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Comparative study of some physico-chemical characteristics of osteoporotic and normal human femur heads
Clinical Biochemistry 40 (2007) 907 – 912
Comparative study of some physico-chemical characteristics of osteoporotic and normal human femur heads T.P. Sastry a,⁎, A. Chandrsekaran b , J. Sundaraseelan a , M. Ramasastry a , R. Sreedhar b a
b
Bio-products Laboratory, Central Leather Research Institute, Chennai-600020, India Department of orthopedics, Sri Ramachandra Medical College, Porur, Chennai-600085, India Received 1 February 2007; received in revised form 18 April 2007; accepted 18 April 2007 Available online 27 April 2007
Abstract Objective: To compare some of the physico-chemical properties of osteoporotic and normal femur heads — a sample study. Materials and methods: The organic and inorganic parts of human normal (healthy) (N), osteoporotic (OP) femur heads and were separated using conventional methods and their physico-chemical characteristics were compared using infrared spectroscopy (IR), thermogravimetric analysis (TGA), X-ray diffraction (XRD) and scanning electron microscopy (SEM). Results: The data revealed that the extra crosslinking had taken place between the intramolecular alpha chains of collagen of OP bone. This was confirmed by IR spectroscopy and TGA studies. XRD data of the inorganic part of N have shown well-resolved peaks compared to OP revealing the decreased crystallinity in the osteoporotic bone. Conclusions: The extra intramolecular crosslinking of OP bone collagen molecules increases its fragility. The crystallinity of inorganic phase is less in OP and this may be the reason for its brittleness. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Osteoporosis; Collagen; Crosslinking; Crystallinity; Fragility
Introduction Bone is a living, growing tissue. It is made mostly of collagen, a protein that provides soft framework, and hydroxyapatite, a mineral that adds strength and hardens the framework. Osteoporous bone is a disease characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures of the hip, spine and wrist. Many authors have studied the structure and physico-chemical properties of normal and osteoporotic bone. Raif and Harmand [1] characterized the interfaces of calcified tissues and demonstrated that collagen is not directly linked to the mineralized phase. Paschalis et al. [2] performed FTIR micro-spectroscopic analysis of human iliac biopsies from untreated osteoporotic bone and stated that osteoporotic bone mineral is monotonically different in its
⁎ Corresponding author. Fax: +91 44 24911589. E-mail address: [email protected] (T.P. Sastry).
properties expressed as crystallinity/maturity than the normal bone. Johnson and Slemenda [3] studied the pathogenesis of osteoporosis. Their clinical outcome associated with osteoporosis have a complex pathogenesis involving in most cases both trauma to the bone and increased skeletal fragility. Baily et al. [4] observed the changes in the collagen of human osteoporotic bone matrix. Their analysis of collagen types revealed little change in the proportion of type III collagen, but in some cases, there was a significant loss of type VI. Dickenson et al. [5] observed that osteoporotic bone showed less strength and less stiffness than the normal bone. Grynpas et al. [6] indicated that non-collagenous protein content was considerably reduced in osteoporotic bone when compared with age-matched young controls, whereas the collagen content is unaffected. Burr [7] observed that in osteoporosis, there was a decrease in reducible collagen crosslinks without an alteration in collagen concentration and this would tend to increase in bone fragility. Hiller et al. [8] subjected human bones to controlled heating at 500–900 °C for 15–45 min and found that bone crystals alter in the first 15 min of heating to 500 °C or above, then appear to stabilize
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.04.011
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T.P. Sastry et al. / Clinical Biochemistry 40 (2007) 907–912
after prolonged heating. Using EDTA extraction procedure cancellous bones of femur heads and osteoporotic subjects were analyzed by Mbuyi et al. [9], in terms of their content in collagen, sialoprotein, proteoglycan and carbohydrate. The percentage of extracted matrix proteins of the osteoporotic bone in EDTA was significantly decreased, as was the collagenaseresistant fraction. Evans et al. [10] recorded lower bone mass in the relatives of osteoporotic patients compared to control subjects. The crosslinks in the collagen molecules are obviously intra- and intermolecular in nature. Intramolecular crosslinks
occur due to aldol condensation of allysine/hydroxy allysine residues, whereas intermolecular crosslinks occur due to aldimine formation between the same residues. Mansel and Bailey [11] demonstrated that residual collagen in osteoporotic bone was not normal but possessed higher levels of lysine hydroxylation and modified crosslinking. Koger et al. [12] measured the bone density at various sites in men and women with osteoporotic fractures of spine and hip and found the bone density reduction. Bechamou et al. [13] developed and validated a trabecular texture analysis from radiographic images. Their
Fig. 1. (A) IR spectrum of normal (healthy) bone (N) showing amide absorption bands at 1660 cm− 1, 1550 cm− 1 and 1240 cm− 1. (B) IR spectrum of collagen of osteoporotic bone (OP) showing amide absorption bands at 1660 cm− 1, 1550 cm− 1 and 1240 cm− 1 and aldehydic carbonyl group at 1740 cm− 1. (C) IR spectrum of inorganic part of normal (healthy) bone showing broad peaks at 915 to 1180 cm− 1 and 550 to 650 cm− 1 representing phosphate groups. (D) IR spectrum of inorganic part of osteoporotic bone showing broad peaks at 915 to 1180 cm− 1 and less intensity peaks between 550 to 650 cm− 1 representing phosphate groups.
