**4.6 Other methods for the tissue stabilization**

Others non-aldehydic crosslinking procedures have been proposed with the aims of prevent or mitigate tissue calcification. The disuccinimidyl glutarate (DSG) is another non- aldehyde alternative to tissue crosslinking. The process is carried out by the reaction between primary amino groups of tissue and NHS ester groups of DSG forming amide bonds with a length of five-carbon crosslinking and releasing NHS. The DSG crosslinked tissue was resistant to enzymatic degradation, exhibited low tendency to calcify and high temperature of denaturation. However, it was necessary to use dimethyl sulfoxide due to the insolubility of crosslinking agent in water (Pathak et al., 2000). In response to this drawback, a water soluble crosslinking agent has been used, i. e., the disuccinimidyl suberate. The presence of sulfonyl groups at the ends of the molecule conferred water solubility while retaining reactivity with amino groups by crosslinking chemistry similar to DSG, but with a length of 8 carbon intermediates. The tissue crosslinked under these conditions showed very low levels of calcium (0.2 mg/g of tissue) after 90 days of implantation in rats (Pathak et al., 2001). The crosslinking of collagen type I proposed for cartilage regeneration has also been achieved by the diimidoesters—dimethyl suberimidate (DMS). In this procedure, collagen amino groups react with DMS imidoester groups to form amidine groups and a length crosslinking of 8 carbons (Charulatha & Rajaram, 2003).

The stabilization of bioprosthetic tissue by filling the tissue interstitial spaces with polyacrylamide hydrogel resulted in the mitigation of tissue calcification in a rat study (Oosthuysen et al., 2006). Physical methods such as photo-oxidation (Khorn et al., 1997) or the use of ultraviolet radiation (Pfau et al., 2000) have also been proposed for the crosslinking of collagen-rich biomaterials. However, despite the increase in tissue shrinkage temperature, in some case the treated tissue did not show resistance to the proteins extraction (Moore et al., 1996).

#### **4.7 Masking reactions**

At this point it is important to distinguish between the effective formation of crosslinking sites, i. e., two reactive sites in collagen linked by a same molecule of crosslinking agent, and the masking of crosslinking, i.e., the reaction between a single end of bifunctional crosslinking agent and one reactive site of collagen. Table 6 shows the possible reactions of crosslinking and masking between collagen and difunctional crosslinking agents.

#### **4.8 Glycosaminoglycans stabilization**

Glycosaminoglycans present in both aortic valves and perichardium have been fixed to prevent the loss of these polysaccharides during the fixation of bioprosthetic valves. The sodium metaperiodate has been used for the stabilization of glycosaminoglycans in porcine aortic valves with the subsequent glutaraldehyde crosslinking (Vyavahare & Lovekamp, 2001). The stabilized porcine aortic valve showed compatibility and reduced calcification

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

Others non-aldehydic crosslinking procedures have been proposed with the aims of prevent or mitigate tissue calcification. The disuccinimidyl glutarate (DSG) is another non- aldehyde alternative to tissue crosslinking. The process is carried out by the reaction between primary amino groups of tissue and NHS ester groups of DSG forming amide bonds with a length of five-carbon crosslinking and releasing NHS. The DSG crosslinked tissue was resistant to enzymatic degradation, exhibited low tendency to calcify and high temperature of denaturation. However, it was necessary to use dimethyl sulfoxide due to the insolubility of crosslinking agent in water (Pathak et al., 2000). In response to this drawback, a water soluble crosslinking agent has been used, i. e., the disuccinimidyl suberate. The presence of sulfonyl groups at the ends of the molecule conferred water solubility while retaining reactivity with amino groups by crosslinking chemistry similar to DSG, but with a length of 8 carbon intermediates. The tissue crosslinked under these conditions showed very low levels of calcium (0.2 mg/g of tissue) after 90 days of implantation in rats (Pathak et al., 2001). The crosslinking of collagen type I proposed for cartilage regeneration has also been achieved by the diimidoesters—dimethyl suberimidate (DMS). In this procedure, collagen amino groups react with DMS imidoester groups to form amidine groups and a length

The stabilization of bioprosthetic tissue by filling the tissue interstitial spaces with polyacrylamide hydrogel resulted in the mitigation of tissue calcification in a rat study (Oosthuysen et al., 2006). Physical methods such as photo-oxidation (Khorn et al., 1997) or the use of ultraviolet radiation (Pfau et al., 2000) have also been proposed for the crosslinking of collagen-rich biomaterials. However, despite the increase in tissue shrinkage temperature, in some case the treated tissue did not show resistance to the proteins

At this point it is important to distinguish between the effective formation of crosslinking sites, i. e., two reactive sites in collagen linked by a same molecule of crosslinking agent, and the masking of crosslinking, i.e., the reaction between a single end of bifunctional crosslinking agent and one reactive site of collagen. Table 6 shows the possible reactions of

Glycosaminoglycans present in both aortic valves and perichardium have been fixed to prevent the loss of these polysaccharides during the fixation of bioprosthetic valves. The sodium metaperiodate has been used for the stabilization of glycosaminoglycans in porcine aortic valves with the subsequent glutaraldehyde crosslinking (Vyavahare & Lovekamp, 2001). The stabilized porcine aortic valve showed compatibility and reduced calcification

crosslinking and masking between collagen and difunctional crosslinking agents.

collagen (Chau et al., 2005).

extraction (Moore et al., 1996).

**4.8 Glycosaminoglycans stabilization** 

**4.7 Masking reactions** 

**4.6 Other methods for the tissue stabilization** 

crosslinking of 8 carbons (Charulatha & Rajaram, 2003).

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

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