Gene-supplemented collagen scaffolds for non-viral gene delivery for brain tissue engineering

Abstract

Recent advances in tissue engineering, combining an extracellular matrix (ECM)-like vehicle with therapeutic molecules, cells and/or genes has yielded promising results for brain injury repair. The purpose of this thesis was to develop a collagen scaffold for the non-viral delivery of the gene encoding for Glial Cell-Derived Neurotrophic Factor (GDNF); hence to provide a local, long-term release and overexpression of GDNF via transfection of cells seeded into the scaffold or endogenous cells. The first part of the thesis aimed to investigate the in vitro transfection of marrow stromal stem cells (also referred to as mesenchymal stem cells, MSCs) in monolayer with plasmid GDNF ([mu]gDNF). Several parameters were evaluated: the choice of a transfer reagent (GenePorter2 versus Lipofectamine 2000), the doses of plasmid incorporated in the liposomes (ranging from 0.2[mu]g to 2[mu]g), the post-transfection medium (Medium 1: DMEM low glucose, 20% FBS and 1% antibiotic versus Medium 2: DMEM low glucose, 20% FBS, 1% antibiotic and 10ng/ml FGF-2) and the culture environment during transfection (static versus dynamic). The objective of the second part was to determine the conditions, including the design of the scaffold and the method of seeding, under which MSCs could attach and grow on the scaffold.

(cont.) Collagen scaffolds were made by a freeze-drying technique and prepared with various amounts of collagen, cross-link densities, and freezing temperatures. The effect of gene supplementation on the cross-link density was evaluated using the swelling ratio. Finally, the aim of the third part was to evaluate different parameters to optimize the transfection of cells grown in the scaffolds. The profile of production of GDNF was studied for different cross-link density, initial plasmid dose (2 and 10 [mu]g) and plasmid-transfection reagent ratio. Finally the effect of the pore diameter and static and dynamic culture environments were tested to optimize the in vitro conditions for the plasmid uptake and expression by the MSCs. The results demonstrated the possibility of using non-viral transfection conditions in vitro to enable MSCs to express a selected neurotrophic factor, GDNF, in therapeutic doses. MSCs were shown to over-express GDNF for at least a two-week period of time. Lipoplexes loaded with as little as 0.2 [mu]g could result in a significant production of GDNF by MSCs for several days, before falling off to control levels after one week. For the highest loading of plasmid (2 [mu]g), the level of GDNF production was still above the control after 2 weeks.

(cont.) Dynamic transfection had a dramatic effect on the production of GDNF. The accumulated amount of GDNF during the 2-week period reached 65 ng/ml compared to 20 ng/ml produced in static conditions. The growth factor bFGF, which is used in transdifferentiation of MSCs for a neuronal phenotype, was shown to promote a high level of cell death when used in the post-transfection medium. Collagen scaffolds can be prepared to incorporate the plasmid DNA-lipid complexes for subsequent release. Also, gene and subsequent cross-link density have an effect on the mechanical behavior of scaffolds. Finally, the gene-supplemented collagen scaffolds can serve as a carrier for lipoplexes and modified MSCs and provide a long-term overexpression of GDNF. The level of gene expression in the collagen constructs was lower than those obtained in MSC monolayers but high enough to result in therapeutic doses previously found in vitro. The cross-linking treatment did not affect significantly the release profile of GDNF. The application of orbital shaking during the 4 hours of transfection had a positive effect on the production of GDNF but not as strong as reported in monolayer studies. The load of plasmid DNA is a prominent parameter in the three-dimensional (3-D) transfection.

(cont.) In this study, the highest level of GDNF expression was observed for 10 [mu]g of plasmid DNA and 6 days after transfection. Overall, these results demonstrated that the combination of tissue engineering and non-viral transfection of MSCs for the over-expression of GDNF was a promising approach for the long-term production of selected neurotrophic growth factors. This approach could provide benefits in the treatment of conditions involving the loss of brain tissue.