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Michael T Longaker - One of the best experts on this subject based on the ideXlab platform.
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absence of endochondral ossification and craniosynostosis in posterior Frontal cranial Sutures of axin2 mice
PLOS ONE, 2013Co-Authors: Bjorn Behr, Michael T Longaker, Natalina QuartoAbstract:During the first month of life, the murine posterior-Frontal Suture (PF) of the cranial vault closes through endochondral ossification, while other Sutures remain patent. These processes are tightly regulated by canonical Wnt signaling. Low levels of active canonical Wnt signaling enable endochondral ossification and therefore PF-Suture closure, whereas constitutive activation of canonical Wnt causes PF-Suture patency. We therefore sought to test this concept with a knockout mouse model. PF-Sutures of Axin2−/− mice, which resemble a state of constantly activated canonical Wnt signaling, were investigated during the physiological time course of PF-Suture closure and compared in detail with wild type littermates. Histological analysis revealed that the architecture in Axin2−/− PF-Sutures was significantly altered in comparison to wild type. The distance between the endocranial layers was dramatically increased and Suture closure was significantly delayed. Moreover, physiological endochondral ossification did not occur, rather an ectopic cartilage appeared between the endocranial and ectocranial bone layers at P7 which eventually involutes at P13. Quantitative PCR analysis showed the lack of Col10α1 upregulation in Axin2−/− PF-Suture. Immunohistochemistry and gene expression analysis also revealed high levels of type II collagen as compared to type I collagen and absence of Mmp-9 in the cartilage of Axin2−/− PF-Suture. Moreover, TUNEL staining showed a high percentage of apoptotic chondrocytes in Axin2−/− PF-Sutures at P9 and P11 as compared to wild type. These data indicated that Axin2−/− PF-Sutures lack physiological endochondral ossification, contain ectopic cartilage and display delayed Suture closure.
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craniosynostosis of coronal Suture in twist1 mice occurs through endochondral ossification recapitulating the physiological closure of posterior Frontal Suture
Frontiers in Physiology, 2011Co-Authors: Bjorn Behr, Michael T Longaker, Natalina QuartoAbstract:Craniosynostosis, the premature closure of cranial Suture, is a pathologic condition that affects 1/2000 live births. Saethre-Chotzen syndrome is a genetic condition characterized by craniosynostosis. The Saethre-Chotzen syndrome, which is defined by loss-of-function mutations in the TWIST gene, is the second most prevalent craniosynostosis. Although much of the genetics and phenotypes in craniosynostosis syndromes is understood, less is known about the underlying ossification mechanism during Suture closure. We have previously demonstrated that physiological closure of the posterior Frontal (PF) Suture occurs through endochondral ossification. Moreover, we revealed that antagonizing canonical Wnt signaling in the sagittal Suture leads to endochondral ossification of the Suture mesenchyme and sagittal synostosis, presumably by inhibiting Twist1. Classic Saethre-Chotzen syndrome is characterized by coronal synostosis, and the haploinsufficient Twist1+/- mice represents a suitable model for studying this syndrome. Thus, we seeked to understand the underlying ossification process in coronal craniosynostosis in Twist1+/- mice. Our data indicate that coronal Suture closure in Twist1+/- mice occurs between postnatal day 9 to 13 by endochondral ossification, as shown by histology, gene expression analysis and immunohistochemistry. In conclusion, this study reveals that coronal craniosynostosis in Twist1+/- mice occurs through endochondral ossification. Moreover, it suggests that haploinsufficency of Twist1 gene, a target of canonical Wnt-signaling, and inhibitor of chondrogenesis, mimics conditions of inactive canonical Wnt-signaling leading to craniosynostosis.
