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Endocrinology Vol. 142, No. 1 430-436
Copyright © 2001 by The Endocrine Society

ARTICLES

Regulation of Growth Plate Chondrogenesis by Bone Morphogenetic Protein-21

Francesco De Luca, Kevin M. Barnes, Jennifer A. Uyeda, Stacy De-Levi, Veronica Abad, Teresa Palese, Veronica Mericq and Jeffrey Baron

Division of Pediatric Endocrinology, Department of Pediatrics, University of Maryland School of Medicine (F.D.L., T.P.), Baltimore, Maryland 21201-1595; and Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (K.M.B., J.A.U., S.D.-L., V.A., V.M., J.B.), Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Francesco De Luca, Division of Pediatric Endocrinology, 22 South Greene Street, Room N5E13, Baltimore, Maryland 21201-1595. E-mail: fdeluca{at}peds.umaryland.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone morphogenetic proteins (BMPs) regulate embryonic skeletal development. We hypothesized that BMP-2, which is expressed in the growth plate, also regulates growth plate chondrogenesis and longitudinal bone growth. To test this hypothesis, fetal rat metatarsal bones were cultured for 3 days in the presence of recombinant human BMP-2. The addition of BMP-2 caused a concentration-dependent acceleration of metatarsal longitudinal growth. As the rate of longitudinal bone growth depends primarily on the rate of growth plate chondrogenesis, we studied each of its three major components. BMP-2 stimulated chondrocyte proliferation in the epiphyseal zone of the growth plate, as assessed by [3H]thymidine incorporation. BMP-2 also caused an increase in chondrocyte hypertrophy, as assessed by quantitative histology and enzyme histochemistry. A stimulatory effect on cartilage matrix synthesis, assessed by 35SO4 incorporation into glycosaminoglycans, was produced only by the highest concentration of BMP-2. These BMP-2-mediated stimulatory effects were reversed by recombinant human Noggin, a glycoprotein that blocks BMP-2 action. In the absence of exogenous BMP-2, Noggin inhibited metatarsal longitudinal growth, chondrocyte proliferation, and chondrocyte hypertrophy, which suggests that endogenous BMPs stimulate longitudinal bone growth and chondrogenesis. We conclude that BMP-2 accelerates longitudinal bone growth by stimulating growth plate chondrocyte proliferation and chondrocyte hypertrophy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BONE MORPHOGENETIC proteins (BMPs) appear to play an important role in skeletal development (1, 2, 3, 4, 5). These glycoproteins were initially characterized by their ability to induce ectopic cartilage and bone formation in soft tissues (6). Expression of BMP-2 and BMP-4 in specific areas of the limb bud suggests a regulatory role for these factors during early skeletal development (7). Consistent with this concept, overexpression of BMP-2 in embryonic limbs causes dysplastic changes in the cartilaginous structures (8). Because BMP-2 is also expressed in the growth plates of long bones (9, 10), we hypothesized that this growth factor also plays a role at a later stage of skeletal development, after morphogenesis is completed.

In long bones, morphogenesis is followed by an extended period of growth. Longitudinal growth occurs at the growth plate by endochondral ossification (11, 12), a process in which cartilage is formed and then remodeled into bone. Growth plate chondrocyte proliferation, hypertrophy, and extracellular matrix secretion lead to the formation of new cartilage, chondrogenesis (13). Simultaneously, the growth plate is invaded from the metaphysis by blood vessels and bone cell precursors, which remodel the cartilage into bone tissue (14). The rate of longitudinal bone growth depends primarily on the rate of growth plate chondrogenesis.

To study the role of BMP-2 in growth plate chondrogenesis, we assessed the effects of recombinant human BMP-2 (rhBMP-2) on cultured fetal rat metatarsal bone rudiments. Unlike isolated cells in culture, this organ culture system preserves the histological architecture of the growth plate and thus the intercellular interactions and local microenvironments found in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Organ culture
Metatarsal bone rudiments were dissected from Sprague Dawley rat fetuses on embryonic day 20 (e20) and cultured separately in 24-well culture dishes (15). Each well contained 0.5 ml MEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 0.2% BSA (Sigma, St. Louis, MO), 0.3 mg/ml L-glutamine (Life Technologies, Inc.), 0.05 mg/ml ascorbic acid (Life Technologies, Inc.), 1 mM sodium glycerophosphate (Sigma), 100 U/ml penicillin, and 100 |gmg/ml streptomycin (Life Technologies, Inc.).

