**4. Perspectives of the decellularized ECM-based materials in immunomodulation**

Biomaterials with immunomodulatory activity are being studied in the context of the repair/ regeneration of soft tissues, such as diabetic chronic wounds. Evidences indicate the effect of the characteristics of biomaterials and their (released/biodegraded) by-products over promoting of required immunological responses that could support the wound healing. Moreover, the residual components remaining the animal source as well as the modification of ECMbased materials can elucidate an undesirable response.

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

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

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

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].

**4. Perspectives of the decellularized ECM-based materials in** 

based materials can elucidate an undesirable response.

Biomaterials with immunomodulatory activity are being studied in the context of the repair/ regeneration of soft tissues, such as diabetic chronic wounds. Evidences indicate the effect of the characteristics of biomaterials and their (released/biodegraded) by-products over promoting of required immunological responses that could support the wound healing. Moreover, the residual components remaining the animal source as well as the modification of ECM-

optical microscopy [42] or using electron microscopy as previously explained [48].

modulus becomes higher than the loss modulus (G´ > G´´) [68].

**3.5. Thermal stability test**

12 Hydrogels

**immunomodulation**

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

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

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 proliferation, death, secretion of the immunomodulatory factors [78]. Altogether, reports suggest that decellularization, as the first step in the development of ECM-based hydrogels, and scaffolds, impact the cellular and extracellular components within biomaterials. Consequently, these components become in a key player in the mechanisms of tissue regeneration observed when decellularized ECM materials are used. The ability to support the proliferation and migration of different cells [82], to allow the differentiation of mesenchymal cells [83, 84], and to transit from the inflammatory first steps to a regenerative action [75, 85] are among the mechanisms by which the ECM biomaterials participate. Once animal tissues are decellularized, they are cross-linked to increase their stability, reduce degradation, and immunogenicity. However, the reconstruction of functional tissue appears to be compromised after cross-linking as discussed below.

**4.4. Immunomodulation with decellularized ECM-based hydrogels**

**5. Final remarks**

The performance of hernia standard surgical grafts, manufactured from polypropylene, has been improved by the coating of them with decellularized ECM-based hydrogels. This was attributed to the polarization of alternatively-activated and constructive M2Ms macrophages induced by the degradation products from ECM materials, which in turn facilitates migration and myogenesis of skeletal muscle progenitor cells [84]. The migration and proliferation of perivascular stem cells are influenced by the structural components (include a number of partially digested proteoglycans and proteins such as collagens, elastin, laminin, fibronectin, hyaluronan, and heparan) as well as soluble components of hydrogels derived from urinary bladder matrix (include cryptic peptide fragments generated from partial proteolysis of scaffold resident growth factors, and matricellular proteins, e.g., tenascin, osteopontin, and thrombospondin) [76]. The mechanism through the soluble and structural components of ECM-based hydrogels contribute to the host response appears to be different. Both components altered the macrophage behavior but with different fingerprints according to the cytokines secretion profiles [76]. A hernia rodent model study revealed that the implantation of polypropylene meshes coated with ECM hydrogel for a period of 14 days decreased the inflammatory response, which was characterized by the number and distribution of M1Ms around polypropylene fibers, compared to the uncoated devices. After a period of 180 days, the density of mature type I collagen deposited between mesh synthetic fibers was decreased with the coating of ECM hydrogel was used [76]. The coating based on ECM-based hydrogel suggested a low scar tissue deposition on the synthetic mesh, which can be associated with a mitigated chronic inflammatory response, an attenuated M1M response, and an increased M2M/M1M ratio to abdominal defect polypropylene standard grafts [91]. The use of decellularized amniotic membrane tissue combined with poly (urethane-ester) showed a better biocompatibility compared to polypropylene meshes when implanted into abdomen of rabbits over a period of 10 months [92]. Results of in vitro cytocompatibility tests demonstrated that this composite can support primary smooth muscle cells to grow and differentiate, with high proliferation, mitochondrial activity, and special protein expression (α-smooth muscle actin).

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Mammalian tissues from various sources can be used as biomaterials after modification by decellularization and cross-linking processes. Among these materials, the ECM-based hydrogels seem promising alternatives to modulate the required properties in applications related to biomedicine and tissue engineering. Current approaches usually affect the network structure, physicochemical properties, and biocompatibility of natural ECMbased scaffolds. Thus, a balance between the mechanical and degradation properties and immunology response is a present challenge. In this respect, methodologies based on the combination of the ECM with natural and synthetic polymers, minimizing the removal of the characteristics of the natural ECM, seem to be the best alternatives for this purpose. The structural modification of the natural ECM is related to the variation of its properties; this process can be monitored by a variety of physicochemical techniques, which could provide direct evidence of the structure-property relationship in ECM-based biomaterials. A direct evidence of the ECM properties is definitely a challenge, because some of the most common

