Arch. histol. jap., Vol. 42, No. 5 (1979) p. 517-531
Scanning
Electron
Microscope
Studies
on the
Synovial
Membrane
Kazutomo DATE Department of Anatomy (Prof. K. TANAKA)and Department of Orthopedic Surgery (Prof. I. MAEYAMA), Tottori
University
School of Medicine,
Yonago,
Japan
Received March 15, 1979
Summary. Synovial membranes from human and rabbit joints were observed by scanning electron microscopy. 1. The surfaces of synovial membranes present locally variable appearances. In some parts cytoplasmic processes of lining cells extend long and flat causing an appearance like overlapping renal podocytes, whereas in other parts the cells protrude in cauliflower-like or more smooth-surfaced round bodies. 2. In cracked surfaces of synovial membranes, two types of lining cells are distinguished. One is the cell which has more surface processes and numerous granules in the cytoplasm, the other is the cell which has fewer processes and better developed endoplasmic reticulum without granules. 3. Fibroblasts apparently forming collagen fibers are observed in the subsynovial tissue. Two types of fibrogenesis are found. In the first type microfibrils seem to be formed extracellularly, whereas in the second type bundles of filaments are preformed in the cytoplasm and they appear to be extruded directly from the cell surface.
There have been many studies on the histologic structure of the joint capsule, which is microscopically divided into the inner and outer layer. The outer layer is the fibrous layer which is continuous with the periosteum or perichondrium and consists of bundles of dense connective tissue, and the inner layer is the synovial layer which consists of loose connective tissue, with epithelial-like or epithelioid cells lying innermost. Recent electron microscope investigation (BARLANDet al., 1962) has revealed occurrence of two types of cells, type A and type B, in the epithelioid cells of the synovial membrane, and these cells clearly differ from the ordinary epithelial cells in their shape and relation to the adjacent cells. On the other hand, scanning electron microscopy has developed during the past ten years, and has been applied to the field of biology along with progress in specimen preparation technique using the critical point drying method (ANDERSON, 1951) and various kinds of cracking methods (HAGGIS, 1970; TANAKA, 1972; HAMANOet al., 1973). of the
Among numerous surface structures
reports on the scanning electron of tissues and cells, there have 517
microscope observations been papers also on the
518
K. DATE:
synovial
membrane
of the joint capsule (FUJITA et al., 1968; WOODWARD etal., 1971;
HAYASHI, 1976). The findings obtained, however, have been insufficient in information on the cellular and subcellular level because the above-mentioned techniques in specimen preparation have not been applied in most of the previous studies. In this paper the lining cells and the fibroblasts in the subsynovial tissue in styrene-cracked and critical point-dried specimens are observed using a field emission scanning electron microscope whose resolving power is superior to that of conventional scanning electron microscopes.
MATERIALS
AND METHODS
The synovial membrane of humans and rabbits was used for this study. Human synovial specimens were obtained from legs amputated because of malignant bone disease (12-year-old boy, 13-year-old girl, 16-year-old boy and girl). Rabbit synovial specimens were obtained from the knee joints under ether anesthesia. The specimens were immediately washed with physiological saline solution, and fixed in 2.5% glutaraldehyde buffered with 0.1M phosphate (pH. 7.4) for 2 days. Then, they were prepared for surface and cracked surface observation, and were treated respectively as follows. 1) The specimens for surface observation were immersed in 2% tannic acid solution for 3hrs, and washed in distilled water. They were post-fixed in 1% osmium tetroxide for 12hrs. After dehydration in a graded series of ethanol, they were critical point-dried using dry ice (TANAKAand IINO, 1974). 2)
The
specimens
approximately
for cracked
2×2×10mm
surface
in size.
Then,
observation after
similar
were
cut
into
dehydration,
small
pieces
styrene
resin
cracking (TANAKA and IINO, 1974) was applied. The specimens were embedded in small gelatin capsules filled with styrene monomer containing 3% benzoyle peroxide. Polymerization
critical
was
point-dried,
made
at 60℃
and then
within
followed
24hrs.
by ion-etching
After
removal
carried
of
resin,
faces for 3min (Eiko-Engineering IB-2 Ion Coater). All specimens were spatter-coated with platinum (Eiko-Engineering Coater) and examined by a field emission scanning electron microscope FSH-2ST)
with
25kV
accelerating
they
out on the cracked
were
sur-
IB-3 Ion (Hitachi
voltage.
RESULTS A. 1.
Surface
of the synovial
membrane
Human synovial membrane
The surface of the synovial membrane appears furrowed and is covered by slender processes of lining cells running in all directions; fibrillar substances are observed between them. The cell processes are partly flat and smooth in surface but partly covered by numerous irregular protrusions which cause a cauliflower-like appearance (Fig. 1). Under high magnification, the cauliflower-like structures consist of cytoplasmic processes attached by microvilli and small round projections; no ruffle-like protrusions can be observed (Fig. 2). These structures are frequently observed,
SEM
Fig.
1.
Human
synovial
cauliflower-like
Fig.
2.
Human consist to them.
surface. protrusions.
Cytoplasmic
Studies
processes
of Synovial
of lining
cells show
519
smooth
or
×4,700
synovial surface. Under high magnification, of irregular cytoplasmic processes, microvilli ×7,800
Membrane
cauliflower-like protrusions and round processes attached
520
K. DATE:
whereas be
occasionally
dispersed
diameter) 2.
all
round, over
the
larger synovial
are observed attaching
cytoplasmic surface
processes (Fig.3).
