**4.3 Tissue crosslinking with epoxy compounds**

The chemistry of epoxy groups, cyclic ethers of three members, has also been explored and applied in the fixation of tissue. Polyepoxide compounds or epoxy bifunctional polyether react with amino groups from collagen opening the terminal epoxide ring (Tu et al., 1993; Lee at al., 1994; Khorn, 1997). This reaction is nucleophilic and can be carried out under acidic conditions (highly reactive protonated epoxy) or alkaline (amine at its most nucleophilic). In this case, the modification of swine tendons with ethylene glycol diglycidyl ether has been reported for the repair of cruciate ligaments (Sung et al., 1996). The 1,4 butanediol diglycidyl ether (BD) has been reported as a crosslinking agent in the preparation of bioprosthetic valves (Zeeman et al., 2000). However, the fixation of porcine valves with BD caused immune response, foreign body reaction (proliferation of lymphocytes and macrophages) and calcification of implanted tissue using rats as animal model to levels similar to glutaraldehyde-fixed tissue, although low levels of cytotoxicity were reported (van Wachem et al., 2000). The combined treatment of BD and EDACdicarboxylic acid or detergents led to a reduction in calcification (implantation in rats) but not at significant levels (van Wachem et al., 1994). Therefore, it was concluded that the treatment with BD did not represents an alternative to glutaraldehyde to reduce the calcification of bioprosthetic valves (van Wachem et al., 1994). However, in another report the crosslinking of bovine perichardium and porcine aortic valves with triglycidylamine, a molecule of high polarity and solubility in water, resulted an improvement in biocompatibility (assessed using bovine aortic valve interstitial cells, human umbilical endothelial cells and rats artery smooth muscle cells) and resistance to calcification (subcutaneous implantation in rats) compared with glutaraldehyde-fixed tissues (Connolly et al., 2005). Furthermore, triglycidylamine-fixed tissues showed stable mechanical properties (Sacks et al., 2007) and optimal reduction of calcification when treatments included mercapto-aminobisphosphonate (Rapoport et al., 2007). It was hypothesized that the difference between these two results, which explored the chemistry of epoxy in the crosslinking of tissue, may be due to differences in water solubility, chemical heterogeneity and contamination with used epoxy residual reactants (Connolly et al., 2005).

Decellularization, Stabilization and Functionalization of

(Chang et al., 2004).

polypeptide chains.

tendency to calcify (Isenburg et al., 2006).

**4.5 Tissue crosslinking with naturally-derived compounds** 

Collagenous Tissues Used as Cardiovascular Biomaterials 173

Crosslinking agents of natural origin have also been explored in the tissue crosslinking. Such is the case of genipin, an aglycone or an iridoid glycoside, which can be obtained by enzymatic hydrolysis of the glucoside previously isolated from gardenia fruit. The stabilization of porcine perichardium (Sung et al., 1999) and acellular bovine perichardium (Sung et al., 2000) with genipin probably was achieved through cyclic structures. The crosslinking density for genipin-fixed tissue was similar to glutaraldehyde and ethylene glycol diglycidyl ether -crosslinked tissues. Moreover, the genipin-crosslinked porcine perichardium was less cytotoxic (fibroblasts) than glutaraldehyde-crosslinked tissue, whereas exhibiting the same tensile strength and resistance to enzymatic degradation (Chang et al., 2002). Furthermore, acellular bovine perichardium fixed with genipin showed capacity of angiogenesis (microvessel infiltration) after implantation in rats (Liang et al., 2004). Moreover, cell extraction with solutions of Triton™X-100 and the crosslinking with different concentrations of genipin were used to establish a relationship between the crosslinking degree and the degradation rate or the model of acellular tissue regeneration

Polyphenolic compounds have also been investigated as natural agents of tissue stabilization, such as the proanthocyanidins from the family known as condensed tannins, which are essentially oligomers of flavonoids available in several fruits and vegetables. The stabilization of collagen with proanthocyanidins may involve the formation of hydrogen bond type interactions between the phenolic hydroxyl and amide carbonyls of the

The proanthocyanidin has a high affinity for proline-rich proteins, because this amino acid is a good hydrogen bond acceptor (Zhai et al., 2006). The proanthocyanidins can be used to crosslink collagen sponges with similar density and efficiency to glutaraldehyde but with reduced calcification after 6 weeks implantation in rats and it was reported to be 120 times less toxic to fibroblasts direct contact (Han et al., 2003). The proanthocyanidin crosslinking procedure was repeated in decellularized porcine aortic valves resulting in low toxicity to bovine aortic valve interstitial cells and in the stimulation of cell proliferation to low

The stabilization of elastin in porcine aortas has been achieved by treatment with polyphenolic tannins, which is composed of a central molecule of glucose (hydrophobic core) and one or more galoil residues (hydrophilic shell) (Isenburg et al., 2006). Polyphenolic compounds were acetylated tannic acid, pentagaloil glucose, gallic acid and glucose. In this study, pentagaloil glucose treatment was the least toxic to fibroblasts (Isenburg et al., 2004). Also, the study revealed that polyphenolic hydroxyl groups are essential for the interaction between the tannic acid and elastin. The combination of tannic acid and glutaraldehyde rendered a biostable tissue with high resistance toward elastase and collagenase and low

The reuterin (-hydroxypropionic acid) produced by *Lactobacillus reuteri* has been used in the fixation of porcine perichardium (Sung et al., 2002). The reuterin is soluble in water, with antimicrobial and antifungal activity. The properties of reuterin-fixed tissue are comparable to glutaraldehyde-fixed tissue in terms of amino group content, denaturation temperature,

Microbial (mTG; *Streptoverticillium mobaraense*) and tissue (TG2; tTG) transglutaminases (protein-glutamine -glutamyltransferase, EC 2.3.2.13) have been explored in the

concentrations of this stabilization agent in the culture media (Zhai et al., 2009).

tensile strength and collagenase digestion resistance (Sung et al, 2003).

#### **4.4 Tissue crosslinking with diisocyanate**

Bifunctional molecules capable of crosslinked proteins by urea bond formation after reaction between terminal isocyanate groups and -amino group of lysine residue have been explored. Such is the case of crosslinking of extracellular matrix proteins (elastin and fibronectin) with hexamethylene diisocyanate in dimethyl sulfoxide (Tirrell and Nowatzki, 2004). Similarly, the crosslinking of ovine skin collagen with hexamethylene diisocyanate has been reported. This crosslinking procedure was carried out in an aqueous medium including surfactants to increase solubility and promote the penetration of diisocyanate into the tissue (Olde Damink et al., 1995). Futhermore, the effects of the tissue crosslinking with hexamethylene diisocyanate and the effects of mixtures of water/isopropanol (50/50 and 0/100) as solvent on the thermal and biomechanical properties of bovine perichardium have been reported (Naimark et al., 1995). On the other hand, the stabilization of porcine perichardium has been achieved by the interaction of polyurethane oligomers containing isocyanate end groups (Loke et al., 1996). The interaction in organic media between perichardial tissue and polyurethane oligomers resulted in the increase of the denaturation temperature, a reduction in the content of lysine and a poor diffusion of polyurethane oligomers into the tissue (H&E staining). The crosslinking of bovine perichardium with polyurethane oligomers, EDAC and diphenyl phosphoric azide showed less cytotoxicity (assessed by a direct cytotoxicity test or Homsy test) than the tissue crosslinking with glutaraldehyde (Jorge-Herrero et al., 2005).

After these results, it is clear that diisocyanates are an alternative to glutaraldehyde in the preparation of bioprostheses. However, protein fixation with isocyanates has the disadvantage of using organic solvents. In addition, during the fixation in aqueous media, the crosslinking degree can be reduced due to competition of hydrolysis reactions. Therefore, the blocking reaction of isocyanate with bisulphite salts is an alternative in the preparation of water soluble isocyanates (Petersen, 1949). The protein crosslinking process with blocking isocyanates has the advantages of the use of aqueous media and reduced isocyanate toxicity (Mata-Mata et al., 2008). In this regard, the treatment of perichardial tissue with the carbamoylsulphonate blocked polyurethane prepolymers resulted in an increase of the in vitro tissue biostability (Mendoza-Novelo, 2011). The coating of collagen fiber network of perichardial tissue with polyurethane is shown in figure 8.

Fig. 8. SEM micrographs for bovine perichardium (a) native and treated with polyurethane prepolymers

Bifunctional molecules capable of crosslinked proteins by urea bond formation after reaction between terminal isocyanate groups and -amino group of lysine residue have been explored. Such is the case of crosslinking of extracellular matrix proteins (elastin and fibronectin) with hexamethylene diisocyanate in dimethyl sulfoxide (Tirrell and Nowatzki, 2004). Similarly, the crosslinking of ovine skin collagen with hexamethylene diisocyanate has been reported. This crosslinking procedure was carried out in an aqueous medium including surfactants to increase solubility and promote the penetration of diisocyanate into the tissue (Olde Damink et al., 1995). Futhermore, the effects of the tissue crosslinking with hexamethylene diisocyanate and the effects of mixtures of water/isopropanol (50/50 and 0/100) as solvent on the thermal and biomechanical properties of bovine perichardium have been reported (Naimark et al., 1995). On the other hand, the stabilization of porcine perichardium has been achieved by the interaction of polyurethane oligomers containing isocyanate end groups (Loke et al., 1996). The interaction in organic media between perichardial tissue and polyurethane oligomers resulted in the increase of the denaturation temperature, a reduction in the content of lysine and a poor diffusion of polyurethane oligomers into the tissue (H&E staining). The crosslinking of bovine perichardium with polyurethane oligomers, EDAC and diphenyl phosphoric azide showed less cytotoxicity (assessed by a direct cytotoxicity test or Homsy test) than the tissue crosslinking with

After these results, it is clear that diisocyanates are an alternative to glutaraldehyde in the preparation of bioprostheses. However, protein fixation with isocyanates has the disadvantage of using organic solvents. In addition, during the fixation in aqueous media, the crosslinking degree can be reduced due to competition of hydrolysis reactions. Therefore, the blocking reaction of isocyanate with bisulphite salts is an alternative in the preparation of water soluble isocyanates (Petersen, 1949). The protein crosslinking process with blocking isocyanates has the advantages of the use of aqueous media and reduced isocyanate toxicity (Mata-Mata et al., 2008). In this regard, the treatment of perichardial tissue with the carbamoylsulphonate blocked polyurethane prepolymers resulted in an increase of the in vitro tissue biostability (Mendoza-Novelo, 2011). The coating of collagen

Fig. 8. SEM micrographs for bovine perichardium (a) native and treated with polyurethane

fiber network of perichardial tissue with polyurethane is shown in figure 8.

**4.4 Tissue crosslinking with diisocyanate** 

glutaraldehyde (Jorge-Herrero et al., 2005).

prepolymers
