Synchrotron micro-FT-IR spectroscopic evaluation of normal paediatric human bone

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Abstract

Cortical and cancellous normal bone biopsies were obtained intra-operatively from the inter-trochanteric region of an 11-year old boy and were studied with FT-IR spectroscopy. Undecalcified cortical bone sections, in which the micro-structure of the tissue had been preserved, were studied with synchrotron micro-FT-IR spectroscopy. The Amide I, II and III bands showed peaks near 1650, 1550 and 1240 cm−1, respectively. The v1, v2 and v3 phosphate and carbonate groups absorbed in the regions 1200–900 and 900–850 cm−1, respectively. Calculation of the ratio of the integrated areas under the v1, v3 phosphate and Amide I peaks, as a function of changing distance from the centre of the osteon, was used as an index of the relative mineral to matrix presence at each site of micro-FT-IR spectroscopic analysis. The ratio of the integrated areas under the v2 carbonate and v1, v3 phosphate peaks, as a function of changing distance from the centre of the osteon was used as an index of the relative carbonate to phosphate groups presence at each site of analysis. Both ratios were found to consistently increase with increasing distance from the centre of the osteon, for all osteons examined. In some fresh osteons, a peak near 1125 cm−1, characteristic of newly precipitated apatites (PO43−) was identified for the first time in the FT-IR spectra obtained from the inner lamellae of each Haversian system. This may be attributed to crystal effects of in vivo environments of fresh paediatric osteons.

Introduction

Human bone is a composite material consisting, in decreasing proportions, of mineral, collagen, water, non-collagenous proteins, lipids, vascular elements and cells [1]. Fourier Transform Infrared (FT-IR) spectroscopy and Micro Fourier Transform Infrared spectroscopy (Micro-FT-IR) are powerful techniques in the study of biomaterials [2], [3]. FT-IR spectroscopy has been used extensively in the study of the mineral phase in both synthetic apatites and homogenised calcified tissues from animals and humans. With micro-FT-IR spectroscopy one can analyse the chemical composition of materials without altering morphology. This powerful technique permits the study of spatially inhomogeneous systems such as bone, providing information from all tissue components (both organic and inorganic). The use of synchrotron IR beam for micro-spectroscopy offers the great advantage of 100–1000 times greater brightness, compared to the IR beam of a globar source. The long wavelengths of IR radiation limits the spatial resolution that can be achieved, and spatial resolution is of paramount importance for the study of inhomogeneous biological tissues, such as bone. The use of a synchrotron IR source permits the micro-spectroscopic study of very small surface areas of biological samples, with acceptable signal-to-noise ratio (S/N), at apertures even smaller than the wavelength of light used [3].

Cortical human bone is mainly composed of densely packed Haversian systems (osteons). The osteons are cylinders, measuring approximately 200–250 μm in diameter, which run longitudinally and spiral around the diaphyses of long bones, irregularly branching and anastomosing with each other. They are formed from concentric lamellae of bone surrounding central canals. The central canals of osteons, referred as haversian canals, contain blood vessels, lymphatic vessels, cells and occasionally nerves. The cells occupying the vessel-filled central tunnel are osteoblasts. The osteoblasts deposit concentric layers of organic bone matrix (osteoid), which subsequently mineralise, and the most recently deposited mineral is found near the centre of the osteon [1], [4].

The inorganic component of bone contributes approximately 65% of the wet weight of bone. It is not pure hydroxyapatite, Ca10(PO4)6(OH)2, but a poorly crystalline, calcium hydroxide in deficient biological apatite, containing numerous trace ions, the most abundant of which are carbonate (CO32−) and acid phosphate (HPO42−), fluoride (F) and citrate (C6H5O73−) ions and magnesium (Mg2+), potassium (K+) cations being also common substitutes of calcium and hydroxide (OH) of the hydroxyapatite.

The second most abundant component of bone is collagen, predominantly type I, which provides the bone with elasticity and flexibility and directs the organisation of matrix. The collagen fibres are organised circumferentially in the osteonal bone. Water accounts for 5–10% of the weight of bone tissue. Hydrogen bonds between water and collagen contribute to the stabilisation of the collagen fibril, and there have been suggestions that dehydration of the collagen may take place during mineralisation of bone [1], [4].

In this work of micro-FT-IR spectroscopic studies of normal human bone we have shown that the bone mineral component undergoes reproducible changes in its composition as a function of increasing distance from the centre of the osteon. The protein (Amide I) to mineral (phosphate group) ratio has been found to be higher in the younger mineral near the centre of the osteon. The carbonate to phosphate ratio has been found to decrease in the more mature, aged mineral, near the periphery of the osteon in accord with previous studies [5], [6], [7].

The chemical composition of paediatric normal bone in homogenised cancellous bone samples and in undecalcified cortical bone sections in which the microstructure of the tissue has been preserved, was also evaluated in three dimensions.

Section snippets

Materials and methods

Cortical and cancellous bone biopsies were obtained intra-operatively from the inter-trochanteric region of a male 11-year-old patient, undergoing various derotation and shortening femoral osteotomy. Informed consent was signed pre-operatively.

The cortical section of the bone biopsies was studied with micro-FT-IR spectroscopy and the adjacent cancellous sections were studied with FT-IR spectroscopy. The anatomic location and size of the biopsies and the young age of the patient did not allow

FT-IR spectra

The FT-IR spectra of fresh homogenised cancellous bone sample and homogenised cancellous bone sample that had been processed with hydrogen peroxide and acetone, in the region 4000–400 cm−1, are shown in Fig. 1. Band assignments are shown in Table 1.

Hydrogen peroxide and acetone processing is known to reduce the fat tissue and blood chromophores of fresh bone [8], but it does not remove the organic bone components completely, as it is observed in the FT-IR spectra of deproteinised bone samples.

In

Conclusions

The use of micro FT-IR spectroscopy can overcome some of the difficulties encountered with other methods of evaluation. The micro-FT-IR spectroscopic studies up today, have shown that in adult as well as in paediatric normal human bone, the ageing process of bone mineral around the centre of any individual osteon follows a similar pattern. The younger mineral is found near the centre of the osteon, whereas the older mineral is found closer to its periphery. The mineral to matrix ratio has been

Acknowledgements

The Central Oxford Research Ethics Committee supported this study (Grant 7154, Rec. Ref. C01.009). The authors would like to thank Professor N. Athanasou (Nuffield Department of Orthopaedic Surgery, Oxford, UK) for the histological evaluation of bone samples. The FT-IR and micro-FT-IR spectroscopic evaluation of bone samples was done at the Brookhaven National Laboratory, Upton, NY, USA. The authors would like to thank Dr L. Miller and N. Marincovic (Brookhaven National Laboratory, Upton, NY,

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