Journal of Orthopaedic Research 9 4 4 5 4 5 1 Raven Press, Ltd., New York 0 1991 Orthopaedic Research Society

Experimental Transplantation of the Swarm Rat Chondrosarcoma into Bone: Radiological and Pathological Studies "Samuel Kenan and *?German C. Steiner *Department of Pathology and Laboratory Medicine, Hospital for Joint Diseases Orthopaedic Institute, and #New York University School of Medicine, New York, New York, U.S.A.

Summary: The Swarm rat chondrosarcoma has been the subject of extensive biochemical studies. However, to our knowledge, there are no previous reports in the literature on transplantation of this tumor into bone. This article describes the natural history of the tumor when implanted into the bone of the rat, and correlates its histological growth pattern with its radiological appearance. Our results showed that the tumor grows slowly in the bone. The rate of intramedullary growth, however, was variable and was not the same in all the animals. Its growth pattern resembles human chondrosarcoma, with extensive invasion of the marrow and cortex. In the first few weeks after implantation, the only radiological changes noted were mild medullary radiopacities. At a later stage, 12-14 weeks postimplantation, as the tumor infiltrated the bone, significant radiological abnormalities were observed in the medullary cavity and cortex. Periosteal reaction was seen after the tumor invaded the cortex with the production of a soft-tissue mass. Distant dissemination was rare; only 1 of 24 rats developed pulmonary metastases. The Swarm rat chondrosarcoma is a well-differentiated malignant tumor that histologically resembles welldifferentiated human chondrosarcoma. Transplanted into bone, it may be useful as an experimental model for comparative studies with human chondrosarcoma. Key Words: Transplantation-Swarm rat chondrosarcoma.

chondrosarcoma into bone. The purpose of our experiment was to determine the growth potential of this tumor after it was implanted into the medullary cavity of bone, to study the natural history of the tumor, and to evaluate and correlate the histological pattern of invasion with the radiological changes that were observed. Because there are few histological descriptions of the Swarm rat chondrosarcoma in the literature, the morphology of this tumor is briefly reviewed before the experiment is presented.

The Swarm rat chondrosarcoma is derived from a tumor that originally arose spontaneously in a female Sprague-Dawley rat. It has been maintained for several years by serial subcutaneous transplantation every 4-6 weeks (16). The tumor was originally composed of both bone and cartilage elements. With successive transplantations, the bony elements disappeared and only cartilaginous elements remained (1). The composition of the tumor has been stable for the past several years (9,12). A search of the literature has revealed no experimental studies on transplantation of the Swarm rat

MORPHOLOGICAL DESCRIPTION OF SWARM RAT CHONDROSARCOMA

Received August 12, 1987; accepted September 26, 1990. Address correspondence and reprint requests to Dr. G. C. Steiner at Hospital for Joint Diseases Orthopaedic Institute, 301 East 17th Street, New York, NY 10003, U.S.A.

Most tumors are recognized on gross examination, usually 2-3 weeks later after subcutaneous 445

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transplantation of tumor tissue into female SpragueDawley rats. The incidence of tumor growth is >90% of injected animals. The tumor grows rapidly by local expansion, reaching several centimeters in subsequent weeks (1). On cut section, the tumor is well circumscribed, grayish-translucent, and nodular. Central degeneration and necrosis of the larger nodules are usually present and granular calcification sometimes is seen in these areas. Histologically, the tumor is a well-differentiated chondrosarcoma containing prominent nuclei of uniform size with mild atypia (Fig. 1A). Cellular pleomorphism, binucleation, and giant cells are infrequent and mitoses are rare (1). Calcification is rarely seen between viable cartilage cells. No reactive ossification is noted. The matrix reacts positively with alcian blue at pH 1, and S-100 protein is positive in the cells. In our experience, this tumor grows locally in the subcutaneous tissue and does not metastasize. Electron microscopy studies reveal that the chondrocytes of this tumor have features similar to those of normal hyaline cartilage (9). The extracellular matrix consists of fine, electron-dense granules separated by amorphous material with fine filamentous structures. The density of the matrix is decreased around the cells (Fig. 1B). MATERIALS AND METHODS

The Swarm rat chondrosarcoma has been maintained in our laboratory for many generations by serial subcutaneous transplantation. For the present study, we used fresh, viable tumor tissue from the extraperitoneal growth, taken under sterile conditions. After the rat was sacrificed, the tumor was immediately minced as finely as possible. Crystalline penicillin was the only solution added to the tumor, and this was done with the purpose of reducing the risk of contamination. The experiment used 24 Sprague-Dawley adult female rats (age 3 months and approximate weight 250-270 g) that were put under general anesthesia by means of an intraperitoneal injection of phenoFIG. l.The Swarm rat chondrosarcoma (subcutaneous implant). A: Photomicrograph shows well-differentiated chondrosarcoma with a lobular pattern. The cells contain nuclei of uniform size and there is mild pleomorphism (H&E x66). B: Electron micrograph of a chondrocyte from the chondrosarcoma. The nucleus is eccentric and the rough endoplasmic reticulum is often dilated. There is scalloping of the cell membrane with short cell projections. The extracellular matrix is sparse and contains fine granules and fibrils (~4,300).

