Experimental Research Of The Effect Of Resveratrol And Umbilical Cord Mesenchymal Stem Cells To Mices,Bone Defect Repair

Objectives Explore whether resveratrol(RSV)and human umbilical cord mesenchymal stem cells(h UCMSCs)has the promoting repair effect on nude mice with skull defect.Methods Experiment in vitro:After family members agree,returning the umbilical cord tissue 15-20 cm from the full term pregnant women in the course of the plane palace production operation under aseptic conditions,using the method of collagenase and trypsin digestion to separate h UCMSCs from the umbilical cord,then culture and extend the h UCMSCs,observe the morphology of h UCMSCs under inverted phase contrast microscope.Useing the method of flow cytometry instrument to identify its surface markers.Using absorbability gelatin sponge(referred to as the gelatin sponge hereinafter)as the scaffold material to let the h UCMSCs and scaffold materials cultivate 3 d,then they are observed under scanning electron microscopy(SEM)to see whether h UCMSCs grow well with gelatin sponge.Experiment in vivo: 18 SPF nude mice,after 1w,under aseptic conditions,each nude mice was made the defect model of 4×4 mm in the right side of the cranial parietal skull,They were randomly divided into 3 groups.gelatin sponge control group: the stent materials are separately implanted into the bone defect model,no any intervention;h UCMSCs group: the complex of h UCMSCs and gelatin sponge scaffolds are planted into the bone defect model,this group are given the contrast solvent DMSO+0.9% physiological saline 8 w,30 mg/kg/d intervention;RSV + h UCMSCs group: the complex of h UCMSCs and gelatin sponge scaffolds are planted into the bone defect model,this group is given RSV 8 w,30 mg /kg/d drug intervention.At the end of the experimental,the end of 8 w,puting the nude mice to death,using Micro CT for imaging evaluation;Using HE staining,improved Masson trichromatic staining and immunohistochemical(IHC)method to detect the expression of SIRT1、RUNX2、OCN for histological evaluation.Statistical analysis: the Image Pro Plus image analysis software was used to detect the expression of SIRT1,RUNx2,OCN staining semi-quantitatively.the results with the Integral optical density,(Integral optical density,IOD)the Sum of the value.The results were expressed by sum value of the IOD.Experimental data obtained was expressed as mean ± standard deviation(`x±s),then was analysised with SPSS19.0 for one-way ANOVA analysis,with P<0.05 indicating a statistically significant difference.Results 1 Under the inverted phase contrast microscope showed that h UCMSCs are uniform spindle,parallel or vortex pattern.2 The text of flow cytometry instrument show that h UCMSCs highly express the mesenchymal stem cells markers: CD73-APC(99.9%),CD90-FITC(99.8%)of CD105-APC(98.5%);do not express hematopoietic cells markers: HLA-DR-FITC(0.1%),CD45-FITC(0.1%)and CD34-FITC(0.0%),CD11b-FITC(0.0%),CD19-FITC(0.0%).3 Scanning electron microscopy(SEM)results show that gelatin sponge is a three-dimensional structure,the pore connect together and interwove into the nets,the aperture is 200-400 um,the porosity is 98%,h UCMSCs grow well on the surface of gelatin sponge.4 Micro CT results show that BV、BV/TV of the group of the gelatin sponge control,h UCMSCs and RSV+h UCMSCs aresignificant,the difference is statistically significant(P < 0.05).5 HE staining: the new bone formation is more,the gelatin sponge control group have a certain amount of new bone formation,the h UCMSCs group with a small amount of new bone formation,the RSV+h UCMSCs group in new bone formation volume increased obviously than h UCMSCs group and the gelatin sponge control group.6 Improved Masson trichromatic dyeing results show that the RSV+h UCMSCs group :collagen volume increased obviously than the h UCMSCs group and the gelatin sponge control group.7 Immun ohistochemistry(IHC)staining results show that the RSV+h UCMSCs group the h UCMSCs group SIRT1 、 RUNX2 positive cells was significantly greater than the gelatin sponge control group,the difference is statistically significant(P < 0.05).the RSV+h UCMSCs group OCN positive cells was significantly greater than the gelatin sponge control group,the difference is statistically significant(P < 0.05).Conclusions 1 Absorbable gelatin sponge and umbilical cord mesenchymal stem cells are successfully build as the cell scaffold,and has proved that it can be used in the skull defect repair of the nude mice.2 Resveratrol joint between umbilical cord mesenchymal stem cells can promote skull defect repair of the nude mice.3 Resveratrol joint between umbilical cord mesenchymal stem cells can increase the SIRT1 expression and osteogenesis iconic RUNX2 gene and the expression of OCN,which may be to promote to be one of h UCMSCs bone repair mechanisms.