**4.2 Tissue crosslinking after carboxylic group activation**

Due to the problems associated with the use of glutaraldehyde, various non-aldehyde alternative methods have been developed to stabilize and post-treat tissues. The crosslinking agents used in collagen-rich biomaterials can use both primary amino groups and acid groups of polypeptide chains. Historically, a water soluble carbodiimide (1-ethyl-3- (3-dimethyl amino propyl) carbodiimide / EDAC) was first used for the modification of carboxylic groups in proteins for peptide synthesis (Sheehan & Hlavka, 1956) and to promote crosslinking in gelatin (Sheehan & Hlavka, 1957).

The mechanism for the reaction between carboxylic groups and EDAC leading to amide bond formation is as follows: The addition of a carboxylic acid diimide produces an isourea ester, an O-acyl isourea. The intermediate O-acyl isourea is an activated carboxylic acid derivative with similar reactivity to an anhydride or acyl halide, and can be subjected to a subsequent nucleophilic substitution by an amine yielding a dialkyl amide and urea (Carraway & Khosland, 1972). Because carbodiimide is just a coupling agent, when used to crosslink collagen in the absence of agents with dual functionality, only promotes the formation of an amide bond between carboxylic acid and amino reactive groups present in the tissue, as depicted in figure 6.

Fig. 6. Schematic representation of tissue crosslinking with EDAC and NHS

Fig. 7. Bovine perichardial tissue crosslinked with EDAC (rectangules) or glutaraldehyde (circles)

This requires that the activated carboxylic groups be close enough to the amino groups to achieve direct bonding (amide bond formation). The carbodiimides are hydrolyzed rapidly

Due to the problems associated with the use of glutaraldehyde, various non-aldehyde alternative methods have been developed to stabilize and post-treat tissues. The crosslinking agents used in collagen-rich biomaterials can use both primary amino groups and acid groups of polypeptide chains. Historically, a water soluble carbodiimide (1-ethyl-3- (3-dimethyl amino propyl) carbodiimide / EDAC) was first used for the modification of carboxylic groups in proteins for peptide synthesis (Sheehan & Hlavka, 1956) and to

The mechanism for the reaction between carboxylic groups and EDAC leading to amide bond formation is as follows: The addition of a carboxylic acid diimide produces an isourea ester, an O-acyl isourea. The intermediate O-acyl isourea is an activated carboxylic acid derivative with similar reactivity to an anhydride or acyl halide, and can be subjected to a subsequent nucleophilic substitution by an amine yielding a dialkyl amide and urea (Carraway & Khosland, 1972). Because carbodiimide is just a coupling agent, when used to crosslink collagen in the absence of agents with dual functionality, only promotes the formation of an amide bond between carboxylic acid and amino reactive groups present in

Fig. 6. Schematic representation of tissue crosslinking with EDAC and NHS

Fig. 7. Bovine perichardial tissue crosslinked with EDAC (rectangules) or glutaraldehyde

This requires that the activated carboxylic groups be close enough to the amino groups to achieve direct bonding (amide bond formation). The carbodiimides are hydrolyzed rapidly

**4.2 Tissue crosslinking after carboxylic group activation** 

promote crosslinking in gelatin (Sheehan & Hlavka, 1957).

the tissue, as depicted in figure 6.

(circles)

in aqueous solution and the intermediate O-acyl isourea is extremely unstable producing a low crosslinking.

The crosslinking density and the shrinkage temperature of bovine perichardium treated with EDAC had values lower that a control of bovine perichardium fixed with glutaraldehyde (Mendoza-Novelo & Cauich-Rodríguez, 2009). However, the use of the Nhydroxysuccinimide (NHS) during crosslinking with EDAC improved the stabilization of tissue due to the formation of a stable intermediate compound after reaction of the NHS with carboxylic groups or isourea O-acyl intermediate (Lee et al., 1996). Such is the case reported for porcine aortic valves crosslinked by a two-step method. These steps included the blocking of the free primary amino groups of collagen with butanal and the crosslinking with JeffaminesTM of different molecular weights by activating the carboxylic acid groups with EDAC and NHS. This process led to a decrease in calcification (subcutaneous implantation in rats) of engineered tissue (Everaerts et al., 2004).

The appearance of bovine perichardial tissue crosslinked with glutaraldehyde and EDAC is shown in figure 7.