#### **4.3. Impact of the collagen cross-linking on the immune response**

The cross-linking process of ECM-based biomaterials is commonly associated with a detrimental effect on the ultrastructure and composition of the ECM and consequently the biological response [78]. The ability of the decellularized ECM materials to interact with cells is modified by the altered surface chemistry after cross-linking. As discussed above, distinct methods for cross-linking collagen biomaterials have been studied. The understanding and control of cell fate in modified chemically collagen materials is a matter of study. For instance, the cell membrane morphology, cell adhesion and enzymatic activity of the acid phosphatase and esterase of U937 macrophage-like cells have shown to be differentially influenced by the glutaraldehyde cross-linking, and EDAC coupling methods. Glutaraldehyde cross-linking induced an increase in the release of the proinflammatory cytokines IL-1ra, IL-6, IL-10, and TNF-α, unlike to EDAC-cross-linked materials and uncross-linked tissues [86]. Differences in the microenvironment of ECM-based implants cross-linked with glutaraldehyde and diisocyanate (aliphatic) cross-linking methods modified the infiltration of neutrophils and the function of macrophages [87]. A strong proinflammatory milieu was observed in glutaraldehyde-crosslinked materials, while in diisocyanate-cross-linked materials, an anti-inflammatory milieu was seen. The proliferation of immune cell subpopulations was found stronger on both porcine nondecellularized and decellularized materials than on the glutaraldehyde-cross-linked ones [79]. This observation has been reported in the case of cross-linking of tissue-derived heart valves [88]. The integration of this implant type has been associated with a reduced antigenicity by masked immunogenicity [88]. A lack of acute inflammation in dermis-derived implants (fixed with glutaraldehyde at low concentration) both in animal models and humans was observed. Thus, the high concentrations of aldehyde employed in the processing of ECM biomaterials appear to induce a more pronounced and sustained inflammatory response [89]. The dermis-derived implants cross-linked with diisocyanates showed a low chronic inflammatory response after a 20-week period of implantation with both limited collagen degradation and vascular ingrowth [89]. Non–cross-linked ECM materials showed earlier cell infiltration, extracellular matrix (ECM) deposition, scaffold degradation, and neovascularization compared with cross-linked materials, after a 1-month period of implantation. However, after 6 and 12 months, diisocyanate-cross-linked materials showed comparable results compared with the non–cross-linked materials [90]. The cross-linked collagen-derived implants showing an acceptable performance in diverse applications would seem to suggest a degree of tolerance to these materials [90]. However, the tissue remodeling associated with the ECM constituents is yet a challenge to be addressed in the development of new cross-linked ECM biomaterials.

#### **4.4. Immunomodulation with decellularized ECM-based hydrogels**

The performance of hernia standard surgical grafts, manufactured from polypropylene, has been improved by the coating of them with decellularized ECM-based hydrogels. This was attributed to the polarization of alternatively-activated and constructive M2Ms macrophages induced by the degradation products from ECM materials, which in turn facilitates migration and myogenesis of skeletal muscle progenitor cells [84]. The migration and proliferation of perivascular stem cells are influenced by the structural components (include a number of partially digested proteoglycans and proteins such as collagens, elastin, laminin, fibronectin, hyaluronan, and heparan) as well as soluble components of hydrogels derived from urinary bladder matrix (include cryptic peptide fragments generated from partial proteolysis of scaffold resident growth factors, and matricellular proteins, e.g., tenascin, osteopontin, and thrombospondin) [76]. The mechanism through the soluble and structural components of ECM-based hydrogels contribute to the host response appears to be different. Both components altered the macrophage behavior but with different fingerprints according to the cytokines secretion profiles [76]. A hernia rodent model study revealed that the implantation of polypropylene meshes coated with ECM hydrogel for a period of 14 days decreased the inflammatory response, which was characterized by the number and distribution of M1Ms around polypropylene fibers, compared to the uncoated devices. After a period of 180 days, the density of mature type I collagen deposited between mesh synthetic fibers was decreased with the coating of ECM hydrogel was used [76]. The coating based on ECM-based hydrogel suggested a low scar tissue deposition on the synthetic mesh, which can be associated with a mitigated chronic inflammatory response, an attenuated M1M response, and an increased M2M/M1M ratio to abdominal defect polypropylene standard grafts [91]. The use of decellularized amniotic membrane tissue combined with poly (urethane-ester) showed a better biocompatibility compared to polypropylene meshes when implanted into abdomen of rabbits over a period of 10 months [92]. Results of in vitro cytocompatibility tests demonstrated that this composite can support primary smooth muscle cells to grow and differentiate, with high proliferation, mitochondrial activity, and special protein expression (α-smooth muscle actin).

