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The Journal of Bone & Joint Surgery, Volume 83, Issue 5

Articles | May 01, 2001

Histopathologic Changes in Growth-Plate Cartilage Following Ischemic Necrosis of the Capital Femoral Epiphysis : An Experimental Investigation in Immature Pigs

Harry K.W. Kim, MD, FRCS(C); Phi-Huynh Su, BSc; Yu-Shan Qiu, MD

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Investigation performed at the Center for Research in Skeletal Development and Pediatric Orthopaedics, Shriners Hospitals for Children, Tampa, Florida
Harry K.W. Kim, MD, FRCS(C) Phi-Huynh Su, BSc Yu-Shan Qiu, MD Shriners Hospitals for Children, 12502 North Pine Drive, Tampa, FL 33612. E-mail address for H.K.W. Kim: hkim@shrinenet.org. Please address requests for reprints to H.K.W. Kim.
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding source was Shriners Hospitals for Children.

The Journal of Bone & Joint Surgery. 2001; 83:688-697

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Abstract

Background: The developing capital femoral epiphysis consists of a secondary center of ossification surrounded by epiphyseal cartilage. Between the epiphyseal cartilage and the secondary center of ossification is a growth plate, which contributes to the circumferential increase in size of the secondary center of ossification during development. The main objective of this study was to describe the histopathologic changes that occur in the growth plate surrounding the secondary center of ossification during the early and reparative phases following the induction of ischemic necrosis of the capital femoral epiphysis in immature pigs.

Methods: Ischemic necrosis of the capital femoral epiphysis was induced in eighteen piglets by placing a nonabsorbable suture ligature around the femoral neck following a capsulotomy and transection of the ligamentum teres. The animals were killed three days to eight weeks following the induction of ischemia, and visual, radiographic, and histologic assessments were performed.

Results: Two to four weeks after the induction of ischemic necrosis, the growth plate surrounding the secondary center of ossification became necrotic. The observed histopathologic changes included chondrocyte death, loss of safranin-O staining of the matrix of the necrotic growth-plate cartilage, an absence of vascular invasion of terminal hypertrophic chondrocytes, and a decrease in the amount of primary spongiosa, indicating cessation of endochondral ossification. In the reparative phase, at four to eight weeks postoperatively, chondrocyte clusters and intense safranin-O staining were observed in the epiphyseal cartilage around the necrotic growth-plate cartilage. In the peripheral region of the femoral head, necrotic growth-plate cartilage surrounding the secondary center of ossification was resorbed by a fibrovascular tissue from the marrow space. By six weeks, new accessory centers of ossification with restored endochondral ossification were observed in the peripheral epiphyseal cartilage. New ossification centers contributed to the fragmented radiographic appearance of the secondary center of ossification. The physis appeared essentially normal in most animals, although five of the eighteen piglets showed mild or moderate histopathologic changes.

Conclusions: In this model, ischemic necrosis of the capital femoral epiphysis resulted in necrosis of the growth plate surrounding the secondary center of ossification. Small new ectopic centers of ossification appeared in the epiphyseal cartilage, explaining in part the fragmented radiographic appearance of the secondary center of ossification.

Clinical Relevance: This immature swine model may facilitate systematic study of the sequence of cellular and structural events that follow ischemic injury to the capital femoral epiphysis. Better understanding of the injury and repair processes that follow ischemia may lead to novel treatment strategies to stimulate the repair of the infarcted capital femoral epiphysis and to restore normal growth of the secondary center of ossification.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

Ischemic necrosis of the capital femoral epiphysis can be caused by a variety of conditions affecting the hip in children, including traumatic and iatrogenic injuries and Legg-Calvé-Perthes disease. Invariably, one of the earliest signs of ischemic necrosis of the capital femoral epiphysis is cessation of the growth of the secondary center of ossification, suggesting that the growth-plate cartilage surrounding the secondary center of ossification is affected by the ischemic process. The extent of the injury to the growth-plate cartilage and the repair process that follows this injury have not been clearly defined. A model of ischemic necrosis of the capital femoral epiphysis was used to determine the histopathologic changes in the growth-plate cartilage following the induction of ischemic necrosis in the immature pig. Particular attention was paid to the fate of the growth-plate cartilage surrounding the secondary center of ossification and to how endochondral ossification is restored following the ischemic injury.

