**3. Methods for physicochemical characterization of collagen-based hydrogels**

in the case of TEM is essential a good handle of the available staining techniques to avoid "artifacts" in the images. However, when those measurements are the reference for wet properties of the scaffold it can be taken only as a guide and other techniques on wet materials are needed

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Using atomic force microscopy (AFM) is possible to observe collagen fibers with different shape and structures on different surfaces [48, 54, 60] as well as to determine micromechanical properties of a collagen hydrogel; both in wet and dry formulations. AFM is microscopy based on the movement of a microtip, in the range of micro- and nanometers, that interacts with the surface at the microscopic level and sense its shape and roughness. The movements of the tip are followed by a laser on it; forming an image of deflection laser intensity. This image, if the

Another important set of characterization techniques are the X-ray techniques; although their availability depends strongly on the level of development of each particular scientific community in specific countries due to the extensive facilities and economic resources that they need. The more accessible are the X-ray photoelectron spectroscopy (XPS) and the small angle X-ray scattering (SAXS). XPS is able to measure the carbonyl, C═O, and C─C interaction on a collagen surface [53], as well as traces of silicon commonly used now in collagen-based scaffolds [11], for instance. It is in general, the right technique to obtain the gross chemical composition on the collagen surface and a definitive indication for traces of impurities of other elements [54]. Depending on the instrumentation available, wet samples can be measured, since X-rays must work under light vacuum. In addition, it is not expected to see under the surface because the X-ray source is weak and cannot penetrate the sample. On the other hand, scattering X-ray techniques can be used to get the shape, length and width, pore size, and fiber orientation directly from the sample, wet or dry, without further manipulation [55–57]. Depending on the distance to determine, normal X-ray diffraction equipment can also be used [58]. Those techniques are based on the scattering of the X-rays from the sample: a scattering vector is an inverse function of the X-ray wavelength and proportional to the sine of the half the scattering angle. A characteristic distance in the material scatters X-rays of a specific wavelength proportional to the scattering vector and with a different scattering intensity. This intensity is a function of the scattering angle, and it is from where the different properties of the material can be extracted. In general, using smaller scattering angles, it is possible to obtain information about larger distances, as those observed in the collagen when X-rays are used [59].

Mechanical properties (determined at microscopic or macroscopic level) of the collagen are those of a gel or an entangled polymer: basically, oscillatory rheology shows a plateau of the storage modulus (G´) in a frequency ranging between hertz and kilohertz, which can be considered as its Young's modulus, and three times this value can be determined by extensional and compressional experiments of strain versus stress [52]. The convergence of micro- and macromechanical moduli values are not common in the literature [42, 61], although it is

deflection force of the tip is known, forms also a map of micromechanical properties.

to confirm what is being observed.

**3.3. X-ray techniques**

**3.4. Mechanical tests**

A collagen scaffold is a hierarchical, protein-based fibrillary network: after the triple helix formation that conforms tropocollagen, it forms fibrils that align themselves in microfibers and finally, in collagen fibers of a tissue [41]. To correlate the different physicochemical properties observed in a collagen derivative scaffold with its hierarchical network is an attractive challenge partially explored. It is important to notice that the chemical modification of plain tropocollagen is a current practice to tune some properties as the mechanical ones. In general, the characterization methods for the collagen-based materials do not vary when it is chemically modified as we will see in this chapter. On the other hand, for composites that contain collagen, it is intuitive to imagine that the methods to determine their properties could be different depending on the other components of the materials.

