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The stromal cell reaction of pigmented villonodular synovitisAn electron microscopic study.

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The Stromal Cell Reaction of Pigmented Villonodular Synovitis:
An Electron Microscopic Study
By
JOHN
C. WYLLIE,M.D.
Synovium obtained from a patient with
pigmented villonodular synovitis was
studied with the electron microscope.
Attention was directed to the stromal cell
hyperplasia, a histological feature of this
disease. It was shown that the participating cells were of two main types. The
P
a
curious disease of joints, bursae, and
tendon sheaths, is characterized by villous
and nodular synovial outgrowths, increased
vascularity, hyperplasia of stromal cells
within the synovium, infiltrates of hemosiderin-containing macrophages, and a serosanguinous joint effusion.13 The etiology
and pathogenesis of this disorder are unknown. The nature of the proliferating
stromal ce.lls is also uncertain; they have
been regarded as potential or actual macrophages,l as connective tissue-forming cells
similar to fibroblasts? and as neoplastic
cells of vasculals or synoviale origin.
This paper describes an electron microscopic study of a case of pigmented villonodular synovitis. Particular attention was
paid to the stromal cell reaction in order to
determine the nature of the participating
cells.
IGMENTED
VZLLONODULAR SYNOVITIS,
more abundant cell resembled the fibroblast of connective tissue in general, and
appeared capable of collagen and mucopolysaccharide synthesis. The second cell
type was similar t o a macrophage and
contained numerous hemosiderin inclusions.
joint, but there was no limitation of active and
passive movement. The other joints were normal.
A chest radiogram was negative; a radiogram
of the right knee showed soft tissue swelling. The
hemoglobin was 12.8 Gm. per cent, white blood
cell count 8750, sedimentation rate 21, C-reactive
protein negative, anti-streptolysin 0 titer 166 (subsequently within normal limits), latex fixation
negative, and serum protein electrophoresis normal. Serosanguinous fluid, aspirated from the right
knee joint, was sterile. A diagnosis of pigmented
villonodular synovitis was made, and a surgical
synovectomy was performed. At operation, the
joint cavity contained viscid serosanguinous fluid,
detached villi, and fibrin. The thickened, reddishbrown synovial membrane was thrown into numerous villous and nodular folds. The patient made
an uneventful recovery. Twelve months later she
was symptom-free; there was slight residual swelling of the soft tissues about the right knee joint.
MATERIALSAND METHODS
The patient, a 13-year-old girl, was admitted to
hospital with pain, recurrent swelling, and instability of her right knee of 10 months’ duration. She
gave no history of trauma, infection, or other joint
involvement, and her general health was excellent.
An effusion was demonstrated in the right knee
Specimens of synovium, for light and electron
microscopy, were obtained during the surgical
synovectomy. For light microscopy, the synovium
was &xed for 18 hours in 10 per cent neutral
buffered Formalin, dehydrated, and embedded in
paraffin. Sections approximately 5 /.in
i thickness
were stained with hematoxylin, phloxine, and
saffron (H.P.S. ), and phosphotungstic acidhematoxylin, and tested for Perls’ Prussian blue
reaction for ferric salts.
For electron microscopy, representative samples
From the Department of Pathology, Queen’s
University, and the Kingston General Hospital,
Kingston, Ontario, Canada.
Supported b y a research grant from the Canadian Arthritis and Rheumatism Society.
JOHN C . WYLLIE, M.D.: Associate Professor of
Pathology, Queen’s University, Kingston, Ontario,
Canada.
Reprint requests should be addressed to Dr.
Wyllie.
ARTHRITISAND RHEUMATISM,VOL. 12, No. 3
1969)
CASEREPORT
(JUNE
205
206
JOHN C. WYLLIE
Fig. 1.-Photomicrograph showing the thickened synovial membrane infiltrated by
hemosiderin-containing macrophages (arrows) and stromal cells. Note multinucleated
giant cells near the synovial lining cell layer (S). JS, joint space. H.P.S. X 380 (original
size reduced).
Fig. 2.-Photomicrograph showing proliferating stromal cells in vascular synovial
tissue. H.P.S. X 150 (original size reduced).
of the diseased synovium were cut into 2 X 2 X 1
mm. blocks, fixed for 90 min. in cold (4OC) 2 per
cent buffered (pH 7.4) osmium tetroxide containing 0.045 Gm. of sucrose per milliliter,7 dehy-
drated in ascending concentrations of ethanol, and
embedded in Maraglas 655-Cardolite NC-513.8
Material from 20 blocks was examined. The blocks
were cut on a Huxley Ultramicrotome with glass
STROMAL CELL RGACTION OF PIGMENTED VJLLONODULAR SYNOVITIS
207
Fig. 3.-Photomicrograph showing widespread fibrosis within a synovial villus. There
are scattered cells in the fibrous tissue. Note the well-formed small blood vessels. H.P.S.
