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Skeletal Development in Fetal Pig Specimens: MR Imaging of Femur with Histologic Comparison¹

¹ From the Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, Mass (S.A.C., D.J.); and Department of Radiology (S.A.C., J.K.H.) and Orthopaedic Surgery-Orthopaedic Research Laboratory (F.S.), Children’s Hospital and Harvard Medical School, 300 Longwood Ave, Boston, MA 02115. Received January 22, 2003; revision requested April 11; final revision received November 26; accepted January 29, 2004. Supported by a Children’s Hospital Research Council Award and by NIH grant AR42396–05. Address correspondence to S.A.C. (e-mail: susan.connolly@childrens.harvard.edu).

	ABSTRACT

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PURPOSE: To evaluate the magnetic resonance (MR) imaging features of the developing femur in fetal pig specimens.

MATERIALS AND METHODS: MR images of 15 fetal pig femurs, which were categorized into three groups of specimens representing each third of the gestational period, were used to compare increasing femoral length (as an indication of gestational age) with epiphyseal growth in multiple dimensions by using Pearson product moment correlation. Physeal-epiphyseal demarcation, visibility of the secondary ossification center and its physis, prominence of the perichondrial structures (ie, groove of Ranvier and bone bark), metaphyseal undulation, and corticomedullary differences were evaluated qualitatively. These features were also evaluated on histologic sections.

RESULTS: With femoral length measurements used as indications of gestational age, there were three, five, and seven fetal pig specimens in each gestation group. During fetal development, the cartilaginous epiphysis of the distal femur transformed from an oval to a bicondylar structure, with most of the growth occurring sagittally (P < .001). Physeal-epiphyseal demarcation, visibility of the secondary ossification center and its physis, and metaphyseal undulation increased later in gestation. Detection of perichondrial structures, however, was greatest during the middle third of gestation and decreased thereafter. During the fetal period, the perichondrial groove of Ranvier and the bone bark were easily identifiable at MR imaging. Marrow cavitation increased with gestation.

CONCLUSION: MR imaging depicts fetal pig skeletal features that can be confirmed histologically and that may prove to be useful at human prenatal skeletal imaging.

© RSNA, 2004

Index terms: Animals • Experimental study • Femur, MR, 451.121411, 451.121412, 451.121416 • Fetus, growth and development • Fetus, skeletal system

	INTRODUCTION

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Congenital disorders of the skeleton are frequent—with an incidence of one disorder in every 3000 births (1)—and are an important cause of lifelong deformity, disability, and early degenerative disease. A common pathway to these conditions is a deranged structure of the growth cartilage, the epiphysis, and/or the growth plate. The prenatal diagnosis of congenital skeletal anomalies, like that of central nervous system malformations, could be of great prognostic importance, and when the diagnosis is relevant, it could help in the planning of postnatal evaluations and in the treatment of these disorders. In addition, with increasing use of fetal magnetic resonance (MR) imaging, it has become important to define the characteristics and developmental changes of normal fetal structures so that early epiphyseal and physeal formation abnormalities can be detected.

The long bones undergo multiple changes before birth. The epiphyseal cartilage grows, the physis becomes progressively differentiated from the epiphyseal cartilage, and a secondary center of ossification develops. The perichondrial structures develop to provide support and axial growth. The metaphysis changes shape and undergoes marrow transformation, and the diaphyseal cortex thickens (2).

The skeletal changes that are observable prenatally have been well documented histologically and morphologically (3,4). The embryologic development of the limbs, however, has not been well studied with imaging. Ultrasonographic (US) study results have established correlations between femoral length and gestational age (5) and between gestational age and the appearance of the ossification center of the distal femur (6). US visualization of the fetal skeleton, however, is limited.

The technical considerations involved in performing MR imaging of live fetuses are different from those involved in performing MR imaging of fetal tissue specimens. As a first step toward performing prenatal skeletal MR imaging in humans, this study was undertaken to evaluate the MR imaging features of the developing femur in fetal pig specimens.

	MATERIALS AND METHODS

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Study Design
We performed high-spatial-resolution MR imaging of the femurs of 15 fetal pig specimens, with the intent to have five representative specimens from each third of the gestational period (hereafter referred to as third of gestation). The final study sample included 29 femurs, because one femur of a piglet in the first third of gestation was damaged during preparation for imaging. The mean gestational period for pigs is 114.7 days ± 6 (standard error of the mean), or 4 months and 3 days (7); thus, each third of gestation lasts 1 month 10 days. For the fetal pig, there is a linear relationship between gestational age and measured femoral length (8), so femoral length can be used as an indication of gestational age. One millimeter of femoral growth after the first third of gestation is roughly equivalent to 2.4 days of gestation.

Fetal pig specimens were purchased from a specialized vendor (Pel-Freez, Rogers, Ariz), which provided fetuses that were at each third of gestation. The specimens had been frozen immediately after being removed from the sows. The animal care and use committee of Children’s Hospital, Boston approved the study.

