Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 117
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biomolecules ; 11(8)2021 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-34439795

RESUMO

The cranial bones constitute the protective structures of the skull, which surround and protect the brain. Due to the limited repair capacity, the reconstruction and regeneration of skull defects are considered as an unmet clinical need and challenge. Previously, it has been proposed that the periosteum and dura mater provide reparative progenitors for cranial bones homeostasis and injury repair. In addition, it has also been speculated that the cranial mesenchymal stem cells reside in the perivascular niche of the diploe, namely, the soft spongy cancellous bone between the interior and exterior layers of cortical bone of the skull, which resembles the skeletal stem cells' distribution pattern of the long bone within the bone marrow. Not until recent years have several studies unraveled and validated that the major mesenchymal stem cell population of the cranial region is primarily located within the suture mesenchyme of the skull, and hence, they are termed suture mesenchymal stem cells (SuSCs). Here, we summarized the characteristics of SuSCs, this newly discovered stem cell population of cranial bones, including the temporospatial distribution pattern, self-renewal, and multipotent properties, contribution to injury repair, as well as the signaling pathways and molecular mechanisms associated with the regulation of SuSCs.


Assuntos
Regeneração Óssea/genética , Suturas Cranianas/citologia , Células-Tronco Mesenquimais/citologia , Osteócitos/citologia , Fraturas Cranianas/genética , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Catepsina K/genética , Catepsina K/metabolismo , Diferenciação Celular , Proliferação de Células , Suturas Cranianas/crescimento & desenvolvimento , Suturas Cranianas/lesões , Suturas Cranianas/metabolismo , Craniossinostoses/genética , Craniossinostoses/metabolismo , Craniossinostoses/patologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteócitos/metabolismo , Transdução de Sinais , Fraturas Cranianas/metabolismo , Fraturas Cranianas/patologia , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
2.
Nat Commun ; 12(1): 4797, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376651

RESUMO

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.


Assuntos
Suturas Cranianas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Osteogênese/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Acrocefalossindactilia/embriologia , Acrocefalossindactilia/genética , Acrocefalossindactilia/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Suturas Cranianas/citologia , Suturas Cranianas/embriologia , Dura-Máter/citologia , Dura-Máter/embriologia , Dura-Máter/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA-Seq/métodos , Crânio/citologia , Crânio/embriologia , Crânio/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo
3.
Nat Commun ; 12(1): 4640, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330896

RESUMO

Cranial sutures are major growth centers for the calvarial vault, and their premature fusion leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1+/- mouse model of coronal craniosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co-transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1+/- mice. Our study reveals that decrease and/or imbalance of skeletal stem/progenitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis.


Assuntos
Suturas Cranianas/metabolismo , Craniossinostoses/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Células-Tronco/metabolismo , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Suturas Cranianas/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Sistema Musculoesquelético/citologia , Sistema Musculoesquelético/metabolismo , Células-Tronco/citologia , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Via de Sinalização Wnt/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo
4.
Mol Cell Biol ; 41(8): e0014921, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-33972395

RESUMO

ETS2 repressor factor (ERF) haploinsufficiency causes late-onset craniosynostosis (CRS) (OMIM entry 600775; CRS4) in humans, while in mice Erf insufficiency also leads to a similar multisuture synostosis phenotype preceded by mildly reduced calvarium ossification. However, neither the cell types affected nor the effects per se have been identified so far. Here, we establish an ex vivo system for the expansion of suture-derived mesenchymal stem and progenitor cells (sdMSCs) and analyze the role of Erf levels in their differentiation. Cellular data suggest that Erf insufficiency specifically decreases osteogenic differentiation of sdMSCs, resulting in the initially delayed mineralization of the calvarium. Transcriptome analysis indicates that Erf is required for efficient osteogenic lineage commitment of sdMSCs. Elevated retinoic acid catabolism due to increased levels of the cytochrome P450 superfamily member Cyp26b1 as a result of decreased Erf levels appears to be the underlying mechanism leading to defective differentiation. Exogenous addition of retinoic acid can rescue the osteogenic differentiation defect, suggesting that Erf affects cranial bone mineralization during skull development through retinoic acid gradient regulation.


