The Effect Of Laponite Concentration On The Osteogenic Effect Of Composite Hydrogel

In the process of clinical diagnosis and treatment,large bone defects are difficult to self-repair.The vulnerability of defect sites and tissues to infection and the poor tolerance of patients will affect the progress of the disease.Due to the shortage of donor and the limitation of immune risk of traditional treatment methods,biological replacement materials for bone regeneration have become a hot topic in research.Hydrogels are often used as biological materials for filling defects because of their rich water content and good biocompatibility.Laponite has thixotropy,and the combination with hydrogel can improve its mechanical properties to make up for the deficiency of strength.Previous studies had found that laponite could promote osteogenic differentiation of BMSCs and promote bone repair in hydrogels.However,there were few studies on the concentration of laponite to optimize its osteogenic effect,and the biological pathway through which it exerts its osteogenic effect.In this study,we prepared composite hydrogel scaffolds containing different concentrations of laponite and implanted them into bone defects to observe bone repair,to investigate the effect of laponite content in hydrogels on promoting bone regeneration.By comparing the RNA sequencing of BMSCs cultured with and without laponite,we analyzed the related signal pathways of promoting bone repair with laponite.ObjectiveTo investigate the effects of laponite concentration on the bone-enhancing repair of composite hydrogel scaffolds,hydrogel scaffolds containing different concentrations of laponite were prepared,and the osteogenic properties of different concentrations of laponite were compared through in vivo and in vitro experiments.MethodsIn the first part,the composite hydrogel scaffold was prepared by injection molding.According to the concentration of laponite,the scaffolds were divided into four groups(0%Lap,1%Lap,2%Lap,3%Lap).Scanning electron microscopy was used to observe the internal pore structure of each group of hydrogels,energy spectrum analysis was used to determine the composition and proportion of scaffold elements,hydration performance of each scaffold was measured by lyophilized weight loss,and compression modulus of each scaffold was measured by universal material testing machine.The scaffolds were immersed in simulated body fluids to observe and measure the degradation and ion release of scaffolds at different time points.In the second part,the effects of hydrogels containing different concentrations of laponite on osteogenic differentiation of BMSCs were examined at the cellular level.BMSCs were extracted and cultured from SD rats and identified by flow cytometry.Then the cells were cultured with hydrogel scaffold extracts from each group.The cell activity was detected by flow cytometry and CCK8 assay,and the adhesion of cell plaque protein was determined by immunofluorescence staining.Finally,the expressions of Runx2,ALP,Col1 a and OCN were determined by q RT-PCR,and ALP staining and activity were determined.In the third part,a rat model of femoral condyle defect was established.Composite hydrogel scaffolds were implanted in each group,and the osteogenic repair of each group was measured after 4,8 and 12 weeks.Firstly,new bone production was measured by Xray and micro-CT 3D reconstruction.Then,sequential fluorescence double labeling was applied to calculate the mineralization deposition rate at each time period to measure the rate of new bone formation.Van Gieson staining was performed to observe the new bone formation in the bone defect,and HE staining was performed to observe the material degradation and bone formation.Finally,collagen generation and collagen typing ratio were determined by masson staining and Sirius scarlet staining.In the fourth part,the mechanism of laponite promoting osteogenic differentiation of BMSCs was studied.The genes of BMSCs cultured with and without hydrogel extracts of laponite were sequenced by RNA,and the enrichment pathway was analyzed by KEGG.Then the phosphorylation levels of key proteins in the enrichment pathway were detected by protein western blotting.Finally,the pathway was inhibited and verified by the expression of osteoblast proteins and the formation of mineralized nodules.ResultsPart Ⅰ.The preparation of composite hydrogel scaffolds with different contents of laponiteComposite hydrogel scaffolds containing different concentrations of laponite can be prepared conveniently by molds.Scanning electron microscope found that laponite made the internal pores of hydrogel more regular and increased the pore size.The content of magnesium silicate in each group of hydrogels was detected by EDS,which indicated that laponite existed and distributed evenly in hydrogels.When the content of laponite is above2%,the water content of hydrogel is less than 90%.The addition of laponite improves the mechanical strength of hydrogel,and the content of laponite increases the most when the content of laponite is 2%.The hydrogels degrade slowly in simulated body fluids and are basically stable at 21 days.The hydrogels containing laponite can release magnesium silicate ions slowly.Part Ⅱ.Effects of composite hydrogel extracts on the growth and differentiation of BMSCsThe BMSCs extracted and cultured by the adherent method grew well.Flow cytometry and CCK8 showed that the cell activity of 3%Lap group was lower than that of other groups,indicating that the content of laponite in hydrogel had a certain effect on cell growth and proliferation at 3%.The results of immunofluorescence showed that the amount of adhesive patches produced by 1%Lap,2%Lap and 3%Lap was more than that in Blank group and0%Lap group,but there was no difference in 1%Lap,2%Lap and 3%Lap groups.The results of q RT-PCR showed that the expressions of Runx2,ALP,Col1 a and OCN in 2%Lap and3%Lap groups were higher than those in other groups.ALP staining showed that the ALP activity of 1%Lap,2%Lap and 3%Lap was higher than that of Blank group and 0%Lap group with the increase of laponite content.Part Ⅲ.Repair of critical bone defect of femoral condyle in rats by hydrogel with different concentrations of laponiteAfter 8 weeks of implantation,more callus filled the defect in the 1%Lap,2%Lap,and3%Lap groups.Micro-CT results showed that the BV/TV of the 1%Lap,2%Lap and 3%Lap groups were all higher than those of the Blank and 0%Lap groups,and the BV/TV of the 2%group was higher than that of the 1%Lap and 3%Lap groups.The comparison of mineral deposition rate showed by fluorescence double labeling was consistent with CT results.VanGieson staining showed that there was more new bone formation in the defect of 1%Lap,2%Lap,and 3%Lap groups.Masson staining showed that the 2%Lap and 3%Lap groups produced more collagen,while the proportion of type I/type III collagen in the 1%Lap,2%Lap and 3%Lap groups was higher,and there was no difference among the three groups.Part Ⅳ.The mechanism of laponite promoting osteogenic differentiation of BMSCsPrincipal component analysis showed that the samples were well repetitive.KEGG showed the most significant difference in the MAPK pathway,and protein western blot showed that the phosphorylation level of Erk protein increased.The expression levels of osteoblast proteins Col1 a and Osterix decreased after the inhibition of Erk pathway,and the ALP staining became light and alizarin red staining showed reduced mineralization.ConclusionThe composite hydrogel scaffold was prepared by injection molding method,which was simple and convenient.Laponite improved the internal pore arrangement structure of the hydrogel and increased the pore size.The addition of laponite could improve the mechanical strength of the composite hydrogel,and the best effect was achieved when the content was 2%.The hydrogel scaffolds can degrade slowly in vitro,and the scaffolds adding laponite can release silicon and magnesium for a long time.The activity of BMSCs was not affected when the content of laponite in hydrogel was less than 2%,but the adhesion ability of BMSCs was improved.Moreover,BMSCs can promote directed osteogenic differentiation,and the best promotion effect is when the content is 2%.In vivo studies can show that hydrogels containing laponite can improve the repair ability of bone defects,and the repair ability is strongest when the content of laponite in hydrogels is 2%.RNA transcriptome sequencing analysis showed that laponite in hydrogel promoted osteogenic differentiation mainly by activating MAPK-Erk pathway in BMSCs.