate (1%), organic matter (4%) and magnesium carbonate (1%) and egg pigment (14). There are also nearly as many as 8000 microscopic pores in the shell itself.
Therefore, aim of this study concerns to synthesize pure and biocompatible hydroxyapatite (HA) powder with Ca/P ratio 1.67 through wet chemical method by using hen's eggshell as the Ca-source (15,16,17). In the present work, we report the results of structural character of hydroxyapatite obtained from egg-shell using XRD, Fourier transform infrared (FT-IR), Scanning electrom microscopy (SEM) and Transmission electron microscopy (TEM). The HAP sample is also characterized by TG-DTA analysis.
Experimental and Analytical Procedure
Synthesis of HAP from Egg-Shell
At first the raw egg shells were cleaned thoroughly with water followed by boiling for V hr. It was then dried overnight in an oven and then crushed and powdered using an agate mortar. This egg shell powder was calcined at 950[degrees]C to burn out the organic and other substances. The eggshells transformed into calcium oxide evolving carbon dioxide and then converted to calcium nitrate [Ca[(N[O.sub.3]).sub.2]] by treating with requisite amount of concentrated nitric acid followed by dilution with distilled water. A requisite amount of 0.10 M [Na.sub.2]HP[O.sub.4].12[H.sub.2]O in ammonia solution (pH~10.0) was added drop wise to the egg shell solution with continuous stirring condition. White gelatinous precipitate of HA was formed which was filtered through a Buchner funnel and thoroughly washed with plenty of distilled water. At this stage, the filtered precipitate was dried in oven to remove any trace of water and then crushed to achieve fine powder (18).
Chemical and crystallographic analysis
Chemical and crystallographic characterizations were done to establish the micro architecture, phase purity, crystallinity, composition and the functional groups of hydroxyapatite (19).
The morphology and particle size analysis of the synthesized powder was studied using scanning electron microscope (SEM JEOL Japan Model-JSM 5200, installed in USIC of JU). The exposed surfaces of the specimen were made conductive by coating with nano-size (5-20nm) gold film with an accelerating voltage of 15 kV by sputtering and examined under the SEM after mounting in suitable metallic stub.
Crystalline properties of synthetic nanostructured hydroxyapatite (n-HA) was studied using high-resolution transmission electron microscopy. Nanostructured HA samples for TEM analyses were prepared by dropping suspended n-HA powder in ethanol on copper grids with a thin amorphous carbon film approximately between 3 and 5 nm.
Among many properties, crystallographic changes directly affect the bioactivity of HAP; thus, it is necessary to study its phase purity and crystallography before intending for in vitro and in vivo trials (20). The non destructive XRD technique was employed to assess the phase purity and crystallographic changes (21). Sintered HAP granules were powdered in a fine scale using ball mill and the X-ray diffraction was performed in Bruker D8 Advance powder diffractometer, Germany. Fixed V degree divergence slits (0.6mm) and scintillation counters as detector were used in these experiments. Diffraction measurements were performed using Cu K radiation with (primary beam germanium monochromator). Measurements were taken from 100-400 20 with 10 second count time. Continuous scans were acquired at 500C temperature intervals. X-ray diffraction analyzer was operated at 40KV and 40 mA using copper K^ target as X-ray source. Data obtained from these tests were used to determine whether a specific sample matched standard card files for HA and what other calcium phosphate phases were present; whether the lattice parameters of that sample matched with standard values, and the crystalline perfection of the coating. Lattice parameters were determined from peak positions (28 values) obtained during the scan. Finally, in order to determine the crystalline perfection of the coating, the full band-width at half-height of the peaks was determined and compared to that of a 100% hydroxyapatite sample (22).
