**4.2.1 Chitosan-based hydrogels**

Polysaccharides, e.g. chitosan, represent a class of hydrogels used as would healing materials. Native chitosan has low solubility above pH 6. Modifications on chitosan can improve its solubility and make it suitable as in-situ forming materials. Ono et al. reported potocrosslinked chitosan as a dressing for wound occlusion (Ono et al., 2000). The modified chitosan (Az-CH-LA) containing both lactose moieties and azide groups exhibited a good

In-Situ Forming Biomimetic Hydrogels for Tissue Regeneration 51

reported on TGF-β1-loaded-gelatin particles which were incorporated in oligo(poly(ethylene glycol) fumarate) hydrogels (Holland et al., 2003). In vitro release experiments showed a suppressed burst release and prolonged delivery of TGF-β1. Besides, when chondrocytes were embedded, an increased cellular proliferation and enhanced chondrocyte-specific gene expression was observed for the hydrogels containing TGF-

Fig. 8. Schematic representation of methods for encapsulating growth factors either by

Another example to control the release of growth factors is to use heparin-containing hydrogels. It is known that heparin is able to bind basic fibroblast growth factor (bFGF) by the formation of a stable complex (Yayon et al., 1991). bFGF can be prevented from denaturation and proteolysis meanwhile maintaining its biological activity after its release. Besides, the use of hparin can efficiently control the release rate of bFGF. However, large quantity use of heparin induces side effects such as thrombocytopenia, thrombosis, and hemorrhage (Silver et al., 1983). To solve this problem, Prestwich et al. proposed an in-situ forming glycosaminoglycan (GAG) hydrogel based on hyaluronan (HA) and chondroitin sulfate (CS) (Cai et al., 2005). Crosslinking occur between thiol-modified HA or CS (HA-DTPH or CS-DTPH), thiol-modified heparin (HP) and poly(ethylene glycol) diacrylate (PEG-DA) to generate copolymers, containing only a small percentage of co-crosslinked thiol-modified HP, which is capable of controlled release of basic fibroblast growth factor. Notably, the diffusion of bFGF in the hydrogels can be substantially slowed down only with 1% (w/w) covalently bound heparin (relative to total glycosaminoglycan content). In vivo studies, carried out on the Balb/c mice using the hydrogels (HA-DTPH+HP-DTPH and CS-DTPH+HP-DTPH) with/without bFGF, showed that the implanted hydrogels containing bFGF enhanced the production of new blood vessels to a high extent than equal amount of injected free bFGF, indicating that covalently crosslinked HP was necessary to enhance

Novel crosslinking methods provide significant opportunities for the design of in-situ forming hydrogels with multifunctional properties on demand for tissue regeneration. The fast progress in molecular biology inspires researchers to design biomimetic in situ forming hydrogels. Polymer composition and structures, hydrogel forming methods, degradation properties, mechanical strength and biocompatibility are of significant importance. Artificial extracellular matrices combining in situ forming hydrogel scaffolds, cells and growth factors

hold great promise for tissue engineering, and pave the way for regenerated tissue.

loaded-gelatin particles (H. Park et al., 2005).

(a) direct incorporation or (b) preloading into microparticles

bFGF activity and promote neovascularization.

**5. Conclusion** 

solubility at neutral pH. Application of ultraviolet light (UV) irradiation to Az-CH-LA produced an insoluble hydrogel within 60 s. The results showed that the chitosan hydrogels could completely stop bleeding from a cut mouse tail within 30 s and firmly adhere two pieces of sliced skins of mouse to each other. The wound healing efficacy of hydrogel was evaluated in experimental full-thickness-round wounds of skin using a mouse model and it is showed that chitosan hydrogels could significantly induce wound contraction and accelerate wound closure and healing in both db and db+ mice (Ishihara et al., 2002). Incorporation of fibroblast growth factor-2 further accelerated wound closure in db mice, however, not in db+ mice (Obara et al., 2003).

## **4.2.2 Alginate-gelatin hydrogels**

Gelatin is a degraded form of collagen and alginate is derived from brown seaweed, both of which are biocompatible. Balakrishnan *et al.* reported on the evaluation of alginate-gelatin hydrogels for wound dressing (Balakrishnan et al., 2005). Hydrogels were prepared from oxidized alginate and gelation in the presence of borax. It was found that the hydrogel have the ability to prevent accumulation of exudates on the wound bed due to fluid uptake. In vivo experiment showed the wound covered with hydrogel was completely filled with new epithelium after two weeks using a rat model. In addition, incorporation of dibutyryl cyclic adenosine monophosphate (DBcAMP) into the in-situ forming hydrogels and sustained release of DBcAMP led to the enhancement in the rate of healing as well as reepithelialization of the wounds (Balakrishnan et al., 2006). Complete healing was achieved within 10 days associated with mild contracture of some of the wounds.

#### **4.3 Delivery system**

From the clinical point of view, the success of a scaffold for tissue engineering is judged by its ability to regenerate tissue in both the onset and completion of tissue defect repair. During this process, the presence of growth factors in the hydrogel-based scaffolds usually helps to govern neo-tissue formation and organization. However, growth factors generally have short half-life time and are easy to lose their bioactivity(Edelman et al., 1991). Moreover, some unexpected adverse effects may occur which could be caused by initial burst release of growth factors. Therefore, the appropriate mode to deliver growth factors, make them available at the site of action and effectively controlled release of them to exert their maximum efficacy is of great important.

