*3.2. Proteins/Peptides*

Polypeptides are compounds composed of α-amino acids connected by peptide bonds. They are also intermediate products in protein hydrolysis. Amino acids form polypeptides via dehydration condensation. After winding and folding, polypeptides can form macromolecules with certain spatial structures, namely proteins. Proteins and peptides play important roles in the growth and development of life. Because of their widespread sources, nontoxic degradation products, and even benefits to the human body, proteins and polypeptide products are widely used in the biomedical field. Because the amino acids that make up proteins and peptides have free side-chain groups, they can be endowed with new functions through chemical modification, such as photocrosslinking groups. Common proteins used for modification with photocrosslinking groups include collagen, gelatin, keratin, silk fibroin, and albumin. Their application scope is greatly expanded through chemical modification, which gives them wide influence in the biomedical field.

Collagen is a protein formed by three peptide super-chain helices; it accounts for 25–30% of all proteins in mammals and is the most abundant protein [76,77]. Common collagens include type I, type II, type III, type V, and type XI. Collagen is widely used in foods, medicines, tissue engineering, cosmetics, and other fields because of its good biocompatibility, biodegradability, and biological activity [78]. Photocrosslinking of collagen hydrogel can be realized with chemical modifications and photocrosslinking. The degradation rate after the collapse of collagen hydrogels is slow, and they can be maintained for a long time to achieve specific functions. For example, Yang et al. photocrosslinked methacrylated type II collagen and used it for encapsulation of bone-marrow mesenchymal stem cells (BMSCs) (Figure 3a). The crosslinked collagen maintained its triple-helix structure, which provides a good microenvironment for proliferation and differentiationof BMSCs [79].

**Figure 3.** Part of the synthetic route maps or related schematic diagrams for crosslinked hydrogels. (**a**) Chemical modification of type II collagen and photocrosslinking hydrogel for bone-marrow mesenchymal stem-cell culture [79]. (**b**) Synthetic route of methacryloylated gelatin and its gelling diagram [80]. (**c**) Synthesis roadmap of two-step modified silk fibroin [81]. (**d**) Schematic diagram for crosslinking between keratin and 2,2-dimethoxy-2-phenylacetophenone [82]. (**e**) Schematic diagram of free-radical formation in the rib/L-Arg system [83]. **Figure 3.** Part of the synthetic route maps or related schematic diagrams for crosslinked hydrogels. (**a**) Chemical modification of type II collagen and photocrosslinking hydrogel for bone-marrow mesenchymal stem-cell culture [79]. (**b**) Synthetic route of methacryloylated gelatin and its gelling diagram [80]. (**c**) Synthesis roadmap of two-step modified silk fibroin [81]. (**d**) Schematic diagram for crosslinking between keratin and 2,2-dimethoxy-2-phenylacetophenone [82]. (**e**) Schematic diagram of free-radical formation in the rib/L-Arg system [83].

Gelatineis formed by partial hydrolysis of collagen, and it can be dissolved in hot water and forms a gel after cooling. This excellent feature provides flexible application scenarios. However, in most cases, a more stable hydrogel structure is desired, and the temperature range for gelatine gels is relatively narrow, which makes it difficult to meet the demand. Therefore, researchers have modified gelatinewith methacryloyl to enable photocrosslinking. Methacryloylated gelatine (GelMA) has good biocompatibility and structural stability after photocrosslinking. Therefore, methacryloylated gelatine shows great application value in the biomedical field [84]. The Khademhosseini team used gelatineas a raw material to synthesize GelMA, and used it in cell culture and microchips to verify its application value in cell-responsive microengineered hydrogels (Figure 3b) [80]. Fu et al. prepared structural colored microspheres with GelMA and used them for cell culture. They found that they had good biocompatibility and are expected to be used to construct liver chips [85]. Gelatineis formed by partial hydrolysis of collagen, and it can be dissolved in hot water and forms a gel after cooling. This excellent feature provides flexible application scenarios. However, in most cases, a more stable hydrogel structure is desired, and the temperature range for gelatine gels is relatively narrow, which makes it difficult to meet the demand. Therefore, researchers have modified gelatinewith methacryloyl to enable photocrosslinking. Methacryloylated gelatine (GelMA) has good biocompatibility and structural stability after photocrosslinking. Therefore, methacryloylated gelatine shows great application value in the biomedical field [84]. The Khademhosseini team used gelatineas a raw material to synthesize GelMA, and used it in cell culture and microchips to verify its application value in cell-responsive microengineered hydrogels (Figure 3b) [80]. Fu et al. prepared structural colored microspheres with GelMA and used them for cell culture. They found that they had good biocompatibility and are expected to be used to construct liver chips [85].

