The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 84, Issue 8

Scientific Article | August 01, 2002

Development of Flattening and Apparent Fragmentation Following Ischemic Necrosis of the Capital Femoral Epiphysis in a Piglet Model

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

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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, MSc, FRCS(C)
Phi-Huynh Su, BSc
Shriners Hospitals for Children, 12502 North Pine Drive, Tampa, FL 33612. E-mail address for H.K.W. Kim: hkim@shrinenet.org

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Shriners Hospitals for Children. Neither of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

J Bone Joint Surg Am, 2002 Aug 01;84(8):1329-1334

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Abstract

Background: The repair response that follows ischemic necrosis of the immature femoral head and the biological processes that are responsible for the development of femoral head deformity and fragmentation have not been clearly defined. A piglet model was used to study the radiographic and histopathologic changes that occur prior to and during the development of femoral head deformity and fragmentation following ischemic necrosis.

Methods: Twenty-five male piglets were studied. A nonabsorbable ligature was placed tightly around the femoral neck to disrupt the blood supply to the capital femoral epiphysis. The animals were killed three days to eight weeks following the induction of ischemia. Radiographs of whole and sectioned femoral heads were made, and the radiographic findings were correlated with the histopathologic changes observed in the specimens.

Results: Mild femoral head flattening was observed by four weeks after the induction of ischemia, and severe flattening and fragmentation were observed by eight weeks. The predominant repair response observed following revascularization was osteoclastic bone resorption. Prior to the development of flattening, a large area of osteoclastic bone resorption was observed in the central region of the femoral head. Many osteoclasts were present along the revascularization front, which we believe were responsible for active resorption of the necrotic trabecular bone. Appositional new-bone formation, the hallmark of the repair response in adult ischemic necrosis, was not observed in the area of bone resorption. Instead, the areas of resorbed bone were replaced with a fibrovascular tissue that persisted for up to eight weeks. Appositional new-bone formation was observed, but it was limited to small areas in which revascularization was not followed by osteoclastic bone resorption and in which necrotic trabecular bone was still present. The simultaneous presence of the areas of bone resorption and new-bone formation contributed to the fragmented radiographic appearance of the femoral head.

Conclusions: The predominant repair response observed in the piglet model of ischemic necrosis was osteoclastic bone resorption. The early bone loss, the lack of new-bone formation, and the persistence of fibrovascular tissue in the areas of bone resorption compromised the structural integrity of the femoral head and produced progressive femoral head flattening over time. The repair response was different from that observed in femoral heads removed from adult patients with ischemic necrosis and from that observed in the adult rabbit model of ischemic necrosis.

Clinical Relevance: The piglet model of ischemic necrosis may be useful for the investigation of the biological processes that lead to the development of femoral head deformity following ischemic necrosis of the immature femoral head.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

Appositional new-bone formation on the surfaces of the necrotic trabeculae is the predominant repair response observed in the cancellous portion of the adult femoral head following ischemic necrosis and idiopathic osteonecrosis in human beings ¹ . Extensive studies performed by Glimcher and Kenzora ^1-3 on adult femoral heads removed at the time of surgery and studies involving adult rabbit models of ischemic necrosis ^4,5 have clearly supported this observation. Following the invasion of the marrow spaces of the necrotic portion of the femoral head by proliferative mesenchymal cells and capillaries from adjacent living bone, the mesenchymal cells nearest the surfaces of the necrotic trabeculae differentiate into osteoblasts ⁴ . The osteoblasts then form appositional bone on the surface of the necrotic bone trabeculae, eventually covering their entire surface. Subsequently, the central, necrotic portion of the trabeculae is resorbed by osteoclasts and replaced by new bone. Overall, these biological events produce thickening of the trabeculae, increased bone mass per volume, and increased radiodensity in the areas of repair.

