**2.1.4 Peptide ligation**

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.

modulated.

**2.2.2 Hydrophobic interaction** 

the break of micelle structure.

In-Situ Forming Biomimetic Hydrogels for Tissue Regeneration 41

and the water content of dex-lactate solutions, the properties of the hydrogels can be well

The hydrophobic interaction provides another driving force of physical gelation. Some amphiphilic copolymers can undergo a sol-gel transition via this mechanism. Typical amphiphilic polymers are block copolymers based on poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), also called as Pluronics® (Fusco et al., 2006). It was found that, when increasing temperature, the PEO-PPO polymers can undergo a dehydration process. This leads to the formation of hydrophobic domains and, in turn, transition of an aqueous liquid to a hydrogel. The drawbacks of PEO-PPO hydrogels include rapid erosion, potential cytotoxicity (Khattak et al., 2005), and non-biodegradability. Alternatively, polyester-based copolymers that are biodegradable received much attention. For example, Jeong and coworkers described thermosensitive, biodegradable hydrogels based on poly(ethylene oxide) and poly(lactic acid) (Jeong et al., 1997). Solutions of the diblock copolymers were shown to be in solution state at 45°C, but gel state at body temperature. However, the encapsulation of drugs at an elevated temperature might lead to denaturation of bioactive agents such as therapeutic proteins or growth factors. The same group subsequently reported on a series of triblock copolymers of poly(ethylene oxide) and poly(lactic acid)/poly(glycolic acid) (Jeong et al., 1999). This thermosensitive hydrogel system is inverse to the hydrogel based on poly(ethylene oxide)-co-poly(lactic acid) diblock polymers, that is, poly(ethylene oxide)-b- (D,L-lactic acid-co-glycolic acid)-b-poly(ethylene oxide) triblock copolymers were found to be in a solution at room temperature, but form a hydrogel when the temperature is increased to 37°C. This makes the gel system easy to handle and favourable for tissue engineering applications (Jeong et al., 2000). Moreover, the sol–gel transition temperature and degradation properties can be adjusted by the polymer concentration, molecular weight of poly(ethylene oxide) and the lactic acid/glycolic acid ratio in the poly(lactic acid-coglycolic acid) blocks. In another study, Ding *et al.* reported that the end groups have a surprising effect on the hydrogel formation (L. Yu et al., 2006). The results showed that the transition temperature increased with a decreasing hydrophobicity of the end groups. Importantly, it is noted that sol-gel transition takes place only when the hydrophobic interactions are strong enough to induce the large-scale self-assembly of micelles. However, over hydrophobicity and higher temperature lead to precipitation of polymers as a result of

Polypeptide or peptide-conjugated polymers is another type of polymers that can be used to form hydrogels via hydrophobic interactions. The formation of hydrogels from polypeptides are based on coil-coil interaction, triggered by the self-assembly of peptide sequences. The coiled-coil interaction is one of the basic folding patterns of native proteins and consists of two or more helices winding together to form a superhelix (Y.B. Yu, 2002). A series of hydrogels based on peptides or peptide/synthtic polymer hydrids were made. Typical examples are synthetic N-(2-hydroxypropyl)methacrylamide (HPMAm) copolymer grated with coiled-coil protein motifs (C. Wang et al., 1999; J. Yang et al., 2006b). The gelation time can be adjusted by the length and the number of coiled-coil grafts per chain an ranged from a few minutes to several days (J. Yang et al., 2006a). Besides, it was found that at least 4 heptads were needed to achieve hydrogels formation. In another study, Xu *et al.* 

Fig. 4. Preparation of in-situ forming hydrogel via peptide ligation

#### **2.2 Physical crosslinking**

Much attention has been paid in the preparation of in-situ forming physical hydrogels. The advantages of physical crosslinking are relatively good biocompatibility and less toxicity since toxic crosslinking reagent or initiators are not used during crosslinking. However, physically-crosslinked hydrogels are gnerally unstable and mechanically weak. The changes in the environment such as pH, temperature and ionic strength may lead to the disruption of the gel network. Typical physical crosslinking methods include stereocomplexation, hydrophobic interactions and ionic interaction.

