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In vitro cell proliferation and proteoglycan synthesis of rabbit meniscal fibrochondrocytes as a function of age and sex.

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1010
IN VITRO CELL PROLIFERATION AND
PROTEOGLYCAN SYNTHESIS O F
RABBIT MENISCAL FIBROCHONDROCYTES
AS A FUNCTION OF AGE AND SEX
RICHARD J. WEBBER, TERESA ZITAGLIO, and AUBREY J. HOUGH, JR.
Meniscal fibrochondrocytes from male and female New Zealand white rabbits, ages 6, 12, and 24
months, were grown in primary and secondary
monolayer cell culture. Neither age nor sex affected the
majority of their cell culture characteristics. Cells from
young males (6 months old) synthesized greater amounts
of sulfated proteoglycans than did those from young
females, but by 2 years of age, this result was reversed.
All age groups synthesized 2 classes of proteoglycans,
based on hydrodynamic size, but the ratio of the 2
classes changed as a function of age. Overall, the
meniscal fibrochondrocytes from both skeletally immature and skeletally mature rabbits of both sexes were
capable of proliferation and matrix synthesis in vitro.
One of the most frequently performed orthopedic operations is knee joint arthrotomy with meniscectomy. The frequent and widespread use of this procedure was encouraged by early reports of successful
postoperative results (1-4). However, longer-term
studies indicate that, very often, there are complications after meniscectomy (5-12). The most frequent
and debilitating of such complications is precocious
osteo,arthritis in the joint (10,12,13). In an attempt to
_____
From the Musculoskeletal Research Laboratory, the Departments of Pathology and Biochemistry, University of Arkansas
for Medical Sciences, Little Rock.
Supported by NIH grant AM-34349-02.
Richard J. Webber, PhD: Departments of Pathology and
Biochemistry; Teresa Zitaglio, BA: Department of Pathology;
Aubrey J. Hough, Jr., MD: Department of Pathology.
Address reprint requests to Richard J. Webber, PhD,
Musculoskeletal Research Laboratory, Department of Pathology,
Slot 51’7, University of Arkansas for Medical Sciences, 4301 West
Markha,m, Little Rock, AR 72205.
Submitted for publication October 3, 1985; accepted in
revised form February 10, 1986.
Arthritis and Rheumatism, Vol. 29, No. 8 (August 1986)
avoid some of the morbid sequelae associated with
total meniscectomy, partial meniscectomy has recently come into vogue (1616). Experimental studies
in both dogs and rabbits (15,17) have demonstrated
that no articular changes are found in areas that remain
covered by meniscal tissue, although degenerative
changes are found in the articular cartilage areas
where meniscus has been removed. It would, therefore, be more desirable to devise a means of salvaging
the entire injured meniscus, rather than excising even
a portion of it. Several investigators have attempted to
encourage meniscal repair in situ (1&21), but the
consensus is that lesions of the meniscal fibrocartilage
cannot repair themselves except when they extend
into the peripheral, vascularized synovial attachment.
We have recently demonstrated that the failure
of the fibrocartilage to repair itself is not due to a lack
of intrinsic growth capabilities (22). In that study, we
showed that meniscal fibrochondrocytes were capable
of cell proliferation and synthesis of matrix molecules,
when given the proper stimuli. However, the cells
were isolated from the menisci of 6-month-old rabbits
that were skeletally immature. Thus, the fibrochondrocytes from such animals would be expected to be
mitotically and synthetically active, since they were
still contributing to the growth of the organ. As the
meniscus reaches its adult size and the animal begins
to mature, the fibrochondrocytes might, at least in
part, lose their ability to carry out those activities
needed for wound repair. It is known, for instance,
that the ability of central nervous system cells to
communicate with each other (23), the capacity to
launch an immune response (24), the cellular control
and biofeedback of bone cells (25), as well as protein
synthesis and function of transfer RNA in liver cells
AGE AND SEX EFFECT ON FIBROCHONDROCYTE CULTURE
(26,27) decrease as a function of age. Therefore, there
is a possibility that the ability of meniscal fibrochondrocytes to proliferate and synthesize matrix molecules in culture might also be an age-dependent process. This study was undertaken to determine whether
the age of the donor rabbit had any effect on the
phenotypic expression of fibrochondrocytes in cell
culture. The effect of sex on culture characteristics
was also studied.
