PURPOSE: Current bone regeneration strategies include delivery of recombinant proteins and the use of viral mediated gene therapy. Limitations to these strategies include the instability, high-dosing, and expense of delivering and retaining recombinant proteins at the regeneration site. In addition, viral mediated gene therapy can elicit host inflammatory responses and insertional mutagenesis. Thus, enhancing non-viral mediated gene therapy by improving uptake and localized expression has high therapeutic potential. We hypothesized that using a sustained delivery system of polylactic acid (PLA) porous scaffolds embedded with a circular DNA plasmid encoding green fluorescent protein (GFP) will be equivalent to direct application of various DNA-polymer complexes.
METHODS: Five types of DNA-polymer complexes with and without the PLA were evaluated in vitro for their ability to transfect and express a circular plasmid DNA construct containing eGFP into BHK cells (baby hamster kidney fibroblasts). Negative control was the BHK cell only group. (n=3 per group) Group 1: GFP/FuGene 6 (Roche) (positive control), Group 2: GFP/20mM DOTAP:cholesterol (1:1) liposome (Sigma), Group 3: GFP/PLA, Group 4: GFP/PLA/FuGene6, and Group 5: GFP/PLA/liposome. Several days before transfection, BHK cells were plated at a seeding density of 5000/well in 24-well plates. In the groups using PLA, the naked plasmid was mixed with collagen(1mg/ml) and loaded into porous pre-made 3mm PLA scaffolds. The collagen was allowed to set for 3h prior to the scaffold being placed onto the cells. The other groups had the specific treatment directly applied to the cells. The DNA-polymer complexes and controls were exposed to the BHK cells for 2-4d. Thereafter, the cells were removed by trypsin, washed and scanned for transfection efficiencies by a fluorescences-activated cell sorter (Bectin-Dickinson FACScan instrument). Cut-off times were based on negative controls. The cell sorter results are seen as a distribution curve based on intensity of expression. Data are presented as mean % +/- SD of cells expressing GFP vs. total cell number. Statistical analyses were conducted using single-factor ANOVA and Fisher’s PLSD post hoc analysis with significance established at p<0.05.
RESULTS: The mean % of BHK cells expressing GFP for the negative control and Groups 1-5 respectively are: 0.74(+/-0.17), 30.9(+/-4.85), 21.9(+/-5.17), 1.9(+/-0.46), 27.9(+/-4.46), and 28.1(+/-3.86) Significance was established between all groups over the negative control and Group 3, and also Groups 1, 4, and 5 over Groups 2 and 3. No significance was seen between the negative control and Group 3. Group 2 exhibited the highest toxicity with ~50% cell death.
CONCLUSION: The sustained delivery system described in the present studies exhibited equivalent expression to direct application of the DNA-polymer complexes. In addition, with the added benefit of these PLA scaffolds to preserve, protect, and release DNA as well as serve as matrices for bone regeneration, transfection efficiencies of non-viral mediated gene therapy and bone regeneration can be further improved with such a sustained delivery system. Further studies are needed to elucidate the precise scaffold DNA-polymer combination that will optimize bone regeneration for ultimate therapeutic application.