T.P. Sastry et al. / Clinical Biochemistry 40 (2007) 907–912
909
Fig. 1 (continued).
objective was to determine if the fractal analysis of texture was able to distinguish osteoporotic fracture groups, either in vertebrae, hip or wrist fractures. Rubin and Jasiuk [14] characterized the lamellar structure of osteoporotic human trabecular bone using TEM. In the present study the organic and inorganic portions of the normal, osteoporotic femur heads and trabecular femur bones were separated by using conventional methods and some of their physico-chemical characteristics were compared using IR, TGA, XRD and SEM techniques.
died in a road traffic accident (healthy) (N), and five from male osteoporotic (OP) subjects (aged between 66 and 74 years) heads retrieved during prosthetic replacement for fractured neck of femur bones were collected from the hospital after obtaining proper permission from the ethical committee. Pepsin (3200–4500 U/mg protein) was purchased from Sigma-Aldrich Company, USA.
Materials and methods
N and OP bones were crushed to 1 × 1-in. pieces and heated at 800 °C for 2 h using Muffle furnace. The resultant portion of the bone is powdered and used for further analysis.
Cancellous bone femur heads from five normal, retrieved from a young men (age between 35 and 45 years) who
Isolation of inorganic part of bone
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T.P. Sastry et al. / Clinical Biochemistry 40 (2007) 907–912
Isolation of collagen Collagen was isolated using modified method of Takeshi et al. [15]. N, OP and TB bones were crushed and pulverized. The pulverized bone powders were treated with 0.5 M sodium acetate solution to remove the bloodstains. Later the samples were demineralized, separately, using 1 M HCl solution for 2 days. The demineralized samples were treated with a mixture of 0.1 M Tris–HCl 9 (pH 8), 0.5 M NaCl, 0.05 M EDTA, 0.2 M Bmercaptoethanol for 3 days under stirring. The residues were filtered with the help of cheesecloth, washed with water, suspended in 0.5 M acetic acid and digested with 10%, 100:10 (bone tissue:pepsin) at 4 °C for 24 h. The pepsin soluble collagen was centrifuged at 10,000 rpm for 1 h. The supernatant was salted out using 0.9 M NaCl, further dissolved in 0.5 M acetic acid, dialyzed with water and subsequently lyophilized to yield pepsin soluble collagen. This collagen was used for further analysis.
Fig. 2. XRD spectrum of N and OP, showing well resolved peaks in N indicating crystallinity and decreased crystallinity is observed in OP.
The samples were coated with gold ions using an ion coater (Fisons sputter coater) under the following conditions: 0.1-Torr pressure, 20-mA current and 70-s coating time. The morphology of the samples was visualized by scanning electron microscope (SEM model LEICA Stereoscn 440) using a 15-kV accelerating voltage.
