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Endocrinology, doi:10.1210/en.2003-1435
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Endocrinology Vol. 145, No. 5 2478-2486
Copyright © 2004 by The Endocrine Society

Insulin-Like Growth Factor-I Augments Chondrocyte Hypertrophy and Reverses Glucocorticoid-Mediated Growth Retardation in Fetal Mice Metatarsal Cultures

T. Mushtaq, P. Bijman, S. F. Ahmed and C. Farquharson

Bone and Endocrine Research Group (T.M., S.F.A.), Royal Hospital for Sick Children, Glasgow G3 8SJ; and Bone Biology Group (T.M., P.B., C.F.), Roslin Institute, Edinburgh EH25 9PS, United Kingdom

Address all correspondence and requests for reprints to: Dr. C. Farquharson, Bone Biology Group, Roslin Institute, Edinburgh EH25 9PS, United Kingdom. E-mail: Colin.Farquharson{at}bbsrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The study aims were to improve our understanding of the mechanisms of glucocorticoid-induced growth retardation at the growth plate and determine whether IGF-I could ameliorate the effects. Fetal mouse metatarsals were cultured for up to 10 d with dexamethasone (Dex; 10–6 M) and/or IGF-I and GH (both at 100 ng/ml). Both continuous and alternate-day Dex treatment inhibited bone growth to a similar degree, whereas IGF-I alone or together with Dex caused an increase in bone growth. GH had no effects. These observations may be explained at the cellular level; cell proliferation within the growing bone was decreased by Dex and increased by IGF-I and these effects were more marked in the cells of the perichondrium than those in the growth plate. However, the most prominent observation was noted in the hypertrophic zone where all treatments containing IGF-I significantly increased (3-fold) the length of this zone, whereas Dex alone had no significant effect. In conclusion, Dex impaired longitudinal growth by inhibiting chondrocyte proliferation, whereas IGF-I stimulated chondrocyte hypertrophy and reversed the growth-inhibitory Dex effects. However, the IGF-I-mediated improvement in growth was at the expense of altering the balance between proliferating and hypertrophic chondrocytes within the metatarsal.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CLINICAL STUDIES BY our own group, as well as others, have shown that growth and skeletal development are impaired during treatment with prednisolone and dexamethasone (Dex) (1, 2). Most children who require systemic glucocorticoids (GCs) also suffer from chronic inflammatory disease, and in the clinical scenario, it can be difficult to clearly assess the relative contribution of disease and drugs on growth. In these children, maintenance of growth is a complex process that is influenced by a number of different mechanisms that influence the GH/IGF-1 axis by disrupting GH secretion or altering GH/IGF-1 sensitivity (3, 4). Although catch-up growth often follows cessation of GC therapy, children with systemic chronic inflammatory diseases who are on long-term GCs may have reduced final height (5, 6). Concomitant high-dose recombinant GH therapy may prevent a further deterioration in height velocity, but there is no evidence that it can normalize height in this group of children (7, 8, 9). Alternate-day GC treatment may have a lesser impact on childhood growth velocity than continuous GC treatment, but permanent growth impairment has also been noted in children receiving this form of therapy (10, 11). Optimization of growth-promoting therapy requires an improved understanding of the biological effects of GCs and GH/IGF-1 at the level of the growth plate.

The dual-effector theory of GH/IGF-1 action at the growth plate proposes that GH acts directly on germinal zone precursors of the growth plate to stimulate the differentiation of chondrocytes and then amplify local IGF-I synthesis, which, in turn, induces the clonal expansion of chondrocyte columns in an autocrine/paracrine manner (12). Although liver-derived IGF-I is the main determinant of serum IGF-I levels, it is not as important for postnatal growth as locally derived IGF-1 (13, 14).

In the ATDC5 chondrogenic cell line, our group’s recent in vitro studies show that GC effects may be dependent on the stage of chondrocyte maturation with maximal effects during chondrogenesis and minimal effects during terminal differentiation (15). It also seems that, although the progenitor cells may become quiescent when exposed to GC, their capacity to undergo chondrogenesis is maintained and the program is reactivated when the GC is removed (16). These data are consistent with the in vivo model of catch-up growth that is observed after cessation of GC administration directly into the growth plate (17).

