**3. Results and discussion**

#### **3.1 FT-IR spectra**

The FT-IR spectra of homogenized bone samples before and after interaction with EDTA solution is shown in Figs. 2a and 2b, respectively.

Fig. 2. FT-IR spectra of a) bone and b) bone after the first week of demineralization with EDTA

be studied by FT-IR. A small section of the bone slices was also studied with SEM. It must be

Fourier Transform Infrared (FT-IR) spectra were recorded in a frequency range of 4000-400 cm-1 using an FTS 3000 MX BioRad, Excalibur Series spectrophotometer and were processed with the Bio-Rad Win-IR Pro 3.0 Software. Twenty mg of fresh bone were mixed with 200 mg of KBr powder in a pestle and mortar and compressed into a pellet. Typically, 32 scans

The morphologic and chemical composition of the compounds was obtained by Scanning Electron Microscopy (SEM) with a Quanta 200, (FEI, Hillsboro, Or, Usa) apparatus equipped with an Χ-ray detector ΕDS, Saphire CDU, (Edax Int, Mawhaw, NJ, USA). The spectra were obtained with acceleration of 10 kV and beam light 100 μÅ was applied. The samples were covered with graphite with an SCD 004 Sputter-Coater and OCD 30 attachment (Bal-Tec, Vaduz, Liechtenstein). The SEM spectral maps were processed with the Gemin (3.5 version,

The FT-IR spectra of homogenized bone samples before and after interaction with EDTA

Fig. 2. FT-IR spectra of a) bone and b) bone after the first week of demineralization with

were collected at 4 cm-1 resolution over the wavenumber range of 400-4000 cm-1.

noticed that both compounds A and B did not interact with EDTA.

**2.2 FT-IR spectroscopy** 

Edax Int) Software.

**3.1 FT-IR spectra** 

EDTA

**3. Results and discussion** 

**2.3 Scanning Electron Microscopy (SEM)** 

solution is shown in Figs. 2a and 2b, respectively.


The infrared absorption bands (cm-1) and their assignments are given in Table 1.

Table 1. Peak assignments of the FT-IR spectra of homogenized bone before and after a week of demineralization with EDTA.

Significant differences are shown in the spectra of the bone (Fig. 1b) after interaction with EDTA. The broad band which appears at 3420 cm-1in the bone spectrum shifts to 3407 cm-1 after decalcification of the bone. This band is dominated by absorptions from stretching vibration of *v*OH and *v*NH functional groups of hydroxyapatite and proteins, respectively and is particularly sensitive by decalcification of the bone. This band shows that the OH groups of HA are reduced, while there are other free NH groups, which do not give neither inter- nor intra-molecular hydrogen bonds, leading to the result that the decalcification changes the secondary structure of proteins. The band at 3067 cm-1 is attributed to *v*=CH stretching vibration of oxidized lipids (Petra et al., 2005; Mamarelis et al., 2010).

The bands in the spectra between 3000 and 2800 are characteristic of the antisymmetric and symmetric stretching vibrations of methyl (CH3) and methylene (CH2) groups. The bands near 2925 cm-1 and near 2852 cm-1 correspond to antisymmetric and symmetric stretching vibrations of *v*CH2, respectively. These bands do not shift after demineralization, but increase in intensity. These changes show that the secondary structure of proteins changed and their environment became less lipophilic (Mamarelis et al., 2010; Anastassopoulou and Theophanides, 1990). The characteristic peak at 1746 cm-1 due to the stretching vibration of

The Role of β-Antagonists on the Structure of Human Bone – A Spectroscopic Study 265

In order to study the role of demineralization on the bone structure and to extrapolate the results to possible various bone diseases, we used calcium antagonists during demineralization with EDTA. The FT-IR spectra of bone, which were recorded after demineralization with EDTA in the presence of β- (timolol) and α- (atenolol) blockers after one week of reaction, are shown in Figs. 4b and 4c, respectively, in comparison with the

spectra of untreated bone (Fig. 4a). The band assignments are given in Table 2.

