title: Gene Delivery of TGF-β3 and BMP2 in an MSC-Laden Alginate Hydrogel for Articular Cartilage and Endochondral Bone Tissue Engineering.
abstract: Incorporating therapeutic genes into three-dimensional 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 nonviral 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 transforming growth factor-beta 3 (pTGF-β3), bone morphogenetic protein 2 (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 were achieved. Confocal microscopy demonstrated that complexing the plasmid with nHA before 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 sulfated glycosaminoglycan and collagen production, particularly in the pTGF-β3-pBMP2 codelivery 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 codelivery group. In contrast, greater levels of calcium deposition were observed in the pTGF-β3- and pBMP2-only groups compared to codelivery, with a 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 toward either a chondrogenic or an osteogenic phenotype depending on whether TGF-β3 and BMP2 were delivered in combination or isolation.
authors.affiliation: 1 Trinity Centre for Bioengineering (TCBE), Trinity Biomedical Sciences Institute , Trinity College Dublin, Dublin, Ireland .|2 Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin , Dublin, Ireland .|3 Advanced Materials and Bioengineering Research Centre (AMBER), Royal College of Surgeons in Ireland and Trinity College Dublin , Dublin, Ireland .|4 Tissue Engineering Research Group (TERG), Department of Anatomy, Royal College of Surgeons in Ireland , Dublin, Ireland .
authors.forename: Tomas
authors.initials: T
authors.lastname: Gonzalez-Fernandez
authors.affiliation: 4 Tissue Engineering Research Group (TERG), Department of Anatomy, Royal College of Surgeons in Ireland , Dublin, Ireland .
authors.forename: Erica G
authors.initials: EG
authors.lastname: Tierney
authors.affiliation: 1 Trinity Centre for Bioengineering (TCBE), Trinity Biomedical Sciences Institute , Trinity College Dublin, Dublin, Ireland .|2 Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin , Dublin, Ireland .|3 Advanced Materials and Bioengineering Research Centre (AMBER), Royal College of Surgeons in Ireland and Trinity College Dublin , Dublin, Ireland .
authors.forename: Grainne M
authors.initials: GM
authors.lastname: Cunniffe
authors.affiliation: 1 Trinity Centre for Bioengineering (TCBE), Trinity Biomedical Sciences Institute , Trinity College Dublin, Dublin, Ireland .|3 Advanced Materials and Bioengineering Research Centre (AMBER), Royal College of Surgeons in Ireland and Trinity College Dublin , Dublin, Ireland .|4 Tissue Engineering Research Group (TERG), Department of Anatomy, Royal College of Surgeons in Ireland , Dublin, Ireland .
authors.forename: Fergal J
authors.initials: FJ
authors.lastname: O'Brien
authors.affiliation: 1 Trinity Centre for Bioengineering (TCBE), Trinity Biomedical Sciences Institute , Trinity College Dublin, Dublin, Ireland .|2 Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin , Dublin, Ireland .|3 Advanced Materials and Bioengineering Research Centre (AMBER), Royal College of Surgeons in Ireland and Trinity College Dublin , Dublin, Ireland .|4 Tissue Engineering Research Group (TERG), Department of Anatomy, Royal College of Surgeons in Ireland , Dublin, Ireland .
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Kelly
mesh_terms: D000464:Alginates / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000096:biosynthesis / Q000235:genetics; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D005269:Femur / Q000166:cytology / Q000378:metabolism; D018014:Gene Transfer Techniques; D020723:Glucuronic Acid / Q000737:chemistry / Q000494:pharmacology; D006603:Hexuronic Acids / Q000737:chemistry / Q000494:pharmacology; D020100:Hydrogels / Q000737:chemistry / Q000494:pharmacology; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D013552:Swine; D023822:Tissue Engineering; D053782:Transforming Growth Factor beta3 / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000464:Alginates; D055396:Bone Morphogenetic Protein 2; D006603:Hexuronic Acids; D020100:Hydrogels; D053782:Transforming Growth Factor beta3; D020723:Glucuronic Acid