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Henry M. Kronenberg - One of the best experts on this subject based on the ideXlab platform.
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the transcriptional cofactor lbh regulates angiogenesis and Endochondral Bone formation during fetal Bone development
2009Co-Authors: Katrin Conen, Sylvain Provot, Shigeki Nishimori, Henry M. KronenbergAbstract:Lbh is thought to act as a transcriptional cofactor and is highly conserved among species. Here we show that Lbh is expressed in chondrocytes, cells of the perichondrium, and the primary spongiosa in fetal growth plates of mice and chickens. Lbh overexpression in chick wings, using the RCAS-retroviral vector strategy, results in shortened skeletal elements and delayed hypertrophic chondrocyte maturation and Bone formation. Additionally, osteoclast and endothelial cell invasion are delayed in the Lbh-overexpressing Bones. Finally, we find a dramatic suppression of Runx2 and VEGF mRNAs in chondrocytes and osteoblasts that overexpress Lbh. Strikingly, this abnormal Bone development in infected limbs can be rescued by concurrent overexpression of Runx2. These results suggest that during Endochondral Bone formation, Lbh may negatively regulate vascular invasion and formation of the early ossification center at least in part by interfering with Runx2 and/or VEGF expression.
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adamts 7 a direct target of pthrp adversely regulates Endochondral Bone growth by associating with and inactivating gep growth factor
2009Co-Authors: Henry M. Kronenberg, Xiaohui Bai, Dawei Wang, Li Kong, Yan Zhang, Yi Luan, Tatsuya Kobayashi, Chuanju LiuAbstract:ADAMTS-7, a metalloproteinase that belongs to ADAMTS family, is important for the degradation of cartilage extracellular matrix proteins in arthritis. Herein we report that ADAMTS-7 is upregulated during chondrocyte differentiation and demonstrates the temporal and spatial expression pattern during skeletal development. ADAMTS-7 potently inhibits chondrocyte differentiation and Endochondral Bone formation, and this inhibition depends on its proteolytic activity. The cysteine-rich domain of ADAMTS-7 is required for its interaction with the extracellular matrix, and the C-terminal four-thrombospondin motifs are necessary for its full proteolytic activity and inhibition of chondrocyte differentiation. ADAMTS-7 is an important target of canonical PTHrP signaling, since (i) PTHrP induces ADAMTS-7, (ii) ADAMTS-7 is downregulated in PTHrP null mutant (PTHrP-/-) growth plate chondrocytes, and (iii) blockage of ADAMTS-7 almost abolishes PTHrP-mediated inhibition of chondrocyte hypertrophy and Endochondral Bone growth. ADAMTS-7 associates with granulin-epithelin precursor (GEP), an autocrine growth factor that has been implicated in tissue regeneration, tumorigenesis, and inflammation. In addition, ADAMTS-7 acts as a new GEP convertase and neutralizes GEP-stimulated Endochondral Bone formation. Collectively, these findings demonstrate that ADAMTS-7, a direct target of PTHrP signaling, negatively regulates Endochondral Bone formation by associating with and inactivating GEP chondrogenic growth factor.
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a raf and b raf are dispensable for normal Endochondral Bone development and parathyroid hormone related peptide suppresses extracellular signal regulated kinase activation in hypertrophic chondrocytes
2008Co-Authors: Sylvain Provot, Gregory Nachtrab, Jennifer L Paruch, Adele Pin Chen, Alcino J Silva, Henry M. KronenbergAbstract:Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in Endochondral Bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in chondrocytes. Though B-Raf is expressed in proliferative chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with chondrocytes deficient in both A-Raf and B-Raf exhibited normal Endochondral Bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for Endochondral Bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.
