**2. The biomaterial choice**

The replacement of damaged organs or tissues is one of the objectives of the biomaterials science. For this, natural or synthetic materials can be used for example in the cardiovascular field in the manufacture of heart valves and vascular grafts. The success of a device depends not only on the type of biomaterials but also on a set of acceptable characteristics such as biocompatibility, biostability, haemocompatibility, antitrombogenecity, resistance to degradation and calcification.

Among those biomaterials that can fulfil these requirements, natural tissues are good candidates and that is why they have been under investigation in the past fifty years.

#### **2.1 Composition and sources of natural tissue**

Natural tissue biomaterial can be obtained from either animal-derived tissue (xenograft) or human-derived tissue (homograft). However, due to the limited availability of autografts, animal-derived tissues are, in many cases, the first choice for cardiovascular biomaterials. Animal derived tissues widely used as biological biomaterials include perichardial tissue from various sources such as cows, calves and ostrich in addition to pig aortas.

Tissue-derived biomaterials are mainly comprised of collagen in addition to the tissue extracellular matrix (ECM) which is a complex mixture of structural and functional proteins such as collagen, proteoglycans, glycoproteins, elastin, metalloproteins, etc. Collagen, being the main structural protein, is a polypeptide that contain amino (-NH2), carboxylic acid (- COOH) and hydroxyl (-OH) functional groups as substituents, and together with the amide bonds in the polymer backbone form the reactive centers. The repetitive unit in the polymer backbone of collagen and the amino acid residues as side group are depicted in figure 1.

Fig. 1. Representation of the repetitive unit of collagen and some side group R of amino acid residue

linked to the tissue crosslinking to increase durability. However, due to some complications in the stabilized tissue, several post-crosslinking protocols have been proposed to address these complications. More recently, biological scaffolds derived from acellular tissue has

Therefore, this chapter deals with the processing of collagenous tissue for the preparation of cardiovascular biomaterials. The processing techniques include the extraction of cellular and nuclear material by various decellularization methods, the preservation of tissue through of crosslinking reactions, hydrogen-bond interactions or interstitial space filling, and the functionalization or the blocking of free groups with various low molecular and

The replacement of damaged organs or tissues is one of the objectives of the biomaterials science. For this, natural or synthetic materials can be used for example in the cardiovascular field in the manufacture of heart valves and vascular grafts. The success of a device depends not only on the type of biomaterials but also on a set of acceptable characteristics such as biocompatibility, biostability, haemocompatibility, anti-

Among those biomaterials that can fulfil these requirements, natural tissues are good

Natural tissue biomaterial can be obtained from either animal-derived tissue (xenograft) or human-derived tissue (homograft). However, due to the limited availability of autografts, animal-derived tissues are, in many cases, the first choice for cardiovascular biomaterials. Animal derived tissues widely used as biological biomaterials include perichardial tissue

Tissue-derived biomaterials are mainly comprised of collagen in addition to the tissue extracellular matrix (ECM) which is a complex mixture of structural and functional proteins such as collagen, proteoglycans, glycoproteins, elastin, metalloproteins, etc. Collagen, being the main structural protein, is a polypeptide that contain amino (-NH2), carboxylic acid (- COOH) and hydroxyl (-OH) functional groups as substituents, and together with the amide bonds in the polymer backbone form the reactive centers. The repetitive unit in the polymer backbone of collagen and the amino acid residues as side group are depicted in figure 1.

Fig. 1. Representation of the repetitive unit of collagen and some side group R of amino acid

candidates and that is why they have been under investigation in the past fifty years.

from various sources such as cows, calves and ostrich in addition to pig aortas.

been used in tissue engineering and regenerative medicine.

trombogenecity, resistance to degradation and calcification.

**2.1 Composition and sources of natural tissue** 

macromolecular substances.

