**2.1.2 Radical polymerization**

36 Biomedicine

In-situ forming hydrogels are referred to as hydrophilic polymer networks that are in-situ formed in the body after the injection of liquid gel precursors. They are typically categorized into chemical hydrogels and physical hydrogels according to the mechanism underlying the network formation. Chemical hydrogels are those that are prepared by chemically covalent crosslinking of polymers. On the other hand, physical hydrogels are obtained by physical interactions of polymers, such as stereocomplex formation, hydrophobic interactions, and ionic interactions. So far, different crosslinking methods have been developed to prepare in-

Chemical crosslinking produces irreversible, also called permanent hydrogels. Generally, the hydrogels have robust mechanical properties and chemcial stability, which are favorable as supportive scaffolds for tissue engineering. Furthermore, covalent cross-linking is a good means to precisely control the cross-linking density of chemical hydrogels, thus controlling

Michael-type addition reaction is one of the commonly used approaches for the preparation of hydrogels, especially, in-situ forming hydrogels. By this approach, in-situ forming hydrogels can be obtained by mixing aqueous solutions of polymers bearing nucleophilic (amine or thiol) and electrophilic groups (vinyl, acrylate or maleimide) (Mather et al., 2006). For example, Feijen *et al.* prepared in-situ forming hydrogels based on vinyl sulfoneconjugated dextran and poly(ethylene glycol) (PEG) thiols through Michael-type addition (Hiemstra et al., 2007a). The gelation times can be tailored from 7 to 0.5 min when the drgree of vinyl sulfone subsititution increased from 4 to 13. Additionally, by varying the degrees of substitution, dextran molecular weights and polymer concentrations, the storage moduli of the hydrogels can be adjusted from 3 to 46 kPa, and the degradation time from 3 to 21 days. In another study, Hubbell et al. reported on smart hydrogels that were formed in-situ by the addition of thiol-containing oligopeptides to multi-arm vinyl sulfone-terminated PEG (Fig. 1) (Lutolf et al., 2003). Rheology test showed the pH condition plays an important role in the gel formation. With the increasing pH value from 7 to 8, the gelation time decreased from 24 to 4 min. Also, different thiol-bearing peptides (e.g., cysteine-bearing peptides) could be

Fig. 1. In-situ forming cell-responsive hydrogels prepared from vinyl sulfone-functionalized

4-arm poly(ethylene glycol) and the MMP-sensitive bis-cysteine peptide.

**2. Strategies to design in-situ forming hydrogels** 

situ forming hydrogels, which are described in detail as follows.

the hydrogels properties such as degradation time and mechanical strength.

**2.1 Chemical crosslinking** 

**2.1.1 Michael-type addition** 

Radical polymerization is one of the most frequently used crosslinking methods to prepare robust and stable in-situ forming hydrogels. Radicals are created from initiator molecules through thermal, redox or photointiated mechanisms. Then, the radicals propagate through unreacted double bonds during polymerization to form long kinetic chains, and the chains react further with each other to form crosslinked polymeric networks (Ifkovits & Burdick, 2007). In general, macromers bearing vinyl groups are relatively biocompatible and more favourable as compared to monomers. Since the reaction takes place in aqueous solutions, the conversion of double bonds is high due to the high mobility of reacting species during gel formation. This also decreases the potential toxicity of the materials. PEG and PEG-based copolymers are commonly used synthetic biomaterials (Nguyen & West, 2002) (Fig. 2). They can be functionalized with acryl chloride and further crosslinked in the presence of freeradical initiators under a physiological environment to form hydrogels. Multi-arm polymers such as 4-arm and 8-arm PEG were also employed to increase the crosslinking density because of their increased functionality as compare to linear analogues. Other types of polymers are natural polymers such as dextran, hyaluronic acid and collagen (Dong et al., 2005; S.H. Kim et al., 1999; Y.D. Park et al., 2003). As compared to synthetic polymers, they have different functional groups (hydroxyl, amine or carboxylic groups) on their polymer backbones and are amenable to various chemical modifications. The number of double bonds introduced can be precisely controlled on demand. Thus, the properties of freeradical polymerized hydrogel such as gelation time, mechanical properties and degradation profiles can be adjusted for use in different tissue engineering.

Fig. 2. Commonly used poly(ethylene glycol)-based polymers for in-situ forming hydrogels

#### **2.1.3 Enzymatic crosslinking**

In-situ hydrogels formation using enzymes have emerged recently. Enzymes are known to exhibit a high degree of substrate specificity, which potentially avoids side reactions during crosslinking. Another advantage of the enzymatic crosslinking is of mild gelation conditions (e.g. physiological conditions), favourable for tissue regeneration.