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Fig. 1:: A bisected normal capital femoral epiphysis of a six-week-old piglet. The capital femoral epiphysis consists of a secondary center of ossification (SCO) surrounded by epiphyseal cartilage (EC). The metaphyseal physis (M) is located on the base of the secondary center of ossification. In the immature animal, the proximal part of the femur contains two growth plates, the metaphyseal physis and the growth plate (x) surrounding the secondary center of ossification.

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Fig. 2:: A microangiogram of the capital femoral epiphysis following placement of a suture ligature around the femoral neck (right). Following the perfusion of Microfil contrast medium, the specimens were rendered transparent with use of the modified Spalteholz technique. The untreated side (left) shows Microfil contrast medium filling the small vessels in the epiphyseal cartilage and the secondary center of ossification. The side on which the operation was performed (right) shows a suture ligature around the femoral neck (arrow) and a complete absence of contrast medium in the vessels of the secondary center of ossification and the epiphyseal cartilage. The contrast medium is present in the proximal metaphysis just distal to the metaphyseal physis.

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Fig. 3-A:: Section (3 mm thick), made in the coronal plane, through the middle of a capital femoral epiphysis six weeks following the induction of ischemia (right). The transected suture ligature is shown in the superior and inferior aspects of the femoral neck as black dots (arrows). The contralateral, untreated side is shown on the left. In comparison with the untreated side, the epiphyseal cartilage is thicker and the secondary center of ossification is much smaller on the infarcted side.

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Fig. 3-B:: Radiographs of the capital femoral epiphyses shown in Figure 3-A. The secondary center of ossification on the infarcted side (right) is small, irregular, and radiodense, and it has a fragmented appearance in comparison with the secondary center of ossification on the untreated side (left). Photomicrographs of the same femoral head are shown in Figures 7 through 8-C.

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Fig. 4-A:: A photomicrograph of normal growth-plate cartilage surrounding the secondary center of ossification. The growth-plate cartilage contains the same cellular zones that are found in the metaphyseal physis. The reserve (R), proliferative (P), and hypertrophic (H) zones are shown. Vascular invasion of terminal hypertrophic chondrocytes and new-bone formation, indicating endochondral ossification, are observed. The primary spongiosa is shown (safranin O-fast green, ×330).

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Fig. 4-B:: A photomicrograph of the growth-plate cartilage surrounding the secondary center of ossification two weeks following induction of ischemia. Many empty lacunae are observed in the proliferative and hypertrophic zones. There is a complete absence of vascular invasion of hypertrophic chondrocytes at the chondro-osseous junction. Only a few remnants of the primary spongiosa are seen, and there is an absence of osteoblasts lining these structures (safranin O-fast green, ×330).

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Fig. 5:: A low-magnification photomicrograph of a section from a capital femoral epiphysis four weeks following induction of ischemia. The area of loss of safranin O (A) and the area of fibrovascular tissue invasion (B) are magnified in Figures 6-A and 6-B, respectively. LT represents the ligamentum teres, and SCO represents the infarcted secondary center of ossification (safranin O-fast green, ×10).

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Fig. 6-A:: A higher-magnification photomicrograph of area A in Figure 5 shows further progression of ischemic damage to the growth-plate cartilage. The growth-plate cartilage is necrotic, with an absence of chondrocytes and a loss of safranin-O staining. Cellular debris is observed in the lacunae. A cleft is seen in the necrotic cartilage. Epiphyseal cartilage above the necrotic growth-plate cartilage shows chondrocyte-clustering. The extracellular matrix around the chondrocyte clusters stained strongly with safranin O (safranin O-fast green, ×100).

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Fig. 6-B:: A higher-magnification photomicrograph of area B in Figure 5. Invasion of the necrotic growth-plate cartilage by fibrovascular tissue is observed. The invading tissue is composed of many small vessels, fibroblasts, and loose connective tissue (safranin O-fast green, ×100).