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microarray analysis of the role of regional dura mater in cranial Suture fate
Plastic and Reconstructive Surgery, 2008Co-Authors: Matthew Kwan, Bethany J Slater, Deepak M. Gupta, Zhen Wang, Michael T LongakerAbstract:Background: Craniosynostosis, the premature fusion of cranial Sutures, results in serious neurologic and morphologic abnormalities when left untreated. Surgical excision of the fused Sutures and remodeling of the skull remains the standard therapy. Development of novel, minimally invasive therapies for craniosynostosis will undoubtedly be dependent on a more thorough understanding of the molecular mechanisms underlying this abnormality. Significant evidence suggests the influence of regional dura mater on the behavior of the overlying Suture complex. The mouse model has been instrumental in investigating this observation because of the natural juxtaposition of the posterior Frontal Suture, which fuses early in life, with the other cranial Sutures, which remain patent. Methods: The authors used microarray analysis to compare genomic changes in the dura mater underlying the posterior Frontal and sagittal Sutures of mice. Suture-associated dura mater was harvested from mice before (postnatal day 5), during (postnatal day 10), and after (postnatal day 20) posterior Frontal Suture fusion (n = 20 mice for each of the three time points). Results: Microarray results confirmed differential regulation of genes involved in paracrine signaling, extracellular matrix, and bone remodeling between the dura mater underlying the fusing posterior Frontal Suture and the patent sagittal Suture. Conclusions: These data confirm global differences in gene expression between regional dura mater underlying fusing and patent Sutures. These results provide further insight into potential molecular mechanisms that may play a role in cranial Suture biology.
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Expression and possible mechanisms of regulation of BMP3 in rat cranial Sutures.
Plastic and Reconstructive Surgery, 2005Co-Authors: Randall P. Nacamuli, Kelly A. Lenton, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Michael T LongakerAbstract:BACKGROUND: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of Suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial Sutures and the factors regulating its expression in vitro. METHODS: Microarray analysis was performed on rat posterior Frontal and sagittal cranial Sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonSuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: BMP3 expression in the posterior Frontal Suture decreased over the time course analyzed, whereas it increased in the sagittal Suture. Notably, BMP3 expression was higher in the patent sagittal Suture during the window of posterior Frontal Suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). CONCLUSIONS: BMP3 is expressed in rat cranial Sutures in a temporal pattern suggesting involvement in cranial Suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial Suture biology.
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Expression and possible mechanisms of regulation of BMP3 in rat cranial Sutures.
Plastic and reconstructive surgery, 2005Co-Authors: Randall P. Nacamuli, Kelly A. Lenton, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Michael T LongakerAbstract:Background: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of Suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial Sutures and the factors regulating its expression in vitro. Methods: Microarray analysis was performed on rat posterior Frontal and sagittal cranial Sutures at 5, 10, 15, 20, and 30 days of life ( n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor β1, or cultured with primary rat nonSuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. Results: BMP3 expression in the posterior Frontal Suture decreased over the time course analyzed, whereas it increased in the sagittal Suture. Notably, BMP3 expression was higher in the patent sagittal Suture during the window of posterior Frontal Suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). Conclusions: BMP3 is expressed in rat cranial Sutures in a temporal pattern suggesting involvement in cranial Suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial Suture biology.
Hanjoon M Song - One of the best experts on this subject based on the ideXlab platform.
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Expression and possible mechanisms of regulation of BMP3 in rat cranial Sutures.
Plastic and Reconstructive Surgery, 2005Co-Authors: Randall P. Nacamuli, Kelly A. Lenton, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Michael T LongakerAbstract:BACKGROUND: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of Suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial Sutures and the factors regulating its expression in vitro. METHODS: Microarray analysis was performed on rat posterior Frontal and sagittal cranial Sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonSuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: BMP3 expression in the posterior Frontal Suture decreased over the time course analyzed, whereas it increased in the sagittal Suture. Notably, BMP3 expression was higher in the patent sagittal Suture during the window of posterior Frontal Suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). CONCLUSIONS: BMP3 is expressed in rat cranial Sutures in a temporal pattern suggesting involvement in cranial Suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial Suture biology.
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Expression and possible mechanisms of regulation of BMP3 in rat cranial Sutures.
Plastic and reconstructive surgery, 2005Co-Authors: Randall P. Nacamuli, Kelly A. Lenton, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Michael T LongakerAbstract:Background: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of Suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial Sutures and the factors regulating its expression in vitro. Methods: Microarray analysis was performed on rat posterior Frontal and sagittal cranial Sutures at 5, 10, 15, 20, and 30 days of life ( n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor β1, or cultured with primary rat nonSuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. Results: BMP3 expression in the posterior Frontal Suture decreased over the time course analyzed, whereas it increased in the sagittal Suture. Notably, BMP3 expression was higher in the patent sagittal Suture during the window of posterior Frontal Suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). Conclusions: BMP3 is expressed in rat cranial Sutures in a temporal pattern suggesting involvement in cranial Suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial Suture biology.
