A new histomorphometric method to assess growth plate chondrodysplasia and its application to the toothless (tl, Csf1(null)) osteopetrotic rat

The proliferation and hypertrophy of growth plate chondrocytes set the pace and pattern for growth of endochondral bones. Complex signaling pathways regulating chondrocyte differentiation during development and growth have been discovered in recent years, but as yet little is known about how chondrocytes are able to orient themselves to align properly with respect to the direction of bone growth. Histomorphometric methods developed for analysis of growth plates rely to a significant extent on assessments of the relative heights of the zones of proliferating and hypertrophic chondrocytes. In a growing number of osteopetrotic mutations, however, it is becoming apparent that growth plates lack clearly demarcated zones of chondrocyte differentiation, and they show other notable histological abnormalities that cannot be measured with standard approaches. This is particularly true of mutations in which osteoclasts are altogether absent. We therefore developed a new approach that measures the salient features of this type of chondrodysplasia and have applied it to the toothless (tl) rat. The tl rat has a frameshift mutation in the Csf-1 gene that renders it null, resulting in severe osteopetrosis. An accompanying pathology is a severe, progressive growth plate chondrodysplasia. We measured cell orientation, cell area, and local columnar organization as functions of distance from the upper margin of the growth plate, in addition to growth plate thickness and cell density. All these parameters were markedly abnormal in the tl rats, thus implicating Csf-1 not only in its well-established role in regulating osteoclastic bone resorption, but also in endochondral ossification. This approach should prove useful in distinguishing among growth plate chondrodysplasias, most especially in the growing number of osteopetrotic mutations having growth plates that lack the normal zonal organization and in which the chondrocytes are mis-oriented. In turn, detailed assessments of chondrocyte misorientation may give insights into how they normally are able to arrange themselves with such precision.     

Avian dyschondroplasia: Local deficiencies in growth factors are integral to the aetiopathogenesis

Rabbit polyclonal antibodies to chicken transforming growth factor-beta 3 (TGF-/J3) and to insulin-like growth factor-I (IGF-I) were used to detect the two growth factors in normal and dyschondroplastic broiler growth plates (physes). Histo-morphometry was used to estimate the percentage of chondrocytes containing IGF-I and TGF-beta3 in the lower proliferative, transitional and hypertrophic zones of the growth plate. In the normal chick growth plates IGF-I was present in 63% of transitional chondrocytes and in 67% of hypertrophic chondrocytes and TGF-beta3 was present in 81% of transitional chondrocytes and in 93% of hypertrophic chondrocytes. Both growth factors were found to be deficient within transitional chondrocytes at sites of dyschondroplasia, a condition in which there is a local defect in chondrocyte differentiation and the subsequent replacement of the cartilage by bone. In addition, both growth factors were identified in chondrocytes within areas of repair, where chondrocyte differentiation and endochondral ossification have resumed. This supports the hypothysis that the reduction in TGF-beta3 and IGF-I in dyschondroplasia is integral to the aetiopathogenesis and indicative that both these growth factors are part of the cascade of events associated with chondrocyte differentiation during endochondral ossification.     

QRICH1 mutations cause a chondrodysplasia with developmental delay

In many children with short stature, the etiology of the decreased linear growth remains unknown. We sought to identify the underlying genetic etiology in a patient with short stature, irregular growth plates of the proximal phalanges, developmental delay, and mildly dysmorphic facial features. Exome sequencing identified a de novo, heterozygous, nonsense mutation (c.1606C>T:p.R536X) in QRICH1. In vitro studies confirmed that the mutation impaired expression of the QRICH1 protein. SiRNA-mediated knockdown of Qrich1 in primary mouse epiphyseal chondrocytes caused downregulation of gene expression associated with hypertrophic differentiation. We then identified an unrelated individual with another heterozygous de novo nonsense mutation in QRICH1 who had a similar phenotype. A recently published study identified QRICH1 mutations in three patients with developmental delay, one of whom had short stature. Our findings indicate that QRICH1 mutations cause not only developmental delay but also a chondrodysplasia characterized by diminished linear growth and abnormal growth plate morphology due to impaired growth plate chondrocyte hypertrophic differentiation.     

