**4.1.1 Factors influencing cartilage regeneration**

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, and correspondingly play an important role in cartilage tissue engineering.

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

In-Situ Forming Biomimetic Hydrogels for Tissue Regeneration 47

al., 2007) (Figure 7a). In another approach, methacrylated chondroitin sulfate (CSMA) was functionalized to endow aldehyde moieties which was covalently attached to collagen via Schiff-base formation (D.A. Wang et al., 2004) (Figure 7b). The CSMA layer was further polymerized by photo-crosslinking of PEGDA to give a gel/cartilage integrated scaffold.

Understanding of the tissue structure and composition can lead to a rational material design, targeted towards mimicking the underlying biological cues and specific chemistry of cartilage. Generally, in-situ forming hydrogels have been prepared from synthetic polymers, natural polymers or their hybrids. The latter has gained increasing attention in recent years because they combine the advantage of both synthetic and natural polymers, that is, tightly defined physical, chemical and biological properties. Table 1 lists typical examples of in-situ

Natural moiety Comments Ref.

aggrecan

production

production

Table 1. Typical examples of in-situ forming hybrid hydrogels for cartilage regeneration

During the cartilage regeneration process growth factors play a crucial role in regulating cellular proliferation, differentiation, migration, and gene expression. Besides, they have large influences on the communication between cells and their microenvironment. A number of growth factors have been studied and include bone morphogenetic protein (BMP), transforming growth factor (TGF), insulin-like growth factor (IGF) and basic fibroblast growth factor (bFGF). Their main functions in cartilage regeneration are summarized in Table 2. For example, the BMP family can stimulate mitosis and matrix production by chondrocytes and induce chondrogenesis of mesenchymal cells, triggering them to differentiate and maintain a chondrogenic phenotype (Yuji et al., 2004). TGF-β not only enhances chondrocyte proliferation, but also increases the synthesis of proteoglycans

PEG Heparin Promoted chondrocyte proliferation,

Proteolytic degradability, enhanced gene expression of type II collagen and

of hydrogels, enhanced matrix

Improved cellular adhesion and proliferation, increased matrix

Balance between modulus and swelling

Retention of ECM production inside hydrogel via collagen binding, enhanced chondrogenesis

while maintaining chondrogenic nature

(Y. D. Park et al., 2004)

(Bryant et al., 2005)

(H.J. Lee et al., 2008)

2009)

(M. Kim et al., 2010)

(H. Lee & Park,

**4.1.2 Hybrid hydrogels for cartilage regeneration** 

forming hybrid hydrogels for cartilage tissue regeneration.

Synthetic polymer

Pluronic F127

**4.1.3 Growth factor** 

(H. Park et al., 2005).

PEG MMP-sensitive peptide

PVA Chondroitin sulfate

PEG Collagen-mimic peptide

> Hyaluronic acid, RGD

0.012% to 0.036% or higher. In another study, Chung *et al.* noticed that a higher macromer concentration potentially compromised cell viability and growth (Chung et al., 2006). Besides, a higher polymer concentration also resulted in a decreased accumulation of matrix components such as proteoglycans and collagen type II (Sontjens et al., 2006).

Recent studies showed that the degradation properties of the gels may have a significant influence on the matrix production and distribution as well. Degradable hydrogels induced a more homogenous distribution of GAG than non-degradable hydrogels (Bryant & Anseth, 2002, 2003; Bryant et al., 2003; Martens et al., 2003). However, in fast degrading hydrogels void spaces are generally present before new matrix formation has taken place (Bryant & Anseth, 2003; Martens et al., 2003). Therefore, the degradation rate of hydrogels needs to be tailored by the combination of degradable main chain linkages and crosslinks.

Fig. 7. Hydrogel-cartilage integration by (a) tissue-initiated photopolymerization or (b) Schiff-base formation**.** 

A major problem for cartilage regeneration is poor integration of neocartilage with native cartilage tissue. To solve this problem, the gel precursor molecules were modified with functional groups that can react with collagen type II, a molecule present in native cartilage. For example, improved tissue adhesion and integration was achieved by tissue-initiated polymerization between acrylate groups in polymerizable PEGDA macromers and tyrosine groups in collagen when exposed to light and an oxidative reagent like H2O2 (D.A. Wang et

0.012% to 0.036% or higher. In another study, Chung *et al.* noticed that a higher macromer concentration potentially compromised cell viability and growth (Chung et al., 2006). Besides, a higher polymer concentration also resulted in a decreased accumulation of matrix

Recent studies showed that the degradation properties of the gels may have a significant influence on the matrix production and distribution as well. Degradable hydrogels induced a more homogenous distribution of GAG than non-degradable hydrogels (Bryant & Anseth, 2002, 2003; Bryant et al., 2003; Martens et al., 2003). However, in fast degrading hydrogels void spaces are generally present before new matrix formation has taken place (Bryant & Anseth, 2003; Martens et al., 2003). Therefore, the degradation rate of hydrogels needs to be

> Collagen exposed at interface

**CSMA adhesive layer in defect**

**Interface**

Fig. 7. Hydrogel-cartilage integration by (a) tissue-initiated photopolymerization or

A major problem for cartilage regeneration is poor integration of neocartilage with native cartilage tissue. To solve this problem, the gel precursor molecules were modified with functional groups that can react with collagen type II, a molecule present in native cartilage. For example, improved tissue adhesion and integration was achieved by tissue-initiated polymerization between acrylate groups in polymerizable PEGDA macromers and tyrosine groups in collagen when exposed to light and an oxidative reagent like H2O2 (D.A. Wang et

UV

**PEGDA hydrogel filled in defect**

**Interface**

**Interface**

**PEGDA hydrogel with CSMA adhesive in defect**

**PEGDA**

UV

**PEGDA**

components such as proteoglycans and collagen type II (Sontjens et al., 2006).

tailored by the combination of degradable main chain linkages and crosslinks.

Remove proteoglycan

**(a)**

Remove proteoglycan

**CSMA**

Collagen

Collagen exposed at interface

(b) Schiff-base formation**.** 

**(b)**

**Cartilage defect**

al., 2007) (Figure 7a). In another approach, methacrylated chondroitin sulfate (CSMA) was functionalized to endow aldehyde moieties which was covalently attached to collagen via Schiff-base formation (D.A. Wang et al., 2004) (Figure 7b). The CSMA layer was further polymerized by photo-crosslinking of PEGDA to give a gel/cartilage integrated scaffold.
