Zone Of Growth Plate Cartilage Research Articles

The role of chondrocyte-to-osteoblast trans-differentiation in fetal bone dysplasia of mice caused by prenatal exposure to dexamethasone.

Maternal exposure to dexamethasone can cause developmental toxicity of long bones in offspring. However, the effect of dexamethasone on the trans-differentiation of growth plate chondrocytes into osteoblasts and its role in bone dysplasia of fetuses caused by prenatal dexamethasone exposure (PDE) remains unclear. In this study, pregnant mice were treated with different doses, stages, and courses of dexamethasone according to clinical practice to reveal the phenomenon. Further, growth plate chondrocytes were treated with dexamethasone in vitro to clarify the phenomenon and mechanism. The results showed that PDE caused dysplasia of fetal long bones in female and male mice, accompanied by the delayed formation of the primary ossification center and the widening hypertrophic zone of growth plate cartilage. Meanwhile, PDE increased the number of hypertrophic chondrocytes at growth plate cartilage and decreased the number of osteoblasts at the primary ossification center. Moreover, PDE significantly decreased the expression of osteogenic transcription factor Runx2 but increased the expression of hypertrophic chondrocytes marker Col10. These above phenomena were more significant in the high dose, early stage, and double courses of dexamethasone exposure groups, and the male fetal mice showed more obvious than the female fetal mice. In vitro, dexamethasone significantly inhibited the trans-differentiation of growth plate chondrocytes into osteoblasts, accompanied by a decrease in Runx2 expression and an increase in Col10 expression. In conclusion, this study revealed the phenomenon and mechanism of fetal bone dysplasia caused by PDE from the new perspective of trans-differentiation disorder of growth plate chondrocytes to osteoblasts.

Spatial regulation of bone morphogenetic proteins (BMPs) in postnatal articular and growth plate cartilage.

Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers—the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may contribute to the spatial differentiation of chondrocytes in the postnatal endochondral skeleton.

Gene expression profiling reveals similarities between the spatial architectures of postnatal articular and growth plate cartilage.

Articular and growth plate cartilage are discrete tissues but arise from a common cartilaginous condensation and have comparable spatial architectures consisting of distinct layers of chondrocytes. To investigate similarities and differences between articular and growth plate cartilage and to explore transcriptional changes that occur during the onset of their divergence, we performed manual microdissection of 10-day-old rat proximal tibias, microarray analysis, bioinformatics, and real-time PCR to compare gene expression profiles in individual cartilage layers. We found that many genes that were spatially upregulated in the intermediate/deep zone of articular cartilage were also spatially upregulated in the resting zone of growth plate cartilage (overlap greater than expected by chance, P<0.001). Interestingly, the superficial zone of articular cartilage showed an expression profile with similarities to both the proliferative and hypertrophic zones of growth plate cartilage (P<0.001 each). Additionally, significant numbers of known proliferative zone markers (3 out of 6) and hypertrophic zone markers (27 out of 126) were spatially upregulated in the superficial zone (more than expected by chance, P<0.001 each). In conclusion, we provide evidence that the intermediate/deep zone of articular cartilage has a gene expression profile more similar to that of the resting zone of growth plate cartilage, whereas the superficial zone has a gene expression profile more similar to those of the proliferative and hypertrophic zones. These findings suggest that the superficial zone chondrocytes of articular cartilage differentiate according to a program that is not completely different from but instead has distinct similarities to the hypertrophic differentiation program of growth plate chondrocytes. We also present functional signaling pathways implicated by differential gene expression between articular and growth plate cartilage during their initial separation by the secondary ossification center.