#### **3.1. Spectroscopy techniques**

Distinct kinds of techniques have been used to characterize the physical and chemical structure of the collagen. Confocal microscopy using the second-harmonic generation [42] and Raman effect [43] have proved to be valuable techniques to determine the presence of the plain collagen in different tissues. The generation of the second harmonic signal in collagen scaffolds is due to the fiber alignment, and it is poor in ECM hydrogels, but it can be enhanced once the collagen is aligned (in natural collagen tissues) or stained. Staining, however, is not recommended because can affect the conformation and interaction among the different components of the scaffold. On the other hand, Raman spectroscopy does not have this limitation and can be used to determine and map collagen in dry and wet samples. In addition, it is sensible to the relative composition of amino acids that confirm the collagen and as a consequence, can be used to determine different kinds of collagen or collagen degradation in time during a disease as cancer for instance [43]. Infrared spectroscopy (IR) can be seen as a complement of the Raman spectroscopy because they are sensitive to the same organic groups. The technical difference is that in Raman, we observe the energy of photons scattered from the sample after excitation using a single wavelength, and in IR, we observe the absorption of photons in a range of wavelengths [44]. In general, IR is used when the sample is not extremely complicated, and signals in the spectra can be assigned to specific interactions in the gel as a chemical modifier as a cross-linker for instance [45, 46]. In such a way, another classical technique that determines chemical interactions among distinct parts of a composite, as 1 H-NMR can be used also in ECMs [47].

#### **3.2. Microscopy techniques**

Scanning and transmission electronic microscopy (SEM and TEM) are also important techniques for collagen characterization and a first easy access to get the pore size [48], length and width of the fibers as well as shape [49], amount and location of nano- and microstructures of different materials added to the scaffold as can be inorganic salts [50] or nanoparticles [51]. SEM and TEM are excellent characterization techniques for lyophilized scaffolds [52], although in the case of TEM is essential a good handle of the available staining techniques to avoid "artifacts" in the images. However, when those measurements are the reference for wet properties of the scaffold it can be taken only as a guide and other techniques on wet materials are needed to confirm what is being observed.

Using atomic force microscopy (AFM) is possible to observe collagen fibers with different shape and structures on different surfaces [48, 54, 60] as well as to determine micromechanical properties of a collagen hydrogel; both in wet and dry formulations. AFM is microscopy based on the movement of a microtip, in the range of micro- and nanometers, that interacts with the surface at the microscopic level and sense its shape and roughness. The movements of the tip are followed by a laser on it; forming an image of deflection laser intensity. This image, if the deflection force of the tip is known, forms also a map of micromechanical properties.

#### **3.3. X-ray techniques**

**3. Methods for physicochemical characterization of collagen-based** 

different depending on the other components of the materials.

A collagen scaffold is a hierarchical, protein-based fibrillary network: after the triple helix formation that conforms tropocollagen, it forms fibrils that align themselves in microfibers and finally, in collagen fibers of a tissue [41]. To correlate the different physicochemical properties observed in a collagen derivative scaffold with its hierarchical network is an attractive challenge partially explored. It is important to notice that the chemical modification of plain tropocollagen is a current practice to tune some properties as the mechanical ones. In general, the characterization methods for the collagen-based materials do not vary when it is chemically modified as we will see in this chapter. On the other hand, for composites that contain collagen, it is intuitive to imagine that the methods to determine their properties could be

Distinct kinds of techniques have been used to characterize the physical and chemical structure of the collagen. Confocal microscopy using the second-harmonic generation [42] and Raman effect [43] have proved to be valuable techniques to determine the presence of the plain collagen in different tissues. The generation of the second harmonic signal in collagen scaffolds is due to the fiber alignment, and it is poor in ECM hydrogels, but it can be enhanced once the collagen is aligned (in natural collagen tissues) or stained. Staining, however, is not recommended because can affect the conformation and interaction among the different components of the scaffold. On the other hand, Raman spectroscopy does not have this limitation and can be used to determine and map collagen in dry and wet samples. In addition, it is sensible to the relative composition of amino acids that confirm the collagen and as a consequence, can be used to determine different kinds of collagen or collagen degradation in time during a disease as cancer for instance [43]. Infrared spectroscopy (IR) can be seen as a complement of the Raman spectroscopy because they are sensitive to the same organic groups. The technical difference is that in Raman, we observe the energy of photons scattered from the sample after excitation using a single wavelength, and in IR, we observe the absorption of photons in a range of wavelengths [44]. In general, IR is used when the sample is not extremely complicated, and signals in the spectra can be assigned to specific interactions in the gel as a chemical modifier as a cross-linker for instance [45, 46]. In such a way, another classical technique that determines chemical interactions among distinct parts of a composite,

