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The effect of beta-aminoproprionitrile on the periodontal ligamentII. Radioautographic study of collagen secretion from fibroblasts

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THE ANATOMICAL RECORD 209:41-52 (1984)
The Effect of Beta-Aminoproprionitrile on the
Periodontal Ligament: II. Radioautographic
Study of Collagen Secretion From
Fibroblasts
MOON-IL CHO AND PHILIAS R. GARANT
Department of Oral Biology and Pathology, School of Dental Medicine, State
University ofNew York at Stony Brook, Stony Brook, NY 11794
ABSTRACT
Fibroblasts are distributed evenly throughout the periodontal
ligament IPDL) of normal mice. In mice fed beta-aminoproprionitrile (0-APN)
the fibroblasts undergo aggregation to form palisades of closely juxtaposed
cells abutting pools of acellular collagenous matrix. Individual fibroblasts
within these aggregates retain their polarized cytoplasmic organization and
continue to synthesize and secrete collagen. However, unlike normal PDL
fibroblasts, the P-APN-treated cells appear immobilized by well-developed cellto-cell adherens-type junctions along their lateral surfaces. We studied collagen
secretion from P-APN-treated fibroblasts by light and electron microscopic
radioautography after injection of 3H-proline. Newly synthesized collagen was
secreted from the distal ends of the P-APN-aggregated fibroblasts as a distinct
band of labeled material, resembling the pattern of matrix deposition seen in
osteogenesis and dentinogenesis. The radioactive band of collagenous matrix
was displaced further away from the fibroblasts at 2 and 4 days after 3Hproline injection a s more collagen was secreted.
This pattern of radiolabeled collagen secretion confirmed previous observations that PDL fibroblasts are highly polarized and that collagen secretory
granules are extruded from the distal or secretory pole of the cell. In normal
PDL the even distribution of fibroblasts and the complex interrelationship of
their distal cell processes leads to a diffuse pattern of silver grain deposition,
masking the oriented flow of new collagen from the distal ends of individual
fibroblasts.
Analysis of electron microscopic radioautographs revealed that newly synthesized collagen was packaged and secreted from p-APN-treated fibroblasts
via the normal cytoplasmic pathways but a t a slower rate.
Periodontal ligament (PDL) fibroblasts are
highly polarized cells with a n extensive network of microtubules which function in collagen secretion-granule translocation (Cho
and Garant, 1981b,c,d). The appearance of
the PDL fibroblast Golgi apparatus and the
formation and maturation of collagen secretion granules is closely related to the macromolecular assembly and cross-linking of
the procollagen chains (Cho and Garant,
1981a). On the basis of the above findings,
one could predict that agents that interfere
with collagen cross-linking inside the cell
would produce changes in secretory granule
maturation and perhaps alter the rate of col-
0 1984 ALAN R. LISS, INC.
lagen secretion. Furthermore, interference
with collagen cross-linking in the extracellular environment could cause changes in cell
shape or cell distribution, since cell-to-matrix
interactions are important factors in determining cellular behavior (Bellairs et al.,
1982).
Beta-aminoproprionitrile (p-APN) interferes with lysyl oxidase, preventing the formation of lysyl-derived aldehyde groups on
the collagen alpha chains (Deshmukh and
Nimmi, 1968; Bornstein, 1970). Because of
Received September 22,1983; accepted November 16, 1983.
42
M.-I. CHO AND P.R. GARANT
the reduced number of aldehyde groups, the
collagen alpha chains fail to become fully
cross-linked, and, therefore, the tensile
strength of the collagen fibers is greatly reduced (Fry et al., 1962).
The pathologic consequences of this molecular defect are most pronounced in connective tissues exposed to increased stress such
as in articulations, muscle insertions, and in
the periodontal ligament of functional teeth
(Gardner et al., 1958; Ponseti and Shepard,
1954; Krikos, 1959; Barrington and Meyer,
1966; Sciaky and Ungar, 1961; Levene, 1973).
The classic histologic lesion that develops
in the periodontal ligament within 1-2 weeks
after the onset of 6-APN administration is
called the “lathyritic body.” It is characterized by an aggregation or palisading of the
PDL fibroblasts in rows bordering on acellular amorphous matrix (Sciaky and Ungar,
1961; Barrington and Meyer, 1966; Yoshikawa, 1971; Sims, 1977; Baden and Bouissou, 1983; Krikos et al., 1958; Gardiner et al.,
1958).
