Programmed Sustained Release Of RhBMP-2 And Inorganic Ions Composite Hydrogel As Artificial Periosteum

Part 1.Preparation of composite hydrogel artificial periosteal membrane of and characterization of material performance[Objective]Fabricate mesoporous bioactive glass nanoparticles loaded with BMP-2 by chemical grafting method and evaluate the properties of the nanoparticles in Gelma hydrogel membrane.[Methods]Mesoporous bioactive glass nanoparticles MBGNs were firstly prepared and characterized for their morphology and physicochemical properties.Then the EDC/NHS chemical grafting method was used to adsorb and graft BMP-2 onto the surface of MBGNS to obtain MBGNS-BMP-2.The microstructure,physicochemical properties,and release of MBGNS-BMP-2 were observed.Finally,the photosensitive GelMA hydrogel was prepared by gelatin and methacrylic acid.Then,the above MBGNS-BMP-2 nanoparticles were mixed into GelMA aqueous solution and solidified by adding photoinitiator to obtain GelMA/MBGNS-BMP-2 artificial periosteum.The structure of the hydrogels were characterized The mechanical properties of the hydrogels were tested by a universal mechanical tester.The BMP-2 release behavior and ion release behavior of the hydrogels were characterized by ELISA and inductively coupled plasma emission spectroscopy,respectively.[Results]The results of SEM and TEM analysis showed that the mesoporous bioactive glass particles prepared by the improved buffer solution-cationic surfactant solution-gel method in this study had good monodispersion and uniform morphology.The size of the nanoparticles was about 272nm±80nm,the diameter of the nanopore was about 5.3nm and the specific surface area was 78.87m2/g.After the grafting of rhBMP-2,MBGNS-rhBMP-2 showed blurring edges and adhesion among particles,and the dispersion decreased,and the average particle size increased to 401nm±180nm due to agglomeration.FTIR results showed that MBGNS-rhBMP-2 showed a new characteristic peak associated with C-N bond compared with pure MBGNS,indicating the successful graft of rhBMP-2.After ELISA test and calculation,the drug loading rate of rhBMP-2 directly adsorbed was 64.7%±8.8%,while the drug loading rate of grafting with EDC/NHS reaction assisted enhanced was 72.1%±11.4%.However,the release behavior of rhBMP-2 adsorbed by MBGNS was also different from that of rhBMP-2 enhanced by MBGNS.In the direct adsorption group,a burst release rate of 31.5%±5.5%of the total drug load was produced on the first day,and rhBMP2 could not be detected in the supernatant solution after 14 days.In the enhanced grafting group,the cumulative release rate on the first day was only 20.2%±2.1%,and the cumulative release rate was still 72.7%±6.8%at 28 days.The results of inductively coupled plasma emission spectroscopy(ICP-AES)showed that the chemical modification and grafting had little effect on the ion release behavior of MBGNs.SEM results of hydrogels showed that there was no statistical significance in the effect of adding MBGNS on the pore size of Gelma hydrogel.However,the mechanical strength of hydrogel film increased with the increase of MBGNS content.However,the flexibility of hydrogel film decreased significantly when the added content reached 5%.The FTIR results of the hydrogel also showed that GelMA hydrogel and MBGNS-RhBMP-2 could be mixed successfully.[Conclusion]MBGNs prepared in this experiment have good dispersibility and highly ordered mesoporous structure,and have good hydrophilicity and modifiable potential.Through APTES,we successfully aminated MBGNs to make MBGNs become a more reliable and efficient graft load carrier for protein factors.Through the water-soluble EDC/NHS grafting method,rhBMP-2 was successfully strengthened into the inner and surface of MBGNS channels,which improved the loading efficiency of MBGNS on rhBMP2,significantly extended the release period,and reduced the initial burst release.At the same time,drug-loaded MBGNs can be added to Gelma hydrogel and use Gelma hydrogel as the loading mechanism to form membrane coating material.The addition of MBGNS-RhBMP2 not only enhanced the structural stability of GelMA hydrogel membrane,but also further restricted the release rate of RhBMP-2,while the release rate of various ions of MBGNS was not significantly affected.Part 2 Programmable sustained-release BMP-2 and inorganic ion composite hydrogel artificial periosteum to promote cell adhesion and osteogenesis in vitro[Objective]To investigate the ability of composite hydrogel artificial periosteum to promote the adhesion,proliferation and differentiation of BMSCs and the adhesion and angiogenesis of HUVECs in rats.[Methods]The cells were inoculated on the surface of the hydrogel artificial periosteum material for 1,3 and 5 days,then the cells were removed and fixed,dehydrated by ethanol gradient,dried at critical point,and then the cell adhesion and proliferation were observed by SEM.The cells were inoculated on artificial periosteum for 1,3,5 and 7 days,and the cell proliferation was detected by CCK-8 method.The toxicity of the hydrogel material was detected by live staining 14 days after BMSCs inoculation.The early osteogenic property of BMSCs under the influence of composite artificial periosteum was evaluated by ALP staining,and the late osteogenic property of BMSCs under the influence of composite artificial periosteum was evaluated by alizarin red staining and quantitative analysis.Immunofluorescence staining and qPCR were used to verify the effect of artificial periosteum on osteogenic differentiation of BMSCs.The HUVECs cells were inoculated in the matrix glue on the surface of the hydrogel artificial periosteum for 6 hours and then the cytoskeleton was dyed and photographed to evaluate the ability of promoting angiogenesis in vitro.