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The effect of ascorbic acid on the growth of chick bone rudiments in chemically defined medium
Experimental
158
THE GROWTH
EFFECT
OF CHICK
OF ASCORBIC BONE
Strangeways
PREVIOUS
Research
42, 178-188
(1966)
ON THE IN CHEMICALLY
MEDIUM
REYNOLDS
Research Laboratory, Cambridge, U.K. Received
ACID
RUDIMENTS
DEFINED J. J.
Cell
October
Wads
Causeway,
4, 1965
studies have shown that cartilaginous bone rudiments from embryonic chicks, when cultured in chemically defined media, become excessively hydrated after about four days in vitro [2, 6, 25, 291. This phenomenon has been related to either cellular degeneration [2], or the synthesis of an abnormal extracellular matrix [25]. It has also been shown that it can be retarded by the addition of hydrocortisone to the medium [6, 25, 291, but that the hormone also inhibits growth even at levels below that necessary to prevent hydration [25]. Although the hydration can be retarded by the steroid, the composition of the extracellular matrix synthesised by the rudiment is still abnormal, with a much higher ratio of polysaccharide/collagen than in corresponding material ez ouo [25]. A continued search has been made for compounds that would maintain the normal ratio of the components of the matrix and thus possibly prevent the terminal hydration. It has long been accepted that ascorbic acid deficiency reduces collagen synthesis in viuo, and a number of reviews on this subject have appeared [14, 15, 271. Hitherto the results of testing the effect of ascorbic acid on collagen production in tissue culture systems have been equivocal [15, 261, but recently Jeffrey and Martin [ 191 conclusively showed that the addition of the vitamin (50 pg/ml) to defined medium in which 8-g-day chick tibiae were growing, much increased the collagen content of the rudiments; without ascorbic acid collagen synthesis ceased after about four days in uitro. The present paper records observations on the effect of ascorbic acid in chemically defined medium on explanted bone rudiments from 7-day chick embryos. The results confirm those of Jeffrey and Martin [19]; in addition to collagen synthesis, the production of polysaccharide and the effect of ascorbic acid on the hydration of the explants have been studied. Since this work began Experimental
Cell Research
42
Ascorbic
acid and chick bone rudiments
179
two reports on the effect of ascorbic acid on cell cultures have appeared [16, 301, both of which have shown that ascorbic acid greatly stimulates the synthesis of collagen. MATERIALS
AND
METHODS
Explants.-Paired sets of three bone rudiments from 7-day-old chick embryos were used; usually two sets, each containing tibiotarsus, femur and humerus, were taken from each chick. The selection of suitable embryos and the isolation of the explants have been described previously [25]. Culture technique.-The culture dishesand conditions of incubation were similar to those described by Fell and Weiss [12] with minor modifications. The disheswere incubated at 38.5”C. The chemically defined medium, which was changed every second day, was similar to that used in earlier work [25], except that the concentration of phosphate was halved. For the experiments with ascorbic acid, the vitamin (British Drug Houses) was dissolved in the medium, usually at a concentration of 50 yg/ml, before sterilisation by passingthrough a Millipore filter. Morphology and measurement of growth.-The lengths of the explanted rudiments were measured at the beginning and end of each experiment. Wet and dry weights were determined at the end of the experiment by methods previously described [25]. Burton’s modification of the diphenylamine reaction [5] was used to estimate the DNA content of the rudiments. Composition of extracellular matrix.-The total amount of hydroxyproline in sets of rudiments and their culture media were determined asbefore [25]. The hexosamine content of the explants and media were estimated as previously described, except that all hydrolyses were carried out for 4 hr at 100°C with 4.5 N hydrochloric acid. Histology.-Rudiments were fixed for 45 min in Zenker’s fluid containing 3 per cent glacial acetic acid followed by 1 hr in Zenker’s fluid alone. Serial sections were cut from paraffin blocks at a thickness of 7 ,Uand stained either with toluidine blue [12] or with celestine blue, Mayer’s acid haemalum and Van Gieson’s stain. RESULTS
Effect
of
ascorbic acid on the growth of embryonic chick bones in uitro
Sets of 7-day-old chick bone rudiments were cultured either in chemically defined medium alone, or in medium to which ascorbic acid at a concentration of 50 ,ng/ml of medium had been added. The usual culture period was 6 days, but in a few experiments explants were also grown for 4 and 8 days. Growth in weight.-The wet and dry weights of paired sets of chick bone rudiments cultured with and without ascorbic acid for various periods of time are presented in Table I. For an in vitro period of 4 days or less, ascorbic acid has little effect on either the wet or dry weights of the rudiments. The data for 6 or 8 days’ growth in vitro show that explants without the vitamin 12*-681803
Experimental
Cell Research
42
J. J. Reynolds
180
have progressively higher wet weights and lower dry weights than paired rudiments with ascorbic acid. To illustrate this difference the ratio of the dry weight/wet weight of sets of rudiments grown for various periods of time are shown graphically in Fig. 1; the data recorded in this figure refer to one experiment only in which as nearly identical sets of rudiments as possible TABLE
various
I. Wet and dry weights of sets of chick bone rudiments grown for times in either synthetic medium alone, A, or in synthetic medium with 50 pglml ascorbic acid, B. A Wet
Days in vifro
weight m3
B Dry
weight mi3
Wet
weight mg
Dry
weight mg
4 days
9.2 11.3
0.86 1.11
9.2 11.4
0.87 1.12
6 days
15.6 16.7 16.8
1.25 1.41 1.34
14.1 15.3 15.3
1.42 1.64 1.59
8 days
28.7 24.9 22.4
1.53 1.41 1.39
18.5 20.1 19.1
2.03 1.93 1.90
were cultured for the period of time indicated. After 8 days in vitro the dry weight/wet weight ratio of explants with ascorbic acid is twice that of controls lacking the vitamin, and is similar to that of normal rudiments ez ovo [4, 251. Growth in size.-Bone rudiments cultured in medium with ascorbic acid for 6 days attain a length 10 per cent above that of paired explants in synthetic medium [25]; their morphology more nearly resembles that of rudiments e2 ovo, and the abnormal swelling of the epiphyses that is usually observed without the vitamin is prevented. Growth in DNA content.-No significant differences were observed between the DNA contents of sets of rudiments cultured for 6 days in the presence or absence of ascorbic acid, and the values obtained are similar to those previously reported for explants in defined medium for 6 days [25]. After 8 days in vitro, the DNA content of ascorbic acid-treated rudiments is usually slightly higher than that of untreated controls (lo-15 per cent in three experiments) but more experiments are needed to establish the significance of this difference. Experimental
Cell
Research
42
Ascorbic The effect of ascorbic chick bones in vitro
acid and chick
acid on the extracellular
bone rudiments
181
components
of embryonic
Hydroxyproline content.-Sets of rudiments were cultured for 6 days in either chemically defined medium alone, or in medium to which 50 ,ug/ml ascorbic acid had been added; their contents of hydroxyproline are given in
0
I a
I Days
Fig. I.-Percentage times in synthetic
dry weight/wet medium alone
I 6
I a
1”’ culture
weight of sets of chick bone rudiments grown (---), and in medium with 50 pg/ml ascorbic
for acid
various (-).