Fetal age was determined in consensus (by S.A.C. and J.K.H.) on the basis of the length of the femoral bone measured on MR images. We also recorded the fetal weight and compared it with the femoral length measurement. The left femur was measured. The left femur was decided on arbitrarily and was used consistently throughout the study.

MR Imaging
Approximately 6 hours before MR imaging, the frozen fetal pig specimens were thawed by means of immersion in normal saline at room temperature. Once each fetal carcass had reached room temperature—that is, after approximately 6 hours—the specimen was removed from the saline and placed in the isocenter of the magnet to maximize the signal-to-noise ratio. MR imaging was performed with a 1.5-T system (Horizon; GE Medical Systems, Milwaukee, Wis) by using a dedicated wrist coil (Medical Advances, Milwaukee, Wis). The coil enabled simultaneous examination of both femurs, regardless of the gestational age of the fetal specimen.

An initial intermediate-weighted fast spin-echo (1300/11 [repetition time msec/echo time msec], echo train length of eight) localizer MR imaging sequence was performed. Then, oblique longitudinal MR images encompassing the proximal and distal portions of the femoral epiphysis simultaneously were obtained in the coronal and sagittal planes. These images were obtained by using T1-weighted spin-echo (300/25), intermediate-weighted fast spin-echo (2000/15, echo train length of eight), T2-weighted (2000/80, echo train length of eight), and gradient-recalled-echo (300/13, 30° flip angle) sequences.

MR imaging parameters included a 7-cm field of view, a 2.0-mm section thickness, no intersection gap, a matrix size of 512 x 512, and four signals acquired (with an in-plane spatial resolution of 137 µm). The imaging time for each of the high-spatial-resolution intermediate- and T2-weighted sequences was 9 minutes. The imaging time for the T1-weighted sequences was 6 minutes. Initial examinations of two fetal pig specimens—one in the middle third and one in the last third of gestation—that were performed by using single-shot fast spin-echo MR imaging (echo time, 120 msec) failed to yield adequate contrast between the epiphysis and the adjacent structures, so this sequence was not included in the protocol.

Histologic Analysis
Histologic examinations of the femurs of six fetal pig specimens—two from each third-of-gestation group—that were randomly chosen were performed by using a technique similar to that used in a prior study of epiphyseal and long-bone embryology (3). The histologic specimens were prepared by F.S. at the Orthopaedic Research Laboratory of Children’s Hospital, Boston. After the MR imaging examinations, the six excised femurs were fixed in 10% neutral buffered formalin (Stephens Scientific, Riverdale, NJ) for 2 weeks and decalcified in 25% formic acid (Fisher Scientific, Fair Lawn, NJ) for an additional 3 weeks. Intact femurs from the first-third-of-gestation group were sectioned. In the older-fetus femurs, the proximal and distal epiphyseal-metaphyseal regions, once soft, were removed from the diaphysis by using a sharp scalpel and sectioned in the midcoronal or midsagittal planes. The remaining metaphyseal-diaphyseal-metaphyseal segment was also processed for sectioning in the coronal plane. The tissues were then embedded in JB4 plastic (Polysciences, Warrington, Pa), cut into 5-µm-thick sections, and stained with 1% toluidine blue (Sigma-Aldrich, St Louis, Mo).

MR Image Analyses
Quantitative and qualitative analyses of the MR images were performed by observers who were blinded to the gestational ages. During the initial reading session, two pediatric radiologists (S.A.C. and D.J.), who, respectively, had 11 and 15 years experience in MR imaging, and a medical student (J.K.H.) jointly measured the structures on one specimen from each third of gestation. Subsequent measurements of all of the fetal pig femur specimens were then performed by the medical student and one of the pediatric radiologists (S.A.C.) in consensus. On the MR images, the observers jointly measured the femoral bone length from the proximal to the distal portion of the metaphysis, and the cartilaginous epiphyseal dimensions (ie, midsagittal plane, midcoronal plane, and proximal-to-distal height) of the proximal and distal portions of the epiphysis. Growth in the proximal and distal regions of the femur was plotted against femoral bone length.

Qualitative analysis of the MR images was performed by one pediatric radiologist (S.A.C.) with experience in MR imaging. The proximal and distal portions of the femoral epiphysis, physis, and metaphysis, as well as the diaphysis, were evaluated according to the shape, visibility, or demarcation of various structures, as outlined in Table 1.

Histologic sections were compared with corresponding MR images of femoral specimens from the three gestational period groups. For each main developmental feature seen, the third of gestation in which the structure was best seen was determined independently by the orthopedic surgeon and bone histologist (F.S.), who analyzed the histologic sections, and by the pediatric radiologist (S.A.C.) and the medical student (J.K.H.), who analyzed the MR images.

