Electrospinning using natural proteins or synthetic polymers is a promising technique for the fabrication of fibrous scaffolds for various tissue engineering applications. However, one limitation of scaffolds electrospun from natural proteins is the need to cross-link with glutaraldehyde for stability, which has been postulated to lead to many complications in vivo including graft failure. In this study, we determined the characteristics of hybrid scaffolds composed of natural proteins including collagen and elastin, as well as gelatin, and the synthetic polymer poly(ε-caprolactone) (PCL), so to avoid chemical cross-linking. Fiber size increased proportionally with increasing protein and polymer concentrations, whereas pore size decreased. Electrospun gelatin/PCL scaffolds showed a higher tensile strength when compared to collagen/elastin/PCL constructs. To determine the effects of pore size on cell attachment and migration, both hybrid scaffolds were seeded with adipose-derived stem cells. Scanning electron microscopy and nuclei staining of cell-seeded scaffolds demonstrated complete cell attachment to the surfaces of both hybrid scaffolds, although cell migration into the scaffold was predominantly seen in the gelatin/PCL hybrid. The combination of natural proteins and synthetic polymers to create electrospun fibrous structures resulted in scaffolds with favorable mechanical and biological properties.

Flock house virus (FHV), a bipartite RNA virus of insects and a member of the Nodaviridae family, shares viral replication features with the tripartite brome mosaic virus (BMV), an RNA virus that infects plants and is a member of the Bromoviridae family. In BMV and FHV, genome packaging is coupled to replication, a widely conserved mechanism among positive-strand RNA viruses of diverse origin. To unravel the events that modulate the mechanism of replication-coupled packaging, in this study, we have extended the transfer DNA (T-DNA)-based agroinfiltration system to express functional genome components of FHV in plant cells (Nicotiana benthamiana). Replication, intracellular membrane localization, and packaging characteristics in agroinfiltrated plant cells revealed that T-DNA plasmids of FHV were biologically active and faithfully mimicked complete replication and packaging behavior similar to that observed for insect cells. Complementation with homologous replicase (with respect to CP) failed to enhance packaging specificity. Taken together, we propose that the transcription of CP mRNA from homologous replication and its translation must be synchronized to confer packaging specificity.Flock house virus (FHV), a member of the family Nodaviridae, and brome mosaic virus (BMV), the type member of the Bromovirus genus, are multicomponent positive-strand RNA viruses of animals and plants, respectively (44, 52). FHV was first isolated from the New Zealand grass grub Costelytra zealandica (52). The 4.5-kb single-strand positive-sense RNA genome of FHV is divided between two capped and nonpolyadenylated RNAs copackaged into a single nonenveloped icosahedral virion with a T ϭ 3 symmetry (50). Genomic RNA 1 (F1) is a 3,107-nucleotide (nt) sequence that encodes a 112-kDa RNA-dependent RNA polymerase (RdRp or protein A) that is necessary and sufficient for FHV RNA replication (8,27,42). In addition, F1 also encodes a 387-nt subgenomic RNA 3 (sgF3) sequence that corresponds to that of the 3Ј terminus of F1 (16,22). sgF3 encodes two proteins, b1 and b2. Protein b1 has no recognized function, and b2 is the designated suppressor of RNA silencing activity in Drosophila melanogaster S2 cells (33). Genomic RNA 2 (F2) is a 1,400-nt sequence that encodes the 43-kDa viral capsid protein (CP) precursor ␣ required for the assembly of FHV provirions (49). Each provirion consists of 180 subunits of protein ␣ arranged with a T ϭ 3 quasi-equivalent symmetry and the two genomic RNAs.Provirions are not infectious unless they undergo an autocatalytic maturation process, which results in the cleavage of protein ␣ into protein ␤ (38 kDa) and protein ␥ (5 kDa) (23, 51). sgF3 has been shown to trans activate F2 replication (20). The replication of FHV occurs on the outer mitochondrial membranes (36). An unusual and remarkable feature associated with FHV is its ability to cross the kingdom barrier and to infect a wide variety of cells, including cells of insects (23), mammals (8), yeasts (41), and several species of monocotyledonous and dicotyledon...

Synthetic polymers or naturally-derived extracellular matrix (ECM) proteins have been used to create tissue engineering scaffolds; however, the need for surface modification in order to achieve polymer biocompatibility and the lack of biomechanical strength of constructs built using proteins alone remain major limitations. To overcome these obstacles, we developed novel hybrid constructs composed of both strong biosynthetic materials and natural human ECM proteins. Taking advantage of the ability of cells to produce their own ECM, human foreskin fibroblasts were grown on silicon-based nanostructures exhibiting various surface topographies that significantly enhanced ECM protein production. After 4 weeks, cell-derived sheets were harvested and histology, immunochemistry, biochemistry and multiphoton imaging revealed the presence of collagens, tropoelastin, fibronectin and glycosaminoglycans. Following decellularization, purified sheet-derived ECM proteins were mixed with poly(ε-caprolactone) to create fibrous scaffolds using electrospinning. These hybrid scaffolds exhibited excellent biomechanical properties with fiber and pore sizes that allowed attachment and migration of adipose tissue-derived stem cells. Our study represents an innovative approach to generate strong, non-cytotoxic scaffolds that could have broad applications in tissue regeneration strategies.

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