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1 Department of Radiology, Harvard Medical School and Massachusetts General
Hospital, 32 Fruit St., Boston, MA 02114.
2 Department of Radiology, Harvard Medical School and Children's Hospital
Boston, 300 Longwood Ave., Boston, MA 02115.
3 Department of Orthopaedic Surgery and Orthopaedic Research Laboratory, Harvard
Medical School and Children's Hospital Boston, Boston MA 02115.
Received June 17, 2003;
accepted after revision August 20, 2003.
Supported by Children's Hospital Boston Research Council and National
Institutes of Health grant AR42396-06.
Abstract
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MATERIALS AND METHODS. We quantitatively and qualitatively analyzed the normal changes on sequential T1-weighted images after the IV administration of gadoteridol. In an investigation approved by the research animal care committee at our hospital, we studied the proximal and distal femurs of 26 piglets 1–6 weeks old and correlated the enhanced images with findings on intermediate-weighted, T2-weighted, and gradient-recalled echo images and at histologic examination.
RESULTS. We observed early enhancement of the epiphyseal vascular canals, the main physis, the physis of the secondary ossification center, and a metaphyseal band adjacent to the physis. Enhancement of the epiphyseal and metaphyseal marrow and of the epiphyseal cartilage was slower. In the epiphyseal cartilage, we saw three phases of enhancement: vascular, canalicular, and cartilaginous. As the piglets matured, enhancement of the epiphyseal cartilage decreased, and the epiphyseal vascular canals were less conspicuous. Physeal enhancement was greatest during the first week of life, declined at 3 weeks, and subsequently increased again as the physis came to lie adjacent to a larger segment of the epiphyseal ossification center.
CONCLUSION. Gadoteridol-enhanced MRIs showed multiple cartilaginous and vascular structures of the growing skeleton. With maturity and progressive epiphyseal ossification, epiphyseal cartilage enhancement decreased, and physeal cartilage enhancement increased.
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Gadolinium-enhanced MRI depicts the vascular canals of the cartilaginous epiphysis and the enhancement of the physeal and epiphyseal cartilage [2], as well as the vascularity of the marrow. The temporal sequence of and the changes in gadolinium enhancement that occur with age are still poorly understood. To clarify the patterns of enhancement in the developing cartilage and bone, we studied gadolinium-enhanced images of the femurs of healthy piglets of various ages and compared them with histologic findings. Our purpose was to evaluate the normal anatomy and enhancement patterns of the growing epiphysis, physis, and metaphysis and the age-related changes in enhancement, as determined on gadolinium-enhanced MRI.
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The second goal was to evaluate the age-related variation in enhancement in 24 piglets (the two previously discussed piglets were not included). These piglets were evaluated weekly for between 1 and 6 weeks, beginning at the first week of life. They were sacrificed at the following ages for histologic correlation. Twelve piglets, healthy controls from another study on epiphyseal ischemia [2], were imaged and sacrificed at 1 week of life. Of the remaining 12 piglets, two each were sacrificed at ages 2, 3, and 4 weeks of life, and six were sacrificed at 6 weeks of life. In this group of 24 piglets, 50 MR examinations were performed. The distribution of examinations was as follows: week 1, n = 17; week 2, n = 7; week 3, n = 7; week 4, n = 7; week 5, n = 6; and week 6, n = 6. In every piglet, we evaluated the right and left proximal and distal femurs for a total of 200 epiphyseal studies.
The animals were divided into two age groups according to the degree of skeletal development. Piglets younger than 3 weeks (group 1) had recently ossified epiphyses at both ends of the femur and easily identifiable cartilage surrounding the ossification center; their skeletal development at this age is comparable to that of children 1–2 years old. Piglets 3–6 weeks old (group 2) had increasingly ossified epiphyses; their skeletal development is comparable to that of children 2–5 years old. The piglets had normal activity and diet. At the end of the study period, the animals were sacrificed for histologic correlation. The animal care and use committee at our hospital approved the study, and the authors complied with the National Institutes of Health guidelines for use of laboratory animals [3].
MR Evaluation
For imaging, the piglets were anesthetized with a single intramuscular
injection of 20 mg/kg of ketamine hydrochloride (Ketalar, Parke-Davis, Morris
Plains, NJ) and 5 mg/kg of xylazine (Rompun, Miles, Shawnee, KS) followed by a
continuous IV infusion of 1% diprivan (Propofol, Stuart Pharmaceuticals,
Wilmington, DE) diluted in normal saline at a dose of 0.002 mg/kg per
minute.
MRI was performed using a 1.5-T system (Signa or Horizon, General Electric Medical Systems, Milwaukee, WI). Piglets younger than 4 weeks were imaged in the lateral decubitus position, using a pair of 10-cm receive-only surface coils (General Electric Medical Systems) placed simultaneously over each hip. Because the older piglets were too large for the smaller coils, the animals were imaged using a linear transmit–receive coil (General Electric Medical Systems). An axial T1-weighted (TR/TE, 300/25) sequence was followed by imaging in the sagittal plane of the femur. The following sagittal sequences were obtained: T1-weighted conventional spin-echo (300/25); intermediate-weighted (4,000/15; echo-train length, 8) fast spin-echo; T2-weighted (4,000/80; echo-train length, 8) fast spin-echo; and gradient-recalled echo (300/13; flip angle, 30°) images. Imaging parameters were field of view, 12–14 cm; section thickness, 3.0 mm; interslice gap, 1 mm; and matrix size, 256 x 192 for an in-plane resolution of 469 µm.
Gadoteridol (Prohance, Squibb, Princeton, NJ) was then injected manually at a dose of 0.2 mmol/kg in a rapid bolus into an ear or upper extremity vein. T1-weighted sequential spin-echo (300/25) images (sequence length, 2 min) were obtained in the plane of the femoral shaft immediately before the injection; during the injection (completed 1 min after injection); and at 3, 5, 10, and 20 min after the injection. Two excitations were obtained for the T1-weighted sequential images and one or two for the other sequences.
Histologic Studies
Immediately after completion of the final imaging session, the animals were
sacrificed in accordance with the schedule described previously via an
intracardiac injection of pentobarbital sodium (Somlethol, Webster, Sterling,
MA) at a dose of 1 mL/5 kg. To harvest the epiphyses, one of two orthopedic
surgeons dissected the hip region carefully down to the joint capsule. The
capsule was incised to allow gross inspection of the joint and of the femoral
head, after which the entire femur was excised. The femurs were fixed in 10%
neutral buffered formalin. After 2 weeks of fixation, each femur was
decalcified in 25% formic acid until soft. The femur was sectioned
transversely at the proximal metaphyseal–diaphyseal junction and the
head–trochanter–neck segment was then sectioned in the mid coronal
plane. Photographs of specimens of the mid coronal anatomy are displayed in
Figure 1A,
1B,
1C. Tissue preparation for
histologic examination continued, using plastic-embedded sections. After 2
weeks of infiltration in JB4 plastic solution (Polysciences, Warrington, PA),
segments of tissue were embedded in the plastic, cut into 5-µm-thick
slices, and stained with 1% toluidine blue.
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Image Analysis
Enhancement ratios.—In the 200 epiphyseal studies, we
obtained enhancement ratios of the main physis, physis of the secondary center
of ossification, epiphyseal vascular canals, epiphyseal cartilage, and
epiphyseal and metaphyseal marrow. All the researchers performing the
measurements had been trained with at least 10 supervised measurements of
enhancement ratios. The main landmarks for measurement were the physis and
structures such as the metaphysis and secondary center of ossification.
Physeal cartilage was easily identifiable as an enhancing band at the junction
with the metaphyseal bone. The physis of the secondary center of ossification
was identified as a discrete, thin, enhancing band surrounding the secondary
ossification center; the vascular canals were discrete, linear, enhancing
structures oriented radially around the ossification center. Epiphyseal
cartilage was measured in areas of cartilage around the secondary ossification
center that had no discrete enhancing structures.
Enhancement ratios were derived from signal intensity (SI) measurements according to the following formula: enhancement ratio = (contrast-enhanced SI – unenhanced SI) / unenhanced SI. For each structure, we calculated the enhancement ratio at each of the five time intervals and graphed the changes over time. In two animals, enhancement during the first 2 hr was assessed by changes in the enhancement ratio at 15-min intervals.