Statistical Analyses
We calculated the Pearson product moment correlation coefficient to assess the correlations between changes in femoral length and various piglet features, including weight and cartilaginous dimensions of the proximal and distal portions of the epiphysis (midsagittal plane, midcoronal plane, and superior-to-inferior height). The R² value obtained and the significance of this value were calculated by using STATA statistical software (STATA, College Station, Tex). P < .05 was considered to indicate a significant difference. The R² value indicates the proportion of the variance in epiphyseal characteristics that is attributable to the variance in femoral length.

	RESULTS

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MR Imaging
Measurements.—According to femoral length measurements, three fetal pigs had been younger than 40 gestational days (femur shorter than 6 mm), seven had been aged between 40 and 80 gestational days (femur shorter than 20 mm), and five had been aged between 80 and 100 gestational days (femur shorter than 32 mm) (7). For the femur, there was a direct linear relationship between bone length and fetal weight and between bone length and total femoral length (ie, the bone plus the cartilaginous ends). The sagittal (anterior-to-posterior) epiphyseal dimension increased the most with increasing femoral length; the coronal (right-to-left) and then the proximal-to-distal dimensions followed (Fig 1). Similarly, in the proximal epiphysis, the coronal dimension increased more than the proximal-to-distal dimension. The sagittal dimension increase was considerably greater in the distal portion than in the proximal portion of the epiphysis. Epiphyseal dimensions changed linearly with femoral length (Table 2).

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Results of quantitative analysis of changes in epiphyseal dimensions with increasing femoral length—specifically, growth of the distal femoral epiphysis. Graph data demonstrate that size change along sagittal (anterior-to-posterior) plane is much greater than that along other dimensions. The smallest change is that in total epiphyseal height (proximal-to-distal).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Coronal and sagittal outlines of the left distal femoral epiphysis created by tracing the maximal epiphyseal perimeter on T2-weighted fast spin-echo MR images (2000/80; echo train length of eight). The outlines are grouped by thirds of gestation. The femoral epiphysis is nearly spherical early in gestation and is hemispheric during the middle of the second third of gestation. Note the presence of the intercondylar notch late in gestation.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 3a. Graphs illustrate visibility of distal femoral structures with increasing femoral length (as indication of gestational age). (a) Comparison between development of secondary ossification center and visibility of surrounding spherical growth plate. Graph shows that as the ossification center increases in size, the spherical growth plate becomes more evident. (b) Visibility of physeal-epiphyseal demarcation with increasing gestational age. Graph shows that the demarcation increases to a peak level during the second third of gestation and either plateaus or decreases slightly thereafter. (c) Comparison between development of secondary ossification center and undulation of adjacent metaphysis. Graph shows that as the ossification center increases and abuts the physis to become hemispheric, the contour of the adjacent metaphysis becomes progressively undulated. Both the ossification center grade and the metaphyseal shaping grade increase with bone length (ie, gestational age). (d) Visibility of perichondrial structures with increasing gestational age. Graph shows that perichondrial structures are most detectable during the second third of gestation and decrease in visibility thereafter.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 3b. Graphs illustrate visibility of distal femoral structures with increasing femoral length (as indication of gestational age). (a) Comparison between development of secondary ossification center and visibility of surrounding spherical growth plate. Graph shows that as the ossification center increases in size, the spherical growth plate becomes more evident. (b) Visibility of physeal-epiphyseal demarcation with increasing gestational age. Graph shows that the demarcation increases to a peak level during the second third of gestation and either plateaus or decreases slightly thereafter. (c) Comparison between development of secondary ossification center and undulation of adjacent metaphysis. Graph shows that as the ossification center increases and abuts the physis to become hemispheric, the contour of the adjacent metaphysis becomes progressively undulated. Both the ossification center grade and the metaphyseal shaping grade increase with bone length (ie, gestational age). (d) Visibility of perichondrial structures with increasing gestational age. Graph shows that perichondrial structures are most detectable during the second third of gestation and decrease in visibility thereafter.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 3c. Graphs illustrate visibility of distal femoral structures with increasing femoral length (as indication of gestational age). (a) Comparison between development of secondary ossification center and visibility of surrounding spherical growth plate. Graph shows that as the ossification center increases in size, the spherical growth plate becomes more evident. (b) Visibility of physeal-epiphyseal demarcation with increasing gestational age. Graph shows that the demarcation increases to a peak level during the second third of gestation and either plateaus or decreases slightly thereafter. (c) Comparison between development of secondary ossification center and undulation of adjacent metaphysis. Graph shows that as the ossification center increases and abuts the physis to become hemispheric, the contour of the adjacent metaphysis becomes progressively undulated. Both the ossification center grade and the metaphyseal shaping grade increase with bone length (ie, gestational age). (d) Visibility of perichondrial structures with increasing gestational age. Graph shows that perichondrial structures are most detectable during the second third of gestation and decrease in visibility thereafter.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 3d. Graphs illustrate visibility of distal femoral structures with increasing femoral length (as indication of gestational age). (a) Comparison between development of secondary ossification center and visibility of surrounding spherical growth plate. Graph shows that as the ossification center increases in size, the spherical growth plate becomes more evident. (b) Visibility of physeal-epiphyseal demarcation with increasing gestational age. Graph shows that the demarcation increases to a peak level during the second third of gestation and either plateaus or decreases slightly thereafter. (c) Comparison between development of secondary ossification center and undulation of adjacent metaphysis. Graph shows that as the ossification center increases and abuts the physis to become hemispheric, the contour of the adjacent metaphysis becomes progressively undulated. Both the ossification center grade and the metaphyseal shaping grade increase with bone length (ie, gestational age). (d) Visibility of perichondrial structures with increasing gestational age. Graph shows that perichondrial structures are most detectable during the second third of gestation and decrease in visibility thereafter.