Bone rudiments were cultured for 3 days in a humidified incubator with 5% CO2 in air at 37 C. The medium was changed daily. For the first set of experiments, rhBMP-2 (donated by Genetics Institute, Cambridge, MA) was dissolved in PBS and further diluted into the culture medium to reach a final concentration of 10, 100, or 1000 ng/ml. Control cultures received the same volume of PBS.

In the second set of experiments, the bone rudiments were cultured in the presence of recombinant human Noggin (a gift from Regeneron Pharmaceuticals, Inc., Tarrytown, NY); dissolved in 50 mM NaCl, 1 mM magnesium acetate, and 20% glycerol; and diluted into the culture medium to reach a final concentration of 10 µg/ml with or without rhBMP-2 (100 ng/ml). Control cultures contained equal amounts of vehicle.

Measurement of longitudinal bone growth
The length of each bone rudiment was measured under a dissecting microscope, using an eyepiece micrometer (Carl Zeiss, Thornwood, NY) at 20 x magnification (16). Length measurements were performed at 0, 1, 2, and 3 days of culture. Culture medium was briefly removed before each measurement.

Histology
Metatarsal rudiments were fixed in 10% neutral buffered formalin, embedded in plastic, cut in 5-µm longitudinal sections, and stained with toluidine blue. From each bone rudiment, we obtained three sections parallel to the long axis of the bone and taken 30 µm apart. We measured the heights (microns) of the proliferative zone, the hypertrophic zone, and the ossification center, determined the number of hypertrophic cells in both growth plates in each of the three sections, and calculated the average value. Hypertrophic chondrocytes were operatively defined by a height along the longitudinal axis greater than 9 µm (16). All measurements were performed by two observers blinded to the treatment regimen.

Enzyme histochemistry
The activity of alkaline phosphatase was localized by enzyme histochemistry (17). Bone rudiments were embedded in OCT compound (Sakura Finetek, Torrance, CA) and frozen. Ten-micrometer cryostat sections were cut and mounted onto poly-L-lysine-coated slides. Mounted sections were treated at room temperature with 4% formaldehyde in PBS for 10 min, rinsed in PBS, and then placed in 0.1 M triethanolamine-HCl, pH 8.0, for 10 min. Slides were rinsed in diethylpyrocarbonate-treated water and then stained for alkaline phosphatase for 3 min at room temperature using the simultaneous coupling azo dye method. In the working solution (0.5% N,N-dimethylformamide and Tris buffer, pH 9.1), 0.03% napthol AS phosphate (Sigma) was used as substrate, with 0.1% Fast Blue BB salt (Sigma) as the azo dye. Slides were rinsed and then counterstained with 0.25% safranin O (Sigma) for 3 sec, followed by several rinses in distilled water. Sections were then dried and mounted in Faramount aqueous mounting medium (DAKO Corp., Carpinteria, CA).

[3H]Thymidine incorporation
Cell proliferation was assessed in the cultured bone rudiments by measuring [3H]thymidine incorporation into the bones (18). [3H]thymidine (Amersham Pharmacia Biotech, Arlington Heights, IL; SA, 25 Ci/mmol) was added to the culture medium 3 h before the end of the incubation period, at a concentration of 5 µCi/ml. All rudiments were then washed three times for 10 min each time with MEM and solubilized with NCS-II Tissue Solubilizer (0.5 N) solution (Amersham Pharmacia Biotech). The amount of incorporated isotope was determined by liquid scintillation counting.

To analyze the sites of the growth plate where DNA synthesis occurred, we incubated bone rudiments with [3H]thymidine as described above. At the end of the incubation, all bones were fixed in 10% buffered formalin and processed for autoradiography. Autoradiography was performed by dipping the slides in Kodak NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposing them for 1 wk, and developing with Kodak D-19 developer. Sections were counterstained with hematoxylin and eosin. The labeling index (number of labeled cells per total cells) was determined separately for the epiphyseal region (comprising round cells irregularly embedded in extracellular matrix) and for the proliferative zone (the area of the growth plate formed of columns of flat cells). Cells in the perichondrium and in the primary center of ossification were not included in either analysis. All determinations were made by the same observer, who was blinded to the treatment category.