X 155 (original size reduced).
knives. Thick sections, of approximately 1 p, were
stained with toluidine blue for orientation purposes. Blocks containing foci of stromal cell hyperplasia were selected and thin sections prepared.
Thin sections were picked up on uncoated copper
grids, stained with uranyl acetate9 for 30 min.,
followed by lead citratelo for 2 min., and examined in an Hitachi HU-11 or RCA EMU3D
electron microscope. Electron micrographs were
taken at original magnifications of 2500 to 12,000
and enlarged photographically to the desired size.
hSULTS
Light Microscopy
The typical changes of pigmented villonodular synovitis were observed in the
synovial membrane. Synovial villi, covered
by a layer of synovial lining cells two or
three cells in depth, were infiltrated by
clusters of macrophages containing granules
of golden-brown pigment (Fig. 1).A similar pigment was present in some of the
synovial lining cells. This pigment proved
to be iron-positive and was regarded as
hemosiderin. There was a striking hyperplasia of stromal cells, resulting in the formation of masses of closely packed cells in
the loose, well-vascularized connective tissue of the villi (Fig. 2). These cells had
large oval nuclei and moderately abundant
weakly acidophilic cytoplasm; some contained granules of hemosiderin. The deposition of variable amounts of collagen accompanied the stromal cell reaction; in some
areas, fibrosis was marked, partially obliterating the cellular and vascular reaction
(Fig. 3). Well-formed small blood vessels
( arterioles, venules, and capillaries) were
present in regions of stromal cell hyperplasia and also in the more fibrotic areas
(Figs. 1, 2, and 3). Multinucleated giant
cells with abundant strongly acidophilic
cytoplasm were observed immediately below the synovial lining cell layer ( Fig. 1).
There were a few “foam cells,” with finely
vacuolated cytoplasm, scattered among the
stromal cells.
Electron Microscopy of the Stromul Cell
HYPerpM
The stromal cells were dispersed randomly in a matrix of low electron density
208
JOHN C. WYLLIE
Fig. 4.-Electron micrograph from an area of stromal cell hyperplasia showing loosely
dispersed stromal cells containing electron-dense inclusions. There are collagen fibers
(C) in the intercellular space. m, marginal fold; P, cell process. Uranyl acetate and lead
citrate X 3080 (original size reduced).
containing fibrillar material and small
groups of collagen fibers (Fig. 4).The cells
were frequently closely apposed, but lacked
a definite orientation and did not form vascular sprouts or channels (Fig. 4). Two
principal cell types could be distinguished
on the basis of their fine structure.
The more abundant cell, which resembled the fibroblast of connective tissues
in general, was elongated, with a large
nucleus and abundant fibrillar cytoplasm
(Figs. 5 and 6). The Golgi complex was
highly developed, consisting of the usual
parallel arrays of saccules and accompanying vacuoles (Figs. 5 and 6), and was
widely dispersed throughout the cell. A
moderate quantity of rough-surfaced endoplasmic reticulum was also present, chiefly
distributed in the peripheral cytoplasm
(Figs. 5 and 6). The cisternae of the roughsurfaced endoplasmic reticulum were
mildly dilated, containing granular and fibrillar material. Large mitochondria were
numerous (Fig. 5 ) , and a centriole was
often present (Fig. 5 ) . These cells contained a few membrane-bound vesicles,
some filled with finely granular electrondense material (Figs. 5 and 6). Lipid droplets were also observed (Figs. 5 and 6),
but were sparse.
The less abundant cell type was similar
to a macrophage and had a large indented
eccentrically placed nucleus with a moderate quantity of cytoplasm (Fig. 7). This
cell frequently contained numerous membrane-bound vesicles (similar in appearance to those observed in fibroblast-like
cells), the majority filled with finely granular electron-dense material (Figs. 7 and
8 ) . These cellular inclusions corresponded
with the golden-brown iron-positive granules which had been observed in various
synovial cells by light microscopy and regarded as hemosiderin. I n addition, these
inclusions had an identical fine structure to
that of hemosiderin as described by Bessis
and Breton-Gorius.ll A few lipid droplets
were noted in the cytoplasm. Rough-sur-
STROMAL CELL REACIlON OF PIGMENTED VILLONODULAR SYNOVITIS
Fig. 5.-Electron micrograph showing a stromal cell with part of its nucleus (N) and
an elongated cell body. The Golgi complex (G) is well-developed, and rough-surfaced
endoplasmic reticulum (r) is moderate in amount. Note delicate fibrils ( f ) in the cytoplasm, electron-dense inclusions (H), and lipid droplets (L). c, centriole; C, collagen;
M, mitochondrion. Uranyl acetate and lead citrate X 10,000 (original size reduced).
