Combination of simvastatin, calcium silicate/gypsum, and gelatin and bone regeneration in rabbit calvarial defects

Abstract The present study was performed to determine whether simvastatin improves bone regeneration when combined with calcium silicate/gypsum and gelatin (CS-GEL). The surface morphology was determined using field-emission scanning electron microscopy (FSEM). Degradation in vitro was evaluated by monitoring the weight change of the composites soaked in phosphate buffered saline (PBS). Drug release was evaluated using high-performance liquid chromatography (HPLC). Cytotoxicity testing was performed to assess the biocompatibility of composites. Four 5 mm-diameter bone defects were created in rabbit calvaria. Three sites were filled with CS-GEL, 0.5 mg simvastatin-loaded CS-GEL (SIM-0.5) and 1.0 mg simvastatin-loaded CS-GEL (SIM-1.0), respectively, and the fourth was left empty as the control group. Micro-computed tomography (micro-CT) and histological analysis were carried out at 4 and 12 weeks postoperatively. The composites all exhibited three-dimensional structures and showed the residue with nearly 80% after 4 weeks of immersion. Drug release was explosive on the first day and then the release rate remained stable. The composites did not induce any cytotoxicity. The results in vivo demonstrated that the new bone formation and the expressions of BMP-2, OC and type I collagen were improved in the simvastatin-loaded CS-GEL group. It was concluded that the simvastatin-loaded CS-GEL may improve bone regeneration. Introduction Bone defects are often seen in clinical situations. Autografts, allografts, and artificial bone substitutes have been widely used in bone repair. Autografts have some specific drawbacks, such as the limited source material and risks of unpredictable resorption and morbidity at the donor site1. One alternative to autografts, allografts may transmit disease and induce immune responses if not pretreated appropriately2. A variety of artificial bone substitutes are also being widely used in practice. These include metals, synthetic polymers such as poly lactic acid (PLA) and polyurethane (PU), and ceramics such as hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP)3,4,5,6. Among the numerous bone substitutes, calcium sulfate dihydrate (CaSO4∙2H2O), called gypsum, has been used to repair bone defects for over 100 years7. Gypsum is formed by the hydration of calcium sulfate hemihydrate (CaSO4∙0.5H2O), which undergoes in situ setting after filling bone defects. Due to its considerable biocompatibility, gypsum has already been approved by the FDA for clinical use as a bone graft substitute8. However, because of the poor bioactivity of pure gypsum, it is difficult to form an effective chemical bond with the newly formed bone during the early healing stage9. It has been reported that composite biomaterials may be more suitable than pure biomaterials10. In a study by Wang, calcium silicate/gypsum was found to induce apatite formation on the surface of cement after incubation in simulated body fluid, which means the calcium silicate/gypsum composite has good bioactivity11. This shows that the addition of calcium silicate to form a composite biomaterial sidesteps the disadvantages of pure gypsum. Gelatin has good biocompatibility and efficient hemostatic properties. It is also completely biodegradable in vivo and its physicochemical characteristics can be appropriately modulated12,13. Because of its strong adhesive and plasticity properties, gelatin can form a suitable matrix in calcium silicate/gypsum composites. An early study indicated the potential of the calcium sulfate and gelatin composite as a biodegradable bone substitute and in the promotion of new bone ingrowth14. In order to increase the bioactivity of calcium silicate/gypsum and gelatin composite (CS-GEL), growth factors and drugs supporting bone regeneration should be incorporated into the composite. Simvastatin, a cholesterol-lowering drug, has been shown to have a positive effect on bone formation and bone mineral density in vivo15. Local application of simvastatin has been shown to promote fracture healing in ovariectomized rats16. Mukozawa et al. demonstrated that simvastatin with hydrogel and atelocollagen sponge (ACS) enhanced the bone growth of critical-sized nasal defects in rabbits17. Sukul et al. manufactured a simvastatin-loaded gelatin-nanofibrillar cellulose-beta tricalcium phosphate hydrogel scaffold, and reported that the scaffold could release the optimum concentration of simvastatin to enhance osteogenesis18. It has also been reported that the combination of simvastatin and gypsum can stimulate bone regeneration19. It has been demonstrated that gypsum has potential as a carrier for local release of antibiotics, growth factors, and drugs20,21,22. All these findings show that the combination of simvastatin, calcium silicate/gypsum, and gelatin has promise as a bone substitute in the promotion of bone growth. In this study, the characteristics of this composite material were determined and its effects on the (...truncated)