Incorporating therapeutic genes into 3D biomaterials is a promising strategy for enhancing tissue regeneration. Alginate hydrogels have been extensively investigated for cartilage and bone tissue engineering, including as carriers of transfected cells to sites of injury, making them an ideal gene delivery platform for cartilage and osteochondral tissue engineering. The objective of this study was to develop gene-activated alginate hydrogels capable of supporting nanohydroxyapatite (nHA)-mediated non-viral gene transfer to control the phenotype of mesenchymal stem cells (MSCs) for either cartilage or endochondral bone tissue engineering. To produce these gene-activated constructs, MSCs and nHA complexed with plasmid DNA (pDNA) encoding for TGF-β3 (pTGF-β3), BMP2 (pBMP2), or a combination of both (pTGF-β3/pBMP2), were encapsulated into alginate hydrogels. Initial analysis using reporter genes showed effective gene delivery and sustained overexpression of the transgenes was achieved. Confocal microscopy demonstrated that complexing the plasmid with nHA prior to hydrogel encapsulation led to transport of the plasmid into the nucleus of MSCs, which did not happen with naked pDNA. Gene delivery of TGF-β3 and BMP2 and subsequent cell-mediated expression of these therapeutic genes resulted in a significant increase in sGAG and collagen production, particularly in the pTGF-β3/pBMP2 co-delivery group in comparison to the delivery of either pTGF-β3 or pBMP2 in isolation. In addition, stronger staining for collagen type II deposition was observed in the pTGF-β3/pBMP2 co-delivery group. In contrast, greater levels of calcium deposition were observed in the pTGF-β3 and pBMP2 only groups compared to co-delivery, with strong staining for collagen type X deposition, suggesting these constructs were supporting MSC hypertrophy and progression along an endochondral pathway. Together these results suggest that the developed gene-activated alginate hydrogels were able to support transfection of encapsulated MSCs and directed their phenotype towards either a chondrogenic or osteogenic phenotype depending on whether TGF-β3 and BMP2 were delivered in combination or in isolation.
Royal College of Surgeons in Ireland ->