35SO4 incorporation
As a measure of cartilage matrix synthesis, we assessed 35SO4 incorporation into glycosaminoglycans (15). Na235SO4 (Amersham Pharmacia Biotech; SA, up to 100 mCi/mmol), at a concentration of 5 µCi/ml, was added to the culture medium 3 h before the end of the 3-day culture period. The rudiments were washed three times for 10 min with Puck’s saline solution and then digested in 1.5 ml MEM with 0.3% papain at 60 C for 16 h. They were then incubated with 0.5 ml of 10% cetylpyridinium chloride (Sigma) in 0.2 M NaCl at room temperature for 18 h. At the end of the incubation, each precipitate was collected by vacuum filtration through filter paper (Whatman, Clifton, NJ; catalogue no. 1001090), washed three times with 1 ml 0.1% cetylpyridinium chloride in 0.2 M NaCl, and dissolved in 0.5 ml 23 N formic acid. The amount of radioactivity incorporated into glycosaminoglycans was measured by liquid scintillation.

Statistics
Data were expressed as the mean ± SEM. Statistical evaluation was performed by ANOVA and post-hoc Tukey tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of BMP-2 on growth plate chondrogenesis
Metatarsal bones were dissected from rat fetuses (e20) and cultured in serum-free medium containing 0, 10, 100, or 1000 ng/ml rhBMP-2 for 3 days. BMP-2 at concentrations of 100 and 1000 ng/ml caused a significant acceleration of longitudinal bone growth (P < 0.001; n = 21/group; Fig. 1Go).



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  		Figure 1. Effects of rhBMP-2 on longitudinal bone growth (mean ± SEM). Fetal rat metatarsals (e20; n = 21/group) were cultured for 3 days in serum-free medium containing 0, 10, 100, or 1000 ng/ml BMP-2. The lengths of the bones were measured using an eyepiece micrometer in a dissecting microscope.

 
Histological studies were performed on the metatarsal rudiments after 3 days in culture. Treatment with BMP-2 increased the height of the hypertrophic zone [110 ± 5.4 vs. 72.3 ± 5.4 µm; BMP-2 (100 ng/ml) vs. control, P < 0.001] and caused a concentration-dependent increase in the number of hypertrophic chondrocytes (P < 0.001; n = 4–7/group; Figs. 2AGo and 3Go). In the growth plate hypertrophic zone, BMP-2 also increased the expression of alkaline phosphatase, a marker for hypertrophic chondrocytes (Fig. 3Go). BMP-2 did not significantly affect the heights of the proliferative zone or the ossification center (data not shown). To assess chondrocyte proliferation, we measured [3H]thymidine incorporation into the metatarsal bones. Doses of 100 and 1000 ng/ml BMP-2 both produced a significant increase in total [3H]thymidine incorporation, which is the sum of thymidine incorporation in the epiphyseal region, proliferative zone, ossification center, and perichondrium (P < 0.001; n = 6–12/group; Fig. 2BGo). At the lowest effective concentration (100 ng/ml), BMP-2 increased the proliferation rate only in the epiphyseal region (P < 0.01; n = 4/group; Figs. 4Go and 6CGo). The effect of BMP-2 on cartilage matrix synthesis was determined by measuring the incorporation of 35SO4 into newly synthesized glycosaminoglycans. At the highest concentration (1000 ng/ml), BMP-2 significantly enhanced glycosaminoglycan synthesis (P < 0.001; n = 6/group; Fig. 2CGo).



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  		Figure 2. Effects of rhBMP-2 on growth plate chondrogenesis. Fetal rat metatarsals (e20) were cultured for 3 days in serum-free medium with the indicated concentrations of BMP-2 (control = 0 ng/ml BMP-2). A, Quantitation of hypertrophic chondrocytes (mean ± SEM). After routine histological processing, bones were embedded in plastic, and 5-µm longitudinal sections were obtained. Hypertrophic cells were operationally defined by a height along the longitudinal axis greater than 9 µm. Quantitation was performed by two observers blinded to the treatment category. B, Total [3H]thymidine incorporation (mean ± SEM). During the last 3 h of the 3-day culture period, [3H]thymidine was added to the culture medium. Bones were washed and solubilized. Total [3H]thymidine incorporation was then measured by liquid scintillation counting. C, Sulfate incorporation into glycosaminoglycans (mean ± SEM). Bones were labeled with 5 µCi/ml Na235SO4 for the last 3 h of culture, then rinsed and digested with papain. Glycosaminoglycans were precipitated with 10% cetylpyridinium chloride, and the precipitate radioactivity was counted by liquid scintillation. *, P < 0.01; **, P < 0.001 (vs. control)

