ABSTRACT
The growth of the tibia and radius from 6-to 10-day-old embryos was estimated from the increase in the total nitrogen content of the rudiments. A comparison of the specific growth rates of eight limb bones showed that the bones could be arranged in the following order of diminishing specific growth rate: tibia, third metatarsus, femur, ulna, humerus, third metacarpus, radius, fourth metacarpus.
If the small bones were arranged in order of the percentage growth of their rudiments in vitro, the sequence found in various media was the same as that of their specific growth rates in vivo.
A comparison of the growth-promoting properties of various biological media showed that the best growth and differentiation occurred on a plasma and embryo extract clot. The growth-promoting properties of the extract were greatly enhanced in a plasma clot with which the fibroblasts of the explant, through their abundant outgrowth, were in intimate contact. In the absence of extract there was no difference between the growth of rudiments in contact with the clot or supported by lens paper floating on serum.
The order of a series of bones arranged according to their growth response to T3 in a medium of plasma and embryo extract was the same as their order when arranged according to their specific growth rate in vivo: the growth of potentially fast-growing bones was reduced by T3, that of slowly growing bones was increased.
The growth rates in vitro of different rudiments were varied by altering either the composition of the medium or the incubation temperature, but the rudiments continued to respond differentially to T3. The characteristic differences in the relative growth of the different bones when not treated with T3 were maintained under these conditions.
INTRODUCTION
The maturation, of the cartilage of embryonic chick long-bone rudiments growing in tissue culture is accelerated by addition of the thyroid hormones, thyroxine and triiodothyronine, but the growth in length of different long bones is not uniformly affected (Fell & Mellanby, 1955, 1956). Thus the growth of the hormone-treated tibia is less than that of a normal tibia, while the effect of thyroid hormone on the radius is to increase its growth. This differential response is not determined either by the stage of development at which the limb-bone rudiments are exposed to hormone, or by the size of the explant (Lawson, 1961).
Investigations to determine whether the differential response of limb-bone rudiments to triiodothyronine (T3) is due to differences in the growth rates of different bones are described in this paper. The work was divided into three parts.
Data on the relative growth in weight of limb bones from the sixth to tenth day of embryonic development were obtained to supplement the data on the relative growth in length which were analysed by Landauer (1934) and Lerner (1936).
Growth in vitro can be altered by changing either the temperature at which the explants are incubated (Bucciante, 1926; Martinovitch, 1939), or the composition of the nutrient medium. The extent to which the growth and differentiation of chick skeletal explants are affected by the composition of the medium cannot be easily ascertained from previous studies on various media of biological origin (plasma and embryo extract: Strangeways & Fell, 1926; Miszurski, 1939; serum and embryo extract: Ito & Endo, 1956; plasma: Chen, 1954; serum: Hay, 1958) and on chemically defined media (Wolff, Haffen, Kieny, & Wolff, 1953; Biggers, Webb, Parker, & Healy, 1957; Kieny, 1958) since most of these studies have been made under different experimental conditions and with embryos of different ages. The results of a controlled comparison of the effects of different biological media on the growth of limb-bone rudiments are described in the second part of the paper.
These studies of growth in vivo and in vitro provide a basis for the third part of the paper which consists of a description of the effect of T3 on rudiments cultured under different growth-promoting conditions.
MATERIALS AND METHODS
Tissue culture
Media
Plasma and embryo extract
Limb-bone rudiments were grown on fowl plasma and chick embryo extract in watch-glasses (Fell & Mellanby, 1952) as previously described (Lawson, 1961).
Serum
For the study of growth in different biological media, serum was separated from cock plasma clotted with thrombin by centrifuging through a Hemming filter as described by Hay (1958). It was not necessary to incubate the plasma clot at 37° C. before or after breaking it up. For the other experiments, serum was obtained from a clot of cock blood which had been incubated at 37° C. overnight.
Broken clot
Plasma was clotted with thrombin or embryo extract in the upper bottle of a Hemming filter. The glass beads and rayon net were omitted from the upper side of the steel joint and the clot was broken as it passed through the perforated plate of the joint during centrifugation. This medium was transferred to the culture dishes with a 1-ml. syringe.
Chemically defined medium
CMRL 858 (Healy, Fisher, & Parker, 1955) or a similar medium, BLj (Biggers & Lucy, 1960), was used. The glucose concentration of both media was raised to 2·5 g./l.
