| 1.BackgroundBone defects have always been a difficult problem in orthopedics.The research on its mechanism mainly focuses on the correlation between osteogenesis and angiogenesis.Recent reports have found that a special vascular subtype H(CD31hiEmcnhi)blood vessel exists in mouse bone tissue,which maintains the uniqueness of bone The metabolic microenvironment actively regulates the formation of bones.Studies have found that a large number of H-type blood vessels appear in the early stage of bone injury repair(14 days),and related osteogenic factors are closely related to it.However,is there a role for intra-bone H-type blood vessels during the entire injury repair cycle,especially the healing process of large bone defects? There is no literature report yet.Therefore,it is very important to further study the role of H-type vascular mechanism,and it has important guiding significance for the treatment of segmental femoral defects.2.Objective2.1 Bone Marrow Mesenchyml Stem Cells(BMSCs)and endothelial Cells(ECs)were used with Desferrioxamine(DFO)in different concentrations to determine the best concentration of the drugs and to study the effects of DFO on osteogenesis and angiogenesis.2.2 Using Gelatin Microspheres(Gelatin Microspheres,GMs)in vitro for method records drug slow-release,further gel carrier drug microsphere scaffolds in vivo evaluation the feasibility of repair the large defect;2.3 Building bone defect model in rats,the defect gel carrier drug microsphere stents implanted into different groups,observation defect repair,H blood vessels,Hif factor 1 alpha(Hypoxia Inducer-1 alpha,alpha Hif-1)and osteogenesis related transcription factor(Osterix,Osx)space-time distribution characteristics,and discussed H blood vessels in the femoral condyle period of sexual role in the process of defect.3.Method3.1 DFO was designed at three gradient concentrations(low concentration30 mol/L,medium concentration 60 mol/L,and high concentration 120 mol/L).The control group(PBS)with Phosphate Buffer Saline(60 mol/L)and the group(60 mol/L)with DFO+ PG-478(HIF-1 inhibitor)were set to observe the effect of DFO at different concentrations on osteogenic induction and differentiation of BMSCs.3.2 DFO was designed into three gradient concentrations,and PBS control group and DFO+ PX-478 group were set with the same concentration.To observe the in vitro angiogenesis of ECs by DFO at different concentrations.3.3 GMs was prepared by a series of steps using gelatin and olive oil.Three groups of gradient dose DFO microspheres were loaded(DFO-L-GMS group 50 mg,DFO-M-GMS group 100 mg,and DFO-H-GMS group 200mg).Meanwhile,the Empty microspheres and DFO+ PX-478-GMS group 100 mg were prepared.The feasibility and efficacy characteristics of the drug sustained release system were analyzed.3.4(1)Computed Tomography(Micro-CT)was used to analyze the repair of defects in the 5 groups of rats,including the number of new trabeculae and bone density;(2)Hematoxylin-Eosin staining(HE),Masson staining(Masson)and Goldner staining(Goldner)were used to analyze the defect and new bone tissue of the 5 groups of rats.(3)The number of H-type blood vessels in five groups of rats was analyzed by Immunofluorescence,with growth conditions including morphology and distribution.The potential association between HiF-1 and Osx was also analyzed.(4)The expression levels of HIF-1 mRNA and Osx mRNA in the five groups of rats were analyzed by using the Reverse transcription-polymerase chain reaction(RT-PCR),and the h-type blood vessels and their regulatory mechanism were analyzed.4.Result4.1 Comparison of concentrations of DFO group and PBS group showed that BMSCs osteogenic induction and differentiation were promoted,among which the concentration of 60 mol/L was the highest,and the concentration of Empty group was the lowest.4.2 Compared with the number and total length of the vessels composed of PBS,the concentrations of DFO in the three groups were better,among which the number and total length of the vessels formed by the medium concentration of 60 mol/L were the largest,and the two indexes of the Empty group were the worst in the five groups.4.3 The compound DFO and PX-478 gelatin microspheres were successfully prepared.The drug sustained release efficiency was good,and the final release time averaged at least 19 d.The drug sustained release showed four obvious stages: explosive release of the drug within the first day of release,nearly 35% of the total amount.After the initial burst is released,the release rate slows until it reaches a near steady state 2days later.The steady state release from day 3 to day 13 accounted for about 8% of the total release.Eventually,after a sustained release period,the rate of drug release begins to increase dramatically,and about 50 percent of the drug is released in less than five days.4.4(1)X-ray images showed that the femoral defect of the rats at 12 weeks was healed in all three groups of DFO-GMS,and defects were still visiblein the Empty group and the DFO+ PX-478-GMS group,with the largest defect in the Empty group.(2)Micro-CT reconstruction in line with the results of X-ray,which dose(100 mg),almost completely repair,low dose group(50 mg)and high dose group(200 mg)defect end connections,there are still defects,surface DFO + PX-478-GMs group defect area visible tiny defect of discontinuity,the Empty group showed large discontinuity,the defects of new bone trabecular number corresponds with the reconstruction result,DFO-the number of trabecular bone GMs group was obviously more than the other two groups(P < 0.05),The number of the Empty group was significantly lower than that of the DFO+ Px-478-GMS group(P<0.05),and that of the DFO-M-GMS group was significantly higher than that of the DFO-L-GMS group and DFO-H-GMS group(P<0.05).(3)Histological staining results showed that new bone tissue in the DFO-GMS group was significantly higher than that in the other two groups(P<0.05),and the amount of Empty was significantly lower than that in the DFO+ Px-478-GMS group(P<0.05),and that in the medium-dose group was significantly higher than that in the low-dose and high-dose groups(P<0.05).(4)Immunofluorescence results showed that:4 weeks DFO-H blood vessel number GMs group was obviously higher than that of the rest of the two groups(P < 0.05),one of the Empty group had obviously less than DFO + PX-478-GMs group(P< 0.05),the dose of DFO-M-GMs significantly more than the low dose and high dose group(P < 0.05),and 8 weeks DFO-H blood vessel number GMs group was obviously higher than that of the rest of the two groups(P < 0.05),one of the Empty group had obviously less than DFO + PX-478-GMs group(P < 0.05),the dose significantly more than the low dose and high dose group(P < 0.05);At 12 weeks,the number of H vessels in DFO-GMS group was significantly higher than that in the other two groups(P<0.05),and the Empty group was significantly lower than that in DFO+ Px-478-GMS group(P<0.05).Fluorescence colocalization showed that HIF-1 and Osx were mainly distributed around the H-type vessels.Rt-pcr showed that HIF-1mRNA and Osx mRNA in the DFO-GMS group were significantly higher than those in the other two groups(P<0.05).The Empty group was significantly lower than the DFO+ PARx-478-GMS group(P<0.05),and the medium dose group was significantly higher than the low dose and high dose group(P<0.05).5.Conclusion5.1 DFO at a certain concentration has the effect of promoting osteogenic differentiation of BMSCs and angiogenesis of ECs in vitro,and PX-478 has an inhibitory effect.5.2 Gelatin microspheres can slowly release drugs for a long time,and it is feasible to implant compound drugs to repair bone defects,and the repair effect is better than traditional bone tissue engineering.5.3 DFO can stimulate the proliferation of H-type blood vessels by stimulating the expression of Hif-1?,and generate a large amount of osteogenic differentiation regions centered on H-type blood vessels to generate a large amount of osteogenic transcription factor Osterix,which can effectively promote the repair of segmental bone defects. |
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