4.3. BIOMATERIAL CLASSIFICATION 43

protect the seeded or recruited cells and maintain their structure under mechanical perturbations existing during cultivation and at implant site. At the same time, the scaffold mechanical properties should be compatible with the host tissue to allow its integration without interfering with the normal function of the organ. This is especially critical when biomaterial is used as ECM replacement of damaged myocardium.

Biodegradation/bioresorption – Ideally, the scaffold should disappear from the host when tissue regeneration has been accomplished and normal function was restored. Biodegradable scaffolds can do so via polymer backbone degradation (e.g., hydrolysis, enzymatic cleavage) or by dissolution of the matrix. It is fundamental that the products of this process would be biocompatible and be resorbed by the body or removed from it via excretion from the urine.

Scaffold fabrication – Ideally, this process should be mild, using safe reagents and not affecting material properties, such as its cell recognition motifs. For example, cross-linking between polymer chains is often used in hydrogel fabrication from natural materials such as alginate, collagen, hyaluronan, and others. Cross-linking can be physical, where the polymer chains self-assemble due to electrostatic interactions, response to temperature and irradiation, or chemical, where covalent bonds are introduced between the polymer chains. Chemical crosslinking often changes the material properties (degradability, mechanical strength, and cell recognition) due to the lack of precise control over the position where the crosslink linkages are formed. In addition, chemical cross-linking often involves the use of harsh reagents, thus raising concerns about the material biocompatibility.

Scaffold internal morphology – When used as scaffolding for cells, the matrix should be porous with interconnecting pore structure and pore size larger than 50 μm, to enable cell-cell interactions and construct vascularization after implantation.

4.3BIOMATERIAL CLASSIFICATION

Polymers commonly used for scaffold fabrication can be categorized by their source origin (natural or synthetic) and by their chemical structure (peptides/proteins, polysaccharides, polyesters, and others).

Natural polymers are usually biocompatible, easy to be chemically and physically modified and to be processed into various structures. Most of them, such as hyaluronan and collagen, possess cell recognition patterns enabling them to stimulate cell response. There are, however, concerns over their use in human therapy because of the risks of pathogen transmission and immune rejection associated with natural polymers that are produced from animal and cadaver sources.

Synthetic polymers can be designed with versatile properties, such as mechanical strength and biodegradation rate, and can be tailored with functional groups. A disadvantage that can be found in synthetic polymers, with respect to natural polymers, is the lack of the biological cues for promoting cell responses.

44 4. BIOMATERIALS – POLYMERS, SCAFFOLDS, AND BASIC DESIGN CRITERIA

Table 4.1 lists the main materials used in cardiac tissue engineering strategies, as components in cellular constructs or standalone biomaterials treatments, and presents their main characteristics according to the design criteria listed above, namely, biocompatibility, mechanical strength, biodegradability/bioresorption, fabrication/cross-linking, and interactions with cells.

Table 4.1: Main biomaterials applied in cardiac tissue engineering [3, 4, 5, 6, 7, 8, 9]

-

4.3.1NATURAL PROTEINS

Collagen is a fibrous protein and the main component of ECM of mammalian tissues.About 25 types of collagen different in their chemical composition and molecular structure have been identified. Among the different collagen types, the fibrillar Type I collagen is the most abundant in nature and easy to produce. The biocompatibility, biodegradability, and cell-adhesive properties of the collagen type I matrix attributed to its selection by most researchers as the candidate scaffold for tissue growth and support. Collagen can be fabricated in many forms, such as hydrogel or macroporous scaffold; its fabrication frequently requires chemical cross-linking, which may affect its biological recognition by cells, biocompatibility, and degradability. Collagen is already commercialized as injectable product, thus it has been recognized as safe material by regulatory agencies. Two collagen-based products containing bone morphogenic protein (BMP)-2 or BMP-7 have been approved by the Food and

4.3. BIOMATERIAL CLASSIFICATION 45

Drug Administration (FDA) in recent years for human clinical use: Infuse Bone Graft (Medtronik, US; Wyeth, UK), containing rhBMP-2, and Osigraft (Stryker Biotech) containing BMP-7.

In cardiac tissue engineering and myocardial repair after MI, collagen type I has been widely used as hydrogels or macroporous scaffolds for reconstructing the cardiac patch, either with or without seeded cells, as will be described in detail in upcoming chapters [8].

