Investigation On The Effect Of Simvastatin Sustained-Released From PLGA Nanofibrous Scaffolds On Bone Regeneration

Millions of people are suffering from bone defects arising from trauma, tumor or bone related diseases every year. Current therapies for bone defects or bone substitutes include autografts, allografts and rehabilitation, etc. However, these substitutes have their own shortcomings. With the development of tissue engineering, bone regeneration has become the new therapy of bone defect in recent years,Objective: many researchers pay close attention to the microenvironment of bone tissue, and investigate the osteoblasts and growth factors. In this paper, We prepared poly (lactic -co-glycolic acid)(PLGA)/ Hydroxyapatite(HA)/ simvastatin composite nano-fiber scaffolds. When the PLGA slowly degraded, HA which is the main component of bone, and simvastatin were released from scaffolds into the microenvironment to promote the bone regeneration.Method: Various types of nanofibers such as pure PLGA and PLGA/HA blended, PLGA/HA/Simvastatin (PLGA/HA/S) were fabricated using electrospinning technique. The morphology of the scaffolds is characterized by SEM instrument. The relationship of time vs dose of released S was obtained as PLGA/HA/S scaffolds degraded in simulated body fluid (SBF). The vitro experiments included the attachment and proliferation of the pre-osteoblast (MC3T3-E1). The effects of simvastatin on cells'differentiation were evaluated by ALP activity assay (pNPP method). Bone repair was examined by a rat with critical-sized (5mm) calvarial defect which was implanted with scaffolds. Animals were randomly divided into 4 groups with 4 for each group (blank control group, PLGA, PLGA/HA, and PLGA/HA/S). Specimens of each group were harvested at 4 weeks after implantation and animals in each group were sacrificed. The crania were carefully separated and fixed in 10% neutral formalin for 24 hours, and then used to soft X-ray examinations and histological analysis.Result: We prepared PLGA, PLGA/HA, PLGA/HA/S nanofibrous scaffolds. The diameters of nanofibers were 100nm-1μm. The simvastatin released from the scaffold could be measured at different time. The effective dose of drug could also be detected at the sixtieth-day. The biocompatibility of the scaffolds was evaluated in vitro by MC3T3 cultured in close contact with the scaffold. Experimental results showed the micrographs of cell attachment onto the scaffold after 1 and 3 days cell culture. Cells secreted mount of extracellular matrix and attregated with each other to form stratified cell layers. The cell proliferation in each group was evaluated using MTT test. In 1-day cell culture, the cell number of PLGA/HA/S increased dramatically. Statistical anlysis indicated significant difference in the cell number between PLGA/HA/S and PLGA/HA as well as PLGA. ALP activity shows the ALP activity of PLGA/HA/S group at 14 days is higher than that of other groups. This indicated that cells on the PLGA/HA/S group obviously differentiated to some extent. The calvarial samples were shown by soft X-ray analyses. For blank control group, no high density was observed and margins of the defects were smooth. There are few osteoblasts and mesenchymal stem cells on the margin of the defects. There is little fibrous connective tissue. For PLGA/HA/S group, obvious high density was observed, the diameter reduced. Histological changes of defect regions for each group are obvious. For blank control group, no bone formation was shown in defect regions. PLGA/HA/S group showed more osteoid formation than other groups, new bone covered 1/3 of the defects, osteoblasts and mesenchymal stem cells are like cube on the margin of the defects.Conclusion: We prepared the nanofibrous scaffolds including pure PLGA scaffolds, PLGA/HA scaffolds and PLGA/HA/S scaffolds, which are similar to the extrocellar matrix.The vitro experiment proved that simvastatin could be released from the degraded PLGA into the microenvironment and promoted the attachment, proliferation and differentiation of the cells. PLGA/HA/S scaffolds have positive effect on the bone regeneration. The composite scaffolds provide a new approach to the bone regeneration. The study will become one of the potential theories of treating the bone defects.