Abstract
Massive segmental defects arising from trauma or tumor resection remain a challenging clinical problem. To heal massive, segmental bone defects using adipose-derived stem cells (ASCs), which alone cannot heal large defects, we hypothesized that sustained expression of factors promoting bone regeneration (BMP2) and angiogenesis (VEGF) provides continuous stimuli to augment the healing. Insect baculovirus (BV) holds promise for gene therapy and efficiently transduces stem cells, but it only mediates transient transgene expression. Therefore we developed a dual BV system whereby one BV expressed FLP recombinase (BacFLP) while the other hybrid BV harbored an Frt-flanking transgene cassette. The New Zealand White (NZW) rabbit ASCs were transduced with the BacFLP and hybrid BV or conventional BV, seeded to scaffolds and transplanted into critical-sized (10 mm) femoral segmental defects in NZW rabbits. The bone regeneration was assessed by radiography, Positron Emission Tomography/Computer Tomography (PET/CT) at 2, 4, 8 and 12 weeks post-transplantation (wpt), and μCT, histological staining, biomechanical torsional testing at 12 wpt, respectively. Within the ASCs transduced with BacFLP and the hybrid BV, the transduction efficiency reached 98% while the FLP/Frt-mediated recombination efficiency approached 46%, leading to cassette excision off the BV genome, enabling transgene persistence in episomal form and prolonging the expression to >28 days. Transduction of ASCs with the BMP2-encoding hybrid BV led to prolonged BMP2 expression and augmented ASCs osteogenesis even without other osteogenic supplements. ASCs engineered by conventional BV transiently expressing BMP2/VEGF (S group) only healed the critical-sized femoral bone defects in 40% of NZW rabbits at 12 wpt, but ASCs engineered by the hybrid vectors persistently expressing BMP2/VEGF (L group) healed the critical-sized defects in 12 out of 12 animals in 8 weeks. Compared with the S group, the L group not only accelerated the healing, but also ameliorated the bone metabolism, bone volume, bone density, angiogenesis and mechanical properties. These data confirmed our hypothesis that persistent BMP2/VEGF expression is essential. Use of the hybrid BV vector for ASCs engineering represents a novel approach to treating massive segmental bone defects necessitating concerted ossification and vascularization.