Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Cellular heterogeneity and insulin-like growth factor I immunoreactivity among epiphysial growth plate chondrocytes in the pig

The localization of insulin-like growth factor I (IGF-I, also called somatomedin C) production in porcine epiphysial growth plates of the distal humerus was studied by immunohistochemistry. Counterstaining with Alcian blue-van Gieson demonstrated two cell types (blue and red cells) in the germinal (reserve), proliferating and hypertrophic zones; only those chondrocytes of the proliferative and hypertrophic zones that stained red were also immunoreactive to the antibody to IGF-I. The results indicate that there exists a functional heterogeneity among the chondrocytes of both the proliferative and hypertrophic zones of growth cartilage and that IGF-I is locally produced in only the red cells of these zones. Because the red cells of the germinal zone were not immunoreactive, the results suggest that the red cells of the germinal zone and the red cells of the proliferative and hypertrophic zones are also functionally distinct.     

Immunolocation analysis of glycosaminoglycans in the human growth plate.

Monoclonal antibodies were used in this study to immunolocate glycosaminoglycans throughout the human growth plate. Chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate were observed in the extracellular matrix of all zones of the growth plate and persisted into the cartilage trabeculae of newly formed metaphyseal bone. Also present in the extracellular matrix was an oversulfated chondroitin/dermatan sulfate glycosaminoglycan which appeared to be specific to the proliferative and hypertrophic zones of the growth plate. As with the other extracellular matrix molecules, this epitope persisted into the cartilage trabeculae of the metaphyseal bone. Zonal differences between the extracellular and pericellular or lacunae matrix were also observed. The hypertrophic chondrocytes appeared to synthesize chondroitin sulfate chains containing a non-reducing terminal 6-sulfated disaccharide, which were located in areas immediately adjacent to the cells. This epitope was not found to any significant extent in the other zones. The pericellular region around hypertrophic chondrocytes also contained a keratan sulfate epitope which was also observed in the resting zone but not in the proliferative zone. These cell-associated glycosaminoglycans were not found in the cartilage trabeculae of metaphyseal bone, indicating their removal as the terminal hypertrophic chondrocytes and their lacunae are removed by invading blood vessels. These changes in matrix glycosaminoglycan content, both in the different zones and within zones, indicate constant subtle alterations in chondrocyte metabolic products as they proceed through their life cycle of proliferation, maturation, and hypertrophy.     

Differentiation and mineralization in chick chondrocytes maintained in a high cell density culture: A model for endochondral ossification

Summary Chondrocytes isolated from the proliferative and differentiating zones of 3-wk-old chick growth plates were cultured in the presence of 10% fetal bovine serum (FBS) and ascorbic acid for up to 21 d in a high cell density culture within Eppendorf tubes. The proliferative, differentiating, and calcification properties of the chondrocytes were examined by immunolocalization and by enzyme histochemical and biochemical methods. The cells maintained a chondrocyte phenotype throughout culture: they were round in shape and synthesized both collagen type II and proteoglycans. The expression of a hypertrophic phenotype was evident by Day 3 of culture and from this time onwards characteristics of terminal differentiation were observed. The cells were positive for both alkaline phosphatase (ALP) activity and c-myc protein and the surrounding matrix stained strongly for collagen type X. Small foci of mineralization associated with individual chondrocytes were first evident by Day 6 and more widespread areas of mineralization occupying large areas of matrix were present by Day 15. Mineralization occurred without the addition of exogenous phosphate to the medium. This culture system displays characteristics that are similar in both morphological and developmental terms to that of chick chondrocyte differentiation and calcification in vivo and therefore offers an excellent in vitro model for endochondral ossification.     

