**7. Porosity**

thickened epidermis during 15 day of healing of impaired wounds [114]. In the same way, acrylic acid (AA) grafted onto poly(ethylene terephthalate) (PET) film through gamma-ray induced graft copolymerization with silver nanoparticles on the surface showed strong and

It is highly desired to have a hydrogel material bearing the excellent antifouling property/biocompataiblity to prolong the lifetime of implanted materials, switchable antimicrobial property to eliminate infection and inflammation, and good mechanical properties to avoid the failure of the implanted material. We hypothesize derivatives of zwitterionic carboxybetaine with hydroxyl group(s) can switch between the lactone form (anti-microbial) and the zwitterionic form (anti-fouling) and the intramolecular hydrogen bonds will enhance the mechanical property of the zwitterionic hydrogel. It is highly desired to have a hydrogel material bearing the excellent antifouling property/biocompataiblity to prolong the lifetime of implanted materials, switchable antimicrobial property to eliminate infection and inflammation, and good mechanical properties to avoid the failure of the implanted material. We hypothesize derivatives of zwitterionic carboxybetaine with hydroxyl group(s) can switch between the lactone form (antimicrobial) and the zwitterionic form (anti-fouling) and the intramolecular hydrogen bonds will

On the other hand, the surface of hydrogels must be modified to make it resistant to protein adsorption and cell adhesion to avoid fouling. Thus, there is a need for coatings with antifouling properties that are able to improve the performances of implanted biomedical devices. Thus, the antifouling properties of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) hydrogels were improved by the surface grafting of a brush of poly(oligoethylene glycol methyl ether acrylate) [poly(OEGA)] [116]. Novel antifouling highly wettable hydrogels with superior mechanical and self-healing properties have also been developed by UV-initiated copolymerization of non-fouling zwitterionic carboxybetaine methacrylamide (CBMAA-3) and 2-hydroxyethyl methacrylate (HEMA) in the presence of uniformly dispersed clay nanoparticles (Laponite

Therefore, it would be highly desired to have a hydrogel material bearing the excellent antifouling property/biocompataiblity to prolong the lifetime of implanted materials, switchable antimicrobial property to eliminate infection and inflammation, and good mechanical properties to avoid the failure of the implanted material. Thus, derivatives of zwitterionic carboxybetaine have been developed with hydroxyl group(s), which can switch between the lactone form (antimicrobial) and the zwitterionic form (anti-fouling) [118]. Besides, the intramolecu-

Nevertheless, the rapid emergence of antibiotic resistance in pathogenic microbes is becoming an imminent global public health problem because they are highly prone to develop resistance through mutation and the treatment with conventional antibiotics often leads to resistance development leaving the bacterial morphology intact. Therefore, much research is currently being done in the development of new antimicrobial hydrogels because they have been demonstrated to be very effective in preventing and treating multidrug-resistant

lar hydrogen bonds enhance the mechanical property of the zwitterionic hydrogel.

stable antibacterial activity [115].

104 Hydrogels

XLG) in water [117].

infections.

enhance the mechanical property of the zwitterionic hydrogel.

Porous polymers have received an increased level of research interest because of their potential to merge the properties of both porous materials and polymers [119]. Porous polymers have potential applications in many fields such as gas storage and separation materials [120, 121], drug delivery [122], catalysts [123], supports for electrochemical sensing [124], low-dielectric constant materials [125], packing materials in chromatography [126], scaffolds or three-dimensional porous matrices for tissue engineering in regenerative medicine [5, 41, 127, 128] and many others. These high value applications have driven much emphasis on development of reliable methods for preparation of porous polymers with designed pore architectures in the last decades (see **Figure 6**).

Tissue engineering holds great promise for regeneration and repair of diseased tissues, making the development of new porous supports as scaffolds for tissue regeneration a topic of great interest in biomedical research. Hydrogels have emerged as leading candidates for engineered tissue scaffolds due to their good biocompatibility and similarities to native extracellular

**Figure 6.** Different porous polymers prepared by different preparation methods to obtain diverse pore architectures. Reprinted with permission from Ref [12, 41, 48, 128–130].

matrix. However, precise control of hydrogel properties, such as high porosity, remains a challenge. Traditional techniques for creating bulk porosity in polymers have demonstrated success in hydrogels for tissue engineering. However, some problems related to direct cell encapsulation often occur. Thus, emerging technologies have demonstrated the ability to control porosity and morphology in hydrogels, creating engineered tissues with structure and function similar to native tissues [131]. The interconnection and geometry of pores, which depends on the tissue to regenerate, physicochemical properties, and mechanical resistance of the material play a major role in these biomedical applications. Thus, there are several methods to produce *scaffolds*, which include gas foaming [132], sintering fiber meshes [133], solvent casting [134], polymerization in solution [86, 135], porogen technique [129, 136], freeze-drying techniques [137, 138], electrospinning [139], 3D printing [140], 3D bioplotting of scaffold with cells [141], etc. For example, scaffolds with interconnected spherical pores and controlled hydrophilicity with interconnected porous structure were synthesized using a template of sintered PMMA microspheres of controlled size. In these scaffolds, the geometric characteristics (pore size, connectivity and porosity) and the physico-chemical properties of the resulting material can be controlled in an independent way. Copolymerization of hydrophobic ethyl acrylate (EA) and hydrophilic hydroxyethyl methacrylate comonomers in the free space of the template and subsequent solution of the PMMA microspheres gave rise to the scaffold with the designed pore

