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Animal Models |
From the Institute of Pathology,*
Johannes Gutenberg
University, Mainz, Germany; Biomatech,
Chasse-sur-Rhône, France; and the Institute of Clinical
Engineering,
University of Liverpool,
Liverpool, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By comparison, sarcomas arise much less frequently in humans. This, coupled with the absence of a clearly defined preneoplastic lesion for human sarcomas, has hampered progress in understanding sarcoma tumorigenesis. This in turn has prompted the search for suitable animal models of sarcoma. A variety of models exists to create sarcomas, including the subcutaneous implantation of methylcholanthrene-induced sarcoma in Fischer rats9 and the induction of visceral angiosarcomas in C57B1/6 mice using dimethyl hydrazine.10 Newborn Fischer-344 rats have also been used to establish a dimethylnitrosamine-induced model of a malignant mesenchymal nephroma with similarities to the atypical mesoblastic nephroma of infancy.11 Moreover, sarcomas are also known to arise in p53-deficient mice.12
Foreign body-induced sarcomas were extensively studied by KG Brand, especially in the 1970s and early 1980s. A variety of mouse strains, such as the CBA/H and CBA/H-T6, were found to be of particular use in eliciting sarcomas following subcutaneous implantation of copolymer films of vinyl chloride/vinyl acetate,13 although a broad spectrum of other mouse strains was also tested to determine the role of sex and strain in sarcoma incidence.14 Using transfer of preneoplastic reactive tissue from one mouse strain to another, it was shown how the size, material composition, and surface properties of the implants modulated tumorigenesis.15,16
In the present paper we describe a rat model of sarcoma using the subcutaneous implantation of eight different biomaterials, including metals and various synthetic polymers. This gives not only a reproducibly high yield of sarcomas but also a spectrum of lesions, beginning with connective tissue hyperplasia through dysplasia to sarcoma, predominantly malignant fibrous histiocytoma, and pleomorphic sarcoma. This relatively simple model should open up the field of sarcoma tumorigenesis to the molecular biologist and allow the multistage hypothesis to be tested, analogous to what has already been performed for carcinoma.
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Materials and Methods |
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Nine different types of biomaterial (5 polymers, 3 metals, and 1 ceramic) were chosen, all of which are in routine clinical use. These standard medical grade biomaterials were as follows: ultrahigh molecular weight polyethylene (PE), aliphatic polyurethane (PU), polyvinyl chloride (PVC), polymethylmethacrylate (PMMA), silicone (Si), 99% purity titanium (Ti), nickel chromium (NiCr, 78% nickel, 20% chromium), cobalt-chromium alloy (65% cobalt, 27% chromium), and aluminum oxide (Al2O3).
Material Characterization
The biomaterial disks were prepared in such a way as to give a smooth surface, which was confirmed by scanning electron microscopy (Hitachi S 800 SEM). The purity of the medical grade biomaterials was confirmed by a combination of conventional physicochemical surface characterization techniques. These were energy-dispersive X-ray analysis, Fourier-transformed infrared spectroscopy, and X-ray photoelectron spectroscopy.
Animals and Implantation Protocol
The Fischer rat, which has a low natural incidence of soft tissue tumor development, was selected as experimental animal. The rats were individually caged and maintained under controlled conditions of temperature, humidity, and lighting. Animals were anesthetized using intramuscular injection of tiletamine-zolazepam (50 mg/kg body weight). Implantation with 3 identical implants per animal was performed in the subcutaneous tissue of the back in young rats 7 to 8 weeks old. Disks of 15 mm diameter and a total surface area of 350 mm2 per disk were used. Before implantation, metal samples were sterilized by steam autoclaving, and polymer samples were sterilized by ethylene oxide gas. Controls consisted of sham-operated animals, in which subcutaneous incisions were made without implantation. A maximum follow-up of 24 months included general evaluation of the animal and specific investigation of the implantation site every 2 weeks, when animal weight was recorded.
To increase the likelihood of detecting early stages in the development of tumors around the implants, two principal studies were carried out, ie, at 8 months and 24 months. For the 8-month study each group consisted of 10 rats (5 male, 5 female), each with 3 implantation sites. In addition, from a preliminary study, tissue was also available from a PU and PE group after 3 months of implantation (10 animals per group, 90 animals in total). In the 24-month study, each group consisted of either 30 or 68 rats (50% female), again with 3 implantation sites per animal (490 animals in total).