View larger version (68K): [in this window] [in a new window] [Download PPT slide]   Figure 4a. Features of the porcine femur during first third of gestation (as indicated by 4-mm femoral length). (a) Coronal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) shows very low signal intensity in ossified shaft. The bone bark is well seen as a spicule (arrow) extending beyond the shaft. There is no evidence of demarcation between the cartilage of the physis and that of the epiphysis, and the muscle is isointense to the cartilage. (b) Coronal histologic section of the distal femur shows nonossified epiphyseal femoral cartilage (e), poor physeal-epiphyseal demarcation, and clearly discernible bone bark (arrows). (Toluidine blue stain; original magnification, x60.)

View larger version (101K): [in this window] [in a new window] [Download PPT slide]   Figure 4b. Features of the porcine femur during first third of gestation (as indicated by 4-mm femoral length). (a) Coronal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) shows very low signal intensity in ossified shaft. The bone bark is well seen as a spicule (arrow) extending beyond the shaft. There is no evidence of demarcation between the cartilage of the physis and that of the epiphysis, and the muscle is isointense to the cartilage. (b) Coronal histologic section of the distal femur shows nonossified epiphyseal femoral cartilage (e), poor physeal-epiphyseal demarcation, and clearly discernible bone bark (arrows). (Toluidine blue stain; original magnification, x60.)

View larger version (83K): [in this window] [in a new window] [Download PPT slide]   Figure 5a. Porcine femoral changes during second third of gestation (as indicated by 15-25-mm femoral length). (a) Coronal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) shows entire diaphysis has become ossified. The perichondrial structures are depicted much better: The bone bark (straight arrow) is readily identified laterally, and the perichondrial groove (curved arrow) is seen medially. (b) Sagittal fast spin-echo T2-weighted image (2000/80, echo train length of eight) obtained in another fetal pig shows further physeal (straight arrow)-epiphyseal (e) demarcation. The perichondrium is very distinct distally and can also be identified in the proximal femur (curved arrow). (c) Histologic section of anteromedial aspect of proximal femur of same fetal pig as in b shows prominent bone bark (white arrows) and deepest region of the perichondrial groove (black arrow), with multiple osteocytes surrounding the growth plate. (Toluidine blue stain; original magnification, x60.)

View larger version (110K): [in this window] [in a new window] [Download PPT slide]   Figure 5b. Porcine femoral changes during second third of gestation (as indicated by 15-25-mm femoral length). (a) Coronal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) shows entire diaphysis has become ossified. The perichondrial structures are depicted much better: The bone bark (straight arrow) is readily identified laterally, and the perichondrial groove (curved arrow) is seen medially. (b) Sagittal fast spin-echo T2-weighted image (2000/80, echo train length of eight) obtained in another fetal pig shows further physeal (straight arrow)-epiphyseal (e) demarcation. The perichondrium is very distinct distally and can also be identified in the proximal femur (curved arrow). (c) Histologic section of anteromedial aspect of proximal femur of same fetal pig as in b shows prominent bone bark (white arrows) and deepest region of the perichondrial groove (black arrow), with multiple osteocytes surrounding the growth plate. (Toluidine blue stain; original magnification, x60.)

View larger version (117K): [in this window] [in a new window] [Download PPT slide]   Figure 5c. Porcine femoral changes during second third of gestation (as indicated by 15-25-mm femoral length). (a) Coronal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) shows entire diaphysis has become ossified. The perichondrial structures are depicted much better: The bone bark (straight arrow) is readily identified laterally, and the perichondrial groove (curved arrow) is seen medially. (b) Sagittal fast spin-echo T2-weighted image (2000/80, echo train length of eight) obtained in another fetal pig shows further physeal (straight arrow)-epiphyseal (e) demarcation. The perichondrium is very distinct distally and can also be identified in the proximal femur (curved arrow). (c) Histologic section of anteromedial aspect of proximal femur of same fetal pig as in b shows prominent bone bark (white arrows) and deepest region of the perichondrial groove (black arrow), with multiple osteocytes surrounding the growth plate. (Toluidine blue stain; original magnification, x60.)