Fig. 6.-Electron micrograph showing a stromal cell with a Golgi complex (G) and
a moderate quantity of rough-surfaced endoplasmic reticulum (r). Electron-dense inclusions (H) and lipid droplets (L) are present. c, centriole; C, collagen; M, mitochondrion.
Uranyl acetate and lead citrate X 10,000 (original size reduced).
209
210
JOHN C. WYLLIE
Fig. 7.-Electron micrograph showing a stromal macrophage. There are numerous
membrane-bound inclusions with electron-dense finely granular contents. Rough-surfaced
endoplasmic reticulum (r) is sparse. N, nucleus; f, filopodium. Uranyl acetate and lead
citrate X 20,000 (original size reduced).
Fig. S.-Electron micrograph showing hemosiderin inclusions enclosed within membrane-bound vesicles in a stromal macrophage. The electron-dense particles are randomly
dispersed or arranged in a crystal-like pattern (arrow). Uranyl acetate and lead citrate
X 70,000 (original size reduced).
STROMAL CELL REACTION OF PIGMENTED VILLONODULAR SYNOVITIS
Fig. 9.-Electron
micrograph showing a “foam cell” with large numbers of lipid
droplets (L) in its cytoplasm. N, nucleus. Uranyl acetate and lead citrate X 8320 (original size reduced).
Fig. 10.-Electron micrograph showing a multinucleated giant cell adjacent to the
synovial lining cell layer (S). Note the large numbers of mitochondria (M) and small
vacuoles (v). Uranyl acetate and lead citrate X 5700 (original size reduced).
211
212
JOHN C.
Fig. 11.-Electron micrograph showing an area of marked fibrosis with wide separation of stromal cell processes (P). Collagen fibers (C) are accompanied by many smaller
fibrils (s). Uranyl acetate and lead citrate X 7500 (originalsize reduced).
faced endoplasmic reticulum was sparse deposited, resulting in wide separation of
and the Golgi complex small and confined the stromal cells (Fig. 11).Fibrillar mater(Fig. 7). Short filopodia extended from the ial was often abundant in these sites (Fig.
cell surface into the intercellular space 11).
(Fig. 7).
DISCUSSION
The stromal cell reaction included two
The stromal cell hyperplasia of pigadditional cell types. There were a few
cells with large numbers of lipid droplets mented villonodular synovitis was examin their cytoplasm (Fig. 9). It was not pos- ined by electron microscopy. It was shown
sible to determine whether these “foam that the principal cell involved in this reaccells” were macrophages, fibroblasts, or tion had a well-developed and extensive
some other cell type because their fine Golgi complex and a moderate quantity of
structure was obscured by the lipid. Multi- rough-surfaced endoplasmic reticulum. This
nucleated giant cells were noted on the cell had thecfine structure of a fibrobla~t’~J~
edges of the stromal cell masses, usually rather than ‘&at of a macrophagef4or endoadjacent to the synovial lining cell layer. thelial cell.15 The second cell type observed
These cells, in addition to their large size was similar to a macrophage,14 and freand increased number of nuclei, were char- quently contained numerous inclusions of
acterized by a high content of mitochondria hemosiderin, an indication of its phagocytic
(Fig. 10). A Golgi complex and numerous capabilities.