Explants grown on serum, broken clot, or chemically defined medium were supported by lens paper (Chen, 1954).
3,5,34triiodo-L-thyronine (T3)
This substance was dissolved in 0·1 per cent. Na2CO3 or in 10 per cent, propylene glycol. The stock solution was added to plasma, serum, or the chemically defined medium to give a concentration of 1·6 × 10−4 g. T3/l. of final medium, unless otherwise stated.
Volume of medium
The volume of medium and the proportions of explant to medium have already been described (Lawson, 1961). The medium used in the two factorial experiments in which the growth of bones on different biological media was measured, and in any experiment in which embryo extract was present, was obtained by mixing three volumes of plasma or serum with one volume of embryo extract or thrombin in 1 per cent, glucose Tyrode. In other experiments 80 volumes of serum were diluted with 1 volume of 16 per cent, glucose in distilled water.
Culture period
The explants were changed to fresh medium every other day and most experiments ended after 6 or 8 days.
Age of embryos
Rudiments were usually obtained from 6-day-old embryos (stage 28-29, Hamburger & Hamilton, 1951). In one experiment (see p. 545) rudiments were explanted ‘at the same histogenetic stage’: for convenience rudiments were selected at the stage when their ends were easily visible and their hypertrophic zones barely visible under the dissecting microscope, which in practice meant taking the femur and humerus from 5- or 6-day-old embryos, the tibia from 6-day-old embryos, the radius, ulna, and third metatarsus from 6
-day-old embryos, and the third and fourth metacarpals from 7-day-old embryos.
Histological techniques
The rudiments were fixed in 3 per cent, acetic Zenker or in Rossman’s fluid at 0° C. Paraffin sections were stained with Delafield’s haematoxylin and chromatrope 2R or by the periodic acid/Schiff technique.
Measurement of the rudiments
Length, wet weight, and total nitrogen content were measured by methods previously described (Lawson, 1961). Total nitrogen was estimated on samples containing from 1 to 6 rudiments, according to size.
Experimental design
Paired samples
In experiments in which the effect of only one treatment was tested against a control, the rudiment from one side of the embryo was treated and compared with the untreated, corresponding rudiment from the other side of the same embryo.
Factorial experiments
The effects of several treatment combinations were compared in factorial experiments. The rudiments were allotted to the treatment combinations in a randomized block design. The data were analysed by analysis of variance procedures (Goulden, 1952) after transformation of the measurements to the common logarithmic scale. Significant reduction in experimental error was achieved by eliminating the effect of variations in the initial lengths of the bones from different embryos by an analysis of covariance (Biggers, Webb, Parker, & Healy, 1957).
Percentage growth in vitro
Total nitrogen was measured at the end of the culture period and compared with the initial total nitrogen of the corresponding rudiment from the same embryo. The observations were transformed to logarithms so that the percentage increase (I per cent.) of any one rudiment was given by log I per cent. = log Nf=log Ni+2, where Nf= total nitrogen content of the rudiment at the end, and Nt= total nitrogen content at the beginning of the culture period. The mean log I per cent, for each type of bone was calculated and these values were used in statistical comparisons of the percentage growth of different bones. This procedure was adopted to equalize the variances of the percentage growth of the different bones. The theoretical aspects of the comparison of ratios have been discussed by Biggers (1961).
Estimation of response to T3
The effect of T3 was estimated from its effect on the final length and wet weight of the cultured rudiments, and on their growth in length (Lawson, 1961).
In experiments on paired samples, response was expressed as log T— log C, where T and C are the final length or wet weight of the treated and control rudiments. The significance of the difference in response of different bones was tested by the Z-test for unpaired samples.
When the effect of T3 was evaluated in a factorial experiment the data were transformed to the logarithmic scale, and the statistical significance of the treatments was tested by analysis of variance and covariance of the final length or wet weight of the rudiments and their initial length (see above).
RESULTS
The normal growth of limb-bone rudiments in vivo
The tibia and radius were chosen for the initial study of growth during the mainly cartilaginous phase of limb-bone development (6 to 10 days of incubation) because they show the largest difference in growth response to T3in vitro, and any growth differences that were related to their differential response might be more easily demonstrated. Furthermore, because these bones represent the two extremes in response, generalizations about their growth are likely to be valid for the growth of other limb bones, intermediate in response. The total nitrogen content of tibiae from 7- to 10-day-old embryos is highly correlated with the wet weight of the rudiments (r = 0·987, n = 25); increase in total nitrogen was chosen as the criterion of growth of the whole rudiment because total nitrogen content can be determined more accurately than the weight of small samples. Seventy-four samples of tibiae and 64 samples of radii, obtained from 95 embryos, were estimated.
