In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects.
To examine a retroviral gene transfer to chondrocytes in vitro and in vivo in tissue-engineered cell-collagen constructs articular chondrocytes from rabbits and humans were isolated and transduced with VSV.G pseudotyped murine leukemia virus-derived retroviral vectors. Viral supernatants were generated by transient transfection of 293T cells using the pBullet retroviral vector carrying the nlslacZ gene, a Moloney murine leukemia virus gag/pol plasmid and a VSV.G coding plasmid. Transduction efficiency was analyzed by fluorescence-activated-cell-sorter analysis and transduced autologous chondrocytes from rabbits were seeded on collagen-scaffolds and implanted into osteochondral defects in the patellar groove of the rabbit's femur (n=10). LacZ-expression was analyzed by X-gal staining on total knee explants and histological sections. Retroviral transduction efficiency exceeded 92.3% (SEM+/-3.5%) in rabbit articular chondrocytes, 74.7% (SEM+/-1.8%) in human articular chondrocytes and 52.7% (SEM+/-5.8%) in osteoarthritic human chondrocytes. Reporter gene expression remained high after 15 weeks in 75.7% (SEM+/-8.2%) of transduced rabbit articular chondrocytes. In vivo, intraarticular beta-galactosidase activity could be determined in the majority of implanted chondrocytes in the osteochondral defects after 4 weeks.
Ueblacker
Peter
P
Department of Trauma, Hand and Reconstructive Surgery, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Wagner
Bettina
B
Vogt
Stephan
S
Salzmann
Gian
G
Wexel
Gabi
G
Krüger
Achim
A
Plank
Christian
C
Brill
Thomas
T
Specht
Karin
K
Hennig
Tilla
T
Schillinger
Ulrike
U
Imhoff
Andreas B
AB
Martinek
Vladimir
V
Gansbacher
Bernd
B
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology; D018929:Cell Culture Techniques; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D024042:Collagen Type I / Q000378:metabolism*; D005260:Female; D005269:Femur; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D020007:Implants, Experimental; D007668:Kidney / Q000166:cytology; D007763:Lac Operon; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D011817:Rabbits; D012190:Retroviridae / Q000235:genetics; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods; D014161:Transduction, Genetic; D014182:Transplantation, Autologous; D001616:beta-Galactosidase / Q000235:genetics
D024042:Collagen Type I; D001616:beta-Galactosidase