Table II together with values for the hydroxyproline containing material released into the medium during the culture period. The corresponding values for two sets of rudiments grown in vitro for 4 and 8 days respectively are also recorded. The table clearly shows that the formation of hydroxyproline containing material is greatly increased by the addition of ascorbic acid, and that in the absence of the vitamin there is little synthesis of this material after 4 days in vitro. These observations may be correlated with the onset of hydration between the 4th and 6th days, reported above. Hexosamine content.-The hexosamine contents of rudiments cultured for 6 days in vitro are not significantly different whether ascorbic acid is present or not [25]; only small amounts of such material are found in the culture media in either case. After eight days’ growth, the hexosamine content of explants cultured without the vitamin is lower (approx. 15 per cent) than in those grown with ascorbic acid. Experimental
Cell
Research
42
J. J. Reynolds
182
Ratio of collagen/hexosamine.-The percentage of dry weight that is hexosamine-containing material or collagen is given in Table III, in which data are shown for rudiments cultured for six days in vitro with and without ascorbic acid. The ratio of 2.0 for collagen/hexosamine is similar to that of embryonic bones ex ovo [25], and clearly demonstrates that ascorbic acid II. Hydroxyproline content of chick bone rudiments cultured for various times in synthetic medium alone, A, and medium with ascorbic acid, 50 ,uglml, B.
TABLE
Days in vitro
A Hydroxyproline
4 days
Explants Medium
9.9 9.8
6 days
Explants Medium
13.8 15.8
8 days
Explants Medium
11.4 16.1
B Hydroxyproline
(pg)
(pg)
12.5 7.8 12.6 15.4
11.2 14.8
31.0 19.1
31.5 11.0
30.1 12.4
38.0 27.4
is necessary for the maintenance of a normal ratio of extracellular components. Time of addition of ascorbic acid.-The data recorded in Tables I and II show that during the first 4 days in vitro the addition of ascorbic acid has little effect on the composition of the matrix; this probably indicates that the explants’ endogenous supply lasts for about four days [see also ref. 191. After six days in vitro a progressive hydration has begun in the rudiments without ascorbic acid and their collagen content is abnormally low, As expected, when explants are cultured for 2 or 4 days without the vitamin and the medium is then replaced with medium containing ascorbic acid, at 6 or 8 days the weights and collagen contents of the explants are similar to those of rudiments grown continuously in medium containing the vitamin. When explants cultured for 6 days in vitro without ascorbic acid, which have become hydrated in appearance, are changed to ascorbic acid containing medium, synthesis of collagen is resumed and the hydration retarded; at eight days such rudiments have a collagen content (24 ,ug OH-proline) and a dry weight/wet weight ratio (7.2) intermediate between those observed for cultures grown with and without ascorbic acid for eight days. It seems possible that a further period of cultivation with ascorbic acid might lead Experimental
Cell Research
42
Ascorbic
acid and chick bone rudiments
183
to, a complete reversal of the hydration noted at 6 days, but it is unlikely that the morphology (large swollen epiphyses) would be improved. Nature and distribution of hydroxyproline-containing material.-It has been assumed above that the hydroxyproline containing material indicates collagen production. Although usually the determinations were made on III. Percentage composition of extracellular components of chick bone rudiments cultured for six days in synthetic medium alone, A, and medium with 50 ,ug/ml ascorbic acid, B.
TABLE
A
B
7.5
7.1
7.1
14:2
0.9
2.0
Hexosamine (% of dry
weight)
Collagen’ (% of dry
weight)
Collagen/Hexosamine a Collagen
calculated
from
hydroxyproline
x 7.46.
total hydrolysates of rudiments, some parallel cultures were extracted with 5 per cent trichloracetic acid (at 4°C) before hydrolysis. These experiments show that the amount of free hydroxyproline present in the rudiments is never more than 5 per cent of the total. No further attempts to fractionate the collagen have yet been made. The fact that the addition of ascorbic acid to synthetic medium both increases collagen synthesis and inhibits hydration of the rudiments, suggests that the two effects may be correlated, but before this possibility is seriously considered it is necessary to establish that the increase in collagen occurs throughout the explants. Ascorbic acid-treated rudiments have a thicker ossified collar (see below for histology), so that it was just possible that the increase in collagen was related solely to this ossified layer. To investigate this point, rudiments were cultured for 8 days in the presence of ascorbic acid, dried, and cut into three parts, roughly corresponding to the (ossified) diaphyseal and two (unossitied) epiphyseal regions; the diaphyseal and epiphyseal fragments were collected in two pools. Hydroxyproline determinations on these pools show that about 85 per cent of this material is associated with the epiphyseal fraction; similar proportions were observed in corresponding material ex ouo, so it may be conclused that the increased collagen synthesis is not confined to the diaphyseal region. Experimental
Cell Research
42
.I. J. Reynolds
184
Fig. 2.-Zone of flattened medium for 6 days. Note Fig. ?I.-Zone medium with
cells in the epiphyseal the bubbly appearance
of flattened cells in the epiphyseal 50 pg/ml ascorbic acid for 6 days;
Fig. I.-Diaphyseal the thin ossified
region of a 7-day of the matrix. region of a 7-day the matrix appears
region of a ‘I-day chick femur grown collar. Celestin blue, Mayer’s haemalum
Fig. 5.-Diaphyseal region of a 7-day chick femur ascorbic acid for 6 days. Note ossified collar and haemalum and van Gieson’s stain, x 100. Experimental
Cell
Research
42
chick femur grown in synthetic Toluidine blue, x 540. chick femur grown normal. Toluidine
in synthetic blue, x 540.