The results are plotted in Text-fig. 1A. The simplest expression relating size to time was sought which described the growth of different bones within the limits of experimental error and which could be expected to reveal growth differences between different bones. Linear regression analysis was done on the transformed values which, when plotted, appeared to lie approximately on a straight fine and in which there was no significant heterogeneity of variance.
A, total nitrogen content of the tibia (dots) and the radius (open circles) from 6-to 10-day-old embryos. The number of samples estimated for each mean is indicated in brackets, B, the data from A plotted on a log-log scale and regression lines fitted. The vertical line through each mean represents twice the standard deviation. There is no significant heterogeneity in the variances.
A, total nitrogen content of the tibia (dots) and the radius (open circles) from 6-to 10-day-old embryos. The number of samples estimated for each mean is indicated in brackets, B, the data from A plotted on a log-log scale and regression lines fitted. The vertical line through each mean represents twice the standard deviation. There is no significant heterogeneity in the variances.
There was an approximately linear relationship when log nitrogen content (log N) was plotted against time (t), and also when log N was plotted against log t (Text-fig. 1B). Regression analysis, however, revealed significant deviation from linear regression for both bones when log N was analysed with t (Table la), but no significant deviation for either bone when log N was analysed with log t (Table lb). In the latter analysis the value of the regression coefficient for the tibia, bT, was 4·86, standard error 0·14, and this was significantly greater (P< 0·001) than that of the radius (bR = 3·64, standard error 0·16). The difference between the regression coefficients is indicated by the difference in the slope of the two fitted regression lines in Text-fig. 1B.
Results of analyses of variance and linear regression of total nitrogen of the tibia and radius from 6- to 10-day-old embryos

Thus the specific growth rate decreases with time, but the ratio of the specific growth rates of the tibia and radius is constant, and equal to bT/bR. From the observations bT/bR = 1·33.
The values of b for eight limb-bone rudiments—femur, tibia, third metatarsus, humerus, radius, ulna, third metacarpus, and fourth metacarpus, were therefore estimated from their total nitrogen content at 6 and 10 days. The total nitrogen content of the fourth metacarpus was measured also at 7 and 11 days, since at 6 days the rudiments consisted only of an ill-defined mass of procartilage. All the observations on 6-day-old embryos were made on samples containing several rudiments, because of the small size of individual rudiments; samples from 10-day-old embryos contained one or two rudiments. The regression analysis was weighted accordingly (Quenouille, 1958); the method is illustrated by the data for the humerus (Table 2).
Data for weighted regression analysis of total nitrogen content of the humerus from 6- and 10-day-old embryos

The results of the nitrogen analysis and the calculated values of b are given in Table 3. The values of b for the tibia and radius in this series of eight bones are slightly lower than those found when the whole of the 6- to 10-day period was sampled (bT = 4·75, cf. 4·86; bR = 3·59, cf. 3·64). The earlier analyses covered two years, and the measurements on the eight bones together were made during January and February; during these months the embryos are often smaller than later in the year and it may be inferred that growth is slower. Since the ratio of bT/bR in the original investigation was 1 ·33 and in the January/February results it was 1·32, it may be concluded that skeletal proportions were not affected.
Growth of limb-bone rudiments in vitro
The effect of different media on growth and differentiation
Tibiae and radii from 6-day-old embryos were grown on plasma or serum, with and without embryo extract. Four replicates each of eight bones were set up in a 22 factorial experiment, thus providing 32 samples of each type of bone for statistical analysis.
At the time of explantation each rudiment consisted of a cartilaginous rod with a crescent-shaped arrangement of cells towards the ends. The perichondrium had formed, but there was no osteoid tissue, and the central cells had not begun to hypertrophy, although they contained a little glycogen. At the end of the culture period all rudiments, except one on thrombin-clotted plasma, had developed the usual three zones of epiphysis, flattened cell zone, and hypertrophic shaft. Differentiation appeared to be more advanced in rudiments grown in the presence of embryo extract : the hypertrophic cells were larger and more osteoid tissue had formed than in rudiments grown in the absence of embryo extract. The periosteum of the rudiments grown on cock serum without embryo extract remained intact and healthy, unlike that of femora from 9-day-old embryos grown on horse serum without embryo extract (Endo, 1960). There were no obvious histological differences between rudiments grown on plasma with embryo extract and those grown on serum with embryo extract, except that slightly more osteoid tissue was produced in the plasma cultures.
