Preparation Of Melatonin-loaded Composite Barrier Membrane And Its Effect On Osteogenic Differentiation Of MC3T3-E1

Objective: The SP/MT complex biofilms loaded with different concentrations of melatonin(MT)were prepared by electrospinning.The physical and chemical properties of the biofilms and their effects on the proliferation and osteogenic differentiation of mouse embryonic osteoblast progeners(MC3T3-E1)were investigated.The aim is to provide a new idea for the development of barrier membrane in the field of bone regeneration.Methods: The SF/PLCL(SP)mixture with a mass volume fraction of 8% was matched with the mass ratio of 3:7 in hexafluoroisopropyl alcohol.SP/MT electrospinning solution containing(0,0.01,0.05,0.1 M)melatonin was obtained by adding different concentrations of melatonin into SP mixture overnight.Four groups of SP/MT biofilms were electrospun under the conditions of humidity of 25-20%,applied voltage of 10 k V,advance speed of 0.02 m L/h and receiving distance of 15 cm.Then,the physicochemical properties and related functions of the biofilm and the proliferation and osteogenic differentiation of MC3T3-E1 were analyzed.(1)Physical and chemical properties: the microscopic morphology of biofilm was observed by scanning electron microscope(SEM);Fourier transform infrared spectroscopy(FTIR)and X-ray diffraction(XRD)were used to characterize the chemical bonds and crystal structures of biofilms.The tensile tester was used to test the mechanical strength of the sample.Contact Angle measuring instrument to detect biofilm surface hydrophilicity;The degradation cycle of biofilm was studied by in vitro degradation experiment.(2)Functional study: UV spectrophotometer was used to detect the drug sustainedrelease ability of SP biofilm loaded with melatonin.DPPH method was used to evaluate the free radical scavenging ability of SP/MT composite biofilm to determine its antioxidant properties.(3)In vitro study: The effect of biofilm on the proliferative activity of MC3T3-E1 was evaluated by using CCK-8 and cell adhesion assay.Key enzymes of osteogenic differentiation were qualitatively and quantitatively analyzed by ALP,and the expression levels of ALP,OCN,OPN and Runx2 osteogenic m RNA were detected by RT-q PCR.Results:(1)SEM results showed that the biofilm surface of each group was smooth without beading and the fiber diameter distribution was uniform.The chemical bond and crystal structure changes were observed by FTIR and XRD,which confirmed that melatonin was successfully loaded into SP fiber film.The mechanical properties test results showed that each group of fiber membrane had ideal mechanical strength,fully meet the clinical application.The results of contact Angle test showed that the hydrophilicity of biofilm surface also varied with the different melatonin loading concentration,and all showed a positive correlation trend.The results of in vitro degradation of 15 W biofilm showed that the degradation rate accelerated gradually with the increase of drug loading concentration.(2)In vitro drug release curve demonstrated that melatonin could be released from biofilm effectively and sustainably for 27 days.DPPH assay showed that the antioxidant activity of SP/MT was positively correlated with melatonin concentration,and the SP/ MT-0.1m group was close to the positive control.(3)The results of CCK-8 and adhesion test showed no cytotoxicity,and MC3T3-E1 proliferate well on the biofilm.The results of ALP staining and activity detection showed that the key enzymes of osteogenic differentiation in the drug loading group were higher than those in the SP group at 14 days,in a concentration-dependent manner.RT-q PCR results showed that the m RNA expression level of SP/ MT-0.1M biofilm was significantly higher than that of the control group(P < 0.05).Conclusions: The SP/MT composite biofilm was successfully prepared by electrospinning technology.The SP/MT drug-loaded biofilm not only had sustained drug release rate and good antioxidant activity,but also promoted the adhesion and proliferation of MC3T3-E1 cells on the fiber membrane,and up-regulated the expression of related osteogenic genes in a concentration-dependent manner.It promoted the early osteogenic differentiation of MC3T3-E1 cells.