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barbital. The right leg was prepared and draped in the usual sterile fashion. Through a longitudinal incision at the anteromedial aspect of the midleg, a 2-mm drill hole was made through the cortex of the tibia into the medullary cavity. A cannulated needle was inserted proximally into the medullary cavity and approximately 0.015 cc (0.015 g) of minced tumor was carefully injected. After injection, the area was thoroughly irrigated with saline solution. After the implantation, the drill hole was plugged with bone wax and the wound was closed. Because of the time required for the procedure, the tumor was implanted into the animals on different occasions. Younger rats were originally used in our studies, but the tibia often fractured during the drilling of the bone and for that reason we chose adult rats. Radiographs of the entire animal were taken at different intervals after implantation of the tumor tissue. In this way any changes occurring in the right tibia could be observed as the tumor developed. Two rats were sacrificed at intervals of 2, 4, 6,8,10,12, 14,16,18,20,22, and 24 weeks after the tumor was implanted. Autopsies were performed on all the animals. The right leg of every animal was dissected out, and after fixation, the bone was decalcified, embedded in paraffin, cut longitudinally, and stained with hematoxylin and eosin. RESULTS Intramedullary tumor was detected in the right tibia of 20 of the 24 rats used in the experiment.

Two cases were rejected because most of the tumor cells were necrotic (Fig. 2). Therefore, definite evidence of tumor growth was present in 18 of 24 rats (75%). The degree of tumor growth was variable and did not occur at the same rate (Fig. 2). (The size of the tumor represents the longest single measurement of the lesion). At 2 weeks after implantation, no radiological changes were noted other than minimal central radiopacities. A few millimeters of tumor was identified histologically in the medullary cavity. Mild reactive calcification and ossification were noted around the tumor lobules. In addition, foci of inflammatory reaction as well as foreignbody reaction to wax were seen adjacent to the drill hole. Minimal periosteal reaction also was observed at the site of the drill hole. At 4-6 weeks, mild central radiopacities were noted in the implanted right tibia in 19 of 22 rats. They were not present in the normal left tibia (Fig. 3A). Only one rat showed cortical thickening in the tibia. Histologically, the tumor was seen infiltrating proximally the medullary cavity of the tibia (Fig. 3B). At 8-10 weeks, progressive tumor infiltration of the tibia could be seen histologically. Radiographs of 18 rats showed central radiopacities in 17 and soft-tissue infiltration in 2 rats. In one rat, there was cortical thickening and scalloping. No periosteal reaction was noted in any of the animals (Fig. 4A and B). At 12-14 weeks, histologically, the tumor permeated the cortex through the haversian canals. Radiographs of 11 rats showed soft-tissue extent in 6

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FIG. 2. Rate of tumor growth in the tibia of 24 rats with transplanted Swarm chondrosarcoma. x, one rat showed no tumor; tumor cells were mostly necrotic; 0,tumor with lung metastases.

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FIG. 3. Six weeks postimplantation. A: Radiograph of the right tibia shows mild radiopacity within the bone (arrow). No periosteal reaction is seen. A defect from the drill hole is noted in the cortex. B: Photomicrograph demonstrates tumor infiltration of the medullary cavity, with extension to the inner cortex. Reactive ossification is seen surrounding the tumor lobules (arrow); this correlates with the radiopacities noted in the radiograph (A) (H&E x29).

rats and periosteal reaction in 3 rats (Fig. 5A and B). From 16 to 24 weeks, progressive bone infiltration was seen associated with a large soft-tissue mass (Fig. 6A and B). Autopsy examination revealed pulmonary metastases in only one rat, 14 weeks after implantation, in the form of multiple nodules measuring 0.10.4 cm in both lungs. The histological appearance of the primary and metastatic tumor in this case was similar to that of the other tibia1 tumors (Fig. 7). No other metastases were seen. Tumor contamination at the site of injection in the soft tissue occurred in 3 of the 24 rats. Soft-tissue infection was noted in one rat. Only one animal died during the experiment, 18 weeks after bone implantation. It died of natural causes and no tumor was found. DISCUSSION Chondrosarcoma is an uncommon tumor in all species of mammals (2,23). Kirsten et al., in 1962, produced an osteochondrosarcoma by injection of polyoma virus into neonatal Wistar rats (15). Stan-