Keratin is the main protein constituting hair, horn, claws, and the outer layers of animal skin. Because keratin contains cystine, it has a high proportion of disulfide bonds and plays acrosslinking role in protein peptide chains. Therefore, keratin has particularly stable chemical properties and high mechanical strength [86,87]. Studies have shown that keratin extracted from human hair fibers contains a leucine–aspartate–valine (LDV) cell-adhesion motif. Therefore, keratin has great application potential for cell culture and tissue engineering. To provide more application scenarios for keratin, it can be modified. Keratin is the main protein constituting hair, horn, claws, and the outer layers of animal skin. Because keratin contains cystine, it has a high proportion of disulfide bonds and plays acrosslinking role in protein peptide chains. Therefore, keratin has particularly stable chemical properties and high mechanical strength [86,87]. Studies have shown that keratin extracted from human hair fibers contains a leucine–aspartate–valine (LDV) cell-adhesion motif. Therefore, keratin has great application potential for cell culture and tissue engineering. To provide more application scenarios for keratin, it can be modified.

For example, Yu and Hu et al. successfully realized photocrosslinking of keratin by us-

For example, Yu and Hu et al. successfully realized photocrosslinking of keratin by using click chemistry and introducing 2,2-dimethoxy-2-phenylacetophenone (Figure 3c) [81,88].

Silk fibroin is a natural high-molecular fibrin extracted from silk. It has good mechanical and physicochemical properties and a long application history. To improve plasticity, many researchers have tried a variety of methods to modify it chemically (Figure 3d) [82]. Qi and others used silk fibroin modified by methacryl groups to obtain an injectable silkfibroin hydrogel after photocrosslinking [89]. However, the lower side chains of silk fibroin decreased the content of methacryl groups, so the photocrosslinked silk-fibroin hydrogel had lower mechanical strength. Therefore, it was necessary to modify other active groups on silk fibroin to improve the mechanical strength. For example, Ju et al. obtained silkfibroin hydrogels with good biocompatibility and mechanical properties by modifying the silk-fibroin hydroxyl with methacrylic acid and then performing crosslinking [90].

Albumin is a protein in plasma that maintains body nutrition and osmotic pressure, and it accounts for approximately 50% of all plasma proteins. It has good biocompatibility and solubility. Therefore, when preparing biomedical materials with albumin as a raw material, the inherent defects of synthetic materials can be avoided. For example, Chiriac and collaborators used riboflavin and arginine as natural initiators to crosslink bovine serum albumin (BSA) and obtain BSA hydrogels (Figure 3e) [83]. In vitro and in vivo experiments showed that the hydrogel had good biocompatibility.

## **4. Preparation of Photocrosslinked Hydrogels with Different Morphologies**

In biomedical applications, different applications have different morphological requirements for common hydrogels. Therefore, it is crucial to prepare a hydrogel with a specific morphology. Common photocrosslinked hydrogel morphologies mainly include fibers, microspheres, thin films, microneedles, amorphous shapes, and so on. At present, many researchers have effectively explored processing methods for hydrogels with different morphologies, which has greatly expanded the application potential of hydrogels in biomedicine.

### *4.1. Fiber*

Fibrous products such as gauze are widely used in the life science and biomedical fields. Inspired by this, many researchers have developed new hydrogel-fiber products. Due to their high surface-to-volume ratios and high porosities, they exhibit good water absorbance and air permeability and can replace some functions, such as those of traditional gauze in the biomedical field [91,92]. Usually, a hydrogel precursor solution is converted into fibers, photocrosslinked, and finally accumulated in woven-fiber products. At present, the main method used for fiber preparation is electrospinning [93–95]. When the solution is squeezed out under a high-pressure electric field, the liquid becomes filamentous due to the electric field. After the solvent evaporates, the polymer, polymer mixture, composite materials, and other molecules form fiber shapes. Electrospun fibers have many remarkable properties, such as high surface-to-volume ratios and functional tunability; they can be applicable in many areas including drug delivery, wound dressings, tissue engineering, membranes or filters, electronics, sensors, and energy [92]. Furthermore, when photocrosslinking technology is applied, the mechanical performance and stability of the fibercan be improved further [96,97]. For example, Tang et al. developed a novel high-throughput nanofiber-composite ultrafiltration membrane (Figure 4a) [98]. They first prepared chemically crosslinked polyvinyl-alcohol (PVA) nanofiberscaffolds on a nonwoven substrate, and then prepared a polyvinyl-alcohol (UV-PVA) barrier layer via UV crosslinking. The results showed that the 5 wt% UV-PVA solution coating provided an ultrafiltration membrane with high throughput and high retention rate after UV curing for 20 s. It had good pollution resistance and could be used for the separation of oil and water emulsions.

**Figure 4.** Schematic diagram of the preparation and application of hydrogels with fiber and microspherical morphologies. (**a**) Electron micrograph of ahigh-flux nanofiber-composite ultrafiltration membrane [98]. (**b**) Schematic diagram of the preparation and application of photocrosslinked GelMA-hydrogel microsphere [99]. (**c**) Enzyme-functionalized antiproteolytic-hydrogel microspheres were used for biocatalysis [100]. (**d**) Schematic diagram for preparation and application of ALP microcapsules [101]. **Figure 4.** Schematic diagram of the preparation and application of hydrogels with fiber and microspherical morphologies. (**a**) Electron micrograph of ahigh-flux nanofiber-composite ultrafiltration membrane [98]. (**b**) Schematic diagram of the preparation and application of photocrosslinked GelMA-hydrogel microsphere [99]. (**c**) Enzyme-functionalized antiproteolytic-hydrogel microspheres were used for biocatalysis [100]. (**d**) Schematic diagram for preparation and application of ALP microcapsules [101].

