Gelatin Release Microspheres As Cell Microcarriers Preparation And Properties

A major problem in tissue engineering is the availability of a sufficient number of cells with the appropriate phenotype for tissue repair and restoration of function. The microcarrier technique of cell cultivation offers an attractive method for cell amplification and enhancement of phenotype expression, which has been proven to be more effective than that of cell culture on flat substrates such as culture dishes. Microcarriers have the advantage of providing a large surface area for monolayer cell growth during propagation in a homogenous suspension culture system. So, the design of microcarriers plays an important role in the success of cell expansion.Gelatin is a kind of natural polymer obtained by the partial hydrolysis of animal collagen. It is a biodegradable, biocompatible, nontoxic, and noncarcinogenic biomaterial, which has been largely investigated for the preparation and application of the gelatin materials. Gelatin presumably retains informational signals, which makes it mimic the natural extra cellular matrix (ECM). Moreover, in previous studies on gelatin-based wound dressing, it was shown that gelatin particles containing growth factors have potential applications in medicine. Growth factors may improve survival and differentiation of the cells, and may also affect the immediate environment, thus allowing better graft integration. Nevertheless, the administration of these factors still remains a technological challenge, due to their short half-life. To overcome these difficulties, we prepared a site-specific delivery approach, using implantation of biodegradable gelatin microspheres and allowing a controlled and sustained release of a growth factor. The thesis includes three parts as follows:(1) Used natural biodegradable gelatin materials as raw material and glutaraldehyde as cross-linking agent, micron-level gelatin microspheres were prepared by an improved emulsified cold-condensation method. The results indicated that the optimal condition to prepare gelatin microspheres was the gelatin solution 25% (w/w), water phase and oil phase ratio 3 : 20 (v/v), stirring rate 300 r/min, glutaraldehyde 0.1 mL (25%) and surfactant span80 0.1 g. The average diameter of microspheres was 250μm. The gelatin microspheres were spherical and had more uniform size. The surface of microspheres was smooth, without porous and cracks. Gelatin microspheres had good release ability: the released process for BSA from the BSA-impregnated gelatin microsphere can be sustained for 30 days, and there was no sudden release phenomenon in the released process for BSA. For the cultured cartilage cells by suspension culture technology on gelatin microspheres, we found that the gelatin microcarriers were suitable for cartilage cell growth. Cartilage cells on the microcarrier surface grew well and had uniform distribution. (2) Reference to the most optimum condition to prepare gelatin microspheres. We used three different post processing ways respectively to process the microspheres and got gelatin microspheres with the different surface structures. The first approach was to wash the gelatin microspheres with acetone and deionized water respectively, the smooth surface gelatin microspheres were obtained; the second one was to wash the gelatin microspheres with acetone and isopropanol respectively, the crapy gelatin microspheres were gained; the third one was that the gelatin microspheres were reprocessed by freeze-drying, the foveolar gelatin microspheres were acquired. After swelling 40 min, all the gelatin microspheres could attain the swelling balance, but the swelling degree and swelling rate of the reprocessed microspheres by freeze-drying were significantly higher than that of the other two gelatin microspheres. There was no obvious difference in the in vitro degradation for the three gelatin microspheres. All the microspheres could be biodegradable completely, and the degradation rate was related to the microspheres size. The larger the microspheres were, the longer the degradation time. Used the three kind gelatin microspheres to culture fibroblast, it was found that the fibroblast grew well on the three kind gelatin microspheres surface, and had obviously proliferation phenomenon. Thereinto, the use effect as micropheres of the micropheres reprocessed by freeze-drying was the best one.(3) Reference to the most optimum condition to prepare uncrosslinked gelatin microspheres. Used glutaraldehyde, UV-irradiation and thermal denaturation to crosslink the uncrosslinked gelatin microspheres respectively, three typical gelatin microspheres GGMs, UVGMs, and TGMs were obtained. Observed the surface of these microspheres under SEM, it was found completely different results. The GGMs showed a very smooth and uniform surface. The surface of TGMs had abundant inhomogeneous particles, and the UVGMs were unfairly round particles with an accidented surface. In the in vitro study, the degradation, growth factor release, cell proliferation, and cell viability for the three microspheres were evaluated respectively. The GGMs had a long time of degradation and bFGF release due to the highest crosslinking degree. Fibroblasts attached, spread, and proliferated well on the GGMs. Although the TGMs had the least bFGF accumulative release, the fibroblasts had good adhesion and proliferation in a short time. Because of the fast degradation, UVGMs' changeable shape make against to cell culture. According to the practical applications, above results will bring us some help in scaffold materials in tissue engineering.