| | Cell characterization of mononuclear and giant cells constituting pigmented villonodular synovitis☆☆☆Accepted 20 September 2002. Abstract The aim of this study was to determine the histologic and cellular characteristics of 2 cell types, mononuclear cells (Mos) and multinuclear giant cells (GCs), that predominantly constitute pigmented villonodular synovitis (PVS). Synovial tissues examined in this study were obtained from 10 patients with PVS. Five methods were used for cell analysis: (1) enzyme-histochemistry for tartrate-resistant acid phosphatase (TRAP); (2) immunohistochemistry using antibodies for CD68, macrophage colony–stimulating factor (M-CSF), MIB-1, p53, p21, p16, and cathepsin-L (cath L); (3) TdT-mediated deoxyuridine triphosphate-biotin terminal end labeling (TUNEL) as a measure of apoptosis; (4) fluorescence-based polymerase chain reaction single-strand conformation polymorphism analyses (FPCR-SSCP) to detect p53 gene mutations; and (5) in situ hybridization using gene-specific oligoprobes for matrix metalloproteinase (MMP)-2, MMP-9, receptor activator of nuclear factor κB ligand (RANKL), and calcitonin receptor (CTR). Both Mos and GCs were shown to express the macrophage/histiocyte marker CD68. In GCs, TRAP and CTR, both of which are known as characteristic phenotype markers of osteoclasts, were expressed. M-CSF and RANKL, which are together essential for osteoclast differentiation, were expressed in both Mos and GCs. Mos were shown to express MIB-1, but GCs were not. Although proliferation-suppressor proteins p53, p21, and p16 were expressed in both Mos and GCs, little apoptotic phenomenon of lining Mos was detected by TUNEL. In our study, p53 gene mutations for exons 5, 7, and 8 in PVS synovial tissues were not detected by FPCR-SSCP analysis. Furthermore, both types of cells demonstrated the proteolytic enzymes MMP-2 and MMP-9 mRNA, and cath L protein. These results suggest that PVS has a hyperplastic property consisting of the CD68-positive monocytic cell lineage with differentiation of osteoclastic giant cells from monocyte and probably controlled against proliferation by wild-type p53, p21, and p16. HUM PATHOL 34:65-73. Copyright 2003, Elsevier Science (USA). All rights reserved.
Abbreviations:
AP
, alkaline phosphatase,
cath L
, cathepsin L,
CTR
, calcitonin receptor,
FPCR-SSCP
, fluorescence-based polymerase chain reaction single-strand conformation polymorphism,
GC
, giant cell,
M-CSF
, macrophage colony–stimulating factor,
MMP
, matrix metalloproteinase,
Mo
, mononuclear cell,
PBS
, phosphate-buffered saline,
PCR
, polymerase chain reaction,
PVS
, pigmented villonodular synovitis,
RANKL
, receptor activator of nuclear factor κB ligand,
SSC
, standard saline citrate,
TUNEL
, TdT-mediated deoxyuridine triphosphate-biotin terminal end labeling,
TRAP
, tartrate-resistant acid phosphatase
Pigmented villonodular synovitis (PVS) is a rare disorder characterized by monoarticulation, a proliferative process in synovial tissues with hemoarthrosis.1 Histologically, PVS is a neoplastic-like villonodular hyperplasia of synovial tissue composed of proliferating mononuclear cells (Mos) and multinuclear giant cells (GCs) with hemosiderin deposition.2 Jaffe et al first introduced the term “PVS” in 1941. They suggested that the pathogenesis of this peculiar disorder may be the result of an inflammatory reaction to uncertain trauma of the joint.1 On the other hand, other authors have suggested that clinically, PVS has a neoplastic-like character because recurrence following surgical synovectomy was often encountered, occasionally accompanied with severe destruction of articular cartilage or bone.3 As of yet, there is no consensus on whether PVS is an inflammatory reaction or a true neoplasm.4, 5, 6, 7 The aim of this study was to define the cellular characteristics (especially of the Mos and GCs) predominantly constituting PVS, so as to gain a better insight into the properties of this peculiar joint lesion.