The complex effects and physiological mechanisms of GC on growth plate chondrocytes are difficult to study solely in live animals where effects cannot be localized to specific cell types. The fetal mouse metatarsal explant culture is a highly physiological model for studying growth as the growth rate of fetal bones in culture is similar to that found in vivo, whereas bones harvested postnatally from 2-d-old rats arrest in culture after 2 d in vitro (18, 19). In addition, the metatarsal culture model maintains cell-cell and cell-matrix interactions, and the direct assessment of bone growth and histological architecture can be determined. By using the fetal mouse metatarsal assay, the aims of the present study were to obtain a clearer understanding of the cellular events underlying GC-induced growth retardation and, in addition, determine whether IGF-I can ameliorate the effects of GC on bone growth. This model has also allowed a comparison of the effect of continuous vs. alternate-day GC exposure. Our studies reveal that the fetal mouse metatarsal model can replicate in vivo bone growth, and these experiments represent the first in vitro study to demonstrate the prohypertrophic effects of IGF-I and reversibility of Dex-induced growth retardation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Fetal metatarsal organ culture
The middle three metatarsals were aseptically dissected from 18-d-old embyronic Swiss mice. Bones were cultured at 37 C in a humidified atmosphere of 95% air/5% CO2 individually in 24-well plates (Costar, High Wycombe, UK) for up to 10 d. Each well contained 300 µl of {alpha}-MEM without nucleosides (Invitrogen, Paisley, UK) supplemented with 0.2% BSA Cohn fraction V (Sigma, Dorset, UK), 0.1 mmol/liter ß-glycerophosphate (Sigma), 0.05 mg/ml L-ascorbic acid phosphate (Wako, Fukuoka, Japan), 0.292 mg/ml L-glutamine (Invitrogen), 0.05 mg/ml gentamicin (Invitrogen), and 1.25 µg/ml fungizone (amphotericin B) (Invitrogen). Dex (Sigma), IGF-I (Bacham, St. Helens, UK), and GH (Bacham) were added at a final concentration of 10–6 M, 100 ng/ml, and 100 ng/ml, respectively, to the cultured bones. In addition, the effects of continuous Dex 10–6 M vs. alternate-day Dex 10–6 M exposure on total metatarsal length was also studied. The control and experimental groups contained six metatarsals each, and the experiment was repeated at least two times.

Morphometric analysis
Images were taken of the metatarsals every second day of culture using a digital camera (COHU, San Diego, CA) attached to an Olympus MO81 microscope. The total length of the bone and width through the center of the mineralizing zone was determined using Image Tool (Image Tool version 3.00, University of Texas Health Life Science Centre, San Antonio, TX). All results are expressed as a percentage change from harvesting length, which was regarded as baseline to demonstrate the rate of growth over time. For the determination of the size (in the direction of longitudinal growth) within the growth region of the distinct chondrocyte maturational zones, the 4- and 10-d-old metatarsals were fixed in 70% ethanol, dehydrated, and embedded in paraffin wax (20). Wax sections (10 µm in thickness) were reacted for alkaline phosphatase (ALP) activity (21) for the demarcation of the hypertrophic and proliferating zones. Serial sections were stained with von Kossa and hematoxylin and eosin using standard protocols to identify the zone of cartilage mineralization. Images of the stained metatarsals were captured using Image Tool (University of Texas), and the size of the combined (distal and proximal) ALP-negative proliferating zone was determined: proliferating zone = total length – (hypertrophic zone + mineralizing zone. Similarly, the size of the combined ALP-positive hypertrophic zone located at either side of the mineralizing zone was determined: hypertrophic zone = (hypertrophic zone + mineralizing zone) – mineralizing zone. The size of the mineralizing zone was determined directly from the von Kossa-stained sections.

ALP enzyme activity
At the end of the culture period (d 10), ALP activity within the metatarsals was determined as previously described (22). Briefly, each metatarsal was permeabilized in 100 µl of 10 mmol/liter glycine (pH 10.5) containing 0.1 mmol/liter MgCl2, 0.01 mmol/liter ZnCl2, and 0.1% Triton X-100 by freeze-thawing three times. The extract was assayed for ALP activity by measuring the rate of cleavage of 10 mM p-nitrophenyl phosphate. Total ALP activity was expressed as nanomoles p-nitrophenyl phosphate hydrolyzed per minute per metatarsal. Each group contained three metatarsals, and the experiment was repeated at least twice.