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absence of transcription factor c maf causes abnormal terminal differentiation of hypertrophic chondrocytes during Endochondral Bone development
2003Co-Authors: Helen E Maclean, Henry M. Kronenberg, James I Kim, Melvin J Glimcher, Jinxi Wang, Laurie H GlimcherAbstract:Abstract In this study, we report that the transcription factor c-Maf is required for normal chondrocyte differentiation during Endochondral Bone development. c-maf is expressed in hypertrophic chondrocytes during fetal development (E14.5–E18.5), with maximal expression in the tibia occurring at E15.5 and E16.5, in terminally differentiated chondrocytes. In c-maf -null mice, fetal Bone length is decreased ∼10%, and hypertrophic chondrocyte differentiation is perturbed. There is an initial decrease in the number of mature hypertrophic chondrocytes at E15.5 in c-maf -null tibiae, with decreased expression domains of collagen X and osteopontin, markers of hypertrophic and terminal hypertrophic chondrocytes, respectively. By E16.5, there is an expanded domain of late hypertrophic, osteopontin -positive chondrocytes in the c-maf −/−. This accumulation of hypertrophic chondrocytes persists and is still observed at 4 weeks of age. These data suggest that c-Maf facilitates the initial chondrocyte terminal differentiation and influences the disappearance of hypertrophic chondrocytes. BrdU and TUNEL analyses show normal proliferation rate and apoptosis in the c-maf -null. There is a specific decrease in MMP-13 expression at E15.5 in the c-maf -null. MMP-13 is known to be regulated by AP-1 and may also be a target of c-Maf. Thus, cartilage is a novel system in which c-Maf acts during development, where c-Maf is required for normal chondrocyte differentiation.
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developmental regulation of the growth plate
2003Co-Authors: Henry M. KronenbergAbstract:Vertebrates do not look like jellyfish because the Bones of their skeletons are levers that allow movement and protect vital organs. Bones come in an enormous variety of shapes and sizes to accomplish these goals, but, with few exceptions, use one process — Endochondral Bone formation — to generate the skeleton. The past few years have seen an enormous increase in understanding of the signalling pathways and the transcription factors that control Endochondral Bone development.
Zena Werb - One of the best experts on this subject based on the ideXlab platform.
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complementary interplay between matrix metalloproteinase 9 vascular endothelial growth factor and osteoclast function drives Endochondral Bone formation
2010Co-Authors: Nathalie Ortega, Ke Wang, Napoleone Ferrara, Zena WerbAbstract:Long Bone development depends on Endochondral Bone formation, a complex process requiring exquisite balance between hypertrophic cartilage (HC) formation and its ossification. Dysregulation of this process may result in skeletal dysplasias and heterotopic ossification. Endochondral ossification requires the precise orchestration of HC vascularization, extracellular matrix remodeling, and the recruitment of osteoclasts and osteoblasts. Matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and osteoclasts have all been shown to regulate Endochondral ossification, but how their function interrelates is not known. We have investigated the functional relationship among these regulators of Endochondral ossification, demonstrating that they have complementary but non-overlapping functions. MMP-9, VEGF and osteoclast deficiency all cause impaired growth plate ossification resulting in the accumulation of HC. VEGF mRNA and protein expression are increased at the MMP-9-/- growth plate, and VEGF activity contributes to Endochondral ossification since sequestration of VEGF by soluble receptors results in further inhibition of growth plate vascularization and ossification. However, VEGF bioavailability is still limited in MMP-9 deficiency, as exogenous VEGF is able to rescue the MMP-9-/- phenotype, demonstrating that MMP-9 may partially, but not fully, regulate VEGF bioavailability. The organization of the HC extracellular matrix at the MMP-9-/- growth plate is altered, supporting a role for MMP-9 in HC remodeling. Inhibition of VEGF impairs osteoclast recruitment, whereas MMP-9 deficiency leads to an accumulation of osteoclasts at the chondro-osseous junction. Growth plate ossification in osteoclast-deficient mice is impaired in the presence of normal MMP-9 expression, indicating that other osteoclastic functions are also necessary. Our data delineate the complementary interplay between MMP-9, VEGF and osteoclast function that is necessary for normal Endochondral Bone formation and provide a molecular framework for investigating the molecular defects contributing to disorders of Endochondral Bone formation.