Assuntos
Suturas Cranianas/metabolismo , Craniossinostoses/metabolismo , Osteogênese/fisiologia , Tretinoína/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Craniossinostoses/genética , Camundongos , Osteogênese/genética , Fenótipo , Células-Tronco/metabolismo
5.
Drug Des Devel Ther ; 15: 905-917, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33688165

RESUMO

Background: The potential of relapse of craniofacial disharmony after trans-sutural distraction osteogenesis is high due to the failure to produce a stable bone bridge in the suture gap. The aim of this study is to evaluate whether hydroxyapatite nanoparticles (nHAP) have the effect of promoting osteoblast differentiation of suture-derived stem cells (SuSCs) and bone formation in sagittal suture during expansion. Methods: SuSCs were isolated from sagittal sutures and exposed to various concentrations of nHAP (0, 25, 50, and 100 µg mL-1) to determine the optimal concentration of nHAP in osteoblast differentiation via performing Western Blotting and RT-qPCR. Twenty 4-week-old male Sprague-Dawley rats were randomly assigned into 4 groups: SHAM (sham-surgery), distraction, ACS (absorbable collagen sponge) and ACS+nHAP groups. In the ACS and ACS+nHAP groups, saline solution and nHAP suspended in a saline solution were delivered by ACS placed across the sagittal suture, respectively. In the latter three groups, the suture was expanded for 14 days by 50 g of constant force via a W shape expansion device. Suture gap area, bone volume fraction (BV/TV) and bone mineral density (BMD) of sagittal sutures were assessed via micro-CT, while the mechanical properties of sagittal sutures were evaluated via nanoindentation test. The efficacy of nHAP on bone formation in sagittal suture was also evaluated via BMP-2 immunohistochemistry staining. Results: The expression of osteoblast related genes and proteins induced by 25µg mL-1 nHAP were significantly higher than the other groups in vitro (p<0.05). Furthermore, treating with 25µg mL-1 nHAP in vivo, the suture gap area was significantly reduced when compared with the distraction group. Correspondingly, the BV/TV, BMD, hardness and modulus of sagittal sutures were significantly increased in the ACS+nHAP group (p<0.05). Conclusion: The 25µg mL-1 dose of nHAP delivered by ACS can facilitate bone formation into the sagittal suture during expansion via inducing osteoblast differentiation of SuSCs.


Assuntos
Suturas Cranianas/efeitos dos fármacos , Durapatita/farmacologia , Nanopartículas/química , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Suturas Cranianas/metabolismo , Relação Dose-Resposta a Droga , Durapatita/química , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade
6.
Cell Rep ; 32(1): 107871, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32640236

RESUMO

Craniofacial abnormalities often involve sutures, the growth centers of the skull. To characterize the organization and processes governing their development, we profile the murine frontal suture, a model for sutural growth and fusion, at the tissue- and single-cell level on embryonic days (E)16.5 and E18.5. For the wild-type suture, bulk RNA sequencing (RNA-seq) analysis identifies mesenchyme-, osteogenic front-, and stage-enriched genes and biological processes, as well as alternative splicing events modifying the extracellular matrix. Single-cell RNA-seq analysis distinguishes multiple subpopulations, of which five define a mesenchyme-osteoblast differentiation trajectory and show variation along the anteroposterior axis. Similar analyses of in vivo mouse models of impaired frontal suturogenesis in Saethre-Chotzen and Apert syndromes, Twist1+/- and Fgfr2+/S252W, demonstrate distinct transcriptional changes involving angiogenesis and ribogenesis, respectively. Co-expression network analysis reveals gene expression modules from which we validate key driver genes regulating osteoblast differentiation. Our study provides a global approach to gain insights into suturogenesis.