Cell proliferation and dry weight determination
[3H]Thymidine uptake.
On d 4 and 10 of culture [3H]thymidine (Amersham Biosciences, Little Chalfont, UK) was added (final concentration, 10 µCi/ml) to each metatarsal culture for the last 6 h of culture. After washing in PBS, the metatarsals were extracted in trichloroacetic acid (twice for 30 min), acetone (twice for 30 min), and ether (three times for 30 min) and air dried overnight at room temperature. After the determination of dry weight (Sartorious Micro, Gottingen, Germany) the tissue was solubilized (NCS-II tissue solubilizer, Amersham) and the DNA incorporating [3H]thymidine was determined using a scintillation counter (20). The cell proliferation data were expressed as [3H]thymidine (dpm) per metatarsal. Each group contained three metatarsals, and the experiment was repeated at least two times.

Histological assessment of bromodeoxyuridine (BrdU) uptake.
BrdU (Sigma) was added (final concentration, 1 mg/ml) to the culture medium of the metatarsals for the last 6 h of culture on d 4 and 10 as described previously (20). At the end of the incubation period, the tissue was washed in PBS and fixed in 70% ethanol, dehydrated, and embedded in paraffin wax. Sections, 10 µm in thickness, were cut along the longitudinal axis, and chondrocyte nuclei with incorporated BrdU were detected using an indirect immunofluorescence procedure as detailed previously (23). Briefly, sections were denatured with 1.5 M HCl for 30 min before incubation with an antibody to BrdU (Dako, Ely, UK) diluted 1:50 in PBS for 1 h. After washing, the sections were incubated for an additional 1 h in fluorescein isothiocyanate-labeled goat antimouse IgG (Sigma) diluted 1:50 in PBS. The sections were finally mounted in PBS/glycerol (Citifluor, Agar Scientific, Essex, UK). Sections were examined using a Leica BMRB fluorescent microscope, and the total number of BrdU-positive chondrocytes within both the proximal and distal growth regions was determined. BrdU-labeled cells located to the perichondrium were also counted. Three sections from each of six bones from each treatment group at both time points were examined to obtain an aggregate value.

Statistics
All data are expressed as the mean ± SEM, and statistical analysis was performed using an ANOVA (GenStat, sixth edition, VSN International Ltd., Hemel Hempstead, UK). P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
All fetal mice metatarsals grew in culture and displayed a central core of mineralized cartilage juxtaposed on both sides to a translucent area representing the hypertrophic chondrocytes (Fig. 1Go, B–D). The localization of ALP reactivity within metatarsal sections was restricted to the mineralizing and hypertrophic chondrocytes and thus clearly delineated the boundary between the proliferating and hypertrophic zones (Fig. 1EGo) whereas von Kossa staining was specific to the mineralizing zone (not shown).



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  		FIG. 1. Measurements of digital images of fetal mouse metatarsal bones in culture with clearly delineated mineralizing zones (B– D) were taken using a calibrated ruler (A). These images demonstrate the harvesting day length (B) and the increased longitudinal growth at d 4 (C). An IGF-I-exposed metatarsal at d 10 is illustrated in D. Section of an IGF-I-treated metatarsal at d 10 reacted for ALP activity showing staining within both the mineralizing and hypertrophic zone. The proliferating zone is negative (E). The location of the proliferating (PZ), mineralizing (MZ), and hypertrophic (HZ) zones are also illustrated in E.

 
Longitudinal bone growth
All experiments were done on metatarsals from 18-d-old embryos that were cultured for intervals for up to 10 d. Dex-treated bones paralleled control bone growth rate until d 8 when their rate of growth decreased resulting in a total length that was significantly reduced from controls at d 8 (P < 0.05) and 10 (P < 0.05), (Fig. 2AGo). IGF-I and combined IGF-I plus Dex (IGF-I+Dex)-treated bones showed a rapid acceleration in growth from d 2 that was significantly higher than the control group (P < 0.05), and this increased growth rate was maintained throughout the duration of the experiment. At d 10, mean increase from baseline in total length of control, Dex, IGF-1, and IGF1+Dex bones was 50 ± 3%, 42 ± 2% (P < 0.05), 99.3 ± 5%, (P < 0.05), and 87 ± 4% (P < 0.05), respectively. Compared with the IGF-I-treated bones, the length of the bones treated with IGF-I+Dex was also significantly lower at d 8 (P < 0.05) and 10 (P < 0.05). The ability of GH to directly influence bone growth in this model system was also studied (Fig. 2BGo). In contrast to the growth-promoting effects of IGF-I (Fig. 2AGo), GH was found to have no significant effects on total bone length compared with control metatarsals. We also determined whether alternate-day Dex had a lesser deleterious impact on growth rate than continuous Dex treatment (Fig. 2BGo). Both modes of Dex treatments resulted in a significant reduction in total length from d 8 (P < 0.05). By d 10 in the continuously treated Dex group there was a 27 ± 2% (P < 0.05) reduction in total length vs. a 21 ± 3% decrease in the alternate-day Dex group (P < 0.05) when compared with the control metatarsals. No significant differences were detected between the continuous and alternate-day Dex lengths.