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galectin 3 is a downstream regulator of matrix metalloproteinase 9 function during Endochondral Bone formation
2005Co-Authors: Nathalie Ortega, Danielle J Behonick, Celine Colnot, Douglas N W Cooper, Zena WerbAbstract:Endochondral Bone formation is characterized by the progressive replacement of a cartilage anlagen by Bone at the growth plate with a tight balance between the rates of chondrocyte proliferation, differentiation, and cell death. Deficiency of matrix metalloproteinase-9 (MMP-9) leads to an accumulation of late hypertrophic chondrocytes. We found that galectin-3, an in vitro substrate of MMP-9, accumulates in the late hypertrophic chondrocytes and their surrounding extracellular matrix in the expanded hypertrophic cartilage zone. Treatment of wild-type embryonic metatarsals in culture with full-length galectin-3, but not galectin-3 cleaved by MMP-9, mimicked the embryonic phenotype of Mmp-9 null mice, with an increased hypertrophic zone and decreased osteoclast recruitment. These results indicate that extracellular galectin-3 could be an endogenous substrate of MMP-9 that acts downstream to regulate hypertrophic chondrocyte death and osteoclast recruitment during Endochondral Bone formation. Thus, the disruption of growth plate homeostasis in Mmp-9 null mice links galectin-3 and MMP-9 in the regulation of the clearance of late chondrocytes through regulation of their terminal differentiation.
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altered Endochondral Bone development in matrix metalloproteinase 13 deficient mice
2004Co-Authors: Dominique Stickens, Nathalie Ortega, Danielle J Behonick, Babette Heyer, Bettina Hartenstein, Amanda J Fosang, Marina Schorppkistner, Peter Angel, Zena WerbAbstract:The assembly and degradation of extracellular matrix (ECM) molecules are crucial processes during Bone development. In this study, we show that ECM remodeling is a critical rate-limiting step in Endochondral Bone formation. Matrix metalloproteinase (MMP) 13 (collagenase 3) is poised to play a crucial role in Bone formation and remodeling because of its expression both in terminal hypertrophic chondrocytes in the growth plate and in osteoblasts. Moreover, a mutation in the human MMP13 gene causes the Missouri variant of spondyloepimetaphyseal dysplasia. Inactivation of Mmp13 in mice through homologous recombination led to abnormal skeletal growth plate development. Chondrocytes differentiated normally but their exit from the growth plate was delayed. The severity of the Mmp13- null growth plate phenotype increased until about 5 weeks and completely resolved by 12 weeks of age. Mmp13-null mice had increased trabecular Bone, which persisted for months. Conditional inactivation of Mmp13 in chondrocytes and osteoblasts showed that increases in trabecular Bone occur independently of the improper cartilage ECM degradation caused by Mmp13 deficiency in late hypertrophic chondrocytes. Our studies identified the two major components of the cartilage ECM, collagen type II and aggrecan, as in vivo substrates for MMP13. We found that degradation of cartilage collagen and aggrecan is a coordinated process in which MMP13 works synergistically with MMP9. Mice lacking both MMP13 and MMP9 had severely impaired Endochondral Bone, characterized by diminished ECM remodeling, prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular Bone formation (resulting in drastically shortened Bones). These data support the hypothesis that proper ECM remodeling is the dominant rate-limiting process for programmed cell death, angiogenesis and osteoblast recruitment during normal skeletal morphogenesis.
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vegf couples hypertrophic cartilage remodeling ossification and angiogenesis during Endochondral Bone formation
1999Co-Authors: Hanspeter Gerber, Zena Werb, Thiennu H Vu, Anne M Ryan, Joe Kowalski, Napoleone FerraraAbstract:Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in Endochondral Bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular Bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of Bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and Bone formation in the growth plate.
Frank Beier - One of the best experts on this subject based on the ideXlab platform.