Generation and Activity of a Humanized Monoclonal Antibody That Selectively Neutralizes the Epidermal Growth Factor Receptor Ligands Transforming Growth Factor-αand Epiregulin

At least seven distinct epidermal growth factor (EGF) ligands bind to and activate the EGF receptor (EGFR). This activation plays an important role in the embryo and in the maintenance of adult tissues. Importantly, pharmacologic EGFR inhibition also plays a critical role in the pathophysiology of diverse disease states, especially cancer. The roles of specific EGFR ligands are poorly defined in these disease states. Accumulating evidence suggests a role for transforming growth factor α (TGFα) in skin, lung, and kidney disease. To explore the role of Tgfa, we generated a monoclonal antibody (mAb41) that binds to and neutralizes human Tgfa with high affinity (KD = 36.5 pM). The antibody also binds human epiregulin (Ereg) (KD = 346.6 pM) and inhibits ligand induced myofibroblast cell proliferation (IC50 values of 0.52 and 1.12 nM for human Tgfa and Ereg, respectively). In vivo, a single administration of the antibody to pregnant mice (30 mg/kg s.c. at day 14 after plug) or weekly administration to neonate mice (20 mg/kg s.c. for 4 weeks) phenocopy Tgfa knockout mice with curly whiskers, stunted growth, and expansion of the hypertrophic zone of growth plate cartilage. Humanization of this monoclonal antibody to a human IgG4 antibody (LY3016859) enables clinical development. Importantly, administration of the humanized antibody to cynomolgus monkeys is absent of the skin toxicity observed with current EGFR inhibitors used clinically and no other pathologies were noted, indicating that neutralization of Tgfa could provide a relatively safe profile as it advances in clinical development.

Gla-Rich Protein Is a Novel Vitamin K-Dependent Protein Present in Serum That Accumulates at Sites of Pathological Calcifications

Mineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization.

Trps1 deficiency enlarges the proliferative zone of growth plate cartilage by upregulation of Pthrp

We have reported that elongation of the columnar proliferative zone of long bone growth plates in Trps1−/− mice during the late fetal stage in the previous study [1]. Since expression of Trps1 protein was found to overlap with that of mRNAs for Indian hedgehog (Ihh), PTH/PTHrP receptor (PPR), and PTHrP, we hypothesized that Trps1 may inhibit the hypertrophic differentiation of chondrocytes by interacting with the Ihh/PTHrP feedback loop. To investigate whether Trps1 has a role in this Ihh/PTHrP feedback loop, we compared the growth plates of Trps1−/− mice and wild-type (Trps1+/+) mice. Immunohistochemistry showed that Trps1 protein was strongly expressed in the periarticular and prehypertrophic zones of the fetal growth plate in wild-type mice on embryonic day 18.5 (E18.5). On the other hand, Ihh, PPR, and PTHrP mRNAs were predominantly expressed in the prehypertrophic zone at this stage of development. While expression of Ihh and PPR by prehypertrophic chondrocytes was unaffected in the growth plates of Trps1−/− mice, the range of PTHrP expression was expanded toward the proliferating zone in these mice. Quantitative real-time PCR analysis demonstrated upregulation of PTHrP in the epiphyseal growth plates of Trps1−/− mice. Furthermore, promoter analysis combined with the chromatin immunoprecipitation (ChIP) assay demonstrated that direct binding of Trps1 to the PTHrP promoter suppressed the transcription of PTHrP. Finally, organ culture of E14.5 tibiae in the absence or the presence of Pthrp revealed that the proliferative zone of the tibial growth plate was elongated by culture with Pthrp compared to that of control tibiae. Taken together, these data provide the first genetic evidence that lack of Trps1 leads to overexpression of PTHrP, and that Trps1 is required to maintain the normal organization of chondrocytes in the growth plate.

Collagens at a glance

Collagens are a large family of triple helical proteins that are widespread throughout the body and are important for a broad range of functions, including tissue scaffolding, cell adhesion, cell migration, cancer, angiogenesis, tissue morphogenesis and tissue repair. Collagen is best known as the

Structural changes in the large proteoglycan, aggrecan, in different zones of the ovine growth plate.