(3-4μ Small
in
dialneter)
granules
may
(0.3-0.6μ
in
to these round processes.
Rabbit synovial membrane
There are considerable variations in the surface structure of the synovial membrane in each examined specimen. One of them shows slender cytoplasmic processes protruding from lining cells like the overlapping cytoplasmic processes of renal podocytes (Fig. 4, 5). In this variation round processes are not visible; the cytoplasmic processes are flat and smooth in general, with only a few small granules attached to them. Many fibrillar substances are seen between these processes. Another variant is characterized by cytoplasmic processes expanding like the shape of a potbelly and possessing sparse microvilli (Fig. 6A), or by slender cytoplasmic processes with sparse microvilli projected into the joint cavity (Fig. 6B). On the other hand, in the rabbit synovial membrane one week after experimentally induced hemarthrosis, ruffle-like protrusions and round processes are seen, and they appear cauliflower-like in shape where they come together (Fig. 7). B.
Cracked
surface
of the synovial membrane
Observation of the cracked surfaces reveals the synovial membrane composed of the synovial and subsynovial layers. The synovial layer consists of lining cells, whereas the subsynovial may be either fibrous (Fig. 8) or adipose. 1.
Lining cell
The lining cells which face the joint cavity are from one to several cells in thickness.
Fig.
3.
Human diameter)
synovial attach
surface. are
seen
Round spreading
processes all over
to the
which synovial
small
granules
surface.
(0.3-0.6μ ×6,600
in
SEM Studies
Fig.
Fig.
4.
5.
Rabbit
synovial
lapping.
×800
High like
surface.
magnification overlapping
Cytoplasmic
of the synovial processes
of
renal
processes
surface podocytes.
of Synovial
of lining
in Figure ×3,100
Membrane
cells are smooth
4. Cytoplasmic
521
and over-
processes
look
522
K. DATE:
A
B Fig.
6.
Rabbit synovial surface. A. Cytoplasmic processes expand like the shape of a potbelly and are covered by sparse microvilli. B. Slender cytoplasmic processes with sparse microvilli
Fig.
7.
protrude
into
the
joint
cavity.
A:
Rabbit synovial surface one week after Ruffle-like protrusions are seen, and they they
come
together.
×4,900
×8,700;
B:
×1,600
experimentally induced appear cauliflower-like
hemarthrosis. in shape where
SEM Studies
of Synovial
Membrane
523
Owing to the relatively wide intercellular space, they are not in close contact with each other. Fibrillar substances are seen in the intercellular space. As to the lining cells, two cell types are distinguishable. One is relatively large in size and possesses surface processes and numerous intracytoplasmic granules (0.3-1.3μ
in diameter).
The
other
has
numerous
cisternae
of endoplasmic
reticulum
instead of intracellular granules (Fig. 9, 10). Contacting surface between the cells is very small, and cell junctions such as desmosome or adhesion plate are not discerned. By observing both the synovial surface and its cracked surface simultaneously, it is confirmed that cytoplasmic processes projecting into the joint cavity are derived from lining cells (Fig. 11). 2. Subsynovial tissue The bundles of collagen fibers in the subsynovial tissue are small and loose in comparison with those of the fibrous layer (Fig. 8). Many fibroblasts and wandering cells are observed. a. Fibroblast and fibrogenesis The
fibroblast
is
about
20×8×5μ
in
size,
often
appears
spindle-shaped
and
possesses
variously bulbous processes and sparse microvilli on the cell surface (Fig. 12). Their cracked surface shows a fairly large nucleus in the cytoplasm, and the nucleus is elliptic in shape. As regards the relation between the fibroblast and extracellular microfibrils, two types of fibrogenesis are observed. In the first microfibrils appear on the cell surface as if they surround the cell like a basket. They are gradually oriented in one direction,
and
keeping
come
to
form
some distance
Fig. 8.
a
bundle
of
collagen
fibers
(approximately
1μ in
diameter),
from the cell (Fig. 13). In the second type of fibrogenesis
Cracked surface of the human synovial membrane.
The joint capsule is composed
of the
FL
synovial
and
fibrous
layer.
SL
synovial
layer,
fibrous
layer.
×400
524
K. DATE:
Fig.
9.
The lining 1-3μ
Fig.
10.
in
The lining reticulum.
cells which
diameter).
cells which ×4,500
are relatively
large
in size contain
numerous
granules
(0.3-
×4,500
are relatively
small
in size have well developed
endoplasmic
SEM Studies
of Synovial
Membrane
525
bundles of collagen fibers protrude directly from the cell surface (Fig. 14). In the cracked surface of the cell, bundles of filaments which can be seen in the cytoplasm are generally parallel with each other and with the extracellular collagen bundles (Fig. 15).
Fig. 11.
Cracked surface of the human synovial membrane. ing
Fig.
12.
into
Stereopair appears surface.
the
joint
of
the
cavity
fibroblast.
spindle-shaped. ×1,900
are
derived
The Bundles
from
cell of
lining
is approximately collagen
Cytoplasmic processes project-
cells.
fibers
×6,100
20×8×5μ protrude
in size
directly
from
and
often the
cell
526
K. DATE:
Fig.
13.
Microfibrils collagen
Fig.
14.
Bundles ×10,000
which fibers
some
of collagen
surround
the
distance
from
the
seen
to protrude
fibers
are
fibroblast cell.
are CM
seen cell
directly
to
grow
into
membrane.
from
a
bundle
×11,800
the
cell
surface.
of
SEM Studies
Fig.
