Based On Three Ternary Regulation To Explore The Effect Of Total Flavones Of Rhizoma Drynariae On Masquelet Technique’s Induced Membrane Formation

ObjectiveTo evaluate the clinical efficacy of Masquelet technique in the treatment of segmental bone defects,and explore its advantages and disadvantages via reviewing clinical cases retrospectively.To establish a proven experimental model for studying Masquelet induced membrane of the rat’s femur,which lays the foundation for the later study on the effect of traditional Chinese medicine on induced membrane formation.To explore the effect of total flavones of Rhizoma Drynariae on induced membrane formation by analyzing the expression of key factors of the three ternary regulation and VEGF,HMGB1 in the induced membranes.MethodsMasquelet technique case review:A total of 9 cases treated with Masquelet technique between January 2014 and July 2018 in the department of trauma orthopedic of the First Affiliated Hospital of Guangzhou University of Chinese Medicine were retrospectively analyzed.To evaluate the clinical efficacy of Masquelet technique in the treatment of segmental bone defects by observing the length of bone defects,bone healing,graft and the number of operations,etc.The establishment of the Masquelet induced membrane experimental model:A total of 24 male Sprague-Dawley rats（8-10weeks old and weighing approximately 260–280 g）were randomised to observation group and control group.After receiving a 4 mm segment defect of the femur,it was stabilised by a stain-less steel plate osteosynthesis.The space was filled with PMMA cement spacers in the observation group,while the control group not.Routine nursing was given after surgery.Four weeks after surgery,the induced membranes wereharvestedfortheanalysisofgeneralobservation,x-rayand characterization of histology.The experimental study on the effect of total flavones of Rhizoma Drynariae on induced membrane formation based on three ternary regulation:After stratified by weight,a total of 60 male Sprague-Dawley rats were randomised to control group,model group,low dose traditional Chinese medicine group,middle dose traditional Chinese medicine group and high dose traditional Chinese medicine group,twelve in each group.After the successful establishment of the model by the method in the second chapter,beginning with the second day after surgery with different concentrations of naringin gavage to each traditional Chinese medicine group;model group and control group were given equal volume of pure water until 28 days postoperative.28 days after surgery,the induced membranes were harvested for relevant analysis.It was stained with haematoxylin and eosin（HE）for the analysis of histology;in addition,the X-ray of surgical limb was performed to observe the alignment of the osteotomy site,as well as the position of plate and screw.Immunohistochemistry and western blot were used to analyze the expression of proteins in induced membranes of each group.Q-PCR was adopted to analyze the mRNA expression of key factors of the three ternary regulation and VEGF,HMGB1.ResultsMasquelet technique case review:All 9 patients had bony union at the last followed up.The average healing time was 6.3±1.2 months,the average numbers of surgery was 2.9±1.4.One case had pain in the graft harvested area,one case suffered ankle stiffness,one case experienced a loosening of external fixator and intramedullary cavity infection recurrence,one case underwent knee stiffness and proximal intramedullary cavity infection recurrence.The establishment of the Masquelet induced membrane experimental model:In both of the two groups,no rats had postsurgery symptoms of dysporia and eating disorder;rats walked normally 2 days postsurgery;no wound problem and infection were founded.In the control group,the plate of two rats experienced loosening;one rat’s surgical limb had sustained swelling and knee movements restrictions in the observation group,however,the internal fixation and osteotomy site were stable without loosening.In the observation group,the induced membrane was richly cellular,and the formation of large vascular network as well as fibres oriented parallel to the PMMA could easily be identified.Histology analysis showed significantly decreased amount of vascular in the control group.The experimental study on the effect of total flavones of Rhizoma Drynariae on induced membrane formation based on three ternary regulation:Four weeks after the model establishment,X-ray of each group showed that the position of the plate,screw and PMMA were good,the internal fixation and alignment of osteotomy site were stable.Overall,q-PCR analysis showed that compared with the control group and model group,traditional Chinese medicine group up-regulate the gene expression of key factors of the three ternary regulation（PDGF-BB、CD31、Endomucin）and VEGF,HMGB1.However,some factors were significantly influenced by the concentration of traditional Chinese medicine.Western blot analysis suggested that traditional Chinese medicine group markedly up-regulate the protien expression of key factors of the three ternary regulation（PDGF-BB、CD31、Endomucin）and VEGF,HMGB1 when comparing with the control group and model group,only individual factors was influenced by the concentrationoftraditionalChinesemedicine.Theresultof immunohistochemistry showed that the protien expression of CD31 in the induced membrane was strongly positive,while the expression of Endomucin was weakly positive.ConclusionMasquelet technique case review:Masquelet technique for the treatment of segmental bone defects leaded to satisfactory clinical results.However,the delayed graft vascularization and cortical causes prolonged treatment period,and complications such as the recurrence of infection,especially in patients with intramedullary nail fixation initially.The establishment of the Masquelet induced membrane experimental model:The study demonstrates an exact,simple,effective and reproducible method for establishing the induced membrane of the rat femur model.The experimental study on the effect of total flavones of Rhizoma Drynariae on induced membrane formation based on three ternary regulation:Naringin can up-regulate the proteins and gene expression of key factors of the three ternary regulation（PDGF-BB、CD31、Endomucin）and VEGF,HMGB1.However,the scientific hypothesis of three ternary regulatory mechanism does not exist in the Ma-squelet technique’s induced membrane formation period due to the absence of CD31^hiEmcn^hivessels in the induced membrane,relevant molecular mechanisms need to be further explored.