Effect of expansion medium on ex vivo gene transfer and chondrogenesis in type II collagen-glycosaminoglycan scaffolds in vitro.
OBJECTIVE
Growth factors can profoundly affect the behaviour of chondrocytes during expansion and subsequent growth in three-dimensional (3-D) scaffolds. Prolonging such effects has stimulated investigation of the transfer of growth factor genes to chondrocytes. This study evaluated the effects of the monolayer expansion medium on the proliferation and cartilage matrix molecule synthesis of chondrocytes in 3-D pellet culture and in type II collagen-glycosaminoglycan (CG) scaffolds, and on ex vivo insulin-like growth factor-1 (IGF-1) gene transfer to articular chondrocytes in monolayer. The possibility of transfecting cells in 3-D culture using CG scaffolds was also investigated and the resulting effect of IGF-1 overexpression on glycosaminoglycan (GAG) biosynthesis in 3-D culture was assessed.


METHODS
Two expansion media were compared-one that has been widely used for growing chondrocytes (Medium 1) and one that has been found to increase chondrocyte proliferation rates and preserve the redifferentiation potential of monolayer-expanded chondrocytes when subsequently placed in pellet cultures (Medium 2). Chondrocytes were expanded in monolayer culture and then 1) redifferentiated in 3-D culture, or 2) infected with the IGF-1 gene in monolayer or in type II CG scaffolds.


RESULTS
The cell count for first passage chondrocytes was more than 3-fold higher when using Medium 2. In 3-D culture, cells expanded with Medium 2 and seeded in CG scaffolds produced more total GAG/DNA and displayed more intense immunohistochemical staining for collagen type II. Gene transfer and IGF-1 release kinetics from infected cells in monolayer were significantly affected by the composition of the expansion medium, the gene transfer method and time. IGF-1 gene transfer in CG scaffolds resulted in a 35-fold elevation in accumulated IGF-1 released from transfected Medium 2-expanded chondrocytes over controls, and resulted in a 40% increase in accumulated GAG/DNA.


CONCLUSIONS
The composition of the expansion medium significantly affects monolayer proliferation of adult canine chondrocytes, GAG synthesis when the cells are subsequently grown in CG scaffolds, and ex vivo IGF-1 gene transfer.
Capito
R M
RM
Tissue Engineering, VA Boston Healthcare System, Boston, MA 02130, USA.
Spector
M
M
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism; D049109:Cell Proliferation; D048430:Cell Shape; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D020219:Chondrogenesis / Q000502:physiology*; D024043:Collagen Type II / Q000378:metabolism; D003470:Culture Media; D004285:Dogs; D018014:Gene Transfer Techniques; D006025:Glycosaminoglycans / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000235:genetics; D023822:Tissue Engineering / Q000379:methods*
D024043:Collagen Type II; D003470:Culture Media; D006025:Glycosaminoglycans; D007334:Insulin-Like Growth Factor I