MATERIALS AND METHODS
Chemicals, reagents, and media. The following materials were purchased: Gey’s balanced salt solution (GBSS)
and Dulbecco’s modified Eagle’s medium (DMEM; regular
and sulfate-free), from Gibco (Grand Island, NY);
hyaluronidase, lyophilized trypsin, and clostridial collagenase (CLS-II), from Cooper Biomedical (Malvern, PA);
NuSerum, from Collaborative Research (Lexington, MA);
and pituitary fibroblast growth factor (FGF), from Bethesda
Research Laboratory (Gaithersburg, MD). Penicillin/
streptomycin was purchased from Irvine Scientific (Santa
Ana, CA). All plastic cultureware was obtained from
Becton-Dickinson (Oxnard, CA). 3H-thymidine and
Na235S04(carrier free) were purchased from ICN Biochemicals (Irvine, CA). Sephacryl S-500 was purchased from
Pharmacia (Piscataway, NJ); CsCl, guanidinium chloride
(GuCI), sodium acetate (NaAc), and phenylmethylsulfonyl
fluoride (PMSF) were obtained from Sigma (St. Louis, MO).
EDTA, benzamidine hydrochloride, and aminohexanoic
acid were purchased from Aldrich (Milwaukee, WI).
Fibrocartilage and fibrochondrocyte isolation. Male
and female New Zealand white rabbits, 6, 12, and 24 months
of age, were killed by COz euthanasia. The knee joints were
exposed under aseptic conditions. The lateral and medial
menisci were surgically removed and placed in a beaker of
sterile GBSS. Extraneous tissue, such as adherent synovium
and joint capsule, was dissected away. The remaining
fibrocartilage was pooled and finely minced. The cells were
isolated according to the method of Green (28). Briefly, this
involved: (a) sequential treatment of the minced tissue with
0.05% hyaluronidase, 0.2% trypsin, and 0.2% clostridial
collagenase to release the cells from the tissue; (b) filtration
of the cells through a fine nylon mesh to separate them from
undigested tissue and debris; and (c) washing the cells
several times by gently suspending them in GBSS and
centrifuging the suspension at low speed (1,OOOg)to collect
the cells. The cells were then counted in a hemacytometer.
Monolayer cell culture. Primary cultures were started
by inoculating 5 x lo5 fibrochondrocytes into 75-cm2 Falcon
plastic culture flasks containing 12 ml of DMEM supplemented with 10% NuSerum and 0.1% penicillin/streptomycin (l0,OOO units and 10,000 pg/ml, respectively). Cultures were maintained at 37°C in a humidified atmosphere of
10% C02 and 90% air.
Secondary cultures were established from cells obtained by trypsin treatment of confluent primary cultures.
Fibrochondrocytes were inoculated into either 25-cm2 Falcon plastic culture flasks (2 x lo5 cells/flask) or 9.6-cm2
101 1
Falcon multiwell culture plates (1 x lo4 cells/well) containing DMEM supplemented as described above. The cells
were allowed to attach to the flask or well surface for 24
hours before any assay was begun.
Primary and secondary cultures were used in this
study because the cells had spent a relatively short period of
time in vitro and therefore had a greater probability of
retaining many of their in vivo phenotypic characteristics.
The relatively small number of cells obtainable from each
rabbit precluded using all primary cultures.
Measurement of population doubling time. The population doubling time was measured from the accumulation of
DNA in secondary culture flasks at 0,8, 16,24, and 32 hours,
beginning 1 day after initiation of cultures. To this end, a
total of 30 flasks were used. At each of the stated intervals,
6 flasks were harvested, and the DNA content of the
trypsinized cell pellet from each flask was determined by a
slight modification of Burton’s diphenylamine method (29).
Thus, each point on the growth curve from which the
doubling time was computed represented the mean of 2
groups of 3 culture flasks.