#### **5. Final remarks**

proliferation, death, secretion of the immunomodulatory factors [78]. Altogether, reports suggest that decellularization, as the first step in the development of ECM-based hydrogels, and scaffolds, impact the cellular and extracellular components within biomaterials. Consequently, these components become in a key player in the mechanisms of tissue regeneration observed when decellularized ECM materials are used. The ability to support the proliferation and migration of different cells [82], to allow the differentiation of mesenchymal cells [83, 84], and to transit from the inflammatory first steps to a regenerative action [75, 85] are among the mechanisms by which the ECM biomaterials participate. Once animal tissues are decellularized, they are cross-linked to increase their stability, reduce degradation, and immunogenicity. However, the reconstruction

of functional tissue appears to be compromised after cross-linking as discussed below.

The cross-linking process of ECM-based biomaterials is commonly associated with a detrimental effect on the ultrastructure and composition of the ECM and consequently the biological response [78]. The ability of the decellularized ECM materials to interact with cells is modified by the altered surface chemistry after cross-linking. As discussed above, distinct methods for cross-linking collagen biomaterials have been studied. The understanding and control of cell fate in modified chemically collagen materials is a matter of study. For instance, the cell membrane morphology, cell adhesion and enzymatic activity of the acid phosphatase and esterase of U937 macrophage-like cells have shown to be differentially influenced by the glutaraldehyde cross-linking, and EDAC coupling methods. Glutaraldehyde cross-linking induced an increase in the release of the proinflammatory cytokines IL-1ra, IL-6, IL-10, and TNF-α, unlike to EDAC-cross-linked materials and uncross-linked tissues [86]. Differences in the microenvironment of ECM-based implants cross-linked with glutaraldehyde and diisocyanate (aliphatic) cross-linking methods modified the infiltration of neutrophils and the function of macrophages [87]. A strong proinflammatory milieu was observed in glutaraldehyde-crosslinked materials, while in diisocyanate-cross-linked materials, an anti-inflammatory milieu was seen. The proliferation of immune cell subpopulations was found stronger on both porcine nondecellularized and decellularized materials than on the glutaraldehyde-cross-linked ones [79]. This observation has been reported in the case of cross-linking of tissue-derived heart valves [88]. The integration of this implant type has been associated with a reduced antigenicity by masked immunogenicity [88]. A lack of acute inflammation in dermis-derived implants (fixed with glutaraldehyde at low concentration) both in animal models and humans was observed. Thus, the high concentrations of aldehyde employed in the processing of ECM biomaterials appear to induce a more pronounced and sustained inflammatory response [89]. The dermis-derived implants cross-linked with diisocyanates showed a low chronic inflammatory response after a 20-week period of implantation with both limited collagen degradation and vascular ingrowth [89]. Non–cross-linked ECM materials showed earlier cell infiltration, extracellular matrix (ECM) deposition, scaffold degradation, and neovascularization compared with cross-linked materials, after a 1-month period of implantation. However, after 6 and 12 months, diisocyanate-cross-linked materials showed comparable results compared with the non–cross-linked materials [90]. The cross-linked collagen-derived implants showing an acceptable performance in diverse applications would seem to suggest a degree of tolerance to these materials [90]. However, the tissue remodeling associated with the ECM constituents is yet a challenge to be addressed in the development of new cross-linked ECM biomaterials.

**4.3. Impact of the collagen cross-linking on the immune response**

14 Hydrogels

Mammalian tissues from various sources can be used as biomaterials after modification by decellularization and cross-linking processes. Among these materials, the ECM-based hydrogels seem promising alternatives to modulate the required properties in applications related to biomedicine and tissue engineering. Current approaches usually affect the network structure, physicochemical properties, and biocompatibility of natural ECMbased scaffolds. Thus, a balance between the mechanical and degradation properties and immunology response is a present challenge. In this respect, methodologies based on the combination of the ECM with natural and synthetic polymers, minimizing the removal of the characteristics of the natural ECM, seem to be the best alternatives for this purpose. The structural modification of the natural ECM is related to the variation of its properties; this process can be monitored by a variety of physicochemical techniques, which could provide direct evidence of the structure-property relationship in ECM-based biomaterials. A direct evidence of the ECM properties is definitely a challenge, because some of the most common techniques give only approximations to them. A sample preparation that could include denaturation, drying, staining, etc., can completely change a parameter as the pore size or the fiber alignment. In such a way, new or revised techniques that can be used on undamaged and functional ECM are desirable [93, 94]. Those new techniques where the cellular function is not compromised, will give not only more reliable information about the way ECM interacts in the body, but will open new perspectives on the way to study and prepare ECMs for future applications.

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