**2.4. Chemical cross-linking methods**

response of the ECM hydrogels have shown a direct relationship with the ECM remaining composition [11]. The understanding of the formation process of collagen gels is of relevant importance for the development of strategies capable to synthesize them successfully. The

The physical methods for the modification of the ECM hydrogels are related to the physical cross-linking of collagen fibers caused by pH, temperature, electrical fields or other physical stimuli, as schematized in **Figure 3** [22]. The advantages of this type of process are the relatively easy manufacture, and the absence of exogenous cross-linking agents, which could reduce the toxicity risks [23]. The variation of pH and temperature of the collagen solution during the in vitro fibrillogenesis produces the collagen cross-linking and increases the fiber size [11, 24]. The temperature-dependent process is reversible [25]. Commonly, the physical methods are not associated with a significant improvement of the mechanical properties of ECM hydrogels,

An interesting physical method to improve the mechanical properties of ECM hydrogels is to apply lyophilization cycles. In this methodology, extracted collagen is incubated at 37°C during 24 h to induce the collagen polymerization, later the hydrogel is frozen at −20°C for 3 h, −80°C for 3 h, and in liquid nitrogen, and then lyophilized. The resulting collagen network demonstrated highly aligned fibrillar features along the scaffold surface, decreased pore size, and increased mechanical properties [27]. However, a major disadvantage related to the

**2.2. Collagen gel formation in response to change of pH and temperature**

**Figure 2.** The ECM composition as source for the preparation of collagen-based hydrogels.

limiting the use of these methods in the preparation of biomedical hydrogels [26].

next subsections are focused on those strategies.

6 Hydrogels

The search for an ideal procedure to stabilize the structure of collagen maintaining its physical integrity and natural conformation has led to the evaluation of diverse strategies to form covalent bonds. As shown in **Figure 5**, this takes advantage of the conjugation of reactive groups of collagen molecule such as carboxylate (─COO─) and amine (─NH<sup>2</sup> ) with reactive cross-linkers. Among the most studied processes are the glutaraldehyde (a pentadialdehyde) cross-linking,

**Figure 4.** Preparation of hydrogels derived from polymeric IPNs.

**Figure 5.** Chemical cross-linking to generate biomedical collagen-based hydrogels.

and the use of carbodiimide 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water-soluble carbodiimide), genipin, polyethylene glycol diacrylate (PEGDA), and aqueous polyurethane prepolymers. These methods increase the resistance of the hydrogel toward both chemical and enzymatic degradation, reduce its immunogenicity, sterilize and improve its mechanical properties. **Table 1** summarizes the main characteristics, advantages and disadvantages of the

**Advantages Disadvantages Ref.**

Decellularized ECM-Derived Hydrogels: Modification and Properties

Drastic reduction of the biocompatibility.

http://dx.doi.org/10.5772/intechopen.78331

The cross-linking reaction is not taken out at physiological conditions.

Generation of blue residues during the preparation of biomaterials, limiting their transparence and use as 3D

Limitations of use of UV irradiation for applications related to gelation in situ or cell encapsulation.

Higher concentrations of Pp inhibit the collagen polymerization, and decrease its biocompatibility.

culture systems.

[29]

9

[33–35]

[36–38]

[39]

[40]

The cross-linking reaction is relatively fast; reacting with most ε-amine groups, improving both mechanics and degradation resistance.

The degradation products of these biomaterials do not show cytotoxic

character.

The structure and properties of hydrogels show a direct relationship with the genipin concentration.

irradiation.

conditions. The structure and properties of collagen hydrogels show a direct relationship with the chemical structure of Pp. Pp accelerates the polymerization.

Hydrogels show enhanced hydrolytic stability, susceptibility to collagen enzymatic degradation. Mechanical properties depend on time of UV

The cross-linking process is taken out at physiological

The elucidation of the impact of the modification upon the structure and properties of the hydrogels derived from decellularized ECM requires the use of a combination of distinct techniques. A forthright correlation between modification and properties is key to balance stability and bioactivity. This chapter thus discusses some aspects of the methods used to discern the characteristics of collagen hydrogels and scaffolds and the implications of their

covalent chemical cross-linking methods.

**Collagen cross-linker Main characteristic of** 

Glutaraldehyde (GA) The ε-amine groups of

1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide (EDAC)

Poly(ethylene glycol) diacrylate (PEGDA)

Polyurethane prepolymers (Pp) **the process**

collagen yield an imine bond (so-called as Schiff base), when they react with a GA molecule.

Effective catalyst in the condensation of collagen carboxylic acids with alcohols and amines, without presence of the carbodiimide (so-called

zero-length).

linking by formation of Schiff base is produced. A Michael reaction is involved in this process.

Photo cross-linking based on the formation of covalent linkages among the functional groups acrylamide with the collagen-amines.

Pp based on PEG and aliphatic diisocyanates cross-links the collagen chains. The process involves the formation of urea linkages between end-blocked isocyanate of Pp and collagen-amines.

Genipin Spontaneous cross-

use as safe biomaterials with immunomodulatory properties.

**Table 1.** Chemical cross-linkers for the preparation of collagen-based hydrogels.


**Table 1.** Chemical cross-linkers for the preparation of collagen-based hydrogels.

and the use of carbodiimide 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water-soluble carbodiimide), genipin, polyethylene glycol diacrylate (PEGDA), and aqueous polyurethane prepolymers. These methods increase the resistance of the hydrogel toward both chemical and enzymatic degradation, reduce its immunogenicity, sterilize and improve its mechanical

**Figure 5.** Chemical cross-linking to generate biomedical collagen-based hydrogels.

**Figure 4.** Preparation of hydrogels derived from polymeric IPNs.

8 Hydrogels

properties. **Table 1** summarizes the main characteristics, advantages and disadvantages of the covalent chemical cross-linking methods.

The elucidation of the impact of the modification upon the structure and properties of the hydrogels derived from decellularized ECM requires the use of a combination of distinct techniques. A forthright correlation between modification and properties is key to balance stability and bioactivity. This chapter thus discusses some aspects of the methods used to discern the characteristics of collagen hydrogels and scaffolds and the implications of their use as safe biomaterials with immunomodulatory properties.
