Monday, December 26, 2011

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.


The initial site of bone deposition, called a primary ossification center, appears in human fetus from the 8th week of gestation. Later on, secondary ossification centers occur for instance in the epiphyses of long bone. The first bone tissue formed in the embryo is an immature bone, called woven bone or non- lamellar bone. Woven bone can be formed very rapidly and may also be found in adults when bone has been remodeled or in fracture healing. In this tissue, the collagen fibers of the matrix are arranged irregularly in the form of a meshwork. Woven bone is gradually converted to lamellar bone. Woven bone may be recognized from mature bone by appropriate staining in histology.

At the cell levels, osteoblasts and osteoclasts are respectively responsible for bone deposition or bone removal. Another type of cells, osteocytes, which are osteoblasts that have been trapped in bone matrix during tissue production, communicate through a network of canaliculi and serve as sensors of mechanical stimuli within bone tissue. Bone growth involves rapid changes in shape and in size. The dynamic of bone activity may be described by bone modeling or remodeling. The cellular interactions associated with remodeling cycles follow a sequence of four main stages, activation, resorption, reversal and formation, well described by Parfitt. In bone remodeling the activity of osteoblasts and osteoclasts is coupled, bone resorption and formation occurring at the same locations on bone surfaces. After a remodeling cycle, bone mass is preserved in normal young adult bone and tends to decline with aging. In contrast, during bone modeling, the activity of osteoblasts and osteoclasts is not coupled, formation and resorption occurring on different surfaces. In the growing fetus, modeling is supposed to be the main mechanism governing the rapid changes in bone mass and structure.

Bone formation involves a number of chemical, cellular and morphological processes, the investigation of which requires various techniques at different scales. From a chronologic point of view, histology using light microscopy has been the main technique used to describe the different stages in bone growth and the cellular mechanism involved in bone formation. Histology with appropriate staining enables the appreciation of different type of bones (woven bone, lamellar bone, calcified cartilage...) and examinations at the cell level. The characterization of the crystalline structure and the chemical composition of bone tissue requires techniques at the chemical level, such as x-ray diffraction, electron or infrared microscopy, and infrared or NMR spectroscopy. In, the ultrastructure of the initial stages of ossification in human embryos has been studied by light microscopy and scanning electron microscopy. In long bones, matrix vesicles were found amongst maturing and hy- pertrophic chondrocytes already by the 6th week of gestation. Mineralization of cartilage in long bone started in the form of hydroxyapatite (HAP) crystals within or around the matrix vesicles. The calcification rate in lumbar vertebrae in fetuses aged 17-41 weeks has been documented in. However most studies concerning the structural composition and the maturation of early calcium phosphate are still investigated and mostly in vitro or on animal models.

Nowadays, various modalities of medical imaging allow to get insight into bone formation. X-ray radiography, and Dual Energy X-ray Absorptiometry, relying on the attenuation properties of bone, were used to sequence the ossification centers and evaluate bone mineral density. Quantitative Computed Tomography (QCT) allowed to study the mineral density in developing vertebral bodies, showing an increase of bone mineral density with gestational age. However due to x-ray dose limitation, QCT cannot be used to image bone formation in vivo. In contrast, ultrasonic imaging methods which are non invasive are well adapted to the follow up of fetus development in vivo. The improvement in the quality of echographic examinations allows to use this technique to control the normality of fetuses. In particular the length of various bone may be measured from ultrasound and normal reference plots of growth are now available. Unfortunately, these techniques do not provide information on bone microarchitecture in vivo. Don't let the pharmacy companies beat you. Buy  cialis 200mg online

Quantitative data of bone microarchitecture during development are thus limited to ex-vivo analysis on post-mortem bodies and are relatively scarce due to ethical and legal reasons. Histomorphometry was used to assess trabecular microarchitecture in iliac crest bone to investigate osteopenia in preterm neonate. The development of the femoral metaphysis has been quantitatively reported in two major papers by the groups of Salle and Glorieux. These data are relatively unique in the literature and constitute a reference for architectural parameters in growing human femurs. More recently three-dimensional microtomography (micro-CT) has been used for the evaluation of trabecular bone microarchitecture in human fetal vertebral bodies. Micro-CT was recently used to study vascular invasion during the development of the secondary centres of ossification in a rabbit model but no quantification was provided.

In the second section, we shall particularly recall the quantification of bone microarchitecture based on 3D micro-CT and review the differences with respect to histomorphometry. In the third section, the results available for the femoral metaphysis and vertebral body bone microarchitecure will be reported. New data obtained from 3D quantitative analysis of micro-CT images on human femurs during gestation will be presented. Finally, the different results will be summarized and discussed in a last section.

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