Preparation And Application Of Bacterial Cellulose Tissue Engineering Scaffold

Tissue engineering is an emerging integrated science. It applies the principles of cell biology and engineering,research and develop substitutes that repair organizations damage and improve their function. A small number of cells are amplified in vitro, reach to a certain number, and plant them onto a certain space of the scaffold, restruct a life of vitality composite of cells and scaffold, transplant it into body to meet the purposes of repair or replacement of tissue injury. Therefore, tissue engineering is the research direction of human tissues and organs functional decline and loss.Scaffold is the bridge and link in tissue engineering. It is the survival and dependent three-dimensional structure for cells prior to formation of organizations. It provide spaces for cells physiological activities, such as proliferation, differentiation, exchange of nutrition, metabolism, and extracellular matrix secretion. Addition to general non-toxic biological materials, no adverse reactions, sources adequate, stable, easy to store and disinfection, and other characteristics, tissue engineering scaffolds must have good biocompatibility and tissue compatibility, biologically degradation, plasticity, certain porosity, pore size and certain mechanical strength.As a new type of biological material, bacterial cellulose has been widely used in the biomedical field. It has unique biological compatibility, biological adaptablity, biodegradation, high water holding capacity and the degree of crystallinity, high tension and intensity, good nanofiber network, as well as good machanical toughness, non-toxic, non-irritating, non-immunogenic, non-thermal original reaction, non-hemolysis, non-mutagenic. These characteristics determine its good biocompatibility and cell adsorption, so it is feasible for bacterial cellulose as a cell culture scaffold in tissue engineering technology.In this study, we prepared bacterial cellulose (BC) scaffold of a certain pore size and pore rate. Reconstruct tissue-engineered dermis and cartilage by culturing of FB and chondrocyte with BC. Transplant the tissue-engineered dermis which marked by GFP gene into the back subcutis of nude mice. By examining attachment, growth, proliferation and extracellular matrix secretion of FB on the scaffold, as well as histocompatibility of FB and BC in subcutis of nude mice, to explore the feasibility for use of bacterial cellulose as tissue engineering scaffold.This study include three elements:1.Based a large number of research on bacterial cellulose of domestic and foreign summary, we use of Acetobacter xylinum M12 static culture, extract BC membrane, with the alkali treatment and freeze-drying, prepare BC scaffold with a certain pore size and pore rate. By light microscope and electron microscope, we found there are uniform pore with diameter of 0.6 to 2.8μm. With the help of float weight and wet weight we measured the volume of porosity of approximately 90%. AFM observation indicates that cellulose belt is with a width 50 to 80 nm. Compared with PE and PVC, BC have high water holding capacity. It tips that BC is natural polymer nano-material and suitable for tissue engineering applications.2.Based on the previous work in our lab, we successfully isolated and cultured fibroblasts (FB), rendered growth curve to observe the growth of FB. Marked FB with GFP gene, composited bacterial cellulose and transfected FB to culture tissue-engineered dermis, and transplanted to nude mice. From general form, light microscope, scanning electron microscopy and laser scanning confocal microscope, the results showed that the transfected FB were spindle-shaped bracket and attached to BC. revealed green fluorescence under the Blu-ray excitation. It convinced that the transfected FB expressed GFP. The color around graft was rosy, and there were new capillaries obviously at the junction of the graft and normal skin. It fuse good around wound with the normal skin. Laser scanning confocal microscope showed that there diffused large number of green fluorescent protein, mostly distribution in the dermis and hair follicles, epidermis also distributed. Under normal skin there obviously was the porous scaffold. It meant the GFP gene transfected FB well attach and grow on BC and subcutaneously in nude mice. The results shows that BC is suitable for FB attachment, growth and proliferation, reveals its good cytocompatibility and histocompatibility3.Isolated and cultured rabbit chondrocytes, rendered growth curve to observe the cell growth and proliferation, with toluidine blue and immunocytochemical staining to identify the cell phenotype. Composited chondrocytes with BC, constructed into tissue-engineered cartilage. HE staining showed mesh uniform distribution culturing for 1 week, framed layer formed on the surface of scaffold. Chondrocytes were round. There was no cell in the hole of scaffold. Scanning electron microscope showed that cells firmly attached to the surface of BC, extending the edge of a number of synapases. The second week, cell layer become obviously thicke, and the density of cells is large on the surface. And the cell layer of the third and fourth week become more thicken. That means that BC is suitable for chondrocytes attachment, growth, proliferation, and it has good cytocompatibility.The results shows that FB and chondrocytes not only can be attached to the BC scaffold, , but also can normally grow and proliferate. It means that BC has excellent cytocompatibility and histocompatibility. It is feasible for BC as tissue engineering scaffold. This suggests that there is a vast range of potential applications for BC in the field of tissue engineering. And it is expected to become a new type of scaffold in tissue engineering.