To determine the cellular characteristics of Mos and GCs, we examined the expression of CD68, a histiocytic/macrophage marker,8, 9 and tartrate-resistant acid phosphatase (TRAP), a specific marker for cells with an osteoclast-like character,10 using immunohistochemical and enzyme-histochemical staining. Furthermore, using in situ hybridization, we examined the mRNA expression of calcitonin receptor (CTR), which has been shown to be a definitive osteoclast marker.10, 11 To differentiate Mo and GC cells, we examined protein and mRNA expression, as determined by immunohistochemistry and in situ hybridization, respectively, of macrophage colony–stimulating factor (M-CSF), a cytokine derived from monoctytes essential for osteoclast differentiation,9, 10, 11, 12 and the receptor activator of nuclear factor κB ligand (RANKL), a recently described cytokine critical in osteoclast differentiation.11, 13 In an effort to describe the proliferative status of PVS, we examined the immunohistochemical expression of proliferation-suppressor proteins p53, p21, and p1614, 15, 16 and the proliferating marker MIB-117 in Mos and GCs.
Missense mutations in exons 5, 7, and 8 of the p53 gene is one of the most common abnormalities in neoplasia lacking the ability to arrest the cell cycle.18 Therefore, we sequenced exons 5, 7, and 8 of the p53 gene by fluorescence-based polymerase chain reaction single-strand conformation polymorphism (FPCR-SSCP) analysis to detect missense gene mutations. We observed apoptotic phenomena using a TdT-mediated deoxyuridine triphosphate-biotin terminal end-labeling (TUNEL) technique19 in Mos and GCs. Finally, in an attempt to visualize the expression of proteolytic enzymes related with bony destruction in PVS, we used in situ hybridization to localize matrix malleoproteinase (MMP)-2 and MMP-9 mRNAs and immunohistochemical staining to localize cathepsin L (cath L) protein.
Materials and methods  Synovial tissues examined in this study were taken from the knee and ankle joints during surgery from 10 patients with PVS (8 from knee joints and 2 from ankle joints). All cases were clinically and histologically diagnosed as diffuse-type PVS without invasion. This study was performed in accordance with the guidelines of the Declaration of Helsinki. The following methods were used for cell analysis: (1) immunohistochemical staining using antibodies for CD68, M-CSF, MIB-1, p53, p21, p16, and cath L; (2) the TUNEL technique, to investigate cell apoptosis; (3) enzyme-histochemistry for TRAP; (4) FPCR-SSCP analyses to detect p53 gene mutations; and (5) in situ hybridization using gene-specific oligoprobes for MMP-2, MMP-9, RANKL, and CTR. Immunohistochemical staining Synovial tissues from 10 cases of PVS were embedded in paraffin, sliced into 2-μm sections, and subsequently immunohistochemically stained with 1 of 7 antibodies, including monoclonal antibodies CD68 (1:80 dilution; Dako, Glostrup, Denmark), anti-MIB-1 (1:100; Immunotech, Luminy, France), anti-p53 (DO-7, 1:100; Dako), anti-p21 (1:100; PharMingen, San Diego, CA), anti-p16 (1:50; PharMingen), and polyclonal antibodies anti-cath L (1:10; a gift from Dr. Eiki Kominami, Juntendo University, Tokyo, Japan) and anti-M-CSF (1:10; a gift from Otsuka Pharmaceutical, Tokyo, Japan). All sections were deparaffinized in xylene, rehydrated in ethanol, and washed with phosphate-buffered saline (PBS), followed by blocking of endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 30 minutes. Sections immunostained with p53, p21, and p16 antibodies were autoclaved for 5 minutes at 121°C with 10 mmol/L sodium citrate (pH 6.0) to activate these antigens before the blocking of endogenous peroxidase activity. Sections immunostained with CD68, cath L, and M-CSF antibodies, however, were incubated at 37°C for 30 minutes in distilled water containing trypsin and calcium carbide before the reaction with primary antibodies. After washing with PBS, 10% normal goat serum was applied to all sections for 30 minutes and