Etymologically, ischemia means lack of blood flow. Decreased flow interferes with the supply of oxygen and nutrients. The starved cells initially malfunction, later die, and as a result, tissue structure is destroyed. In children, ischemia of the proximal femoral epiphysis occurs frequently and can lead to growth disturbance.1–5 As we know, ischemia of the proximal femoral epiphysis occurring as a result of therapy for hip dysplasia results from immobilization in extreme abduction.6–9 The previous study10,11 has demonstrated that this ischemia is reversible if the hip is repositioned within a brief period (within 6 hours) and the conventional gadoteridol (Gd)enhanced SE T1WI can detect this ischemia at least one hour after hyperabduction. Without a doubt, earlier diagnosis of ischemia leads to more effective treatment. Therefore, it is of value to identify the non-invasive means that can accurately reflect the blood supply of epiphysis and is more sensitive in detection of early ischemia of epiphysis than the conventional Gd-enhanced SE T1WI.
In this study, the distal femurs and proximal tibias of piglets were subjected to dynamic Gd-enhanced MR imaging that involved use of fast spoiled gradient-recalled echo images (FSPGR) with a high temporal resolution. By observing the continuous fast imaging with multi-sequences, the enhancements of different anatomic regions of epiphysis were quantitatively evaluated and compared with the histological findings. In this research, the hips of piglets were abducted extremely to mimic the position in therapy for hip dysplasia in children to determine whether changes in blood perfusion of femoral head epiphysis can be detected within 30 minutes after persistent hip hyperabduction by using dynamic Gd-enhanced MRI. The purpose of this study was to determine whether the blood supply characteristics of the end of normal growing long bone could be revealed by dynamic Gd-enhanced MR imaging techniques and to show if it is more sensitive than the conventional Gd-enhanced SE imaging in the detection of early epiphyseal ischemia.
Experimental design and animals
In this study, 27 two-week-old piglets were used (weighing 5–7 kg, with a mean weight of 6.1 kg). For the study of the end of normal growing long bone, eleven unilateral knees including distal femurs and proximal tibias were studied. The piglets at this age have newly ossified femoral epiphyses and their skeletal development is comparable with that of a 2–4 year old human child. For the examination of the ischemic hip, 16 piglets were randomly divided into two groups, with the control group having 8 piglets (involving 16 normal hips) and an ischemic group including 8 piglets (involving 16 hips in abduction). In the control group, an MR scan was performed on the bilateral hips in a neutral position. In the ischemic group, MR imaging was performed on the bilateral hips with persistent immobilization in the maximal achievable abduction position, ranging from 75° to 90° (average, 83±5°) for 30 minutes. After MRI, piglets in the ischemic group were allowed to ambulate freely for 1 day and the identical MR scanning was then repeated in a neutral position. Prior to MR scanning, anesthesia was induced by intramuscular injection of 40 mg midazolam hydrochloride (Baxter, Healthcare Co., Deerfield, IL, USA), followed by intravenous infusion of 20 mg/kg ketamine hydrochloride (Ketalar, Parke-Davis Co. Inc., Morris Plains, NJ, USA) and 20 mg/kg xylazine hydrochloride (Rompun, Miles, Shawnee, Kan, USA). After the anesthesia, 1% propofol (Diprivan, Stuart Pharmaceuticals, Wilmington, Del) was intravenously and continuously given at 0.002 mg·kg-1· min-1 in the process of the scanning. All the animals were imaged under the intubation and received "blow-by" oxygen at 3–4 L/min. The piglets were sacrificed immediately after the MR imaging for histological study. The study was conducted upon the approval by the Animal Care and Use Committee of our hospital and efforts were made to fully comply with the Health Guidelines for Use of Laboratory Animals introduced by China National Institutes.
All the piglets were examined using a 3.0 Tesla Signa MR Scanner (GE Medical Systems, Milwaukee, USA) with a receive-only surface coil. For the imaging of the knee, MR sequences, including unenhanced sagittal SE T1WI (TR/TE, 350/30 ms), FSE T2WI (TR/TE, 3000/80 ms) and dynamic Gd-enhanced MR imaging were performed on distal femurs and proximal tibias. For the imaging of the hip, besides all the sequences performed for the normal knees, the conventional Gd-enhanced SE T1WI, which was performed following dynamic Gd-enhanced MRI, was also conducted.