The different media affected the growth of the tibia and radius to the same extent. The growth of the rudiments was greatly enhanced by the presence of embryo extract in the medium and the effect of the embryo extract was greater when combined with plasma than with serum (Text-fig. 2). When embryo extract was replaced by an equivalent amount of Tyrode and thrombin there were no significant differences between the growth of rudiments supported by lens paper floating on serum and that of rudiments growing on the surface of the plasma clot (Text-fig. 2). It is therefore unlikely that the difference in the growth-promoting action of embryo extract in serum and in plasma is connected with mechanical differences in the two substrates. The most obvious explanation is that additional nutrients are provided by the proteolytic action of the embryo extract on the fibrin.
Adjusted means from the analysis of variance of the wet weight and length of the tibia (A and c) and of the radius (B and D) after 7 days in different biological media. In each figure the two columns on the left (−) show the size of rudiments cultivated without embryo extract, the columns on the right (+) show the size of rudiments cultivated in the presence of embryo extract. Open columns, serum; striped columns, plasma. Vertical lines represent 5 per cent, fiducial limits.
Adjusted means from the analysis of variance of the wet weight and length of the tibia (A and c) and of the radius (B and D) after 7 days in different biological media. In each figure the two columns on the left (−) show the size of rudiments cultivated without embryo extract, the columns on the right (+) show the size of rudiments cultivated in the presence of embryo extract. Open columns, serum; striped columns, plasma. Vertical lines represent 5 per cent, fiducial limits.
This hypothesis was tested by comparing the growth of the tibia on three different media containing embryo extract: serum, plasma clot, and broken plasma clot (see section on Materials and Methods). Growth-promoting substances liberated from fibrin by the action of embryo extract would be expected to increase growth on the broken clot as compared with that on serum. Rudiments grown on the three media with-out extract served as controls. All components of the media were incubated at 38° C. for 1 –2 hours before use, either as intact plasma clots in watch-glasses or as clots in the Hemming filter bottles before centrifugation. In the latter situation the plasma was either in contact with embryo extract (broken clot with embryo extract) or apart from it (serum with extract and the media without embryo extract). The six treatment combinations formed a 3 × 2 factorial experiment. A randomized block design of 18 units was used and the experiment was replicated once giving a total of 36 observations.
The final wet weight of the tibia was not enhanced by fibrin in a liquid medium of serum and embryo extract (broken clot) (Text-fig. 3, columns 4 and 5); there was increased growth (P< 0·001) only when the extract-containing clot was intact (Text-fig. 3, last column). The weight of the control rudiments grown on the media without embryo extract did not differ significantly. These results indicate that interaction of embryo extract with the fibrin of the plasma does not enhance the growth of the cartilage rudiments; any additional growth-promoting substances liberated by digestion of the clot by embryo extract would be present in both the intact and the broken clot, but growth on the broken clot was no greater than on serum and embryo extract.
Adjusted means from the analysis of variance of the wet weight of the tibia after 6 days in different biological media. The three columns on the left (−) show the size of the rudiments cultured without embryo extract, those on the right (+) the size of the rudiments in the presence of embryo extract. Open columns, serum; dotted columns, broken plasma clot; striped columns, intact plasma clot. Vertical lines represent 5 per cent, fiducial limits.
Adjusted means from the analysis of variance of the wet weight of the tibia after 6 days in different biological media. The three columns on the left (−) show the size of the rudiments cultured without embryo extract, those on the right (+) the size of the rudiments in the presence of embryo extract. Open columns, serum; dotted columns, broken plasma clot; striped columns, intact plasma clot. Vertical lines represent 5 per cent, fiducial limits.
The fibroblastic outgrowth from the rudiments varied in different media and on different substrates and was removed when the medium was changed and before the rudiments were weighed. The outgrowth in the living cultures was scored on an arbitrary system (Table 4). By the end of the experiment there was little or no connective tissue outgrowth on lens paper when embryo extract was absent from the medium, although there was considerable outgrowth on plasma clotted with thrombin. Embryo extract maintained connective tissue outgrowth for a longer period when fibrin was present in the liquid medium (Table 4, columns 4 and 5); this suggests that fibroblastic outgrowth, but not cartilage growth (see above), is enhanced by the proteolysis of fibrin by embryo extract. There was abundant outgrowth on the intact plasma clot with embryo extract.