FT-IR spectral analysis
The vibrational spectrum has long been used as a technique for evaluating calcium phosphates, particularly hydroxyapatite. IR spectroscopy complements characterization by x-ray diffraction because it identifies the chemical composition of crystalline as well as non-crystalline phosphates. While x-ray diffraction identifies a calcium phosphate as apatite, infrared spectroscopies will identity some elements of the composition, group substitution and presence of carbonate and hydroxyl groups. The formation of the HAP phase was tested by FT-IR spectral analysis. FT-IR transmittance spectra of the crushed samples were obtained on infrared spectrometer (IRPRESTIGE-21 SHIMADZU model installed in Dept. of Metallurgy JU). The functional groups present in HA were ascertained by Fourier transform infrared spectroscopy (FT-IR). The FT-IR spectra were obtained over the region 400-4,000 cm-1 using KBr pellet technique with spectral resolution of 4 cm-1 (23). The chemistry of the hydroxyapatite powders obtained from egg-shell was determined by peak locations and comparison was made with the standard data (24). The amorphous content was determined by taking the 2nd derivative of the 900-1200 cm' 1 phosphate range in order to determine exact peak locations, which was followed by de-convolution in order to determine estimates of peak height and width. These numbers were then used to curve fit the spectra. The half-widths of the residual peaks used to curve fit the spectra were then compared to those of the starting powders.
TGA-DTA thermal behavior
The weight loss and thermal stability of the samples were also evaluated from the thermogravimetric analysis graphics. The thermogravimetric (TG) analysis and differential thermal analysis (DTA) of hydroxyapatite was studied using a Perkin-Elmer TG analyzer (Pyris Diamond TG/DTA Model installed in Dept. of Metallurgy) and measurements were recorded from 0[degrees] to 800[degrees]C at 10[degrees]C/min heating rate under nitrogen flow of 150 mL/min.
Results and discussion
Analysis of the SEM micrographs presented in figure 1 shows that the egg-shell synthesized HAP consisted of agglomerates. From the SEM pictures (Fig 1), it is clearly observed that the precipitated crystallites of was spherulite morphology with narrow size distribution of about few micron in diameter, the spherulite is composed of tiny nanosize platelets of loosely aggregated stabilized structure. The shapes of which were almost the same and the size was always between 2qm and 5qm, built up from fine particles about 40-100 nm in size. Individual fine particles with spherical and semi-spherical shapes were observed as seen in figure (1). There are many spherical agglomerations and crystallites of nano-sized with a tendency to agglomerates leaving pores in between.
To characterize the morphology of the powder, TEM analysis was done on ultrasonically dispersed HA in ethanol. The bright field TEM image of HA powder is shown in figure 2. The morphology clearly indicates that the particles are platelets, mostly agglomerated and of dimensions: length 33-50 nm and width 814 nm. This result thus clearly confirms the nanosize nature of HA.
X-ray diffraction analysis
The XRD technique was employed to assess the phase purity and crystallographic changes. Among many properties, crystallographic changes directly affect the bioactivity of HAP; thus, it is necessary to study its phase purity and crystallography prior to in vitro and in vivo trials. The observed diffractogram of the hydroxyapatite powder synthesized from egg-shell by wet chemical method has been shown in figure-3. The data were analyzed in the 20 range from 10[degrees] to 80[degrees] with a scanning step of 2[degrees] per min. The XRD spectrum of HA defined the crystallinity of the material with major peaks assigned for hydroxyapatite (Fig-3). The XRD phase analysis has been performed using Reference Card No. 09-0432 Quality: I. The peaks of XRD found matched with pure HA. This confirms that the sample produced was pure HA and also minor quantity of tri-calcium phosphate with no other chemical impurities. Mean crystalline size of hydroxyapatite powder was calculated from XRD graph and presented in Table 1.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
The size of crystallites varies from approx. 10-16 nm. It is found to be in the nanocrystalline range.