Fig. 4. FT-IR spectra of a) homogenized cancellous bone, b) homogenized and

C14H22O2N2, for 1 week, in the region 4000-400 cm-1.

animals (Mano et al., 2010).

demineralized, cancellous bone with EDTA in the presence of timolol, C13H24N4O3S, and c) homogenized and demineralized cancellous bone with EDTA in the presence of atenolol,

By comparison of the spectra it was observed that the presence of β-blocker differentiates the reaction between EDTA and the bone. Especially, in the region 1700-1500 cm-1, where the bands of Amide I and Amide II are located, the spectra show that the collagen loses partly the α-helix structure. On the other hand, in demineralized samples and in the presence of αblocker the secondary structure of collagen changed from α-helix to β-pleated sheet. Interesting are also the results of the spectra in the region of the phosphate groups between 1110 cm-1 and 870 cm-1 are characteristic. Furthermore, from the spectral data (Fig.3) and Table 2 it results again that there is a competition reaction between EDTA and β-blocker for calcium cations (Ca2+). Taking into account the chemical structure of the two calcium antagonists it was suggested that they should bind to hydroxyapatite of the bones with hydrogen bonds and thus inhibit the demineralization. These results are in accordance with the data of other investigators who reported that β-adrenoblockers prevented bone loss in

*ν*C=O of the non-ionized carboxyl group –COOH (Petra et al., 2005; Kolovou and Anastassopoulou, 2007; Anastassopoulou et al., 2008; Mythili et al., 2000) is absent in the spectrum of the demineralized bone. In the spectrum of the unprocessed cancellous bone, an intense band is seen near 1031 cm-1, which is characteristic of stoichiometric biological apatites, with two shoulders, one at 1097 cm-1, which is assigned to non-stoichiometric hydroxyapatite (HA), containing HPO42- and/or CO3 2- groups3 and one at 960 cm-1, which is assigned to the symmetric stretching vibration of the PO43- groups (Petra et al., 2005; Graham et al., 2008). The bands near 1452, 1406 and 871 cm-1, are *v3* and *v2* carbonate groups and the bands near 605 and 561 cm-1, are due to the *ν*4PO43- vibrational modes. These are clearly seen in the spectrum of the unprocessed cancellous bone and are absent in the spectrum of the decalcified or demineralized bone, since decalcification eliminates the calcium phosphates, Ca3(PO4)2.

Significant changes were observed in the region of 1670 - 1540 cm-1, where the Amide I and Amide II absorb. In many biological samples the Amide I band arises from the C=O stretching vibration with contribution of bending *δ*NH of peptide bond of proteins. These Amide bands shifted to lower frequencies and are found at 1653 cm-1 and 1546 cm-1, respectively in demineralized bones. This observation leads to the conclusion that the decalcification changes the secondary structure of collagen matrix from α-helix to β-sheet formation (Anastassopoulou et al., 2008, 2011; Kolovou and Anastassopoulou, 2007; Pissaridi et al., 2011; Mamarelis et al., 2010). This observation shows also that the hydrogen bonds between proteins and hydroxyl apatite have been broken and that the proteins have much more freedom in vibrational movements. This is also in agreement with the increase of the bands, which were observed in the region of 3000-2850 cm-1, where as we noticed, the antisymmetric and symmetric stretching vibrations of methyl *v*CH3 and methlylen *v*CH2 groups absorb. The above results were expected, since EDTA subtracts the Ca2+ cations from the bone to form complexes (Fig. 3) leading to its demineralization.

Fig. 3. EDTA-Ca complexes A. Tetrahedral coordination and B. Octahedral coordination

The non-intense band at 1239 cm-1 is assigned to Amide III, which arises from the in-phase *δ*N-H in plane bending and *v*C-N stretching vibrations, which almost disappeared after demineralization. This band is also sensitive to protein structural changes.

*ν*C=O of the non-ionized carboxyl group –COOH (Petra et al., 2005; Kolovou and Anastassopoulou, 2007; Anastassopoulou et al., 2008; Mythili et al., 2000) is absent in the spectrum of the demineralized bone. In the spectrum of the unprocessed cancellous bone, an intense band is seen near 1031 cm-1, which is characteristic of stoichiometric biological apatites, with two shoulders, one at 1097 cm-1, which is assigned to non-stoichiometric hydroxyapatite (HA), containing HPO42- and/or CO32- groups3 and one at 960 cm-1, which is assigned to the symmetric stretching vibration of the PO43- groups (Petra et al., 2005; Graham et al., 2008). The bands near 1452, 1406 and 871 cm-1, are *v3* and *v2* carbonate groups and the bands near 605 and 561 cm-1, are due to the *ν*4PO43- vibrational modes. These are clearly seen in the spectrum of the unprocessed cancellous bone and are absent in the spectrum of the decalcified or demineralized bone, since decalcification eliminates the