then reacted with primary antibodies at 4°C for 48 hours. After washing with PBS, all sections were reacted at room temperature for 30 minutes with rabbit immunoglobulins conjugated to a peroxidase-labeled amino acid polymer (Histofine Simplestain Multi Po; Nichirei, Tokyo, Japan), and then finally treated with 3,3'-diaminobenzidine (DAB; Sigma, St. Louis, MO). Negative control sections were reacted with normal mouse, rabbit, and sheep serum instead of the primary antibody. All sections were examined for iron staining to detect hemosiderin deposition after the DAB reaction. TdT-mediated deoxyuridine triphosphate-biotin terminal end labeling To confirm the apoptotic phenomenon in Mos and GCs, TUNEL was performed using an Apoptag Peroxidase kit (Oncor, Gaithersburg, MD). Briefly, deparaffinized and rehydrated 2-μm sections of synovial tissue sections, obtained from 10 cases of PVS, were subjected to treatment with proteinkinase K for 15 minutes at room temperature. After washing with distilled water and blocking endogenous peroxidase, all sections were immersed in an equilibration buffer and incubated at 37°C for 1 hour. Antidigoxigenin peroxidase conjugate was applied to these sections, which were then incubated at room temperature for 30 minutes. The reaction products were visualized with DAB and iron staining. Tartrate-resistant acid phosphatase staining TRAP staining was performed using an acidic phosphotase (ACP) staining kit (Muto Pure Chemicals, Tokyo, Japan). Deparaffinized and rehydrated 2-μm tissue sections were incubated at 37°C for 6 hours in the reactive solution, with an attachment of the kit dissolved with tartaric acid and fast red violet LB salt, then dribbled with 10% manganese chloride. After washing with distilled water, all sections were counterstained with hematoxylin. Fluorescence-based polymerase chain reaction single-strand conformation polymorphism analysis We commissioned Otsuka Assay Laboratories to detect p53 gene mutations in PVS tissues using FPCR-SSCP analysis, as described previously.18 Primers Oligonucleotide primers were synthesized for polymerase chain reaction (PCR) based on the published p53 gene sequence for each region flanking the intron–exon boundaries of exons 5, 7, and 8. The sequence for each primer were as follows: EX-05f, 5'-TCTGTCTCCTTCCTCTTCCT-3', EX-05r, 5'-TCTCCAGCCCCAGCTGCT-3'; EX-F05r, F-5'-TCTCCAGCCCCAGCTGCT-3'; EX-06f, 5'-TGATTCCTCACTGATTGCTCT-3'; EX-06r, 5'-GAGACCCCAGTT-GCCAAACC-3'; EX[font“Osaka”]8722μ[/font“Osaka”]F06r, F-5'-GAGACCCCAGTTGCCAAACC-3'; EX-07f, 5'-TCT-TGGGCCTGTGTTATCTC-3'; EX-07, 5'-AGGGTGGCAAGTGGCTCC-3'; EX-F07r, 5'-AGGGTGGCAAGTGGCTCC-3'; EX-08f, 5'-GCTTCTCTTTTCCTATCCTGA-3'; EX-08r, 5'-CGCTTCTTGTCCTGCTTGC-3'; and EX-F08r, F-5'-CGCTTCTTGTCCTGCT-TGC-3'. Here “f” and “r” indicate forward and reverse primer, respectively. The reverse primers were labeled at their 5' ends with fluorescein derivatives by the Fluore primer method (Pharmacia). “F” in the labeled sequences indicates carboxyfluorescein linked to the 5' end of the nucleotide via a linker and a phosphate. Analysis Genomic DNA (100 ng) was amplified in a total volume of 50 μL buffer as recommended by Perkin-Elmer Cetus (Boston, MA), containing forward and 5' fluorescein-labeled reverse primers. The PCR products were diluted 50-fold by a stop solution (Pharmacia), heated to 95°C for 5 minutes, and then placed on ice for 5 minutes. Then 4 μL of this solution was subsequently applied to each lane of an FSSCP gel fitted to an ALF II automated DNA sequencer (Pharmacia). The FSSCP gel used to resolve PCR products was a 7% polyacrylamide gel containing 5% glycerol. Electrophoresis was performed at 30 W for 3 to 4 hours, depending on the length of the amplified nucleotide. The temperature of the gel was maintained at 25°C with a built-in water jacket connected to an external thermostat–regulated water circulation system. In situ hybridization Probes Complementary DNA probes for mRNA in situ hybridization. The 24-base MMP-2 