Histology and Immunohistochemistry
Animals were sacrificed by a lethal intraperitoneal injection of barbiturates once a tumor had reached a mass of approximately 50 g or before this from ethical considerations. This endpoint was determined by estimating from the first sarcomas obtained the tumor size corresponding to a weight of 50 g. A complete autopsy was performed on each animal. All local tumors at the implantation site, as well as any remote tumors, were fixed for histological examination. In addition, all capsules around the implants were preserved for microscopy, irrespective of the presence or absence of a tumor. Tissues were fixed immediately after removal from the sacrificed animals in 10% buffered formalin, embedded in paraffin, and cut into sections 5 µm thick. Conventional staining was performed with hematoxylin and eosin (HE) and the elastica-van Gieson stain for connective tissue components.
A total of 193 tumors and 312 capsules surrounding the biomaterials was investigated. Buffered formalin-fixed tissues obtained from biomaterial implant sites were sectioned from paraffin blocks in the conventional manner at 5 µm thickness. Immunohistochemical reactions were performed with a variety of antibodies. Macrophage-detecting antibodies (ED1, diluted 1:50, ED2, diluted 1:50, ED3, diluted 1:200, and KiM2R, diluted 1:100) were supplied by BMA Biomedicals AG (Augst, Switzerland) and used after trypsin pretreatment. Visualization was achieved using either avidin-biotin complex (ABC) or alkaline phosphatase-anti-alkaline phosphatase protocols. T cells were stained with the antibodies MAS 010 and MAS 041 (each diluted 1:150), supplied by Sera Feinbiochemica GmbH (Heidelberg, Germany) and visualized using the ABC protocol. B lymphocytes were detected with the antibody MAS 258 (KiB 1R, Sera), diluted 1:200, using the ABC method after trypsinization. Finally, the proliferating cell nuclear antigen (PCNA) antibody (DAKO Diagnostika GmbH, Hamburg, Germany) was used with an ABC protocol after microwave treatment of the sections. Controls consisted of the use of an irrelevant antibody or omission of the primary (specific) antibody.
Statistical Analysis
The weight change between a particular implant group and the sham-operated controls was tested for statistical significance using the one-way analysis of variance parametric test, followed by the Scheffé test a posteriori. Statistical significance was placed at the 5% confidence level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At 2 years (week 104), 340 tumors had developed from a total of 1266 implantation sites, with 48 animals bearing more than one tumor. The control (sham-operated) animals had no tumor development at any incision site. Generally, once a tumor became visible, rapid growth was observed over a period of 3 to 4 weeks.
Figure 1
presents a typical curve of
weight evolution, exemplified by the rats in the NiCr implant group,
compared with their sham-operated controls. No statistically
significant differences could be detected. This applied also for most
implant groups.
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presents a
summary of the most important data, namely the time of first appearance
of a sarcoma and the percentage of implantation sites in animals
(irrespective of gender) bearing macroscopic soft tissue tumors at the
end of the 2-year observation period. Figure 2
illustrates a typical cumulative
display of sarcoma development with time, as well as a survival curve
for the PU group.
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Histological Tumor Type
All tumors at biomaterial implantation sites were of mesenchymal
origin. However, no correlation could be established between
biomaterial groups and a specific histological type of tumor, although
the PU group did exhibit a tendency to form hemangiosarcomas. The most
frequently encountered tumor was the malignant fibrous histiocytoma
(MFH), followed by pleomorphic sarcomas. Many cases exhibited mixed
differentiation patterns. Table 2
gives
the absolute incidence of the various histological types in a total
population of 193 tumors from all groups. These sarcomas were seen to
be locally invasive, with infiltration of fatty connective tissue and,
in many cases, of skeletal muscle. Multiple foci of necrosis were
frequently observed.
