**2. Methods of preparation of natural ECM-based hydrogels**

The versatility of collagen to generate biomedical hydrogels is primarily associated with its complex hierarchical structure originated from its amino acid molecular sequence and the formation of triple helical structures [8]. The collagen polymerization is a self-assembly process of long fibrillar structures, regulated by both electrostatic and hydrophobic interactions that promote the collagen fibers cross-linking [9]. This process is influenced by parameters such as temperature, pH, collagen concentration, and the presence of other biomolecules or polymers [10]. The macroscopic result of the in vitro collagen fibrillogenesis is a 3D water-swollen network.

ECM hydrogels are very suitable materials for biomedical applications due to their good interaction with living tissues, biocompatibility, soft and elastic consistency, high water content, and ECM remaining composition [11]. The swelling in liquid medium gives them the capacity to absorb, retain and release under controlled conditions amounts of water; regulating their structural conformation [12]. The ECM residual composition and the methods of modification of the hydrogels determinate the water uptake capacity and influence their biological and physicochemical properties. This section is dedicated to the discussion of the main characteristics of strategies for the modification of collagen in hydrogel state.

#### **2.1. Importance of the tissue source in the natural ECM-based hydrogels**

**Figure 1.** Schematic representation of the biomedical applications of collagen-based hydrogels.

of platelets, and the absorption of fluid, and regulating the deposition of other ECM's proteins such as fibrin, laminin, elastin, and fibronectin [2]. Besides, collagen can induce processes of the cell signalization involved in the growth, proliferation, migration, and differentiation of cells. Low inflammatory and cytotoxic responses and high biodegradability are other attractive properties of collagen [3]. The collagen can be extracted from diverse ECMs using acid hydrolysis assisted by proteolytic enzymes. The extracted collagen can be subsequently polymerized under physiological conditions (pH 7, 37°C) to generate a highly hydrated 3D network [4].

4 Hydrogels

The ECM-based hydrogels maintain the biocompatibility and biodegradability associated with the collagen. Diverse authors use to refer ECM hydrogels like collagen hydrogels, as the collagen is the major component inside ECM. However, these biomaterials have poor mechanical properties and fast degradation rate, limiting the range of use in applications such as the loading, encapsulating and controlled delivery of cells or drugs, or as wound care dressings [5]. The structure and mechanics of the ECM hydrogels can be modified by chemical cross-linking (using glutaraldehyde, genipin, carbodiimides, acrylates, oligourethanes, and among others); and/or by physical cross-linking (using freeze-drying cycles, forming interpenetrated networks (IPN) with other proteins or polymers). The selection of the cross-linking strategy has to consider the impact upon the structure-property relationships. After modification, several advances have been reported in the design of ECM-based hydrogels. The delivery of cells and biomolecules, the enhancing of the stiffness, the regulation of the cell-material interactions, the control of the cell fate and function, and the modulation of the environment of both normal and injured/diseased tissues are among them [6]. As shown in **Figure 1**, ECM hydrogels have been studied as substrates for ophthalmology, sponges for burns/wounds, systems for controlled delivery of functional molecules or

> The ECM is the noncellular component present within all tissues and organs that provides not only essential physical scaffolding for the cellular constituents but also initiates crucial biochemical and biomechanical cues, which are required for tissue morphogenesis, differentiation, and homeostasis [13]. As shown in **Figure 2**, this matrix is composed of a variety of proteins and polysaccharides that are locally secreted and assembled into an organized network in close association with the surface of the cells that produced them [14].

> Collagen is the main component of the ECM [15]. The collagen is extracted from diverse ECM by multistep processes including the tissue decellularization and acid hydrolysis assisted with proteases. Among others, collagen has been extracted from porcine dermis [16], bovine pericardium [17], porcine urinary bladder [18], porcine small intestine submucosa [19], bovine Achilles tendon [20], and rat tail tendon [21]. The polymerization of the extracted collagen under physiological conditions (37°C, pH 7) has allowed to develop biomedical hydrogels mimicking the structure and function of the ECM in vitro. The polymerization kinetics and the structural characteristics of the fibrillar collagen gel network are influenced by the residual composition of the ECM. Consequently, the swelling, mechanics, degradation, and biological

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

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 next subsections are focused on those strategies.

execution of freeze-drying cycles is the decrease in water uptake of the collagen scaffold [11]. Physical methods do not allow controlling the rate of degradation of collagen-based hydrogels [28]. Therefore, it is still necessary to investigate methods to regulate the characteristics and to expand the use of collagen scaffolds and hydrogels in the medical biotechnology field.

Decellularized ECM-Derived Hydrogels: Modification and Properties

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

7

IPN hydrogels are based on the physicochemical interactions between the collagen polymeric chains and chains of another type of polymer, as shown in **Figure 4**. The hydrophobic, ionic or hydrogen bonding inside the IPN is responsible for the improved mechanics and degradation behavior. Two examples are the IPNs formed between collagen and chitosan [29], and collagen and polyethylene oxide (PEG) [30]. In these approaches, the ECM extracted collagen is combined with different mass concentrations of polymers, and later this mixture is incubated at 37°C to induce the collagen polymerization. The polymerization process is influenced by the presence of the exogenous polymeric chains altering the collagen fiber size and the physical cross-linking. The IPN hydrogels show poor stability with the change of the temperature and pH [31]; but the enhanced mechanical properties of these biomaterials are adequate for

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

Among the most studied processes are the glutaraldehyde (a pentadialdehyde) cross-linking,

) with reactive cross-linkers.

collagen molecule such as carboxylate (─COO─) and amine (─NH<sup>2</sup>

**2.3. Interpenetrated networks (IPN) based on collagen and other polymers**

**Figure 3.** Physical methods for preparation of collagen-based hydrogels.

the cell and drug encapsulation [32].

**2.4. Chemical cross-linking methods**

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

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

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

**Figure 3.** Physical methods for preparation of collagen-based hydrogels.

execution of freeze-drying cycles is the decrease in water uptake of the collagen scaffold [11]. Physical methods do not allow controlling the rate of degradation of collagen-based hydrogels [28]. Therefore, it is still necessary to investigate methods to regulate the characteristics and to expand the use of collagen scaffolds and hydrogels in the medical biotechnology field.
