**4.1 Tissue crosslinking with glutaraldehyde**

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 immobilization of enzymes or cell fixation.

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 suggested to solve them.

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).

Decellularization, Stabilization and Functionalization of

acid -

Heparin-

Poly(ethylene glycol)-

RGD peptides-

L-arginine, Lglutamine, Llysine, Lglutamic acid, L-cysteine -

cell adhesion after the fixation treatment.

Macromolecules Hyaluronic

Acids Homocysteic

coupling of L-cysteine

acid -

Amino oleic acid -

Table 5. Molecules grafted on crosslinked bovine perichardial tissue

Amino acids

Collagenous Tissues Used as Cardiovascular Biomaterials 169

acids have been suggested in order to provide non-cytotoxic tissue biomaterial and biomaterial with reduced calcification, as it is shown in table 5. In this table, it is also included molecules of biological importance as well as peptide sequences used to improve

Reduce the calcification of

Inhibit the platelet surface attachment and spreading and decrease the calcification of glutaraldehyde-treated tissue

Enhace the adhesion and proliferation of human mesemchymal stem cells on

Reduce the protein adsorption and platelet adhesion of glutaraldehyde treated tissue. However, BP treatment with amino acids does not effectively prevent

calcification. Incorporation of thiol moieties to the tissue

Reduce toxicity but does not

glutaraldehyde-treated tissue

glutaraldehyde-treated tissue Chen et al., 1994

Inhibit the calcification of

affect the stability of

Fig. 5. Schematic representation of tissue crosslinking with glutaraldehyde and chemical

acellular tissue

and the cytotoxicity of glutaraldehyde-treated

Reduce the calcium deposition

glutaraldehyde-treated tissue Ohri et al., 2004

Lee et al., 2000

Dong et al., 2009

Jorge-Herrero et al., 1996; Jee et al., 2003; Mendoza-Novelo & Cauich-

Rodríguez, 2009

Stacchino et al.,

1998

Vasudev & Chandy, 1999; Park et al., 1997

Type of molecule Effects on biomaterial References

The fixation reaction was carried out by the exposure of the tissue balanced with glutaraldehyde acetals solutions to triethylamine vapours. This process allowed the diffusion of the non-reactive glutaraldehyde into the tissue, minimized the formation of polymeric glutaraldehyde and reduced the waterproofing (hydrophobicity) at the tissue surface (Yoshioka & Giossis, 2008).

The conditions of the crosslinking reaction (pressure for instance) have been varied with the aim of improving the biomechanical properties of bovine perichardium. The crosslinking of bovine perichardium with glutaraldehyde at a pressure of 4 mm Hg (low pressure) both statically and dynamically (1.2 Hz) has been reported. By comparing the properties of crosslinked bovine perichardium, the dynamically crosslinked tissue showed a very similar extensibility to native biomaterial (non-crosslinked) in contrast to statically crosslinked tissue, which showed a higher extensibility, while no differences were reported in other mechanical properties (Duncan & Boughner, 1998). The bovine perichardial fixation with glutaraldehyde under biaxial static pressures (~225 and ~1875 mmHg) has been proposed. The bovine perichardium treated at high pressure showed an increase in stiffness and almost isotropic behaviour, while low pressure-treated bovine perichardium preserved the anisotropy exhibited by the native tissue (Langdon, et al., 1999). Porcine valves have also been subjected to crosslinking at high pressure (80 mm Hg), low and zero pressure. In this case, it was reported an increase in the rigidity of the leaflets fixed under low pressure and the preservation of geometric corrugations and undulations of the native tissue when the leaflet were fixed without pressure (Lee et al., 1984).

Heat treatment during glutaraldehyde fixation has also been reported. The thermal treatment at 50ºC showed an anti-calcifying effect which was attributed to structural changes in collagen or lipid extraction by heat treatment (Carpentier et al., 2001).

#### **4.1.1 Post-treatments after glutaraldehyde fixation**

The residual unbounded aldehyde groups that remain in the tissue after glutaraldehyde fixation process have been associated with degenerative phenomenum on different bioprosthesis. The grafting of different molecules on collagenous tissues treated with glutaraldehyde has been an answer to these disadvantages.

The grafted molecules are incorporated in order to block free aldehyde groups and thus to reduce or to neutralize both cytotoxicity and calcification. Some surface modification procedures of crosslinked collagenous tissues are described in table 5.

It is known that nitric oxide releasing compounds can improve the biocompatibility of blood-contacting medical devices (Frost et al., 2005; Masters et al., 2005). Two common nitric oxide generating substances immobilized on synthetic polymers are diazeniumdiolates and S-nitrosothiols (Frost et al., 2005). In the same line of thought, surface modification of polymeric materials, such as PET or PU, with thiol compounds is interesting as it might exchange nitric oxide with endogenous donors such as S-nitrosothiols that already circulate in blood (Gappa-Fahlenkamp et al., 2004; Gappa-Fahlenkamp & Lewis, 2005).