Perspective. Osteoclastogenesis and growth plate chondrocyte differentiation: emergence of convergence

A "bone" is really a dynamic and highly interactive complex of many cell and tissue types. In particular, for the majority of skeletal elements to develop and grow, the process of endochondral ossification requires a constantly moving interface between cartilage, invading blood vessels, and bone. A great deal has been learned in recent years about the regulation of chondrocyte proliferation and differentiation by hormones, growth factors, and physiologic stimuli during skeletal development and growth. Likewise, the discovery that colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-kappaB ligand (RANKL, a tumor necrosis factor superfamily member also called TRANCE, ODF, OPGL, and TNFSF11) are pivotal in communicating from osteoblasts to osteoclasts has led to deeper insights into bone growth, turnover, and maintenance. Little is known, however, about how these two quite different systems communicate to solve the problem of providing integrated, continuous mechanical support during the dynamic invasion of cartilage by bone that characterizes endochondral bone growth. Evidence has accumulated in recent years that provides insight into the communication between growing bone and cartilage in the form of a subset of osteopetrotic mutations, which share a lack of osteoclasts and an accompanying chondrodysplasia of the growth plate. These mutations thus implicate some of the same gene products in regulating chondrocyte differentiation and bone resorption. We also consider expression studies of some known growth plate regulators, such as parathyroid hormone-related protein (PTHrP) and Indian hedgehog (Ihh), in light of this and propose a model in which the osteoclastogenic factors act also on chondrocytes, but downstream of PTRrP and Ihh in regulating proliferation and differentiation, and after early morphogenic patterns are established.     

Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18

Gain of function mutations in fibroblast growth factor (FGF) receptors cause chondrodysplasia and craniosynostosis syndromes. The ligands interacting with FGF receptors (FGFRs) in developing bone have remained elusive, and the mechanisms by which FGF signaling regulates endochondral, periosteal, and intramembranous bone growth are not known. Here we show that Fgf18 is expressed in the perichondrium and that mice homozygous for a targeted disruption of Fgf18 exhibit a growth plate phenotype similar to that observed in mice lacking Fgfr3 and an ossification defect at sites that express Fgfr2. Mice lacking either Fgf18 or Fgfr3 exhibited expanded zones of proliferating and hypertrophic chondrocytes and increased chondrocyte proliferation, differentiation, and Indian hedgehog signaling. These data suggest that FGF18 acts as a physiological ligand for FGFR3. In addition, mice lacking Fgf18 display delayed ossification and decreased expression of osteogenic markers, phenotypes not seen in mice lacking Fgfr3. These data demonstrate that FGF18 signals through another FGFR to regulate osteoblast growth. Signaling to multiple FGFRs positions FGF18 to coordinate chondrogenesis in the growth plate with osteogenesis in cortical and trabecular bone.     

MORPHOLOGICAL AND BIOCHEMICAL EVIDENCE FOR APOPTOSIS IN THE TERMINAL HYPERTROPHIC CHONDROCYTES OF THE GROWTH PLATE

The purpose of this study was to investigate the mechanism of cell death in chondrocytes of the growth plate. In the degenerative chondrocyte zone of the growth plate, apoptotic chondrocytes were defeated by the in situ nick end labelling method, by DNA analysis in agarose gel, and by electron microscopy. The results of the in situ nick end labelling method and the occurrence of a ladder pattern of DNA in agarose gel analysis indicated the activation of endogenous endonucleases, resulting in DNA fragmentation. Electron micrographs showed the early morphological changes associated with apoptosis. This report presents both morphological and biochemical evidence for apoptosis in the terminal hypertrophic chondrocytes of the growth plate. These data suggest that apoptosis of degenerative chondrocytes may play an important role in the control of normal and pathological endochondral ossification.     