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  		FIG. 2. A, Continuous Dex at 10–6 M caused a significant decrease in linear growth from 8 d, whereas IGF-I and IGF-I+Dex had significant stimulatory effects from 2 d. B, Effects of GH (100 ng/ml) and continuous and alternate-day Dex on total length. GH had no significant effects on total length. However, both continuous and alternate-day Dex caused a significant decrease in length from d 8 (P < 0.05). C, Effects of Dex, IGF-I, and IGF-I+Dex on the length of the mineralized zone. In the control metatarsals, mineralization increased from 4 d. All treatments caused a significant reduction in mineralization from d 6. IGF-I-treated bones were the least mineralized, whereas Dex and IGF-I+Dex effects were intermediate. D, Effects of Dex, IGF-I, and IGF-I+Dex on metatarsal thickness. Both IGF-I and IGF-I+Dex caused a significant increase in the metatarsal thickness from d 4 and 6, respectively. Results shown in A, B, and D were obtained from the same cultures, whereas the data shown in B were from a separate experiment. All data are expressed as the mean ± SEM; *, P < 0.05 compared with control; {dagger}, significance of IGF-I compared with IGF-I/Dex (P < 0.05); {triangleup}, continuous Dex; {blacktriangleup}, alternate-day Dex; {blacksquare}, IGF-I (100 ng/ml); {square}, combined IGF-I+Dex; {circ}, GH (100 ng/ml); {bullet}, control cultures.

 
In control bones, there was a significant increase in the length of the mineralizing zone by d 6, and by d 10 the mean increase in length from baseline was 122 ± 2% (P < 0.05) (Fig. 2CGo). The mineralizing zone length of the IGF-I-treated bones changed little throughout the culture period and by d 10 it had increased from baseline by only a mean of 10 ± 2% (P < 0.05). Also by d 10 the length of the mineralizing zone in the Dex-treated metatarsals had increased by a mean of 79 ± 19% (P < 0.05) from baseline, significantly less than in the control metatarsals (P < 0.05). The growth rate of the mineralizing zone in the IGF-I+Dex-treated metatarsals was also less than control, and Dex-treated metatarsals with the mineralizing zone length significantly decreased at d 6 (11 ± 5%; P < 0.05), d 8 (32 ± 8%; P < 0.05), and d 10 (33 ± 10%; P < 0.05) from the control bones. Overall, these data suggest the existence of an inverse relationship between the length of the mineralization zone and total bone length (Fig. 2Go, A and C).

The thickness of the control and Dex-treated metatarsals did not change with time in culture and were not significantly different from each other at any of the time points examined (Fig. 2DGo). In comparison with controls, both the IGF-I and IGF-I+Dex-treated bones were significantly thicker from d 4 (P < 0.05) and 6, respectively (P < 0.05). At d 4, the thickness of the IGF-I-treated bones was significantly different from the IGF-I+Dex-treated bones. At d 10, the thickness of the IGF-I and IGF-I+Dex-treated bones were, respectively, 51 ± 10% (P < 0.05) and 35 ± 14% (P < 0.05) greater than that of their harvesting lengths.

With the exception of the results shown in Fig. 2BGo, the data presented in Fig. 2Go, A, C, and D (and all subsequent results), were obtained from metatarsals of embryos from the same mother. The differing growth rates shown in Fig. 2Go, A and B, are likely to be due to variability between the embryos selected for each experiment. The inhibition of growth rate by Dex was observed in both studies.