Scanning and transmission electronic microscopy (SEM and TEM) are also important techniques for collagen characterization and a first easy access to get the pore size [48], length and width of the fibers as well as shape [49], amount and location of nano- and microstructures of different materials added to the scaffold as can be inorganic salts [50] or nanoparticles [51]. SEM and TEM are excellent characterization techniques for lyophilized scaffolds [52], although

**hydrogels**

10 Hydrogels

as 1

**3.1. Spectroscopy techniques**

H-NMR can be used also in ECMs [47].

**3.2. Microscopy techniques**

Another important set of characterization techniques are the X-ray techniques; although their availability depends strongly on the level of development of each particular scientific community in specific countries due to the extensive facilities and economic resources that they need. The more accessible are the X-ray photoelectron spectroscopy (XPS) and the small angle X-ray scattering (SAXS). XPS is able to measure the carbonyl, C═O, and C─C interaction on a collagen surface [53], as well as traces of silicon commonly used now in collagen-based scaffolds [11], for instance. It is in general, the right technique to obtain the gross chemical composition on the collagen surface and a definitive indication for traces of impurities of other elements [54]. Depending on the instrumentation available, wet samples can be measured, since X-rays must work under light vacuum. In addition, it is not expected to see under the surface because the X-ray source is weak and cannot penetrate the sample. On the other hand, scattering X-ray techniques can be used to get the shape, length and width, pore size, and fiber orientation directly from the sample, wet or dry, without further manipulation [55–57]. Depending on the distance to determine, normal X-ray diffraction equipment can also be used [58]. Those techniques are based on the scattering of the X-rays from the sample: a scattering vector is an inverse function of the X-ray wavelength and proportional to the sine of the half the scattering angle. A characteristic distance in the material scatters X-rays of a specific wavelength proportional to the scattering vector and with a different scattering intensity. This intensity is a function of the scattering angle, and it is from where the different properties of the material can be extracted. In general, using smaller scattering angles, it is possible to obtain information about larger distances, as those observed in the collagen when X-rays are used [59].

#### **3.4. Mechanical tests**

Mechanical properties (determined at microscopic or macroscopic level) of the collagen are those of a gel or an entangled polymer: basically, oscillatory rheology shows a plateau of the storage modulus (G´) in a frequency ranging between hertz and kilohertz, which can be considered as its Young's modulus, and three times this value can be determined by extensional and compressional experiments of strain versus stress [52]. The convergence of micro- and macromechanical moduli values are not common in the literature [42, 61], although it is expected a similar trend of increment or decrement of the properties toward the same change in a particular variable as can be the cross-linking degree. The Young's modulus can easily vary between cross-linked and uncross-linked collagen one order of magnitude [62, 63]. An important physical parameter directly correlated to the value of the storage modulus is the pore size of the collagen network: the size of the pore is simply the cubic root of the thermal energy (3kT) over the Young's modulus [64]. Alternatively, the pore size distribution of a scaffold can be obtained by analyzing images of thin sections of paraffin-embedded samples obtained by optical microscopy [42] or using electron microscopy as previously explained [48].

**4.1. Macrophage polarization in decellularized ECM-based materials**

Macrophages are cells of the innate immune system with a dominant effector activity in the injury site after biomaterial implantation. Cross-talk between immune cells activates macrophages after which, they release a variety of signaling molecules. Signaling molecules secreted by macrophages such as cytokines (as interleukins), growth factors as the basic fibroblast growth factor, the vascular endothelial growth factor and the transforming growth factor-beta 1 (bFGF, VEGF and TGF-β1 respectively); and tumor necrosis factor (TNF-α) influence the development of other cell types [70]. In fact, the profile of signaling molecules secretion is commonly evaluated to study the polarization of the macrophage response from an inflammation and tissue injury process to a repair process [71, 72] or to study angiogenesis and scaffold vascularization [73]. Macrophages mediate the healing responses to implanted biomaterials, fundamentally by two outcomes: scar tissue formation (M1M pathway) or regeneration (M2M pathway) [70]. The modulation of the inflammatory response by the physical and chemical properties of biomaterials represents a hypothesis currently

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assessed in the design of biomaterials intended to the repair/regeneration of soft tissue.