In a recent publication (Cho and Garant,
1983) we described the ultrastructural appearance of 0-APN-treated fibroblasts of the
periodontal ligament. We noted that although the fibroblasts became aggregated to
form typical “lathyritic bodies,” individual
fibroblasts maintained their polarity and appeared to contain a normal complement of
cytoplasmic organelles. In fact, the polarity
of the cells was easier to detect in the pAPN-induced fibroblast aggregates, a feature which we thought could be used to advantage to study collagen secretion from PDL
fibroblasts. Furthermore, we noted changes
in the appearance of the Golgi apparatus,
which suggested a decreased rate of collagen
secretion.
In this paper we report our findings on
collagen secretion from 0-APN-treated PDL
fibroblasts and compare these results to those
published for normal PDL fibroblasts and
colchicine-treated PDL fibroblasts (Cho and
Garant, 1981c,d).
MATERIALS AND METHODS
Ultrastructural Study
Five male Balb-C mice weighing 15 to 17
gm were fed a powdered Purina rat chow diet
containing 0.4% P-APN for 2 weeks. The mice
were then anesthetized by i.p. injection of
Nembutal (1mg/lO g m body weight) prior to
intracardiac perfusion of 70-80 ml of 2% glutaraldehyde in a modified Tyrode solution,
pH 7.4 (Sjostrand, 1967). The maxillae were
then dissected free of the surrounding tissues
in a dish containing 4% glutaraldehyde and
5% paraformaldehyde in 0.1 M cacodylate
buffer, pH 7.4 (Karnovsky, 1965).The cleaned
maxillae were fixed in Karnovsky’s fixative
for a n additional 3 4 h, rinsed in 0.1 M cacodylate buffer, pH 7.4, for 15 min, and decalcified in 0.1 M EDTA containing 3% glutaraldehyde, pH 7.4, for about 2-3 weeks a t
4°C. The first interdental areas were then dissected out and cut into two identical pieces
in a dish containing 0.1 M cacodylate buffer,
pH 7.4. After washing in the same buffer
with several changes for 45-60 min, the
pieces of tissue were postfixed in 1%Osmium
tetroxide in S-collidine buffer, pH 7.4, for 1.5
h. En-block staining was carried out for 1 h
a t room temperature in 1% uranyl acetate in
0.1 M maleate buffer, pH 6.2, after rinsing in
the same buffer, pH 5.2, for 15 min. Tissues
were then dehydrated in a graduated series
of cold ethanols and propylene oxide prior to
infiltration with Epon mixture at room temperature for 4 h and placed in flat embedding
molds containing fresh Epon mixture. Polymerization was accomplished a t 60°C for 48 h.
For ultrastructural study, ultrathin sections (60-80 nm in thickness) were cut on a
Porter-Blum MT2 ultramicrotome equipped
with a DuPont diamond knife. The sections
were doubly stained for 25 min with freshly
prepared saturated uranyl acetate in 50%
ethanol and for 7 min with lead citrate (Reynolds, 1963). The stained sections were then
examined and photographed with a JEMlOOB electron microscope.
For light and electron microscopic radioautography 11 male Balb-C mice weighing 1517 gm were fed powdered Purina rat chow
containing 0.4% 0-APN for 2 weeks. They
were then injected with L-(2, 3-3H)proline(SA
= 40 Ciimmol; New England Nuclear, Inc.,
Boston, MA) a t a concentration of 1mCi per
0.1 ml of sterile saline solution per mouse.
All injections were made via the jugular vein,
in animals anesthetized with ether, a t a
standard time of between 8:30 and 9:30 A.M.
The mice were killed, one per time period, a t
3, 10,20, 30 min, 1 , 2 , 4 , 8 , 2 4 h, 2 and 4 days
after injection of the tritiated proline, by proline, by intracardiac perfusion of 2% glutaraldehyde in Tyrode’s solution. The tissues
were subsequently processed as described
above, except for the omission of en-block
staining with uranyl acetate.