in synthetic medium for 6 days. and van Gieson’s stain, x 100.
grown in synthetic medium hypertrophied cells. Celestin
Note
with 50 ,ug/ml blue, Mayer’s
Ascorbic Histology
of rudiments
acid and chick bone rudiments
cultured
in the presence of ascorbic
185 acid
The histological appearance of chick bone rudiments cultured in chemically defined medium has been described previously [25] and agrees with the accounts of Biggers [3] and Schryver [29] for explants grown in medium BGJ. Briefly, the rudiments appear to have a sparse foamy matrix (Fig. 2) and in all areas the cells lie in swollen lacunae; the hypertrophy that normally occurs in the cells of the diaphyseal region (Fig. 4) at this stage of development [lo] does not advance very much in culture. Addition of ascorbic acid to the synthetic medium greatly increases the intensity of the metachromasia throughout the rudiments (Fig. 3) and the matrix has a dense structure similar to that of bone rudiments ex ouo; in appearance the matrix resembles that of rudiments treated with hydrocortisone [6, 25, 291. The cells of the diaphyseal region undergo more hypertrophy than control cultures without the vitamin (contrast Fig. 4 and Fig. 5) and the ossified collar becomes considerably thicker. DISCUSSION
Previous reports [2, 6, 25, 291 have shown that chick bone rudiments cultured in chemically defined media lacking ascorbic acid become excessively hydrated after a short period in uitro. The results described in this paper have demonstrated that the addition of 50 ,ug/ml of ascorbic acid to synthetic medium completely inhibits this terminal hydration. Unlike hydrocortisone which also prevents hydration [6, 25, 291, ascorbic acid does not inhibit .growth and produces a more normal morphology of the explants. Unpublished experiments by the present author indicate that concentrations of ascorbic acid below 50 ,ug/ml do not completely inhibit the hydration. Jeffrey and Martin [ 191 have described the rapid decrease in collagen synthesis of B-g-day chick tibiae grown in vitro without ascorbic acid. From their results it appeared that collagen synthesis ceased after about 4 days unless the vitamin were present; normal synthesis of collagen could be restored upon addition of ascorbic acid to the depleted cultures. These observations are confirmed by the closely similar results on 7 day bone rudiments reported in this paper; here again, collagen synthesis decreased rapidly after the fourth day in vitro without ascorbic acid, and could be restored by addition of the vitamin. Jeffrey and Martin [19] found that other cellular activities were relatively less impaired; the growth measurements recorded in this paper agree well with their observations, since no difference in DNA or hexosamine content could be measured in rudiments cultured Experimental
Cell
Research
42
186
J. J. Reynolds
for 6 days, whether ascorbic acid was present or not. Only a small depression in the synthesis of either DNA or hexosamine was noted in rudiments cultured for 8 days without the vitamin. In a previous publication [25] the present author suggested that the terminal hydration of chick bone rudiments cultured in synthetic medium was correlated with the synthesis of an abnormal extracellular matrix which was shown to be low in the ratio of collagen/hexosamine. This view is supported by the present observations that the addition of ascorbic acid to the medium produces a more normal ratio of collagen/hexosamine and at the same time completely prevents the abnormal uptake of water; the increased collagen synthesis takes place throughout the rudiments. The addition of the vitamin to already hydrated cultures reduces the hydration, concurrently with resumption of collagen synthesis. On the other hand experiments in which hydrocortisone was added to rudiments at different times after explantation showed that the hormone does not affect the hydration once it has occurred [see also refs. 25 and 291; this is to be expected since the steroid causes no significant increase in collagen synthesis, and it was thought to inhibit hydration mainly by depressing polysaccharide synthesis [25]. It seems probable that the water of hydration is associated in some way with the chondromucoprotein of the matrix, and that the abnormal hydration cannot take place if the polysaccharide is associated with a normal amount of collagen. The histological appearance of rudiments grown with and without ascorbic acid is consistent with the interpretation of the biochemical data presented above. The abnormal foamy matrix of explants cultivated without the vitamin is in sharp contrast to the more normal material synthesised in the presence of ascorbic acid, in which the metachromasia is increased throughout the rudiments. Hydrocortisone-treated explants show a similar improvement in metachromasia [6, 251; the diaphyseal cells are abnormally small, however, and not much periosteal bone is formed [ll, 311, whereas the diaphyseal cells of ascorbic acid-treated rudiments hypertrophy more than those of untreated controls and the ossified collar increases in thickness considerably during cultivation. A recent survey by Fabro and Rinaldini [9] of the ability of various embryonic cells of the chick to synthesise ascorbic acid, showed that many tissues quickly lose this capacity and the loss comes at different times during embryonic life depending on the particular organ. This is interesting in relation to the observation that collagen synthesis is stimulated in embryonic bones of the chick by exogenous ascorbic acid, and might explain some previous Experimental
Cell Research
42
Ascorbic
acid and chick bone rudiments
187