Direct administration of growth factors is commonly associated with problems such as a short biological half-life and easy diffusion. In-situ forming hydrogels offer significant opportunities for controlled local delivery of such biomolecules. These bioactive agents can be easily incorporated into hydrogels prior to gelation and their release kinetics can be adjusted on demand by the crosslinking density and stability of the networks. The disadvantages of in situ incorporation (Fig. 8a), however, is the potential damage of proteins during the gelation process (Sperinde & Griffith, 1997) or the occurrence of an initial burst release. To circumvent these disadvantages, growth factors can be incorporated into microparticles which can be added to the hydrogel precursor solutions (Fig. 8b). Microparticles can be prepared either from synthetic polymers (e.g. PGA, PLA, and PLGA) or from natural polymers (e.g. gelatin) (S.H. Lee & Shin, 2007). For example, Holland et al.

solubility at neutral pH. Application of ultraviolet light (UV) irradiation to Az-CH-LA produced an insoluble hydrogel within 60 s. The results showed that the chitosan hydrogels could completely stop bleeding from a cut mouse tail within 30 s and firmly adhere two pieces of sliced skins of mouse to each other. The wound healing efficacy of hydrogel was evaluated in experimental full-thickness-round wounds of skin using a mouse model and it is showed that chitosan hydrogels could significantly induce wound contraction and accelerate wound closure and healing in both db and db+ mice (Ishihara et al., 2002). Incorporation of fibroblast growth factor-2 further accelerated wound closure in db mice,

Gelatin is a degraded form of collagen and alginate is derived from brown seaweed, both of which are biocompatible. Balakrishnan *et al.* reported on the evaluation of alginate-gelatin hydrogels for wound dressing (Balakrishnan et al., 2005). Hydrogels were prepared from oxidized alginate and gelation in the presence of borax. It was found that the hydrogel have the ability to prevent accumulation of exudates on the wound bed due to fluid uptake. In vivo experiment showed the wound covered with hydrogel was completely filled with new epithelium after two weeks using a rat model. In addition, incorporation of dibutyryl cyclic adenosine monophosphate (DBcAMP) into the in-situ forming hydrogels and sustained release of DBcAMP led to the enhancement in the rate of healing as well as reepithelialization of the wounds (Balakrishnan et al., 2006). Complete healing was achieved

From the clinical point of view, the success of a scaffold for tissue engineering is judged by its ability to regenerate tissue in both the onset and completion of tissue defect repair. During this process, the presence of growth factors in the hydrogel-based scaffolds usually helps to govern neo-tissue formation and organization. However, growth factors generally have short half-life time and are easy to lose their bioactivity(Edelman et al., 1991). Moreover, some unexpected adverse effects may occur which could be caused by initial burst release of growth factors. Therefore, the appropriate mode to deliver growth factors, make them available at the site of action and effectively controlled release of them to exert

Direct administration of growth factors is commonly associated with problems such as a short biological half-life and easy diffusion. In-situ forming hydrogels offer significant opportunities for controlled local delivery of such biomolecules. These bioactive agents can be easily incorporated into hydrogels prior to gelation and their release kinetics can be adjusted on demand by the crosslinking density and stability of the networks. The disadvantages of in situ incorporation (Fig. 8a), however, is the potential damage of proteins during the gelation process (Sperinde & Griffith, 1997) or the occurrence of an initial burst release. To circumvent these disadvantages, growth factors can be incorporated into microparticles which can be added to the hydrogel precursor solutions (Fig. 8b). Microparticles can be prepared either from synthetic polymers (e.g. PGA, PLA, and PLGA) or from natural polymers (e.g. gelatin) (S.H. Lee & Shin, 2007). For example, Holland et al.

within 10 days associated with mild contracture of some of the wounds.

however, not in db+ mice (Obara et al., 2003).

their maximum efficacy is of great important.

**4.2.2 Alginate-gelatin hydrogels** 

**4.3 Delivery system** 

reported on TGF-β1-loaded-gelatin particles which were incorporated in oligo(poly(ethylene glycol) fumarate) hydrogels (Holland et al., 2003). In vitro release experiments showed a suppressed burst release and prolonged delivery of TGF-β1. Besides, when chondrocytes were embedded, an increased cellular proliferation and enhanced chondrocyte-specific gene expression was observed for the hydrogels containing TGFloaded-gelatin particles (H. Park et al., 2005).

Fig. 8. Schematic representation of methods for encapsulating growth factors either by (a) direct incorporation or (b) preloading into microparticles

Another example to control the release of growth factors is to use heparin-containing hydrogels. It is known that heparin is able to bind basic fibroblast growth factor (bFGF) by the formation of a stable complex (Yayon et al., 1991). bFGF can be prevented from denaturation and proteolysis meanwhile maintaining its biological activity after its release. Besides, the use of hparin can efficiently control the release rate of bFGF. However, large quantity use of heparin induces side effects such as thrombocytopenia, thrombosis, and hemorrhage (Silver et al., 1983). To solve this problem, Prestwich et al. proposed an in-situ forming glycosaminoglycan (GAG) hydrogel based on hyaluronan (HA) and chondroitin sulfate (CS) (Cai et al., 2005). Crosslinking occur between thiol-modified HA or CS (HA-DTPH or CS-DTPH), thiol-modified heparin (HP) and poly(ethylene glycol) diacrylate (PEG-DA) to generate copolymers, containing only a small percentage of co-crosslinked thiol-modified HP, which is capable of controlled release of basic fibroblast growth factor. Notably, the diffusion of bFGF in the hydrogels can be substantially slowed down only with 1% (w/w) covalently bound heparin (relative to total glycosaminoglycan content). In vivo studies, carried out on the Balb/c mice using the hydrogels (HA-DTPH+HP-DTPH and CS-DTPH+HP-DTPH) with/without bFGF, showed that the implanted hydrogels containing bFGF enhanced the production of new blood vessels to a high extent than equal amount of injected free bFGF, indicating that covalently crosslinked HP was necessary to enhance bFGF activity and promote neovascularization.