Assessment of chondrocyte maturational zone sizes
In many of the metatarsal rudiments, the boundary between the proliferating and hypertrophic zone of chondrocytes was difficult to delineate while in culture; therefore, measurements of the size of these individual maturational zones was performed on histological sections of 4- and 10-d-old metatarsals. The lengths of the proliferating, mineralizing, and hypertrophic zones are shown in Table 1Go.


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  		TABLE 1. Lengths of the proliferating, mineralizing and hypertrophic zones

 
Although Dex decreased and IGF-I increased the length of the proliferating zone, these changes did not reach statistical significance. However, IGF-I+Dex treatment resulted in a significant increase in the length of the proliferating zone at d 4 (P < 0.05), which was not sustained by d 10. The length of the mineralizing zone was significantly reduced with all treatments at both time points (P < 0.05) compared with the controls. At d 4, there was a 10% reduction with all treatments; this decrease became larger by d 10, with Dex, IGF-I, and IGF-I+Dex causing a 16, 51, and 42% reduction, respectively, in the length of the mineralizing zone compared with the control bones (P < 0.05). Dex caused a nonsignificant increase in the length of the hypertrophic zone at d 4 and 10. In contrast, IGF-I led to a marked increase in the length of the hypertrophic zone at d 4 (98% increase; P < 0.05), which became more pronounced by d 10 (346% increase; P < 0.05) (Table 1Go; Fig. 3Go, A and B). The combined effects of IGF-I+Dex were similar to IGF-I exposure alone, resulting in a 74% and 233% increase in length at d 4 and d 10, respectively (P < 0.05) (Table 1Go). The size of the individual hypertrophic chondrocytes in the 10-d IGF-I-treated metatarsals was also much larger than those of the control metatarsals (Fig. 3Go, C and D).



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  		FIG. 3. Histological assessment of chondrocyte hypertrophy (A–D) and proliferation (E–J) in metatarsals treated with Dex and IGF-I. A–D, Hematoxylin- and eosin-stained sections of 10-d-old cultures of control (A and C) and IGF-I-treated (B and D) metatarsals. There is an increase in the size of the hypertrophic zone of the IGF-I-treated metatarsals (B) compared with controls (A). The chondrocytes of the hypertrophic zone of the metatarsals in A and B are shown in higher magnification in C and D. The chondrocytes juxtaposed to the von Kossa-positive mineralized cartilage are larger in the IGF-I-treated (D) than in the control metatarsals (C). Note the micrographs shown in A and B are taken at different magnifications to accommodate the increased length of the IGF-I-treated metatarsals. P, Proliferating chondrocytes; H, hypertrophic chondrocytes. The dashed line marks the boundary between the proliferating and hypertrophic zones. E–J, BrdU-labeled cells in control (E and H), Dex-treated (F and I), and IGF-I-treated (G and J) metatarsals cultured for 4 d (E–G) and 10 d (H–J). Note the decreased number of proliferating cells in the Dex-treated metatarsals and in particular the lack of staining within the perichondrium (F and I). Increased perichondrial staining is observed in the 4-d-old IGF-I-treated cultures. Bars, 100 µm (A and E–J), 200 µm (B), and 25 µm (C and D).

 
Metatarsal ALP enzyme activity
The enlargement of the hypertrophic zone with IGF-I treatment after 10 d in culture was further studied by determining ALP activity in the metatarsals at the end of the culture period The ALP activity (nmoles hydrolyzed per minute per metatarsal) expressed as mean ± SEM was as follows: control, 0.0187 ± 0.009; Dex, 0.0117 ± 0.009; IGF-I, 0.038 ± 0.0061 (P < 0.05); and IGF-I+Dex, 0.026 ± 0.0044. In agreement with the increase in hypertrophic zone length (Table 1Go; Fig. 3BGo), IGF-I treatment resulted in significantly elevated levels of ALP activity within the metatarsals (103% increase; P < 0.05) compared with the control bones. Combined IGF-I+Dex caused an increase and Dex treatment alone a reduction in ALP activity, although these results were not significantly different from the control values. It was of interest to note that the data from the IGF+Dex-treated cultures are consistent with the metatarsal length data (Fig. 2AGo) where the presence of Dex partially reduced the effects of IGF-I.