Differential growth in vitro
The growth rate throughout the culture period was not analysed, but the percentage increase in total nitrogen of various rudiments growing in different media was determined at the end of the culture period (Table 5) and the significance of the differences in percentage increase between bones was tested (Table 6).
The significance of differences in percentage increase of total nitrogen of different bones in culture

The percentage increase in the total nitrogen content of small bones (ulna, third metatarsus, radius, third metacarpus, and fourth metacarpus) varied after cultivation in plasma and embryo extract (Tables 5,a, b\ 6 a, b). If these bones were arranged in order of their percentage growth, the sequence was the same as that of their specific growth rates in vivo (Table 3). Differential growth was also shown by the metatarsus and radius in a chemically defined medium during cultivation for 48 hours (Table 5e).
The percentage growth of the tibia, femur, and humerus in plasma and embryo extract (Table 5b) was rather less than expected from the specific growth rates in vivo. Indeed, radii from 7-day-old embryos had a greater percentage increase; than the corresponding tibiae when cultivated on this medium (Table 5c), though not when cultivated on serum (Table 5d). The growth of large explants may be limited by the ratio of surface area to volume, so that the more growth is promoted by the medium and the larger the initial size of the bone, the sooner a limiting value is reached. The variations in surface : volume ratio should not affect the relative growth of small bones, however, and differences in the percentage increase in size of such explants at the end of the culture period are considered to be a valid indication that bones differ in specific growth rate in vitro as well as in vivo.
Growth and the differential response to T3 in vitro
Relative growth and the differential response to T3
The effect of T3 was measured on different limb-bone rudiments explanted at the same histogenetic stage, because the response is modified by the stage of development at which the rudiment is first exposed to the hormone (Fell & Mellanby, 1956; Lawson, 1961). The rudiments were cultivated on clots of plasma and embryo extract. The percentage increase in total nitrogen was measured on similar rudiments from the same batch of embryos cultivated on control medium under the same conditions as the T3-treated rudiments and their controls.
When the small bones, that is third metatarsus, ulna, radius, third metacarpus, and fourth metacarpus, were arranged in order of their response in T3, the sequence was the same as when the bones were arranged in order of their percentage growth in normal medium, i.e. bones with the greatest percentage increase in control medium were most retarded by T3, while the growth of bones with a small percentage increase was stimulated by T3 (Table 5b, cf. Table 7).
The series of bones arranged in order of decreasing specific growth rate in vivo compared with their response to T3 after 8 days in vitro

The sequence of eight limb-bones when arranged in order of their response to T3, was the same as their sequence when arranged in order of their specific growth rate in vivo (Table 7), i.e. T3 retarded the growth in vitro of potentially fast-growing bones, such as the tibia and metatarsus, and increased the growth of bones, such as the radius and fourth metacarpus, which grow slowly in vivo.
Growth rate and the differential response to T3
Since the differential response to T3 appears to be associated with the specific growth rate in vivo and also with differential growth in vitro, the question arises whether the growth rate itself determines the response to T3. If so, a bone that is retarded by T3 in a medium in which it normally grows fast should be stimulated by the hormone when it is cultivated under conditions less likely to promote growth.
The effect of the medium on the response to T3
The response of tibiae from 7-day-old embryos to two levels of T3 (4× 10−5 g./l. and l·6× 10−4 g./l.) was measured in four different media: plasma with embryo extract, serum with embryo extract, serum alone, and CMRL 858. The twelve treatment combinations made a 3 ×34 factorial experiment; this was replicated twice to give a total of 36 observations.
The final length of the bones treated with l·6× 10−4 g./l. was less than that of their controls in all the media tested (Text-fig. 4). The statistical interaction between the media and levels of T3 did not approach the 5 per cent, level of significance; the apparent difference in response to the lower level of T3 in different media was therefore a chance effect. Similar results were found when the experiment was repeated with the femur and when the response was estimated on the wet weight of the rudiments.
