In vitro cytocompatibility evaluation of alginate dialdehyde for biological tissue fixation
Highlights
► We used alginate dialdehyde for biological tissue fixation. ► Its cytocompatibility for biological tissue fixation was evaluated thoroughly. ► It is a study which has not been reported elsewhere. ► Both glutaraldehyde and genipin were used as experimental controls. ► The alginate dialdehyde is an excellently cytocompatible crosslinking reagent.
Introduction
Naturally derived biological tissues have been used extensively to fabricate various bioprostheses such as heart valves (Zhai et al., 2006), vascular grafts (Yu, Liu, Xu, & Wan, 2010), ligament substitutes (Sung, Shih, & Hsu, 1996) and pericardial patches (Jayakrishnan & Jameela, 1996), because that they can offer better constructions for adhesion and growth of cells than synthetic materials and possess mechanical properties similar to those of native tissues. However, due to the immediate degradation and presence of antigenicity, these naturally derived tissues must be chemically fixed before they can be implanted in humans (Schmidt & Baier, 2000). In recent years, various synthetic crosslinking reagents including formaldehyde, glutaraldehyde (Cheung, Perelman, Ko, & Nimni, 1985), polyepoxy compound (Sung et al., 1996) and cyanimide, 1-ethyl-3(-3 dimethyl aminopropyl) carbodiimide hydrochloride (EDC) (Lee, Edwards, & Pereira, 1996), have been widely used in fixing biological tissues, however, these synthetic crosslinking reagents are all highly (or relatively highly) cytotoxic (Nishi et al., 1995, Yu et al., 2008). Therefore, to overcome the cytotoxic effect of the aforementioned synthetic crosslinking reagents, a novel crosslinking agent, alginate dialdehyde (ADA), was developed to fix biological tissues by our group.
Alginate (ALG), an important naturally occurring carbohydrate polymer derived from brown algae, has been widely used in a variety of biomedical applications, mainly due to its high biocompatibility and nontoxicity (Goh et al., 2011, Isiklan et al., 2010, Knill et al., 2004, Li et al., 2005, Wang et al., 2010, Yang et al., 2011). In recent years, it was reported that alginate can be oxidized with periodate to produce entity with multiple functional aldehyde groups (aldehyde alginate, ADA) (Bouhadir et al., 2001). As with glutaraldehyde, there exist multiple functional aldehyde groups in the chemical structure of ADA, which can produce crosslinking within biological tissues. In our previous study, the feasibility of using ADA as a crosslinking agent in fixing biological tissues was evaluated. Glutaraldehyde and polyepoxy compound were used as controls. It was found that ADA was in possession of the fixation index and mechanical strength comparable to glutaraldehyde and significantly superior to polyepoxy. Histological examination of the tissues after the ADA fixation process also showed intact total framework. This indicated that ADA is an effective crosslinking agent in the fixation of biological tissues (Xu, Li, Yu, Gu, & Zhang, 2012).
The present study was conducted to further evaluate and analysis the cytocompatibility of ADA for biological tissue fixation in vitro. In the study, fresh porcine aortas procured from a slaughterhouse were used as raw materials. Glutaraldehyde (GA) and genipin (GP)-fixed counterparts were used as controls. Glutaraldehyde is the most commonly used crosslinking reagent for tissue fixation (Jayakrishnan & Jameela, 1996) and genipin is the most popularly investigated low-cytotoxicity crosslinking reagent in recent years (Huang, Sung, Tsai, & Huang, 1998). The chemical structures of these crosslinking reagents are shown in Fig. 1.
Section snippets
Materials
Sodium alginate (viscosity: 495 cps at 25 °C) was obtained from Qingdao Jingyan Biotechnology Co. LTD (China). Glutaraldehyde and diphenyl tetrazolium bromide (MTT) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Genipin was purchased from Linchuanzhixin Biotechnology Co. LTD (China). Triton X-100 was obtained from Amresco Co. (USA). DNaseI and RNseA were obtained from Aladdin Co. (Shanghai, China). Fetal bovine serum was obtained from Hyclone Laboratories (Logan, UT, USA). DMEM, trypsin,
Decellularization and crosslinking of the tissues
As shown in Fig. 2(a) and (b), the morphology of the porcine aortas demonstrated an intact total framework and excellent porous micro architecture after the decellularization treatment. It was obvious that this decellularization processes could result in porous scaffolds without apparent disruptions of histoarchitecture, which had prominent influence on cell intrusion, proliferation, and function in tissue engineering. Additionally, there was no cellular components observed in the decelluarized
Conclusion
In summary, this study demonstrated the cytocompatibility to use ADA as a novel crosslinking reagent in the fixation of biological tissues. The result suggested that the cytotoxicity of ADA crosslinking reagent was significantly lower than that of glutaraldehyde and genipin. Additionally, in the cytotoxicity of fixed tissue itself and the residues from the fixed tissues, as well as the cell adhesion property, ADA-fixed tissue was significantly superior to its glutaraldehyde counterpart and
Acknowledgement
The authors would like to thank the Scientific and Technological Project of Sichuan Province (2012SZ0015) for financial support.
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