Abstract
To assess the potential of baculovirus as a gene delivery vector for cartilage tissue engineering, a recombinant baculovirus expressing enhanced green fluorescent protein (EGFP) was constructed to transduce rat articular chondrocytes and human mesenchymal stem cells (MSCs). Using the traditional transduction protocol (incubating the cells with concentrated virus at a multiplicity of infection (MOI) 200 for 1 h), the gene delivery efficiency into chondrocyte was low (<5 %). Therefore, a modified protocol was adopted (incubating cells with unconcentrated virus for 8 h with PBS as surrounding solution), which markedly enhanced the efficiency (88 %) and prolonged the duration of expression (21 days). Using the transduction condition we developed, MSCs were transduced at efficiencies up to 70 %. The elevated efficiency, gene expression level and duration correlated well with the increased virus uptake upon extended incubation time at 25 C. Although the virus transduction somewhat hindered the cell proliferation due to EGFP overexpression and the accompanying metabolic burden, the growth rate could restore when the burden was relived in the long-term culture. More importantly, the virus transduction imposed no perceptible cytotoxicity and the chondrocytes could secrete articular cartilage-specific type II collagen and glycosaminoglycan as well as mock-transduction cells did. Additionally, baculovirus-transduced MSC remained capable of differentiating into adipocytes. Furthermore, baculovirus could transduce MSC progenitor cells differentiating into different lineages (adipogenic, chondrogenic and osteogenic) at different differentiation stages (1, 2, 3 and 4 weeks post-induction). For MSC induced into adipogenic pathway, the transduction at 1 week post-induction resulted in an efficiency of up to 90 % and a duration of transgene expression for up to 41 days. Notably, the transduction efficiencies and transgene expression durations varied with the differentiation lineage and differentiation stages. The variation in transgene expression was independent of the cell cycle and virus entry, but resulted from the differential efgp transcription in the progenitors differentiating along different pathways. The differentiation capacities of MSC progenitors were not influenced by baculovirus transduction, as demonstrated by histochemistry staining and reverse transcription PCR (RT-PCR). These data collectively confirmed that baculovirus enables highly efficient gene transfer into rat articular chondrocytes and human MSCs, but does not impair the normal proliferation and differentiation capability of the transduced cells, suggesting that baculovirus expressing growth factors may be used to genetically modify, and modulate the differentiation status of chondrocytes and MSCs.