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**Figure 8.** Morphology of the gelatin-PHEMA porous scaffolds as obtained through μ-CT (panels (a)–(c): (a)—top view, (b)—bottom view, and (c)—side view) and SEM (panel (d)) analyses: panel (I)—C0; panel (II)—C1; panel (III)—C2; panel

architecture (see **Figure 7**) [129].

(IV)—C3. *Reprinted with permission from Ref.* [138].

**Figure 7.** Scanning electron micrographs of EA/HEMA copolymer scaffolds (30% HEMA) at different magnifications. *Reprinted with permission from Ref.* [129].

[137, 138], electrospinning [139], 3D printing [140], 3D bioplotting of scaffold with cells [141], etc. For example, scaffolds with interconnected spherical pores and controlled hydrophilicity with interconnected porous structure were synthesized using a template of sintered PMMA microspheres of controlled size. In these scaffolds, the geometric characteristics (pore size, connectivity and porosity) and the physico-chemical properties of the resulting material can be controlled in an independent way. Copolymerization of hydrophobic ethyl acrylate (EA) and hydrophilic hydroxyethyl methacrylate comonomers in the free space of the template and subsequent solution of the PMMA microspheres gave rise to the scaffold with the designed pore architecture (see **Figure 7**) [129].

matrix. However, precise control of hydrogel properties, such as high porosity, remains a challenge. Traditional techniques for creating bulk porosity in polymers have demonstrated success in hydrogels for tissue engineering. However, some problems related to direct cell encapsulation often occur. Thus, emerging technologies have demonstrated the ability to control porosity and morphology in hydrogels, creating engineered tissues with structure and function similar to native tissues [131]. The interconnection and geometry of pores, which depends on the tissue to regenerate, physicochemical properties, and mechanical resistance of the material play a major role in these biomedical applications. Thus, there are several methods to produce *scaffolds*, which include gas foaming [132], sintering fiber meshes [133], solvent casting [134], polymerization in solution [86, 135], porogen technique [129, 136], freeze-drying techniques

**Figure 7.** Scanning electron micrographs of EA/HEMA copolymer scaffolds (30% HEMA) at different magnifications.

*Reprinted with permission from Ref.* [129].

106 Hydrogels

**Figure 8.** Morphology of the gelatin-PHEMA porous scaffolds as obtained through μ-CT (panels (a)–(c): (a)—top view, (b)—bottom view, and (c)—side view) and SEM (panel (d)) analyses: panel (I)—C0; panel (II)—C1; panel (III)—C2; panel (IV)—C3. *Reprinted with permission from Ref.* [138].

A novel preparation of gelatin-PHEMA porous scaffolds by freeze-drying technique was developed recently [138]. Their morphology was assessed by SEM and μ-CT (**Figure 8**). In this study, four types of novel hydrogels using different methacrylamide-modified gelatin/2-hydroxyethyl methacrylate ratios between 1/0 and 1/2 (w/w) (samples from C0 to C3) were prepared and the results indicated that the HEMA content in the initial polymerization mixtures modulate the architecture of the porous scaffolds from straightforward, top-to-bottom oriented channels for hydrogels possessing the lowest HEMA content to a complex and dense internal porosity of the channels the case of higher HEMA loaded materials. Besides, the covalently bound gelatin sequences significantly improve the biocompatibility of PHEMA based hydrogels, which is very desirable for tissue engineering purposes.

exhibit even poorer mechanical properties when they are porous and hydrated. Therefore, it is usually necessary to enhance the mechanical properties of these porous structures by means of the methods, shown in chapter 2, with nanomaterials or other techniques. For example, the use of a hybrid hydrogel nanocomposite of silica/PHEA as scaffold material matrix greatly

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Other modifications of scaffolds such as those of PHEMA with cholesterol methacrylate (CHLMA) and laminin have been developed in the presence of ammonium oxalate crystals to introduce interconnected superpores in the matrix in order to design superporous scaffolds that promote cell-surface interaction [146]. PHEMA has also been modified with laminin-derived Ac-CGGASIKVAVS-OH peptide sequences to construct scaffolds that promote cell adhesion and neural differentiation. With the same goal, nanofiber scaffolds of poly (L-lactide) (PLLA) prepared by electrospinning were treated with oxygen plasma and then simultaneously *in situ* grafted with hydrophilic acrylic acid to obtain PLLA-g-PAA with a modified surface, which significantly improved cell adhesion and