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quantitative transcriptional analysis of fusing and nonfusing cranial Suture complexes in mice
Plastic and Reconstructive Surgery, 2004Co-Authors: Randall P. Nacamuli, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Jonathan A Mathy, Michael T LongakerAbstract:: Previous studies have documented the differences in expression of various genes associated with the process of osteogenesis in fusing and nonfusing cranial Sutures, including growth factors, growth factor receptors, and extracellular matrix molecules. Most of these studies were performed in rats, and although the biology regulating cranial Suture fusion in mice and rats is presumed to be similar, studies are needed to verify these expression patterns as mice become increasingly utilized for scientific inquiry into the molecular biology of Suture fusion and patency. The purpose of this study was to determine the differences in expression of several genes known to be critical to osteoblast biology. Posterior Frontal and sagittal Suture complexes (including the associated dura mater, Suture mesenchyme, and osteogenic fronts) were isolated from 5-, 15-, 25-, 35-, and 45-day-old male CD-1 mice (n = 8 per age; n = 40 total). Total cellular RNA was extracted and converted to cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction was then performed for the following genes: transforming growth factor beta1 and beta3, fibroblast growth factor receptor 1, Runx2,Osteopontin, and Osteocalcin. Expression of all genes examined was increased significantly in the posterior Frontal Suture as compared with the sagittal Suture. Peak expression for all genes was observed on day 25. These data demonstrate that the expression of osteogenic growth factors, growth factor receptors, transcription factors, and extracellular matrix molecules is increased in the fusing posterior Frontal Suture in mice.
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apoptosis in a rodent model of cranial Suture fusion in situ imaging and gene expression analysis
Plastic and Reconstructive Surgery, 2004Co-Authors: Kenton Fong, Randall P. Nacamuli, Hanjoon M Song, Tony D Fang, Stephen M Warren, Benjamin L Franc, Carina Mari, Christopher H Contag, Francis G Blankenberg, Michael T LongakerAbstract:Craniosynostosis, the premature fusion of cranial Sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial Sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-Frontal Suture fusion and sagittal Suture patency to globally examine apoptosis in cranial Sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-Frontal Suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent Sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-Frontal and sagittal Suture complexes during the period of predicted posterior-Frontal Suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the Suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-Frontal and sagittal Sutures during the period of predicted posterior-Frontal Suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-Frontal Suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal Suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing Sutures is similar, the pathways mediating apoptosis within each Suture are distinct.
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mechanisms of murine cranial Suture patency mediated by a dominant negative transforming growth factor beta receptor adenovirus
Plastic and Reconstructive Surgery, 2004Co-Authors: Hanjoon M Song, Randall P. Nacamuli, Kenton Fong, Tony D Fang, Oliver Aalami, Catherine M Cowan, Jonathan A Mathy, Stephen M Warren, Michael T LongakerAbstract:: Using a physiologic model of mouse cranial Suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the Suture complex of the fusing posterior Frontal compared with the patent sagittal Suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior Frontal Suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial Suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial Suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial Suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the Suture mesenchyme and subjacent dura mater during the early period of predicted posterior Frontal Suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior Frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production.
Randall P. Nacamuli - One of the best experts on this subject based on the ideXlab platform.
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Expression and possible mechanisms of regulation of BMP3 in rat cranial Sutures.
Plastic and Reconstructive Surgery, 2005Co-Authors: Randall P. Nacamuli, Kelly A. Lenton, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Michael T LongakerAbstract:BACKGROUND: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of Suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial Sutures and the factors regulating its expression in vitro. METHODS: Microarray analysis was performed on rat posterior Frontal and sagittal cranial Sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonSuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: BMP3 expression in the posterior Frontal Suture decreased over the time course analyzed, whereas it increased in the sagittal Suture. Notably, BMP3 expression was higher in the patent sagittal Suture during the window of posterior Frontal Suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). CONCLUSIONS: BMP3 is expressed in rat cranial Sutures in a temporal pattern suggesting involvement in cranial Suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial Suture biology.
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Expression and possible mechanisms of regulation of BMP3 in rat cranial Sutures.
Plastic and reconstructive surgery, 2005Co-Authors: Randall P. Nacamuli, Kelly A. Lenton, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Michael T LongakerAbstract:Background: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of Suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial Sutures and the factors regulating its expression in vitro. Methods: Microarray analysis was performed on rat posterior Frontal and sagittal cranial Sutures at 5, 10, 15, 20, and 30 days of life ( n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor β1, or cultured with primary rat nonSuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. Results: BMP3 expression in the posterior Frontal Suture decreased over the time course analyzed, whereas it increased in the sagittal Suture. Notably, BMP3 expression was higher in the patent sagittal Suture during the window of posterior Frontal Suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). Conclusions: BMP3 is expressed in rat cranial Sutures in a temporal pattern suggesting involvement in cranial Suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial Suture biology.