View larger version (82K): [in this window] [in a new window] [Download PPT slide]   Figure 6a. Porcine femoral changes during last third of gestation (as indicated by >25-mm femoral length). (a) Sagittal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) of distal femur in region of lateral condyle shows further growth of secondary center of ossification (*). There is good physeal-epiphyseal demarcation (straight arrows). The contour of the ossification center is parallel with the adjacent metaphysis. The bone bark (curved arrow) has become less prominent. In the proximal femur, the femoral epiphysis and the greater trochanter are now separate. (b) On histologic section, the proximal femur shows differential staining between epiphyseal cartilage (e) and physeal cartilage (p). (Toluidine blue stain; original magnification, x60.)

View larger version (103K): [in this window] [in a new window] [Download PPT slide]   Figure 6b. Porcine femoral changes during last third of gestation (as indicated by >25-mm femoral length). (a) Sagittal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) of distal femur in region of lateral condyle shows further growth of secondary center of ossification (*). There is good physeal-epiphyseal demarcation (straight arrows). The contour of the ossification center is parallel with the adjacent metaphysis. The bone bark (curved arrow) has become less prominent. In the proximal femur, the femoral epiphysis and the greater trochanter are now separate. (b) On histologic section, the proximal femur shows differential staining between epiphyseal cartilage (e) and physeal cartilage (p). (Toluidine blue stain; original magnification, x60.)

View larger version (82K): [in this window] [in a new window] [Download PPT slide]   Figure 7a. Coronal fast spin-echo T2-weighted MR images (2000/80, echo train length of eight) show changes in distal femoral contour with gestational age (at 4-36-mm femoral length). (a) Image of femur at first third of gestation shows straight metaphyseal (m) contour with a bone bark (arrow) but not a well-defined perichondrial groove. There is no distal femoral secondary ossification center, and the contour of the distal femoral epiphysis is smooth. (b) Image of femur at second third of gestation shows well-defined perichondrial groove (curved arrow), which outlines the contour of the physis. The perichondrial structures are well defined. (c) Image of another femur at second third of gestation shows metaphyseal undulation (m) concurrently with secondary ossification center (*). The perichondrial structures are still well seen, and there is good physeal-epiphyseal demarcation. Bone bark is less apparent. (d) Image of femur at last third of gestation shows distal femur ossification now occupying a substantial portion of the epiphysis and abutting the metaphysis, which has a central undulation. The perichondrial structures are no longer apparent.

View larger version (85K): [in this window] [in a new window] [Download PPT slide]   Figure 7b. Coronal fast spin-echo T2-weighted MR images (2000/80, echo train length of eight) show changes in distal femoral contour with gestational age (at 4-36-mm femoral length). (a) Image of femur at first third of gestation shows straight metaphyseal (m) contour with a bone bark (arrow) but not a well-defined perichondrial groove. There is no distal femoral secondary ossification center, and the contour of the distal femoral epiphysis is smooth. (b) Image of femur at second third of gestation shows well-defined perichondrial groove (curved arrow), which outlines the contour of the physis. The perichondrial structures are well defined. (c) Image of another femur at second third of gestation shows metaphyseal undulation (m) concurrently with secondary ossification center (*). The perichondrial structures are still well seen, and there is good physeal-epiphyseal demarcation. Bone bark is less apparent. (d) Image of femur at last third of gestation shows distal femur ossification now occupying a substantial portion of the epiphysis and abutting the metaphysis, which has a central undulation. The perichondrial structures are no longer apparent.

View larger version (88K): [in this window] [in a new window] [Download PPT slide]   Figure 7c. Coronal fast spin-echo T2-weighted MR images (2000/80, echo train length of eight) show changes in distal femoral contour with gestational age (at 4-36-mm femoral length). (a) Image of femur at first third of gestation shows straight metaphyseal (m) contour with a bone bark (arrow) but not a well-defined perichondrial groove. There is no distal femoral secondary ossification center, and the contour of the distal femoral epiphysis is smooth. (b) Image of femur at second third of gestation shows well-defined perichondrial groove (curved arrow), which outlines the contour of the physis. The perichondrial structures are well defined. (c) Image of another femur at second third of gestation shows metaphyseal undulation (m) concurrently with secondary ossification center (*). The perichondrial structures are still well seen, and there is good physeal-epiphyseal demarcation. Bone bark is less apparent. (d) Image of femur at last third of gestation shows distal femur ossification now occupying a substantial portion of the epiphysis and abutting the metaphysis, which has a central undulation. The perichondrial structures are no longer apparent.

