The Effect Of SA Coating Loaded With DMOG And Strontium On The Biomimetic Porous BCP Composite

Part one:Effect of SA coating of different calcium-strontium crosslinking solution on the performance of BCP compositeObjective:Using biomimetic porous BCP as scaffold,sodium alginate（SA）sustained release coating was coated in the lamellar pores of the scaffold by dipping method,and cross-linked with different calcium-strontium ratio,as to select the best cross-linking agent to construct SA coating.Methods:1.Lamellar graded porous BCP scaffolds were prepared by improved ice template method,and SA coatings were prepared in the pores of stents by dipping method,and cross-linked with cross-linking solution with different ratios of calcium ions to strontium ions.Scanning electron microscope（SEM）and energy dispersive spectrum analysis（EDS）were used to observe the microstructure of the stent,analyze the surface elements,and evaluate whether the coating was successfully coated.2.The weight loss rate and surface morphology of each stent at different time points were detected by immersion method,and the effect of coating on the degradation of BCP stent was evaluated,and the strontium ion precipitation concentration of each stent was detected by inductively coupled plasma spectrometer（ICP）.3.The scaffolds of each group were co-cultured with mouse embryonic cell progenitor cells（MC3T3-E1 cells）,and the effects of scaffolds on the proliferation of MC3T3-E1 cells were detected by CCK8method,and the effects of drug-loaded BCP scaffolds on the expression of ALP,OPN,COL and RUNX2m RNA in MG63 cells were evaluated by RT-PCR.Results:1.SEM showed that the sodium alginate coating uniformly covered the surface of the laminates of each porous BCP scaffold,the gap and thickness of the laminates did not change significantly,the pores at all levels were clear and unobstructed,and a few areas showed that the SA coating was flaky or wire-drawn connecting between the two layers.EDS analysis of the coating group contains C,Sr,Cl and other elements,which proves that the sodium alginate coating has been successfully coated.2.It can be seen that in the early stage of immersion（7 days）,the weight of stents in BCP group increased,and the degradation of stents accelerated with the extension of immersion time（14 and 21 days）.With the extension of soaking time,the quality of each coating group decreased,and the weight loss curve was close.When the scaffolds were immersed in PBS solution for 14 days,the SEM images showed that there were a little HA particles on the surface of the stents in each group,and the sodium alginate coating was covered in a grid after degradation in the coating group,and no obvious cracking or exfoliation of the coating was observed.The sudden release of Ca²⁺and Sr²⁺in each coating group appeared on the 1st day,and then the release curve tended to be stable.3.The extract of different scaffolds was co-cultured with MC3T3-E1cells for 1、3、7 days,and the effect of coating on the proliferation of MC3T3-E1 cells was better than that of the control group and BCP group,and the Sr1 group with Ca Cl₂:Sr Cl₂=1:1 as cross-linking medium showed the best effect.Real-time fluorescence quantitative PCR was used to detect the expression of osteogenesis-related genes（RUNX2,COL-I,OPNm RNA）co-cultured with MC3T3-E1 cells in each group:on the 7th day,RUNX2,COL-I and OPNm RNA in Sr1 group were higher than those in other groups,and the difference was statistically significant.Conclusions:1.SA crosslinked with Ca²⁺and Sr²⁺can be used as a coating for BCP scaffolds,which does not affect the layer gap and thickness of BCP scaffolds,increases the roughness of scaffolds surface as well as the traffic between the layer of scaffolds and can release Ca²⁺,Sr²⁺smoothly.2.The leaching solutions of different scaffolds were co-cultured with MC3T3-E1 cells,among which the Sr1 group with calcium:strontium ratio of 1:1 as crosslinking liquid exhibited the best pro proliferative effect.Co-culture of each group of scaffolds with MC3T3-E1 cells Sr1 group can promote the upregulation of RUNX2,COL-I and OPN m RNA expression.Part 2 Effect of concentration of DMOG on vascularization of BCP compositeObjective:Using biomimetic porous BCP as scaffold,dip-coating method was used to construct SA sustained-release coating with different concentrations of dimethyloxalylglycine（DMOG）in the lamellar pores of the scaffold,and the best concentration of DMOG was selected by cross-linking solution with the best ratio of calcium to strontium.Methods:1.the optimal calcium to strontium ratio crosslinking solution（Ca Cl₂:Sr Cl₂=1:1）in the first part was selected to prepare scaffolds,and the drug sustained-release ability of scaffolds loaded with different concentrations of dmog was detected by immersion method and UV spectrophotometer;2.Different concentrations of DMOG loaded scaffolds were co-cultured with human umbilical vein endothelial cells（HUVECs）and the effects of scaffolds on cell proliferation of HUVECs were detected by CCK8 assay;The