The goal of the decellularization process of mammalian tissues is to remove its cellular and nuclear components. This aim must be balanced with retention of both the extracellular composition and microstructural characteristics, as much as possible. As result, an incomplete removal of nuclear components has been reported in diverse ECM biomaterials, even in commercial biological implants [74]. The intensity of the host immune response after implantation is heavily influenced by the residual material, which acts as like cell signals [74, 75]. For instance, a decrement in the DNA amount in small intestine submucosa tissue provoked a shift of the M1M proinflammatory macrophage phenotype to the M2M anti-inflammatory one [76]. On the other hand, the tissue regeneration induced by the ECM-based biomaterials has been associated with extracellular residual components such as collagen type I, polysaccharides or basal membrane complex components [77]. Glycosaminoglycans such as hyaluronic acid extracted from brain and urinary bladder have been associated with an up-regulated secretion of anti-inflammatory factors and suppressed secretion of proinflammatory factors, consistent with M2M phenotype macrophages [76]. Moreover, studies revealed that the anionic detergent sodium dodecyl sulfate and nonionic detergent TritonX-100 produce a different impact over the stability of ligands and proteins in the basal membrane complex [80]. The decellularization method and tissue source thus influence the retention of the basal membrane complex components within ECM materials and the bioactivity of them. The bioactivity of ECM-based materials was also evidenced by the differentiation of human monocytes differentiated to macrophages. The higher amounts of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1, but lower amounts of interleukin-10 (IL-10) and interleukin-1 receptor antagonist (IL-1ra) were detected on decellularized pericardium matrix, in comparison with polydimethylsiloxane or polystyrene surfaces [81]. Cellular residual components such as damage-associated molecular patterns (DAMPs, proteins that are retained within the ECM scaffolds) have been considered as bioinductive molecules with a key role in the macrophage polarization [78]. High-mobility group box 1 (a DAMP that functions intracellularly as a DNA binding nuclear protein), detected in ECM biomaterials derived from small intestinal submucosal, and urinary bladder matrix, was correlated with differences in cell

**4.2. Impact of the residual composition on the immune response**

Shear flow experiments are useful to obtain the viscosity of the collagen hydrogel precursors, the concentration of the proto-collagen present in a solution, and an estimation of the molecular weight of the minimal structured collagen in solution [65]. It has been also suggested that collagen denaturation can be determined by viscosimetric measurements [66]. Those experiments become important in the case of development of injectable systems because parameters as viscosity [47, 67] and compressibility [52] are important during extrusion. Rheological methods described previously are also convenient to measure the formation of the gel in time: storage (G´) (colloquially speaking, how much the viscoelastic material looks like a solid) and loss modulus (G´´) (how much the viscoelastic material looks like a liquid) can be determined in an oscillatory rheological measurement to get the gel formation point: where the storage modulus becomes higher than the loss modulus (G´ > G´´) [68].

#### **3.5. Thermal stability test**

The denaturation heat and denaturation temperature of a collagen scaffold are obtained from calorimetric experiments commonly using a differential scanning calorimeter (DSC) [67]. Since the technique is based on calorimetric differences sensed by an extremely sensitive electronic device, it is important to consider that minimal differences in the medium concentration (buffer concentration, conductivity of the water used as a solvent, etc.) or during the preparation of the samples (pH, size and shape of the particles, etc.), are observed [48, 69]. Thermal denaturation peak of wet collagen occurs around 50°C, although the heat absorption peak is broad and could start under 20°C before the peak; a straightforward evidence that the collagen has distinct levels of structure. The integral under this endothermal process, that is, energy versus temperature, gives the denaturation heat of the collagen. In general, it has been reported that both denaturation heat and temperature are higher for cross-linked collagen that for uncross-linked collagen [50].
