| BackgroundWith the development of modern society,the incidence of bone defects is increasing gradually.Although bone tissue has a certain self-repair function,the bone defect will not be able to self-repair when the size of the bone defect is too large.The causes of large segmental bone defects are varied and the treatment methods are complicated.This is a challenge and a difficult problem for orthopedic surgeons.With the development of BTE,a variety of biomaterials have a good performance in the treatment of bone defects.Among them,mineralized collagen is a good biomimetic material.Mineralized collagen is a biomimetic material prepared by simulating the components in bone tissue and has good biocompatibility.However,it has a fast degradation rate and does not have a good drug-release ability,so it is not suitable as a drug carrier.The results show that alginate can improve the mineralization efficiency of mineralized collagen and optimize the degradation and drug release performance of mineralized collagen.In addition,drugs with good osteogenic activity also play an important role in the treatment of large segmental bone defects in BTE.In recent years,Chinese medicine has been extensively studied in BTE.Many traditional Chinese medicines contain a variety of bioactive ingredients,which can play a variety of biological functions.ICA,as one of the traditional Chinese medicines used in orthopedic diseases for many years,has a wide range of functions and is beneficial to the repair of bone defects.ICA can promote osteogenic differentiation,inhibit the expression of osteoclast-related genes,and promote angiogenesis.Therefore,developing an improved mineralized collagen scaffold with slow release of ICA may be an effective treatment for large segment bone defects.ObjectiveICA is a flavonoid glycoside isolated from Epimedium,a Chinese herb,and can promote osteogenesis and angiogenesis.Previous studies have shown that ICA can promote the formation of osteoblasts and angiogenesis in vitro by inducing the expression of BMP-2 and the production of NO.ICA also can reduce the formation of osteoclasts by increasing OPG/RANKL to reduce bone resorption and treat osteoporosis.However,due to the half-life of the drug and the systemic mode of drug delivery,ICA cannot be consistently maintained at appropriate concentrations in bone defects.The rapid development of BTE in recent years provides convenience for local drug treatment of bone defects.Therefore,in this study,AMC hydrogel with good biocompatibility was used as the ICA release system,and the Ti scaffold was combined to provide mechanical support for bone defects.The purpose of this study was to verify the compatibility and biological activity of AMCI/PTi scaffold in vitro and the therapeutic effect of this system on large segmental bone defects.Method1.At room temperature,the appropriate concentration of ICA was mixed with mineralized collagen solution and the alginate was added to obtain AMCI hydrogel.The AMCI/PTi scaffold was obtained by filling the micropores of the 3D-printed microporous titanium alloy scaffold with AMCI hydrogel.The micropore structure of the scaffold was observed by scanning electron microscope.The drug release and hydrogel degradation performance of AMCI/PTi scaffold were studied by drug release experiment and degradation experiment.2.The biocompatibility of AMCI/PTi scaffolds was verified by cell proliferation experiments and Calcein-AM/PI staining after co-culture with BMSCs in vitro.The effect of the AMCI/PTi scaffold on the osteogenic differentiation of BMSCs and the expression of osteogenic genes was verified by alkaline phosphatase staining and realtime quantitative polymerase chain reaction.The effect of AMCI/PTi scaffolds on angiogenesis of HUVECs was verified by tube formation assay,wound healing assay and RT-q PCR.3.The vivo studies were conducted to verify the effect of AMCI/PTi scaffold on bone regeneration.We established a large segmental bone defect model of the rabbit radius by radial osteotomy and the rabbits were divided it into three groups:microporous titanium scaffold(Ti),microporous titanium scaffold filled with AMC hydrogel(AMC/PTi)and microporous titanium scaffold filled with the AMCI hydrogel(AMCI/PTi).Each scaffold was implanted into the bone defect of the rabbit radius,and the bone regeneration effect of the composite system in vivo was studied by Micro-CT,X-ray,histological staining analysis and immunohistochemical analysis.Results1.In this study,the AMCI/PTi composite scaffold was successfully prepared by the above method.The scanning electron microscope results showed that the pore size of the 3D-printed microporous titanium alloy scaffold was about 600μm and the porosity was 70%.The results of drug release experiment showed that AMCI/PTi scaffold could release ICA continuously within 4 weeks,and could maintain appropriate drug concentration locally.The results of degradation experiment showed that the degradation cycle of AMC hydrogel in vivo was about 50 days,which was suited for the regeneration time of bone tissue.2.In the cell proliferation experiment and Calcein-AM/PI staining experiment,all groups showed good biocompatibility and AMCI/PTi scaffold could significantly promote the proliferation of BMSCs.In the osteogenic experiment,the AMC/PTi scaffold group and the AMCI/PTi scaffold group significantly promoted the osteogenic differentiation of BMSCs and up-regulated the expression of osteogenic-related genes,and the AMCI/PTi scaffold group had the best effect.The experiment of angiogenesis showed that the AMCI/PTi scaffold group could promote the migration of endothelial cells,promote angiogenesis and up-regulate the expression of angiogenesis-related genes.3.In vivo experiment,we successfully established the large segmental bone defect model of the rabbit radius.The X-ray,Micro-CT and histological staining results of each group at 12 weeks after surgery showed that compared with the titanium scaffold group,the AMCI/PTi scaffold group and the ANC/p Ti scaffold group promoted significantly bone regeneration at the bone defect site.And the effect of the AMCI/PTi scaffold group was better than that of the AMC/PTi scaffold group.In addition,immunohistochemical analysis showed that the number of positive cells in the AMCI/PTi scaffold group and the AMC/PTi scaffold group was significantly more than that in the Ti scaffold group.Moreover,compared with the AMC/PTi scaffold group,the number of positive cells in the AMCI/PTi scaffold group was higher,indicating that the AMCI/PTi scaffold group had a stronger role in promoting angiogenesis and bone repair.ConclusionIn this study,AMCI/PTi was filled into the microporous structure of a 3D-printed microporous titanium alloy scaffold to promote large segmental bone defect repair.The vitro experiments showed that the composite system not only had good biocompatibility,degradability and drug slow-release ability,but also significantly promoted angiogenesis and osteoblast differentiation.The vivo experiments show that the composite system can promote the bone repair effect of large segmental bone defects,and can enhance the growth of bone tissue and improve the local blood environment by promoting local blood vessel formation.Therefore,the above results indicated that AMCI/PTi scaffolds can significantly promote the repair of large segmental bone defects in rabbits and provide a new method for the clinical treatment of large segmental bone defects. |
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