Parathyroid hormone-related peptide expression in the epiphyseal growth plate of the juvenile chicken: evidence for the origin of the parathyroid hormone-related peptide found in the epiphyseal growth plate

Parathyroid hormone-related peptide (PTHrP) has been shown to be essential for normal endochondral bone formation. Along with Indian hedgehog (Ihh), it forms a paracrine regulatory loop that governs the pace of chondrocyte differentiation. However, the source of PTHrP for this regulatory loop is not clear. While one hypothesis has suggested the periarticular perichondrium as the source of PTHrP for growth plate regulation, other data utilizing immunohistochemistry and in situ hybridization would indicate that growth plate chondrocytes themselves are the source of this peptide. The data described in this report supports the view that postnatal growth plate chondrocytes have the ability to synthesize this important regulatory peptide. Immunohistochemistry of tissue sections showed that PTHrP protein was evident throughout the chick epiphysis. PTHrP was seen in chondrocytes in the periarticular perichondrium, the perichondrium adjacent to the growth plate, the prehypertrophic zone of the growth plate, and the hypertrophic zone of the growth plate. However, cells in the proliferative zone, as well as some chondrocytes in the deeper layers of articular cartilage were predominantly negative for PTHrP. PTHrP was detected by Western blotting as a band of 16,400 Da in extracts from hypertrophic chondrocytes, but not from proliferative cells. RT-PCR detected PTHrP mRNA in both proliferative and hypertrophic growth plate chondrocytes, as well as in articular chondrocytes. PTH/PTHrP receptor mRNA was detected by Northern blotting in growth plate, but not articular chondrocytes. Thus, we conclude that most of the PTHrP present in the epiphyseal growth plate of the juvenile chick originates in the growth plate itself. Furthermore, the presence of large amounts of PTHrP protein in the hypertrophic zone supports the concept that PTHrP has other functions in addition to regulating chondrocyte differentiation.     

PTHrP,PTHr, and FGFR3 are involved in the process of endochondral ossification in human osteophytes

To elucidate the process of endochondral ossification in human osteophytes we have studied the expression of parathyroid hormone-related protein (PTHrP), its receptor (PTHr), and fibroblast growth factor receptor 3 (FGFR3). Osteophytes from patients undergoing total knee replacement ( n=13), and fetal growth plate cartilages ( n=4) were processed for safranin O staining and immunohistochemistry. Chondrocytes and their matrix were preferentially stained for PTHrP in the middle and deep zones of the osteophytes examined. Ossified areas did not show a positive staining. In fetal joints the cartilaginous surface and the perichondrium as well as the osteoblasts in the trabecular bone were positive. PTHr was expressed at large in chondrocytes and osteoblasts of all osteophytes and fetal joints. Cells of the perichondrium were also positive. The FGFR3 antibody stained only single chondrocytes in some osteophytes, and groups of cells in others. In fetal samples, chondrocytes of the proliferating and the hypertrophic zone showed staining for FGFR3. This is the first report on the expression of PTHrP, PTHr, and FGFR3 in human osteophytes. As in fetal joints these mediators might regulate proliferation and differentiation of chondrocytes playing an important role in osteo(chondro)phyte growth.     

Estrogen receptor-alpha and estrogen receptor-beta are present in the human growth plate in childhood and adolescence,in identical distribution

OBJECTIVE: To localize estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) within the growth plate and adjacent bony tissue of children in the prepubertal and pubertal age period. METHODS: Tissue was taken during orthopedic surgery (epiphysiodesis) for correction of congenital or traumatic leg length difference in 2 prepubertal females and 2 adolescent males. Immunohistochemistry was performed on paraffin-embedded or cryostat sections by using commercially available rabbit polyclonal antibodies for ER-alpha and ER-beta. RESULTS: Both ER-alpha and ER-beta were detected within the growth plate in all sections investigated. Immunostaining was restricted to hypertrophic chondrocytes. In the bony tissue adjacent to the growth plate, osteoblasts stained positive for both ER-alpha and ER-beta, whereas osteocytes and osteoclasts were negative. Staining with ER-alpha was mainly nuclear but some cells also showed cytoplasmic signals, while ER-beta staining was predominantly cytoplasmic, only few nuclei stained positive. There was no difference in the local distribution of both ERs between tissue from prepubertal and pubertal patients. CONCLUSION: Our findings indicate that the hypertrophic chondrocyte is the main target cell for estrogen action within the growth plate. The presence of ER in prepubertal children suggests that estrogens play a role in skeletal maturation under physiological conditions also in this age-group.     