View larger version (69K): [in this window] [in a new window] [Download PPT slide]   Figure 7d. Coronal fast spin-echo T2-weighted MR images (2000/80, echo train length of eight) show changes in distal femoral contour with gestational age (at 4-36-mm femoral length). (a) Image of femur at first third of gestation shows straight metaphyseal (m) contour with a bone bark (arrow) but not a well-defined perichondrial groove. There is no distal femoral secondary ossification center, and the contour of the distal femoral epiphysis is smooth. (b) Image of femur at second third of gestation shows well-defined perichondrial groove (curved arrow), which outlines the contour of the physis. The perichondrial structures are well defined. (c) Image of another femur at second third of gestation shows metaphyseal undulation (m) concurrently with secondary ossification center (*). The perichondrial structures are still well seen, and there is good physeal-epiphyseal demarcation. Bone bark is less apparent. (d) Image of femur at last third of gestation shows distal femur ossification now occupying a substantial portion of the epiphysis and abutting the metaphysis, which has a central undulation. The perichondrial structures are no longer apparent.

Physeal-epiphyseal demarcation was evident in conjunction with well-defined perichondrial structures (Figs 4b, 7b). The perichondrial groove of Ranvier and a low-signal-intensity linear area representing the combination of the bone bark and the overlying fibrous layer of the perichondrium were seen on MR images during the second third of gestation. The physis of the secondary center of ossification was detectable on MR images as a thin layer of uniform thickness around the secondary center of ossification (Fig 7c).

In the fetal pig specimens that were imaged before the appearance of the epiphyseal ossification center, high-signal-intensity changes in the center of the epiphyseal cartilage, which is sometimes referred to as the preossification center, were seen. The physis of the secondary center was detectable approximately 2 weeks (femoral length, 5 mm) (Fig 3a) after the appearance of this preossification center during the second third of gestation, and it became more distinct throughout the remainder of the gestational period.

During the first and second thirds of gestation, the medullary space of the femur was hypointense relative to muscle at all MR imaging sequences and became progressively less hypointense and eventually isointense relative to muscle during the later stages of gestation. The medullary space, which had homogeneous low signal intensity during the first third of gestation, showed low-signal-intensity cones in the metaphyses, between the lower signal intensity diaphyseal cortex, with the cone apices directed toward the diaphysis during the second third of gestation (Fig 8a). During the latter part of the last third of gestation, at the cone apices, a marrow cavity with slightly higher signal intensity than the cones was seen on T1- and T2-weighted MR images (Fig 8b).

View larger version (56K): [in this window] [in a new window] [Download PPT slide]   Figure 8a. Medullary space of the shaft. (a) Sagittal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) of femur at second third of gestation (as indicated by 17-mm femoral length) shows conal appearance of medullary space (*) between the diaphyseal cortices, which have lower signal intensity. (b) Sagittal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) of femur at last third of gestation (as indicated by 32-mm femoral length) shows central cavitation at apices of the cones (arrows). (c) Sagittal histologic section obtained at last third of gestation (as indicated by 32-mm femoral length) from the same femur as in b shows metaphysis-diaphysis-metaphysis complex, with metaphyseal cones (m) (ie, enchondral bone), diaphyseal cortices (d), and central cavitation (*). (Toluidine blue stain; original magnification, x10.)

View larger version (94K): [in this window] [in a new window] [Download PPT slide]   Figure 8b. Medullary space of the shaft. (a) Sagittal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) of femur at second third of gestation (as indicated by 17-mm femoral length) shows conal appearance of medullary space (*) between the diaphyseal cortices, which have lower signal intensity. (b) Sagittal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) of femur at last third of gestation (as indicated by 32-mm femoral length) shows central cavitation at apices of the cones (arrows). (c) Sagittal histologic section obtained at last third of gestation (as indicated by 32-mm femoral length) from the same femur as in b shows metaphysis-diaphysis-metaphysis complex, with metaphyseal cones (m) (ie, enchondral bone), diaphyseal cortices (d), and central cavitation (*). (Toluidine blue stain; original magnification, x10.)

View larger version (93K): [in this window] [in a new window] [Download PPT slide]   Figure 8c. Medullary space of the shaft. (a) Sagittal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) of femur at second third of gestation (as indicated by 17-mm femoral length) shows conal appearance of medullary space (*) between the diaphyseal cortices, which have lower signal intensity. (b) Sagittal fast spin-echo T2-weighted MR image (2000/80, echo train length of eight) of femur at last third of gestation (as indicated by 32-mm femoral length) shows central cavitation at apices of the cones (arrows). (c) Sagittal histologic section obtained at last third of gestation (as indicated by 32-mm femoral length) from the same femur as in b shows metaphysis-diaphysis-metaphysis complex, with metaphyseal cones (m) (ie, enchondral bone), diaphyseal cortices (d), and central cavitation (*). (Toluidine blue stain; original magnification, x10.)