reports of lack of stimulation in other fetal tissues [ 151. It is also interesting to note that some of the cell lines used by Green and Goldberg [ 161 synthesised appreciable amounts of collagen without the addition of ascorbic acid, and might indicate that some cells in culture are able to form the vitamin. The most recent evidence seems to suggest that ascorbic acid functions in the hydroxylation of proline to hydroxyproline [23], but it may also have activities in the cells less directly related to synthesis of extracellular material
WI. The conflicting evidence for the effect of ascorbic acid on polysaccharide synthesis in vivo has been reviewed recently [15, 26, 271. Some authors claim that sulphation diminishes in ascorbic acid deficiency [20, 241 but Friberg [ 131 noted no change in sulphation in healing wounds of scorbutic animals. Hughes and Kodicek [18] found that the concentration of galactosamine-containing polysaccharides was much lower in scorbutic than in normal granulomas from pair-fed animals. Studies on cell cultures in vitro have been hampered by the fact that cell cultures, unlike organ cultures, rarely continue to synthesise their tissue specific mucopolysaccharide but seem to secrete hyaluronic acid instead [15, 17, 22, 301. In the organ culture system employed in the present investigation, there was a small depression of polysaccharide synthesis in rudiments grown without ascorbic acid, but only after 8 days in vitro, which indicates that the effect may be secondary. The sulphation of the polysaccharide synthesised by chick bone rudiments cultured either in synthetic medium [7], or partly natural medium [8] is normal. Unpublished experiments [ 1 ] have shown that the ratio of galactosamine/glucosamine in 7-day chick bone rudiments cultured for 6 days was approximately 20: 1 whether the vitamin was present or not, and a similar ratio for rudiments ex ovo was observed. So far there is little evidence to indicate that ascorbic acid has any effect on the mucopolysaccharides synthesised by chick bone rudiments in vitro, though it is of course possible that a low level of ascorbic acid remains in the rudiments, which is sufficient to support normal mucopolysaccharide synthesis after the formation of collagen has ceased. A similar conclusion that ascorbic acid has little effect on the synthesis of sulphated mucopolysaccharides in vivo has been reached by Kofred and Robertson [al]. SUMMARY
The effect of the addit ion of ascorbic acid (50 fig/ml) to synthetic medium has been investigated in 7-day chick limb bone rudiments in culture. The Experimental
Cell Research
42
J. J. Reynolds
188
vitamin completely prevented the terminal waterlogging usually observed in explants grown in chemically defined medium. Ascorbic acid had little effect on the DNA content or synthesis of hexosamine-containing material, but the rudiments attained a higher dry weight and a lower wet weight than untreated controls; part of the increased dry weight was due to a greatly stimulated synthesis of collagen. It is suggested that the inhibition of hydration is correlated with the production of a normal extracellular matrix under the influence of ascorbic acid. Grateful acknowledgement is made to Professor Dame Honor Fell, F.R.S., for her advice and encouragement. This work has been generously supported by funds from the Nuffield Foundation.
REFERENCES 1. BARRETT, 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21.
22. 23. 24.
25. 26. 27. 28. 29.
30. 31.
A. J. and REYNOLDS, J. J., Unpublished observations. J. D., Nat1 Cancer Inst. Monograph, No. 11, 1 (1963). J. D., J. Expfl Zool. 144, 233 (1960). J. D., LAWSON, K. A., LUCY, J. A. and WEBB, M., Biochim. Biophys. Acta 54, . I 236 (1961). BURTON, K., Biochem. J. 62, 315 (1956). DINGLE, J. T., FELL, H. B. and LUCY, J. A., Biochem. J. 98, 173 (1966). DINGLE, J. T., SLEDGE, C. B. and BARRETT, A. J., In preparation. ENDO, H. and MUROTA, S., Abstracts of Tissue Culture Seminar, Baroda, India, Jan., 1965. FABRO, S. P. and RINALDINI, L. M., Deuelop. BioI. 11, 468 (1965). FELL, H. B., J. Morphol. 40, 417 (1925). FELL, H. B. and THOMAS, L., J. Expfl Med. 114, 343 (1961). FELL, H. B. and WEISS, L., J. Exptl Med. 121, 551 (1965). FRIBERG, U., Arkio Kemi 12, 501 (1958). GOULD, B. S., Ann. N.Y. Acad. Sci. 92, 168 (1961). ~ Intern. Reu. Cyfol. 15, 301 (1963). GREEN, H. and GOLDBERG, B., Proc. Sot. Exptl Biol. Med. 117, 258 (1964). GREEN, H. and HAMERMAN, D., Nature 201, 710 (1964). HUGHES, R. E. and KODICEK, E., Biochem. J. 77, 3P (1960). JEFFREY, J. J. and MARTIN, G. R., Abstracts of the 6th International Congress of Biochemistry, New York, July 1964; Collagen Currents 5, 114 (1964). KODICEK, E. and LOEWI, G., Proc. Roy. Sot. London 144B, 100 (1955). KOFRED, J. A. and ROBERTSON, W. VAN B., Fed. Proc. 24, 354 (1965). MORRIS, C. C., Ann. N. Y. Acad. Sci. 86, 878 (1960). PETERKOFSKY, B. and UDENFRIEND, S., Proc. Nat1 Aead. Sci. U.S. 53, 335 (1965). REDDI, K. K. and NORSTROM, A., Nafure 173, 1232 (1954). REYNOLDS, J. J., Exptl Cell Res. In press. RINALDINI, L. M., in E. N. WILLMER (ed.), Cells and Tissues in Culture, Vol. 1, p. 680. Academic Press, London and New York, 1965. ROBERTSON, W. VAN B., Ann. N. Y. Acad. Sci. 92, 159 (1961). SCHNEIDER, W. and STAUDINGER, HJ., Biochim. Biophys. Acta 96, 157 (1965). SCHRYVER, H. F., Expil Cell Res. 37, 327 (1965). SCHIMIZU, Y., MCCANN, D. S. and KEECH, M. K., J. Lab. Clin. Med. 65, 286 (1965). SOBEL, H. and FREUND, O., Experientia 14, 421 (1958). BIGGERS, BIGGERS, BIGGERS,
Experimental
Cell Research
42
158
THE GROWTH
EFFECT
OF CHICK
OF ASCORBIC BONE
Strangeways
PREVIOUS
Research
42, 178-188
(1966)
ON THE IN CHEMICALLY
MEDIUM
REYNOLDS
Research Laboratory, Cambridge, U.K. Received
ACID
RUDIMENTS
DEFINED J. J.
