A Study To Explore The Effect Of Total Flavones Of Rhizoma Drynariae On Masquelet Technique In The Treatment Of Critical-Size Bone Defect Base On The Notch Signaling Pathway

Objective1.To evaluate the therapeutic effect of Masquelet technique in the application of critical-size bone defect via reviewing clinical cases retrospectively,which provided the basis for further optimization of treatment plan.2.To establish an animal model of Masquelet technique for critical-size bone defect repair,which lay an experimental foundation for the following study on drug intervention and evaluation of its effect on Masquelet technique for critical size bone defect.3.To investigate the effects of TFRD on Masquelet technique for repairing criticalbone defect of femur in rats based on the expression of Notch signaling pathway.Methods1.Retrospective study of Masquelet technique in the treatment of critical-size bone defectsA total of 20 cases with critical-bone defects treated by Masquelet technique from January 2014 to December 2020 in the First Affiliated Hospital of Guangzhou University of Chinese Medicine were retrospectively analyzed.The clinical data of the case series were collected and analyzed,and the observation indicators included: bone defect length,operation time,fixation method,bone grafting method,second-stage operation timing,operation times,surgical complications,bone defect healing time,weight-bearing time。2.Establishment and evaluation of Masquelet technique model for repairing criticalsize bone defects of SD ratsTwenty SPF male SD rats were selected to undergo surgical osteotomy on the right femur to create a 4mm bone defect,which was fixed by steel plate and implanted with PMMA(Polymethyl methacrylate)bone cement spacer in the defect area.After 4 weeks,10 rats were randomly selected for sampling,and the gross appearance,imaging and histological characteristics of the induced membrane were observed.The remaining 10 rats underwent secondary surgery.After the PMMA spacer was removed in the secondly stage,autologous coccyx was used for autograft,and the induced membrane was carefully sutured.12 weeks after the secondary operation,specimens were collected.The healing and repair situation of bone defect were evaluated by gross observation,imaging observation and histomorphology.3.Experimental study to explore the effect of total flavones of Rhizoma Drynariae on the repair of critical-size bone defects by Masquelet technique base on the Notch signaling pathwayA total of 96 SPF male Sprague-Dawley(SD)rats were randomly divided into 4groups according to body weight: Traditional Chinese Medicine group(TFRD),control group(CON),Traditional Chinese Medicine combined with inhibitor group(TFRD+DAPT)and inhibitor group(DAPT),with 24 rats in each group.After the successful establishment of the model useing the modeling method described in Part II,drugs intervention were initiated on the second day postoperatively.The rats in the Traditional Chinese Medicine group(TFRD)and Traditional Chinese Medicine combined with inhibitor group(TFRD+DAPT)were given total flavones of Rhizoma Drynariae by intragastric administration,while the control group(CON)and the inhibitor group(DAPT)group was given the same amount of pure water by intragastric administration.Intragastric administration began on the first day after modeling,once a day until sampling.Rats in the Traditional Chinese Medicine combined with inhibitor group(TFRD+DAPT)and the inhibitor group(DAPT were subcutaneously injected useing the Notch signal pathway inhibitor DAPT.While the the Traditional Chinese Medicine group(TFRD)and the control group(CON)were subcu-taneously injected using same dose Of dimethyl sulfoxide.Subcutaneous injection was started on the first day after modeling and continued for 5 days.4 weeks after the first surgery,12 rats were randomly selected from each group to obtain induced membrane tissue for detection.The position of plate,screw,PMMA spacer and osteotomy site were observed by X-ray.HE staining was used to observe the gross morphological characteristics of induced membrane tissues in each group.The expressions of Notch signaling pathway related proteins BMP-2,VEGFA,Notch1 and Hes-1 in the induced membrane tissues were detected by immunehistochemistry and Western-blot.The m RNA expression of the above factors in the induced membrane tissues of each group was analyzed by Q-PCR.The remaining 48 rats underwent second stage surgery.After the PMMA spacer was removed in the second stage operation,autologous coccyx was used for bone grafting in the defect.The healing and repair of bone defects were observed by gross observation,imageology and histomorphology 12 weeks after the second stage.The expression of Notch signaling pathway related factors mentioned above were detected