antisense oligonucleotide probe used for mRNA in situ hybridization analysis had a sequence of 5'-TGG GCT ACG GCG CGG CGG CGT GGC. The sense oligonucleotide probe 5'-GCC ACG CCG CCG CGC CGT AGC CCA was used as a negative control. The sequence of the 26-base MMP-9 antisense oligonucleotide probe used for mRNA in situ hybridization analysis was 5'-CCG GTC CAC CTC GCT GGC GCT CCG GU. The sense oligonucleotide probe 5'-AC CGG AGC GCC AGC GAG GTG GAC CGG was used as a negative control.20 The sequence of the 20-base RANKL antisense RNA probe used for mRNA in situ hybridization analysis was 5'-ATC AGA CAG CAC TCA CT. The sense oligonucleotide probe sequence used as a negative control for RANKL in situ hybridization was 5'-ATC TAG GAC ATC CAT GCT AAT GTT C 3'.13 The sequence of the 40-base CTR antisense RNA probe was 5'-ATG GTC GCA ACA AAG AAG CCC TGG AAA TGA ATC AGA GAG T, and the sense probe sequence used as negative control was 5'-ACT CTC TGA TTC ATT TCC AGG GCT TCT TTG TTG CGA CCA T.21 These probes were synthesized with a 3'-biotinylated tail (Brigati tail; 5'-probe-biotin-biotin-biotin-TAG-TAG-biotin-biotin-biotin-3').22, 23 Hybridization mRNA in situ hybridization was performed with a manual capillary action system (MicroProbe Staining System, Fisher Scientific, Fairlawn, NJ) using a modification of previously reported methods.22, 24 Then 2-μm issue sections applied to Probe On Plus slides (Fisher Scientific) were rapidly dewaxed, cleared with alcohol, rehydrated with a Tris-based buffer, pH 7.4 (Universal Buffer; Research Genetics, Huntsville, AL) and digested with pepsin (2.5 mg/mL; Research Genetics) for 3 minutes at 105°C. The probe was dissolved in formamide-free diluent. Slides were immersed in the probe diluent for 3 minutes at 105°C, cooled for approximately 1 minute at room temperature, and then allowed to hybridize at 45°C for 60 minutes. The sections were then washed twice with 2×standard saline citrate (SSC) at 45°C (3 minutes per wash) and incubated with alkaline phosphatase (AP)-conjugated streptavidin (Research Genetics). After washing twice in AP chromogen buffer, pH 9.5 (Research Genetics), at room temperature, hybridization products were visualized with fast red salt. Slides were subsequently counterstained with hematoxylin, air-dried, and cover-slipped for microscopic examination. Results The results are summarized in Tables 1, 2, 3, and 4.
All tables show the ratio of positive cases with respect to each of the markers. We defined the specimen that indicated that immunopositive cells accounted for >30% of the field of view under high-power fields (except in TUNEL, positive cells accounted for >10% of the field of view under high-power fields) as th-e positive case in the light of difference condition in each sample caused by various durations of formalin fixation. | | |  | | CD68 | TRAP | CTR | M-CSF | RANKL | |
|---|
| Mo | 100 | 0 | 0 | 100 | 100 | |
| GC | 100 | 100 | 100 | 100 | 100 | |
| | | | | |
| | | | | MIB-1 | p53 | p21 | p16 | |
|---|
| Mos | 100 | 70 | 80 | 80 | |
| GCs | 0 | 70 | 80 | 80 | |
| | | | | |
| | | | | MMP-2 | MMP-9 | cath L | |
|---|
| Mos | 100 | 100 | 100 | |
| GCs | 100 | 100 | 100 | |
| | | | | |
Cellular characteristics Both Mos and GCs were shown to have a strong and wide distribution of positive immunostaining for CD68 (Fig 1A), with immunopositive cells accounting for >30% of all cells under high-power fields in each section (Table 1).
Most GCs in the PVS synovium were immunopositive for TRAP ( Fig 1B), whereas almost all Mos were immunonegative ( Table 1). To examine the expression of CTR mRNA, we performed in situ hybridization analysis in PVS synovial tissues. CTR mRNA was detected in GCs ( Fig 1C), but not in Mos. GCs expressing CTR mRNA were also immunopositive for TRAP. Cell differentiation Mos and GCs were almost all positive for M-CSF and distributed widely, with immunopositive cells accounting for much more than 30% of all cells under high-power fields in each section (Fig 2A; Table 1).
