Construction Of Nanofiber Tissue Engineering Scaffolds With A Rough Surface By Supercritical Fluid Technology

Tissue engineering scaffold is the foundation of tissue engineering, as well as the key factor in tissue engineering technology. The properties of the scaffold play a pivotal significance in repairing damage tissue or regenerating function. However, conventional preparation methods such as compression molding-particulate leaching et al., suffer various limitations, such as complicated operating processes, high organic solvent residues, poor interconnected pores or using the salt leaching process for removal of solid porogen, easy deactivation of loaded growth factors and difficulty in obtaining nanotopography which mimics the architecture of natural extracellular. Therefore, the objective of this study was to fabricate poly(L-lactic acid)(PLLA) nanofiber scaffold with a rough surface via phase inversion using supercritical carbon dioxide(SC-CO2) by choosing ammonium bicarbonate(AB) particles and menthol as dual porogens. In addition, the biocompatibility, the interactions of different nanotopographies with cells and the chondrogenic differentiation potential of chondrogenic precursor cell C5.18 in vitro were also investigated. The main contents of this dissertation are as follows:1. Preparation and structure characterization of PLLA nanofiber tissue engineering scaffoldsThe effects of AB size, menthol/PLLA weight ratio and PLLA concentration on the porosity and compressive strength of the PLLA scaffolds were studied in a factorial experiment designed by Minitab. The results demonstrated that, when the PLLA concentration was kept in constant, a proper menthol/PLLA weight ratio produced the PLLA scaffolds with a good compressive strength and a high porosity. The effects of pressure, temperature, polymer concentration and menthol/PLLA weight ratio on the morphology of scaffold were studied. The results indicated that the phase separation process could be effectively controlled by changing pressure, and the tissue engineering scaffolds with different nanotopography structures were obtained. At 15 MPa, the pressure could promote the liquid-liquid demixing and solid-liquid demixing process, thus the rough surface around the nanofibers scaffold was formed. The temperature and polymer concentration had less effect on scaffold morphology. The porogen of menthol could improve the mass transfer environment of nonsolvent and promote the formation of pores during the process of polymer precipitation, thus the interconnectivity between the pores of the nanofibers was increased.Gas chromatograph analysis confirmed that the method was removed thoroughly, and the dioxane residue was below 3 ppm, which is much less than the limits of USP 467 Pharmacopeia(380 ppm). These results indicated the advantages caused by the characteristic properties of SC-CO2. The surface microstructure of the nanofiber was further characterized by atomic force microscopy(AFM). Differential scanning calorimetry(DSC), attenuated total reflectance Fourier transform infrared spectroscopy(ATR-FTIR) and wide-angle X-ray diffraction(WAXD) analysis confirmed that the SC-CO2 promoted the recrystallization of the PLLA and the menthol might affect the mutual transition between the ?' and ? crystals of PLLA during the phase separation process.2. The biocompatibility evaluation of the PLLA scaffoldThe L929 as cell model was used to perform cytotoxicity experiment. The results of Alamar Blue assay and acridine orange/ethidium bromide(AO/EB) staining indicated that the cytotoxicity of the scaffold was low with a cell relative growth rate of above 80%, which belongs to qualified level. Injected the scaffold in the form of extracts into mice, the status of the mice had no difference, namely the scaffold showed no acute toxicity. Cell hemolysis test showed that the hemolysis ratio of the scaffold was lower than 1.5% and this was in accordance with regulation in hemolysis test, which demonstrated the scaffold has good blood compatibility.3. Study of interaction between the surface topology of PLLA scaffold and cellChoosing three kinds of scaffolds with different structural characteristics, the interactions of these scaffolds with the protein adsorption, cell adhesion, cell proliferation and protein secretion were investigated. The results of the adsorption experiments indicated that the special surface properties of nanofiber increased the surface area, and provided more binding sites for protein absorption that enhanced the adsorption of protein. The cell adhesion results showed that the nanofiber scaffold promoted cell adhesion faster than the other groups. After 7 h of culture, cells adhered on the nanofiber scaffold almost reached the highest cell adhesion rate of 94%. The results of cell proliferation and protein secretion experiments further confirmed that these scaffolds had a good biocompatibility and were suitable for the growth and proliferation of cells. Specifically, the nanofiber scaffold has the similar structural characteristics to the natural extracellular matrix, and this can provide more suitable microenvironment for cell fate.4. In vitro assessment of the chondrogenic differentiation potential of C5.18 on PLLA scaffoldsThe results of C5.18 chondrogenic differentiation conditions demonstrated that the high concentrations(10-6 M) of dexamethasone can make the capacity of chondrogenesis of C5.18 maximized. Given the same culture conditions, the differences between C5.18 cells into cartilage differentiation potential on different topological structure of the scaffolds were further investigated. The results indicated that the cells covered the scaffolds uniformly. Particularly, the cells clustered were observed on the nanofiber scaffold. As the increasing in chondrogenic differentiation induction time, the productions of glycosaminoglycans and collagen protein showed significant differences between cells cultured on different groups after 7 days of incubation. The C5.18 cells had the highest expression on the nanofiber scaffold.