Cell
October
Wads
Causeway,
4, 1965
studies have shown that cartilaginous bone rudiments from embryonic chicks, when cultured in chemically defined media, become excessively hydrated after about four days in vitro [2, 6, 25, 291. This phenomenon has been related to either cellular degeneration [2], or the synthesis of an abnormal extracellular matrix [25]. It has also been shown that it can be retarded by the addition of hydrocortisone to the medium [6, 25, 291, but that the hormone also inhibits growth even at levels below that necessary to prevent hydration [25]. Although the hydration can be retarded by the steroid, the composition of the extracellular matrix synthesised by the rudiment is still abnormal, with a much higher ratio of polysaccharide/collagen than in corresponding material ez ouo [25]. A continued search has been made for compounds that would maintain the normal ratio of the components of the matrix and thus possibly prevent the terminal hydration. It has long been accepted that ascorbic acid deficiency reduces collagen synthesis in viuo, and a number of reviews on this subject have appeared [14, 15, 271. Hitherto the results of testing the effect of ascorbic acid on collagen production in tissue culture systems have been equivocal [15, 261, but recently Jeffrey and Martin [ 191 conclusively showed that the addition of the vitamin (50 pg/ml) to defined medium in which 8-g-day chick tibiae were growing, much increased the collagen content of the rudiments; without ascorbic acid collagen synthesis ceased after about four days in uitro. The present paper records observations on the effect of ascorbic acid in chemically defined medium on explanted bone rudiments from 7-day chick embryos. The results confirm those of Jeffrey and Martin [19]; in addition to collagen synthesis, the production of polysaccharide and the effect of ascorbic acid on the hydration of the explants have been studied. Since this work began Experimental
Cell Research
42
Ascorbic
acid and chick bone rudiments
179
two reports on the effect of ascorbic acid on cell cultures have appeared [16, 301, both of which have shown that ascorbic acid greatly stimulates the synthesis of collagen. MATERIALS
AND
METHODS
Explants.-Paired sets of three bone rudiments from 7-day-old chick embryos were used; usually two sets, each containing tibiotarsus, femur and humerus, were taken from each chick. The selection of suitable embryos and the isolation of the explants have been described previously [25]. Culture technique.-The culture dishesand conditions of incubation were similar to those described by Fell and Weiss [12] with minor modifications. The disheswere incubated at 38.5”C. The chemically defined medium, which was changed every second day, was similar to that used in earlier work [25], except that the concentration of phosphate was halved. For the experiments with ascorbic acid, the vitamin (British Drug Houses) was dissolved in the medium, usually at a concentration of 50 yg/ml, before sterilisation by passingthrough a Millipore filter. Morphology and measurement of growth.-The lengths of the explanted rudiments were measured at the beginning and end of each experiment. Wet and dry weights were determined at the end of the experiment by methods previously described [25]. Burton’s modification of the diphenylamine reaction [5] was used to estimate the DNA content of the rudiments. Composition of extracellular matrix.-The total amount of hydroxyproline in sets of rudiments and their culture media were determined asbefore [25]. The hexosamine content of the explants and media were estimated as previously described, except that all hydrolyses were carried out for 4 hr at 100°C with 4.5 N hydrochloric acid. Histology.-Rudiments were fixed for 45 min in Zenker’s fluid containing 3 per cent glacial acetic acid followed by 1 hr in Zenker’s fluid alone. Serial sections were cut from paraffin blocks at a thickness of 7 ,Uand stained either with toluidine blue [12] or with celestine blue, Mayer’s acid haemalum and Van Gieson’s stain. RESULTS
Effect
of
ascorbic acid on the growth of embryonic chick bones in uitro
Sets of 7-day-old chick bone rudiments were cultured either in chemically defined medium alone, or in medium to which ascorbic acid at a concentration of 50 ,ng/ml of medium had been added. The usual culture period was 6 days, but in a few experiments explants were also grown for 4 and 8 days. Growth in weight.-The wet and dry weights of paired sets of chick bone rudiments cultured with and without ascorbic acid for various periods of time are presented in Table I. For an in vitro period of 4 days or less, ascorbic acid has little effect on either the wet or dry weights of the rudiments. The data for 6 or 8 days’ growth in vitro show that explants without the vitamin 12*-681803
Experimental
Cell Research
42
J. J. Reynolds
180
have progressively higher wet weights and lower dry weights than paired rudiments with ascorbic acid. To illustrate this difference the ratio of the dry weight/wet weight of sets of rudiments grown for various periods of time are shown graphically in Fig. 1; the data recorded in this figure refer to one experiment only in which as nearly identical sets of rudiments as possible TABLE
various
I. Wet and dry weights of sets of chick bone rudiments grown for times in either synthetic medium alone, A, or in synthetic medium with 50 pglml ascorbic acid, B. A Wet
Days in vifro
weight m3
B Dry
weight mi3
Wet
weight mg
Dry
weight mg
4 days
9.2 11.3
0.86 1.11
9.2 11.4
0.87 1.12
6 days
15.6 16.7 16.8
1.25 1.41 1.34
14.1 15.3 15.3
1.42 1.64 1.59
8 days
28.7 24.9 22.4
1.53 1.41 1.39
18.5 20.1 19.1
2.03 1.93 1.90
were cultured for the period of time indicated. After 8 days in vitro the dry weight/wet weight ratio of explants with ascorbic acid is twice that of controls lacking the vitamin, and is similar to that of normal rudiments ez ovo [4, 251. Growth in size.-Bone rudiments cultured in medium with ascorbic acid for 6 days attain a length 10 per cent above that of paired explants in synthetic medium [25]; their morphology more nearly resembles that of rudiments e2 ovo, and the abnormal swelling of the epiphyses that is usually observed without the vitamin is prevented. Growth in DNA content.-No significant differences were observed between the DNA contents of sets of rudiments cultured for 6 days in the presence or absence of ascorbic acid, and the values obtained are similar to those previously reported for explants in defined medium for 6 days [25]. After 8 days in vitro, the DNA content of ascorbic acid-treated rudiments is usually slightly higher than that of untreated controls (lo-15 per cent in three experiments) but more experiments are needed to establish the significance of this difference. Experimental
Cell
Research
42
Ascorbic The effect of ascorbic chick bones in vitro
acid and chick
acid on the extracellular
bone rudiments
181
components
of embryonic
Hydroxyproline content.-Sets of rudiments were cultured for 6 days in either chemically defined medium alone, or in medium to which 50 ,ug/ml ascorbic acid had been added; their contents of hydroxyproline are given in
0
I a
I Days
Fig. I.-Percentage times in synthetic
dry weight/wet medium alone
I 6
I a
1”’ culture
weight of sets of chick bone rudiments grown (---), and in medium with 50 pg/ml ascorbic
for acid
various (-).
