the use of bifunctional polyethyleneglycol derivatives for coupling of proteins to and cross-linking...

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JOURNAL OF MATERIALS SCIENCE: MATERIALS IN MEDICINE 13 (2002) 1029±1035 The use of bifunctional polyethyleneglycol derivatives for coupling of proteins to and cross-linking of collagen matrices J.-S. CHEN 1,2 , E. M. NOAH 2 , N. PALLUA 2 , G. C. M. STEFFENS 1 * 1 Institute of Biochemistry, Aachen University of Technology, Aachen, Germany 2 Department of Plastic, Hand-, and Burn Surgery, Aachen University of Technology, Aachen, Germany E-mail: [email protected] The realization of three-dimensional (3D) degradable matrices which slowly release bio- active components represents a major challenge in the ®eld of tissue engineering. In this paper we report on the usage of commercially available bifunctional agents for both the covalent coupling of proteins to and the cross-linking of collagen matrices. Proteins ± horse radish peroxidase (HRP) was used as a model protein ± were cross-linked with either a homobifunctional (disuccinimidyldisuccinatepolyethylene-glycol) or a heterobifunctional (N-hydroxysuccinimidylvinylsulfonepolyethyleneglycol) agent. In the case of the heterobifunctional cross-linking agent the collagen matrices were previously modi®ed with succinimidylacetylthioacetate in order to introduce sulfhydryl groups. As compared with control experiments a 10-fold and 50-fold increase of immobilized proteins were achieved with the homobifunctional and heterobifunctional cross-linker resp. The HRP±PEG conjugates demonstrated a better long-term stability as compared to the non-treated HRP. The effects of the cross-linking agents and the thiolation reagent succinimidylacetylthio acetate on the in vitro degradation of the collagen matrices by collagenase were also investigated. In particular the reaction with succinimidylacetylthio acetate appears to offer interesting opportunities both for coupling active proteins and modulating the degradation times of collagen matrices. # 2002 Kluwer Academic Publishers 1. Introduction One of the aims of the interdisciplinary ®eld of research termed tissue engineering is the regeneration or replacement of lost cells and tissue by applying modern bioengineering techniques [1±3]. Regeneration of desired functional tissue requires regulation of cellular adhesion, modulation of the direction and speed of cell migration, encouragement of cell proliferation, maintenance of cell viability and the differentiated function [1, 4]. Soluble signaling proteins, such as growth factors, have proven to be valuable tools for the control of many of the cellular behaviors associated with tissue regenera- tion and formation [5]. The great advances in biotechnology, such as recombinant technologies, have made it possible to produce large quantities of highly puri®ed polypeptides and proteins. However, in general, direct injection of bio- active proteins such as growth factors ± even with extremely high doses ± into the target site is ineffective, since the in vivo stability of many of these agents are short, normally of the order of minutes, a time scale which is shorter than the time usually required for a tissue response [6]. The main goal of the present study is to develop a pro- tein delivery system, which releases bioactive proteins in a controlled manner at the site of action over a longer period of time. A controlled and sustained release may be achieved by many different approaches [1]. One of the approaches comprises the tethering of proteins to natural or synthetic matrices via a speci®c linkage strategy. The advantage of this procedure is that covalently bound proteins will be released simultaneously with the degradation of matrix, whereas admixed and non-cross- linked proteins will be washed out on a much faster time scale. In the present study, matrices made of collagen were chosen as the carriers of the bioactive proteins. Collagen represents a suitable substrate for cell attach- ment, it is biocompatible and degrades into harmless products, that are metabolized or excreted [7]. Collagen can be formed into three-dimensional (3D) matrices which may be applied as a tissue substitutes and scaffolds for tissue regeneration. Therefore, the main focus of the *Author to whom all correspondence should be addressed: Institute of Biochemistry, Aachen University of Technology, Pauwelsstr. 30, 52057 Aachen, Germany. 0957±4530 # 2002 Kluwer Academic Publishers 1029

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