Studies On Protein Loaded Open Porous Biodegradable Injectable Microspheres Scaffold

Tissue engineering is designed to regenerate natural tissues or to create biological substitutes for defective,lost tissues and organs through the use of cells. In addition to cells and their scaffolds, growth factors are required and play a very important role in cell culture, cells proliferation and tissue regeneration process. Indeed, growth factor-induced vascularization is effective in supplying the oxygen and nutrients necessary for the survival of transplanted cells in organ substitution. In the early 1990s it was first shown that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) proteins could actually stimulate collateral flow. However, additional studies also demonstrated that the duration of exposure of the vessels to angiogenesis factors was critical, and that the administration of proteins, with their relatively brief half-lives and poor stability in vivo, may pose important practical limitations. So the biological effects are often unpredictable unless the delivery system is contrived. Drug delivery systems are of great importance in tissue engineering. The most widely used delivery form of protein and peptide drugs is as injectables. As these therapeutics have short half-lives due to proteolysis and rapid clearance from the bloodstream, patients often require daily injections. The high concentration often induce some of severe side effects. Encapsulation of proteins within biodegradable polymers has been shown to obtain sustained release effect and to enhance drug stability in vitro and in vivo, These also meet the requirements of drug delivery in tissue engineering.In this study, BSA were used as a model protein drug and fabricated protein loaded gas foamed highly open porous PLGA microspheres were prepared for injectable cell delivery used as tissue engineering scaffolds (microspheres scaffold).Water-in-oil-in-water (W1/O/W2) double emulsion solvent evaporation method was modified to produce protein loaded porous microspheres scaffold. An effervescent salt was used as a gas foaming agent. Ammonium bicarbonate was incorporated in the primary W1 droplets to generate carbon dioxide and ammonia gas bubbles during the solvent evaporation process. Based on the result of single-factor selecting experiment, the formulations and the preparation methods were evaluated by orthogonal experiment method and central composite design (CCD). The microspheres scaffold was spongy and spherical with a mean diameter of 451μm; the mean diameter of surface pores is 20.30μm, it is suggested that stirring rate, PVA and PLGA concentration, homogenizing power and the concentration of gas forming reagent have effects on the surface pore diameter, density and particle size of microspheres scaffold. The encapsulation efficiency was 60.08%, and the cumulated releases of BSA exceed 80%.BSA concentration in release medium was determined by BCA (Bicinchoninic acid) protein determination method and the 30 days in vitro release behavior of microspheres was studied. The release kinetics fitted the Riger-peppas mode. By dissolving the BSA loaded open porous PLGA microspheres into different solvents, the BSA remaining and denatured protein concentration was also detected. Results showed that the concentration of BSA remaining is nearly 10%,non-covalent bonded BSA aggregates is lower than 2% and disulfide-bonded BSA aggregates is lower than 1%. The method provides a relatively easy and accurate measurement of the concentrations of dissolvable BSA and BSA in other bonded ways.Then the potentials of the optimized microspheres scaffold for cell cultivation and delivery were explored by the 3T3 cell culture test. Attached cells on the microspheres scaffold were stained with Molecular Probes for cytological observation while the unstained microspheres scaffold were used as blank control. The confocal and SEM images showed that the adhered cells were well spread on the surface. After prolonged suspension cultivation of 7 days, however, it can be observed that spherical cells were densely present within the pores of the microspheres, not only on the surface. This suggests that initially attached cells on the surface gradually migrated. Open porous microspheres can be utilized as injectable and biodegradable scaffold microcarriers.Conclusion: Tissue engineering microspheres scaffold is a good controlled release system for protein. Microspheres Scaffold is a better choice for a better immobilization and sustained release of the drug to enhance tissue regeneration or to cure the correlated disease.