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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.
Plast Reconstr Surg ; 145(3): 552e-562e, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32097313

RESUMO

BACKGROUND: Several studies have verified that bone morphogenetic proteins (BMPs) may be involved in the development of craniosynostosis; little attention has been focused on the role of BMP9 in cranial suture biology. The authors investigated the role of BMP9 in suture progenitor cells. METHODS: The authors isolated and cultured prematurely fused and internal control patent suture progenitor cells from patients with nonsyndromic craniosynostosis. Overexpression of BMP9 was mediated by adenoviral vectors. Osteoblast and osteoclast differentiation-related markers were evaluated by staining techniques and touchdown quantitative polymerase chain reaction analysis. In vivo analysis of BMP9-induced suture progenitor cell osteogenesis was performed in an ectopic bone formation model. RESULTS: The authors demonstrated that the prematurely fused sutures have a higher endogenous expression of the osteogenic differentiation-related genes than patent sutures, whereas the same pattern of gene expression exists between fused and patent suture progenitor cells. Importantly, both patent and fused suture progenitor cells undergo osteogenic differentiation and express multiple lineage regulators and NELL-1 on BMP9 stimulation, whereas fused suture progenitor cells have a higher basal osteogenic potential than patent suture progenitor cells. BMP9 regulates the expression of osteoclast differentiation-related genes in suture progenitor cells. Forced BMP9 expression enhances the mineralization and maturity of ectopic bone formation of suture progenitor cells implanted in vivo. CONCLUSIONS: The authors' findings suggest that fused suture progenitor cells have elevated osteogenic potential. BMP9 could regulate the expression of multiple osteoblast and osteoclast differentiation-related genes, and NELL-1, in both suture progenitor cells, indicating that BMP9 may play a role in craniosynostosis.


Assuntos
Suturas Cranianas/patologia , Craniossinostoses/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Células-Tronco Mesenquimais/patologia , Osteogênese/genética , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/genética , Suturas Cranianas/citologia , Suturas Cranianas/cirurgia , Craniossinostoses/patologia , Craniossinostoses/cirurgia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Lactente , Masculino , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Cultura Primária de Células , Procedimentos Cirúrgicos Reconstrutivos
5.
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
6.
Keio J Med ; 68(2): 42, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31243185

RESUMO

Development of the skeleton is mediated through two distinct ossification mechanisms. Craniofacial bones are formed mainly through intramembranous ossification, a mechanism different from endochondral ossification required for development of the body skeleton. The skeletal structures are quite distinct between the two, thus they are likely to have their unique stem cell populations. The sutures serve as the growth center critical for healthy development of the craniofacial skeleton. Defects in suture morphogenesis cause its premature closure, resulting in development of craniosynostosis, a devastating disease affecting 1 in ~2,500 individuals. The suture mesenchyme has been postulated to act as the niche of skeletal stem cells essential for calvarial morphogenesis. However, very limited knowledge is available for suture biology and suture stem cells (SuSCs) have yet to be isolated. Here we report the first evidence for identification and isolation of a stem cell population residing in the suture midline. Genetic labeling of SuSCs shows their ability to self-renew and continually give rise to mature cell types over a 1-year monitoring period. They maintain their localization in the niches constantly produce skeletogenic descendants during calvarial development and homeostastic maintenance. Upon injury, SuSCs expand drastically surrounding the skeletogenic mesenchyme, migrate to the damaged site and contribute directly to skeletal repair in a cell autonomous fashion. The regeneration, pluripotency and frequency of SuSCs are also determined using limiting dilution transplantation. In vivo clonal expansion analysis demonstrates a single SuSC capable of generating bones. Furthermore, SuSC transplantation into injured calvaria facilitates the healing processes through direct engraftments. Our findings demonstrate SuSCs are bona fide skeletal stem cells ideally suited for cell-based craniofacial bone therapy as they possess abilities to engraft, differentiate.(Presented at the 1980th Meeting, April 16, 2019).


