Bone microarchitecture in human fetuses: Discussion

The morphometric parameters obtained on fetal vertebrae and femurs evidenced a dense trabecular structure as compared to that of young adults. The histomorphometric analysis of femoral metaphysis and the 3D micro-CT analysis of vertebral bodies were consistent and showed a significant increase of trabecular bone volume BVTV with gestational age. Three-dimensional analysis of anisotropy of cancellous bone in vertebra and in femoral metaphysis did not exhibit the same behavior at these two bone sites. The isotropy of trabecular bone in vertebral bodies demonstrated a growth that expands radially, while the anisotropy of the femoral metaphysis was related to the growth of long bone which spreads out longitudinally. In the vertebral body, cancellous bone was characterized by an inner core made of trabeculae about 100 jm in thickness, and a peripheral layer with thinner trabeculae of about 9 jm. The peripheral region is supposed to correspond to newly formed bone struts which allows the vertebral body to expand radially. In the inner core, trabecular thickness is of the same order of magnitude than in adults.

Bone microarchitecture in human fetuses: Result part 2

We present here additional unpublished results obtained on fetal femoral metaphysis. Three femurs of respective gestational age (17, 20 and 29 weeks) were obtained from the same fetuses as discussed in the previous section. They were embedded in methylmetacrylate and imaged with 3D SR micro-CT at an energy of 20 keV, and a voxel size of 10 jm. The upper limit of each reconstructed volume was chosen at the frontier between mineralized tissue bone and cartilage, the later having little contrast. Smaller VOIs avoiding periostal bone, with a total height between 2.5 and 4.5 mm, were selected in the cancellous bone region to get quantitative parameters of trabecular microarchitecture. Figure 4 illustrates a 2D transverse slice and a 3D display of the three femoral images. The 2D slices were taken at 3 mm below the top of the volumes. The 3D displays were made on the half 3D image to show the core of femurs. The 2D slices show the anisotropic elliptic shape of the sections perpendicular to the main direction of the femurs, with a large axis approximately 1,6 times larger than the small axis length. We may also note an increase of this length between 17 and 29 weeks (2.3 and 4.4 mm, respectively). The 3D displays clearly show the increase in size of the section as it goes towards the growth plate. The top of the 3D images corresponds to the cartilaginous epiphysis of the femur, which has little contrast in x- ray attenuation. Close to this region, bone trabeculae are very small, irregular, and less mineralized. Cancellous bone may clearly be seen either on the transverse sections as well as in the 3D rendering.

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.

Role of hypogonadism in development of bone alterations in thalassemic patients: HRT effects on bone mass deficiency

Because this important role of hypogonadism, we should expect that hormonal replacement therapy (HRT) corrects or prevents bone mass deficiency in adult TM patients. Conflicting results have been presented with some studies indicating a clear improvement and other giving doubtful results. In our recent study, we found that female TM patients treated with HRT still have reduced bone mass and increased bone turnover compared to normal women of similar age. However, patients who started the HRT in younger age had better results than patients who started the treatment later. Maybe, it is important to start the treatment early, at the pubertal age. On the other hand, it cannot be excluded that HRT is not sufficient because other factors contribute to the bone mass deficiency of TM patients. Consistent with our results, Lasco et al. found that bone mass, measured by DEXA, was lower in all TM patients than in controls, but the difference was more marked in patients who didn't receive HRT. We can conclude that HRT is an important 
part of the treatment of bone mass deficiency in adult TM patients but that the treatment has to be started early and that often alone is not sufficient to normalize bone mass. Because of the disappointing results of HRT on bone mass in adult TM patients, it has been suggested to associate HRT and bisphosphonates. Limited experience is available but the results of our group and of other authors suggest that the results may be better than with HRT alone.

Role of hypogonadism in development of bone alterations in thalassemic patients: Causes of hypogonadism in adult TM patients

It has been suggested that in TM patients the hypothalamus and the pituitary are damaged by the iron overload. In fact the pituitary gland is very sensitive to iron and also a modest deposition may impair its functionality. Histological studies have confirmed the damage of the pituitary gland by iron overload in TM patients and MRI has shown a signif-icantly smaller anterior pituitary volume. Other studies have shown that iron overload may also directly damage the gonads. Therefore, it may be assumed that the hypogo- nadism of adult TM patients is mainly a consequence of the iron deposit.

Some authors have reported higher ferritin levels in subjects who had hypogonadism compared to those with normal go- nadal function. Ferritin is the protein that stores iron intra- cellularly and when its capability is exceeded an excess of active iron is released and catalyses the formation of free radicals. Free radicals may damage membrane lipids, leading to mitochondrial and lysosomal damage and finally to cell death. Because of these findings it has been suggested that improvement of chelation treatments may prevent or reduce the appearance of hypogonadism in TM patients. While it may be probable, other studies did not find such correlations and our normogonadic patients had the same transfusion and chelation treatment than hypogonadic patients. Moreover, while hypogonadic patients had slightly higher ferritin circulating levels, the difference with normogonadic patients was not significant.