Tissue and cellular morphological changes in growth plate explants under compression

The mechanisms by which mechanical loading may alter bone development within growth plates are still poorly understood. However, several growth plate cell or tissue morphological parameters are associated with both normal and mechanically modulated bone growth rates. The aim of this study was to quantify in situ the three-dimensional morphology of growth plate explants under compression at both cell and tissue levels. Growth plates were dissected from ulnae of immature swine and tested under 15% compressive strain. Confocal microscopy was used to image fluorescently labeled chondrocytes in the three growth plate zones before and after compression. Quantitative morphological analyses at both cell (volume, surface area, sphericity, minor/major radii) and tissue (cell/matrix volume ratio) levels were performed. Greater chondrocyte bulk strains (volume decrease normalized to the initial cell volume) were found in the proliferative (35.4%) and hypertrophic (41.7%) zones, with lower chondrocyte bulk strains (24.7%) in the reserve zone. Following compression, the cell/matrix volume ratio decreased in the reserve and hypertrophic zones by 24.3% and 22.6%, respectively, whereas it increased by 35.9% in the proliferative zone. The 15% strain applied on growth plate explants revealed zone-dependent deformational states at both tissue and cell levels. Variations in the mechanical response of the chondrocytes from different zones could be related to significant inhomogeneities in growth plate zonal mechanical properties. The ability to obtain in situ cell morphometry and monitor the changes under compression will contribute to a better understanding of mechanisms through which abnormal growth can be triggered.     

Expression of Bcl-2 protein in the epiphyseal plate cartilage and trabecular bone of growing rats

 The protooncogene protein, Bcl-2, protects cells from apoptosis and ensures their survival in vitro by inhibiting the action of the apoptosis-inducer, Bax. Its expression in proliferative and long-lived cells in vivo also indicates that it protects against cell death. The chondrocytes of the epiphyseal plate cartilage undergo a series of maturation steps and deposit mineral in the cartilage matrix before dying. The possibility that Bcl-2 helps protect chondrocytes until mineral deposition is completed was investigated by determining the distribution of Bcl-2 immunoreactivity in the epiphyseal plate cartilage of growing rats and its subcellular localization, using a specific antibody. The involvement of Bax in the triggering of chondrocyte death was checked by immunocytochemistry. Bcl-2 expression in the osteoblasts and the final result of their evolution, the osteocytes, was also examined in trabecular bone. Bcl-2 immunoreactivity was non-uniformly distributed throughout the epiphyseal cartilage. It was maximal in proliferative chondrocytes, decreased in mature chondrocytes, and low in hypertrophic chondrocytes, whereas there was Bax immunoreactivity in all chondrocytes examined. Immunolabeling was intense in osteoblasts but considerably lower in fully differentiated osteocytes. Bcl-2 immunoreactivity was mainly in the cytoplasm of chondrocytes, osteoblasts, and early osteocytes; the nuclei appeared clear. The subcellular distribution of Bcl-2 immunolabeling in chondrocytes, revealed by gold particles in the electron microscope, showed that gold particles were frequently concentrated in the mitochondria in all the cartilage zones and lay mainly within the organelles, not at their periphery. The endoplasmic reticulum contained moderate immunoreactivity and there were few gold particles in the cytoplasm and nuclei. The number of gold particles decreased in all the subcellular compartments from proliferative to hypertrophic chondrocytes. In contrast, Bax immunoreactivity changed little during chondrocyte terminal evolution, and its subcellular distribution mirrored that of Bcl-2. These immunocytochemical data indicate that Bcl-2 helps maintain chondrocytes and osteoblasts until their terminal maturation. Accepted: 19 February 1997     

Use of microarray analysis to study gene expression in the avian epiphyseal growth plate