#### **2.2.1 Stereocomplexation**

A typical polymer used for stereocomplex formation is poly(lactide) (PLA). It is a kind of aliphatic polyesters, which are known to be biocompatible and render PLA-based hydrogels biodegradable. Lactide has three possible configurations, which refer to D-lactide, L-lactide and meso-lactide according to the arrangement of substituents around the chiral carbon. The corresponding polymers are defines as poly(L-lactide) (PLLA), poly(D-lactide) (PDLA) and poly(D,L-lactide) (PDLLA). The formation of stereocomplexes when mixing PLLA and PDLA was first reported by Ikada *et al.* (Ikada et al., 1987). The stereocomplexation not only occurs in the blends of PLLA and PDLA homopolymers, but also in water-soluble PLA and poly(ethylene glycol) (PEG) block copolymers, such as linear (Hiemstra et al., 2005) and multiarm PEG-PLLA and PEG-PDLA block copolymers (Hiemstra et al., 2006). This gives the possibility to design different PLA-conjugated materials in hydrogel preparation. For example, Hennink *et al.* reported on physical hydrogels based on the stereocomplexation of PLA-dextran conjugates (de Jong et al., 2000). L- and D-lactic acid oligomers were coupled to dextran to yield dex-(L)lactate and dex-(D)lactate. It was found that the degree of polymerization of lactic acid oligomers must be at least 11 to obtain the hydrogels. The stereocomplex crosslinking can be detected by X-ray diffraction (de Jong et al., 2002). Varying the degree of polymerization of oligomer, the degree of substitution of dex-lactate and the water content of dex-lactate solutions, the properties of the hydrogels can be well modulated.

#### **2.2.2 Hydrophobic interaction**

40 Biomedicine

Much attention has been paid in the preparation of in-situ forming physical hydrogels. The advantages of physical crosslinking are relatively good biocompatibility and less toxicity since toxic crosslinking reagent or initiators are not used during crosslinking. However, physically-crosslinked hydrogels are gnerally unstable and mechanically weak. The changes in the environment such as pH, temperature and ionic strength may lead to the disruption of the gel network. Typical physical crosslinking methods include stereocomplexation,

A typical polymer used for stereocomplex formation is poly(lactide) (PLA). It is a kind of aliphatic polyesters, which are known to be biocompatible and render PLA-based hydrogels biodegradable. Lactide has three possible configurations, which refer to D-lactide, L-lactide and meso-lactide according to the arrangement of substituents around the chiral carbon. The corresponding polymers are defines as poly(L-lactide) (PLLA), poly(D-lactide) (PDLA) and poly(D,L-lactide) (PDLLA). The formation of stereocomplexes when mixing PLLA and PDLA was first reported by Ikada *et al.* (Ikada et al., 1987). The stereocomplexation not only occurs in the blends of PLLA and PDLA homopolymers, but also in water-soluble PLA and poly(ethylene glycol) (PEG) block copolymers, such as linear (Hiemstra et al., 2005) and multiarm PEG-PLLA and PEG-PDLA block copolymers (Hiemstra et al., 2006). This gives the possibility to design different PLA-conjugated materials in hydrogel preparation. For example, Hennink *et al.* reported on physical hydrogels based on the stereocomplexation of PLA-dextran conjugates (de Jong et al., 2000). L- and D-lactic acid oligomers were coupled to dextran to yield dex-(L)lactate and dex-(D)lactate. It was found that the degree of polymerization of lactic acid oligomers must be at least 11 to obtain the hydrogels. The stereocomplex crosslinking can be detected by X-ray diffraction (de Jong et al., 2002). Varying the degree of polymerization of oligomer, the degree of substitution of dex-lactate

Fig. 4. Preparation of in-situ forming hydrogel via peptide ligation

**2.2 Physical crosslinking** 

**2.2.1 Stereocomplexation** 

hydrophobic interactions and ionic interaction.

The hydrophobic interaction provides another driving force of physical gelation. Some amphiphilic copolymers can undergo a sol-gel transition via this mechanism. Typical amphiphilic polymers are block copolymers based on poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), also called as Pluronics® (Fusco et al., 2006). It was found that, when increasing temperature, the PEO-PPO polymers can undergo a dehydration process. This leads to the formation of hydrophobic domains and, in turn, transition of an aqueous liquid to a hydrogel. The drawbacks of PEO-PPO hydrogels include rapid erosion, potential cytotoxicity (Khattak et al., 2005), and non-biodegradability. Alternatively, polyester-based copolymers that are biodegradable received much attention. For example, Jeong and coworkers described thermosensitive, biodegradable hydrogels based on poly(ethylene oxide) and poly(lactic acid) (Jeong et al., 1997). Solutions of the diblock copolymers were shown to be in solution state at 45°C, but gel state at body temperature. However, the encapsulation of drugs at an elevated temperature might lead to denaturation of bioactive agents such as therapeutic proteins or growth factors. The same group subsequently reported on a series of triblock copolymers of poly(ethylene oxide) and poly(lactic acid)/poly(glycolic acid) (Jeong et al., 1999). This thermosensitive hydrogel system is inverse to the hydrogel based on poly(ethylene oxide)-co-poly(lactic acid) diblock polymers, that is, poly(ethylene oxide)-b- (D,L-lactic acid-co-glycolic acid)-b-poly(ethylene oxide) triblock copolymers were found to be in a solution at room temperature, but form a hydrogel when the temperature is increased to 37°C. This makes the gel system easy to handle and favourable for tissue engineering applications (Jeong et al., 2000). Moreover, the sol–gel transition temperature and degradation properties can be adjusted by the polymer concentration, molecular weight of poly(ethylene oxide) and the lactic acid/glycolic acid ratio in the poly(lactic acid-coglycolic acid) blocks. In another study, Ding *et al.* reported that the end groups have a surprising effect on the hydrogel formation (L. Yu et al., 2006). The results showed that the transition temperature increased with a decreasing hydrophobicity of the end groups. Importantly, it is noted that sol-gel transition takes place only when the hydrophobic interactions are strong enough to induce the large-scale self-assembly of micelles. However, over hydrophobicity and higher temperature lead to precipitation of polymers as a result of the break of micelle structure.

