| PART1THE PREPARATION OFNANO-HYDROXYAPATITE/POLYAMID66/GLASSFIBRE COMPOSITE AND IN VITRO STUDY ONEFFECTS OF THE COMPOSITE ON MC3T3-E1OSTEOBLAST BEHAVIORObjectiveTo evaluate the effect of a new nano-hydroxyapatite/polyamide66/glass fiber (n-HA/PA66/GF) biomaterials used for new bioactive bone screwon MC3T3-E1osteoblast behavior, hoping to provide experiment basis forthe following study.MethodsThe nano-hydroxyapatite/polyamid66/glass fiber (n-HA/PA66/GF) discs were prepared by co-precipitation method and their extracts wereprepared according to the ISO10993. The MC3T3-E1osteoblasts wereco-cultured with n-HA/PA66/GF disc or its extracts, respectively. Thedirect-contact test, scanning electron microscope observation and CCK-8assay were performed to assess the effects of n-HA/PA66/GF biomaterial oncell adhension, growth and proliferation. The the effects of n-HA/PA66/GFbiomaterial on synthesis of total protein was tested by BCA method. Elisaassay was performed to test the osteocalcin secretion of MC3T3-E1cells.The cell cycle and cell apoptosis were analysed by flow cytometryand Annexin V-FITC/PI double-label flow cytometry, respectively.Transwell migration test was performed to test effects of n-HA/PA66/GFbiomaterial on cell migration. Double labeling cells indirectimmunofluorescence staining was used to observe the cytoskeleton anddistribution of actin filaments using a laser scanning confocal microscope.ResultsThe MC3T3-E1cells grew well around n-HA/PA66/GF in the directcontact experiments, which meant that the biomaterial had no obviouscytotoxicity. Cells had a normal long spindle shapes witout shrinking orvacuolation. CCK-8assay revealed that the MC3T3-E1cells proliferatedgradually with the increasing of culture time and cells cultured with then-HA/PA66/GF sample exhibited the greatest proliferation among the threegroups, showing a significant difference from the negative and positivecontrol groups after5days of co-culture (p<0.05).The total protein contentswere （2.21±0.48）mg/ml,（1.68±0.25）mg/ml in the experiment group andthe control group. The biomaterial could promote synthesis of total protein（p<0.05）.Flow cytometry test results confirmed n-HA/PA66/GF couldmake more cells enter S period without obvious influence on cell apoptosis rate. The cell apoptosis rates of the two groups are7.93%±2.37%and8.31%±2.84%, respectively. The n-HA/PA66/GF biomaterial could alsopromote secretion of osteocalcin after culturing for10and14day（sp<0.05）.Scanning electron microscopy observation found that the MC3T3-E1cellswith predominantly long spindle shapes connected with each other andbecame anchored to the surface of the scaffold via their pseudopodia. Sevendays after culturing, cells had proliferated dramatically and formed stratifiedcell layers, accompanied by filamentous fibres on the surface of the scaffold.However, no obvious influence on cytoskeleton, actin filament distributionand transmembrane cell number was observed between the experimentgroup and control group (p>0.05). The n-HA/PA66/GF composite had goodcytobiocompatibility and is suitable for cell adhension and proliferation.ConclusionThe n-HA/PA66/GF biomaterial has excellent cytocompatibility, whichhas positive regulatory effects on the cell growth, proliferation, secretion,adhesion, cycle and migration.PART2IN VIVO STUDY ON A NEWNANO-HYDROXYAPATITE/POLYAMID66/GLASSFIBRE(N-HA/PA66/GF) BIOACTIVE SCREW FOR FIXINGINTERCONDYLAR FRACTURE OF FEMUR IN DOGObjectiveTo investigate the in vivo biocompatibility, internal fixation properties and osteogenesis of the nano-hydroxyapatite/polyamid66/glass fiber(n-HA/PA66/GF) bioactive screw, hoping to provide experiment basis forclinical applicationMethodsThe n-HA/PA66/GF bioactive screws were prepared by using injectionmolding method. The coating surface and cross sections of screws wereevaluated by Scanning electron microscope (SEM). Energy-dispersive X-rayspectroscopy (EDS) on the SEM was used to analyse the elements of thecoating layer. Then the coating was analysed by X-ray diffraction (XRD)with Cu Kα radiation at40kV and25mA with the2θ values increasing from10°to70°at a rate of0.05°s-1.Also,the fourier transform infrared (FTIR)technique was used to characterise the coating layer with a wave numberrange of400cm-1to4000cm-1. Twenty-four healthy and mature dogs wereselected and divided randomly into two groups: the bioactive bone screwexperimental group and the metallic screw control group. In each group anintercondylar fracture model of femur was established and the fracture wasfixed with bioactive screws and metallic screws separately. Grossexamination, histological staining, CT examination，biomechanical test，blood routine and serum biochemical index were performed4,8,12,24weeks after operation. Histological staining of the liver, kidney and spleenwas conducted24weeks after operation.ResultsSEM images showed that there was no apparent interval or phaseseparation between the bulk matrix and n-HA coating layer, and the coatingwas distributed evenly around