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

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

## **2.1 Chemical crosslinking**

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 the hydrogels properties such as degradation time and mechanical strength.

#### **2.1.1 Michael-type addition**

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.

incorporated to yield biofunctional or bioreponsive hydrogels, enhancing cell adhesion and matrix production (Seliktar et al., 2004). This indicates that Michael-type addition is an ideal method for the preparation of in-situ crosslinked hybrid hydrogels.