[Results]SEM results showed BMSCs were good in adhesion and proliferation on the surface of hydrogel artificial periosteum.CCK-8 quantification results showed that the number of cells in GelMA/MBGNs group and GelMA/MBGNs-rhBMP-2 group was significantly higher than that in the pure GelMA group at the 3rd,5th and 7th day of culture,while the number of cells in GelMA/MBGNs/rhBMP-2 group and blank control group(P<0.05),and there was no significant difference in quantity between blank control group,GelMA/rhBMP-2 group and GelMA group(P<0.05),there was no significant difference between GelMA/MBGNS group and GelMA/MBGNs-rhBMP-2 group(P<0.05),the above results proved that the addition of MBGNs was the main factor promoting cell adhesion and proliferation.Osteogenesis ALP staining and alizarin red staining and quantitative differentiation,immunofluorescence staining and rt-per experiments have confirmed that contain MBGNs osteogenetic differentiation levels of groups were higher than GelMA group and the blank group,at the same time in the early osteogenesis induced with rhBMP-2 GelMA/MBGNs/rhBMP-2 group and GelMA/MBGNs-rhBMP-2 bone activity that is better than does not contain rhBMP-2 categories,and direct the adsorption load rhBMP GelMA/MBGNs/rhBMP-2 the optimal set of early osteogenesis activity,Stronger than the other groups.However,at the late stage of osteogenic induction,the GelMA/MBGNSrhBMP-2 group with chemically grafted rhBMP-2 showed better osteogenic activity.The results of HUVECs in the matrix gel on the hydrogel surface showed that MBGNS also had a positive effect on angiogenic differentiation,and rhBMP-2 also showed a positive effect on angiogenic activity,but the difference was not statistically significant(p<0.05).[Conclusion]he addition of MBGNs-rhBMP-2 as a photocrosslinked GelMA hydrogel fiber material can provide a better adhesion surface for BMSCs,and has good biocompatibility,which is conducive to the proliferation and differentiation of cells.After the release of rhBMP-2 in the late stage of osteogenic activity,the dissolved ions in MBGNS still have certain osteogenic ability to promote matrix mineralization.At the same time,the synergistic effect of the two can also promote the formation of new blood vessels.Part 3 Programmable sustained-release BMP-2 and inorganic ion composite hydrogel artificial periosteum to promote osteogenesis and vascularization in vivo[Objective]To investigate the osteogenic properties of GelMA/MBGNs-rhBMP-2 composite hydrogel artificial periosteum in vivo.[Methods]Rat models of skull defect were established and divided into control group,GelMA group,GelMA/MBGNs group,GelMA/MBGN s/rhBMP-2 group and GelMA/MBGNs-rhBMP-2 group.Four weeks and eight weeks after implantation,the skull specimens were collected and fixed.The volume of new bone tissue in total defect(BV/TV)and bone trabecular density(Tb.Th)were analyzed.Then the specimens were decalcified and sectioned.After HE staining,Masson staining and CD31 immunohistochemical staining,the sections were photographed and quantified to evaluate the ability of artificial periosteum to promote bone tissue repair and vascularization in vivo.[Results]after operation,the rats recovered well,had normal diet and activities,and had no infection or abnormal behavior.Micro CT quantitative results showed that there was almost no new bone growth in the white group,only a small amount of high-density burr around the defect site,and the volume of new bone only accounted for 7.44%±1.48%of the total defect volume.There was a small amount of fragmentary new bone growth in the gelma group,accounting for 9.6%±1.88%of the total bone volume,nearly half of the area in GelMA/MBGNs,GelMA/MBGNs/rhbmp-2 group and GelMA/MBGNs-rhbmp-2 group was covered by new bone,and the volume proportions were 12.7%±2.18%,10.97%±2.24%and 19.55%±2.76%respectively.At the 8th week,only a small amount of new bone was formed in the white group,accounting for 11%± 1.49%of the total defect volume.The content of new bone in the gelma group was about 22.95%± 2.98%,which was lower than that in the group containing mbgns.The content of new bone in the gelma/mbgns-rhbmp2 group grafted with rhBMP-2 was the highest,and the difference was statistically significant.The bone trabecular density of gelma/mbgns group and gelma/mbgns-rhbmp-2 group was significantly higher than that of the other three groups at 4 weeks.At 8 weeks,the new bone volume and trabecular density of gelma/mbgns-rhbmp-2 group were higher than those of gelma/mbgns group.HE staining showed that the hydrogel materials were not completely degraded by 4W,and the trend of new bone area was consistent with Micro-CT.When 8W,all the hydrogel materials were completely degraded,and the defect sites were replaced by connective tissue or new bone tissue.The cross sectional new bone tissue was also consistent with Micro-CT.Masson staining showed that the maturity of bone formation in gelma/mbgns/rhBMP-2 group was higher than that in other groups in the early stage,but in the late stage of bone repair,the results were consistent with the in vitro experiment.Gelma/mbgns-rhbmp-2 group had more advantages in promoting bone tissue maturation.The immunofluorescence results of CD31 neovascularization were similar to those in vitro.The group containing mbgns had a higher ability to promote neovascularization,and the diameter and number of neovascularization gradually decreased with time.[Conclusion]The 5mm rat skull defect model has the significance of a critical bone defect model,and the biological factors and ion release function of GelMA/MBGNsrhBMP2 artificial periosteum can play an ideal programmed long-term osteogenesis in the body,while the ion release function It also provides early vascularization properties for artificial periosteal materials.