Controllable Preparation Of Magnetically Oriented Chitosan Fiber-reinforced Hydroxyapatite/gelatin Porous Scaffolds And Their Application In Bone Defect Repair

Bone tissue defect is a serious threat to human health,and traumatic bone grafting needs a lot of bone tissue repair and reconstruction.Autologous bone has always been an excellent method for treating bone defects,mainly because of its stable structure,low immunogenicity and natural osteogenic ability.However,due to the limited source of autologous bone,the incidence of complications in the process of collection is high.As a bone graft substitute,allogeneic bone has good bone conduction ability and biomechanical properties,especially can avoid the occurrence of complications.The limited quantity and quality of bone available limits its use in large defects.It is these disadvantages of autograft and allograft that make the development of new alternative bone repair materials especially urgent.It is always an important research direction to imitate the orientation structure of ossein fibers and it is full of great challenges.Orientation fiber has been applied in bone repair materials by some people,but no significant progress has been made.In this paper,a method to promote fiber orientation was developed.The magnetic orientation chitosan fiber reinforced hydroxyapatite/gelatin（CSF/Fe₃O₄/HA/GEL）porous scaffold was successfully prepared by combining magnetic field and freeze drying.The magnetic nanoparticles ferric oxide were loaded on the surface of the fiber to make the fiber magnetic,and then combined with the magnetic field to make the fiber aligned in the scaffold.After freeze-drying,the magnetic orientation chitosan fiber reinforced hydroxyapatite/gelatin（CSF/Fe₃O₄/HA/GEL）porous scaffold was successfully prepared.Chitosan was modified several times by loading Fe₃O₄,depositing calcium carbonate,and then converting to hydroxyapatite.Each step of the composite fiber was analyzed by SEM、XRD and FTIR to ensure that the successfully loaded material was pure and free of impurities.After that,the orientation fiber scaffold was prepared by combining magnetic field and freeze drying.The surface morphology,XRD and FTIR phase analysis were carried out to prove that the composition of the scaffold was pure and free of impurities.The mechanical properties of the scaffold in different directions of the fiber were also tested,namely tensile and compression properties.The results show that,in terms of tensile properties,the average maximum tensile strength of the scaffold in the vertical fiber direction is 8.6 MPa,the average maximum tensile strength of the scaffold in the parallel fiber direction is 4.7 MPa,and the average maximum tensile strength of the scaffold without fiber is 2.0 MPa.In terms of compression performance,the average maximum compressive strength of the scaffolds in the vertical fiber direction is 1.6 MPa,the average maximum compressive strength of the scaffolds parallel to the fiber direction is 0.9 MPa,and the average maximum compressive strength of the scaffolds without fiber is 0.6 MPa.These results show that the addition of orientation fibers enhances the mechanical properties of the scaffold and has anisotropy.The swelling and degradation of the scaffold were tested later,and the results showed that the swelling performance of each group was 9 times of its own weight,and the mass loss was less than 15%after 14 days of degradation test.The above results proved that scaffold materials can exist in the body for a long time,meeting the effects of sustained drug release and administration,and meeting the characteristics of long bone repair cycle.In addition,drug release is relatively rapid in the first 24 hours,then the rate slows down and gradually reaches equilibrium.These results indicated that CSF/Fe₃O₄/HA/GEL/LCD stent was a good drug delivery carrier with excellent drug delivery performance.This effect is inseparable from the special structure of the large hole and orientation of the bracket.In order to study the application effects of CSF/Fe₃O₄/HA/GEL and CSF/Fe₃O₄/HA/GEL/LCD scaffolds in bone defect repair,we conducted in vitro and in vivo experiments to explore their effects on biocompatibility,osteogenesis and angiogenesis.CCK8 test and cell adhesion scan showed that the two scaffolds not only had good biocompatibility,but also could further promote cell adhesion.The results show that CSF/Fe₃O₄/HA/GEL and CSF/Fe₃O₄/HA/GEL/LCD groups showed better expression of osteogenic related proteins,which compared with GEL group.The results were better than those without LCD.In addition,the inhibition of osteoclast differentiation was similar,and the inhibition of GEL group,CSF/Fe₃O₄/HA/GEL and CSF/Fe₃O₄/HA/GEL/LCD group were improved successively.These results indicate that the introduction of orientation fiber and the loading of LCD enhance the expression of osteogenic related proteins and enhance the inhibitory effect of osteoclast.In addition,we also conducted endothelial cell invasion and tube-forming experiments,and the results were consistent with the above.The introduction of orientation fiber and the loading of LCD both enhanced the invasive ability and tube-forming ability of the scaffold,and the stent with both orientation fiber and LCD had the best invasive ability and tube-forming ability.Subsequently,Micro CT scanning was performed on the skull implanted in rats after 12 weeks of skull defect,and bone density was calculated and new bone formation was analyzed qualitatively and quantitatively.The results showed that,compared with the GEL group,the CSF/Fe₃O₄/HA/GEL and CSF/Fe₃O₄/HA/GEL/LCD groups have better osteogenesis promotion ability,and the scaffolds with both orientation fiber and LCD have the best effect.In order to further verify the osteogenic and angiogenic abilities of the scaffold,immunofluorescence and immunohistochemistry experiments were performed.The results showed that the expression of osteogenic related proteins in the CSF/Fe₃O₄/HA/GEL/LCD group was the best,and the effect of the CSF/Fe₃O₄/HA/GEL group and GEL group decreased successively.In conclusion,CSF/Fe₃O₄/HA/GEL and CSF/Fe₃O₄/HA/GEL/LCD scaffolds have good biocompatibility,cell adhesion,excellent ability to promote bone and angiogenesis,and inhibit osteoclast differentiation,so these two scaffolds are good bone repair materials.It provides a new idea for the study of biomaterials and has profound significance.