Adenoviral transduction is more efficient in alginate-derived chondrocytes than in monolayer chondrocytes.
Gene transfer into cultured chondrocytes by using adenoviral vectors has potential applications in treating cartilage disorders. The present study was undertaken to compare and optimize two chondrocyte culture conditions for adenoviral transduction efficacy by using primary human articular chondrocytes cultivated either directly in a monolayer condition or as outgrowths from alginate-stored chondrocyte cultures. Isolated primary chondrocytes from human articular cartilage were either immediately transduced with an EGFP (enhanced green fluorescent protein)-gene-bearing adenoviral vector (1,000 and 3,000 virus particles/cell) or cultured in alginate before transduction. Immunohistochemistry and flow cytometric analysis were employed to determine the expression of extracellular matrix proteins and of the alphavbeta5 integrin receptor involved in adenoviral cell entry. Monolayer chondrocytes exhibited moderate transduction rates (mean 22.2% and 46.9% EGFP-positive cells at 1,000 and 3,000 virus particles/cell by 72 h post-transduction), whereas alginate-derived chondrocytes revealed significantly higher transduction efficacies (95.7% and 99%). Both monolayer and alginate-derived chondrocytes expressed alphavbeta5 integrin, type II collagen and cartilage proteoglycans. The mean fluorescence intensity of type II collagen was significantly higher in the alginate-derived chondrocytes, whereas that of alphavbeta5 integrin was higher in the monolayer chondrocytes. Our results indicate that transduction efficacy is independent of alphavbeta5 integrin expression levels in chondrocytes. Moreover, adenoviral transduction of alginate-derived chondrocytes is more efficient than that for monolayer chondrocytes and may be a suitable tool to achieve sufficient numbers of transduced and differentiated chondrocytes for experimental applications and cartilage repair.
Oberholzer
A
A
Department of Trauma and Reconstructive Surgery, Charité Medical Schools Berlin, Campus Benjamin Franklin, FEM, Krahmerstrasse 6-10, Berlin, Germany.
John
T
T
Kohl
B
B
Gust
T
T
Müller
R D
RD
La Face
D
D
Hutchins
B
B
Zreiqat
H
H
Ertel
W
W
Schulze-Tanzil
G
G
D000256:Adenoviridae / Q000235:genetics*; D000464:Alginates / Q000378:metabolism*; D002356:Cartilage / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D024043:Collagen Type II / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors; D020723:Glucuronic Acid / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000378:metabolism; D019030:Receptors, Vitronectin / Q000378:metabolism; D014161:Transduction, Genetic
D000464:Alginates; D024043:Collagen Type II; D006603:Hexuronic Acids; D016023:Integrins; D019030:Receptors, Vitronectin; C485184:enhanced green fluorescent protein; C064660:integrin alphaVbeta5; D049452:Green Fluorescent Proteins; D020723:Glucuronic Acid