* Corresponding author. Tel.: 886-3-422-7151-x4228; fax: # 886-3- 425-2296. E-mail address: hwsung@cc.ncu.edu.tw (H.-W. Sung). Biomaterials 21 (2000) 1353}1362 In vitro surface characterization of a biological patch "xed with a naturally occurring crosslinking agent Hsing-Wen Sung*, Chiun-Nan Chen, Rong-Nan Huang, Jer-Chen Hsu, Wen-Hsiang Chang Department of Chemical Engineering, National Central University, Chung-Li, 32054 Taiwan Institute of Life Science, National Central University, Chung-Li, 32054 Taiwan Received 26 July 1999; accepted 15 December 1999 Abstract The study was designed to characterize the surface properties (including water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility) of a biological patch "xed with genipin, a naturally occurring crosslinking agent. Fresh and glutaraldehyde-"xed counterparts were used as controls. It was found that both glutaraldehyde and genipin are e!ective crosslinking agents for biological tissue "xation. Fixation of biological tissue with glutaraldehyde or genipin signi"cantly increased its hydrophilicity and surface tension and reduced its mol ratio of adsorbed "brinogen to adsorbed albumin as well as the amount of adhered platelet. There were no signi"cant di!erences in hydrophilicity, surface tension, the mole ratio of adsorbed "brinogen to adsorbed albumin, and the amount of platelet adhesion between the glutaraldehyde- and genipin-"xed tissues. However, the cellular compatibilities of fresh and the genipin-"xed tissues were signi"cantly superior to the glutaraldehyde-"xed tissue.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Biological patch; Genipin; Glutaraldehyde; Surface characterization 1. Introduction Biological patches, made from mammalian pericardia, are often used in cardiovascular surgery for pericardial substitute [1], intracardiac defect repair [2], stenosis enlargement [3], and change of blood stream direction [4]. Additionally, they have been used in the construc- tion of lea#ets for heart value bioprostheses [5]. To increase its resistance against enzymatic degradation and reduce its antigenicity, the mammalian pericardium has to be "xed and subsequently sterilized before it can be used clinically [6]. Various crosslinking agents have been used in "xing biological tissues. The most common are synthetic chem- icals such as formaldehyde [6], glutaraldehyde [6], dialdehyde search [7], epoxy compound [8], etc. However, these synthetic chemical agents are all highly (or relatively highly) cytotoxic [9,10]. It is therefore desir- able to provide a crosslinking agent suitable for used in biomedical applications that is of low cytotoxicity and that forms stable and biocompatible crosslinked products. To achieve this goal, a naturally occurring crosslinking agent, genipin, was used by out group to "x biological tissues [10}13], Genipin can be obtained from its parent compound geniposide, which may be isolated from the fruits of Gardenia jasminoides ELLIS [14,15]. In herbal medicine, genipin and its related iridoid glucosides have been widely used as an antiphlogistic and cholagogue [16]. The in vitro cytotoxicity of genipin was previously studied by our group using 3T3 "broblasts [10]. Glutaraldehyde was used as a control. The results ob- tained in the MTT assay implied that genipin was about 10 000 times less cytotoxic than glutaraldehyde. Addi- tionally, the colony-forming assay suggested that the proliferative capacity of cells after exposure to genipin was approximately 5000 times greater than that after exposure to glutaraldehyde. The feasibility of using this naturally occurring cross- linking agent to "x biological tissue was investigated in vitro by our group [11]. Glutaraldehyde and an epoxy 0142-9612/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 0 1 7 - X