 


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  		Figure 3. Representative photomicrographs demonstrating the effects of BMP-2 and Noggin on growth plate histology (upper panels) and alkaline phosphatase activity (lower panels). Metatarsal bones from e20 rat fetuses were cultured for 3 days in serum-free medium only (A; control), or with 100 ng/ml BMP-2 (B), 1000 ng/ml BMP-2 (C), or 10 µg/ml Noggin (D). Upper panels, After routine histological processing, bones were embedded in plastic, and 5-µm longitudinal sections were obtained and stained with toluidine blue. ER, Epiphyseal region; PZ, proliferative zone; HZ, hypertrophic zone; OC, primary ossification center. Lower panels, Enzyme histochemistry was performed on frozen sections using the simultaneous coupling azo dye method. Alkaline phosphatase activity produces blue stain. Separation of the perichondrium/bone collar from the interior of the bone rudiments is an artifact that occurs in frozen sections. BC, Bone collar; HZ, hypertrophic zone; OC, primary ossification center.

 


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  		Figure 4. Autoradiography of [3H]thymidine incorporation. Fetal rat metatarsals were cultured for 3 days in serum-free medium only (A; control), or with 100 ng/ml BMP-2 (B), 10 µg/ml Noggin (C), or 100 ng/ml BMP-2 plus 10 µg/ml Noggin (D). [3H]Thymidine at a concentration of 5 µCi/ml was added to the culture medium 3 h before the end of the incubation period. All bones were fixed in 10% buffered formalin and after routine processing were embedded in paraffin, then 5-µm longitudinal sections were prepared. Autoradiography was performed by standard techniques using a 1-week exposure time. Sections were counterstained with hematoxylin and eosin. Representative sections are shown in darkfield (upper panels) and brightfield (lower panels) views. ER, Epiphyseal region; PZ, proliferative zone.

 


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  		Figure 6. Effects of Noggin on growth plate chondrogenesis. Fetal rat metatarsals (e20; n = 5–6/group) were cultured for 3 days in serum-free medium with or without 100 ng/ml BMP-2 and/or 10 µg/ml Noggin. A, Number of hypertrophic chondrocytes per histological section (mean ± SEM). After routine histological processing, bones were embedded in plastic, and 5-µm longitudinal sections were obtained. Hypertrophic cells were operationally defined by a height along the longitudinal axis greater than 9 µm. Quantitation was performed by a single observer, who was blinded to the treatment category. B, Total [3H]thymidine incorporation (mean ± SEM). During the last 3 h of the 3-day culture period, [3H]thymidine was added to the culture medium. Bones were washed and solubilized. Total [3H]thymidine incorporation was measured by liquid scintillation counting. C and D, [3H]Thymidine labeling indexes. [3H]Thymidine was added to the culture medium as described above. At the end of the 3-h incubation, bone rudiments were fixed in 10% formalin and embedded in paraffin. Autoradiography was performed on 5-µm longitudinal sections by standard techniques using a 1-week exposure time. The labeling index (number of labeled nuclei/total nuclei) in the epiphyseal region (C) and in the proliferative zone (D) of the growth plate was determined by a single observer, who was blinded to the treatment category.