The Effect of Mechanical Loading on the Metabolism of Growth Plate Chondrocytes

It is well known that mechanical loading influences the endochondral bone formation essential for the growth and development of longitudinal bones. The question was, however, asked whether the effect of mechanical loading on the chondrocyte metabolism is dependent on the loading frequency. This study was aimed at evaluating the effect of tensile loadings with various frequencies on the proliferation of growth plate chondrocytes and extracellular matrix synthesis. The chondrocytes obtained from rib growth plate cartilage of 4-week-old male Wistar strain rats were cultured by day 4 and day 11 and used as proliferating and matrix-forming chondrocytes, respectively. Intermittent tensile stresses with different frequencies were applied to each stage chondrocyte. DNA syntheses were examined by measuring the incorporation of [³H]thymidine into the cells. Furthermore, the rates of collagen and proteoglycan syntheses were determined by measuring the incorporation of [2,3-³H]proline and [³⁵S]sulfate into the cells, respectively. At the proliferating stage, intermittent tensions with the frequencies of 30 cycles/min and 150 cycles/min significantly (p < 0.05) upregulated the syntheses of DNA, which indicates the promotion of chondrocyte proliferation. At the matrix-forming stage, collagen, and proteoglycan syntheses also enhanced with increase of the loading frequency. In particular, the intermittent tension with the frequencies of 30 cycles/min and 150 cycles/min increased significantly (p < 0.05 or p < 0.01) both the collagen and proteoglycan syntheses. These results suggest that the proliferation and differentiation of growth plate chondrocytes are regulated by the mechanical loading and that the chondrocyte metabolism enhanced with increase of loading frequency. These may give more insight into the possible mechanism leading to endochondral bone formation.     

Septoclast Deficiency Accompanies Postnatal Growth Plate Chondrodysplasia in the Toothless (tl) Osteopetrotic,Colony-Stimulating Factor-1 (CSF-1)-Deficient Rat and Is Partially Responsive to CSF-1 Injections