1740 cm− 1, which represents aldehyde carbonyl group (Fig. 1B). In the IR spectrum of inorganic part of sample N (Fig. 1C), broad peaks at 915–1180 cm− 1 and 550–650 cm− I represent vibration of various phosphate groups in the hydroxyapatite. Vibrational modes of carbonyl groups were observed at 1420 and 1500 cm− 1. The peak that represents TCP is seen at 3550 cm− 1 in OP (Fig. 1D) samples. Fig. 2 shows the XRD pattern of the samples. The intensity of the peaks is indexed according to the standard pattern based on hexagonal lattice (JCPDS 9-432). The XRD pattern of sample N exhibits well-resolved peaks with increased crystallinity of β-TCP and hydroxyapatite (HA). In the case of the OP sample the peaks are not well resolved and the crystallinity is further decreased. The peaks, which are indexed, are β-TCP, marked as (•) are the HA peaks. In thermogravimetry, the losses of weight due to evolution of water, CO, CO2 and evaporation of other pyrolysis products are collectively measured as percentage of original weight. Collagen sample of N has shown a single-step weight loss. Sample N lost 78% of its weight between 191 and 430 °C. A two-step weight loss was observed in the case of osteoporotic bone, first being from 194 to 272 °C and the second from 273 to 469 °C. The first loss may be explained due to the loss of bound water and the second loss due to protein loss and total loss was about 69% between 194 and 469 °C. Figs. 3A and B show the scanning electron micrographs of inorganic portions of N and OP bones. The inorganic phase of N has shown continuity in the crystal structure. The SEM of the OP bone mineral exhibited discontinuous crystal structure, which may be due to leaching or defect in the crystal structure.
Results
Discussion
The IR spectrum of the collagen part of sample N exhibited amide I, amide II and amide III bands at around 1660, 1550 and 1240 cm− 1 respectively. The hydroxyl bonds and associated hydrogen bonds are seen as broadband between 3600 and 3100 cm− 1 (Fig. 1A). In the collagen sample of OP, apart from the peaks of amide groups a sharp band was observed around
The IR spectrum of collagen parts of N is more or less similar and represents typical protein IR spectrum [16,17]. However, the carbonyl group formed in the OP clearly represents the extra crosslinking in the intramolecular ∝ chains of its collagen. The sharp peak at 1740 cm− 1 clearly represents the carbonyl group, which may be explained by the following reaction [18]. Lysine
IR spectroscopy The IR spectra of samples were recorded Using Nicolet 300 Fourier Transform Infrared Spectra (FTIR) spectroscope using KBr pellet containing 2–6 mg of sample. X-ray diffraction (XRD) XRD analysis of the samples [50 mg each] deposited on glass substrates was carried out on Phillip ph 1830 instrument operating at a voltage of 40 kV and a current of 50 mA with Cu Kα radiation. Scans were performed in step mode between 2θ values of 4° and 120° with a step size of 0.2° and 10 s per step. Thermogravimetric analysis (TGA) TGA was carried out using a Seiko SSC 5200 H in nitrogen atmosphere (80 mL/min) at a heating rate of 10 °C/min. Primary weight change of the samples as a function of temperature was recorded using this study. Scanning electron microscopy (SEM)
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instead aldimine bonds are noticed [20]. Hence the crosslinks formed in the osteoporotic collagen are intramolecular in nature. Normally, increase in the crosslinking of proteins leads to brittleness or fragility [21] and thus may be the reason for the fragility of the OP bone. In the IR spectrum of inorganic part of N higher content of HA is observed and TCP is less. Whereas in OP the TCP is higher and this is evident as the hydroxyl band is not having intensity at 630 cm− 1. The well-resolved peaks observed in the N indicate the crystallinity of the healthy bones, whereas in OP the crystallinity is decreased. The decrease in crystallinity might be an indication of the weak structure of the inorganic phase and hence weakness in the bone. The discontinuous crystal structure observed in the SEM images of OP may be one of the reasons for the brittleness of the bone. TGA studies revealed that there was a single-step weight loss in N, a two-step weight loss was observed in the OP. N lost 78% and 81% of weight up to 430 and 450 °C respectively. Whereas OP lost only 69% of its weight up to 469 °C, this reduction in weight loss may be due to the extra intramolecular crosslinking of ∝ chains in its collagen. As it may require more energy to break the crosslinks, the percentage of weight loss was less at 469 °C, whereas more weight loss was observed in the case of N bone at around the same temperature. Conclusions
Fig. 3. (A) SEM of inorganic part of normal (healthy) bone (N) showing continuity in crystal structure. (B) SEM of inorganic part of osteoporotic bone (OP) showing discontinuous crystal structure.
or hydroxy lysine residues are oxidized to reactive allysine or hydroxy allysine respectively by the enzyme lysyloxydase.