To our knowledge, a similar investigation on the repair response of the immature femoral head following ischemic necrosis (as in patients with Legg-Calvï¿½-Perthes disease) has not been performed because of the impossibility of obtaining immature femoral heads for histopathologic examination. In the early stages of Legg-Calvï¿½-Perthes disease, the secondary center of ossification appears flattened and fragmented and has both osteolytic and osteosclerotic areas, suggesting that appositional new-bone formation alone cannot account for the radiographic changes observed. The radiographic changes associated with Legg-Calvï¿½-Perthes disease are different from those associated with ischemic necrosis and idiopathic osteonecrosis of the femoral head in the adult ^2,6-8 , and we hypothesized that the repair response that follows ischemic necrosis of the immature femoral head is different from the repair process in the adult. The purpose of the present investigation was to determine the repair response of the immature femoral head in piglets following ischemic necrosis and to study the biological processes responsible for the development of femoral head flattening and fragmentation. The piglet model of ischemic necrosis of the capital femoral epiphysis, as described by Salter, was used because it is thought to be a clinically relevant model ^9,10 and has been reported to produce femoral head deformity ¹¹ .

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Fig. 1-A:: Radiograph of central, coronal sections of femoral heads from a piglet killed two weeks after the induction of ischemic necrosis. The sections are 4 mm thick. An osteolytic area is clearly seen on the infarcted side (right). The area of bone resorption appeared before the development of the femoral head flattening and apparent fragmentation. The secondary center of ossification on the infarcted side is smaller than that on the contralateral side (left) because of cessation of endochondral ossification at the chondro-osseous junction.

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Fig. 1-B:: Photomicrograph of the central region of the osteolytic area seen in Fig. 1-A. The necrotic trabecular bone has been resorbed and replaced with a fibrovascular tissue. Many small blood vessels, proliferative mesenchymal cells, and fibroblast-like cells are seen. There is no evidence of new-bone formation in this area (hematoxylin and eosin, ×250).

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Fig. 1-C:: Photomicrograph of the peripheral region of the osteolytic area seen in Fig. 1-A. This area represents the area of active bone resorption at the revascularization front. Osteoclasts (multinucleated cells) are seen around the trabeculae, which show scalloping and decreased size, indicating active resorption (hematoxylin and eosin, ×660).

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Fig. 2:: Composite radiograph of central, coronal sections of femoral heads from piglets killed three, four, six, and eight weeks after the induction of ischemic necrosis. An osteolytic area is seen within the secondary center of ossification at three weeks. Progressive flattening and apparent fragmentation of the femoral head begins at four weeks. Severe flattening is observed by eight weeks. It is important to note that the appearance of the osteolytic area preceded the development of the femoral head flattening. A large area of bone loss was still present at eight weeks. Some areas of patchy sclerosis are seen within the secondary center of ossification, where appositional new-bone formation occurred.

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Fig. 3-A:: Radiograph of central sections of the femoral heads from a piglet killed six weeks after the induction of ischemic necrosis. The infarcted femoral head on the right appears flattened and fragmented compared with the normal femoral head on the left.

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Fig. 3-B:: Photomicrograph of the infarcted femoral head shown in Fig. 3-A. Areas of bone resorption (R) and new-bone formation (square) are observed within the secondary center of ossification, giving the radiographic appearance of fragmentation (hematoxylin and eosin, ×25). P = metaphyseal physis.

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Fig. 4-A:: Photograph of coronally bisected femoral heads of a piglet killed eight weeks after the induction of ischemic necrosis. The infarcted femoral head on the right is flattened, and the secondary center of ossification shows the presence of vascular granulation tissue (arrowhead). The contralateral, normal femoral head is shown on the left.

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Fig. 4-B:: Radiograph of central, coronal sections of femoral heads from a piglet killed eight weeks after the induction of ischemic necrosis. The infarcted femoral head on the right demonstrates severe flattening and apparent fragmentation. A large osteolytic area is observed on the infarcted side. There is an absence of new-bone formation within the area of bone loss.

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Experimental Design

The experimental investigation was approved by the institutional Animal Care and Use Committee. Twenty-five male piglets, three to five weeks old and weighing 6 to 9 kg, were obtained from a commercial breeder. Ischemic necrosis was induced in twenty-one animals by placing a nonabsorbable ligature tightly around the femoral neck as described previously ¹² . Three animals each were killed arbitrarily at three days and at one, two, three, four, six, and eight weeks following the induction of ischemic necrosis. The remaining four animals served as sham controls. In these animals, a capsulotomy and transection of the ligamentum teres was performed but the ligature was not placed around the femoral neck. Two animals each were killed at four and eight weeks following the sham operation. In the experimental group, the contralateral side (that is, the side not operated on) was also used as a control.

Radiographic and Histologic Assessments

After the animal had been killed, the proximal part of the femur was dissected, examined visually, and radiographed as a whole and as 4-mm-thick sections. Specimens were then prepared for histologic assessments as described earlier ¹² .

