(251g) Gene Activated Matrix Transfection of Human Mesenchymal Stem Cells by Chitosan-DNA Nanoparticles In Chitosan-Based Scaffolds
AIChE Annual Meeting
Tuesday, October 18, 2011 - 10:40am to 11:00am
Human mesenchymal stem cells (hMSCs) are a potent osteogenic cell source, but hMSC based bone constructs require a scaffold that provides both mechanical stability and the capability to promote hMSC proliferation and to direct osteogenic differentiation. Gene activated matrices (GAMs) create a singular environment where hMSCs can adhere, proliferate, be continuously transfected with plasmid, and exhibit sustained protein expression. We are investigating a scaffold that incorporates BMP2 plasmid DNA for sustained delivery. Naked BMP2 pDNA or chitosan-BMP2 pDNA nanoparticles inside a bulk material of hydroxyapatite-chitosan-gelatin (HCG) hydrogel will serve as the GAMs. This composite material has many advantages, including similarity to natural bone ECM, biocompatibility and long term protection of plasmid DNA from nucleases.
Our previous studies have demonstrated that HCG hydrogel absorbs a mediating ECM protein layer that supports hMSC attachment and proliferation, and possesses intrinsic osteoinductive properties (1). In the current study, hMSC transfection in the HCG GAM is achieved by directly incorporating BMP2-GFP pDNA and by incorporating chitosan encapsulated BMP2-GFP pDNA. This fusion vector provides both the functional BMP2 gene that promotes osteogenic differentiation and the GFP reporter gene that allows direct evaluation of transfection efficiency. Using bolus administration of chitosan-DNA nanoparticles as controls, hMSCs exhibit a dose dependent transfection to bolus chitosan-DNA nanoparticles. Confocal analysis revealed uniform distribution of chitosan-pDNA nanoparticles in the HCG scaffolds with comparable bulk morphology to the HCG hydrogel controls. hMSC exhibited extensive proliferation in both the HCG GAMs and control during first three weeks but the rate decreased in the GAMs from weeks 3 to 5. Protein expression kinetics revealed that HCG hydrogel matrix is continuously enriched with bone marker proteins throughout the culture period and that this enrichment is more extensive in the GAM hydrogels. PCR results confirm expression of the GFP reporter protein after 35 days in culture, indicating the sustained delivery of DNA from the HCG hydrogel GAM. Ongoing experiments focus on elucidating hMSC’s dose-dependent response to bolus transfection kinetics, and evaluating the impact of nanoparticle encapsulation on the scaffold mediated hMSC transfection.
1. Zhao F, Grayson W, Ma T, Bunnell B, Lu W. Effects of hydroxyapatite in 3-D chitosan gelatin polymer network on human mesenchymal stem cell construct development. Biomaterials, 2006, Vol. 27, pp. 1859-1867.