RANKL mRNA was found to be expressed in both Mos and GCs, with >30% of all cells under high-power fields in each section ( Fig 2B). Mos and GCs expressing RANKL mRNA were also immunopositive for M-CSF. Status of proliferation Results from our immunohistochemical staining analysis appear to suggest that cells in a proliferative state, as marked by an immunopositive reaction for MIB-1, were only those having the cellular characteristics of Mos but not of GCs (Fig 3A).
When observed under high-power fields, MIB-1–immunopositive Mos were diffusely distributed in the synovial tissue, accounting for >30% of all cases ( Table 2). Immunoreactive expression of p53, p21, and p16 was identified in all cases examined in both Mos and GCs in terms of intensity and the number of positive cells ( Fig 3B, C, and D). In 70% of cases, p53-immunopositive cells accounted for >30% of the field of view, and in 80% of cases, p21- and p16-immunopositive cells accounted for >30% of the field of view. MIB-1 was not detected in these cells, however ( Table 2). To detect mutations in exons 5, 7, and 8 of the p53 gene, we performed FPCR-SSCP analysis on PVS tissues. As shown in Fig 4, DNA from PVS tissue had 3 peaks for exon 5, 2 peaks for exon 7, and 4 peaks for exon 8.
All of the peaks for PVS tissue DNA migrated to the same positions as those for normal DNA. Positive controls on FPCR-SSCP analysis are shown in Fig 4D. These results suggest that the p53 gene has no mutations in these exons. Apoptosis TUNEL results indicated that apoptosis was detected only in Mos of the synovial lining layer, in <1% of cells under high-power fields. Apoptosis was not identified in GCs in any case (Table 3). Cellular function Results from in situ hybridization demonstrated that sections from each patient exhibited a similarly high level of mRNA expression for MMP-2 and MMP-9 in both Mos and GCs (Fig 5A and B; Table 4).
Immunoreactive cath L staining was strong in Mos and GCs in all cases examined ( Fig 5C; Table 5). This proteolytic enzyme was diffusely expressed in the synovium. Discussion Jaffe et al1 described PVS as an inflammatory and reactive proliferative disease of synovial tissues caused by uncertain trauma of the joint. A study by Young et al25 reported that repeated injections of autologous blood into the joints of dogs produced hypertrophy of the synovia with hemosiderin deposition similar to that of PVS synovial tissue. They suggested that trauma was an important factor in the pathogenesis of PVS.25 Some investigators, however, have suggested that PVS is a neoplasm arising in the joint synovial tissue. Rao and Vigortia3 put forward the idea that PVS may represent a benign neoplastic proliferation of synovial fibroblasts and histiocytes. Focusing on the proliferating cells extending into the dense connective tissue layer beneath the lining cells, they noted increased mitotic activity of proliferating cells in recurrent lesions. Yudoh et al26 reported that synoviocytes isolated from PVS synovial tissues expressed a high level of telomerase activity, suggesting that the synovial hyperplasia in PVS was due to an up-regulated activity of telomerase in the PVS synovium, a finding commonly demonstrated in most tumor cells. Alguacil-Garcia et al27 observed in their ultrastructural study that PVS was composed mainly of 2 types of cells, A and B synovial cells. Histologically, O'Connell et al8 observed in their immunohistochemical studies that in all cases of PVS, histiocytic markers, including CD68, consistently and strongly stained both Mos and GCs. In the present study, an intense, positive immunoreaction against CD68 was confirmed in both Mos and GCs. This finding clearly shows the histiocyte/macrophage properties of Mos and GCs. PVS synovial tissue is composed of several cell types, including myofibroblasts, fibroblasts, and lymphocytes.3 We examined for these cells using immunohistochemistry. Although the presence of these cells in PVS synovial tissues was confirmed, they were not the predominant cell type compared with CD68-positive Mos and GCs (data not shown). CTR mRNA and immunoreactive TRAP were strongly detected only in GCs. Similarly, Darling et al2 reported mRNA expression for CTR and positive immunostaining for TRAP in GCs. TRAP is a characteristic phenotypic marker of osteoclasts and CTR, which has been suggested as a definitive marker for identifying a fully functional osteoclast.2, 11 In the present study, GCs were immunopositive for TRAP and CTR in all cases, suggesting that GCs in PVS are fully differentiated osteoclastic giant cells. M-CSF, a known factor related to GC differentiation of monocytes,10, 11, 12, 13 and RANKL, which