| Replacement of missing teeth with implants and implant-supported prostheses are currently routine procedures for the oral rehabilitation of partially or fully edentulous patients.Osseointegration is an important criterion to evaluate the success of implants in implant surgery.Successful osseointegration refers to the direct contact between the implant and bone tissue without any fibrous connective tissue attachment.Since the concept of osseointegration was proposed,bone-related researchs have mainly focused on two important issues:how to improve the process of osseointegration and how to maintain long-term stable osseointegration.The material characteristics of the implant,the osteogenic differentiation of stem cells and the microenvironment of the implant area were all important factors for good osseointegration to support the implants and increase their efficacy.Titanium and titanium alloy materials have become the first choice for dental implant restoration because of their excellent mechanical properties and good biocompatibility.However,with the wide application of metal implants in clinic,some disadvantages of metal implants have been exposed,and non-metallized prostheses have become the important direction of oral restoration.Zirconia is widely used in the field of dental restoration because of its excellent mechanical properties,good aesthetic effects and biocompatibility.However,zirconia is a biologically inert material which makes it difficult for it to form an effective bond with bone tissue.Surface modification is needed to improve its surface activity and form a good bone bonding effect.Based on the above,this research was based on previous study of the modified polymer materials,the purpose of this study was to modify the surface of zirconia materials by carbon and nitrogen plasma immersion implantation,and to construct high potential nitrogen-containing functional groups on the materials surface,which provided a suitable extracellular microenvironment for the osteogenic differentiation of cells,in order to improve the bone binding effect of zirconia.The research content of this topic was divided into the following three parts.Part Ⅰ:The isolation,culture and identification of rat bone marrow mesenchymal stem cellsMethods:1.4-week-old SD rats were killed with cervical vertebra dislocation,and BMSCs were isolated,cultured and passaged.The molecular phenotype of BMSCs was identified,and the ability of clone formation,cell cycle,apoptosis and cell proliferation were detected.2.The multi-directional differentiation ability of BMSCs was detected,including alkaline phosphatase staining,alizarin red staining and oil red O staining.Results:1.The isolated and cultured rat BMSCs had active growth,strong ability of clone formation and proliferation,exuberant cell growth and small number of apoptosis.Phenotypic identification showed that BMSCs had low expression of hematopoietic stem cell surface markers CD31,CD34 and CD45,and high expression of mesenchymal stem cell surface markers CD44,CD73,CD105,CD29 and CD90.2.After osteogenic induction,the number of cells increased and showed overlapping arrangement,alkaline phosphatase staining showed positive reaction,alizarin red staining showed red calcium nodules,adipocytes induced by adipogenesis accumulated lipids,lipid droplets formed,and oil red O staining showed red,indicating that the expanded BMSCs could differentiate into osteoblasts and adipocytes.Part Ⅱ:Preparation and Surface characteristics of zirconia samples modified by plasma immersion implantationMethods:1.Zirconia disk specimens with a diameter of 15mm and thickness of 1.8mm were prepared and polished,and the surface was required to be smooth and uniform.Plasma implantation and grouping of zirconia samples were completed according to different treatment parameters.The specimens were divided into four groups:untreated control group（Blank）;carbon plasma treatment group for 60 minutes（C60N0）,carbon plasma treatment for 60 minutes followed by nitrogen plasma treatment for 6 minutes（C60N6）,and carbon plasma treatment for 60minutes followed by nitrogen plasma treatment for 18 minutes（C60N18）.2.The surface morphology of zirconia specimens treated under different conditions was observed,the surface roughness and surface relative potential were measured,the crystal phase structure was analyzed by X-ray diffraction analysis,and the surface chemical composition and chemical construction was analyzed by X-ray photoelectron spectroscopy and the surface wettability of the specimen was measured by contact angle.Results:1.The results of SEM and AFM showed that there was no significant difference in the surface morphology of zirconia specimens in each group,although the Ra values of zirconia specimens in the plasma treated group were slightly changed,they all belonged to the category of smooth surface in the roughness classification,the surface roughness of the plasma treated zirconia specimens did not change obviously.However,the surface relative potential changed obviously.Compared with Blank,the surface potential of the plasma treated zirconia specimen was significantly higher,and the C60N6 surface relative potential was the highest.2.The results of XPS energy spectrum analysis showed that compared with the untreated group,C and N elements were implanted into the surface of zirconia