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Abstract 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.

Abstract Background RT-qPCR is a common tool for quantification of gene expression, but its accuracy is dependent on the choice and stability (steady state expression levels) of the reference gene/s used for normalization. To date, in the bone field, there have been few studies to determine the most stable reference genes and, usually, RT-qPCR data is normalised to non-validated reference genes, most commonly GAPDH, ACTB and 18 S rRNA. Here we draw attention to the potential deleterious impact of using classical reference genes to normalise expression data for bone studies without prior validation of their stability. Results Using the geNorm and Normfinder programs, panels of mouse and human genes were assessed for their stability under three different experimental conditions: 1) disease progression of Crouzon syndrome (craniosynostosis) in a mouse model, 2) proliferative culture of cranial suture cells isolated from craniosynostosis patients and 3) osteogenesis of a mouse bone marrow stromal cell line. We demonstrate that classical reference genes are not always the most ‘stable’ genes and that gene ‘stability’ is highly dependent on experimental conditions. Selected stable genes, individually or in combination, were then used to normalise osteocalcin and alkaline phosphatase gene expression data during cranial suture fusion in the craniosynostosis mouse model and strategies compared. Strikingly, the expression trends of alkaline phosphatase and osteocalcin varied significantly when normalised to the least stable, the most stable or the three most stable genes. Conclusion To minimise errors in evaluating gene expression levels, analysis of a reference panel and subsequent normalization to several stable genes is strongly recommended over normalization to a single gene. In particular, we conclude that use of single, non-validated “housekeeping” genes such as GAPDH, ACTB and 18 S rRNA, currently a widespread practice by researchers in the bone field, is likely to produce data of questionable reliability when changes are 2 fold or less, and such data should be interpreted with due caution.

Abstract Background Craniosynostosis, the premature fusion of calvarial sutures, is a common craniofacial abnormality. Causative mutations in more than 10 genes have been identified, involving fibroblast growth factor, transforming growth factor beta, and Eph/ephrin signalling pathways. Mutations affect each human calvarial suture (coronal, sagittal, metopic, and lambdoid) differently, suggesting different gene expression patterns exist in each human suture. To better understand the molecular control of human suture morphogenesis we used microarray analysis to identify genes differentially expressed during suture fusion in children with craniosynostosis. Expression differences were also analysed between each unfused suture type, between sutures from syndromic and non-syndromic craniosynostosis patients, and between unfused sutures from individuals with and without craniosynostosis. Results We identified genes with increased expression in unfused sutures compared to fusing/fused sutures that may be pivotal to the maintenance of suture patency or in controlling early osteoblast differentiation (i.e. RBP4, GPC3, C1QTNF3, IL11RA, PTN, POSTN). In addition, we have identified genes with increased expression in fusing/fused suture tissue that we suggest could have a role in premature suture fusion (i.e. WIF1, ANXA3, CYFIP2). Proteins of two of these genes, glypican 3 and retinol binding protein 4, were investigated by immunohistochemistry and localised to the suture mesenchyme and osteogenic fronts of developing human calvaria, respectively, suggesting novel roles for these proteins in the maintenance of suture patency or in controlling early osteoblast differentiation. We show that there is limited difference in whole genome expression between sutures isolated from patients with syndromic and non-syndromic craniosynostosis and confirmed this by quantitative RT-PCR. Furthermore, distinct expression profiles for each unfused suture type were noted, with the metopic suture being most disparate. Finally, although calvarial bones are generally thought to grow without a cartilage precursor, we show histologically and by identification of cartilage-specific gene expression that cartilage may be involved in the morphogenesis of lambdoid and posterior sagittal sutures. Conclusion This study has provided further insight into the complex signalling network which controls human calvarial suture morphogenesis and craniosynostosis. Identified genes are candidates for targeted therapeutic development and to screen for craniosynostosis-causing mutations.

Up until late in the third trimester of gestation and through to adulthood, the healing response acts more to regenerate than to repair a wound. The mechanisms underlying this 'scar-free' healing remain unknown although the actin cytoskeleton has a major role. Flightless I (Flii), an actin-remodelling protein and essential developmental regulator, negatively affects wound repair but its effect on scar-free fetal healing is unknown. Using fetal skin explants from E17 (regenerate) and E19 (repair) rats, the function of Flii in fetal wound repair was determined. Expression of Flii increased between E17 and E19 days of gestation and wounding transiently increased Flii expression in E17 but not E19 wounds. However, both confocal and immunofluorescent analysis showed E17 keratinocytes immediately adjacent to the wounds downregulated Flii. As a nuclear coactivator and inhibitor of proliferation and migration, the absence of Flii in cells at the edge of the wound could be instrumental in allowing these cells to proliferate and migrate into the wound deficit. In contrast, Flii was strongly expressed within the cytoplasm and nucleus of keratinocytes within epidermal cells at the leading edge of E19 wounded fetal skin explants. This increase in Flii expression in E19 wounds could affect the way these cells migrate into the wound space and contribute to impaired wound healing. Neutralising Flii protein improved healing of early- but not late-gestation wounds. Flii did not colocalise with actin cables formed around E17 wounds suggesting an independent mechanism of action distinct from its actin-binding function in scar-free wound repair. [ABSTRACT FROM AUTHOR]