| Objective: In this study,a biodegradable PPF/β-TCP scaffold was prepared by thermal crosslinking using wax mold 3D printing technology,and VA-PPF/PLGA microspheres were loaded on it.The cytotoxicity,biocompatibility,bacteriostatic and osteogenic properties of the microspheres and the composite scaffold were preliminarily evaluated.To explore a new therapeutic method for bone infection and bone defect.Methods: PPF with different molecular weight was prepared by two-step synthesis method,and VA-PPF/PLGA microspheres were prepared by emulsion solvent evaporation method.The optimal preparation process of the microspheres was screened and characterized by central point design-response surface method.The in vitro drug release behavior of microspheres was evaluated by dialysis bag method,and the cytotoxicity of microspheres was evaluated by CCK-8 method.The MIC and MBC of microspheres against S.aureus were determined by double dilution method.Porous scaffolds were formed by adding β-TCP to PPF after 3D printing wax mold.The cable extractor was used to remove the toxicity of scaffolds,and the VA-PPF/PLGA microspheres were prepared by chitosan coated electrostatic adsorption method.The degradation rate of scaffolds,water absorption rate and pH value of the materials during degradation were evaluated.The cytotoxicity of the scaffolds was evaluated by CCK-8method,and the biocompatibility of MC3T3-E1 cells cultured on the surface of the scaffolds was determined by scanning electron microscope morphologies at different times.After co-culture of Staphylococcus aureus and drug-containing scaffolds in sterile AGAR plates,the antimicrobial effect of drug-loaded scaffolds was evaluated according to the diameter of bacteriostatic zone.Osteocalcin(OCN)and collagen type I(COL1)were determined to evaluate the osteogenic properties of the drug loaded materials.Results: The optimal prescription of microspheres was screened by central design-response surface method: PPF:PLGA mass ratio =2.41;PPF/PLGA: Drug mass ratio =3.56;The concentration of in PPF/PLGA was 129.73mg/ml,and the mean encapsulation rate was 83.38%.Compared with the predicted value,the deviation was0.63%.The average measured drug load was 18.19%,and the deviation was 0.55%compared with the predicted value.The microsphere is smooth and round,and the uniformity is good.The mean particle size was 103.902μm and the ζ potential was-21.5m V.The in vitro drug release of the microspheres prepared by the optimal prescription showed obvious sudden release on the first day,and then continued to release slowly from the microspheres,reaching(22.90±0.55)% after 3 days,(43.57±1.02)% after 28 days,and(97.89±1.39)% after 42 days.The RGR value of microspheres was 86.79%,and the CTS grade was 1.The calculated shrinkage rates of spherical diameter of OP and QD scaffolds were 8.07% and 0.85%,respectively,19.87% and 2.6% of bore diameter,and19.63% and 4.5% of rod diameter,respectively.The printing accuracy of ball diameter,pore diameter and rod diameter of the two structures is more than 90%,and the actual porosity of OP and QD structures are 55.3% and 55.58%,respectively.Mold optimization design to determine the final design of the size of the bracket aperture 0.75 mm,6.5mm ball diameter,rod diameter 0.6mm.During the degradation process,the pH value was close to 7.4,and the water absorption rate in PBS was(14.50±0.52)% at the first week of degradation,then slowly increased to(19.35±0.29)% at the 10 th week,and then remained stable at about 19%,and was(19.35±0.11)% at the 20 th week.The weight loss rate was(7.68±0.69)% in the first week,(20.28±0.59)% in the 14 th week,and then the degradation rate slowed down,and the weight loss rate was(22.25±0.78)% in the 20 th week.The mass loss was(7.82±2.75)% at 2 days,(25.55±0.91)% at 7 days,and(41.39±1.84)% at 30 days.When the maximum compressive strength of the stent reaches about 5.82 Mpa,when the pressure reaches 12.42 Mpa,the stent breaks.Scanning electron microscopy showed that MC3T3-E1 adhered to the scaffold surface and grew well.The drug loading of OP and QD were similar,which were(10.1±0.316)% and(10.5±0.290)%,respectively.The cytotoxicity of scaffolds was determined as grade 1.The inhibition zone experiment showed that the VA release curve of QD scaffold was slower than that of OP structure,and it still showed antibacterial effect at 28 days.The OCN level and COL1 concentration of MC3T3-E1 cells cultured in osteogenic α-MEM increased 7-fold and 10-fold and 7-fold and 11-fold,respectively,at week 2 and week 3,respectively,compared with that at week 1.The number of calcium nodules in the scaffold group was significantly higher than that in the control group after 4 weeks of culture with mouse osteogenic induction medium.Conclusion: In conclusion,the optimized preparation process of microspheres by central point design-effector surface method has good predictability,and the prepared microspheres have good in vitro sustained release characteristics and biocompatibility.The 3D-printed PPF/β-TCP scaffolds have low water absorption and slow degradation.The degradation process has little effect on pH,small cytotoxicity and good biocompatibility,and can promote osteogenesis.Drug-loaded scaffolds have good antibacterial and osteogenic properties in vitro. |
