Electrospun Fibrous Scaffolds With Iron-doped Hydroxyapatite Induce Osteogenic Differentiation Of Mesenchymal Stem Cells With Static Magnetic Field Exposure

Inflammation, trauma, or tumor can result in large skeletal defects, which are common and urgent problems in oral medicine. At present, bone graft is the most common way for bone restoration. An increasing number of researches are focused on obtaining substitutes for bone graft, which possess function to induce tissue regeneration.In recent years, the function of magnetic field was found out, which showed an ability of inducing new bone formation. Researchers fabricated a series of superparamagnetic biomaterials for the purpose to use the synergistic effect of magnetic nanoparticles and magnetic field. It is discovered that hydroxyapatite in nano-scale is distributed in collagen stroma uniformly in bone. Therefore, the modification of HA to obtain a superparamagnetic HA is a good idea for bone regeneration. With the Fe-HA incorporated in polymer nanofibers via electrospinning can fabricate a magnetic scaffold. In this study, we embedded Fe-HA in PLGA to produce magnetic scaffold with a structure conducive to the proliferation, adhesion and osteogenic differentiation of rat MSCs. This magnetic fibrous scaffold might be innovatively applied in bone regeneration.[Objective] To fabricate the magnetic PLGA/Fe-HA scaffold and analyze its physical and chemical properties, further more, to observe the biocompatibility and osteoinductive ability of PLGA/Fe-HA under static magnetic field exposure.[Methods] First of all, Fe species were added into the reaction solution during the nucleation of HA to obtain iron-doped hydroxyapatite (Fe-HA). The morphology of Fe-HA nanoparticles were detected by transmission electron microscope (TEM). The PLGA/Fe-HA scaffold was fabricated by electronspinning. X-ray diffraction was used to evaluate the crystal form of nanoparticles and scaffolds. The characterization of scaffolds were analyzed by scanning electron microscope (SEM), energy-dispersive X-ray fluorescence analysis (EDX), water contact angle analysis (WCA) and magnetic analysis.Rat mesenchymal stem cells (RMSCs) were isolated by explant method, and the third passage was identified by flow cytometry. The morphology, adhesion and growth situation of RMSCs cell seeded on scaffold with or without SMF were examined by SEM and cell counting kit (CCK-8). Alkaline phosphatase assay kit and q-PCR were used to detect the differentiation of cells on scaffold with or without SMF.[Results] Fe-HA particles were needle-like crystals, with 5-20nm in width and 50-80nm in length. XRD demonstrated that the synthesized particles were HA crystals with a little amount of magnetite as second phase. The scaffolds had a highly porous network, with interconnected pores and a smooth surface. Fe-HA nanoparticles were seen embedded in fibers. The results of XRD and EDX evidenced the existence of Ca, P and Fe. The hydrophilicity of scaffold was improved with the incorporation of Fe-HA. The result of magnetic analysis demonstrated that Fe-HA and PLGA/Fe-HA scaffold showed typical characterization of superparamagetic-like behaviour. Three days after primary culture, RMSCs gradually climbed out from the organizations and were seen as short fusiform or starlike. At day 7, cells proliferated stably and present like radial arrangement. The results of flow cytometry showed that CD90 and CD 105 were positive, while CD45 and CD11b were negative, which identified there origin. RMSCs adhered to the PLGA/Fe-HA scaffolds with obvious cilium and filopodia, and they proliferated stably on scaffolds. When applying static magnetic field to the cultural environment, RMSCs on PLGA/Fe-HA scaffold were seen stepping over the fibers and growing into the internal space of scaffold. CCK8 assay showed that both scaffolds provided a good support for cell proliferation without any significant difference. ALP kit showed that ALP activity was upregulated on PLGA/Fe-HA, which upregulated more under SMF. Real-time quantitative PCR analysis showed that expressions of Runx2 and OPN, were upregulated by the synergistic effect of PLGA/Fe-HA scaffold and SMF.[Conclusions] The PLGA/Fe-HA scaffold produced by electronspinning showed superparamagnetic performance, moreover, had good porous structure and hydrophilicity with the incorporation of Fe-HA. RMSCs of primary culture could adhere to and proliferate on PLGA/Fe-HA scaffold. Under static magnetic effect, magnetic PLGA/Fe-HA scaffold could induce osteogenic differentiation of RMSCs.