Length of the tibia after treatment with T3 in different media for 8 days. I, plasma and embryo extract; II, serum and embryo extract; III, serum; IV, CMRL 858. Open columns, no added T3; dotted columns, 4× 10−5g. T3/l. medium; striped columns, 1·6 × 10−4g. T3/l. medium. Vertical lines represent 5 per cent, fiducial limits.
Length of the tibia after treatment with T3 in different media for 8 days. I, plasma and embryo extract; II, serum and embryo extract; III, serum; IV, CMRL 858. Open columns, no added T3; dotted columns, 4× 10−5g. T3/l. medium; striped columns, 1·6 × 10−4g. T3/l. medium. Vertical lines represent 5 per cent, fiducial limits.
Histological examination of the rudiments cultivated in the chemically defined medium showed a localized necrosis in the flattened cell zones of the T3-treated rudiments. The formation of matrix was deficient throughout the rudiments in both control and treated explants.
The response to T3 in serum was examined in more detail. Twenty-four pairs of tibiae and radii from 7-day-old embryos were cultivated on serum from whole blood for 6 days. The concentration of T3 added to the treated cultures was T6x IO-4 g./l. of medium. The growth in length of the tibia was slightly stimulated by T3 during the first 24 hours (P<0-01) (Text-fig. 5a). The characteristic retardation appeared in the T3-treated rudiments after the third day in culture.
The final wet weights and total nitrogen contents of the T3-treated tibiae were also less than those of the controls (Table 8). Thus, although the percentage growth of the control tibia in serum was less than that of the control radius in plasma and embryo extract (Table 5,c, d) the growth of the tibia was retarded by T3 in both media. The growth in length of the radius was stimulated by T3 during the first 4 days in culture. During the last 2 days the length of the T3-treated rudiments increased at the same rate as that of the controls (Text-fig. 5b). Both the final wet weight and total nitrogen content were greater in the T3-treated radii than in the controls (Table 8).
The effect of temperature on the response to T3
Tibiae, third metatarsi, and radii from 6-day-old embryos were grown in plasma and embryo extract for 8 days. The response to T3 at different temperatures was tested on 24 bones of each type in a 22 factorial experiment in which the factors were T3 (control and 1·6×10−4 g. T3/l.) and incubation temperature (34° C. and 38-5° C.).
The rudiments appeared healthy and of normal shape after growth at both temperatures.
At 34° C. the retardation of growth in length, characteristic of the leg bones, cultivated in T3 medium at 38·5° C., did not occur, but there was no stimulation of growth (Text-fig. 6 A, B). The growth in length of the radius, however, was stimulated both at 34° C. and at 38·5° C. (Text-fig. 6c). This stimulation was significant at the 1 per cent, level.
The growth in length of the control bones was considerably reduced at 34° C. when compared with growth at 38·5° C. (P<0·001), but relative differences in growth were maintained. For example, the metatarsus grew 3·8 mm. at 38-5° C., but only 2·5 mm. at 34° C.; the radius, which was the same initial length as the metatarsus, grew 3·3 mm. at 38·5° C. compared with 1·9 mm. at 34°. Thus even though the metatarsus grew more slowly at 34° C. than the radius at 38-5°, its growth was not stimulated by T3.
DISCUSSION
The normal growth of limb-bone rudiments in vivo and in culture
No biological significance can be attached to the exact analytical form of the growth equation (Gray, 1929; Kavanagh & Richards, 1934; Medawar, 1940, 1945); it is useful in so far as it makes the experimental results available in an algebraic form from which information concerning the specific growth rate, which is considered to have biological significance, can be extracted easily. The power law N = atb was chosen because it fitted the data of both the tibia and the radius within the limits of experimental error; these limits were sufficiently narrow for statistical control to reject the exponential formula, N = atf. Since a significant proportion of the total nitrogen of the rudiments must be contributed by the intercellular matrix which is not self-reproducing, this result is satisfactory. The growth equation implies that the specific growth rates of chick limb-bone rudiments during a restricted period of their normal development, decrease with time; this finding is neither original nor unexpected, but the formula indicated a convenient method for comparing the specific growth rates of different bones.