15.
of Synovial
Membrane
527
Cracked surface of the fibroblast. Bundles of filaments (arrows) exist in the cytoplasm, while a bundle of collagen fibers (double arrows) protrudes from the cell surface.
N
nucleus
×7,600
Fig. 16. Mast cell in the subsynovial tissue. The cell appears amoeba-shaped. The cytoplasm
is
filled
is a fibroblast.
with N
numerous nucleus.
granules ×5,400
(0.4-0.6μ
in
diameter).
The
adjacent
cell
528
K. DATE:
Fig.
17.
Cracked pits
surface
of the human
(approximately
30μ
in
synovial
diarneter)
in
membrane. the
Lipocytes
subsynovial
tissue.
are seen as large ×1,500
b. Mast cell As there are various kinds of cells in the subsynovial tissue, it is often difficult to identify many of them. In this study the mast cell is defined as an irregularly amoeba-shaped cell with an eccentrically placed nucleus, and is filled with round granules
c.
(0.4-0.6μ
in
diameter)
(Fig.
16).
Lipocyte
Lipocytes
are
of dissolution
seen
as
of their
large
pits
(30μ
fat in specimen
in
diameter)
preparation
in
the
subsynovial
tissue,
because
(Fig. 17).
DISCUSSION The lining
cells have
tron microscopy.
been hitherto
divided
into two cell types
BARLANDet al. (1962) regard
by transmission
one cell as type A, which
elec-
includes
more cytoplasmic processes, a prominent Golgi apparatus, numerous vesicles, vacuoles and mitochondria, and the other as type B, which has well developed rough endoplasmic reticulum and fewer vesicles, vacuoles and mitochondria. These two cell types are found also in the synovial membrane of the rabbit (GHADIALLY and ROY.
1966) and rat (ROY and GHADIALLY, 1967). HIROHATAet al. (1963) name the cell F-type which resembles
the fibroblast,
the
cell M-type which resembles the macrophage, and the cell F-M-type which is an intermediate type between both. In this observation of the cracked surfaces, two cell types are distinguished. One is relatively large in size, and has more cytoplasmic processes and numerous granules
(0.3-1.3μ
in
diameter),
whereas
the
other
is characterized
by
well
developed
SEM Studies
of Synovial
Membrane
529
endoplasmic reticulum instead of granules. The former is considered to correspond to type A cell and the latter to type B cell. Scanning electron microscope observations on the synovial membrane have been
reported 1971). imens
previously But, were
(FUJITA et al., 1968; REDLERand ZIMMY,1970; WOODWARD etal.,
none of the authors has given air-dried. By the use of the
sufficient information critical point drying
because method,
the specIHAYASHI
(1976) could distinguish under the scanning electron microscope cauliflower-like cells covered with many cytoplasmic processes and spindle-shaped cells with relatively smooth surface in the normal synovial membrane. Also in this study, slender cytoplasmic processes appear to overlap each other, and some of them present a cauliflower-like shape (Fig. 1). By observing some of the synovial membranes, there appear
to be
synovial
many
surface,
round
processes
to which
small
of granules
a
large
size
(0.3-0.6μ
(3-5μ
in
in diameter)
diameter)
all
attach
(Fig.
over 3).
the The
synovial surfaces thus show considerably variable appearances from specimen to specimen and from part to part of a specimen. As human materials were operatively obtained from patients, the problem is what the normal synovial membrane is. In this respect it is thought that the most basic surface structure of the synovial membrane in a static phase has no ruffle-like protrusions, but relatively smooth cytoplasmic processes (Fig. 4, 5). The reason is that the surface structure in a static phase is often seen in the normal synovial membrane of rabbit, and that the cauliflower-like protrusions are found in the rabbit hemarthrotic synovial membrane which is experimentally induced (Fig. 7). It is speculated that the cauliflower-like protrusions are seen only when stimulus is provided or some function is activated to the synovial membrane. As round processes (Fig. 6A) and slender cytoplasmic processes projecting into the joint cavity (Fig. 6B) are also observed in the normal synovial membrane, it can not be always said that the cauliflower-like protrusions represent an abnormal phase. Further observation is necessary in the future. The problem whether collagen fiber formation by fibroblasts might occur intracellularly has been intensively debated since the late nineteenth century. Owing to the
progress
precursors
of
electron
of collagen
microscope
fibrils
have
studies,
microfibrils
been observed
(50-100Å
in close proximity
in
diameter)
as
to fibroblasts
(HAMANO,1966), whereas the existence of cytoplasmic fibrils has also been elucidated (HAUST and MORE, 1966). By electron microscopic autoradiography it is generally thought that soluble collagen which is synthesized in the cell might be released into the extracellular space, where it is gathered into bundles of microfibrils after cyclic polymerization,
assuming
gradually
a characteristic
increasing
in diameter
and
length,
and
simultaneously
cross banding (Ross and BENDITT, 1963; REVEL and HAY,
1965).
In the present observation many microfibrils enclosing the cell surface are seen to grow into bundles of fibrils some distance from the cell (Fig. 13). Noteworthily, this study demonstrates bundles of collagen fibrils with a cross banding projecting directly from the cell surface. Furthermore, the cracked surface of the cell reveals that many bundles of filaments as precursors of collagen fibers are formed intracellularly and may be extruded as such into the extracellular space. At any rate it is worthy of attention that two different types of fibrogenesis can be observed in the subsynovial tissue.
