**3.1 Effect of decellularization treatment on tissue properties**

A biomaterial or scaffold for tissue engineering should provide not only mechanical support for the cell proliferation but also they must be versatile to give the required anatomical shape (Kidane et al., 2009). The decellularization of collagenous tissues has been explored as the ECM may serve as appropriate biological scaffold for cell attachment and proliferation. However, alterations both in the structural composition and in the mechanical properties of the remaining ECM can be induced during the decellularization protocols. The mechanical integrity can be affected and it may be associated either to the denaturation of the collagen triple helix or to the loss of macromolecular substances such as glycoproteins.

The efficiency of a given decellularization method and their effects on the properties of animal tissues must be studied in a specific manner due to compositional and structural differences (Gilbert et al., 2006). For example, the decellularization of porcine heart valve with sodium dodecyl sulphate, an anionic detergent, appeared to maintain the critical mechanical and structural properties of the valves leaflets (Liao et al., 2008) while decellularization of bovine perichardium with sodium dodecyl sulphate caused irreversible swelling, resulted in a reduction of the denaturation temperature (Courtman et al., 1994; García-Páez et al, 2000) and caused a reduction of almost 50% on tensile strength when compared to native tissue and tissue treated with Triton X100, a non-ionic detergent (Mendoza-Novelo & Cauich-Rodríguez, 2009).

Decellularization, Stabilization and Functionalization of

calcification in the cell membrane (Schmidt & Baier, 2000).

Alteration in the collagen

Removal of Cardiolipin

Alcohols Removal of phospholipids and cholesterol

> conformation Cellular death

calcification (Jorge-Herrero et al., 1994).

**4. Stabilization of tissues** 

(Mendoza-Novelo et al., 2011).

Pretreatment

Collagenous Tissues Used as Cardiovascular Biomaterials 165

However, the alkaline treatment altered the perichardial tissue stress relaxation behaviour

Bovine perichardium undergoes several treatments prior to crosslinking with the aim to improve its biocompatibility, to reduce immunogenicity, to decrease its tendency to calcification, to promote neo-vascularization and infiltration, and to increase cell adhesion and proliferation. Some of the pre-treatments proposed in the literature to reduce calcification of cardiovascular bioprostheses are showed in the table 2. It has been reported that with the treatment of bioprostheses with sodium dodecyl sulphate and Triton™X-100 most of the acidic phospholipids are extracted resulting in the initial suppression of

**3.2 Pre-treatments (pre-crosslinking) methods to reduce tissue calcification** 

Anti-calcification action mode Reference

Table 2. Tissue pretreatment in order to reduce the bioprostheses calcification

Surfactants Removal of acidic phospholipids Schmidt & Baier, 2000; Chang et al.,

The pretreatment of collagen-rich biomaterials with different concentrations of ethanol may prevent calcification through the extraction of phospholipids and cholesterol but causes a permanent alteration in the collagen conformation (Schmidt & Baier, 2000). Additionally, this treatment affected the interaction of the tissue with water and lipids and increased the resistance of the tissue to the action of collagenase. Several high molecular weight alcohols have been used in order to remove cellular components that contain elements responsible for the calcification (Pathak et al., 2002). The pretreatment with 50% ethanol for 5 min reduces fibrosis of bovine perichardium implanted in the aorta of sheep as a result of cell death and cardiolipin removal more than the phospholipids extraction (Vyavahare et al., 1997). Mixtures of chloroform/methanol have also been effective in reducing tissue

The stability of tissues is increased by physical or chemical crosslinking. The fixation enhances tissue stability, inhibits autolysis, allows a prolonged shelf-life, and allows a surgeon to have medical devices of various sizes readily available for implantation (Schoen & Levy, 1999). The chemical treatments also mitigate immunogenicity while maintaining both thromboresistance and antimicrobial sterility but greatly influence their degradation and calcification. However, tissue calcification is multifactorial phenomenum where chemical crosslinking is considered just one of these factors. In fact, the alteration in the electrical charge that exists in the perichardial tissue surface has been associated to the

2004

Pathak et al., 2002

Vyavahare et al. 1997; Pfau et al. 2000;

Fig. 4. Histological micrographs for native (a),(c) and decellurized (b),(d) perichardial tissue in H&E (top) and alcian blue (bottom) staining