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loss of Bone sialoprotein leads to impaired Endochondral Bone development and mineralization
2015Co-Authors: Erik Holm, Frank Beier, Jane E Aubin, Graeme K Hunter, Harvey A GoldbergAbstract:Abstract Bone sialoprotein (BSP) is an anionic phosphoprotein in the extracellular matrix of mineralized tissues, and a promoter of biomineralization and osteoblast development. Previous studies on the Bsp-deficient mouse (Bsp−/−) have demonstrated a significant Bone and periodontal tissue phenotype in adulthood. However, the role of BSP during early long Bone development is not known. To address this, early Endochondral ossification in the Bsp−/− mouse was studied. Embryonic day 15.5 (E15.5) wild-type (WT) tibiae showed early stages of ossification that were absent in Bsp−/− mice. At E16.5, mineralization had commenced in the Bsp−/− mice, but staining for mineral was less intense and more dispersed compared with that in WT controls. Tibiae from Bsp−/− mice also demonstrated decreased mineralization and shortened length at postnatal day 0.5 (P0.5) compared to WT Bones. There was no detectable difference in the number of tartrate-resistant acid phosphatase-positive foci at P0.5, although the P0.5 Bsp−/− tibiae had decreased Vegfα expression compared with WT tissue. Due to the shortened tibiae the growth plates were examined and determined to be of normal overall length. However, the length of the resting zone was increased in P0.5 Bsp−/− tibiae whereas that of the proliferative zone was decreased, with no change in the hypertrophic zone length of Bsp−/− mice. A reduction in cells positive for Ki-67, an S-phase cell-cycle marker, was noted in the proliferative zone. Decreased numbers of TUNEL-positive hypertrophic chondrocytes were also apparent in the Bsp−/− tibial growth plates, suggesting decreased apoptosis. Expression of the osteogenic markers Alp1, Col1a1, Sp7, Runx2, and Bglap was reduced in the Endochondral Bone of the neonatal Bsp−/− compared to WT tibiae. These results suggest that BSP is an important and multifaceted protein that regulates both chondrocyte proliferation and apoptosis as well as transition from cartilage to Bone during development of Endochondral Bone.
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choline kinase beta is required for normal Endochondral Bone formation
2014Co-Authors: Roger B Sher, Frank Beier, Gregory A Cox, Zohreh Khavandgar, Martin Hermansson, Michael R Doschak, Monzur Murshed, Dennis E VanceAbstract:Abstract Background Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb−/− mice display neonatal forelimb Bone deformations. Methods To understand the mechanisms underlying the Bone deformations, we compared the biology and biochemistry of Bone formation from embryonic to young adult wild-type (WT) and Chkb−/− mice. Results The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb−/− mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb−/− mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb−/− mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb−/− mice contained fewer osteoclasts along the cartilage/Bone interface. Conclusions Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice. General Significance Our data indicate that choline kinase beta plays an important role in Endochondral Bone formation by modulating growth plate physiology.
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role of interleukin 10 in Endochondral Bone formation in mice anabolic effect via the Bone morphogenetic protein smad pathway
2013Co-Authors: Younkwan Jung, Frank Beier, Gunwoo Kim, H Park, Eunju Lee, Jeyong Choi, Seungwoo HanAbstract:Objective Interleukin-10 (IL-10) is a pleiotropic immunoregulatory cytokine with a chondroprotective effect that is elevated in cartilage and synovium in patients with osteoarthritis. However, the role of IL-10 during Endochondral Bone formation and its mechanism of action have not been elucidated. Methods IL-10–/– mice and IL-10–treated tibial organ cultures were used to study loss and gain of IL-10 functions, respectively, during Endochondral Bone formation. Primary chondrocytes from the long Bones of mouse embryos were cultured with and without IL-10. To assess the role of IL-10 in chondrogenic differentiation, we conducted mesenchymal cell micromass cultures. Results The lengths of whole skeletons from IL-10–/– mice were similar to those of their wild-type littermates, although their skull diameters were smaller. The tibial growth plates of IL-10–/– mice showed shortening of the proliferating zone. Treatment with IL-10 significantly increased tibial lengths in organ culture. IL-10 also induced chondrocyte proliferation and hypertrophic differentiation in primary chondrocytes in vitro. Mechanistically, IL-10 activated STAT-3 and the Smad1/5/8 and ERK-1/2 MAP kinase pathways and induced the expression of Bone morphogenetic protein 2 (BMP-2) and BMP-6 in primary chondrocytes. Furthermore, the blocking of BMP signaling attenuated the IL-10–mediated induction of cyclin D1 and RUNX-2 in primary chondrocytes and suppressed Alcian blue and alkaline phosphatase staining in mesenchymal cell micromass cultures. Conclusion These results indicate that IL-10 acts as a stimulator of chondrocyte proliferation and chondrogenic or hypertrophic differentiation via activation of the BMP signaling pathway.