Significant changes were observed in the region of 1670 - 1540 cm-1, where the Amide I and Amide II absorb. In many biological samples the Amide I band arises from the C=O stretching vibration with contribution of bending *δ*NH of peptide bond of proteins. These Amide bands shifted to lower frequencies and are found at 1653 cm-1 and 1546 cm-1, respectively in demineralized bones. This observation leads to the conclusion that the decalcification changes the secondary structure of collagen matrix from α-helix to β-sheet formation (Anastassopoulou et al., 2008, 2011; Kolovou and Anastassopoulou, 2007; Pissaridi et al., 2011; Mamarelis et al., 2010). This observation shows also that the hydrogen bonds between proteins and hydroxyl apatite have been broken and that the proteins have much more freedom in vibrational movements. This is also in agreement with the increase of the bands, which were observed in the region of 3000-2850 cm-1, where as we noticed, the antisymmetric and symmetric stretching vibrations of methyl *v*CH3 and methlylen *v*CH2 groups absorb. The above results were expected, since EDTA subtracts the Ca2+ cations from

Fig. 3. EDTA-Ca complexes A. Tetrahedral coordination and B. Octahedral coordination

demineralization. This band is also sensitive to protein structural changes.

The non-intense band at 1239 cm-1 is assigned to Amide III, which arises from the in-phase *δ*N-H in plane bending and *v*C-N stretching vibrations, which almost disappeared after

the bone to form complexes (Fig. 3) leading to its demineralization.

calcium phosphates, Ca3(PO4)2.

In order to study the role of demineralization on the bone structure and to extrapolate the results to possible various bone diseases, we used calcium antagonists during demineralization with EDTA. The FT-IR spectra of bone, which were recorded after demineralization with EDTA in the presence of β- (timolol) and α- (atenolol) blockers after one week of reaction, are shown in Figs. 4b and 4c, respectively, in comparison with the spectra of untreated bone (Fig. 4a). The band assignments are given in Table 2.

Fig. 4. FT-IR spectra of a) homogenized cancellous bone, b) homogenized and demineralized, cancellous bone with EDTA in the presence of timolol, C13H24N4O3S, and c) homogenized and demineralized cancellous bone with EDTA in the presence of atenolol, C14H22O2N2, for 1 week, in the region 4000-400 cm-1.

By comparison of the spectra it was observed that the presence of β-blocker differentiates the reaction between EDTA and the bone. Especially, in the region 1700-1500 cm-1, where the bands of Amide I and Amide II are located, the spectra show that the collagen loses partly the α-helix structure. On the other hand, in demineralized samples and in the presence of αblocker the secondary structure of collagen changed from α-helix to β-pleated sheet. Interesting are also the results of the spectra in the region of the phosphate groups between 1110 cm-1 and 870 cm-1 are characteristic. Furthermore, from the spectral data (Fig.3) and Table 2 it results again that there is a competition reaction between EDTA and β-blocker for calcium cations (Ca2+). Taking into account the chemical structure of the two calcium antagonists it was suggested that they should bind to hydroxyapatite of the bones with hydrogen bonds and thus inhibit the demineralization. These results are in accordance with the data of other investigators who reported that β-adrenoblockers prevented bone loss in animals (Mano et al., 2010).

The Role of β-Antagonists on the Structure of Human Bone – A Spectroscopic Study 267

This pattern of the infrared spectra, which correspond to amorphous structure of hydroxyapatite was also observed upon irradiation of bones (Anastassopoulou et al., 2008; Pissaridi et al., 2011) as well as in cancerous bones (Anastassopoulou et al., 2011), which suggest that under oxidative stress the bone loses its native molecular structure, in the same

The spectra of the quantitative analysis of all the bone samples, obtained from the scanning

electron microscope are shown in Fig. 6 and the results are shown in Table 3.

Fig. 6. Spectra of quantitative analysis of the bone samples.

**with EDTA** 

Table 3. Quantitative analysis data of the bone samples (% wt composition)

**Ο** 59.01 69.71 64.24 76.90 **Νa** - 21.33 19.20 15.16 **P** 11.70 02.54 07.56 01.70 **S** - 05.31 01.38 02.31 **Ca** 29.29 01.11 07.62 03.94

From the percentage data it is clear that the concentration of calcium has the highest value in the non-demineralized bone. The reaction of the bone tissue with EDTA leads to the disappearance of calcium of the bone. The presence of timolol during the reaction of the bone with EDTA seems to inhibit partially the decalcification of the bone tissues, since the

**Decalcified bone EDTA+C13H24N4O3S** 

**Decalcified bone EDTA+C14H22O2N2**

**Element bone Decalcified bone** 

way as under artificial demineralization.

**3.2 SEM spectroscopy** 


Table 2. Band assignments of the FT-IR spectra of homogenized decalcified bones with EDTA in the absence and presence of 0.5 M antagonists C13H24N4O3S and C14H22O2N2 for 1 week.