For light microscopic radioautographs,
COLLAGEN SECRETION FROM 0-APN-TREATED FIBROBLASTS
three to four 1-pm sections from each block
(four blocks per animal) were mounted on
glass slides, coated with Kodak NTM-3 liquid
emulsion, exposed for 3 to 8 days at 4"C, and
developed as previously described (Garant
and Cho, 1979b). The radioautographs were
subsequently stained with 1% toluidine blue
in sodium veronal acetate buffer.
For electron microscopic radioautography
the loop method of Car0 and Van Tubergen
(1962) was employed. Thin sections, about
100 nm in thickness from four blocks for each
time period, were coated with a crystalline
monolayer of Ilford L4 emulsion, exposed a t
4"C, and developed with either Microdal-X
or a physical developer as described by
Kopriwa (1973).
The simple quantitative analysis of EM radioautographs described by Nadler (1971)was
used. Silver grains were counted or. micrographs with a final magnification of x 30,000.
The following compartments were analyzed:
RER, Golgi complex (cisternae, saccules, intermediate vesicles, and secretory granules),
intracellular collagen fibrils, extracellular
collagen fibrils, nuclei, mitochondria, and cell
periphery. One point was recorded when the
grain was clearly encompassed by the organelle in question. When a grain was shared by
two organelles, each compartment received a
half point.
RESULTS
Light Microscopic Radioautography
In P-APN-treated PDL, radioautographs
obtained 10 min after 3H-proline injection
showed silver grains a t the periphery of the
cell bodies where the RER is located (Fig. 1).
The juxtanuclear Golgi complex, observed as
a pale staining zone, and the extracellular
matrix were relatively free of silver grains
(Fig. 1).At 30 min, most of the silver grains
were concentrated over the supranuclear cytoplasm, i.e., the region of the Golgi complex
(Fig. 2). A small number of grains were 10cated over distal cell processes or the adjacent extracellular matrix at 30 min (Fig. 2).
By 8 h, the majority of silver grains were
concentrated over the extracellular collagen
matrix close to the distal cell processes (Fig.
3). The grain distribution in Figure 3 is reminiscent of that observed adjacent to a n osteoblast or odontoblast layer, and thus it
demonstrates a similar oriented flow of secretory material from the secretory pole (distal
end) of the polarized fibroblasts. At 2 days
(Fig. 4)and 4 days (Fig. 5) the concentrated
43
band of radioactive collagen was located progressively farther away from the fibroblasts,
presumably pushed away by newly secreted
collagenous matrix. As not all fibroblasts
were oriented in the same direction, one band
formed on each side of the palisaded fibroblasts. There were few grains over the lateral
intercellular spaces or the cell bodies a t these
later time periods. The difference in grain
distribution over the extracellular matrix can
be appreciated by examination of Figure 6,
which is a radioautograph of normal PDL 2
h after injection of 3H-proline. The difference
in grain distribution is presumably due to
the difference in the fibroblast distribution
pattern and not to differences in mobility of
the newly synthesized collagen precursors.
Electron Microscopic Radioautography
The percentage distribution of silver grains
over various organelles with time is summarized in Table 1 and presented in graphic
form in Figure 13.
At 3 min after injection of 3H-proline the
great majority of radioactivity was concentrated over the RER (Fig. 7). At 10 min most
silver grains (72.5%) were still located over
the RER, but a small number of grains
(15.5%) were present over the immature side
of the Golgi apparatus, near the RER (Fig.
8). The RER was still heavily labeled a t 20
min (58.1% of silver grains), while the Golgi
complex reached its maximum labeling of
34.8% (Fig. 9). Presecretory granules (Cho
and Garant, 1981a) were still unlabeled a t
20 min. The disappearance of silver grains
over the RER was slower than that previously observed in normal PDL fibroblasts
At 30 min,
(Fig. 13)(Cho and Garant, 1981~).