Results

Introduction | Materials and Methods | Results | Discussion | References

None of the animals that had undergone the sham operation demonstrated any evidence of ischemic necrosis or femoral head deformity. Nineteen of the twenty-one experimental animals had histopathologic changes consistent with ischemic necrosis. The two experimental animals without ischemic changes had had improper placement of the ligature.

Less than two weeks after the induction of ischemia, the infarcted femoral heads revealed no evidence of deformity or change in the radiodensity of the secondary center of ossification, either grossly or radiographically. Histologically, empty lacunae were observed in some regions of the trabecular bone and extensive cell death was observed in the marrow space. At the chondro-osseous junction, a cessation of endochondral ossification was observed.

At two weeks, the secondary center of ossification did not show any gross or radiographic evidence of deformity, but it was smaller than that on the contralateral side because of the cessation of growth. The most striking change, however, was the presence of an osteolytic area within the infarcted secondary center of ossification ( Fig. 1-A ). Histologically, the osteolytic area correlated with an area of revascularization of the necrotic marrow space. In the revascularized area, a fibrovascular tissue consisting of blood vessels, proliferative mesenchymal cells, and fibroblastic cells was observed ( Fig. 1-B ). In the perimeter of the revascularized area, designated as the revascularization front, many osteoclasts were present on the trabeculae bordering the invading fibrovascular tissue ( Fig. 1-C ). There was scalloping and shortening of the trabeculae, indicating active bone resorption. The osteoclastic bone resorption was the predominant early repair response observed following revascularization of the necrotic marrow space. New-bone formation was not observed in the area of bone resorption.

At four weeks, mild flattening of the infarcted femoral head was observed both grossly and radiographically ( Fig. 2 ). A larger area of the infarcted secondary center of ossification showed osteolysis with osteoclastic resorption of necrotic trabeculae and replacement by fibrovascular tissue. No new-bone formation was observed within the area of bone resorption; however, in adjacent areas where revascularization of the necrotic marrow spaces was not followed by osteoclastic bone resorption, appositional new-bone formation was observed. New-bone formation was not as pronounced as bone resorption in the early stage (that is, at two to three weeks) and occurred in the areas where the necrotic trabeculae were still present. Over time, appositional bone formation on the surfaces of the necrotic trabeculae produced trabecular thickening and decreased marrow space. The areas of bone resorption and the areas of thickened trabeculae combined to produce a fragmented appearance of the femoral head on radiographs ( Figs. 3-A and 3-B ). Assessment of the newly formed bone with use of polarized light microscopy revealed a random distribution of the collagen matrix, indicating woven-bone formation.

At eight weeks, severe flattening and fragmentation of the femoral head was observed grossly and radiographically ( Figs. 4-A and 4-B ). The fibrovascular tissue that had replaced the necrotic bone persisted at this time-period. No new-bone formation was observed within the area of bone resorption.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

In the present study, the predominant response observed in the areas of revascularization in the piglet model was osteoclastic bone resorption. Appositional new-bone formation was not observed in the areas of bone resorption. The necrotic bone was replaced by fibrovascular tissue rather than new bone. The appearance of the osteolytic area preceded the development of femoral head flattening, suggesting that the replacement of bone by fibrovascular tissue compromised the structural integrity of the femoral head and that the repair process plays an important role in the pathogenesis of femoral head deformity. Further studies are required to investigate why the resorption of necrotic bone is not closely followed by new-bone formation in the piglet model and why fibrovascular tissue persists in the areas of bone resorption.

The repair response observed in the piglet model is different from the repair response reported by Kenzora et al. ⁴ and others ⁵ in the adult rabbit model of ischemic necrosis and in femoral heads obtained at the time of surgery from adult patients with ischemic necrosis and idiopathic osteonecrosis ¹ . In the adult rabbit model of ischemic necrosis, osteoblastic differentiation of mesenchymal cells and appositional new-bone formation are the predominant early repair responses observed ⁴ . It is only when the appositional new bone has surrounded the entire surface of the necrotic bone that resorption of the central, dead trabecular bone takes place. Since new bone initially forms on the surfaces of the necrotic bone and the central, necrotic bone is subsequently replaced by additional new bone, one does not observe areas of osteolysis in the adult rabbit model. Instead, increased radiodensity is observed in the areas of revascularization in that model. We believe that this difference in the repair process not only produces different radiographic changes in the two animal models but also has important implications for the mechanical properties of the femoral head and the development of femoral head flattening following ischemic necrosis.

