**3. Biomimetic hydrogels**

The success of tissue engineering depends on biomimetic hydrgoel scaffolds that possess controlled structures and on-demand properties to modulate specific cellular behaviors. The development of suitable synthetic methods encompassing chemistry and molecular biology open a new way for the design of biomimetic hydrogels mimicking basic processes of living systems. In general, biomimetic hydrogels can be categorized into bioactive, bioresponsive, and biofunctional hydrogels.

### **3.1 Bioactive hydrogels**

42 Biomedicine

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

Ionic interaction is another route to construct in-situ forming hydrogels. For example, the hydrogels can be formed by ionic interactions between water-soluble charged polymers and their di- or multi-valent counter-ions. As a typical example, alginate, a naturally occurring polysacchride, can form a hydrogel network in the presence of calcium ions under physiological conditions. The mechanism underlying the ionic crosslinking is ion exchange of sodium ions by calcium ions in the carboxylic groups and subsequent formation of an egg-box structure (Gombotz & Wee, 1998). The hydrogel is degradable slowly with the diffusion of calcium ions out of the hydrogels and finally excreted from the kidney. Fur ther studies showed that alginate-based hydrogels with CaSO4 usually reveal a heterogeneous structure due to the difficulty in the control of gelation kinetics (Kuo & Ma, 2001). This phenomenon also occured for the hydrogels using CaCl2 (Skjak-Brvk et al., 1989). In contrast, CaCO3 can give homogeneous alginate hydrogel, while its low solubility is unfavourable for further biomedical application. Ma et al. reported on crosslinked alginate hydrogels using CaCO3-GDL (D-glucono-d-lactone) and CaSO4-CaCO3-GDL systems (Kuo & Ma, 2001). Gelation rates and mechanical properties of the alginate hydrogels could be controlled by varying the composition of calcium compound systems and alginate

In-situ forming hydrogels can also be prepared by ionic interactions between polycations and polyanions. For example, Hennink et al. reported on self-gelling hydrogels based on oppositely charged dextran microspheres (Tomme et al., 2005). These charged dextranmicrospheres were prepared by radical polymerization of hydroxyethyl methacrylatederivatized dextran (dex-HEMA) with methacrylic acid (MAA) or dimethylaminoethyl methacrylate (DMAEMA). Hydrogels could be formed as a result of ionic interactions between oppositively-charged microspheres. The networks of hydrogels were disrupted either by applied stress, low pH or high ionic strength. Reversible yield point from rheological analysis indicated that this hydrogel system can be applied for controlled delivery of pharmaceutically active proteins and tissue engineering. However, a main disadvantage of ionically-crosslinked hydrogels is that their mechanical strength is far from

The success of tissue engineering depends on biomimetic hydrgoel scaffolds that possess controlled structures and on-demand properties to modulate specific cellular behaviors. The development of suitable synthetic methods encompassing chemistry and molecular biology open a new way for the design of biomimetic hydrogels mimicking basic processes of living systems. In general, biomimetic hydrogels can be categorized into bioactive, bioresponsive,

concentration, thereby giving rise to structurally uniform hydrogels.

satisfactory when they are served as scaffolds for tissue regeneration.

**3. Biomimetic hydrogels** 

and biofunctional hydrogels.

with different stability.

**2.2.3 Ionic interaction** 

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.

#### **3.2 Bioresponsive hydrogel**

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

In-Situ Forming Biomimetic Hydrogels for Tissue Regeneration 45

hydrogels can be postcrosslinked by UV-irradiation (Fig. 6). These double-crosslinked hydrogels showed increased mechanical moduli and prolonged degradation times compared to the hydrogels that were formed only by stereocomplexation. The photopolymerization takes place at much lower initiator concentrations (0.003 wt%) than conventional photocrosslinking systems (0.05 wt%), which greatly reduces the possibility of

Second, robust hydrogels are produced that consist of two interpenetrated polymeric networks. The hydrogels with double networks contain a subset of interpenetrating networks (IPNs) formed by two hydrophilic networks, one highly crosslinked, the other loosely crosslinked. The double network structure can be obtained by pre- and postcrosslinking through exploiting the disparity of their reaction times. For example, a double netwok composed of two mechanically weak hydrophilic networks based on N, Ndimethylacrylamide and glycidyl methacrylated hyaluronan, provides a hydrogel with outstanding mechanical properties (Weng et al., 2008). Hydrogels containing more that 90% water possessed a compressive modulus and a fracture stress over 0.5 MPa and 5.2 MPa, respectively, demonstrating both hardness and toughness. Besides, it is found that both the concentrations of monomers and crosslinkers are important parameters related to the mechanical strength of double network gels. Therefore, it is easy to control the mechincal properties such as hardness and toughness independently by adjusting the compositions of

Cartilage is a flexible, connective tissue in which chondrocytes are sparsely distributed in the extracellular matrixes rich in proteoglycans (PGs) and collagen fibers. Cartilage has a limited capacity for self-repair due to its avascular nature and low mitotic activity of chondrocytes. In articular cartilage, chondrocytes are the only cell type and responsible for the synthesis and maintenance of resilient extracellular matrix. Chondrocytes may undergo a dedifferentiation process during monolayer culturing and lose their phenotype. However, once cultured in hydrogels, dedifferentiated chondrocytes are able to redifferentiate (Benya & Shaffer, 1982), as indicated by their rounded morphology and the production of ECM

In-situ forming hydrogels enable a perfect match with irregular cartilage defects and good alignment with the surrounding tissues. Therefore, they are promising materials that can function as scaffolds for chondrocyte culturing and cartilage regeneration. Several factors may influence the cell viability, recovery or the maintenance of the chondrocytic phenotype,

Chemical compositions of hydrogels have been studied to explore their influence on cartilage regeneration. For example, Elisseeff *et al.* studied the cellular toxicity of transdermal photopolymerization on chondrocytes (Elisseeff et al., 1999). There was a significant decrease in the cell viability when the initiator concentration was increased from

molecules such as type II collagen and sulfated glycosaminoglycans.

and correspondingly play an important role in cartilage tissue engineering.

**4.1.1 Factors influencing cartilage regeneration** 

heating effects that can damage cells.

of the gels for practical applications.

**4.1 Cartilage tissue regeneration** 

**4. Tissue engineering applications** 

peptide crosslinker which contained the sequence sensitive to matrix metalloproteinases (Lutolf et al., 2003). The hydrogels were proteolytically degraded via the invasion of primary human fibroblasts. The invasion process depended on MMP substrate activity, adhesion ligand concentration, and network crosslinking density. By mimicking the MMPmediated invasion of the natural provisional matrix, the hydrogels were shown to assist tissue regeneration. These results indicate potential applications of the cell-responsive hydrogels in tissue engineering and regenerative medicine.