The septoclast is a specialized, cathepsin B-rich, perivascular cell type that accompanies invading capillaries on the metaphyseal side of the growth plate during endochondral bone growth. The putative role of septoclasts is to break down the terminal transverse septum of growth plate cartilage and permit capillaries to bud into the lower hypertrophic zone. This process fails in osteoclast-deficient, osteopetrotic animal models, resulting in a progressive growth plate dysplasia. The toothless rat is severely osteopetrotic because of a frameshift mutation in the colony-stimulating factor-1 (CSF-1) gene (Csf1^tl). Whereas CSF-1 injections quickly restore endosteal osteoclast populations, they do not improve the chondrodysplasia. We therefore investigated septoclast populations in Csf1^tl/Csf1^tl rats and wild-type littermates, with and without CSF-1 treatment, at 2 weeks, before the dysplasia is pronounced, and at 4 weeks, by which time it is severe. Tibial sections were immunolabeled for cathepsin B and septoclasts were counted. Csf1^tl/Csf1^tl mutants had significant reductions in septoclasts at both times, although they were more pronounced at 4 weeks. CSF-1 injections increased counts in wild-type and mutant animals at both times, restoring mutants to normal levels at 2 weeks. In all of the mutants, septoclasts seemed misoriented and had abnormal ultrastructure. We conclude that CSF-1 promotes angiogenesis at the chondroosseous junction, but that, in Csf1^tl/Csf1^tl rats, septoclasts are unable to direct their degradative activity appropriately, implying a capillary guidance role for locally supplied CSF-1.The majority of skeletal elements, including limb bones, the vertebral column, ribs, pelvis, digits, and the posterior mandible, grow via the process of endochondral ossification in which a cartilage anlage, or model, is first formed and subsequently replaced by bone.^1,2 This is necessitated by the fact that bone, which is rigid, cannot expand interstitially, but can only grow at its surface by bone deposition. To protect the growing cartilage from the rigors of excessive mechanical loading, a secondary ossification center develops to support the articular cartilage of the joint, and the growth cartilage forms into a growth plate across the end of the bone shaft, separating the epiphysis from the metaphysis.^3,4 Chondrocytes in the growth plate proliferate, undergo expansion, or hypertrophy, and drive bone elongation. This process has been well studied and is regulated by complex interactions among many growth factors and morphogens, including Indian Hedgehog, parathyroid hormone-related protein, insulin-like growth factors, and other regulatory pathway components (reviewed in 5,6). The growth cartilage is ultimately resorbed and replaced by bone. This conversion from cartilage to bone takes place along the chondroosseous junction (COJ), and it requires that cartilage, which is avascular, be invaded by blood vessels that carry with them bone-resorbing and bone-forming cells.Chondrocytes of the growth plate are normally highly organized spatially with respect to cell differentiation. The uppermost cell population is called the resting zone. Its cells are nonproliferative and serve as a reserve pool for future growth. The cells of the proliferating zone are immediately subjacent, typically occupy roughly the upper half of the growth plate, and are active in the cell cycle and at the same time are secreting significant amounts of cartilage matrix. After several days, the cells cease proliferating, switch from expression of type II to type X collagen, and begin to swell, or become hypertrophic, expanding up to 10 times in height within 1 to 2 days.^7,8,9 They also secrete matrix vesicles, which mineralize the matrix. Ultimately, capillaries invade from the metaphyseal side, and osteoclasts (also called “chondroclasts” at this location) attach to and consume roughly two of three of the longitudinal cartilage septa, the vertical walls that divide the chondrocyte columns. The remaining longitudinal septa comprise the substrate onto which osteoblasts deposit the bone of the primary spongiosa. During rapid growth, this area is the site of intense metabolic activity, with bone resorption and formation taking place within a few micrometers of each other. Added to these complex metabolic processes are the growth of vasculature and establishment of hematopoietic marrow.The septoclast is a specialized, perivascular cell that facilitates capillary invasion of the chondroosseous junction.¹⁰ Septoclasts are rich in the protease cathepsin B, and they are located such that the cell body and nucleus lie just behind the budding end of the capillary. Their cytoplasm extends toward the growth plate and takes on a ruffled border-like appearance adjacent to the terminal transverse septum of the growth plate. It has been inferred that this cell type is required for the continued growth of blood vessels into the growth plate, thus permitting normal bone elongation to occur.¹⁰In some animal models of osteopetrosis, a condition in which defective bone resorption by osteoclasts leads to a sclerotic skeleton, we and others have noted an accompanying pathological change in the growth plate. The toothless (Csf1^tl/Csf1^tl) osteopetrotic rat has a recessive, loss-of-function frameshift mutation in the gene for colony-stimulating factor-1 (CSF-1 or macrophage-CSF).^11,12 The loss renders the Csf1^tl/Csf1^tl rat severely osteopetrotic because of profound osteoclastopenia. In this strain, as in a growing list of osteoclast-deficient mutant mice,¹³ there is a progressive dysplasia in which chondrocytes fail to form their normal columns and differentiation zones, the central region of the growth plate thickens with time, and vascular invasion at the COJ is deficient.^14,15,16,17 Injections of CSF-1 restore osteoclast populations and activity in the Csf1^tl/Csf1^tl rat and rescue some, but not all, aspects of the mutant phenotype.^15,18 Notably, the growth plate dysplasia remains unimproved despite daily CSF-1 injections beginning at birth, before the dysplasia develops, and continuing for 6 weeks.¹⁵ The lower margin of the growth plate, rather than being a site for vessel budding and bone and cartilage remodeling, becomes covered with a layer of bone and the vessels are blunted and enlarged and do not penetrate the cartilage. Despite this lack of effect on the chondrodysplasia, it was shown that CSF-1 injections have a stimulatory effect on angiogenesis in the metaphysis of Csf1^tl/Csf1^tl rats as measured by vascular casts and electron microscopy,¹⁴ at least in the short term.CSF-1 is known to promote angiogenesis, primarily through the stimulation of VEGF secretion.¹⁹ Depletion of VEGF has been shown to block vascularization of the growth plate.²⁰ Because of the association between septoclasts and vascular invasion of the growth plate, we wished to investigate septoclast populations in Csf1^tl/Csf1^tl rats and their possible response to CSF-1 treatment. We also wished to further investigate the septoclasts themselves in normal animals and to document their relation to other cell types at the COJ, because relatively little is presently known about them. We report here the results of histological, immunohistochemical, and electron microscopic investigations of this specialized perivascular cell population in wild-type and Csf1^tl/Csf1^tl mutant rats with and without CSF-1 treatments.     

