The Effect Of Psoralen Micro/nano Morphology Sustained Release System On Osseointegration In Osteoporotic Rats

Objective:In this study,psoralen micro/nano（MN-Pso-CHI）morphology sustained-release system was established by micro/nano processing of titanium implant surface,psoralen loading and chitosan multilayer coating.Then,the effect of MN-Pso-CHI morphology sustained release system on MC3T3-E1osteoblasts was evaluated in vitro by CCK-8 cytotoxicity assay,and the release behavior of psoralen in MN-Pso-CHI morphology sustained release system was studied in vitro by drug release assay to find the release regularity.Then the rat model of osteoporosis was constructed,and the possibility of psoralen in combination with micro-nano bionic morphology to promote early bone bonding and enhance implant stability was studied in vivo.Methods:After polishing,acid etching,anodic oxidation,vacuum-assisted physical adsorption and spin coating,the following five groups of surface morphologies were established:Smooth group（ST）morphology,psoralen loaded smooth group（ST-Pso）morphology,micro/nano group（MN）morphology,psoralen loaded micro/nano group（MN-Pso）morphology,chitosan coated psoralen loaded micro/nano group（MN-Pso-CHI）morphology.The surface morphology of the above five groups of titanium plates was observed by scanning electron microscope,and the surface contact angles of the above five groups of titanium plates were measured by contact Angle measuring instrument.The release of psoralen in vitro in the morphologies of ST-Pso,MN-Pso and MN-Pso-CHI groups was determined by UV8000-visible spectrophotometer,and the cumulative release amount and cumulative release percentage were calculated.MC3T3-E1 cells were inoculated on the surface of the above five morphologic titanium plates for 1,4 and 7 days to conduct CCK-8 cytotoxicity test.Thirty-six specific pathogen free（SPF）,3-month-old female SD rats were randomly divided into osteoporosis group（n=33）and sham operation group（n=3）.In the osteoporosis group,both ovaries were removed,and the same amount of fat tissue around ovaries was removed from sham operation group.After 3 months of feeding,3 rats in each group were randomly selected for micro-CT scanning and 3D reconstruction to confirm the successful establishment of osteoporosis model.Furthermore,the above five morphologic titanium implants were respectively implanted into the bilateral femoral epiphysis of 30 female SD osteoporosis model rats.After 28 days,samples were taken for toluidine blue staining,micro-CT scanning and 3D reconstruction.Results:（1）Surface morphology analysis results:Scanning electron microscopy showed that there were only a few scratches on the surface of ST titanium.The surface of MN titanium exhibits irregular stepped structure at 10.00k×microscope,and regular and uniform nanotube structure at 100.00k×microscope,with a diameter of about 78～80nm.The Ti O₂ nanotube structure on the titanium surface of MN-Pso group was partially blocked.In MN-Pso-CHI group,chitosan multilayer formed a uniform surface covering the titanium surface of the nanotube structure.The contact Angle of the morphologic surface of ST group is about 80.8±1.7°,ST-Pso group is about 87.5±3.2°,MN group is about 47.3±2.7°,MN-Pso group is about 73.5±2.0°.The contact Angle of MN-Pso-CHI formation surface is about 64.8±1.8°.（2）Psoralen loading amount and release rule de tection results:The cumulative release amount of ST-Pso,MN-Pso and MN-Pso-CHI groups were5.18,10.05 and 10.78μg,respectively.ST-Pso group had the lowest drug loading amount and release time was only 1 day.In MN-Pso group,the percentage of drug release in the first 24 hours was 89.33%,and the maximum release was 3 days.In the MN-Pso-CHI group,the percentage of drug release at the first 24 hours was56.68%,and the release time was 15 days.（3）CCK-8 cytotoxicity test results:All groups showed continuous proliferation within 7 days.MN-Pso-CHI group had the highest proliferation on day 7,which was related to the continuous relea se of psoralen.These data indicate that MN-Pso-CHI surface morphology can be used as biocompatible biomaterials.（4）In vivo animal experimental results:Toluidine blue staining and micro-CT 3D reconstruction showed that MN-Pso-CHI group formed more new bone and was more dense than the other four groups.Bone contact rate（BIC）,bone volume fraction（BV/TV）,bone trabecular thickness（Tb.Th）and bone trabecular number（TB.N）were ST<MN<MN-Pso-CHI（P<0.05）,and bone trabecular separation（Tb.Sp）was ST>MN>MN-Pso-CHI（P<0.05）.Animal experiments verified the possibility of MN-Pso-CHI morphology psoralen in combination with micro-nano morphology to promote early bone bonding and enhance implant stability.Conclusion:The micro/nano（MN-Pso-CHI）morphology sustained release system loaded with psoralen was successfully constructed.MN morphology has higher drug loading than ST morphology,and MN-Pso-CHI group has the best sustained release performance.CCK-8 cytotoxicity test showed that the surface morphology of MN-Pso-CHI had no cytotoxicity.Psoralen in MN-Pso-CHI group could promote early bone bonding and enhance the stability of implants in coordination with micro-nano morphology.