Biomaterials ] (]]]]) ]]]–]]] Crosslinked hyaluronic acid hydrogels: a strategy to functionalize and pattern Tatiana Segura a , Brian C. Anderson a , Peter H. Chung b , Rebecca E. Webber c , Kenneth R. Shull c , Lonnie D. Shea a,b, * a Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, E156 Evanston, IL 60208-3120, USA b Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, E156 Evanston, IL 60208-3120, USA c Department of Materials Science and Engineering, Northwestern University, 2145 Sheridan Road, E156 Evanston, IL 60208-3120, USA Received 17 February 2004; accepted 20 February 2004 Abstract The physiological activity of hyaluronic acid (HA) polymers and oligomers makes it a promising material for a variety of applications. The development of HA–hydrogel scaffolds with improved mechanical stability against degradation and biochemical functionality may enhance their application to tissue engineering. In this report, a crosslinking strategy targeting the alcohol groups via a poly(ethylene glycol) diepoxide crosslinker was investigated for the generation of degradable HA hydrogels. To provide support for cell adhesion in vitro, collagen was incorporated into the HA solution prior to the crosslinking process. The hydrogels have a continuous exterior and a porous interior, with pore diameters ranging from 6 to 9 mm. HA and HA–collagen hydrogels degrade in the presence of hyaluronidase and collagenase enzymes, indicating that the chemical modification does not prevent biodegradation. Complete degradation of the hydrogels occurred within 14 days in hyaluronidase (100U/ml) and 3 days in collagenase (66U/ml). Pattern transfer was employed to introduce a surface topography onto the hydrogel, which was able to orient cell growth. Furthermore, the hydrogels could be functionalized with the biomolecule neutravidin by incorporation of biotin along the HA backbone. This biotinylation approach may allow attachment of bioactive molecules that are conjugated to avidin. r 2004 Elsevier Ltd. All rights reserved. Keywords: Collagen; Biotin; Avidin; Orientation; Topographical control 1. Introduction Polymer scaffolds serve a central role in the field of tissue engineering by directing cellular processes based on the structural and biochemical properties of the scaffold. The materials used for scaffold fabrication can determine many physical properties, such as biocompat- ibility, biodegradability, and mechanical stability. Im- portantly, the scaffold must provide the appropriate signals to direct cellular processes that lead to tissue formation. The surface of the scaffold provides a substrate for cell adhesion and migration, which can influence the survival of transplanted cells or the invasion of cell from the surrounding tissue. These surfaces can be designed to present specific cell adhesion sequences at controlled densities [1], or can be functio- nalized with growth factors [2–4], and DNA [5]. Additionally, topographical patterns can be introduced into the scaffold, which can orient cell growth [6–8], alter gene expression [9], regulate the structure of the resulting tissue [7], and affect the mechanical properties of the developing tissue [10]. The fabrication of scaffolds from natural materials, such as hyaluronic acid (HA) and collagen, can impart intrinsic signals within the structure that can enhance tissue formation. HA is a glycosaminoglycan copolymer of d-glucuronic acid and n-acetyl-d-glucosamine, and is a major intracellular component (IC) of connective tissues such as the synovial fluid of joints, vitreous fluid of the eye, and the scaffolding within cartilage and the umbilical cord. HA has been shown to play an important role in lubrication, cell differentiation and cell growth [11–13]. Cellular interactions with HA occur ARTICLE IN PRESS *Corresponding author. Department of Chemical Engineering, Northwestern University, 2145 Sheridan Road/E156, Evanston, IL 60208-3120, USA. Tel.: +1-847-491-7043; fax: +1-847-491-3728. E-mail address: l-shea@northwestern.edu (L.D. Shea). 0142-9612/$-see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.02.067