Phases of vascular enhancement.—Qualitative analyses of the time that the enhancement appeared and that maximal conspicuity of each structure was achieved were made by one radiologist who was unaware of length of time that had elapsed since contrast material administration. Of the piglets whose skeletal structures were studied for conspicuity, 12 were 1 week old, and the distribution of the ages of the rest of the piglets was spread relatively evenly from 2 to 6 weeks old. We also quantitatively analyzed the change in enhancement ratio over time for each region.
Size of vascular structures (epiphyseal vessels vs canals).—To determine whether the multiple linear structures seen in the epiphyseal cartilage on enhanced MRIs represented epiphyseal vascular canals or epiphyseal vessels, a single radiologist took 50 measurements of these structures and compared them with the diameter of the vessels and vascular canals on histologic sections.
Changes in physeal enhancement with age and rate of growth.—We measured the mean peak enhancement ratios of the physis for each of the age groups and compared the enhancement in the proximal and distal femoral physes to assess whether the greater growth rate of the distal femoral physis resulted in increased enhancement. In the piglets in which enhancement was measured sequentially, we compared these measurements with the growth of the corresponding bone (in millimeters, as determined from MRIs) over time.
Comparison of sequences.—In 12 proximal epiphyses and 12 distal epiphyses selected randomly from the group of 200, we evaluated the conspicuity of the physis, epiphyseal vascular canals, and physis surrounding the ossification center. One observer graded the structures in a binary fashion as present or absent.
Statistical Analysis
Preliminary assessment of normality of the data revealed that enhancement
ratios were distributed normally. We evaluated differences in age-related
changes in peak enhancement in each anatomic area using one-way analysis of
variance. We compared enhancement in the proximal and distal femurs of the
1-week-old piglets (the largest group, which allowed us to make the most
accurate comparisons) using an unpaired, two-tailed Student's t test.
Statistical evaluation was performed using STATA statistical software (STATA,
College Station, TX). Differences with a p value of less than 0.05
were considered significant.
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Within 1 min of the administration of gadoteridol, enhancement of multiple linear vascular structures within the epiphyseal cartilage was observed. These structures were arranged radially around the secondary center of ossification and presumably represent vessels within the epiphyseal vascular canals (Fig. 2A, 2B, 2C, 2D). There was also bandlike enhancement of the main physis and of the physis of the secondary center of ossification. In the juxtaphyseal metaphysis, a second band of enhancement simultaneously appeared in the expected location of the metaphyseal spongiosa, parallel to the enhancing physis (Fig. 1A, 1B, 1C). No enhancement in the epiphyseal cartilage was detectable.
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Three minutes after the administration of contrast material, the band of enhancement of the physis became thicker and more intense. Similar but less striking changes occurred in the physis of the secondary center of ossification. Enhancement in the marrow moved as a poorly defined wave from metaphysis to diaphysis.
Epiphyseal vascular structures were larger and better defined on the 3-min images than on the 1-min images and achieved maximal conspicuity on the 5-min images, after which their margins began to blur. The vascular structures became less apparent subsequently and were not visible 20 min after the administration of contrast material.
Enhancement and Time After Injection
The mean enhancement ratios in proximal and distal femoral epiphyseal and
physeal cartilage of piglets younger than 3 weeks and those 3–6 weeks
old are shown in Figures 3,
4,
5,
6. Enhancement in the physis
peaked at 3–5 min after injection and declined slowly thereafter. The
epiphyseal cartilage enhanced more gradually; epiphyseal signal intensity
continued to increase during the 20 min of the contrast-enhanced imaging
session. Twenty minutes after the administration of contrast material, all the
structures of the cartilage had similar signal intensity, which was much
greater than that of the adjacent marrow. Enhancement ratios were similar in
the epiphyseal and metaphyseal marrow, but the marrow of the metaphysis
enhanced faster than that of the epiphysis.
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Enhancement and Age
In the physis, peak enhancement decreased in the piglets from the first to
the second week. In piglets 4–6 weeks old, physeal enhancement tended to
gradually increase (Figs. 7
and 8). Enhancement of the
cartilaginous epiphysis decreased after the first week and continued to
diminish thereafter. The epiphyseal vascular canals were conspicuous on images
acquired during the first week of life but became less conspicuous as the
piglets aged. By the time the piglets reached the age of 4 weeks, the
epiphyseal vascular canals were barely detectable in the proximal femur.
