The effect of continuous culture on the growth and structure of tissue‐engineered cartilage

Abstract

The use of bioreactors for cartilage tissue engineering has become increasingly important as traditional batch-fed culture is not optimal for in vitro tissue growth. Most tissue engineering bioreactors rely on convection as the primary means to provide mass transfer; however, convective transport can also impart potentially unwanted and/or uncontrollable mechanical stimuli to the cells resident in the construct. The reliance on diffusive transport may not necessarily be ineffectual as previous studies have observed improved cartilaginous tissue growth when the constructs were cultured in elevated volumes of media. In this study, to approximate an infinite reservoir of media, we investigated the effect of continuous culture on cartilaginous tissue growth in vitro. Isolated bovine articular chondrocytes were seeded in high density, 3D culture on Millicell filters. After two weeks of preculture, the constructs were cultivated with or without continuous media flow (5-10 microL/min) for a period of one week. Tissue engineered cartilage constructs grown under continuous media flow significantly accumulated more collagen and proteoglycans (increased by 50-70%). These changes were similar in magnitude to the reported effect of through-thickness perfusion without the need for large volumetric flow rates (5-10microL/min as opposed to 240-800 microL/min). Additionally, tissues grown in the reactor displayed some evidence of the stratified morphology of native cartilage as well as containing stores of intracellular glycogen. Future studies will investigate the effect of long-term continuous culture in terms of extracellular matrix accumulation and subsequent changes in mechanical function.

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold‐free cartilage

“…Dynamic compression stimulates chondrocyte metabolism and increases matrix synthesis, thus improving the mechanical properties of tissue engineered cartilage (Chowdhury et al, 2003;Davisson et al, 2002a;LeBaron and Athanasiou, 2000;Lima et al, 2006;Mauck et al, 2002;Sah et al, 1989;Waldman et al, 2002). Perfusion has been linked to an accumulation of ECM in tissue engineered cartilage by inducing shear stress on constructs and allowing for constant nutrient supply and waste removal (Darling and Athanasiou, 2003;Davisson et al, 2002b;Khan et al, 2009). We expected to see an increase in biochemical and biomechanical properties between the static and perfused groups and further enhancement between the perfused and loaded groups.…”

Effects of Perfusion and Dynamic Loading on Human Neocartilage Formation in Alginate Hydrogels

“…Perfusion of medium has been used to enhance cell seeding of porous scaffolds, [17][18][19][20] and, in comparison with static cultures, perfusion can increase glycosaminoglycan (GAG) synthesis and deposition by chondrocytes and mesenchymal stem cells. 1,17,19,[21][22][23][24] Similar to compressive load, reports of reduced ECM accumulation have been observed as a consequence of perfusion, which appears to move newly synthesized proteins into the medium. 25,26 Few studies examined the combined effects of loading and medium perfusion.…”

“…A delayed loading regime has less effect on stimulating anabolic and catabolic gene expression in comparison to using an immediate application of an intermittent loading regime. However, Khan et al 21 showed that following 2 weeks of continuous perfusion of bovine articular chondrocytes increased collagen and proteoglycan syntheses. Our FS condition showed a more uniform aggrecan and link-protein deposition; thus, a preculture approach should be tested with the system used in this current study as a possible means to improve ECM accumulation.…”

Development of Scaffold-Free Elastic Cartilaginous Constructs with Structural Similarities to Auricular Cartilage

“…28,32,33 Briefly, the reactor consisted of multichannel vented polypropylene chambers to house single constructs (3 cm 2 containing a maximum media volume of 4 mL). A constant flow of fresh media (from aerated reservoirs) at 10 mL/min was provided by a peristaltic pump (Ismatec; Cole Parmer Canada, Anjou, Canada) with waste media collected in a vented reservoir.…”