Optimization of dynamic culture conditions: effects on biosynthetic activities of chondrocytes grown in collagen sponges.
Application of mechanical stimulation, using dynamic bioreactors, is considered an effective strategy to enhance cellular behavior in load-bearing tissues. In this study, two types of perfusion mode (direct and free flow) are investigated in terms of the biosynthetic activities of chondrocytes grown in collagen sponges by assessment of cell proliferation rate, matrix production, and tissue morphology. Effects of the duration of preculture and dynamic conditioning are further determined. Our results have demonstrated that both bovine and human-derived chondrocytes demonstrate a dose-dependent response to flow rate (0-1 mL/min) in terms of cell number and glycosaminoglycan (GAG) content. This may reflect the weak adhesion of cells to the sponge scaffolds and the immature state of the constructs even after 3 weeks of proliferative culture. Our studies define an optimal flow rate between 0.1 and 0.3 mL/min for direct perfusion and free flow bioreactors. Using fresh bovine chondrocytes and a lower flow rate of 0.1 mL/min, a comparison was made between free flow system and direct perfusion system. In the free flow bioreactor, no cell loss was observed and higher GAG production was measured compared with static cultured controls. However, as with direct perfusion, the enhancement effect of free flow perfusion was strongly dependent on the maturation and organization of the constructs before the stimulation. To address the maturation of the matrix, preculture periods were varied before mechanical conditioning. An increase in culture duration of 18 days before mechanical conditioning resulted in enhanced GAG production compared with controls. Interestingly, additional enhancement was found in specimens that were further subjected to a prolonged duration of perfusion (63% increase after an additional 4 days of perfusion) after prematuration. The free flow system has an advantage over the direct perfusion system, especially when using sponge scaffolds, which have lower mechanical properties; however, mass transfer of nutrients is still more optimal throughout the scaffolds in a direct perfusion system as demonstrated by histological analysis.
Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-Université Lyon I and IFR 128-Biosciences Lyon-Gerland, Lyon, France.
A-M
AM
Freyria
Y
Y
Yang
H
H
Chajra
C F
CF
Rousseau
M-C
MC
Ronzière
D
D
Herbage
A J
AJ
El Haj
D000818:Animals; D019149:Bioreactors; D002417:Cattle; D019902:Chondrocytes / Q000502:physiology*; D003094:Collagen; D006801:Humans; D046509:Tissue Culture Techniques / Q000295:instrumentation; D023822:Tissue Engineering / Q000295:instrumentation
D003094:Collagen