Secondary palate fusion requires adhesion and epithelial-to-mesenchymal transition (EMT) of the epithelial layers on opposing palatal shelves. This EMT requires transforming growth factor b3 (TGFb3), and its failure results in cleft palate. Ephrins, and their receptors, the Ephs, are responsible for migration, adhesion, and midline closure events throughout development. Ephrins can also act as signal-transducing receptors in these processes, with the Ephs serving as ligands (termed ''reverse'' signaling). We found that activation of ephrin reverse signaling in chicken palates induced fusion in the absence of TGFb3, and that PI3K inhibition abrogated this effect. Further, blockage of reverse signaling inhibited TGFb3-induced fusion in the chicken and natural fusion in the mouse. Thus, ephrin reverse signaling is necessary and sufficient to induce palate fusion independent of TGFb3. These data describe both a novel role for ephrins in palate morphogenesis, and a previously unknown mechanism of ephrin signaling. Developmental Dynamics 240:357-364,

The development of suitable scaffolds for bone tissue engineering requires an in-depth understanding of the interactions between osteoblasts and scaffolding biomaterials. Although there have been a large amount of knowledge accumulated on the cell-material interactions on two-dimensional (2D) planar substrates, our understanding of how osteoblasts respond to a biomimetic nano-structured three-dimensional (3D) scaffold is very limited. In this work, we developed an approach to use confocal microscopy as an effective tool for visualizing, analyzing and quantifying osteoblast-matrix interactions and bone tissue formation on 3D nanofibrous gelatin scaffolds (3D-NF-GS). Integrin β1, phosphor-paxillin, and vinculin were used to detect osteoblasts responses to the nanofibrous architecture of 3D-NF-GS. Unlike osteoblasts cultured on 2D substrates, osteoblasts seeded on 3D-NF-GS showed less focal adhesions for phospho-paxillin and vinculin and the integrin β1 was difficult to detect after the first 5 days. Bone sialoprotein (BSP) expression on the 3D-NF-GS was present mainly in the cell cytoplasm at 5 days and inside secretory vesicles at 2 weeks, whereas most of the BSP on the 2D gelatin substrates was concentrated either in cell interface towards the periphery or at focal adhesion sites. Confocal images showed that osteoblasts were able to migrate throughout the 3D matrix within 5 days. By 14 days, osteoblasts were organized as nodular aggregations inside the scaffold pores and a large amount of collagen and other cell secretions covered and remodeled the surfaces of the 3D-NF-GS. These nodules were mineralized and were uniformly distributed inside the entire 3D-NF-GS after being cultured for 2 weeks. Taken together, these results give insight into osteoblast-matrix interactions in biomimetic nanofibrous 3D scaffolds and will guide the development of optimal scaffolds for bone tissue engineering.

An in-depth understanding of the interactions between cells and three-dimensional (3D) matrices (scaffolds) is pivotal to the development of novel biomaterials for tissue regeneration. However, it remains a challenge to find suitable biomimetic substrates and tools to observe cell-material and cell-cell interactions on 3D matrices. In the present study, we developed biomimetic nanofibrous 3D gelatin scaffolds (3D-NF-GS) and utilized confocal microscopy combined with a quantitative analysis approach to explore cell-matrix and cell-cell interactions on the 3D-NF-GS. Human gingival fibroblasts (HGFs) migrated throughout the 3D-NF-GS by 5 days and formed stable focal adhesions by 14 days. The focal adhesions were detected using integrin-β1, phospho-paxillin and vinculin expression, which were quantified from specific wavelength photon data generated using a spectral separation confocal microscope. As the cells became more confluent after 14 days of culture, cell-cell communication via gap junctions increased significantly. Collagen I matrix production by HGFs on 3D-NF-GS was visualized and quantified using a novel approach incorporating TRITC label in the scaffolds. Based on confocal microscopy, this study has developed qualitative and quantitative methods to study cell-matrix and cell-cell interactions on biomimetic 3D matrices, which provides valuable insights for the development of appropriate scaffolds for tissue regeneration.

Cleft palate is one of the most common birth defects. Understanding of the molecular events in palate development is a prerequisite to more effective treatment of this condition. The hard palate of humans and mice forms from shelves of mesenchyme covered with a layer of epithelial cells. These shelves adhere at the midline to form the midline epithelial seam (MES). It has been well understood that epithelial to mesenchymal transition (EMT) and apoptosis in the MES require Transforming Growth Factor β3 (TGFβ3). Eph receptor tyrosine kinases and their ephrin ligands are responsible for many contact‐mediated developmental processes. Binding of ephrins causes receptor activation in Eph‐bearing cells (forward signaling), and intracellular signaling inside ephrin‐bearing cells (reverse signaling). We discovered that ephrin‐B reverse signaling is required for palate fusion and is sufficient to do so in the absence of TGFβ3 in chicken palates. This leads us to propose that ephrin reverse signaling directs palatal EMT and fusion. To find out, we dissected palatal shelves from chicken and mice at different embryonic stages of fusion. We isolated RNA from these tissues and performed reverse transcription‐PCR for a panel of Ephs and ephrins. We found that all but a few of those genes were present in both chicken and mouse palatal tissues. Our future work will spatially locate the mRNA to specific cell layers.Grant Funding Source : March of Dimes 6‐FY06‐321

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