Assuntos
Suturas Cranianas/embriologia , Suturas Cranianas/metabolismo , Redes Reguladoras de Genes , Transcriptoma/genética , Processamento Alternativo/genética , Animais , Diferenciação Celular , Linhagem Celular , Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Osteogênese/genética , RNA-Seq , Análise de Célula Única , Fatores de Tempo , Transcrição Genética
7.
J Cell Physiol ; 235(9): 5972-5984, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-31970784

RESUMO

Mechanical force across sutures is able to promote suture osteogenesis. Orthodontic clinics often use this biological characteristic of sutures to treat congenital cranio-maxillofacial malformations. However, the underlying mechanisms still remain poorly understood. Craniofacial sutures provide a special growth source and support primary sites of osteogenesis. Here, we isolated rat sagittal suture cells (rSAGs), which had mesenchymal stem cell characteristics and differentiating abilities. Cells were then subjected to mechanical tension (5% elongation, 0.5 Hz; sinusoidal waveforms) showing that mechanical tension could enhance osteogenic differentiation but hardly affect proliferation of rSAGs. Besides, mechanical tension could increase Rho-associated kinase (ROCK) expression and enhance transcriptional coactivator with PDZ-binding motif (TAZ) nuclear translocation. Inhibiting ROCK expression could suppress tension-induced osteogenesis and block tension-induced upregulation of nuclear TAZ. In addition, our results indicated that TAZ had direct combination sites with runt-related transcription factor 2 (Runx2) in rSAGs, and knock-downed TAZ simultaneously decreased the expression of Runx2 no matter with or without mechanical tension. In summary, our findings demonstrated that the multipotency of rSAGs in vitro could give rise to early osteogenic differentiation under mechanical tension, which was mediated by ROCK-TAZ signal axis.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Suturas Cranianas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Osteogênese/genética , Transativadores/genética , Quinases Associadas a rho/genética , Animais , Diferenciação Celular/genética , Suturas Cranianas/crescimento & desenvolvimento , Suturas Cranianas/patologia , Fenômenos Mecânicos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Ratos , Transdução de Sinais/genética
8.
Cell Prolif ; 53(1): e12697, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31713930

RESUMO

OBJECTIVES: miR-21 can promote osteoblast differentiation of periodontal ligament stem cells. However, the effect of miR-21 on bone remodelling in the midpalatal suture is unclear. This study aimed to elucidate the effects of miR-21 on the midpalatal suture bone remodelling by expanding the palatal sutures. MATERIALS AND METHODS: miR-21 deficient (miR-21-/- ) and wild-type (WT) mice were used to establish animal models by expanding the palatal sutures. Micro-CT, haematoxylin-eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, fluorescence labelling and immunohistochemistry were used to investigate the function of miR-21 in midpalatal suture bone remodelling. Besides, bone mesenchymal stem cells (BMSCs) derived from both miR-21-/- and WT mice were cultured. The MTT, CCK8, EdU analysis, transwell and wound healing test were used to assess the effects of miR-21 on the characteristics of cells. RESULTS: The expression of ALP was suppressed in miR-21-/- mice after expansion except 28 days. The expression of Ocn in WT mice was much higher than that of miR-21-/-  mice. Besides, with mechanical force, miR-21 deficiency downregulated the expression of Opg, upregulated the expression of Rankl, and induced more osteoclasts as TRAP staining showed. After injecting agomir-21  to miR-21-/- mice, the expression of Alp, Ocn and Opg/Rankl were rescued. In vitro, the experiments suggested that miR-21 deficiency reduced proliferation and migration ability of BMSCs. CONCLUSIONS: The results showed that miR-21 deficiency reduced the rate of bone formation and prolonged the process of bone formation. miR-21 regulated the bone resorption and osteoclastogenesis by affecting the cell abilities of proliferation and migration.


Assuntos
Células da Medula Óssea/metabolismo , Remodelação Óssea , Suturas Cranianas/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Palato/metabolismo , Estresse Mecânico , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Proliferação de Células , Suturas Cranianas/citologia , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Osteoprotegerina/biossíntese , Osteoprotegerina/genética , Palato/citologia , Ligante RANK/biossíntese , Ligante RANK/genética , Fosfatase Ácida Resistente a Tartarato/biossíntese , Fosfatase Ácida Resistente a Tartarato/genética
9.
J Oral Rehabil ; 47 Suppl 1: 19-28, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31461788

