Infoscience

Thesis

Studies on scaffold design for tissue engineering applications: contribution of synthetic, natural and recombinant materials

The aim of tissue engineering is to regenerate tissue for the purpose of repairing or replacing diseased or injured tissue. Therefore, cells that are able to proliferate are seeded on scaffolds that provide mechanical stability and direct the three-dimensional structure of the newly formed tissue. This work focused on scaffolds made of synthetic and natural polymers. Solutions are proposed to efficiently monitor cell growth within three-dimensional scaffolds and to overcome the lack of cell recognition sites in synthetic materials. In vitro scaffold-cell interactions are used to estimate the bioactivity of a given scaffold. Conventional cell enumeration techniques are not adapted to monitor the number of viable cells on and within three-dimensional scaffolds. To address this problem, we developed a cell-based assay using green fluorescent protein (GFP) as a surrogate marker for cell number. Suspension-adapted, GFP-expressing CHO cells conferred flexibility and scalability to the scaffold screening assay. Adherence of CHO cells to synthetic polymers under agitation was shown. Comparison with established quantification assays validated the GFP assay. The use of GFP-expressing CHO cells allowed the identification of the fiber diameter (< 20 µm) and fiber drawing (e.g. raugher fiber surface) as beneficial parameters of polyvinylidene fluoride (PVDF) scaffolds for enhanced cell attachment and growth. These observations were confirmed by fibroblasts. It would be advantageous to analyze the scaffolds selected by the GFP assay in vitro and in vivo using the primary target cells combined with GFP-specific fluorescence. Since primary cells are resistant to most common chemical transfection methods, nucleofection was used for gene delivery to generate primary human bladder fibroblasts expressing GFP. Plasmids bearing a piggyBac (PB) transposon and the PB transposase were co-transfected. As an alternative approach, fibroblasts were transduced with a lentivirus vector carrying the GFP gene. PB-mediated gene delivery resulted in 30% and lentiviral transduction in 60% GFP-positive cells. Cell populations originating from viral transduction showed a stable GFP-expression over 2 months. In contrast, a stable GFP expression was not found in all cell pools generated by transfection of the PB-based plasmids. The results demonstrated the feasibility of generating GFP-expressing human bladder fibroblasts using either lentiviral vectors or transposon-mediated transfection. Synthetic polymer scaffolds can be manufactured in a defined and reproducible way; however, they suffer from a lack of cell recognition sites. In principle the combination of synthetic with natural polymers overcomes this problem and enhances the bioactivity of synthetic polymer scaffolds. The combination of warp knitted polylactic acid-ε-caprolactone (PLAC) scaffolds and collagen type I were explored. Collagen was supplied either as a recombinant protein or as plastic compressed (pc) collagen gels. Recombinant collagen represents a safe alternative to the use of animal-derived proteins, assuring large protein amounts independently of their natural occurrence and the ability to extract it from natural tissue. Human collagenI was expressed transiently and stably in CHO DG44 cells and titers up to 50 µg/mL were reached. Triple helical conformation of the collagen was shown by pepsin digestion. CHO cells overexpressing human collagen were seeded on PLAC scaffolds to mediate collagen deposition and were afterwards removed using osmotic shock, resulting in a collagen-PLAC scaffold. It was observed that cell-mediated collagen deposition was not efficient. Most of the collagen was associated with cell membranes and not with the polymer surface, and it was not possible to differentiate between endogenous and recombinant collagen. Nevertheless, bladder smooth muscle cells (SMCs) seeded on collagen-PLAC scaffolds proliferated more than those seeded on PLAC scaffolds. The combination of PLAC scaffolds with pc collagen gels resulted in PLAC-pc collagen hybrid scaffolds. Hybrid scaffolds were seeded with human bladder SMCs and urothelial cells (UCs) to evaluate their potential as scaffolds for bladder tissue regeneration. Cells were analyzed by scanning electron microscopy, conventional histology, immunohistochemistry, and a proliferation assay over a period of 14 days in vitro. Both cell types proliferated on the construct surface, forming dense cell layers after two weeks. However, SMCs seeded within the construct assessed with the alamarBlue assay had a lower level of proliferation than did surface-seeded ones. After seeding SMCs remained positive for smooth muscle alpha-actin and UCs kept their cytokeratin 7- and 17-positive phenotype over the 14-day period in vitro. The obtained data encourage the use of PLAC-pc collagen hybrids as scaffolds for bladder tissue regeneration. The combination of synthetic with natural polymers resulted in scaffolds with enhanced bioactivity and good mechanical properties.

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