The large cartilage proteoglycan, aggrecan, was found to vary throughout the ovine physis corresponding to the maturational state of the resident chondrocytes. Two populations of proteoglycan monomer were observed in articular, epiphyseal, and in the resting zone of growth plate cartilage. These proteoglycans contained chondroitin sulfate glycosaminoglycan chains sulfated predominantly in the 4 position along with lesser amounts of chondroitin-6-sulfate and keratan sulfate. In the proliferative zone of the growth plate, chondrocytes synthesize one population of proteoglycan monomer which was significantly larger than monomer populations in articular, epiphyseal, or resting zone and this size increase could be attributed to an increase in its constituent chondroitin sulfate side chains. As these chondrocytes progress through their life cycle they continue to modify the structural characteristics of the aggrecan molecule they synthesize. Thus, in the hypertrophic region of the growth plate, the proteoglycan monomer is larger again than in the proliferative region. Variation in sulfation pattern on aggrecan chondroitin sulfate side chains is also observed in the hypertrophic region with an increasing proportion of unsulfated residues present, which may play a role in the initiation of mineralization. In addition, increasing amounts of the carbohydrate sequence recognized by monoclonal antibody 7-D-4 are observed in the hypertrophic zone.

Regulation of rat interstitial collagenase gene expression in growth cartilage and chondrocytes by vitamin D3, interleukin-1β, and okadaic acid

The interstitial collagenase produced by the rat growth plate chondrocytes is the homologue of the human collagenase-3, or matrix metalloproteinase-13. This enzyme is responsible for the loss of collagen during hypertrophy of chondrocytes and for the degradation of transverse septa in long bone growth. Rachitic rats (42 days, male Sprague-Dawley) had an 8-fold higher level of collagenase mRNA in the hypertrophic versus proliferative zone of growth plate cartilage. Intramuscular injection of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3; 1.0 micrograms/kg body weight) in rachitic rats increased collagenase mRNA another 1.5-fold in the hypertrophic zone. The regulation of collagenase gene by 1,25-(OH)2D3 and interleukin (IL)-1 beta in cultured proliferative chondrocytes was studied by means of steady-state mRNA and half-life determination of mRNA using the transcriptional inhibitor actinomycin D, and nuclear run-on transcription analyses. Treatment of cells with 1,25-(OH)2D3 (10(-6) M) and IL-1 beta (2 ng/ml) increased collagenase mRNA 8- and 13-fold, respectively. Additionally, the collagenase mRNA half-life was increased by 1,25-(OH)2D3 and IL-1 beta. In the presence of a protein kinase C inhibitor, staurosporine, 1,25-(OH)2 D3 induction of collagenase mRNA was blocked. Here the addition of phorbol 12-myrisate 13-acetate (PMA) to activate protein kinase C increased collagenase mRNA 10-fold. However, in the presence of staurosporine (50 nM), PMA induction was blocked, whereas IL-1 beta was not. IL-1 beta is known to activate several phosphorylation pathways. Okadaic acid (500 nM), a protein phosphatase inhibitor, increased the relative collagenase mRNA abundance 10-fold. The rate of the rat collagenase gene transcription in nuclei was increased with 1,25-(OH)2D3, IL-1 beta and okadaic acid. In separate experiments, the collagenase promoter was ligated to a reporter plasmid and the plasmid was transfected into chondrocytes. The results showed that 1,25-(OH)2D3, IL-1 beta, and PMA increased reporter activity 2.5-, 2.8-, and 3.27-fold, respectively. Thus, there are multiple nuclear and cytoplasmic mechanisms by which cartilage modulators regulate rat interstitial collagenase gene expression.

Regulation of rat interstitial collagenase gene expression in growth cartilage and chondrocytes by vitamin D3, interleukin-1 beta, and okadaic acid.

