AbstractObjectives: Gene active matrix (GAM) has wide potential utilization in tissue engineering. It may genetically modify cells with plasmid DNA endcoding therapeutic genes and release of the protein to surrounding tissues. In this study, we assessed the feasibility of the local gene release from collagen and fibronectin substrates (GAS) and the efficacy of local release of stromal cell-derived factor1 (SDF-1) gene on c-kit positive cells homing.
Methods: Non-viral poly-ethyleneimine (25kDa PEI)/DNA complexes were mixed with rat tail collagen (type I) or human fibronectin substrates to form the GASs. The released profiles from the substrates of the complex were studied. The PEI/DNA N/P ratio of the complex and the substrates dose were optimized by luciferase expression and cell viability assay. Localized gene expression in COS7 cells cultured on the GASs was assessed by green fluorescent protein (GFP) and LacZ marker genes.
Results: Both fibronectin and collagen allowed sustained DNA release over two months. Both the optimal transfection conditions for collagen and fibronectin GASs were 4 of complex N/P ratio, while the optimal DNA dose was 7.5 g/cm2 for fibronectin-GAS and 10.0 g/cm2 for collagen-GAS. Comparing with fibronectin-GAS, collagen-GAS can promote cells proliferation. In vitro, both fibronectin and collagen GASs could locally deliver gene at the defined area and the expression of SDF-1 from the transfected rat mesenchymal stem cells or COS7 cells could attract c-kit positive cells to the GAS defined patterns. In vivo expressions of both GFP and SDF-1 in quadriceps were detected in a mouse model by coating the collagen-GAS onto the mixed cellulose ester membranes and transplanting the membranes into the quadriceps. RT-PCR result showed that the c-kit expression had a significant enhancement induced by collagen-GAS containing GFP-N3-SDF1 gene.
Conclusion: Collagen and fibronectin GASs can induce localized stem cells migration and homing and could be employed to guide tissue formation.