The faster rate of growth in length of the long bones of the leg as compared with the growth rate of the wing bones (Landauer, 1934) is reflected in the whole limb by the faster rate of growth in weight (Schmalhausen, 1926) and in DNA content in the hind limb (Nowinski & Yushok, 1953). Lerner (1936) derived values from Landauer’s (1934) data for the growth ratio of bone length to body weight in the allometry equation y = bxα where y and x are magnitudes relating to part and whole respectively, and a is the growth ratio (Huxley & Teissier, 1936). The descending order of growth ratios was metatarsus, tibiotarsus, femur, humerus, radius, ulna. The distal-proximal growth gradient in the leg was also found in Creeper embryos and in other experiments (Lerner & Gunns, 1938). Since the allometry formula implies that the ratio of the specific growth rates of the parts compared is constant, the sequence of bones arranged in order of their specific growth rates, inferred from the values of b calculated from total nitrogen content, should be the same as the sequence of their specific growth ratios. Although the total nitrogen content of the three leg bones which were investigated increased faster than that of any of the wing bones, there was no obvious growth gradient in the leg. It is possible that the measurement by Lerner of the combined tibia and tarsus and the relative thickness of different bones are factors in the difference in sequence. For example, Lerner found that the value of a for the radius was slightly greater than that for the ulna, but in the present work the reverse was found. Similarly, the value of b for the third metacarpus was considerably higher than that of the fourth metacarpus.
Relative growth in vitro was inferred from differences in the percentage increase of total nitrogen of explanted small rudiments; the pattern of relative growth in various media was the same as in vivo. The difference in the relative growth in length of the metatarsus and radius was also maintained when the growth rate was reduced by lowering the incubation temperature. These results agree with the findings of Lerner & Gunns (1938) who compared the relative growth rates in the leg bones in 11-to 18-day-old embryos incubated at 98°, 101°, and 104° F. There was increased growth at the higher temperatures, but the values of a were unaffected and so normal proportions were maintained. It is concluded that the relative growth of different bones during normal development is intrinsic by 6 days of incubation, when the bones have differentiated as distinct cartilaginous rudiments, and is not imposed by environmental factors in the limb. The metabolic basis of the proportionate growth of the skeleton remains unexplored; the establishment of regional differences within the limb occurs at an early stage in the development of the limb-bud, and may be controlled by the apical cap (Saunders, Cairns, & Gasseling, 1957).
The experiments on the growth of rudiments in different biological media showed that growth was greater when embryo extract was added to plasma than when it was added to serum, but that growth was similar on plasma and serum in the absence of embryo extract (Text-figs. 2, 3). Peptic digests of fibrin slightly enhance the growth of fibroblasts on plasma clots (Carrel & Baker, 1926; Willmer & Kendal, 1932), and in this laboratory the exudate from an incubated plasma-embryo extract clot is used as a better growth-promoting medium for fibroblasts in hanging drop cultures than serum and embryo extract. The results of the experiment with broken plasma clots show that any substance which may be produced by the proteolysis of fibrin by embryo extract, if present in addition to serum and embryo extract, do not increase the growth of the whole rudiment. The enhanced organized growth of the bone rudiments on the plasma and embryo extract medium as compared with that on serum and embryo extract, appears to be associated with the abundant fibroblastic outgrowth in these cultures; the reason for this is not clear.
The differential response to T3
The differential growth response of different rudiments to T3 is not dependent on the absolute growth rate of the rudiments in vitro, whether this is controlled by the composition of the medium or by the incubation temperature. Neither of these methods of controlling growth rate disrupt the pattern of relative growth and the differential response to T3in vitro appears to be closely associated with the relative growth of the rudiments in vivo (Table 7).
Of the bones studied, the tibia and radius show the greatest difference in their growth response to the same concentration of T3in vitro. The drastic reduction in the growth of the tibia was modified in three situations. The growth in length of T3-treated tibiae from 5-to -day-old embryos was slightly stimulated during the first 2 days in culture (Lawson, 1961); this was interpreted as a precocious onset of hypertrophy. A transitory stimulation of growth in length also occurred in tibiae that were cultivated on serum (Text-fig. 5); these rudiments were taken from 7-day-old embryos and hypertrophy had already begun in the shafts. T3 did not modify the growth in length of tibiae cultivated on plasma and embryo extract at 34° C. (Text-fig. 6). The increase in the growth in length of the T3-treated radius, both in serum and at 34° C., was proportionately greater than that produced at 38° C. by the same concentration of hormone added to plasma and embryo extract. The present results are not necessarily inconsistent in view of the differences in sensitivity of different bones to T3 which will be demonstrated in a future paper. The effective concentration of T3 added to serum may have been lower than in plasma and embryo extract as the natural thyroid hormone of the serum may have been destroyed during the incubation of the blood-clot overnight at 37° C. (see section on Materials and Methods); in the experiments at subnormal temperature, T3 added to plasma and embryo extract may have been less active at 34° C. than at 38° C.
