Bone microarchitecture in human fetuses: Quantification of fetal bone microarchitecture in femurs and vertebal body

Results from histomorphometry

Before reporting microarchitectural parameters on femurs, we recall the evolution of femur size during gestation. Femur lengths at different gestational ages are relatively well known, since they can be examined in vivo using obstetrical echography. Various formulas relating gestational age from femur lengths have been proposed and are used to evaluate gestational age. Among those we report, the predication proposed by Doubilet, which reduces the mean errors to less than 0.6 week in the 14 to 42 weeks period. The relationship between gestational age (GA) and femur length (L) is based on logarithmic regression, GA = exp (a + в L ) with a = 2.45132, and в = 0.016590. The variation of femur length as a function of the gestational age, obtained by inverting this formula is illustrated in Figure 1. The dots indicate the characteristics of the femur samples that will be presented in the next section. We report here quantitative results obtained from the works of Salle, Glorieux et al.. Histomorphometry was performed on a collection of samples taken in thirty-five fetuses and newborns with gestational ages ranging between 16 and 41 weeks. Histomorphometric parameters were evaluated from frontal cuts in femurs, which were divided in several regions of interest of 0.56 mm2. The data were organized so that parameters could be evaluated as a function of their distance to the metaphysis growth cartilage junction. Derived histomorphometric parameters such as recalled in section II.2 were computed. Parameters of bone formation (osteoid surface, osteoid thickness, osteoid volume), as well as parameters of bone resorption were also reported. Some parameters from this study useful to the scope of this paper are reproduced in Table I. Osteoid thickness and partial bone volume (BV/TV) increased with gestional age. The increase of BV/TV between 20 and 40%, was due to an increase of the mean trabecular thickness evaluated to 71 jim in the first period (16-27 weeks) and 93 jim in the last period of gestation (34-41 weeks). Interestingly there was a spatial evolution between partial bone volume, trabecu- lar thickness, trabecular number, and cartilage volume with the distance to the growth plate. Partial bone volume and trabecu- lar thickness were found to increase respectively of about 10% and 100% whereas trabecular number, and cartilage volume decreased. From the variation of trabecular thickness within the femoral metaphysis, the authors estimated the dynamics of trabecular thickening to about 3 jim/day. Indices of bone re- sorption decreased with gestional age, and were found to decrease with the distance from the growth plate. The authors emphasized that the changes involved for the femoral metaphyseal cancellous bone development were related to modeling.

Bone microarchitecture in human fetuses: Investigation of bone microarchitecture

Imaging techniques

The reference technique for the investigation of bone microar-chitecture has for long been histomorphometry, which consists in analyzing histological slices. The bone sample has to be embedded in a resina and a slice with a thickness of a few micrometer is cut. This slice is then examined under a light microscope, and processed using specific quantification methods as described in the next section.

However, since the last decade, quantification based on x-ray microtomography (micro-CT) has considerably increased due to the amazing progresses made by this technique, and the availability of commercial systems. Micro-CT is a high resolution version of CT, used in clinical routine at the hospital. Its principle is to measure the attenua-tion of x-rays in a slice (or a volume) under different angles of view; then these measures are numerically processed to reconstruct a digital image (or volume). Conversely to histomorphometry which requires the cutting of the bone sample to be analyzed, micro-CT is a non destructive technique requiring no special preparation for the sample. In addition, micro-CT may provide three-dimensional images that are difficult to obtain by using serial histological slices due to slice deformations or deteriorations during cutting. The quantification of bone mor- phometry may then be directly performed from three-dimen-sional images, which presents some advantages over bi-di- mensional analysis, as will be highlighted in the next section. Though the accuracy of quantitative microarchitecture parameters is strongly related to image quality in terms of spatial resolution and signal to noise ratio. Spatial resolution refers to the size of the smallest detail that can be observed in the image. It is admitted that for the analysis of adult human trabecular bone a spatial resolution of 10-15 |jm is sufficient to get accurate quantification. Spatial resolution is not necessarily equal to the pixel (picture element) size in the image, although there is often some confusion in these terms. The signal to noise ratio in the micro-CT image is another important parameter with respect to the quantification accuracy since a noisy image makes it difficult the separation of bone from background. However, this segmentation is crucial since it is the first step of quantification and strongly influences the subsequent measurements. Keeping the same signal to noise ratio when spatial resolution increases is a technical difficulty to which micro-CT is confronted. A solution to get high signal to noise ratio in limited acquisi-tion time, is to use x-rays with high photon fluxes. X-rays with such characteristics may be produced by synchrotron sources, and synchrotron radiation (SR) micro-CT systems have been developed in a few synchrotron facilities in the world. A SR micro-CT system has been developed on beam-line ID19 at the ESRF (European Synchrotron Radiation Facility). The system provides three-dimensional images with spatial resolution between 15 and 0.5 |jm, this last resolution being still unachieved by micro-CT systems based on standard x-ray sources. The system has been used for the quantification of bone microarchitecture in human adults, animal models and human fetal bone. A significant advantage of this system over standard micro-CT is that it enables the simultaneous quantification of bone microarchitecture and tissue mineralization, which is possible thanks to the use of monochromatic x-ray beams with sufficient photon fluxes.

Bone microarchitecture in human fetuses

Introduction

Bone microarchitecture is receiving increasing attention in the assessment of the biomechanical properties of bone. If it is well characterized in normal and pathologic human subjects, few quantitative data are available in human fetal development. The different stages of bone formation in human embryo have been extensively described in histological textbooks. Ossification begins as mesenchymal condensations during the embryonic period. Bone formation is typically classified in in- tramembranous and endochondral ossification. In intramem- branous (or dermal) ossification, the mesenchymal tissue is directly converted into bone, while in endochondral ossification, the mesenchymal cells differentiate into a cartilage model, which is later replaced by bone. Intramembranous ossification concerns flat bones of the skull and face, the mandible and the clavicle. Endochondral ossification concerns most bone of the skeleton, and in particular bones of the axial skeleton and long bones.

Endochondral ossification involves several steps:

1) Chondrocytes in the centre of the cartilage model hypertrophy. The matrix is reduced to a series of small struts that soon begin to calcify. After they initiate matrix changes, the enlarged chondrocytes degenerate and disintegrate leaving cavities within cartilage.

2) Blood vessels grow into the perichondrium surrounding the shaft of the cartilage. The cells in the inner layer of the peri- chondrium differentiate into osteoblasts. The perichondrium is now a periosteum, and a thin layer of bone is produced around the shaft of the cartilage.

3) Blood invasion increases by capillaries penetrating in the space left by the disintegrating chondrocytes. Osteoblasts begins producing spongy bone. This primary center of ossification expands towards both ends of the cartilage model.

4) As the bone enlarges, trabeculae in the center of the shaft region are resorbed and form a marrow cavity. The bone of the shaft becomes thicker and the cartilage between each epiphyses is replaced by shafts of bone. Further growth involves increase in length and diameter.

5) Capillaries and osteoblasts migrate into the epiphyses creating secondary ossification centers. The epiphyses become filled with spongy bone. At each metaphysis an epiphyseal cartilage separates the epiphyses from the diaphysis.