Peter J. Anderson

Unravelling the molecular control of calvarial suture fusion in children with craniosynostosis

BackgroundCraniosynostosis, 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.ResultsWe 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.ConclusionThis 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.

One Pathway Can Incorporate either Adenine or Dimethylbenzimidazole as an α-Axial Ligand of B12 Cofactors in Salmonella enterica

Corrinoid (vitamin B12-like) cofactors contain various alpha-axial ligands, including 5,6-dimethylbenzimidazole (DMB) or adenine. The bacterium Salmonella enterica produces the corrin ring only under anaerobic conditions, but it can form complete corrinoids aerobically by importing an incomplete corrinoid, such as cobinamide (Cbi), and adding appropriate alpha- and beta-axial ligands. Under aerobic conditions, S. enterica performs the corrinoid-dependent degradation of ethanolamine if given vitamin B12, but it can make B12 from exogenous Cbi only if DMB is also provided. Mutants isolated for their ability to degrade ethanolamine without added DMB converted Cbi to pseudo-B12 cofactors (having adenine as an alpha-axial ligand). The mutations cause an increase in the level of free adenine and install adenine (instead of DMB) as an alpha-ligand. When DMB is provided to these mutants, synthesis of pseudo-B12 cofactors ceases and B12 cofactors are produced, suggesting that DMB regulates production or incorporation of free adenine as an alpha-ligand. Wild-type cells make pseudo-B12 cofactors during aerobic growth on propanediol plus Cbi and can use pseudo-vitamin B12 for all of their corrinoid-dependent enzymes. Synthesis of coenzyme pseudo-B12 cofactors requires the same enzymes (CobT, CobU, CobS, and CobC) that install DMB in the formation of coenzyme B12. Models are described for the mechanism and control of alpha-axial ligand installation.

Testing an extended theory of planned behaviour to predict young people's sun safety in a high risk area

OBJECTIVESnThe present research examined the sun protection intentions and behaviours of young people in a high risk skin cancer area using an extended theory of planned behaviour (TPB) incorporating additional social influences of group and image norms.nnnDESIGNnThe study employed a prospective design to examine young peoples sun protection intentions and behaviour.nnnMETHODnParticipants (N=1,134), aged 12-20 years, were students (school, university, TAFE) and young employees living in Queensland, Australia. Participants completed a questionnaire assessing the TPB predictors (attitude, subjective norm, perceived behavioural control) and additional social influences (group norm, image norm) of sun protection intentions. Two weeks later, participants (N=734) reported their sun protection behaviour for the previous fortnight.nnnRESULTSnResults revealed that the TPB variables of attitude, subjective norm and perceived behavioural control and the additional social influence variable of group norms, but not image norms, emerged as significant predictors of intentions to engage in sun protection. The extended TPB variables accounted for 36% of the variance in intentions. For behaviour, the extended TPB variables accounted for 27% of the variance with both intention and, unexpectedly, group norm as the significant direct predictors of sun protective behaviours.nnnCONCLUSIONSnResults of this study provide support for the application of the TPB in the sun safety context and highlight the importance of considering the influence of group norms in the development of future interventions to increase young peoples sun protection intentions and behaviour.

A three-dimensional computed tomographic analysis of the cervical spine in unoperated infants with cleft lip and palate

Objective: To investigate anatomical variations and abnormalities of cervical spine morphology in unoperated infants with cleft lip and palate. Design: Retrospective cross-sectional investigation of infants born with nonsyndromic cleft lip and palate using computed tomography scans acquired for investigation of a spectrum of clinical conditions. Setting: Computed tomography scan data were obtained from 29 unoperated cleft lip and palate infants and 12 noncleft infants of Malay origin, ages 0 to 12 months. Methods: Observational study of cervical spine computed tomography scans. Heights of cervical vertebral bodies (C2-C7) and intervertebral spaces were measured from landmarks identified from computed tomography reformats and three-dimensional computed tomography reconstructions. Linear modeling of heights and spaces, with age as a covariate, was undertaken to identify differences between the samples. Results: Anomalous features observed in the cleft lip and palate sample included short posterior arch of C1 (2/29), abnormal development of the anterior arch of C1 (2/29), and fusions of the posterior arch of C2 and C3 (2/29). No anomalies of the cervical spine were observed in the noncleft sample. Although the heights of three cervical vertebral bodies were significantly smaller and two intervertebral spaces were significantly larger in infants with cleft lip and palate compared with noncleft infants (p < .05), overall length of the cervical spine did not differ significantly between the samples. Conclusion: There was evidence for subtle upper spinal anomalies in the infant cleft lip and palate population. Our finding of reduced size of some cervical vertebral bodies may reflect delayed upper spinal development in infants with cleft lip and palate.

Dental Findings in Parents of Children with Cleft Lip and Palate

The incidence of dental abnormalities in the cleft lip and palate population has been reported to be much higher than in the normal population. The role of genes in the production of a cleft lip and palate, and dental anomalies is thought to be complex, with autosomal dominant, recessive, and x-linked genes all playing a role. Noncleft parents can carry some of the cleft lip and palate genes, which produce clinically subtle manifestations in their facial skeleton. The purpose of this study was to look for evidence of increased dental anomalies in the non-cleft parents of cleft lip and palate children. The dentitions of the parents of 60 children with different types of cleft lip and palate were examined prospectively to see whether or not they exhibited features found more readily in the cleft lip and palate rather than did the normal population. Their dentitions were studied to record the following dental features: congenitally missing teeth, supernumerary teeth, or morphologic changes of the crowns of the permanent teeth. The number and position of any frenal attachments were also recorded. The results of this study did not show any differences in incidence of dental anomalies from the noncleft population. There was no evidence to support the hypothesis that congenital absence of lateral incisors is a microform of cleft lip and palate. Further, these results also failed to reveal any consistent pattern in the number and position of frenal attachments.

