Microenvironment-responsive Release BMP-2 Peptide From MnO₂/GelMA Hydrogel For Bone Repair

Bone injury is one of the common organ tissue damage.After injury,a series of changes will occur in the internal microenvironment,including:1,the pH of the injury site changed from normal alkalinity to acidity,and then gradually changed back to alkalinity with injury healing;2,the content of hydrogen peroxide(H2O2)in the damaged site will also be increased due to vascular rupture;3.oxygen tension at the injury site decreases with insufficient blood supply.Acidic conditions can promote absorption of the end of bone for salt and calcium and can also cause acidosis at the same time,finally affecting the bone healing process.H2O2 is the small molecules participating in induced inflammation,can cause hematoma and delay the bone healing process,even can also cause the failure of bone healing.After injury,reactive oxygen species(ROS)can restrain activity of bone marrow cells by affecting injury microenvironment,thereby inhibiting bone healing.Numerous studies have shown that improving the microenvironment in the process of bone healing can accelerate bone repair or bone regeneration.Based on the bone injury microenvironment,we have prepared hollow manganese dioxide(H-MnO2)nanoparticles which is responsive to acid and high content of H2O2,using it as a drug carrier to load bone morphogenetic protein-2 peptide(BMP-2),and then dispersed in methyl acrylic acid anhydride gelatin(GelMA)to prepare BMP-2/MnO2/GelMA hydrogel which is responsive to bone injury microenvironment.After implantation of BMP-2/MnO2/GelMA hydrogel into the skull defect site of rats,H-MnO2 in the composite hydrogel will gradually degrade and release BMP-2 peptide to promote bone repair in response to the acidic and high content of H2O2 in the microenvironment of injury site.At the same time,the reaction of H-MnO2 in the composite hydrogel with H2O2 can generate oxygen(O2),so that it can reduce the probability of inflammatory reaction by consuming H2O2,improve the activity of osteoblasts and accelerate the healing of bone tissue.In addition,manganese ions(Mn2+)produced by degradation of H-MnO2 can also promote bone repair.In the first part of this paper,we fabricated MnO2/GelMA composite hydrogels and their properties were characterized.At first,using silicon dioxide(SiO2)nanoparticles as template,SiO2@MnO2 nanoparticles with core-shell structure were prepared by redox reaction of potassium permanganate(KMnO4)and hydroxyl on the surface of SiO2.Then,SiO2 in SiO2@MnO2 nanoparticles was removed by sodium carbonate(Na2CO3)to obtain H-MnO2 nanoparticles.Then,H-MnO2 nanoparticles were added to GelMA hydrogel to prepare MnO2/GelMA composite hydrogel.The structure of H-MnO2 nanoparticles were characterized by transmission electron microscopy(TEM).The cross section morphology of MnO2/GelMA composite hydrogel was characterized by scanning electron microscope(SEM).Through mechanical experiments,the mechanical properties of composite hydrogels containing different H-MnO2 nanoparticles were tested.The biocompatibility of MnO2/GelMA composite hydrogels was investigated by cytoskeleton staining,CCK-8 and Live/Dead staining.The results showed that H-MnO2 nanoparticles were successfully prepared by template method with uniform particle size distribution of 250?300 nm.With the content of H-MnO2 nanoparticles in GelMA increased from 0.25%to 1%,the Young's modulus of the composite hydrogel decreased from about 3.74±0.37 kPa to about 1.82 ±0.18 kPa,but still had a certain strength and could maintain a certain shape.Moreover,MnO2/GelMA hydrogel had good biocompatibility and can promote the adhesion and proliferation of bone marrow mesenchymal stem cells(BMSC)in rats.In the second part of this paper,the osteogenic properties of MnO2/GelMA hydrogels loaded with BMP-2 peptide(BMP-2/MnO2/GelMA)were studied.Firstly,BMP-2 peptide were loaded on H-MnO2 nanoparticles to study the release of BMP-2 peptide in MnO2/GelMA composite hydrogel in the presence of acidic environment(pH 5.5,pH 6.5)and H2O2.Then,through alkaline phosphatase(ALP)staining,alizarin red staining and detection of the expression of osteogenic genes ALP,Col-1,OPN,OCN and Runx 2,the in vitro osteogenic performance of BMP-2/MnO2/GelMA composite hydrogel was investigated.A rat model of skull defect was used,BMP-2/MnO2/GelMA composite hydrogel was implanted,and Micro-CT and Hematoxylin-Eosin(H&E)staining were used to analyze the bone volume and bone morphology of newborn bone.The results showed that H-MnO2 was degraded and BMP-2 peptide was released in acidic environment,and the release rate was up to 95%at 28 days.ALP and alizarin red staining results showed that BMP-2/MnO2/GelMA hydrogel highly expressed alkaline phosphatase and formed a large amount of calcium deposition during the same period.Moreover,PCR results showed that the expression levels of ALP,Col-1,OPN,OCN and Runx 2 in BMP-2/MnO2/GelMA composite hydrogels were higher than those in other groups,indicating excellent in vitro osteogenesis.Animal experiments showed that BMP-2/MnO2/GelMA composite hydrogel could well repair the skull defects of rats,and nearly 90%of the defects had been repaired by 8 weeks.It could be used as an excellent osteogenic material for bone tissue engineering.In a word,using the template method,we have successfully fabricated H-MnO2 nanoparticles,the particle can response acidic environment and be degraded,H-MnO2 can react with hydrogen peroxide,consume hydrogen peroxide and produce oxygen at the same time.Thus protect cells against damage under hypoxia environment.Not only that H-MnO2 nanoparticles can load BMP-2 peptides.The fabricated BMP-2/MnO2/GelMA hydrogel,have excellently osteogenesis function both in vivo and in vitro,can be used as bioactive materials for bone tissue engineering.