In Vitro And In Vivo Evaluation Of Chitosan Scaffolds Combined With Simvastatin-loaded Nanoparticles For Guided Bone Regeneration

Objective:Bone resorption or trauma can cause bone defects,which may lead to significant morbidity.Bone restoration is necessary to prevent further functional and aesthetic losses.Currently,more studies are being conducted that are focused on bone tissue engineering,which holds great promise as an important therapeutic strategy to restore bone defects.But topical application of simvastatin without control of the optimal release concentration may have a negligible desired effect or induce inflammation.Chitosan(CS)has been extensively studied in medical applications due to its good biocompatibility,biodegradability,low toxicity,and antimicrobial activity.Chitosan can be fabricated into membranes for guided bone regeneration(GBR),and drugs with the ability to promote bone formation may improve CS efficiency in GBR.The aim of this study was to prepare and characterize chitosan composite with different doses of simvastatin nanospheres and to prepare chitosan scaffolds loaded with simvastatin nanospheres,so then to discuss its application prospect in guiding bone regeneration in vivo and in vitro.Methods:The study encapsulated different amounts of simvastatin in CSNs through an ionic gelation and subsequent lyophilization method.The SIM-CSNs were incorporated into a chitosan scaffold via another lyophilization process.The chitosan scaffold combined with simvastatin-loaded nanoparticles(SIM-CSNs-S)was evaluated for GBR by investigating scaffold morphology,biodegradation,drug release profile and effect on BMSCs in vitro.The critical-size cranial defect model and ectopic osteogenesis in SD rats were used to evaluate the influence of SIM-CSNs-S on bone regeneration in vivo.Results:As shown in scanning electron microscopy,the SIM-CSNs-S exhibited a porous 3-dimensional structure with a multilevel internal network,and the diameter of the pores varied from 5 ?m to 150 ?m.The CSNs-S/SIM-CSNs-S showed a high degradation rate during the first three weeks,but degradation slowed in the subsequent two weeks.The higher the simvastatin dose added to the SIM-CSNs-S the greater the amount of simvastatin released from the sample in the same period.All the cells were primarily stained green,and appreciable levels of red staining were not evident.The overall cell proliferation trend for the four lines were nearly parallel,similar to an “S”.The ALP activity of the 4 mg SIM formulation was significantly higher than that of the other two SIM-CSNs-S groups.The tissue specimens from ectopic bone formation demonstrated that the four scaffold groups had the potential to induce new osteoid tissue formation,and the 4 mg formulation exhibited significantly greater and earlier osteoid tissue production than the other groups.The result of skull defect model indicated that the 4 mg SIM-CSNs-S samples show superior bone regeneration in the defect healing compared with the other groups.Conclusions:In this study,the poorly soluble drug simvastatin was successfully loaded into chitosan nanoparticles through an ionic gelation and subsequent lyophilization method.Then,the nanoparticles were combined into a chitosan membrane through another lyophilization process.The resulting SIM-CSNs-S showed sustained simvastatin release.The CSNs-S showed excellent biodegradability and biocompatibility.In this way,the osteoconductivity of the chitosan scaffold was significantly improved.In an in vivo study,the 4 mg formulation exhibited significant potential for GBR.