ADENOVIRAL BMP-2 GENE TRANSFER INTO MESENCHYMAL STEM CELLS FOR BONE FORMATION (2003)

Ex vivo gene transfer of osteogenic factors into multipotential stem cells offers potentially important therapeutic implications in a variety of musculoskeletal diseases. One possible approach is the development of a cellular vehicle, namely bone morphogenetic protein (BMP)-producing bone marrow cells, created using adenoviral gene transfer. These transduced cells provide local delivery of BMP for bone formation. The aims of this study were to study the feasibility of gene transfer to human bone osteoprogenitor cells, using adenoviral vectors. Specifically, the aims were to study the efficacy of transduction with an adenoviral vector expressing BMP-2 and then to determine the ability of the transduced cells to produce active BMP-2 and to generate bone ex vivo. Primary human bone marrow osteoprogenitor cells were expanded in culture and infected with AxCALacZ, a replication-deficient adenoviral vector carrying the E. coli lacZ gene, with a range of multiplicity of infection (MOI) of 6.25 to100. Transduced cells showed positive staining for β-galactosidase using X-Gal with an efficiency close to 100%. Uninfected cells showed no β-galactosidase activity. Efficiency was independent from MOI, however cells infected at the lower MOIs expressed lower levels of β-galactosidase. Following confirmation that primary bone marrow cells could be infected by adenoviral constructs, additional osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene, at a multiplicity of infection of 10–20. In order to determine BMP-2 activity, conditioned media from bone marrow cells expressing BMP-2 was added to promyoblast C 2 C 12 cells. The promyoblast C 2 C 12 cells are exquisitely sensitive to BMP-2 with induction of alkaline phosphatase activity (ED 50 20 nM) in a dose-dependant manner. Alkaline phosphatase activity was induced following culture with conditioned media from BMP-2 expressing cells, in a dose dependant manner, confirming successful secretion of active BMP-2. Immunohistochemical staining for alka- line phosphatase in C 2 C 12 cells also confirmed the bio-chemical observations. Media from uninfected control human bone marrow cells failed to produce a similar effect. The concentration of BMP-2 in the media was estimated to be 5–10 nM/10 7 cells. To examine whether adenoviral transfection affected the osteoblast phenotype and their ability to mineralise in vitro, adenovirally-transduced bone marrow cells expressing BMP-2 were seeded onto poly(-lactic acid co ÷glycolic acid) (75:25) porous scaffolds (provided by K. Shakesheff and S. Howdle; Nottingham University) and cultured for up to 6 weeks. Expression of alkaline phosphatase activity, type I collagen formation, as well as the synthesis of osteoblast stimulating factor-1 confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks. These results indicate the ability to deliver active BMP-2 using human bone marrow osteoprogenitor cells following adenoviral infection. The maintenance of osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs offers a realistic approach to tissue engineer bone for orthopaedic applications.