ABSTRACT
The purpose of the presented work is to examine the response of engineered cartilage to a transient, 2-week application of anabolic growth factors compared to continuous exposure in in vitro culture. Immature bovine chondrocytes were suspended in agarose hydrogel and cultured for 28 days (Study 1) or 42 days (Study 2) in chondrogenic media with TGF-ß1, TGF-ß3, or IGF-I either added for only the first 14 days in culture or added to the media for the entire study period. In both studies, there were no statistical differences in tissue mechanical or biochemical properties between the growth factors on day 14. In Study 1, growth factor removal led to a significant and drastic increase in Young's modulus and glycosaminoglycans content compared to continuously exposed controls on day 28. In Study 2, both TGF-ß1 and ß3 led to significantly higher mechanical properties and collagen content vs. IGF-I on day 42. These results indicate that the rapid rise in tissue properties (previously observed with TGF-ß3 only) is not dependent on the type but rather the temporal application of the anabolic growth factor. These findings shed light on possible techniques to rapidly develop engineered cartilage tissue for the future treatment of osteoarthritis.
Subject(s)
Cartilage, Articular , Chondrocytes , Intercellular Signaling Peptides and Proteins/metabolism , Tissue Engineering/methods , Animals , Cartilage, Articular/cytology , Cartilage, Articular/metabolism , Cattle , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Chondrogenesis/physiology , Collagen/analysis , Collagen/metabolism , Compressive Strength , Elastic Modulus , Glycosaminoglycans/analysis , Glycosaminoglycans/metabolism , Hydrogel, Polyethylene Glycol Dimethacrylate , Insulin-Like Growth Factor I/metabolism , Sepharose , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta3/metabolismABSTRACT
Media supplementation with collagen hydrolysate was hypothesized to increase the collagen content in engineered cartilage. By d28, hydrolysate at 0.5 mg/mL increased type II collagen content and 1 mg/mL increased mechanical properties, total collagen content, and type II collagen content over controls. By d42, however, controls possessed the highest GAG content and compressive Young's modulus. Real-time PCR found that 1 mg/mL increased type II collagen gene expression in d0 constructs, but increased MMP expression with no effect on type II collagen on d28. A 10 mg/mL concentration produced the lowest tissue properties, the lowest type II collagen gene expression on d0, and the highest MMP gene expression on d28. These results indicate that the duration of culture modulates the response of chondrocytes to collagen hydrolysate in 3D culture, transforming the response from positive to negative. Therefore, collagen hydrolysate as a media supplement is not a viable long-term method to improve the collagen content of engineered cartilage tissue.