Preparation Of Functionalized Porous Titanium Scaffolds With Nano-surface And Their Osteogenesis And Antibacterial Activity

BackgroundWith the development of our society,various means of transportation are widely used and people's travel is more convenient.However,accidents caused by various powered vehicles are frequent and the number of casualties caused by them is increasing day by day.Among them,there are many patients with large segment bone defect and severe soft tissue pollution caused by accidents,and the resulting mixed wound infection can lead to prolonged infection and concurrent necrosis,etc.,which has brought great difficulties to the treatment of orthopedic surgeons,and has become a major difficulty and research hotspot in the field of orthopedic trauma treatment at present.At present,the treatment of segmental bone defect with severe contamination is mainly for early thorough debridement,removal of contaminated and necrotic soft tissue and bone segment,first,external fixation,and further treatment after local tissue condition is good and infection control.In the later treatment,the large bone defect is mainly filled with autologous,allograft or allograft?pig,cow,etc.?.But they are in the process of the clinical practical application is more or less defects,such as autogenous bone materials limited.Material extraction will be accompanied by trauma and pain in the material collection site.Allogeneic bone and xenogeneic bone may have more severe rejection due to the autoimmune reaction.Even if no rejection occurs,the possibility of being absorbed by the body after implantation in the body cannot be completely ruled out.In the clinical treatment of infectious bone defects,higher requirements have been put forward for bone defect repair materials,mainly including:?1?the surface is loaded with antibacterial factors,local continuous antibacterial,reducing the risk of postoperative infection;?2?proper pore size and sufficient porosity can facilitate the growth of surrounding bone tissue;?3?it has compressive strength close to that of surrounding bone tissue,so as to avoid poor bone healing caused by stress shielding;?4?the surface of the material was modified to promote osteoblast differentiation and adhesion.Methods1.The porous titanium scaffold was prepared by the pore-forming method,The mechanical properties of porous titanium scaffolds prepared with different proportions of titanium slurry?concentration gradient of 30%,50%,70%?were tested.SEM scanning was used to observe the structure of porous titanium with different titanium paste concentrations.2.The surface of porous titanium was modified by anodic oxidation,then copper-doped hydroxyapatite microspheres were synthesized by hydrothermal method.They were loaded on different porous titanium supports and divided into three groups:?1 Porous titanium?PTi?,?2 Porous titanium loaded copper-doped hydroxyapatite microspheres?PTi-HA-Cu?,?3 Anodized porous titanium loaded copper-doped hydroxyapatite microspheres?APTi-HA-Cu?.3.SEM scanning was used to observe the changes of surface morphology of porous titanium scaffolds before and after anodic oxidation,and the microstructure of porous titanium scaffolds loaded with copper hydroxyapatite before and after oxidation was observed and compared.4.In the simulated humoral environment?PBS?,the release concentrations of calcium and copper ions on the surface of loaded copper-doped hydroxyapatite microspheres were detected?electroinductively coupled plasma mass spectrometer?.5.BMSCs cells were cultured on porous titanium scaffolds,and the proliferation of BMSCs on the surface of porous titanium scaffolds was detected by enzyme-marker,and the morphology of nuclei and skeletons of BMSCs on the surface of porous titanium scaffolds was observed by immunofluorescence microscopy.6.BMSCs cells were cultured on porous titanium scaffolds for 7 days and 14 days,and the expression of osteogenic genes on the scaffold surfaces was determined by real-time PCR quantitative analysis.7.Porous titanium scaffolds were inserted into 1ml staphylococcus aureus and 1ml escherichia coli?1×10⁶CFU/ml?,respectively,and the antibacterial rates of the scaffolds against the two bacteria were measured 24 hours later.ResultsThrough the above experimental research,we obtained the following research results:1.Through mechanical property test and SEM observation on porous titanium scaffolds,it is found that the three-dimensional porous titanium scaffolds prepared with50%titanium slurry have relatively good pore structure and permeability.At the same time,the pore size is also suitable for the growth of osteoblasts,which will eventually make the porous titanium scaffold more closely combine with bone tissue,which is expected to be used in clinical applications.2.After anodic oxidation,porous titanium loaded with copper-doped hydroxyapatite microspheres was characterized by SEM,EDS,XRD and other physical and chemical characteristics,indicating that a layer of TiO₂ nanotubes was generated on the surface of three-dimensional porous titanium support,which improved its hydrophilicity and histocompatibility.At the same time,the presence of calcium and copper elements was detected on its surface,which also indicated that copper-doped hydroxyapatite microspheres were successfully loaded onto the scaffold.3.In vitro ion release experiments of two porous titanium scaffolds loaded with copper-doped hydroxyapatite?PTi-HA-Cu?APTi-HA-Cu?showed that the titanium scaffolds after anodic oxidation had higher release of copper ions and calcium ions,mainly because more copper-doped hydroxyapatite microspheres could be loaded after anodic oxidation.4.After immunofluorescence staining and real-time fluorescence quantitative PCR analysis of BMSCs,we learned that the anodized porous titanium,loaded with copper-doped hydroxyapatite,had no significant cytotoxicity to BMSCs,had no significant effect on the proliferation of MSCs,and contributed to the differentiation of BMSCs into osteoblasts.5.The results of bacterial experiments showed that the porous titanium scaffold loaded with copper-doped hydroxyapatite microspheres after anodic oxidation had a certain antibacterial ability against gram-negative bacteria and gram-positive bacteria.Conclusion1.The APTi-HA-Cu porous titanium scaffold has good structural connectivity,suitable pore size,the compressive strength is comparable to that of human cancellous bone,which is also helpful for bone tissue growth.Therefore,it has certain clinical use value.2.In vitro cell experiments,APTi-HA-Cu porous titanium scaffold showed no significant cytotoxicity to BMSCs,the nanometer surface morphology can promote the osteogenic differentiation of BMSCs.3?In vitro bacterial experiments,APTi-HA-Cu porous titanium scaffold had certain inhibitory effects on staphylococcus aureus and escherichia coli within 24 hours.Thus,the scaffold can reduce bacterial infection and promote the repair of bone defects during bone tissue repair.