Growth in length of the tibia (A) and the radius (B) during cultivation for 6 days on serum. Broken lines, the length of the control bones; continuous lines, the length of the equivalent rudiments grown in the presence of l·6× 10−4 g. T3/l.
Growth in length of the tibia (A), third metatarsus (B), and radius (c) during 8 days cultivation at 38·5° C. (upper figures) and at 34° C. (lower figures). Broken lines, the length of control bones; continuous lines, the length of bones treated with T3.
Fell & Mellanby (1955, 1956) found that the length of the flattened cell zone was reduced by treatment with thyroid hormone; the amount of reduction varied, but was greatest in the leg bones. They interpreted this as precocious hypertrophy in the flattened cell zone without an accompanying increase in cell-division. Experiments on chemically defined media (Kieny, 1958) showed that the process of hypertrophy and lengthening of the shaft occur without any increase in dry weight of the rudiment; it is possible that the increased growth in length of the radius in response to T3 may have been due to such a process. On the other hand, the few measurements that were made of the effect of T3 on the total nitrogen content of the rudiments (Table 8) indicate that T3 increases the synthesis of nitrogenous compounds in the radius. The ratio of nitrogen to wet weight of the radius is reduced by treatment with T3, however, so an increase in water content cannot be excluded.
The warning of Weiss (1949) against considering growth and differentiation as separate phenomena in embryogenesis is relevant; during the course of skeletal differentiation the speed of a synthetic process varies. For example, the young hypertrophic cells are the most active incorporators of S35 (Amprino, 1954; Fell, Mellanby, & Pelc, 1956); hastening the maturation of these cells, therefore, would limit chondroitin sulphate synthesis and might result in an apparent retardation of the growth of the whole cartilaginous rudiment. The results of the present experiments on the growth of the rudiment as a whole, serve to emphasize the differences in behaviour between various rudiments and to indicate that the response to T3 probably depends on differences in metabolism that are reflected in the relative growth of different rudiments; the study provided no indication of the nature of the metabolic basis of these differences.
RÉSUMÉ
Action de la triiodothyronine sur la croissance des ébauches cartilagineuses de différents os longs
II. Vitesse de croissance
Pour estimer la croissance du tibia et du radius d’embryons de poulets de 6 à 10 jours d’incubation, on a étudié la teneur en azote total de ces ébauches. En comparant la vitesse de croissance (1/N. dN/dt) d’ébauches cartilagineuses de 8 os longs on peut les classer, selon un ordre décroissant : tibia, 3ème métatarsien, fémur, cubitus, humérus, 3ème métacarpien, radius, 4ème métacarpien.
On retrouve la même séquence lorsqu’on cultive in vitro les ébauches de ces différents os sur des milieux variés.
Les meilleures croissance et différenciation se réalisent sur des coagula à base de plasma et d’extrait embryonnaire. Les propriétés stimulatrices de l’extrait embryonnaire sont nettement renforcées lorsque les fibroblastes de l’explant, à la suite d’une abondante prolifération, sont en contact intime avec le coagulum. En l’absence d’extrait embryonnaire il n’y a aucune différence entre la croissance des ébauches cartilagineuses cultivées en contact direct avec le coagulum ou déposées sur du ‘lens paper’ flottant dans du sérum.
L’addition de triiodothyronine (T3) dans le milieu qui contient du plasma et de l’extrait embryonnaire, d’une part, inhibe la croissance des os à vitesse de croissance élevée in vivo, d’autre part, stimule la croissance des os à vitesse de croissance basse in vivo.
Les différences caractéristiques de la vitesse de croissance des différents os longs sont maintenues lorsqu’on modifie la composition du milieu ou la température d’incubation. Mais, même dans ces conditions les ébauches cartilagineuses continuent de répondre différemment à la présence de T3 dans le milieu.
ACKNOWLEDGEMENTS
I am indebted to Dr. H. B. Fell, F.R.S., for her encouragement and helpful discussion, and to Dr. J. D. Biggers for advice and guidance with experimental design and statistical analysis. I am grateful to the Sir Halley Stewart Trust for a Research Studentship. The triiodothyronine was a gift from Dr. R. Pitt-Rivers, F.R.S.