For dynamic Gd-enhanced MRI, FSPGR with fat suppression was used before, during and after the gadoteridol injection (over a time of 5 seconds). FSPGR parameters included TR=30 ms, TE=4.5 ms, flip angle=30°, slice thickness=2 mm, FOV=150 mm, and voxel=1.2 mm × 0.6 mm × 2.0 mm. A total of 22 continuous FSPGR sequences were obtained in dynamic Gd-enhanced MRI. Each sequence included 3 slices and a total of 66 images were acquired. Each sequence lasted 9 seconds, with a total scanning time lasting 3.3 minutes. Gadoteridol (a nonionic contrast agent, Bracco) was injected into the ear vein at 0.2 mmol/kg.
With the knees, regions of interest (ROI) in physeal cartilage, epiphyseal cartilage, metaphyseal spongiosa (a metaphyseal band adjacent to the physis), the secondary ossification center, and metaphysis at the end of distal femurs and proximal tibias on dynamic MR images were chosen. The size of ROI was decided on the basis of anatomic features of tissue structures. Physis and the spongiosa were shown on the images as thin and belt-like structures, and accordingly, ROI in physis and the spongiosa was semi-automatically selected at workstation (AW4.1), with each ROI having 9–15 pixels. ROI of epiphyseal cartilage surrounding the second center of ossification was also semi-automatically chosen, and each ROI included 45–60 pixels because the size of epiphyseal cartilage was larger than that of physeal cartilage or the spongiosa. The secondary ossification center and metaphysis were relatively regular in shape and large in size, and they were chosen automatically and each ROI included 50–65 pixels.
With the hips, ROI in physeal cartilage, the anterior part and posterior part of the femoral head were chosen semi-automatically on Gd-enhanced MR images. Each ROI in the anterior or posterior part of the femoral head had 20–25 pixels, and ROI in physeal cartilage had 5–9 pixels. In the collection of the average signal intensity (SI) of ROI of the same anatomic region, location and size of ROI were consistent on the images of each continuous sequence.
The enhancement ratio (ER) of each anatomic area was calculated by using the formula: ER=(SI at each time point after Gd injection - SI before Gd injection) /SI before Gd injection. With the knee, the ER of distal femur was compared with that of proximal tibia from the same anatomic structure. The comparison of the ER between the different tissues was made 30, 66 and 198 seconds after injection of Gd, respectively. The ER of the same tissue at various time points (including 30, 66 and 198 seconds after Gd injection) was also compared.
With the hips, the differences in the ER of each anatomic region between normal control and ischemia at the same time points were compared. In the ischemic hips, the ER of the anterior part of capital femoral epiphysis was compared with that of the posterior part. After ambulation, the comparison of the ER of each anatomic region between the normal control and ischemia at the same time points was repeated.
According to enhancement features of various tissues, we used the slope of the enhancement curve to reflect the enhancement speed (ES) of tissue. Because the slope of enhancement curve changed with the different time period, we selected the slope of the segment of enhancement curve from the beginning of Gd injection to the 30th second after Gd injection to reflect the ES of the tissue. With the knees, the slope of distal femur was compared with that of proximal tibia, and the difference in the slope between the various tissues was evaluated. With the hips, the difference in the slope for the same anatomic structure between the normal control and ischemia group was evaluated.
All of the piglets were sacrificed after MR scanning. Complete femurs and tibias were taken out, and kept in 10% formalin for 2 weeks for fixation, and then put in 25% formic acid for decalcification. Afterwards, they were embedded in JB4 plastic fluid, cut into 5 μm slices and stained with 1% toluidine blue.