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quantitative transcriptional analysis of fusing and nonfusing cranial Suture complexes in mice
Plastic and Reconstructive Surgery, 2004Co-Authors: Randall P. Nacamuli, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Jonathan A Mathy, Michael T LongakerAbstract:: Previous studies have documented the differences in expression of various genes associated with the process of osteogenesis in fusing and nonfusing cranial Sutures, including growth factors, growth factor receptors, and extracellular matrix molecules. Most of these studies were performed in rats, and although the biology regulating cranial Suture fusion in mice and rats is presumed to be similar, studies are needed to verify these expression patterns as mice become increasingly utilized for scientific inquiry into the molecular biology of Suture fusion and patency. The purpose of this study was to determine the differences in expression of several genes known to be critical to osteoblast biology. Posterior Frontal and sagittal Suture complexes (including the associated dura mater, Suture mesenchyme, and osteogenic fronts) were isolated from 5-, 15-, 25-, 35-, and 45-day-old male CD-1 mice (n = 8 per age; n = 40 total). Total cellular RNA was extracted and converted to cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction was then performed for the following genes: transforming growth factor beta1 and beta3, fibroblast growth factor receptor 1, Runx2,Osteopontin, and Osteocalcin. Expression of all genes examined was increased significantly in the posterior Frontal Suture as compared with the sagittal Suture. Peak expression for all genes was observed on day 25. These data demonstrate that the expression of osteogenic growth factors, growth factor receptors, transcription factors, and extracellular matrix molecules is increased in the fusing posterior Frontal Suture in mice.
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apoptosis in a rodent model of cranial Suture fusion in situ imaging and gene expression analysis
Plastic and Reconstructive Surgery, 2004Co-Authors: Kenton Fong, Randall P. Nacamuli, Hanjoon M Song, Tony D Fang, Stephen M Warren, Benjamin L Franc, Carina Mari, Christopher H Contag, Francis G Blankenberg, Michael T LongakerAbstract:Craniosynostosis, the premature fusion of cranial Sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial Sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-Frontal Suture fusion and sagittal Suture patency to globally examine apoptosis in cranial Sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-Frontal Suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent Sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-Frontal and sagittal Suture complexes during the period of predicted posterior-Frontal Suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the Suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-Frontal and sagittal Sutures during the period of predicted posterior-Frontal Suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-Frontal Suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal Suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing Sutures is similar, the pathways mediating apoptosis within each Suture are distinct.
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mechanisms of murine cranial Suture patency mediated by a dominant negative transforming growth factor beta receptor adenovirus
Plastic and Reconstructive Surgery, 2004Co-Authors: Hanjoon M Song, Randall P. Nacamuli, Kenton Fong, Tony D Fang, Oliver Aalami, Catherine M Cowan, Jonathan A Mathy, Stephen M Warren, Michael T LongakerAbstract:: Using a physiologic model of mouse cranial Suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the Suture complex of the fusing posterior Frontal compared with the patent sagittal Suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior Frontal Suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial Suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial Suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial Suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the Suture mesenchyme and subjacent dura mater during the early period of predicted posterior Frontal Suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior Frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production.
Kenton Fong - One of the best experts on this subject based on the ideXlab platform.
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Expression and possible mechanisms of regulation of BMP3 in rat cranial Sutures.
Plastic and Reconstructive Surgery, 2005Co-Authors: Randall P. Nacamuli, Kelly A. Lenton, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Michael T LongakerAbstract:BACKGROUND: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of Suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial Sutures and the factors regulating its expression in vitro. METHODS: Microarray analysis was performed on rat posterior Frontal and sagittal cranial Sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonSuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: BMP3 expression in the posterior Frontal Suture decreased over the time course analyzed, whereas it increased in the sagittal Suture. Notably, BMP3 expression was higher in the patent sagittal Suture during the window of posterior Frontal Suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). CONCLUSIONS: BMP3 is expressed in rat cranial Sutures in a temporal pattern suggesting involvement in cranial Suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial Suture biology.
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Expression and possible mechanisms of regulation of BMP3 in rat cranial Sutures.
Plastic and reconstructive surgery, 2005Co-Authors: Randall P. Nacamuli, Kelly A. Lenton, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Michael T LongakerAbstract:Background: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of Suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial Sutures and the factors regulating its expression in vitro. Methods: Microarray analysis was performed on rat posterior Frontal and sagittal cranial Sutures at 5, 10, 15, 20, and 30 days of life ( n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor β1, or cultured with primary rat nonSuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. Results: BMP3 expression in the posterior Frontal Suture decreased over the time course analyzed, whereas it increased in the sagittal Suture. Notably, BMP3 expression was higher in the patent sagittal Suture during the window of posterior Frontal Suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). Conclusions: BMP3 is expressed in rat cranial Sutures in a temporal pattern suggesting involvement in cranial Suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial Suture biology.
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quantitative transcriptional analysis of fusing and nonfusing cranial Suture complexes in mice
Plastic and Reconstructive Surgery, 2004Co-Authors: Randall P. Nacamuli, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Jonathan A Mathy, Michael T LongakerAbstract:: Previous studies have documented the differences in expression of various genes associated with the process of osteogenesis in fusing and nonfusing cranial Sutures, including growth factors, growth factor receptors, and extracellular matrix molecules. Most of these studies were performed in rats, and although the biology regulating cranial Suture fusion in mice and rats is presumed to be similar, studies are needed to verify these expression patterns as mice become increasingly utilized for scientific inquiry into the molecular biology of Suture fusion and patency. The purpose of this study was to determine the differences in expression of several genes known to be critical to osteoblast biology. Posterior Frontal and sagittal Suture complexes (including the associated dura mater, Suture mesenchyme, and osteogenic fronts) were isolated from 5-, 15-, 25-, 35-, and 45-day-old male CD-1 mice (n = 8 per age; n = 40 total). Total cellular RNA was extracted and converted to cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction was then performed for the following genes: transforming growth factor beta1 and beta3, fibroblast growth factor receptor 1, Runx2,Osteopontin, and Osteocalcin. Expression of all genes examined was increased significantly in the posterior Frontal Suture as compared with the sagittal Suture. Peak expression for all genes was observed on day 25. These data demonstrate that the expression of osteogenic growth factors, growth factor receptors, transcription factors, and extracellular matrix molecules is increased in the fusing posterior Frontal Suture in mice.
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apoptosis in a rodent model of cranial Suture fusion in situ imaging and gene expression analysis
Plastic and Reconstructive Surgery, 2004Co-Authors: Kenton Fong, Randall P. Nacamuli, Hanjoon M Song, Tony D Fang, Stephen M Warren, Benjamin L Franc, Carina Mari, Christopher H Contag, Francis G Blankenberg, Michael T LongakerAbstract:Craniosynostosis, the premature fusion of cranial Sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial Sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-Frontal Suture fusion and sagittal Suture patency to globally examine apoptosis in cranial Sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-Frontal Suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent Sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-Frontal and sagittal Suture complexes during the period of predicted posterior-Frontal Suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the Suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-Frontal and sagittal Sutures during the period of predicted posterior-Frontal Suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-Frontal Suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal Suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing Sutures is similar, the pathways mediating apoptosis within each Suture are distinct.
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mechanisms of murine cranial Suture patency mediated by a dominant negative transforming growth factor beta receptor adenovirus
Plastic and Reconstructive Surgery, 2004Co-Authors: Hanjoon M Song, Randall P. Nacamuli, Kenton Fong, Tony D Fang, Oliver Aalami, Catherine M Cowan, Jonathan A Mathy, Stephen M Warren, Michael T LongakerAbstract:: Using a physiologic model of mouse cranial Suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the Suture complex of the fusing posterior Frontal compared with the patent sagittal Suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior Frontal Suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial Suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial Suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial Suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the Suture mesenchyme and subjacent dura mater during the early period of predicted posterior Frontal Suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior Frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production.
Tony D Fang - One of the best experts on this subject based on the ideXlab platform.
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Expression and possible mechanisms of regulation of BMP3 in rat cranial Sutures.
Plastic and Reconstructive Surgery, 2005Co-Authors: Randall P. Nacamuli, Kelly A. Lenton, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Michael T LongakerAbstract:BACKGROUND: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of Suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial Sutures and the factors regulating its expression in vitro. METHODS: Microarray analysis was performed on rat posterior Frontal and sagittal cranial Sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonSuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: BMP3 expression in the posterior Frontal Suture decreased over the time course analyzed, whereas it increased in the sagittal Suture. Notably, BMP3 expression was higher in the patent sagittal Suture during the window of posterior Frontal Suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). CONCLUSIONS: BMP3 is expressed in rat cranial Sutures in a temporal pattern suggesting involvement in cranial Suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial Suture biology.