Expression of type X collagen,Indian hedgehog and parathyroid hormone related-protein in normal and tibial dyschondroplastic chick growth plates

Tibial dyschondroplasia (TD) is a form of aberrant endochondral ossification in chickens, in that a plug of avascular cartilage (TD lesion) is formed within the growth plate. Histologically, the lesion is filled with apparently transitional chondrocytes that have been unable to differentiate to hypertrophic chondrocytes. We have examined the spatial expression of mRNAs for type X collagen, Indian hedgehog (Ihh) and Parathyroid Hormone-related protein (PTHrP) in the TD growth plate by in situ hybridization in order to ascertain at which stage chondrocyte differentiation is arrested in TD. In the normal growth plate, type X collagen mRNA was expressed by both prehypertrophic and hypertrophic chondrocytes. Indian Hedgehog mRNA was detected in a band of prehypertrophic chondrocytes and PTHrP expression was localized to a narrow band of prehypertrophic chondrocytes and in osteoblasts within the diaphysis. In TD sections, collagen X expression was seen within differentiating cells, within a small number of lesion cells, and within hypertrophic chondrocytes on the diaphyseal side of the lesion. Ihh expression was also seen within the differentiating cells and throughout the lesion. These data indicate that chondrocyte differentiation is arrested at the transitional stage just prior to hypertrophy. Contrary to the previously reported PTHrP expression patterns in TD chicks by immunohistochemistry, PTHrP mRNA was not detected in the TD lesion. This observation probably reflects the cessation of PTHrP gene expression by chondrocytes in the more severe TD lesions. The results from the present study also imply that the arrest of cell differentiation in TD is independent of PTHrP and that endochondral ossification in the post-hatch avian growth plate may involve additional regulatory pathways.     

Peripheral chondrosarcoma progression is associated with increased type X collagen and vascularisation

Endochondral bone formation requires a cartilage template, known as the growth plate, and vascular invasion, bringing osteoblasts and osteoclasts. Endochondral chondrocytes undergo sequences of cell division, matrix secretion, cell hypertrophy, apoptosis, and matrix calcification/mineralisation. In this study, two critical steps of endochondral bone formation, the deposition of collagen X-rich matrix and blood vessel attraction/invasion, were investigated by immunohistochemistry. Fourteen multiple osteochondromas and six secondary peripheral chondrosarcomas occurring in patients with multiple osteochondromas were studied and compared to epiphyseal growth plate samples. Mutation analysis showed all studied patients (expect one) to harbour a germ-line mutations in either EXT1 or EXT2. Here, we described that homozygous mutations in EXT1/EXT2, which are causative for osteochondroma formation, are likely to affect terminal chondrocyte differentiation and vascularisation in the osteocartilaginous interface. Contrastingly, terminal chondrocyte differentiation and vascularisation seem to be unaffected in secondary peripheral chondrosarcoma. In addition, osteochondromas with high vascular density displayed a higher proliferation rate. A similar apoptotic rate was observed in osteochondromas and secondary peripheral chondrosarcomas. Recently, it has been shown that cells with functional EXT1 and EXT2 are outnumbering EXT1/EXT2 mutated cells in secondary peripheral chondrosarcomas. This might explain the increased type X collagen production and blood vessel attraction in these malignant tumours.     

Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype

We generated transgenic mice that express a constitutively active mutant of MEK1 in chondrocytes. These mice showed a dwarf phenotype similar to achondroplasia, the most common human dwarfism, caused by activating mutations in FGFR3. These mice displayed incomplete hypertrophy of chondrocytes in the growth plates and a general delay in endochondral ossification, whereas chondrocyte proliferation was unaffected. Immunohistochemical analysis of the cranial base in transgenic embryos showed reduced staining for collagen type X and persistent expression of Sox9 in chondrocytes. These observations indicate that the MAPK pathway inhibits hypertrophic differentiation of chondrocytes and negatively regulates bone growth without inhibiting chondrocyte proliferation. Expression of a constitutively active mutant of MEK1 in chondrocytes of Fgfr3-deficient mice inhibited skeletal overgrowth, strongly suggesting that regulation of bone growth by FGFR3 is mediated at least in part by the MAPK pathway. Although loss of Stat1 restored the reduced chondrocyte proliferation in mice expressing an achondroplasia mutant of Fgfr3, it did not rescue the reduced hypertrophic zone, the delay in formation of secondary ossification centers, and the achondroplasia-like phenotype. These observations suggest a model in which Fgfr3 signaling inhibits bone growth by inhibiting chondrocyte differentiation through the MAPK pathway and by inhibiting chondrocyte proliferation through Stat1.     

Skeletal biology in the toothless (osteopetrotic) rat

The toothless (tl) rat is a nonlethal osteopetrotic mutation characterized by the presence of few osteoclasts and the failure to be cured by bone-marrow transplantation. We examined the skeletal biology of tl rats and normal littermates up to 6 weeks after birth. Osteoclasts in tl rats were small, reduced 25-fold in number, and had greatly reduced concentrations of acid hydrolases. Bone shape internally and externally reflected reduced bone resorption, and tl rats were hypophosphatemic and mildly hypocalcemic at 2 weeks. These data indicate that the basic defect in tl rats is one of differentiation of osteoclasts and, coupled with the observation that normal bone-marrow cells cannot develop into osteoclasts in the tl skeleton, suggest that the defect lies in the skeletal microenvironment.     

Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage

Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate.     

Transforming growth factor beta: an autocrine regulator of chondrocytes

Transforming growth factor beta (TGF-beta) is a ubiquitous regulator of cellular growth and differentiation. The present study investigated the effects of TGF-beta on chick growth plate chondrocyte proliferation, matrix synthesis, and alkaline phosphatase activity in short term cultures. TGF-beta markedly stimulated DNA synthesis in a dose-dependent manner, while collagen synthesis and cellular and matrix vesicle alkaline phosphatase activity were inhibited. Biologic effects of TGF-beta were correlated with binding to specific receptors, and both high and low affinity receptors were identified. Countercurrent centrifugal elutriation was used to fractionate growth plate chondrocytes to obtain populations of cells in different stages of maturation (effectively from different zones of the growth plate). TGF-beta showed increasing mitogenicity with increasing cellular maturation in the growth plate, with maximal stimulation in the proliferating and early hypertrophic cells. The smallest cells expressed only the high affinity receptor, while with hypertrophy there was increasing expression of the low affinity receptor and a progressive increase in the number of both receptors per cell. Furthermore, the dose-response curves for TGF-beta-stimulated DNA synthesis were not biphasic in the smaller cells, but became progressively more biphasic with cellular hypertrophy and expression of the low affinity receptor. Finally, TGF-beta activity was identified in partially purified chondrocyte conditioned medium by specific bioassay, indicating TGF-beta production by growth plate chondrocytes. The data suggests a potentially important autocrine function for TGF-beta in modulating chondrocyte proliferation and matrix synthesis in endochondral calcification.     