The interstitial collagenase produced by the rat growth plate chondrocytes is the homologue of the human collagenase-3, or matrix metalloproteinase-13. This enzyme is responsible for the loss of collagen during hypertrophy of chondrocytes and for the degradation of transverse septa in long bone growth. Rachitic rats (42 days, male Sprague-Dawley) had an 8-fold higher level of collagenase mRNA in the hypertrophic versus proliferative zone of growth plate cartilage. Intramuscular injection of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3; 1.0 micrograms/kg body weight) in rachitic rats increased collagenase mRNA another 1.5-fold in the hypertrophic zone. The regulation of collagenase gene by 1,25-(OH)2D3 and interleukin (IL)-1 beta in cultured proliferative chondrocytes was studied by means of steady-state mRNA and half-life determination of mRNA using the transcriptional inhibitor actinomycin D, and nuclear run-on transcription analyses. Treatment of cells with 1,25-(OH)2D3 (10(-6) M) and IL-1 beta (2 ng/ml) increased collagenase mRNA 8- and 13-fold, respectively. Additionally, the collagenase mRNA half-life was increased by 1,25-(OH)2D3 and IL-1 beta. In the presence of a protein kinase C inhibitor, staurosporine, 1,25-(OH)2 D3 induction of collagenase mRNA was blocked. Here the addition of phorbol 12-myrisate 13-acetate (PMA) to activate protein kinase C increased collagenase mRNA 10-fold. However, in the presence of staurosporine (50 nM), PMA induction was blocked, whereas IL-1 beta was not. IL-1 beta is known to activate several phosphorylation pathways. Okadaic acid (500 nM), a protein phosphatase inhibitor, increased the relative collagenase mRNA abundance 10-fold. The rate of the rat collagenase gene transcription in nuclei was increased with 1,25-(OH)2D3, IL-1 beta and okadaic acid. In separate experiments, the collagenase promoter was ligated to a reporter plasmid and the plasmid was transfected into chondrocytes. The results showed that 1,25-(OH)2D3, IL-1 beta, and PMA increased reporter activity 2.5-, 2.8-, and 3.27-fold, respectively. Thus, there are multiple nuclear and cytoplasmic mechanisms by which cartilage modulators regulate rat interstitial collagenase gene expression.

Preferential accumulation in vivo of 24R,25-dihydroxyvitamin D3 in growth plate cartilage of rats

Vitamin D(3) is metabolized in vivo through 25-(OH)D(3) (25D) to both 1α,25-(OH)(2)D(3) (1,25D) and 24R,25-(OH)(2)D(3) (24,25D). Whereas it is assumed that this metabolism occurs primarily in the kidney, recent studies show that there are extrarenal 1α-and 24R-hydroxylase activities as well, and in chondrocytes, these enzymes are regulated by hormones and growth factors. Furthermore, chondrocytes from the resting zone of growth plate cartilage are a target cell population for 24,25D action, suggesting that this vitamin D metabolite may be targeted to this tissue in vivo. To test this hypothesis, 30 normal male Sprague Dawley rats (120 ±20 g) were divided into three groups of eight animals each, and a control group of six animals, and fed ad libitum for 2 wk, a standard rat chow (Teklad LM-485), which contained 3 IU vitamin D(3)/g. The rats were then injected im daily at 9:00AM: , for 4 consecutive d, with 0.1 mL of either [(3)H]-25D, [(3)H]-1,25D or [(3)H]-24,25D. Each dose contained 13 pmol of hormone (0.36 μCi/dose). The distribution of these metabolites was assessed in tibial bone (B) following ablation of the bone marrow, articular cartilage from the tibia (AC), costochondral growth plate cartilage (GC), serum (S), small intestine (I), and kidney (K). The use of high specific activity tritiated vitamin D metabolites facilitated determining tissue localization and further metabolism without perturbation of the body pools of each major metabolite. Accumulation of [(3)H]-1,25D or [(3)H]-24,25D in each tissue was compared to circulating serum levels. In rats dosed with [(3)H]-25D, the tissue:serum ratios for 1,25D were 4.1 (AC), 35.4 (GC), 1.3 (B), 0.7 (K), and 3.0 (I); and tissue:serum ratios for 24,25D were 1.6 (AC), 9.9 (GC), 0.04 (B), 0.2 (K), and 0.4 (I). In rats dosed with [(3)H]-24,25D alone, GC was the only tissue to accumulate the administered metabolite at a concentration significantly higher than that of serum. Similarly, in rats dosed with [(3)H]-1,25D alone, GC was the only tissue to accumulate 1,25D at a concentration higher than that of serum. These results demonstrate, for the first time, that under in vivo conditions, GC specifically accumulates 24,25D and 1,25D. This suggests that growth plate may be a target organ for these two hormones.