55.2% of all silver grains were still located
over the RER. Despite the slower decline of
labeling of the RER and Golgi complex, a
small number of presecretory granules and
collagen secretion granules appeared to be
labeled a t 30 min (Fig. 10). The later observation was in agreement with a decrease in
the number of silver grains over the Golgi
complex from 34.8% a t 20 min to 28.7% at 30
min. Thus, at about 30 min after 3H-proline
injection, labeled collagen precursors were
secreted from PDL fibroblasts. At 1 h the
number of silver grains over the extracellular matrix increased to 29.5% up from 8.4%
a t 30 min. At 1 and 4 h, silver grains positioned over the extracellular matrix were associated with striated collagen fibrils near
the distal cell processes (Figs. 11, 12). The
44
M.4. CHO AND P.R. GARANT
45
COLLAGEN SECRETION FROM p-APN-TREATED FIBROBLASTS
TABLE 1. Distribution of silver grains at various times over PAPN-treated PDL fibroblasts in electron microscopic
radioautographs of 3H-proline uptake
10 min
20 min
30 min
l h
2h
8h
24 h
RER
Golgi
ICC
ECC
Nuclei
Mito.
Autoph.
Cell
periph.
8781
72.i!i2
54.83
577
58.1
44.9
588
55.2
29.2
378
34.2
16.6
188
19.2
3.6
86
12.0
3.8
102
11.5
3.4
188
15.5
36.9
346
34.8
48.9
306
28.7
36.8
178
16.1
9.4
89
9.1
4.80
49
6.8
0.4
38
4.3
0.1
0.0
0.0
0.0
0.0
0.0
0.1
1.0
0.1
0.5
0.0
0.0
0.3
0.0
0.0
0.2
0.0
0.0
0.0
0.0
0.0
0.1
37
3.0
4.0
48
4.0
1.4
18
1.8
0.8
14
1.3
1.4
34
3.1
1.2
24
2.4
1.4
13
1.8
1.2
14
1.6
0.6
40
3.3
1.5
27
2.7
1.4
22
2.1
2.2
16
1.4
2.2
6
0.6
0.8
7
1.0
0.3
7
0.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.3
0.0
0.0
0.1
0.0
0.0
0.3
0.0
0.0
21
1.7
1.7
19
1.9
3.0
45
4.2
4.8
174
15.7
17.2
149
15.2
15.6
71
9.9
11.7
93
10.5
7
0.7
0.9
89
8.4
25.1
326
29.5
52.9
524
53.5
68.5
491
68.5
82.3
631
71.3
84.8
0.0
Total
1210
994
1065
1106
980
717
885
11.o
'Number of silver grains in 8-APN-treated cells.
'Percentage of total grains at each time period in 8-APN-treated cells.
3Percentage of total grains at each time period in normal cells (Cho and Garant, 1981~)
delayed movement of labeled collagen pre- tained in normal cells (3.4% and 0.1%) 24 h
cursors from RER and Golgi complex was after 3H-proline injection.
The collagenous matrix adjacent to the ficlearly evident at 1h, for 34.2% and 16.1% of
all silver grains were still located over the broblasts consisted of 640-banded fibrils of
RER and Golgi complex. By 24 h the number uniform width plus a n amorphous material
of silver grains over RER and the Golgi com- with fine filaments (Figs. 11,12). At 1h most
plex fell to a minimal value of 11.5% and silver grains were located over the banded
4.3%, respectively (Table 1). These values collagen fibrils (Fig. 111, while at 4 h both
were still higher, however, than those ob- banded fibrils and amorphous material were
labeled (Fig. 12).
DISCUSSION
Fig. 1. Li ht microscopic radioautograph 10 min after
injection of H-proline. The majority of silver grains are
located at the periphery of cell, while few grains are
found in the Golgi area (g). x 1,200.
P
Fig. 2. Lifht microscopic radioautograph 30 min after
injection of H-proline. In addition to localization of silver grains at the periphery of cell, numerous grains are
now observed in the Golgi area (g). Most of the grains
which appear to be over the extracellular matrix are
observed to be associated with cell processes upon close
examination (arrows). x 1,100,
Fig. 3. Light microscopic radioautograph 8 h after
injection of 3H-proline. The majority of silver grains are
located in the extracellular matrix close to cell processes
(arrows). A few silver grains are associated with the
Golgi area (9) and very few are located between cells.
x 1,350.
