The Nanoscale Geometry Of TiO2 Nanotubes Promote Osteogenic Differentiation Of JMMSCs Via KAT2A

BackgroundThe performance of biomedical materials strongly depends on the first interactions occurring when the material’s surface comes into contact with a biological environment.At the same time,cell-nanotopography interaction is convinced to represent a promising management to precisely control function of seed cell and differentiation in bone tissue engineering,because bone itself has a structural hierarchy at the first level in the nanometer range,the research of application between seed cells and nanoscale materials is popular.In the process of dental clinical treatment,implant materials are one of the most common biological materials,which are frequently used for missing teeth and bone defect repair,academics and companies also keep exploring and improving the technology of surface treatment about implants.It has been known that epigenetics and microenvironments are closely linked to which is deserved to discuss in the study of implants.Yeling and other studies showed that the histone demethylases KDM4 B and KDM6 B play critical roles in osteogenic commitment of MSCs in 2012.Yong-sheng zhou observed that TiO₂ nanotubes promoted the osteogenic differentiation of hASCs by upregulating methylation level of histone H3 atlysine 4（H3K4）in the promoter regions of osteogenic genes Runx2 and osteocalcin,by inhibiting demethylase retinoblastoma binding protein 2（RBP2）.However,the role of acetyltransferase in the process of interaction between the TiO₂ nanotubes and bone marrow mesenchymal stem cells.Therefore,understanding epigenetic enzymes in the interaction between cells and materials can offer us a new insight into the repairing materials in our clinical application and a new train of treatment options for patients.PurposeThe effects of epigenetic enzymes on the development of osteogenic differentiation in the JMMSCs were observed in vitro.It is important to explore whether epigenetic genetics plays an important role in the biological performance of implant surface and the mechanism of interaction between materials and cells.It is also necessary to provide theoretical support for the technology of electrochemical processing on the surface of implant,improve a new idea for improving the ability of bone bonding,and give a basic support to epigenetic enzymes in the process of implant combined with bone restoration.Methods1.JMMSCs separation,purification,culture and identificationJMMSCs were isolated from orthognathic surgical sites and cultured by limited dilution assay for single cell clone.Flow cytometry instrument to detect cell surface markers;Thiazole blue（methylthiazolyl tetrazolium,determined by MTT）to detect cell proliferation performance;we determine their properties of stem cells through investigating multi-directional differentiation capacity such as osteogenesis and adipogenesis.2.The nanoscale morphology of TiO₂ nanotubes influences the osteogenic differentiation of JMMSCsTiO₂ nanotubes arrays with well-regulatedly different morphologies could be produced in organic electrolytes by anodic oxidation process.Furthermore,TiO₂ nanotubes were characterized by scanning electron microscopy（SEM）so that we can make sure wheher nanotubes are manufactured;cell morphology was observed under electron microscope nanotubes;the ability of cell proliferation on the TiO₂ nanotubes was detected by cell count kit 8,comparing ability of osteogenetic differentiation of JMMSCs on the TiO₂ nanotubes by alizarin red staining after osteogenesis induced.3.The key role of KAT2A in the program of nanotubes produing JMMSCs osteogenic differentiationThe specific genes expression of epigenetic enzymes were screened by using the TiO₂ nanotubes cultured with JMMSCs in vitro at different times,then measuring influences of osteogenic genes in 14 d afer interference with small RNA.During the same period,JMMSCs on TiO₂ nanotubes will be induced by osteogenic differentiation with 7d and 14 d,to study JMMSCs osteogenetic differentiation capacity through Alkaline phosphatase staining（ALP）after specific gene interference.4.Statistical analysisAll the data for the statistical analysis by SPSS 17.0 software system.Comparison between the two groups using two sample t test;Multiple sets of comparison between the single factor analysis of variance.Measurement data using mean + /-standard deviation,said with a double side（P < 0.05）for the difference is statistically significant.The experimental results1.All samples can get the original generation of JMMSCs;Long spindle cells under a microscope,Flow cytometry instrument testing resμlts show that JMMSC mesenchymal stem cell surface markers（CD105 and CD90）are all positive expression,and hematopoietic stem cell surface markers（CD34,CD14,CD45）are all negative expression;Determined by MTT experiment results show that proliferation ability of JMMSCs;A accompanied induction detection of visible fat droplets,osteogenesis induction detection to calcified nodul edeposits,show that JMMSCs has a multi-directional differentiation capacity.2.TiO₂ nanotubes morphology was successfully produced by observing through scanning electron microscopy（SEM）,which was fabricated under the conditon of anodic oxidationanodize with the featured electrolyte.The ability of proliferation for JMMSCs on TiO₂ nanotubes have a certain role in promoting osteogenesis measured by the CCK8 kit,results of alizarin red after induction of 28 d showed that the group of JMMSCs on TiO₂ nanotubes have a remarkable promoting effect on osteogenetic differentiation capacity.3.The gene expression of acetyltransferase was detected by qRT-PCR after the culture between Ti O2 nanotubes and JMMSCs at 3d,7d.With complete analysis,we conclude that KAT2A was particularly expressed in the family of acetyltransferas;after using small interference RNA,osteogenic genes present down-regulated expression;Alkaline phosphatase staining detection related results of histone acetylation illuminate that osteogenic differentiation ability of JMMSCs cultured with TiO₂ nanotubes manifestates down-regulation after the interference.The results also reveal that KAT2A is a promoter of the osteogenic differentiation of the JMMSCs in the interaction with TiO₂ nanotubes topography.Conclusion1.The acquisition of JMMSCs has the characteristics of stem cells in vitro culture,which can be applied in vitro.2.The nanoscale morphology of TiO₂ nanotubes influences the osteogenic differentiation of JMMSCs.3.The nanoscale geometry of TiO₂ nanotubes promote osteogenic differentiation of JMMSCs via KAT2A.