Role of hypogonadism in development of bone alterations in thalassemic patients

Beta thalassemia major (TM) is an important health concern in many countries, mostly in the Mediterranean area, in the Middle East, India, East Asia. In 1988 a report from the WHO documented that almost 50000 children per year were born with TM.

In the last decades, improved programs of transfusional and chelation therapy have permitted to TM patients to survive until their forties or fifties and to get a better quality of life. However, several complications progressively arise and in particular endocrine alterations are common with diabetes, hypothyroidism, hypoparathyroidism, and hypogonadism being well known complications of adult thalassemia major. More recently, in adult TM patients, bone mass deficiency has been recognised as a major problem that may cause pathologic fractures and limb deformities and greatly worsen the quality of life of these patients. The etiology of bone mass deficiency in thalassemia is still unclear and many factors have been suggested as possible causes, including IGF-I deficiency, low vitamin D levels, alterations of genes related to collagen synthesis, bone cortical thinning because of bone marrow expansion and altered levels of some oligoelements such as zinc and copper. However, a main role is probably played by hypogonadism that is a hallmark of adult TM patients. In this brief review, we will examine the characteristics of hypogonadism in TM patients and the possible link between hypogonadism and bone mass deficiency. Finally, we will discuss the role of the hormonal substitutive therapy in the prevention and treatment of bone mass deficiency in TM.

Low bone mineral density and high bone turnover in adult subjects with thalassemia major: Discussion

Discussion

In the general population osteoporosis is less common in men than in women, with the incidence of vertebral fractures being one sixth of that in women.

However, the sex difference was reversed in the thalassemia patients studied here, with men being both more commonly and more severely affected with low bone mass. This striking observation is difficult to explain. Clinical experience indicates that male thalassemia patients, particularly during their adolescent years, are less compliant than females with DFX therapy. The impact of this on peak bone mass, even with subsequent improvement in chelation compliance as they improve chela- tion therapy, may contribute to the sex difference in observed bone mineral density. However, we could find no significant correlation between severely low bone mass and serum ferritin levels, measured at the time of this study. In a study of 17 transfusion-dependent children with thalassemia also no apparent association between iron overload and vitamin D deficiency and bone disease was found. The most common endocrine disorder among our patients is hypogonadotrophic hypogonadism. In the general population hypogonadism is a well-recognized cause of overt osteoporosis and of asymptomatic osteopenia. Oestrogen replacement therapy for women is the most effective preventative measure against postmenopausal osteoporosis. The exact mechanism of action of oestrogen on bone, calcium and phosphorus metabolism has not been determined, but oestrogens appear primarily to inhibit osteoclastic activity and bone resorption. It is important to note that therapeutic correction of hypogonadism with appropriate HRT in these thalassemia patients has failed to protect them from low bone mass. In spite of regular blood transfusions the ineffective erythro- poiesis is not fully suppressed in thalassemia major. Expansion of the marrow may contribute to the decreased bone mineral density. It is also possible that excess iron in the bone may influence osteoblast number and activity and lead to the development of osteoporosis.
In the general population, osteoporosis is associated with a sedentary lifestyle, but no association with current exercise habits was apparent in this study. However, parental anxiety may have limited participation in sporting activities during childhood and it is possible that this contributed to the development of severely low bone mass.

Low bone mineral density and high bone turnover in adult subjects with thalassemia major : Patients and methods

38 TM patients (recruited from the thalassemia centre of Palermo) were studied (14 male and 24 female), age range 20-40 years old. Osteoporosis was defined using the standard World Health Organization criteria (z-score of BMD lower than 25). The BMD of the lumbar spine (L1-L4), the femoral neck and the forearm was determined by Dual Energy X-Ray Absorptiometry (DEXA; Lunar DPX plus).The PTH was studied by ELISA (Biosource, Belgium), osteocalcin, C-telopeptide and bone alkaline phosphatase by Elisa (Beckmann-Coulter USA). Analisys of variance and U test of Mann-Whitney were used for TM patients and controls. The correlation was obtained with Pearson index. P value < 0.05 was considered statistically sig¬nificant. The calculated z-scores for males and females were evaluated by normal ranges.

Low bone mineral density and high bone turnover in adult subjects with thalassemia major

Introduction

Beta Thalassemia Major (TM) affects a significant number of the population in certain areas of the world. TM is an inherited blood disorder in which the body is unable to make adequate hemoglobin. This is due to an inborn error of metabolism that leads to absence or reduced synthesis of one or more types of globin polypeptide chains of the hemoglobin molecule; is a hereditary disorder of haemoglobin synthesis re­sulting in severe anemia.