Modified Chitosan-Gelatin Networks And Their Application In Tissue Engineering

Tissue engineering utilizes the theories and methods of life sciences and engineering sciences, researching and developing new clinic substitute for human tissue and apparatus. Its typical method is seeding cells in the artificial extracellar matrices (ECMs) to form cells/artificial ECMs constructs. Here the manual ECMs are three-dimensional biomaterial scaffolds with excellent biocompatibility. The functions of biomaterial scaffolds act as analogues to the natural ECMs found in tissues, which provide information for cells expressing their functions, e.g. adhesion, proliferation, differention etc. Therefore, the aim for biomaterial scaffolds design is to mimic the natural ECMs, from the compositions to the microstructure. From the point of view of mimic, the work incorporated hyaluronic acid (HA), a component of the ECMs, in chitosan (Cs)-gelatin (Gel) network, fabricating two-dimensional Cs-Gel-HA surface modification membranes and polyblend membranes. HA not only obviously improved the hydrophilicity properties of the membranes obtained but also made them more flexible and extended their degradation time. The concentration of HA had a significant influence on the biocompatibility of Cs-Gel-HA membranes. Results demonstrated that it was only the concentrations of HA in a certain range (0.01%～0.1%) could promote the cell adhesion, migration and proliferation, while the concentration of HA was larger than 0.1% it would reduce or even inhibit these behavior. Three-dimensional Cs-Gel-HA scaffolds for tissue engineering were fabricated by freezing and lyophilizing methods. To improve the tensile strength of the scaffolds, Cs-Gel scaffolds were soaked in different concentrations of composite biomaterial poly (D,L-lactic acid) (PDLLA) solutions. Moreover, the microspheres containing tetrandrine were incorporated in the Cs-Gel scaffolds. The physicochemical properties and the biocompatibility of mimic three-dimensional scaffolds were studied. The structure of the scaffolds obtained was heterogeneous and they had two layer with different pore size structure. Hydrophilicity propertie of the Cs-Gel scaffold was greatly improved by the incorporation of HA. Soaking in different concentrations of PDLLA solutions obviously increased tensile strength of Cs-Gel scaffold. The biocompatibilities of Cs-Gel-HA scaffolds and Cs-Gel scaffolds containing tetrandrine microspheres were better than Cs-Gel scaffolds. When the concentration of PDLLA was 0.1%, the biocompatibility of Cs-Gel-PDLLA scaffolds was almost the same with Cs-Gel scaffolds. Keratinocytes were co-cultured with fibroblasts in chitosan-gelatin-hyaluronic acid scaffolds to construct a tissue engineered artificial bilayer skin in vitro. After three weeks of co-culture, the keratinocytes of the artificial skin obtained could differentiate into several stratums and were demonstrated to have basement membrane, which was similar to normal human skin. The tissue engineered artificial skin was used to repair full-thickness skin defects in pig model. The results demonstrated that Cs-Gel-HA scaffolds had good biocompatibility and possibly could be used to repair large skin defects in clinic.