RESUMO

Craniosynostosis, a severe craniofacial developmental disease, can only be treated with surgery currently. Recent studies have shown that proteoglycans are involved in the suture development. For the bone matrix protein, dentin matrix protein 1 (DMP1), glycosylation on the N-terminal of it could generate a functional proteoglycan form of DMP1 during osteogenesis. We identified that the proteoglycan form of DMP1 (DMP1-PG) is highly expressed in mineralisation front of suture. But, the potential role of DMP1-PG in suture fusion remain unclear. To investigate the role of DMP1-PG in cranial suture fusion and craniofacial bone development. By using a DMP1 glycosylation site mutation mouse model, DMP1-S89G mice, we compared the suture development in it with control mice. We compared the suture phenotypes, bone formation rate, expression levels of bone formation markers in vivo between DMP1-S89G mice and wild-type mice. Meanwhile, cell culture and organ culture were performed to detect the differences in cell differentiation and suture fusion in vitro. Finally, chondroitin sulphate (CHS), as functional component of DMP1-PG, was employed to test whether it could delay the premature suture fusion and the abnormal differentiation of bone mesenchymal stem cells (BMSCs) of DMP1-PG mice. DMP1-S89G mice had premature closure of suture and shorter skull size. Lack of DMP1-PG accelerated bone formation in cranial suture. DMP1-PG maintained the essential stemness of BMSCs in suture through blocking the premature differentiation of BMSCs to osteoblasts. Finally, chondroitin sulphate, a major component of DMP1-PG, successfully delayed the premature suture fusion by organ culture of skull in vitro. DMP1-PG could inhibit premature fusion of cranial suture and maintain the suture through regulating the osteogenic differentiation of BMSCs.


Assuntos
Suturas Cranianas , Osteogênese , Animais , Suturas Cranianas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicosilação , Humanos , Camundongos , Osteoblastos/metabolismo , Crânio
10.
Biochem Biophys Res Commun ; 512(2): 145-149, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30853186

RESUMO

Cdc42 (cell division cycle 42) is ubiquitously expressed small GTPases belonging to the Rho family of proteins. Previously, we generated limb bud mesenchyme-specific Cdc42 inactivated mice (Cdc42 conditional knockout mice; Cdc42 fl/fl; Prx1-Cre), which showed short limbs and cranial bone deformities, though the mechanism related to the cranium phenotype was unclear. In the present study, we investigated the role of Cdc42 in cranial bone development. Our results showed that loss of Cdc42 caused a defect of intramembranous ossification in cranial bone tissues which is related to decreased expressions of cranial suture morphogenesis genes, including Indian hedgehog (Ihh) and bone morphogenetic proteins (BMPs). These findings demonstrate that Cdc42 plays a crucial role in cranial osteogenesis, and is controlled by Ihh- and BMP-mediated signaling during cranium development.


Assuntos
Desenvolvimento Ósseo , Suturas Cranianas/crescimento & desenvolvimento , Osteogênese , Proteína cdc42 de Ligação ao GTP/genética , Animais , Suturas Cranianas/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Proteína cdc42 de Ligação ao GTP/metabolismo
11.
J Neurosurg Pediatr ; 22(6): 620-626, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30215585

RESUMO

OBJECTIVECranial suture patterning and development are highly regulated processes that are not entirely understood. While studies have investigated the differential gene expression for different sutures, little is known about gene expression changes during suture fusion. The aim of this study was to examine gene expression in patent, fusing, and fused regions along sagittal suture specimens in nonsyndromic craniosynostosis patients.METHODSSagittal sutures were collected from 7 patients (average age 4.5 months) who underwent minimally invasive craniotomies at the Children's Hospital of Richmond at VCU under IRB approval. The sutures were analyzed using micro-CT to evaluate patency. The areas were classified as open, fusing, or fused and were harvested, and mRNA was isolated. Gene expression for bone-related proteins, osteogenic and angiogenic factors, transforming growth factor-ß (TGF-ß) superfamily, and Wnt signaling was analyzed using quantitative polymerase chain reaction and compared with normal sutures collected from fetal demise tissue (control).RESULTSMicro-CT demonstrated that there are variable areas of closure along the length of the sagittal suture. When comparing control samples to surgical samples, there was a significant difference in genes for Wnt signaling, TGF-ß, angiogenic and osteogenic factors, bone remodeling, and nuclear rigidity in mRNA isolated from the fusing and fused areas of the sagittal suture compared with patent areas (p < 0.05).CONCLUSIONSIn nonsyndromic sagittal craniosynostosis, the affected suture has variable areas of being open, fusing, and fused. These specific areas have different mRNA expression. The results suggest that BMP-2, FGFR3, and several other signaling pathways play a significant role in the regulation of suture fusion as well as in the maintenance of patency in the normal suture.