**2. The biomaterial choice** 

residue

The crystal lattice of collagen fibers are embedded in an amorphous matrix. The amorphous matrix is composed mainly by glycosaminoglycans as proteoglycans (sulphated glycosaminoglycans bound to proteins). In this matrix, in addition to the fibers, tissue cells and interstitial fluid (water or electrolytes) are embedded. The glycosaminoglycans are negatively charged polysaccharides of varying degrees of complexity. The glycosaminoglycan polymers consist of repeating disaccharide units, usually consisting of a hexosamine and an uronic acid (Yeung et al., 2001). The charged negatively units contribute to the elasticity and hydration of the tissues (Mavrilas et al., 2005), but may attract counter-ions, which could intervene in the processes of calcification of bioprostheses. The repetitive disaccharide unit of glycosaminoglycans mainly presents in native bovine perichardium is shown in figure 2.

Fig. 2. Repetitive disaccharide units of common glycosaminoglycans in bovine perichardial tissue

The different soft tissues including cartilage, tendons, ligaments, skin and perichardium have the capacity of support mechanical load of variable magnitude. Therefore, the properties of the tissue depend on the number and the arrangement of collagen fibers, which can be parallel or perpendicular to the surface or randomly distributed in the matrix. The hierarchical nature of collagen confers to the tissue its structural complexity. The fibrous nature of bovine perichardial tissue is revealed in figure 3. In perichardial tissue, a multi-laminate structure is observed with difference in both serosa (Fig. 3b) and rugosa surface (Fig. 3a).

Fig. 3. SEM micrographs for the fibrosa (a) and the serosa surface (b) of native bovine perichardium

Decellularization, Stabilization and Functionalization of

biomaterials as scaffolds for tissue engineering.

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

(Mendoza-Novelo & Cauich-Rodríguez, 2009).

triple helix or to the loss of macromolecular substances such as glycoproteins.

**3. Decellularization of tissues** 

decellularization process.

Collagenous Tissues Used as Cardiovascular Biomaterials 163

inflammatory degradation, mechanical damage and pannus overgrowth (Zilla et al., 2008). In general, the stabilization of collagen-rich tissue is achieved by direct binding of functional groups to amino acid residues from collagen by coupling agents or by the linkage between the functional groups on collagen and various chemical agents. Both processes are referred in literature as the fixation or crosslinking processes. While the crosslinking agents make durable, stable and resistant tissues, the crosslinking density and the chemical process seems to have an effect on some of the major disadvantages of bioprostheses, such as calcification (Zilla et al., 2008). For this reason, a large number of crosslinking agents have been suggested with the aim of obtaining bioprosthesis that fulfill successfully its function. In addition to this treatment, there are reports on the post-crosslinking and pre-crosslinking treatments in order to reduce the calcification of biomaterial and in order to prepare porous

The concept of decellularization is referred as the extraction of cellular components from natural tissues of human or animal origin. Different approaches have been reported as effective procedures to remove cells from xenogeneic and allogeneic collagenous tissue with the aim of removing cellular antigens and procalcifying remnants while the extracellular matrix (ECM) integrity is preserved as much as possible (Schmidt & Baier, 2000). The combination of chemical, physical and enzymatic methods destroys the cell membrane and removes nuclear and cellular material (Gilbert et al., 2006). The remaining acellular ECM will be a complex mixture of structural and functional proteins, glycoproteins and glycosaminoglycans arranged in a three-dimensional architecture. However, some mechanical and structural alterations on the ECM can be induced during the

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

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

#### **2.2 Properties of collagenous tissues**

Collagen-rich tissues are composed of 75% of collagen, 20% of mucopolysaccharides and water, although elastin can be found in less than 5% (Cauich-Rodríguez, 2008). All these tissue components maintain the structural and functional integrity of the composite tissue. Some mechanical properties of collagenous tissues are shown in table 1.