Aldol condensation product can be derived by condensation of two allysine or hydroxy allysine residues in two separate α chains of one single molecule of collagen giving rise to α–β unsaturated aldehyde (aldol) [19]. In the case of intermolecular crosslinking aldol condensation products are not observed,
The collagenous part of the osteoporotic bone has exhibited extra intramolecular crosslinks in the ∝ chains of the collagen. Due to this crosslinking the thermal stability of OP collagen was increased. The extent of crystallinity and continuity in the crystal structure was less in the case of OP compared to those of N. Extra crosslinking in the collagen and less crystallinity in the inorganic phase of OP bone might be the cause for brittleness in the bones of osteoporotic patients. References [1] Raif EM, Harmand MF. Molecular interface characterization in human bone matrix: I. Biochemical and IR spectroscopic studies. Biomaterials 1993;14:978–84. [2] Paschalis EP, Betts F, DiCarlo E, Mendelsohn R, Boskey AL. FTIR microspectroscopic analysis of human iliac crest biopsies from untreated osteoporotic bone. Calcif Tissue Int 1997;61:487–92. [3] Johnson CC Jr., Slemanda CW. Pathogenesis of osteoporosis. Bone 1995;17:19S–22S. [4] Bailey Aj, Wotton SF, Sims TJ, Thompson PW. Biochemical changes in the collagen of human osteoporotic bone matrix. Connect Tissue Res 1993;29:119–32. [5] Dickenson RP, Hutton WC, Stott JR. The mechanical properties of bone in osteoporosis. J Bone Joint Surg Br 1981;63-B:233–8. [6] Grynpas MD, Tupy JH, Sodek J. The distribution of soluble, mineralbound, and matrix-bound proteins in osteoporotic and normal bones. Bone 1994;15:505–13. [7] Burr DB. Bone material properties and mineral matrix contributions to fracture risk or age in women and men. J Musculoskelet Neuronal Interact 2002;2:201–4. [8] Hiller JC, Thompson TJ, Evison MP, Chamberlain AT, Wess TJ. Bone mineral change during experimental heating: an X-ray scattering investigation. Biomaterials 2003;24:5091–7. [9] Mbuyi-Muamba JM, Gevers G, Dequeker J. Studies on EDTA extracts and
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[11]
[12]
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T.P. Sastry et al. / Clinical Biochemistry 40 (2007) 907–912 collagenase digest from osteoporotic cancellous bone of the femoral head. Clin Biochem 1987;20:221–4. Evans RA, Marel GM, Lancaster EK, Kos S, Evans M, Wong SY. Bone mass is low in relatives of osteoporotic patients. Ann Intern Med 1988;109:870–3. Mansell JP, Bailey AJ. Increased metabolism of bone collagen in postmenopausal female osteoporotic femoral heads. Int J Biochem Cell Biol 2003;35:533–9. Kroger H, Lunt M, Reeve J, Dequeker J, Adams JE, Birkenhagar JC, et al. Bone density reduction in various measurement sites in men and women with osteoporotic fractures of spine and hip: the European quantitation of osteoporosis study. Calcif Tissuse Int 1999;64:191–9. Bechamou CL, Poupon S, Lespessailles E, Loiseau S, Jennane R, Siroux V, et al. Fractal analysis of radiographic trabecular bone texture and bone mineral density: two complementary parameters related to osteoporotic fractures. J Bone Miner Res 2001;16:697–704. Rubin MA, Jasiuk I. The TEM characterization of the lamellar structure of osteoporotic human trabecular bone. Micron 2005;36:653–64. Nagai Takeshi, Izumi Masami, Ishii Masahide. Fish scale collagen. Preparation and partial characterization. Int J Food Sci Tech 2004;39:239–44.
[16] Jesurietta Sathian, Sastry, Lakshminarayana Y. Ganga Radhakrishnan preparation and characterization of degelatinised bone and grafting of poly (methyl methacrylate). J Polym Mater 2002;19:23–8. [17] Kavitha A, Kamala Boopalan B, GangaRadhakrishnan S, Sankaran B, Das NT, Sastry P. Preparation of feather keratin hydrolyzate-gelatin composites and their graft copolymers. J Macromol Sci 2005;42:1703–13. [18] Howard B, Bensusan A novel hypothesis for the mechanism of crosslinking in collagen. 1965; Chapter 1.8, 42–46. In: Structure and function of connective and skeletal tissues. Proceedings of an advanced study Institute organized under auspices of NATO St. Andrews, Ed: S. F. R. Jackson, D. S. Harkness, M. G. Partridge, R. Teistram, Publisher: Butterworth London. [19] Gay S, Miller E, J Biochemistry and metabolism of collagen 1978, Chapter 1 19–24. In: Collagen in physiology and pathology of connective tissue, Publisher: Gustav Fischer Verlag, New York. [20] Bailey AJ, Robins S, Balian P. Biological significance of the intermolecular crosslinks of collagen. Nature 1974;251:105–9. [21] Sastry TP, Rao K. Chemically modified collagenous amniotic layer as a wound dressing material. Chapter 11. In: Szycher Michael, editor. High performance biomaterials. Switzerland: Technomic Pub; 1991.
Comparative study of some physico-chemical characteristics of osteoporotic and normal human femur heads T.P. Sastry a,⁎, A. Chandrsekaran b , J. Sundaraseelan a , M. Ramasastry a , R. Sreedhar b a
b
Bio-products Laboratory, Central Leather Research Institute, Chennai-600020, India Department of orthopedics, Sri Ramachandra Medical College, Porur, Chennai-600085, India Received 1 February 2007; received in revised form 18 April 2007; accepted 18 April 2007 Available online 27 April 2007
Abstract Objective: To compare some of the physico-chemical properties of osteoporotic and normal femur heads — a sample study. Materials and methods: The organic and inorganic parts of human normal (healthy) (N), osteoporotic (OP) femur heads and were separated using conventional methods and their physico-chemical characteristics were compared using infrared spectroscopy (IR), thermogravimetric analysis (TGA), X-ray diffraction (XRD) and scanning electron microscopy (SEM). Results: The data revealed that the extra crosslinking had taken place between the intramolecular alpha chains of collagen of OP bone. This was confirmed by IR spectroscopy and TGA studies. XRD data of the inorganic part of N have shown well-resolved peaks compared to OP revealing the decreased crystallinity in the osteoporotic bone. Conclusions: The extra intramolecular crosslinking of OP bone collagen molecules increases its fragility. The crystallinity of inorganic phase is less in OP and this may be the reason for its brittleness. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Osteoporosis; Collagen; Crosslinking; Crystallinity; Fragility
Introduction Bone is a living, growing tissue. It is made mostly of collagen, a protein that provides soft framework, and hydroxyapatite, a mineral that adds strength and hardens the framework. Osteoporous bone is a disease characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures of the hip, spine and wrist. Many authors have studied the structure and physico-chemical properties of normal and osteoporotic bone. Raif and Harmand [1] characterized the interfaces of calcified tissues and demonstrated that collagen is not directly linked to the mineralized phase. Paschalis et al. [2] performed FTIR micro-spectroscopic analysis of human iliac biopsies from untreated osteoporotic bone and stated that osteoporotic bone mineral is monotonically different in its
⁎ Corresponding author. Fax: +91 44 24911589. E-mail address: [email protected] (T.P. Sastry).
properties expressed as crystallinity/maturity than the normal bone. Johnson and Slemenda [3] studied the pathogenesis of osteoporosis. Their clinical outcome associated with osteoporosis have a complex pathogenesis involving in most cases both trauma to the bone and increased skeletal fragility. Baily et al. [4] observed the changes in the collagen of human osteoporotic bone matrix. Their analysis of collagen types revealed little change in the proportion of type III collagen, but in some cases, there was a significant loss of type VI. Dickenson et al. [5] observed that osteoporotic bone showed less strength and less stiffness than the normal bone. Grynpas et al. [6] indicated that non-collagenous protein content was considerably reduced in osteoporotic bone when compared with age-matched young controls, whereas the collagen content is unaffected. Burr [7] observed that in osteoporosis, there was a decrease in reducible collagen crosslinks without an alteration in collagen concentration and this would tend to increase in bone fragility. Hiller et al. [8] subjected human bones to controlled heating at 500–900 °C for 15–45 min and found that bone crystals alter in the first 15 min of heating to 500 °C or above, then appear to stabilize
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.04.011
908
T.P. Sastry et al. / Clinical Biochemistry 40 (2007) 907–912
after prolonged heating. Using EDTA extraction procedure cancellous bones of femur heads and osteoporotic subjects were analyzed by Mbuyi et al. [9], in terms of their content in collagen, sialoprotein, proteoglycan and carbohydrate. The percentage of extracted matrix proteins of the osteoporotic bone in EDTA was significantly decreased, as was the collagenaseresistant fraction. Evans et al. [10] recorded lower bone mass in the relatives of osteoporotic patients compared to control subjects. The crosslinks in the collagen molecules are obviously intra- and intermolecular in nature. Intramolecular crosslinks
occur due to aldol condensation of allysine/hydroxy allysine residues, whereas intermolecular crosslinks occur due to aldimine formation between the same residues. Mansel and Bailey [11] demonstrated that residual collagen in osteoporotic bone was not normal but possessed higher levels of lysine hydroxylation and modified crosslinking. Koger et al. [12] measured the bone density at various sites in men and women with osteoporotic fractures of spine and hip and found the bone density reduction. Bechamou et al. [13] developed and validated a trabecular texture analysis from radiographic images. Their
Fig. 1. (A) IR spectrum of normal (healthy) bone (N) showing amide absorption bands at 1660 cm− 1, 1550 cm− 1 and 1240 cm− 1. (B) IR spectrum of collagen of osteoporotic bone (OP) showing amide absorption bands at 1660 cm− 1, 1550 cm− 1 and 1240 cm− 1 and aldehydic carbonyl group at 1740 cm− 1. (C) IR spectrum of inorganic part of normal (healthy) bone showing broad peaks at 915 to 1180 cm− 1 and 550 to 650 cm− 1 representing phosphate groups. (D) IR spectrum of inorganic part of osteoporotic bone showing broad peaks at 915 to 1180 cm− 1 and less intensity peaks between 550 to 650 cm− 1 representing phosphate groups.
T.P. Sastry et al. / Clinical Biochemistry 40 (2007) 907–912
909
Fig. 1 (continued).
objective was to determine if the fractal analysis of texture was able to distinguish osteoporotic fracture groups, either in vertebrae, hip or wrist fractures. Rubin and Jasiuk [14] characterized the lamellar structure of osteoporotic human trabecular bone using TEM. In the present study the organic and inorganic portions of the normal, osteoporotic femur heads and trabecular femur bones were separated by using conventional methods and some of their physico-chemical characteristics were compared using IR, TGA, XRD and SEM techniques.
died in a road traffic accident (healthy) (N), and five from male osteoporotic (OP) subjects (aged between 66 and 74 years) heads retrieved during prosthetic replacement for fractured neck of femur bones were collected from the hospital after obtaining proper permission from the ethical committee. Pepsin (3200–4500 U/mg protein) was purchased from Sigma-Aldrich Company, USA.
Materials and methods
N and OP bones were crushed to 1 × 1-in. pieces and heated at 800 °C for 2 h using Muffle furnace. The resultant portion of the bone is powdered and used for further analysis.
Cancellous bone femur heads from five normal, retrieved from a young men (age between 35 and 45 years) who
Isolation of inorganic part of bone
910
T.P. Sastry et al. / Clinical Biochemistry 40 (2007) 907–912
Isolation of collagen Collagen was isolated using modified method of Takeshi et al. [15]. N, OP and TB bones were crushed and pulverized. The pulverized bone powders were treated with 0.5 M sodium acetate solution to remove the bloodstains. Later the samples were demineralized, separately, using 1 M HCl solution for 2 days. The demineralized samples were treated with a mixture of 0.1 M Tris–HCl 9 (pH 8), 0.5 M NaCl, 0.05 M EDTA, 0.2 M Bmercaptoethanol for 3 days under stirring. The residues were filtered with the help of cheesecloth, washed with water, suspended in 0.5 M acetic acid and digested with 10%, 100:10 (bone tissue:pepsin) at 4 °C for 24 h. The pepsin soluble collagen was centrifuged at 10,000 rpm for 1 h. The supernatant was salted out using 0.9 M NaCl, further dissolved in 0.5 M acetic acid, dialyzed with water and subsequently lyophilized to yield pepsin soluble collagen. This collagen was used for further analysis.
Fig. 2. XRD spectrum of N and OP, showing well resolved peaks in N indicating crystallinity and decreased crystallinity is observed in OP.