Response to pituitary fibroblast growth factor. The
growth activity of the fibrochondrocytes, with and without
24 hours of exposure to pituitary FGF (10 ng/ml) in secondary culture, was measured by the incorporation of 3Hthymidine into DNA. This concentration of FGF was used
because it has previously been shown to elicit a nearmaximal response with this cell type (22). Cells grown in
multiwell culture dishes, as described above, were labeled
for 1 hour with ’H-thymidine (5 pCi/ml, specific activity 64
CilmM). The wells were washed twice for 15 minutes with
chilled thymidine (W 5 M ) in GBSS and then hydrolyzed
with 1 ml of 0.1NNaOH. An aliquot (500 pl) from each well
was assayed for radioactivity by liquid scintillation spectrometry. The results were expressed as a “A %,” which
represents the percent change in the counts per minute of
treated cells compared with the control cells. Each value
represents the mean cpm of 6 wells.
35S04 incorporation. The effect of age and sex on
sulfated proteoglycan synthesis was measured by the incorporation of 3sS04.Secondary cultures (5 flaskdgroup) were
grown to confluence. The cultures were labeled for 20 hours
with 2 pCi of Na235S04/mlin DMEM (sulfate-free) with 10%
NuSerum and 0.1% penicillin/streptomycin. The amount of
radiosulfate incorporated into the medium and trypsin wash
in each culture flask was measured separately, as described
elsewhere (30). Results were expressed as mean cpm per
microgram of DNA. The percent radiolabel in the trypsin
wash was considered to represent newly synthesized
proteoglycan remaining in the pericellular coat.
Proteoglycan analysis. The hydrodynamic size distribution of the 35S04-labeled proteoglycans synthesized in
secondary culture by the fibrochondrocytes from each animal was determined by chromatography on Sephacryl S-500
after isopyknic CsCl density gradient ultracentrifugation
under dissociative conditions, as described in detail elsewhere (31). Briefly, this entailed: (a) collection of the culture
medium and addition (1 :1) of 4M GuCl plus proteinase
inhibitors (PI; 0.lM EDTA, 5 mM benzamidine HCI, 0.2M
6-aminohexanoic acid, and 1 mM PMSF); (b) exhaustive
dialysis against distilled water with PI; (c) lyophilization and
WEBBER ET AL
Table 1. Effect of age and sex on cell culture Characteristics of rabbit meniscal fibrochondrocytes*
Primary culture
llge
(mas.)/
sex
6/M
6/F
12/M
12/F
24M
24/F
Weight
(gm)
2,270
2,330
2,800
2,800
3,400
4,160
No.
cellsl4
menisci
Time
to
attach
( x ~ O - ~ ) (hours)
1.7
2.3
2.3
2.7
2.2
2.7
Time
to
spread
out
(days)
<24
<24
<24
<24
<24
<24
3
2
3
3
4
3
Secondary culture
"SO4 incorporation
(% in fraction)
Time to
confluence
(days)
10
9
12
11
11
10
PDT
(hours)
Response
to FGF
(A %)
Medium
19
19
18
16
17
16
48.8
44.4
27.7
66.6
62.2
20.1
1,980 ? 61.5 (87)
529 ? 10.0 (85)
1,263 2 60.9 (81)
901 ? 29.4 (81)
811 2 11.2 (82)
1,570 t 116.6 (77)
Trypsin
wash
298 2
95 2
304 2
207 +182 +482 2
10.4 (13)
3.5 (15)
17.3 (19)
7.9 (19)
23.9 (18)
23.9 (23)
* 35SC)4incorporation is expressed as the mean c p d p g of DNA 2 SEM (n = 5). PDT = population doubling time; FGF
factor; A % = percent change in treated cells versus control cells. Total = medium + trypsin wash.
reconstitution with dissociative CsCl buffer (4M GuCl in
0.1M NaAc, pH 5.8, plus PI; po [starting density of solution
at 21"CI = 1.50 g d m l ) ; and (d) centrifugationat 100,000g at
12°C for 48 hours in a Beckman L5-75 ultracentrifuge using
an SW-65Ti rotor. The resulting gradients were divided into
4 equal fractions and labeled D1 (most dense) through D4.