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the role of akt1 in terminal stages of Endochondral Bone formation angiogenesis and ossification
2009Co-Authors: Veronica Ulici, Hanga Agoston, Katie D Hoenselaar, David D Mcerlain, J Umoh, Subrata Chakrabarti, David W Holdsworth, Frank BeierAbstract:Longitudinal Bone growth is the result of Endochondral Bone formation which takes place in the growth plate. The rate of chondrocyte proliferation and hypertrophy, vascular invasion with the formation of primary ossification centers and cartilage replacement by Bone tissue are all important processes required for normal growth. We have shown a role for the PI3K signaling pathway in chondrocyte hypertrophy and Bone growth in tibia explant cultures. In this current study, we aimed to investigate the role of Akt1, an important target of PI3K, in Endochondral ossification. Akt1 KO mice showed reduced size compared to their littermates throughout life, but the largest difference in body size was observed around 1 week of age. Focusing on this specific developmental stage, we discovered delayed secondary ossification in the long Bones of Akt1 KO mice. A delay in formation of a structure resembling a secondary ossification center was also seen in tibia organ cultures treated with the PI3K inhibitor LY294002. The expression of matrix metalloproteinase-14 (MMP-14), the main protease responsible for development of secondary ossification centers, was decreased in the epiphysis of Akt1 KO mice, possibly explaining the delay in secondary ossification centers seen in the Akt1 KO mice. Bone mineral density (BMD) and Bone mineral content (BMC) measured in the proximal tibia of 1-year-old mice were decreased in Akt1 KO mice, suggesting that the original delay in ossification might affect Bone quality in older animals.
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nitric oxide c type natriuretic peptide and cgmp as regulators of Endochondral ossification
2008Co-Authors: Cristina C Teixeira, Hanga Agoston, Frank BeierAbstract:Coordinated proliferation and differentiation of growth plate chondrocytes is required for Endochondral Bone growth, but the mechanisms and pathways that control these processes are not completely understood. Recent data demonstrate important roles for nitric oxide (NO) and C-type natriuretic peptide (CNP) in the regulation of cartilage development. Both NO and CNP stimulate the synthesis of cGMP and thus the activation of common downstream pathways. One of these downstream mediators, cGMP-dependent kinase II (cGKII), has itself been shown to be essential for normal Endochondral Bone formation. This review summarizes our knowledge of the roles and mechanisms of NO, CNP and cGKII signaling in cartilage and Endochondral Bone development.
Carsten Mullertidow - One of the best experts on this subject based on the ideXlab platform.
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abstract b03 the Endochondral Bone protein chm1 sustains an undifferentiated invasive phenotype promoting lung metastasis in ewing sarcoma
2018Co-Authors: Kristina Von Heyking, David Schirmer, Oxana Schmidt, Julia Calzadawack, Stefanie Gollner, Tim Hensel, Annette Fasan, Carsten Mullertidow, Poul H Sorensen, Stefan BurdachAbstract:Background: Ewing sarcoma (ES), an osteogenic malignancy that mainly affects children and young adults, is characterized by early metastasis to lung and Bone. In the clinical setting, prognosis for patients with metastatic ES at diagnosis is clearly worse than for those without metastases (5-year survival > 30%). Hence, there is an urgent need to understand the fundamental molecular mechanisms of ES differentiation, invasion, and metastasis to possibly identify novel therapeutic strategies to prevent metastasis. The purpose of this study was to shed further light into the function of Chondromodulin 1 (CHM1) on ES pathogenesis, especially on metastasis, and at best to establish new therapeutic targets. Material and Methods: Expression of CHM1 