Table II together with values for the hydroxyproline containing material released into the medium during the culture period. The corresponding values for two sets of rudiments grown in vitro for 4 and 8 days respectively are also recorded. The table clearly shows that the formation of hydroxyproline containing material is greatly increased by the addition of ascorbic acid, and that in the absence of the vitamin there is little synthesis of this material after 4 days in vitro. These observations may be correlated with the onset of hydration between the 4th and 6th days, reported above. Hexosamine content.-The hexosamine contents of rudiments cultured for 6 days in vitro are not significantly different whether ascorbic acid is present or not [25]; only small amounts of such material are found in the culture media in either case. After eight days’ growth, the hexosamine content of explants cultured without the vitamin is lower (approx. 15 per cent) than in those grown with ascorbic acid. Experimental
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J. J. Reynolds
182
Ratio of collagen/hexosamine.-The percentage of dry weight that is hexosamine-containing material or collagen is given in Table III, in which data are shown for rudiments cultured for six days in vitro with and without ascorbic acid. The ratio of 2.0 for collagen/hexosamine is similar to that of embryonic bones ex ovo [25], and clearly demonstrates that ascorbic acid II. Hydroxyproline content of chick bone rudiments cultured for various times in synthetic medium alone, A, and medium with ascorbic acid, 50 ,uglml, B.
TABLE
Days in vitro
A Hydroxyproline
4 days
Explants Medium
9.9 9.8
6 days
Explants Medium
13.8 15.8
8 days
Explants Medium
11.4 16.1
B Hydroxyproline
(pg)
(pg)
12.5 7.8 12.6 15.4
11.2 14.8
31.0 19.1
31.5 11.0
30.1 12.4
38.0 27.4
is necessary for the maintenance of a normal ratio of extracellular components. Time of addition of ascorbic acid.-The data recorded in Tables I and II show that during the first 4 days in vitro the addition of ascorbic acid has little effect on the composition of the matrix; this probably indicates that the explants’ endogenous supply lasts for about four days [see also ref. 191. After six days in vitro a progressive hydration has begun in the rudiments without ascorbic acid and their collagen content is abnormally low, As expected, when explants are cultured for 2 or 4 days without the vitamin and the medium is then replaced with medium containing ascorbic acid, at 6 or 8 days the weights and collagen contents of the explants are similar to those of rudiments grown continuously in medium containing the vitamin. When explants cultured for 6 days in vitro without ascorbic acid, which have become hydrated in appearance, are changed to ascorbic acid containing medium, synthesis of collagen is resumed and the hydration retarded; at eight days such rudiments have a collagen content (24 ,ug OH-proline) and a dry weight/wet weight ratio (7.2) intermediate between those observed for cultures grown with and without ascorbic acid for eight days. It seems possible that a further period of cultivation with ascorbic acid might lead Experimental
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Ascorbic
acid and chick bone rudiments
183
to, a complete reversal of the hydration noted at 6 days, but it is unlikely that the morphology (large swollen epiphyses) would be improved. Nature and distribution of hydroxyproline-containing material.-It has been assumed above that the hydroxyproline containing material indicates collagen production. Although usually the determinations were made on III. Percentage composition of extracellular components of chick bone rudiments cultured for six days in synthetic medium alone, A, and medium with 50 ,ug/ml ascorbic acid, B.
TABLE
A
B
7.5
7.1
7.1
14:2
0.9
2.0
Hexosamine (% of dry
weight)
Collagen’ (% of dry
weight)
Collagen/Hexosamine a Collagen
calculated
from
hydroxyproline
x 7.46.
total hydrolysates of rudiments, some parallel cultures were extracted with 5 per cent trichloracetic acid (at 4°C) before hydrolysis. These experiments show that the amount of free hydroxyproline present in the rudiments is never more than 5 per cent of the total. No further attempts to fractionate the collagen have yet been made. The fact that the addition of ascorbic acid to synthetic medium both increases collagen synthesis and inhibits hydration of the rudiments, suggests that the two effects may be correlated, but before this possibility is seriously considered it is necessary to establish that the increase in collagen occurs throughout the explants. Ascorbic acid-treated rudiments have a thicker ossified collar (see below for histology), so that it was just possible that the increase in collagen was related solely to this ossified layer. To investigate this point, rudiments were cultured for 8 days in the presence of ascorbic acid, dried, and cut into three parts, roughly corresponding to the (ossified) diaphyseal and two (unossitied) epiphyseal regions; the diaphyseal and epiphyseal fragments were collected in two pools. Hydroxyproline determinations on these pools show that about 85 per cent of this material is associated with the epiphyseal fraction; similar proportions were observed in corresponding material ex ouo, so it may be conclused that the increased collagen synthesis is not confined to the diaphyseal region. Experimental
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.I. J. Reynolds
184
Fig. 2.-Zone of flattened medium for 6 days. Note Fig. ?I.-Zone medium with
cells in the epiphyseal the bubbly appearance
of flattened cells in the epiphyseal 50 pg/ml ascorbic acid for 6 days;
Fig. I.-Diaphyseal the thin ossified
region of a 7-day of the matrix. region of a 7-day the matrix appears
region of a ‘I-day chick femur grown collar. Celestin blue, Mayer’s haemalum
Fig. 5.-Diaphyseal region of a 7-day chick femur ascorbic acid for 6 days. Note ossified collar and haemalum and van Gieson’s stain, x 100. Experimental
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Research
42
chick femur grown in synthetic Toluidine blue, x 540. chick femur grown normal. Toluidine
in synthetic blue, x 540.