Assuntos
Desenvolvimento Ósseo/fisiologia , Suturas Cranianas/citologia , Ossos Faciais/citologia , Mesoderma/citologia , Células-Tronco/citologia , Animais , Regeneração Óssea/fisiologia , Suturas Cranianas/crescimento & desenvolvimento , Suturas Cranianas/fisiologia , Craniossinostoses/patologia , Ossos Faciais/crescimento & desenvolvimento , Ossos Faciais/fisiologia , Humanos , Mesoderma/fisiologia , Morfogênese/fisiologia , Osteogênese/fisiologia , Base do Crânio/citologia , Base do Crânio/crescimento & desenvolvimento , Base do Crânio/fisiologia , Coluna Vertebral/citologia , Coluna Vertebral/crescimento & desenvolvimento , Coluna Vertebral/fisiologia , Células-Tronco/fisiologia
7.
J Histochem Cytochem ; 67(2): 107-116, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30113872

RESUMO

We investigated the influence of mechanical stretching on the genetic expression pattern of non-collagenous bone matrix proteins in osteoblasts. The cranial sutures of neonatal mice were subjected to ex vivo mechanical stretching. In the non-stretched control group, as osteoblast differentiation progressed, the successive genetic expression of bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) was detected using in situ hybridization, in that order. In the stretched group, the sutures were widened, and after 24 hr of cultivation, a large number of osteoblasts and abundant new osteoid were observed on the borders of the parietal bones. All new osteoblasts expressed BSP and some of them expressed OPN, but very few of them expressed OCN. After 48 hr, more extensive presence of osteoid was noted on the borders of the parietal bones, and this osteoid was partially mineralized; all osteoblasts on the osteoid surface expressed BSP, and more osteoblasts expressed OPN than those after 24 hr cultivation. Surprisingly, many of the osteoblasts that did not express OPN, expressed OCN. This suggests that when osteoblast differentiation is stimulated by mechanical stress, the genetic expression pattern of non-collagenous proteins in the newly differentiated osteoblasts is affected.


Assuntos
Suturas Cranianas/citologia , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Fenômenos Mecânicos , Osteoblastos/metabolismo , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos , Camundongos
8.
Hum Mol Genet ; 28(6): 896-911, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445456

RESUMO

Cleidocranial dysplasia (CCD, #119600), which is characterized by hypoplastic clavicles, open fontanelles, supernumerary teeth and a short stature, is caused by heterozygous mutations in RUNX2. However, it currently remains unclear why suture closure is severely impaired in CCD patients. The closure of posterior frontal (PF) and sagittal (SAG) sutures was completely interrupted in Runx2+/- mice, and the proliferation of suture mesenchymal cells and their condensation were less than those in wild-type mice. To elucidate the underlying molecular mechanisms, differentially expressed genes between wild-type and Runx2+/- PF and SAG sutures were identified by microarray and real-time reverse transcription polymerase chain reaction analyses. The expression of hedgehog, Fgf, Wnt and Pthlh signaling pathway genes, including Gli1, Ptch1, Ihh, Fgfr2, Fgfr3, Tcf7, Wnt10b and Pth1r, which were directly regulated by Runx2, was reduced in the sutures, but not the calvarial bone tissues of Runx2+/- mice. Bone formation and suture closure were enhanced in an organ culture of Runx2+/- calvariae with ligands or agonists of hedgehog, Fgf, Wnt and Pthlh signaling, while they were suppressed and suture mesenchymal cell proliferation was decreased in an organ culture of wild-type calvariae with their antagonists. These results indicate that more than a half dosage of Runx2 is required for the proliferation of suture mesenchymal cells, their condensation and commitment to osteoblast-lineage cells, and the induction of hedgehog, Fgf, Wnt and Pthlh signaling pathway gene expressions in sutures, but not in calvarial bone tissues, and also that the activation of hedgehog, Fgf, Wnt and Pthlh signaling pathways is necessary for suture closure.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Animais , Biomarcadores , Proliferação de Células , Displasia Cleidocraniana/diagnóstico , Displasia Cleidocraniana/etiologia , Displasia Cleidocraniana/metabolismo , Suturas Cranianas/citologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Microtomografia por Raio-X
9.
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
10.
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
11.
Orthod Craniofac Res ; 19(4): 181-189, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27405789