by immunohistochemistry and Western-blot.Results1.Retrospective clinical cases review.Of the 20 patients,6 cases had bone defects due to open fractures and the other 14 cases were cause by osteomyelitis.Of the 14 cases with osteomyelitis,6 were infected with Staphylococcus aureus,3 with pseudomonas aeruginosa,1 with Staphylococcus ludeng,1with Enterobacter aeriformis,and the other 3 were negative in culture.After treatment with Masquelet technique,the clinical symptoms of all patients were improved.The average length of bone defect was 5.8±2.2cm,and the mean time of cement spacer retained for5.4±1.4 weeks.Sixteen patients achieved bone healing within 1 year after treatment,with a mean time of 6.8±2.2 months.The reinfection rate of osteomyelitis patients with external fixation in the first stage was lower than that of other fixation.The weight bearing time of patients with intramedullary nail fixation in the second stage was earlier than that of other fixation.Four patients with bone defects of more than 8cm were treated with autogenous bone combined with allogeneic bone,and 2 patients were treated with autogenous iliac bone combined with platelet-rich plasma(PRP).Plate brokened in 1 patient 6 months after the second operation,another patient had loosening of the external fixation,these two cases were replaced by intramedullary nail in subsequent operations.Reinfection occurred in 4patients,and infection could be controlled by debridement and cement spacer implantation again.According to Pely bone and function rating,4 cases of limb function were excellent,9 cases were good,6 cases were fair and 1 case was poor.2.Establishment and evaluation of experimental model for repairing critical-size bone defects of SD rats.A total of 20 rats underwent operation in this experiment,and all rats could eat and defecate normally after surgery,and could walk normally on the third day after surgery.Surgical incision infection occurred in 1 rat.The other rat had sustained limb swelling and limited knee movement at the operative side,and X-ray examination confirmed screw loosening and PMMA cement displacement.The 2 rats were replenished after withdrawal.All the other animal models had good alignment of osteotomy site,and the plates and screws were in place without loosening.PMMA spacer has no shift and reversal.A constant4 mm gap was maintained at the osteotomy site.Induced membranes formed around the cement spacer 4 weeks after primary surgery.The induced membrane tissue was stratified,with inner tissue was rich in neovascularization,while the outer layer was mainly fibroblast and collagen tissue.A large number of inflammatory cells were infiltrated in the induced membrane,gathered in the membrane near the cement spacer margin.X-ray examination showed new bone formation at the bone defect site 12 weeks after the second stage protocol.3.The effect of total flavones of Rhizoma Drynariae on Masquelet technique base on the Notch signaling pathway.4.weeks after modeling surgery,X ray of each group confirmed that the plate,screw and PMMA spacer were in good position and fixed firmly,and the osteotomy site was in good alignment.Western-blot showed that compared with other groups,the protein expressions of Notch signaling pathway related factors BMP-2,VEGFA,Notch1 and Hes-1in TFRD group were significantly increased(P<0.05).The results of q-PCR showed that compared with other groups,the gene expression of the above mentioned factors were increased in TFRD group(P<0.05),while the gene expression was down-regulated in the inhibitor group(P<0.05).Immunohistochemistry showed that BMP-2,VEGFA,Notch1 and Hes-1 were strongly expressed in the induced membrane tissue of TFRD group.Eight weeks after the second stage,X-ray examination and histomorphology showed that the TFRD group had a good repair effect on bone defects,and micro-CT showed that the TFRD group had a promoting effect on bone mineralization.Immunohistochemistry showed that TFRD could promote the expression of BMP-2,VEGFA,Notch1,Hes-1.The TFRD group was superior to other groups in vascular formation,bone mineralization,bone plasticity and histomorphology.Conclusion1.Masquelet technique is a reliable surgical technique for the treatment of critical-size bone defect.Better clinical result could be expected by reasonable operation indication,deliberate pre-operation plan and carefully manipulation.2.In this study,an experimental model for repairing critical-size bone defects useing Masquelet technique of SD rats was established with high success rate,stability,feasibility and repeatability.3.TFRD may promote the formation of induced membrane and new bone tissue in Masquelet technique by up-regulating the Notch signaling pathway,thus promoting the repair of critical-size bone defects in a certain extent.