Assuntos
Suturas Cranianas/metabolismo , Craniossinostoses/genética , Regulação da Expressão Gênica no Desenvolvimento , Osteogênese/genética , Suturas Cranianas/cirurgia , Craniossinostoses/metabolismo , Craniossinostoses/cirurgia , Craniotomia , Feminino , Humanos , Lactente , Masculino , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
12.
Plast Reconstr Surg ; 141(2): 250e-260e, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29369995

RESUMO

BACKGROUND: The pathophysiology of nonsyndromic craniosynostosis remains poorly understood. The authors seek to understand the cause of this condition with a specific focus on how osteoclasts may contribute to craniosynostosis. Here, the authors characterize proteins differentially expressed in patent and fused cranial sutures by comparing their respective proteomes. METHODS: Fused and patent suture samples were obtained from craniosynostotic patients undergoing surgery at a single academic medical center. Extracted protein from samples was interrogated using mass spectrometry. Differential protein expression was determined using maximum likelihood-based G-test with a q-value cutoffs of 0.5 after correction for multiple hypothesis testing. Immunolocalization of lead protein candidates was performed to validate proteomic findings. In addition, quantitative polymerase chain reaction analysis of corresponding gene expression of proteins of interest was performed. RESULTS: Proteins differentially expressed in patent versus fused sutures included collagen 6A1 (Col6A1), fibromodulin, periostin, aggrecan, adipocyte enhancer-binding protein 1, and osteomodulin (OMD). Maximum likelihood-based G-test suggested that Col6A1, fibromodulin, and adipocyte enhancer-binding protein 1 are highly expressed in patent sutures compared with fused sutures, whereas OMD is up-regulated in fused sutures compared with patent sutures. These results were corroborated by immunohistochemistry. Quantitative polymerase chain reaction data point to an inverse relationship in proteins of interest to RNA transcript levels, in prematurely fused and patent sutures that potentially describes a feedback loop mechanism. CONCLUSIONS: Proteome analysis validated by immunohistochemistry may provide insight into the mechanism of cranial suture patency and disease from an osteoclast perspective. The authors results suggest a role of inflammatory mediators in nonsyndromic craniosynostosis. Col6A1 may aid in the regulation of suture patency, and OMD may be involved in premature fusion. Additional validation studies are required.


Assuntos
Suturas Cranianas/metabolismo , Craniossinostoses/patologia , Osteoclastos/metabolismo , Proteoma/metabolismo , Adolescente , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão/métodos , Colágeno Tipo VI/metabolismo , Suturas Cranianas/fisiopatologia , Craniossinostoses/etiologia , Craniossinostoses/cirurgia , Proteínas da Matriz Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Proteoglicanas/metabolismo , Proteômica/métodos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Espectrometria de Massas em Tandem/métodos , Regulação para Cima
13.
Sci Rep ; 7(1): 11494, 2017 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-28904366

RESUMO

Craniosynostosis is a bone developmental disease where premature ossification of the cranial sutures occurs leading to fused sutures. While biomechanical forces have been implicated in craniosynostosis, evidence of the effect of microenvironmental stiffness changes in the osteogenic commitment of cells from the sutures is lacking. Our aim was to identify the differential genetic expression and osteogenic capability between cells from patent and fused sutures of children with craniosynostosis and whether these differences are driven by changes in the stiffness of the microenvironment. Cells from both sutures demonstrated enhanced mineralisation with increasing substrate stiffness showing that stiffness is a stimulus capable of triggering the accelerated osteogenic commitment of the cells from patent to fused stages. The differences in the mechanoresponse of these cells were further investigated with a PCR array showing stiffness-dependent upregulation of genes mediating growth and bone development (TSHZ2, IGF1), involved in the breakdown of extracellular matrix (MMP9), mediating the activation of inflammation (IL1ß) and controlling osteogenic differentiation (WIF1, BMP6, NOX1) in cells from fused sutures. In summary, this study indicates that stiffer substrates lead to greater osteogenic commitment and accelerated bone formation, suggesting that stiffening of the extracellular environment may trigger the premature ossification of the sutures.