Neural crest cell-derived VEGF promotes embryonic jaw extension

Significance Craniofacial development is a complex morphogenic event that relies on highly orchestrated interactions between multiple cell types. Since the first description of Meckel’s cartilage in the lower jaw more than 180 years ago, we have come to realize that expansion of this specialized structure underpins correct mandible development. Here we demonstrate that an intricate association between neural crest cells and blood vessels plays an important role in promoting chondrocyte proliferation and expansion of Meckel’s cartilage as a prerequisite of correct mandibular morphogenesis. These findings provide direct insight into the origins and potential treatments of highly prevalent disorders affecting the mandible. Jaw morphogenesis depends on the growth of Meckel’s cartilage during embryogenesis. However, the cell types and signals that promote chondrocyte proliferation for Meckel’s cartilage growth are poorly defined. Here we show that neural crest cells (NCCs) and their derivatives provide an essential source of the vascular endothelial growth factor (VEGF) to enhance jaw vascularization and stabilize the major mandibular artery. We further show in two independent mouse models that blood vessels promote Meckel’s cartilage extension. Coculture experiments of arterial tissue with NCCs or chondrocytes demonstrated that NCC-derived VEGF promotes blood vessel growth and that blood vessels secrete factors to instruct chondrocyte proliferation. Computed tomography and X-ray scans of patients with hemifacial microsomia also showed that jaw hypoplasia correlates with mandibular artery dysgenesis. We conclude that cranial NCCs and their derivatives provide an essential source of VEGF to support blood vessel growth in the developing jaw, which in turn is essential for normal chondrocyte proliferation, and therefore jaw extension.

Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3

BackgroundAchieving 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.ResultsA 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.ConclusionsA 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.

In vitro differentiation of human calvarial suture derived cells with and without dexamethasone does not induce in vivo‐like expression

Osteogenic supplements are a requirement for osteoblastic cell differentiation during in vitro culture of human calvarial suture‐derived cell populations. We investigated the ability of ascorbic acid and β‐glycerophosphate with and without the addition of dexamethasone to stimulate in vivo‐like osteoblastic differentiation. Cells were isolated from unfused and prematurely fused suture tissue from patients with syndromic and non‐syndromic craniosynostosis and cultured in each osteogenic medium for varying lengths of time. The effect of media supplementation was investigated with respect to the ability of cells to form mineralised bone nodules and the expression of five osteodifferentiation marker genes (COL1A1, ALP, BSP, OC and RUNX2), and five genes that are differentially expressed during human premature suture fusion (GPC3, RBP4, C1QTNF3, WIF1 and FGF2). Cells from unfused sutures responded more slowly to osteogenic media but formed comparable bone nodules to fused suture‐derived cells after 16 days of culture in either osteogenic media. However, gene expression differed between unfused and fused suture‐derived cells, as did expression in each osteogenic medium. When compared to expression in the explant tissue of origin, neither medium induced a level or profile of gene expression similar to that seen in vivo. Overall, our results demonstrate that cells from the same suture that are isolated during different stages of morphogenesis in vivo, despite being de‐differentiated to a similar level in vitro, respond uniquely and differently to each osteogenic medium. Further, we suggest that neither cell culture medium recapitulates differentiation via activation of the same genetic cascades as occurs in vivo. J. Cell. Physiol. 218: 183–191, 2009.

Biological response of human suture mesenchymal cells to Titania nanotube-based implants for advanced craniosynostosis therapy

Titania nanotubes (TNTs) engineered on titanium (Ti) surfaces (i.e. TNT/Ti) and loaded with specific drugs have been recognised as a promising solution for localised therapeutic delivery to address several medical problems not feasible with conventional drug administration. We propose the use of TNT/Ti protein-releasing implants to treat paediatric craniofacial abnormality in craniosynostosis caused by premature fusion of cranial sutures. In this study, we have analysed the biological response of human suture mesenchymal cells (SMCs), extracted from two different patients undergoing craniofacial reconstruction surgery, at the TNT/Ti implant surface. The experimental groups included large-diameter TNT/Ti implants, with and without biopolymer surface coating (Chitosan and Pluronic-F127) while the controls comprised of flat Ti disc and tissue culture plastic. The non-loaded implant surfaces and the cellular interactions at the implant-cell interface were characterised using scanning electron microscopy (SEM). The SMC adhesion, viability and proliferation were determined by MTT assay and manual cell counting at day 1 and day 3 of cell incubation. SEM showed significant reduction in initial attachment and adhesion of SMCs at TNT-cell biointerface compared with the control Ti discs. Subsequent cell proliferation results also revealed a decrease in the number of viable cells on the TNT surfaces. The nanotopography and structural features along with the surface chemistry dictated the cellular response, with nanotubular surfaces (with and without polymer coating) impeding cell adhesion and proliferation. Our findings hold promise for the use of TNT-based cranial implants as a delivery system to prevent sutural bone growth for advanced craniosynostosis therapy.

Long‐term visual outcomes in patients with orbitotemporal neurofibromatosis

The study aimed to review the presentation and long‐term visual outcomes of patients with orbitotemporal neurofibromatosis.