Molecular basis of cranial suture biology and disease:Osteoblastic and osteoclastic perspectives

The normal growth and development of the skull is a tightly regulated process that occurs along the osteogenic interfaces of the cranial sutures.Here,the borders of the calvarial bones and neighboring tissues above and below,function as a complex.Through coordinated remodeling efforts of bone deposition and resorption,the cranial sutures maintain a state of patency from infancy through early adulthood as the skull continues to grow and accommodate the developing brain’s demands for expansion.However,when this delicate balance is disturbed,a number of pathologic conditions ensue;and if left uncorrected,may result in visual and neurocognitive impairments.A prime example includes craniosynostosis,or premature fusion of one or more cranial and/or facial suture(s).At the present time,the only therapeutic measure for craniosynostosis is surgical correction by cranial vault reconstruction.However,elegant studies performed over the past decade have identified several genes critical for the maintenance of suture patency and induction of suture fusion.Such deeper understandings of the pathogenesis and molecular mechanisms that regulate suture biology may provide necessary insights toward the development of non-surgical therapeutic alternatives for patients with cranial suture defects.In this review,we discuss the intricate cellular and molecular interplay that exists within the suture among its three major components:dura mater,osteoblastic related molecular pathways and osteoclastic related molecular pathways.

Fibroblast growth factor(FGF)signaling in development and skeletal diseases

Fibroblast growth factors(FGF)and their receptors serve many functions in both the developing and adult organism.Humans contain 18 FGF ligands and four FGF receptors(FGFR).FGF ligands are polypeptide growth factors that regulate several developmental processes including cellular proliferation,differentiation,and migration,morphogenesis,and patterning.FGF-FGFR signaling is also critical to the developing axial and craniofacial skeleton.In particular,the signaling cascade has been implicated in intramembranous ossification of cranial bones as well as cranial suture homeostasis.In the adult,FGFs and FGFRs are crucial for tissue repair.FGF signaling generally follows one of three transduction pathways:RAS/MAP kinase,PI3/AKT,or PLCg.Each pathway likely regulates specific cellular behaviors.Inappropriate expression of FGF and improper activation of FGFRs are associated with various pathologic conditions,unregulated cell growth,and tumorigenesis.Additionally,aberrant signaling has been implicated in many skeletal abnormalities including achondroplasia and craniosynostosis.The biology and mechanisms of the FGF family have been the subject of significant research over the past 30 years.Recently,work has focused on the therapeutic targeting and potential of FGF ligands and their associated receptors.The majority of FGF-related therapy is aimed at age-related disorders.Increased understanding of FGF signaling and biology may reveal additional therapeutic roles,both in utero and postnatally.This review discusses the role of FGF signaling in general physiologic and pathologic embryogenesis and further explores it within the context of skeletal development.

Sustained high level transgene expression in mammalian cells mediated by the optimized piggyBac transposon system

Sustained,high level transgene expression in mammalian cells is desired in many cases for studying gene functions.Traditionally,stable transgene expression has been accomplished by using retroviral or lentiviral vectors.However,such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression.The piggyBac transposon has emerged as a promising nonviral vector system for efficient gene transfer into mammalian cells.Despite its inherent advantages over lentiviral and retroviral systems,piggyBac system has not been widely used,at least in part due to their limited manipulation flexibilities.Here,we seek to optimize piggyBac-mediated transgene expression and generate a more efficient,user-friendly piggyBac system.By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase(PBase),we demonstrate that adenovirusmediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells,compared to that obtained from co-transfection of the CMV-PBase plasmid.We further determine the drug selection timeline to achieve optimal stable transgene expression.Moreover,we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors.Using the engineered tandem expression vector,we show that three transgenes can be simultaneously expressed in a single vector with high efficiency.Thus,these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained,high transgene expression.