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Fig. 7:: A low-magnification photomicrograph of the capital femoral epiphysis six weeks following the induction of ischemia. A radiograph of this capital femoral epiphysis is shown in Fig. 3-B. The epiphyseal cartilage is markedly thickened, and the secondary center of ossification is small. There is a prominence of cartilage canals in the epiphyseal cartilage (arrowheads), and two new accessory centers of ossification (O) are seen in the periphery of the femoral head. Complex tissue changes are observed with the area of restoration of endochondral ossification (A), the area of fibrovascular tissue invasion (B), and the area of necrotic cartilage (C) magnified in Figures 8-A, 8-B, and 8-C, respectively. LT represents the ligamentum teres, M represents the metaphyseal physis, and SCO represents the infarcted secondary center of ossification (safranin O-fast green, ×10).

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Fig. 8-A:: A higher-magnification photomicrograph of area A in Figure 7 shows restored endochondral ossification. Chondrocyte differentiation, zones of proliferating and hypertrophic chondrocytes, and vascular invasion of terminal hypertrophic chondrocytes are observed. The primary and secondary spongiosa with osteoblasts lining these structures are seen (safranin O-fast green, ×100).

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Fig. 8-B:: A higher-magnification photomicrograph of area B in Figure 7 shows invasion of the necrotic bone and cartilage by fibrovascular tissue in the central region of the capital femoral epiphysis (safranin O-fast green, ×40).

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Fig. 8-C:: A higher-magnification photomicrograph of area C in Figure 7. In contrast to the cartilage in area A, the cartilage in area C is necrotic. A severe loss of safranin-O staining is observed except in the calcified cartilage at the bottom. There is an absence of vascular invasion of cartilage and of new-bone formation. No live cells are observed in the marrow space, which is filled with amorphous debris (safranin O-fast green, ×100).

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Experimental Model

The experimental investigation was approved by the local Animal Care and Use Committee. The piglet model of ischemic necrosis of the capital femoral epiphysis was chosen because the hip anatomy of piglets is similar to that of humans and because studies of ischemic necrosis of the capital femoral epiphysis have previously been performed with use of this model^1,2.

Twenty-seven male Yorkshire piglets, two to four weeks old and weighing 5 to 8 kg, were used. Ischemic necrosis of the capital femoral epiphysis was surgically induced in eighteen piglets as follows. After induction of general anesthesia, a longitudinal incision was made over the right hip joint under sterile conditions. The gluteus muscle and the underlying short abductor muscles were split and retracted to expose the hip joint capsule. A partial capsulotomy was performed, and longitudinal traction was applied to subluxate the capital femoral epiphysis. The ligamentum teres was visualized and was transected to facilitate the placement of double suture ligatures around the femoral neck. With use of a curved instrument, two number-one silk sutures were placed around the femoral neck and hand-tied as tightly as possible to disrupt the ascending cervical vessels supplying the capital femoral epiphysis. The wound was closed in layers. No immobilization was used postoperatively.

Radiographic and Histologic Assessments

Two animals each were killed with an overdose of pentobarbital at three days and then weekly from one to eight weeks after the induction of ischemia. The proximal part of the femur from each of these eighteen animals was dissected, examined grossly, and radiographed with use of the Faxitron Cabinet X-Ray System (Hewlett-Packard, McMinville, Oregon). Then, 3-mm-thick sections were made through the femoral heads with use of a band saw in the coronal plane. Each section was examined grossly, radiographed, and fixed in 10% neutral buffered formalin. The specimens were decalcified, embedded in paraffin, and sectioned at a thickness of 6 mm. The mounted sections were stained with hematoxylin and eosin or with safranin O-fast green. The sections were examined and photographed with use of a Vanox microscope (Olympus America, Melville, New York).

The capital femoral epiphysis from the contralateral side (not operated on) was used as a control. In five additional piglets, exactly the same procedure was performed as described above, except that a suture ligature was not placed around the femoral neck. We used these five animals as sham controls to determine the effects of capsulotomy and transection of the ligamentum teres on the capital femoral epiphysis.

