In vitro osteogenic induction of bone marrow stromal cells with encapsulated gene-modified bone marrow stromal cells and in vivo implantation for orbital bone repair.
Osteogenic induction with either growth factors or genetic modification has limitations due to the short half-life and cost of the former, or safety concerns regarding the latter. The objective of this study was to employ a microcapsulation technique to separate genetically modified and nonmodified bone marrow stromal cells (BMSCs) to establish a cost-effective and biosafe osteogenic induction methodology with functional evaluation in vitro and in vivo in a canine model. Autologous BMSCs were isolated and transduced with adenoviral vectors containing either BMP-2 or vascular endothelial growth factor (VEGF) or were dual transduced followed by encapsulation in alginate microcapsules using an electrostatic bead generator. After cocultured with encapsulated cells, normal autologous BMSCs were analyzed for osteogenic differentiation and seeded onto tricalcium phosphate (TCP) scaffolds for in vivo implantation to repair orbital wall bone defects (12 mm in diameter) in a canine model. In vitro assays showed that the expression of the transduced genes was significantly upregulated, with significantly more transduced proteins released from the transduced cells compared with control cells. Importantly, examination of the BMSCs induced by soluble factors released from the encapsulated cells revealed a significant upregulation of expression of osteogenic markers Runx2, BSP, OPN, and OCN in dual-transduction or induction groups. In addition, dual transduction and induction resulted in the highest increase of alkaline phosphatase activity and mineralization compared with other experimental groups. In vivo assays using CT, micro-CT, and histology further supported the qPCR and western blot findings. In conclusion, encapsulation of genetically modified BMSCs was able to release a sufficient amount of BMP-2 and VEGF, which effectively induced osteogenic differentiation of normal-cultured BMSCs and demonstrated bone repair of the orbital wall defect after implantation with β-TCP in vivo.
Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, People's Republic of China .
Yuan
Y
Deng
Huifang
H
Zhou
Chenxi
C
Yan
Yefei
Y
Wang
Caiwen
C
Xiao
Ping
P
Gu
Xianqun
X
Fan
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001861:Bone Regeneration / Q000187:drug effects; D002130:Calcium Phosphates / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002463:Cell Membrane Permeability / Q000187:drug effects; D018914:Cells, Immobilized / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018920:Coculture Techniques; D004285:Dogs; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D009915:Orbit / Q000000981:diagnostic imaging / Q000473:pathology*; D010012:Osteogenesis / Q000187:drug effects; D019919:Prosthesis Implantation; D014057:Tomography, X-Ray Computed; D014161:Transduction, Genetic; D055114:X-Ray Microtomography
D002130:Calcium Phosphates; C485817:beta-tricalcium phosphate; D000469:Alkaline Phosphatase