Non-enzymatic Glycation of Chondrocyte-Seeded Collagen Gels for Cartilage Tissue Engineering Rani Roy, 3 Adele L. Boskey, 1 Lawrence J. Bonassar 2,3 1 Musculoskeletal Integrity Program, Hospital for Special Surgery, New York, New York, 2 Department of Biomedical Engineering, Cornell University, Ithaca, New York, 3 Sibley School of Mechanical and Aerospace Engineering, Cornell University, 218 Upson Hall, Ithaca, New York 14853 Received 25 October 2007; accepted 11 February 2008 Published online 12 May 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20662 ABSTRACT: Collagen glycated with ribose (250 mM) in solution (pre-glycation) and as a gel (post-glycation) was seeded with chondrocytes and the effects of glycation on chondrocyte matrix assembly in culture were determined. Pre-glycation enhanced GAG accumulation significantly over controls at both 2 and 4 weeks (p < 0.05), although at both time points there were no statistical differences in cell number between pre-glycated and control gels. The increased proteoglycan accumulation was shown to be in part due to significantly increased GAG retention by the pre-glycated constructs (p < 0.05). Total collagen content in these pre-glycated gels was also significantly higher than unglycated gels at 4 weeks (p < 0.05). With post-glycation of collagen gels, chondrocyte number and GAG accumulation were all significantly lower than controls (p < 0.05). Post-glycation also inhibited GAG retention by the constructs (p < 0.05). Given these results, pre-glycation may be an improved processing method for collagen gels for tissue engineering techniques. ß 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1434– 1439, 2008 Keywords: cartilage; tissue engineering; nonenzymatic glycation; collagen type I In tissue regeneration techniques, the delivery of cells to the site of cartilage defects has been shown to enhance tissue repair and growth. 1,2 The injectable delivery of chondrocytes to defects in articular cartilage was first shown to be a method to increase the healing response in the early 1970s. 3 Since then, a number of non- invasive, injectable delivery methods have been used to support chondrocyte growth and integration with the surrounding native tissue. Although some techniques involve seeding cells under a physical barrier, such as a periosteal flap, 4 others use an injectable scaffold that may form a solid in vivo. For cartilage tissue engineering, these scaffolds include polymers such as alginate, 5 fibrin glue, 6,7 polyethylene oxide, 8 and PLG microspheres. 9 Central to these injectable techniques are the requirements that these materials have a low viscosity for homogeneous delivery of cells, are bio- compatible, act as a template for cartilage repair by providing some initial mechanical support, and support chondrocyte growth and biosynthesis. Collagen gels have not been previously used for injectable for cartilage tissue engineering, but have been proven as a useful injectable dermal filler, 10,11 and have been propos- ed as an injectable for other tissue engineering applications. 12–15 Type I collagen gels have been shown to be a viable scaffold for tissue-engineered cartilage constructs grown in vitro and when implanted into an in vivo surface, focal, and critical size defects in rabbit articular cartilage. 16–19 The main limitation of collagen gels as a scaffold for the tissue engineering of cartilage is that they may not provide sufficient structural support due to their lack of stiffness. Although a number of crosslinkers have been used for increasing the mechanical properties of these gels, the majority of these agents are cytotoxic or may induce an immune response when constructs are implanted in vivo. 20–22 Non-enzymatic glycation, the crosslinking of proteins via reducing sugars, has been shown to change the mechanical properties of proteins both in vivo and in vitro, 23–29 and is a recognized method for in situ processing of cell-seeded tissue constructs. 23,30 This mechanism of crosslinking has been developed to process collagen gels both in their solid state and in solution. 31 Both of these processing methods result in addition of ribose to the collagen gels, accumulation of advanced glycation endproducts (AGEs), and in- creased compressive modulus of the gels. 31 Chondrocytes have been shown to regulate activities such as proliferation and matrix biosynthesis due to interactions with the surrounding matrix. 32,33 Because glycation changes the mechanical and biochemical proper- ties of the type I collagen scaffold, it may also affect chondrocyte matrix assembly in culture. The objective of this study was to characterize effects of glycation on the assembly of extracellular matrix by chondrocytes in vitro. Chondrocytes were grown in glycated type I collagen gels for 2 and 4 weeks, and the effects of glycation on ex- tracellular matrix (ECM) accumulation, cell proliferation, and mechanical properties of the constructs were inves- tigated. Two different methods of glycation were used— glycation in solution (pre-glycation) and glycation in the solid state of the gel (post-glycation)—with the hypothesis that these two processing methods affect chondrocyte biosynthesis differently. MATERIALS AND METHODS Chondrocyte Isolation and Gel Culture Cartilage was harvested from the diarthroidal joints of 4–6- week-old calves and digested with 0.3% collagenase (Wor- thington Biochemical, Lakewood, NJ) in DMEM (Invitrogen, Carlsbad, CA) with 100 units/mL of penicillin, 100 mg/mL of streptomycin, and 25 ng/mL of amphotericin B for 14 h at 378C. This solution was then strained through a 100-mm cell strainer (Fisher Scientific, Pittsburgh, PA) to obtain chondro- cytes. Chondrocytes were washed with PBS three times prior to resuspension in DMEM supplemented with 10% FBS 1434 JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2008 Correspondence to: Lawrence J. Bonassar (T: 607-255-9381; F: 607- 255-1222; E-mail: lb244@cornell.edu) ß 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.