We report here the self-assembly of macroscopic sacs and membranes at the interface between two aqueous solutions, one containing a megadalton polymer and the other, small self-assembling molecules bearing opposite charge. The resulting structures have a highly ordered architecture in which nanofiber bundles align and reorient by nearly 90 degrees as the membrane grows. The formation of a diffusion barrier upon contact between the two liquids prevents their chaotic mixing. We hypothesize that growth of the membrane is then driven by a dynamic synergy between osmotic pressure of ions and static self-assembly. These robust, self-sealing macroscopic structures offer opportunities in many areas, including the formation of privileged environments for cells, immune barriers, new biological assays, and self-assembly of ordered thick membranes for diverse applications.

Microscale topographical features have been known to affect cell behavior. An important target in this area is to integrate top down techniques with bottom up self-assembly to create three-dimensional (3D) patterned bioactive mimics of extracellular matrices. We report a novel approach toward this goal and demonstrate its use to study the behavior of human mesenchymal stem cells (hMSCs). By incorporating polymerizable acetylene groups in the hydrophobic segment of peptide amphiphiles (PAs), we were able to micro-pattern nanofiber gels of these bioactive materials. PAs containing the cell adhesive epitope arginine-glycine-aspartic acid-serine (RGDS) were allowed to self-assemble within microfabricated molds to create networks of either randomly oriented or aligned ~30 nm diameter nanofiber bundles that were shaped into topographical patterns containing holes, posts, or channels up to 8 μm in height and down to 5 μm in lateral dimensions. When topographical patterns contained nanofibers aligned through flow prior to gelation, the majority of hMSCs aligned in the direction of the nanofibers even in the presence of hole microtextures and more than a third of them maintained this alignment when encountering perpendicular channel microtextures. Interestingly, in topographical patterns with randomly oriented nanofibers, osteoblastic differentiation was enhanced on hole microtextures compared to all other surfaces.

This study investigated the use of a type II collagenglycosaminoglycan (CG) scaffold as a nonviral gene delivery vehicle for facilitating gene transfer to seeded adult articular chondrocytes to produce an elevated, prolonged and local expression of insulin-like growth factor (IGF)-1 for enhancing cartilage regeneration. Gene-supplemented CG (GSCG) scaffolds were synthesized by two methods: (1) soaking a pre-cross-linked CG scaffold in a plasmid solution followed by a freeze-drying process, and (2) chemically cross-linking the plasmid DNA to the scaffold. Two different plasmid solutions were also compared: (1) naked plasmid IGF-1 alone, and (2) plasmid IGF-1 with a lipid transfection reagent. Plasmid release studies revealed that cross-linking the plasmid to the CG scaffold prevented passive bolus release of plasmid and resulted in vector release controlled by scaffold degradation. In chondrocyte-seeded GSCG scaffolds, prolonged and elevated IGF-1 expression was enhanced by using the cross-linking method of plasmid incorporation along with the addition of the transfection reagent. The sustained level of IGF-1 overexpression resulted in significantly higher amounts of tissue formation, chondrocyte-like cells, GAG accumulation, and type II collagen production, compared to control scaffolds. These findings demonstrate that CG scaffolds can serve as nonviral gene delivery vehicles of microgram amounts of IGF-1 plasmid (o10 mg per scaffold) to provide a locally sustained therapeutic level of overexpressed IGF-1, resulting in enhanced cartilage formation.