530
K. DATE:
Acknowledgement. technical
advice
The
author
wishes to thank
Mr. H. OSATAKE and Mr. Y. KASHIMA for their
in this investigation.
滑 膜 の 走 査 電 子 顕 微鏡 的 研 究 伊 達 和 友 ヒ トお よび カ イ ウ サ ギ の関 節 滑 膜 を走 査 電 子顕 微 鏡 に よ り観察 した. 1.
滑 膜 の表 面 は 部 位 に よ っ て か な り形 態 が 異 な る. す な わ ち, 滑 膜 表層 細 胞 の 細 胞 質
突 起 が ち ょ う ど腎臓 の タ コ足 細 胞 の 突 起 の よ うに 長 く扁 平 に のび た も の, あ るい は 花 野 菜 状 か, よ り表 面 が 平 滑 な 球 形 の突 起 を 出 し て い る も の な どで あ る. 2.
滑 膜 の 割 断 面 で,
2種 類 の表 層 細 胞 が 区別 さ れ る. 一 つ は 多 くの 突 起 を 持 ち, 細 胞
内 に 多 数 の 顆 粒 を 含 む 細 胞 で あ り, 他 は 突 起 に 乏 し く, 顆 粒 を 含 まな い で よ く発 達 した小 胞 体 を もつ 細 胞 で あ る. 3.
滑 膜 下組 織 に お い て膠 原 線 維 を形 成 中 と 見 られ る線 維 芽 細 胞 が 観察 され る. 線 維 形
成 の形 式 に は 二 通 りが 見 られ る. 一 つ は細 胞 外 で線 維 が 形成 され る もの と, 他 は細 胞 内 に す で に微 細 な 線 維 束 が見 られ, 細 胞表 面 か らそ れ らが 直 接 突 出 す る もの で あ る.
REFERENCES Anderson, T. F.: Techniques for the preservation of three dimensional structure in preparing specimens for the electron microscope. Trans. Acad. Sci. Ser. II. 13: 130-134 (1951). Barland, P., A. B. Novikoff and D. Hamerman: Electron microscopy of the human synovial membrane. J. Cell Biol. 14: 207-220 (1962). Fujita, T., H. Inoue and T. Kodama: Scanning electron microscopy of the normal and rheumatoid synovial membranes. Arch. histol. jap. 29: 511-522 (1968). Ghadially, F. N. and S. Roy: Ultrastructure of rabbit synovial membrane. Ann. rheum. Dis. 25: 318-326 (1966). Haggis, G. H.: Cryofracture of biological material. In: (ed. by) O. Johari and I. Corvin: Scanning electron microscopy 1970. IIT Res. Inst., Chicago, 1970. (p. 99-104). Hamano, S., S. Otaka, T. Nagatani and K. Tanaka: Freeze liquid cracking method of biological materials for scanning electron microscopy. (In Japanese). In: Proceedings Electron Microscopy Society of Japan, 29th Annual Meeting, 1973 (p. 91). Hatano, S.: Studies on the fine structure of intercellular components of the connective tissue. (In Japanese). J. Juzen Med. Soc. 73: 496-516 (1966). Haust, D. and R. H. More: Electron microscopy of connective tissue and elastogenesis. In: (ed. by) M. W. Bernard and D. E. Smith: The connective tissue. Williams & Wilkins, Baltimore, 1967. (p. 352-376). Hayashi, K.: Three-dimensional observations of rheumatoid synovial membrane. (In Japanese). Ryumachi (Tokyo) 16: 35-56 (1976). Hirohata, K., K. Mizuhara, A. Fuziwara, A. Sato, S. Imura and I. Kobayashi: Electron
SEM Studies
of Synovial
Membrane
531
microscopic studies on the joint tissue (1st report). (In Japanese). J. Jap. Orthoped. Ass. 36: 871-883 (1963). Redler, I. and M. L. Zimmy: Scanning electron microscopy of normal and abnormal articular cartilage and synovium. J. Bone Joint Surg. 52-A: 1395-1404 (1970). Revel, J. P. and E. D. Hay: An autoradiographic and electron microscopic study of collagen synthesis in differentiating cartilage. Z. Zellforsch. 61: 110-144 (1963). Ross, R. and E. P. Benditt: Wound healing and collagen formation. V. Quantitative electron microscope radiographic observations of proline-H3 utilization by fibroblast. J. Cell Biol. 27: 83-106 (1965). Roy, S. and F. N. Ghadially: Ultrastructure of normal rat synovial membrane. Ann. rheum. Dis. 26: 26-37 (1967). Tanaka, K.: Frozen resin cracking method for scanning electron microscopy of biological materials. Naturwissenschaften 59: 77 (1972). Tanaka, K. and A. Iino: Critical point drying method using dry ice. Stain Technol. 49: 203-206 (1974). Tanaka, K., A. Iino and T. Naguro: Styrene resin cracking method for observing biological materials by scanning electron microscopy. J. Electron Microsc. 23: 313-315 (1974). Woodward, D. H., A. Gryfe and D. H. Gardner: Comparative study of scanning electron microscopy of synovial surfaces of four mammalian species. Experientia 25: 1301-1303 (1971).