effect of drug loaded BCP scaffolds on the expression of vascular endothelial growth factor（VEGF）in HUVECs cells was evaluated by RT-PCR;The effect of loading different concentrations of DMOG scaffolds on the cell migration of HUVECs was assessed by Transwell assay.Results:1.The sustained-release profiles of the stent materials loaded with different concentrations of DMOG were close,and the sustained-release rates of the drug were 22.4%,29.3%and 23.6%on day 1,respectively,while showing a better sustained-release effect.The sustained release rates of the drugs were 31.3%,38.2%,and 27.7%on day 3,and 56.9%,58.6%,and 44.1%on day 7,respectively.The DMOG in the 12.5 mm group achieved more than 90%sustained-release on day 16,and the sustained release was almost complete by 21 days.Whereas the 25 mm group and 50 mm group achieved 90%of the sustained-release only on days 18-19 and 24-25,respectively,and the sustained-release basically ended on days 28 and 33,respectively.2.Effects of different concentrations of DMOG scaffold on cell proliferation of HUVECs results:at 1D,only Sr+D2 group promoted cell proliferation of HUVECs（P<0.05）;By the third day,all four experimental groups exerted a promoting effect on the cell proliferation of HUVECs（P>0.05）,and by the seventh day,a statistically significant difference in OD was observed between the Sr+D2 group and the Sr+D3 group and the control group（P<0.05）,and the addition of DMOG and Sr²⁺could promote the cell proliferation of HUVECs.The SA-coated BCP scaffolds loaded with different concentrations of SA were co-cultured with HUVECs cells,and the expression of VEGF in the Sr+D2 group on days 3 and 7 was significantly higher than that in the other groups,while the Sr+D2 group could promote HUVECs cell migration.Conclusion:1.The calcium-strontium cross-linked SA coating prepared on BCP scaffolds can slow release DMOG and has good slow-release performance.2.The DMOG released by the coating can promote the proliferation and migration of HUVECs cells,and can up-regulate the expression of VEGF,among which the Sr+D2 group has the best promoting effect.Part 3 Effect of slow-release of strontium and dmog from BCP composite on biological effectsObjective:The scaffolds were co-cultured with MC3T3-E1 cells and HUVECs cells with or without strontium,with or without DMOG,to explore whether the sustained release of Sr²⁺and DMOG from the scaffold was better than that from single sustained release.Methods:1.The experimental groups were Sr0,Sr0+DMOG,Sr1 and Sr1+DMOG.The scaffolds were co-cultured with MC3T3-E1 cells.The effects of drug-loaded BCP scaffolds on the expression of OPN,COL-I and RUNX2m RNA in MC3T3-E1 cells were evaluated by RT-PCR,and the effect of scaffolds on osteogenesis was detected by alkaline phosphatase（ALP）and alizarin red staining.2.The experimental groups were co-cultured with HUVECs cells,and the effects of scaffolds on the expression of vascular endothelial growth factor（VEGF）in HUVECs cells were evaluated by real-time fluorescence quantitative PCR,and the effects of stents on the expression of HIF-1αand VEGF were detected by Western Blot.Results:1.At 3d and 7d,the expression of RUNX2,COL-I and OPN in Sr0group and Sr1 group after loading DMOG was higher than that in unloaded Sr0 group and Sr1 group.There was significant difference between Sr0+D group and Sr1+D group（P<0.05）.The results of Western Bolt showed that the expressions of COL-Ⅰ,RUN2 and HIF-1αin Sr1 and Sr1+D groups were significantly higher than those in Sr0group,and the expression of osteogenesis related genes in Sr1+D group was significantly higher than that in other groups.2.At 3d and 7d,the expression of VEGF in Sr0 group and Sr1 group after loading DMOG was higher than that in unloaded Sr0 group and Sr1group,and there was significant difference in VEGF expression between Sr0+D group and Sr1+D group.The results of Western Bolt showed that the expression of HIF-1αand VEGF protein in Sr1 and Sr1+D groups was significantly higher than that in Sr0 group,and the expression of HIF-1αin Sr1+D group was significantly higher than that in other groups.3.Compared with the blank group,the scaffold materials of each group could induce new bone formation at 4,8 and 12 weeks.The new bone formation was the least in the BCP group and better in the Sr0 group and Sr1 group,but there was still less low density between the scaffold and the new bone,and the new bone formation in the Sr1 group was more than that in the Sr0 group,and the new bone was the most in the Sr0+D group and Sr1+D group,and new bone could be seen around the scaffold in the Sr1+D group at 12 weeks.Conclusion:1.Both Sr²⁺and DMOG are beneficial to the expression of osteogenesis-related genes and proteins in MC3T3-E1 cells and angiogenesis-related genes and proteins in HUVECs cells.The sustained release of Sr²⁺and DMOG at the same time is better than that of sustained release alone.2.The amount of new bone formation of both Sr²⁺and DMOG in the microenvironment of the scaffold was more than that of Sr²⁺and DMOG alone.