The samples were coated with gold ions using an ion coater (Fisons sputter coater) under the following conditions: 0.1-Torr pressure, 20-mA current and 70-s coating time. The morphology of the samples was visualized by scanning electron microscope (SEM model LEICA Stereoscn 440) using a 15-kV accelerating voltage.
1740 cm− 1, which represents aldehyde carbonyl group (Fig. 1B). In the IR spectrum of inorganic part of sample N (Fig. 1C), broad peaks at 915–1180 cm− 1 and 550–650 cm− I represent vibration of various phosphate groups in the hydroxyapatite. Vibrational modes of carbonyl groups were observed at 1420 and 1500 cm− 1. The peak that represents TCP is seen at 3550 cm− 1 in OP (Fig. 1D) samples. Fig. 2 shows the XRD pattern of the samples. The intensity of the peaks is indexed according to the standard pattern based on hexagonal lattice (JCPDS 9-432). The XRD pattern of sample N exhibits well-resolved peaks with increased crystallinity of β-TCP and hydroxyapatite (HA). In the case of the OP sample the peaks are not well resolved and the crystallinity is further decreased. The peaks, which are indexed, are β-TCP, marked as (•) are the HA peaks. In thermogravimetry, the losses of weight due to evolution of water, CO, CO2 and evaporation of other pyrolysis products are collectively measured as percentage of original weight. Collagen sample of N has shown a single-step weight loss. Sample N lost 78% of its weight between 191 and 430 °C. A two-step weight loss was observed in the case of osteoporotic bone, first being from 194 to 272 °C and the second from 273 to 469 °C. The first loss may be explained due to the loss of bound water and the second loss due to protein loss and total loss was about 69% between 194 and 469 °C. Figs. 3A and B show the scanning electron micrographs of inorganic portions of N and OP bones. The inorganic phase of N has shown continuity in the crystal structure. The SEM of the OP bone mineral exhibited discontinuous crystal structure, which may be due to leaching or defect in the crystal structure.
Results
Discussion
The IR spectrum of the collagen part of sample N exhibited amide I, amide II and amide III bands at around 1660, 1550 and 1240 cm− 1 respectively. The hydroxyl bonds and associated hydrogen bonds are seen as broadband between 3600 and 3100 cm− 1 (Fig. 1A). In the collagen sample of OP, apart from the peaks of amide groups a sharp band was observed around
The IR spectrum of collagen parts of N is more or less similar and represents typical protein IR spectrum [16,17]. However, the carbonyl group formed in the OP clearly represents the extra crosslinking in the intramolecular ∝ chains of its collagen. The sharp peak at 1740 cm− 1 clearly represents the carbonyl group, which may be explained by the following reaction [18]. Lysine
IR spectroscopy The IR spectra of samples were recorded Using Nicolet 300 Fourier Transform Infrared Spectra (FTIR) spectroscope using KBr pellet containing 2–6 mg of sample. X-ray diffraction (XRD) XRD analysis of the samples [50 mg each] deposited on glass substrates was carried out on Phillip ph 1830 instrument operating at a voltage of 40 kV and a current of 50 mA with Cu Kα radiation. Scans were performed in step mode between 2θ values of 4° and 120° with a step size of 0.2° and 10 s per step. Thermogravimetric analysis (TGA) TGA was carried out using a Seiko SSC 5200 H in nitrogen atmosphere (80 mL/min) at a heating rate of 10 °C/min. Primary weight change of the samples as a function of temperature was recorded using this study. Scanning electron microscopy (SEM)
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instead aldimine bonds are noticed [20]. Hence the crosslinks formed in the osteoporotic collagen are intramolecular in nature. Normally, increase in the crosslinking of proteins leads to brittleness or fragility [21] and thus may be the reason for the fragility of the OP bone. In the IR spectrum of inorganic part of N higher content of HA is observed and TCP is less. Whereas in OP the TCP is higher and this is evident as the hydroxyl band is not having intensity at 630 cm− 1. The well-resolved peaks observed in the N indicate the crystallinity of the healthy bones, whereas in OP the crystallinity is decreased. The decrease in crystallinity might be an indication of the weak structure of the inorganic phase and hence weakness in the bone. The discontinuous crystal structure observed in the SEM images of OP may be one of the reasons for the brittleness of the bone. TGA studies revealed that there was a single-step weight loss in N, a two-step weight loss was observed in the OP. N lost 78% and 81% of weight up to 430 and 450 °C respectively. Whereas OP lost only 69% of its weight up to 469 °C, this reduction in weight loss may be due to the extra intramolecular crosslinking of ∝ chains in its collagen. As it may require more energy to break the crosslinks, the percentage of weight loss was less at 469 °C, whereas more weight loss was observed in the case of N bone at around the same temperature. Conclusions
Fig. 3. (A) SEM of inorganic part of normal (healthy) bone (N) showing continuity in crystal structure. (B) SEM of inorganic part of osteoporotic bone (OP) showing discontinuous crystal structure.
or hydroxy lysine residues are oxidized to reactive allysine or hydroxy allysine respectively by the enzyme lysyloxydase.