was analyzed using microarrays and its function was examined by RNA interference (RNAi). To analyze resulting changes qRT-PCR, ELISA, FACS, IHC, proliferation and invasion assays, as well as a xeno-transplant model in immune deficient mice were applied. Results: In this study, we investigated the role of the BRICHOS chaperon domain containing Endochondral Bone protein chondromodulin I (CHM1) in ES pathogenesis. CHM1 is significantly overexpressed in ES and ChIP data demonstrate CHM1 to be directly bound by EWS-FLI1. Using RNA interference we demonstrate that CHM1 enhanced contact-dependent as well as independent proliferation and the invasive potential of ES cells in vitro. This invasiveness was in part mediated via CHM1-regulated MMP9 expression. In a xenograft mouse model CHM1 was essential for the establishment of lung metastases, which is in line with the observed increased CHM1 expression in patient specimens with ES lung metastases. Mechanistically, CHM1 promoted chondrogenic differentiation capacity of ES cells but suppressed endothelial differentiation. Further, CHM1 suppressed the number of TRAP + osteoclasts in an orthotopic model of tumor growth in line with suppression of osteolytic genes such as HIF1A, IL6, JAG1 , and VEGF , indicating that CHM1-blocked osteomimicry might play a role in homing, colonization, and invasion into Bone tissues. Conclusions: Our results suggest that CHM1 is an important player suppressing endothelial differentiation capacity and seems essential for the invasive and metastatic capacities of ES. Citation Format: Kristina von Heyking, Julia Calzada-Wack, Stefanie Gollner, Oxana Schmidt, Tim Hensel, David Schirmer, Annette Fasan, Carsten Muller-Tidow, Poul Sorensen, Stefan Burdach, Gunther H.S. Richter. The Endochondral Bone protein CHM1 sustains an undifferentiated, invasive phenotype promoting lung metastasis in Ewing sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B03.
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the Endochondral Bone protein chm1 sustains an undifferentiated invasive phenotype promoting lung metastasis in ewing sarcoma
2017Co-Authors: Kristina Von Heyking, David Schirmer, Oxana Schmidt, Julia Calzadawack, Stefanie Gollner, Tim Hensel, Annette Fasan, Frauke Neff, Irene Esposito, Carsten MullertidowAbstract:Ewing sarcomas (ES) are highly malignant, osteolytic Bone or soft tissue tumors, which are characterized by EWS–ETS translocations and early metastasis to lung and Bone. In this study, we investigated the role of the BRICHOS chaperone domain-containing Endochondral Bone protein chondromodulin I (CHM1) in ES pathogenesis. CHM1 is significantly overexpressed in ES, and chromosome immunoprecipitation (ChIP) data demonstrate CHM1 to be directly bound by an EWS–ETS translocation, EWS-FLI1. Using RNA interference, we observed that CHM1 promoted chondrogenic differentiation capacity of ES cells but decreased the expression of osteolytic genes such as HIF1A, IL6, JAG1, and VEGF. This was in line with the induction of the number of tartrate-resistant acid phosphatase (TRAP+)-stained osteoclasts in an orthotopic model of local tumor growth after CHM1 knockdown, indicating that CHM1-mediated inhibition of osteomimicry might play a role in homing, colonization, and invasion into Bone tissues. We further demonstrate that CHM1 enhanced the invasive potential of ES cells in vitro. This invasiveness was in part mediated via CHM1-regulated matrix metallopeptidase 9 expression and correlated with the observation that, in an xenograft mouse model, CHM1 was essential for the establishment of lung metastases. This finding is in line with the observed increase in CHM1 expression in patient specimens with ES lung metastases. Our results suggest that CHM1 seems to have pleiotropic functions in ES, which need to be further investigated, but appears to be essential for the invasive and metastatic capacities of ES.
Wouter J A Dhert - One of the best experts on this subject based on the ideXlab platform.
