The effects of oyster shell/alpha-calcium sulfate hemihydrate/platelet-rich plasma/bone mesenchymal stem cells bioengineering scaffold on rat critical-sized calvarial defects

摘要

Engineering scaffolds combining natural biomineral and artificially synthesized material hold promising potential for bone tissue regeneration. We fabricated a bioengineering scaffold, oyster shell (OS) and alpha-calcium sulfate hemihydrate (alpha-CSH) as scaffold, platelet-rich plasma (PRP) as provider of growth factors and bone mesenchymal stem cells (BMSCs) as seed cells, and determined it could be applied as a new type of bone graft substitutes by rat calvarial defects repairing experiment in vitro and in vivo. SEM showed that the mean diameter of the pores was about 150 mu m with a range of 50-200 mu m, and scaffold's porosity was similar to 27.4% by Archimedes' Principle. In vitro, Scaffold+BMSCs+PRP group presented a higher ALP activity compared with other groups by ELISA (P0.05). But the expression of OC was not detectable on day 4 or 8. The MTT assay showed that the relative cell number of BMSCs+PRP group increased significantly (P0.05). In vivo, the smallest defect area of skull and highest volume of regenerated new bone were observed in Scaffold+PRP+BMSCs group by X-ray and Micro-CT analysis (P0.05). And the similar results also were observed in HE and Masson staining. The immunohistochemistry staining for osteogenic marker proteins ALP and OC showed that the most obvious positive staining was observed in Scaffold+PRP+BMSCs group (P0.05). The expression of inflammatory markers IL-6 and TNF-alpha was the lowest in control group (P0.05). In conclusion, a bioengineering scaffold based on OS, created by simply combining alpha-CSH and PRP and implanting with BMSCs, could be clinically useful and has marked advantages as a targeted, off-the-shelf, cell-loaded treatment option for the bone healing of critical-size calvarial defects.