To investigate the use of a scaffold seeded with genetically modified meniscal cells or mesenchymal stem cells (MSCs) for the healing of meniscal lesions, primary meniscus cells and bone marrow-derived MSCs were isolated from bovine calves and transduced with first-generation adenoviral vectors encoding green fluorescent protein, luciferase, or transforming growth factor (TGF)-beta1 complementary deoxyribonucleic acid (cDNA). The genetically modified cells were seeded in type I collagen-glycosaminoglycan (GAG) matrices and transplanted into tears of the avascular zone of bovine menisci. After 3 weeks of in vitro culture, constructs and repair tissues were analyzed histologically, biochemically, and using reverse transcriptase polymerase chain reaction. Recombinant adenovirus readily transduced meniscal cells and MSCs, and transgene expression remained high after the cells were incorporated into collagen-GAG matrices. Transfer of TGF-beta1 cDNA increased cellularitiy and the synthesis of GAG/DNA [microg/microg]. It also led to stronger staining for proteoglycans and type II collagen and enhanced expression of meniscal genes. Transplantation of the TGF-beta1 transduced constructs into meniscal lesions of the avascular zone resulted in filling of the lesions with repair tissue after 3 weeks of in vitro culture. These results indicate that TGF-beta1 cDNA delivery may affect cell-based meniscus repair approaches in vivo.

The objective of the present study was to investigate the use of gelatin and cationized-gelatin nanoparticles for the nonviral delivery of the plasmid DNA encoding for insulin-like growth factor (IGF)-1 to adult canine articular chondrocytes in vitro; plasmid for enhanced green fluorescence protein (EGFP) was used as a marker gene. The spherical cationized gelatin nanoparticles were on average 172 nm in diameter, compared with the often ellipsoid-shaped unmodified (noncationized) gelatin particles that generally appeared to be 10 mum to greater than 20 mum in length. The zeta potential of the positively charged cationized gelatin nanoparticles containing the plasmid was around 20 mV compared with about 2 mV for the unmodified gelatin particles. There was no noticeable fluorescence from the cells treated with the nanoparticles prepared with the original (noncationized) gelatin particles containing the pEGFP. In contrast, numerous cells in the group transfected with the cationized gelatin-pEGFP nanoparticles were found to fluoresce demonstrating the transfection of the cells. There was five-fold elevation in the amount of IGF-1 produced by the cells treated with the cationized gelatin nanoparticles containing the IGF-1 plasmid compared with the unmodified (noncationized) gelatin particles. There was a clear effect of varying the weight ratio of plasmid IGF-1 in the cationized gelatin nanoparticles on the IGF-1 in the medium of cells exposed to the nanoparticles for 5 h. A peak in the amount of released IGF-1 was detected at a gelatin:IGF-1 weight ratio of 250:1.

The objective of the present study was to prepare chitosan nanoparticles incorporating a relatively large plasmid encoding for osteogenic protein (OP)-1 and to determine the ability of these nanoparticles to transfect adult canine articular chondrocytes in vitro. The positive charge of chitosan acted to condense the relatively large negatively-charged OP-1 plasmid such that it could be incorporated into nanoparticles. Incorporation of the plasmid into the chitosan nanoparticles did not affect the structural integrity of the plasmid as demonstrated by gel electrophoresis. The morphology and size of the nanoparticles were found to vary with the chitosan:plasmid weight ratio. Nanoparticles formulated with a chitosan:plasmid ratio of 10:1 were of uniformly small size (less than 250 nm) and spherical shape. These nanoparticles had a positive charge of about 20 mV. FITC-labeled chitosan nanoparticles were found in virtually all of the cells after 24 h of incubation with the nanoparticles, and confocal microscopy revealed FITC-related fluorescence in the nucleus of the chondrocytes. Although transfection of the chondrocytes was demonstrated by the fluorescence of cells treated with chitosan nanoparticles containing the plasmid for the enhanced green fluorescence protein, cells transfected with nanoparticles incorporating the larger OP-1 plasmid did not show OP-1 expression measured by ELISA for up to 2 weeks in culture. These results indicate that although a large plasmid can be successfully incorporated within chitosan nanoparticles, the size of the plasmid incorporated within the nanoparticles may still significantly affect gene transfer to cells.

No abstract

The composition of the expansion medium significantly affects monolayer proliferation of adult canine chondrocytes, GAG synthesis when the cells are subsequently grown in CG scaffolds, and ex vivo IGF-1 gene transfer.