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, which demonstrates not only the
most frequently encountered soft tissue tumors, such as MFH (Figure 3A)
, pleomorphic sarcoma (Figure 3B)
, and fibrosarcoma (Figure 3C)
, but
also some of the rarer tumors, such as leiomyosarcoma (Figure 3D)
and
osteosarcoma (Figure 3E)
. In addition, based on a distinctive
cytological appearance of the tumor cells, we recognized individual
cases that we termed epithelioid sarcoma (Figure 3F)
and round cell
sarcoma (Figure 3G)
. Ranked third in incidence were the sarcomas that
could not be placed in one category because substantial parts of the
tumor mass exhibited more than one differentiation pathway. These
tumors were classified as sarcomas with mixed differentiation and often
showed the combination of MFH with a vascular element, usually
hemangiosarcoma or hemangiopericytoma (Figure 4A)
. The pure hemangiosarcomas often gave
the appearance on low-power microscopy of a spongy tissue (Figure 4B)
.
High-power examination revealed the clearly atypical nuclei of the
cells lining these sponge-like spaces and various degrees of vessel
branching (Figure 4C)
. One vascular tumor that developed around a NiCr
implant showed some features reminiscent of papillary
hemangioendothelioma (Figure 4D)
. Occasionally, hemangiosarcomas were
highly cellular and, in one case around a titanium implant,
demonstrated a high level of apoptosis (Figure 4, E and F)
. Sarcomas
that presented no identifiable differentiation pathways were classified
as sarcoma not otherwise specified (NOS). This group accounted for the
fourth highest incidence among all tumors (Table 2
and Figure 3H
).
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The time after implantation at which capsule tissue became
available was generally determined by the decision to sacrifice, based
on the presence of a macroscopically manifest tumor. This meant that a
broad spectrum of times of exposure to the biomaterials became
available for histopathological evaluation. In general, a thin fibrous
connective tissue capsule of approximately 300 µm formed around the
entire circumference of the subcutaneous implant (Figure 5A)
. Histopathological examination
revealed a variety of morphological entities, which were clearly
distinguishable from each other. As was the case for the histological
type of tumor developing around a biomaterial, no association could be
established between the biomaterial type and the capsule reaction. In
addition to paucicellular fibrous tissue capsules, focal or
extensive proliferative lesions were observed (Figure 5B)
. These
proliferative lesions consisted of groups of polygonal and/or spindle
cells, usually in the inner portion of the capsule, adjacent to the
biomaterial (Figure 5C)
. A further morphological variation took the
form of a proliferative lesion, but with the additional feature that
discrete cellular atypia were present, which could not be definitively
categorized as preneoplastic. These lesions were therefore termed
proliferative lesions, possibly preneoplastic (Figure 5D)
. In many
cases, however, foci of proliferation were observed with marked
cellular atypia, such as cellular and nuclear pleomorphism, coupled
with hyperchromasia and well-defined nucleoli (Figure 5E)
. These
capsules were designated as preneoplastic lesions. A few capsules
revealed a further stage in the development of a sarcoma and showed
usually a larger area of markedly atypical cells with an organoid
pattern. These lesions could be clearly identified as malignant, but as
they were macroscopically unable to be detected, we termed them
incipient sarcoma (Figure 5F)
.
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shows the histology of a manifest tumor which developed on
the deeper surface of a PU implant. This tumor was classified as a
rhabdomyosarcoma and clearly demonstrated large tumor cells with
abundant eosinophilic cytoplasm. The superficial (ie, overlying the
implant) capsule layer was macroscopically a thin fibrous capsule.
However, histologically this layer contained some foci with abnormal
connective tissue cells, showing a rhabdomyoblastic phenotype (Figure 5H)
. These cells were clearly atypical, were topographically completely
isolated from the rhabdomyosarcoma on the deeper capsule layer, and
were therefore regarded as a preneoplastic lesion.
Concerning the incidence of the various capsule lesions, Table 3
shows data from the investigation of
the short-term implant capsules, that is, after 8 months (after 3 and 8
months in the case of PU, and after 3 months in the case of PE).
Extensive proliferative lesions characterized the PU group, which along
with the Si group gave the highest incidence of proliferative lesions
with possible preneoplastic change (33 and 50%, respectively).
However, in the short-term study no unequivocal diagnosis of a
preneoplastic lesion could be made. An important observation was that
the Ti group revealed a high incidence (70%) of focal proliferative
lesions but, in the long term, the lowest incidence of sarcoma
development.
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, which shows the
percentage of incidence of capsule lesions in the various implantation
groups studied from month 8 onward. It must be stressed that the
sarcoma cases listed in this table represent tumors that were
macroscopically not evident. The incidence of these small sarcomas and
incipient sarcomas was particularly high in the PE and NiCr groups (34
and 24%, respectively). Concerning the interesting group of clearly
diagnosable preneoplastic lesions, the highest incidence was observed
in the PMMA and Si groups, with values of 39 and 25%, respectively.