In the case of the presence of atenolol during demineralization, the pattern of the spectrum in the region 1097-960 cm-1 has changed to that of the characteristic amorphous structure of hydroxyapatite (Kolovou and Anastassopoulou, 2007;Anastassopoulou et al., 2008) as shown in Fig. 4.

Fig. 5. FT-IR spectra of a) amorphous and b) biological hydroxyapatite.

This pattern of the infrared spectra, which correspond to amorphous structure of hydroxyapatite was also observed upon irradiation of bones (Anastassopoulou et al., 2008; Pissaridi et al., 2011) as well as in cancerous bones (Anastassopoulou et al., 2011), which suggest that under oxidative stress the bone loses its native molecular structure, in the same way as under artificial demineralization.

#### **3.2 SEM spectroscopy**

266 Infrared Spectroscopy – Life and Biomedical Sciences

3420 3414 3397 *v*Ν-Η, *v*OH 3067 3067 3067 *v*=CH 2925 2924 2924 *v*asCH2 2852 2855 2857 *v*sCH2

1452 1450 1447 *δas*CH3 + ν3CO3

1337 1335 1335 *ρw*-CH2

1406 1410 1394 *ν*COO- & ν3CO32-,

1239 1241 1233 *ν*CN + δΝΗ in-plane,

1097 sh 1113 *ν3*-PO43-,non stoich. ΗΑ 1031 1028 *ν3*-PO43-, stoich. HA 960 sh 960 *ν1*-PO43-, stoich. HA 871 870 *ν2*CO32-, Β carbonate-605 602 *ν4*-PO43-, ΗΑ 561 557 *ν4*-PO43-, ΗΑ Table 2. Band assignments of the FT-IR spectra of homogenized decalcified bones with EDTA in the absence and presence of 0.5 M antagonists C13H24N4O3S and C14H22O2N2 for 1

In the case of the presence of atenolol during demineralization, the pattern of the spectrum in the region 1097-960 cm-1 has changed to that of the characteristic amorphous structure of hydroxyapatite (Kolovou and Anastassopoulou, 2007;Anastassopoulou et al., 2008) as

Fig. 5. FT-IR spectra of a) amorphous and b) biological hydroxyapatite.

**Decalcified bone C14H22O2N2**

1746 1741 1741 *v*C=O non-ionised -COOH 1653 1653 *ν*C=O + δΝ-Η Amide I 1648 1649 *ν*C=O + δΝ-Η Amide I 1546 1550 1540 *δ*Ν-Η in-plane + *ν*C-N,

**Assignments** 

Amide II

AB carbonate

AB carbonate

Amide III

2- ,

**Decalcified bone C13H24N4O3S** 

**Unprocessed cm-1**

week.

shown in Fig. 4.

The spectra of the quantitative analysis of all the bone samples, obtained from the scanning electron microscope are shown in Fig. 6 and the results are shown in Table 3.

Fig. 6. Spectra of quantitative analysis of the bone samples.


Table 3. Quantitative analysis data of the bone samples (% wt composition)

From the percentage data it is clear that the concentration of calcium has the highest value in the non-demineralized bone. The reaction of the bone tissue with EDTA leads to the disappearance of calcium of the bone. The presence of timolol during the reaction of the bone with EDTA seems to inhibit partially the decalcification of the bone tissues, since the

The Role of β-Antagonists on the Structure of Human Bone – A Spectroscopic Study 269

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concentration of calcium increases up to 07.62. A similar result was obtained in the presence of atenolol, C14H22O2N2, but in both cases the calcium concentration was less than normal concentration.

In Fig. 7 are given the SEM images of cancellous bone sections with enlargement of X80. From the architecture and morphology of the images it is shown that after the reaction of bone with EDTA the sample does not show any bright regions, since the density of the bone is minimized (Fig. 7b) after the elimination of minerals of bone tissue.

Fig. 7. SEM images of bone a) without any penetration, b) after demineralization with EDTA, c) after demineralization in the presence of C13H24N4O3S and d) after demineralization in the presence of C14H22O2N2.

Significant changes in the brightness of the image are observed, when the demineralization takes place in the presence of C13H24N4O3S. It is observed an increase in bone density and the deposition of calcium on bone tissue is obvious (Fig. 7c). The image in Figure 6d corresponds to the result, which was obtained after the reaction of bone with EDTA in the presence of C14H22O2N2. The picture shows that the bone gets a more amorphous structure. These results are in agreement with the FT-IR data, which led to the suggestion that the biological hydroxyapatite changed from low crystallinity to amorphous state.