Four additional animals were used to determine the effect of suture ligatures on the blood supply to the femoral head. Immediately following the placement of suture ligatures on one side, the animals were killed with an overdose of pentobarbital and microangiography was performed, as described by Visco et al.³. Heparin was administered intravenously (1000 U/kg) prior to the animal’s death to prevent blood clotting and to facilitate the clearing of blood from the microvasculature. A laparotomy was performed to isolate and cannulate the distal aorta and the inferior vena cava. Saline solution (1 to 2 L) was infused through the inflow tubing in the distal aorta to clear the blood in the lower extremities. When a clear return was obtained from the outflow tubing in the inferior vena cava, a low-viscosity perfusion contrast medium (Microfil; Flow Tech, Carver, Massachusetts) was infused into the inflow tubing in the distal aorta. When the contrast medium was observed exiting through the outflow tube in the inferior vena cava, the tubes were clamped and the animal was kept at 4°C for twenty-four hours to allow the contrast medium to set. The femoral head from the side of the operation and the contralateral (untreated) femoral head were then removed and fixed in 10% formalin. The femoral heads were made transparent with methylsalicylate with use of the modified Spalteholz clearing technique to visualize the microvasculature in the capital femoral epiphysis⁴.

Results

Introduction | Materials and Methods | Results | Discussion | References

The capital femoral epiphysis of the two to six-week-old Yorkshire piglet consists of a roughly hemispherical secondary center of ossification surrounded by epiphyseal cartilage (SCO and EC on Fig. 1). Between the epiphyseal cartilage and the secondary center of ossification is a growth plate (x on Fig. 1), which is responsible for the circumferential growth of the secondary center of ossification. The metaphyseal physis (M on Fig. 1) covers the proximal femoral metaphysis and is responsible for the longitudinal growth of the proximal part of the femur.

None of the animals subjected to the sham operative procedure (capsulotomy and transection of the ligamentum teres) had any gross, radiographic, or histologic evidence of ischemic necrosis or other abnormality. None of the animals had hip dislocation or instability at the time of death.

Placement of the suture ligature around the femoral neck immediately disrupted the blood flow to the capital femoral epiphysis (Fig. 2). No contrast medium was observed in the epiphyseal cartilage or the secondary center of ossification proximal to the ligature. Contrast medium was observed in the proximal metaphysis just distal to the metaphyseal physis.

Examination of the coronally transected femoral heads revealed a smaller secondary center of ossification and thicker epiphyseal cartilage on the infarcted side compared with the untreated side (Fig. 3-A). The difference in size was noticeable at two weeks and increased with time as the secondary center of ossification on the infarcted side ceased to grow (Fig. 3-A). There was also radiographic evidence of the cessation of growth of the secondary center of ossification at two weeks. By six weeks, the difference between the diameters of the secondary centers of ossification on the infarcted and untreated sides was clearly visible, and the secondary center of ossification on the infarcted side appeared irregular and fragmented (Fig. 3-B). A detailed description of the gross, radiographic, and histopathologic changes in the secondary center of ossification will be presented in a separate article.

Histopathologic Changes in the Growth Plate Surrounding the Secondary Center of Ossification

Evidence of an ischemic effect on the growth-plate cartilage surrounding the secondary center of ossification was first observed at two weeks. There was an absence of vascular invasion of terminal hypertrophic chondrocytes and a decrease in the amount of primary spongiosa at the chondro-osseous junction, indicating cessation of endochondral ossification (Figs. 4-A and 4-B). Osteoblasts were absent from the surface of the few remaining primary spongiosa. Many empty lacunae were noted in the proliferative and hypertrophic zones of the growth-plate cartilage surrounding the secondary center of ossification. Chondrocytes in the growth-plate cartilage revealed absent nuclear staining with condensed eosinophilic cytoplasm suggesting cell death. The epiphyseal cartilage surrounding the growth-plate cartilage appeared normal.