 
Effects of Noggin on growth plate chondrogenesis
To determine whether endogenous BMPs affect growth plate chondrogenesis, we cultured the bone rudiments in the presence of 10 µg/ml rhNoggin, a glycoprotein that binds BMP-2 and blocks its action (19). In the absence of exogenous BMP-2, Noggin reduced the metatarsal growth rate (P < 0.001; n = 22/group; Fig. 5Go), the height of the hypertrophic zone (39.9 ± 13.2 vs. 72.3 ± 5.4 µm; Noggin vs. control, P < 0.05) the number of hypertrophic cells (P < 0.001; n = 5–6; Fig. 6AGo), alkaline phosphatase expression (Fig. 3Go), and the proliferation rate in the epiphyseal region (assessed by [3H]thymidine autoradiography; P < 0.05; n = 6; Figs. 4Go and 6CGo). Total [3H]thymidine incorporation (Fig. 6BGo) and proliferation rate in the proliferative zone (Figs. 4Go and 6DGo) were also lower in Noggin-treated metatarsals than in controls, but differences did not achieve statistical significance. Noggin did not significantly affect the heights of the proliferative zone or the ossification center (data not shown). Cotreatment with 100 ng/ml BMP-2 (the lowest effective concentration) and Noggin abolished the stimulatory effects of BMP-2 on longitudinal bone growth (P < 0.001; n = 21; Fig. 5Go), chondrocyte hypertrophy (P < 0.001; n = 6; Fig. 6AGo), total thymidine incorporation (P < 0.01; n = 6; Fig. 6BGo), and proliferative rate in the epiphyseal region (P < 0.001; n = 6; Figs. 4Go and 6CGo).



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  		Figure 5. Effects of Noggin on longitudinal bone growth (mean ± SEM). Fetal rat metatarsals (e20; n = 22/group) were cultured for 3 days in serum-free medium with or without 100 ng/ml BMP-2 and/or 10 µg/ml Noggin. The lengths of the bones were measured using an eyepiece micrometer in a dissecting microscope.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In cultured fetal rat metatarsal bones, rhBMP-2 caused a concentration-dependent acceleration of longitudinal bone growth. The rate of longitudinal bone growth depends primarily on the rate of growth plate chondrogenesis. Therefore, to determine the mechanisms underlying the BMP-2-mediated growth acceleration, we studied the three principal components of growth plate chondrogenesis: chondrocyte proliferation, chondrocyte hypertrophy, and cartilage matrix synthesis.

BMP-2 caused a concentration-dependent increase in cell proliferation, as assessed by [3H]thymidine incorporation. BMP-2 also dramatically stimulated hypertrophic differentiation of growth plate chondrocytes. Addition of BMP-2 to the culture medium greatly increased the height of the hypertrophic zone, the number of hypertrophic cells, and the expression of alkaline phosphatase, an enzyme expressed by hypertrophic zone chondrocytes. The increased number of hypertrophic cells in the BMP-2-treated bones could be due either to an increase in the number of proliferative zone chondrocytes undergoing hypertrophy or a decrease in the number of hypertrophic zone chondrocytes that are replaced by bone tissue. The latter possibility appears less likely, because BMP-2 did not significantly affect the size of the ossification center. BMP-2 also did not significantly affect the size or proliferation rate of the proliferative zone, suggesting that the BMP-2-mediated increase in the number of hypertrophic cells is not due to premature chondrocyte hypertrophy, but, rather, to an increased number of epiphyseal chondrocytes entering the proliferative-hypertrophic pathway.

A stimulatory effect on glycosaminoglycan synthesis was also observed. Glycosaminoglycans represent a major component of cartilage matrix, suggesting that BMP-2 may also stimulate growth plate chondrogenesis by increasing cartilage matrix formation. However, this effect was observed only at the highest concentration of BMP-2 and thus may represent a pharmacological, rather than a physiological, effect.

To determine whether BMPs endogenous to the growth plate physiologically regulate chondrogenesis, we exposed fetal rat metatarsal bones to rhNoggin, a glycoprotein that binds BMP-2 with high affinity and blocks its activity (19, 20). In the absence of BMP-2, rhNoggin caused inhibition of bone growth, chondrocyte hypertrophy, and chondrocyte proliferation in the epiphyseal region, an area of the growth plate that contributes to longitudinal bone growth at this stage of development. These findings are consistent with our hypothesis that endogenous BMP-2 (or another member of the BMP family) acts as a positive regulator of chondrogenesis in the growth plate. As a control, we showed that rhNoggin was also able to abolish the effects of exogenous BMP-2 in this system, thus confirming that Noggin was capable of binding and neutralizing the growth factor in the concentrations and conditions present in this system. The lack of a significant effect of Noggin and exogenous BMP-2 on proliferation in the proliferative zone may suggest differential expression of BMP receptors within different regions of the growth plate.