In-Situ Forming Biomimetic Hydrogels for Tissue Regeneration 39

Tyrosinase is another enzyme used to form in-situ forming hydrogels. Unlike HRP, the tyrosinase crosslinks phenol-containing polymers in the presence of oxygen instead of hydrogen peroxide (Fig. 3b). Besides, tyrosinase is an oxidative enzyme present in the animal or human body. These features imply milder gelation conditions and better cytobiocompatibility of tyrosinase-crosslinked hydrogels as compared to HRP-crosslinked hydrogels. So far, only few studies have been conducted to construct hydrogels using tyrosinase. For example, Payne *et al.* reported on the hydrogels from the composites of chitosan and gelatin (Chen et al., 2003). The strength of tyrosinase-catalyzed gels could be adjusted by altering the gelatin and chitosan compositions. The author speculated that the

Transglutaminase (TGase) is an enzyme frequently used in protein crosslinking. Recently, it has been employed in the preparation of in-situ forming hydrogels. TGase catalyzes an acyltransfer reaction between the γ-carboxamide group of protein bound glutaminyl residues and the amino group of ε-lysine residues, resulting in the formation of ε-(γ-glutamyl)lysine isopeptide side chain bridges (Sperinde & Griffith, 1997) (Fig. 3c). McHale *et al.* designed and synthesized engineered elastin-like polypeptide (ELPs) hydrogels that are capable of undergoing enzyme-initiated gelation via tissue TGase (McHale et al., 2005). Two kinds of polymer solutions ELP[KV6-112] and ELP[QV6-112] were first mixed and the gels were formed within an hour after enzymes and CaCl2 were subsequently added. In another study, Sanborn *et al.* investigated TGase-catalyzed gelation of peptide-modified PEG and the results showed that gelation times ranged from 9 to 30 min (Sanborn et al., 2002). To shorten the gelation time, Messersmith *et al.* attempted to rationally design the peptide substrates by increasing their specificity (Hu & Messersmith, 2003). It was found that the introduction of an N-terminal L-3,4-dihydroxylphenylalanine (DOPA) residue into the tripeptides resulted in ca. 2.4-fold increment in specificity. This facilitated the gel formation with shorter gelation

Peptide ligation is often employed in the synthesis of proteins and enzymes, which is based on chemoselective reaction of two unprotected peptide segments. Recently, this reaction was explored by Grinstaff *et al* for the preparation of in-situ forming hydrogels due to the mild chemical reaction conditions*.* A typical peptide ligation reaction is based on the reaction of aldehyde groups in poly(ethylene glycol) derivatives and NH2-terminal cysteine moieties in peptide dendrons, which can form thiazolidine rings (Wathier et al., 2004) (Fig.4a). The gelation process took place within a few minutes. However, these hydrogels were intact for short periods of time (about 1 week) due to the reversible thiazolidine ring formation. To overcome the problem, the same group developed stable hydrogels prepared from poly(ethylene glycol) with endcapped ester-aldehyde groups instead of aldehyde groups (Wathier et al., 2006) (Fig.4b). The ester-aldehyde groups firstly reacted with the NH2 terminal cysteine moieties to form the thiazolidine ring, which then can undergo a rearrangement to give chemically stable pseudoproline ring. The mechanical properties of the hydrogels depend on the concentrations of the polymer solutions and different ratios of aldehyde to cysteine reactive functionality. Degradation studies demonstrated that the pseudoproline ring was more stable than the thiazolidine ring and the hydrogels retained

their shape and size with less than 10% weight loss for more than 6 months.

gel system may be useful as emergency dressings for burns and wounds.

time of 2 min.

**2.1.4 Peptide ligation** 

Horseradish peroxidase (HRP) has been recently employed in the preparation of in situ forming hydrogels. HRP is a single-chain b-type hemoprotein that catalyzes the coupling of phenols or aniline derivatives in the presence of hydrogen peroxide (Kobayashi et al., 2001). Crosslinking reaction takes place via a carbon-carbon bond at the ortho positions and/or via a carbon-oxygen bond between the carbon atom at the ortho position and the phenoxy oxygen in the phenol moieties (Fig. 3a). For example, Feijen *et al.* reported on HRP-mediated in-situ forming dextran-tyramine hydrogels for cartilage tissue engineering (Jin et al., 2007). Tyramine was conjugated to dextran by first activation of the hydroxyl groups in dextran using p-nitrophenyl chloroformate and then treatment with tyramine by aminolysis. The gelation rates induced by enzyme-mediated crosslinking can be readily adjusted from minutes to seconds by varying the HRP concentrations. By the same approach, Jin and Lee *et al.* prepared the chitosan-phloretic acid and hyaluronic acid-tyramine hydrogels for cartilage tissue engineering (Jin et al., 2009) and protein delivery applications (F. Lee et al., 2009), respectively. The disadvantage of this approach is the use of hydrogen peroxide. It is reported that high concentration of hydrogen peroxide (>0.2 mM) may induce cell apoptosis (Asada et al., 1999). Therefore, it is important to control the amount of hydrogen peroxide used in a cell-favourable range.