Alquots (200 jd) of the fractions were chromatographed on
Sephacryl S-500 and eluted with 4M GuCl in 0.1M NaAc, pH
5.8. The void volume (V,) and total volume (V,) of the
Sephacryl S-500column were determined with Blue Dextran
2000 and Na235S04,respectively. Column effluents were
assayed for radioactivity by liquid scintillation spectrometry.
RESULTS
Isolation of fibrochorildrocytes. Table 1 lists the
effect of age and sex on culture characteristics of 6
anim,als that were representative of the 18 animals
studied. The number of fibrochondrocytes obtained
from the 4 menisci of each rabbit ranged from 1.7 x lo6
to 2.'7 X lo', depending on the age and sex of the
animal. Menisci from females consistently yielded 4 x
lo5 to 6 x lo5 more cells than did those from males,
which generally meant that an additional primary
cultuire flask could be established with cells from
females.
Primary culture characteristics. Neither age nor
sex appeared to make any difference in the time
required for the majority of the cells to attach to the
surface of the culture flask (<24 hours), the time
requiired for the cells to flatten or "spreadout" once
attached ( 2 4 days), or the time necessary to reach a
confluent monolayer in primary culture (9-12 days)
(Table 1). Although there was some variation between
culture characteristics of the cells from different rabbits, all values were within the limits previously found
for 6-month-old females (22).
Total
=
2,274
624
1,567
1,108
944
2,051
t 69.1
20.9
74.9
34.4
t 15.2
t 138.5
2
2
2
fibroblast growth
Secondary culture characteristics. As in primary
culture, sex and age showed little effect on population
doubling time (16-19 hours) or proliferative response
to FGF (2046%) in secondary culture (Table 1).
However, incorporation of 3'S04 into sulfated proteoglycans in secondary culture appeared to be related to
both sex and age. Cells from 6-month-old males incorporated 4 times as much radiosulfate per pg of DNA
(2,274 cpm) as did those from females of the same age
(624 cpm). By 12 months of age, male cells incorporated only 1.5 times as much 3'S04 as did those from
females (1,567 cpm versus 1,108 cpm). By 24 months
of age, the situation had reversed, and cells from the
females incorporated twice as much radiosulfate as did
cells from the males (2,051 cpm versus 944 cpm). The
percent of 35S04-labeledproteoglycans retained in the
pericellular coat increased slightly as the age of the
animals increased.
Proteoglycan distribution and size. Overall, the
distributions of 35S radioactivity within the various
fractions of the CsCl density gradient were similar for
both sexes and all ages. Approximately 2630% radioactivity was found in the D1 fraction and also in the D4
Table 2. Density gradient distribution of 35S0,-labeled proteoglycans from New Zealand white rabbits, under dissociative
conditions
% %04radioactivity
Average
6 months old 12 months old 24 months old
density
Fraction (gm/mi) Male Female Male Female Male Female
D1
D2
D3
D4
1.62
1.52
1.45
1.39
30.1
22.3
21.9
25.7
29.5
21.2
21.2
28.1
24.7
23.7
23.7
28.0
24.2
23.5
22.9
29.5
24.1
23.1
24.0
28.9
34.5
21.2
16.5
27.8
1013
AGE AND SEX EFFECT ON FIBROCHONDROCYTE CULTURE
fraction, with the remainder being equally split between the D2 and D3 fractions (Table 2). One exception was the 24-month-old female, which had a slightly
higher percentage of the counts in the D1 fraction
(34%) and slightly less in the D3 fraction (16%). The
magnitude of the difference, however, was slight.
After chromatography on Sephacryl S-500under dissociative conditions, the newly synthesized
35S-labeledproteoglycans had similar elution profiles
for both sexes within all age groups. Figure 1 shows
the elution profiles of the D1 proteoglycans €or males
and females ages 6 months (Figure lA), 12 months
(Figure lB), and 24 months (Figure 1C). In all cases,
there were 2 major peaks having average partition
coefficients (KaJ of 0.24 and 0.56, which represent,
respectively, a relatively large and a relatively small
proteoglycan. Fibrochondrocytes from the 6-monthold rabbits synthesized both classes of proteoglycan in
approximately equal amounts. Cells from the l-yearold and 2-year-old rabbits synthesized mainly the
larger proteoglycan, in approximately a 2:l ratio to
synthesis of the smaller proteoglycan. The elution
profiles for the D2-D4 fractions (data not shown) were
more heterogeneous, but generally followed the same
pattern as those in the D1 fraction (Figure 1) and as
those seen in organ culture (31).
