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

J. Anastassopoulou1, P. Kolovou1,

P. Papagelopoulos2 and T. Theophanides1 *1National Technical University of Athens, Chemical Engineering Department, Radiation Chemistry and Biospectroscopy, Zografou Campus, Zografou, Athens 2National and Kapodistrian University of Athens, School of Medicine, Department of Orthopaedic Surgery and Traumatology, Athens Greece* 

#### **1. Introduction**

Human bones are inherently complex materials consisting of minerals, collagen, water, noncollagenous proteins, lipids, vascular elements and cells. The bone is a physiologically active and reactive tissue (Petra et al., 2005). Through hormonal or mechanical signals the osteoblasts and osteoclasts are forming the bones. It is known that the role of osteoblasts is to create a collagen-rich extracellular matrix, which will become mineralized (bone formation) with calcium. On the other hand, the main role of osteoclasts is to degrade calcified bone tissue (resorption) (Shier et al., 1996). In the bone microenvironment, there is a dynamic balance between resorption and formation that maintains skeletal homeostasis. This process between bone formation and bone resorption is called remodelling. Bone remodelling and bone loss, is in function of age, external mechanical loads originating from physical activity and diseases.

The inorganic component of bone accounts approximately to 65% of the wet weight of bone and it is not pure hydroxyapatite, Ca10(PO4)6(OH)2, but a poorly crystalline calcium hydroxide in a deficient biological apatite, containing numerous trace anions, the most abundant of which is carbonate (CO3 2-) and acid phosphate (HPO4 2-), fluoride (F- ) and citrate (C6H5O7 3-) anions, as well as, magnesium (Mg2+), potassium (K+) cations. These anions and cations are common substitutes of calcium and hydroxide (OH) in hydroxyapatite (Petra et al., 2005; Maguire and Cowan, 2002). The major organic component of bone is collagen, predominantly type I, which provides the bone with elasticity and flexibility and directs the organisation of matrix. Water accounts for 5-10% and collagen with proteins are about 25-30% of the weight of bone tissue. Hydrogen bonds between water and collagen contribute to the stabilisation of a triple helix, and there have been suggestions that dehydration of the collagen may take place during demineralization of bone (Petra et al., 2005; Buckwalter et al., 2000).

In the last decade, there is an increasing interest in using infrared spectroscopy to evaluate and study biological systems (Petra et al., 2005; Mantsch et al., 1986; Anastassopoulou et al., 2008, 2009, 2011; Conti et al., 2008; Kolovou and Anastassopoulou, 2007;Pissaridi etal., 2011;

The Role of β-Antagonists on the Structure of Human Bone – A Spectroscopic Study 261

Ethylene Diamino Tetracetic Acid (EDTA), (C10H14O8N2Na2).2H2O [CH2N(CH2COOH) CH2COO].2H2O and benzeneacetamide [4-[2'-hydroxy-3'-[(1-methyl-ethyl)amino] propoxyl]

The bones were obtained intra-operatively from heads of femur of 65-75 year patients undergoing osteotomy. The samples were prepared in slices in order to preserve better the natural characteristics of the bones. The bone was cut into slices of 2 mm perpendicular to the longitudinal axis of the head (Fig. 1). The histological evaluation of representative sections of the biopsies showed no evidence of any metabolic disease, osteopenia, or bone

The bone sections were immersed successively in hydrogen peroxide solution (H2O2) and in acetone, according to a modification of the method described (Petra et al., 2005). Another bone section was reacted with aqueous EDTA solution and was left for a week in 4 oC (Veis and Schlueter, 1964). Twenty mg of bone were mixed with 200 mg of KBr powder in a pestle and was grownded in a mortar and compressed into a pellet. Two more sections were reacted with 2ml aqueous solutions of Νa2EDTA 0,5 Μ in the presence of timolol 0,5 Μ and

Fresh cancellous bone was immersed successively in hydrogen peroxide solution (H2O2) and in acetone, according to a modification method (Petra et al., 2005). Hydrogen peroxide and acetone processing is known to reduce blood chromophores of fresh bones and the fat tissues of the bone (Petra et al., 2005), but it does not remove the organic components completely. The processed bone sample was cut with microtome in multiple slices of 2 mm

One dry slice was demineralized. The demineralization of the bone was carried out by extraction of calcium ions with 0.5 M EDTA at 4oC, where the pH was adjusted to pH 7.4 with potassium hydroxide (Veis and Schlueter, 1964). Then the bone slice was washed

Two bone slices were left to become demineralized in the same way by extraction at 4oC with 0.5 M EDTA adjusted to pH 7.4 with potassium hydroxide in the presence of 0.25 M timolol or atenolol, for one week. Twenty milligrams of the cancellous section of the slices were mixed with 200 mg KCl powder in a pestle and mortar and compressed into a pellet to

Fig. 1. The anatomic location and the size of the bone sections.

