Purpose: The ultimate goal of this study was to develop a coating for titanium surfaces that contained polyplexes of polyethylenimine (PEI)and genes encoding platelet-derived growth factor B (PDGF-B) such that the polyplex-containing coating enabled the transfection of native cells with PDGF-B genes. The secreted PDGF-BB protein in turn will enhance local proliferation and spreading of native cells. The soft tissue seal around dental implants is critical to the maintenance of peri-implant health. Transfection of local gingival cells with PDGF-B and the resulting enhancement of proliferation and spreading of these cells could be useful in mitigating peri-implant complications by promoting rapid formation of a soft tissue seal around the implant. Here, as an early proof of principle, plasmids encoding for enhanced green fluorescent protein (peGFP) were complexed with PEI and coated onto discs in a sucrose solution to transfect local HEK 293 cells.
Methods: Titanium discs were polished and then sandblasted to yield a uniform, roughened surface. PEI polyplex solutions containing 100µg/mL of peGFP were prepared using a nitrogen to phosphate ratio (N:P) of 10. Forty percent sucrose was added to the resulting polyplex solution to reach a final sucrose concentration of 1% (w/v). Aliquots of the 1% sucrose, 100µg/mL peGFP polyplex solution were placed atop the rough side of the titanium discs and frozen at -80oC, after which they were lyophilized. The resulting coated discs were then suspended above HEK 293 cells in serum-free media for 4 hours, after which the cells were cultured in complete medium for 3 days before quantitation of the transfection efficiency using flow cytometry. The negative control consisted of cells exposed to an uncoated disc, and the “lyophilized control” consisted of cells exposed to a 2.5µg peGFP dose created by lyophilizing excess 100µg/mL peGFP polyplex solution, resuspending it at time of administration, and adding the correct volume directly to the serum-free media. Coated and uncoated discs were also analyzed with XPS to confirm the presence of the coating and uncoated discs were imaged with SEM to visualize the rough surface.
Results: The transfection efficiency, expressed as a proportion of the average transfection efficiency of the lyophilized control samples, showed a dose-dependent increase as the amount of pDNA coated onto the titanium discs increased from 1.5 to 6.5 µg. XPS analysis showed a clear difference in the amount of nitrogen on the coated disc, confirming the presence of the coating. SEM images showed a micro-textured surface that appears uniform in its texture.
Conclusion: Coating titanium discs with PEI-gene polyplexes is a promising method of delivering genes to local cells for gene therapy, and may be able to be used as a coating for dental implants to enhance soft tissue seal formation and maintenance.
Jaidev Chakka– Post-Doctorate Researcher, University of Iowa
Satheesh Elangovan– Professor, College of Dentistry, University of Iowa
Aliasger Salem– Professor, College of Pharmacy, University of Iowa, Iowa City, Iowa