Polysaccharide hydrogels have become increasingly studied as matrices in soft tissue engineering due to their known cytocompatibility. For example, cross-linkable dextran methacrylates and hyaluronan methacrylate hydrogels, which are candidates as matrices for soft tissue reconstruction, were synthesized showing that the *in vitro* degradation behavior of these types of hydrogels could be controlled by the polysaccharide structure and the cross-linking density. Furthermore, under *in vitro* conditions, these novel materials had no cytotoxic effects against fibroblasts and the use of composite gels improved the adherence

Therefore, great advances have been achieved so far in scaffold design of new advanced porous hydrogels for tissue engineering applications. Nevertheless, much research has to be conducted still in order to find new ways and methods capable of providing suitable materi-

This work was supported by the 2017-231-001UCV grant from the Universidad Católica de

Bioengineering and Cellular Therapy Group, Facultad de Veterinaria y Ciencias Experimentales, Universidad Católica de Valencia San Vicente Mártir, Valencia, Spain

als able to fulfill all the necessary requirements of this biomedical field.

improves the mechanical properties [41].

proliferation [130].

of cells [147].

**Acknowledgements**

**Author details**

Ángel Serrano-Aroca

Valencia "San Vicente Mártir".

Address all correspondence to: angel.serrano@ucv.es

Superporous scaffolds can be also prepared by the salt-leaching technique using NaCl or (NH4)2 SO<sup>4</sup> as a porogen [142] or with many other porogenic agents such as ammonium oxalate crystals [143].

By submitting carbon dioxide to supercritical conditions after certain time and then rapidly depressurized is also possible to fabricate porous structures that are related to the supercritical parameters and to the polymer blend composition [131]. The use of CO<sup>2</sup> to create such scaffolds has received some attention in the past but many researchers believe that there is limited interconnectivity between the pores, which is required for tissue engineering. However, highly porous (greater than 85%) and well interconnected scaffolds with very promising applications for cartilage repair have been obtained in a blend of poly(ethyl methacrylate) and tetrahydrofurfuryl methacrylate [144].

Probably the most sophisticated techniques to produce scaffolds are electrospinning, 3D printing and bioprinting. Electrospinning is composed of a high-voltage DC power supply, an infusion pumps and a syringe with a needle tip usually with a diameter of 0.5 mm. For example, a three-dimensional aligned nanofibers-collagen type I hydrogel scaffold for controlled non-viral drug/gene delivery to direct axon regeneration in spinal cord injury treatment has been reported very recently [145].

3D printing promises to produce complex biomedical devices according to computer design using patient-specific anatomical data. This 3D printing technique has slowly evolved to create one-of-a-kind devices, implants, scaffolds for tissue engineering, and drug delivery systems among other important applications. However, several technological limitations, related to the kind of commercially printable materials available and other technical printing aspects such as printing speed, must still be overcome. The common 3D printing technologies are three-dimensional printing, fused deposition modeling, selective laser sintering, stereolithography, and 3D plotting/direct-write/bioprinting, and are still under deep research for the progress of each technology applied in tissue engineering. Bioprinting is the more advanced 3D printing technology because it consists of printing cells combined with custom 3D scaffolds for personalized regenerative medicine [140].

Mechanical resistance depends both on the material properties and on the interconnected pore structure of the scaffold. This problem is more important in polymer hydrogels, which exhibit even poorer mechanical properties when they are porous and hydrated. Therefore, it is usually necessary to enhance the mechanical properties of these porous structures by means of the methods, shown in chapter 2, with nanomaterials or other techniques. For example, the use of a hybrid hydrogel nanocomposite of silica/PHEA as scaffold material matrix greatly improves the mechanical properties [41].

Other modifications of scaffolds such as those of PHEMA with cholesterol methacrylate (CHLMA) and laminin have been developed in the presence of ammonium oxalate crystals to introduce interconnected superpores in the matrix in order to design superporous scaffolds that promote cell-surface interaction [146]. PHEMA has also been modified with laminin-derived Ac-CGGASIKVAVS-OH peptide sequences to construct scaffolds that promote cell adhesion and neural differentiation. With the same goal, nanofiber scaffolds of poly (L-lactide) (PLLA) prepared by electrospinning were treated with oxygen plasma and then simultaneously *in situ* grafted with hydrophilic acrylic acid to obtain PLLA-g-PAA with a modified surface, which significantly improved cell adhesion and proliferation [130].

Polysaccharide hydrogels have become increasingly studied as matrices in soft tissue engineering due to their known cytocompatibility. For example, cross-linkable dextran methacrylates and hyaluronan methacrylate hydrogels, which are candidates as matrices for soft tissue reconstruction, were synthesized showing that the *in vitro* degradation behavior of these types of hydrogels could be controlled by the polysaccharide structure and the cross-linking density. Furthermore, under *in vitro* conditions, these novel materials had no cytotoxic effects against fibroblasts and the use of composite gels improved the adherence of cells [147].

Therefore, great advances have been achieved so far in scaffold design of new advanced porous hydrogels for tissue engineering applications. Nevertheless, much research has to be conducted still in order to find new ways and methods capable of providing suitable materials able to fulfill all the necessary requirements of this biomedical field.
