Abstract: Objective · To prepare chitosan-gelatin porous microspheres by high voltage electrostatic method combined with freeze-drying and ionic cross-linking method and investigate the factors that influence the formation of porous medium.  Methods · Porous chitosan microspheres and chitosangelatin porous microspheres were prepared using high voltage electrostatic method combined with freeze-drying and ionic cross-linking method, with sodium tripolyphosphate (STPP) as crosslinking agent. Factors that affect the porous structure and pore size of porous microspheres were compared, such as different chitosan-gelatin ratio, freezing temperature, curing time with saturated STPP in 85% ethanol solution. The morphology, surface and internal structure, particle size of the porous chitosan microspheres and chitosan-gelatin porous microspheres were observe by using light microscope, scanning electron microscope and hematoxylin-eosin staining.  Results · Microspheres prepared by freeze-drying with an electrostatic and ionic cross-linking method have open, interconnected and highly macroporous, with good spherical surface. Saturated STPP ethanol solution (85% ethanol) was chosen as the crosslinking agent to prevent destruction of the porous structure. The order of freeze and crosslinking, cross-linking time and the second freezing temperature, can influence the pore size of porous microspheres. Gelatin and chitosan can form polyelectrolyte complexes, and can also be used as porogen in porous structure.  Conclusion · The preparation of porous chitosan-gelatin microspheres via this method has a large pore size (diameter 100-200 µm), suitable for cell growth and the migration.

Key words:  porous microspere, chitosan, three-dimensional culture, tissue engineering