Assuntos
Craniossinostoses/etiologia , Craniossinostoses/metabolismo , Osteogênese , Transdução de Sinais , Calcificação Fisiológica , Células Cultivadas , Colágeno/metabolismo , Biologia Computacional/métodos , Suturas Cranianas/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Osteogênese/genética , Transcriptoma
14.
Development ; 144(21): 4026-4036, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28947535

RESUMO

Craniosynostosis is a prevalent human birth defect characterized by premature fusion of calvarial bones. In this study, we show that tight regulation of endogenous PDGFRα activity is required for normal calvarium development in the mouse and that dysregulated PDGFRα activity causes craniosynostosis. Constitutive activation of PDGFRα leads to expansion of cartilage underlying the coronal sutures, which contribute to suture closure through endochondral ossification, in a process regulated in part by PI3K/AKT signaling. Our results thus identify a novel mechanism underlying calvarial development in craniosynostosis.


Assuntos
Cartilagem/embriologia , Suturas Cranianas/embriologia , Suturas Cranianas/metabolismo , Craniossinostoses/metabolismo , Morfogênese , Osteogênese , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Alelos , Animais , Cartilagem/anormalidades , Cartilagem/metabolismo , Linhagem da Célula , Condrogênese , Suturas Cranianas/patologia , Regulação da Expressão Gênica no Desenvolvimento , Ligantes , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Crista Neural/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais , Crânio/anormalidades , Crânio/patologia
15.
J Craniofac Surg ; 28(6): 1620-1625, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28692512

RESUMO

BACKGROUND: The mutations in a Notch signaling ligand, jagged 1, are associated with unilateral coronal craniosynostosis in humans. However, the underlying mechanisms of Notch signaling in cranial suture biology still remain unclear. METHODS: The temporal and spatial patterns of Notch signaling expression were examined in the posterofrontal and sagittal sutures of Sprague-Dawley rats by real-time quantitative reverse-transcription polymerase chain reaction at postnatal ages of 2, 15, and 25 days. The role of Notch signaling in the proliferation and differentiation of osteoblasts isolated from calvarial was examined in vitro by EdU incorporation assays and real-time quantitative reverse-transcription polymerase chain reaction after activating and inhibiting Notch signaling. RESULTS: The mRNA levels of Notch family members (including Jagged 1, Delta 1, 3, 4, Notch 1-4, Hes 1, and Hes 5) decreased during the posterofrontal cranial suture fusion in rat. However, in the patent sagittal sutures, the mRNA levels of Notch family members (Jagged 2, Delta 1, Notch 1, Notch 3, Hes 5, and Hey 1) increased during suture development. The EdU incorporation assays revealed that the induction of Notch signaling in calvaria osteobalsts using Jagged 1 promoted the proliferation rates in those cells in vitro. Further studies showed that activation of Notch signaling calvaria osteobalsts using Jagged 1 led to the suppression of late osteogenetic markers such as type I collagen and osteocalcin. CONCLUSIONS: The regulation of Notch signaling is of crucial importance during the physiological patterning of posterofrontal and sagittal cranial sutures. Thus, targeting this pathway may prove significant for the development of future therapeutic applications in craniosynostosis.


Assuntos
Suturas Cranianas , Osteoblastos , Receptores Notch , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Suturas Cranianas/citologia , Suturas Cranianas/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptores Notch/análise , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia
16.
J Cell Mol Med ; 21(11): 2782-2795, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28470873