DISCUSSION
We have studied the effect of age and sex on the
culture characteristics of rabbit meniscal fibrochondrocytes. The ages (6, 12, and 24 months) were so chosen
because, with regard to the limbs, the youngest were
skeletally immature, the intermediate group had just
matured skeletally, and the oldest had been skeletally
mature €or some time (based on epiphyseal closure). It
would have been preferable to study even older animals, but most rabbit breeders kill animals as reproduction declines, and we have difficulty obtaining
older animals.
There are few published data regarding age
comparisons between humans and rabbits. However,
based on data published by Heikel (32), it can be
estimated that a 6-month-old rabbit would have the
approximate skeletal age of a 9-10-year-old human,
and a 12-month-old rabbit would have that of a 20year-old human. A 2-year-old rabbit is probably equivalent to about a 30-year-old human. By the age of 4
years, rabbits are considered to be “aged” (33).
Although it is of little consequence in the context of this particular paper, the number of cells
obtained from the menisci of the animals should be
noted. The 6-month-old animals of both sexes yielded
fewer cells than did the older animals, probably because they were not yet skeletally mature and their
menisci had not yet attained adult size. Female
12
8
4
0
B
12
8
4
0
li
I
L
I
1 28
V.
I
I
38
40
,
I
VI
I
58
FRACTION NO.
Figure 1. Sephacryl S-500 chromatography of fibrochondrocyte
proteoglycans from New Zealand white rabbits at ages 6 months (A),
12 months (B), and 24 months (C). Secondary cultures were grown
to confluence and labeled with 35S04as detailed in Materials and
Methods. After CsCl isopyknic density gradient ultracentrifugation,
the D1 fractions were chromatographedon Sephacryl S-500, and the
35Sradioactivity of the eluent was measured by liquid scintillation
spectrometry. The average partition coefficient (Kay) of the first
peak is 0.24, that of the second peak is 0.56. Note the increased ratio
of the first peak to the second peak as the age of the animal
increases. Vo = void volume; VT = total volume.
1014
rabbits consistently yielded more cells than did male
rabbits. This is an interesting corollary because the
organs of female rabbits are larger than those of male
rabbits of the same age (33-35). Thus, if one is
concerned with obtaining the maximum number of
fibrochondrocytes, it is advisable to use mature female
rabbits.
Overall, the innate capability of rabbit meniscal
fibrochondrocytes to proliferate and synthesize matrix
macromolecules appears to be a property of skeletally
mature, as well as skeletally immature, animals of both
sexes. No discernible differences in primary culture
characteristics could be found between the sexes or at
any age of the animals studied. The growth activity in
secondary culture also seemed to be affected little by
sex or age. Although at first glance, the population
doubtling time appeared to decrease slightly with age,
this is probably artefactual, since in previous studies
(22), we found the population doubling time to be as
short as 15 hours for 6-month-old fibrochondrocytes
grown under similar conditions. Similar variations in
population doubling time have been found with articular chondrocytes in monolayer culture (36). Likewise., the differences in response to FGF did not seem
to be substantial. Although the response ranged between 20.1% and 66.6% among the various animals,
there was no sex- or age-based pattern. Therefore, the
differences are most likely the result of biologic variation between animals. Again, other investigators
have found a similar range of responses to FGF in
articular chondrocyte cultures (37).
Unlike growth activity, substantial differences
in raidiosulfate incorporation were found between
males and females at the different ages. The differences between the sexes at any age could be interpreted in at least one of two ways: (a) one sex was
producing greater quantities of sulfated proteoglycans
per clell than the other produced, or (b) the amount of
proteoglycan produced by both sexes was equal, but
the proteoglycans of one sex were more highly sulfated than were those of the other. The latter explanation appears untenable in view of the data presented in
Table 2.