Aldol condensation product can be derived by condensation of two allysine or hydroxy allysine residues in two separate α chains of one single molecule of collagen giving rise to α–β unsaturated aldehyde (aldol) [19]. In the case of intermolecular crosslinking aldol condensation products are not observed,
The collagenous part of the osteoporotic bone has exhibited extra intramolecular crosslinks in the ∝ chains of the collagen. Due to this crosslinking the thermal stability of OP collagen was increased. The extent of crystallinity and continuity in the crystal structure was less in the case of OP compared to those of N. Extra crosslinking in the collagen and less crystallinity in the inorganic phase of OP bone might be the cause for brittleness in the bones of osteoporotic patients. References [1] Raif EM, Harmand MF. Molecular interface characterization in human bone matrix: I. Biochemical and IR spectroscopic studies. Biomaterials 1993;14:978–84. [2] Paschalis EP, Betts F, DiCarlo E, Mendelsohn R, Boskey AL. FTIR microspectroscopic analysis of human iliac crest biopsies from untreated osteoporotic bone. Calcif Tissue Int 1997;61:487–92. [3] Johnson CC Jr., Slemanda CW. Pathogenesis of osteoporosis. Bone 1995;17:19S–22S. [4] Bailey Aj, Wotton SF, Sims TJ, Thompson PW. Biochemical changes in the collagen of human osteoporotic bone matrix. Connect Tissue Res 1993;29:119–32. [5] Dickenson RP, Hutton WC, Stott JR. The mechanical properties of bone in osteoporosis. J Bone Joint Surg Br 1981;63-B:233–8. [6] Grynpas MD, Tupy JH, Sodek J. The distribution of soluble, mineralbound, and matrix-bound proteins in osteoporotic and normal bones. Bone 1994;15:505–13. [7] Burr DB. Bone material properties and mineral matrix contributions to fracture risk or age in women and men. J Musculoskelet Neuronal Interact 2002;2:201–4. [8] Hiller JC, Thompson TJ, Evison MP, Chamberlain AT, Wess TJ. Bone mineral change during experimental heating: an X-ray scattering investigation. Biomaterials 2003;24:5091–7. [9] Mbuyi-Muamba JM, Gevers G, Dequeker J. Studies on EDTA extracts and
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[16] Jesurietta Sathian, Sastry, Lakshminarayana Y. Ganga Radhakrishnan preparation and characterization of degelatinised bone and grafting of poly (methyl methacrylate). J Polym Mater 2002;19:23–8. [17] Kavitha A, Kamala Boopalan B, GangaRadhakrishnan S, Sankaran B, Das NT, Sastry P. Preparation of feather keratin hydrolyzate-gelatin composites and their graft copolymers. J Macromol Sci 2005;42:1703–13. [18] Howard B, Bensusan A novel hypothesis for the mechanism of crosslinking in collagen. 1965; Chapter 1.8, 42–46. In: Structure and function of connective and skeletal tissues. Proceedings of an advanced study Institute organized under auspices of NATO St. Andrews, Ed: S. F. R. Jackson, D. S. Harkness, M. G. Partridge, R. Teistram, Publisher: Butterworth London. [19] Gay S, Miller E, J Biochemistry and metabolism of collagen 1978, Chapter 1 19–24. In: Collagen in physiology and pathology of connective tissue, Publisher: Gustav Fischer Verlag, New York. [20] Bailey AJ, Robins S, Balian P. Biological significance of the intermolecular crosslinks of collagen. Nature 1974;251:105–9. [21] Sastry TP, Rao K. Chemically modified collagenous amniotic layer as a wound dressing material. Chapter 11. In: Szycher Michael, editor. High performance biomaterials. Switzerland: Technomic Pub; 1991.