0,5 M atenolol. The mixture was left for a week at 40 C.

thoroughly with repeated changes of distilled water and acetone.

**2.1 Preparation of bone samples** 

thickness each.

were products of Sigma.

cancer.

Mamarelis et al., 2010, Theophanides, 1979; Theophanides et al., 1993). FT-IR spectroscopy is a powerful non destructive technique and easy to investigate complex systems as bones with this technique. The absorption of infrared radiation excites the vibrations of the chemical bonds to higher energy levels, by changing the dipole moment of the molecule with which it interacts. This change and interaction gives the absorption spectrum with the characteristic absorption bands based on the total vibrational modes of biological molecules within the sample, such as hydroxyapatite. This powerful technique permits the study of homogenous and inhomogeneous systems, such as, bone, providing information from all tissue components, both organic and inorganic.

Adrenergic receptors are well known to be present in osteoblastic cells, and it is known that the α- and β-receptors could activate pharmacologically the proliferation of these cells (Suzuki et al., 1998). It is also known that adrenergic agonists efficiently activate βadrenoreceptors on osteoblasts and can stimulate bone resorption in intact mouse calvaria (Moore et al., 1993). Furthermore, it has been reported that propranolol inhibited cAMP formation induced by β–adrenergic agonists in bone organ cultures (Takeuchi et al., 2000) increased bone strength and the rates of endochondral bone formation in rats (Dietrich et al., 1979; Minkowitz et al., 2005).

The purpose of this *in vitro* work is to study the role of the non-selective beta-adrenergic receptor and beta1-selective adrenoreceptor blocking agents on demineralization of human bones, which was induced by EDTA.

#### **2. Materials and methods**

The chemical compounds:

Timolol, (S)-1-[(1,1-dimethylethyl)amino]-3-[(4-4(morpholinyl)-1,2,5-thiadiazol-3-yl] oxy] -2 propanol (Z) –butenedioate, with the empirical formula C13H24N4O3S and chemical structure, A:

And Atenolol, 4-[2'-hydroxy-3'-[(1-methyl-ethyl) amino] propoxyl, C14H22N2O2 with chemical structure, B:

Ethylene Diamino Tetracetic Acid (EDTA), (C10H14O8N2Na2).2H2O [CH2N(CH2COOH) CH2COO].2H2O and benzeneacetamide [4-[2'-hydroxy-3'-[(1-methyl-ethyl)amino] propoxyl] were products of Sigma.

The bones were obtained intra-operatively from heads of femur of 65-75 year patients undergoing osteotomy. The samples were prepared in slices in order to preserve better the natural characteristics of the bones. The bone was cut into slices of 2 mm perpendicular to the longitudinal axis of the head (Fig. 1). The histological evaluation of representative sections of the biopsies showed no evidence of any metabolic disease, osteopenia, or bone cancer.

Fig. 1. The anatomic location and the size of the bone sections.

The bone sections were immersed successively in hydrogen peroxide solution (H2O2) and in acetone, according to a modification of the method described (Petra et al., 2005). Another bone section was reacted with aqueous EDTA solution and was left for a week in 4 oC (Veis and Schlueter, 1964). Twenty mg of bone were mixed with 200 mg of KBr powder in a pestle and was grownded in a mortar and compressed into a pellet. Two more sections were reacted with 2ml aqueous solutions of Νa2EDTA 0,5 Μ in the presence of timolol 0,5 Μ and 0,5 M atenolol. The mixture was left for a week at 40 C.

