Journal of Non-Crystalline Solids 351 (2005) 29352939 www.elsevier.

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Dierential scanning calorimetry and temperature dependence of electric conductivity in studies on denaturation process of bone collagen
Leszek Kubisz
a

a,*

, Slawomir Mielcarek

Department of Biophysics, Poznan University of Medical Sciences, Fredry 10, 61-701 Poznan, Poland b Institute of Physics, Adam Mickiewicz University, Poznan, Poland Available online 1 August 2005

Abstract Temperature induced structural transformation of bone collagen has been studied by electric conductivity measurements and differential scanning calorimetry. The process of collagen denaturation and structural transformation was manifested as an endothermic peak on the thermogram and as an increase in the electric conductivity, within the range of 400455 K. The temperatures of denaturation assessed by both methods were 429 K and 436 K respectively. The experiments were carried out on dry bovine bone heated in the temperature range of 295520 K. In order to remove free water absorbed by bone samples, they were pre-heated at 380 K prior to the measurements. 2005 Elsevier B.V. All rights reserved.
PACS: 72.80.Le; 74.25.Fy; 81.05.Qk; 87.14.g

1. Introduction Bone material is used in medicine as auto- and allografts in order to ll a bone gap or reinforce mechanical resistance. In industry bone components are used in many products. In physical terms bone is a natural composite material consisting of two phases. The heterogeneous structure of bone is formed by hydroxyapatite (HAP) the mineral component, and collagen the organic component. The physico-chemical properties of bone dier from those of each separate constituent. The important component of bone is water and in collagen and materials consisting of collagen, water content aects the temperature of denaturation. A decrease in water content usually leads to higher temperature of denaturation. Denaturation temperature of collagen de-

Corresponding author. Tel.: +48 61 8546227; fax: +48 61 8520455. E-mail address: kubisz@main.amu.edu.pl (L. Kubisz).

pends also on the presence of other compounds. It is known that the presence of HAP increases the denaturation temperature of collagen [13]. The process of thermal denaturation of protein involves its structural changes. Therefore, structural studies of biological materials usually have been carried out by means of X-ray diraction and electron microscopy. Also other experimental techniques such as thermal, electric and mechanical have been widely used [312]. Besides denaturation, heating of biological materials leads to such processes as water release and thermal decomposition. All the above mentioned processes can be studied during monotonic temperature increase. There are two methods that can be applied to study materials under the monotonic temperature increase: dierential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). DSC has recently been successfully applied to study thermal properties of such biological materials as leather, dentin, DNA, bones and collagen, particularly in the studies of thermal

0022-3093/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jnoncrysol.2005.05.041

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denaturation, water removal and thermal decomposition of amino acids [1317]. Also electric conductivity temperature relationship (rT) applied in bone study by Behari, Libo and Shamos along with the study of other biological materials such as hemoglobin, elastin and nucleic acids can provide useful information. Usually a DC voltage is applied to a sample and the current owing through it is measured [1822]. Measurements of rT can be carried out also during monotonic temperature changes and it is applied in studies on polymers and complexes [23,24]. Monotonic heating of the sample induces changes in its dimensions. Thus, the measured current is a sum of both conduction and displacement current, and the latter is caused by a change in capacitance of the sample. Electric properties of biological solid-state materials usually depend on the water content. Electric conductivity increases with increasing water content and the activation energy of charge conduction process is reduced [22,25]. Bone properties are deeply affected by the water content. Traditionally, water associated with proteins is divided into three types: structural, bound and free water released subsequently during heating. The structural water, about 00.07 g/g, is incorporated in the collagen structure. Its liberation is possible when collagen undergoes thermal denaturation. The term bound water, 0.070.25 g/g, refers to water molecules tightly bound to specic sites in collagen chains lling in the spaces between molecules. The bound waterprotein interaction is not as strong as in that of structural water. The term free water refers to the water content higher than 0.45 g/g. According to Nomura, in the range 0.250.45 g/g both, free and bound water are absorb. The release of both free and bound water occurs below denaturation [26]. Solid-state proteins are characterized by relatively high temperature of denaturation. Usually denaturation of collagen is observed in the temperature range of 450 520 K but the denaturation of bone collagen was also found at 428 K [2,27,28]. The range of denaturation temperatures is an outcome of the degree of collagen crystallinity, water content and the presence of other substances such as HAP. Changes in the thermal stability of collagen can be shown either by an increase or a decrease in the temperature of denaturation [2932]. Bone, prior to any application, usually is mechanically processes which often results in changes in its physicochemical properties. In this paper attention is drawn to a comparison of the rT relationship of bone, measured during monotonic temperature increase, with its DSC thermogram. Although the measurements of electric conductivity carried out at monotonic heating are limited to the solidstate biological materials, their advantages over other mentioned above methods is simplicity and low costs. The aim of this work is to show the application of the DSC method and rT relationship analysis in the stud-

ies of thermal denaturation process of bone collagen and to establish the temperature of denaturation and the temperature range of the denaturation process.