Table 1. Mechanical properties of some collagenous tissue

The thermal transitions experienced by materials with amorphous and/or crystalline regions are also observed in the collagenous tissue. When the biomaterial is heated, its specific volume increases, exhibiting the glass transition of amorphous regions and the fusion of crystalline collagen fibers to a temperature higher than the glass transition temperature (Li, 2007). The melting temperature of collagen fibers is an irreversible process and is often referred in the literature as the denaturation temperature (Td) or shrinkage temperature (Ts). In fact, the denaturation temperature is widely used as an indicator of the tissue stabilization.

The collagenous tissues require chemical or physical treatments in order to be preserved or stabilized. In fact, the introduction of cardiovascular bioprostheses in 1960s was linked to the chemical fixation of porcine aortic valves or bovine perichardial tissue with glutaraldehyde. This process produces a non-living material without the capability of intrinsic repair as native tissue does after some structural injury (Flanagan & Pandit, 2003). The processed tissue tends to fail in modes related to the remnant immunogenicity,

Collagen-rich tissues are composed of 75% of collagen, 20% of mucopolysaccharides and water, although elastin can be found in less than 5% (Cauich-Rodríguez, 2008). All these tissue components maintain the structural and functional integrity of the composite tissue.

> 10.9 MPa

33.0 %

58.2 MPa

18.4 MPa

21.4 %

198 MPa

2.51 MPa

34.9 %

20.4 MPa

6.25 MPa

30.8 %

54.6 MPa

The thermal transitions experienced by materials with amorphous and/or crystalline regions are also observed in the collagenous tissue. When the biomaterial is heated, its specific volume increases, exhibiting the glass transition of amorphous regions and the fusion of crystalline collagen fibers to a temperature higher than the glass transition temperature (Li, 2007). The melting temperature of collagen fibers is an irreversible process and is often referred in the literature as the denaturation temperature (Td) or shrinkage temperature (Ts). In fact, the denaturation temperature is widely used as an indicator of the

The collagenous tissues require chemical or physical treatments in order to be preserved or stabilized. In fact, the introduction of cardiovascular bioprostheses in 1960s was linked to the chemical fixation of porcine aortic valves or bovine perichardial tissue with glutaraldehyde. This process produces a non-living material without the capability of intrinsic repair as native tissue does after some structural injury (Flanagan & Pandit, 2003). The processed tissue tends to fail in modes related to the remnant immunogenicity,

1981

Lee at el., 1989

Lee & Boughner, 1985

Lee at el., 1984

Lee & Boughner, 1981; Wiegner & Bing,

Tissue Property Value Reference

Some mechanical properties of collagenous tissues are shown in table 1.

strength

Strain at rupture

Tissue modulus

strength

Strain at rupture

Tissue modulus

strength

Strain at rupture

Tissue modulus

Tensile strength

Strain at rupture

Tissue modulus

Table 1. Mechanical properties of some collagenous tissue

**2.2 Properties of collagenous tissues** 

Bovine perichardium Tensile

Canine perichardium Tensile

Human perichardium Tensile

Porcine aortic valve

tissue stabilization.

leaflet

inflammatory degradation, mechanical damage and pannus overgrowth (Zilla et al., 2008). In general, the stabilization of collagen-rich tissue is achieved by direct binding of functional groups to amino acid residues from collagen by coupling agents or by the linkage between the functional groups on collagen and various chemical agents. Both processes are referred in literature as the fixation or crosslinking processes. While the crosslinking agents make durable, stable and resistant tissues, the crosslinking density and the chemical process seems to have an effect on some of the major disadvantages of bioprostheses, such as calcification (Zilla et al., 2008). For this reason, a large number of crosslinking agents have been suggested with the aim of obtaining bioprosthesis that fulfill successfully its function. In addition to this treatment, there are reports on the post-crosslinking and pre-crosslinking treatments in order to reduce the calcification of biomaterial and in order to prepare porous biomaterials as scaffolds for tissue engineering.