FT-IR spectral analysis
In the FT-IR analysis, mainly the peaks from PO43'' and OH" groups in the hydroxyapatite can be identified (Fig-4) The IR band at 3564 [cm.sup.1] belongs to the vibration of hydroxyl, the bands observed at 1031 and 962 [cm.sup.1] are characteristic of the phosphate stretching vibration, and the bands observed at 601, 565, and 476 [cm.sup.1] are due to the phosphate bending vibration. The bands appearing at 873 [cm.sub.1] reveal the presence of carbonate ions in the resultant HA crystallites due to the interaction between HA and ambient C[O.sub.2] in the processing. FT-IR band positions and their corresponding assignments were shown in Table 2.
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
The thermogram and its differential thermogravimetric plots were shown in figure 5. There is an amount of 5% weight loss around 135[degrees]C. It is due to weakly entrapped moisture in the material of which major part of weight loss of 50% occurred between 200[degrees]C and 420[degrees]C. It is assigned to the dehydration of calcium hydroxide. However, there was no further weight loss on heating up to 800[degrees]C, which indicates the high thermal stability of the sample.
[FIGURE 5 OMITTED]
In this present work, an attempt has been made to synthesize Stoichiometric, pure and thermally stable hydroxyapatite (HAP) powder by using hen's eggshell as the Ca-source. After preparation the HA powder was characterized by XRD, FT-IR, TEM and SEM analyses. FT-IR and XRD analyses indicated the phase purity and crystallinity of the HAP powder. The XRD data clearly demonstrate the structural analogy between the hydroxyapatite obtained from natural teeth and waste egg-shell. TG-DTA results showed the thermal stability of HAP powder obtained from hen's egg-shell. The present study suggests the eggshell as a possible material- recycling technology for future waste management and ecology. Also, eggshell-originated HAP is a potential ceramic, which could be useful as an inexpensive bioceramic for biomedical applications.
This work is funded by UGC, Govt. of India to the 1st author. The authors are thankful to JIS College of Engineering and the Department of Material Science & Metallurgy and USIC of Jadavpur University Kolkata for their immense help.
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S. Bag , K. Ganguly , B.K. Biswas 
 JIS College of Engineering, Kalyani, India
 Avinash Dental Laboratory & Research Institute Pvt. Ltd, Kolkata, India
* Coresponding author: Dr. Sandip Bag; E-mail: firstname.lastname@example.org
Received 26 January 2016; Accepted 7 June 2016; Published online 10 June 2016
Table 1: The crystallite sizes were calculated using Scherrer's relationship, d = K[lambda]/[beta] cos[theta] (where A be the wavelength of [lambda] rays = 1.54056 A[degrees]); value for K is 0.94 for FWHM of spherical crystals with cubic symmetry was assumed Peak No 2[theta] "[theta]" "[beta]" Full angle the Bragg's Width at Diffraction half max angle 5 21.73 10.85 0.071 6 21.84 10.92 0.106 7 22.84 11.42 0.106 13 31.78 15.89 0.106 14 32.23 16.11 0.176 15 32.92 16.46 0.176 26 48.13 24.06 0.176 27 49.3 24.65 0.071 28 49.5 24.75 0.176 Peak No Crystallite Mean crystallite size 'd' size 'd' (in nm) (in nm) 5 20.59473 16.07629 6 13.79458 7 13.83955 13 14.06451 10.3623 14 8.511202 15 8.511202 26 8.956561 13.37177 27 22.20218 28 8.956561 Table 2: FT-IR band positions and their corresponding assignments Observed band positions Corresponding assignments ([cm.sup.-1]) HA sample 476.41 P[O.sub.4] bending 565.14 P[O.sub.4] bending 601.79 P[O.sub.4] bending 632.65 OH liberation (Structural OH-) 873.75 C[O.sub.3] group 962.47 P[O.sub.4] stretching 1031.91 P[O.sub.4] bending 1419.5 C[O.sub.3] group 1635.63 [H.sub.2]0 adsorbed 3564.45 Structural OH-
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|Title Annotation:||Original Article|
|Author:||Bag, S.; Ganguly, K.; Biswas, B.K.|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Jan 1, 2016|
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