Development of genetically modified cells for bone tissue regeneration
Blum, Jeremy Scott
Mikos, Antonios G.
Doctor of Philosophy
Bone regeneration through transplantation of genetically modified cells provides an opportunity to combine the fields of tissue engineering and gene therapy. The ex vivo modification of cells for overproduction of proteins can be used for therapeutic and/or diagnostic applications in tissue regeneration. The aim of this work was to develop genetically modified cells for enhanced osteogenic potential and for non-destructive diagnostic evaluation of cell behavior on biomaterials. Prior to modification of cells, a luminescent based assay was developed for the evaluation of osteodifferentiation of progenitor cells. A chemiluminescent substrate was used for detection of the early osteodifferentiation marker, alkaline phosphatase (AP). The results of this work demonstrated a faster, simpler, and more sensitive method of monitoring changes in AP levels during osteodifferentiation using the chemiluminescent substrate relative to the traditional colorimetric substrate. The ability of primary rat marrow stromal cells (MSCs) to be genetically modified by adenoviral, retroviral, and cationic lipid gene therapy vectors was investigated. All vectors delivered the reporter transgene luciferase to MSCs. We also explored the influence of MSC differentiation induced by different culture conditions on the vectors' gene delivery efficiency. Only adenovirus demonstrated a substantial increase in transgene expression when cells were cultured in the presence of osteogenic supplements. Moreover, it was determined that increased transgene expression was specific to the supplement concentration of dexamethasone in the culture medium. For therapeutic bone regeneration the osteogenic transgene bone morphogenetic protein 2 (BMP-2) was delivered to MSCs. Adenovirus, retrovirus, and cationic lipids delivering this transgene were used and the osteogenic potential of the MSCs was evaluated. Results both in vitro and in vivo indicated that MSCs genetically modified with adenovirus had an enhanced osteogenic response compared to unmodified control MSCs and MSCs modified by the other vectors. For diagnostic non-destructive evaluation of cells on biomaterials, two fibroblastic cell lines were developed to stably express the fluorescent transgene enhanced green fluorescent protein (EGFP) and the luminescent transgene luciferase. Cells were able to be visualized both microscopically and macroscopically on two separate biomaterials. In addition, quantitative growth of cells on the materials was assessed non-destructively both in vitro and in vivo.