With the knees, depending on the vascular density and distribution at various anatomic regions, two different techniques were employed to reflect the blood supply of the tissues. (1) In epiphyseal and physeal cartilage—where vessels are sparse—a mid-coronal plane of the femoral head was chosen to count the vessel number. (2) In the metaphyseal spongiosa, the second ossification center and the metaphysis—where it is difficult to precisely count the vessels in bone marrow tissue—we calculated the density of red blood cells (RBC/mm2) by using a software package (Image-Proplus 5.1) to reflect the blood supply. The difference in RBC/mm2 was compared between the various tissues. With the hips, the difference in the histological findings was evaluated between the control and ischemia groups.
We evaluated differences in the ER, slope and RBC/mm2 by using two-way analysis of variance (ANOVA) with the General Linear Model when three or more groups were compared, and a post-hoc test was performed by utilizing the Student-Newman-Keuls comparison (SNK) when the difference was statistically significant among the groups. We compared the difference in the ER or slope by using a paired t-test when two groups were compared. Pearson correlation coefficients between the ER and RBC/mm2 and those between slope and RBC/mm2 were obtained by bivariate correlation. The significant level was set at P <0.05. Statistical analysis was conducted by using SPSS 12.0 software for Windows (SPSS Inc., Chicago, Illinois, USA).
Dynamic Gd-enhanced MR images
The end of normal growing long bone
The consecutive images of dynamic Gd-enhanced MRI of distal femurs and proximal tibias in piglets (FSPGR images of the same anatomic slice at different time points in a row) showed that SI of each anatomic region changed with time (Figure 1). The enhancement of distal femur seemed to be similar to that of proximal tibia. The metaphyseal spongiosa was enhanced the most and fastest. Compared to the surrounding tissues, epiphyseal cartilage was enhanced the least and most slowly. No apparent difference in enhancement was found between the second center of ossification and metaphysis. The metaphyseal spongiosa in the secondary ossification center presented an enhancement similar to that of the corresponding main metaphyseal spongiosa.
The ischemic femoral head
A series of dynamic Gd-enhancement MR images of bilateral hips with persistent hyperabduction (88°) for 30 minutes showed that as with the enhancement in the control, enhancement at various anatomic regions of the femoral head increased with time (Figure 2), however, the enhancement of each tissue seemed to be less and slower than that in the control. In the meantime, no apparent difference in the enhancement between normal and ischemic hips was found on conventional Gd-enhancement SE T1WI (Figure 3). No abnormalities were found on unenhanced SE T1WI and FSE T2WI.
ER and ES
There was no significant difference in the ER or ES between distal femur and proximal tibia (P=0.98). The ER of the spongiosa was highest (P <0.001) and the ER of physeal cartilages was higher than that of epiphyseal cartilages (P <0.001), which was the lowest among all the tissues (P <0.001). Although the ER of metaphysis was higher than that of the second ossification center, no significant difference existed between them (P >0.05). The differences in the ER of the same tissue between the various time points (30, 66 and 198 seconds after Gd injection) were significant (P <0.05) (Table and Figure 4).
As to the slope of the enhancement curve, which reflects the ES, the slope of the spongiosa was greater than that of physis, but there was no significant difference between them (P >0.05). The slope of physis was greater than that of epiphyseal cartilage (P <0.05), which was the lowest among all the tissues (P <0.05). The slope of the spongiosa was greater than that of metaphysis and the second ossification center (P <0.05). No significant difference in slope was found between the second center of ossification and metaphysis (P=0.52) (Figure 4). The ERs of all the tissues, except for epiphyseal cartilage, increased rapidly during the first minute after Gd injection and subsequently rose slowly. However, the ER of epiphyseal cartilage gradually and slowly increased with time.
The ERs of physeal cartilage, the anterior part and posterior part of capital femoral epiphysis in ischemic hips were significantly lower than those of normal hips (P <0.05) and all the tissues were enhanced more slowly than those in normal hips (P <0.05) (Figure 5). In ischemic hip, the ERs of the anterior part of capital femoral epiphysis were lower than those of the posterior part (P <0.05). In normal hips, however, the ERs of the anterior part were the same as those of the posterior part of capital femoral epiphysis. On conventional Gd-enhanced SE T1WI, although the ER of the anterior part of capital femoral epiphysis in 5 of the 16 ischemic hips were lower than that of normal femoral head, no statistically significant difference in the ER between the normal and ischemic hips was found (P >0.05). The MR scan revealed that the ER and ES of various regions of the femoral head in ischemic hips after 1-day free ambulation were similar to those in the control hips (P >0.1).