has recently been described as a cytokine and a very important osteoclast differentiation factor,11, 13, 28 were strongly and diffusely expressed in Mos and GCs. In osteclastogenesis, both M-CSF and RANKL are essential for osteoclast production and are involved in promoting multinucleation of prefusion osteoclasts and the survival of nascent osteoclasts.13 In in vitro coculture systems, RANKL is sufficient to induce osteoclast differentiation in the presence of M-CSF and also has the capability to directly activate multinucleated osteoclasts.29, 30 Our results suggest that Mos mediate the differentiation of CTR- and TRAP-positive osteoclasts from Mos via an autocrine mechanism that ivolves M-CSF and RANKL. Furthermore, GCs also contribute to osteoclast differentiation via the expression of these factors. Tashiro et al31 reported that MIB-1 was present in Mos but not in GCs. Only Mos appear to reveal a proliferative character via the expression of MIB-1. GCs in our study were found to not express this protein. Results from the present study suggest that in PVS, MIB-1–positive proliferating cells are restricted to CD68-positive histiocyte/macrophage Mos, which in turn transform into TRAP- and CTR-positive osteoclastic giant cells when induced by M-CSF and RANKL from Mos and GCs. In the present study, proliferation-suppressor proteins p53, p21, and p16 were strongly expressed in Mos and GCs without MIB-1 expression. When cellular DNA is damaged, p53 arrests the cell cycle at the G1/S phase boundary via the expression of p21. Moreover, p53 can induce apoptosis if DNA damage is severe.32 These functions of p53 control cell proliferation.33 Many types of neoplasm express mutant p53, which lacks the suppressive functions of cell proliferation.15, 34 Anti-p53 antibody DO7 used in the present study recognizes both wild-type and mutant p53.35, 36 The p53 gene has a high incidence of mutation in many types of malignancy.34, 37 Therefore, we also examined the p53 gene for the presence of missense mutations in exons 5, 7, and 8 by means of FPCR-SSCP. The FPCR-SSCP technique is a rapid and efficient method for detecting mutations and polymorphisms in genomic or cDNA sequences.18 No mutations were found in exons 5, 7, and 8 by FPCR-SSCP, suggesting that the p53 protein detected by immunohistochemistry was of the wild type. Thus, expression of both wild-type p53 and p21 without apoptosis in Mos and GCs may be considered a normal cell reaction to pathologic cell proliferation in PVS. Overexpression of p16 may also be secondary via other pathways and have suppressive effects on the proliferation of the PVS synovium. These results suggest that PVS has a regulating property in proliferation and might have some of the hyperplastic character, rather than the true neoplastic nature, of MIB-1–immunopositive histiocytes Mos, which show ordinary differentiation toward osteoclasts and have the normal signaling pathway toward proliferation. A proliferated synovial tissue in PVS occasionally leads to local bone invasion and cartilage erosion. Darling et al38 have suggested that the pathogenesis of damage and destruction of joint structures in PVS may be caused by proteolytic enzymes, mainly MMP-1 and MMP-3, both of which are expressed in the synovial lining cells.38 Expression of MMP-2, MMP-9, and cath L has been found in the synovial tissues of patients with rheumatoid arthritis with progressive cartilage and bone destruction.39, 40 MMP-2 and MMP-9 were widely confirmed in Mos and GCs by in situ hybridization and immunohistochemical staining. These results suggest that MMP-2, MMP-9, and cath L participate and play a key role in joint destruction in PVS. We conclude that PVS consists of a histiocytic cell lineage with osteoclastic giant cell differentiation, and that it might have a hyperplastic character rather than a true neoplastic nature, having proteolytic activity. Acknowledgements The authors are very grateful to Dr. Masami Hosaka and Yuji Shibata for providing materials, methods, and helpful discussion. 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Department of Pathology and Department of Orthopedic Surgery, Iwate Medical University School of Medicine, Morioka, Japan ☆ Supported in part by a grant-in-aid for Advanced Medical Science Research by the Ministry of Science, Education, Sports and Culture, Japan. ☆☆ Address correspondence and reprint requests to Takashi Sawai, MD, PhD, Department of Pathology, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan. PII: S0046-8177(03)00007-8 doi:10.1053/hupa.2003.52 © 2003 Elsevier Science (USA). All rights reserved. | |
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