Thus, taken together, our findings suggest that BMP-2 (or other members of the BMP family) acts as a positive regulator of growth plate chondrogenesis. This concept is supported by two other lines of evidence. First, BMP-2 is expressed in the growth plate. In the costochondral cartilage of adult rats, epiphyseal and proliferative chondrocytes express BMP-2 (21). In the mouse long bones, BMP-2 is expressed in growth plate prehypertrophic (22) and hypertrophic (23) chondrocytes. Second, implantation of BMP-2 in mammalian soft tissues results in de novo bone formation (24). The cellular events induced by the implanted BMP-2 mimic the process of endochondral ossification in the growth plate.

BMP function in the skeleton has also been studied using genetic approaches. Unfortunately, targeted deletion of the BMP-2 gene in mice provides little information on skeletogenesis, because it results in early embryonic lethality (25). Overexpression of Noggin in chicken embryonic limb buds caused shortening of the developing bones and decreased cartilage differentiation (26). These effects in the avian skeleton are consistent with the proposed stimulatory role for endogenous BMPs.

The effects of BMP-2 on chondrocytes have also been previously studied in isolated cells in culture. Although BMP-2 has consistently promoted a chondrogenic phenotype in undifferentiated mesenchymal cells (27, 28), it has produced variable effects on cells that already show a chondrocytic phenotype. In rat costochondral chondrocytes, BMP-2 consistently increased the expression of alkaline phosphatase, a marker of chondrocyte differentiation, but did not induce physical cell hypertrophy or incorporation of 35SO4. BMP-2 increased thymidine incorporation in cells cultured in serum-containing medium, but not in serum-free medium (29). In chick sternal chondrocytes, BMP-2 did not stimulate thymidine incorporation (30). In the same system, BMP-2 promoted hypertrophic differentiation only in the more mature cells, which were those derived from the cephalic portion of the sternum (30, 31). Thus, these discrepancies may arise from differences in specific culture conditions, such as medium composition, cell density, and cell origin. A cell culture system does not maintain the architecture and the cell-cell and cell-matrix interactions seen in the intact growth plate. The organ culture system employed in the current study may better reflect the activity of BMP-2 within the growth plate in vivo.

BMP-2 might affect chondrogenesis directly or indirectly through other paracrine factors expressed in the growth plate. In cultured chondrocytes, BMP-2 down-regulates the expression of PTHrP and PTH/PTHrP receptor (32, 33). Because PTHrP inhibits chondrocyte hypertrophy (34), this down-regulation of the ligand or receptor may explain the stimulatory effect of BMP-2 on hypertrophic differentiation. However, other studies suggest that BMPs may act downstream from PTHrP, with PTHrP effects on chondrocyte hypertrophy being mediated by BMPs (35, 36). In osteoblasts, BMP-2 and osteogenic protein-1 stimulate the expression of insulin-like growth factors I and II (37, 38). If similar effects occur in chondrocytes, these effects could explain the observed BMP-2-mediated stimulation of chondrocyte proliferation and hypertrophy.

Although BMPs often exhibit functional redundancy, our findings suggest a differential effect of the BMPs expressed in the growth plate on the cellular events characterizing chondrogenesis. In our study, BMP-2 stimulated both chondrocyte proliferation and chondrocyte hypertrophy. In a similar organ culture system, osteogenic protein-1 (BMP-7) inhibited cartilage hypertrophy (39). BMP-5 deficiency in short ear mice prevents age-related decelerations in chondrocyte proliferation and hypertrophy (40), suggesting a role for BMP-5 in these processes. In chick epiphyseal chondrocytes, although BMP-6 stimulated hypertrophy, it did not significantly affect proliferation (41). These different effects may reflect not only different experimental systems, but also the existence of multiple receptors (2), which appear to have distinct roles in chondrogenesis (42, 43, 44), and the spatial expression of these receptors and their ligands within the growth plate.

We conclude that BMP-2 stimulates longitudinal bone growth by increasing growth plate chondrocyte proliferation, hypertrophy, and, at high concentrations, cartilage matrix synthesis. The inhibition of bone growth, chondrocyte proliferation, and chondrocyte hypertrophy caused by a functional BMP antagonist, Noggin, suggests a stimulatory role for endogenous BMPs on growth plate chondrogenesis.


   Acknowledgments
 
We thank Genetics Institute, Inc., for providing rhBMP-2, and Regeneron Pharmaceuticals, Inc., for providing rhNoggin.


   Footnotes
 
1 Supported in part by a grant from Pharmacia & Upjohn. Back

Received May 11, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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