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.
Table I - Histomorphometric parameters in femoral metaphysis
16-27 weeks | 28-33 weeks | 34-41 weeks | |
Gestational Age | 20.9 ± 3.4 | 29.2± 1.7 | 38.1± 2.3 |
BV/TV (%) | 24.0 ± 4.5 | 32.2 ± 6.3 | 33.6 ± 5.1 |
Tb.Th (jim) | 71 ± 14 | 98 ± 15 | 93 ± 9 |
Tb.N (/mm) | 3.4 ± 0.4 | 3.3 ± 0.5 | 3.6 ± 0.4 |
The only study, to our knowledge, related to the three-dimensional evaluation of bone microarchitecture in fetal bone was reported in one of our previous works. The microarchitecture in vertebral body was evaluated by using 3D SR mi- cro-CT at the ESRF (European Synchrotron Radiation Facility). Twenty-two lumbar vertebral bodies from preterm human stillborns with gestational age ranging between 16 to 26 weeks were studied. Cubic bone samples with a side of 7 and 2 mm were cut, embedded in methylmetacrylate and imaged using SR micro-CT. The samples were imaged both with a pixel size on the detector of 10.13 jim and 1.8 jim. The highest spatial resolution required to image the samples with a smaller transverse section (diameter < 2 mm) to avoid local tomography. The x-ray beam was monochromatic with an energy of 20 keV.
Figure 1 - Evolution of femur length with gestational ages. The dots correspond to the age of femurs examined in section III.2.
A 2D slice and a 3D display of the image of a vertebra sample (23 weeks) at the 10 jim scale are illustrated in Figure 2. The 2D slice in Figure 2, left, has a thickness of 10 jim and is similar to what may be seen in histology textbooks. When observed in three-dimensional space (Figure 2, right), a dense and isotropic 3D structure, with a peripheral part made of very thin bone struts, is clearly seen. These bone struts are not well rendered in these images, and were accurately studied in the images acquired at higher spatial resolution (voxel size 1.8 jm). Figure 3, left, illustrates a slice in the 3D micro-CT image acquired at the 2 jim scale. On the left side of this section, the small struts are well resolved, and on the right side, osteoctye lacunae may be observed in the bone structure. These observations were confirmed by comparing the micro-CT image to an histological slice. For this purpose, a 8 jim-thick section of the sample was stained with solochrome cyanin R and observed under light microscopy. The histological image matched to the micro-CT slice is presented in Figure 3 right (magnification 2). It shows organic tissue (cartilage) in pink, mineral tissue in blue, and calcified cartilage in light blue. At higher magnification, a high density of irregular osteocyte lacunae generally related to immature bone was observed. The examination of the histological slice under polarized light did not exhibit the presence of lamellar bone.
Figure 2 - Micro-CT image (voxel size: 10 jim) of cancellous bone in the vertebral body of a human fetus (GA = 23 weeks). Left: 2D slice (thickness = 10 jim), right: 3D display.
Table II - 3D morphometric parameters in vertebral bodies.
Internal Bone Region | External Bone Region | ||||
BV/TV (%) | TbTh* (jim) | TbSp* (jim) | TbTh* (jim) | TbSp* (jim) | |
Min/Max | 29.8 / 54 | 84.2 / 118.2 | 155.2 / 321.2 | 9.47 / 9.82 | 23.3 / 29.9 |
Mean (Std) | 40.1 (10.1) | 102.3 (11.3) | 224.5 (63.8) | 9.64 (1.17) | 25.8 (3.1) |
Quantitative morphologic parameters were then computed from volumes of interest (VOIs) extracted from the 3D images. The 10 jim scale was used to quantify the inner region as seen in the center of Figure 2, and the 2 jim scale for the quantification of the peripheral region. The main results are recalled in Table II. Partial bone volume, BV/TV, ranged between 30% and 55%, and a significant increase with gestational age was found. The mean trabecular thickness, in the inner region was around 100 jim, and no significant variation with aging in 16-26 weeks period was found. In the peripheral region, the bone thickness was around 9 jm, corresponding to forming trabecu- lae. The presence of crystalline form of HAP was also assessed by synchrotron radiation diffraction. The size of crystal was found to increase significantly with gestational age. Femoral metaphysis.
Figure 3 - Left: 2D slice of a micro-CT image (voxel size: 1.8 jim) of cancellous bone in the vertebral body of a human fetus (GA = 19 weeks), right: corresponding histologic section stained with solochrome cyanin R. The same features may be recognized.
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