Histologic Analysis
First third of gestation.—Histologic sections of the entire femur showed development of the primary ossification center and its extension toward a well-defined physis at the proximal and distal ends. Metaphyseal endochondral bone was present at both ends, but marrow cavitation was slight. Secondary ossification centers had not yet developed in the epiphysis. In the distal epiphysis, only a few cartilage canals were seen primarily posteriorly; in the proximal epiphysis, there was very early development of the lateral epiphyseal vessels passing into the cartilage mass of the proximal femoral epiphysis. There was no demarcation of cartilage staining between the physeal and the adjacent epiphyseal cartilages. A prominent perichondrial bone bark was seen proximally and distally as a continuation of the intramembranous bone formation from periosteal tissues. The perichondrial bone ensheathed the physeal cartilage beyond the hypertrophic zone and was adjacent to the zone of proliferating cartilage and sometimes to the reserve zone (Fig 4b).

Second third of gestation.—Both portions of the epiphysis were larger, and the cartilage canals were abundant in the distal epiphysis but slightly less abundant in the proximal epiphysis. The physeal cartilage was more densely stained with toluidine blue than was the adjacent epiphyseal cartilage. The perichondrial bone bark was thinner and shorter, particularly at the lower end, being primarily present adjacent to the hypertrophic zone of the physis. The bone bark and the groove of Ranvier were covered by the thick outer fibrous layer of the perichondrium, which was composed of parallel fibrous bundles that inserted beyond the groove into the epiphyseal cartilage (Fig 5c). The diaphyseal cortices were well developed, and marrow cavitation was prominent. The secondary ossification center had formed distally but not proximally.

Last third of gestation.—The perichondrial bone bark became even thinner and shorter and was markedly less prominent than it was in the first third of gestation. There was increasingly clear physeal-epiphyseal cartilage delineation at histochemical analysis, with the physeal cartilage showing much greater uptake of the toluidine blue stain (Fig 6b). In the section of the proximal femur that included the adjacent developing pelvis, this physeal-epiphyseal cartilage demarcation was seen not only at the proximal femoral epiphysis but also in the developing bone centers of the acetabular bone–cartilage interface. A well-formed secondary ossification center had developed in the distal femur, but no ossification center had developed in the proximal femur. The diaphyseal cortices were well developed, and marrow cavitation was seen in the diaphysis and the metaphysis (Fig 8c).

Comparison between Histologic and MR Image Findings
Data in Table 3 show correlations between the MR imaging and histologic depictions of porcine fetal femur development. Although histologically, the perichondrial structures were largest relative to the epiphysis during the first third of gestation, they were depicted best at MR imaging during the second third of gestation. Physeal-epiphyseal demarcation improved with gestation histologically but not at MR imaging. The preossification center (ie, hypertrophic cartilage), the secondary center of ossification, and the spherical growth plate all were seen well with MR imaging, but the cartilage canals could not be detected on MR images. No cortical-medullary demarcation was depicted at MR imaging during the first third of gestation. The bone signal intensity was uniform, and histologically it showed cortical and endochondral metaphyseal bone but little marrow cavitation. By the second and last thirds of gestation, MR imaging depicted a distinct cortex and marrow cavity; these features correlated exactly with the histologic findings.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Given the linear correlation between ossified femoral diaphyseal-metaphyseal length and gestational age (8), we considered it appropriate to use femoral length as the main indicator of gestational age, although weight would have been adequate (Fig 1). The three linear measurements illustrated in Figure 1 demonstrate that the growth is not circumferential and that some dimensions grow faster than others. The development of the femur in pigs, as compared with that in humans, is substantially more complete at birth. For example, the proximal femoral ossification center, which appears in humans at approximately the 5th month after birth, is present in newborn piglets. Therefore, evaluation of porcine prenatal skeletal development includes assessment of the developmental features that are seen in humans several months after birth.

T2-weighted MR imaging sequences depicted changes in the contour and dimensions of the epiphysis. MR imaging results showed that the fetal cartilaginous epiphysis evolves from a rounded structure to a specialized joint structure that has a shape similar to that in adults (10,12). As the epiphyseal ossification center expands, the growth of the adjacent central part of the physis slows down. This leads to a hemispheric secondary center adjacent to a concavity in the center of the metaphysis. The perichondrial structures are most prominent in the first and second thirds of gestation and later become less apparent at the proximal and distal ends. The marrow cavity becomes more distinct as the pregnancy progresses.