in synthetic medium for 6 days. and van Gieson’s stain, x 100.
grown in synthetic medium hypertrophied cells. Celestin
Note
with 50 ,ug/ml blue, Mayer’s
Ascorbic Histology
of rudiments
acid and chick bone rudiments
cultured
in the presence of ascorbic
185 acid
The histological appearance of chick bone rudiments cultured in chemically defined medium has been described previously [25] and agrees with the accounts of Biggers [3] and Schryver [29] for explants grown in medium BGJ. Briefly, the rudiments appear to have a sparse foamy matrix (Fig. 2) and in all areas the cells lie in swollen lacunae; the hypertrophy that normally occurs in the cells of the diaphyseal region (Fig. 4) at this stage of development [lo] does not advance very much in culture. Addition of ascorbic acid to the synthetic medium greatly increases the intensity of the metachromasia throughout the rudiments (Fig. 3) and the matrix has a dense structure similar to that of bone rudiments ex ouo; in appearance the matrix resembles that of rudiments treated with hydrocortisone [6, 25, 291. The cells of the diaphyseal region undergo more hypertrophy than control cultures without the vitamin (contrast Fig. 4 and Fig. 5) and the ossified collar becomes considerably thicker. DISCUSSION
Previous reports [2, 6, 25, 291 have shown that chick bone rudiments cultured in chemically defined media lacking ascorbic acid become excessively hydrated after a short period in uitro. The results described in this paper have demonstrated that the addition of 50 ,ug/ml of ascorbic acid to synthetic medium completely inhibits this terminal hydration. Unlike hydrocortisone which also prevents hydration [6, 25, 291, ascorbic acid does not inhibit .growth and produces a more normal morphology of the explants. Unpublished experiments by the present author indicate that concentrations of ascorbic acid below 50 ,ug/ml do not completely inhibit the hydration. Jeffrey and Martin [ 191 have described the rapid decrease in collagen synthesis of B-g-day chick tibiae grown in vitro without ascorbic acid. From their results it appeared that collagen synthesis ceased after about 4 days unless the vitamin were present; normal synthesis of collagen could be restored upon addition of ascorbic acid to the depleted cultures. These observations are confirmed by the closely similar results on 7 day bone rudiments reported in this paper; here again, collagen synthesis decreased rapidly after the fourth day in vitro without ascorbic acid, and could be restored by addition of the vitamin. Jeffrey and Martin [19] found that other cellular activities were relatively less impaired; the growth measurements recorded in this paper agree well with their observations, since no difference in DNA or hexosamine content could be measured in rudiments cultured Experimental
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186
J. J. Reynolds
for 6 days, whether ascorbic acid was present or not. Only a small depression in the synthesis of either DNA or hexosamine was noted in rudiments cultured for 8 days without the vitamin. In a previous publication [25] the present author suggested that the terminal hydration of chick bone rudiments cultured in synthetic medium was correlated with the synthesis of an abnormal extracellular matrix which was shown to be low in the ratio of collagen/hexosamine. This view is supported by the present observations that the addition of ascorbic acid to the medium produces a more normal ratio of collagen/hexosamine and at the same time completely prevents the abnormal uptake of water; the increased collagen synthesis takes place throughout the rudiments. The addition of the vitamin to already hydrated cultures reduces the hydration, concurrently with resumption of collagen synthesis. On the other hand experiments in which hydrocortisone was added to rudiments at different times after explantation showed that the hormone does not affect the hydration once it has occurred [see also refs. 25 and 291; this is to be expected since the steroid causes no significant increase in collagen synthesis, and it was thought to inhibit hydration mainly by depressing polysaccharide synthesis [25]. It seems probable that the water of hydration is associated in some way with the chondromucoprotein of the matrix, and that the abnormal hydration cannot take place if the polysaccharide is associated with a normal amount of collagen. The histological appearance of rudiments grown with and without ascorbic acid is consistent with the interpretation of the biochemical data presented above. The abnormal foamy matrix of explants cultivated without the vitamin is in sharp contrast to the more normal material synthesised in the presence of ascorbic acid, in which the metachromasia is increased throughout the rudiments. Hydrocortisone-treated explants show a similar improvement in metachromasia [6, 251; the diaphyseal cells are abnormally small, however, and not much periosteal bone is formed [ll, 311, whereas the diaphyseal cells of ascorbic acid-treated rudiments hypertrophy more than those of untreated controls and the ossified collar increases in thickness considerably during cultivation. A recent survey by Fabro and Rinaldini [9] of the ability of various embryonic cells of the chick to synthesise ascorbic acid, showed that many tissues quickly lose this capacity and the loss comes at different times during embryonic life depending on the particular organ. This is interesting in relation to the observation that collagen synthesis is stimulated in embryonic bones of the chick by exogenous ascorbic acid, and might explain some previous Experimental