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) showed focal
accumulations of macrophages and B lymphocytes without giving any
pattern which could be defined as typically associated with a specific
lesion. Thus, a specific leukocyte reaction could not be documented,
either in the stable (fibrous) capsules or in the capsules with
proliferative or preneoplastic lesions. Of greater importance, the
immunohistochemical staining pattern of the preneoplastic lesions gave
data that were inconsistent with the available information on
specificity of reaction of the antibodies. This became apparent in a
detailed immunohistochemical study carried out on 7 randomly selected
cases of diagnosed preneoplastic lesion (capsules from 2 cases of PMMA,
2 cases of Ti, and 1 case each of PVC, PE, and NiCr). In all cases the
atypical cell population, whether of spindle cell or polygonal cell
morphology, gave a strongly positive reaction with PCNA in 90 to 95%
of cells (Figure 6, A and B)
. This also
applied to such lesions, as illustrated in Figure 6C
, in which the
cellular focus was only 5 to 6 cell layers thick. The atypical nuclei
stained intensely with PCNA (Figure 6D)
. Figure 6D
also demonstrates an
important phenomenon, namely the definite gradient of positivity from
the dense staining of the atypical nuclei adjacent to the site of
the biomaterial to complete negativity of quiescent fibroblasts in the
deep part of the capsule. The problematic data derive from the
immunohistochemical examination using the inflammatory cell markers.
Figure 6E
shows the reaction of a PMMA capsule to the B lymphocyte
marker, KiB1R. This antibody recognizes an epitope on a subpopulation
of the atypical cells. A similar biphasic staining pattern, ie, some
cells positive, some negative, was obtained for the KiM2R antibody,
which should identify activated macrophages (Figure 6F)
.
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As a result of ethical considerations, animals were sacrificed
once tumors had become manifest. This meant that no further possibility
existed in the experimental model to study metastatic behavior of these
tumors over a defined period of time. Nevertheless, in 8 animals, 4 of
which were in the polyurethane group, distant metastasis of the
sarcomas did occur. Figure 7
illustrates
this for a sarcoma around a PU implant. At the time of sacrifice (87
weeks) multiple pulmonary metastases were observed, the phenotype being
that of a round cell sarcoma.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The F-344 rat model has also been used to study the early cellular response to subcutaneous implants at 1 week and 2 months postimplantation.21 Silicone elastomer and impermeable cellulose acetate filters (pore size <0.02 µm) were taken as two positive groups, known to induce sarcoma after about 11 months, and compared with porous cellulose acetate filters (pore size >0.65 µm), which are noncarcinogenic. This latter group was found to have less cell proliferation, apoptosis, and fibrosis, but more extensive inflammation than the two tumorigenic implants, which could not be distinguished from each other on the basis of the parameters chosen.
A study of the literature makes it abundantly clear that there are relatively few models available which present early stages of sarcoma development. This is in marked contrast to the situation for carcinomas, in which both human and animal tissues are available for study and which include clearly defined premalignant stages.3-8 Expression of the human immunodeficiency virus tat gene in transgenic mice has been shown to induce leiomyosarcomas and early stages of Kaposi’s sarcoma in the skin.22 The transgenic mouse model has also been used to express the bovine papillomavirus type 1 genome, leading to development of dermal fibromatosis and fibrosarcomas.23 Chromosomal studies revealed that only in the latter group could aberrations be detected, which in this sarcoma involved chromosome 8 (trisomy or duplication) and chromosome 14 (monosomy or translocation). As early as 1983, Rachko and Brand used subcutaneous implantation of a copolymer of vinyl chloride acetate in two mouse strains to induce sarcomas.24 They described 6 cases of preneoplasia derived from the peri-implant tissue taken at 4, 6, 9, and 16 months postimplantation. Using their mouse model, Brand and colleagues established a hypothetical model to describe the stages of foreign body tumorigenesis.25 Their proposed sequence involved an initial phase of proliferation with the acute foreign body inflammatory reaction, followed by capsule fibrosis, quiescence of phagocytic activity, and, finally, direct contact between material and clonal preneoplastic cells. Further investigations by this group documented [3H]-thymidine labeling of predominantly macrophages attached to the copolymer film used as implant26 as well as a temporary repression of fibronectin synthesis in the early stages of preneoplasia.27 In addition, aromatic amines have been used to induce splenic sarcomas in F-344 rats.28 In that model splenic fibrosis and capsule hyperplasia were described as preneoplastic lesions.
