title: Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.
abstract: The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering.
authors.affiliation: Departments of Orthopaedic Surgery and Cell Biology, Duke University Medical Center, Durham, NC 27710.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Brunger
authors.forename: Nguyen P T
authors.initials: NP
authors.lastname: Huynh
authors.forename: Caitlin M
authors.initials: CM
authors.lastname: Guenther
authors.forename: Pablo
authors.initials: P
authors.lastname: Perez-Pinera
authors.forename: Franklin T
authors.initials: FT
authors.lastname: Moutos
authors.forename: Johannah
authors.initials: J
authors.lastname: Sanchez-Adams
authors.forename: Charles A
authors.initials: CA
authors.lastname: Gersbach
authors.forename: Farshid
authors.initials: F
authors.lastname: Guilak
mesh_terms: D000704:Analysis of Variance; D001696:Biomechanical Phenomena; D002454:Cell Differentiation / Q000502:physiology*; D020219:Chondrogenesis / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D005109:Extracellular Matrix / Q000502:physiology*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D011091:Polyesters; D011107:Polylysine; D044968:Regenerative Medicine / Q000379:methods; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000821:virology*; D014161:Transduction, Genetic / Q000379:methods*; D053782:Transforming Growth Factor beta3 / Q000235:genetics
chemical_list: D017931:DNA Primers; D011091:Polyesters; D053782:Transforming Growth Factor beta3; C016240:polycaprolactone; D011107:Polylysine
keywords: biomaterials; chondrocyte; gene therapy; genetic engineering; regenerative medicine