Modulation of collagen II fiber formation in 3-D porous scaffold environments Guak-Kim Tan a , Donna Lee M. Dinnes c , Justin J. Cooper-White a,b,⇑ a Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Bld 75, Cnr Cooper and College Rds, QLD 4072, Australia b School of Chemical Engineering, The University of Queensland, Bld 75, Cnr Cooper and College Rds, QLD 4072, Australia c Centre for Vascular Research, Faculty of Medicine, University of New South Wales, Kensington, NSW 2052, Australia article info Article history: Received 10 December 2010 Received in revised form 18 February 2011 Accepted 17 March 2011 Available online 23 March 2011 Keywords: Collagen II Fibrillogenesis EDAC/NHS crosslinking 2-D and 3-D environments Surface modification abstract Collagen II, a major extracellular matrix component in cartilaginous tissues, undergoes fibrillogenesis under physiological conditions. The present study explored collagen II fiber formation in solution and in two- (coverslip) and three-dimensional (scaffold) environments under different incubation conditions. These conditions include variations in adsorption buffers, the presence of 1-ethyl-3-(3-dimenthylamino- propyl) carbodiimide/N-hydroxysuccinimide crosslinker and the nature of the material surfaces. We extend our observations of collagen II fiber formation in two dimensions to develop an approach for the formation of a fibrillar collagen II network throughout surface-modified polylactide-co-glycolide por- ous scaffolds. Morphologically, the collagen II network is similar to that present in native articular carti- lage. Biological validation of the resultant optimized functional scaffold, using rat bone marrow-derived mesenchymal stem cells, shows appreciable cell infiltration throughout the scaffold with enhanced cell spreading at 24 h post-seeding. This economic and versatile approach is thus believed to have significant potential in cartilage tissue engineering applications. Crown Copyright Ó 2011 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. All rights reserved. 1. Introduction Collagen, a major structural protein of the extracellular matrix (ECM) in the human body, has been widely used in tissue engineer- ing for promoting cell adhesion, growth and differentiation [1,2]. To date, 28 types of collagens have been identified [3]. Fibrillar col- lagens, collagen I and II, are derived in vivo from a precursor named procollagen. As procollagen is secreted into the extracellu- lar space, it is converted to collagen via proteolytic cleavage of ami- no and carboxyl propeptides from both ends of the molecule. These molecules were subsequently self-assemble into a D-periodic banding pattern to form microfibrils and fibrils through hydropho- bic and electrostatic interactions, which are stabilized by covalent crosslinks [1,2,4]. In native tissue, fibrillar collagen is typically or- ganized in a three-dimensional (3-D) fiber network composed of closely packed, parallel aligned fibrils that vary in diameter from 30 to 300 nm [5]. This multistep process, termed collagen fibrillogenesis, also takes place in vitro. When subjected to physiological conditions, native and recombinant collagen II spontaneously self-assemble into short tapered tactoids with a broad range of diameters up to 2 lm [6,7]. Collagen fibrillogenesis can be regulated by a number of factors, including pH [8], temperature [9] and the presence of other macromolecules [6,10]. It is usually monitored in vitro using a solution turbidity assay, atomic force microscopy (AFM) or trans- mission electron microscopy (TEM) on 2-D surfaces. However, observations obtained from these assays may not accurately reflect what occurs in a 3-D porous scaffold environment, which is com- monly utilized for tissue engineering applications. Furthermore, many investigations have focused on in vitro reconstitution and structure of collagen I fibers with little attention to collagen II, which is predominantly found in the ECM of cartilaginous tissues and has potential applications for cartilage tissue engineering [11–13]. Crosslinking of collagen-based matrices using the water-soluble crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) has been reported as an effective means for further improv- ing their mechanical properties and resistance against enzymatic degradation [14,15], a critical consideration for biomedical implants. The addition of N-hydroxysuccinimide (NHS) to the reaction further enhances the coupling efficiency by minimizing hydrolysis of the unstable ester intermediates. EDAC contains hete- robifunctional groups that are able to form amide linkages between collagen molecules (Fig. 1) [16]. The pK a values for the carboxylic acid and amine functional groups of the collagen are 4.1 and 9.3, respectively [17]. In the pH 5.5 buffer that is typically used for EDAC/NHS crosslinking, both of the functional groups are in a dissociated state. 1742-7061/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. All rights reserved. doi:10.1016/j.actbio.2011.03.022 ⇑ Corresponding author at: Australian Institute for Bioengineering and Nano- technology (AIBN), The University of Queensland, Bld 75, Cnr Cooper and College Rds, QLD 4072, Australia. Tel.: +61 7 3346 3858; fax: +61 7 3346 3973. E-mail address: j.cooperwhite@uq.edu.au (J.J. Cooper-White). Acta Biomaterialia 7 (2011) 2804–2816 Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat