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

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 (Chang et al., 2004).

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 polypeptide chains.

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

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

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

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

Decellularization, Stabilization and Functionalization of

Masking Crosslinking

crosslinking structures

**5. Conclusions** 

Collagenous Tissues Used as Cardiovascular Biomaterials 175

Crosslinking agent Collagen

Intramolecular crosslinks

Short range intermolecular crosslinks

Large range intermolecular crosslinks

Table 6. Schematic representation of presumable masking and intra- or inter-molecular

metalloproteinase at the implantation site (Arenaz et al., 2004).

rates. Also it was reported the effectiveness of EDAC and neomycin (an inhibitor of the enzyme hyaluronidase) for the prevention of glycosaminoglycans loss (Ragharan et al., 2007; Shah & Vyavahare, 2008). The addition of exogenous glycosaminoglycans and the stabilization of endogenous glycosaminoglycans in ostrich perichardium reduced tissue calcification after implantation in rats, but slightly increased the presence of matrix-

Natural tissues from various sources can be used as biomaterials in the cardiovascular field after decellularization and fixation with various crosslinking agents. However, the current

crosslinking of collagen type I due to their ability to crosslink proteins through the -amino group of lysine and -carboxamide group of glutamine residue (Chen et al., 2005; Chau et al., 2005). The results indicated the efficiency of this crosslinking agents in terms of denaturation temperature, mechanical strength, low toxicity to fibroblasts (Chen et al., 2005) and an increase in osteoblasts and fibroblasts adhesion and proliferation compared to native collagen (Chau et al., 2005).
