Formation of Chitosan-Based Hydrogels Network - Chitosan Based Hydrogels: Functions and Applications - page 196

of gelatin although there is no clear trend [125]. The chitosan/gelatin cross-linking net-
work has excellent cytocompatibility [126]. For example, the cross-linked network contain-
ing 80 wt% gelatin well supports neuroblastoma cell adhesion and proliferation, which
results in optimal candidates for future trials in the field of peripheral nerve regeneration
[127]. In addition, it is more efficient in inducing fibrin formation and vascularization at the
implant-host interface. However, the inflammatory reactions for the gelatin/chitosan
network gel are significantly stronger than those for the gelatin gel [128].
Chitosan/gelatin network scaffolds are developed via their PEC formation, freeze-
drying, and postcross-linking with GA. The average pore sizes of chitosan/gelatin scaf-
folds can be controlled within the range of 30-100 μm, and pore size can be modulated via
prefreezing temperatures. Li and coworkers [129] have reported a novel method to fabri-
cate chitosan/gelatin network scaffolds according to multilevel internal architectures via
solid free form fabrication, microreplication, and lyophilization techniques. The porosity,
pore size, and morphology can be easily controlled. The compression modulus of chitosan/
gelatin scaffolds can be controlled within the range of 10-100 kPa [130,131]. Yao and
coworkers [132] prepared the bilayer chitosan/gelatin scaffold via contacting with −56°C
lyophilizing plate directly and then lyophilized. First, fibroblasts are seeded in the loose
layer of the bilayer scaffold. The cell/scaffold constructs are cultured for 4 weeks.
Afterward, keratinocytes were cocultured on the thick layer of fibroblast locating scaf-
folds, where cells proliferate well for 7 days. The results revealed that the chitosan/gelatin
scaffold with multilevel internal architectures has the potential for being used in artificial
skin [133].
Chitosan/gelatin microspheres can be prepared by the water-in-oil emulsion method
and postcross-linking with GA. The human recombinant basic FGF (bFGF) is loaded
within the microspheres by adsorption in its PBS solution. Incorporation of bFGF micro-
spheres into the chitosan/gelatin scaffold significantly augmented the proliferation and
glycosaminoglycan synthesis of human fibroblasts [134].
4.3.1.2 Chitosan/GAG Cross-Linking Network
GAGs can be cross-linked on the chitosan scaffold. The efficiency of GAG cross-linking is
limited by the amounts of available amine groups on the scaffold for cross-linkage [135].
Different cross-linkers and cocross-linkers are used, such as GA, EDC, N- hydroxysuccini-
mide (NHS) and 2-morpholinoethane (MES), and Ca 2+ , according to different GAGs.
4.3.1.2.1 Chitosan/Chondroitin Sulfate Cross-Linking Network
The tensile strength of chitosan/chondroitin sulfate, which was prepared via immobiliz-
ing chondroitin sulfate on the chitosan nonwoven scaffold using GA as a cross-linker,
increases to over 200% of that of chitosan, but the elongation of chitosan/chondroitin sul-
fate decreased to less than 20% of that of chitosan. A possible reason is that the immobili-
zation of chondroitin sulfate can also cause the fibers to bind with each other [136]. Chen
et al. [137] augmented the GAGs (chondroitin-4-sulfate (CSA), chondroitin-6-sulfate, der-
matan sulfate (DS), and heparin) onto chitosan films via cross-linking using EDC/NHS.
The results suggested that the chitosan/GAG network (containing low CSA levels) leads
to the maintenance of proper chondrocyte phenotype, as judged by the chondrocyte-like
morphology, modest cell expansion, higher GAG and collagen production, and proper car-
tilage marker gene expression. The pore morphology and size agree with those of other
chitosan-only scaffolds and exceed 70-120 μm, which is sufficient for uniform cell penetra-
tion and migration and improves GAG and collagen production [138].