RESUMO

The cranial suture complex is a heterogeneous tissue consisting of osteogenic progenitor cells and mesenchymal stem cells (MSCs) from bone marrow and suture mesenchyme. The fusion of cranial sutures is a highly coordinated and tightly regulated process during development. Craniosynostosis is a congenital malformation caused by premature fusion of cranial sutures. While the progenitor cells derived from the cranial suture complex should prove valuable for studying the molecular mechanisms underlying suture development and pathogenic premature suture fusion, primary human cranial suture progenitors (SuPs) have limited life span and gradually lose osteoblastic ability over passages. To overcome technical challenges in maintaining sufficient and long-term culture of SuPs for suture biology studies, we establish and characterize the reversibly immortalized human cranial suture progenitors (iSuPs). Using a reversible immortalization system expressing SV40 T flanked with FRT sites, we demonstrate that primary human suture progenitor cells derived from the patent sutures of craniosynostosis patients can be efficiently immortalized. The iSuPs maintain long-term proliferative activity, express most of the consensus MSC markers and can differentiate into osteogenic and adipogenic lineages upon BMP9 stimulation in vitro and in vivo. The removal of SV40 T antigen by FLP recombinase results in a decrease in cell proliferation and an increase in the endogenous osteogenic and adipogenic capability in the iSuPs. Therefore, the iSuPs should be a valuable resource to study suture development, intramembranous ossification and the pathogenesis of craniosynostosis, as well as to explore cranial bone tissue engineering.


Assuntos
Suturas Cranianas/metabolismo , Craniossinostoses/genética , Efeito Fundador , Fatores de Diferenciação de Crescimento/genética , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Linhagem Celular Transformada , Proliferação de Células , Suturas Cranianas/patologia , Craniossinostoses/metabolismo , Craniossinostoses/patologia , Expressão Gênica , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento/metabolismo , Humanos , Lactente , Masculino , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/metabolismo , Transformação Genética
17.
J Bone Miner Metab ; 35(1): 40-51, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26825658

RESUMO

Sutures are fibrous tissues that connect bones in craniofacial skeletal complexes. Cranio- and dentofacial skeletal deformities in infant and adolescent patients can be treated by applying tensile force to sutures to induce sutural bone formation. The early gene expression induced by mechanical stress is essential for bone formation in long bones; however, early gene expression during sutural bone formation induced by tensile force is poorly characterized. In vivo studies are essential to evaluate molecular responses to mechanical stresses in heterogeneous cell populations, such as sutures. In this paper we examined in vivo early gene expression and the underlying regulatory mechanism for this expression in tensile-force-applied cranial sutures, focusing on genes involved in vascularization. Tensile force upregulated expression of vascular factors, such as vascular endothelial growth factor (Vegf) and endothelial cell markers, in sutures within 3 h. The expression of connective tissue growth factor (Ctgf) and Rho-associated coiled-coil containing protein kinase 2 (Rock2) was also upregulated by tensile force. A CTGF-neutralizing antibody and the ROCK inhibitor, Y-27632, abolished tensile-force-induced Vegf expression. Moreover, tensile force activated extracellular signal-related kinase 1/2 (ERK1/2) signaling in sagittal sutures, and the ERK1/2 inhibitor, U0126, partially inhibited tensile-force-induced Ctgf expression. These results indicate that tensile force induces in vivo gene expression associated with vascularization early in tensile-force-induced sutural bone formation. Moreover, the early induction of Vegf gene expression is regulated by CTGF and ROCK2.


Assuntos
Suturas Cranianas , Regulação da Expressão Gênica/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Neovascularização Fisiológica/fisiologia , Resistência à Tração/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Adolescente , Animais , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Suturas Cranianas/irrigação sanguínea , Suturas Cranianas/metabolismo , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estresse Mecânico , Quinases Associadas a rho/metabolismo
18.
Eur J Orthod ; 39(3): 227-234, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27141932

RESUMO

Background and objectives: Relaxin (RLN) is an insulin-like hormone associated with extracellular matrix degradation, osteoclastogenesis, and osteoblast differentiation. This study aimed to assess the effect of RLN during and after lateral expansion of murine calvarial sagittal sutures. Materials and methods: RLN was injected topically using a nano-sized liposome carrier into the sagittal sutures of 8- to 10-week-old wild type mice just before lateral expansion. Suture morphology, bone mineral density (BMD), and bone volume were analysed by micro-computed tomography. Collagen deposition and osteoclast differentiation were observed by Verhoeff-Van Gieson (VVG) and tartrate-resistant acid phosphatase (TRAP) staining, respectively. Results: Less collagen staining and higher tissue-specific relaxin/insulin-like family peptide receptor (Rxfp)-1 and -2 expression were observed in the RLN-treated samples after 48 hours. Increased BMD and volume, and thick well-organised osteoid tissue, with multinucleated TRAP-positive cells, were observed in RLN-treated samples after 1 week. Increased Rxfp-1 expression was observed in the sagittal sutures in the mid-suture fibrous tissue following RLN treatment. Rxfp-2 was only expressed in the calvarial bone under tensile stimulation and RLN treatment further increased its expression. Limitations: RLN-liposomes were not detected at any instance under the current experimental conditions. This is a preliminary study and the sample number limits the power of its results. VVG staining cannot quantify collagen contents but can provide preliminary information on the presence of collagen fibres. Conclusions: RLN treatment may modify bone remodelling and collagen metabolism during and after suture expansion.