By four weeks, a loss of safranin-O staining and the presence of intracartilaginous clefts were observed in the necrotic growth-plate cartilage surrounding the secondary center of ossification (Figs. 5 and 6-A). Chondrocyte clusters and intense safranin-O staining were seen in the epiphyseal cartilage around the necrotic growth plate. Early evidence of invasion of the necrotic cartilage by fibrovascular tissue from the marrow space was also observed (Fig. 6-B). The tissue was composed of small vessels, fibroblasts, and loose connective tissue. More extensive resorption of the necrotic cartilage by the fibrovascular tissue was seen over time.

In animals killed at six and eight weeks, other striking histopathologic changes were observed in different regions of the capital femoral epiphysis (Fig. 7). In the peripheral region of the epiphyseal cartilage, areas of new-bone formation and restoration of endochondral ossification were seen. Small, new ossification centers were observed with chondrocyte differentiation, zones of proliferating and hypertrophic chondrocytes, and vascular invasion of terminal hypertrophic chondrocytes (Fig. 8-A). The presence of small ossification centers contributed to the irregular and fragmented appearance of the femoral head, as seen radiographically in Figure 3-B. In the area of the necrotic cartilage, there was more extensive resorption of the cartilage by the fibrovascular tissue invading from the marrow space (Fig. 8-B). The epiphyseal cartilage above the fibrovascular tissue showed more proliferative and hypertrophic chondrocytes and more cartilage canals. Some vessels in the cartilage canals contained red blood cells, suggesting blood flow through them. In the central region of the capital femoral epiphysis, the necrotic growth plate was still present, with no evidence of restoration of endochondral ossification (Fig. 8-C).

Histologic Changes in the Physis

The physis was abnormal in five of the eighteen animals. The histopathologic changes in the physis were variable and included cessation of endochondral ossification, chondrocyte death, loss of safranin-O staining, resorption of necrotic physeal cartilage by fibrovascular tissue, and complete disruption of the central portion of the physis by fibrovascular tissue invasion.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

The piglet model of ischemic necrosis of the capital femoral epiphysis was originally described in an abstract by Salter¹. Although this model was used to develop some of the current concepts regarding the pathogenesis of Legg-Calvé-Perthes disease^5,6, little has been written about the model itself, including the ischemic changes in the growth-plate cartilage surrounding the secondary center of ossification. It is only recently that those changes have received some attention^7,8. Since ischemic necrosis of the capital femoral epiphysis produces a cessation of growth of the secondary center of ossification, we sought to understand the cellular basis for this effect. Our study shows that ischemic necrosis of the capital femoral epiphysis not only leads to a cessation of endochondral ossification at the growth plate but also produces extensive, irreversible damage to the growth plate surrounding the secondary center of ossification. The piglet model of ischemic necrosis also produced histopathologic changes in the physis, but the changes were not as consistently observed as those seen in the growth plate surrounding the secondary center of ossification.

Our study shows that the necrotic growth-plate cartilage surrounding the secondary center of ossification is resorbed by fibrovascular tissue invading from the marrow space prior to restoration of endochondral ossification. The fibrovascular tissue invasion and the transformation of the epiphyseal cartilage above the necrotic growth-plate cartilage appear to be closely linked. In association with the fibrovascular tissue invasion and resorption of the necrotic growth-plate cartilage, an increase of proliferative and hypertrophic chondrocytes was observed in the epiphyseal cartilage in this region.

It is important to note that revascularization and restoration of endochondral ossification does not occur globally in the femoral head, but rather they occur in the peripheral region of the femoral head first. The asymmetric establishment of growth of the secondary center of ossification contributes to the flattened appearance of the femoral head as the peripheral region of the secondary center of ossification grows but the central region does not.

The formation of new ossification centers in the epiphyseal cartilage contributes to the irregular and fragmented radiographic appearance of the femoral head during the reparative phase. Although it is generally believed that the fragmented appearance of the femoral head on radiographs is due to disintegration of the original secondary center of ossification, our study shows that the new ossification centers that form during the reparative phase of ischemic necrosis explain, at least in part, the fragmented and irregular appearance of the secondary center of ossification.