RESUMO

OBJECTIVES: To evaluate and compare the cellular morphologic changes and proliferating cell nuclear antigen (PCNA) expression within craniofacial sutures in growing Rhesus monkeys treated with a Class III functional appliance. MATERIALS AND METHODS: Six Rhesus monkeys in the mixed dentition stage were divided into three groups: a 45-day experimental group, a 90-day experimental group, and a control group. Monkeys in the experimental groups were fitted with a Class III magnetic twin-block appliance. Cellular changes in six craniofacial sutures-the zygomaticomaxillary, zygomaticotemporal, transverse palatine, pterygopalatine, zygomaticofrontal, and frontomaxillary sutures were qualitatively and quantitatively evaluated by means of histomorphologic analysis, TEM, and immunohistochemical test of PCNA. RESULTS: Obvious and altered bone remodeling combined with bone deposition and resorption was present in craniofacial sutures in the experimental groups. Increased activity of enlarged fibroblasts with abundant organelles was revealed. PCNA expression increased in the 45-day group compared with the control group, followed by the 90-day group. The highest percentage of PCNA-positive cells was found in the pterygopalatine suture in the 45-day group and the zygomaticomaxillary suture in the 90-day group. CONCLUSIONS: The pterygopalatine and zygomaticomaxillary sutures are more active among the craniofacial sutures in the craniofacial complex remodeling during Class III treatment. The magnetic twin-block appliance effectively promoted suture remodeling by enhancing the activity and proliferation of osteoblasts, osteoclasts, and fibroblasts, especially in the early phase.


Assuntos
Remodelação Óssea/fisiologia , Suturas Cranianas/química , Suturas Cranianas/citologia , Suturas Cranianas/crescimento & desenvolvimento , Aparelhos Ortodônticos Funcionais , Antígeno Nuclear de Célula em Proliferação/química , Animais , Proliferação de Células/fisiologia , Dentição Mista , Ossos Faciais/química , Ossos Faciais/citologia , Ossos Faciais/crescimento & desenvolvimento , Fibroblastos/citologia , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Macaca mulatta , Imãs , Má Oclusão Classe III de Angle/terapia , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteoblastos/ultraestrutura , Osteoclastos/citologia , Osteoclastos/fisiologia , Osteoclastos/ultraestrutura
12.
J Neurosurg Pediatr ; 18(1): 83-91, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27035551

RESUMO

OBJECTIVE The aim of this study was to examine messenger RNA (mRNA) levels of bone morphogenetic protein (BMP) ligands, receptors, and soluble inhibitors in cells isolated from single-suture synostoses from fused coronal, metopic, sagittal, and lambdoid sutures. METHODS Cells were isolated from bone collected from patients undergoing craniotomies at Children's Healthcare of Atlanta. Real-time polymerase chain reaction was used to examine mRNA levels in cells isolated from fused sutures or patent sutures in comparison with levels in normal bone from the same patient. RESULTS Cells isolated from fused sutures in cases of sagittal and coronal synostosis highly expressed BMP2, while cells isolated from fused metopic or lambdoid synostosis expressed high BMP4. Noggin, a BMP inhibitor, was lower in fused sutures and had high expression in patent sutures. CONCLUSIONS These results suggest that BMPs and inhibitors play a significant role in the regulation of suture fusion as well in the maintenance of patency in the normal suture.


Assuntos
Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 4/biossíntese , Suturas Cranianas/metabolismo , Craniossinostoses/diagnóstico , Craniossinostoses/metabolismo , Células Cultivadas , Estudos de Coortes , Suturas Cranianas/citologia , Feminino , Humanos , Lactente , Masculino , RNA Mensageiro/biossíntese
13.
PLoS One ; 10(10): e0139719, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26431045

RESUMO

The use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression during pregnancy is suggested to increase the incidence of craniofacial abnormalities including craniosynostosis. Little is known about this mechanism, however based on previous data we propose a mechanism that affects cell cycle. Excessive proliferation, and reduction in apoptosis may lead to hyperplasia within the suture that may allow for differentiation, bony infiltration, and fusion. Here we utilized in vivo and in vitro analysis to investigate this proposed phenomenon. For in vivo analysis we used C57BL-6 wild-type breeders treated with a clinical dose of citalopram during the third trimester of pregnancy to produce litters exposed to the SSRI citalopram in utero. At post-natal day 15 sutures were harvested from resulting pups and subjected to histomorphometric analysis for proliferation (PCNA) and apoptosis (TUNEL). For in vitro studies, we used mouse calvarial pre-osteoblast cells (MC3T3-E1) to assess proliferation (MTS), apoptosis (Caspase 3/7-activity), and gene expression after exposure to titrated doses of citalopram. In vivo analysis for PCNA suggested segregation of effect by location, with the sagittal suture, showing a statistically significant increase in proliferative response. The coronal suture was not similarly affected, however there was a decrease in apoptotic activity at the dural edge as compared to the periosteal edge. No differences in apoptosis by suture or area due to SSRI exposure were observed. In vitro results suggest citalopram exposure increased proliferation and proliferative gene expression, and decreased apoptosis of the MC3T3-E1 cells. Decreased apoptosis was not confirmed in vivo however, an increase in proliferation without a concomitant increase in apoptosis is still defined as hyperplasia. Thus prenatal SSRI exposure may exert a negative effect on post-natal growth through a hyperplasia effect at the cranial growth sites perhaps leading to clinically significant craniofacial abnormalities.


