Regenerating Cartilages by Engineered ASCs: Prolonged TGF-β3/BMP-6 Expression Improved Articular Cartilage Formation and Restored Zonal Structure

Abstract

Adipose-derived stem cells (ASCs) hold promise for cartilage regeneration but their chondrogenesis potential is inferior. Here, we used a baculovirus (BV) system that exploited FLPo/Frt-mediated transgene recombination and episomal minicircle formation to genetically engineer rabbit ASCs (rASCs). The BV system conferred prolonged and robust TGF-β3/BMP-6 expression in rASCs cultured in porous scaffolds, which critically augmented rASCs chondrogenesis and suppressed osteogenesis/hypertrophy, leading to the formation of cartilaginous constructs with improved maturity and mechanical properties in 2-week culture. Twelve weeks after implantation into full-thickness articular cartilage defects in rabbits, these engineered constructs regenerated neocartilages that resembled native hyaline cartilages in cell morphology, matrix composition and mechanical properties. The neocartilages also displayed cartilage-specific zonal structures without signs of hypertrophy and degeneration, and eventually integrated with host cartilages. In contrast, rASCs that transiently expressed TGF-β3/BMP-6 underwent osteogenesis/hypertrophy and resulted in the formation of inferior cartilaginous constructs, which after implantation regenerated fibrocartilages. These data underscored the crucial role of TGF-β3/BMP-6 expression level and duration in rASCs in the cell differentiation, constructs properties and in vivo repair. The BV-engineered rASCs that persistently express TGF-β3/BMP-6 improved the chondrogenesis, in vitro cartilaginous constructs production and in vivo hyaline cartilage regeneration, thus representing a remarkable advance in cartilage engineering.

Characterization of Chondrogenic Gene Expression and Cartilage Phenotype Differentiation in Human Breast Adipose-Derived Stem Cells Promoted by Ginsenoside Rg1 In Vitro

“…Cell-based approaches for chondrogenesis and cartilage regeneration are widely considered to be the most effective, as they are able to efficiently sustain the physiological osteogenic process in vivo. Indeed, the most promising field for the application of ASCs is cartilage reconstruction/regeneration [42,43].…”

Gene therapy for chondral and osteochondral regeneration: is the future now?

“…For example, the induction of Stem Cells to chondrogenesis is often followed by osteogenesis and hypertrophy, and the number of chondrocytes or chondral progenitors that can be obtained is limited [101]. Recent works suggest that the combination of appropriate delivery vectors, genes, target cells and scaffolds might offer the possibility to obtain hyaline cartilage, even though the type of lesion (size, localization, structure) determines a variation of the combination of these factors [82].…”

“…This is observed for IGF-1, co-transfected with FGF-2, in osteochondral defects [16,18] and in MSCs [43], or with BMP2 and BMP7 in chondrocytes [20]. In addition, TGF-β, co-transfected with BMP2 or BMP6, is used to treat fullthickness defects through MSC infection [46,82].…”

“…antibiotic resistance) and consequently irradiate them to prevent further in vivo growth and minimize tumour development [70,81]. Finally, the viral vector least employed is baculovirus, a virus that infects insect cells, but that is also able to transduce numerous mammalian cells, including chondrocytes and ADSCs for TGF-β [82] and BMPs [82,83] in vitro and in vivo. However, this virus is not able to replicate and integrate its DNAs into the chromosomes of transduced mammalian cells, determining a transient transgene expression (<7 days).…”

“…The combination of gene therapy and scaffolds seems to greatly enhance both the efficiency and duration of transfected genes, leading to systems able to promote bone, cartilage, and osteochondral regeneration [22,23,26]. Scaffolds can be natural such as DBM, gelatine, alginate, fibrinogen and collagen based [16-18, 21, 27, 46, 52, 62, 71, 73, 84], or synthetic such as polyglycolic acid (PGA), polylactic acid (PLA) and poly(lactic-co-glycolide) (PLGA) [14,42,54,[82][83][84] (Table 1). Besides favouring cell engineering within the scaffold, their design can be used as guide for the correct integration and directional localization of cells in the defect.…”