Combination of baculovirus-expressed BMP-2 and rotating-shaft bioreactor culture synergistically enhances cartilage formation

Abstract

Baculovirus is an emerging gene delivery vector, thanks to a number of unique advantages. Herein, we genetically modified the rabbit articular chondrocytes with a recombinant baculovirus (Bac-CB) encoding bone morphogenetic protein-2 (BMP-2), which conferred high level BMP-2 expression and triggered the re-differentiation of dedifferentiated third passage (P3) chondrocytes in the monolayer culture. The transduced and mock-transduced P3 cells were seeded into porous scaffolds and cultured in either the dishes or the rotating-shaft bioreactor (RSB), a novel bioreactor imparting a dynamic, two-phase culture environment. Neither mocktransduced constructs in the RSB culture nor the Bac-CBtransduced constructs in the static culture grew into uniform cartilaginous tissues. Only the Bac-CB-transduced constructs cultured in the RSB for 3 weeks resulted in cartilaginous tissues with hyaline appearance, uniform cell distribution, cartilage-specific gene expression and considerably enhanced cartilage-specific extracellular matrix deposition, as determined by histological staining, reverse transcription-PCR analyses and biochemical assays. This is the first study demonstrating that combination of baculovirusmediated growth factor expression and RSB culture synergistically enhanced in vitro creation of cartilaginous tissues from dedifferentiated chondrocytes. Since baculovirus transduction is generally considered safe, this approach represents a viable alternative to stimulate the formation of engineered cartilage in a more cost-effective way than the growth factor supplementation.

Development of hybrid baculovirus vectors for artificial MicroRNA delivery and prolonged gene suppression

“…Since overexpression of the pro-inflammatory cytokine TNF-a contributes to chronic inflammation in the joint and ultimately to cartilage degeneration and osteoarthritis (Ghivizzani et al, 2008), the effective TNF-a knockdown implicates the feasibility of this baculovirus miRNA shuttle to treat chronic inflammatory diseases such as osteoarthritis. Given that baculovirus efficiently transduces cells within the joint including chondrocytes (Chen et al, 2008) and synoviocytes (Fig. 3A), one may inject the recombinant baculovirus into the joint for sustained miRNA expression and TNF-a inhibition to alleviate the symptom (Khoury et al, 2010).…”

“…Finally, recombinant baculovirus can be readily constructed and propagated to high titers in biosafety level 1 facilities by infecting its natural host insect cells . These attributes have prompted researchers to employ baculovirus for a plethora of applications, ranging from protein production (Liu et al, 2010), virus vector production (Huang et al, 2007;Lesch et al, 2008Lesch et al, , 2011, surface display of proteins (Grabherr and Ernst, 2010), delivery of vaccine immunogens Madhan et al, 2010), cancer therapy (Suzuki et al, 2010;Wang and Balasundaram, 2010), cell-based assay development (Kost et al, 2010) to tissue regeneration (Chen et al, 2008;Chuang et al, 2007;Lin et al, 2010). One disadvantage of baculovirus, however, is that it only mediates transient expression, thus impeding its application in complications requiring long-term transgene expression.…”

Defective Antiviral Responses of Induced Pluripotent Stem Cells to Baculoviral Vector Transduction

“…Additionally, BV effectively transduces chondrocytes (12), human (20) and rabbit (27) mesenchymal stem cells (MSCs), and adult and embryonic stem cells (16,50) as well as neural stem cells derived from iPSCs (26). Transduction of MSCs and chondrocytes with BV expressing growth factors stimulates cellular differentiation in vitro.…”

Concanavalin a affinity chromatography for efficient baculovirus purification

“…These features have inspired expanding baculovirus applications to eucaryotic protein display, 4,5 cell-based assay development, 6,7 virus vector production, 8,9 stem cell engineering, 10,11 and tissue engineering. 12,13 Furthermore, baculovirus has been employed for in vivo gene delivery, 14,15 cancer therapy, 16 and has emerged as a novel vaccine vector for antigen display 17,18 or in vivo antigen expression. 19 The wide spectrum of in vitro and in vivo applications entails the development of a powerful method for baculovirus purification.…”