2. Materials The bone samples were obtained from the central part of the diaphyses of adult bovine femurs [33]. The bones were mechanically cleaned and washed in a 0.1 M solution of NaCl soon after the 2 year old animals had been slaughtered. The samples were machine-cut using a diamond saw with continuous water irrigation to minimize thermal damage during the machining process and washed in distilled water. Next the samples were dried at 294 K in air. For electric measurements, the samples as plano-parallel plates, 1 mm thick, were cut at 90 to the longitudinal axis of the bone. The samples were powdered for DSC measurements.

3. Methods The rT relationship was measured for the bone samples studies in the temperature range of 295520 K. The DC voltage was applied to the sample and the electric eld strength was within the range of voltagecurrent linearity where Ohms law is obeyed [18]. The generated electric led strength was in the range of 1.2 0.2 kV/ m. Intensity of the current was measured by an electrometer (W7-30 or Keithley 6514). Prior the measurement of electric conductivity, each sample was maintained at 380 K for a period of 1 h. At this time the release of free water took place [27]. Then, the sample was cooled to room temperature and heated again from 295 K to 530 K. All experiments were performed at the heating rate of 1 K/min in air under atmospheric pressure. In order to show the eect of water release at 380 K, measurements were also carried out for nondried samples in the range 295380 K. All the DSC measurements were carried out using a Perkin-Elmer DSC 7. About 20 mg of bone powder was packed into a stainless steel DSC cell. The heating rate was 10 K/min. Each sample was heated from 300 K to 530 K. In order to release free water, prior to the measurement each sample was kept in an open pan at 385 K for an hour. This procedure helped to avoid the large endothermic peak, related to the release of absorbed water, usually observed in the range of 290 440 K [15]. According to the earlier results for bone, the hydration level was reduced to residual bound water and structural water [27]. In order to prevents the occurrence of phenomena related to structural changes in HAP, taking place above 673 K, DSC studies were carried out in the range of 300530 K [34].

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Because water is an essential bone component inuencing electric conductivity [22] the hydration level was measured by thermogravimetric method. The hydration level w is dened as w mi mf 100%; mi

where mi and mf stand for the sample mass at the initial and nal temperatures, respectively. These measurements were performed by means of the Mettler-Toledo halogen hydration analyzer HR 73. In order to assess the contents of free water, bound water and structural water [26,35] the samples were heated isothermally at 293 K, 383 K and 473 K respectively. The heating was continued for 1 h to a constant mass of the sample. The samples that had been kept in air of relative humidity of 70% and 55% prior the measurement were labelled as HH and LH respectively.

4. Results Assessed hydration levels of the bone sample are collected in Table 1. In the experiment the bone hydration level was limited to bound (w1) and structural water (w3). Fig. 1 shows the electric conductivitytemperature relationship for the non-dried samples. For the HH sample the relationship showed a peak at 327 K whereas a

monotonic increase in r with temperature was observed for LH samples. The temperature dependence of electric conductivity of the dry bone sample and its DSC themogram are shown in Fig. 2. As a result of heating the electric conductivity increased. A change in the slope of r(T) was observed at 380 K and a local maximum at 455 K was followed by a local minimum at about 500 K. Further heating led to an increase in the electric conductivity. A comparison of the results of electric measurements, shown in Figs. 1 and 2 indicates that water release lead to a decrease in electric conductivity of bone. The calculated values of conductivity, lower than 106 X1 m1 and a low level of hydration, imply that the electrode eect can be neglected [36]. The relative error of the electric conductivity determination was less than 5%. The obtained DSC curve (Fig. 2) of dry bone shows two endothermic peaks at 395 K and 429 K respectively. The peak at 429 K was broader and had a higher amplitude than at 395 K.