It is known from the work of Ito et a1 (38) that
the buoyant density of a proteoglycan is directly
proportional to its degree of sulfation. Therefore, if
within any 1 age group the proteoglycans of one sex
were more highly sulfated than those of the other,
there should be a proportionate increase in the buoyant density of their proteoglycans. This, however, was
not tlhe case. Fibrochondrocytes from 6-month-old
males incorporated 4 times as much 35S04than did
WEBBER ET AL
those of females of that age, yet the percentage of
sulfated proteoglycans in any particular fraction of the
density gradient was practically indistinguishable between the sexes (Table 2). Even in the 24-month-old
group, in which 10% more of the proteoglycans of the
females were contained in the D1 fraction, this could
not explain a twofold increase in their 3sS04incorporation. Thus, the most plausible explanation is that the
increased radiosulfate incorporation represents increased proteoglycan synthesis.
It is not quite clear why males produce more
proteoglycans than females at the younger ages and
then produce less as they mature. It is possible that the
reversal is caused by changes in hormonal homeostasis over time. It is not simply due to pubertal changes,
however, since this phenomenon occurs in rabbits at
approximately 4 months of age (39), which is quite
some time before the changes noted in this study.
All rabbits synthesized 2 classes of proteoglycans, based on hydrodynamic size. The proteoglycans
of males and females exhibited similar elution profiles;
however, synthesis of the smaller proteoglycan decreased (compared with the larger proteoglycan) as the
age of the animals increased. Thus, although the cells
from both younger and older animals are equally
capable of synthesizing proteoglycans, the major type
of proteoglycan synthesized varies with age. It is
possible that the smaller proteoglycan represents a
more immature type of proteoglycan, the synthesis of
which decreases as the animal ages. This, however,
would be in direct contrast to articular cartilage
proteoglycans of several species, in which the hydrodynamic size decreases with age (40).
For meniscal repair to occur, subsequent to an
injury, cell proliferation and matrix synthesis must
take place adjacent to and within the lesion. These
processes are believed to be initiated in vivo by
endogenous modulators, which are termed “competence factors” (e.g., FGF and platelet-derived growth
factor) and ‘‘progression factors” (e.g., somatomedinC), that are found in serum (41,42). For reasons that
are obscure at present, meniscal fibrochondrocytes do
not, in most instances, initiate a reparative response.
This might be due to the tissue’s avascularity (43) and
a consequent lack of contact between the cells and
these serum factors (22). However, despite the several
differences found between males and females and
between animals of different ages, this study demonstrates that the fibrochondrocytes from both skeletally
immature and skeletally mature rabbits retain their
ability to proliferate and synthesize matrix macromolecules when stimulated with progression factors and
AGE AND SEX EFFECT ON FIBROCHONDROCYTE CULTURE
competence factors. This indicates that even the menisci of skeletally mature individuals are innately
capable of initiating a reparative response and that
conditions exogenous to the cell prevent wound
healing.
We have previously demonstrated that although
FGF stimulates DNA synthesis in meniscal fibrochondrocytes, it decreases total 35S04 incorporation
when these cells are grown in 10% fetal bovine
serum-containing culture medium (22). This effect is
similar to that seen under equivalent conditions with
rabbit articular chondrocytes (unpublished data) and
growth plate chondrocytes (30). On the other hand,
Prins et a1 (44) have shown that in 1% fetal bovine
serum-containing culture medium, FGF increases
35S04 incorporation and DNA synthesis by rabbit
articular chondrocytes. It therefore appears that the
chondrocyte’s response to 1 growth factor may be
dependent on the concentration of other serumderived factors (e.g., platelet-derived growth factor) in
the extracellular milieu. The reparative response in
wound healing would then appear to be finely tuned by
the amount and type of growth factors present. Thus,
the various factors necessary for modulating the phenotypic expression of the meniscal fibrochondrocyte
should be sought, so that it may be possible someday
to stimulate, in situ, meniscal repair and obviate the
necessity of even a partial meniscectomy.
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sex, proteoglycans, synthesis, rabbits, proliferation, age, fibrochondrocytes, function, meniscal, vitro, cells
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