RESUMO
Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage-specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm-like cells, lateral plate mesoderm-like cells, and neural crest-like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest-derived OPs-a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes.
Assuntos
Diferenciação Celular , Linhagem da Célula , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Crista Neural/citologia , Osteogênese , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Análise de Componente Principal , Transcriptoma/genéticaRESUMO
In this case report, we focus on Muenke syndrome (MS), a disease caused by the p.Pro250Arg variant in fibroblast growth factor receptor 3 (FGFR3) and characterized by uni- or bilateral coronal suture synostosis, macrocephaly without craniosynostosis, dysmorphic craniofacial features, and dental malocclusion. The clinical findings of MS are further complicated by variable expression of phenotypic traits and incomplete penetrance. As such, unraveling the mechanisms behind MS will require a comprehensive and systematic way of phenotyping patients to precisely identify the impact of the mutation variant on craniofacial development. To establish this framework, we quantitatively delineated the craniofacial phenotype of an individual with MS and compared this to his unaffected parents using three-dimensional cephalometric analysis of cone beam computed tomography scans and geometric morphometric analysis, in addition to an extensive clinical evaluation. Secondly, given the utility of human induced pluripotent stem cells (hiPSCs) as a patient-specific investigative tool, we also generated the first hiPSCs derived from a family trio, the proband and his unaffected parents as controls, with detailed characterization of all cell lines. This report provides a starting point for evaluating the mechanistic underpinning of the craniofacial development in MS with the goal of linking specific clinical manifestations to molecular insights gained from hiPSC-based disease modeling.
RESUMO
The aim of this study is to investigate the morphological and chemical changes of demineralized dentin collagen-matrix and resin/dentin interface associated with chitosan/riboflavin modification. Dentin disc specimens were prepared from sound molars, acid-etched with 35% phosphoric acid and modified with either 0.1% riboflavin or chitosan/riboflavin (Ch/RF ratios 1:4 or 1:1) and photo-activated by UVA. Morphological and chemical changes associated with surface modification were characterized by SEM and micro-Raman spectroscopy. Dentin surfaces of sound molars were exposed, acid-etched, and modified as described before. Etch-and-rinse dentin adhesive was applied, light-cured, and layered with resin-restorative composite. The resin infiltration and resin/dentin interface were characterized by micro-Raman spectroscopy and SEM. An open-intact collagen network-structure, formation of uniform hybrid-layer and higher resin infiltration were found with 0.1%RF and Ch/RF 1:4 modifications. Raman analysis revealed chemical changes and shifts in Amide bands with the modification of dentin collagen-matrix. The use of riboflavin and chitosan/riboflavin formulations to modify dentin-collagen matrix, with the defined ratios, stabilizes the collagen fibrillar network and enhances resin infiltration and hybrid layer formation. These preliminary results are encouraging for subsequent consideration of chitosan/riboflavin modification in adhesive dentistry.
Assuntos
Quitosana/química , Dentina/química , Resinas Sintéticas/química , Riboflavina/química , Colágeno/química , Reagentes de Ligações Cruzadas , Reparação de Restauração Dentária , Microscopia Eletrônica de Varredura , Soluções , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
In this study, the effect of high-intensity focused ultrasound (HIFU) on Enterococcus faecalis on both planktonic suspensions and biofilms was investigated. E. faecalis persist in secondary dental infections as biofilms. Glass-bottom Petri dishes with biofilms were centered at the focal point of the HIFU wave generated by a 250-kHz transducer. Specimens were subjected to HIFU exposure at different periods of 30, 60 and 120 s. The viable bacteria, removal effect and bacterial viability of biofilms attached to the Petri dish surface were studied by colony-forming units (CFUs), scanning electron microscopy and confocal microscopy, respectively. The removal and bactericidal effects of HIFU are dependent on the exposure time. A significant reduction in biofilm thickness and CFU was found with the increase in HIFU exposure. The removal or bactericidal effect of HIFU was more significant starting from 60 s of exposure. This study highlighted the potential application of HIFU as a novel method for root canal disinfection.
Assuntos
Biofilmes/crescimento & desenvolvimento , Biofilmes/efeitos da radiação , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/efeitos da radiação , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Doses de RadiaçãoRESUMO
Previous studies have suggested different approaches to modify dentin collagen for potential improvement in bonding to dentin. Here, we are proposing a new approach to reinforce dentin collagen fibrils network by chitosan as a reinforcement phase and UVA-activated riboflavin as crosslinking agent within clinically acceptable time-frame as potential substrate for bonding. The effect of modifying demineralized dentin substrates with chitosan/riboflavin, with a gradual increase in chitosan content, was investigated by SEM, nano-indentation, conventional-mechanical testing and hydroxyproline (HYP) release at collagenolytic and/or hydrolytic challenges. The resin/dentin interface morphology, immediate bond strength and short-term bond durability were also investigated using etch-and-rinse dentin adhesive. Modification with chitosan/riboflavin increased the mechanical properties, enhanced the mechanical stability of demineralized dentin substrates against hydrolytic and/or collagenolytic degradation challenges and decreased HYP release with collagenase exposure. When chitosan was added to riboflavin at 20%v/v ratio, significant improvement in bond strength at 24 h and 6 months in distilled water was found indicating the positive dual effect on bonding to dentin. With the gradual increase in chitosan content, obliteration of interfibrillar-spaces that might adversely affect bonding to dentin was found. Although it has a synergetic effect, chitosan content is crucial for any subsequent application in adhesive dentistry.
Assuntos
Calcificação Fisiológica , Quitosana/metabolismo , Colagem Dentária/métodos , Dentina/metabolismo , Riboflavina/metabolismo , Adesivos/química , Adolescente , Adulto , Bactérias/enzimologia , Colágeno/metabolismo , Colagenases/metabolismo , Humanos , Hidroxiprolina/metabolismo , Fenômenos Mecânicos , Resinas Sintéticas/química , Fatores de Tempo , Adulto JovemRESUMO
OBJECTIVES: To investigate the effect of photo-activation of riboflavin either by ultraviolet (UVA) or visible blue light (BL) on the biodegradation resistance, strength of demineralized dentin matrix, bond strength to dentin and resin/dentin interface morphology. METHODS: Dentin beams were demineralized, treated with 0.1% or 1% riboflavin solution for 5min and photo-activated with UVA or BL for 20s. The ultimate tensile strength (UTS) and hydroxyproline (HYP) release were assessed after 24h collagenase challenge. For micro-tensile bond strength (µTBS) testing and resin/dentin interface morphology investigation, dentin was acid-etched, crosslinked with riboflavin and bonded with an etch-and-rinse adhesive system. Riboflavin was photo-activated separately with UVA or BL followed by photo-polymerization of the bonding resin with BL (two-step) or both riboflavin photo-activation and bonding resin photo-polymerization were done in one-step using BL. RESULTS: Significant improvement in the UTS and biodegradation resistance against collagenase challenge was found when riboflavin was photo-activated either with UVA or BL. However, UVA showed more significant improvement compared to BL. After 4months of water-storage, both UV and BL two-step photo-activation methods significantly preserved higher values of the µTBS compared to the non-crosslinked control group, where UVA showed significantly higher µTBS than BL. SIGNIFICANCE: Although UVA most effectively activated riboflavin, visible blue light showed to be a promising substitute for UVA as it is clinically more applicable and acceptable, and still managed to increase the biodegradation resistance, enhance the mechanical properties of dentin collagen and improve and maintain the bond strength and interface integrity after short-term water storage.