Abstract: BACKGROUND: The number of patients with periodontitis increases year by year, and the use of traditional periodontal treatment cannot restore the periodontal soft and hard tissue. Therefore, it is necessary to prepare a drug sustained-release material to assist the treatment of periodontitis and restore the damaged soft and hard tissues of periodontitis.
OBJECTIVE: To prepare bovine serum albumin microspheres composite hydrogel material, detect the dual sustained release effect on simvastatin, and further study the effect of the composite material on the adhesion and proliferation of osteoblasts. 
METHODS: (1) Bovine serum albumin microspheres loaded with simvastatin were prepared by solvent removal method. The morphology of microspheres was observed by transmission electron microscopy and scanning electron microscopy and the particle size of microspheres was measured. The encapsulation rate, drug loading rate and in vitro release of microspheres were detected by a microplate analyzer. (2) Simvastatin albumin microspheres were loaded into the prepared hydrogel, and in vitro drug release from hydrogels was detected using a microplate reader. The morphology of the composite material was observed by scanning electron microscopy. The hydrogel extract was used to culture MC3T3-E1 cells. The proliferation of osteoblasts was detected by CCK-8 assay. The alkaline phosphatase expression in osteoblasts was detected by alkaline phosphatase kit. 
RESULTS AND CONCLUSION: (1) Transmission electron microscopy showed that bovine serum albumin microspheres and blank microspheres loaded with simvastatin were smooth, spherical and dispersed, with no obvious aggregation. The particle size of bovine serum albumin microspheres was uniform, and 80% of the microspheres had a particle size between 0.2 μm and 0.8 μm. Scanning electron microscopy showed that the microspheres were smooth and spherical with good dispersion and uniform particle size distribution, ranging from 0.2 μm to 0.8 μm. (2) The encapsulation rate and drug loading rate of simvastatin microspheres were 68.9%-87.5% and 0.95%-1.21%, respectively. (3) Drug-loaded microspheres had the performance of gentle and sustained release of simvastatin. The release curve of simvastatin in the drug-loaded hydrogel is a fast release in the early stage and a slow sustained release process in the later stage, in which the drug-loaded hydrogel can release faster than the drug-loaded microspheres and reach the effective concentration of the drug. The slow release in the later stage maintains the concentration of the drug. (4) Scanning electron microscopy showed that the drug-loaded hydrogel had a porous strip-like structure, which was suitable for the adhesion and growth of osteoblasts, and spherical drug-loaded microspheres could be seen on the surface of the hydrogel. (5) The drug-loaded hydrogel can promote the proliferation and alkaline phosphatase expression of MC3T3-E1 cells. (6) Simvastatin microspheres hydrogel composite had good biocompatibility and could promote the adhesion and proliferation of osteoblasts. 

Key words: simvastatin, sustained drug release, microsphere, hydrogel, osteoblasts, proliferation and differentiation