伊 達 和友 〒683米 子市西町86 鳥取 大学 医学 部 第二解剖学 教室
Dr. Kazutomo DATE Department of Anatomy Tottori University School of Medicine Yonago, 683 Japan
Scanning
Electron
Microscope
Studies
on the
Synovial
Membrane
Kazutomo DATE Department of Anatomy (Prof. K. TANAKA)and Department of Orthopedic Surgery (Prof. I. MAEYAMA), Tottori
University
School of Medicine,
Yonago,
Japan
Received March 15, 1979
Summary. Synovial membranes from human and rabbit joints were observed by scanning electron microscopy. 1. The surfaces of synovial membranes present locally variable appearances. In some parts cytoplasmic processes of lining cells extend long and flat causing an appearance like overlapping renal podocytes, whereas in other parts the cells protrude in cauliflower-like or more smooth-surfaced round bodies. 2. In cracked surfaces of synovial membranes, two types of lining cells are distinguished. One is the cell which has more surface processes and numerous granules in the cytoplasm, the other is the cell which has fewer processes and better developed endoplasmic reticulum without granules. 3. Fibroblasts apparently forming collagen fibers are observed in the subsynovial tissue. Two types of fibrogenesis are found. In the first type microfibrils seem to be formed extracellularly, whereas in the second type bundles of filaments are preformed in the cytoplasm and they appear to be extruded directly from the cell surface.
There have been many studies on the histologic structure of the joint capsule, which is microscopically divided into the inner and outer layer. The outer layer is the fibrous layer which is continuous with the periosteum or perichondrium and consists of bundles of dense connective tissue, and the inner layer is the synovial layer which consists of loose connective tissue, with epithelial-like or epithelioid cells lying innermost. Recent electron microscope investigation (BARLANDet al., 1962) has revealed occurrence of two types of cells, type A and type B, in the epithelioid cells of the synovial membrane, and these cells clearly differ from the ordinary epithelial cells in their shape and relation to the adjacent cells. On the other hand, scanning electron microscopy has developed during the past ten years, and has been applied to the field of biology along with progress in specimen preparation technique using the critical point drying method (ANDERSON, 1951) and various kinds of cracking methods (HAGGIS, 1970; TANAKA, 1972; HAMANOet al., 1973). of the
Among numerous surface structures
reports on the scanning electron of tissues and cells, there have 517
microscope observations been papers also on the
518
K. DATE:
synovial
membrane
of the joint capsule (FUJITA et al., 1968; WOODWARD etal., 1971;
HAYASHI, 1976). The findings obtained, however, have been insufficient in information on the cellular and subcellular level because the above-mentioned techniques in specimen preparation have not been applied in most of the previous studies. In this paper the lining cells and the fibroblasts in the subsynovial tissue in styrene-cracked and critical point-dried specimens are observed using a field emission scanning electron microscope whose resolving power is superior to that of conventional scanning electron microscopes.
MATERIALS
AND METHODS
The synovial membrane of humans and rabbits was used for this study. Human synovial specimens were obtained from legs amputated because of malignant bone disease (12-year-old boy, 13-year-old girl, 16-year-old boy and girl). Rabbit synovial specimens were obtained from the knee joints under ether anesthesia. The specimens were immediately washed with physiological saline solution, and fixed in 2.5% glutaraldehyde buffered with 0.1M phosphate (pH. 7.4) for 2 days. Then, they were prepared for surface and cracked surface observation, and were treated respectively as follows. 1) The specimens for surface observation were immersed in 2% tannic acid solution for 3hrs, and washed in distilled water. They were post-fixed in 1% osmium tetroxide for 12hrs. After dehydration in a graded series of ethanol, they were critical point-dried using dry ice (TANAKAand IINO, 1974). 2)
The
specimens
approximately
for cracked
2×2×10mm
surface
in size.
Then,
observation after
similar
were
cut
into
dehydration,
small
pieces
styrene
resin
cracking (TANAKA and IINO, 1974) was applied. The specimens were embedded in small gelatin capsules filled with styrene monomer containing 3% benzoyle peroxide. Polymerization
critical
was
point-dried,
made
at 60℃
and then
within
followed
24hrs.
by ion-etching
After
removal
carried
of
resin,
faces for 3min (Eiko-Engineering IB-2 Ion Coater). All specimens were spatter-coated with platinum (Eiko-Engineering Coater) and examined by a field emission scanning electron microscope FSH-2ST)
with
25kV
accelerating
they
out on the cracked
were
sur-
IB-3 Ion (Hitachi
voltage.
RESULTS A. 1.
Surface
of the synovial
membrane
Human synovial membrane
The surface of the synovial membrane appears furrowed and is covered by slender processes of lining cells running in all directions; fibrillar substances are observed between them. The cell processes are partly flat and smooth in surface but partly covered by numerous irregular protrusions which cause a cauliflower-like appearance (Fig. 1). Under high magnification, the cauliflower-like structures consist of cytoplasmic processes attached by microvilli and small round projections; no ruffle-like protrusions can be observed (Fig. 2). These structures are frequently observed,
SEM
Fig.
1.
Human
synovial
cauliflower-like
Fig.
2.
Human consist to them.
surface. protrusions.
Cytoplasmic
Studies
processes
of Synovial
of lining
cells show
519
smooth
or
×4,700
synovial surface. Under high magnification, of irregular cytoplasmic processes, microvilli ×7,800
Membrane
cauliflower-like protrusions and round processes attached
520
K. DATE:
whereas be
occasionally
dispersed
diameter) 2.
all
round, over
the
larger synovial
are observed attaching
cytoplasmic surface
processes (Fig.3).