Gelatin (the irreversibly hydrolyzed form of collagen) has also been used in myocardial tissue engineering, especially in the format of a hydrogel prepared by chemical cross-linking. Gelatin is a biodegradable material, but under various conditions it can provoke an unspecific inflammatory response [10].

Fibrin(ogen) is a natural scaffold protein that is used extensively for medical applications (e.g., surgical adhesive sealant) and for myocardial tissue engineering. It is FDA-approved due to its favorable wound healing properties. Fibrinogen circulates as an inactive precursor in circulation and is recruited to injury sites where it becomes activated by proteolytic cleavage; the covalent crosslinking of fibrin by thrombin and factor XIIIa form the fibrin clot, a complex network, composed of fibrils. Fibrin clots provide a natural wound healing matrix that can be remodeled via cellular activities to form the tissue-specific mature ECM. Fibrin scaffolds were prepared as hydrogels and in injectable form, wherein its components (fibrinogen and thrombin) are mixed during injection into the tissue. Their application as 3D scaffolds for in-vitro cell culture is limited in time (4–7 days) due to their rapid degradation by enzymes secreted by the seeded cells. In vivo, they have been shown to increase cell retention after transplantation into infarcted hearts and in some studies, fibrin alone caused improvement in cardiac function, as will be described in Chapter 9 [8].

4.3.2NATURAL POLYSACCHARIDES

Alginate is an anionic polysaccharide extracted from brown algae, composed of 1→4 linked β- D-mannuronic acid (M) and α-L-guluronic acid (G) (Fig. 4.2). Divalent cations, such as calcium ions, interact with high affinity with the G monomer blocks to form ionic bridges between different alginate chains (“egg box” model) eventually leading to hydrogel formation (Fig. 4.2).

The physical cross-linking of alginate represents a significant advantage, as the use of various chemical agents for gelation is eliminated. Since there is no known mammalian enzyme which degrades the alginate backbone, it is assumed that alginate is not degradable in mammals. Yet, the calcium-cross-linked hydrogel is readily erodible with time due to exchange of calcium ions by sodium ions in physiological milieu, leading to hydrogel dissolution. The water-soluble alginate chains are excreted through the kidney if the molecular weight is below 50 kDa [11].

The alginate matrix is inert and resistant to cell adhesion, thus it can be used as a “blank canvas” to investigate specific cell-matrix interactions by attaching biological cues, as will be described in detail in Chapter 6. The long experience with alginate as cell matrix and implant indicates its biocompatibility.

Chitosan is a biodegradable cationic polysaccharide made of D-glucosamine and N -acetyl-D- glucosamine linked by β(1,4) glycoside bonds. Due to its positive charge, it can ionically interact with

46 4. BIOMATERIALS – POLYMERS, SCAFFOLDS, AND BASIC DESIGN CRITERIA

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Figure 4.2: Alginate structure and the egg-box model of hydrogel formation.

negatively charged polymers and/or ions and molecules. Chitosan is abundant in nature; it derives from the deacetylation of chitin that forms the crustaceous skeleton. The polymer is soluble in water at acidic pH, but it can be chemically modified and/or salified to make it soluble in physiological pH. It is degraded by enzymes, such as lysozymes. Due to its origin, a matrix made of chitosan does not support specific and high affinity interactions with mammalian cells, and attachments of recognition peptide to the polymer are required to enable such interactions. Scaffolds made of chitosan and/or its modified forms have been applied for cardiac tissue engineering, as further described in Chapter 9.

Hyaluronan (HA) is a linear un-sulfated polysaccharide, composed of repeating disaccharides [(1→3)-β-N-acetyl-D-glucosamine-(1→4)-β-D-glucoronic acid]. In the human body, HA is found primarily in the extracellular and pericellular matrix; its degradation occurs by hyaluronidases. The HA has versatile biological functions, such as a lubricant material and numerous receptormediated roles in different cell processes. The processing of HA as scaffolds for tissue engineering requires chemical modification of the material to achieve cross-linking, such as by photopolymerization. The un-cross-linked HA is not effective as an injectable material due to its poor mechanical properties, rapid degradation, and clearance in vivo [12, 13].

4.3.3SYNTHETIC PEPTIDES AND POLYMERS

Among the recently developed synthetic biomaterials, self-assembling peptides have become a favorable option as scaffold materials since they are composed of natural building blocks, they can be synthesized with the appropriate features, and the manufacturing of scaffolds from them is simple and mild. These peptides are usually 8-16 amino acids long and composed of alternating hydrophobic and hydrophilic residues. They form stable β-sheets in water, and upon exposure to physiological