Experimental Study On The Repair Of Bone Defects From Rabbits With A Novel Mineralized Collagen-guided Tissue Regeneration Membrane

Background:Bone defect is a common clinical disease in orthopedics,and the treatment of bone defect has been troubling clinicians.Guided bone tissue regeneration（GBR）is a safe,effective and reliable bone augmentation technology which is widely used in orthopedics and stomatology.As a mature GBR technology,collagen membrane has been widely used in clinic for its good biocompatibility,plasticity,tissue regeneration ability and suitable degradation rate.However,at present,the collagen membrane widely used in clinical is mainly imported,which is not only expensive but also monopolized by foreign enterprises.Objective:In this study,type I collagen was used to construct mineralized collagen membrane in vitro and a layer of type I collagen was compounded to form a new bionic mineralized collagen/collagen（Col/MC）bilayer membrane.The characterization of new bionic mineralized collagen artificial bilayer periosteum and Haio absorbable biofilm（marked as Z membrane,which is a domestic collagen membrane that has been commercialized）was studied by means of X-ray diffraction（XRD）,infrared spectroscopy（IR）and scanning electron microscopy（SEM）.The model of radial truncation defect in New Zealand rabbits was established to study the repairing effect of radial truncation defect in order to develop a domestic collagen membrane with low-cost and good effect of guiding bone regeneration.Method:The new mineralized collagen artificial bone membrane is made of the composite of mineralized collagen membrane formed by in vitro mineralization and collagen membrane.The characterization feature of new bionic biomimetic mineralized collagen artificial bilayer periosteum and Z membrane were compared by XRD,IR and SEM,including:（1）Elements analysis of mineralized collagen artificial bilayer periosteum and Z membrane was carried on by X-ray diffraction pattern.（2）Chemical composition of mineralized collagen artificial bilayer periosteum and Z membrane was detected of by infrared spectroscopy;（3）The micromorphology of mineralized collagen artificial bilayer periosteum and Z membrane was observed by scanning electron microscope.The effect of biomimetic mineralized collagen artificial bilayer periosteum and Z membrane on the repairing of bone truncation defect was studied by establishing the model of New Zealand radial truncation defect in rabbits,including:（1）Gross observation of New Zealand rabbits was carried on in 6^th and 12^th week after operation;（2）The radius was separated after sacrificed at the 12^th week after operation,and pathological observation was made via HE staining,Golden staining and Masson staining after the sections of bone specimens were prepared.（3）In the 6^th and 12^th week after operation,X-ray and CT scanning was used to evaluate the bone defects of New Zealand rabbits in each group.Results:Results of study on the characterization feature:（1）New bionic mineralized collagen artificial bilayer periosteum and Z membrane had characteristic peaks of collagen around 9°,21°and 31°,respectively,and the mineralized collagen artificial bilayer periosteum had characteristic diffraction peaks of hydroxyapatite around 25.9°,31.6°and 39.8°.（2）Both new bionic mineralized collagen artificial bilayer periosteum and Z membrane had collagen co-absorption peaks at 3312 cm^-1（Amide A）,1653 cm^-1（Amide I）,1550 cm^-1（Amide II）,and 1222 cm^-1（Amide III）,but the absorption peaks of Z membrane were relatively strong,while the above-mentioned absorption peaks of new bionic mineralized collagen artificial bilayer periosteum were weak,and the absorption peaks of hydroxyapatite appeared at 631 cm^-1 and 1045 cm^-1.（3）Under scanning electron microscope,the surface of new bionic mineralized collagen artificial bilayer periosteum was smooth,the cross section was multi-layered,and the thickness was moderate.The surface of Z membrane was smooth.Gross findings:（1）The results of gross examination in 6^th week after operation showed that the periosteum implanted in new bionic mineral collagen artificial bilayer periosteum treatment group and Z membrane treatment group were bound by some fibrous callus,and new bone formation occurred;The results of gross examination in 12^th week after operation showed that the periosteum implanted in new bionic mineral collagen artificial bilayer periosteum treatment group and Z membrane treatment group were bound by the fibrous callus,and there were different degrees of new bone formation,and the bone repair effect of the mineral collagen artificial bilayer periosteum treatment group was better than that of Z membrane treatment group.Pathological findings:（1）The results of HE staining showed that the bone defect area of mineral collagen artificial bilayer periosteum treatment group and Z membrane treatment group was mainly filled with new bone tissue,but the amount of new bone tissue in mineral collagen artificial bilayer periosteum treatment group was significantly higher than that in Z membrane treatment group,and bone marrow-like tissue formation could be seen in the center of new bone formation in mineral collagen artificial bilayer periosteum treatment group.（2）The results of Goldner three-color staining showed that the mineralized bone（green）was visible in the bone defect area of New Zealand rabbits in mineralized collagen artificial bilayer periosteum treatment group and Z membrane treatment group,and the mineralized bone（green）was significantly increased in mineralized collagen artificial bilayer periosteum treatment group compared with that of Z membrane treatment group.（3）The results of Masson staining showed that there was a large amount of immature（light blue）bone tissue in the bone defect area in the mineralized collagen artificial bilayer periosteum treatment group and Z membrane treatment group,and a small amount of mature（dark red）bone tissue could be found in mineralized collagen artificial bilayer periosteum treatment group.Imaging findings:（1）The results of X-ray examination showed that a large amount of new bone formation was observed in the bone defect area in both groups in the 6^th week after operation.Significant cortical bone formation was observed in mineralized collagen artificial bilayer periosteum treatment group,and some cortical bone formation was observed in Z membrane treatment group.The new bone regeneration and remodeling of bone defect area and reconstruction of bone marrow cavity in mineralized collagen artificial bilayer periosteum group were superior to those in Z membrane group.（2）The results of CT showed that the medullary cavity in mineralized collagen artificial bilayer periosteum treatment group was basically formed,and the medullary cavity in Z membrane treatment group was not formed in the 6^th week after operation.The bone defect medullary cavity was completely formed in mineralized collagen artificial bilayer periosteum treatment group and basically formed in Z membrane treatment group in the12^th week after operation.（3）The study found that at 6^th week after surgery,the bone volume in the bone defect area was significantly increased in the novel biomimetic mineralized collagen and Z-membrane group compared to the blank group（p<0.05）.Additionally,the bone volume of the bone defect area in the new bionic mineralized collagen double-layer artificial periosteal treatment group was significantly increased compared with that in the Z-membrane treatment group（p<0.05）.At 12^th week after surgery,the bone volume in the bone defect area was significantly increased in the new bionic mineralized collagen double-layer artificial periosteal treatment group compared to the Z-membrane treatment group and the blank group（p<0.01）.the bone volume of the bone defect area in the Z-membrane treatment group was significantly increased compared with that in the blank group（p<0.05）.（4）In the 6^th and 12^th week after operation,the bone mineral density in the bone defect area in new bionic mineralized collagen artificial bilayer periosteum treatment group and Z membrane treatment group tended to increase compared with the model control group,but the difference was not statistically significant（P>0.05）.Conclusion:The new bionic mineralized collagen artificial bilayer periosteum developed by our research group has a good effect on repairing the truncated bone defect of New Zealand rabbit radius,and its effect is superior to that of the imported Haio absorbable biofilm,which has a potential application prospect.