(3-4μ Small
in
dialneter)
granules
may
(0.3-0.6μ
in
to these round processes.
Rabbit synovial membrane
There are considerable variations in the surface structure of the synovial membrane in each examined specimen. One of them shows slender cytoplasmic processes protruding from lining cells like the overlapping cytoplasmic processes of renal podocytes (Fig. 4, 5). In this variation round processes are not visible; the cytoplasmic processes are flat and smooth in general, with only a few small granules attached to them. Many fibrillar substances are seen between these processes. Another variant is characterized by cytoplasmic processes expanding like the shape of a potbelly and possessing sparse microvilli (Fig. 6A), or by slender cytoplasmic processes with sparse microvilli projected into the joint cavity (Fig. 6B). On the other hand, in the rabbit synovial membrane one week after experimentally induced hemarthrosis, ruffle-like protrusions and round processes are seen, and they appear cauliflower-like in shape where they come together (Fig. 7). B.
Cracked
surface
of the synovial membrane
Observation of the cracked surfaces reveals the synovial membrane composed of the synovial and subsynovial layers. The synovial layer consists of lining cells, whereas the subsynovial may be either fibrous (Fig. 8) or adipose. 1.
Lining cell
The lining cells which face the joint cavity are from one to several cells in thickness.
Fig.
3.
Human diameter)
synovial attach
surface. are
seen
Round spreading
processes all over
to the
which synovial
small
granules
surface.
(0.3-0.6μ ×6,600
in
SEM Studies
Fig.
Fig.
4.
5.
Rabbit
synovial
lapping.
×800
High like
surface.
magnification overlapping
Cytoplasmic
of the synovial processes
of
renal
processes
surface podocytes.
of Synovial
of lining
in Figure ×3,100
Membrane
cells are smooth
4. Cytoplasmic
521
and over-
processes
look
522
K. DATE:
A
B Fig.
6.
Rabbit synovial surface. A. Cytoplasmic processes expand like the shape of a potbelly and are covered by sparse microvilli. B. Slender cytoplasmic processes with sparse microvilli
Fig.
7.
protrude
into
the
joint
cavity.
A:
Rabbit synovial surface one week after Ruffle-like protrusions are seen, and they they
come
together.
×4,900
×8,700;
B:
×1,600
experimentally induced appear cauliflower-like
hemarthrosis. in shape where
SEM Studies
of Synovial
Membrane
523
Owing to the relatively wide intercellular space, they are not in close contact with each other. Fibrillar substances are seen in the intercellular space. As to the lining cells, two cell types are distinguishable. One is relatively large in size and possesses surface processes and numerous intracytoplasmic granules (0.3-1.3μ
in diameter).
The
other
has
numerous
cisternae
of endoplasmic
reticulum
instead of intracellular granules (Fig. 9, 10). Contacting surface between the cells is very small, and cell junctions such as desmosome or adhesion plate are not discerned. By observing both the synovial surface and its cracked surface simultaneously, it is confirmed that cytoplasmic processes projecting into the joint cavity are derived from lining cells (Fig. 11). 2. Subsynovial tissue The bundles of collagen fibers in the subsynovial tissue are small and loose in comparison with those of the fibrous layer (Fig. 8). Many fibroblasts and wandering cells are observed. a. Fibroblast and fibrogenesis The
fibroblast
is
about
20×8×5μ
in
size,
often
appears
spindle-shaped
and
possesses
variously bulbous processes and sparse microvilli on the cell surface (Fig. 12). Their cracked surface shows a fairly large nucleus in the cytoplasm, and the nucleus is elliptic in shape. As regards the relation between the fibroblast and extracellular microfibrils, two types of fibrogenesis are observed. In the first microfibrils appear on the cell surface as if they surround the cell like a basket. They are gradually oriented in one direction,
and
keeping
come
to
form
some distance
Fig. 8.
a
bundle
of
collagen
fibers
(approximately
1μ in
diameter),
from the cell (Fig. 13). In the second type of fibrogenesis
Cracked surface of the human synovial membrane.
The joint capsule is composed
of the
FL
synovial
and
fibrous
layer.
SL
synovial
layer,
fibrous
layer.
×400
524
K. DATE:
Fig.
9.
The lining 1-3μ
Fig.
10.
in
The lining reticulum.
cells which
diameter).
cells which ×4,500
are relatively
large
in size contain
numerous
granules
(0.3-
×4,500
are relatively
small
in size have well developed
endoplasmic
SEM Studies
of Synovial
Membrane
525
bundles of collagen fibers protrude directly from the cell surface (Fig. 14). In the cracked surface of the cell, bundles of filaments which can be seen in the cytoplasm are generally parallel with each other and with the extracellular collagen bundles (Fig. 15).
Fig. 11.
Cracked surface of the human synovial membrane. ing
Fig.
12.
into
Stereopair appears surface.
the
joint
of
the
cavity
fibroblast.
spindle-shaped. ×1,900
are
derived
The Bundles
from
cell of
lining
is approximately collagen
Cytoplasmic processes project-
cells.
fibers
×6,100
20×8×5μ protrude
in size
directly
from
and
often the
cell
526
K. DATE:
Fig.
13.
Microfibrils collagen
Fig.
14.
Bundles ×10,000
which fibers
some
of collagen
surround
the
distance
from
the
seen
to protrude
fibers
are
fibroblast cell.
are CM
seen cell
directly
to
grow
into
membrane.
from
a
bundle
×11,800
the
cell
surface.
of
SEM Studies
Fig.
