| Purposes1.Reverse design of a new nickel-titanium memory alloy anchor nail with graphene coating to compare its mechanical properties with those of common titanium alloy anchor nails and the effects of both materials on osteoblast proliferation and osteogenic activity in mice。2.To compare the effects of graphene-coated Ni Ti memory alloy anchor nails,uncoated Ni Ti memory alloy anchor nails and plain titanium alloy anchor nails on local osseointegration,bone formation and tendon-bone healing strength after rotator cuff injury with bone loss,as assessed at biomechanical,histological and imaging levels.Methods1.A new nickel-titanium memory alloy anchor was designed and prepared,and its surface was modified with graphene coating using the film transfer method.The surface morphology,hydrophilicity and the success of the coating on the graphene-coated Ni Ti alloy material were characterized using scanning electron microscopy,contact angle meter and confocal Raman spectroscopy.To further analyze the bioactivity of graphenecoated Ni Ti materials,we examined the effects of graphene-coated Ni Ti materials on the proliferation and viability of mouse osteoblasts using CCK-8 and Alkaline phosphatase(ALP)kits.The maximum extraction force of the new anchor nails was examined on polyurethane models with different densities compared to the normal titanium alloy anchor nails,and we compared the mechanical properties of the two anchor nails in normal and bone loss bone.2.The rabbit bone loss model was constructed by ovariectomy + dexamethasone injection.3 months later,Micro-CT was taken to measure the trabecular thickness(Tb Th),trabecular number(Tb N)and trabecular space(Tb Sp)in the specified area.Tb Sp)to verify successful modeling.3.After confirming the successful establishment of the bone loss rabbit model,the acute supraspinatus tendon tear model in rabbits with bone loss was established and divided into three groups: experimental group 2(graphene-coated new anchor nail repair group),experimental group 1(uncoated new anchor nail repair group)and control group(plain titanium alloy anchor nail repair group),respectively.The specimens were taken at 6W and 12 W postoperatively,and the histological evaluations were performed by HE staining,pink-solid green staining and Masson staining to observe the bone trabeculae,cartilage production and collagen arrangement.Biomechanical tests were performed to compare the maximum tensile load and stiffness between the different groups.micro-CT was performed to assess the local osteogenesis of the different groups.Results1.Characterization: Scanning electron microscopy observations revealed that the graphene-coated treated Ni Ti alloy material was visible with a clear lamellar-like film covering the surface and partially overlapped;while the lamellar-like structure could not be observed on the surface of the uncoated Ni Ti alloy material.The contact angle measurements showed that the graphene coating significantly improved the hydrophilicity of the Ni Ti alloy material.Confocal Raman spectroscopy results confirmed that we have successfully coated graphene on the surface of Ni Ti alloy material.2.Bioactivity: CCK-8 and ALP assay,the results showed that the graphene-coated Ni Ti material had the strongest ability to enhance osteoblast proliferation and osteogenic activity,which was significantly higher than the blank control group,titanium alloy group and uncoated group(P<0.05).3.Anchor nail mechanics: In the normal bone model,the maximum extraction force of the Ni Ti anchor nail at 45° was higher than that at 90°(P<0.05);in the bone loss model,regardless of the angle of extraction,the maximum extraction force of the Ni Ti anchor nail was higher than that of the titanium anchor nail at the same angle(P<0.05),and the maximum extraction force of both anchor nails at 45° was higher than that of the same anchor nail at 90°(P<0.05).The maximum pull-out force at 45° was higher than that at 90° for the same anchor(P<0.05).4.Results of bone loss rabbit model modeling: local Tb Th and Tb N in the bone loss modeling group were significantly lower than those in the unmodeled control group,while Tb Sp was the opposite(P<0.05),which verified the success of modeling.5.Histological results: 6W after surgery,HE staining showed that the bone tissue of experimental group 2 was normal in structure,with more bone marrow cells in the bone marrow cavity,more bone trabeculae,and thicker bone trabeculae than the other two groups;fuchsin-fixed green staining showed that experimental group 2 had a larger heterostaining area and more chondrocyte production than the other two groups;Masson staining showed that the collagen fibers in experimental group 2 were more dense and neatly arranged than the other two groups.Masson staining showed that the collagen fibers in experimental group 2 were more dense and neatly arranged than the other groups.At 12 W postoperatively,HE staining showed that the bone trabeculae in experimental group 2 were abundant,evenly distributed,and thicker overall,and the osteogenesis was better than that of the remaining groups,as well as better than that of the bone groups at 6W;the staining with red-solid green showed that the heterostaining area of experimental group 2 increased,and the chondrocyte production increased,which was significantly better than that of the remaining two groups and the groups at6 W.Masson staining showed that the collagen in experimental group 2 was oriented,and the amount of collagen production was increased and the structure was more complete,and the collagen production was better than that of the remaining groups and the groups at 6W.6.Biomechanical results: the maximum tensile strength and stiffness of each group showed an increasing trend with time.At 6W postoperatively,the stiffness of experimental group 2 was better than that of the remaining groups(P<0.05);at 12 W postoperatively,the maximum tensile strength and stiffness of experimental group 2were better than those of the remaining two groups(P<0.05).7.Imaging results: 6W postoperatively,Tb Th and Tb N of experimental group 2were significantly higher than those of experimental group 1 and control group(P<0.05),on the contrary,Tb Sp of control group was significantly higher than those of both experimental groups,and experimental group 1 was higher than experimental group 2(P<0.05);12W postoperatively,Tb Th and Tb N of both experimental groups were significantly higher than those of control group,of which experimental group 2was the highest(P<0.05).Tb Th and Tb N in both experimental groups were significantly higher than those in the control group,with the highest in experimental group 2(P<0.05),while Tb Sp in the control group was significantly higher than those in both experimental groups,and the lowest in experimental group 2(P<0.05),and with the prolongation of anchor implantation time,Tb Th and Tb N in experimental group 2showed an increasing trend,while Tb Sp gradually decreased.Conclusions1.Graphene-coated nickel-titanium memory alloy anchors have good hydrophilic,biocompatible,osteogenic and mechanical properties.2.Graphene-coated Ni Ti memory alloy anchors can effectively promote local osteogenesis,improve the microenvironment of tendon-bone healing,increase anchor pullout resistance,and enhance the quality of tendon-bone healing. |
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