The epiphysis and the physis are composed of hyaline cartilage. Chondrocytes are embedded in a matrix that is composed of water, collagen fibrils, proteoglycans, and glycoproteins. In the developing fetus, the physis is more cellular and has poorly defined cell columns. When the physis is first identified on MR images, it has a slightly lower signal intensity than the epiphysis on T2-weighted images (Fig 7b). The signal intensity of the physis changes once the secondary center forms, and the physis becomes slightly hyperintense compared with the epiphyseal cartilage on T2-weighted images (Fig 7c). T2 values in cartilage have been shown experimentally (13) to change according to collagen content. As cartilage matures, there is a global increase in collagen and a decrease in proteoglycans, with the cartilage matrix becoming more fibrotic (13). This phenomenon may account for the changes in signal intensity with time.

The fetal physis also decreases in thickness as a result of cell and matrix losses. The postnatal physis contains more matrix and has a highly organized columnar structure (14).

The appendicular and axial skeletons have a major component formed by means of endochondral ossification. MR imaging and histologic analysis reveal that the shaft of the fetal femoral bone is made up of two cones joined together at the apex. Ogden (2) found that these cones represent endochondral bone that is formed by the physis. These cones are best seen in aquatic, non–weight-bearing mammals; perhaps the fetal environment of humans resembles the fetal environment of these mammals. An envelope of bone formed by means of membranous ossification surrounds the cones and gives the bone its cylindric shape (2). Eventually there is cavitation at the apices of the cones and formation of a marrow cavity.

In children, MR imaging readily depicts maturational changes in the gross morphologic features of the epiphyseal cartilage. The demonstration of such changes in the human fetus is much more challenging, but if successful, it may contribute substantially to the evaluation of normal prenatal skeletal development (15) and congenital musculoskeletal disorders. For example, MR imaging is becoming useful in the prenatal evaluation of skeletal dysplasias (16) and congenital limb deficiencies and in the quantification of fetal bone marrow (17). The demonstration of changes in the cartilage structure and in newly formed bone requires higher spatial resolution at MR imaging than is currently used in routine clinical examinations. The perichondrial structures are easily recognizable in the pig fetus at MR imaging. The depiction of these structures may become relevant in infants with disorders involving the perichondrium, including hereditary multiple exostoses, intrauterine fractures in osteogenesis imperfecta, and other skeletal dysplasias.

Our study data are limited because the number of fetal pig specimens was small and reader agreement was not assessed. The experiments were performed in dead pig fetuses. Although this protocol yielded images with fewer artifacts and much greater spatial resolution, the MR imaging characteristics of some structures may change after death. There is evidence, however, that reliable postmortem data can be obtained from MR images of the immature musculoskeletal system (18).

Our MR imaging protocol yielded limited spatial resolution on the images obtained in very small pig fetuses in the first third of gestation, and it was extremely difficult to determine the sex of the fetuses reliably. The MR imaging sequences used in the present study were designed to maximize spatial resolution at the expense of increased imaging time; clearly, these sequences are not appropriate for imaging fetuses in utero. We had no success in visualizing musculoskeletal structures with the single-shot fast spin-echo MR imaging sequence, the pulse sequence most frequently used for prenatal imaging. Single-shot fast spin-echo MR imaging yields minimal contrast between cartilage and surrounding structures such as bone and muscle. It will be necessary to develop sequences other than single-shot fast spin-echo that are fast and yet better at depicting cartilaginous and bone detail. Levine et al (19) observed preliminarily that use of a water excitation MR imaging sequence led to substantially improved depiction of skeletal structures. Other promising techniques may be those involving the use of total body MR imaging, such as turbo short inversion time inversion-recovery or turbo fast spin-echo imaging (20,21).

In summary, MR imaging enables visualization of maturational changes in the fetal pig skeleton that previously were demonstrable primarily at histologic analysis. MR imaging evaluation of these changes may be useful in improving the determination of gestational age, evaluating fetal maturity, and enabling the early detection of focal or generalized abnormalities of the fetal skeleton.

Practical applications: MR imaging of fetal pig specimens reveals prenatal developmental features of the epiphysis, physis, perichondrium, and metaphysis. These structures at the ends of bones undergo multiple changes during pregnancy; when they develop abnormally, lifelong skeletal deformities can ensue. Although current technologies do not enable the visualization of these structures in vivo, in the future, optimized MR imaging techniques may enable the detection of these structures in live fetuses. Prenatal diagnosis of skeletal abnormalities may be important for establishing the prognosis and planning the therapy of patients who have generalized diseases such as skeletal dysplasias or focal anomalies such as club feet, hip dysplasias, and limb deficiencies.

	FOOTNOTES

 
Author contributions: Guarantors of integrity of entire study, S.A.C., D.J.; study concepts and design, S.A.C., D.J.; literature research, S.A.C., D.J., F.S.; experimental studies, S.A.C., D.J.; data acquisition, S.A.C., D.J., F.S.; data analysis/interpretation, all authors; manuscript preparation, editing, and revision/review, S.A.C., D.J., F.S.; manuscript definition of intellectual content and final version approval, S.A.C., D.J.

Authors stated no financial relationship to disclose.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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