Reduced cell proliferation and increased apoptosis are significant pathological mechanisms in a murine model of mild pseudoachondroplasia resulting from a mutation in the C-terminal domain of COMP

Pseudoachondroplasia (PSACH) is one of the more common skeletal dysplasias and results from mutations in cartilage oligomeric matrix protein (COMP). Most COMP mutations identified to date cluster in the TSP3 repeat region of COMP and the mutant protein is retained in the rough endoplasmic reticulum (rER) of chondrocytes and may result in increased cell death. In contrast, the pathomolecular mechanism of PSACH resulting from C-terminal domain COMP mutations remain largely unknown. This study describes the generation and analysis of a murine model of mild PSACH resulting from a p.Thr583Met mutation in the C-terminal globular domain (CTD) of COMP. Mutant animals are normal at birth, but grow slower than their wild-type littermates and by 9 weeks of age they have mild short-limb dwarfism. Furthermore, by 16 months of age mutant animals exhibit severe degeneration of articular cartilage, which is consistent with early onset osteoarthritis seen in PSACH patients. In the growth plates of mutant mice the chondrocyte columns are sparser and poorly organized. Mutant COMP is secreted into the extracellular matrix, but its localization is disrupted along with the distribution of several COMP-binding proteins. Although mutant COMP is not retained within the rER there is an unfolded protein/cell stress response and chondrocyte proliferation is significantly reduced, while apoptosis is both increased and spatially dysregulated. Overall, these data suggests a mutation in the CTD of COMP exerts a dominant-negative effect on both intra- and extracellular processes. This ultimately affects the morphology and proliferation of growth plate chondrocytes, eventually leading to chondrodysplasia and reduced long bone growth.     

Transforming Growth Factor Beta: An Autocrine Regulator of Chondrocytes

Transforming growth factor beta (TGF-β) is a ubiquitous regulator of cellular growth and differentiation. The present study investigated the effects of TGF-β on chick growth plate chondrocyte proliferation, matrix synthesis, and alkaline phosphatase activity in short term cultures. TGF-β markedly stimulated DNA synthesis in a dose-dependent manner, while collagen synthesis and cellular and matrix vesicle alkaline phosphatase activity were inhibited. Biologic effects of TGF-β were correlated with binding to specific receptors, and both high and low affinity receptors were identified. Countercurrent centrifugal elutriation was used to fractionate growth plate chondrocytes to obtain populations of cells in different stages of maturation (effectively from different zones of the growth plate). TGF-β showed increasing mitogenicity with increasing cellular maturation in the growth plate, with maximal stimulation in the proliferating and early hypertrophic cells. The smallest cells expressed only the high affinity receptor, while with hypertrophy there was increasing expression of the low affinity receptor and a progressive increase in the number of both receptors per cell. Furthermore, the dose-response curves for TGF-β-stimulated DNA synthesis were not biphasic in the smaller cells, but became progressively more biphasic with cellular hypertrophy and expression of the low affinity receptor. Finally, TGF-β activity was identified in partially purified chondrocyte conditioned medium by specific bioassay, indicating TGF-β production by growth plate chondrocytes. The data suggests a potentially important autocrine function for TGF-B in modulating chondrocyte proliferation and matrix synthesis in endochondral calcification.     

Longitudinal bone growth is impaired by direct involvement of caffeine with chondrocyte differentiation in the growth plate

