# Cellular Engineeering: Tissue Engineering and Biomaterials Characterisation of elastin and collagen in aortic bioprostheses V. Samouillan I A. Lamure I E. Maurel I J. Dandurand 1 C. Lacabanne I F. Ballarin 2 M. Spina 2 1Laboratoire de Physique des Polym&es, Universit6 Paul Sabatier, Toulouse, France 21stituto di Istologia, Universita degli Studi di Padova, Padova, Italy Abstract--Porcine aortic valves used as cardiac valve bioprostheses are well adapted to physiological functions in the short term, but they lack long-term durability. Several multi-step extractions have been performed to obtain a perfectly acellular matrix. A new physical methodology is proposed to evaluate the resulting fibrous protein damage after biochemical extraction (TRI-COL and SDS). Thermal analysis techniques are adapted to collagen and elastin characterisation in the solid state. The aortic tissue thermal transitions are determined by differential scanning calorimetry (DSC): elastin glass transition is observed around 200°C, and collagen denaturation is observed around 230 ° C. These parameters are characteristic of the elastin network arrangement and of collagen triple-helix stability. The technique of thermostimulated currents (TSC) is well suited to specify the chain dynamics of proteins. The low- temperature relaxations observed in both collagen and elastin are associated with Iocalised motions, whereas the high-temperature modes are attributed to more delocalised motions of the chains. Therefore TSC and DSC spectrometries allow physical parameters specific to collagen and elastin to be obtained and their interaction in aortic tissues to be determined. According to the significant evolution of these parameters on SDS samples, the destabilising effect of this detergent is highlighted. Keywords--Porcine aortic valves, Bioprostheses, Collagen, Elastin, Differential scan- ning calorimetry, Thermostimulated currents Med. Biol. Eng. Comput., 2000, 38, 226-231 J 1 Introduction A NEW approach for the replacement of cardiac valves consists of obtaining a free cell matrix from a porcine cardiac valve (because of the similarity in size and shape between human and porcine aortic valves) that could be replanted with the patient's own cells. Therefore the aim is the modelling of an active and non antigenic bioprosthesis, free from calcification and fibroblast proliferation induced by both glutaraldehyde treatment and cell remnants (VALENTE et al., 1985; COLOMB et al., 1987). Dunng recent years, several enzymatic and detergent multi- step extractions have been developed for cell removal (VESELY et al., 1991; COURTMAN et al., 1994; WILSON et al., 1995); in this work, we focus on the characterisation of elastin and collagen in detergent-treated aortic tissue (triton X-100 and cholate, sodium dodecyl sulphate). In this way, we will be able to determine whether the resulting fibrous proteins are structurally preserved. Correspondence should be addressed to Dr V. Samouillan; emaih vsamou@cict.fr First received 26 March 1999and in final form 8 November 1999 © IFMBE:2000 226 2 Materials and methods 2.1 Materials Aortic roots comprising the terminal part of the aortic wall and corresponding leaflets were freshly dissected from the hearts of young pigs. One set of samples was suspended in a degased physiological buffer (50mM HEPES (4-(2-hydroxyethyl)-l- piperazine ethanesulfonic acid), 0.1 M NaC1, pH 7.4) containing protease inhibitors (5mM ethylene diamine tetra-acetic acid (EDTA), 2mM phenylmethylsulphonyl fluoride (PMSF), 5 mM N-ethylmaleimide (NEM), 5 mM benzamidine, 1 mM iodoacetamide), 10 mM sodium ascorbate and 10% dimethyl- sulphoxide (DMSO). The surrounding solution was gently stirred at 4:C for 3 h under N2 atmosphere. Then samples were extracted under the same conditions by replacing DMSO with 1% (w/w) sodium dodecyl sulphate (SDS) at 37:C for 16h and for three further 16-h periods in the absence of protease inhibitors. Another set of samples was treated initially under the same conditions as the first set, but with the physiological buffer being replaced with hypotonic, 10 times diluted, phosphate buffered saline (PBS), pH 7.4. Then DMSO was replaced by 1% (w/w) triton X-100 (t- octylphenoxy poly-ethoxyethanol) in NaC1 0.6 M, and samples were extracted for 10h at 4:C in the same conditions. After a Medical & Biological Engineering & Computing 2000, Vol. 38