Assuntos
Remodelação Óssea/efeitos dos fármacos , Suturas Cranianas/efeitos dos fármacos , Técnica de Expansão Palatina , Relaxina/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Colágeno/metabolismo , Suturas Cranianas/metabolismo , Suturas Cranianas/cirurgia , Avaliação Pré-Clínica de Medicamentos/métodos , Lipossomos , Masculino , Camundongos Endogâmicos C57BL , Osteogênese/efeitos dos fármacos , Relaxina/administração & dosagem , Microtomografia por Raio-X
19.
Cleft Palate Craniofac J ; 54(1): 109-118, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-26954032

RESUMO

OBJECTIVE: Fusion of the cranial sutures is thought to depend on signaling among perisutural tissues. Mapping regional variations in gene expression would improve current models of craniosynostosis. Laser capture microdissection (LCM) isolates discrete cell populations for gene expression analysis. LCM has rarely been used in the study of mineralized tissue. This study sought to evaluate the potential use of LCM for mapping of regional gene expression within the cranial suture. DESIGN: Coronal sutures were isolated from 10-day-old wild-type and craniosynostotic (CS) New Zealand White rabbits, and LCM was used to isolate RNA from the sutural ligament (SL), osteogenic fronts (OF), dura mater, and periosteum. Relative expression levels for Fibroblast Growth Factor 2 (FGF2), Fibroblast Growth Factor Receptor 2 (FGFR2), Transforming Growth Factor Beta 2 (TGFß-2), Transforming Growth Factor Beta 3 (TGFß-3), Bone Morphogenetic Protein 2 (BMP-2), Bone Morphogenetic Protein 4 (BMP-4), and Noggin were determined using quantitative real-time PCR. RESULTS: A fivefold increase in TGFß2 expression was detected in the CS SL relative to wild type, whereas 152-fold less TGFß-3 was detected within the OF of CS animals. Noggin expression was increased by 10-fold within the CS SL, but reduced by 13-fold within the CS dura. Reduced expression of FGF2 was observed within the CS SL and dura, whereas increased expression of FGFR2 was observed within the CS SL. Reduced expression of BMP-2 was observed in the CS periosteum, and elevated expression of BMP-4 was observed in the CS SL and dura. CONCLUSIONS: LCM provides an effective tool for measuring regional variations in cranial suture gene expression. More precise measurements of regional gene expression with LCM may facilitate efforts to correlate gene expression with suture morphogenesis and pathophysiology.


Assuntos
Suturas Cranianas/cirurgia , Craniossinostoses/genética , Perfilação da Expressão Gênica , Microdissecção e Captura a Laser , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Suturas Cranianas/metabolismo , Coelhos , Reação em Cadeia da Polimerase em Tempo Real
20.
PLoS One ; 11(11): e0165775, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27829009

RESUMO

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.


Assuntos
Suturas Cranianas/citologia , Osso Frontal/citologia , Regulação da Expressão Gênica no Desenvolvimento , Osso Occipital/citologia , Osteogênese/genética , Osso Parietal/citologia , Animais , Colágeno/genética , Colágeno/metabolismo , Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Suturas Cranianas/crescimento & desenvolvimento , Suturas Cranianas/metabolismo , Osso Frontal/crescimento & desenvolvimento , Osso Frontal/metabolismo , Humanos , Osso Occipital/crescimento & desenvolvimento , Osso Occipital/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Osso Parietal/crescimento & desenvolvimento , Osso Parietal/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição Twist/genética , Fatores de Transcrição Twist/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...