It has been proposed that an anionic detergent binds to proteins, increases negative charges and results in tissue irreversible swelling (Courtman et al., 1994). In addition, a highly negative charged perichardial tissue has been associated to a higher tendency to tissue calcification (Jorge-Herrero et al., 2010). Due to these adverse effects, non-ionic detergents are preferred over ionic surfactants in the decellularization process of perichardial tissue. However, there are some issues related to the use of aromatic (phenolic) or non-aromatic (non-phenolic) non-ionic detergents used in the decellularization process. For example, the biodegradation products of derivatives of non-ionic detergents such as alkylphenol ethoxylates have been associated to toxicity (Argese et al., 1994) and estrogenic effects (Soto et al., 1991; Jobling & Sumpter et al., 1993). Figure 4 shows the histological results for bovine perichardial tissue decellularized with a non-aromatic non-ionic detergents. In this case, a reduction in the cell nuclei present in bovine perichardial tissue and a decrease in the glycosaminoglycan content after decellularization treatment were observed (Mendoza-Novelo et al., 2011).

In addition to tissue decellularization with nonionic surfactants, reversible swelling has also been studied. In this case, the reversible alkaline swelling did not change the threedimensional architecture of native bovine perichardium. This means that the laminar structure and fibrous nature of the native perichardial tissue were maintained after decellularization although the opening of the interfibrilar spaces was observed. The reversible alkaline swelling cause a reversible change in the tissue thickness i.e. increased 45% after swelling step, but the tissue original thickness was regained after deswelling step.

Fig. 4. Histological micrographs for native (a),(c) and decellurized (b),(d) perichardial tissue

It has been proposed that an anionic detergent binds to proteins, increases negative charges and results in tissue irreversible swelling (Courtman et al., 1994). In addition, a highly negative charged perichardial tissue has been associated to a higher tendency to tissue calcification (Jorge-Herrero et al., 2010). Due to these adverse effects, non-ionic detergents are preferred over ionic surfactants in the decellularization process of perichardial tissue. However, there are some issues related to the use of aromatic (phenolic) or non-aromatic (non-phenolic) non-ionic detergents used in the decellularization process. For example, the biodegradation products of derivatives of non-ionic detergents such as alkylphenol ethoxylates have been associated to toxicity (Argese et al., 1994) and estrogenic effects (Soto et al., 1991; Jobling & Sumpter et al., 1993). Figure 4 shows the histological results for bovine perichardial tissue decellularized with a non-aromatic non-ionic detergents. In this case, a reduction in the cell nuclei present in bovine perichardial tissue and a decrease in the glycosaminoglycan content after decellularization treatment were observed (Mendoza-

In addition to tissue decellularization with nonionic surfactants, reversible swelling has also been studied. In this case, the reversible alkaline swelling did not change the threedimensional architecture of native bovine perichardium. This means that the laminar structure and fibrous nature of the native perichardial tissue were maintained after decellularization although the opening of the interfibrilar spaces was observed. The reversible alkaline swelling cause a reversible change in the tissue thickness i.e. increased 45% after swelling step, but the tissue original thickness was regained after deswelling step.

in H&E (top) and alcian blue (bottom) staining

Novelo et al., 2011).

However, the alkaline treatment altered the perichardial tissue stress relaxation behaviour (Mendoza-Novelo et al., 2011).

### **3.2 Pre-treatments (pre-crosslinking) methods to reduce tissue calcification**

Bovine perichardium undergoes several treatments prior to crosslinking with the aim to improve its biocompatibility, to reduce immunogenicity, to decrease its tendency to calcification, to promote neo-vascularization and infiltration, and to increase cell adhesion and proliferation. Some of the pre-treatments proposed in the literature to reduce calcification of cardiovascular bioprostheses are showed in the table 2. It has been reported that with the treatment of bioprostheses with sodium dodecyl sulphate and Triton™X-100 most of the acidic phospholipids are extracted resulting in the initial suppression of calcification in the cell membrane (Schmidt & Baier, 2000).


Table 2. Tissue pretreatment in order to reduce the bioprostheses calcification

The pretreatment of collagen-rich biomaterials with different concentrations of ethanol may prevent calcification through the extraction of phospholipids and cholesterol but causes a permanent alteration in the collagen conformation (Schmidt & Baier, 2000). Additionally, this treatment affected the interaction of the tissue with water and lipids and increased the resistance of the tissue to the action of collagenase. Several high molecular weight alcohols have been used in order to remove cellular components that contain elements responsible for the calcification (Pathak et al., 2002). The pretreatment with 50% ethanol for 5 min reduces fibrosis of bovine perichardium implanted in the aorta of sheep as a result of cell death and cardiolipin removal more than the phospholipids extraction (Vyavahare et al., 1997). Mixtures of chloroform/methanol have also been effective in reducing tissue calcification (Jorge-Herrero et al., 1994).