Cell proliferation: [3H]thymidine incorporation and BrdU staining
The incorporation of [3H]thymidine into the metatarsals was determined at d 4 and 10, representing two distinct phases of varying growth rates. There was a tailing off in the linear growth curve from d 6 in all bones (Fig. 2AGo), and this was reflected in a lower [3H]thymidine incorporation rate in the control metatarsals at d 10 (75131 ± 5864 dpm) compared with the control bones at d 4 (98608 ± 6732 dpm) (Table 2Go). In comparison with control bones, Dex treatment for 4 d resulted in a significant reduction (50%; P < 0.05) in [3H]thymidine incorporation, whereas both IGF-I and IGF-I+Dex treatment resulted in significant increases of 43 and 57%, respectively (P < 0.05). After 10 d, there was a significant reduction in [3H]thymidine incorporation in all treatment groups compared with the control cultures (Table 2Go). However, this reduction, from control bone values, was greater with Dex (80%; P < 0.05) than that observed with IGF-I (64%; P < 0.05) or IGF-I+Dex (53%; P < 0.05).


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  		TABLE 2. Cell proliferation: effect of Dex, IGF-I, and IGF-I + Dex on [3H]thymidine uptake at d 4 and 10

 
To further refine the [3H]thymidine uptake data we determined the localization of the proliferating cells (BrdU positive) and quantified their number in both the growth plate and the perichondrium (Figs. 3Go and 4Go). The total number of proliferating cells in all control metatarsal groups was higher at d 4 than d 10, which is in agreement with the [3H]thymidine incorporation data and indicative of slower linear growth with time in culture (Table 2Go and Fig. 2AGo). Compared with d 4 control metatarsals, Dex significantly reduced the number of BrdU-positive cells located in the growth plate (42% decrease; P < 0.05) and perichondrium (76% decrease; P < 0.05) and therefore also the total number of dividing cells within the whole metatarsal (56% decrease; P < 0.05), (Figs. 3Go, E and F, and 4A). In contrast, IGF-I treatment significantly increased the number of BrdU-positive cells in the perichondrium (76% increase; P < 0.05) but not those within the growth plate (Figs. 3GGo and 4AGo). Combined IGF-I+Dex treatment had no significant effect on BrdU incorporation in the perichondrium and growth plate compared with the control metatarsals at d 4.



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  		FIG. 4. Effect of Dex, IGF-I, and IGF-I+Dex on the number of BrdU-positive cells within the growth plate (black bars), the perichondrium (white bars), and the combined number within the growth plate and perichondrium (hatched bars) at d 4 (A) and d 10 (B). Cell proliferation is higher at d 4 than d 10 with all treatments. At d 4, Dex causes a significant reduction in cell proliferation in the growth plate and perichondrium (P < 0.05), whereas IGF-I increases the number of proliferating perichondrial cells (P < 0.05). By d 10, Dex sustains the decrease in cell proliferation, which is significant in the perichondrium (P < 0.05). Both IGF-I and IGF-I+Dex also cause a decrease in the number of the positive immunofluorescent cells at this time point. All data are expressed as the mean ± SEM; *, P < 0.05 compared with controls.

 
Compared with d 10 control metatarsals (Fig. 3HGo), treatment with Dex alone (Fig. 3IGo) or in combination with IGF-I, significantly reduced the number of BrdU-positive cells within the perichondrium (Dex, 96% decrease, P < 0.05; IGF-I+Dex, 71% decrease, P < 0.05) (Fig. 4BGo). Similarly, treatment with IGF-I alone (Fig. 3JGo) or in combination with Dex resulted in a reduction in BrdU-positive cells within the growth plate chondrocytes (IGF-I, 63% decrease, P < 0.05; IGF-I+Dex, 57%, P < 0.05) (Fig. 4BGo). No cells outside of the perichondrium and growth plates showed any BrdU-positive staining. These results indicate that in comparison with chondrocytes within the growth plate, the cells within the perichondrium are more sensitive to stimulation by IGF-I during rapid growth (d 4) and inhibition by Dex at both time points.

Dry weights
At d 4 and 10, there was no significant difference between the weights of the control and Dex-treated metatarsals They were, however, significantly lighter (P < 0.05) than the IGF-I and IGF-I+Dex-treated bones, which were themselves similar in weight to each other at both time points (Table 3Go).