Longitudinal bone growth depends upon the execution of an intricate series of cellular activities by epiphyseal growth plate chondrocytes. In order to better understand these coordinated events, microarray analysis was used to compare gene expression in chondrocytes isolated from the proliferative and hypertrophic zones of the avian growth plate. RT-PCR was used to confirm the identity of a select number of genes. The expression of 745 genes was found to differ 3-fold or greater at the 0.05 level of probability. Transferrin was the most highly up-regulated (321-fold) gene associated with chondrocyte hypertrophy. Immunohistochemistry localized this peptide adjacent to the penetrating blood vessels in the growth plate of 3-week-old chicks. Fibulin, OC-116, DMP-1 and PHEX were among the expanded number of genes associated with extracellular matrix metabolism. The presence of NELL2, ATOH8 and PLEXIN suggests a neuronal involvement in growth plate physiology. In addition, the expression of a large number of genes associated with angiogenesis and cellular stress was up-regulated. These processes are important to the physiology and survival of chondrocytes in the unique and stressful environment of the epiphyseal growth plate.     

Sexual dimorphism of growth plate prehypertrophic and hypertrophic chondrocytes in response to testosterone requires metabolism to dihydrotestosterone (DHT) by steroid 5-alpha reductase type 1

Rat costochondral growth plate chondrocytes exhibit sex-specific and cell maturation dependent responses to testosterone. Only male cells respond to testosterone, although testosterone receptors are present in both male and female cells, suggesting other mechanisms are involved. We examined the hypothesis that the sex-specific response of rat costochondral cartilage cells to testosterone requires further metabolism of the hormone to dihydrotestosterone (DHT). Resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) chondrocytes from male and female Sabra strain rats exhibited sex-specific responses to testosterone and DHT: only male cells were responsive. Testosterone and DHT treatment for 24 h caused a comparable dose-dependent increase in [3H]-thymidine incorporation in quiescent preconfluent cultures of male GC cells, and a comparable increase in alkaline phosphatase specific activity in confluent cultures. RC cells responded in a differential manner to testosterone and DHT. Testosterone decreased DNA synthesis in male RC cells but DHT had no effect and alkaline phosphatase specific activity of male RC cells was unaffected by either hormone. Inhibition of steroid 5alpha-reductase activity with finasteride (1, 5, or 10 microg/ml), reduced the response of male GC cells to testosterone in a dose-dependent manner, indicating that metabolism to DHT was required. RT-PCR showed that both male and female cells expressed mRNAs for steroid 5alpha-reductase type 1 but lacked mRNAs for the type 2 form of the enzyme. Male cells also exhibited 5alpha-reductase activity but activity of this enzyme was undetectable in female cells. These observations show that sex-specific responses of rat growth zone chondrocytes to testosterone requires the further metabolism of the hormone to DHT and that the effect of DHT in the male growth plate is maturation-state dependent. Failure of female chondrocytes to respond to testosterone may reflect differences in testosterone metabolism, since these cells possess greater ability to aromatize the hormone to estradiol.     

Development of the terminally differentiated state sensitizes epiphyseal chondrocytes to apoptosis through caspase-3 activation

The maturation of epiphyseal chondrocytes is accompanied by dramatic changes in energy metabolism and shifts in proteins concerned with the induction of apoptosis. We evaluated the role of mitochondria in this process by evaluating the membrane potential (Delta psi m) of chondrocytes of embryonic tibia and the epiphyseal growth plate. We observed that there was a maturation-dependent change in fluorescence, indicating a fall in the Delta psi m. The level of mitochondrial Bcl-2 was decreased during maturation, while in the same time period there was an obvious increase in Bax levels in the mitochondrial fraction of the terminally differentiated chondrocytes. Bcl(xL), another anti-apoptotic protein, was also robustly expressed in the mitochondrial fraction, but its expression was not dependent on the maturation status of the chondrocytes. We found that caspase-3 was present throughout the growth plate and in hypertrophic cells in culture. We blocked caspase-3 activity and found that alkaline phosphatase staining and mineral formation was decreased, and the cells had lost their characteristic shape. Moreover, we noted that the undifferentiated cells were insensitive to elevated concentrations of inorganic phosphate (Pi). It is concluded that during hypertrophy, the change in membrane potential, the increased binding of a pro-apoptotic protein to mitochondria, and the activation of caspase-3 serve to prime cells for apoptosis. Only when the terminally differentiated chondrocytes are challenged with low levels of apoptogens there is activation of apoptosis.     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ--prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.