15.
of Synovial
Membrane
527
Cracked surface of the fibroblast. Bundles of filaments (arrows) exist in the cytoplasm, while a bundle of collagen fibers (double arrows) protrudes from the cell surface.
N
nucleus
×7,600
Fig. 16. Mast cell in the subsynovial tissue. The cell appears amoeba-shaped. The cytoplasm
is
filled
is a fibroblast.
with N
numerous nucleus.
granules ×5,400
(0.4-0.6μ
in
diameter).
The
adjacent
cell
528
K. DATE:
Fig.
17.
Cracked pits
surface
of the human
(approximately
30μ
in
synovial
diarneter)
in
membrane. the
Lipocytes
subsynovial
tissue.
are seen as large ×1,500
b. Mast cell As there are various kinds of cells in the subsynovial tissue, it is often difficult to identify many of them. In this study the mast cell is defined as an irregularly amoeba-shaped cell with an eccentrically placed nucleus, and is filled with round granules
c.
(0.4-0.6μ
in
diameter)
(Fig.
16).
Lipocyte
Lipocytes
are
of dissolution
seen
as
of their
large
pits
(30μ
fat in specimen
in
diameter)
preparation
in
the
subsynovial
tissue,
because
(Fig. 17).
DISCUSSION The lining
cells have
tron microscopy.
been hitherto
divided
into two cell types
BARLANDet al. (1962) regard
by transmission
one cell as type A, which
elec-
includes
more cytoplasmic processes, a prominent Golgi apparatus, numerous vesicles, vacuoles and mitochondria, and the other as type B, which has well developed rough endoplasmic reticulum and fewer vesicles, vacuoles and mitochondria. These two cell types are found also in the synovial membrane of the rabbit (GHADIALLY and ROY.
1966) and rat (ROY and GHADIALLY, 1967). HIROHATAet al. (1963) name the cell F-type which resembles
the fibroblast,
the
cell M-type which resembles the macrophage, and the cell F-M-type which is an intermediate type between both. In this observation of the cracked surfaces, two cell types are distinguished. One is relatively large in size, and has more cytoplasmic processes and numerous granules
(0.3-1.3μ
in
diameter),
whereas
the
other
is characterized
by
well
developed
SEM Studies
of Synovial
Membrane
529
endoplasmic reticulum instead of granules. The former is considered to correspond to type A cell and the latter to type B cell. Scanning electron microscope observations on the synovial membrane have been
reported 1971). imens
previously But, were
(FUJITA et al., 1968; REDLERand ZIMMY,1970; WOODWARD etal.,
none of the authors has given air-dried. By the use of the
sufficient information critical point drying
because method,
the specIHAYASHI
(1976) could distinguish under the scanning electron microscope cauliflower-like cells covered with many cytoplasmic processes and spindle-shaped cells with relatively smooth surface in the normal synovial membrane. Also in this study, slender cytoplasmic processes appear to overlap each other, and some of them present a cauliflower-like shape (Fig. 1). By observing some of the synovial membranes, there appear
to be
synovial
many
surface,
round
processes
to which
small
of granules
a
large
size
(0.3-0.6μ
(3-5μ
in
in diameter)
diameter)
all
attach
(Fig.
over 3).
the The
synovial surfaces thus show considerably variable appearances from specimen to specimen and from part to part of a specimen. As human materials were operatively obtained from patients, the problem is what the normal synovial membrane is. In this respect it is thought that the most basic surface structure of the synovial membrane in a static phase has no ruffle-like protrusions, but relatively smooth cytoplasmic processes (Fig. 4, 5). The reason is that the surface structure in a static phase is often seen in the normal synovial membrane of rabbit, and that the cauliflower-like protrusions are found in the rabbit hemarthrotic synovial membrane which is experimentally induced (Fig. 7). It is speculated that the cauliflower-like protrusions are seen only when stimulus is provided or some function is activated to the synovial membrane. As round processes (Fig. 6A) and slender cytoplasmic processes projecting into the joint cavity (Fig. 6B) are also observed in the normal synovial membrane, it can not be always said that the cauliflower-like protrusions represent an abnormal phase. Further observation is necessary in the future. The problem whether collagen fiber formation by fibroblasts might occur intracellularly has been intensively debated since the late nineteenth century. Owing to the
progress
precursors
of
electron
of collagen
microscope
fibrils
have
studies,
microfibrils
been observed
(50-100Å
in close proximity
in
diameter)
as
to fibroblasts
(HAMANO,1966), whereas the existence of cytoplasmic fibrils has also been elucidated (HAUST and MORE, 1966). By electron microscopic autoradiography it is generally thought that soluble collagen which is synthesized in the cell might be released into the extracellular space, where it is gathered into bundles of microfibrils after cyclic polymerization,
assuming
gradually
a characteristic
increasing
in diameter
and
length,
and
simultaneously
cross banding (Ross and BENDITT, 1963; REVEL and HAY,
1965).
In the present observation many microfibrils enclosing the cell surface are seen to grow into bundles of fibrils some distance from the cell (Fig. 13). Noteworthily, this study demonstrates bundles of collagen fibrils with a cross banding projecting directly from the cell surface. Furthermore, the cracked surface of the cell reveals that many bundles of filaments as precursors of collagen fibers are formed intracellularly and may be extruded as such into the extracellular space. At any rate it is worthy of attention that two different types of fibrogenesis can be observed in the subsynovial tissue.
