Huang-Chi Chen

Baculovirus as a new gene delivery vector for stem cell engineering and bone tissue engineering

Baculovirus has emerged as a novel vector for in vitro and in vivo gene delivery due to its low cytotoxicity and non-replication nature in mammalian cells, but the applications of baculovirus in the genetic modification of human mesenchymal stem cells (hMSCs) and tissue engineering are yet to be reported. In this study, we genetically engineered hMSCs with a baculovirus (Bac-CB) expressing bone morphogenetic protein-2 (BMP-2). Bac-CB transduction of hMSCs at a multiplicity of infection of 40 triggered effective differentiation of hMSCs into osteoblasts. Supertransduction at day 6 after initial transduction enhanced the BMP-2 expression and further accelerated the in vitro osteogenesis, as confirmed by alkaline phosphatase assay, Alizarin red staining and reverse transcription-polymerase chain reaction analysis of osteoblastic genes. Implantation of the supertransduced cells at ectopic sites in the nude mice resulted in efficient cell differentiation into osteoblasts at week 2 and induced progressive mineralization and partial bone formation at week 6, as confirmed by hematoxylin and eosin, immunohistochemical and Alizarin red staining. These data collectively demonstrated, for the first time, the potential of baculovirus in hMSCs engineering and implicated its use in bone tissue engineering.

The repair of osteochondral defects using baculovirus-mediated gene transfer with de-differentiated chondrocytes in bioreactor culture

Baculovirus has emerged as a promising gene delivery vector. Hereby de-differentiated rabbit chondrocytes were transduced ex vivo with a recombinant baculovirus expressing BMP-2 (Bac-CB), seeded to scaffolds and cultured statically for 1 day (Bac-w0 group) or in a rotating-shaft bioreactor (RSB) for 1 week (Bac-w1 group) or 3 weeks (Bac-w3 group). Mock-transduced constructs were cultured statically for 1 day to serve as the control (Mock-w0 group). We unraveled that Bac-CB transduction and increasing culture time in the RSB yielded more mature cartilaginous constructs in vitro. Eight weeks after implanting into the rabbit osteochondral defects, Mock-w0 constructs failed to repair the lesion while Bac-w0 constructs resulted in augmented, yet incomplete, repair. Bac-w1 constructs yielded neocartilage layers rich in glycosaminoglycans and collagen II, but the integration between the graft and host cartilages was not complete. In contrast, Bac-w3 constructs led to the regeneration of hyaline cartilages as characterized by cartilage-like appearance, improved integration, chondrocytes clustered in lacunae, smooth and homogeneous matrix rich in collagen II and glycosaminoglycans but deficient in collagen I. In conclusion, combining baculovirus-modified de-differentiated chondrocytes and RSB culture creates constructs that repair osteochondral defects, and in vitro culture time dictates the construct maturation and subsequent in vivo repair.

Baculovirus transduction of human mesenchymal stem cell-derived progenitor cells: variation of transgene expression with cellular differentiation states.

We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem cells (MSCs). In this study, we further demonstrated, for the first time, that baculovirus can transduce adipogenic, chondrogenic and osteogenic progenitors originating from MSCs. The transduction efficiency (21–90%), transgene expression level and duration (7–41 days) varied widely with the differentiation lineages and stages of the progenitors, as determined by flow cytometry. The variation stemmed from differential transgene transcription (as revealed by real-time reverse transcription-polymerase chain reaction), rather than from variability in virus entry or cell cycle (as determined by quantitative real-time PCR and flow cytometry). Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic, osteogenic and chondrogenic pathways. The susceptibility to baculovirus transduction was higher for adipogenic and osteogenic progenitors, but was lower for chondrogenic progenitors. In particular, the duration of transgene expression was prolonged in the transduced adipogenic and osteogenic progenitors (as opposed to the MSCs), implicating the possibility of extending transgene expression via a proper transduction strategy design. Taken together, baculovirus may be an attractive alternative to genetically modify adipogenic and osteogenic progenitors in the ex vivo setting for cell therapy or tissue engineering.

A Novel Rotating-Shaft Bioreactor for Two-Phase Cultivation of Tissue-Engineered Cartilage

A novel rotating‐shaft bioreactor (RSB) was developed for two‐phase cultivation of tissue‐engineered cartilage. The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter × 3.5 mm thickness) were fixed and a reactor vessel half‐filled with medium. The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0–0.32 dyn/cm2 at 10 rpm), as revealed by computer simulation. Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 × 106 chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition. The 4‐week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type II collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage‐like constructs in the RSB cultures. In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading. With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage‐like constructs.

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

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 mock-transduced constructs in the RSB culture nor the Bac-CB-transduced 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 baculovirus-mediated 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.

Co-Conjugating Chondroitin-6-Sulfate/Dermatan Sulfate to Chitosan Scaffold Alters Chondrocyte Gene Expression and Signaling Profiles

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‐β1, Sox9, BMP2, PTHrP and Ihh (as confirmed by qRT‐PCR). These data suggested that the CSC/DS/chitosan scaffold potentiated the TGF‐β 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. Biotechnol. Bioeng. 2008;101: 821–830.

Baculovirus-mediated growth factor expression in dedifferentiated chondrocytes accelerates redifferentiation: effects of combinational transduction.

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.

Baculovirus transduction of chondrocytes elicits interferon-α/β and suppresses transgene expression

Baculovirus is an effective vector for gene delivery into primary chondrocytes and repeated baculovirus transduction (i.e. supertransduction) appears to be promising for prolonging transgene expression, but how supertransduction may influence baculovirus‐mediated gene delivery is unknown.

Eggshell as a Novel Biomimetic Reactor for in vitro Tissue Culture

In this study, a biomimetic eggshell reactor capable of highly efficient gas transfer was developed for in vitro tissue cultures. The reactor consisted of a natural eggshell (working volume≈50-60mL) for the containment of medium and a chamber-stirrer assembly for the containment of tissue constructs and for agitation. The pores on the eggshell allowed for gas transfer; thus, no air sparging was required. Compared with commercial spinner flasks, the eggshell reactor resulted in slightly better oxygen transfer rates at low agitation speeds, but significantly higher volumetric mass transfer coefficients at high agitation speeds. The highly efficient gas transfer in the eggshell reactor enabled the in vitro cultivation of actively growing E. coli and long-term cultivation of tissue engineered cartilage-like constructs. Taken together, our results implicate the potential application of this reactor as a low-cost alternative for cell and tissue cultures.