Of the mesenchymal collagen secreting
cells, the odontoblasts and osteoblasts are
clearly polarized with all collagen secretion
granules being released from that portion of
the cell in contact with predentin and osteoid, respectively. We recently showed that
PDL fibroblasts are also highly polarized and
that microtubules play a n important role in
collagen secretion from the distal end of the
cell, i.e., the end of the cell furthest away
from the nuclear pole (Cho and Garant,
1981b,c). We suggested that the distal cell
processes of normal PDL fibroblasts were
specialized for secretion of collagen precursors on the basis of the following morphologic
observations: 1) secretory granules labeled
with 3H-proline are formed in the Golgi com-
Fig. 4. Light microscopic radioautograph 2 days after
administration of 3H-proline. Silver grains deposited as
a loosely arranged band (arrows) are now pushed away
from the cell processes. Only a few silver grains are
observed over the Golgi area (g) and other portions of
cell. x 1,300.
Fig. 5. Light microscopic radioautograph 4 days after
administration of 3H-proline. Bands of silver grains (arrows) are pushed farther away as new unlabeled extra-
cellular collagen matrix (*) is formed between cell
processes and the silver grains. The cell body, including
the Golgi area (g),is free of silver grains. X 1,300.
Fig. 6. Light microscopic radioauto aphy of normal
H-proline.
' Silver
fibroblasts, after administration of P
grains are evenly distributed over the extracellular collagen matrix among the cells. Heavy labeling appears to
be associated with thin cytoplasmic processes (arrowheads) of the fibroblasts. x 1,280.
COLLAGEN SECRETION FROM 8-APN-TREATED FIBROBLASTS
Fig. 7. Electron microscopic radioautograph 3 min
after injection of 3H-proline. Most silver grains are located over RER, while the Golgi area (G) remains unlabeled. RBC, red blood cell. X12,600.
47
Fig. 8. Electron microscopic radioautograph 10 min
after administration of 3H-proline. The majority of silver
grains are still present over RER but labeling of the
Gold area (GI begins. Extravasation of red blood cells
(RBC) was a common finding in the PDL of 8-APNtreated animals. ~ 8 , 4 0 0 .
48
M.-I. CHO AND P.R. GARANT
COLLAGEN SECRETION FROM b-APN-TREATED FIBROBLASTS
plex and translocated in association with microtubules to the distal processes; 2) secretory
granules are found in higher concentration
in the distal processes than elsewhere in the
cell; 3) fusion of secretory granules with the
cell membrane of the distal cell processes
was occasionally observed; and 4) tritiated
proline-labeled collagen is visualized in secretory granules in distal cell processes and
the adjacent extracellular matrix a t 30 to 60
min after intravascular injection of the
radioisotope.
However, because of the apparent random
distribution of fibroblasts in the normal PDL
and the ramification of the distal cell processes, it was not possible to observe in radioautographs an “oriented” flow of secretory
product from the distal end of the cell (see
Fig. 6). The palisading of polarized fibroblasts in the PDL of 0-APN-treated animals
provided a situation where the deposition of
new collagen could be observed as a “band”
of radiolabeled matrix after injection of 3Hproline intravascularly. The most distinct
“bands” were located adjacent to the distal
cytoplasm at 24 h and were subsequently
pushed away during a 3-day observation period, as new collagen matrix continued to be
deposited from the distal ends of the cells.
This process appears similar to the formation
of osteoid in bone. Thus, the PDL fibroblasts
are functionally, as well as morphologically,
polarized. These results suggest that PDL
fibroblasts function like the odontoblasts and
osteoblasts, in that collagen precursors are
released from a specialized region of the cell.
Fig. 9. Electron microscopic radioautograph 20 min
after administration of 3H-proline. Increased labeling
over the Golgi area is observed. However, RER still
demonstrates heavy labeling. ~21,000.
Fig. 10. Electron microscopicradioautography 30 min
after injection of ‘H-proline. Numerous silver grains are
located in the Golgi area (Gj as well as over the rer.
Some silver grains are now associated with secretory
granules (psg; inset: high magnification of Golgi complex
labelled with arrow), secretory granules (sg), cell processes, and extracellular collagen fibrils (colj near the
cell processes. However, intercellular spaces (*) remain
relatively unlabeled. ~ 7 , 2 0 0inset,
;
x 17,680.
49
That this process is difficult to visualize in
normal PDL is due to the way in which the
cells are distributed in the matrix, the variation in the number and size of the distal cell
processes, and may be further obscured by
cell migration.
