Abstract
Massive segmental bony defects often occur following trauma or tumor resection which may result in non-union and even physical impairment, and over 15 billion dollars are spent annually in the US for the bone regeneration related therapies. Recently, mesenchymal stem cells (MSCs)-based therapy and virus-based gene therapy have converged and hold promise in assisting and accelerating bone healing. To explore whether BMSCs engineered by baculovirus can heal large bone defects, the New Zealand White (NZW) rabbit BMSCs were transduced with the BMP2-expressing baculovirus or VEGF-expressing baculovirus, co-seeded to scaffolds and co-implanted into critical-sized (10 mm) femoral segmental defects in NZW rabbits. X-ray analysis revealed that the baculovirus-engineered BMSCs not only bridged the defects at as early as week 2, but also healed the defects in 100% of rabbits at week 4. When compared with other control groups, the BMP2/VEGF-expressing BMSCs remarkably enhanced the segmental bone repair and mechanical properties, as evidenced by positron emission tomography (PET), micro-computed tomography (□CT), histochemical staining and biomechanical testing. The immunohistochemical staining further attested that the ameliorated bone healing concurred with the augmented angiogenesis. Additionally, adipose-derived stem cells (ASCs) also hold great promise for tissue regeneration because, unlike BMSCs requiring bone marrow harvest, ASCs are easy to isolate through liposuction and are more applicable in the future clinical application. But, ASCs were reported to failed in femoral segmental bone defect healing. To solve this problem, we hypothesized that sustained expression of factors promoting osteogenesis (BMP2) and angiogenesis (VEGF) in ASCs may provide continuous stimuli to augment the bone healing. Therefore we developed a hybrid baculovirus system and attested the ASCs transduced with the hybrid baculovirus can leading to cassette excision off the baculovirus genome, enabling transgene persistence in episomal form and prolonging the expression to >28 days. Compared with the ASCs engineered by the conventional baculovirus transiently expressing BMP2/VEGF only healed the critical-size segmental femoral bone defects in 40% of rabbits at 12 weeks post-implantation, whereas ASCs engineered by the hybrid vectors persistently expressing BMP2/VEGF healed the critical-size defects in 100% of animals in 8 weeks. Thereby attesting our hypothesis that persistent BMP2/VEGF expression is essential in use of ASCs to treating massive segmental defects necessitating sustained stimuli. Moreover, the in vivo immunological evaluations are evaluated after the hybrid baculovirus-transduced ASCs are implanted into the femoral critical defects. The activation of immune cells (e.g. macrophage and T cells) are observed by immuno histological staining and the recipient lymphocytes against donor-ASCs are evaluated by spleen derived mixed lymphocyte cytotoxic assays and peripheral blood differential count. These data demonstrated that the transplantation of ASCs engineered by hybrid baculovirus will not elicit severe immune rejection and antigen specific cellular immune response. These in vivo experiments will shed light on whether baculovirus transduction and transgene expression trigger unwanted immune responses and change immunocharacteristics of ASCs in vivo, which will benefit future baculovirus-mediated gene therapy.