NEXAFS study of carbonate substituted bioapatite

The investigation of unique carbonate substituted bioapatite of Champsocephalus gunnari icefish jaw and skull bones was carried out using NEXAFS spectroscopy. It has been established that these bones contain the B-type carbonate substituted hydroxyapatit with a content [CO3]2- anion of about 0.79-3.07 wt.%.

. Thus the carbonate concentration and the ratio of carbonate [CO 3 ] 2to phosphate [PO 4 ] 3anion concentration are important parameters that are characterized biological apatite's with functional differences [2]. Moreover these parameters are differ with the age of biomaterial and play an important role in the study of fossil bone remains [10][11][12].
Due to the complexity of the [CO 3 ] 2anions quantitative determination, the B-type carbonate substituted hydroxyapatite is characterized by calcium to phosphorus atomic proportion (apatite module) [1,11]. However, the NEXAFS spectroscopy allow reliably to determine the presence of anion [CO 3 ] 2in the hard tissue bioapatite by the characteristic high-intensity π*-resonance in the absorption C1s-spectrum and determine the [CO 3 ] 2and [PO 4 ] 3anions concentration and its ratio. Moreover NEXAFS sensitive to atomic and chemical composition and allow to indicate the correlation between biapatite hierarchical organization and local electronic structure of its mineralized phase [4,12,13]. Our preliminary studies provide strong evidence that NEXAFS spectroscopy is the most suitable method to examine structural changes and differences of organic and inorganic functional groups within biomaterials [14][15][16].

Sample preparation
For the investigation of bioapatite structure and composition selected jaw and skull bones of C. gunnari icefish together with the calcite CaCO 3 as a control sample were taken. The detailed description of C. gunnari icefish bone formation and structure peculiarities was published earlier in [3,12]. The preliminary treatment of selected icefish bones were carried out into 6 % H 2 O 2 solution for 24 hours with subsequent mechanical separation from muscles. For finalize cleaning of the hard tissues from attached soft tissues the samples, the ultrasonic bath with 15 % aqueous solution of NaOH has been used for 2-4 hours at room temperature. Finally the samples were washed in distilled water and then dried at 50 o C on air. The samples for NEXAFS measurements were prepared ex situ in air by pressing of powdered investigated matter into the clean copper plate surface.

NEXAFS spectroscopy study
NEXAFS of the powdered samples under study were carried out using monochromatic synchrotron radiation at the RGBL BESSY-II [17]. All absorption measurements were performed under vacuum conditions 10 −9 torr in the experimental chamber. NEXAFS-spectra were measured by the total electron yield mode (TEY) with an energy resolution about 0.01eV. NEXAFS spectra were collected by TEY-signal monitoring by the drain current detection with Keithley 617 microammetr. The spectra were normalized to the incident photon flux, which was defined as a ratio between TEY-signal from a clean Au-plate. The suppression and estimation of the nonmonochromatic background in TEY-signal was carried out by absorption Ti-filter method [18,19].

Results and discussion
The [CO 3 ] 2anions concentration in C. gunnari icefish bones were examined by NEXAFS C1sspectrum analysis. It is known that some of fish species hard tissue contain mineralized phase in the form of fluoroapatite. However the neither of measured NEXAFS F1s spectra of investigated icefish bones indicates the presence of fluoride. This fact suggests that C. gunnari icefish include bioapatite in the form of hydroxylapatite. Moreover this fact was confirmed by NEXAFS P2p and Ca2p spectra analysis discussed in our previous studies [3,12].
NEXAFS C1s-spectra of selected C. gunnari icefish bones and CaCO 3 are presented in Figures 1  and 2. All bone spectra demonstrate identical features. The peaks at 285.0 eV and 285.4 eV correspond with transitions from C1s-edge to π*-unoccupied orbitals of benzol and phenol aromatic  [20]. The structure between 287.3 eV and 288.6 eV is presumably connected with single, double and epoxy bonds of carbon and oxygen atoms [16]. The presence of mentioned spectral features assume the small amount of contamination in the samples.   [21], although it is known that the specific gravity of the carbonate substituted biapatite is less than one used in calculation. More other the α and β ratio depend not only on the specific gravity but also on the content of amorphous calcium phosphate and aqueous solutionof OH -, [PO 4 ] 3and [CO 3 ] 2anions and Ca 2+ dications.

Conclusion
The comparative analysis of NEXAFS C1s-adsorption spectra of selected C. gunnari icefish jaw and skull bones shows that the bones contain the B-type carbonate substituted hydroxyapatite. The presence of anion [CO 3 ] 2was clearly determined by the characteristic π-resonance in the absorption C1s-spectrum of bones. The quantitative assessment of ratio of peaks area associated with [CO 3 ] 2in bones and CaCO 3 NEXAFS C1s-spectra allow to determine the relative concentration of [CO 3 ] 2and [PO 4 ] 3anions and the content of [CO 3 ] 2anion in carbonate substituted hydroxyapatit of C. gunnari icefish jaw and skull bones. The estimated content is 0.79 and 1.85 wt.% for jaw and skull bone samples, respectively. The obtained data are well correlated with literature, but they can be slightly distorted due to the usage of the average specific gravity of apatite in the calculation.