We showed previously that caffeine adversely affects longitudinal bone growth and disrupts the histomorphometry of the growth plate during the pubertal growth spurt. However, little attention has been paid to the direct effects of caffeine on chondrocytes. Here, we investigated the direct effects of caffeine on chondrocytes of the growth plate in vivo and in vitro using a rapidly growing young rat model, and determined whether they were related to the adenosine receptor signaling pathway. A total of 15 male rats (21 days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180 mg kg⁻¹ day⁻¹ for 4 weeks. After sacrifice, the tibia processed for the analysis of the long bone growth and proliferation of chondrocytes using tetracycline and BrdU incorporation, respectively. Caffeine‐fed animals showed decreases in matrix mineralization and proliferation rate of growth plate chondrocytes compared with the control. To evaluate whether caffeine directly affects chondrocyte proliferation and chondrogenic differentiation, primary rat chondrocytes were isolated from the growth plates and cultured in either the presence or absence of caffeine at concentrations of 0.1–1 mm , followed by determination of the cellular proliferation or expression profiles of cellular differentiation markers. Caffeine caused significant decreases in extracellular matrix production, mineralization, and alkaline phosphatase activity, accompanied with decreases in gene expression of the cartilage‐specific matrix proteins such as aggrecan, type II collagen and type X. Our results clearly demonstrate that caffeine is capable of interfering with cartilage induction by directly inhibiting the synthetic activity and orderly expression of marker genes relevant to chondrocyte maturation. In addition, we found that the adenosine type 1 receptor signaling pathway may be partly involved in the detrimental effects of caffeine on chondrogenic differentiation, specifically matrix production and mineralization, as evidenced by attenuation of the inhibitory effects of caffeine by blockade of this receptor. Thus, our study provides novel information on the integration of caffeine and adenosine receptor signaling during chondrocyte maturation, extending our understanding of the effect of caffeine at a cellular level on chondrocytes of the growth plate.     

The Muenke syndrome mutation (FgfR3P244R) causes cranial base shortening associated with growth plate dysfunction and premature perichondrial ossification in murine basicranial synchondroses

Muenke syndrome caused by the FGFR3(P250R) mutation is an autosomal dominant disorder mostly identified with coronal suture synostosis, but it also presents with other craniofacial phenotypes that include mild to moderate midface hypoplasia. The Muenke syndrome mutation is thought to dysregulate intramembranous ossification at the cranial suture without disturbing endochondral bone formation in the skull. We show in this study that knock-in mice harboring the mutation responsible for the Muenke syndrome (FgfR3(P244R)) display postnatal shortening of the cranial base along with synchondrosis growth plate dysfunction characterized by loss of resting, proliferating and hypertrophic chondrocyte zones and decreased Ihh expression. Furthermore, premature conversion of resting chondrocytes along the perichondrium into prehypertrophic chondrocytes leads to perichondrial bony bridge formation, effectively terminating the postnatal growth of the cranial base. Thus, we conclude that the Muenke syndrome mutation disturbs endochondral and perichondrial ossification in the cranial base, explaining the midface hypoplasia in patients.     

Proliferation and Differentiation of Osteoblasts in Osteopetrotic Rats: Modification in Expression of Genes Encoding Cell Growth and Extracellular Matrix Proteins

Osteopetrosis is characterized by a congenital defect in osteoclast differentiation and/or activity. The unresorbed matrix produces dense and sclerotic bone with the absence of a marrow cavity. Osteoblasts function in both the production and degradation of bone. However, the potential contribution of an osteoblast abnormality in the etiology of osteopetrosis has not been explored. We examined expression of cell growth-related genes (Core H1 histones and c myc) and genes related to osteoblast differentiation (Type I collagen, osteopontin and osteocalcin, an osteoblast-specific marker) in calvarial bone from the 3 osteopetrotic mutations in the rat (ia/ia, op/op and tl/tl) and normal littermates. mRNA preparations from these bones showed up to a 5-fold increase in all cell growth related genes in tl/tl and op/op rats, compared to normal littermates, suggesting a stimulation of proliferative activity of bone cells. The matrix genes also exhibited 2 to 10+fold increases in these two mutations. In contrast ia/ia rats showed no significant changes in expression of proliferation or matrix genes (except for osteopontin) which is consistent with the greatly reduced skeletal sclerosis in this mutation at the time (4 wk) when tissues were analyzed. Since the tl and op mutations have greater elevations in serum 1,25(OH)2D3 than found in the ia mutation, these results may reflect a stimulatory effect on cell proliferation and osteoblast activity by 1,25(OH)2D3. These data suggest that, in addition to osteoclast abnormalities, certain osteopetroses may also have aberrations of osteoblast function.