In comparing the radioautographs from
normal animals (Fig. 6) with those obtained
from the P-APN-treated animals (Figs. 3-5),
one may also conclude that the apparent random distribution of labeled newly secreted
collagen in normal PDL is not due to the free
diffusion of procollagen prior to its polymerization as collagen, but due to the underlying
organization of the cells. If procollagen was
able to diffuse freely within the extracellular
matrix of the PDL, one would not expect to
obtain the bands of newly labeled collagen as
shown in Figures 3-5. Furthermore, in PAPN-treated animals with a lesser potential
for cross-linking, one would expect higher
mobility of procollagen. That new collagen
polymerized a s a band adjacent to the distal
end of the fibroblast in /3-APN-treated animals is highly suggestive that new collagen
is also polymerized close to the secretory pole
of fibroblasts in the normal PDL. This observation supports our previous conclusion that
collagen fibrillogenesis occurs rapidly at the
distal cell process (Garant and Cho, 1979a,
Cho and Garant, 1981b,c). This concept is
also in agreement with the observations of
Trelstad and Hayashi (1975, 1979) that the
conversion of procollagen to collagen with
elongation of collagen fibrils occurs near the
fibroblastic cell surface, perhaps within a
deep cytoplasmic recess.
Comparison of the results of a previous
study of 3H-proline utilization by normal
PDL fibroblasts (Cho and Garant, 1981~)
with
those obtained in this study (Table 1) revealed a pattern of delayed translocation of
newly synthesized collagen through the 0APN-treated fibroblasts. Just as in normal
fibroblasts, the radiolabeled secretory product in @-APN-treated fibroblasts moved sequentially from RER to Golgi, to secretory
granule, to distal processes, and to the extracellular space. However, there was a slower
decline in labeling for each cytoplasmic compartment in the P-APN-treated group (Fig.
13).
At 30 min after 3H-proline injection, 55.2%
and 28.7% of all silver grains were superimposed over RER and Golgi in the 0-APNtreated group, while in normal cells the RER
50
M.-I. CHO AND P.R. GARANT
Fig. 11. Electron microscopic radioautograph 1 h after
administration of 'H-proline. Silver grains are observed
over 640 A banded extracellular collagen fibrils located
adjacent to cytoplasmic processes (arrows). The labeled
bundles of collagen fibrils are short in length and few in
number. G, Golgi area; M, mitochondria. X 13,500.
Fig. 12. Electron microscopic radioautograph 4 h after
administration of 'H-proline. Silver grains are still located over collagen fibrils adjacent to cell processes (arrows). However, labeled collagen fibrils are much shorter
in length and surrounded by disorganized abnormal collagen matrix (*I, which is also labeled. sg, Secretory
granules. x 13,500.
COLLAGEN SECRETION FROM (3-APN-TREATEDFIBROBLASTS
51
Fig. 13. Graphic representation of silver grain distribution over the normal and the P-APN-treated periodontal ligament at various time periods after injection of
3H-proline. ECC: extracellular matrix collagen fibrils;
Golgi: Golgi complex; RER rough endoplasmic
reticulum.
and Golgi counts were 29.2% and 36.8%.
These results indicate that 0-APN caused
slower release and translocation of secretory
product from the RER to the Golgi. These
observations reinforce our previous impression that fl-APN-treated fibroblasts contain
more RER and that their Golgi region contained a greater number of transitional elements of RER origin (Cho and Garant, 1983).
The slower translocation of secretory product
to the Golgi apparatus was also reflected in
the delay of collagen secretion denoted in
Figure 13. In normal PDL fibroblasts secretion of labeled, newly synthesized collagen
occurs approximately 30 min after injection
of 3H-proline (Cho and Garant, 1981~).Although some labeled collagen was secreted
from fl-APN-treated fibroblasts a t 30 min
after injection of the radioisotope, there was
much less. In fact, the analysis of radioautographs showed a 50% reduction of collagen
secretion at 1h in /3-APN-treated fibroblasts.
LITERATURE CITED
ACKNOWLEDGMENTS
This research was supported by the National Institute for Dental Research, National Institutes of Health, research grants
#ROlDE03745 and #ROlDE06165.
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beta, effect, periodontal, aminoproprionitrile, stud, radioautography, ligament, secretion, collagen, fibroblasts
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