Construction Of Bioactive Scaffold With Growth Factors And Gene For Angiogenesis

One strategy for regenerative medicine is to induce tissue regeneration at damaged tissues or organs with either transplanted cells or host cells. This cell induced tissue regeneration is achieved by providing a local environment which enables cells to promote proliferation and differentiation. It has been recognized that the environment is naturally composed of biological signal molecules, extracellular matrix (ECM) molecules, mechanical stress, and cell-cell interactions. Thus, "bioactive" scaffold with an appropriate combination of the biological cues may modify cell activities for tissue regeneration.Initially, we tried to realize bioactive scaffold by incorporating growth factors. A class of thin film with bioactivity was constructed by using the layer-by-layer self-assembly technique. Acid fibroblast growth factor (aFGF) in the presence of heparin was used as negatively charged polyelectrolytes, while poly(ethyleneimine) (PEI) was chosen as a positively charged counterpart. The self deposition process and surface morphology of the resultant multilayers were monitored and detected by UV-Vis absorbance spectroscopy, advanced contact angle measurements and scanning force microscopy (SFM) observations, respectively. Cell culture was performed to assess the efficiency of the growth factors. The fibroblasts proliferated faster on surface assembled with 5 bilayers of (aFGF/heparin)/PEI with apparent higher cytoviability than on those surfaces modified by 1 bilayer of (aFGF/heparin)/PEI, 5 bilayers of aFGF/PEI or 5 bilayers of heparin/PEI, and tissue culture polystyrene. Enhanced secretion of collagen type I and interleukin 6 (IL-6) by the fibroblasts seeded on the 5 bilayers of (aFGF/heparin)/PEI was also verified by immunohistochemical examination. The bioactivity of the (aFGF/heparin)/PEI multilayers could be largely preserved when stored at -20℃. Using the same method, a bioactive collagen scaffold was constructed by (aFGF/heparin)/PEI assembly. By monitoring the linear increase of FITC-labeled aFGF fluorescence intensity with the assembled layer, the self-assembled process was proved. Cell viability test showedthat the assembled aFGF has positive effect on the cell viability.As growth factors typically have half-lives only on the order of minutes and easy to denature during preparation, we intended to promote cell proliferation and differentiation or the secretion of ECM components for tissue regeneration by the transfection of genes encoding growth factors.N,N,N-trimethyl chitosan chloride (TMC) with different quaternization degree and molecular weight was synthesized. Their molecular structure was characterized by ¹HNMR. The size and the morphology of TMC/DNA particles were observed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The particle size ranged from 150nm to 600nm depending on the N/P ratio and was less influenced by the quaternization degree. The zeta potential of these particles was increased along with the N/P ratio, while higher quaternization degree TMC resulted in particles with higher zeta potential. The affinity between DNA and TMC was examined by ethidium bromide competitive binding assay. Those vectors with higher positive charge strength have stronger ability to combine with DNA. HEK293 cell line was chosen as a model to study the cytotoxicity, cellular uptake of particles, and gene transfection. The short term contact experiments showed good biocompatibility of TMC, but long term contact experiments revealed the high toxicity of TMC. The higher quaternization degree and molecular weight lead to higher toxicity of TMC. The higher quaternization degree leaded to higher cellular uptake of TMC/DNA particles, yet exhibited lower transfection efficiency of delivered gene. The optimized gene transfection efficiency achieved is comparable to PEI, but still lower than Lipofectamine 2000.N,N,N-trimethyl chitosan-g-poly(N-isopropylacrylamide) (TMC-g-PNIPAAm), a thermoresponsive copolymer, was synthesized by coupling PNIPAAm-COOH to TMC. Their molecular structures were characterized by ¹HNMR. The lower critical solution temperature (LCST) of TMC-g-PNIPAAm in PBS was measured as 32℃ by DLS, regardless of the grafting ratios. Upon mixing with DNA, TMC/DNA particles were formed, whose size and morphology were investigated by DLS and TEM, respectively. The particle size ranged from 200nm to 900nm depending on the N/Pratio and was less influenced by the temperature variation. The zeta potentials of these particles were increased along with the N/P ratio. At a given N/P ratio, the zeta potentials were almost constant at 25℃ regardless of the existence of serum proteins. However, the values were significantly decreased at 37℃ in a solution containing serum protein. TMC-g-PNIPAAm has stronger ability to combine with DNA at 40℃ when the PNIPAAm chain is collapsed. The grafting of PNIPAAm won't affect cellular uptake of the particles at 37℃. The level of gene transfection could be thermally controlled. By using a temperature variation protocol, i.e. incubation of the cultured cells at 25℃ for a while, the gene transfection efficiency was significantly improved. Finally, the optimized gene transfection efficiency achieved by TMC-g-PNIPAAm is comparable to Lipofectamine 2000. No obvious cytotoxicity was detected for the TMC-g-PNIPAAm/DNA particles.In order to increase cellular uptake and nuclear targeting, Tat peptide was used to modify SiO₂ particles as a model. The grafting amount of Tat peptide could be controlled by feeding amount. The morphology and zeta potential of the modified particles were similar to the original ones. Grafting of Tat peptide would largely increase the cellular uptake of the particles at 4℃ and 37℃, and affect the subcellular distribution of particles, leading to enter of the particles in the nucleus. According to these results, Tat peptide was grafted to TMC/DNA particles via glutaraldehyde crosslinking. The size and zeta potential of modified particles were similar to the original particles. The grafting of Tat peptide would largely increase the cellular uptake of the particles and gene transfection efficiency at 37℃. No obvious cytotoxicity was detected for the Tat modified TMC/DNA particles. Finally, a bioactive collagen scaffold was realized by combination of vector/DNA particles. The in vitro releasing test showed that the DNA had a faster releasing rate in the initial stage, with subsequent slower release lasted for 96h. Plasmid DNA encoding human vascular epithelial growth factor (VEGF) was used to induce angiogenesis of scaffold in vivo. After embedded in SD mice, the pDNA loaded scaffolds showed good tissue compatibility, and had the ability to enhance the angiogenesis of scaffold in vivo.