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ton, in 1967, induced chondrosarcoma by direct introduction of cupric-chelated N-hydroxy-Nacetylaminofluorene into the medullary cavity of the femur in rats (21). Gregson and Offer, in 1981, reported two cases of spontaneous chondrosarcoma with lung metastases in Sprague-Dawley and Wistar rats, noting that these were the first documented instances of spontaneous chondrosarcoma in rats (8). The Swarm rat chondrosarcoma is a tumor that originated spontaneously in the female SpragueDawley rat. It has been a useful model for morphological and biochemical studies, because it has histological features similar to those of well-differentiated (grade I) human chondrosarcoma such as moderate cellularity and mild nuclear hyperchromasia (1,5,9-13,19,22). Binucleated cells, cell pleomorphism, and giant cells are infrequent, and mitoses are rare. S-100 protein, which is a useful immunohistochemical marker for cartilage (24), is positive in Swarm rat chondrosarcoma, although the reaction is weaker than it is in human chondrosarcoma. The matrix of the rat chondrosarcoma re-

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FIG. 4. Ten weeks postimplantation. A: Increased areas of radiopacity and radiolucency can be seen within the bone. Minimal cortical changes are noted. B: Most of the medullary cavity is infiltrated by the tumor, which has destroyed the preexisting trabeculae. The tumor has reached the growth plate (arrow) (H&E x30).

FIG. 5. Fourteen weeks postimplantation. A: The cortex is destroyed and there is periosteal reaction associated with a soft-tissue mass. B: Chondrosarcoma is seen eroding and infiltrating the cortex. The tumor is histologically similar to the subcutaneous implants (A) (H&E x67). J Orthop Res, Vol. 9. No. 3, 1991

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FIG. 6. Eighteen weeks postimplantation. A: Extensive bone changes are evident with widening of the medullary cavity, destruction of the cortex, periosteal reaction, and the development of a soft-tissue mass. B: Low-power photomicrograph shows marked cortical destruction of the bone. and soft-tissue invasion. Codman’s triangle is seen in the cortex at the periphery of the tumor (arrows) (H&E x29).

acts positively with alcian blue at pH 1 in the same fashion as hyaline cartilage and human chondrosarcoma do, indicating the presence of sulphated glycosaminoglycans (14,20). The proteoglycans from the rat chondrosarcoma, however, differ from those in mature hyaline cartilage and human chondrosarcoma, mainly because they contain only condroitin 4-sulphate and hyaluronic acid (3,17,18,22). Our experimental studies demonstrated that Swarm rat chondrosarcoma grows when it is implanted in the tibial bone of the rat. However, as shown in Fig. 2, the rate of intramedullary growth was variable and it was not the same in all the rats. Histologically, the degree of cellularity and the appearance of the tibial tumor are similar to those of the subcutaneous implants. The tumor infiltrates the intertrabecular spaces and cortex of the tibia in a manner resembling that of human chondrosarcoma, and this is one of the most important criteria for the histological diagnosis of chondrosarcoma, as emphasized by Sanerkin (19). Like human chondrosarcoma, the Swarm rat chondrosarcoma grows slowly in the bone. It takes approximately 8-10

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weeks for the tumor to extensively infiltrate the medullary cavity of the tibia. In contrast, in experimentally implanted carcinoma and induced osteosarcoma in bone, the tumor infiltrates the medullary cavity in several days and readily metastasizes (4,6,7). In our experiment, the earliest radiological changes seen after tumor implantation were mild increased radiopacities within the bone, which were probably due to reactive ossification seen histologically in the tumor. However, the radiopacities were also seen in the four rats in which no tumor was found. Therefore, these early radiological changes are nonspecific and may also indicate reactive ossification due to disruption of the medullary bone at the time of tumor implantation. Periosteal changes were noted in several cases after the tumor invaded the cortex and extended into the soft tissues. This usually occurred approximately 12-14 weeks postimplantation. Only 1 of 24 rats demonstrated pulmonary lesions at the time of autopsy 14 weeks after tumor implantation. This correlates with the rare incidence of

SWARM RAT CHONDROSARCOMA

FIG. 7. Photomicrograph of the lung reveals metastatic chondrosarcoma. The tumor is partly necrotic and is histologically similar to the tibial implant (H&E x59).

metastases in well-differentiated human chondrosarcoma (19). Some technical difficulties were encountered during the experiment, such as tumor contamination of the surrounding soft tissue, and failure of tumor growth after implantation and infection. However, with greater care, those problems can be minimized in future experiments. In conclusion, we have demonstrated for the first time the natural history of the Swarm rat chondrosarcoma when implanted into the tibial bone of the rat, and correlated the histological pattern of tumor growth with radiological changes in the bone. We believe that this transplantable chondrosarcoma in bone may be useful as an experimental model for comparison with human chondrosarcoma. Acknowledgment: We gratefully acknowledge the technical assistance of Mr. A1 Can.

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