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  		TABLE 3. Dry weights (microgram) of all bones at d 4 and 10

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The rate of longitudinal bone growth is determined by a complex interplay of proliferative kinetics, size of the proliferative pool, matrix synthesis, and hypertrophic chondrocyte enlargement (24, 25). The control of these processes is still a matter of debate, and the individual contribution of each variable to bone growth differs with growth rate and is not uniform for all bones (26). Any perturbation of these synchronized variables may underlie the growth-modulatory effects of both Dex and IGF-I. Therefore, the identification of such changes in this present study will provide a better understanding of the mechanisms underlying Dex-induced growth retardation.

Our results unequivocally show that Dex and IGF-I have major and opposite effects on longitudinal bone growth with IGF-I clearly reversing the growth-inhibitory effects of Dex. However, the potential for Dex to inhibit bone growth was still present in the IGF-I+Dex cultures where the growth rates did not match those of bones cultured with IGF-I alone. The Dex-induced reduction in total metatarsal length from d 8 was due to reduced chondrocyte proliferation and a reduction in the growth of the mineralizing zone. Similar effects with other GCs have previously been reported by Picherit et al. (27) who demonstrated that hydrocortisone induced growth retardation in fetal rat metatarsals. Interestingly, our present data did not reveal a significant growth-sparing effect of alternate-day glucocorticoids. The growth-sparing effect of alternate-day steroids is not a universal observation, and it may be influenced not only by the duration of therapy but also by the underlying disease process and the sex of the patient (4). In addition, most clinical reports refer to the use of prednisolone or hydrocortisone, whereas our studies employed the use of Dex, which has markedly more potent effects on growth in vivo and in vitro (2, 15).

In contrast to the effects of Dex, IGF-I rapidly stimulated linear bone growth by increasing both the size of the hypertrophic zone and the chondrocyte proliferation rate during the early phase of bone growth. Stimulation of bone growth by IGF-I has also been reported by Scheven and Hamilton (18), but they further demonstrated that the stimulation of cell proliferation in cultured rat metatarsals by IGF-I was not sustained with IGF-I over time. This may indicate the rapid use of endogenous growth factors needed to support longitudinal growth. However, these workers (18) reported GH stimulatory effects on metatarsal length, which is in contrast to the data of this present study. Although GH is well recognized to stimulate longitudinal bone growth in vivo (28, 29) its effects in vitro are less clear (30, 31). Other studies have strongly suggested that GH effects in vivo may be indirect and that IGF-I effects are more pervasive in vitro (32, 33).

To understand the cellular mechanisms underlying the opposite effects of Dex and IGF-I on bone length we analyzed the distribution of BrdU-positive cells within metatarsals treated by both Dex and IGF-I alone and in combination. The number of dividing cells within the perichondrium was greatly reduced by Dex at both 4 and 10 d of culture. In contrast, at 4 d, the number of BrdU-positive cells was greater in the perichondrium of IGF-I-treated bones. Stimulation of cell proliferation was not observed in the IGF-I-treated 10-d-old metatarsals, and this may be due to the observed slowing of growth in these rapidly growing bones. In 4-d-old rapidly growing metatarsals, IGF-I completely reversed the inhibitory effects of Dex on cell proliferation within the perichondrium and growth plate. This reversal of the negative effects of Dex by IGF-I coincubation was also observed, albeit to a lesser extent, in the perichondrial cells of 10-d-old cultures. These results extend the [3H]thymidine incorporation data and also confirm the ability of IGF-I to reverse the deleterious effects of Dex on cell proliferation. Our observation that cell proliferation within the perichondrium was more sensitive to inhibition by Dex and stimulation by IGF-I than chondrocytes within the growth plate has previously not been recognized. The perichondrium is vital to the endochondral process through its role in mediating the parathyroid hormone-related peptide-indian hedgehog (PTHrP-Ihh) signaling cascade, and it is possible that the marked Dex-induced inhibition of proliferation within cells of the perichondrium has a more direct effect on the bone growth process (34, 35, 36). The differential sensitivity of cells to Dex treatment within the perichondrium and within the growth plate requires additional study.

