**4. Stabilization of tissues**

The stability of tissues is increased by physical or chemical crosslinking. The fixation enhances tissue stability, inhibits autolysis, allows a prolonged shelf-life, and allows a surgeon to have medical devices of various sizes readily available for implantation (Schoen & Levy, 1999). The chemical treatments also mitigate immunogenicity while maintaining both thromboresistance and antimicrobial sterility but greatly influence their degradation and calcification. However, tissue calcification is multifactorial phenomenum where chemical crosslinking is considered just one of these factors. In fact, the alteration in the electrical charge that exists in the perichardial tissue surface has been associated to the

Decellularization, Stabilization and Functionalization of

**4.1 Tissue crosslinking with glutaraldehyde** 

immobilization of enzymes or cell fixation.

suggested to solve them.

Collagenous Tissues Used as Cardiovascular Biomaterials 167

Table 3. Some chemical agents used for the stabilization and fixation of biological tissues

The procedure most studied and exploited in the manufacture of tissue valve includes the crosslinking with glutaraldehyde, which is also widely used as tanning agent in the leather industry. Glutaraldehyde is an important reagent in the biomedical field and has been used as crosslinking agent in the preparation of collagen-rich biomaterials or for the

Glutaraldehyde is an efficient agent for the crosslinking of collagen matrix because it react relatively quickly and because is able to join separate protein molecules by means of the amino groups abundantly present in collagen. Glutaraldehyde is a cheap and water soluble five-carbon bifunctional aldehyde that in aqueous solution consists of a mixture of free aldehyde, mono and dihydrated monomeric glutaraldehyde, monomeric and polymeric cyclic hemiacetals and various α, β unsaturated polymers (Whipple & Ruta, 1974). This means that glutaraldehyde itself forms a number of different reactive species and that these species may also react in different ways, rendering a highly crosslinked network. Glutaraldehyde crosslinking has been and is still applied to most of the experimental and clinical bioprostheses. This process consists in blocking the ε-amino groups of lysine in the protein through imino bond formation. The contribution of the glutaraldehyde as sterilization and crosslinking agent is partly due to its hydrophobicity and hydrophilicity, allowing it to penetrate both aqueous media and in the cell membrane. However, in the manufacture of bioprostheses, the use of glutaraldehyde has led to many disadvantages associated with the residual free aldehyde groups. Table 4 shows some of the problems associated with glutaraldehyde tissue crosslinking and some solutions that have been

In aqueous solution, the glutaraldehyde is presented as a mixture of free aldehyde, mono and dihydrate glutaraldehyde monomer, monomeric and polymeric cyclic hemiacetals, and several alpha or beta unsaturated polymers (Monsan et al., 1975). In turn, this heterogeneity of chemical species leads to a heterogeneous crosslinking. In addition, high concentration of glutaraldehyde promotes rapid surface crosslinking in the tissue (Olde-Damink et al., 1995), creating a barrier that impedes or prevents the diffusion of more glutaraldehyde within the biomaterial. In order to avoid this, the use of low concentrations has been suggested (Khor, 1997). It has also been proposed glutaraldehyde protection as a monomer by the formation of di-acetals, between glutaraldehyde and alcohols in acidic medium (Giossis et al., 1998).

Tannic acid Isenburg et al., 2004;

Isenburg et al., 2006

calcification (Jorge-Herrero et al., 2010). Several crosslinking techniques have been suggested as the ideal procedure to stabilize the collagen structure while maintaining their physical and natural shaping. The structure and name of some chemicals used as crosslinking agents for collagenous tissue are shown in table 3.


calcification (Jorge-Herrero et al., 2010). Several crosslinking techniques have been suggested as the ideal procedure to stabilize the collagen structure while maintaining their physical and natural shaping. The structure and name of some chemicals used as

Glutaraldehyde

Ethyl-aminopropyl carbodiimide

(EDAC) e hydroxysuccinimide

Ethylene glycol diglycidyl ether, n=1; Poly(ethylene oxide) diglycidyl ether, n=22

Triglycidylamine

Hexamethylene diisocyanate

Proanthocyanidin, Procyanidins

Glycerol diglycidyl ether Lee et al., 1994

Olde Damink et al., 1995; Duncan & Boughner, 1998; Langdon et al., 1999

Lee et al., 1996; Everaerts et al., 2004; Mendoza-Novelo & Cauich-Rodríguez, 2009

Tu et al., 1993; Sung et al., 1996; Zeeman et al., 2000

Conolly et al., 2005; Sack et al., 2007; Rapoport et al.,

Naimark et al., 1995; Olde Damink et al., 1995; Nowatzki & Tirrel, 2004

Han et al., 2003; Zhai et al.,

2007

Genipin Sung et al. 1999; Sung et al., 2000

2009

Reuterin Sung et al., 2002; Sung et al., 2003

Disuccinimidyl suberate Pathak et al., 2001

Structure of stabilization agents Name Reference

(NHS)

crosslinking agents for collagenous tissue are shown in table 3.

Table 3. Some chemical agents used for the stabilization and fixation of biological tissues
