Requirement for Serum in Medium Supplemented with Insulin-Transferrin-Selenium for Hydrodynamic Cultivation of Engineered Cartilage

Abstract

Achievement of viable engineered tissues through in vitro cultivation in bioreactor systems requires a thorough understanding of the complex interplay between hydrodynamic forces and biochemical cues such as serum. To this end, chondrocyte-seeded constructs were cultured under continuous fluid-induced shear forces with reduced serum content (0%-2%, v/v), which was partially or completely replaced by a potential substitute, insulin-transferrin-selenium, to minimize deleterious effects associated with the use of culture media containing high levels of serum (10%-20%). Low-serum cultures yielded constructs with similar biochemical properties to those cultivated with high-serum supplements, whereas the serum-free constructs exhibited poor cell proliferation, insufficient extracellular matrix production, and rapid degradation of and/or shear-induced damage to polyglycolic acid scaffolds. A fibrous outer capsule typically observed in hydrodynamic cultures and characterized by increased cell density and decreased (virtually none) glycosaminoglycan deposition was eliminated when serum concentration was equal to or <0.2% in the presence of hydrodynamic stimuli. Our findings suggest that serum is a requirement in insulin-transferrin-selenium-supplemented cultures in order for constructs to exhibit improved properties in response to hydrodynamic forces, and that mechanical and biochemical stimuli may synergistically modulate tissue properties and morphology through shear-responsive signals.

Differential Morphology and Homogeneity of Tissue-Engineered Cartilage in Hydrodynamic Cultivation with Transient Exposure to Insulin-Like Growth Factor-1 and Transforming Growth Factor-β1

“…45,46 We also reported that sufficient fibrosis-promoting factors are necessary for the capsule formation even in hydrodynamic cultivation. 18 However, the present study demonstrates that treatment with TGF-b1 eliminated the capsule in hydrodynamic constructs. One explanation is that elongated cells at the periphery of TGF-b1 constructs expressed a minimal level of TGF-bRII (Fig.…”

“…The dose of ITS (1%) employed here has previously been shown by our group to be essential to support the WWB cultivation of neocartilage in the presence of a minimal content of serum. 18 Taken together, the suppressed influence of continuous exposure to IGF-1 or TGF-b1 on construct development can be attributed to massive growth factor leftovers in the environments, which elicit a negative feedback response that inhibits cellular expression of specific surface receptors and probably accelerates subsequent catabolic activities. 19,40 Furthermore, transient exposure to IGF-1 or TGF-b1 is likely to maintain chondrocyte homeostasis, and thus promotes their anabolic functions under hydrodynamic stimuli.…”

“…18,42 TGF-b is thought to be one of the molecules capable of facilitating fibrosis of different tissues and organs. [42][43][44] A solid capsule is commonly recognized in cultures under hydrodynamic stimuli probably because chondrocytes located at the edge of constructs have a flattened, elongated shape, and thus become fibroblast-like 6 such that more collagen I, but less GAG are produced.…”

“…Polyglycolic acid (PGA)(Biomedical Structures, Warwick, RI) discs (Ø10 · 2 mm thick, 85 mg/mL bulk density) were sterilized by a series of rinses in sterile 70% ethanol and deionized water, followed by a 30-min exposure to ultraviolet light. 18 Two of the sterilized PGA discs were threaded onto each of the 21-gauge needles and separated by a silicone spacer, resulting in totally eight scaffolds per bioreactor. The WWBs were then filled with 120 mL of the Dulbecco's modified Eagle's medium (DMEM) and allowed to stabilize overnight with a stir bar spinning at 50 rpm inside a humidified, 37°C, 5% CO 2 incubator.…”

“…17 To resolve this issue, we recently developed a low-serum (2%) medium for the WWB cultivation of chondrocyte-seeded constructs that are morphologically and functionally similar to the high-serum samples. 18 Another solution to this problem could be the shortened supplementation of exogenous growth factors, which has recently been utilized to facilitate chondrogenesis of chondrocyte- 19,20 or mesenchymal stem cell-laden 21,22 hydrogels under static or dynamically compressive conditions. As Byers et al suggested, it may be that cultured cells require substantial time of *2 weeks to adapt themselves to signaling cascades resulting from growth factor removal from the surroundings before exhibiting stronger anabolic potential for ECM synthesis.…”