Assuntos
Citalopram/farmacologia , Suturas Cranianas/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Suturas Cranianas/citologia , Camundongos , Camundongos Endogâmicos C57BL
14.
Int J Clin Exp Pathol ; 8(3): 2946-54, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26045803

RESUMO

BACKGROUND: Understanding the pathophysiological process of calvarial bones development is important for the treatments on relative diseases such as craniosynostosis. While, the role of fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) and how they interacted in osteoblast differentiation remain unclear. METHODS: we digested bone fragments around the coronal and sagittal sutures from newborn rats to harvest suture cells. Markers expression at different osteoblast differentiation stage was analyzed by increasing FGF2 concentration and BMP2 blocking in these cells. RESULTS: BMP2 expression could be stimulated by FGF2 in a dose and time dependent manner. FGF2 stimulation may decrease early marker of osteoblast differentiation (collagen type-1, COL-1) and increase the expression of continuously-expressed or late markers (alkaline phosphatase, ALP; osteocalcin, OC and bone sialoprotein, BSP) to accelerate mineralization. Inhibition of BMP2 signaling by Noggin weakens the effect of FGF2 on induction of later-stage osteoblastic differentiation of cranial suture cells. CONCLUSION: Our data suggest that BMP2 signaling is required for FGF2-dependent induction of later-stage of cranial suture cell osteoblastic differentiation.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Suturas Cranianas/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Osteoblastos/citologia , Osteogênese/fisiologia , Animais , Animais Recém-Nascidos , Suturas Cranianas/citologia , Osteoblastos/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia
15.
Nat Cell Biol ; 17(4): 386-96, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25799059

RESUMO

Bone tissue undergoes constant turnover supported by stem cells. Recent studies showed that perivascular mesenchymal stem cells (MSCs) contribute to the turnover of long bones. Craniofacial bones are flat bones derived from a different embryonic origin than the long bones. The identity and regulating niche for craniofacial-bone MSCs remain unknown. Here, we identify Gli1+ cells within the suture mesenchyme as the main MSC population for craniofacial bones. They are not associated with vasculature, give rise to all craniofacial bones in the adult and are activated during injury repair. Gli1+ cells are typical MSCs in vitro. Ablation of Gli1+ cells leads to craniosynostosis and arrest of skull growth, indicating that these cells are an indispensable stem cell population. Twist1(+/-) mice with craniosynostosis show reduced Gli1+ MSCs in sutures, suggesting that craniosynostosis may result from diminished suture stem cells. Our study indicates that craniofacial sutures provide a unique niche for MSCs for craniofacial bone homeostasis and repair.


Assuntos
Suturas Cranianas/citologia , Craniossinostoses/genética , Fatores de Transcrição Kruppel-Like/genética , Células-Tronco Mesenquimais/citologia , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genética , Anilidas/farmacologia , Animais , Diferenciação Celular/genética , Suturas Cranianas/irrigação sanguínea , Suturas Cranianas/crescimento & desenvolvimento , Consolidação da Fratura/genética , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Osteoporose/genética , Piridinas/farmacologia , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened , Proteína GLI1 em Dedos de Zinco
16.
Eur J Orthod ; 37(3): 308-13, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25312980