530
K. DATE:
Acknowledgement. technical
advice
The
author
wishes to thank
Mr. H. OSATAKE and Mr. Y. KASHIMA for their
in this investigation.
滑 膜 の 走 査 電 子 顕 微鏡 的 研 究 伊 達 和 友 ヒ トお よび カ イ ウ サ ギ の関 節 滑 膜 を走 査 電 子顕 微 鏡 に よ り観察 した. 1.
滑 膜 の表 面 は 部 位 に よ っ て か な り形 態 が 異 な る. す な わ ち, 滑 膜 表層 細 胞 の 細 胞 質
突 起 が ち ょ う ど腎臓 の タ コ足 細 胞 の 突 起 の よ うに 長 く扁 平 に のび た も の, あ るい は 花 野 菜 状 か, よ り表 面 が 平 滑 な 球 形 の突 起 を 出 し て い る も の な どで あ る. 2.
滑 膜 の 割 断 面 で,
2種 類 の表 層 細 胞 が 区別 さ れ る. 一 つ は 多 くの 突 起 を 持 ち, 細 胞
内 に 多 数 の 顆 粒 を 含 む 細 胞 で あ り, 他 は 突 起 に 乏 し く, 顆 粒 を 含 まな い で よ く発 達 した小 胞 体 を もつ 細 胞 で あ る. 3.
滑 膜 下組 織 に お い て膠 原 線 維 を形 成 中 と 見 られ る線 維 芽 細 胞 が 観察 され る. 線 維 形
成 の形 式 に は 二 通 りが 見 られ る. 一 つ は細 胞 外 で線 維 が 形成 され る もの と, 他 は細 胞 内 に す で に微 細 な 線 維 束 が見 られ, 細 胞表 面 か らそ れ らが 直 接 突 出 す る もの で あ る.
REFERENCES Anderson, T. F.: Techniques for the preservation of three dimensional structure in preparing specimens for the electron microscope. Trans. Acad. Sci. Ser. II. 13: 130-134 (1951). Barland, P., A. B. Novikoff and D. Hamerman: Electron microscopy of the human synovial membrane. J. Cell Biol. 14: 207-220 (1962). Fujita, T., H. Inoue and T. Kodama: Scanning electron microscopy of the normal and rheumatoid synovial membranes. Arch. histol. jap. 29: 511-522 (1968). Ghadially, F. N. and S. Roy: Ultrastructure of rabbit synovial membrane. Ann. rheum. Dis. 25: 318-326 (1966). Haggis, G. H.: Cryofracture of biological material. In: (ed. by) O. Johari and I. Corvin: Scanning electron microscopy 1970. IIT Res. Inst., Chicago, 1970. (p. 99-104). Hamano, S., S. Otaka, T. Nagatani and K. Tanaka: Freeze liquid cracking method of biological materials for scanning electron microscopy. (In Japanese). In: Proceedings Electron Microscopy Society of Japan, 29th Annual Meeting, 1973 (p. 91). Hatano, S.: Studies on the fine structure of intercellular components of the connective tissue. (In Japanese). J. Juzen Med. Soc. 73: 496-516 (1966). Haust, D. and R. H. More: Electron microscopy of connective tissue and elastogenesis. In: (ed. by) M. W. Bernard and D. E. Smith: The connective tissue. Williams & Wilkins, Baltimore, 1967. (p. 352-376). Hayashi, K.: Three-dimensional observations of rheumatoid synovial membrane. (In Japanese). Ryumachi (Tokyo) 16: 35-56 (1976). Hirohata, K., K. Mizuhara, A. Fuziwara, A. Sato, S. Imura and I. Kobayashi: Electron
SEM Studies
of Synovial
Membrane
531
microscopic studies on the joint tissue (1st report). (In Japanese). J. Jap. Orthoped. Ass. 36: 871-883 (1963). Redler, I. and M. L. Zimmy: Scanning electron microscopy of normal and abnormal articular cartilage and synovium. J. Bone Joint Surg. 52-A: 1395-1404 (1970). Revel, J. P. and E. D. Hay: An autoradiographic and electron microscopic study of collagen synthesis in differentiating cartilage. Z. Zellforsch. 61: 110-144 (1963). Ross, R. and E. P. Benditt: Wound healing and collagen formation. V. Quantitative electron microscope radiographic observations of proline-H3 utilization by fibroblast. J. Cell Biol. 27: 83-106 (1965). Roy, S. and F. N. Ghadially: Ultrastructure of normal rat synovial membrane. Ann. rheum. Dis. 26: 26-37 (1967). Tanaka, K.: Frozen resin cracking method for scanning electron microscopy of biological materials. Naturwissenschaften 59: 77 (1972). Tanaka, K. and A. Iino: Critical point drying method using dry ice. Stain Technol. 49: 203-206 (1974). Tanaka, K., A. Iino and T. Naguro: Styrene resin cracking method for observing biological materials by scanning electron microscopy. J. Electron Microsc. 23: 313-315 (1974). Woodward, D. H., A. Gryfe and D. H. Gardner: Comparative study of scanning electron microscopy of synovial surfaces of four mammalian species. Experientia 25: 1301-1303 (1971).
伊 達 和友 〒683米 子市西町86 鳥取 大学 医学 部 第二解剖学 教室
Dr. Kazutomo DATE Department of Anatomy Tottori University School of Medicine Yonago, 683 Japan
