Healing of massive segmental femoral bone defects in minipigs by allogenic ASCs engineered with FLPo/Frt-based baculovirus vectors.
Adipose-derived stem cells (ASCs) hold promise for bone regeneration but possess inferior osteogenesis potential. Allotransplantation of ASCs engineered with the BMP2/VEGF-expressing baculoviruses into rabbits healed critical-size segmental bone defects. To translate the technology to clinical applications, we aimed to demonstrate massive bone healing in minipigs that more closely mimicked the clinical scenarios, using a new hybrid baculovirus system consisting of BacFLPo expressing the codon-optimized FLP recombinase (FLPo) and the substrate baculovirus harboring the transgene flanked by Frt sequences. Co-transduction of minipig ASCs (pASCs) with BacFLPo and the substrate baculovirus enabled transgene cassette excision, recombination and minicircle formation in ≈73.7% of pASCs, which substantially prolonged the transgene (BMP2 and VEGF) expression to 28 days. When encoding BMP2, the FLPo/Frt-based system augmented the pASCs osteogenesis. Allotransplantation of the BMP2/VEGF-expressing pASCs into minipigs healed massive segmental bone defects (30 mm in length) at the mid-diaphysis of femora, as evaluated by computed tomography, positron emission tomography, histology, immunohistochemical staining and biochemical testing. The defect size was ≈15% of femoral length in minipigs and was equivalent to ≈60-70 mm of femoral defect in humans, thus the healing using pASCs engineered with the FLPo/Frt-based baculovirus represented a remarkable advance for the treatment of massive bone defects.
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
Chin-Yu
CY
Lin
Department of Nursing, Yuanpei University, Hsinchu 300, Taiwan.
Yao-Horng
YH
Wang
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
Kuei-Chang
KC
Li
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
Li-Yu
LY
Sung
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
Chia-Lin
CL
Yeh
Department of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan 333, Taiwan.
Kun-Ju
KJ
Lin
Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan 333, Taiwan; College of Medicine, Chang Gung University, Taoyuan 333, Taiwan.
Tzu-Chen
TC
Yen
Center for Tissue Engineering, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; Department of Orthopaedic, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan. Electronic address: hardbone2002@yahoo.com.
Yu-Han
YH
Chang
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan. Electronic address: ychu@mx.nthu.edu.tw.
Yu-Chen
YC
Hu
D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D001483:Base Sequence; D001696:Biomechanical Phenomena; D001861:Bone Regeneration; D002478:Cells, Cultured; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D005269:Femur / Q000098:blood supply / Q000000981:diagnostic imaging / Q000473:pathology*; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000378:metabolism*; D010012:Osteogenesis; D049268:Positron-Emission Tomography; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013552:Swine; D013556:Swine, Miniature; D014057:Tomography, X-Ray Computed; D019076:Transgenes; D014184:Transplantation, Homologous; D014945:Wound Healing
D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase
Adipose-derived stem cells; Baculovirus; Gene therapy; Massive bone defects; Minipig; Pre-clinical