5. Discussion The temperature dependence of electric conductivity of bone resembles the results obtained by Libo and Shamos. They reported that heating of dry bone

Table 1 Hydration level of bone Air humidity 70% w1 11.1 2.1% w2 13.4 2.1% w3 2.6 2.5% Air humidity 55% w1 9.8 1.3% w2 12.6 1.4 w3 3.1 1.6%

Fig. 1. rT relationship for non-annealed bone, LH bone stored at air of 55% relative humidity, HH bone stored at air of 70% relative humidity.

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Fig. 2. The DSC and the rT thermograms of dry bovine bone.

brought about an increased electric conductivity and caused a change in classication of bone from insulating to semiconducting materials [20]. Values of electric conductivity obtained in the present study are consistent with results reported by Behari et al. and Libo et al. [18,20]. Also a decrease in the electric conductivity observed on two subsequent heating run (Figs. 1 and 2) had been reported earlier by Libo and Shamos [20]. The explanation of the observed changes in the rT and DSC curves requires exclusion of eects of water content and HAP presence. The eect of HAP structural changes was excluded because up to 673 K, HAP does not change its physical form [34]. On the basis of the assessed hydration level of bone (Table 1) it was concluded, according to Nomura, that bound water was released on the sample annealing at 380 K. Therefore, further heating of the bone up to 530 K led to the release of structural water, which, according to the Ramachandran model of collagen macromolecule [37], takes place when collagen changes its structure during denaturation. Thus, the results of thermogravimetric measurements of hydration level permitted a conclusion that the denaturation process occurred in the temperature range of 385 530 K. The observed changes in the rT relationship and the DSC curve were connected with transformation of organic component of bone. The process of pre-heating permitted elimination of a large endothermic peak which is usually of observed for bone and collagen in the range of 290440 K (Fig. 2) [16]. The origin of the small peak on the DSC curve at 395 K is unclear. It

probably was caused by the release of residual bound water but also another explanation is not excluded. The recorded DSC curve shown in Fig. 2 enabled the assessment of the temperature of denaturation. The peak which appears in the DSC curve at 429 K, according to Kronick and Usha is assigned to the thermal denaturation of bone collagen [2,27,28]. The thermally activated irreversible process of denaturation begins at 400 K and involves uncoupling of the alpha chains leading to the helix-random coil transition and ends at 455 K [3841]. The process of thermal denaturation was observed on the rT curve in the range 418455 K and the temperature of denaturation was assessed as 436 K (Fig. 2). The dierence in the denaturation temperatures determined by the DSC and rT method was probably caused by dierent experimental conditions, namely the heating rate and the form of sample. The DSC thermograms enabled a calculation of the enthalpy of the denaturation process. It was calculated in the range of 400455 K. Table 2 shows the averaged value of enthalpy and the peak temperature estimated on the basis of four independent measurements. The process of thermal denaturation of bone collagen was reected by an increase in electric conductivity and change in the deviation of the DSC curve from the baseline. According to Miles and Bailey [39] the temperature increase caused an increase in the rate of the denaturation process. When the peak temperature had been achieved all molecules were denaturated and a new equilibrium was established. Thermal energy delivered to

Table 2 Enthalpy, temperature range and temperature of thermal denaturation process of dry bone collagen Temperature range (K) DSC rT 400455 418455 Denaturation temperature (K) 429 436 Enthalpy (J/g) 210 40

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bone collagen induced a helix-coil transition and increased mobility of the collagen polypeptide chain. The increased mobility can eect the electric properties by changing the capacitance which could lead to an increase in the displacement current. Simultaneously, the exponential increase in r, which was observed up to about 450 K, suggests the occurrence of thermally assisted hopping [42]. The obtained results and experimental conditions precluded to answer the question unambiguously. The methods described in the paper seem to be useful for studies of thermal denaturation of biological materials. Both methods applied for the same material gave similar results consistent with those reported by Kronick and Cooke [2]. Moreover, the dierence between the denaturation temperature obtained by the DSC method and by the rT relationship made it possible to deduce that dierent values of denaturation temperatures of solid-state collagen, which vary from about 430 K to 520 K in many papers, probably result from dierent hydration level of samples and other factors which occur during sample preparation, generally on individual history of sample and dierent experimental conditions.

6. Conclusion Both applied methods have estimation of the denaturation temperature and the temperature range of the denaturation process, which are important parameters of structural transitions in bone collagen. The experiments described and the results obtained have shown that temperature dependence of electric conductivity can be useful in the study of the process of thermal denaturation. This method can be expected to be applied as a standard method in the study of solid-state biological materials along with the dierential scanning calorimetry.

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