A morphometric analysis was completed to further characterize the response of metatarsals to both Dex and IGF-I with respect to the size of the individual maturational zones within the growth plate. The reduction in length of the mineralization zone with Dex was consistent with metatarsals treated with hydrocortisone (27). However, the absence of an increase in the length of this zone after IGF-I is at variance to others who have demonstrated an increase in mineralization zone length with IGF-I in a rat metatarsal model system (19). Dex also led to a small, nonsignificant increase in the length of the hypertrophic zone, which is similar to the findings of Smink and colleagues (37) who demonstrated an increase in the hypertrophic zone length in mice treated with Dex. They further postulated that this was a likely consequence of an acceleration of the chondrocyte differentiation rate as observed in PTHrP null mice (34, 37). Alternatively, due to the restriction of cartilage mineralization, the increased size of the hypertrophic zone may be in part due to a simple buildup of nonmineralized hypertrophic chondrocytes. A similar, more pronounced process may explain the more marked reduction in the mineralization zone observed in the IGF-I-treated metatarsals.

Within the maturational zones of the growth plate, the major effects of IGF-I were clearly on the length of the hypertrophic chondrocyte zone and also the size of the cells within. This result is in accord with the hypothesis that it is the size of the hypertrophic zone rather than chondrocyte proliferative kinetics that is the single major determinant of bone growth rate (26, 38). Although IGF-I is expressed by chondrocytes situated in all maturational zones of the growth plate, IGF-I mRNA expression is mainly restricted to the hypertrophic zone, and the infusion of IGF-I into hypophysectomized rats showed that IGF-I stimulated growth plate chondrocytes at all stages of differentiation including those in the hypertrophic zone (29, 37, 39). The growth retardation in the IGF-I null mouse is associated with an attenuation of chondrocyte hypertrophy and no significant changes in proliferation (40), and our data further strengthen the hypothesis that the predominant role of IGF-I in growth promotion is in augmenting chondrocyte hypertrophy rather than proliferation. This effect of IGF-I can reverse GC-induced growth retardation, but this apparent ameliorative effect results in an alteration of the relative proportion of proliferative, hypertrophic, and mineralized chondrocytes.

The opposite effects of IGF-I and Dex on cell proliferation and bone growth and the ability of IGF-I to reverse the growth-inhibitory effects of Dex have not been previously reported. Its clinical significance is unclear, but an up-regulation of chondrocytes expressing IGF-I after GC exposure has been reported previously, and it is possible that IGF-I is an important local growth factor that counteracts the effect of GCs at the tissue level (37, 41, 42). Besides GH and IGF-I, GC exposure may also alter the GH and IGF binding proteins that modulate tissue exposure to these growth factors, and this requires additional study, especially now that a complex of IGF-I and IGFBP3 is available for treatment of GH insensitivity (37, 43, 44, 45).

In conclusion, we have shown that Dex and IGF-I have opposite effects on linear bone growth. The effects of Dex were time dependent, whereas IGF-I effects were immediate. During the phase of rapid growth, Dex decreased and IGF-I increased cell proliferation. The alteration in proliferation rate by both Dex and IGF-I were most marked within the cells of the perichondrium. Dex decreased skeletal mineralization, whereas IGF-I markedly stimulated chondrocyte hypertrophy in favor of mineralization and completely reversed Dex-induced growth retardation. However, the potential for Dex to inhibit bone growth was still present in the IGF-I+Dex cultures where growth rates did not match those of bones cultured with IGF-I alone. In addition, the IGF-I-mediated improvement in growth was at the expense of altering the balance between proliferating and hypertrophic chondrocytes within the metatarsal. Alternate-day Dex administration did not have a growth-sparing effect, and GH had no beneficial effect on metatarsal growth at the dose studied. The fetal mouse metatarsal model can replicate in vivo bone growth, and this is the first in vitro study to demonstrate the prohypertrophic effects of IGF-I and reversibility of Dex-induced growth retardation.


   Acknowledgments
 
We are indebted to Dr. P. Veldhuijzen (Vrije Univeriteit, Amsterdam, The Netherlands) who demonstrated to us the metatarsal organ culture system. We are also grateful to Miss Elaine Seawright for her contribution to the experiments.


   Footnotes
 
This study was generously supported by the Chief Scientist Office of Scotland, Novo Nordisk UK Ltd., the Biotechnology and Biological Sciences Research Council (BBSRC), and a research award from the British Society of Pediatric Endocrinology and Diabetes.

Abbreviations: ALP, Alkaline phosphatase; BrdU, bromodeoxyuridine; Dex, dexamethasone; GC, glucocorticoid.

Received October 24, 2003.

Accepted for publication January 15, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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