| Recent studies have found that phosphoprotein plays a very important role in the biomineralization,and the phosvitin(PV)possesses the highest level of phosphorylation of all known proteins in eggs.Phosvitin was speculated to regulate biological mineralization in some studies,but the regulation mechanism in the mineralization and active sites is unclear.In order to explore the regulation mechanism of phosvitin in mineralization,this paper established three mineralized systems to find the promotion of the mineralization by PV in vitro and the regulation mechanism of phosvitin was discussed.First,we constructed a biomimetic mineralization system based on collagen electrospinning membrane,and then researched the action and regulation mechanism of phosvitin in the above system.Secondly,we used MC3T3-E1 osteoblast model to explore the regulation mechanism of phosvitin with different phosphorylation level in cell mineralization.Finally,RAW264.7 osteoclast model was constructed to study the effect of phosvitin on osteoclast activity and function.The mineralization function of phosvitin was studied by above three different mineralization systems,which supported the explanation and application of its mineralization mechanism.In a tenfold-concentrated simulated body fluid,a strategy for rapid deposition of a biomimetic calcium phosphate layer on the scaffolds of electrospun collagen nanofiber membranes was developed.The aim of this study is to explore the effects of mineralization conditions on hydroxyapatite nucleation and growth.The mineralization model,the pH of the environment and the deposition time were optimized.Scanning electron microscopy(SEM)images demonstrated that homogeneous and wel-crystal ized inorganic mineral layers were generated at pH 5.7 with the dynamic mineralization model for 3 hours.The minerals formed on electrospun collagen nanofiber membranes were identified to be from hydroxyapatite by Fourier transform infrared spectroscopy(FTIR)and X-ray diffraction(XRD),the crystals distribution in the electrospun membrane internal and surface.Phosvitin,which possesses the highest level of phosphorylation of egg proteins,and bovine serum albumin(BSA),the most abundant serum protein,were used as model proteins to investigate the contribution of protein in the mineralization process.Through the SEM,the morphology and formation of the minerals at the interface and surface of the membrane were tracked under the control of PV and BSA.The morphological structure and composition of the collagen/calcium phosphate composite nanofibers were also characterized by energy dispersive spectroscopy(EDS),scanning photoelectron spectrometer(XPS)and XRD.The system was proved to be a rapid and effective method to promote the formation of bone-like calcium phosphate coating.The introduction of PV and BSA built a good sequence of biomimetic mineralization system: both phosvitin and BSA significantly promoted the phase transformation from dicalcium phosphate dihydrate(DCPD)to hydroxyapatite(HA),but phosvitin accelerated the transformation process more quickly than BSA.PV adsorbed on the mineral to improve the concentration of Ca2+ in the microcosmic environment by electrostatic interaction.The HPLC is used to track the PV content in the solution at different time.The results show that PV is dissolved in the mineralization solution later.This part is aimed to investigate the effects of phosvitin with different phosphorylation levels on the proliferation,differentiation,mineralization and possible mechanism of osteoblastic MC3T3-E1 cel s.The dephosphorization rates were 22.67%,45.86%,59.24% and 75.13%,respectively.The effects of phosphorylation level on the proliferation and differentiation of MC3T3-E1 cel s were analyzed by CCK-8 method and alkaline phosphatase assay,respectively.The effect of phosphorylation degree on the mineralization of the MC3T3-E1 cell line was monitored using the alizarin red staining.The expressions of BMP-2,RANKL and OPG mRNA in the MC3T3-E1 cell line were analyzed by RT-PCR.The results showed that the phosphorylation of phosvitin was the key factor to promote the proliferation,differentiation and mineralization of MC3T3-E1 cel s.Phosvitin stimulated the proliferation as well as the differentiation of the MC3T3-E1 cell line in a phosphorylation level-dependent manner.Furthermore,the up-regulated ratio of OPG/RANKL mRNA and the obvious mineralization of the cel s were closely related to the phosphorylation level of phosvitin.The effect of 100 ?g/mL PV treatment group was the best,while the PV group with the highest dephosphorization rate was equal to or significantly lower than that of the control group.The effects of different concentrations of PV(0,50,100,200 and 500 ?g/mL)on the proliferation,cell cycle and cell apoptosis of RAW264.7 cel s for different time were studied.It was found that low concentration of PV could promote proliferation and inhibit the apoptosis of RAW264.7 cel s.The effect of 100 ?g/mL PV concentration which can promote the proliferation and inhibit apoptosis is best.CCK-8 method was used to characterize the proliferation of RAW264.7 cel s.It was found that the proliferation of RAW264.7 cel s showed two aspects.The cell proliferation was promoted at low concentration,the cell proliferation was inhibited at high concentration.The results showed that the cell viability was 64%-76% higher than that of the control group at 100 ?g/mL.By analyzing the effect of PV on the cell cycle,found that the percentage of PV in the S phase was significantly higher than that in the control group(P <0.05),100 ?g/mL PV has the best effect.The apoptotic effects of PV group at 50,100 and 200?g/mL were significantly lower than that of control group.When the concentration of PV was 500?g/mL,the apoptotic rate was higher than that of the control group,which indicated that low concentration of PV could inhibit the apoptosis and high concentration of PV could induce apoptosis.In addition,different concentrations of PV can significantly reduce the number of osteoclasts and the activity of TRAP(P <0.05),thus inhibiting the RAW264.7 cell differentiation to form osteoclasts,and in a concentration-dependent manner,the best inhibitory concentration was 200?g/mL.In conclusion,PV was not by inhibiting the growth of RAW264.7 cels,but by inhibiting the differentiation of RAW264.7 cel s significantly reducing the activity of TRAP,thereby inhibiting the formation of osteoclasts. |
PDF Full Text Request