Effect Of New Spherical Nano-bioactive Glass On Osteogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells

PART 1.Study on physical and chemical properties of bioactive glass 45SObjective:To characterize and analyze the surface morphology,size distribution and in vitro mineralization properties of bioactive glass 45S,so as to provide an experimental basis for the further evaluation and optimization of the related properties of new spherical nano bioactive glass.Method:X-ray photoelectron spectroscopy(XPS)was used to detect the element types and relative contents of bioactive glass 45S.The surface morphology,particle size and dispersion of bioactive glass 45S were observed by Scanning electron microscope(SEM)and Transmission electron microscope(TEM).The particle size and distribution of bioactive glass 45S were measured by laser particle size analyzer.The ion concentration in bioactive glass 45S was determined by Inductively Coupled Plasma(ICP-OES)to evaluate the in vitro ion release performance of bioactive glass45S.Simulated body fluid(SBF)bioactive glass 45S after 24 hours,by Fourier Infrared Spectrometer(Fourier Transform Infrared Spectrometer,FTIR)to detect the vibration of the Simulated body fluid before and after processing material band,verify its mineralization in vitro performance.Results:Photoelectron spectroscopy analysis showed that the bioactive glass 45S was composed of Si O₂,Na₂O,Ca O and P₂O₅,and the contents of Si,Na,Ca and P were20.7%,16.5%,11.6%and 2.1%,respectively.SEM results showed that the bioactive glass 45S were irregular micron particles with good dispersion.TEM results showed multifaceted crystals with clear and smooth boundaries.The results of laser particle size analyzer show that the particle size of bioactive glass 45S is about 2?m.ICP-OES assay results showed that when the bioactive glass 45S was co-cultured with?-MEM,with the decrease of P ion concentration in the medium,the concentration of Si ion and Ca ion increased.FTIR results showed that after 24 hours of immersion in SBF,the surface of bioactive glass 45S was formed by carbonated hydroxyapatite(HCA).Conclusion:Bioactive glass 45S is mainly composed of Si O₂,Na₂O,Ca O and P₂O₅.It is a micron particle with good dispersibility,and has certain ion releasing and in vitro mineralization ability.PART 2.Study on physical and chemical properties of new spherical nano bioactive glass Objective: To study the surface morphology,size distribution and in vitro mineralization properties of two types of nano-scale bioactive glass,spherical nano-bioactive glass(SNBG)and radial nano-bioactive glass(RSNBG),so as to provide theoretical basis for further cytological experiments.Method: SEM was used to observe the size and dispersion of the particles.TEM was used to observe the surface morphology of the bioactive glass.Laser particle size analyzer was used to measure the size distribution.The ion concentration of particle extracts was determined by ICP-OES to evaluate the ion release of bioactive glass in vitro.The materials were incubated in SBF for 24 h,the FTIR was used to detect the spectra of particles that before or after the incubation,and the changes in morphology were observed by SEM.SEM was used to observe the size and dispersion of the particles.TEM was used to observe the surface morphology of the bioactive glass.Laser particle size analyzer was used to measure the size distribution.The ion concentration of particle extracts was determined by ICP-OES to evaluate the ion release of bioactive glass in vitro.The materials were incubated in SBF for 24 h,the FTIR was used to detect the spectra of particles that before or after the incubation,and the changes in morphology were observed by SEM.Results: SEM and TEM showed that both types of particles were regular spherical structures with good dispersibility,and comparing to the smooth spherical surface of SNBG,a good amount of branch-like structures were seen on the surface of RSNBG.The average particle sizes of the two were 890.8 nm and 592.3 nm,respectively.The results of ICP-OES showed the two kinds of bioactive glasses were co-cultured with alpha-mem.With the decrease of P ion concentration in the medium,the concentration of Si ion and Ca ion increased.That is to say,both kinds of bioactive glasses could release Si ion and Caion.Furthermore,the ion releasing capacity of RSNBG was stronger than that of SNBG.New absorption bands attributed to bending vibrations of P-O(960 cm-1)and C-O(875 cm-1)bonds are in presence on