Polypeptide or peptide-conjugated polymers is another type of polymers that can be used to form hydrogels via hydrophobic interactions. The formation of hydrogels from polypeptides are based on coil-coil interaction, triggered by the self-assembly of peptide sequences. The coiled-coil interaction is one of the basic folding patterns of native proteins and consists of two or more helices winding together to form a superhelix (Y.B. Yu, 2002). A series of hydrogels based on peptides or peptide/synthtic polymer hydrids were made. Typical examples are synthetic N-(2-hydroxypropyl)methacrylamide (HPMAm) copolymer grated with coiled-coil protein motifs (C. Wang et al., 1999; J. Yang et al., 2006b). The gelation time can be adjusted by the length and the number of coiled-coil grafts per chain an ranged from a few minutes to several days (J. Yang et al., 2006a). Besides, it was found that at least 4 heptads were needed to achieve hydrogels formation. In another study, Xu *et al.* 

In-Situ Forming Biomimetic Hydrogels for Tissue Regeneration 43

Much effort has been made in the design of bioactive hydrogels which can instruct cell behaviors and promote tissue regeneration. A well-know bioactive ligand is cell-adhesive peptides, e.g., Arg-Gly-Asp (RGD). It was revealed that RGD-modified PEG diacrylate hydrogels could induce enhanced cell attachment and mineralized matrix deposition of osteoblasts as compared to RGD-free hydrogels (Burdick & Anseth, 2002). Natural proteins such as collagen and its analogs may also serve as bioactive ligands due to inherent nature of biological recognition. Seliktar *et al.* reported on the preparation of proteins (collagen, albumin and fibrinogen) conjugated with acrylated PEG and subsequent hydrogel formation by photopolymerization (Gonen-Wadmany et al., 2007) (Fig. 5). The modified protein maintained its cell-adhesive properties and supported proteolytic degradability based on the specific characteristics of the protein backbone. In another study, Lee and coworkers reported on the collagen mimetic peptide-conjugated poly(ethylene glycol) hydrogels (H.J. Lee et al., 2006). The collagen mimetic peptide (CMP) with a specific amino acid sequence, -(Pro-Hyp-Gly)x-, forms a triple helix conformation that resembles the native protein structure of natural collagens. CMP was first conjugated with acrylated PEG, which copolymerized with poly(ethylene oxide) diacrylate to create a novel PEG hydrogel. The modified protein can maintain their cell-adhesive properties and support proteolytic degradability based on the specific characteristics of the protein backbone. The biochemical analysis showed that chondrocytes-encapsulated hydrogels revealed an 87% increase in glycosaminoglycan content and a 103% increase in collagen content compared to that of control PEG hydrogels after 2 weeks. These results indicate that the CMP enhances the tissue production of cells encapsulated in the PEG hydrogel by providing cell-manipulated

crosslinks and collagen binding sites that simulate natural extracellular matrix.

Fig. 5. Bioactive hydrogels prepared from poly(ethylene glycol) and proteins/peptide.

Biomimetic hydrogels can response to biological components, such as enzymes, receptors and antibodies. After the hydrogels undergo a macroscopic transition (gelation, enzymatic degradation and swelling/shrinkage), this in turn directly leads to microscopic response of living cells (cell migration, differentiation, cell division and matrix production). For example, Lutolf *et al.* developed cell-responsive hydrogels that can degrade in response to local protease activity such as matrix metalloproteinase (MMP) at the cell surface. MMP is a protease family extensively involved in tissue development and remodeling. The hydrogel systems were made from vinyl sulfone-functionalized multiarmed PEG and the bis-cysteine

**3.1 Bioactive hydrogels** 

**3.2 Bioresponsive hydrogel** 

reported on the hydrogels based on genetically engineered protein block copolymers with 2 coiled-coil domains in a random coil polyelectrolyte (Xu & Kopeček, 2008). The selfassmebly process between coil-coils was influenced by the protein concetration, pH and temperature. Changes in the peptide sequence of the coil-coil domains endow hydrogels with different stability.