Fig. 3. Enzymatic crosslinking method to prepare in-situ forming hydrogels

Horseradish peroxidase (HRP) has been recently employed in the preparation of in situ forming hydrogels. HRP is a single-chain b-type hemoprotein that catalyzes the coupling of phenols or aniline derivatives in the presence of hydrogen peroxide (Kobayashi et al., 2001). Crosslinking reaction takes place via a carbon-carbon bond at the ortho positions and/or via a carbon-oxygen bond between the carbon atom at the ortho position and the phenoxy oxygen in the phenol moieties (Fig. 3a). For example, Feijen *et al.* reported on HRP-mediated in-situ forming dextran-tyramine hydrogels for cartilage tissue engineering (Jin et al., 2007). Tyramine was conjugated to dextran by first activation of the hydroxyl groups in dextran using p-nitrophenyl chloroformate and then treatment with tyramine by aminolysis. The gelation rates induced by enzyme-mediated crosslinking can be readily adjusted from minutes to seconds by varying the HRP concentrations. By the same approach, Jin and Lee *et al.* prepared the chitosan-phloretic acid and hyaluronic acid-tyramine hydrogels for cartilage tissue engineering (Jin et al., 2009) and protein delivery applications (F. Lee et al., 2009), respectively. The disadvantage of this approach is the use of hydrogen peroxide. It is reported that high concentration of hydrogen peroxide (>0.2 mM) may induce cell apoptosis (Asada et al., 1999). Therefore, it is important to control the amount of hydrogen peroxide

Fig. 3. Enzymatic crosslinking method to prepare in-situ forming hydrogels

used in a cell-favourable range.

Tyrosinase is another enzyme used to form in-situ forming hydrogels. Unlike HRP, the tyrosinase crosslinks phenol-containing polymers in the presence of oxygen instead of hydrogen peroxide (Fig. 3b). Besides, tyrosinase is an oxidative enzyme present in the animal or human body. These features imply milder gelation conditions and better cytobiocompatibility of tyrosinase-crosslinked hydrogels as compared to HRP-crosslinked hydrogels. So far, only few studies have been conducted to construct hydrogels using tyrosinase. For example, Payne *et al.* reported on the hydrogels from the composites of chitosan and gelatin (Chen et al., 2003). The strength of tyrosinase-catalyzed gels could be adjusted by altering the gelatin and chitosan compositions. The author speculated that the gel system may be useful as emergency dressings for burns and wounds.

Transglutaminase (TGase) is an enzyme frequently used in protein crosslinking. Recently, it has been employed in the preparation of in-situ forming hydrogels. TGase catalyzes an acyltransfer reaction between the γ-carboxamide group of protein bound glutaminyl residues and the amino group of ε-lysine residues, resulting in the formation of ε-(γ-glutamyl)lysine isopeptide side chain bridges (Sperinde & Griffith, 1997) (Fig. 3c). McHale *et al.* designed and synthesized engineered elastin-like polypeptide (ELPs) hydrogels that are capable of undergoing enzyme-initiated gelation via tissue TGase (McHale et al., 2005). Two kinds of polymer solutions ELP[KV6-112] and ELP[QV6-112] were first mixed and the gels were formed within an hour after enzymes and CaCl2 were subsequently added. In another study, Sanborn *et al.* investigated TGase-catalyzed gelation of peptide-modified PEG and the results showed that gelation times ranged from 9 to 30 min (Sanborn et al., 2002). To shorten the gelation time, Messersmith *et al.* attempted to rationally design the peptide substrates by increasing their specificity (Hu & Messersmith, 2003). It was found that the introduction of an N-terminal L-3,4-dihydroxylphenylalanine (DOPA) residue into the tripeptides resulted in ca. 2.4-fold increment in specificity. This facilitated the gel formation with shorter gelation time of 2 min.