Microspherical hydrogels have excellent mobility and mass delivery, and thus have received considerable attention in fields such as biomedical detection and drug delivery. Specific structural differences can be used to subdivide the micropellets into homogeneous micropellets, antiopal micropellets [32], and nuclear-shell micropellets (microcapsules) [101]. Microspherical hydrogels have excellent mobility and mass delivery, and thus have received considerable attention in fields such as biomedical detection and drug delivery. Specific structural differences can be used to subdivide the micropellets into homogeneous micropellets, antiopal micropellets [32], and nuclear-shell micropellets (microcapsules) [101].

### *4.2. Microballoons 4.2. Microballoons*

### 4.2.1. Homogeneous Micropellets 4.2.1. Homogeneous Micropellets

Homogenous hydrogel microspheres are usually formed by photocrosslinking after the hydrogel precursor solution is dispersed into liquid droplets. There are many ways to disperse solutions into droplets, but the principle is basically consistent; all methods use emulsification to generate droplets, such as with stirring, microfluidics, and electrospraying. Among these techniques, microfluidic technology has obvious advantages in preparing microspheres due to its accurate fluid control [99,102]. For example, Zhao et al. obtained hydrogel microspheres loaded with cells and growth factors by mixing cells and growth factors with GelMA solution, using microfluidics to cut them into droplets, Homogenous hydrogel microspheres are usually formed by photocrosslinking after the hydrogel precursor solution is dispersed into liquid droplets. There are many ways to disperse solutions into droplets, but the principle is basically consistent; all methods use emulsification to generate droplets, such as with stirring, microfluidics, and electrospraying. Among these techniques, microfluidic technology has obvious advantages in preparing microspheres due to its accurate fluid control [99,102]. For example, Zhao et al. obtained hydrogel microspheres loaded with cells and growth factors by mixing cells and growth factors with GelMA solution, using microfluidics to cut them into droplets, and

and then photocrosslinking them. BMSCs in microspheres showed significant osteogenic

then photocrosslinking them. BMSCs in microspheres showed significant osteogenic effects both in vitro and in vivo, and significantly increased mineralization, thus promoting bone regeneration (Figure 4b) [99]. Combining microfluidic technology with electrojet technology allowed the application of an electric field as an additional shear force to simplify the microfluidic device. For example, Zhang et al. successfully used microfluidic electroinjection technology to prepare photocrosslinked chondroitin-sulfate microspheres, which showed good value for drug loading and wound repair [103].

### 4.2.2. Antiopal Micropellets

Opal has a periodic, ordered structure formed by nanoparticles. When the nanoparticles have a specific size, the opal reflects light of a specific wavelength and thus shows a structural color. Negative replication using opal as a template yields an antiopal material. This material has the same structural color characteristics and a continuous porous structure, which provide good prospects for applications in mass transfer (especially in the fields of drug loading and sensing) [100]. For example, Wang and others prepared inverse-opal hydrogel microspheres by using colloidal-crystal microspheres assembled with silicondioxide nanoparticles as templates and AAm and N,N0 -methylenebis-(acrylamide) (Bis) as photocrosslinked hydrogel skeletons to copy the template microspheres [32]. Afterhydrolysis and enzymatic immobilization of the microspheres, the resulting material had biocatalytic functionality and is expected to be used for treatment of complex water bodies (Figure 4c).

### 4.2.3. Nuclear-Shell Microspheres (Microcapsules)

Antiopal hydrogel microspheres were obtained by using corrosion for removal of nanoparticle templates. If the corrosion was incomplete, the prepared microspheres had a nuclear-shell structure consisting of a hydrogel or nanoparticle-composite core and an antiopal hydrogel shell. These nuclear-shell microspheres exhibit responsiveness similar to that of antiopal hydrogel microspheres, and they also have high stability and can be used for encoding [104]. Based on this, Xu et al. developed photocrosslinked nuclear-shell hydrogel microspheres that can be used for miRNA detection by modifying the corresponding aptamer [105].

Homogeneous hydrogel microspheres are generally prepared by single-emulsion devices. Core-shell hydrogel microcapsules can be prepared if a double-emulsion device is used and the inner solute is not crosslinked. For example, Zhao et al. developed microcapsules containing an alkaline phosphatase (ALP) solution by applying microfluidic electrojet technology. The shells of the microcapsule were formed by calcium alginate and photocrosslinked PEGDA, which can preserve the activity of ALP in the digestive tract and thus be used for intestinal endotoxin cleaning (Figure 4d) [101].