The stromal cell reaction of pigmented
small vesicles were present, but rough-surfaced endoplasmic reticulum, hemosiderin- villonodular synovitis has aroused interest
containing inclusions and lipid droplets in the past, but the nature of the participatwere not seen (Fig. 10). In parts of the ing cells has not been clear. Jaffe et al. conlesion, large amounts of collagen had been cluded that the stromal cells were potential
STROMAL CELL REACTION OF PIGMENTED VILLONODULAR SYNOVITIS
or actual macrophages, basing their opinion
on the apparent phagocytic capacity of the
cells for hemosiderin and lipid.l We have
shown that macrophages are a component
of this reaction but are fewer than the
stromal fibroblasts. Stromal fibroblasts also
appear capable of phagocytosis, as indicated by the occurrence of small amounts
of hemosiderin within them. However,
phagocytosis is not limited to the functionally phagocytic cells of the reticuloendothelial system but is a property (to a
lesser degree) of such connective tissue
cells as the fibrobla@ and the type B synovial lining cell.17918
The possible neoplastic nature of pigmented villonodular synovitis has been discussed by Jaf€el9 and by Lichtenstein.*O
Jaffe feels that the histological evidence,
centered around the accumulation of stroma1 cells which he regards as macrophages,
is suggestive of an inflammatory process
rather than a neoplasm. He points out that
the usual tendency of the disease towards
fibrosis would also point to inflammation.
Clark, on the other hand, views the lesion
as a neoplasm of vascular origin with the
histological features of a sclerosing hemangioma.16 We observed well-formed blood
vessels ( arterioles, venules, and capillaries )
in the connective tissue where the stromal
cells were proliferating. But the stromal
cells, though often lying in close proximity
to one another, did not form vascular
sprouts or channels.
Pigmented villonodular synovitis has
been also conside.red a tumor of synovial
213
origin, related to the giant cell tumor of
tendon sheath origin (the benign synovioma) .16 Electron microscopic studies of
synovial tumors are rare. Luse has described the fine structure of a synovial sarcoma.2l The neoplastic synovial cells had a
h e l y vacuolated cytoplasm, an indistinct
Golgi complex, and tended to form acinarlike structures. They differed significantly
from the synovial fibroblasts we observed.
The stromal cell reaction of pigmented
villonodular synovitis appears basically reparative and is similar to fibrosis subsequent
to injury in connective tissue elsewhere, i.e.,
it is characterized by macrophage activity,
proliferation of fibroblasts, increase in vascularity, and collagen deposition. The nature of the injurious agent was not apparent
from our study. The fibroblast of the stroma1 cell reaction is the probable cellular
source of protein required for the formation
of collagen fibers observed nearby, as it possesses the necessary organelles for protein
synthesis ( the rough-surfaced endoplasmic
reticulum) and secretion (the Golgi complex). The degree of development of the
Golgi complex in these cells is noteworthy
in view of the localization of mucopolysaccharide synthesis in this organelle.22
Hence this cell, inferentially, is in addition
a likely source of the mucopolysaccharides
of the surrounding ground substance.
ACKNOWLEDGMENTS
The author wishes to thank Dr. H. G. Kelly and
Dr. M. A. Simurda for providing the material for
this study, and Dr. Nathan Kaufman for his
criticisms and comments.
SUMMAFUO
IN INTERLINGUA
Synovio obtenite ab un patiente con pigmentate synovitis villonodular esseva studiate
per medio del microscopio electronic. Le attention del observator se concentrava super
le stromal hyperplasia cellular que es un characteristica histologic del morbo in question. Esseva monstrate que le cellulas participante esseva de duo typos principal. Le
typo le plus abundante resimilava, a generalmente parlar, le fibroblast0 de tissu conjunctive e pareva capace de synthese de collageno e mucopolysaccharida. Le secunde
typo cellular resimilava un macrophago e contineva numerose inclusiones de hemosiderina.
214
JOHN C. WYLLIE
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and tenosynovitis. A discussion of the synovial and
bursa1 equivalents of the tenosynovial lesion commonly denoted as xanthoma, xanthogranuloma,
giant cell tumor or myeloplaxoma of the tendon
sheath, with some consideration of this tendon
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2. Copeman, W. S . C.: Textbook of the Rheumatic Diseases (ed. 3.). Edinburgh and London,
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3. Gardner, D. L.: Pathology of the Connective
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4. Robbins, S. L.: Textbook of Pathology (ed.
2 ) . Philadelphia and London, W. B. Saunders
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5. Clark, W. S.: Pigmented villonodular synovitis. Bull. Rheum. Dis. 8:161, 1958.
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16. Bloom, W., and Fawcett, D. W.: A Textbook of Histology (ed. 8 ) . Philadelphia and London, W. B. Saunders Co., 1962, p. 93.
17. Ball, J., Chapman, J. A., and Muirden, K.
D.: The uptake of iron in rabbit synovial tissue
following intra-articular injection of iron dextran;
a light and electron microscopic study. J. Cell
Biol. 22:351, 1964.
18. Wyllie, J. C., More, R. H., and Haust, M.
D.: The fine structure of normal guinea pig synovium. Lab. Invest. 13:1254, 1963.
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