15. Viability of Nanotransfection- Based Reprogrammed Vasculogenic Cells in a Mouse Model of Autogenous Cranioplasty

INTRODUCTION: Autologous cranioplasty using CCBG is associated with complication rates as high as 32%, with bone resorption accounting for a significant portion of these CCBG failures. Lack of revascularization is thought to be an underlying cause of this resorption. We hypothesized that a novel nanotransfection-based technology, which enables reprogramming of mouse embryonic fibroblasts to become vasculogenic cells, can be utilized for CCBG revascularization. Herein, we aim to utilize immunohistological studies to demonstrate viability of nanotransfected cells after surgical implantation during cranioplasty with CCBG. METHODS: Adults C56BL/6 mice were divided into experimental (EFF, indicates fibroblasts were nanotransfected with Etv2, Foxc2, and Fli1 (EFF) to drive reprogramming-based vasculogenesis) and control (Sham) group. Hemicraniectomy was performed using standard techniques, and the bone graft cryopreserved at -80C. Cranioplasty was performed 2 weeks later with the autologous CCBG. Mice in the EFF group had EFF-nanotransfected cells implanted at the defect site, while mice in the sham group had sham nano-transfected cells. Immunohistochemical method to identify the nanotransfected cells, assess vascularity and cell density were used. RESULTS: BrdU labeled cells were identified around the cranioplasty site at 1 week post cranioplasty, demonstrating viability of the cells in vivo. In order to evaluate vascularity with lectin stain, we assessed three separate areas at the cranioplasty site 1) the native bone around the perimeter of the defect; 2) the defect site, i.e. the gap between the native bone and the CCBG; and 3) the CCBG. The defect site of EFF mice had a significantly higher mean lectin fluorescence intensity compared to the native bone of both EFF mice (P=0.0044) and sham-treated mice (P=0.0009). Similarly, the defect site of Sham mice had a higher fluorescence intensity compared to the native bone of EFF mice (P=0.02) and sham mice (P=0.007). There was no difference in intensity between the defect site of EFF mice and the defect site of sham mice (P=0.998). When stratified by timepoint, these differences observed between defect site and native bone of both EFF- and sham -treated mice were not significant. On H&E analysis, cell density at the native bone region of EFF-treated mice was significantly higher than bone grafts of both EFF- (P<0.0001) and sham treated mice (P<0.0001). Similarly, cell density at native bone of sham treated mice were significantly higher than the bone grafts of both EFF- (P=0.0012) and sham-treated (P<0.0001) bone grafts. Cell density at the defect sites of EFF treated mice were also significantly higher than the bone graft of both EFF-treated (P=0.0024) mice and Sham treated mice (P=0.0002). Defect site cell density of sham treated mice were also significantly higher than that of bone graft in the same sham group (P=0.045). When stratified by timepoint, the highest difference in cellular density across cranioplasty sites above was observed at Week 2, with all sites compared above reaching a statistical significance. CONCLUSION: These studies demonstrate that EFF-nanotransfected cells are viable at the cranioplasty site and that bone graft is the least active following cranioplasty, with increased cell density and vasculogenesis at the defect site compared to within the CCBG.