In situ hybridization studies on the expression of type X collagen in fetal human cartilage

Type X collagen is a short, non-fibril-forming collagen restricted to the hypertrophic, calcifying zone of growth plate cartilage. It is developmentally regulated and found exclusively in hypertrophic cartilage. Here we report on the structure and distribution of human type X collagen based on the cloning of a PCR fragment covering 292 bp of the carboxyterminal, non-triple-helical domain. Seventy-five percent of the sequence are identical to that of chicken type X collagen at nucleic acid level and 84% at amino acid level. This probe was used for in situ hybridization analyses of type X collagen expression in a human growth plate. Human fetal cartilage, which is different from the avian cartilage-bone transition zone, showed strong type X collagen expression confined to the lower hypertrophic zone of the growth plate. The upper zone of hypertrophic chondrocytes did not contain α1(X) transcripts, indicating that type X collagen expression follows cellular hypertrophy. The distribution of type X collagen mRNA has been previously unreported in chondrocytes from zones of secondary ossification and in chondrocytes associated with endochondral bone trabecules containing calcified cartilage. In situ hybridization analyses with probes for type I and II collagen on consecutive sections indicated a spatial gradient in chondrocyte differentiation in the human epiphysis. Chondrocytes of low type II collagen expression in the resting zone are followed by proliferating columnar chondrocytes with strong type II collagen expression and a zone of hypertrophic chondrocytes synthesizing type X and type II collagen. In contrast to findings in avian growth cartilage in some of our samples of human epiphyseal cartilage hypertrophic chondrocytes continued to strongly express type II collagen down to the chondro-osseous junction. Transcripts of the α2(I) collagen gene, however, were detected only in perichondrium, vascular cavities, and bone, but not in hypertrophic or any other chondrocytes. The above observations demonstrate that the isolation of the human type X collagen DNA will contribute to studies of pathways of chondrocyte differentiation in the mammalian growth plate.

Cellular turnover at the chondro-osseous junction of growth plate cartilage: analysis by serial sections at the light microscopical level.

In the distal hypertrophic cell zone of growth plate cartilage, the penetration of metaphyseal vascular endothelial cells is into the noncalcified territorial and pericellular matrices. Cellular mechanisms that promote metaphyseal vascularization are understood poorly, partly because no study has addressed the question of the time sequence of cellular interactions at the chondro-osseous junction. The purpose of the present study is to make predictions about the relative and the real time duration of cellular events during vascular invasion, including an analysis of the time sequence of death of the terminal hypertrophic chondrocyte. The data from serial section analysis at the light microscopical level of tetracycline-labeled growth plates indicate that death of the terminal hypertrophic chondrocyte occurs in discrete morphological stages characterized by rapid cellular condensation followed, within minutes, by endothelial cell penetration into the vacated lacuna. Cellular condensation lasts approximately 45 min or 18% of the time a cell spends as a terminal chondrocyte. The data also demonstrate that chondrocytic death occurs prior to invasion by vascular endothelial cells and that the chondrocytic lacuna remains empty for as long as 15 min before an endothelial cell or blood vascular cell fills the space.