RESUMO

BACKGROUND/OBJECTIVE: Chondrogenesis is an integral part of endochondral bone formation, by which the midline cranial base is developed. Reactive oxygen species (ROS) are required in chondrogenic differentiation and antioxidant enzymes regulate their levels. The aim of this study was to localize the antioxidant enzyme glutathione peroxidase 1 (Gpx1) at the spheno-occipital synchondrosis, as well as its effect on ROS challenge and its expression pattern in the course of differentiation. MATERIALS AND METHODS: Gpx1 was semiquantified in immunohistochemically stained sections of spheno-occipital synchondroses of rats. The effect of Gpx1 on ROS-induced apoptosis was investigated by manipulating the expression of Gpx1 in ATDC5 cells. The temporal pattern of Gpx1 expression was determined during chondrocyte differentiation for 21 days in vitro. RESULTS: Proliferating chondrocytes exhibited the greatest Gpx1 immunoreactivity and hypertrophic ones the lowest (P = 0.02). Cells transfected with Gpx1-siRNA had the highest apoptotic rate, while cells overexpressing Gpx1 the lowest one (P < 0.001). Gpx1 was significantly increased on days 10 (P = 0.02) and 14 (P = 0.01). CONCLUSIONS: Hypertrophic chondrocytes have the lowest Gpx1 activity in the spheno-occipital synchondrosis. Gpx1 is implicated in the ROS-induced apoptosis in chondrocytes. Its expression was not constitutive during chondrogenic differentiation.


Assuntos
Apoptose/fisiologia , Suturas Cranianas/enzimologia , Glutationa Peroxidase/análise , Osso Occipital/enzimologia , Espécies Reativas de Oxigênio/análise , Osso Esfenoide/enzimologia , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células , Condrócitos/enzimologia , Condrócitos/fisiologia , Condrogênese/fisiologia , Suturas Cranianas/citologia , Técnicas de Silenciamento de Genes , Glutationa Peroxidase/genética , Glutationa Peroxidase/fisiologia , Hipertrofia , Osso Occipital/citologia , Osteogênese/fisiologia , RNA Interferente Pequeno/genética , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/farmacologia , Base do Crânio/citologia , Osso Esfenoide/citologia , Fatores de Tempo
17.
BMC Biotechnol ; 12: 45, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22857382

RESUMO

BACKGROUND: Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function. RESULTS: A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3. CONCLUSIONS: A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 3/farmacologia , Suturas Cranianas/citologia , Células-Tronco Mesenquimais/citologia , Transfecção/métodos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Genes Reporter , Glipicanas/genética , Glipicanas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lipossomos/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Plasmídeos/genética , Plasmídeos/metabolismo
18.
PLoS One ; 7(5): e36789, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22693558

RESUMO

Growth Differentiation Factor-6 (Gdf6) is a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules. Previous studies have shown that Gdf6 plays a role in formation of a diverse subset of skeletal joints. In mice, loss of Gdf6 results in fusion of the coronal suture, the intramembranous joint that separates the frontal and parietal bones. Although the role of GDFs in the development of cartilaginous limb joints has been studied, limb joints are developmentally quite distinct from cranial sutures and how Gdf6 controls suture formation has remained unclear. In this study we show that coronal suture fusion in the Gdf6-/- mouse is due to accelerated differentiation of suture mesenchyme, prior to the onset of calvarial ossification. Gdf6 is expressed in the mouse frontal bone primordia from embryonic day (E) 10.5 through 12.5. In the Gdf6-/- embryo, the coronal suture fuses prematurely and concurrently with the initiation of osteogenesis in the cranial bones. Alkaline phosphatase (ALP) activity and Runx2 expression assays both showed that the suture width is reduced in Gdf6+/- embryos and is completely absent in Gdf6-/- embryos by E12.5. ALP activity is also increased in the suture mesenchyme of Gdf6+/- embryos compared to wild-type. This suggests Gdf6 delays differentiation of the mesenchyme occupying the suture, prior to the onset of ossification. Therefore, although BMPs are known to promote bone formation, Gdf6 plays an inhibitory role to prevent the osteogenic differentiation of the coronal suture mesenchyme.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Suturas Cranianas/embriologia , Fator 6 de Diferenciação de Crescimento/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Suturas Cranianas/citologia , Suturas Cranianas/metabolismo , Osso Frontal/citologia , Osso Frontal/embriologia , Osso Frontal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator 6 de Diferenciação de Crescimento/deficiência , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese , Fatores de Tempo
19.
J Craniofac Surg ; 23(3): 919-24, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22627405