ceramics treated by C and N2 plasma,the percentage content increased and the chemical structure of the material surfaces changed.X-ray diffractometer showed that there was no obvious monoclinic phase on the surface of the treated zirconia specimen,and the crystal phase structure did not change obviously.The results of contact angle measurement were 91.93°±1.5°（Blank）,86.62°±3.29°（C60N0）,92.87°±0.58°（C60N6）and 90.65°±1.70°（C60N18）respectively.There was no significant difference in surface wettability among the four groups.Part Ⅲ:Effect of plasma immersion implantation of zirconia on biological behavior of BMSCsMethods:1.BMSCs were inoculated on zirconia specimens with different treatment conditions,and the early adhesion and cytoskeleton morphology of cells were observed 24 hours later.2.BMSCs were co-cultured with specimens for 1,3,5 and 7 days,and the proliferation ability of cells in different zirconia samples was detected.3.BMSCs were co-cultured with specimens for 7 and 14 days,and alkaline phosphatase（ALP）staining and activity determination were performed.4.After osteogenic induction 21 days after BMSCs inoculation on the surface of zirconia specimens with different treatments,the ability of cell mineralization was detected.5.Real-time quantitative RT-PCR to detect the m RNA expression level of related osteogenic genes Runx2,Col I,OCN and OPN.Results:1.The attached cells on the surface of plasma-modified zirconia materials were closely arranged,and the number of attached cells increased significantly.After the initial adhesion,the cells spread out more and more pseudopodia protruded.The number of adherent cells on the surface of C60N6 was higher than that on Blank（P<0.05）and other groups by Image J software.The cells on the surface of C60N6 stretched most obviously,had more intercellular connections,and adhered closely to the surface of the materials.2.After co-culture for 1 day,there was no significant difference in cell activity among different samples.With the extension of co-culture time,the absorbance of cells on the surface of samples increased gradually.When co-cultured for 3 days,5 days and 7 days,the cell proliferation activity of materials modified by carbon and nitrogen plasma was significantly higher than that of Blank（P<0.05）,and the proliferative activity of C60N6 was the highest（P<0.05）.3.On the 7th day,compared with Blank,the cell membrane and cytoplasmic granules on the surface of the plasma treated group showed positive reaction,stained with blue-black granules and massive dark-stained granules.The positive reaction of cell staining was more obvious at14 d.Compared with the cells on the untreated zirconia specimens,the cells grown on the surface treated by C and N2 plasma had higher alkaline phosphatase activity（P<0.05）,and the difference was statistically significant.4.When co-cultured for 21 days,the results of alizarin red staining showed that on the surface of zirconia material treated by plasma,the growing cells overlapped and formed mineralized nodules containing a large amount of mineral salt deposition in the stroma,and alizarin red staining showed red.There was no obvious nodule formation in Blank.We measured the absorbance of the dissolution and precipitation of mineralized nodules.The results showed that the absorbance of mineralized nodules for plasma treated groups was higher than that of Blank,and the difference in C60N6 was more obvious.5.Results of PCR showed that the expression of osteogenesis-related genes on the surface of materials treated by C and N2plasma increased significantly,and the expression level in C60N6 group was the highest（P<0.05）.The expression levels of Runx2 and Col I were the highest at 7 d and decreased slightly at 14 d,while the expression levels of OCN and OPN increased slightly at 7 d and significantly increased at 14 d.Conclusion:1.In this study,BMSCs were successfully obtained from SD rats and the stem cell characteristics of BMSCs were analyzed,such as morphology and surface marker identification,proliferation ability,osteogenic differentiation ability,adipogenic differentiation ability and so on.It was confirmed that BMSCs with strong proliferation ability and good differentiation function were successfully obtained,which provided seed cells for the subsequent study of biological behavior of cells.2.In this study,When the zirconia materials were modified by carbon and nitrogen plasma implantation,the surface roughness and wettability of the material haven’t changed obviously,but new chemical structure were formed on the material surfaces,and the nitrogen-containing functional groups with high potential were constructed.The stability of zirconia would not be affected if the bulk phase structure of zirconia was not changed.The relative surface potential of C60N6 group was the highest among the four groups of zirconia materials treated differently,3.In the in vitro experiment,we observed the number and morphology of cell adhesion,cell proliferation,alkaline phosphatase activity,mineralization ability,and the expression of Runx2,Col I,OCN and OPN at m RNA level.The results showed that the zirconia materials with high surface potential constructed a suitable extracellular environment and promoted the adhesion,proliferation and osteogenic differentiation of BMSCs. |
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