It should be stressed here that the sarcoma induction mechanism involves direct contact with the biomaterials. Thus, this model is limited to signals that are elicited locally. The role of systemic factors in the induction process remains completely unknown, as does the differentiation between local chemical and physical characteristics as the predominant pathogenetic factor. Nevertheless, it may be speculated that the chemical component might be minimal, as the biomaterials span a vast spectrum of chemical structure (polymers, metals, ceramic), so that to implicate chemical sarcomagenesis as the prime factor would suggest that a number of compounds of entirely different chemical composition can, under the implantation conditions described here, induce tumors with relatively high incidence. Theoretically, it is possible that residual monomers from the polymer synthesis and/or compounds used in the plasticizing process or other leachables could play a role, although the fact that the materials used were of medical grade and thus pretested for negative effects of leachables makes this hypothesis improbable. In the case of the metals the chemical carcinogenesis mechanism is much more feasible, as it has been shown in numerous studies that metallic elements used in surgical alloys, including cobalt, can act as chemical carcinogens.29 The complexity of this topic is indicated by the studies of Meachim and colleagues,30 who were unable to induce sarcoma in rats or guinea pigs with intramuscular implantation of particulate debris of cobalt-chromium-molybdenum (CoCrMo), and Lewis et al,31 who failed to induce sarcomas by intra-articular injection of Fischer-344 rats with CoCrMo or titanium-aluminum-vanadium powders. With respect to possible chemical induction from the aluminum oxide ceramic material, very interesting data are to be found in the veterinary pathology literature, where fibrosarcomas are especially known to arise in cats at vaccination sites after feline leukemia virus and rabies vaccination.32,33 Using electron probe microanalysis, Hendrick and colleagues succeeded in demonstrating that aluminum oxide from the vaccine adjuvant was present in tumor-associated macrophages and suggested that this could be involved in the development of neoplasia.34 Support for a primarily physical pathogenetic factor comes from the fact that the biomaterial implants, although of varied chemistry, were prepared as disks in such a way that the surface roughness did not differ markedly. It has been known for a long time (the so-called Oppenheimer effect35 ) that solid materials that are not degradable and have a relatively smooth continuous surface are tumorigenic when implanted in rodents, as in the present study.
The lack of correlation between biomaterial composition and the histological type of sarcoma induced is also worthy of discussion. Although not statistically significant, there was an observed trend that polyurethane implants were often associated with vascular tumors. Nevertheless, the total tumor bank from the study covered a wide spectrum of histological types, including angiosarcoma, leiomyosarcoma, rhabdomyosarcoma, fibrosarcoma, pleomorphic sarcoma, and MFH. This fact supports the induction of different differentiation pathways in a mesenchymal stem cell of considerable pluripotence. The lack of effective localizing antibodies for rat epitopes will, unfortunately, continue to hamper the identification of this cell type. At this point it is also important to address the vexed question of sarcoma terminology. Although many pathologists still use the term MFH in human pathology, it should be noted that the specialists in soft tissue tumor pathology are highly skeptical about the existence of such an entity. This has been particularly well articulated and argued by Fletcher, who has dubbed the term a "meaningless diagnosis of convenience."36 On this basis there is a strong case to merge the two categories, which we have called MFH and pleomorphic sarcoma, into a group called pleomorphic-storiform sarcoma. We are in agreement with the argument of Fletcher as applied to human sarcomas. However, in our rat study we have chosen to differentiate between these two groups, as those tumors classified as pleomorphic sarcoma contained multinucleated tumor cells with marked hyperchromasia and nuclear pleomorphism, whereas those we termed MFH had, in addition to a spindle cell component, multinucleated cells with much more bland appearance (that is, with only slight hyperchromasia and nuclear pleomorphism). We therefore saw the need to separate these two groups.