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Throughout the period in which the piglets were studied, the marrow remained hypointense in the unenhanced T1-weighted images of the epiphysis and the metaphysis. Both epiphyseal and metaphyseal marrow enhancement tended to decrease over time. Epiphyseal enhancement was less than metaphyseal enhancement but only by a small amount. As the epiphysis enlarged, particularly in the distal femur, there was delayed enhancement in the central part of the epiphyseal ossification center (Fig. 9A, 9B) that appeared to precede marrow transformation. Metaphyseal gadoteridol enhancement remained relatively constant during the period of growth studied.
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Enhancement, Physeal Location, and Growth
Physeal enhancement was similar for the proximal and distal femurs, both
qualitatively and quantitatively (p = 0.51). We did not find a direct
relationship between the change in femoral length by age and the corresponding
differences in enhancement (Figs.
7 and
8).
Vascular Canals
The mean diameter of the enhancing epiphyseal intracartilaginous linear
structures measured on the 3-min MRIs was 1.19 ± 0.23 mm (1 SD). The
diameter of the canals on histologic specimens of the epiphyseal cartilage of
the femoral head measured in several specimens of piglets 1–4 weeks old
varied. The larger canals ranged between 0.2 and 0.6 mm in diameter. The mean
diameter of 40 canals measured from photomicrographic slides was 0.3 mm. Owing
to the unique vascular anatomy of the canals, a single vessel rarely filled
the entire diameter of the canal. Usually two or three distinct
capillary–sinusoidal vessels 0.1–0.16 mm in diameter were seen on
each transverse or longitudinal section interspersed with connective tissue
and many small discrete vessels 0.01–0.08 mm wide (Fig.
10A,
10B).
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Comparison with Other Sequences
Anatomic definition between the physeal and epiphyseal cartilage was
observed in 96% (23/24) of the gadoteridol-enhanced T1-weighted images, 71%
(17/24) of T2-weighted images, 13% (3/24) of the unenhanced T1-weighted
images, and 8% (2/24) of the gradient-recalled echo images. Although
epiphyseal vascular canals were visible in 92% (22/24) of the enhanced images,
they were not visible on images obtained with any other sequences (Fig.
11A,
11B). The physis surrounding
the ossification center was seen in 83% (20/24) of gadoteridol-enhanced
T1-weighted images and 25% (6/24) of the T2-weighted images but not on
unenhanced T1-weighted images or gradient-recalled echo images.
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Epiphyseal cartilage in infants and young children is supplied by the vessels that course through the cartilage within nonanastomotic vascular canals referred to as cartilage canals. The canals contain arterioles, venules, sinusoidal capillaries, and loose perivascular connective tissue [4, 5]. Such canals supply nutrients to the cartilage and may serve as a source of cartilage stem cells for growth of the epiphysis [6]. Vascular canals are important in the development of epiphyseal ossification because the vessels with their associated mesenchymal cells serve as the source for bone-synthesizing osteoblasts on the calcified cartilage [7]. The number and size of the canals decrease with maturity, particularly after the appearance of the secondary center of ossification [8]. The concentration of vascular canals varies throughout the epiphysis. They are particularly numerous in a band adjacent to the reserve zone of the physis and around the secondary center of ossification [1]. Vascular canals crossing the physis are present at birth in both piglets and human infants. In humans, the vascular canals disappear after infancy, but in piglets they persist. Cartilage canals are most prominent in areas of high cellular turnover [4] and are present in great concentration in neonates [6]. As development continues, the epiphyseal canals are incorporated into the growing secondary ossification center or atrophy.
The vascular canals showed rapid and lasting enhancement after the administration of gadolinium, which presumably was diffused from the vessels into the loose perivascular connective tissue of the vascular canals. The greater diameter of the canals measured on MRIs—approximately three times the histologically measured greatest diameter—suggests that even at 3 min after contrast administration, gadolinium was diffusing beyond the vascular canal into the immediate pericanalicular epiphyseal cartilage. Visualization of the canals for nearly 10 min and the progressive fading of their outline 5 min after contrast administration suggest that the gadoteridol remained within the canals and their surrounding cartilage before diffusing into the rest of the epiphyseal cartilage.