the spectra of RSNBG but in absence on that of SNBG after incubation in SBF for 24 h.SEM results showed that a large number of flake dense deposits appeared on the surface of RSNBG after SBF immersion,while the deposits on the surface of SNBG particles were not obvious.Conclusion: Two types of bioactive glasses both have uniform size,good dispersion and have certain ion release and in vitro mineralization ability,and RSNBG is stronger than SNBG.PART 3.Effects of new spherical nano bioactive glass on the biological behavior of rat bone marrow mesenchymal stem cells Objective: To evaluate the effects of two types of nano bioactive glasses on cell proliferation,cell cycle and cell migration of r BMSCs,to evaluate the effect of two types of nano-bioactive glasses on osteogenic differentiation of r BMSCs and to explore their potential as bone tissue repair materials.Method: Incubated with different concentrations of bioactive glass for 1,3,5,and 7days,the proliferation of r BMSCs was determined using the Cell Counting Kit-8(CCK-8).r BMSCs were treated with 50 ?g/m L SNBG or RSNBG,and the cells without treatment were set as control.After incubated for 3 and 7 days,the cell cycle analysis of r BMSCs were performed using flow cytometry.Scratch test was conducted to assess the effect of bioactive glass on cell migration.r BMSCs were treated with 50 ?g/m L SNBG or RSNBG,and the cells without treatment were set as control.After osteogenesis induction for 5 and 7 days,Alkaline phosphatase(ALP)staining and activity assay were performed to evaluate the effect of particles on early stage of osteogenic differentiation.After 14 and 21 days of osteogenesis induction,alizarin red staining and Semi-quantitative assessment of calcium deposits were conducted to evaluate the effect of bioactive glass on late stage of osteogenic differentiation.The expression of osteogenic genes,such as Runx2?Col1 and Osx was detected by reverse transcriptase-polymerase chain reaction(RT-PCR)after 7 and 14 days of osteogenic induction to evaluate the effect of bioactive glass on osteogenic differentiation from the m RNA level.The expression of osteogenic proteins,such as Runx2,COL1,and OSX,was detected by western blot(WB)after 7 and 14 days of osteogenic induction to evaluate the effect of bioactive glass on osteogenic differentiation from the protein level.Results: The CCK-8 results showed that all the cells proliferated within 7 days and the proliferation rate was related to the concentration of bioactive glass.The proliferation of r BMSCs incubated with 50 ?g/m L RSNBG was observed significantly greater compared to those incubated with 50 ?g/m L bioactive glass with higher concentration(80 ?g /m L or more)inhibited the proliferation of cells,and the inhibitory effect of the material on the proliferation was more obvious with the increase of the treatment concentration.The cell cycle analysis demonstrated that the proliferation indices(S%+G2/M%)of r BMSCs cultured with 50 ?g /m L bioactive glass for 3 and 7 days was higher in the RSNBG group than in the SNBG group than in the blank control group.The fastest cell migration was observed in the group that treated with RSNBG.The results of ALP staining and ALP activity analysis indicated that after 5 days of osteogenesis induction,ALP activity was significantly increased in cells incubated with RSNBG compare to SNBG or the control group(P < 0.05),there was no statistical difference in ALP activity between the blank control group and SNBG group,while after osteogenesis induction for 7 days,no difference was found in ALP activity of r BMSCs incubated with RSNBG and SNBG,incubated both of which were significantly higher than the control group(P < 0.05).Alizarin red staining and semi-quantitative results of calcium nodules showed that extracellular matrix mineralization at 14 and 21 days after osteogenic induction was better in the RSNBG group than in the SNBG group than in the blank control group(P<0.05).The results of RT-PCR and WB showed that the expression of osteogenesis related genes and proteins in the RSNBG group was higher than that in the SNBG group and the control after 7 and 14 days of osteogenesis induction(P<0.05).Conclusion: Two types of bioactive glasses have good biocompatibility and both can promote the osteogenic differentiation of r BMSCs,and RSNBG can provide more benefits than SNBG for the proliferation,migration and osteogenic differentiation of r BMSCs.and RSNBG is stronger than SNBG,which has better application prospects.