The question of preneoplastic change in the capsule tissue around implanted biomaterials was only tangentially addressed in the paper by Nakamura et al.19 They described some cells showing tumorous change in one capsule around a PE implant. Our study demonstrates that the capsule tissue often presents a clearly premalignant lesion. In addition, we have been able to show that a spectrum of lesions can be distinguished from a proliferative lesion without identifiable atypical cells through preneoplastic proliferative lesions to incipient sarcoma. Nevertheless, we stress that in this attempted morphological delineation of stages in the progression toward sarcoma an element of subjectivity is present. Thus, another group of pathologists could easily have a different threshold for their diagnosis, so that what we have termed "proliferative lesion, possibly preneoplastic" could be preneoplastic, and some lesions in this latter category could be placed in the sarcoma group. Despite the arbitrary nature of the classification, a spectrum of lesions is clearly discernable in this model, which also raises the question of time course to appearance of the individual lesions. Despite the fact that the experimental design does not lend itself to providing a definitive answer, some generalizations may be attempted. As the time scale was unknown to us, we chose a study which was terminated after a time period of 8 months (short-term, for PE 3 months) and one running over 2 years, whereby because of tumor development, tissue (both tumorous and capsular) became available at various stages between 8 and 24 months. The focal proliferative lesion, that is within a restricted area at the tissue biomaterial interface a proliferate of spindle and polygonal cells, was present at 8 months in all six biomaterial groups studied at this time. Averaged across all groups, this represented 49% (56 of 114 capsules). Unfortunately, at 8 months, those lesions with proliferative foci and on the basis of some clearly atypical cells termed "possibly preneoplastic" were not homogeneously distributed among the biomaterial groups. The frequency ranged from zero in the PVC and PMMA groups to 50% (10 of 20 capsules) in the Si group. The latter, along with the PE and PU groups (22 and 33% of capsules, respectively), could be useful for molecular studies on this atypical cell population. Lesions which we could unequivocally term preneoplastic were found in none of the groups at 3 or 8 months. Deducing the time course of the various capsule lesions from the 2-year study is practically impossible. Nevertheless, some tentative postulates can be made. Thus, for example, in the Si group, although from the short-term study 50% of capsules were questionably preneoplastic, the earliest sarcoma was not observed in the long-term study until week 52, compared with weeks 26 and 36 for PE and PU, respectively. On the basis of these observations it would appear that with silicone atypical cells appear at the biomaterial interface early, but the time to development of definitive sarcomas is long in relation to the other groups. Even if this statement must be made in a guarded fashion, the Si group has also the positive side of providing a 50/50 chance of a preneoplastic or possibly preneoplastic capsule under the conditions of the 2-year study design (8 of 16 capsules). From the viewpoint of statistical probability the PU group is also useful with almost 40% (8 of 21 capsules) showing either of these two lesions. Reviewing the data from the 2-year study with an eye toward the probability of obtaining a capsule lesion with at least questionable preneoplasia (from among the groups proliferative lesion, possibly preneoplastic, preneoplastic lesion, and sarcoma in the capsule) reveals the PMMA group at the top with 67% (12 of 18 capsules). This is followed by PE with 59% (19 of 32 capsules), Si with 56% (9 of 16 capsules), and PU with 52% (11 of 21 capsules). At the end of the probability list is the aluminum oxide ceramic with only 25% (4 of 16 capsules), making it a poor candidate for effective molecular studies.
The high frequency of the described lesions in the subcutaneous peri-implant capsules indicates that this animal model could be useful for tumorigenic studies on the stages of progression from proliferation through atypical proliferation to sarcoma. To our knowledge such a model has not been presented for sarcoma, in which such stages can be distinguished with a high enough incidence to make such studies feasible. An important aspect in ensuring that these preneoplastic lesions can be grasped is the use of three separate implants of similar composition in each animal. This markedly increases the probability of finding the early stages in sarcoma development by compensating for the variability of biological response from animal to animal. By using the ethically determined experimental endpoint, namely the presence of a macroscopically manifest tumor at one site, it was found that the capsules of the remaining two implant sites often revealed preneoplastic alterations.
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Footnotes |
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Supported by a Brite-Euram grant from the European Union, Project 8000 (BRE 2-CT 94.0607) and by the Ministry of Science of the State of Rhineland-Palatinate, Germany.
Accepted for publication December 21, 1999.
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). The number of rats
bearing tumors is shown by the histograms. The latter values are below
those for the number of tumors, as some rats carried more than one
tumor.
