A Study On The Relationship Between Angiogenesis And Osteogenesis During The Treatment Of Gusuibu Total Flavonoids In Induced Membrane Technique

BackgroundThe treatment of critical bone defects caused by trauma,bone tumors,limb deformities and bone infections is a common problem in clinical practice.The incidence of bone deformity and other diseases has a gradual increasing trend,which not only seriously affects the quality of life of patients,but also causes a serious economic and social burden.Therefore,it is particularly important to study the repair and reconstruction of bone defects.Masquelet technique or induction membrane technique is one of the newer surgical methods for the treatment of bone defects in the past ten years.How to improve the osteogenesis efficiency and clinical efficacy of induction membrane technology are the key issues that need to be solved at present.Traditional Chinese medicine believes that the kidney has the relationship of bone.Previous research has shown that use the kidney tonoc can promote fracture repair,bone regeneration and reconstruction.Gusuibu is a traditional Chinese medicine for tonifying the kidney and bone,and total flavonoids of Gusuibu are the main active ingredients,It plays an important role,but it has not been reported in the process of inducing membrane technique in the treatment of bone defects.The coupling mechanism between blood vessels and osteogenesis is not very clear.Our research group has long been committed to the repair of bone defects with traditional Chinese medicine and research on the mechanism of bone reconstruction.A series of previous studies have shown that the total flavonoids of Gusuibu fragmentation play a role in the process of distraction osteogenesis and fracture.This project is part of the National Natural Science Foundation of China’ s youth project"Exploring the Intervening Mechanism of Tonifying the Kidney on Masquelet Technology Induced Membrane Formation and Bone Tissue Reconstruction Based on the Ternary Regulation Theory(Project No.:81603140)" with a view to establishing rat Induced membrane model,the drug intervention of osteoporosis total flavonoids to explore its role in the treatment process of induced membrane technology,as well as clinical case observation,provide scientific basis for clinical application.Part Ⅰ Clinical observation and research on the treatment of large bone defects with induced membrane technologyObject iveThrough the review of clinical cases,evaluate the clinical application effect of induced membrane technique and optimize the clinical treatment plan.MethodsSearch the follow-up of patients diagnosed with osteomyelitis,bone infection,and bone nonunion from September 2016 to December 2019 in the medical record system and imaging system of the First Affiliated Hospital of Guangzhou University of Chinese Medicine,totaling follow-up of outpatients 15 patients.Analyze the general condition of the patients and observe relevant clinical indicators such as bone defect end healing rate,bone healing time,length of hospital stay,weight-bearing time,bone cement placement time,surgical complications,number of operations,and length of bone defect after final debridement,Fixation,bone grafting,etc.ResultsThe 15 patients were infected with Staphylococcus aureus.After treatment,the clinical symptoms were better than before the operation.The bone cement was left for 5.4 ± 1.8 weeks.Most patients obtained bone healing within 1 year after treatment.The average healing time was 8.3 ± 2.4 months.The patients who underwent intramedullary nailing in the second stage underwent ground-bearing time earlier than other fixation methods.Most patients were fixed with external fixators after the first stage of surgery,and some patients were in the second stage Replaced with internal fixation,patients with open fractures were treated with internal fixation after debridement.Bone grafting method:All patients used autologous iliac bone grafting.If the defect was large,allogeneic bones were mixed.Two patients used PRP+fragmentary iliac bone grafting.One patient had a steel plate rupture 6 months after the second operation,with one exception being loose fixation,and all were replaced with intramedullary nails in subsequent operationsConclusionMaster the surgery indications,the appropriate surgical plan,and the induced membrane technique can achieve good results in the treatment of bone defects.Key words:Induced membrane technique;clinical observation;bone defect;infection.Part Ⅱ Establishment and evaluation of Masque let model of large femur bone defect in ratsObject iveTo establish an animal model of induced membrane technique for bone defect,which was easy to implement,and laid the experimental foundation for the development of subsequent experiments and drug intervention for induced membrane technique for bone defect.MethodsTwenty male SD rats underwent osteotomy of 4 mm on the right femur,all of which were fixed with steel plates.They were placed in PMMA bone cement for 4 weeks and left for two weeks,followed by two-stage surgery,coccygeal bone grafting,and gross observation,imaging observation and tissue morphology,to observe the healing and recovery of the osteotomy site.Results:In this experiment,a total of 20 rats were selected for surgery.During the operation,one of the rats died due to injury to the femoral artery.One of the rats continued to have swollen limbs and limited knee joint movements after surgery.All rats were generally in good condition after surgery,with no special conditions such as eating and defecation.They could walk normally on the 3rd day after surgery,move freely in the cage,and the incision healed well without obvious signs of infection.Internal fixation was stable and effective after operation,and had no significant effect on the functional activities of rats.The induced membrane tissue was clearly delaminated 4 weeks after the primary surgery.The inner tissue was rich in neovascular tissue,and the outer layer was mainly fibroblasts and collagen tissues.A large number of inflammatory cells infiltrated in the induced membrane tissue.Induction membrane was near the cement margin.X-ray and Micro-CT examination at 8 weeks after the second operation showed the growth of the bone graft area.Conclusion:The experimentally established Masquelet treatment model of rat femur large segmental bone defect is reliable and practical,and can be repeated.Key words:SD rat;induced membrane technique;animal model;Masquelet technique.Part Ⅲ Effects of Gusuibu Total Flavonoids on Induced Membrane Technique on Vascular and Osteogenic Quality in Bone Defective AreasObject iveTo investigate the effect of Gusuibu total flavonoids on the vascular and osteogenic quality of bone defect area in induced membrane technique.MethodsThirty-six male SD rats were divided into 3 groups(blank group,inducted membrane group,induced membrane+Chinese medicine gavage group),12rats in each group,right femur osteotomy 4 mm,all were fixed with steel plates,and PMMA cement was placed for 4 weeks,and the induced membrane group and the induced membrane+gavage group were use the coccygeal bone grafting to fill the defect area,blank group is untreated for bone grafting.Specimens were collected at 8 weeks after operation,and were observed by gross observation,imaging observation and histomorphology The osteotomy site healing and angiogenesis were observed,and the expression of related proteins was detected by immunohistochemistry and western-blotting.ResultsX-ray results and histomorphology showed that the induced membrane group and the induced membrane+traditional Chinese medicine group had a good effect on the repair of bone defects.Micro-CT showed that the induced membrane+traditional Chinese medicine gavage group had a promoting effect on bone mineralization.Immunohistochemistry It is shown that the total flavones of bone fragment supplement can promote the expression of CD31 and BMP-2 proteins.Immunotracery method has shown that the Gusuibu total flavones supplement,increase the expression of CD31,VEGF and PDGF,also can promote the formation of blood vessels.In the membrane+traditional Chinese medicine group.Bone mineralization,bone plasticity,and histomorphology were better than the other two groupsConclusionGusuibu total flavonoids has a good promotion effect on osteogenesis and blood vessel formation in bone defect areas in the induced membrane technique.