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Expression and possible mechanisms of regulation of BMP3 in rat cranial Sutures.
Plastic and reconstructive surgery, 2005Co-Authors: Randall P. Nacamuli, Kelly A. Lenton, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Michael T LongakerAbstract:Background: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of Suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial Sutures and the factors regulating its expression in vitro. Methods: Microarray analysis was performed on rat posterior Frontal and sagittal cranial Sutures at 5, 10, 15, 20, and 30 days of life ( n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor β1, or cultured with primary rat nonSuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. Results: BMP3 expression in the posterior Frontal Suture decreased over the time course analyzed, whereas it increased in the sagittal Suture. Notably, BMP3 expression was higher in the patent sagittal Suture during the window of posterior Frontal Suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). Conclusions: BMP3 is expressed in rat cranial Sutures in a temporal pattern suggesting involvement in cranial Suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial Suture biology.
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quantitative transcriptional analysis of fusing and nonfusing cranial Suture complexes in mice
Plastic and Reconstructive Surgery, 2004Co-Authors: Randall P. Nacamuli, Hanjoon M Song, Kenton Fong, Tony D Fang, Ali Salim, Jonathan A Mathy, Michael T LongakerAbstract:: Previous studies have documented the differences in expression of various genes associated with the process of osteogenesis in fusing and nonfusing cranial Sutures, including growth factors, growth factor receptors, and extracellular matrix molecules. Most of these studies were performed in rats, and although the biology regulating cranial Suture fusion in mice and rats is presumed to be similar, studies are needed to verify these expression patterns as mice become increasingly utilized for scientific inquiry into the molecular biology of Suture fusion and patency. The purpose of this study was to determine the differences in expression of several genes known to be critical to osteoblast biology. Posterior Frontal and sagittal Suture complexes (including the associated dura mater, Suture mesenchyme, and osteogenic fronts) were isolated from 5-, 15-, 25-, 35-, and 45-day-old male CD-1 mice (n = 8 per age; n = 40 total). Total cellular RNA was extracted and converted to cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction was then performed for the following genes: transforming growth factor beta1 and beta3, fibroblast growth factor receptor 1, Runx2,Osteopontin, and Osteocalcin. Expression of all genes examined was increased significantly in the posterior Frontal Suture as compared with the sagittal Suture. Peak expression for all genes was observed on day 25. These data demonstrate that the expression of osteogenic growth factors, growth factor receptors, transcription factors, and extracellular matrix molecules is increased in the fusing posterior Frontal Suture in mice.
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apoptosis in a rodent model of cranial Suture fusion in situ imaging and gene expression analysis
Plastic and Reconstructive Surgery, 2004Co-Authors: Kenton Fong, Randall P. Nacamuli, Hanjoon M Song, Tony D Fang, Stephen M Warren, Benjamin L Franc, Carina Mari, Christopher H Contag, Francis G Blankenberg, Michael T LongakerAbstract:Craniosynostosis, the premature fusion of cranial Sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial Sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-Frontal Suture fusion and sagittal Suture patency to globally examine apoptosis in cranial Sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-Frontal Suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent Sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-Frontal and sagittal Suture complexes during the period of predicted posterior-Frontal Suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the Suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-Frontal and sagittal Sutures during the period of predicted posterior-Frontal Suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-Frontal Suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal Suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing Sutures is similar, the pathways mediating apoptosis within each Suture are distinct.
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mechanisms of murine cranial Suture patency mediated by a dominant negative transforming growth factor beta receptor adenovirus
Plastic and Reconstructive Surgery, 2004Co-Authors: Hanjoon M Song, Randall P. Nacamuli, Kenton Fong, Tony D Fang, Oliver Aalami, Catherine M Cowan, Jonathan A Mathy, Stephen M Warren, Michael T LongakerAbstract:: Using a physiologic model of mouse cranial Suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the Suture complex of the fusing posterior Frontal compared with the patent sagittal Suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior Frontal Suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial Suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial Suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial Suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the Suture mesenchyme and subjacent dura mater during the early period of predicted posterior Frontal Suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior Frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production.