Our study supports the hypothesis that ischemic necrosis of the capital femoral epiphysis produces selective damage to the growth-plate cartilage surrounding the secondary center of ossification and spares the epiphyseal cartilage^9-12. We observed morphologic changes consistent with cell death along with degradative and reparative changes in the growth-plate cartilage over time. It is interesting to note that surgical and necropsy specimens from patients with Legg-Calvé-Perthes disease also have been found to have cell death in the deep layer of the epiphyseal cartilage, which corresponds to the location of the growth-plate cartilage surrounding the secondary center of ossification. Other histopathologic changes observed in the epiphyseal cartilage and the growth plate surrounding the secondary center of ossification in patients with Legg-Calvé-Perthes disease include chondrocyte-clustering, intracartilaginous clefts, prominent vascularity, fibrovascular granulation tissue invasion, areas of loose cartilage matrix, and small accessory centers of ossification^13-22.

Even though the growth-plate cartilage surrounding the secondary center of ossification was selectively damaged in the piglet model, the extent of the damage to the reserve zone of the growth-plate cartilage was not clearly defined. It is unclear whether the chondrocytes in all three zones (reserve, proliferative, and hypertrophic) of the growth plate are equally affected or whether those in the reserve zone are spared to some degree as the reserve zone blends into the epiphyseal cartilage. Since the morphology of the chondrocytes in the epiphyseal cartilage is similar to that of the chondrocytes in the reserve zone of the growth plate, it is not possible to distinguish a border between the two. In the metaphyseal physis, the chondrocytes in the reserve zone are normally exposed to much lower oxygen tension than are those in the proliferative zone²³. Although we know of no such data for the chondrocytes in the growth plate surrounding the secondary center of ossification, if the same holds true it is likely that the chondrocytes in the reserve zone may be less prone to ischemic injury and may be spared. Sparing of the chondrocytes in the reserve zone provides stem cells for chondrocyte proliferation and differentiation during the reparative phase of ischemic necrosis.

Various experimental models have been used to study the effects of ischemia on the epiphyseal cartilage and the growth-plate cartilage surrounding the secondary center of ossification^9-12. Not all models produce similar histologic changes. The extent of injury to the epiphyseal cartilage ranges from partial-thickness involvement in the central region⁹ to full-thickness involvement¹⁰. In an immature canine model of ischemic necrosis, considerable variability of ischemic changes, ranging from normal to full-thickness abnormalities, was observed in the epiphyseal cartilage¹². In a canine model of ischemic necrosis of the capital femoral epiphysis in which consecutive interruptions of the blood supply to the capital femoral epiphysis were performed¹¹, chondrocyte death was observed selectively in the growth-plate cartilage surrounding the secondary center of ossification. This model, however, was associated with a high rate (fourteen of twenty-five animals) of technical failures. Most of the above studies did not include any observations regarding revascularization or restoration of endochondral ossification following ischemic injury. In comparison with the previously reported canine models of ischemic necrosis, the piglet model used in our study had a low complication rate and it reliably produced ischemic necrosis and histopathologic changes in the growth-plate cartilage surrounding the secondary center of ossification. In this model, there were four failures in the eighteen animals. Septic arthritis of the hip developed in one piglet, and there was no evidence of ischemic necrosis in three piglets. In one of the failures, the suture ligature was not successfully placed around the femoral neck because of a persistently intact ligamentum teres. In the other two animals, the suture ligatures were not tightly placed.

It is important to recognize that the piglet model represents a severe form of ischemic necrosis with whole-head involvement and a prolonged duration of ischemia. The nonabsorbable suture continues to strangulate the femoral neck as the diameter of the neck enlarges through appositional growth from the periosteum. In the animals killed at four weeks, the suture ligatures were found embedded in the femoral neck. Vascular synovial tissue eventually grows over the sutures, and this may be one of the sources of revascularization.

In conclusion, the piglet model of ischemic necrosis of the capital femoral epiphysis produced extensive changes in the growth-plate cartilage surrounding the secondary center of ossification. The model will facilitate the study of the sequence of cellular, structural, and vascular events that follow ischemic injury to the capital femoral epiphysis.

Note: The authors thank Noelle Vallet for making and processing the black-and-white photographs.

References

Introduction | Materials and Methods | Results | Discussion | References

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