| With the increase of the elderly population,tooth loss has become a common oral problem.Traditional removable denture restoration or fixed bridge restoration will bring discomfort to patients or damage adjacent teeth.In recent years,with the development of economic level and the progress of technology,dental implants have become the first choice to repair missing teeth.However,at present,most commercial implants are solid structures and have large elastic modulus.When the patient’s bone quality is poor,there is a risk that the implant will fall off due to bone integration failure.Therefore,in clinical practice,there is a high demand for promoting bone formation and accelerating bone integration,which can shorten the time from implantation to loading and increase the success rate of implantation.Additive manufacturing technology has attracted much attention because it can manufacture scaffolds with different structures,shapes and properties,and can produce implants with complex 3D hierarchical structure by designing models,which provides a new choice for preparing porous implants with low elastic modulus.At the same time,micro-arc oxidation technology can electrochemically treat the surfaces of stents and implants to form relatively stable coatings,thus enhancing their surface activity.In addition,low-energy laser irradiation is more and more widely used in promoting bone repair,wound healing and other fields because of its advantages of painless and convenient use.Therefore,in this study,3D printing technology was used to design and prepare porous scaffolds and implants with different pore sizes and porosities.The optimal pore size was selected through mechanical testing and in vitro experiments,and the pore size group was electrochemically modified by micro-arc oxidation.Combined with the application of low-energy Nd:YAG laser,the osseointegration effect and its influence on the osteogenic behavior of MC3T3-E1 cells were explored from various angles in vivo and in vitro.The specific research contents are as follows:Firstly,porous scaffolds and implants with different pore diameters and porosity were designed and prepared with diamond topological structure.The micro-morphology of porous scaffolds and implants was observed by naked eye and electron microscope,the elemental composition and content were analyzed by X-ray energy spectrometer,and the pore size and porosity were verified by Micro-CT.The results show that the scaffolds and implants in each group have regular structures and uniform pore sizes.3D printing has not changed their element properties,and the aperture of 3D printing is slightly smaller than the designed aperture,which may be related to the limitations of 3D printing technology.Furthermore,the mechanical properties of implants in each group were tested.The results showed that the elastic modulus and compressive strength of the 600μm group were the best match with natural bone tissue compared with the 550μm group and the 650μm group,and the occurrence of"stress shielding"could be avoided.Then,the adhesion state of cells on the porous scaffold was observed by scanning electron microscope and phalloidin stained cells skeleton.Finally,the in vitro biocompatibility of the porous scaffold was detected by living/dead cell staining and CCK-8 method.The results showed that MC3T3-E1 cells had good adhesion and extensibility on the 600μm scaffold.The results of living dead cell staining and CCK-8 showed that the 600μm scaffold had good biocompatibility and was suitable for in vivo experiments.Secondly,the surface of 600μm porous scaffolds and implants was modified by micro-arc oxidation technology.The surface micromorphology was observed by scanning electron microscope,the content and distribution of surface elements were detected by EDS,and the phase composition of the scaffolds before and after modification was analyzed by XRD.The surface roughness of the scaffolds before and after modification was detected by laser confocal scanning and the contact angle of the scaffolds before and after modification was measured by contact angle measuring instrument.The results show that after micro-arc oxidation treatment,the surface of the scaffold with a pore size of 600μm forms a micro-nano scale crater,and the film layer contains calcium and phosphorus bioactive elements.After MAO treatment,the surface roughness and hydrophilicity of porous scaffold increased obviously,which was helpful for the adhesion and proliferation of osteoblasts.Thirdly,Nd:YAG laser with different doses and energy densities were used to co-culture the modified porous scaffolds with MC3T3-E1 cells,and the best low-energy laser group that can promote cell proliferation was selected,and the 600μm porous titanium alloy scaffolds were treated with this energy density Nd:YAG laser combined with micro-arc oxidation.The adhesion of MC3T3-E1 cells was observed by scanning electron microscope and phalloidin stained cells skeleton,the biocompatibility was detected by living dead cell staining,and the effect of promoting bone mineralization was verified by alkaline phosphatase activity and alizarin red staining.The expression of osteogenesis-related genes was detected by RT-q PCR.The results show that Nd:YAG laser irradiation with energy density of11.7 J/cm~2 is most helpful to the adhesion and proliferation of MC3T3-E1 cells,and can promote the expression of genes related to osteogenic differentiation and the formation of minerals.Finally,MAO-treated implants were implanted into the femoral condyle of rabbits combined with Nd:YAG laser LLLT,and the osseointegration around the implants was observed by general observation and Micro-CT,HE staining of histomorphology,Masson staining and VG staining of hard tissue sections.The results show that the porous implant has no inflammatory stimulation to the surrounding tissues,and the implant is not loose,and the combined application of MAO-LA can promote the bone integration around the implant.In addition,the main organs were stained with HE to observe the toxic effect of porous implants on the whole body,and the results of HE staining of various organs showed no abnormality,which indicated that the biocompatibility in vivo was good.In a word,the application of low-energy Nd:YAG laser combined with micro-arc oxidation to 3D printed porous titanium alloy implants can promote bone healing and bone integration,which provides a new idea for the development of new porous implants,the surface modification of porous implants and the clinical application of low-energy Nd:YAG laser. |
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