Infrared imaging of calcified tissue in bone biopsies from adults with osteomalacia

Abstract

Osteomalacia is a pathological bone condition in which there is deficient primary mineralization of the matrix, leading to an accumulation of osteoid tissue and reduced bone mechanical strength. The hypothesis that there are no qualitative or quantitative differences in osteomalacic bone mineral or matrix compared to disease-free bones was tested by examining unstained sections of polymethyl methacrylate (PMMA) embedded iliac crest biopsies using Fourier transform infrared imaging (FTIRI) at approximately 6-μm spatial resolution. Controls were seven female subjects, aged 36–57, without apparent bone disease. The experimental group consisted of 11 patients aged 22–72, diagnosed with osteomalacia. The spectroscopic parameters analyzed in each data set were previously established as sensitive to bone quality: phosphate/amide I band area ratio (mineral content), 1660/1690 cm⁻¹ peak ratio (collagen cross-links), and the 1030/1020 cm⁻¹ peak ratio (mineral crystallinity). The correspondence between spectroscopic mineral content (phosphate/amide I ratio) and ash weight was validated for apatite crystals of different composition and crystallite size. The FTIRI results from the biopsies expressed as color-coded images and pixel population means were compared with the nonparametric Mann–Whitney U test. There were no significant differences in the cortical parameters. Significant difference was found in the mineral content of the trabecular regions with a lower mean value in osteomalacia (P = 0.01) than in controls. Mineral crystallinity tended to be decreased in the trabecular bone (P = 0.09). This study supports the hypothesis that, in osteomalacia, the quality of the organic matrix and of mineral in the center of bone does not change, while less-than-optimal mineralization occurs at the bone surface. This study provides the first spectroscopic evaluation of whole bone mineral and matrix properties in osteomalacia, demonstrating that there are few differences in collagen cross-links between biopsies from patients with osteomalacia and from individuals without histological evidence of bone disease.

Introduction

Osteomalacia is a pathological bone condition in which there is an increase in osteoid thickness, caused by delayed mineralization lag time, due to extrinsic or intrinsic vitamin D deficiency. Extrinsic vitamin D deficiency occurs mainly due to decreased exposure to sun and, to a lesser extent, due to decreased dietary intake, while intrinsic vitamin D deficiency occurs due to impaired absorption or increased catabolism. Intrinsic deficiency is more common in countries that practice fortification of dairy products with vitamin D and is influenced by additional factors such as protein deficiency. Calcium deficiency does not induce osteomalacia in adults, although it might do so in children [1], [2].

The defective primary matrix mineralization in osteomalacia has been associated with reduced mechanical strength in several animal models of dietary-induced osteomalacia [3], [4], [5], [6]. The osteomalacic derangement originates primarily in the subnormal levels of calcium and phosphate and secondarily in the compromised metabolic and transport function of the osteoblasts, leading to production of matrix components that are not mineralized adequately. The mineralization process includes the apposition of extracellular matrix components upon which the mineral crystals are laid. It is well known that physiologic mineralization occurs on a scaffold of collagen fibers [7], but no abnormalities in the collagen type or in the amino acid composition of collagen of patients with osteomalacia have been reported. The levels of collagen hydroxylation, which are part of the posttranslational modifications of collagen fibers, were found to be similar to those in healthy subjects [8].

Vitamin D deficiency in children is expressed slightly different in adults and is referred to as rickets. In this case, the deficiency affects the cartilage tissue as well, causing defective mineralization of the epiphyseal cartilage and thus impairing bone growth. This can lead to skeletal deformities and disturbances in growth.

Diagnosis of osteomalacia in adults is made according to biochemical and radiological criteria [9]. Although bone biopsy is not required for the diagnosis, it is useful in borderline cases, in which a proof of a lag in the mineralization rate is needed [1]. In general, iliac crest biopsies are used because they provide large amounts of trabecular bone, and the procedure is relatively safe [10], [11]. The histomorphometric analysis of these biopsies reflects bone structure and turnover by quantification of mineralized and nonmineralized components of the bone tissue, in dynamic and nondynamic states. However, there is no stain specific for collagen cross-links or mineral crystallinity.

The present study tested the hypothesis that bone mineral and matrix properties in biopsies from osteomalacic individuals are not distinct from that of normal controls, as long as the osteoid was excluded from the analysis. The technique of Fourier transform infrared imaging (FTIRI), which has a spatial resolution of 6–10 μm, enabled this to be accomplished. The parameters evaluated, namely, collagen cross-links, mineral/matrix ratio, and crystal size/perfection, were previously established as related to chemical characteristics examined by X-ray diffraction (XRD) analysis and chromatography [12], [13].