Calcium Sulfate-induced Membrane Of The Masquelet Technique For Bone Repairing In A Large Femoral Defect Rat Model

Clinical management of large segmental bone defects resulting from severe trauma,surgical excision of tumors and debridement after posttraumatic chronic osteomyelitis or infected non-union poses a major challenge.When the wound is severe,followed by extensive soft tissue injury or infection,it is even more difficult to repair the bone defect and to complete the limb reconstruction.Segmental bone defects can be managed by autologous bone graft(ABG)as well as by adequate soft tissue envelope,vascularized free fibular graft,distraction osteogenesis with Ilizarov external fixation technology and Masquelet technique.ABG is mainly applied with bone defect<5cm.Vascularized free fibular graft,Ilizarov technology and Masquelet induced membrane technology are widely used in the treatment of defects exceeding 5 cm.Vascularized free fibular bone graft needs specialized microvascular anastomosis,which is prone to stress fracture,complications at the donor site,incomplete bone healing and risk of resorption.Ilizarov istraction osteogenesis technology also has such disadvantages as pin tract infection,joint stiffness,neurologicalinjury,irregular lines and so on.Masquelet technology combined with the autogenous bone graft and induced membrane to repair the bone defects.But it also has some limitations:? It often requires multiple surgeries,and the interval operative time is uncertain;? The polymethylmethacrylate(PMMA)is often used in Masquelet technology,but it does not degraded,can't be absorbed,unable to repair the bone defects,and need for surgical removal of the non-biodegradable cement before surgical reconstruction of the bone defect.Therefore,extensive research pursuits are targeting alternative,biodegradable materials to replace PMMA,including CaSO4(Calcium sulfate,CS).We hypothesized that PMMA might be replaced by CS since they can both accomplish the important role of inducing membrane and the major disadvantage of PMMA might be overcome and made up for by the major benefit of CS.The degradability of CS may allow one-stage reconstruction of critical-sized bone defect,sparing the necessity of surgical removal of the spacer.Based on the above analysis,our study mainly included the following three sections.The first section was to stimulate the Masquelet technology and to build the large segmental femoral defect in a rat model.Compare the general structural characteristics of the induced membranes and the osteogenesis between the CS group and PMMA group.The second section was to compare the cell components and the level of cytokines between the CS group and PMMA group,reveal the initial mechanism of induced membrane to promote bone formation.The third section focused on observing whether the medical CS can repair the bone defects one-stage.Verify the fact that PMMA might be replaced by CS in Masquelet technology.Part One:Stimulate the Masquelet technology and build the large femoral defect in a rat modelObjectives:The section was to stimulate the Masquelet technology and to build the large bone defect in a rat model.Compare the general structural characteristics of the induced membranes and the osteogenesis between the CS group and PMMA group.Methods:This study was designed as a randomized controlled trial.SD rats(n=60)were randomized into CS,PMMA and control groups equally.CS or PMMA was filled into the bone defects in models of a large femoral bone defect(bone defect is 10mm).Digital radiographs were taken immediately,2,4,6 and 8weeks post-operation for observing the degradation of CS and the stability of plate fixation.So we can confirm the feasibility and reproducibility of the rat model.We also compared the general structure of induced membrane and the histological changes at the broken end of bone defects.Results:The rat models have stability,feasibility and high repeatability.CS and PMMA both can form the induced membranes.CS can also be completely degraded,but PMMA does not occur.Qualitative and quantitative analyses revealed that the new bone tissue obviously occurred at the broken ends of the bone defects in the experimental group than that in control group,but there is no bony connection formed.Conclusions:The rat model has good stability,high success rate and reproducibility.Compared with PMMA,we found that a membrane-like fibrotic capsule formed around the bone defect in CS.So we concluded that CS can also induce the formation of a membrane structure which is similar to that of PMMA-induced membranes.Moreover,CS promoted endochondral ossification at the broken ends of the bone defect at different time points better than PMMA did.Part Two:Investigate the characteristics and the level of cytokines in CS-induced membrane in comparison with the PMMA-induced membraneObjectives:Compare the cell components and the level of cytokines in induced membranes between the CS and PMMA group,reveal the initial mechanism of induced membrane to promote bone formation.Methods:Sprague Dawley rats(n=40)were randomized into CS and PMMA groups equally.CS or PMMA was filled into the bone defects in models of a femoral defect(bone defect is 10mm).Cellular components,histological changes,cytokines expression of IL-6,VEGF,vWF,BMP-2,TGF-01,ALP,SDF-la,CXCR-4 in the CS-induced membranes and PMMA-induced membranes were compared at 2,4,6 and 8 weeks,respectively.Results:The structural characteristics of CS-induced membrane were similar to those of PMMA-induced membrane.However,CS-induced membrane was thicker than that induced by PMMA.Endochondral ossification took place in the CS-induced membrane at 8th week,but not in the PMMA-induced membrane.Levels of VEGF,vWF,BMP-2,TGF-?1 and ALP in CS-induced membrane were insignificantly higher than those in PMMA-induced membrane at different time points.The expression of IL-6 was significantly higher in PMMA-induced membranes than in CS-induced membranes at 2nd week,but insignificantly higher at 4,6 and 8th weeks.In addition,osteogenic and neovascular activities of both CS-and PMMA-induced membranes increased gradually with time and peaked at 6th weeks and declined gradually after that.The expression of levels of SDF-la and CXCR4 showed decreased gradually with time.Conclusions:Compared with PMMA-induced membrane,CS-induced membrane has similar structural characteristics,but a better capacity of generating VEGF,vWF,BMP-2,TGF-?1 and ALP,and with mild inflammatory reaction.CS-and PMMA-induced membranes achieve the highest levels of osteogenic and neovascular activities at 6th weeks.Considering its degradable feature,CS may have the potential to replace PMMA as a novel spacer in Masquelet technique.Moreover,we also preliminarily found that the levels of SDF-1? and CXCR4 in the induced membrane might through the axis of to promote the bone formation.Part Three:One-stage to repair a critical-sized femoral defect in a rat modelObjectives:Observing whether the CS can repair the critical-sized bone defect one-stage.Verify the fact that PMMA might be replaced by CS in Masquelet technology.Methods:This study was also designed as a randomized controlled trial.SD rats(n=30)were randomized into CS,PMMA and control groups equally.CS or PMMA was filled into the bone defects in models of a femoral critical-sized defect(bone defect is 5mm).Digital radiographs were taken immediately,4,8,12,16,and 20 weeks post-operation for observing the degradation of CS and the stability of plate fixation.Micro-CT scan also be taken to determine the change of bone mass at 20th weeks.Confirm that whether the new bone formation occurred in the bone defects and form a bony connection.Results:The new bone tissues occurred in the bone defect and form a bony collection in CS group at 20th weeks.However,the bone defects also appeared in the PMMA and control groups at 20th weeks.Micro-ct revealed that the new bone tissue obviously occurred at the broken ends of the bone defects in the CS group and PMMA groups than that in control group.CS group can form a bony connection at 20th week.PMMA and control groups didn't show the phenomenon.Conclusions:CS can form the new bone tissues and can not be removal,which may allow one-stage reconstruction of small segmental bone defects.