The fragmented appearance of the femoral head, as observed radiographically, is due to a combination of biological processes occurring in the secondary center of ossification. Islands of thickened trabeculae (due to new-bone formation) mixed with areas of bone resorption and replacement by the fibrovascular tissue produce an appearance of fragmentation of the femoral head. Although the term "fragmentation" implies that the secondary center of ossification has been broken up into small parts, the present study shows that the repair process consisting of bone resorption and formation is partly responsible for the fragmented appearance. Another aspect of the repair process that contributes to the fragmented appearance of the secondary center of ossification is the formation of new, small accessory centers of ossification in the epiphyseal cartilage following revascularization and restoration of endochondral ossification. This finding was described in our previous report ¹² .

Our findings are in partial agreement with the concept of biological plasticity of the living bone as proposed by Salter to explain the development of femoral head deformity following ischemic necrosis ¹¹ . Salter observed that the radiographic changes and flattening occurred when the piglets were fully weight-bearing with the involved hip in adduction with varying degrees of subluxation. It has been proposed that the woven bone formed during the repair process leads to gradual deformation of the femoral head because of its inferior ability to withstand mechanical loading when compared with lamellar bone. Our findings suggest that increased bone resorption with very little bone formation in the areas of bone loss compromises the mechanical properties of the femoral head and leads to the loss of structural integrity. Our findings indicate that the femoral heads became deformed because of the lack of new bone formation in the areas of bone resorption and not because of the deformation of new woven bone per se.

It is important to point out that we purposely did not use the term creeping substitution to describe the repair response following ischemic necrosis of the femoral head in this study. Phemister used that term to describe a process in which dead bone is substituted by new bone in the infarcted segment of adult femoral heads following femoral neck fractures ^13,14 . In the piglet model, the term creeping substitution does not describe the repair process observed in the areas of osteoclastic bone resorption. In these areas, the necrotic trabecular bone is resorbed but is replaced not by new bone but rather by fibrovascular tissue. The "substitution" of necrotic bone by new bone does not occur.

In conclusion, the predominant repair response observed in the piglet model of ischemic necrosis was osteoclastic bone resorption. This resorption produced large areas of osteolysis, which compromised the structural integrity of the femoral head. Replacement of resorbed bone by fibrovascular tissue, the lack of new-bone formation, and persistence of the fibrovascular tissue in the areas of bone loss contributed to the development of femoral head deformity.

Note: The authors thank the Shriners Hospitals for Children for funding this research.

References

Introduction | Materials and Methods | Results | Discussion | References

Glimcher MJ,Kenzora JE. Nicolas Andry Award. The biology of osteonecrosis of the human femoral head and its clinical implications: I. Tissue biology. Clin Orthop,1979;138: 284-309. 138284 1979 [PubMed]

Glimcher MJ,Kenzora JE. The biology of osteonecrosis of the human femoral head and its clinical implications: II. The pathological changes in the femoral head as an organ and in the hip joint. Clin Orthop,1979;139: 283-312. 139283 1979 [PubMed]

Glimcher MJ,Kenzora JE. The biology of osteonecrosis of the human femoral head and its clinical implications. III. Discussion of the etiology and genesis of the pathological sequelae; comments on treatment. Clin Orthop,1979;140: 273-312. 140273 1979 [PubMed]

Kenzora JE, Steele RE, Yosipovitch ZH,Glimcher MJ. Experimental osteonecrosis of the femoral head in adult rabbits. Clin Orthop,1978;130: 8-46. 1308 1978 [PubMed]

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Bohr HH. On the development and course of Legg-Calve-Perthes disease (LCPD). Clin Orthop,1980;150: 30-5. 15030 1980 [PubMed]

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Topics

ischemia ; bone resorption ; femur ; head of femur ; necrosis ; femoral epiphysis ; fragmentation procedure

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Harry K.W. Kim, Phi-Huynh Su; Development of Flattening and Apparent Fragmentation Following Ischemic Necrosis of the Capital Femoral Epiphysis in a Piglet Model. The Journal of Bone & Joint Surgery. 2002 Aug;84(8):1329-1334.

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