A gene therapy approach for tissue engineering applications.

摘要

In this work, gene therapy was combined with cell therapy to tackle three tissue engineering applications. The goal of the first project was to promote endothelialization of tissue engineering vascular grafts (TEVGs). We developed a system called the collagen-based gene-activated matrix (GAM) which was able to retain plasmid DNA (pDNA) and allowed smooth muscle cells (SMCs) embedded to gradually take up and express the gene of interest, in this case, vascular endothelial growth factor (VEGF). To obtain better transfection efficiency, pDNA was complexed with polyethyleneimine (PEI) which dramatically improved transfection of SMCs in GAMs. Continual production of VEGF for approximately one month was observed. VEGF produced by SMCs in GAMs was bioactive and induced both enhanced migration and proliferation of endothelial cells (ECs) on collagen which is a common biomaterial for TEVGs. The goal of the second project was to potentiate angiogenesis through overexpression of VEGF in 10T1/2 cells for treatment of ischemic diseases and vascularization of tissue engineered constructs. 10T1/2 cells were transfected with the VEGF transgene successfully via retroviral transfection. VEGF-producing 10T1/2 cells were able to induce enhanced migration, proliferation, as well as invasion of underlying matrix in ECs. Potentiation of angiogenesis was further observed in 3D collagen models when ECs were co-cultured with VEGF-producing 10T1/2 cells. ECs formed extensive network of tubular structures and presence of a lumen in the vessels formed was confirmed by confocal microscopy. VEGF-producing 10TI/2 cells also rescued ECs from starvation and induced them to form organized tubular structures. The goal of the third project was to enhance mechanical strength in dermal wound through increased cross-linking of extracellular matrix (ECM) proteins via overexpression of lysyl oxidase (LO). Using the GAM system we developed and embedding transgene encoding LO with fibroblasts, we obtained enhanced mechanical strength in collagen constructs in vitro. We also demonstrated the same efficacy of these LO-producing GAMs in a dermal wound healing model in vivo.