#### **2.1 Preparation of bone samples**

260 Infrared Spectroscopy – Life and Biomedical Sciences

Mamarelis et al., 2010, Theophanides, 1979; Theophanides et al., 1993). FT-IR spectroscopy is a powerful non destructive technique and easy to investigate complex systems as bones with this technique. The absorption of infrared radiation excites the vibrations of the chemical bonds to higher energy levels, by changing the dipole moment of the molecule with which it interacts. This change and interaction gives the absorption spectrum with the characteristic absorption bands based on the total vibrational modes of biological molecules within the sample, such as hydroxyapatite. This powerful technique permits the study of homogenous and inhomogeneous systems, such as, bone, providing information from all

Adrenergic receptors are well known to be present in osteoblastic cells, and it is known that the α- and β-receptors could activate pharmacologically the proliferation of these cells (Suzuki et al., 1998). It is also known that adrenergic agonists efficiently activate βadrenoreceptors on osteoblasts and can stimulate bone resorption in intact mouse calvaria (Moore et al., 1993). Furthermore, it has been reported that propranolol inhibited cAMP formation induced by β–adrenergic agonists in bone organ cultures (Takeuchi et al., 2000) increased bone strength and the rates of endochondral bone formation in rats (Dietrich et al.,

The purpose of this *in vitro* work is to study the role of the non-selective beta-adrenergic receptor and beta1-selective adrenoreceptor blocking agents on demineralization of human

Timolol, (S)-1-[(1,1-dimethylethyl)amino]-3-[(4-4(morpholinyl)-1,2,5-thiadiazol-3-yl] oxy] -2 propanol (Z) –butenedioate, with the empirical formula C13H24N4O3S and chemical

And Atenolol, 4-[2'-hydroxy-3'-[(1-methyl-ethyl) amino] propoxyl, C14H22N2O2 with

A

B

tissue components, both organic and inorganic.

1979; Minkowitz et al., 2005).

**2. Materials and methods**  The chemical compounds:

structure, A:

chemical structure, B:

bones, which was induced by EDTA.

Fresh cancellous bone was immersed successively in hydrogen peroxide solution (H2O2) and in acetone, according to a modification method (Petra et al., 2005). Hydrogen peroxide and acetone processing is known to reduce blood chromophores of fresh bones and the fat tissues of the bone (Petra et al., 2005), but it does not remove the organic components completely. The processed bone sample was cut with microtome in multiple slices of 2 mm thickness each.

One dry slice was demineralized. The demineralization of the bone was carried out by extraction of calcium ions with 0.5 M EDTA at 4oC, where the pH was adjusted to pH 7.4 with potassium hydroxide (Veis and Schlueter, 1964). Then the bone slice was washed thoroughly with repeated changes of distilled water and acetone.

Two bone slices were left to become demineralized in the same way by extraction at 4oC with 0.5 M EDTA adjusted to pH 7.4 with potassium hydroxide in the presence of 0.25 M timolol or atenolol, for one week. Twenty milligrams of the cancellous section of the slices were mixed with 200 mg KCl powder in a pestle and mortar and compressed into a pellet to

The Role of β-Antagonists on the Structure of Human Bone – A Spectroscopic Study 263

3420 3407 *v*OH*, v*Ν-Η 3067 *v*CH= 2925 2924 *v*asCH2 2852 2851 *v*sCH2

<sup>1452</sup>*δas*CH3 + ν3CO3

<sup>1406</sup>*ν*COO- & ν3CO32-, AB

1337 *ρw*-CH2 wagging <sup>1239</sup>*ν*CN + δΝΗ in-plane,

1031 1031 *ν3*-PO43-, stoich. HA 960 sh *ν1*-PO43-, stoich. HA 871 *ν2*CO32-, Β carbonate-605 609 *ν4*-PO43-, ΗΑ 561 *ν4*-PO43-, ΗΑ Table 1. Peak assignments of the FT-IR spectra of homogenized bone before and after a week

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

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

1097 sh *ν3*-PO4

stretching vibration of oxidized lipids (Petra et al., 2005; Mamarelis et al., 2010).

1746 *v*C=O non-ionised -COOH 1653 1643 *ν*C=O + δΝ-Η Amide I <sup>1546</sup>*δ*Ν-Η in-plane + *ν*C-N,

**Assignment** 

Amide II

carbonate

carbonate

Amide III

3-,non stoich. ΗΑ

2- , AB

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

**After the 1st week of demineralization with EDTA cm-1**

**Unprocessed cm-1**

of demineralization with EDTA.

be studied by FT-IR. A small section of the bone slices was also studied with SEM. It must be noticed that both compounds A and B did not interact with EDTA.

#### **2.2 FT-IR spectroscopy**

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

#### **2.3 Scanning Electron Microscopy (SEM)**

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, Edax Int) Software.