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decellularized cartilage derived matrix as substrate for Endochondral Bone regeneration
2015Co-Authors: Debby Gawlitta, Jetze Visser, Kim E M Benders, Jos Malda, Anja S Van Der Sar, Diederik H R Kempen, L F H Theyse, Wouter J A DhertAbstract:Following an Endochondral approach to Bone regeneration, multipotent stromal cells (MSCs) can be cultured on a scaffold to create a cartilaginous callus that is subsequently remodeled into Bone. An attractive scaffold material for cartilage regeneration that has recently regained attention is decellularized cartilage-derived matrix (CDM). Since this material has shown potential for cartilage regeneration, we hypothesized that CDM could be a potent material for Endochondral Bone regeneration. In addition, since decellularized matrices are known to harbor bioactive cues for tissue formation, we evaluated the need for seeded MSCs in CDM scaffolds. In this study, ectopic Bone formation in rats was evaluated for CDM scaffolds seeded with human MSCs and compared with unseeded controls. The MSC-seeded samples were preconditioned in chondrogenic medium for 37 days. After 8 weeks of subcutaneous implantation, the extent of mineralization was significantly higher in the MSC-seeded constructs versus unseeded controls. The mineralized areas corresponded to Bone formation with Bone marrow cavities. In addition, rat-specific Bone formation was confirmed by collagen type I immunohistochemistry. Finally, fluorochrome incorporation at 3 and 6 weeks revealed that the Bone formation had an inwardly directed progression. Taken together, our results show that decellularized CDM is a promising biomaterial for Endochondral Bone regeneration when combined with MSCs at ectopic locations. Modification of current decellularization protocols may lead to enhanced functionality of CDM scaffolds, potentially offering the prospect of generation of cell-free off-the-shelf Bone regenerative substitutes.
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Endochondral Bone formation in gelatin methacrylamide hydrogel with embedded cartilage derived matrix particles
2015Co-Authors: Jetze Visser, Debby Gawlitta, Kim E M Benders, Selynda M H Toma, Behdad Pouran, Rene P Van Weeren, Wouter J A Dhert, Jos MaldaAbstract:The natural process of Endochondral Bone formation in the growing skeletal system is increasingly inspiring the field of Bone tissue engineering. However, in order to create relevant-size Bone grafts, a cell carrier is required that ensures a high diffusion rate and facilitates matrix formation, balanced by its degradation. Therefore, we set out to engineer Endochondral Bone in gelatin methacrylamide (GelMA) hydrogels with embedded multipotent stromal cells (MSCs) and cartilage-derived matrix (CDM) particles. CDM particles were found to stimulate the formation of a cartilage template by MSCs in the GelMA hydrogel in vitro. In a subcutaneous rat model, this template was subsequently remodeled into mineralized Bone tissue, including Bone-marrow cavities. The GelMA was almost fully degraded during this process. There was no significant difference in the degree of calcification in GelMA with or without CDM particles: 42.5 ± 2.5% vs. 39.5 ± 8.3% (mean ± standard deviation), respectively. Interestingly, in an osteochondral setting, the presence of chondrocytes in one half of the constructs fully impeded Bone formation in the other half by MSCs. This work offers a new avenue for the engineering of relevant-size Bone grafts, by the formation of Endochondral Bone within a degradable hydrogel.
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modulating Endochondral ossification of multipotent stromal cells for Bone regeneration
2010Co-Authors: Debby Gawlitta, Jos Malda, Eric Farrell, Laura B Creemers, Jacqueline Alblas, Wouter J A DhertAbstract:For years it has been recognized that engineering of large Bone constructs will be feasible only if the hurdle of vascularization is overcome. Attempts to engineer Bone tissue have predominantly focused on intramembranous (direct) Bone formation. A relatively new and most likely more physiological approach in this line is Endochondral Bone formation, comprising an intermediate cartilaginous stage. Cartilage in nature is an avascular tissue and its cells are equipped to survive the poor oxygenation and nutritional conditions inherent to implanted tissues. Subsequent terminal differentiation (hypertrophy) of the chondrocytes initiates the formation of a mineralized matrix that will then be converted into Bone. Through this mechanism, our long Bones grow and most fractures heal through the process of secondary fracture healing. The feasibility of the attractive concept of Endochondral Bone tissue engineering has already been shown. Most emphasis has gone to the multipotent stromal cells because of their great potential for expansion and differentiation and immunoprivileged nature. This review will focus on the promises and current status of this new field. Further, potent modulators of Endochondral Bone tissue engineering, including oxygen tension and mechanical stimuli, will be discussed.