The thiol groups on the polymer allowed the exchange reaction with S-nitroso serum albumin and then, the release of nitric oxide to inhibit platelet adhesion on the polymeric surfaces (Duan & Lewis, 2002). This approach has been proposed in perichardial tissue biomaterial by using L-cysteine as thiol compound (Mendoza-Novelo & Cauich-Rodríguez, 2009). One additional advantage of L-cysteine grafting on glutaraldehyde-crosslinked perichardial tissue is that free aldehyde groups will be diminished or even eliminated on the tissue allowing its detoxification. A schematic representation of grafting of collagenous tissue with L-cysteine is described in the figure 5. Similar approaches with other amino

The fixation reaction was carried out by the exposure of the tissue balanced with glutaraldehyde acetals solutions to triethylamine vapours. This process allowed the diffusion of the non-reactive glutaraldehyde into the tissue, minimized the formation of polymeric glutaraldehyde and reduced the waterproofing (hydrophobicity) at the tissue

The conditions of the crosslinking reaction (pressure for instance) have been varied with the aim of improving the biomechanical properties of bovine perichardium. The crosslinking of bovine perichardium with glutaraldehyde at a pressure of 4 mm Hg (low pressure) both statically and dynamically (1.2 Hz) has been reported. By comparing the properties of crosslinked bovine perichardium, the dynamically crosslinked tissue showed a very similar extensibility to native biomaterial (non-crosslinked) in contrast to statically crosslinked tissue, which showed a higher extensibility, while no differences were reported in other mechanical properties (Duncan & Boughner, 1998). The bovine perichardial fixation with glutaraldehyde under biaxial static pressures (~225 and ~1875 mmHg) has been proposed. The bovine perichardium treated at high pressure showed an increase in stiffness and almost isotropic behaviour, while low pressure-treated bovine perichardium preserved the anisotropy exhibited by the native tissue (Langdon, et al., 1999). Porcine valves have also been subjected to crosslinking at high pressure (80 mm Hg), low and zero pressure. In this case, it was reported an increase in the rigidity of the leaflets fixed under low pressure and the preservation of geometric corrugations and undulations of the native tissue when the

Heat treatment during glutaraldehyde fixation has also been reported. The thermal treatment at 50ºC showed an anti-calcifying effect which was attributed to structural

The residual unbounded aldehyde groups that remain in the tissue after glutaraldehyde fixation process have been associated with degenerative phenomenum on different bioprosthesis. The grafting of different molecules on collagenous tissues treated with

The grafted molecules are incorporated in order to block free aldehyde groups and thus to reduce or to neutralize both cytotoxicity and calcification. Some surface modification

It is known that nitric oxide releasing compounds can improve the biocompatibility of blood-contacting medical devices (Frost et al., 2005; Masters et al., 2005). Two common nitric oxide generating substances immobilized on synthetic polymers are diazeniumdiolates and S-nitrosothiols (Frost et al., 2005). In the same line of thought, surface modification of polymeric materials, such as PET or PU, with thiol compounds is interesting as it might exchange nitric oxide with endogenous donors such as S-nitrosothiols that already circulate

The thiol groups on the polymer allowed the exchange reaction with S-nitroso serum albumin and then, the release of nitric oxide to inhibit platelet adhesion on the polymeric surfaces (Duan & Lewis, 2002). This approach has been proposed in perichardial tissue biomaterial by using L-cysteine as thiol compound (Mendoza-Novelo & Cauich-Rodríguez, 2009). One additional advantage of L-cysteine grafting on glutaraldehyde-crosslinked perichardial tissue is that free aldehyde groups will be diminished or even eliminated on the tissue allowing its detoxification. A schematic representation of grafting of collagenous tissue with L-cysteine is described in the figure 5. Similar approaches with other amino

changes in collagen or lipid extraction by heat treatment (Carpentier et al., 2001).

surface (Yoshioka & Giossis, 2008).

leaflet were fixed without pressure (Lee et al., 1984).

**4.1.1 Post-treatments after glutaraldehyde fixation** 

glutaraldehyde has been an answer to these disadvantages.

procedures of crosslinked collagenous tissues are described in table 5.

in blood (Gappa-Fahlenkamp et al., 2004; Gappa-Fahlenkamp & Lewis, 2005).

acids have been suggested in order to provide non-cytotoxic tissue biomaterial and biomaterial with reduced calcification, as it is shown in table 5. In this table, it is also included molecules of biological importance as well as peptide sequences used to improve cell adhesion after the fixation treatment.


Table 5. Molecules grafted on crosslinked bovine perichardial tissue

Fig. 5. Schematic representation of tissue crosslinking with glutaraldehyde and chemical coupling of L-cysteine

Decellularization, Stabilization and Functionalization of

implantation in rats) of engineered tissue (Everaerts et al., 2004).

**4.3 Tissue crosslinking with epoxy compounds** 

low crosslinking.

shown in figure 7.

Collagenous Tissues Used as Cardiovascular Biomaterials 171

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

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

The appearance of bovine perichardial tissue crosslinked with glutaraldehyde and EDAC is

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).