In summary, the pattern of epiphyseal enhancement suggests that during the first minute after contrast administration, the gadoteridol was within the vessels; by 3 min, it had diffused into the perivascular and pericanalicular tissues of the cartilage vascular canals; and after 5 min, the contrast material diffused into the rest of the epiphyseal cartilage. Therefore, we observed three phases of enhancement: vascular, canalicular, and cartilaginous (Fig. 10A, 10B). We measured the widest canals because these were the most likely to be assessed with MRI. A canal was rarely filled with a single vessel but rather with several vessels (capillaries and sinusoids) in a connective tissue bed. A similar pattern of canalicular enhancement has been described in infants and children [9].
The main physis and the smaller physis surrounding the secondary ossification center also enhanced rapidly after administration of contrast material, indicating rapid diffusion of gadolinium. Most of the contrast material appeared to diffuse from the vascular leash just beyond the reserve zone of the physeal cartilage. Some material might have come from the transphyseal vessels directly. Although gadoteridol diffused more slowly into the epiphyseal cartilage, all cartilaginous structures became isointense within 20 min of the contrast injection. After gadoteridol administration, the metaphyseal spongiosa, the area of the metaphysis immediately adjacent to the physis, enhanced intensely as a distinct band. This result was expected because the metaphyseal spongiosa is highly vascularized (it is the site of blood-borne diseases such as leukemic infiltration), metabolically active, and structurally separate from the rest of the metaphysis.
Our data did not support a quantifiable relationship between physeal enhancement and activity of the physis. No difference in enhancement between the physes of the proximal and distal femur was seen, although their contribution to growth is substantially different: 70% of femoral growth occurs at the distal physis and 30% at the proximal. Furthermore, we found no relationship between physeal enhancement and femoral growth (i.e., increased femoral length). The increase in the physeal enhancement with maturity is difficult to explain. It is known that in children treated for hip dysplasia, the risk of avascular necrosis decreases with increasing growth of the ossification center [10]. Perhaps physeal perfusion increases as the adjacent epiphysis ossifies and thus becomes more vascular.
The clinical importance of understanding gadolinium enhancement of epiphyseal and physeal cartilage is becoming more evident. Gadolinium-enhanced images better characterize epiphyseal and physeal abnormalities by improving the definition of the anatomic regions within the cartilage. Preliminary experience in piglets [2] and in humans [11, 12] indicates that gadolinium enhancement can also identify areas of ischemia in the immature proximal femoral epiphysis. Increased enhancement of vascular canals, on the other hand, occurs with epiphyseal inflammation in patients with juvenile rheumatoid arthritis [13]. Analyzing the intracartilaginous diffusion of ionic gadolinium can provide insight into the health of the cartilage, specifically into its glycosaminoglycan content [14, 15]. Further research is needed to compare enhancement patterns of ionic and nonionic gadolinium compounds.
We limited our study to piglets 6 weeks and younger. In the proximal femur, cartilage vascular canals are not identifiable histologically in piglets older than 8 weeks [16] because the canals have been incorporated into the secondary ossification center. Our data for the first week of life were more abundant and more reliable because obtaining high-resolution images in piglets this age was the easiest of all the developmental stages studied. It was technically difficult to obtain comparable high-resolution images of the proximal and distal femoral epiphyses in older animals. Our observations were centered mainly on epiphyseal architecture and development. The window of observation did not allow us to analyze other important developmental changes such as physeal closure and marrow transformation.
Our study was performed in piglets, so our results may differ from those that would be obtained in humans. Although many studies about vascularization of the epiphysis and physis have been performed in animals, there is also considerable knowledge about cartilage vascular canals in children [6], and studies have shown that histologic findings in humans are similar to those in other mammals [17]. Although our own observation shows that the vessels crossing the physis are more persistent in piglets than in humans, we believe that it is safe to assume that our results are applicable to humans as well.
In conclusion, gadoteridol-enhanced T1-weighted images showed multiple anatomic structures within the epiphysis and physis, some of which were not visible on images obtained with other pulse sequences. The appearance of the growing structures changed after injection as the gadolinium diffused through the cartilage and marrow. Enhancement was greatly influenced by maturation of the skeleton of the piglets.
Acknowledgments
We thank Noemi Chavez, Sherry Brec, Elizabeth Olear, and Mauricio Rodriguez
for help with preparation of the manuscript and graphs.
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= proximal femoral physis, 
= distal
femoral physis. Enhancement in piglets 3 weeks and older: 
= proximal
femoral physis, 
= distal femoral physis.
), however, enhancement initially decreases but
gradually increases thereafter. Error bars = standard error of mean.
