Study On The Repair Of Femur Defect In Rats By Collagen Nanometer Ultrathin Film PDPB

Objective and background:At present,with the development of research,tissue-engineered bone is considered as one of the most promising strategies forrepairing large segment bone defects.The scaffold materials withgoodbiocompatibility,degradability,plasticity and the three-dimensional pore structure similar to the natural bone tissue are the focus of the research,but no ideal scaffold materials have been applied in the clinic so far.Studies have shown that difficult cell adhesion and growth,low osteogenic efficiency,poor vascularization,rejection and limited homing mobilization of stem cells in tissue-engineered bone seeds are important factors leading to poor bone defect repair in tissue engineered bone.In the early stage,the research group prepared porcine vertebrae deproteinized bone as a natural bone derivative,which excluded immune factors and had natural pores.However,due to the removal of proteins,the physical strength of porcine vertebrae deproteinized bone decreased significantly.Meanwhile,the adhesion and proliferation of seed cells were affected,and the growth of microvessels and homing of stem cells in vivo were also affected.Therefore,this research adopts the autologous fibroblasts to extract collagen,the PDPB build nano ultrathin membrane material surface,in the treatment of bone defect in clinical build good mechanical strength,noimmunogenicity and high hydrophilicity,is conducive to cell growth and function expression,help new blood vessels form,is conducive to the growth of the stem cell homing adhesion efficient repair scaffold rovides systematic technical support.Methods:(1)SD rats were sacrificed for cervical dislocation,and bilateral femurs were taken.Bone marrow adherent method was used to isolate,purify and culture bone marrow mesenchymal stem cells.The differentiation of bone,lipid and chondrogenesis was identified after cell culture to the third generation.(2)the stent materials and the resuscitation of BMSCs were prepared ingroupsaccording to animal experiments.The previously prepared pure PDPB was ground into bone blocks of 4x2x2mm size.After soaking with 75%ethanol,the freeze-dryer dried the product.And divided into two groups,respectively,PDPB without treatment and PDPB formed by poly lysine electrostatic adsorption of composite collagen nanometer ultrathin film.They were implanted in the following groups.In group A,there was no scaffold in the simple bone defect group.Group B was pure PDPB group.Group C was PDPB of composite collagen nanometer ultrathin film.In group D,PDPB+tail vein BMSCs were injected with composite collagen nano-thin film.(3)48 SD rats were randomly divided into groups A,B,C and D4(group A was A group with bone defects only,and no scaffold material was used.Group B was pure PDPB group.Group C was PDPB of composite collagen nanometer ultrathin film Group D was PDPB+BMSCs injected by tail vein of composite collagen nanometer ultrathin film.Bone defects of 4×2×2mm size were ground out with a small electric drill in the middle of the femur of rats,and the corresponding implants wereimplanted according to the above groups and BMSCs were injected into the tail vein.At 2 weeks,4 weeks,6 weeks and 8 weeks after surgery,the effects of stent materials implanted in different groups in bone defect repair were continuously anddynamically observed through DR,gross specimen observation and HE staining of hard tissue sections.Results:(1)bone marrow mesenchymal stem cells(BMSCs)of SD rats were isolated,purified and cultured by the whole bone marrow adherence method,and high-purity BMSCs could be obtained.The third-generation BMSCs could be differentiated into bone cells,adipocytes and chondrocytes by induction.(2)observation of the general situation after stent implantation:wake up within 2 minutes on average after the surgery,free movement in the feeding cage after waking up,normal diet and drinking water.No redness,bleeding,oozing of the incision,the healing is good.X-ray film was taken for the first time on the fourth day after the operation,and it was found that there were 4 femoral fractures in the rats,and the remaining bone defects were well formed.The success rate is 75%.(3)postoperative X-ray observation:bone defect was still visible under X-ray in group A 2 weeks after surgery,and the defect was gradually healed from 4 weeks to 6-8 weeks.In group B,C and D,the external contour of the graft was clear on X-ray 2 weeks after the surgery,and 4-8 the external contour of the graft was graduallyweakened,the image became blurred,the boundary between the bone block and the defect disappeared,and the bone was fused with the original femoral defect.The transverse comparison of each group showed that group D was more obviously absorbed and fused with the defect,and the healing was better,as compared with group C and group B.(4)gross specimen observation:group A had A large amount of fibrous callus in the defect 2 weeks after surgery,bone repair in 4 weeks,and bone healing in 6-8 weeks.B,C,D group 2 weeks after transplantation of bone by fibrous and soft packages,BingGu block,transplantation of bone and bone defect is fixed firmly,bone block surface pore visible,4 to 8 weeks B,C,D groups of bone defect more dense fibrous tissue package,8 weeks is the bone graft block has been absorbed,smaller volume and fuses gradually with the defect,C,D group is better than that of group B of healing.(5)HE staining results of histological sections showed that group A(group with simple bone defect)could heal spontaneously at 2,4,6 and 8 weeks.A large number of bone marrow cells grew into the pores of the implant materials in group B,C and D,and a large number of blood vessels grew into group D compared with group C and B in the early stage.At 2 weeks,the implant material clearly visible was graduallydegraded with the formation of new bone,and the bone end of the implant wasgradually fused with the bone end of the bone defect.At week 8,group D had the best healing effect.Conclusion:(1)The whole bone marrow adherent method is an ideal method for isolation and amplification of bone marrow mesenchymal stem cells in vitroBy this method,a large number of bone marrow mesenchymal stem cells can be obtained in a short time,and the cells have good activity,stability and high differentiation potential.It provides a basis for further research on the application of bone marrow mesenchymal stem cells in tissue engineering and the treatment of clinical injury repair(2)Through this experiment,an animal model of bone defect with inseparable femur in SD rats was established.It was verified that the 4×2×2mm defect in the middle part of the femur of rats could heal by itself.(3)Compared with the simple PDPB,the composite collagen nano-ultrathin film PDPB has better histocompatibility,which can rapidly form new bone while the scaffold material is degraded,and its degradation and absorption rate are appropriate.The tissue-engineered bone constructed by composite collagen nanometer ultrathin film PDPB combined with BMSC can effectively promote bone defect repair.(4)At the early stage of BMSCs injected by caudal vein,a large number of blood vessels grew into BMSCs,suggesting that collagen nanometer ultrathin film can induce bone marrow mesenchymal stem cells to nest and repair bone tissue damage.