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dc.contributor.authorLi, Ying
dc.contributor.authorTew, Simon R
dc.contributor.authorRussell, Amanda M
dc.contributor.authorGonzalez, Karin R
dc.contributor.authorHardingham, Timothy E
dc.contributor.authorHawkins, Robert E
dc.date.accessioned2009-10-19T15:57:25Z
dc.date.available2009-10-19T15:57:25Z
dc.date.issued2009-10-19T15:57:25Z
dc.identifier.citationTransduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9., 10 (3-4):575-84 Tissue Eng.en
dc.identifier.issn1076-3279
dc.identifier.pmid15165474
dc.identifier.doi10.1089/107632704323061933
dc.identifier.urihttp://hdl.handle.net/10541/84455
dc.description.abstractChondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.
dc.language.isoenen
dc.subject.meshAdenoviridae
dc.subject.meshAlginates
dc.subject.meshCell Division
dc.subject.meshChondrocytes
dc.subject.meshCollagen Type II
dc.subject.meshGenes, Reporter
dc.subject.meshGenetic Vectors
dc.subject.meshGlucuronic Acid
dc.subject.meshHexuronic Acids
dc.subject.meshHigh Mobility Group Proteins
dc.subject.meshHumans
dc.subject.meshLentivirus
dc.subject.meshMicrospheres
dc.subject.meshSOX9 Transcription Factor
dc.subject.meshTranscription Factors
dc.subject.meshTransduction, Genetic
dc.titleTransduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9.en
dc.contributor.departmentUK Centre for Tissue Engineering, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.en
dc.identifier.journalTissue Engineeringen
html.description.abstractChondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.


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