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Ascorbic
acid and chick bone rudiments
187
reports of lack of stimulation in other fetal tissues [ 151. It is also interesting to note that some of the cell lines used by Green and Goldberg [ 161 synthesised appreciable amounts of collagen without the addition of ascorbic acid, and might indicate that some cells in culture are able to form the vitamin. The most recent evidence seems to suggest that ascorbic acid functions in the hydroxylation of proline to hydroxyproline [23], but it may also have activities in the cells less directly related to synthesis of extracellular material
WI. The conflicting evidence for the effect of ascorbic acid on polysaccharide synthesis in vivo has been reviewed recently [15, 26, 271. Some authors claim that sulphation diminishes in ascorbic acid deficiency [20, 241 but Friberg [ 131 noted no change in sulphation in healing wounds of scorbutic animals. Hughes and Kodicek [18] found that the concentration of galactosamine-containing polysaccharides was much lower in scorbutic than in normal granulomas from pair-fed animals. Studies on cell cultures in vitro have been hampered by the fact that cell cultures, unlike organ cultures, rarely continue to synthesise their tissue specific mucopolysaccharide but seem to secrete hyaluronic acid instead [15, 17, 22, 301. In the organ culture system employed in the present investigation, there was a small depression of polysaccharide synthesis in rudiments grown without ascorbic acid, but only after 8 days in vitro, which indicates that the effect may be secondary. The sulphation of the polysaccharide synthesised by chick bone rudiments cultured either in synthetic medium [7], or partly natural medium [8] is normal. Unpublished experiments [ 1 ] have shown that the ratio of galactosamine/glucosamine in 7-day chick bone rudiments cultured for 6 days was approximately 20: 1 whether the vitamin was present or not, and a similar ratio for rudiments ex ovo was observed. So far there is little evidence to indicate that ascorbic acid has any effect on the mucopolysaccharides synthesised by chick bone rudiments in vitro, though it is of course possible that a low level of ascorbic acid remains in the rudiments, which is sufficient to support normal mucopolysaccharide synthesis after the formation of collagen has ceased. A similar conclusion that ascorbic acid has little effect on the synthesis of sulphated mucopolysaccharides in vivo has been reached by Kofred and Robertson [al]. SUMMARY
The effect of the addit ion of ascorbic acid (50 fig/ml) to synthetic medium has been investigated in 7-day chick limb bone rudiments in culture. The Experimental
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J. J. Reynolds
188
vitamin completely prevented the terminal waterlogging usually observed in explants grown in chemically defined medium. Ascorbic acid had little effect on the DNA content or synthesis of hexosamine-containing material, but the rudiments attained a higher dry weight and a lower wet weight than untreated controls; part of the increased dry weight was due to a greatly stimulated synthesis of collagen. It is suggested that the inhibition of hydration is correlated with the production of a normal extracellular matrix under the influence of ascorbic acid. Grateful acknowledgement is made to Professor Dame Honor Fell, F.R.S., for her advice and encouragement. This work has been generously supported by funds from the Nuffield Foundation.
REFERENCES 1. BARRETT, 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21.
22. 23. 24.
25. 26. 27. 28. 29.
30. 31.
A. J. and REYNOLDS, J. J., Unpublished observations. J. D., Nat1 Cancer Inst. Monograph, No. 11, 1 (1963). J. D., J. Expfl Zool. 144, 233 (1960). J. D., LAWSON, K. A., LUCY, J. A. and WEBB, M., Biochim. Biophys. Acta 54, . I 236 (1961). BURTON, K., Biochem. J. 62, 315 (1956). DINGLE, J. T., FELL, H. B. and LUCY, J. A., Biochem. J. 98, 173 (1966). DINGLE, J. T., SLEDGE, C. B. and BARRETT, A. J., In preparation. ENDO, H. and MUROTA, S., Abstracts of Tissue Culture Seminar, Baroda, India, Jan., 1965. FABRO, S. P. and RINALDINI, L. M., Deuelop. BioI. 11, 468 (1965). FELL, H. B., J. Morphol. 40, 417 (1925). FELL, H. B. and THOMAS, L., J. Expfl Med. 114, 343 (1961). FELL, H. B. and WEISS, L., J. Exptl Med. 121, 551 (1965). FRIBERG, U., Arkio Kemi 12, 501 (1958). GOULD, B. S., Ann. N.Y. Acad. Sci. 92, 168 (1961). ~ Intern. Reu. Cyfol. 15, 301 (1963). GREEN, H. and GOLDBERG, B., Proc. Sot. Exptl Biol. Med. 117, 258 (1964). GREEN, H. and HAMERMAN, D., Nature 201, 710 (1964). HUGHES, R. E. and KODICEK, E., Biochem. J. 77, 3P (1960). JEFFREY, J. J. and MARTIN, G. R., Abstracts of the 6th International Congress of Biochemistry, New York, July 1964; Collagen Currents 5, 114 (1964). KODICEK, E. and LOEWI, G., Proc. Roy. Sot. London 144B, 100 (1955). KOFRED, J. A. and ROBERTSON, W. VAN B., Fed. Proc. 24, 354 (1965). MORRIS, C. C., Ann. N. Y. Acad. Sci. 86, 878 (1960). PETERKOFSKY, B. and UDENFRIEND, S., Proc. Nat1 Aead. Sci. U.S. 53, 335 (1965). REDDI, K. K. and NORSTROM, A., Nafure 173, 1232 (1954). REYNOLDS, J. J., Exptl Cell Res. In press. RINALDINI, L. M., in E. N. WILLMER (ed.), Cells and Tissues in Culture, Vol. 1, p. 680. Academic Press, London and New York, 1965. ROBERTSON, W. VAN B., Ann. N. Y. Acad. Sci. 92, 159 (1961). SCHNEIDER, W. and STAUDINGER, HJ., Biochim. Biophys. Acta 96, 157 (1965). SCHRYVER, H. F., Expil Cell Res. 37, 327 (1965). SCHIMIZU, Y., MCCANN, D. S. and KEECH, M. K., J. Lab. Clin. Med. 65, 286 (1965). SOBEL, H. and FREUND, O., Experientia 14, 421 (1958). BIGGERS, BIGGERS, BIGGERS,
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