The Effect Of Cell Adhesion On The Osteogenic Properties Of Magnetic Nanocomposite Scaffolds

Aims:To study the effect of cell adhesion on the osteogenic properties of magnetic nanocomposite scaffolds.Materials and Methods:A porous scaffold polyglycolic acid-hydroxyacetic acid(PLGA)copolymer with dimensions of 4.5 mm × 4.5 mm × 0.36 mm was fabricated by fine direct-write 3D printing technology,and a magnetic nanocomposite scaffold was prepared by assembling iron oxide nanoparticles(IONPs)though the layer-by-layer self-assembly(LBL)method.Firstly,the scaffolds were characterized,the macroscopic morphology of the scaffold was collected by digital camera and the Scanning Electron MicroscopeEnergy Dispersion Spectrum(SEM-EDS),and the microscopic morphology of the scaffold was used to examine by Transmission Electron Microscopy(TEM).Atomic Force Microscope(AFM)and water contact angle meter were investigated to the surface roughness and hydrophilic performance of the scaffold.The magnetic saturation strength and hysteresis loops of the scaffolds were obtained by vibration sample magnetometer(VSM)at room temperature.Secondly,the untreated scaffold was used as a control.In vitro cytology experiments,the effect of magnetic scaffolds on the proliferation of rat bone marrow mesenchymal stem cells(rBMSCs)was investigated by cell proliferation-toxicity(CCK-8)assay.Exploring the changes of alkaline phosphatase(ALP)activity after osteogenic induction of rBMSCs by magnetic scaffolds from three time points.We probed the difference of magnetic scaffolds on rBMSCs after osteogenic differentiation and the effect on cell adhesion by quantitative real-time fluorescence analysis(qRT-PCR)from the mRNA level.Meanwhile,the live/dead viability staining,Laser Scanning Confocal Microscope(CLSM)and SEM assay were used to observe the number of adhesion and morphological changes of BMSCs on the scaffold surface.Finally,in vivo experiment,a SD rat cranial parietal bone critical size defect(CSD)was prepared,and the scaffold was implanted.And the scaffold was not implanted as a blank control.The experiments were divided into three groups: a blank control group,an untreated scaffold group,and the magnetic scaffold group.At last,the osteogenic effect of the magnetic scaffold at the defect was investigated using Micro-CT imaging and histological staining.Results:1.Characterization: Both scaffolds were of the same size,the magnetic scaffold could be picked up by the magnet compared to the untreated scaffold,and the hysteresis lines demonstrated that the magnetic scaffold had magnetic properties.A series of characterization tests confirmed that the magnetic scaffold had a reduced contact angle and a higher roughness value compared to the untreated scaffold,and that the presence of iron was detected.2.In vitro experiment:CCK-8 assay demonstrated that the magnetic scaffold had the potential to promote the proliferation of rBMSCs(p < 0.05),and the ALP activity was significantly higher than that of the untreated scaffold(p < 0.05).The results of qRT-PCR indicated that the expression of osteogenic differentiation genes OCN,Col-Ⅰ,OPN,BMP2 and ALP was elevated in the magnetic scaffold group at14 days(p < 0.05).In addition,there was remarkable expression between adhesionrelated genes integrin α1 and β1 and their downstream signaling molecules FAK and ERK1/2(p < 0.05).After 6h and 12 h,there were more green-stained cells on the magnetic scaffold than on the untreated scaffold under the observation of CLSM(p <0.05),and more cytoskeletons with blue-stained nuclei and spreading red-stained cells were observed on the magnetic scaffold after 24h(p < 0.05),and SEM images also showed that rBMSCs were spreading more widely on the magnetic scaffold.3.In vivo experiment: Micro-CT analysis showed more new bone formation in the magnetic scaffold group at 8 weeks.And the percentages of BV/TV(p < 0.05)and defect closure rates(p < 0.05)of the magnetic scaffold group exceeded the untreated scaffold group and the blank control group.HE and Masson staining results showed that the magnetic scaffold group had a large number of new bone formation in the defect area and a lot of small vessels could be seen,and the amount of new bone production and the rate of defect closure were higher than those of the other two groups(p < 0.05).Conclusion:Enhanced cell adhesion promoted in vitro osteogenic differentiation of rBMSCs by magnetic nanocomposite scaffolds,further promoting rat cranial parietal bone defects regeneration.