Baculovirus is a promising gene delivery vector but its widespread application is impeded as it only mediates transient transgene expression in mammalian cells. To prolong the expression, we developed a dual baculovirus system whereby one baculovirus expressed FLP recombinase while the other harbored an Frt-flanking cassette encompassing the transgene and oriP/EBNA1 derived from Epstein-Barr virus. After cotransduction of cells, the expressed FLP cleaved the Frt-flanking cassette off the baculovirus genome and catalyzed circular episome formation, then oriP/EBNA1 within the cassette enabled the self-replication of episomes. The excision/recombination efficiency was remarkably enhanced by sodium butyrate, reaching 75% in human embryonic kidney-293 (HEK293) cells, 85% in baby-hamster kidney (BHK) cells, 77% in primary chondrocytes, and 48% in mesenchymal stem cells (MSCs). The hybrid baculovirus substantially prolonged the transgene expression to approximately 48 days without selection and >63 days with selection, thanks to the maintenance of replicons and transgene transcription. In contrast to the replicating episomes, the baculovirus genome was rapidly degraded. Furthermore, an osteoinductive growth factor gene was efficiently delivered into MSCs using this system, which not only prolonged the growth factor expression but also potentiated the osteogenesis of MSCs. These data collectively implicate the potential of this hybrid baculovirus system in gene therapy applications necessitating sustained transgene expression.

Baculovirus efficiently transduces human mesenchymal stem cells (hMSCs) and transplantation of hMSCs transduced with a bone morphogenetic protein 2-expressing baculovirus (Bac-CB) into nude mice results in ectopic bone formation. To attest the clinical potential of baculovirus in bone regeneration, hereby we explored whether the hMSCs genetically modified by Bac-CB were tolerant in immunocompetent rats and further healed the critical-sized calvarial bone defect. The histological and computed tomographic studies demonstrated that Bac-CB-engineered hMSCs promoted the cell differentiation and new bone formation in the immunocompetent rats. Immunohistochemical staining revealed that the transplanted human cells remained detectable at 1 and 4 weeks posttransplantation, attesting the immunoprevileged properties of hMSCs. In the recipients, the donor cells aggregated and appeared osteoblast like at later stages, which paralleled the infiltration of macrophages, CD3(+), and CD8(+) T cells into the graft. Administration of immunosuppressive drugs prolonged the cell survival and improved the bone regeneration, yet it failed to entirely abolish the immune response and complete the bone healing. Our data altogether implicate the potential of Bac-CB for hMSCs engineering and calvarial bone repair, but the use of hMSCs cannot overcome the immunological barrier.

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Transduction of partially dedifferentiated rabbit chondrocytes with a baculovirus (Bac-CB) expressing bone morphogenetic protein-2 (BMP-2) reverses dedifferentiation and enhances matrix production. Hereby we examined whether transduction with Bac-CB in combination with another baculovirus expressing transforming growth factor-beta1 (TGF-beta1) or insulin-like growth factor-1 (IGF-1) synergistically augmented chondrogenic differentiation. Passage 3 rabbit articular chondrocytes were transduced by different baculovirus combinations: single transduction with Bac-CB, cotransduction with Bac-CB and Bac-CT (expressing TGF-beta1), cotransduction with Bac-CB and Bac-CI (expressing IGF-1), and transduction with Bac-CB followed by repeated transduction with Bac-CT, Bac-CI, or Bac-CB 5 days later. Transduced cells were encapsulated into alginate beads for culture. Among these strategies, only cotransduction with Bac-CB and Bac-CT led to improved redifferentiation when compared with Bac-CB single transduction, as evidenced by the enhanced expression of aggrecan and collagen IIB (Col IIB), suppressed expression of Col I and Col X, emergence of chondrocyte-specific lacunae, and elevated deposition of matrix molecules. The cotransduction also accelerated the expression of Sox9, Col IIB, and aggrecan. In summary, baculovirus-mediated coexpression of TGF-beta1 and BMP-2 synergistically accelerates the chondrocyte redifferentiation process and improves the maintenance of chondrocyte phenotype and accumulation of cartilage-specific matrix molecules.

Co-conjugating chondroitin-6-sulfate (CSC) and dermatan sulfate (DS) to chitosan scaffolds improves chondrocyte differentiation and extracellular matrix (ECM) production. To further elucidate the cellular responses to CSC/DS conjugation, gene expression profiles for the rat chondrocytes cultured on the CSC/DS/chitosan and chitosan-only scaffolds were compared by reverse-transcription PCR (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR). Our data unraveled that the CSC/DS/chitosan scaffold resulted in low-level expression of collagen I, IIA and X and potentiated the aggrecan, collagen II (including collagen IIB) and TIMP3 expression, but downregulated the decorin expression. Therefore CSC/DS/chitosan scaffold maintained the chondrocyte differentiation while minimized de-differentiation and hypertrophy. Furthermore, CSC/DS conjugation affected the expression of 11 genes implicated in 9 signaling pathways (as unveiled by cDNA microarray) and upregulated the expression of TGF-beta1, Sox9, BMP2, PTHrP and Ihh (as confirmed by qRT-PCR). These data suggested that the CSC/DS/chitosan scaffold potentiated the TGF-beta and Hedgehog pathways, which activated the expression of PTHrP and its downstream Sox9. The signals were transduced to elevate the expression of aggrecan, collagen II and TIMP3, and contributed to the well-differentiated chondrocyte phenotype. Altogether, this study for the first time elucidated the roles of GAGs-conjugated biomaterials in matrix production and breakdown, cellular differentiation and signal transduction at the molecular levels.