International Journal of Biological Macromolecules 47 (2010) 570–577 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Chemical and thermal cross-linking of collagen and elastin hydrolysates A. Sionkowska ∗ , J. Skopinska-Wisniewska, M. Gawron, J. Kozlowska, A. Planecka Nicolaus Copernicus University, Faculty of Chemistry, Gagarin 7, 87-100 Torun, Poland article info Article history: Received 7 July 2010 Accepted 6 August 2010 Available online 14 August 2010 Keywords: Collagen Cross-linking Elastin hydrolysates abstract Chemical and thermal cross-linking of collagen soluble in acetic acid and elastin hydrolysates soluble in water have been studied. Solutions of collagen and elastin hydrolysates were treated using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Moreover, diepoxypropylether (DEPE) has been used as cross-linking agent. Films made of collagen and elastin hydrolysates were also treated with temperature at 60 ◦ C and 100 ◦ C to get additional cross- links. The effect of cross-linking has been studied using FTIR spectroscopy, thermal analysis, AFM and SEM microscopy. Mechanical and surface properties of materials have been studied after cross-linking. It was found that thermal and mechanical properties of collagen and elastin materials have been altered after thermal treatment and after the reactions with EDC/NHS and/or DEPE. Surface properties of colla- gen materials after chemical cross-linking have been modified. Thermal and chemical cross-linking of collagen films lead to alteration of polarity of the surface. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Both, elastin and collagen, are structural proteins and may undergo varied sequences of photochemical and chemical reac- tions [1,2]. Collagen is the main protein of connective tissue and the main component of the skin. As an extracellular matrix protein it is widely used as a biomaterial for tissue regeneration and implanta- tion. Elastin is an extracellular matrix protein in mammals where it is the main component of skin, blood vessels, such as the aorta, and tissues of the lung. Elastin can provide an excellent basis for bioma- terials, such as arterial prosthesis, dermal substitute and hydrogels [3]. It is a highly insoluble structural protein and usually elastin hydrolysates are more useful for biomedical applications. However, the material made of elastin hydrolysates only is very elastic with low mechanical strength. A good way for obtaining new materi- als can be a preparation of a blend containing elastin hydrolysates and collagen [4,5]. In mammals, collagen and elastin are mixed together in appropriate ways depending on the functions [6]. In this context, elastin could represent a valid alternative to synthetic biomaterials, for application in tissue replacement and/or tissue regeneration. For cross-linking of protein materials one can use physical or chemical methods. As physical cross-linking agent gamma radia- tion can be used [7,8]. However, the energy of gamma radiation can destroy the native structure of the protein. Next to gamma radia- tion physical cross-linking agent is UV irradiation. It is less harmful ∗ Corresponding author. E-mail address: as@chem.uni.torun.pl (A. Sionkowska). for proteins than gamma radiation, but UV light can also destroy protein structure [1,9,10]. Chemical cross-linking can give highly cross-linked material in a very short time [11]. There are several chemical compounds capa- ble to cross-link proteins. Covalent cross-linking using 1-ethyl-3-(3-dimethylaminop- ropyl)-1-carbodiimide hydrochloride (EDC) and N-hydroxysuc- cinimide (NHS) is a widely used method [12–14]. The aim of this work was to study the chemical cross-linking of collagen soluble in acetic acid and elastin hydrolysates sol- uble in water. Solutions of elastin hydrolysates and collagen were treated using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Moreover, diepoxypropylether (DEPE) has been used as cross-linking agent. Collagen and elastin hydrolysates have been cross-linked by the temperature to compare chemical cross-linking with physical cross-linking, namely thermal cross-linking. Cross- linked collagen and elastin materials are usually required for the preparation of scaffolds containing hydroxyapatite. Such materials can be applied as bone tissues substitutes. 2. Materials and methods 2.1. Collagen Collagen was obtained in our laboratory from tail tendons of young albinos rats (Medical University, Poznan, Poland). After washing in distillate water, the tendons were dissolved in 0.1 M acetic acid for 3 days at 4 ◦ C. Tendons were blended in a Waring blender in 0.5 M acetic acid, and then spun at 10,000 rpm in a Sorvall 0141-8130/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2010.08.004