Within bone marrow of metaphysis, the density of vessels was similar to that in the second ossification center (Figure 6A), but apparently less than that in the spongiosa. A great number of vessels were found within the red bone marrow of the spongiosa (Figure 6B), especially in the areas adjacent to physeal cartilage. RBC/mm2 in the bone marrow of metaphysis was similar to that in the second ossification center, but significantly less than that in the spongiosa (P <0.001), which was the greatest among the three tissues (P <0.001).
In physeal cartilage, there were merely 7–8 pieces (mean, 7.5) of large vessels that transphyseally connected spongiosa with the second ossification center (Figure 6C) on a mid-coronal plane of the femoral head. In the meantime, small vessels were found to extend to the mineral zone of physis and almost reached the hypertrophic zone of physeal cartilage from the neighboring spongiosa (Figure 6D). The vessels within epiphyseal cartilage were sparse and were located in vascular canals within epiphyseal cartilage, containing arterioles, venules, capillary plexus and connective tissues (Figure 6E). There were 9–11 (mean, 10) vascular canals in epiphyseal cartilage on a mid-coronal plane of the femoral head. Pearson correlation coefficients (R) between the ER and RBC/mm2, and between the slope and RBC/mm2 were all greater than 0.75 (P <0.01). R between the ER and RBC/mm2 of the same tissues 30 seconds after the Gd injection was greater than that at the other two time points (66 seconds and 198 seconds after the Gd injection).
In the hips with abduction, histological studies revealed that no damage of the vessels and any changes in tissue structure were found in the femoral head. No necrosis of cartilage cells or osseous cells was seen in epiphysis of the femoral head.
Although articular cartilage is avascular, epiphyseal and physeal cartilages contain vessels in the early stage of development. The previous studies have confirmed that 1 to 5 minutes after intravenous injection of Gd, conventional SE T1WI can reveal vessel canals within the epiphyseal cartilage and roughly show the difference in enhancement between physeal and epiphyseal cartilages.12,13
In our study, the ER and ES of the spongiosa were highest among all the tissues on dynamic Gd-enhanced MRI with high temporal resolution, and correspondingly, the histological studies revealed12 that the spongiosa is richest in vessels at the end of a developing long bone because cellular metabolism is active during the formation of new bone in this region. Physeal cartilage was enhanced notably and quickly in spite of the fact that cartilage tends to contain only several transphyseal vessels. As we know, the mineral zone of the physis is close to the spongiosa, with some vessels of the spongiosa extending to the mineral zone and almost reaching the hypertrophic zone of physis. We are led to postulate that it might be partially related to the notable and fast enhancement of physeal cartilage. Compared to physeal cartilage, epiphyseal cartilage was enhanced slowly and weakly. Histologically, vessel canals are sparse in epiphyseal cartilage and, in the meantime, the vessels of the epiphyseal cartilage are embedded in the vessel canals. Theoretically, Gd has to diffuse to the matrix of the vessel canals before it enters the cartilaginous matrix. So, Gd takes longer to enter the cartilaginous tissue than it does other anatomical structures. It was reported12 that the Gd could diffuse to the matrix of the epiphyseal cartilage five minutes after medium injection. The density of vessels in metaphysis is histologically comparable to that in the second center of ossification and, as a result, no significant difference in the ER or ES was found between the two areas.
Our results showed that the ER of the same tissue was different at the different time points after Gd administration. We infer that it might have something to do with the diffusion of Gd in the tissues. As we know, gadoteridol can diffuse quickly from vessels to extracellular space of tissues through vascular walls.14–16 We believe that the ER 0.5 minutes after administration of Gd might best reflect the blood perfusion in the tissues because the R value between the ER and RBC/mm2 at this time point was highest among R values at the three time points. In the meantime, we believe that the ER at the time points of 1 and 3 minutes after administration of Gd might be related not only to the density of vessels, but also to the diffusion of Gd into the extracellular spaces, and it was especially true of the ER 3 minutes after the Gd administration. Further study is needed to test this assumption. Generally speaking, our results suggested that dynamic Gd-enhanced MRI can reveal the status of blood supply in various anatomic regions at the end of developing long bone.
Avascular necrosis, the consequence of decreased blood flow to a femoral epiphysis, could not be detected by the plain radiographs or computed tomography, until apparent morphological deformations occur in the subsequent healing stage of a mature ossification capital, such as regions of both osteopenia and osteosclerosis, microfractures of trabeculae, "crescent" sign and eventual collapse of the femoral head.17 Although the early changes of avascular necrosis is detectable by MR imaging, there would probably be no change in the non-enhanced MRI immediately after cessation of blood flow. After sufficient time had destructed for femoral head vascularity, MRI would be expected to demonstrate the changes in signal intensity of the cartilage on T2-weighted images that correlate with cartilaginous destruction in childhood or a focal regions of decreased signal intensity in the sub-articular area of the femoral head in both T1- and T2-weighted images in adulthood. 18 Unfortunately, diagnoses at the advanced stage are too late to prevent the progression of evident abnormal epiphyseal ossification and disturbed growth.
Abduction therapy, an immobilizing method aiming at treating developmental dysplasia of the hip, may cause avascular necrosis.9–11 The femoral head must be held within a "safe" zone: too little abduction results in redislocation, whereas too much abduction leads to avascular necrosis. The vascularity of the capital femoral epiphysis in piglets is similar to that of infants.13 Jaramillo et al10 have confirmed that short-term hyperabduction produces ischemia of the capital femoral epiphysis in piglets, which is reversible within 6 hours. The ischemia is more severe with longer abduction time. Thus, non-invasive techniques for the early detection of changes in blood perfusion of the femoral head are needed to prevent ischemia. Previous research demonstrated that conventional Gd-enhancement SE T1WI could reveal early ischemia caused by persistent hip abduction within at least one hour.10 Our study showed that dynamic Gd-enhancement MRI could detect decreased blood perfusion of the femoral head caused by persistent abduction within 30 minutes while no apparent changes were found on conventional Gd-enhanced SE T1WI, suggesting that dynamic enhanced MR technique is more sensitive than conventional Gd-enhanced SE T1WI in the detection of early epiphyseal ischemia. We intend to make a diagnosis for super-early epiphyseal ischemia that was induced from this preliminary study that is the dynamic Gd- enhanced MRI of the hip is positive, but no abnormality was found in both epiphyseal pathology and conventional Gd- enhanced SE T1WI.
The previous research documented that ischemia of the anterior part of capital femoral epiphysis with hyperabduction is more severe and the area of ischemia is larger than the ischemic area of the posterior part because the ischemia of the anterior part of the femoral head is related to the obstruction of a large vessel and ischemia of the posterior part results from the direct compression of the cartilage of the femoral head against the acetabula. 10 Our study showed that the ER of the anterior part of the femoral head was lower than that of the posterior part, which was consistent with the previously reported findings.
In the clinical setting for hip immobilization, the time of the MR scan seems to be a crucial factor in the prevention of avascular necrosis of the femoral head. We suggest that after immobilization in abduction, patients should be promptly subjected to dynamic Gd-enhanced MR imaging. Typically, the time from the completion of the immobilization to the beginning of the MR scan is more than 30 minutes. If the MR examination yields negative results, the same MR examination should be repeated within 6 hours after the immobilization (since the ischemia is reversible within 6 hours10) for the further verification of absence of ischemia. If the MR examination result is positive in either of the examinations, the hip must be repositioned and the MR exam should be repeated.
In conclusion, this research indicates that dynamic Gd-enhanced MRI can accurately reveal the features of blood supply of the end of normal growing long bone, and can provide information for clinicians to promptly adjust the hip abduction accordingly, which not only helps achieve effective treatment for hip dysplasia, but also avoids the development of ischemia in the immobilization.
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