Hybrid constructs for craniofacial reconstruction: sustained gene delivery using demineralized bone matrix putty

These experiments evaluate the efficacy of a demineralized bone matrix putty engineered as a hybrid construct for sustained, site-directed gene transfer using an adenoviral vector. In vitro experiments were performed to evaluate the optimal dosing for gene transfer to fetal calvarial osteoblasts and dural cells and for the sustainability of gene transfer from the hybrid constructs. In the dosing experiments, hybrid constructs were created by combining 0.5 mL of demineralized bone matrix putty (DBX; SYNTHES Maxillofacial, Monument, CO) with 1 x 10(8), 1 x 10(7), or 1 x 10(6) particle-forming units (PFU) of an adenoviral vector carrying the gene encoding green fluorescent protein (AdGFP). These constructs were then placed in direct contact, or in transwell coculture, with fetal murine calvarial osteoblasts or dural cells at a multiplicity of infection (MOI = viral particle/cell ratio) of 1000, 100, and 10. The sustainability of gene transfer was tested through transfer of the hybrid construct to wells containing untransfected cells every 24 hours for 30 days. In both experiments, gene transfer was determined through the visualization of GFP using fluorescence light microscopy 24 hours after the onset of transfection. Optimal dosing for gene transfer occurred at an MOI of 10 for calvarial osteoblasts and 100 for dural cells. At greater concentrations, toxicity was observed in the majority of samples. Gene transfer to fetal dural cells and calvarial osteoblasts was sustained throughout the 30-day period. These experiments suggest that adenoviral vectors could be successfully incorporated within demineralized bone matrix to provide effective, sustained, site-directed gene transfer.