the screw. The coating particles werenanosized and had a plate-like or leaf-like shape. The EDS graph showedthat the main elements in the coating layer were Ca and P, indicating that the coating layer was n-HA.The XRD test indicated that peaks at2θ=13.26°,31.82°,32.90°,34.25°,38.38°,46.70°,49.52°and53.15°belong to then-HA crystal diffraction peaks, and the two peaks at2θ=20.38°and23.49°are attributed to the phase of the PA66matrix. The wide and strongcharacteristic adsorption peak at3442cm-1represents the-OH vibration.The band at approximately960cm-1represents the symmetrical vibration ofPO43-, while the peaks at1102cm-1and1037cm-1correspond to theasymmetrical vibrations of PO43-. The absorption peaks at605cm-1and565cm-1, which indicate the bending vibration of PO43-, can also be observed.The two kinds of screws could both fix the fracture effectively. The activityof dogs was normal and the cut healed well in both groups after operation.CT examination showed the fracture got osteal healing in all the experimentanimals12weeks after operation. Histological investigation showedn-HA/PA66/GF screws formed tight bonding with the surrounding new bonytissue, which calcified gradually into mature bony tissue. However, therewas an obvious space between the metallic screw and bony tissue and a layerof fibrous tissue proliferation and encapsulation occurred on the interfacebetween bone and metallic screw．After12weeks，representative micro-CTimages showed that cortical bone became continuous with both types ofscrews fixation. The sagittal scanning images showed that the newly formedbone trabecula grew closely around the bioactive screws. The bioactivescrews were tightly integrated with the host bone. However, in the controlgroup, the image of bone trabecula around the metallic screws was vaguedue to the metal artefacts.The mechanic test demonstrated the maximumpush-out load showed no statistically significant difference (p>0.05)between two groups4、8、12weeks after operation,however, it showedstatistically significant difference (p﹤0.05) after24weeks. The maximum push-out load of bioactive screws was larger than that of metallic screws.The blood routine and serum biochemical index were both normal except thelevel of alkaline phosphatase increased [(58.8±14.49)]U/L at24weeks afteroperation. Histological staining of the liver, kidney and spleen was normal at24weeks after operation.ConclusionAs shown by their good biocompatibility, excellent biomechanicalstrength and fast formation and ingrowth of new bone, n-HA/PA66/GFscrews are thus suitable for orthopaedic clinical applications.Part3IN VIVO STUDY ON EFFECTS OFPOLYURETHANE/NANO-HYDROXYAPATITE/POLYAMID66REPAIRINGDEFECTS OF FEMORAL CONDYLE IN DOGObjectiveTo evaluate the effect of polyurethane, nano-hydroxyapatite andPolyamid66(PU/n-HA/PA66) on repairing defects of femoral condyle indog.MethodsThe PU/n-HA/PA66biomaterial was prepared and machined to formthe shape of femoral condyle according to the actual measured value ofdogs. Material surface was observed with scanning electron microscope andthe porosity was measured. Sixteen dogs were selected and divided randomly into two groups: the biological femoral condyle experimentalgroup and the autologous femoral condyle control group. In each grouplateral femoral condyle was cut off and the defects were repaired withbiological femoral condyle and autologous femoral condyle separately.Gross examination, histological staining, collagenⅠimmunohistochemicalstaining，CT examination, blood routine and serum biochemical index wereperformed4,8,12,24weeks after operation. Histological staining of theliver, kidney and spleen was conducted at24weeks after operation.ResultsThe porosity of the composite was80.89%±5.01%and main pore sizereached from300μm to800μm.The size of interconnection pores on the holewall ranged from100μm to300μm. The activity of dogs was normal and thecut healed well in both groups after operation. CT examination disclosed thatPU/n-HA/PA66femoral condyles were closely bonded with autogenousmedial femoral condyles without degradation.The prosthesis matched wellwith tibia platform, articular surface of patella and medial femoral condyle.The medial and lateral knee joint gaps are symmetry witout obviousstenosis. Histological investigation and immunohistochemical stainingshowed trabecular bone in the pores of the biological femoral condylebecame more and more and calcified gradually into mature bony tissue. Thecollagen I expressed positively in the bone newly formed in the pores ofmaterials. Blood routine and serum biochemical index were both normalexcept the level of alkaline phosphatase increased [(62.67±24.04)U/L] afteroperation. Histological staining of the liver, kidney and spleen was normal at24weeks after operation. ConclusionPU/n-HA/PA66biological femoral condyle has good internal bonedefects repairing ability, reconstruction ability and biocompatibility, whichis suitable for orthopedic biomaterials. |
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