RESUMO

BACKGROUND: Cells within the dura mater have been implicated in the determination of suture patency and fusion. Craniosynostosis (CS), the premature fusion of 1 or more of the cranial sutures, could result from abnormal control over the differentiation of osteoprogenitor cells from the dura mater. This study tested whether dura mater cells derived from rabbits with congenital CS were different from cells derived from normal rabbits and investigated the effects that CS dura mater had on osteogenic differentiation in vitro and in vivo. METHODS: Cells were derived from the dura mater from wild-type rabbits (WT; n = 23) or CS rabbits (n = 16). Cells were stimulated with bone morphogenetic protein 4, and alkaline phosphatase (ALP) expression and cell proliferation were assessed. Dura mater-derived cells were also cocultured with primary rabbit bone-derived cells, and ALP was assessed. Finally, interactions between the dura mater and overlying tissues were manipulated in vivo. RESULTS: Craniosynostotic dura mater-derived cells proliferated faster than did WT cells but were not more ALP positive. Coculture experiments showed that CS dura mater cells induced increased ALP activity in CS bone-derived cells, but not in WT bone-derived cells. In vivo experiments showed that a physical barrier successfully inhibited dura mater-derived osteogenesis. CONCLUSIONS: Coculture of CS bone- and CS dura mater-derived cells evoked an abnormal phenotype in vitro. Covering the CS dura mater led to decreased bone formation in vivo. Further investigations will focus on the signaling molecules involved in the communication between these 2 CS tissue types in vitro and in vivo.


Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Suturas Cranianas/citologia , Craniossinostoses/cirurgia , Dura-Máter/citologia , Fosfatase Alcalina/metabolismo , Análise de Variância , Animais , Diferenciação Celular , Proliferação de Células , Técnicas de Cocultura , Suturas Cranianas/metabolismo , Suturas Cranianas/cirurgia , Craniossinostoses/metabolismo , Dura-Máter/metabolismo , Osteogênese/fisiologia , Fenótipo , Politetrafluoretileno , Coelhos
20.
Plast Reconstr Surg ; 125(5): 1352-1361, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20134361

RESUMO

BACKGROUND: In utero retinoid exposure results in numerous craniofacial malformations, including craniosynostosis. Although many malformations associated with retinoic acid syndrome are associated with neural crest defects, the specific mechanisms of retinoid-induced craniosynostosis remain unclear. The authors used the culture of mouse cranial suture-derived mesenchymal cells to probe the potential cellular mechanisms of this teratogen to better elucidate mechanisms of retinoid-induced suture fusion. METHODS: Genes associated with retinoid signaling were assayed in fusing (posterofrontal) and patent (sagittal, coronal) sutures by quantitative real-time polymerase chain reaction. Cultures of mouse suture-derived mesenchymal cells from the posterofrontal suture were established from 4-day-old mice. Cells were cultured with all-trans retinoic acid (1 and 5 muM). Proliferation, osteogenic differentiation, and specific gene expression were assessed. RESULTS: Mouse sutures were found to express genes necessary for retinoic acid synthesis, binding, and signal transduction, demonstrated by quantitative real-time polymerase chain reaction (Raldh1, Raldh2, Raldh3, and Rbp4). These genes were not found to be differentially expressed in fusing as compared with patent cranial sutures in vivo. Addition of retinoic acid enhanced the osteogenic differentiation of suture-derived mesenchymal cells in vitro, including up-regulation of alkaline phosphatase activity and Runx2 expression. Contemporaneously, cellular proliferation was repressed, as shown by proliferative cell nuclear antigen expression. The pro-osteogenic effect of retinoic acid was accompanied by increased gene expression of several hedgehog and bone morphogenetic protein ligands. CONCLUSIONS: Retinoic acid represses proliferation and enhances osteogenic differentiation of suture-derived mesenchymal cells. These in vitro data suggest that retinoid exposure may lead to premature cranial suture fusion by means of enhanced osteogenesis and hedgehog and bone morphogenetic protein signaling.


Assuntos
Suturas Cranianas/citologia , Craniossinostoses/embriologia , Mesoderma/citologia , Osteogênese/efeitos dos fármacos , Retinoides/efeitos adversos , Tretinoína/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Animais Recém-Nascidos , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Expressão Gênica , Camundongos , Tretinoína/metabolismo
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